Re-evaluating therapeutic neovascularization

Journal of Molecular and Cellular Cardiology 36 (2004) 25–32 www.elsevier.com/locate/yjmcc

Review Article

Re-evaluating therapeutic neovascularization E.D. de Muinck a,b, M. Simons a,c,* a

Section of Cardiology, Department of Medicine, Angiogenesis Research Center, Dartmouth Medical School, Lebanon, NH 03756, USA Section of Cardiology, Department of Physiology, Angiogenesis Research Center, Dartmouth Medical School, Lebanon, NH 03756, USA c Section of Cardiology, Department of Pharmacology and Toxicology, Angiogenesis Research Center, Dartmouth Medical School, Lebanon, NH 03756, USA b

Received 14 July 2003; received in revised form 25 September 2003; accepted 7 October 2003

Abstract Numerous animal studies have established that neo-vascularization of ischemic tissue can be enhanced with exogenous growth factors and small clinical studies have shown encouraging results. However, the two largest randomized clinical trials to date were negative. Mechanistically, the major stimuli for neo-vascularization are hypoxia and inflammation. Hypoxia-inducible-factor (HIF-1) is a ‘master switch’ protein that is generated in response to hypoxia and binds to more than 40 hypoxia sensitive genes, inducing a panoply of angiogenic and protective metabolic responses. Inflammatory signals recruit T-lymphocytes and macrophages into areas of neo-vascularization which act as a source of angiogenic and arteriogenic factors. Although hypoxia and inflammation are interdependent in eliciting neo-vascular responses, angiogenesis appears to be hypoxia-dependent, whereas inflammation and hemodynamic factors drive arteriogenesis. The negative outcome of the two largest trials may have many reasons. There are issues relating to patient selection, choice of growth factor therapy, dosing and route administration, concomitant medication, trial design including the efficacy parameters that were selected and a lack of sufficient insight into the mechanisms that are responsible for neo-vascularization. In order to move forward the therapeutic objective should be switched to arteriogenesis although this process is even more poorly understood than angiogenesis. Genetic studies in mice with intrinsically different arteriogenic responses combined with studies in human populations with differences in the extent of collateral development may provide fundamental insight into arteriogenic mechanisms. Attention should also be focused on the way in which arteriogenesis is stimulated and the endpoints of clinical trials should be redefined. © 2003 Elsevier Ltd. All rights reserved. Keywords: Angiogenesis; Arteriogenesis; Hypoxia; Inflammation; Clinical trials; Review

1. Introduction Three distinct mechanisms of new blood vessel formation have been identified to date. Embryonic vascular development is initiated by a process called vasculogenesis [1], while subsequent maturation and expansion of the embryonic vascular bed involve angiogenesis and arteriogenesis [2]. While the extent of vasculogenesis contributing to neovascularization in adult tissues has not been established, both angiogenesis and arteriogenesis are involved in new blood vessel formation during adulthood. During vasculogenesis, cells of mesodermal origin form blood islands outside the embryo and later on, waves of blood islands move progressively * Corresponding author. Section of Cardiology, Dartmouth-Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756, USA. Tel.: +1-603-650-3540; fax: +1-603-650-5171. E-mail address: [email protected] (M. Simons). © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.yjmcc.2003.10.002

inward [3,4]. Simultaneously, intra-embryonic mesodermal cells assemble into peri-endocardial tubes [4]. The blood vessels that arise from these islands and tubes originate from angioblasts and the blood cells develop from hematopoietic cells. They have a common precursor, the hemangioblast [5]. The primary vascular plexus thus formed, matures through angiogenesis [2]. Arteriogenesis describes the formation of muscular arteries, which involves recruitment of smooth muscle cells to sprouting vessels [2]. During adulthood, arteriogenesis denotes the formation of collateral arteries that can arise either de novo or from pre-existing, rudimentary vessels [6]. Most of our knowledge about the growth factors and signaling mechanisms that initiate and regulate new blood vessel formation comes from studies of embryonic vascular development and vessel growth in tumors [7]. Significantly less is known about the regulation of neovascularization in

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adult tissues, which has long been thought to recapitulate embryonic patterns of vascular gene expression [1,2]. However, the need to address the medical issues of the everincreasing incidence of cardiovascular disease worldwide and the large number of patients that remain not amenable to traditional forms of revascularization, i.e. percutaneous intervention or bypass surgery [8], has prompted intense investigations of therapeutic angiogenesis. Numerous animal studies have shown the feasibility of enhancing vessel growth in ischemic tissues and the clinical application of growth factors has shown encouraging results in several small studies [9–12]. However, two large wellcontrolled studies involving vascular endothelial growth factor (VEGF) [13] and basic fibroblast growth factor (bFGF) [14] have not corroborated the results of the smaller studies. They failed to confirm the clinical efficacy of protein-based growth factor therapy and have contributed to the growing realization that attempts to achieve therapeutic neovascularization in ischemic syndromes need to be re-evaluated. In this review, we will address several mechanisms of neovascularization, discuss issues that may have contributed to the inconclusive results of clinical trials and consider future directions for therapeutic angiogenesis.

2. What are the stimuli and mechanisms of neovascularization? Hypoxia and inflammation are the two major stimuli that set in motion the tightly regulated process of neovascularization. However, it is becoming increasingly clear that they are responsible for two distinct neovascular responses. Hypoxia has long been considered a primary stimulus for angiogenesis, but is unlikely to be an important arteriogenic stimulus. Arteriogenesis continues long after tissue ischemia has subsided and occurs in tissues that were never ischemic. For example, observation of extensive neovascularization around the sites of epicardial coronary artery occlusions argues that tissue ischemia per se cannot be the stimulus for vessel growth in these locations [15]. 2.1. Hypoxia Hypoxia leads to enhanced expression of a number of proteins that contribute to the maintenance of homeostasis by increasing circulatory oxygen-carrying capacity and inducing angiogenesis as well as protective metabolic responses. There is increased production of erythropoietin, leading to an increased number of red blood cells. Upregulation of VEGF enhances blood flow through increased production of nitric oxide and angiogenesis, and increased synthesis of glycolitic enzymes maintains intracellular levels of the energy-rich molecule adenosine triphosphate. The increased expression of these and many other proteins depends on the upregulation of the hypoxia-inducible factor (HIF) family of transcription factors [16]. HIF-1 is a tran-

scription factor that binds specifically to a hypoxiaresponsive element (HRE) in more than 40 hypoxia-inducible genes [16]. HIF-1 is a heterodimer composed of HIF-1a and HIF-1b/arylhydrocarbon receptor nuclear transport factor (ARNT) subunits. The b subunit, commonly called ARNT is constitutively expressed, whereas HIF-1a can only be detected under hypoxic conditions, because under normoxia, HIF-1a undergoes continuous proteolysis through the ubiquitin proteasome pathway [17]. The nature of the oxygen sensor that regulates the activity of HIF remains elusive, but it has become clear that binding of the tumor suppressor von Hippel–Lindau protein (pVHL) prevents HIF-1a degradation [18]. Both HIF-a and HIF-b subunits exist as a series of isoforms encoded by distinct genetic loci. The three known isoforms of HIF-a are all induced by hypoxia, whereas the b isoforms are expressed constitutively. HIF-1a and HIF-2a appear to be closely related and each interact with hypoxia responsive elements to induce transcriptional activity [19]. The effects of HIF-1a-induced transcription are far better characterized than those of HIF-2a-induced transcription. However, both transcription factors appear to have at least partially overlapping functions, as demonstrated in hypoxiainduced pulmonary hypertension, where both proteins were implied in the metabolic and vascular changes of the pulmonary vascular bed [20]. HIF-3a is involved in negative regulation of HIF-mediated effects. It exerts these effects through an alternatively spliced transcript called inhibitory PAS protein, which acts as an auxiliary dimerization interface and derives its name from the first three proteins in which it was discovered PER, ARNT and SIM [21]. 2.2. Inflammation Together with hypoxia, inflammation is an essential stimulus of neovascularization. Invasion of macrophages and T-lymphocytes is one of the histological hallmarks of myocardial ischemia and ischemia/reperfusion injury [22]. These blood-borne inflammatory cells are a source of VEGF [23,24] and a host of other angiogenic and arteriogenic factors including FGF2, TGFb1, interleukin-8, PDGF, IGF-1, monocyte chemotactic protein 1, TNF-a and metalloproteinases. These inflammatory mediators in turn attract endothelial and smooth muscle cells, fibroblast, leukocytes and platelets into areas of neovascularization, which stabilize the neovasculature. The presence of neutrophils and macrophages is sufficient to induce a neovascular response in the absence of ischemia [25,26]. Both T-lymphocytes and macrophages are pivotal in initiating and maintaining neovascular responses [22], and conditions that result in impaired recruitment of T-lymphocytes to ischemic areas, such as hypercholesterolemia [23], advanced age [27] and the absence of angiotensin II receptor-type IA [28,29] are characterized by a blunted angiogenic response. Likewise, failure to recruit monocytes/macrophages into areas requiring a neovascular response consistently impairs

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new vessel growth [30]. Both VEGF and its homologue placental growth factor (PlGF) attract monocytes/ macrophages [31,32] and these cells may contribute to neovascularization by their ability to secrete metalloproteinases [33], chemokines and growth factors [34]. While still controversial, the emerging evidence also suggests that monocytes, and not circulating endothelial progenitor cells, may be the ‘precursors’ of the endothelial cells that line new vessels [35]. Finally, studies in which the monocyte population was depleted consistently showed impaired arteriogenic responses [36]. The interdependence of hypoxia and inflammation in eliciting angiogenic responses has recently been confirmed by the demonstration that HIF-1a is essential for myeloid cell-mediated inflammation [37]. Nevertheless, it seems that angiogenesis is hypoxia-dependent, while arteriogenesis is not. Inflammation and hemodynamic factors are the principal drives of arteriogenesis with monocytes as the key cellular component. 2.3. Signals that translate extracellular messages The responses of endothelial cells and vascular smooth muscle cells to soluble mitogens and growth factors depend on the interplay between these soluble factors, the insoluble extracellular matrix (ECM) molecules and membrane receptors that illicit intracellular signaling pathways. A number of molecular mechanisms participate in transmitting this information from the extracellular environment to the cell. The integrin family is a group of cell surface molecules that plays a key role in modulating intracellular responses to extracellular stimuli [38]. Different integrins differ in their ability to mediate ECM signaling. For example, a4b1 engagement enhances cell migration and a5b1 ligation promotes attachment and focal adhesion formation [39,40]. The differences in this behavior likely relate to distinct signaling pathways activated by these integrins. Other transmembrane proteins involved in mediation of ECM signaling and integration of cellular input from various growth factors include the core protein syndecan-4 and the adhesion molecule PECAM-1. Syndecan-4 is found in focal adhesions, frequently in association with the b1 integrin [41]. Its expression is markedly enhanced following arterial injury in smooth muscle [42], following myocardial infarction in myocytes and endothelial cells [43] and in actively growing collateral blood vessels [44]. When oligomerized by binding to fibronectin or by FGF2, syndecan-4 protein has been shown to bind and activate PKCa [45]. Overexpression of syndecan-4 facilitates FGF2-induced cell migration and proliferation while the expression of dominant-negative syndecan-4 constructs selectively inhibits FGF2 signaling [46–49]. The role of PECAM-1 in neovascular responses has been corroborated in antibody studies and in genetic mouse models. Contact between endothelial cells through PECAM is crucial for vessel formation and maintenance. PECAM has

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been shown to regulate endothelial cell migration and tube formation in vitro [50] and more recently lack of PECAM was associated with decreased angiogenesis in a model of inflammation induced neovascularization [51]. The signal transduction pathways that mediate these effects depend upon integrin ligation, where tyrosine phosphorylation of PECAM-1 has been shown to be aIIbb3 and avb3 dependent [52,53].

3. Why have large, randomized clinical trials not corroborated early successes? With hindsight there are several reasons that may explain why the two largest double-blind studies have not shown clinically relevant responses to VEGF and FGF [13,14]. Issues relating to patient selection, choice of growth factor therapy, dosing and route of administration, trial design and assessment of therapeutic effect together with a lack of sufficient insight into the mechanisms that drive new blood vessel formation in response to exogenous growth factors may all have contributed to the inconclusive outcome of these trials. Perhaps the most important among these is the issue of the duration of the presence of growth factor in the myocardium after initiation of therapy. A recent study from the laboratory of Keshet suggested that a prolonged presence of VEGF165 is required to maintain the newly formed vasculature [54]. While the exact timing of the growth factor persistence in the human heart is uncertain, it is likely in the 3–4 months range. Such a duration would effectively rule out any likelihood of success with a single bolus protein approach as attempted with FGF2 and VEGF and would dictate either a sustained-release polymer approach or gene therapy. A small clinical trial of polymer-based FGF2 treatment suggests that this may be effective as clinical benefits persisted for up to 3 years following growth factor administration [10,55]. Several recent gene therapy studies also give reason for some cautious optimism. Among these is the AGENT trial of adenoviral FGF4, the KAT trial of adenoviral and plasmidbased VEGF165 and the plasmid-based VEGF2 trial. The reader is referred to other recent reviews for detailed discussions of these and other clinical trials of therapeutic angiogenesis [56,57]. Finally, it is becoming increasingly clear that the therapeutic goal should be to promote arteriogenesis and not angiogenesis. Clinically, the importance of arteriogenesis in maintaining cardiac function and patient survival is illustrated by the fact that myocardial infarction patients are less prone to develop ventricular aneurysms and show improved survival, if they have collateral arteries [58]. Experimentally, mouse strains that respond to hind limb ischemia with a blunted arteriogenic response developed significantly more severe lower leg ischemia after femoral artery ligation than strains that show more robust arteriogenesis, even though angiogenesis was comparable in both strains [59].

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3.1. Patient selection Typically, the majority of patients that are treated in clinical angiogenesis trials are ‘no option patients’ who have undergone and failed multiple revascularization attempts. These patients may represent failures of native neovascularization and may be particularly resistant to attempts at enhancing this process with exogenous growth factors [60]. Moreover, the animal experiments that laid the foundation for clinical trials were all performed in healthy, young animals which may exhibit a more robust vasculogenic response than old animals. Aged animals manifest both decreased VEGF expression and a decreased endothelial response to VEGF [27]. This age-dependent impairment of VEGF expression is caused at least in part by impaired induction of HIF-1 activity in response to hypoxia or ischemia [61]. The response to other growth factors likely diminishes with age as well. Several observations suggest that genetic differences may play a role in angiogenic responsiveness. A small study showed that the extent of collateral formation visible on coronary angiograms correlated with the extent to which monocytes cultured from these patients responded to hypoxia with an induction of VEGF expression [60]. Different mouse strains demonstrate profound differences in native arteriogenic response to an arterial ligation [59] and show up to a 10-fold range of response to growth factor-stimulated angiogenesis [62]. Current investigations are attempting to unravel these differences but until then trial designs will be hampered by unpredictable differences in biological responses to growth factor therapies. This difficulty is further exacerbated by the lack of a biological marker that correlates with or predicts a response to therapeutic angiogenesis. Medication that many cardiac patients are taking can have a profound inhibitory effect on neovessel formation. This has been shown for captopril and angiotensin receptor blockers, spironolactone, isosorbide dinitrate, lovastatin, bumetanide, and furosemide [63]. Together with age, mentioned earlier, other confounding factors include hypercholesterolemia, smoking, diabetes and the presence of circulating angiogenesis inhibitors [63]. 3.2. Choice of growth factor therapy While it might be possible to initiate neovascularization with a single growth factor, it is not clear that persistence of newly formed vasculature can be achieved this way. Indeed, once formed, vessel survival is dependent upon VEGF and other exogenous survival factors for a critical time during their development [64] and if reductions in VEGF levels are not followed by the recruitment of mural cells and the deposition of basement membrane, the neovessels may regress [65]. Angiopoietin-1 acts after VEGF levels have decreased during remodeling of the vascular plexus and combination growth factor therapy with VEGF and angiopoietin-1 may produce more stable vessels that are less leaky than blood vessels developing in response to VEGF alone [66].

An alternative to combination growth factor therapy is the use of ‘angiogenic master switch’ genes that are capable of initiating multiple neovascularization cascades. For example HIF-1a, as discussed earlier, stimulates expression of multiple angiogenesis-related genes and an increase in HIF-1a levels is one of the first adaptations of the human myocardium to a deprivation of blood [67]. In animal studies HIF-1a gene therapy has been shown to promote significant improvements in perfusion in a rabbit hind limb ischemia model [68]. Another example is a small 39 amino acid proline/argininerich protein PR39. The peptide acts by selectively inhibiting proteasome-mediated degradation of HIF-1a [69]. Adenoviral transfer of the PR39 gene in experimental myocardial ischemia resulted in improved perfusion and function [70]. Hepatocyte growth factor (HGF) is another attractive ‘master switch’, primarily because it induces the transcription factor ets-1 [71]. The ets-1 is expressed in endothelial cells at the initiation of blood vessel formation under normal and pathological conditions [72]. It induces the transcription of matrix-degrading proteins as well as VEGF [71] and its receptor VEGFR-2 (KDR/Flk-1) [73]. In animal studies, HGF has been shown to promote neovascularization in hind limb ischemia and myocardial infarction [74,75]. Importantly, HGF simultaneously promotes endothelial cell and vascular smooth muscle cell migration; and contrary to neovessel formation in response to VEGF, there is no evidence of vascular leaks in vessels that have grown in response to HGF [71]. The VEGF homologue PlGF may also prove to be a clinically relevant ‘master switch’. It affects all modes of neovessel formation: it drives angiogenesis and arteriogenesis [30,76,77], and it recruits endothelial progenitor cells and monocytes from bone marrow to areas of neovascularization [76,78], it recruits vascular smooth muscle cells around nascent vessels contributing to the generation of stable, non-leaky vessels which have been shown to persist up to 1 year [76]. In adulthood, PlGF and VEGFR-1 are only expressed during pathological angiogenesis, contributing to site-specific activity of exogenous PlGF [30]. VEGFR-1 (Flt-1) is the signaling receptor for PlGF whereas most biological functions of VEGF are mediated through VEGFR-2. There is evidence that VEGFR-1 acts as a sink or ‘decoy’ that limits VEGF signaling [30]. PlGF amplifies VEGF signaling by displacing VEGF from VEGFR-1 to VEGFR-2 to induce a stronger vasculogenic response [30] and by promoting intraand intermolecular cross talk between VEGFR-1 and VEGFR-2 [79]. In conclusion, the different effects of PlGF on the process of neovessel formation make it a suitable ‘master switch’ awaiting clinical confirmation of its efficacy. 3.3. Dosing and delivery mode The importance of dosage and duration of expression is poignantly illustrated by the observation that a molecule like VEGF has such a tight dose–response effect that a 50% reduction in expression results in lethality during embryonic

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life [80]. Conversely, continuous local overexpression of VEGF may result in a hemangioma-like vasculature and thus can be deleterious [81]. The combined process of diffusion, consumption of oxygen and nutrients, and production of metabolites establish microenvironmental gradients across tissues that are necessary for vessel network formation [82]. In our ‘black box’ approach towards delivery of growth factors in patients none of the issues of dose, duration of expression, impact of microenvironment on protein activity, as well as temporal and spatial regulation of gene expression relative to other regulators have been adequately addressed. The currently available delivery modes and the pros and cons of protein therapy vs. gene therapy have been discussed extensively elsewhere, and are beyond the scope of this review [57,63]. Cationic nanoparticles coupled to an integrin avb3targeting ligand can deliver genes selectively to angiogenic blood vessels in tumor-bearing mice [83]. The advantages of such an approach are clear; it allows for intravenous delivery of the agent, facilitates repeated administration and sequential treatment with proteins or genes that act at different stages of neovascularization. To further enhance site-specific expression of the gene of interest, its expression can be placed under control of a hypoxia-responsive element that contains the cognate recognition sequences for HIF-1a [84]. This would also ensure cessation of gene expression once adequate tissue oxygenation has been achieved [85]. Homing imaging molecules to angiogenic vessels to monitor the therapeutic effect, is another application of the ‘vesseltargeting approach’ [86]. Proof of principle for this technique has been obtained in a model of myocardial infarction, using homing molecules that target avb3 [87]. 3.4. Safety The safety concerns regarding growth factor therapy focus on two major issues: the enhancement of tumor growth and atherogenesis through the promotion of vessel growth in sub-clinical malignancies and atherosclerotic lesions and ‘bystander’ effects of the growth factor that is being administered (e.g., effects of FGF on the kidney). The clinical data available so far do not show an increased incidence of malignancies nor the progression of atherosclerosis under growth factor therapy [13,14,63]. Experimentally, blockade of the receptor for PlGF, Flt-1 has been shown to reduce atherosclerosis independent of plaque vascularization [76], suggesting a role for PlGF in atherosclerotic lesion progression. Nevertheless, in clinical settings using the routes of administration and doses that are believed to enhance vessel formation in ischemic tissue, growth factors have not been shown to increase plaque size or instability. 3.5. Organization, conduct and interpretation of clinical trials This subject has been reviewed extensively elsewhere [63]. The two largest trials to date were well controlled and

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adequately powered but showed a considerable placebo effect [13,14]. Clearly, future studies should be better designed to account for a placebo effect and include an imaging endpoint that would unequivocally document both improvement in left ventricular function and perfusion. Magnetic resonance imaging, because of its enormous potential to assess myocardial structure, function and blood flow, seems ideally suited for this purpose [63].

4. Conclusions and future directions The translation of neovascularization responses to exogenous growth factors in young healthy animals to old patients with end-stage coronary artery disease, who frequently present with significant co-morbidity, and take multiple medications that affect neovessel growth, has not been successful. Moreover, the therapeutic goal primarily has been to stimulate angiogenesis and not arteriogenesis. Several tasks lay ahead. First, the therapeutic objective should be refocused on arteriogenesis. Healthy animals are of limited value in this setting and more relevant atherosclerotic models should be employed whenever possible. Also, attempts should be made to understand the process in patients. A strategy may be envisaged that operates at a genetic and population level, exploring mouse strains with intrinsically different arteriogenesis rates and distinct human populations demonstrating differences in the extent of collateral development. Second, more consideration should be given to the way in which arteriogenesis is to be stimulated. This includes choice of agent(s), dosing, frequency, timing and route of administration. In view of the paucity of rigorous data that support the concept of physiologically significant angiogenesis by stimulation of intramyocardial production of growth factors and given the appeal of arteriogenesis as the treatment of choice, epicardial perivascular delivery at the site where this process actually takes place is theoretically most attractive [88]. When gene therapy is the modality of choice, advantage should be taken of emerging strategies that enable gene expression to be ‘switched on and off’ [54,84]. Likewise, the concept of utilizing homing molecules to cell surface receptors that are preferentially expressed on angiogenic endothelium should be explored because it may contribute to sitespecific expression and allow for repeated administration of arteriogenic agents. The effects of microenvironment on protein function, and gene expression merit to be investigated in detail. Finally, the end-points of the next clinical trials need to be redefined, taking into account the placebo effect observed in previous studies. While progress in the field of therapeutic neovascularization and tissue regeneration may be characterized as ‘two steps forward, one step backward’, it remains a strategy of great promise for an ever-increasing population of patients with coronary artery disease and heart failure.

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Acknowledgements Supported in part by NIH grants HL-70247, 53793 and 63609 (M.S.).

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