Importance of insulin resistance to vascular repair and regeneration

Free Radical Biology and Medicine 60 (2013) 246–263

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Free Radical Biology and Medicine journal homepage: www.elsevier.com/locate/freeradbiomed

Review Article

Importance of insulin resistance to vascular repair and regeneration Richard M. Cubbon *, Ben N. Mercer, Anshuman Sengupta, Mark T. Kearney Multidisciplinary Cardiovascular Research Centre, LIGHT Laboratories, The University of Leeds, Leeds LS2 9JT, UK

art ic l e i nf o

a b s t r a c t

Article history: Received 29 December 2012 Received in revised form 22 February 2013 Accepted 23 February 2013 Available online 4 March 2013

Metabolic insulin resistance is apparent across a spectrum of clinical disorders, including obesity and diabetes, and is characterized by an adverse clustering of cardiovascular risk factors related to abnormal cellular responses to insulin. These disorders are becoming increasingly prevalent and represent a major global public health concern because of their association with significant increases in atherosclerosis-related mortality. Endogenous repair mechanisms are thought to retard the development of vascular disease, and a growing evidence base supports the adverse impact of the insulin-resistant phenotype upon indices of vascular repair. Beyond the impact of systemic metabolic changes, emerging data from murine studies also provide support for abnormal insulin signaling at the level of vascular cells in retarding vascular repair. Interrelated pathophysiological factors, including reduced nitric oxide bioavailability, oxidative stress, altered growth factor activity, and abnormal intracellular signaling, are likely to act in conjunction to impede vascular repair while also driving vascular damage. Understanding of these processes is shaping novel therapeutic paradigms that aim to promote vascular repair and regeneration, either by recruiting endogenous mechanisms or by the administration of cell-based therapies. & 2013 Elsevier Inc. All rights reserved.

Keywords: Insulin resistance Diabetes mellitus Vascular repair Nitric oxide Reactive oxygen species Growth factor Free radicals

Contents Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247 Vascular repair and regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247 Progenitor nomenclature and function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248 Insulin resistance, vascular repair, and regeneration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248 The metabolic syndrome and adipokines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249 Dyslipidemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249 Hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249 Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249 Adipokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 Coagulation and fibrinolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 Hyperglycemia versus abnormal insulin signaling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 Global versus tissue-specific insulin resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 Unifying molecular mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252 Insulin receptor signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252 PI3K/Akt/eNOS signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253 Oxidative stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254 Hypoxia perception and VEGF activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254 Insulin receptor interactions and signaling cross talk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255 Therapeutic considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256 Pharmacological strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256 Gene-based strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256 Cell-based strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257 Murine cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257 Human cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257

* Corresponding author. Fax: þ 441133437738. E-mail address: [email protected] (R.M. Cubbon). 0891-5849/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.freeradbiomed.2013.02.028

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Safety concerns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257

Introduction

Vascular repair and regeneration

A broad spectrum of overlapping clinical disorders, including obesity, prediabetes, and diabetes mellitus (DM), is associated with impaired metabolic action of the principal glucose-lowering hormone insulin, referred to as “insulin resistance.” Diabetes is associated with a twofold increase in major cardiovascular events, such as myocardial infarction (MI) or stroke; these events occur approximately 15 years earlier than in patients without DM [1,2]. However, cardiovascular risk is also significantly elevated in the prolonged prediabetic phase, before the development of sustained hyperglycemia [3]. The International Diabetes Federation suggest that by 2030 the number of diabetes sufferers globally will rise to 552 million, an increase of 50% compared with current data; these figures rise to approximately 1 billion people if impaired glucose tolerance is also included [4]. In clinical practice we are aware that the prevalence of comorbid DM in patients suffering MI or heart failure is increasing, with contemporary observational studies quoting prevalence in excess of 25% [5,6]; these patients experience mortality double that of those without diabetes [7,8]. Furthermore, even after excluding those with known DM or hyperglycemia, the majority of patients sustaining MI exhibit DM or prediabetes, when comprehensively assessed [9]. Clearly, the impact of insulin resistance is likely to become increasingly important to the primary and secondary prevention of cardiovascular disease, and outcome data suggest current strategies are failing to adequately address this issue. The principal process underlying the cardiovascular risk associated with insulin resistance is atherosclerosis, a phenomenon of arterial wall inflammation and lipid accumulation, progressing toward unstable plaque formation with abrupt vascular occlusion [10]. A clustering of proatherosclerotic factors promoted by insulin resistance and obesity, known as the “metabolic syndrome,” is implicated in atherogenesis even before the onset of established diabetes (marked by sustained hyperglycemia) [11]. Dysfunction and damage to the arterial endothelium is thought to act as a key initiating event in atherogenesis, in part by reducing the bioavailability of the key antiatherosclerotic radical nitric oxide (NO) [10,12]. Indeed, impaired NO-dependent vasomotion is present in patients across the spectrum of insulin resistance [13–15]. Moreover, insulin induces NO generation in the vascular endothelium and glucose uptake in metabolic tissues via analogous signaling cascades, the activity of which is commonly impaired in insulin resistance, linking “endothelial dysfunction” and metabolic insulin resistance [16]. Although our understanding continues to evolve, it is apparent that endogenous vascular repair can mitigate vascular injury, thus retarding atherogenesis and promoting repair of injured tissue in the context of vascular occlusion [17,18]. Given the persistently poor cardiovascular outcomes of patients with insulin-resistant syndromes [8], promotion of cardiovascular repair may therefore represent an attractive therapeutic paradigm. However, it has also emerged that disease processes associated with vascular injury are also commonly linked with diminished indices of vascular repair [19]. This review will detail our current understanding of how insulin resistance adversely influences endogenous vascular repair and regeneration, in particular focusing on molecular mechanisms that may be amenable to novel pharmacologic or gene- or cellbased strategies.

Before discussing the impact of insulin-resistant syndromes upon vascular repair and regeneration, it is important to clarify the distinct, but overlapping, phenomena involved in the repair of injured conduit vessels and the regeneration of vasculature in injured tissue. In this review, vascular repair is predominantly used to denote the reendothelialization of established conduit vessels injured by pathologic processes, such as atherosclerosis, and therapeutic revascularization procedures. The precise mechanisms of vascular repair remain incompletely defined and clearly depend on the context of injury, though relying upon both the direct incorporation and the paracrine activity of multiple populations including local vascular cells, circulating leukocytes, and progenitor cells [20–22]. An emerging body of work suggests that endogenous bone marrow-derived cells do not directly incorporate as endothelial or smooth muscle cells within murine conduit vessel neointima to any meaningful extent [23–25]. However, this is disputed by other authors [18] and by no means discounts an important paracrine contribution of bone marrowderived cells in stimulating and orchestrating local vascular cellmediated repair. Furthermore, a number of reports suggest some contribution of bone marrow-derived cells to human small-vessel endothelium after sex-mismatched cardiac or bone marrow transplantation [26,27]; further work is required to resolve these disparities. The process of vascular regeneration is in many respects more complex, because a hierarchical branching vascular plexus must develop concurrent with the repair or replacement of a wider tissue parenchyma. The term angiogenesis is often incorrectly used as a synonym for vascular regeneration, when in fact it describes the more defined process of sprouting of neovessels from existing vasculature [28]. This is distinct from the process of vasculogenesis, in which neovessels form de novo from circulating stem/progenitor cells; agreement exists as to the role of this process in utero, although there remains no direct evidence of its role in adulthood. Finally, arteriogenesis refers to the maturation of neovessels into conduit vessels, resulting in luminal enlargement and vessel coverage with pericytes and vascular smooth muscle cells [29]. This process is important in achieving sufficient tissue perfusion and remains poorly understood, although endothelial shear sensing and recruitment of circulating leukocyte subsets have been implicated as contributing [30,31]. Importantly, vascular (re)generation is not a homogeneous process, invoking differing mechanisms and mediators according to the context of vessel formation. Angiogenesis is often distinguished as being developmental (occurring in utero) or pathological (in response to an insult to established tissue), although there remains mechanistic overlap between the two [28]; this review focuses on pathological angiogenesis, given its relevance to insulin-resistant diseases. The most profound stimulus for angiogenesis is hypoxia, which activates tissue oxygen-sensing cascades, subsequently altering expression of a host of angiogenesismodulating molecules, including increased transcription of the prototypical proangiogenic mediator vascular endothelial growth factor (VEGF) [32]. It is also clear that inflammation can also promote angiogenesis, though we now recognize there to be very close links between hypoxia and inflammation, such that angiogenesis in the context of inflammation may invoke hypoxic

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mechanisms, and vice versa [33]. The specific tissue context of vascular regeneration may also be important, with qualitative and quantitative differences in the mediators of neovascularization in common disease processes (for example cancer, obesity, and diabetic retinopathy) [34–36]. These insights emphasize an important role of injured tissue, and the ensuing inflammatory cell infiltrate, in signaling appropriately to vascular (and possibly progenitor) cells to stimulate effective vascular regeneration. Once vascular regeneration is initiated, orchestration of the process and maturation of the neovasculature to produce an appropriately branched hierarchy of vessels is also critical. Indeed, disturbance of critical regulatory molecules in vessel branching can result in hypervascular, but poorly perfused tissue, emphasizing the importance of neovascular quality in addition to quantity [28,35]. Exposure of quiescent phalanx endothelial cells, resident in established vessels, to VEGF results in the formation of endothelial sprouts, which comprise leading tip cells, migrating along the VEGF gradient, followed by a column of dividing stalk cells within which the capillary lumen forms. Endothelial cells dynamically switch between these roles as the neovasculature develops and matures, with Notch signaling being recognized as a key modulator of how individual cells interact within the population and so respond differently to their common proangiogenic milieu [37]. Appropriate neovessel coverage with pericytes and/or vascular smooth muscle cells is also important in deriving a stable quiescent neovasculature [28,35].

Progenitor nomenclature and function The potential of circulating cells to directly contribute to endothelial regeneration in adulthood, akin to developmental vasculogenesis, has been postulated for many decades. The more recent work of Asahara et al. [38] led to a resurgence of interest in the potential existence, role, and therapeutic exploitation of circulating vascular progenitor cells. They described “putative progenitor endothelial cells,” derived by magnetic separation of human blood mononuclear cells expressing the hematopoietic progenitor marker CD34, which could express an endothelial-like molecular and functional phenotype in vitro. Moreover, infusion of CD34 þ cells (or those expressing the VEGF receptor-2, KDR) could augment angiogenesis after hindlimb ischemia, with infused cells appearing to form capillary-like structures expressing CD31 6 weeks later. Other authors have subsequently extrapolated these findings to suggest that circulating cells coexpressing CD34 and KDR represent “endothelial progenitor cells” capable of vascular repair and regeneration [39]. However, it is acknowledged that a subset of mature endothelial cells coexpress CD34 and KDR, suggesting circulating CD34 þ /KDR þ cells may simply represent sloughed endothelium. In recognition that mature endothelial cells do not express an additional hematopoietic marker CD133, circulating CD133 þ /CD34 þ /KDR þ cells were proposed to represent a more defined endothelial progenitor/precursor population [40]. Although the abundance of such populations has been shown to independently predict future major cardiovascular events [41], we remain unclear as to the fate or role of such cells in vivo. Indeed, extrapolation of the original conclusions of Asahara et al. and imprecise use of nomenclature have in many respects led to confusion and suspicion regarding the relevance of such cells to (patho)physiology; hence, we refer to such cells as “circulating progenitor cells” or CPC. An alternative means of exploring the potential role of circulating cells in vascular repair involves cell-culture-based manipulation of circulating peripheral blood mononuclear cells (PBMCs) to derive endothelial progenitor cells (EPCs) [42]. Exposure of PBMCs to standard endothelial growth medium over a period of 4–7 days

results in the formation of spindle-shaped cells, akin to those produced by Asahara et al., expressing endothelial surface markers including CD31 and KDR. These “early outgrowth EPCs” (EEPCs) are now recognized to bear myelomonocytic lineage and retain many of the molecular and functional properties of such cells; their possession of some endothelial surface markers is in part due to their ingestion of platelet-derived microparticles in vitro [43]. They exhibit very limited proliferative capacity and it is apparent that they do not directly incorporate into vascular structures when infused in vivo. However, abundant evidence supports their capacity to augment angiogenesis and vascular repair in a paracrine fashion [44]. With modification of cell culture medium and discarding of initially adherent PBMCs, a second subset of early outgrowth EPCs is derived [45]. These “colony-forming units” (or EPC-CFUs) form tight clusters of modified T lymphocytes surrounded by spindle-shaped monocytic cells migrating toward the core [46]. Again, these promote in vitro angiogenesis via paracrine mechanisms, but cannot contribute directly to neovessel endothelium [42]; their abundance has been correlated with Framingham cardiovascular risk score and endothelium-dependent vasomotor function [45]. When culture of PBMCs in endothelial growth medium is sustained over weeks, colonies of “late outgrowth EPCs” (LEPCs) appear. These exhibit the morphologic and molecular characteristics of endothelial cells, a proliferative hierarchy typical of progenitor populations, and can be passaged serially over many population doublings [42]. Importantly, these cells are also able to directly form capillary-like structures in vitro and directly contribute to vascular repair and regeneration in vivo [44]. Hence, this population possesses the characteristics that one might expect an endothelial progenitor to exhibit, and also offer an autologous progenitor population capable of significant ex vivo expansion, which may aid therapeutic translation. However, we remain unclear as to the in vivo existence of any EPC subtype given the current absence of surface markers identified to be specific for such cells. Equally, the relationship between CPCs and EPCs also remains incompletely understood; single-cell plating experiments have suggested that LEPC colonies are derived from cells in the CD34 þ fraction of PBMCs that do not express the panleukocyte marker CD45, nor CD133 [47,48]. However, the circulating CD34 þ / CD45− population remains a heterogeneous mix of mature endothelial cells and LEPCs when studied in cell culture, and even complex polychromatic flow cytometry definitions cannot discriminate between these populations [49]. Hence, all studies of CPCs and EPCs have limitations, which must be borne in mind when interpreting data linking disease states with altered vascular repair and regeneration.

Insulin resistance, vascular repair, and regeneration Insulin resistance is present in a heterogeneous spectrum of disorders, all of which have been linked with altered indices of vascular regeneration. For example, diabetes is associated with impaired coronary reendothelialization after percutaneous intervention and impaired angiogenesis in chronic wounds [50,51]. Equally, prediabetes has been linked with diminished conduit artery repair, and obesity with reduced angiogenesis in the setting of hindlimb ischemia [52,53]. Abundant data also link the spectrum of insulin resistance with reduced CPC numbers and diminished EPC function, both in vitro and when transplanted in the setting of ongoing vascular repair or regeneration [52–57]. However, the heterogeneity of the insulin-resistant phenotype makes it difficult to dissect the contributory roles of its distinct facets (for example, hyperglycemia versus altered vascular insulin signaling), which is important in developing effective therapeutic

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approaches. Therefore, the following discussion presents data on vascular repair and regeneration according to various categorizations of the insulin-resistance phenotype/continuum, which can provide important mechanistic insights. The metabolic syndrome and adipokines As outlined earlier, a clustering of proatherogenic factors is linked with insulin resistance and central obesity, with the term metabolic syndrome being used when a threshold of clustering is exceeded, varying according to the guidelines applied [58]. Each contributing factor has been linked with altered indices of vascular repair and regeneration and may to some extent mediate the association between the insulin-resistant phenotype and diminished vascular renewal. Dyslipidemia A typical pattern of dyslipidemia, with reduced HDL cholesterol, elevated triglycerides, and smaller LDL particle size (not a diagnostic criterion), is present in patients with the metabolic syndrome. Reduced CPC abundance and EPC number/function is associated with low HDL cholesterol [59–61], and the impaired vascular repair noted in HDL-deficient mice can be rescued with exogenous HDL administration [62]. Murine studies have also shown exogenous HDL to promote CPC mobilization and in vitro EPC function in a scavenger receptor B1- and NO-dependent fashion [59,60,63]. Moreover, nicotinic acid-mediated increases in HDL concentration have been linked with augmented angiogenesis and improved functional recovery in a rat model of stroke; augmented phosphatidylinositol 3-kinase (PI3K) and endothelial NO synthase (eNOS) were implicated [64]. Emerging evidence also indicates that cholesterol efflux, a process in which HDL cholesterol is critical, prevents excessive bone marrow stem/progenitor proliferation and modulates leukocyte differentiation, so retarding atherogenesis [65,66]; however, vascular renewal was not specifically addressed in these studies. Investigation of global liver X receptor (LXR) knockout mice indicates that reduced cholesterol efflux may promote angiogenesis, and vice versa, by modulating VEGF receptor-2 (VEGFR2) compartmentalization in lipid rafts [67]. Other investigators have also demonstrated LXR agonism to reduce excess vessel formation in diabetic retinopathy [68]. Hence, modulation of cholesterol efflux may be a legitimate target for future human trials of treatments aiming to alter vascular repair and regeneration. However, the relationship between HDL or cholesterol efflux and vascular renewal seems complex and dependent on the context of modulation [69]; recent atherosclerosis trials with hard clinical endpoints have also raised efficacy concerns [70,71]. The relationship between circulating triglycerides and vascular renewal remains minimally studied, the majority of work only indirectly addressing the issue. EEPC function in vitro is impaired after exposure to remnant lipoproteins from hypertriglyceridemic patients, perhaps via induction of senescence [72]; studies linking EPC-CFU abundance with plasma triglyceride concentration have reached conflicting conclusions [73,74]. The proangiogenic impact of human bone marrow-derived cells in the setting of murine hindlimb ischemia has also been inversely correlated with plasma triglyceride concentration, although no mechanistic studies were performed [75]. Circulating free fatty acids (i.e., no longer esterified to glycerol within triglyceride molecules) also appear to impair EEPC function in vitro via diminished Akt/eNOS signaling [76]. Although the relationship between LDL particle size and vascular renewal has not been studied to date, there are clear links to LDL concentration per se. A number of studies have found an inverse correlation between CPC abundance and LDL cholesterol, whereas

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lifestyle modification-induced reductions in LDL correlate inversely with change in EEPC number [77–79]. Hypercholesterolemic mice exhibit impaired arteriogenesis due to impaired monocyte/macrophage influx [80]. Furthermore, hypercholesterolemia is associated with impaired human monocyte migration to VEGF in vitro [81], a finding that may also be of relevance to EEPCs, given their lineage. Haddad et al. [82] showed a high-cholesterol diet to impair ischemic hindlimb angiogenesis and rescued this using global NADPH oxidase 2 (NOX2) knockout, which reduced oxidized phospholipid-mediated superoxide generation and increased NO bioavailability. Recent data also suggest that reduction of LDL promotes angiogenesis by reducing endothelial expression VEGF receptor 1 (which is thought to act as a decoy for VEGF, reducing VEGFR2 activation) [83]. Oxidation of LDL is noted in atherosclerotic plaque, and oxidized lipoproteins have been shown to induce EEPC apoptosis via impaired PI3K/Akt signal transduction [84]. Overall, it seems that increasing LDL cholesterol has a detrimental impact upon vascular renewal, although the effects are complex and may be difficult to separate from concurrent changes in cholesterol efflux. Hypertension Data linking CPC and EPC abundance with human hypertension are conflicting, although it is difficult to draw firm conclusions because of the differing methods applied, small sample sizes, and confounding of related medical therapy [77,85–87]. Principal mediators of hypertension, including angiotensin II, aldosterone, and endothelin, have all been linked with altered indices of vascular repair and/or regeneration in rodent models. Angiotensin II impairs EEPC function in vitro and retards arterial endothelialization in mice, with rescue of repair noted after transfusion of angiotensin receptor 1 knockout EEPCs (and to a lesser extent wild-type EEPCs); oxidative stress appeared to underlie these effects [88]. Similarly, aldosterone impairs EEPC function both in vitro and in vivo and also impairs angiogenesis in Matrigel plug assays; these findings appeared to be due to oxidative stress, but were blood pressure independent [89]. Studies of salt-sensitive rodents have also implicated endothelin 1-induced (NADPH oxidase-generated) oxidative stress in EEPC dysfunction in vitro and during in vivo hindlimb ischemia studies [90]. However, conflicting data exist, suggesting that aldosterone infusion augments murine hindlimb ischemia-induced angiogenesis in an angiotensin II and angiotensin II receptor 1-dependent manner [91]. More recent data have also suggested a proangiogenic role of angiotensin II, but only under hypoxic conditions and dependent upon the type 2 angiotensin II receptor, along with kinin B2 receptorinduced NO generation [92]. In conclusion, there seem to be complex links between insulin resistance, hypertension, and vascular renewal, which involve alterations in oxidative stress and NO bioavailability, though they remain incompletely defined. Inflammation The term inflammation is applied to describe a diverse spectrum of innate and adaptive immune responses to perceived pathogens; given the complexity and diversity of such responses, it is unsurprising that data relating inflammation to vascular renewal conflict. Available data suggest that acute global inflammatory responses invoked by immunization do not alter CPC abundance/mobilization, whereas illnesses associated with chronic inflammation tend to exhibit reduced CPC and EPC numbers [93,94]. Many studies have addressed the roles of specific inflammatory mediators, which although useful in dissecting potential mechanisms, cannot identify the net contribution of such mediators to altered vascular renewal. Both C-reactive protein and tumor necrosis factor α (TNF-α) have been shown to reduce EPC abundance and function in vitro [95–97]. Equally,

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nuclear factor κB (NF-κB), a transcription factor involved in the modulation of gene transcription by multiple inflammatory mediators, appears detrimental to EEPC function and impedes insulininduced Akt signaling in these cells [98]. Other data show that NFκB activation induces an antiangiogenic phenotype in endothelial cells, which is reversed by its antagonism [99], and that obese transgenic mice with endothelium-specific reduction in NF-κB activity have reduced endothelial senescence [100]. However, the clear role of inflammatory cells (e.g., monocytes) and molecules in vascular renewal and progenitor mobilization [101–104], along with the overlapping transcriptional profiles induced by some inflammatory mediators and VEGF in endothelial cells [105], suggests a more nuanced role of “inflammation” [106]. On balance, the available data suggest that although inflammatory responses are required for normal vascular repair and regeneration, their chronic low-grade activation can be detrimental.

Adipokines The term adipokine is used to refer to a spectrum of adipose tissue-derived cytokines and hormones, which seem to mediate many physiological processes (including vascular renewal) and are dysregulated in the setting of obesity and insulin resistance. Adiponectin is perhaps the most studied adipokine with respect to vascular renewal and is generally less abundant in insulinresistant syndromes. In vitro, this molecule has been shown to augment EEPC differentiation and reduce senescence [107,108]; in LEPCs it has also been shown to protect from the detrimental effects of hyperglycemia via increased Akt and eNOS activity [109]. Available data also suggest adiponectin can promote angiogenesis, both in vitro and in vivo, via Akt and eNOS signaling, which appeared to be mediated by AMP-activated protein kinase (AMPK) [110]. Moreover, a study of global adiponectin knockout mice has revealed that this molecule is important in preserving renal function (an important risk factor for cardiovascular events), potentially by reducing podocyte NOX4-related oxidative stress in an AMPK-dependent manner [111]. Leptin, which circulates in greater concentrations in obesity and insulin resistance, has been reported to have variable effects upon EEPCs and LEPCs, with possibly beneficial functional effects at lower doses, but detrimental effects at higher concentrations [112,113]. The leptin receptor is present on CPCs, and binding of leptin promotes CPC mobilization via induction of NOX2 NADPH oxidase-derived superoxide, which is associated with augmented angiogenesis in the setting of murine hindlimb ischemia [114]. Furthermore, leptin has been linked with augmented angiogenesis in vitro and in murine studies [115,116]. Conflicting data exist regarding a pharmacological analogue of leptin, which was suggested not to alter in vitro angiogenesis or surrogate circulating markers of angiogenesis in humans, although experimental differences may underlie this disagreement [117]. Visfatin, the abundance of which is increased in insulin-resistant states, has been shown to reduce the number and function of EEPCs, in association with increased expression of the proinflammatory transcription factor NF-κB [118]. It has also been shown to induce conduit vessel endothelial dysfunction, although repair was not assessed in this study [119]. Conversely, a number of studies have indicated that visfatin promotes in vitro angiogenesis, via increased endothelial VEGF expression [120,121], and in vivo angiogenesis, implicating PI3K and Notch1 as critical [122,123]. Resistin, which circulates in higher concentrations in obese states, has been linked with augmented in vitro angiogenesis [124]. However, other work has shown the molecule to promote atherogenesis via increased monocyte adhesion to the endothelium, although vascular repair per se was not addressed in this study [125]. Overall, available data suggest that the insulinresistant adipokine milieu may be anticipated to have a net

proangiogenic effect, although with detrimental effects upon EPCs and possibly vascular repair. Coagulation and fibrinolysis Insulin-resistant syndromes are associated with complex alterations in the coagulation and fibrinolysis systems [126], some of which could conceivably alter vascular renewal, although specific studies in insulin-resistant models are lacking. A consistent feature of the metabolic syndrome is elevation of the antifibrinolytic molecule plasminogen activator inhibitor-1 (PAI1) [127], which interferes with tissue plasminogen activator (tPA)induced plasmin generation. Recent work has shown that tPA is important in modulating the bone marrow progenitor mobilization and incorporation into ischemic tissues to promote angiogenesis [128]; the related urokinase-type plasminogen activator also seems essential for angiogenesis [129]. Hence, elevation of PAI1 in insulin resistance might be expected to be antiangiogenic; however, PAI1 knockout mice exhibit impaired tumor angiogenesis [130], suggesting complex links between the plasminogen system and vascular regeneration. Some data also support elevation of von Willebrand factor (vWF), which plays a number of roles in hemostasis, in insulin resistance [127]; available data suggest that vWF possesses antiangiogenic properties [131]. Conversely, other facets of altered coagulation in insulin-resistant diabetes, such as increased tissue factor, factor VII, and fibrinogen [132], might be expected to promote angiogenesis [133,134]. Platelets and EPCs may add a further layer of complexity to these issues by modulating the activity of many of the molecules outlined above [135,136]. Hence, there seem to be complex relationships between coagulation, fibrinolysis, and vascular renewal, although in many cases it seems that either deficiency or excess of factors modulated by insulin resistance could impair vascular regeneration. Hyperglycemia versus abnormal insulin signaling As the insulin-resistant phenotype advances, hyperglycemia becomes more marked and sustained. In vitro studies suggest that pathophysiologically relevant glucose concentrations reduce EPC abundance and function, independent of their osmotic effects, via mechanisms including impaired Akt activity, reduced NO bioavailability, and increased p38 mitogen-activated protein (MAP) kinase activity [57,96,137]. Chorioallantoic membrane angiogenesis is also disturbed in the setting of hyperglycemia, but not osmotic control, and this is associated with endothelial and mural cell apoptosis [138]. Hyperglycemia also disturbs the quality of neovessels, perhaps through imbalance of angiopoietins1/2 [139–142] and reduced PDGF-BB signaling [143,144], resulting in loss of pericyte and vascular smooth muscle coverage, with subsequent vasoregression; this has been implicated in early diabetic retinopathy [36]. Studies of patients across the spectrum of insulin sensitivity also suggest inverse correlation between CPC abundance and indices of glycemia [145]. Further insight into the role of hyperglycemia in vascular renewal is gained from studies of type 1 diabetes (T1DM), which is generally associated with less marked metabolic insulin resistance. Patients with T1DM exhibit reduced CPC abundance and impaired EEPC number and function [146,147], with similar findings noted in murine studies of streptozotocininduced T1DM [148]. Moreover, such murine models of T1DM indicate impaired angiogenesis in the setting of limb ischemia, which may be contributed to by oxidative stress, excess p38 MAP kinase signaling, and reduced proangiogenic paracrine signaling [148,149]. Impaired cutaneous wound healing and angiogenesis in these murine models have also been linked with oxidative stress and reduced NO generation [150,151]; studies of arteriogenesis also suggest that T1DM impairs this process [152]. Comparative

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studies of T1DM and T2DM are sparse and difficult to interpret because of confounding issues and the potential off-target effects of the means used to generate murine models of diabetes (e.g., comparing db/db genetically insulin-resistant diabetes with streptozotocin-induced insulin-deficient diabetes). Acknowledging these methodological difficulties, human studies indicate altered splicing of the prototypical proangiogenic molecule VEGF in retinal tissue, with increased VEGF-A in T1DM and increased VEGF-D in T2DM [153]; the implications of these data are unclear. Murine studies suggest insulin-resistant, compared with insulin-deficient, DM to more profoundly impair ischemia-induced angiogenesis and promote arterial injury-induced neointima formation, as a result of reduced NO bioavailability and altered Akt/MAP kinase signaling, respectively [154,155]. These data collectively support the importance of hyperglycemia to impaired vascular renewal in insulin resistance, but cannot define its net contribution; however, it is possible to conclude that insulin resistance without hyperglycemia is sufficient to impair vascular renewal. Global versus tissue-specific insulin resistance The preceding discussion has highlighted the importance of insulin resistance per se in impaired vascular repair and regeneration, but also emphasizes the complex nature of the insulinresistant phenotype. It is evident that the adverse metabolic profile associated with insulin resistance is likely to contribute to diminished vascular renewal, but the presence of insulin receptors in many nonmetabolic cells suggests that the role of insulin resistance in such tissues warrants attention. Our laboratory has addressed the role of global insulin resistance in vascular repair using mice haploinsufficient for the insulin receptor (IRKO), which are hypertensive, but exhibit normoglycemia and only mild metabolic insulin resistance [52]. These mice demonstrated impaired reendothelialization of the femoral artery after angioplasty wireinduced denudation, compared with wild-type (WT) littermates. This was associated with reductions in basal CPC abundance and blood-derived EEPC number/function, along with absent VEGFinduced CPC mobilization, possibly secondary to reduced bone marrow NO bioavailability. Importantly, vascular repair could be entirely rescued with infusion of WT c-kit þ progenitors, and partially rescued with infusion of IRKO c-kit þ progenitors, implicating deficits in CPC mobilization and function in the impaired vascular repair noted in IRKO. More recently, our laboratory has studied LEPCs derived from insulin-resistant South Asian men, compared with white European controls, finding diminished in vitro function of these cells, linked with reduced Akt and eNOS signaling. When transfused in murine models of vascular repair and regeneration, South Asian LEPCs exhibited marked blunting of reparative and proangiogenic function, which could be rescued with expression of constitutively active Akt [156]. Data from many murine models of cell-specific insulin resistance have now been published, and although very few specifically address vascular renewal, useful insights can be gained, which may guide future studies. Beyond classical metabolic tissues (adipose, skeletal muscle, and liver), the insulin receptor (IR) is expressed in vascular endothelium, vascular smooth muscle, leukocytes, and cardiac myocytes, among many other cell lineages [157]. Gene-targeting technologies have been used to show that endothelium-specific knockout of the insulin receptor, or the related insulin-like growth factor-1 receptor (IGF1R), reduces hypoxia-induced retinal neoangiogenesis, associated with reduced VEGF and eNOS expression [158]. It remains unclear if angiogenesis in response to other stimuli, and in other vascular beds, is influenced similarly; however, studies of identical mice have suggested that neither IR nor IGF1R is required to maintain the integrity of the endothelial blood–brain barrier [159]. Combined

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endothelium-specific knockdown of IR and IGF1R has also been shown to impair neoangiogenesis in wounds, although it had no impact upon basal (and hence developmental) dermal microvascular density or integrity [160]. Studies of mice with cardiac myocyte-specific insulin resistance show reduced (PI3K/Akt-dependent) myocardial VEGF expression, along with diminished basal and postischemic vascular density [161]. A similar phenotype has been noted in diabetic rats, with reductions in myocardial VEGF expression preceding a diminution of myocardial vascular density and subsequent development of dilated cardiomyopathy, which could be prevented with myocardial VEGF gene therapy [162]. Isolated smooth muscle cells and endothelial cells from insulin-resistant models also secrete less VEGF, and impaired PI3K/Akt may underlie this phenomenon [163,164]. Recent studies in mice with renal podocyte-specific insulin receptor knockout have also challenged the concept of diabetic nephropathy being a solely hyperglycemia-related complication. These normoglycemic mice exhibited marked albuminuria and glomerular injury, possibly related to impaired PI3Kand MAP kinase-dependent podocyte cytoskeletal remodeling [165]. These data highlight the importance of cellular insulin resistance within nonvascular tissue and many cell subtypes resident within vasculature in modulating vascular renewal via altered paracrine activity. No data exist to define the role of leukocyte-specific insulin resistance in modulating vascular renewal, although two studies have defined the role of leukocyte insulin resistance in atherogenesis, a process in which altered vascular repair may be relevant. First, transplantation of insulin receptor knockout bone marrow into atherosclerosis-prone mice results in the formation of more complex atherosclerotic plaques; endoplasmic reticulum stressinduced macrophage apoptosis was implicated, but was based on in vitro analyses only [166]. A second study suggested that myeloid-specific insulin resistance reduced atherosclerosis in ApoE mice and was associated with reduced macrophage expression of proinflammatory mediators [167]. Although these studies may seem to offer conflicting conclusions, important experimental differences exist, one of which is the use of bone marrow versus myeloid insulin-resistant models. This may have resulted in progenitor cell insulin resistance in the former study, which might contribute to diminished vascular repair, and so increased atherosclerosis, although this hypothesis requires formal assessment. Although not directly assessing tissue-specific insulin resistance, a number of studies from Waltenberger et al. [168–170] have shown that monocytes isolated from patients with type 2 diabetes exhibit diminished in vitro responses to molecules in angio- and arteriogenesis. Specifically, monocytes from patients with diabetes did not migrate to VEGF, but remained able to migrate toward an alternative stimulus (formylMetLeuPhe), despite intact VEGF receptor 1 phosphorylation in response to VEGF [168]. Further work revealed increased basal phosphorylation of Akt and MAP kinases in diabetic monocytes (which may mask any effect of VEGF signaling), possibly as a result of oxidative species-induced protein tyrosine phosphatase inhibition or receptor for advanced glycosylation end-product signaling [169]. However, diabetic monocyte signaling in response to other chemotactic stimuli that signal via similar cascades seems to be unaffected, highlighting our incomplete understanding of this issue [170]. Furthermore, these studies do not specifically address the phenomenon of insulin resistance and cannot exclude significant contributions from other facets of the insulin-resistant phenotype, such as hyperglycemia. Indeed, animal studies suggest that impaired shear-induced vasodilatation (a stimulus for generation of monocyte chemoattractants), impaired monocyte chemotaxis to VEGF, and reduced arteriogenesis are all noted in streptozotocininduced models of non-insulin-resistant diabetes [152].

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Unifying molecular mechanisms To therapeutically target the impaired vascular renewal associated with insulin resistance, it is important to define its underlying molecular basis. A number of common and interrelated themes have emerged in the preceding discussion, including dysregulation of the expression, activity, and subsequent signaling of molecules critical to vascular repair/regeneration; reduced NO bioavailability; and oxidative stress (Fig. 1). This section expands on these themes. Insulin receptor signaling The insulin receptor is a homodimer, each subunit comprising an α and a β chain; the α chains bind to extracellular insulin, resulting in conformational changes to the intracellular domains of the β subunits, facilitating their autophosphorylation at key tyrosine residues. These changes augment the activity of the β subunit’s tyrosine kinase domain and so phosphorylation of its substrates, such as insulin receptor substrates (IRS), Shc, and Gab1, initiating multiple downstream signaling cascades [157]. Two of the principal signaling cascades activated by the insulin receptor are (Fig. 2): • The PI3K/Akt pathway: binding of IRS to the activated IR facilitates a downstream phosphorylation cascade, first resulting in the activation of PI3K, which then generates phosphatidylinositol trisphosphate (PIP3). PIP3 promotes membrane localization of phosphoinositide-dependent kinase-1 (PDK1), which phosphorylates Akt at serine 473. Activation of Akt is also dependent upon phosphorylation at threonine 308, which is mediated by the mammalian target of rapamycin-2 complex (mTorC2) through less well defined mechanisms. In metabolic tissues, this cascade promotes translocation of GLUT4 to the cell membrane, effecting intracellular glucose disposal, whereas in vascular tissues an important downstream target is eNOS, which catalyzes NO production. • The MAP kinase pathway: Binding of Shc (or IRS) to the activated IR promotes a cascade of activation via GRB2, Sos,

and Ras, and eventually to ERK MAP kinase; such signaling is responsible for insulin’s mitogenic effects. Activation of the IR is also able to promote activation of other classes of MAP kinase, such as c-Jun N-terminal kinase (JNK; via atypical PI3K signaling, as part of a negative feedback mechanism) and p38 (via incompletely defined mechanisms) [171]. The term pathway-specific insulin resistance has been applied to the observation that insulin stimulation of tissues from insulinresistant humans and animals results in impaired PI3K/Akt activation, with unaffected MAP kinase activity, compared to control [16]. The phenomenon is noted both in the classical metabolic targets of insulin and in the vascular endothelium, resulting in the association of metabolic dysregulation with reduced vascular NO bioavailability and hence promotion of atherogenesis. This important paradigm therefore provides a common molecular focus in developing therapeutics to improve the metabolic and vascular abnormalities noted in patients with T2DM and prediabetes. Common themes linked with the development of pathwayspecific insulin resistance are inflammation and oxidative stress, which are driven to some extent by the hyperglycemia and excess circulating free fatty acids that characterize diabetes and prediabetes [16]. However, the precise molecular mechanisms of impaired PI3K/Akt signal transduction in these circumstances remain incompletely understood. Inhibitory phosphorylation of serine residues within IRS1 by a host of protein kinases (e.g., JNK, IKKβ, and NF-κB, activated by inflammatory stimuli, lipotoxicity, hyperglycemia, and oxidative stress, among others) diminishes insulin-mediated PI3K activation, whereas MAP kinase signaling is broadly stable [172–180]. Direct inhibitory phosphorylation of IRS2, and PI3K itself, may also be relevant to pathway-specific insulin resistance [164]. Importantly, mice with global knockout of either IRS1 or IRS2 exhibit increased arterial neointima formation in response to cuff-induced injury, suggesting a role for these molecules in vascular repair [181]; metabolic derangements were also apparent in these studies, confounding mechanistic inference. It is notable that other data, although clearly demonstrating impaired insulin-stimulated Akt phosphorylation with preserved

Fig. 1. Mechanisms of impaired vascular renewal associated with insulin resistance. The schema shows how components of the insulin-resistant phenotype (blue) alter important cellular signaling processes (green), resulting in dysfunction and poor coordination of cells critical to effecting vascular repair and regeneration.

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Fig. 2. Insulin signaling cascade. Simplified schema demonstrating the principal signaling cascades downstream of the ligand-activated insulin (and IGF1) receptor. Insulinresistant syndromes are associated with impaired signal transduction via PI3K/Akt to their downstream targets, which mediate critical functions in vascular and metabolic tissues.

ERK phosphorylation (i.e., pathway-specific insulin resistance) in obese mice, suggest that blunted eNOS phosphorylation is more marked than would be expected from impaired upstream activity [182]. These data suggest that facets of the insulin-resistant phenotype, including free fatty acid toxicity, also directly impair eNOS activation in insulin resistance, highlighting the complex multilevel impact upon vascular signal transduction in insulin resistance. Although the potential impact of pathway-specific insulin resistance has received little attention in the context of vascular renewal, some data have recently emerged, and circumstantial insight can also be gained from the wider literature. Desouza et al. [98] have demonstrated impaired insulin-induced Akt phosphorylation and increased apoptosis in EEPCs derived from Zucker fatty insulin-resistant rats, versus Sprague–Dawley controls. Furthermore, they showed that control EEPCs exposed to the inflammatory cytokine TNF-α exhibited impaired insulin-stimulated Akt phosphorylation, which could be abrogated by pharmacological or genetic reduction in NF-κB activity. NF-κB knockdown also improved insulin signaling and reduced apoptosis in insulinresistant EEPCs and improved the capacity of these EEPCs to reduce neointimal thickening when infused after carotid angioplasty. We have also shown in vitro and in vivo dysfunction of EEPCs and CPCs from mice with global haploinsufficiency for the insulin receptor [52], although we did not define whether such cells exhibit pathway-specific reduction of insulin stimulated PI3K/Akt activity. Although the above data inform us regarding the impact of altered insulin signaling in the context of autologous cell-based vascular renewal therapies, the impact of pathway-specific insulin resistance upon endogenous repair and regeneration is less clear. Studies of obese mice (which would be expected to exhibit dysregulated vascular insulin signaling) suggest impaired perfusion recovery and muscle angiogenesis after induction of hindlimb ischemia [53,183]. Other data from Wang et al. [184] suggest that high-fat diet-induced obesity and insulin resistance are associated with vascular senescence and impaired hindlimb ischemiainduced angiogenesis; these findings were noted in association

with increased nonfasting vascular Akt phosphorylation. In further studies, they noted that chronic constitutive activation of Akt within the endothelium could also induce senescence and impair perfusion recovery after induction of limb ischemia. The impaired vascular regeneration noted in high-fat-fed wild-type mice was finally rescued in experiments administering the mTOR inhibitor, rapamycin, which also reduced vascular Akt phosphorylation. Importantly, rapamycin has many non-Akt-related effects, and so its benefit cannot prove obesity-induced endothelial Akt activation as causal in reduced vascular renewal. However, other data support the detrimental effects of either chronic hyperactivation [185,186], or genetic loss [187,188], of endothelial Akt upon vascular function and regeneration, suggesting that a fine balance of Akt activation seems necessary for endogenous vascular integrity. It is also important to note that a relevant impact of pathwayspecific insulin resistance upon vascular renewal is not incompatible with the data of Wang et al.

PI3K/Akt/eNOS signaling Although there is general agreement that insulin-stimulated PI3K/Akt/eNOS signaling is impaired in insulin resistance (and some data suggest impaired basal vascular signaling in these models [182,189,190]), the relevance of such altered signaling to vascular renewal remains speculative. Increased PI3K signaling augments angiogenesis, and vice versa, suggesting an important role in vascular regeneration [191]; knockout of the PI3K-γ isoform also significantly abrogates the capacity of murine EEPCs and CPCs to promote vascular renewal [192,193]. Equally, Akt1 (but not Akt2) has been shown to be critical for ischemia- and VEGFinduced angiogenesis, EPC mobilization into the circulation, and the proangiogenic function of EEPCs in knockout mice [187]. Moreover, it seems that eNOS critically mediates the downstream proangiogenic effects of Akt1, as mice with eNOS mutation at the Akt1 phosphorylation site exhibit reduced angiogenesis, and rescue of angiogenesis occurs in Akt1 knockout mice crossed with mice expressing constitutively active eNOS [194]. Indeed, eNOS knockout mice also exhibit marked impairment of ischemia- and

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VEGF-induced angiogenesis [195]; it is also apparent that eNOS (or NO bioavailability) is critical to the mobilization and subsequent function of murine CPC [196]. Our own work in insulin-resistant South Asian men has also shown that exercise-induced CPC mobilization is blunted as a result of impaired NO bioavailability, as evidenced by complete abrogation of CPC mobilization after intravenous administration of L-NMMA [54]. We have also demonstrated LEPCs derived from South Asian men to be dysfunctional as a result of reduced Akt/eNOS signaling, with lentiviral introduction of constitutively active Akt rescuing their reparative and proangiogenic potential [156]. Administration of an NO donor has also been demonstrated to augment the migration of CD34 þ CPCs from patients with diabetes, via reduction in cytoskeletal rigidity [197].

Oxidative stress The insulin-resistant phenotype is often characterized by overproduction of chemically reactive oxygen species (ROS), which influence may important cellular signaling cascades and contribute to the development vascular disease [198,199]. Available data support the capacity of oxidative stress to impair indices of angiogenesis, EPC function, and CPC mobilization [200–203]. Moreover, targeted reduction of oxidative stress has been shown to augment angiogenesis in murine models of diabetes [204]. However, some ROS have also been clearly identified as critical mediators of many physiological phenomena and as such cannot be simplistically labeled as detrimental to vascular renewal. For example, NOX2-derived superoxide is critical to ischemia-induced angiogenesis and CPC mobilization [205,206]; NOX2 has also been shown to mediate some proangiogenic signaling downstream of VEGF receptors [207]. NOX4, which predominantly generates hydrogen peroxide (H2O2) [208], has also been implicated in vascular renewal, with global NOX4−/− mice exhibiting impaired angiogenesis after induction of limb ischemia; in vitro studies showed angiogenesis was rescued with H2O2 [209]. Conversely, overexpression of NOX4 within the endothelium has been shown to augment angiogenesis in response to limb ischemia, in an NO-dependent manner [210]. Furthermore, NOX4 has been shown to promote myocardial tolerance of chronic pressure overload by inducing angiogenesis, via induction of HIF1α and VEGF [211]. Recently, NOX4 has been shown to be essential in insulin-induced (HIF1α-dependent) expression of VEGF [212]. However, NOX4 induction of HIF1 and VEGF has been shown by other authors to provoke vascular leakiness in the context of hyperglycemia, which led them to suggest a role in diabetic retinopathy [213]. Other work has implicated NOX4 in endothelial senescence in vitro [214], and neuronal injury after stroke, perhaps due to blood–brain barrier disintegration [215]. Our own work has shown that endothelium-specific insulin resistance is associated with increased NOX2/4 expression and endothelium-dependent vasomotor dysfunction, which is ameliorated by administration of a superoxide dismutase mimetic ex vivo [216]. More recently, we have also shown that in vivo (nonsubtype-specific) pharmacological antagonism of NADPH oxidase normalizes NO-dependent endothelial vasomotor function in the context of endothelium-specific and whole-body insulin resistance [217]. However, given the data suggesting an essential role of NOXderived superoxide in vascular renewal, it remains unclear whether therapeutic targeting of vascular superoxide generation in the context of insulin resistance would promote or retard vascular repair and angiogenesis. Mitochondria-derived ROS (superoxide, which is then predominantly dismuted to hydrogen peroxide [218]) may also be of relevance to vascular renewal, and mitochondrial activity is altered in insulin-resistant syndromes [219]. The inhibition of mitochondrial

ROS generation has been shown to retard HIF activity, via removal of ROS-induced inhibition of prolyl hydroxylase activity, hence linking mitochondrial ROS with hypoxia sensing [220,221]. This observation seems to be relevant in vivo, because pharmacological inhibition of mitochondrial complex III (and hence ROS generation) can reduce tumor angiogenesis in animal models [222]. However, endothelial overexpression of the mitochondrial antioxidant thioredoxin (which reduces mitochondrial ROS generation) has been shown to augment angiogenesis in hindlimb ischemia models [223]. This work suggested that thioredoxin promoted angiogenesis via a reduction in ROS per se, as opposed to indirectly via increased bioavailability of NO. Whether these divergent conclusions relate to methodological issues (e.g., off-target effects of pharmacological inhibitors of mitochondrial ROS generation) or point to an optimal range of mitochondrial ROS in promoting vascular renewal is unclear. More advanced insulin resistance is also associated with progressive hyperglycemia, which further complicates the picture by contributing to oxidative stress, as discussed earlier. It is also important to note the interplay between ROS and NO bioavailability, because superoxide reacts with NO to generate peroxynitrite, itself a free radical capable of modifying vascular biology and renewal [224,225]. Hence, superoxide may also modulate vascular renewal indirectly, via reduction in NO bioavailability or generation of other secondary signaling radicals. Furthermore, superoxide derived from uncoupling of eNOS (related to tetrahydrobiopterin deficiency) has been implicated in the impaired wound healing and angiogenesis in streptozotocin (STZ)-induced diabetes [151]. On balance, it seems that complex temporal and spatial orchestration of many signaling radicals is required for optimal vascular renewal; this may explain why clinical trials of broad-spectrum antioxidants have not proven effective in improving vascular outcomes.

Hypoxia perception and VEGF activity As alluded to in the discussion of cell-specific insulin resistance, VEGF-induced Akt signaling (and subsequent chemotaxis) is impaired in monocytes derived from patients with type 2 DM because of increased basal Akt (and ERK) phosphorylation. Whether these findings can be extrapolated to other cell types and growth factors, and also offer insight into insulin resistance per se, remains unclear. It is also notable from these studies that the insulin-resistant phenotype is associated with increased basal plasma VEGF, potentially supporting the notion of “VEGF resistance” within humans [168]; myocardial VEGF expression is also increased in human DM, whereas VEGF signaling may be impaired [226]. How such alteration in VEGF signaling influences vascular Notch signaling and neovascular network coordination in insulin resistance and diabetes remains unclear. A further layer of complexity is added when considering data demonstrating impaired augmentation of VEGF expression in ischemic tissues from insulin-resistant models versus noninsulin-resistant control [227]. It is apparent that PI3K/Akt signaling can contribute to the expression of VEGF, with reduced VEGF mRNA in models of reduced PI3K signaling, which can be rescued with expression of constitutively active Akt [191]. Moreover, insulin can induce VEGF expression in a PI3K/Akt-dependent fashion [161,163,228], and local delivery of the insulin-sensitizing agent pioglitazone can augment angiogenesis and VEGF expression in an eNOS-dependent manner after induction of hindlimb ischemia in nondiabetic mice [229]. Furthermore, it has been suggested that insulin-stimulated, PI3K-dependent VEGF expression is mediated by increased expression and DNA binding of the hypoxia-sensing transcription factor HIF1α, hence the overlapping gene expression profiles induced by hypoxia and insulin [230,231]. However, these gene expression profiles are not identical, and more recent work has suggested that insulin (via PI3K) induces the

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binding of a HIF-like molecule (in conjunction with the HIFα partner protein ARNT or HIFβ) to hypoxia-responsive elements in the promoter sequence of genes such as VEGF [232]. Other authors have also suggested that hyperglycemia impairs HIF1α DNA binding, thus impairing ischemia-induced VEGF expression in overt diabetes [233]. Overall, the impaired ischemia-induced expression of VEGF in models of insulin resistance may imply wider alterations in the generation of many important proangiogenic mediators; hence, hypoxia sensing/signaling may represent an important therapeutic target. However, the increased basal concentrations of VEGF in insulin-resistant disorders may also imply impaired signaling of VEGF, or possibly inadequate resolution of the underlying hypoxic stimulus due to poor-quality neovascular development (Fig. 3). These issues also need resolving before simply attempting to promote hypoxia-induced proangiogenic mediator activity and expression.

Insulin receptor interactions and signaling cross talk As outlined earlier, the insulin receptor is a homodimer of two α/β subunits and is part of a wider family of receptor tyrosine kinases (RTKs). Alternate splicing results in two isoforms of the IR (IR-A and IR-B), with differing tissue distributions and ligandbinding affinities; IR-A is much more abundant than IR-B within the endothelium, and the two can heterodimerize, although the functional relevance of these observations is unclear [234]. Phylogenetic studies suggest that an IR-like ancestral gene diverged in the evolution of higher order organisms, resulting in the IR and the closely related IGF1R, which binds insulin-like growth factor-1 (IGF1) [157]. Given the significant homology between the IR and the IGF1R, it is unsurprising that they share analogous intracellular signaling properties, principally involving the PI3K/Akt and ERK MAP kinase cascades. Although there is also some functional overlap, the IR is traditionally implicated in metabolism and the IGF1R in modulating growth; how these divergent biological responses arise remains incompletely understood; IGF1R is much more abundant than IR within the vascular endothelium and smooth muscle [234,235]. Importantly, interpretation of in vitro studies of insulin and IGF1 biology are often confounded by the supraphysiological doses of these molecules applied, which facilitates IGF1-induced activation of IR and vice versa; therefore, caution must be applied in the interpretation of such data in isolation. It is evident that IGF1 signaling plays an important role in vascular renewal. Injured conduit vessels markedly increase their expression of IGF1 [236], and infusion of IGF1 in the setting of vascular injury augments reendothelialization and reduces

Fig. 3. VEGF expression in insulin-resistant syndromes. Schema illustrating available data that show elevated basal, but impaired postischemic, expression of VEGF in insulin-resistant syndromes.

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neointimal thickening [237]. Our own work has demonstrated vascular “IGF1 resistance” in the aorta of high-fat-fed mice, with blunting of IGF1-induced eNOS phosphorylation, although vascular renewal was not addressed specifically [238]. Furthermore, we have shown that endothelial overexpression of IGF1R augments reendothelialization of injured conduit vessels, despite concurrent reductions in insulin-stimulated endothelial NO generation [239]. In the context of vascular regeneration, administration of IGF1 has been shown to promote ocular angiogenesis [240], whereas antagonism of IGF1R can retard retinal angiogenesis [241]. Endothelium-specific knockout of the IGF1R also reduces hypoxia-induced retinal angiogenesis [158]. Infusion of IGF1 into atherosclerosis-prone mice has been demonstrated to augment CPC numbers, in association with a reduction in atherosclerosis [242]. In vitro, IGF1 augments EEPC differentiation and function, with similar findings noted in EEPC derived from patients with growth hormone deficiency (upstream of IGF1) after appropriate hormone replacement [243]. Moreover, EEPCs secrete IGF1 and this has been shown to be critical in mediating their paracrine benefits upon vascular renewal [244,245]. The formation of IR/IGF1R heterodimers, or hybrid receptors (HRs), adds further complexity to insulin/IGF1 signaling and their relevance to vascular biology. HRs have greater affinity for IGF1 than for insulin [157] and have been demonstrated within the vascular endothelium and smooth muscle [234,235,246,247]. Although some modeling studies support the generation of HRs simply as a function of the relative abundance of IR and IGF1R subunits, these data are not wholly supported by observational work, suggesting formation may not be passive [157]. Perhaps supporting this notion, recent data suggest that aldosterone signaling can modulate HR formation [248]. Although we remain unclear on the functional relevance of hybrid receptors, recent data from our group suggest that the IGF1R may act as a negative regulator of endothelial insulin signaling, by sequestering IR subunits in hybrid receptors. These data are based upon observations that endothelial haploinsufficiency of IGF1R promotes insulin-induced endothelial NO generation, in association with reduced HRs [249]; conversely, in mice overexpressing IGF1R in the endothelium, HRs were increased and insulin-stimulated eNOS phosphorylation was reduced [239]. Hence, the emerging data suggest HRs are relevant to vascular renewal, although it may prove challenging to mechanistically implicate their role per se, given concurrent changes in IR and IGF1R abundance/signaling. Beyond the close functional links between the ancestrally related IR and IGF1R, some data support wider interactions between IR/IGF1R signaling and less homologous RTKs. The best studied of these links is with VEGF signaling; early work in renal epithelial cells indicated that VEGFR2 can signal to PI3K via the principal IR docking protein IRS1, and knockout of IRS1 altered VEGF-induced protein synthesis [250]. Subsequent work extended this link to show that IRS1 could mediate VEGF-induced eNOS phosphorylation in endothelial cells [251]. This has now been translated to in vivo models, in which antisense oligonucleotides to IRS1 impair corneal angiogenesis [252], and this agent is now in phase III clinical trials of corneal graft rejection [253]. With regard to IGF1R, this also appears to be linked to VEGF signaling, given that in vitro treatment of endothelial cells with an IGF1Rantagonizing antibody reduces VEGF-induced MAP kinase activation by 50% [241]. These data suggest important cross talk between IGF1R/IR and VEGFR due to multiple levels of shared signaling cascades. Similar to data on VEGF discussed earlier, circulating concentrations of other proangiogenic molecules, such as hepatocyte growth factor (HGF), are elevated in insulinresistant humans [254]; whether this association is causal is, of course, unclear. Recent data have shown that HGF also induces IRS phosphorylation and downstream signaling in hepatocytes; this

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was dependent on hybridization of the HGF receptor (Met) with IR (but not IGF1R), and such direct interaction appeared crucial to hepatic insulin responsiveness in vivo [255]. Such interactions have not been probed in vascular cells. In summary, it remains unclear how these observations relate to vascular renewal in human insulin resistance, although they serve to highlight the complex interactions between insulin and proangiogenic RTK signaling.

Therapeutic considerations A principal stimulus for the study of vascular renewal in the setting of insulin resistance is the persistently poor cardiovascular outcomes of these patients, despite the array of evidence-based primary and secondary prevention therapies [8]. Rather than address how these currently applied preventative therapies might modulate endogenous vascular renewal, this section focuses upon some novel pharmacological, gene-based, or cell therapy-based strategies that have targeted the molecular mechanisms outlined earlier. However, this remains an emerging field, and so many of the putative mechanisms have not yet been therapeutically targeted and when investigated tend to use murine or hybrid human/murine systems. Indeed, the field of clinical gene- and cellbased cardiovascular repair remains nascent; hence, this discussion is often speculative, but highlights important therapeutic considerations for future research.

nonspecifically interferes with Notch signaling) augments perfusion recovery in diabetic mice with hindlimb ischemia. However, interfering with Notch signaling can promote the formation of immature neovasculature, with excess sprouting and poor perfusion [35]; indeed, other models suggest interfering with Notch (via Dll4 antagonism) impairs angiogenesis [262]. The local application of other proangiogenic molecules that are expressed in lower concentrations in murine models of diabetes with tissue ischemia has also proven successful in augmenting angiogenesis. For example, sonic hedgehog protein has been shown to augment limb angiogenesis in a PI3K- and NOdependent manner [150], and nerve growth factor therapy has been shown to increase angiogenesis, possibly by promoting VEGF expression [149]. It is unclear whether certain growth factors, or combinations of these, are more efficacious, however. Augmenting the attraction of CPCs and EPCs to ischemic tissues by local antagonism of CXCR4 signaling may also facilitate the recruitment of their wider paracrine activity. Nishimura et al. [263] have recently shown that this strategy improves wound angiogenesis and healing in genetically obese mice, via the paracrine effects of progenitor recruitment. Finally, a modified angiopoietin-1 (Ang1) molecule has been shown to promote endothelial survival, angiogenesis, and neovessel maturation (by stimulating pericyte coverage) in the context of tissue injury in murine models of diabetes [264,265].

Pharmacological strategies

Gene-based strategies

Although the mortality benefits of insulin therapy in the setting of acute cardiovascular events remain debated, murine data support the capacity of acute insulin therapy to promote angiogenesis and endothelial repair after conduit arterial injury [256,257]. Furthermore, similar effects have been attributed to IGF1 [237,244], which may represent a more attractive alternative to acute insulin therapy and has already been applied in clinical trials outside the context of acute cardiovascular events. An early phase clinical trial of IGF1 in the context of primary percutaneous coronary intervention (for ST elevation MI) is currently under way (ClinicalTrials.gov registration NCT01438086). Another agent currently applied for indications other than cardiovascular repair is desferrioxamine; data suggest this normalizes the activity of HIF1α (and so VEGF expression) in diabetic mice with soft-tissue wounding and ischemia [233]. Whether this translates to benefits in human disease is unclear, and no relevant clinical trials are currently registered. Becaplermin, a recombinant human PDGFBB, is currently marketed for clinical use in augmenting diabetic neuropathic (but not ischemic) wound granulation and angiogenesis; however, its efficacy and cost-effectiveness require broader clinical assessment [258]. Moreover, safety concerns have emerged, with the agent receiving an FDA black box warning indicating that use of more than two tubes is associated with an increased risk of mortality due to malignancy. Furthermore, murine data suggest that emerging agents, such as heat shock protein-90 fragments, may be more efficacious in promoting angiogenesis [259]. Therapeutic agents that have not been applied in any human disease setting also offer promise, but clearly are further from translational studies. Whereas some randomized data support improved surrogate secondary (but not primary) outcomes in patients with diabetes and limb ischemia receiving intramuscular VEGF165 gene therapy [260], the phenomenon of VEGF resistance discussed earlier may be problematic. One means of modulating the VEGF response is by interfering with Notch signaling; Cao et al. [261] have shown that coadministration of VEGF and DAPT (which

Although preclinical and early clinical trials suggested angiogenic gene-based therapies to offer potential, larger randomized clinical trials in patients with ischemic diseases have not proven sustained and relevant clinical benefit [266]. Many potential explanations for this failure of translation exist, including the inadequacy of animal models without disease (e.g., diabetes), inadequate dose or duration of effect, targeting of isolated growth factors, and clinical resistance to growth factors. As noted in this section, recent preclinical studies have addressed some of these concerns, although it remains to be seen whether their findings will aid development of effective therapies. As noted earlier, Ang1 can promote neovessel maturation and angiogenesis when delivered as a modified protein. Using adenoviral delivery of Ang1 to db/db mice after induction of myocardial ischemia, Chen and Stinnett [267] demonstrated HIF1α stabilization, increased VEGF and eNOS expression, increased angiogenesis, and normalization of immature vasculature. They also noted a reduction in prolyl hydroxylase-2 (PHD2), the enzyme required for targeting of HIF1α degradation during normoxia, which may underlie many of their observations, although their study cannot define causality. Separate adenoviral administration of Ang1 and VEGF to mice with streptozotocin-induced diabetes and surgically induced myocardial infarction has also been shown to promote angiogenesis/arteriogenesis and reduce adverse myocardial remodeling [268]. A number of groups have also demonstrated that delivery of constitutively active (or stabilized) HIF1α can circumvent the problems with HIF activity in diabetes, augmenting wound healing and ischemic limb angiogenesis [269,270]. These data may also point to a role for pharmacological inhibitors of molecules involved in HIF degradation, such as PHD2, rather than resorting to gene therapies [269]. Although not yet studied, vector targeting of signaling molecules downstream of multiple growth factors may also prove effective in bypassing growth factor resistance and so augmenting vascular renewal; however, more significant side effects might also be anticipated.

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Cell-based strategies

Safety concerns

Clinical trials of diverse autologous and allogeneic cell lines in the context of ischemic heart disease are currently ongoing, with mixed success in terms of surrogate outcomes; adequately powered studies of hard clinical outcomes are awaited [271–273]. These studies have not specifically recruited patients with insulin resistance or diabetes, yet such patients are likely to represent a majority in any cohort with established ischemic heart disease [9]. As with any therapeutic modality, successful cell-based strategies are likely to have to address the specific pathophysiology of altered vascular renewal related to insulin resistance; this may take the form of ex vivo pharmacological or genetic manipulation.

Vascular repair might be expected to reduce major cardiovascular events related to unstable atheroma or recently disrupted conduit vessel intima (e.g., percutaneous coronary intervention). Promoting vascular regeneration may retard tissue loss in the context of acute cardiovascular events (e.g., myocardial infarction), promote tissue function during chronic ischemia (e.g., ischemic cardiomyopathy), and reduce ischemia-related symptoms. However, the risks of any novel therapy must always be considered during development and weighed against the anticipated benefits. In general terms, novel vascular renewal strategies have raised potential concerns with regard to disease processes where excessive angiogenesis is implicated—cancer, proliferative retinopathy, and possibly atherogenesis. Both sides of this equation remain somewhat speculative, although early phase clinical trials of adenoviral growth factor gene therapy and bone marrow mononuclear cells have not highlighted major adverse effects [266,271]. With regard to cancer angiogenesis, a more nuanced appreciation of tumor vascularization is now emerging. Clinical studies of VEGF receptor antagonists have yielded mixed results and also highlighted a potentially increased risk of metastasis; although this remains debated, the loss of tumor vessel integrity has been implicated. Hence, novel molecular targets that promote the normalization of tumor vasculature may in fact repress metastasis [35]. Our appreciation of diabetic retinopathy has evolved in that we now appreciate that the initiating factor is likely to be vasoregression and that the angiogenic response seems excessive because of the poor quality of neovasculature that fails to meet the metabolic needs of the retina and is prone to leakage [36]. Hence, the emerging theme from both cancer and proliferative retinopathy is that neovessel quality is also important in determining pathophysiology—this fact should be borne in mind when assessing novel vascular renewal strategies. However, only carefully conducted clinical trials will be able to define the true scope of adverse effects in patients, who will often be elderly and have complex comorbidity, which cannot easily be modeled in preclinical studies.

Murine cells In db/db mice, autologous EEPCs have been shown to augment wound healing and angiogenesis in a paracrine fashion [274]. However, other work has shown EEPCs from mice with diabetes to be dysfunctional, and one study has implicated a reduction of the antioxidant enzyme manganese superoxide dismutase, showing that adenoviral delivery of the molecule to EEPCs augmented their proangiogenic activity when applied to wounds [275]. Overexpression of peroxisome proliferator-activated receptor-γ coactivator-1α in allogeneic rat bone marrow mesenchymal stem cells has also been shown to augment their proangiogenic capacity after hindlimb ischemia in mice with STZ-induced diabetes [276]. This was associated with enhanced survival and paracrine activity of mesenchymal stem cells in vitro during hypoxic stress, along with greater expression of proangiogenic mediators in the ischemic musculature.

Human cells CD34 þ CPC from patients with diabetes express less eNOS and greater NOX2 NADPH oxidase. Jarajapu et al. [277] have shown that ex vivo pharmacological inhibition of NOX, but not eNOS overexpression, augments the proangiogenic function of these cells in mice with retinal ischemia–reperfusion. Thum et al. [203] have shown that eNOS is uncoupled in EEPCs from patients with diabetes due to hyperglycemia, resulting in the generation of superoxide, which was abrogated by tetrahydrobiopterin administration; their data suggested NOX-derived superoxide was less important. Recent work has also probed the role of ex vivomanipulated bone marrow mononuclear cells from patients with diabetes when injected into immunodeficient mice with STZinduced diabetes 2 weeks before induction of limb ischemia. It was noted that activation of ephrinB2 by an ephrinB2/Fc molecule could promote ischemic limb angiogenesis and perfusion recovery, versus untreated cells, possibly by augmenting the number and function of host bone marrow- and circulating progenitors [278]. Our own work has also addressed the reparative and proangiogenic potential of LEPCs from apparently healthy insulin-resistant South Asian men versus control white European men. We found marked dysfunction of South Asian LEPCs in vitro and when infused in vivo after femoral artery injury or induction of hindlimb ischemia, in conjunction with reduced expression of Akt and eNOS [156]. Lentivirus-mediated expression of constitutively active Akt in South Asian LEPCs restored their reparative phenotype to the magnitude of control LEPCs, suggesting that modulation of Akt activity may be a valid therapeutic target. However, as discussed before, chronic overactivation of Akt may also be detrimental [184,186] and also poses theoretical risks regarding oncogenesis; hence alternative means of modulating LEPC signaling may be required.

Conclusions Insulin-resistant syndromes are increasingly prevalent and associated with poor prognosis due to major cardiovascular events. Vascular damage and dysfunction are noted early in the continuum from preclinical insulin resistance to overt diabetes, possibly as a result of abrogated endogenous vascular repair and regeneration. Many molecular mechanisms have been implicated, although important contributors seem to be reduced NO bioavailability and oxidative stress, which are both linked with abnormal growth factor activity and altered intracellular signaling. A diverse range of pharmacological, gene-based, and cell-based therapeutic strategies have been proposed to address these molecular abnormalities, although most remain at a preclinical or early clinical trial stage. When developing these novel therapies, it will also be important to learn lessons from previously unsuccessful attempts to augment angiogenesis and take note of concepts emerging from cancer and retinopathy therapeutics. If successful, such therapies may represent a paradigm shift in our management of vascular disease in these persistently high-risk patients.

References [1] Seshasai, S. R.; Kaptoge, S., et al. Diabetes mellitus, fasting glucose, and risk of cause-specific death. N. Engl. J. Med. 364:829–841; 2011.

258

R.M. Cubbon et al. / Free Radical Biology and Medicine 60 (2013) 246–263

[2] Booth, G. L.; Kapral, M. K.; Fung, K.; Tu, J. V. Relation between age and cardiovascular disease in men and women with diabetes compared with non-diabetic people: a population-based retrospective cohort study. Lancet 368:29–36; 2006. [3] The DECODE Study Group. Is the current definition for diabetes relevant to mortality risk from all causes and cardiovascular and noncardiovascular diseases? Diabetes Care 26:688–696; 2003. [4] The International Diabetes Federation. URL: 〈http://www.idf.org/diabetesa tlas/5e/the-global-burden〉. Accessed 6 September 2012. [5] From, A. M.; Leibson, C. L.; Bursi, F.; Redfield, M. M.; Weston, S. A.; Jacobsen, S. J., et al. Diabetes in heart failure: prevalence and impact on outcome in the population. Am. J. Med. 119:591–599; 2006. [6] Ovbiagele B., Markovic D., Fonarow G.C. Recent US patterns and predictors of prevalent diabetes among acute myocardial infarction patients. Cardiol. Res. Pract.20111456152011. [7] Cubbon R.M., Adams B., Rajwani A., Mercer B.N., Patel P.A., Gherardi G., et al. Diabetes mellitus is associated with adverse prognosis in chronic heart failure of ischaemic and non-ischaemic aetiology. Diabetes Vasc. Dis. Res. (in press); . [8] Cubbon R.M., Wheatcroft S.B., Grant P.J., Gale C.P., Barth J.H., Sapsford R.J., et al. Temporal trends in mortality of patients with diabetes mellitus suffering acute myocardial infarction: a comparison of over 3000 patients between 1995 and 2003. Eur. Heart J.28540-5452007. [9] Lenzen, M.; Ryden, L.; Ohrvik, J.; Bartnik, M.; Malmberg, K.; Scholte op Reimer, W., et al. Diabetes known or newly detected, but not impaired glucose regulation, has a negative influence on 1-year outcome in patients with coronary artery disease: a report from the Euro Heart Survey on diabetes and the heart. Eur. Heart J. 27:2969–2974; 2006. [10] Ross, R. Atherosclerosis—an inflammatory disease. N. Engl. J. Med. 340: 115–126; 1999. [11] Grundy, S. M.; Brewer Jr. H. B.; Cleeman, J. I.; Smith Jr. S. C.; Lenfant, C., for the Conference Participants. Definition of metabolic syndrome: report of the National Heart, Lung, and Blood Institute/American Heart Association Conference on Scientific Issues Related to Definition. Circulation 109:433–438; 2004. [12] Kuhlencordt, P. J.; Gyurko, R.; Han, F.; Scherrer-Crosbie, M.; Aretz, T. H.; Hajjar, R., et al. Accelerated atherosclerosis, aortic aneurysm formation, and ischemic heart disease in apolipoprotein E/endothelial nitric oxide synthase double-knockout mice. Circulation 104:448–454; 2001. [13] Balletshofer, B. M.; Rittig, K.; Enderle, M. D.; Volk, A.; Maerker, E.; Jacob, S., et al. Endothelial dysfunction is detectable in young normotensive firstdegree relatives of subjects with type 2 diabetes in association with insulin resistance. Circulation 101:1780–1784; 2000. [14] Makimattila, S.; Liu, M. L.; Vakkilainen, J.; Schlenzka, A.; Lahdenpera, S.; Syvanne, M., et al. Impaired endothelium-dependent vasodilation in type 2 diabetes: relation to LDL size, oxidized LDL, and antioxidants. Diabetes Care 22:973–981; 1999. [15] Williams, I. L.; Chowienczyk, P. J.; Wheatcroft, S. B.; Patel, A.; Sherwood, R.; Momin, A., et al. Effect of fat distribution on endothelial-dependent and endothelial-independent vasodilatation in healthy humans. Diabetes Obes. Metab. 8:296–301; 2006. [16] Kim, J.; Montagnani, M.; Koh, K. K.; Quon, M. J. Reciprocal relationships between insulin resistance and endothelial dysfunction: molecular and pathophysiological mechanisms. Circulation 113:1888–1904; 2006. [17] Cimini, M.; Fazel, S.; Zhuo, S.; Xaymardan, M.; Fujii, H.; Weisel, R. D., et al. ckit dysfunction impairs myocardial healing after infarction. Circulation 116: I77–82; 2007. [18] Foteinos, G.; Hu, Y.; Xiao, Q.; Metzler, B.; Xu, Q. Rapid endothelial turnover in atherosclerosis-prone areas coincides with stem cell repair in apolipoprotein E-deficient mice. Circulation 117:1856–1863; 2008. [19] Dimmeler, S. Regulation of bone marrow-derived vascular progenitor cell mobilization and maintenance. Arterioscler. Thromb. Vasc. Biol 30:1088–1093; 2010. [20] Van Belle, E.; Bauters, C.; Asahara, T.; Isner, J. M. Endothelial regrowth after arterial injury: from vascular repair to therapeutics. Cardiovasc. Res. 38:54–68; 1998. [21] Zampetaki, A.; Kirton, J. P.; Xu, Q. Vascular repair by endothelial progenitor cells. Cardiovasc. Res. 78:413–421; 2008. [22] Torsney, E.; Xu, Q. Resident vascular progenitor cells. J. Mol. Cell. Cardiol. 50:304–311; 2011. [23] Hagensen, M. K.; Raarup, M. K.; Mortensen, M. B.; Thim, T.; Nyengaard, J. R.; Falk, E., et al. Circulating endothelial progenitor cells do not contribute to regeneration of endothelium after murine arterial injury. Cardiovasc. Res 93:223–231; 2012. [24] Hagensen, M. K.; Shim, J.; Falk, E.; Bentzon, J. F. Flanking recipient vasculature, not circulating progenitor cells, contributes to endothelium and smooth muscle in murine allograft vasculopathy. Arterioscler. Thromb. Vasc. Biol. 31:808–813; 2011. [25] Hagensen, M. K.; Shim, J.; Thim, T.; Falk, E.; Bentzon, J. F. Circulating endothelial progenitor cells do not contribute to plaque endothelium in murine atherosclerosis. Circulation 121:898–905; 2010. [26] Rupp, S.; Koyanagi, M.; Iwasaki, M.; Bauer, J.; von Gerlach, S.; Schranz, D., et al. Characterization of long-term endogenous cardiac repair in children after heart transplantation. Eur. Heart J. 29:1867–1872; 2008. [27] Jiang, S.; Walker, L.; Afentoulis, M.; Anderson, D. A.; Jauron-Mills, L.; Corless, C. L., et al. Transplanted human bone marrow contributes to vascular endothelium. Proc. Natl. Acad. Sci. USA 101:16891–16896; 2004.

[28] Potente, M.; Gerhardt, H.; Carmeliet, P. Basic and therapeutic aspects of angiogenesis. Cell 146:873–887; 2011. [29] Heil, M.; Eitenmuller, I.; Schmitz-Rixen, T.; Schaper, W. Arteriogenesis versus angiogenesis: similarities and differences. J. Cell. Mol. Med. 10:45–55; 2006. [30] Wedgwood, S.; Mitchell, C. J.; Fineman, J. R.; Black, S. M. Developmental differences in the shear stress-induced expression of endothelial NO synthase: changing role of AP-1. Am. J. Physiol. Lung Cell. Mol. Physiol. 284: L650–L662; 2003. [31] Demicheva, E.; Hecker, M.; Korff, T. Stretch-induced activation of the transcription factor activator protein-1 controls monocyte chemoattractant protein-1 expression during arteriogenesis. Circ. Res 103:477–484; 2008. [32] Coulon, C.; Georgiadou, M.; Roncal, C.; De Bock, K.; Langenberg, T.; Carmeliet, P. From vessel sprouting to normalization. Arterioscler. Thromb. Vasc. Biol. 30:2331–2336; 2010. [33] Eltzschig, H. K.; Carmeliet, P. Hypoxia and inflammation. N. Engl. J. Med. 364:656–665; 2011. [34] Cao, Y. Adipose tissue angiogenesis as a therapeutic target for obesity and metabolic diseases. Nat. Rev. Drug Discovery 9:107–115; 2010. [35] Carmeliet, P.; Jain, R. K. Molecular mechanisms and clinical applications of angiogenesis. Nature 473:298–307; 2011. [36] Hammes, H. P.; Feng, Y.; Pfister, F.; Brownlee, M. Diabetic retinopathy: targeting vasoregression. Diabetes 60:9–16; 2011. [37] Jakobsson, L; Bentley, K; VEGFRs, Gerhardt H. and Notch: a dynamic collaboration in vascular patterning. Biochem. Soc. Trans. 37:1233–1236; 2009. [38] Asahara, T.; Murohara, T.; Sullivan, A.; Silver, M.; van der Zee, R.; Li, T., et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science 275:964–966; 1997. [39] Vasa, M.; Fichtlscherer, S.; Adler, K.; Aicher, A.; Martin, H.; Zeiher, A. M., et al. Increase in circulating endothelial progenitor cells by statin therapy in patients with stable coronary artery disease. Circulation 103:2885–2890; 2001. [40] Peichev, M.; Naiyer, A. J.; Pereira, D.; Zhu, Z.; Lane, W. J.; Williams, M., et al. Expression of VEGFR-2 and AC133 by circulating human CD34 þ cells identifies a population of functional endothelial precursors. Blood 95: 952–958; 2000. [41] Werner, N.; Kosiol, S.; Schiegl, T.; Ahlers, P.; Walenta, K.; Link, A., et al. Circulating endothelial progenitor cells and cardiovascular outcomes. N. Engl. J. Med. 353:999–1007; 2005. [42] Hirschi, K. K.; Ingram, D. A.; Yoder, M. C. Assessing identity, phenotype, and fate of endothelial progenitor cells. Arterioscler. Thromb. Vasc. Biol. 28: 1584–1595; 2008. [43] Prokopi, M.; Pula, G.; Mayr, U.; Devue, C.; Gallagher, J.; Xiao, Q., et al. Proteomic analysis reveals presence of platelet microparticles in endothelial progenitor cell cultures. Blood 114:723–732; 2009. [44] Hur, J.; Yoon, C. H.; Kim, H. S.; Choi, J. H.; Kang, H. J.; Hwang, K. K., et al. Characterization of two types of endothelial progenitor cells and their different contributions to neovasculogenesis. Arterioscler. Thromb. Vasc. Biol. 24:288–293; 2004. [45] Hill, J. M.; Zalos, G.; Halcox, J. P. J.; Schenke, W. H.; Waclawiw, M. A.; Quyyumi, A. A., et al. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N. Engl. J. Med. 348:593–600; 2003. [46] Hur, J.; Yang, H. M.; Yoon, C. H.; Lee, C. S.; Park, K. W.; Kim, J. H., et al. Identification of a novel role of T cells in postnatal vasculogenesis. Circulation 116:1671–1682; 2007. [47] Case, J.; Mead, L. E.; Bessler, W. K.; Prater, D.; White, H. A.; Saadatzadeh, M. R., et al. Human CD34 þ AC133 þ VEGFR-2 þ cells are not endothelial progenitor cells but distinct, primitive hematopoietic progenitors. Exp. Hematol 35:1109–1118; 2007. [48] Timmermans, F.; Van Hauwermeiren, F.; De Smedt, M.; Raedt, R.; Plasschaert, F.; De Buyzere, M. L., et al. Endothelial outgrowth cells are not derived from CD133 þ cells or CD45 þ hematopoietic precursors. Arterioscler. Thromb. Vasc. Biol 27:1572–1579; 2007. [49] Mund, J. A.; Estes, M. L.; Yoder, M. C.; Ingram, D. A.; Case, J. Flow cytometric identification and functional characterization of immature and mature circulating endothelial cells. Arterioscler. Thromb. Vasc. Biol. 32:1045–1053; 2012. [50] Ishigami, K. i.; Uemura, S.; Morikawa, Y.; Soeda, T.; Okayama, S; Nishida, T, et al. Long-term follow-up of neointimal coverage of Sirolimus-eluting stents evaluation with optical coherence tomography. Circ. J. 73:2300–2307; 2009. [51] Rivard, A.; Silver, M.; Chen, D.; Kearney, M.; Magner, M.; Annex, B., et al. Rescue of diabetes-related impairment of angiogenesis by intramuscular gene therapy with adeno-VEGF. Am. J. Pathol. 154:355–363; 1999. [52] Kahn, M. B.; Yuldasheva, N. Y.; Cubbon, R. M.; Smith, J.; Rashid, S. T.; Viswambharan, H., et al. Insulin resistance impairs circulating angiogenic progenitor cell function and delays endothelial regeneration. Diabetes 60:1295–1303; 2011. [53] Chen, Y. L.; Chang, C. L.; Sun, C. K.; Wu, C. J.; Tsai, T. H.; Chung, S. Y., et al. Impact of obesity control on circulating level of endothelial progenitor cells and angiogenesis in response to ischemic stimulation. J. Transl. Med. 10:86; 2012. [54] Cubbon, R. M.; Murgatroyd, S. R.; Ferguson, C.; Bowen, T. S.; Rakobowchuk, M.; Baliga, V., et al. Human exercise-induced circulating progenitor cell mobilization is nitric oxide-dependent and is blunted in South Asian men. Arterioscler. Thromb. Vasc. Biol. 30:878–884; 2010.

R.M. Cubbon et al. / Free Radical Biology and Medicine 60 (2013) 246–263

[55] Fadini, G. P.; Miorin, M.; Facco, M.; Bonamico, S.; Baesso, I.; Grego, F., et al. Circulating endothelial progenitor cells are reduced in peripheral vascular complications of type 2 diabetes mellitus. J. Am. Coll. Cardiol 45:1449–1457; 2005. [56] Ii, M.; Takenaka, H.; Asai, J.; Ibusuki, K.; Mizukami, Y.; Maruyama, K., et al. Endothelial progenitor thrombospondin-1 mediates diabetes-induced delay in reendothelialization following arterial injury. Circ. Res. 98:697–704; 2006. [57] Ingram, D. A.; Lien, I. Z.; Mead, L. E.; Estes, M.; Prater, D. N.; Derr-Yellin, E., et al. In vitro hyperglycemia or a diabetic intrauterine environment reduces neonatal endothelial colony-forming cell numbers and function. Diabetes 57:724–731; 2008. [58] Assmann, G.; Guerra, R.; Fox, G.; Cullen, P.; Schulte, H.; Willett, D., et al. Harmonizing the definition of the metabolic syndrome: comparison of the criteria of the Adult Treatment Panel III and the International Diabetes Federation in United States American and European populations. Am. J. Cardiol. 99:541–548; 2007. [59] Noor, R.; Shuaib, U.; Wang, C. X.; Todd, K.; Ghani, U.; Schwindt, B., et al. Highdensity lipoprotein cholesterol regulates endothelial progenitor cells by increasing eNOS and preventing apoptosis. Atherosclerosis 192:92–99; 2007. [60] Petoumenos, V.; Nickenig, G.; Werner, N. High-density lipoprotein exerts vasculoprotection via endothelial progenitor cells. J. Cell. Mol. Med. 13:4623–4635; 2009. [61] Rossi, F.; Bertone, C.; Montanile, F.; Miglietta, F.; Lubrano, C.; Gandini, L., et al. HDL cholesterol is a strong determinant of endothelial progenitor cells in hypercholesterolemic subjects. Microvasc. Res. 80:274–279; 2010. [62] Seetharam, D.; Mineo, C.; Gormley, A. K.; Gibson, L. L.; Vongpatanasin, W.; Chambliss, K. L., et al. High-density lipoprotein promotes endothelial cell migration and reendothelialization via scavenger receptor-B type I. Circ. Res. 98:63–72; 2006. [63] Feng, Y.; van Eck, M.; Van Craeyveld, E.; Jacobs, F.; Carlier, V.; Van Linthout, S., et al. Critical role of scavenger receptor-B1-expressing bone marrowderived endothelial progenitor cells in the attenuation of allograft vasculopathy after human apo A-I transfer. Blood 113:755–764; 2009. [64] Chen, J.; Cui, X.; Zacharek, A.; Jiang, H.; Roberts, C.; Zhang, C., et al. Niaspan increases angiogenesis and improves functional recovery after stroke. Ann. Neurol 62:49–58; 2007. [65] Murphy, A. J.; Akhtari, M.; Tolani, S.; Pagler, T.; Bijl, N.; Kuo, C. L., et al. ApoE regulates hematopoietic stem cell proliferation, monocytosis, and monocyte accumulation in atherosclerotic lesions in mice. J. Clin. Invest. 121:4138–4149; 2011. [66] Westerterp, M.; Gourion-Arsiquaud, S.; Murphy, A.; Shih, A.; Cremers, S.; Levine, R., et al. Regulation of hematopoietic stem and progenitor cell mobilization by cholesterol efflux pathways. Cell Stem Cell 11:195–206; 2012. [67] Noghero, A.; Perino, A.; Seano, G.; Saglio, E.; Sasso, G. L.; Veglio, F., et al. Liver X receptor activation reduces angiogenesis by impairing lipid raft localization and signaling of vascular endothelial growth factor receptor-2. Arterioscler. Thromb. Vasc. Biol. 32:2280–2288; 2012. [68] Hazra, S.; Rasheed, A.; Bhatwadekar, A.; Wang, X.; Shaw, L. C.; Patel, M., et al. Liver X receptor modulates diabetic retinopathy outcome in a mouse model of streptozotocin-induced diabetes. Diabetes 61:3270–3279; 2012. [69] Besler, C.; Luscher, T. F.; Landmesser, U. Molecular mechanisms of vascular effects of high-density lipoprotein: alterations in cardiovascular disease. EMBO Mol. Med. 4:251–268; 2012. [70] Barter, P. J.; Caulfield, M.; Eriksson, M.; Grundy, S. M.; Kastelein, J. J. P.; Komajda, M., et al. Effects of torcetrapib in patients at high risk for coronary events. N. Engl. J. Med. 357:2109–2122; 2007. [71] AIM-HIGH Investigators; Boden, W. E.; Probstfield, J. L., et al. Niacin in patients with low HDL cholesterol levels receiving intensive statin therapy. N. Engl. J. Med. 365:2255–2267; 2011. [72] Liu, L.; Wen, T.; Zheng, X. y.; Yang, D. G.; Zhao, S. P.; Xu, D. Y., et al. Remnantlike particles accelerate endothelial progenitor cells senescence and induce cellular dysfunction via an oxidative mechanism. Atherosclerosis 202: 405–414; 2009. [73] Miller-Kasprzak, E.; Bogdanski, P.; Pupek-Musialik, D.; Jagodzinski, P. P. Insulin resistance and oxidative stress influence colony-forming unitendothelial cells capacity in obese patients. Obesity 19:736–742; 2011. [74] Xiao, Q.; Kiechl, S.; Patel, S.; Oberhollenzer, F.; Weger, S.; Mayr, A., et al. Endothelial progenitor cells, cardiovascular risk factors, cytokine levels and atherosclerosis: results from a large population-based study. PLoS ONE : e975; 2007. [75] Li, T. S.; Kubo, M.; Ueda, K.; Murakami, M.; Ohshima, M.; Kobayashi, T., et al. Identification of risk factors related to poor angiogenic potency of bone marrow cells from different patients. Circulation 120:S255–S261; 2009. [76] Guo, W. X.; Yang, Q. D.; Liu, Y. H.; Xie, X. Y.; Wang, M.; Niu, R. C. Palmitic and linoleic acids impair endothelial progenitor cells by inhibition of Akt/eNOS pathway. Arch. Med. Res. 39:434–442; 2008. [77] Vasa, M.; Fichtlscherer, S.; Aicher, A.; Adler, K.; Urbich, C.; Martin, H., et al. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circ. Res. :e1–e7; 2001. [78] Pirro, M.; Schillaci, G.; Paltriccia, R.; Bagaglia, F.; Menecali, C.; Mannarino, M. R., et al. Increased ratio of CD31 þ /CD42− microparticles to endothelial progenitors as a novel marker of atherosclerosis in hypercholesterolemia. Arterioscler. Thromb. Vasc. Biol. 26:2530–2535; 2006. [79] Croce, G.; Passacquale, G.; Necozione, S.; Ferri, C.; Desideri, G. Nonpharmacological treatment of hypercholesterolemia increases circulating endothelial progenitor cell population in adults. Arterioscler. Thromb. Vasc. Biol. :e38–e39; 2006.

259

[80] Tirziu, D.; Moodie, K. L.; Zhuang, Z. W.; Singer, K.; Helisch, A.; Dunn, J. F., et al. Delayed arteriogenesis in hypercholesterolemic mice. Circulation 112:2501–2509; 2005. [81] Czepluch, F. S.; Bergler, A.; Waltenberger, J. Hypercholesterolaemia impairs monocyte function in CAD patients. J. Intern. Med. 261:201–204; 2007. [82] Haddad, P.; Dussault, S.; Groleau, J.; Turgeon, J.; Maingrette, F.; Rivard, A. Nox2-derived reactive oxygen species contribute to hypercholesterolemiainduced inhibition of neovascularization: effects on endothelial progenitor cells and mature endothelial cells. Atherosclerosis 217:340–349; 2011. [83] Avraham-Davidi, I.; Ely, Y.; Pham, V. N.; Castranova, D.; Grunspan, M.; Malkinson, G., et al. ApoB-containing lipoproteins regulate angiogenesis by modulating expression of VEGF receptor 1 18:967–973; 2012Nat. Med. 18:967–973; 2012. [84] Tie, G.; Yan, J.; Yang, Y.; Park, B. D.; Messina, J. A.; Raffai, R. L., et al. Oxidized low-density lipoprotein induces apoptosis in endothelial progenitor cells by inactivating the phosphoinositide 3-kinase/Akt pathway. J. Vasc. Res. 47:519–530; 2010. [85] Imanishi, T.; Moriwaki, C.; Hano, T.; Nishio, I. Endothelial progenitor cell senescence is accelerated in both experimental hypertensive rats and patients with essential hypertension. J. Hypertens 23:1831–1837; 2005. [86] Umemura, T.; Soga, J.; Hidaka, T.; Takemoto, H.; Nakamura, S.; Jitsuiki, D., et al. Aging and hypertension are independent risk factors for reduced number of circulating endothelial progenitor cells. Am. J. Hypertens 21:1203–1209; 2008. [87] Delva, P.; Degan, M.; Vallerio, P.; Arosio, E.; Minuz, P.; Amen, G., et al. Endothelial progenitor cells in patients with essential hypertension. J. Hypertens. 25:127–132; 2007. [88] Endtmann, C.; Ebrahimian, T.; Czech, T.; Arfa, O.; Laufs, U.; Fritz, M., et al. Angiotensin II impairs endothelial progenitor cell number and function in vitro and in vivo. Hypertension 58:394–403; 2011. [89] Thum, T.; Schmitter, K.; Fleissner, F.; Wiebking, V.; Dietrich, B.; Widder, J. D., et al. Impairment of endothelial progenitor cell function and vascularization capacity by aldosterone in mice and humans. Eur. Heart J. 32:1275–1286; 2011. [90] Chen, D. D.; Dong, Y. G.; Yuan, H.; Chen, A. F. Endothelin 1 activation of endothelin A receptor/NADPH oxidase pathway and diminished antioxidants critically contribute to endothelial progenitor cell reduction and dysfunction in salt-sensitive hypertension. Hypertension 59:1037–1043; 2012. [91] Michel, F.; Ambroisine, M. L.; Duriez, M.; Delcayre, C.; Levy, B. I.; Silvestre, J. S. Aldosterone enhances ischemia-induced neovascularization through angiotensin II-dependent pathway. Circulation 109:1933–1937; 2004. [92] Munk, V. C.; Sanchez de Miguel, L.; Petrimpol, M.; Butz, N.; Banfi, A.; Eriksson, U., et al. Angiotensin II induces angiogenesis in the hypoxic adult mouse heart in vitro through an AT2-B2 receptor pathway. Hypertension 49:1178–1185; 2007. [93] Padfield, G. J.; Tura, O.; Haeck, M. L. A.; Short, A.; Freyer, E.; Barclay, G. R., et al. Circulating endothelial progenitor cells are not affected by acute systemic inflammation. Am. J. Physiol. Heart Circ. Physiol. 298:H2054–H2061; 2010. [94] Grisar, J.; Aletaha, D.; Steiner, C. W.; Kapral, T.; Steiner, S.; Seidinger, D., et al. Depletion of endothelial progenitor cells in the peripheral blood of patients with rheumatoid arthritis. Circulation 111:204–211; 2005. [95] Fujii, H.; Li, S. H.; Szmitko, P. E.; Fedak, P. W. M.; Verma, S. C-reactive protein alters antioxidant defenses and promotes apoptosis in endothelial progenitor cells. Arterioscler. Thromb. Vasc. Biol. 26:2476–2482; 2006. [96] Seeger, F. H.; Haendeler, J.; Walter, D. H.; Rochwalsky, U.; Reinhold, J.; Urbich, C., et al. p38 mitogen-activated protein kinase downregulates endothelial progenitor cells. Circulation 111:1184–1191; 2005. [97] Verma, S.; Kuliszewski, M. A.; Li, S. H.; Szmitko, P. E.; Zucco, L.; Wang, C. H., et al. C-reactive protein attenuates endothelial progenitor cell survival, differentiation, and function: further evidence of a mechanistic link between C-reactive protein and cardiovascular disease. Circulation 109:2058–2067; 2004. [98] Desouza, C. V.; Hamel, F. G.; Bidasee, K.; O'Connell, K. Role of inflammation and insulin resistance in endothelial progenitor cell dysfunction. Diabetes 60:1286–1294; 2011. [99] Aurora, A. B.; Biyashev, D.; Mirochnik, Y.; Zaichuk, T. A.; Sanchez-Martinez, C.; Renault, M. A., et al. NF-κB balances vascular regression and angiogenesis via chromatin remodeling and NFAT displacement. Blood 116:475–484; 2010. [100] Hasegawa, Y.; Saito, T.; Ogihara, T.; Ishigaki, Y.; Yamada, T.; Imai, J., et al. Blockade of the nuclear factor-κB pathway in the endothelium prevents insulin resistance and prolongs life spans/clinical perspective. Circulation 125:1122–1133; 2012. [101] Stabile, E.; Burnett, M. S.; Watkins, C.; Kinnaird, T.; Bachis, A.; la Sala, A., et al. Impaired arteriogenic response to acute hindlimb ischemia in CD4-knockout mice. Circulation 108:205–210; 2003. [102] Wu, Y.; Ip, J. E.; Huang, J.; Zhang, L.; Matsushita, K.; Liew, C. C., et al. Essential role of ICAM-1/CD18 in mediating EPC recruitment, angiogenesis, and repair to the infarcted myocardium. Circ. Res. 99:315–322; 2006. [103] Yoon, C. H.; Hur, J.; Oh, I. Y.; Park, K. W.; Kim, T. Y.; Shin, J. H., et al. Intercellular adhesion molecule-1 is upregulated in ischemic muscle, which mediates trafficking of endothelial progenitor cells. Arterioscler. Thromb. Vasc. Biol. 26:1066–1072; 2006. [104] Heil, M.; Ziegelhoeffer, T.; Pipp, F.; Kostin, S.; Martin, S.; Clauss, M., et al. Blood monocyte concentration is critical for enhancement of collateral artery growth. Am. J. Physiol. Heart Circ. Physiol 283:H2411–H2419; 2002. [105] Schweighofer, B.; Testori, J.; Sturtzel, C.; Sattler, S.; Mayer, H.; Wagner, O., et al. The VEGF-induced transcriptional response comprises gene clusters at

260

[106] [107]

[108]

[109]

[110]

[111]

[112]

[113]

[114]

[115]

[116] [117]

[118]

[119]

[120]

[121]

[122]

[123]

[124]

[125]

[126]

[127]

[128]

[129]

[130]

R.M. Cubbon et al. / Free Radical Biology and Medicine 60 (2013) 246–263

the crossroad of angiogenesis and inflammation. Thromb. Haemostasis 102:544–554; 2009. Silvestre, J. S.; Mallat, Z.; Tedgui, A.; Levy, B. I. Post-ischaemic neovascularization and inflammation. Cardiovasc. Res. 78:242–249; 2008. Chang, J.; Li, Y.; Huang, Y.; Lam, K. S. L.; Hoo, R. L. C.; Wong, W. T., et al. Adiponectin prevents diabetic premature senescence of endothelial progenitor cells and promotes endothelial repair by suppressing the p38 MAP kinase/p16INK4A signaling pathway. Diabetes 59:2949–2959; 2010. Yang, H.; Zhang, R.; Mu, H.; Li, M.; Yao, Q.; Chen, C. Adiponectin promotes endothelial cell differentiation from human peripheral CD14 þ monocytes in vitro. J. Cell. Mol. Med 10:459–469; 2006. Huang, P. H.; Chen, J. S.; Tsai, H. Y.; Chen, Y. H.; Lin, F. Y.; Leu, H. B., et al. Globular adiponectin improves high glucose-suppressed endothelial progenitor cell function through endothelial nitric oxide synthase dependent mechanisms. J. Mol. Cell. Cardiol. 51:109–119; 2011. Ouchi, N.; Kobayashi, H.; Kihara, S.; Kumada, M.; Sato, K.; Inoue, T., et al. Adiponectin stimulates angiogenesis by promoting cross-talk between AMPactivated protein kinase and Akt signaling in endothelial cells. J. Biol. Chem. 279:1304–1309; 2004. Sharma, K.; RamachandraRao, S.; Qiu, G.; Usui, H. K.; Zhu, Y.; Dunn, S. R., et al. Adiponectin regulates albuminuria and podocyte function in mice. J. Clin. Invest. 118:1645–1656; 2008. Schroeter, M. R.; Leifheit, M.; Sudholt, P.; Heida, N. M.; Dellas, C.; Rohm, I., et al. Leptin enhances the recruitment of endothelial progenitor cells into neointimal lesions after vascular injury by promoting integrin-mediated adhesion. Circ. Res. 103:536–544; 2008. Wolk, R.; Deb, A.; Caplice, N. M.; Somers, V. K. Leptin receptor and functional effects of leptin in human endothelial progenitor cells. Atherosclerosis 183:131–139; 2005. Schroeter, M. R.; Stein, S.; Heida, N. M.; Leifheit-Nestler, M.; Cheng, I. F.; Gogiraju, R., et al. Leptin promotes the mobilization of vascular progenitor cells and neovascularization by NOX2-mediated activation of MMP9. Cardiovasc. Res. 93:170–180; 2012. Cao, R.; Brakenhielm, E.; Wahlestedt, C.; Thyberg, J.; Cao, Y. Leptin induces vascular permeability and synergistically stimulates angiogenesis with FGF-2 and VEGF. Proc. Natl. Acad. Sci. USA 98:6390–6395; 2001. Bouloumie, A.; Drexler, H. C. A.; Lafontan, M.; Busse, R Leptin, the product of Ob gene, promotes angiogenesis. Circ. Res. 83:1059–1066; 1998. Aronis, K.; Diakopoulos, K.; Fiorenza, C.; Chamberland, J.; Mantzoros, C. Leptin administered in physiological or pharmacological doses does not regulate circulating angiogenesis factors in humans. Diabetologia 54:2358–2367; 2011. Sun, Y.; Chen, S.; Song, G.; Ren, L.; Wei, L.; Liu, N., et al. Effect of visfatin on the function of endothelial progenitor cells in high-fat-fed obese rats and investigation of its mechanism of action. Int. J. Mol. Med 30:628; 2012. Vallejo, S.; Romacho, T.; Angulo, J.; Villalobos, L. A.; Cercas, E.; Leivas, A., et al. Visfatin impairs endothelium-dependent relaxation in rat and human mesenteric microvessels through nicotinamide phosphoribosyltransferase activity. PLoS ONE 6; 2011e27299 6; 2011. Adya, R.; Tan, B. K.; Punn, A.; Chen, J.; Randeva, H. S. Visfatin induces human endothelial VEGF and MMP-2/9 production via MAPK and PI3K/Akt signalling pathways: novel insights into visfatin-induced angiogenesis. Cardiovasc. Res. 78:356–365; 2008. Park, J. W.; Kim, W. H.; Shin, S. H.; Kim, J. Y.; Yun, M. R.; Park, K. J., et al. Visfatin exerts angiogenic effects on human umbilical vein endothelial cells through the mTOR signaling pathway. Biochim. Biophys. Acta 1813:763–771; 2011. Bae, Y. H.; Park, H. J.; Kim, S. R.; Kim, J. Y.; Kang, Y.; Kim, J. A., et al. Notch1 mediates visfatin-induced FGF-2 up-regulation and endothelial angiogenesis. Cardiovasc. Res. 89:436–445; 2011. Lovren, F.; Pan, Y.; Shukla, P. C.; Quan, A.; Teoh, H.; Szmitko, P. E., et al. Visfatin activates eNOS via Akt and MAP kinases and improves endothelial cell function and angiogenesis in vitro and in vivo: translational implications for atherosclerosis. Am. J. Physiol. Endocrinol. Metab. 296:E1440–E1449; 2009. Mu, H.; Ohashi, R.; Yan, S.; Chai, H.; Yang, H.; Lin, P., et al. Adipokine resistin promotes in vitro angiogenesis of human endothelial cells. Cardiovasc. Res. 70:146–157; 2006. Cho, Y.; Lee, S. E.; Lee, H. C.; Hur, J.; Lee, S.; Youn, S. W., et al. Adipokine resistin is a key player to modulate monocytes, endothelial cells, and smooth muscle cells, leading to progression of atherosclerosis in rabbit carotid artery. J. Am. Coll. Cardiol. 57:99–109; 2011. Schneider, D. J. Abnormalities of coagulation, platelet function, and fibrinolysis associated with syndromes of insulin resistance. Coron. Artery Dis. 16:473–476; 2005. Mertens, I.; Verrijken, A.; Michiels, J. J.; Van der Planken, M.; Ruige, J. B.; Van Gaal, L. F. Among inflammation and coagulation markers, PAI-1 is a true component of the metabolic syndrome. Int. J. Obes. 30:1308–1314; 2006. Ohki, M.; Ohki, Y.; Ishihara, M.; Nishida, C.; Tashiro, Y.; Akiyama, H., et al. Tissue type plasminogen activator regulates myeloid-cell dependent neoangiogenesis during tissue regeneration. Blood 115:4302–4312; 2010. Heymans, S.; Luttun, A.; Nuyens, D.; Theilmeier, G.; Creemers, E.; Moons, L., et al. Inhibition of plasminogen activators or matrix metalloproteinases prevents cardiac rupture but impairs therapeutic angiogenesis and causes cardiac failure. Nat. Med. 5:1135–1142; 1999. Bajou, K.; Noel, A.; Gerard, R. D.; Masson, V.; Brunner, N.; Holst-Hansen, C., et al. Absence of host plasminogen activator inhibitor 1 prevents cancer invasion and vascularization. Nat. Med. 4:923–928; 1998.

[131] Starke, R. D.; Ferraro, F.; Paschalaki, K. E.; Dryden, N. H.; McKinnon, T. A. J.; Sutton, R. E., et al. Endothelial von Willebrand factor regulates angiogenesis. Blood 117:1071–1080; 2011. [132] Alzahrani, S. H.; Ajjan, R. A. Coagulation and fibrinolysis in diabetes. Diabetes Vasc. Dis. Res. 7:260–273; 2010. [133] Sahni, A.; Khorana, A. A.; Baggs, R. B.; Peng, H.; Francis, C. W. FGF-2 binding to fibrin(ogen) is required for augmented angiogenesis. Blood 107:126–131; 2006. [134] Abe, K.; Shoji, M.; Chen, J.; Bierhaus, A.; Danave, I.; Micko, C., et al. Regulation of vascular endothelial growth factor production and angiogenesis by the cytoplasmic tail of tissue factor. Proc. Natl. Acad. Sci. USA 96:8663–8668; 1999. [135] Nurden, A. T. Platelets, inflammation and tissue regeneration. Thromb. Haemostasis 105:S13–S33; 2011. [136] Nuzzolo, E. R.; Iachininoto, M. G.; Teofili, L. Endothelial progenitor cells and thrombosis. Thromb. Res. 129:309–313; 2012. [137] Chen, Y. H.; Lin, S. J.; Lin, F. Y.; Wu, T. C.; Tsao, C. R.; Huang, P. H., et al. High glucose impairs early and late endothelial progenitor cells by modifying nitric oxide related but not oxidative stress-mediated mechanisms. Diabetes 56:1559–1568; 2007. [138] Larger, E.; Marre, M.; Corvol, P.; Gasc, J. M. Hyperglycemia-induced defects in angiogenesis in the chicken chorioallantoic membrane model. Diabetes 53:752–761; 2004. [139] Chen, J. X.; Stinnett, A. Disruption of Ang-1/Tie-2 signaling contributes to the impaired myocardial vascular maturation and angiogenesis in type II diabetic mice. Arterioscler. Thromb. Vasc. Biol 28:1606–1613; 2008. [140] Yao, D.; Taguchi, T.; Matsumura, T.; Pestell, R.; Edelstein, D.; Giardino, I., et al. High glucose increases angiopoietin-2 transcription in microvascular endothelial cells through methylglyoxal modification of mSin3A. J. Biol. Chem. 282:31038–31045; 2007. [141] Hammes, H. P.; Lin, J.; Wagner, P.; Feng, Y.; vom Hagen, F.; Krzizok, T., et al. Angiopoietin-2 causes pericyte dropout in the normal retina: evidence for involvement in diabetic retinopathy. Diabetes 53:1104–1110; 2004. [142] Singh, H.; Brindle, N. P. J.; Zammit, V. A High glucose and elevated fatty acids suppress signaling by the endothelium protective ligand angiopoietin-1. Microvasc. Res 79:121–127; 2010. [143] Geraldes, P.; Hiraoka-Yamamoto, J.; Matsumoto, M.; Clermont, A.; Leitges, M.; Marette, A., et al. Activation of PKC-[delta] and SHP-1 by hyperglycemia causes vascular cell apoptosis and diabetic retinopathy. Nat. Med. 15:1298–1306; 2009. [144] Tanii, M.; Yonemitsu, Y.; Fujii, T.; Shikada, Y.; Kohno, R. i.; Onimaru, M., et al. Diabetic microangiopathy in ischemic limb is a disease of disturbance of the platelet-derived growth factor-BB/protein kinase C axis but not of impaired expression of angiogenic factors. Circ. Res. 98:55–62; 2006. [145] Fadini, G.; Pucci, L.; Vanacore, R.; Baesso, I.; Penno, G.; Balbarini, A., et al. Glucose tolerance is negatively associated with circulating progenitor cell levels. Diabetologia 50:2156–2163; 2007. [146] Loomans, C. J. M.; de Koning, E. J. P.; Staal, F. J. T.; Rookmaaker, M. B.; Verseyden, C.; de Boer, H. C., et al. Endothelial progenitor cell dysfunction: a novel concept in the pathogenesis of vascular complications of type 1 diabetes. Diabetes 53:195–199; 2004. [147] Sibal, L.; Aldibbiat, A.; Agarwal, S.; Mitchell, G.; Oates, C.; Razvi, S., et al. Circulating endothelial progenitor cells, endothelial function, carotid intimamedia thickness and circulating markers of endothelial dysfunction in people with type 1 diabetes without macrovascular disease or microalbuminuria. Diabetologia 52:1464–1473; 2009. [148] Ebrahimian, T. G.; Heymes, C.; You, D.; Blanc-Brude, O.; Mees, B.; Waeckel, L., et al. NADPH oxidase-derived overproduction of reactive oxygen species impairs postischemic neovascularization in mice with type 1 diabetes. Am. J. Pathol. 169:719–728; 2006. [149] Salis, M.; Graiani, G.; Desortes, E.; Caldwell, R.; Madeddu, P.; Emanueli, C. Nerve growth factor supplementation reverses the impairment, induced by type 1 diabetes, of hindlimb post-ischaemic recovery in mice. Diabetologia 47:1055–1063; 2004. [150] Luo, J. D.; Hu, T. P.; Wang, L.; Chen, M. S.; Liu, S. M.; Chen, A. F. Sonic hedgehog improves delayed wound healing via enhancing cutaneous nitric oxide function in diabetes. Am. J. Physiol. Endocrinol. Metab. 297:E525–E531; 2009. [151] Tie, L.; Li, X. J.; Wang, X.; Channon, K. M.; Chen, A. F.; Endothelium-specific, GTP cyclohydrolase I overexpression accelerates refractory wound healing by suppressing oxidative stress in diabetes. Am. J. Physiol. Endocrinol. Metab. 296: E1423–E1429; 2009. [152] van Golde, J. M.; Ruiter, M. S.; Schaper, N. C.; Voo, S.; Waltenberger, J.; Backes, W. H., et al. Impaired collateral recruitment and outward remodeling in experimental diabetes. Diabetes 57:2818–2823; 2008. [153] Kinnunen, K.; Puustjarvi, T.; Terasvirta, M.; Nurmenniemi, P.; Heikura, T.; Laidinen, S., et al. Differences in retinal neovascular tissue and vitreous humour in patients with type 1 and type 2 diabetes. Br. J. Ophthalmol. 93:1109–1115; 2009. [154] Jonas, M.; Edelman, E. R.; Groothuis, A.; Baker, A. B.; Seifert, P.; Rogers, C. Vascular neointimal formation and signaling pathway activation in response to stent injury in insulin-resistant and diabetic animals. Circ. Res. 725–733; 2005. [155] Yan, J.; Tie, G.; Park, B.; Yan, Y.; Nowicki, P. T.; Messina, L. M. Recovery from hind limb ischemia is less effective in type 2 than in type 1 diabetic mice: roles of endothelial nitric oxide synthase and endothelial progenitor cells. J. Vasc. Surg. 50:1412–1422; 2009.

R.M. Cubbon et al. / Free Radical Biology and Medicine 60 (2013) 246–263

[156] Cubbon, R. M.; Viswambharan, H.; Baliga, V.; Yuldasheva, N.; Stephen, S.; Askham, J., et al. Akt-gene-based restoration of Akt activity in endothelial progenitor cells from human subjects at high cardiovascular risk rescues vascular reparative capacity. Heart 98:A1; 2012. [157] Belfiore, A.; Frasca, F.; Pandini, G.; Sciacca, L.; Vigneri, R. Insulin receptor isoforms and insulin receptor/insulin-like growth factor receptor hybrids in physiology and disease. Endocr. Rev. 30:586–623; 2009. [158] Kondo, T.; Vicent, D.; Suzuma, K.; Yanagisawa, M.; King, G. L.; Holzenberger, M., et al. Knockout of insulin and IGF-1 receptors on vascular endothelial cells protects against retinal neovascularization. J. Clin. Invest. 111:1835–1842; 2003. [159] Kondo, T.; Hafezi-Moghadam, A.; Thomas, K.; Wagner, D. D.; Kahn, C. R. Mice lacking insulin or insulin-like growth factor 1 receptors in vascular endothelial cells maintain normal blood–brain barrier. Biochem. Biophys. Res. Commun. 317:315–320; 2004. [160] Aghdam, S. Y.; Eming, S. A.; Willenborg, S.; Neuhaus, B.; Niessen, C. M.; Partridge, L., et al. Vascular endothelial insulin/IGF-1 signaling controls skin wound vascularization. Biochem. Biophys. Res. Commun. 421:197–202; 2012. [161] He, Z.; Opland, D. M.; Way, K. J.; Ueki, K.; Bodyak, N.; Kang, P. M., et al. Regulation of vascular endothelial growth factor expression and vascularization in the myocardium by insulin receptor and PI3K/Akt pathways in insulin resistance and ischemia. Arterioscler. Thromb. Vasc. Biol. 26:787–793; 2006. [162] Yoon, Y. s.; Uchida, S.; Masuo, O.; Cejna, M.; Park, J. S.; Gwon, H c., et al. Progressive attenuation of myocardial vascular endothelial growth factor expression is a seminal event in diabetic cardiomyopathy. Circulation 111:2073–2085; 2005. [163] Jiang, Z. Y.; He, Z.; King, B. L.; Kuroki, T.; Opland, D. M.; Suzuma, K., et al. Characterization of multiple signaling pathways of insulin in the regulation of vascular endothelial growth factor expression in vascular cells and angiogenesis. J. Biol. Chem 278:31964–31971; 2003. [164] Maeno, Y.; Li, Q.; Park, K.; Rask-Madsen, C.; Gao, B.; Matsumoto, M., et al. Inhibition of insulin signaling in endothelial cells by protein kinase Cinduced phosphorylation of p85 subunit of phosphatidylinositol 3-kinase (PI3K). J. Biol. Chem. 287:4518–4530; 2012. [165] Welsh, G. I.; Hale, L. J.; Eremina, V.; Jeansson, M.; Maezawa, Y.; Lennon, R., et al. Insulin signaling to the glomerular podocyte is critical for normal kidney function. Cell Metab. 12:329–340; 2010. [166] Han, S.; Liang, C. P.; DeVries-Seimon, T.; Ranalletta, M.; Welch, C. L.; CollinsFletcher, K., et al. Macrophage insulin receptor deficiency increases ER stress-induced apoptosis and necrotic core formation in advanced atherosclerotic lesions. Cell Metab. 3:257–266; 2006. [167] Baumgartl, J.; Baudler, S.; Scherner, M.; Babaev, V.; Makowski, L.; Suttles, J., et al. Myeloid lineage cell-restricted insulin resistance protects apolipoproteinE-deficient mice against atherosclerosis. Cell Metab. 3: 247–256; 2006. [168] Waltenberger, J.; Lange, J.; Kranz, A. Vascular endothelial growth factor-A induced chemotaxis of monocytes is attenuated in patients with diabetes mellitus. Circulation 102:185–190; 2000. [169] Tchaikovski, V.; Olieslagers, S.; Bohmer, F.; Waltenberger, J. Diabetes mellitus activates signal transduction pathways resulting in vascular endothelial growth factor resistance of human monocytes. Circulation 120:150–159; 2009. [170] Olieslagers, S.; Pardali, E.; Tchaikovski, V.; ten Dijke, P.; Waltenberger, J. TGFβ1/ALK5-induced monocyte migration involves PI3K and p38 pathways and is not negatively affected by diabetes mellitus. Cardiovasc. Res. 91:510–518; 2011. [171] Gehart, H.; Kumpf, S.; Ittner, A.; Ricci, R. MAPK signalling in cellular metabolism: stress or wellness? EMBO Rep. 11:834–840; 2010. [172] Lee, Y. H.; Giraud, J.; Davis, R. J.; White, M. F. c-Jun N-terminal kinase (JNK) mediates feedback inhibition of the insulin signaling cascade. J. Biol. Chem. 278:2896–2902; 2003. [173] Miele, C.; Riboulet, A.; Maitan, M. A.; Oriente, F.; Romano, C.; Formisano, P., et al. Human glycated albumin affects glucose metabolism in L6 skeletal muscle cells by impairing insulin-induced insulin receptor substrate (IRS) signaling through a protein kinase C-alpha mediated mechanism. J. Biol. Chem. 278:47376–47387; 2003. [174] Gao, Z.; Hwang, D.; Bataille, F.; Lefevre, M.; York, D.; Quon, M. J., et al. Serine phosphorylation of insulin receptor substrate 1 by inhibitor κB kinase complex. J. Biol. Chem. 277:48115–48121; 2002. [175] Gao, Z.; Zhang, X.; Zuberi, A.; Hwang, D.; Quon, M. J.; Lefevre, M., et al. Inhibition of insulin sensitivity by free fatty acids requires activation of multiple serine kinases in 3T3-L1 adipocytes. Mol. Endocrinol. 18:2024–2034; 2004. [176] Kim, J.; Yeh, D. C.; Ver, M.; Li, Y.; Carranza, A.; Conrads, T. P., et al. Phosphorylation of Ser24 in the pleckstrin homology domain of insulin receptor substrate-1 by mouse pelle-like kinase/interleukin-1 receptorassociated kinase. J. Biol. Chem. 280:23173–23183; 2005. [177] Nguyen, M. T. A.; Satoh, H.; Favelyukis, S.; Babendure, J. L.; Imamura, T.; Sbodio, J. I., et al. JNK and tumor necrosis factor-alpha mediate free fatty acid-induced insulin resistance in 3T3-L1 adipocytes. J. Biol. Chem. 280:35361–35371; 2005. [178] Li, Y.; Soos, T. J.; Li, X.; Wu, J.; DeGennaro, M.; Sun, X., et al. Protein kinase C-theta inhibits insulin signaling by phosphorylating IRS1 at Ser1101. J. Biol. Chem 279:45304–45307; 2004. [179] Yuan, M.; Konstantopoulos, N.; Lee, J.; Hansen, L.; Li, Z. W.; Karin, M., et al. Reversal of obesity- and diet-induced insulin resistance with salicylates or targeted disruption of IKK-beta. Science 293:1673–1677; 2001.

261

[180] Hirosumi, J.; Tuncman, G.; Chang, L.; Gorgun, C. Z.; Uysal, K. T.; Maeda, K., et al. A central role for JNK in obesity and insulin resistance. Nature 420:333–336; 2002. [181] Kubota, T.; Kubota, N.; Moroi, M.; Terauchi, Y.; Kobayashi, T.; Kamata, K., et al. Lack of insulin receptor substrate-2 causes progressive neointima formation in response to vessel injury. Circulation 107:3073–3080; 2003. [182] Symons, J. D.; McMillin, S. L.; Riehle, C.; Tanner, J.; Palionyte, M.; Hillas, E., et al. Contribution of insulin and Akt1 signaling to endothelial nitric oxide synthase in the regulation of endothelial function and blood pressure. Circ. Res. 104:1085–1094; 2009. [183] Kito, T.; Shibata, R.; Kondo, M.; Yamamoto, T.; Suzuki, H.; Ishii, M., et al. Nifedipine ameliorates ischemia-induced revascularization in diet-induced obese mice. Am. J. Hypertens 25:401–406; 2012. [184] Wang, C. Y.; Kim, H. H.; Hiroi, Y.; Sawada, N.; Salomone, S.; Benjamin, L. E., et al. Obesity increases vascular senescence and susceptibility to ischemic injury through chronic activation of Akt and mTOR. Sci. Signaling 2:ra11; 2009. [185] Miyauchi, H.; Minamino, T.; Tateno, K.; Kunieda, T.; Toko, H.; Komuro, I. Akt negatively regulates the in vitro lifespan of human endothelial cells via a p53/p21-dependent pathway. EMBO J. 23:212–220; 2004. [186] Nishi, J. i.; Minamino, T.; Miyauchi, H.; Nojima, A.; Tateno, K.; Okada, S., et al. Vascular endothelial growth factor receptor-1 regulates postnatal angiogenesis through inhibition of the excessive activation of Akt. Circ. Res. 103: 261–268; 2008. [187] Ackah, E.; Yu, J.; Zoellner, S.; Iwakiri, Y.; Skurk, C.; Shibata, R., et al. Akt1/ protein kinase B-alpha is critical for ischemic and VEGF-mediated angiogenesis. J. Clin. Invest 115:2119–2127; 2005. [188] Fernandez-Hernando, C.; Ackah, E.; Yu, J.; Suþ írez, Y.; Murata, T.; Iwakiri, Y., et al. Loss of Akt1 leads to severe atherosclerosis and occlusive coronary artery disease. Cell Metab. 6:446–457; 2007. [189] Zhang, Q. J.; Holland, W. L.; Wilson, L.; Tanner, J. M.; Kearns, D.; Cahoon, J. M., et al. Ceramide mediates vascular dysfunction in diet-induced obesity by PP2A-mediated dephosphorylation of the eNOS-Akt complex. Diabetes 61:1848–1859; 2012. [190] Lee, J. H.; Palaia, T.; Ragolia, L. Impaired insulin-mediated vasorelaxation in diabetic Goto-Kakizaki rats is caused by impaired Akt phosphorylation. Am. J. Physiol. Cell Physiol. 296:C327–C338; 2009. [191] Jiang, B. H.; Zheng, J. Z.; Aoki, M.; Vogt, P. K. Phosphatidylinositol 3-kinase signaling mediates angiogenesis and expression of vascular endothelial growth factor in endothelial cells. Proc. Natl. Acad. Sci. USA 97:1749–1753; 2000. [192] Chavakis, E.; Carmona, G.; Urbich, C.; Gottig, S.; Henschler, R.; Penninger, J. M., et al. Phosphatidylinositol-3-kinase-gamma is integral to homing functions of progenitor cells. Circ. Res. 102:942–949; 2008. [193] Madeddu, P.; Kraenkel, N.; Barcelos, L. S.; Siragusa, M.; Campagnolo, P.; Oikawa, A., et al. Phosphoinositide 3-kinase-gamma gene knockout impairs postischemic neovascularization and endothelial progenitor cell functions. Arterioscler. Thromb. Vasc. Biol. 28:68–76; 2008. [194] Schleicher M., Yu J., Murata T., Derakhshan B., Atochin D., Qian L., et al. The Akt1–eNOS axis illustrates the specificity of kinase–substrate relationships in vivoSci. Signaling2ra41; 2009. [195] Murohara, T.; Asahara, T.; Silver, M.; Bauters, C.; Masuda, H.; Kalka, C., et al. Nitric oxide synthase modulates angiogenesis in response to tissue ischemia. J. Clin. Invest. 101:2567–2578; 1998. [196] Aicher, A.; Heeschen, C.; Mildner-Rihm, C.; Urbich, C.; Ihling, C.; TechnauIhling, K., et al. Essential role of endothelial nitric oxide synthase for mobilization of stem and progenitor cells. Nat. Med. 9:1370–1376; 2003. [197] Segal, M. S.; Shah, R.; Afzal, A.; Perrault, C. M.; Chang, K.; Schuler, A., et al. Nitric oxide cytoskeletal-induced alterations reverse the endothelial progenitor cell migratory defect associated with diabetes. Diabetes 55:102–109; 2006. [198] Ceriello, A.; Motz, E. Is oxidative stress the pathogenic mechanism underlying insulin resistance, diabetes, and cardiovascular disease? The common soil hypothesis revisited Arterioscler. Thromb. Vasc. Biol. 24:816–823; 2004. [199] Giacco, F.; Brownlee, M. Oxidative stress and diabetic complications. Circ. Res. 107:1058–1070; 2010. [200] Benndorf, R. A.; Schwedhelm, E.; Gnann, A.; Taheri, R.; Kom, G.; Didie, M., et al. Isoprostanes inhibit vascular endothelial growth factor-induced endothelial cell migration, tube formation, and cardiac vessel sprouting in vitro, as well as angiogenesis in vivo via activation of the thromboxane A2 receptor: a potential link between oxidative stress and impaired angiogenesis. Circ. Res 103:1037–1046; 2008. [201] Ingram, D. A.; Krier, T. R.; Mead, L. E.; McGuire, C.; Prater, D. N.; Bhavsar, J., et al. Clonogenic endothelial progenitor cells are sensitive to oxidative stress. Stem Cells 25:297–304; 2007. [202] Sorrentino, S. A.; Bahlmann, F. H.; Besler, C.; Muller, M.; Schulz, S.; Kirchhoff, N., et al. Oxidant stress impairs in vivo reendothelialization capacity of endothelial progenitor cells from patients with type 2 diabetes mellitus: restoration by the peroxisome proliferator-activated receptor-{gamma} agonist rosiglitazone. Circulation 116:163–173; 2007. [203] Thum, T.; Fraccarollo, D.; Schultheiss, M.; Froese, S.; Galuppo, P.; Widder, J. D., et al. Endothelial nitric oxide synthase uncoupling impairs endothelial progenitor cell mobilization and function in diabetes. Diabetes 56:666–674; 2007. [204] Ceradini, D. J.; Yao, D.; Grogan, R. H.; Callaghan, M. J.; Edelstein, D.; Brownlee, M., et al. Decreasing intracellular superoxide corrects defective ischemiainduced new vessel formation in diabetic mice. J. Biol. Chem. 283:10930–10938; 2008.

262

R.M. Cubbon et al. / Free Radical Biology and Medicine 60 (2013) 246–263

[205] Schroder, K.; Kohnen, A.; Aicher, A.; Liehn, E. A.; Buchse, T.; Stein, S., et al. NADPH oxidase Nox2 is required for hypoxia-induced mobilization of endothelial progenitor cells. Circ. Res. 105:537–544; 2009. [206] Urao, N.; Inomata, H.; Razvi, M.; Kim, H. W.; Wary, K.; McKinney, R., et al. Role of Nox2-based NADPH oxidase in bone marrow and progenitor cell function involved in neovascularization induced by hindlimb ischemia. Circ. Res. 103:212–220; 2008. [207] Ushio-Fukai, M.; Tang, Y.; Fukai, T.; Dikalov, S. I.; Ma, Y.; Fujimoto, M., et al. Novel role of gp91phox-containing NAD(P)H oxidase in vascular endothelial growth factor-induced signaling and angiogenesis. Circ. Res. 91:1160–1167; 2002. [208] Serrander, L.; Cartier, L.; Bedard, K.; Banfi, B.; Lardy, B.; Plastre, O., et al. NOX4 activity is determined by mRNA levels and reveals a unique pattern of ROS generation. Biochem. J. 406:105–114; 2007. [209] Schroder, K.; Zhang, M.; Benkhoff, S.; Mieth, A.; Pliquett, R.; Kosowski, J., et al. Nox4 is a protective reactive oxygen species generating vascular NADPH oxidase. Circ. Res. 110:1217–1225; 2012. [210] Craige, S. M.; Chen, K.; Pei, Y.; Li, C.; Huang, X. Chen, et al. NADPH oxidase 4 promotes endothelial angiogenesis through endothelial nitric oxide synthase activation/clinical perspective. Circulation 124:731–740; 2011. [211] Zhang, M.; Brewer, A. C.; Schroder, K.; Santos, C. X. C.; Grieve, D. J.; Wang, M., et al. NADPH oxidase-4 mediates protection against chronic load-induced stress in mouse hearts by enhancing angiogenesis. Proc. Natl. Acad. Sci. USA 107:18121–18126; 2010. [212] Meng, D.; Mei, A.; Liu, J.; Kang, X.; Shi, X.; Qian, R., et al. NADPH oxidase 4 mediates insulin-stimulated HIF-1a and VEGF expression, and angiogenesis in vitro. PLoS ONE. e48393 ; 2012. [213] Li, J.; Wang, J. J.; Yu, Q.; Chen, K.; Mahadev, K.; Zhang, S. X. Inhibition of reactive oxygen species by lovastatin downregulates vascular endothelial growth factor expression and ameliorates blood–retinal barrier breakdown in db/db mice: role of NADPH oxidase 4. Diabetes 59:1528–1538; 2010. [214] Lener, B.; Koziel, R.; Pircher, H.; Hutter, E.; Greussing, R.; HerndlerBrandstetter, D., et al. The NADPH oxidase Nox4 restricts the replicative lifespan of human endothelial cells. Biochem. J. 423:363–374; 2009. [215] Kleinschnitz, C.; Grund, H.; Wingler, K.; Armitage, M. E.; Jones, E.; Mittal, M., et al. Post-stroke inhibition of induced NADPH oxidase type 4 prevents oxidative stress and neurodegeneration. PLoS Biol :e1000479; 2010. [216] Duncan, E. R.; Crossey, P. A.; Walker, S.; Anilkumar, N.; Poston, L.; Douglas, G., et al. Effect of endothelium-specific insulin resistance on endothelial function in vivo. Diabetes 57:3307–3314; 2008. [217] Sukumar P., Viswambharan H., Imrie H., Cubbon R.M., Yuldasheva N., Gage M., et al. Nox2 NADPH-oxidase has a critical role in insulin resistance-related endothelial cell dysfunction. Diabetes (in press). [218] Kowaltowski, A. J. de Souza-Pinto N.C., Castilho R.F., Vercesi A.E. Mitochondria and reactive oxygen species. Free Radic. Biol. Med. 47:333–343; 2009. [219] Szendroedi, J.; Phielix, E.; Roden, M. The role of mitochondria in insulin resistance and type 2 diabetes mellitus. Nat. Rev. Endocrinol. 8:92–103; 2012. [220] Hamanaka, R. B.; Chandel, N. S. Mitochondrial reactive oxygen species regulate hypoxic signaling. Curr. Opin. Cell Biol. 21:894–899; 2009. [221] Lin, X.; David, C. A.; Donnelly, J. B.; Michaelides, M.; Chandel, N. S.; Huang, X., et al. A chemical genomics screen highlights the essential role of mitochondria in HIF-1 regulation. Proc. Natl. Acad. Sci. USA 105:174–179; 2008. [222] Jung, H. J.; Shim, J. S.; Lee, J.; Song, Y. M.; Park, K. C.; Choi, S. H., et al. Terpestacin inhibits tumor angiogenesis by targeting UQCRB of mitochondrial complex III and suppressing hypoxia-induced reactive oxygen species production and cellular oxygen sensing. J. Biol. Chem. 285:11584–11595; 2010. [223] Dai, S.; He, Y.; Zhang, H.; Yu, L.; Wan, T.; Xu, Z., et al. Endothelial-specific expression of mitochondrial thioredoxin promotes ischemia-mediated arteriogenesis and angiogenesis. Arterioscler. Thromb. Vasc. Biol. 29:495–502; 2009. [224] Guzik, T. J.; West, N. E. J.; Pillai, R.; Taggart, D. P.; Channon, K. M. Nitric oxide modulates superoxide release and peroxynitrite formation in human blood vessels. Hypertension 39:1088–1094; 2002. [225] Platt, D. H.; Bartoli, M.; El-Remessy, A. B.; Al-Shabrawey, M.; Lemtalsi, T.; Fulton, D., et al. Peroxynitrite increases VEGF expression in vascular endothelial cells via STAT3. Free Radic. Biol. Med. 39:1353–1361; 2005. [226] Sasso, F. C.; Torella, D.; Carbonara, O.; Ellison, G. M.; Torella, M.; Scardone, M., et al. Increased vascular endothelial growth factor expression but impaired vascular endothelial growth factor receptor signaling in the myocardium of type 2 diabetic patients with chronic coronary heart disease. J. Am. Coll. Cardiol. 46:827–834; 2005. [227] Kajiwara, H.; Luo, Z.; Belanger, A. J.; Urabe, A.; Vincent, K. A.; Akita, G. Y., et al. A hypoxic inducible factor-1a hybrid enhances collateral development and reduces vascular leakage in diabetic rats. J. Gene Med. 11:390–400; 2009. [228] Doronzo, G.; Russo, I.; Mattiello, L.; Anfossi, G.; Bosia, A.; Trovati, M. Insulin activates vascular endothelial growth factor in vascular smooth muscle cells: influence of nitric oxide and of insulin resistance. Eur. J. Clin. Invest. 34:664–673; 2004. [229] Nagahama, R.; Matoba, T.; Nakano, K.; Kim-Mitsuyama, S.; Sunagawa, K.; Egashira, K. Nanoparticle-mediated delivery of pioglitazone enhances therapeutic neovascularization in a murine model of hindlimb ischemia. Arterioscler. Thromb. Vasc. Biol. 32:2427–2434; 2012. [230] Treins, C.; Giorgetti-Peraldi, S.; Murdaca, J.; Semenza, G. L.; Van Obberghen, E. Insulin stimulates hypoxia-inducible factor 1 through a phosphatidylinositol 3-kinase/target of rapamycin-dependent signaling pathway. J. Biol. Chem. 277:27975–27981; 2002.

[231] Poulaki, V.; Qin, W.; Joussen, A. M.; Hurlbut, P.; Wiegand, S. J.; Rudge, J., et al. Acute intensive insulin therapy exacerbates diabetic blood–retinal barrier breakdown via hypoxia-inducible factor-1a and VEGF. J. Clin. Invest. 109:805–815; 2002. [232] Yim, S.; Choi, S. M.; Choi, Y.; Lee, N.; Chung, J.; Park, H. Insulin and hypoxia share common target genes but not the hypoxia-inducible factor-1a. J. Biol. Chem. 278:38260–38268; 2003. [233] Thangarajah, H.; Yao, D.; Chang, E. I.; Shi, Y.; Jazayeri, L.; Vial, I. N., et al. The molecular basis for impaired hypoxia-induced VEGF expression in diabetic tissues. Proc. Natl. Acad. Sci. USA 106:13505–13510; 2009. [234] Chisalita, S. I.; Nitert, M. D.; Arnqvist, H. J. Characterisation of receptors for IGF-I and insulin; evidence for hybrid insulin/IGF-I receptor in human coronary artery endothelial cells. Growth Horm. IGF Res 16:258–266; 2006. [235] Chisalita, S.; Arnqvist, H. Expression and function of receptors for insulin-like growth factor-I and insulin in human coronary artery smooth muscle cells. Diabetologia 48:2155–2161; 2005. [236] Khorsandi, M. J.; Fagin, J. A.; Giannella-Neto, D.; Forrester, J. S.; Cercek, B. Regulation of insulin-like growth factor-I and its receptor in rat aorta after balloon denudation: evidence for local bioactivity. J. Clin. Invest. 90:1926–1931; 1992. [237] Cittadini, A.; Monti, M.; Castiello, M.; D'Arco, E.; Galasso, G.; Sorriento, D., et al. Insulin-like growth factor-1 protects from vascular stenosis and accelerates re-endothelialization in a rat model of carotid artery injury. J. Thromb. Haemostasis 7:1920–1928; 2009. [238] Imrie, H.; Abbas, A.; Viswambharan, H.; Rajwani, A.; Cubbon, R. M.; Gage, M., et al. Vascular insulin-like growth factor-I resistance and diet-induced obesity. Endocrinology 150:4575–4582; 2009. [239] Imrie, H.; Viswambharan, H.; Sukumar, P.; Abbas, A.; Cubbon, R. M.; Yuldasheva, N., et al. Novel role of the IGF-1 receptor in endothelial function and repair: studies in endothelium-targeted IGF-1 receptor transgenic mice. Diabetes 61:2359–2368; 2012. [240] Grant, M.; Mames, R.; Fitzgerald, C.; Ellis, E.; Aboufriekha, M.; Guy, J. Insulinlike growth factor I acts as an angiogenic agent in rabbit cornea and retina: comparative studies with basic fibroblast growth factor. Diabetologia 36:282–291; 1993. [241] Smith, L. E. H.; Shen, W.; Perruzzi, C.; Soker, S.; Kinose, F.; Xu, X., et al. Regulation of vascular endothelial growth factor-dependent retinal neovascularization by insulin-like growth factor-1 receptor. Nat. Med. 5:1390–1395; 1999. [242] Sukhanov, S.; Higashi, Y.; Shai, S. Y.; Vaughn, C.; Mohler, J.; Li, Y., et al. IGF-1 reduces inflammatory responses, suppresses oxidative stress, and decreases atherosclerosis progression in ApoE-deficient mice. Arterioscler. Thromb. Vasc. Biol. 27:2684–2690; 2007. [243] Thum, T.; Hoeber, S.; Froese, S.; Klink, I.; Stichtenoth, D. O.; Galuppo, P., et al. Age-dependent impairment of endothelial progenitor cells is corrected by growth hormone mediated increase of insulin-like growth factor-1. Circ. Res 100:434–443; 2007. [244] Hynes, B.; Kumar, A. H. S.; O'Sullivan, J.; Klein Buneker, C.; Leblond, A. L.; Weiss, S., et al. Potent endothelial progenitor cell-conditioned media-related antiapoptotic, cardiotrophic, and pro-angiogenic effects post-myocardial infarction are mediated by insulin-like growth factor-1. Eur. Heart J. 34:782–789; 2013. [245] Urbich, C.; Aicher, A.; Heeschen, C.; Dernbach, E.; Hofmann, W. K.; Zeiher, A. M., et al. Soluble factors released by endothelial progenitor cells promote migration of endothelial cells and cardiac resident progenitor cells. J. Mol. Cell. Cardiol 39:733–742; 2005. [246] Johansson, G. S.; Chisalita, S. I.; Arnqvist, H. J. Human microvascular endothelial cells are sensitive to IGF-I but resistant to insulin at the receptor level. Mol. Cell. Endocrinol 296:58–63; 2008. [247] Li, G.; Barrett, E. J.; Wang, H.; Chai, W.; Liu, Z. Insulin at physiological concentrations selectively activates insulin but not insulin-like growth factor I (IGF-I) or insulin/IGF-I hybrid receptors in endothelial cells. Endocrinology 146:4690–4696; 2005. [248] Sherajee, S. J.; Fujita, Y.; Rafiq, K.; Nakano, D.; Mori, H.; Masaki, T., et al. Aldosterone induces vascular insulin resistance by increasing insulin-like growth factor-1 receptor and hybrid receptor. Arterioscler. Thromb. Vasc. Biol. 32:257–263; 2012. [249] Abbas, A.; Imrie, H.; Viswambharan, H.; Sukumar, P.; Rajwani, A.; Cubbon, R. M., et al. The insulin-like growth factor-1 receptor is a negative regulator of nitric oxide bioavailability and insulin sensitivity in the endothelium. Diabetes 60:2169–2178; 2011. [250] Senthil, D.; Ghosh Choudhury, G.; Bhandari, B. K.; Kasinath, B. S. The type 2 vascular endothelial growth factor receptor recruits insulin receptor substrate-1 in its signalling pathway. Biochem. J. 368:49–56; 2002. [251] Feliers, D.; Chen, X.; Akis, N.; Choudhury, G. G.; Madaio, M.; Kasinath, B. S. VEGF regulation of endothelial nitric oxide synthase in glomerular endothelial cells. Kidney Int. 68:1648–1659; 2005. [252] Berdugo, M.; Andrieu-Soler, C.; Doat, M.; Courtois, Y.; BenEzra, D.; BeharCohen, F. Downregulation of IRS-1 expression causes inhibition of corneal angiogenesis. Invest. Ophthalmol. Visual Sci. 46:4072–4078; 2005. [253] Kling, J. Safety signal dampens reception for mipomersen antisense. Nat. Biotech. 28:295–297; 2010. [254] Tsukagawa E., Adachi H., Hirai Y., Enomoto M., Fukami A., Ogata K., et al. Independent association of elevated serum HGF levels with development of insulin resistance in a 10-year prospective study. Clin. Endocrinol.. (in press); . [255] Fafalios, A.; Ma, J.; Tan, X.; Stoops, J.; Luo, J.; DeFrances, M. C., et al. A hepatocyte growth factor receptor (Met)-insulin receptor hybrid governs hepatic glucose metabolism. Nat. Med. 17:1577–1584; 2011.

R.M. Cubbon et al. / Free Radical Biology and Medicine 60 (2013) 246–263

[256] Breen, D. M.; Chan, K. K.; Dhaliwall, J. K.; Ward, M. R.; Al Koudsi, N.; Lam, L., et al. Insulin increases reendothelialization and inhibits cell migration and neointimal growth after arterial injury. Arterioscler. Thromb. Vasc. Biol. 29:1060–1066; 2009. [257] Dong, L.; Kang, L.; Ding, L.; Chen, Q.; Bai, J.; Gu, R., et al. Insulin modulates ischemia-induced endothelial progenitor cell mobilization and neovascularization in diabetic mice. Microvasc. Res. 82:227–236; 2011. [258] Langer, A.; Rogowski, W. Systematic review of economic evaluations of human cell-derived wound care products for the treatment of venous leg and diabetic foot ulcers. BMC Health Serv. Res. 9:115; 2009. [259] Cheng, C. F.; Sahu, D.; Tsen, F.; Zhao, Z.; Fan, J.; Kim, R., et al. A fragment of secreted Hsp90α carries properties that enable it to accelerate effectively both acute and diabetic wound healing in mice. J. Clin. Invest. 121:4348–4361; 2011. [260] Kusumanto, Y.; van Weel, V.; Mulder, N.; Smit, A.; van den Dungen, J.; Hooymans, J., et al. Treatment with intramuscular vascular endothelial growth factor gene compared with placebo for patients with diabetes mellitus and critical limb ischemia: a double-blind randomized trial. Hum. Gene Ther. 17:683–691; 2006. [261] Cao, L.; Arany, P. R.; Kim, J.; Rivera-Feliciano, J.; Wang, Y. S.; He, Z., et al. Modulating Notch signaling to enhance neovascularization and reperfusion in diabetic mice. Biomaterials 31:9048–9056; 2010. [262] Al Haj Zen, A.; Oikawa, A.; Bazan-Peregrino, M.; Meloni, M.; Emanueli, C.; Madeddu, P Inhibition of delta-like-4 mediated signaling impairs reparative angiogenesis after ischemia. Circ. Res. 107:283–293; 2010. [263] Nishimura, Y.; Ii, M.; Qin, G.; Hamada, H.; Asai, J.; Takenaka, H., et al. CXCR4 antagonist AMD3100 accelerates impaired wound healing in diabetic mice. J. Invest. Dermatol. 132:711–720; 2012. [264] Jin, H. R.; Kim, W. J.; Song, J. S.; Piao, S.; Choi, M. J.; Tumurbaatar, M., et al. Intracavernous delivery of a designed angiopoietin-1 variant rescues erectile function by enhancing endothelial regeneration in the streptozotocininduced diabetic mouse. Diabetes 60:969–980; 2011. [265] Cho, C. H.; Sung, H. K.; Kim, K. T.; Cheon, H. G.; Oh, G. T.; Hong, H. J., et al. COMP-angiopoietin-1 promotes wound healing through enhanced angiogenesis, lymphangiogenesis, and blood flow in a diabetic mouse model. Proc. Natl. Acad. Sci. USA 103:4946–4951; 2006. [266] Gupta, R.; Tongers, J.; Losordo, D. W. Human studies of angiogenic gene therapy. Circ. Res. 105:724–736; 2009.

263

[267] Chen, J. X.; Stinnett, A. Ang-1 gene therapy inhibits hypoxia-inducible factor1a (HIF-1a)-prolyl-4-hydroxylase-2, stabilizes HIF-1a expression, and normalizes immature vasculature in db/db mice. Diabetes 57:3335–3343; 2008. [268] Samuel, S. M.; Akita, Y.; Paul, D.; Thirunavukkarasu, M.; Zhan, L.; Sudhakaran, P. R., et al. Coadministration of adenoviral vascular endothelial growth factor and angiopoietin-1 enhances vascularization and reduces ventricular remodeling in the infarcted myocardium of type 1 diabetic rats. Diabetes 59:51–60; 2010. [269] Botusan, I. R.; Sunkari, V. G.; Savu, O.; Catrina, A. I.; Grunler, J.; Lindberg, S., et al. Stabilization of HIF-1a is critical to improve wound healing in diabetic mice. Proc. Natl. Acad. Sci. USA 105:19426–19431; 2008. [270] Sarkar, K.; Fox-Talbot, K.; Steenbergen, C.; Bosch-Marce, M.; Semenza, G. L. Adenoviral transfer of HIF-1a enhances vascular responses to critical limb ischemia in diabetic mice. Proc. Natl. Acad. Sci. USA 106:18769–18774; 2009. [271] Jeevanantham, V.; Butler, M.; Saad, A.; Abdel-Latif, A.; Zuba-Surma, E. K.; Dawn, B. Adult bone marrow cell therapy improves survival and induces long-term improvement in cardiac parameters/clinical perspective. Circulation 126:551–568; 2012. [272] Siu, C. W.; Tse, H. F. Cardiac regeneration: messages from CADUCEUS. Lancet 379:870–871; 2010. [273] Marban, E.; Malliaras, K. Mixed results for bone marrow-derived cell therapy for ischemic heart disease. JAMA 308:2405–2406; 2012. [274] Asai J., Takenaka H., Ii M., Asahi M., Kishimoto S., Katoh N., et al. Topical application of ex vivo expanded endothelial progenitor cells promotes vascularisation and wound healing in diabetic mice. Int. Wound J. (in press). [275] Marrotte, E. J.; Chen, D. D.; Hakim, J. S.; Chen, A. F. Manganese superoxide dismutase expression in endothelial progenitor cells accelerates wound healing in diabetic mice. J. Clin. Invest. 120:4207–4219; 2010. [276] Lu, D.; Zhang, L.; Wang, H.; Zhang, Y.; Liu, J.; Xu, J., et al. Peroxisome proliferator activated receptor-g coactivator-1a (PGC-1a) enhances engraftment and angiogenesis of mesenchymal stem cells in diabetic hindlimb ischemia. Diabetes 61:1153–1159; 2012. [277] Jarajapu, Y. P. R.; Caballero, S.; Verma, A.; Nakagawa, T.; Lo, M. C.; Li, Q., et al. Blockade of NADPH oxidase restores vasoreparative function in diabetic CD34 þ cells. Invest. Ophthalmol. Visual Sci. 52:5093–5104; 2011. [278] Broqueres-You, D.; Lere-Dean, C.; Merkulova-Rainon, T.; Mantsounga, C. S.; Allanic, D.; Hainaud, P., et al. Ephrin-B2-activated peripheral blood mononuclear cells from diabetic patients restore diabetes-induced impairment of postischemic neovascularization. Diabetes 61:2621–2632; 2012.