- Email: [email protected]
Molecular mechanisms of diabetic vasculopathy
Vol. 2, No. 1 2005
Drug Discovery Today: Disease Mechanisms
DRUG DISCOVERY
TODAY
Editors-in-Chief Toren Finkel – National Heart, Lung and Blood Institute, National Institutes of Health, USA Tamas Bartfai – Harold L. Dorris Neurological Research Center and The Scripps Research Institute, USA
DISEASE Cardiovascular diseases MECHANISMS
Molecular mechanisms of diabetic vasculopathy Anne Hamik1, G. Brandon Atkins2, Mukesh K. Jain1,* 1 Program in Cardiovascular Transcriptional Biology, Cardiovascular Division, Brigham and Women’s Hospital, Harvard Medical School, 75 Francis Street, Boston, MA 02115, USA 2 Division of Cardiology, Massachusetts General Hospital, Harvard Medical School, 55 Fruit Street, Boston, MA, USA
Diabetes and its attendant complications constitute a burgeoning global health threat. Hyperglycemia, the
Section Editor: Cam Patterson – Farmal Biomedicines, LLC, USA
sine qua non of diabetes, induces alterations in several signaling pathways that culminate in the clinical manifestations of diabetic vasculopathy (DV). Ultimately, cellular behavior is regulated by gene transcription. Direct manipulation of gene transcription has largely been outside the purview of conventional therapeutics. However, with the sophistication of current molecular approaches, this arena is now poised for rapid development. In this review, we describe key transcription factors that show potential as therapeutic targets for DV.
Introduction The abnormal metabolic milieu created by hyperglycemia or the diabetic state initiates a series of dysfunctional responses which culminate in the development of premature cardiovascular disease [1,2]. This process begins with dysregulation of vascular cell homeostatic mediators, progresses to pathologic anatomic changes in vascular beds, and results in the familiar clinical syndromes of diabetes (outlined in Box 1). At least four major cellular signaling pathways have been implicated in the development of diabetic vasculopathy (DV) (illustrated in Fig. 1). In the first half of this review, we briefly *Corresponding author: M.K. Jain ([email protected]) 1740-6765/$ ß 2005 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddmec.2005.05.030
review these pathways with reference to more extensive reviews. In the second half we focus on nuclear pathways involved in the development of DV. Throughout we highlight novel therapeutic strategies targeting specific pathways or factors (listed in Table 1). Conventional therapies such as aspirin, statins and angiotensin converting enzyme (ACE) inhibitors – known to favorably affect multiple pathways – have been subject to previous reviews and are not included in the table.
Major signaling pathways Oxidative stress pathway The oxidative stress pathway serves as a common element linking all the major pathways implicated in DV [3]. Hyperglycemia induces oxidative stress in vascular cells by enhancing the production of reactive oxygen species (ROS). This results in damage to cellular proteins, reduced nitric oxide (NO) levels and activation of transcription factors such as activated protein-1 (AP-1) and nuclear factor-kB (NF-kB) [1]. Collectively, these effects confer a pro-adhesive, pro-thrombotic phenotype to the vessel wall and serve as the basis of clinical studies aimed at inhibiting oxidant stress. Unfortunately, the use in humans of conventional antioxidants such as Vitamin E to prevent diabetic vascular complications has largely been ineffective [1,4]. However, agents that attenuate the production of ROS (peroxisome proliferator-activated receptor-g ligands, statins, angiotensin converting enzyme (ACE) inhibitors, calcium channel blockers and angiotensin www.drugdiscoverytoday.com
11
Drug Discovery Today: Disease Mechanisms | Cardiovascular diseases
Vol. 2, No. 1 2005
Box 1.
Cellular effects and functional consequences of diabetes. Diabetes alters the homeostasis of the cellular components of the vasculature (including endothelial cells, monocytes and vascular smooth muscle cells) in predictable ways. This leads to dysregulation of the normal anti-thrombotic, anti-adhesive and vasoreactive state of vascular beds and eventually to the macrovascular and microvascular complications of diabetes. DM, diabetes mellitus; EC, endothelial cell; VSMC, vascular smooth muscle cells; phenotypic modulation, change from a quiescent state to one which includes elaboration of extracellular matrix, cell proliferation and migration; TF, tissue factor; PAI-1, plasminogen activator inhibitor-1; eNOS, endothelial nitric oxide synthase; VCAM-1, vascular cell adhesion molecule-1; AT-II, angiotensin II; ET-1, endothelin-1; COX-2, cyclooxygenase-2; TNFa, tumor necrosis factor a; MCP-1, monocyte chemoattractant protein-1; TGFb, transforming growth factor b; CTGF, connective tissue growth factor; CAD, coronary atherosclerotic disease; PAD, peripheral atherosclerotic disease; MI, myocardial infarction.
Table 1. Novel strategies targeting key pathways and transcription factors in diabetic vasculopathy Target pathway or transcription factor
Drug
Who is working on this
Stage
Refs
Oxidative stress pathway Inhibitor of ROS
Vitamin E
Multiple investigators
Phase III clinical trials
[1,4]
AGE–RAGE pathway Blockade of AGE formation Blockade of crosslink formation RAGE blockade Transketolase activator
Aminoguanidine Alagebrium sRAGE Benfotiamine
Multiple investigators Forbes and others Schmidt et al. Brownlee et al.
Phase III clinical trials Phase II clinical trials Preclinical Phase III clinical trials
[8,9] [7,8] [11] [10]
Ruboxistaurin CGP53353 Benfotiamine
King et al. Cosentino et al. Brownlee et al.
Phase III clinical trials Preclinical Phase III clinical trials
[13] [16] [10]
Polyol/aldose reductase pathway Aldose reductase inhibitors
Epalrestat/tolrestat
Srivastava and others
Phase II–III clinical trials
[17,18]
PARP PARP inhibitor
PJ34
Szabo et al.
Preclinical
[35,36]
PPAR PPARa and PPARg agonists
TZDs, fibrates
Plutzky, Staels and others
Phase III clinical trials
[38–41]
Protein kinase C pathway PKCb inhibitor Transketolase activator
Abbreviations: AGE, advanced glycation end product; RAGE, receptor of AGE; sRAGE, soluble RAGE; PKC, protein kinase C; ROS, reactive oxygen species; PPAR, peroxisome proliferatoractivated receptor; TZDs, thiazolidinediones; PARP, poly(ADP ribose) polymerase.
12
www.drugdiscoverytoday.com
Vol. 2, No. 1 2005
Figure 1. Pathways leading to diabetes. Genetic predisposition and environmental factors influence the development of the clinical precursors of diabetes: hyperglycemia, insulin resistance and dyslipidemia. In turn, diabetes leads to the pathological activation of at least four interacting cellular pathways. The net effect is an accelerated vasculopathy – the vasculopathy of diabetes. AGE/RAGE, advanced glycation endproducts/receptor of AGE pathway; PKC, protein kinase C pathway. See text for description of pathways. Modified and reproduced, with permission, from Ref. [2].
II type 1 (AT-1) receptor antagonists) [4] have all been shown to reduce the burden of cardiovascular disease out of proportion to their respective glucose lowering, lipid lowering, or blood pressure lowering effects.
Advanced glycation end products (AGE) pathway AGEs are a heterogenous group of compounds produced as a consequence of the irreversible nonenzymatic glycation of proteins [5]. This process occurs in diabetes as well as in aging and is augmented by oxidative stress and inflammation. AGEs can confer deleterious effects to the vessel wall in two main ways. First, crosslinking of long-lived proteins such as collagen or elastin in the vessel wall can alter structural integrity. Second, binding of AGEs to their receptor (RAGE, GenBank accession no. NM_001136) on vascular cells can activate multiple signaling pathways (e.g. protein kinase C (PKC) and MAPkinase pathways) and activate nuclear factors (e.g. NF-kB and cyclic AMP-responsive element-binding protein; CREB, GenBank accession no. L05515) resulting in an increase in ROS production and elaboration of inflammatory factors [6]. As
Drug Discovery Today: Disease Mechanisms | Cardiovascular diseases
such, ways to limit cross-linking of proteins and inhibit RAGE receptor activation have been the subject of intense study in both animal models and, more recently, human trials. Treatment of diabetic mice with inhibitors of AGE formation (e.g. aminoguanidine) or AGE crosslink breakers such as alagebrium attenuated atherosclerosis and reduced vascular stiffness [7,8]. Clinical trials of aminoguanidine to prevent progression of diabetic nephropathy were terminated owing to lack of efficacy and safety concerns [9]. However, Phase II clinical studies in elderly patients using alagebrium demonstrate improved arterial compliance and systolic blood pressure [7]. Finally, the lipid-soluble thiamine derivative benfotiamine has potential for clinical use; in animal studies it has been demonstrated to inhibit both the hyperglycemia-induced AGE/RAGE and PKC pathways [10]. With respect to RAGE receptor activation, treatment of rodents with sRAGE (a soluble form of the RAGE receptor that blocks activation of cell surface RAGE) reduced atherosclerotic burden, ameliorated lesion complexity and decreased inflammatory gene expression in vascular cells. Furthermore, mice deficient in the RAGE receptor exhibit a decrease in smooth muscle proliferative response and neointimal expansion after vessel injury (reviewed in [11]). Finally, a recent study demonstrated that levels of sRAGE are inversely related to the presence of coronary artery disease in men [12]. These observations provide a compelling argument to assess inhibition of RAGE activation in humans.
Protein kinase C pathway Hyperglycemia can activate the PKC pathway directly via diacylglycerol (DAG) synthesis and indirectly through production of ROS or activation of the AGE/RAGE pathway [1,13, 14]. The observation that preferential activation of PKCb2 (GenBank accession no. NM_212535) occurs in the aorta and hearts of diabetic animals provided the foundation for interest in this field [15]. By virtue of its ability to alter the balance of vasodilatory/vascoconstrictive factors (e.g. endothelial nitric oxide synthase; eNOS, GenBank accession no. NM_000603/endothelin; ET-1, GenBank accession no. NM_001955), increase oxidative stress, induce adhesion molecule expression (e.g. vascular cell adhesion molecule-1; VCAM1, GenBank accession no. NM_001078) and enhance expression of profibrotic factors (e.g. transforming growth factor b1; TGFb1, GenBank accession no. NM_000660 and connective tissue growth factor; CTGF, GenBank accession no. NM_001901), PKCb has emerged as a key regulatory molecule in the pathogenesis of diabetic vasculopathy [1,13,15]. Selective and nonselective PKCb inhibitors have been developed [16]. In humans, infusion of the PKCb inhibitor ruboxistaurin ameliorates endothelial dysfunction induced by glucose infusion. PKC activation has been verified in human pathologic specimens and PKCb inhibitors such as ruboxistaurin are currently undergoing Phase II/III clinical trials [13]. www.drugdiscoverytoday.com
13
Drug Discovery Today: Disease Mechanisms | Cardiovascular diseases
Polyol/aldose reductase pathway Elevation in glucose can activate the enzyme aldose reductase (GenBank accession no. NM_000594) that converts glucose to sorbitol and ultimately to fructose. This pathway alters NAD(P)H/NAD(P) balance in a manner that leads to augmentation of oxidative stress and ultimately vascular disease [14]. Furthermore, as discussed above, redox stress can activate both the PKC and AGE pathways. As such, it is reasonable to consider aldose reductase inhibitors as therapeutic agents. Although studies in animal models using tolrestat and epalrestat have been encouraging, studies in humans have yielded minimal benefit [17,18].
Transcriptional regulatory pathways Promoting vasculopathy NF-kB
Inflammation is a crucial feature in the development of vascular disease, and NF-kB is a central mediator of most inflammatory stimuli. NF-kB proteins exist in unstimulated cells as homo- or hetero-dimers bound to an inhibitory molecule IkB (GenBank accession no. NM_002503). Upon cellular activation by pro-inflammatory stimuli, the IKK complex is activated. Specifically, the beta subunit (IKK-b, GenBank accession no. NM_001556) phosphorylates IkB resulting in its degradation and thereby liberates NF-kB to move into the nucleus, bind DNA and affect target genes. Specific NF-kB inhibitors are being tested for efficacy in other disease states, such as cancer biology. Multiple lines of evidence support a role for NF-kB in diabetes. First, hyperglycemia, oxidative stress, PKC activation, AGE–RAGE interactions and inflammatory cytokines can all activate NF-kB. In animal models, deficiency of one of the NF-kB subunits (p50, GenBank accession no. S76638) can render mice resistant to streptozotocin-induced diabetes [19]. Interestingly, inhibition of IKKb by high dose aspirin reverses hyperglycemia, hyperinsulinemia and dyslipidemia in obese rodents [20]. The effect of IKKb inhibition on the development of vascular disease remains unknown. Finally, in patients with diabetes, a positive correlation exists between insufficient glycemic control and NF-kB activity in peripheral blood mononuclear cells [21]. NF-kB has been shown to regulate gene expression and function of vascular cells in a manner that promotes the development of vascular disease. Hyperglycemia as well as AGE–RAGE interaction can induce NF-kB and endothelial expression of VCAM-1 in cell culture and rodent models [22]. Furthermore, exposure of endothelial cells to hyperglycemic sera from diabetic patients enhanced leukocyte adhesion [23]. A human correlate is suggested by the finding of elevated VCAM-1 levels in the plasma of diabetic patients [22]. In addition, a positive feedback mechanism is suggested by the observation that AGE and TNFa (tumor necrosis 14
www.drugdiscoverytoday.com
Vol. 2, No. 1 2005
factora; GenBank accession no. NM_000594) induce RAGE in an NF-kB-dependent manner [24]. Hyperglycemia can also induce NF-kB activation in vascular smooth muscle cells (VSMCs) resulting in an increase in ROS and VCAM-1 expression [25]. The aldose reductase pathway has also been identified as important in NF-kB activation in VSMCs. Indeed, a recent study demonstrated that both pharmacologic inhibition of aldose reductase as well as small interfering RNA (siRNA)-mediated reduction in the expression of this enzyme prevented the hyperglycemia-mediated activation of NF-kB in VSMCs. Furthermore, treatment of diabetic and wild type rats with the aldose reductase inhibitor tolrestat reduced neointimal proliferation and decreased staining of tissue sections with an antibody against NF-kB [18]. Finally, treatment of VSMCs with AGEs activates NF-kB (as well as AP-1), enhances secretion of cytokines and promotes VSMC proliferation and migration [26]. These data support an important role for NF-kB in diabetic smooth muscle biology. Elegant studies from the Natarajan laboratory support an important role for hyperglycemia-induced NF-kB in monocyte biology. Exposure of monocytes to sustained hyperglycemia resulted in an increase in TNFa and COX-2 (cyclooxygenase-2, GenBank accession no. U04636) production – at least in part owing to NF-kB activation (along with activation of AP-1 and CREB) [27]. Importantly, these investigators demonstrated that hyperglycemia enhanced NF-kB expression while reducing the protein levels of the inhibitory molecule IkBa. Furthermore, chromatin immunoprecipitation assays demonstrate that hyperglycemia increases recruitment of the NF-kB subunit p65 (GenBank accession no. M62399) as well as NF-kB coactivators CREB binding protein (CBP, GenBank accession no. NM_004380) and p300/CBP associated factor (p/CAF, GenBank accession no. U57317) to target promoters while decreasing binding of transcriptional co-repressors such as histone deacetylases (HDACs) [28]. These observations suggest that chromatin modification might be involved in regulating the activation of NF-kB (and perhaps other factors) in diabetes. Others
AP-1: AP-1 complexes are homo- or heterodimers composed of basic region-leucine zipper proteins that belong to the Jun and Fos families of factors that have been implicated in cellular activation, proliferation and survival. Data on this family in the context of diabetes are limited. Studies do show that hyperglycemia and AGE–RAGE interactions can induce AP-1 DNA-binding and, as a consequence, induce expression of aldose reductase in endothelial cells [29]. This observation is of interest as it provides a link between hyperglycemia/AGE– RAGE interactions and the polyol pathway. Finally, activation of AP-1 has also been shown to reduce eNOS expression [30]. Sp1/Egr-1: Sp1 (GenBank accession no. NM_138473) and early growth response protein-1 (Egr-1, GenBank accession
Vol. 2, No. 1 2005
no. NM_001964) are broadly expressed zinc-finger transcription factors. In endothelial and VSMCs the hyperglycemia-mediated increase in plasminogen activator inhibitor-1 (PAI-1) expression is in part Sp1 dependent [31,32]. Egr-1 is known to be involved in activation of transcription of many genes implicated in the development of diabetic vasculopathy, including platelet-derived growth factor (PDGF, GenBank accession no. NM_006206), tissue factor (TF, GenBank accession no. M27436) and PAI-1. Although hyperglycemia and insulin can induce Egr-1 mRNA in endothelial cells [33], the importance of this regulation remains unknown. PARP: Poly(ADP ribose) polymerases (PARPs) are chromatin-associated proteins that catalyze the transfer of ADP-ribose units from NAD+ to target proteins and have been implicated in a variety of cellular processes such as DNA repair, cell cycle control and gene regulation. PARP-1 (GenBank accession no. NM_00168) has also been shown to act as a coactivator for NFkB [34]. Induction of experimental diabetes induces PARP activity in the vessel wall. Furthermore, studies using PARP inhibitors and vessels from PARP-null mice demonstrate resistance against glucose-induced endothelial dysfunction [35]. Furthermore, PARP activation was observed in subjects at risk for developing diabetes [36]. The availability of PARP inhibitors (e.g. PJ34) raises the possibility that these can be used in the treatment of diabetic vasculopathy. Smads: Smads are one of the main mediators of TGFb antiinflammatory and profibrotic effects. In VSMCs, AGEs have been shown to directly activate Smad2/3 (GenBank accession no. NM_005901) leading to increased fibronectin synthesis [37]. In this manner, activation by the AGE pathway might promote vascular sclerosis.
Inhibiting vasculopathy PPAR PPARs are a family of ligand-activated nuclear hormone receptors involved in the regulation of lipid and glucose metabolism. Synthetic compounds that activate PPARa (GenBank accession no. NM_001001928) (e.g. fibrates) and PPARg (GenBank accession no. NM_138712) (e.g. thiazolidinediones, TZDs) are used clinically to decrease lipid abnormalities and as insulin sensitizers, respectively. These receptors are also expressed in vascular cells and macrophages where they have been shown to exhibit potent anti-inflammatory effects – at least in part through inhibition of NF-kB and AP-1 pathways. In models of experimental diabetes, PPARa and PPARg levels are reduced [38]. Activation of PPARs can inhibit adhesion molecule expression, cytokine production, PKC activation, RAGE expression, elaboration of matrix metalloproteinases and cellular proliferation. A recent study comprehensively evaluated the effect of various PPAR agonists in experimental atherosclerosis and demonstrated that PPARa and PPARg ligands can reduce atherogenesis in mice [38]. Finally, clinical observations support an important role for
Drug Discovery Today: Disease Mechanisms | Cardiovascular diseases
PPARa and PPARg ligands in the treatment of vasculopathy. For example, treatment of patients with TZDs has been shown to reduce intimal hyperplasia after coronary stenting [39]. Pilot studies assessing effects of TZDs on carotid artery intimal-medial thickness in patients with type II diabetes are also encouraging and demonstrate a reduction in plaque thickness [40]. Furthermore, treatment of patients with the PPARa agonist gemfibrozil lowered vascular events in patients with type II diabetes [41].
Others KLF2
KLF2 is a zinc-finger protein that is expressed in the endothelial layer of the blood vessel wall. Recent studies from our group indicate that this factor can confer anti-adhesive, antithrombotic and anti-inflammatory properties in endothelial cells ([42], and references therein). The reduction in KLF2 expression by inflammatory stimuli could be a key mechanism by which the endothelium is rendered dysfunctional in disease states such as diabetes. Identification of mechanisms to induce KLF2 in endothelial cells can provide novel approaches in the treatment of diabetic vasculopathy. In fact, multiple statins significantly induce endothelial KLF2 expression, and the statin-dependent induction of eNOS and thrombomodulin requires KLF2 [43]. CREB
The role of this transcription factor in diabetic vasculopathy remains controversial. Studies in monocytes suggest that this factor might cooperate with NF-kB and induce genes such as COX-2 in hyperglycemic conditions. However, in VSMCs, hyperglycemia led to decreased CREB protein and increased enhanced chemokinesis (a sign of phenotypic modulation) [44]. Clearly, additional studies are needed to elucidate the role of CREB in diabetic vasculopathy.
Conclusions The burden of DV on the global population is enormous and growing ever greater. In addition to lifestyle modification, novel therapies are clearly needed in the treatment of diabetes. Over the past two decades, considerable progress has been made in our understanding of the key cellular signaling pathways involved in the pathogenesis of DV. As noted in this review, these studies have provided novel approaches to the treatment of DV. However, given the complexity of this disease process, it is probable that additional therapeutic approaches will be required. Apropos unique approaches, an emerging field that shows great promise – but is beyond the scope of this review – is that concerning the anti-inflammatory activities of adipocytokines such as adiponectin (GenBank accession no. NP_004788). Ultimately, many of these pathways will converge in nuclear events that regulate gene expression and, as a consequence, www.drugdiscoverytoday.com
15
Drug Discovery Today: Disease Mechanisms | Cardiovascular diseases
Figure 2. Transcription factors implicated in regulation of the development of diabetic vasculopathy. NF-kB and PPARa,g have been demonstrated to induce and inhibit, respectively, development of diabetic vasculopathy. Emerging data implicate the additional factors listed under Section ‘Others’; however, current data are most compelling for NF-kB and PPARs. See text for details.
cellular function. As such, an understanding of the nuclear mechanism(s) involved in the development of DV is a crucial but poorly developed area of research, deserving of greater investigation. Integral involvement in DV of the transcriptional regulators NF-kB and the PPARs has been demonstrated experimentally and clinically, respectively (summarized in Fig. 2). However, few studies have been performed to date that take advantage of contemporary molecular techniques (chromatin immunoprecipitation, cofactor identification, analysis of modification of transcription factors and chromatin by acylation and methylation, confirmatory experiments in transgenic mice). It is probable that through such studies we will identify novel therapeutic strategies that will ultimately allow us to curtail the increasing burden of DV and its attendant consequences.
References 1 Sheetz, M.J. and King, G.L. (2002) Molecular understanding of hyperglycemia’s adverse effects for diabetic complications. J. Am. Med. Assoc. 288, 2579–2588 2 Beckman, J.A. et al. (2002) Diabetes and atherosclerosis: epidemiology, pathophysiology, and management. J. Am. Med. Assoc. 287, 2570–2581 3 Nishikawa, T. et al. (2000) Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature 404, 787–790 4 Ceriello, A. and Motz, E. (2004) 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 5 Wautier, J.L. and Schmidt, A.M. (2004) Protein glycation: a firm link to endothelial cell dysfunction. Circ. Res. 95, 233–238 6 Yan, S.F. et al. (2003) Glycation, inflammation, and RAGE: a scaffold for the macrovascular complications of diabetes and beyond. Circ. Res. 93, 1159–1169 7 Bakris, G.L. et al. (2004) Advanced glycation end-product cross-link breakers: a novel approach to cardiovascular pathologies related to the aging process. Am. J. Hypertens. 17 (12 Pt 2), 23S–30S
16
www.drugdiscoverytoday.com
Vol. 2, No. 1 2005
8 Forbes, J.M. et al. (2004) Advanced glycation end product interventions reduce diabetes-accelerated atherosclerosis. Diabetes 53, 1813–1823 9 Thornalley, P.J. (2003) Use of aminoguanidine (Pimagedine) to prevent the formation of advanced glycation endproducts. Arch. Biochem. Biophys. 419, 31–40 10 Hammes, H.P. et al. (2003) Benfotiamine blocks three major pathways of hyperglycemic damage and prevents experimental diabetic retinopathy. Nat. Med. 9, 294–299 11 Naka, Y. et al. (2004) RAGE axis: animal models and novel insights into the vascular complications of diabetes. Arterioscler. Thromb. Vasc. Biol. 24, 1342–1349 12 Falcone, C. et al. (2005) Plasma levels of soluble receptor for advanced glycation end products and coronary artery disease in nondiabetic men. Arterioscler. Thromb. Vasc. Biol. 25, 1032–1037 13 Rask-Madsen, C. and King, G.L. (2005) Proatherosclerotic mechanisms involving protein kinase C in diabetes and insulin resistance. Arterioscler. Thromb. Vasc. Biol. 25, 487–496 14 Brownlee, M. (2001) Biochemistry and molecular cell biology of diabetic complications. Nature 414, 813–820 15 Way, K.J. et al. (2001) Protein kinase C and the development of diabetic vascular complications. Diabet. Med. 18, 945–959 16 Kouroedov, A. et al. (2004) Selective inhibition of protein kinase Cbeta2 prevents acute effects of high glucose on vascular cell adhesion molecule-1 expression in human endothelial cells. Circulation 110, 91–96 17 El-Kabbani, O. et al. (2004) Aldose reductase structures: implications for mechanism and inhibition. Cell. Mol. Life Sci. 61, 750–762 18 Ramana, K.V. et al. (2004) Activation of nuclear factor-kappaB by hyperglycemia in vascular smooth muscle cells is regulated by aldose reductase. Diabetes 53, 2910–2920 19 Mabley, J.G. et al. (2002) NFkappaB1 (p50)-deficient mice are not susceptible to multiple low-dose streptozotocin-induced diabetes. J. Endocrinol. 173, 457–464 20 Yuan, M. et al. (2001) Reversal of obesity- and diet-induced insulin resistance with salicylates or targeted disruption of Ikkbeta. Science 293, 1673–1677 21 Hofmann, M.A. et al. (1998) Insufficient glycemic control increases nuclear factor-kappa B binding activity in peripheral blood mononuclear cells isolated from patients with type 1 diabetes. Diabetes Care 21, 1310–1316 22 Schmidt, A.M. et al. (1995) Advanced glycation endproducts interacting with their endothelial receptor induce expression of vascular cell adhesion molecule-1 (VCAM-1) in cultured human endothelial cells and in mice. A potential mechanism for the accelerated vasculopathy of diabetes. J. Clin. Invest. 96, 1395–1403 23 Morigi, M. et al. (1998) Leukocyte-endothelial interaction is augmented by high glucose concentrations and hyperglycemia in a NF-kB-dependent fashion. J. Clin. Invest. 101, 1905–1915 24 Tanaka, N. et al. (2000) The receptor for advanced glycation end products is induced by the glycation products themselves and tumor necrosis factor-alpha through nuclear factor-kappa B, and by 17 beta-estradiol through Sp-1 in human vascular endothelial cells. J. Biol. Chem. 275, 25781–25790 25 Yerneni, K.K. et al. (1999) Hyperglycemia-induced activation of nuclear transcription factor kappaB in vascular smooth muscle cells. Diabetes 48, 855–864 26 Peiro, C. et al. (2003) Glycosylated human oxyhaemoglobin activates nuclear factor-kappaB and activator protein-1 in cultured human aortic smooth muscle. Br. J. Pharmacol. 140, 681–690 27 Guha, M. et al. (2000) Molecular mechanisms of tumor necrosis factor alpha gene expression in monocytic cells via hyperglycemia-induced oxidant stress-dependent and -independent pathways. J. Biol. Chem. 275, 17728–17739 28 Miao, F. et al. (2004) In vivo chromatin remodeling events leading to inflammatory gene transcription under diabetic conditions. J. Biol. Chem. 279, 18091–18097 29 Nakamura, N. et al. (2000) Induction of aldose reductase in cultured human microvascular endothelial cells by advanced glycation end products. Free Radic. Biol. Med. 29, 17–25
Vol. 2, No. 1 2005
30
31
32
33
34
35
36
Srinivasan, S. et al. (2004) Hyperglycaemia-induced superoxide production decreases eNOS expression via AP-1 activation in aortic endothelial cells. Diabetologia 47, 1727–1734 Chen, Y.Q. et al. (1998) Sp1 sites mediate activation of the plasminogen activator inhibitor-1 promoter by glucose in vascular smooth muscle cells. J. Biol. Chem. 273, 8225–8231 Du, X.L. et al. (2000) Hyperglycemia-induced mitochondrial superoxide overproduction activates the hexosamine pathway and induces plasminogen activator inhibitor-1 expression by increasing Sp1 glycosylation. Proc. Natl. Acad. Sci. USA 97, 12222– 12226 Hasan, R.N. et al. (2003) Differential regulation of early growth response gene-1 expression by insulin and glucose in vascular endothelial cells. Arterioscler. Thromb. Vasc. Biol. 23, 988–993 Hassa, P.O. and Hottiger, M.O. (2002) The functional role of poly(ADPribose)polymerase 1 as novel coactivator of NF-kappaB in inflammatory disorders. Cell. Mol. Life Sci. 59, 1534–1553 Soriano, F.G. et al. (2001) Diabetic endothelial dysfunction: role of reactive oxygen and nitrogen species production and poly(ADP-ribose) polymerase activation. J. Mol. Med. 79, 437–448 Szabo, C. et al. (2002) Poly(ADP-ribose) polymerase is activated in subjects at risk of developing type 2 diabetes and is associated with impaired vascular reactivity. Circulation 106, 2680–2686
Drug Discovery Today: Disease Mechanisms | Cardiovascular diseases
37
38
39
40
41
42 43 44
Li, J.H. et al. (2004) Advanced glycation end products activate Smad signaling via TGF-beta-dependent and independent mechanisms: implications for diabetic renal and vascular disease. FASEB J. 18, 176–178 Kanie, N. et al. (2003) Relationship between peroxisome proliferatoractivated receptors (PPAR alpha and PPAR gamma) and endotheliumdependent relaxation in streptozotocin-induced diabetic rats. Br. J. Pharmacol. 140, 23–32 Takagi, T. et al. (2003) Pioglitazone reduces neointimal tissue proliferation after coronary stent implantation in patients with type 2 diabetes mellitus: an intravascular ultrasound scanning study. Am. Heart J. 146, E5 Koshiyama, H. et al. (2001) Rapid communication: inhibitory effect of pioglitazone on carotid arterial wall thickness in type 2 diabetes. J. Clin. Endocrinol. Metab. 86, 3452–3456 Tonelli, M. et al. (2004) Gemfibrozil for secondary prevention of cardiovascular events in mild to moderate chronic renal insufficiency. Kidney Int. 66, 1123–1130 Lin, Z. et al. (2005) Kruppel-like factor 2 (KLF2) regulates endothelial thrombotic function. Circ. Res. 96, e48–e57 Sen-Banerjee, S. et al. KLF2 as a novel mediator of statin effects in endothelial cells. Circulation (in press) Watson, P.A. et al. (2001) Diabetes-related changes in cAMP response element-binding protein content enhance smooth muscle cell proliferation and migration. J. Biol. Chem. 276, 46142–46150
www.drugdiscoverytoday.com
17
Drug Discovery Today: Disease Mechanisms
DRUG DISCOVERY
TODAY
Editors-in-Chief Toren Finkel – National Heart, Lung and Blood Institute, National Institutes of Health, USA Tamas Bartfai – Harold L. Dorris Neurological Research Center and The Scripps Research Institute, USA
DISEASE Cardiovascular diseases MECHANISMS
Molecular mechanisms of diabetic vasculopathy Anne Hamik1, G. Brandon Atkins2, Mukesh K. Jain1,* 1 Program in Cardiovascular Transcriptional Biology, Cardiovascular Division, Brigham and Women’s Hospital, Harvard Medical School, 75 Francis Street, Boston, MA 02115, USA 2 Division of Cardiology, Massachusetts General Hospital, Harvard Medical School, 55 Fruit Street, Boston, MA, USA
Diabetes and its attendant complications constitute a burgeoning global health threat. Hyperglycemia, the
Section Editor: Cam Patterson – Farmal Biomedicines, LLC, USA
sine qua non of diabetes, induces alterations in several signaling pathways that culminate in the clinical manifestations of diabetic vasculopathy (DV). Ultimately, cellular behavior is regulated by gene transcription. Direct manipulation of gene transcription has largely been outside the purview of conventional therapeutics. However, with the sophistication of current molecular approaches, this arena is now poised for rapid development. In this review, we describe key transcription factors that show potential as therapeutic targets for DV.
Introduction The abnormal metabolic milieu created by hyperglycemia or the diabetic state initiates a series of dysfunctional responses which culminate in the development of premature cardiovascular disease [1,2]. This process begins with dysregulation of vascular cell homeostatic mediators, progresses to pathologic anatomic changes in vascular beds, and results in the familiar clinical syndromes of diabetes (outlined in Box 1). At least four major cellular signaling pathways have been implicated in the development of diabetic vasculopathy (DV) (illustrated in Fig. 1). In the first half of this review, we briefly *Corresponding author: M.K. Jain ([email protected]) 1740-6765/$ ß 2005 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddmec.2005.05.030
review these pathways with reference to more extensive reviews. In the second half we focus on nuclear pathways involved in the development of DV. Throughout we highlight novel therapeutic strategies targeting specific pathways or factors (listed in Table 1). Conventional therapies such as aspirin, statins and angiotensin converting enzyme (ACE) inhibitors – known to favorably affect multiple pathways – have been subject to previous reviews and are not included in the table.
Major signaling pathways Oxidative stress pathway The oxidative stress pathway serves as a common element linking all the major pathways implicated in DV [3]. Hyperglycemia induces oxidative stress in vascular cells by enhancing the production of reactive oxygen species (ROS). This results in damage to cellular proteins, reduced nitric oxide (NO) levels and activation of transcription factors such as activated protein-1 (AP-1) and nuclear factor-kB (NF-kB) [1]. Collectively, these effects confer a pro-adhesive, pro-thrombotic phenotype to the vessel wall and serve as the basis of clinical studies aimed at inhibiting oxidant stress. Unfortunately, the use in humans of conventional antioxidants such as Vitamin E to prevent diabetic vascular complications has largely been ineffective [1,4]. However, agents that attenuate the production of ROS (peroxisome proliferator-activated receptor-g ligands, statins, angiotensin converting enzyme (ACE) inhibitors, calcium channel blockers and angiotensin www.drugdiscoverytoday.com
11
Drug Discovery Today: Disease Mechanisms | Cardiovascular diseases
Vol. 2, No. 1 2005
Box 1.
Cellular effects and functional consequences of diabetes. Diabetes alters the homeostasis of the cellular components of the vasculature (including endothelial cells, monocytes and vascular smooth muscle cells) in predictable ways. This leads to dysregulation of the normal anti-thrombotic, anti-adhesive and vasoreactive state of vascular beds and eventually to the macrovascular and microvascular complications of diabetes. DM, diabetes mellitus; EC, endothelial cell; VSMC, vascular smooth muscle cells; phenotypic modulation, change from a quiescent state to one which includes elaboration of extracellular matrix, cell proliferation and migration; TF, tissue factor; PAI-1, plasminogen activator inhibitor-1; eNOS, endothelial nitric oxide synthase; VCAM-1, vascular cell adhesion molecule-1; AT-II, angiotensin II; ET-1, endothelin-1; COX-2, cyclooxygenase-2; TNFa, tumor necrosis factor a; MCP-1, monocyte chemoattractant protein-1; TGFb, transforming growth factor b; CTGF, connective tissue growth factor; CAD, coronary atherosclerotic disease; PAD, peripheral atherosclerotic disease; MI, myocardial infarction.
Table 1. Novel strategies targeting key pathways and transcription factors in diabetic vasculopathy Target pathway or transcription factor
Drug
Who is working on this
Stage
Refs
Oxidative stress pathway Inhibitor of ROS
Vitamin E
Multiple investigators
Phase III clinical trials
[1,4]
AGE–RAGE pathway Blockade of AGE formation Blockade of crosslink formation RAGE blockade Transketolase activator
Aminoguanidine Alagebrium sRAGE Benfotiamine
Multiple investigators Forbes and others Schmidt et al. Brownlee et al.
Phase III clinical trials Phase II clinical trials Preclinical Phase III clinical trials
[8,9] [7,8] [11] [10]
Ruboxistaurin CGP53353 Benfotiamine
King et al. Cosentino et al. Brownlee et al.
Phase III clinical trials Preclinical Phase III clinical trials
[13] [16] [10]
Polyol/aldose reductase pathway Aldose reductase inhibitors
Epalrestat/tolrestat
Srivastava and others
Phase II–III clinical trials
[17,18]
PARP PARP inhibitor
PJ34
Szabo et al.
Preclinical
[35,36]
PPAR PPARa and PPARg agonists
TZDs, fibrates
Plutzky, Staels and others
Phase III clinical trials
[38–41]
Protein kinase C pathway PKCb inhibitor Transketolase activator
Abbreviations: AGE, advanced glycation end product; RAGE, receptor of AGE; sRAGE, soluble RAGE; PKC, protein kinase C; ROS, reactive oxygen species; PPAR, peroxisome proliferatoractivated receptor; TZDs, thiazolidinediones; PARP, poly(ADP ribose) polymerase.
12
www.drugdiscoverytoday.com
Vol. 2, No. 1 2005
Figure 1. Pathways leading to diabetes. Genetic predisposition and environmental factors influence the development of the clinical precursors of diabetes: hyperglycemia, insulin resistance and dyslipidemia. In turn, diabetes leads to the pathological activation of at least four interacting cellular pathways. The net effect is an accelerated vasculopathy – the vasculopathy of diabetes. AGE/RAGE, advanced glycation endproducts/receptor of AGE pathway; PKC, protein kinase C pathway. See text for description of pathways. Modified and reproduced, with permission, from Ref. [2].
II type 1 (AT-1) receptor antagonists) [4] have all been shown to reduce the burden of cardiovascular disease out of proportion to their respective glucose lowering, lipid lowering, or blood pressure lowering effects.
Advanced glycation end products (AGE) pathway AGEs are a heterogenous group of compounds produced as a consequence of the irreversible nonenzymatic glycation of proteins [5]. This process occurs in diabetes as well as in aging and is augmented by oxidative stress and inflammation. AGEs can confer deleterious effects to the vessel wall in two main ways. First, crosslinking of long-lived proteins such as collagen or elastin in the vessel wall can alter structural integrity. Second, binding of AGEs to their receptor (RAGE, GenBank accession no. NM_001136) on vascular cells can activate multiple signaling pathways (e.g. protein kinase C (PKC) and MAPkinase pathways) and activate nuclear factors (e.g. NF-kB and cyclic AMP-responsive element-binding protein; CREB, GenBank accession no. L05515) resulting in an increase in ROS production and elaboration of inflammatory factors [6]. As
Drug Discovery Today: Disease Mechanisms | Cardiovascular diseases
such, ways to limit cross-linking of proteins and inhibit RAGE receptor activation have been the subject of intense study in both animal models and, more recently, human trials. Treatment of diabetic mice with inhibitors of AGE formation (e.g. aminoguanidine) or AGE crosslink breakers such as alagebrium attenuated atherosclerosis and reduced vascular stiffness [7,8]. Clinical trials of aminoguanidine to prevent progression of diabetic nephropathy were terminated owing to lack of efficacy and safety concerns [9]. However, Phase II clinical studies in elderly patients using alagebrium demonstrate improved arterial compliance and systolic blood pressure [7]. Finally, the lipid-soluble thiamine derivative benfotiamine has potential for clinical use; in animal studies it has been demonstrated to inhibit both the hyperglycemia-induced AGE/RAGE and PKC pathways [10]. With respect to RAGE receptor activation, treatment of rodents with sRAGE (a soluble form of the RAGE receptor that blocks activation of cell surface RAGE) reduced atherosclerotic burden, ameliorated lesion complexity and decreased inflammatory gene expression in vascular cells. Furthermore, mice deficient in the RAGE receptor exhibit a decrease in smooth muscle proliferative response and neointimal expansion after vessel injury (reviewed in [11]). Finally, a recent study demonstrated that levels of sRAGE are inversely related to the presence of coronary artery disease in men [12]. These observations provide a compelling argument to assess inhibition of RAGE activation in humans.
Protein kinase C pathway Hyperglycemia can activate the PKC pathway directly via diacylglycerol (DAG) synthesis and indirectly through production of ROS or activation of the AGE/RAGE pathway [1,13, 14]. The observation that preferential activation of PKCb2 (GenBank accession no. NM_212535) occurs in the aorta and hearts of diabetic animals provided the foundation for interest in this field [15]. By virtue of its ability to alter the balance of vasodilatory/vascoconstrictive factors (e.g. endothelial nitric oxide synthase; eNOS, GenBank accession no. NM_000603/endothelin; ET-1, GenBank accession no. NM_001955), increase oxidative stress, induce adhesion molecule expression (e.g. vascular cell adhesion molecule-1; VCAM1, GenBank accession no. NM_001078) and enhance expression of profibrotic factors (e.g. transforming growth factor b1; TGFb1, GenBank accession no. NM_000660 and connective tissue growth factor; CTGF, GenBank accession no. NM_001901), PKCb has emerged as a key regulatory molecule in the pathogenesis of diabetic vasculopathy [1,13,15]. Selective and nonselective PKCb inhibitors have been developed [16]. In humans, infusion of the PKCb inhibitor ruboxistaurin ameliorates endothelial dysfunction induced by glucose infusion. PKC activation has been verified in human pathologic specimens and PKCb inhibitors such as ruboxistaurin are currently undergoing Phase II/III clinical trials [13]. www.drugdiscoverytoday.com
13
Drug Discovery Today: Disease Mechanisms | Cardiovascular diseases
Polyol/aldose reductase pathway Elevation in glucose can activate the enzyme aldose reductase (GenBank accession no. NM_000594) that converts glucose to sorbitol and ultimately to fructose. This pathway alters NAD(P)H/NAD(P) balance in a manner that leads to augmentation of oxidative stress and ultimately vascular disease [14]. Furthermore, as discussed above, redox stress can activate both the PKC and AGE pathways. As such, it is reasonable to consider aldose reductase inhibitors as therapeutic agents. Although studies in animal models using tolrestat and epalrestat have been encouraging, studies in humans have yielded minimal benefit [17,18].
Transcriptional regulatory pathways Promoting vasculopathy NF-kB
Inflammation is a crucial feature in the development of vascular disease, and NF-kB is a central mediator of most inflammatory stimuli. NF-kB proteins exist in unstimulated cells as homo- or hetero-dimers bound to an inhibitory molecule IkB (GenBank accession no. NM_002503). Upon cellular activation by pro-inflammatory stimuli, the IKK complex is activated. Specifically, the beta subunit (IKK-b, GenBank accession no. NM_001556) phosphorylates IkB resulting in its degradation and thereby liberates NF-kB to move into the nucleus, bind DNA and affect target genes. Specific NF-kB inhibitors are being tested for efficacy in other disease states, such as cancer biology. Multiple lines of evidence support a role for NF-kB in diabetes. First, hyperglycemia, oxidative stress, PKC activation, AGE–RAGE interactions and inflammatory cytokines can all activate NF-kB. In animal models, deficiency of one of the NF-kB subunits (p50, GenBank accession no. S76638) can render mice resistant to streptozotocin-induced diabetes [19]. Interestingly, inhibition of IKKb by high dose aspirin reverses hyperglycemia, hyperinsulinemia and dyslipidemia in obese rodents [20]. The effect of IKKb inhibition on the development of vascular disease remains unknown. Finally, in patients with diabetes, a positive correlation exists between insufficient glycemic control and NF-kB activity in peripheral blood mononuclear cells [21]. NF-kB has been shown to regulate gene expression and function of vascular cells in a manner that promotes the development of vascular disease. Hyperglycemia as well as AGE–RAGE interaction can induce NF-kB and endothelial expression of VCAM-1 in cell culture and rodent models [22]. Furthermore, exposure of endothelial cells to hyperglycemic sera from diabetic patients enhanced leukocyte adhesion [23]. A human correlate is suggested by the finding of elevated VCAM-1 levels in the plasma of diabetic patients [22]. In addition, a positive feedback mechanism is suggested by the observation that AGE and TNFa (tumor necrosis 14
www.drugdiscoverytoday.com
Vol. 2, No. 1 2005
factora; GenBank accession no. NM_000594) induce RAGE in an NF-kB-dependent manner [24]. Hyperglycemia can also induce NF-kB activation in vascular smooth muscle cells (VSMCs) resulting in an increase in ROS and VCAM-1 expression [25]. The aldose reductase pathway has also been identified as important in NF-kB activation in VSMCs. Indeed, a recent study demonstrated that both pharmacologic inhibition of aldose reductase as well as small interfering RNA (siRNA)-mediated reduction in the expression of this enzyme prevented the hyperglycemia-mediated activation of NF-kB in VSMCs. Furthermore, treatment of diabetic and wild type rats with the aldose reductase inhibitor tolrestat reduced neointimal proliferation and decreased staining of tissue sections with an antibody against NF-kB [18]. Finally, treatment of VSMCs with AGEs activates NF-kB (as well as AP-1), enhances secretion of cytokines and promotes VSMC proliferation and migration [26]. These data support an important role for NF-kB in diabetic smooth muscle biology. Elegant studies from the Natarajan laboratory support an important role for hyperglycemia-induced NF-kB in monocyte biology. Exposure of monocytes to sustained hyperglycemia resulted in an increase in TNFa and COX-2 (cyclooxygenase-2, GenBank accession no. U04636) production – at least in part owing to NF-kB activation (along with activation of AP-1 and CREB) [27]. Importantly, these investigators demonstrated that hyperglycemia enhanced NF-kB expression while reducing the protein levels of the inhibitory molecule IkBa. Furthermore, chromatin immunoprecipitation assays demonstrate that hyperglycemia increases recruitment of the NF-kB subunit p65 (GenBank accession no. M62399) as well as NF-kB coactivators CREB binding protein (CBP, GenBank accession no. NM_004380) and p300/CBP associated factor (p/CAF, GenBank accession no. U57317) to target promoters while decreasing binding of transcriptional co-repressors such as histone deacetylases (HDACs) [28]. These observations suggest that chromatin modification might be involved in regulating the activation of NF-kB (and perhaps other factors) in diabetes. Others
AP-1: AP-1 complexes are homo- or heterodimers composed of basic region-leucine zipper proteins that belong to the Jun and Fos families of factors that have been implicated in cellular activation, proliferation and survival. Data on this family in the context of diabetes are limited. Studies do show that hyperglycemia and AGE–RAGE interactions can induce AP-1 DNA-binding and, as a consequence, induce expression of aldose reductase in endothelial cells [29]. This observation is of interest as it provides a link between hyperglycemia/AGE– RAGE interactions and the polyol pathway. Finally, activation of AP-1 has also been shown to reduce eNOS expression [30]. Sp1/Egr-1: Sp1 (GenBank accession no. NM_138473) and early growth response protein-1 (Egr-1, GenBank accession
Vol. 2, No. 1 2005
no. NM_001964) are broadly expressed zinc-finger transcription factors. In endothelial and VSMCs the hyperglycemia-mediated increase in plasminogen activator inhibitor-1 (PAI-1) expression is in part Sp1 dependent [31,32]. Egr-1 is known to be involved in activation of transcription of many genes implicated in the development of diabetic vasculopathy, including platelet-derived growth factor (PDGF, GenBank accession no. NM_006206), tissue factor (TF, GenBank accession no. M27436) and PAI-1. Although hyperglycemia and insulin can induce Egr-1 mRNA in endothelial cells [33], the importance of this regulation remains unknown. PARP: Poly(ADP ribose) polymerases (PARPs) are chromatin-associated proteins that catalyze the transfer of ADP-ribose units from NAD+ to target proteins and have been implicated in a variety of cellular processes such as DNA repair, cell cycle control and gene regulation. PARP-1 (GenBank accession no. NM_00168) has also been shown to act as a coactivator for NFkB [34]. Induction of experimental diabetes induces PARP activity in the vessel wall. Furthermore, studies using PARP inhibitors and vessels from PARP-null mice demonstrate resistance against glucose-induced endothelial dysfunction [35]. Furthermore, PARP activation was observed in subjects at risk for developing diabetes [36]. The availability of PARP inhibitors (e.g. PJ34) raises the possibility that these can be used in the treatment of diabetic vasculopathy. Smads: Smads are one of the main mediators of TGFb antiinflammatory and profibrotic effects. In VSMCs, AGEs have been shown to directly activate Smad2/3 (GenBank accession no. NM_005901) leading to increased fibronectin synthesis [37]. In this manner, activation by the AGE pathway might promote vascular sclerosis.
Inhibiting vasculopathy PPAR PPARs are a family of ligand-activated nuclear hormone receptors involved in the regulation of lipid and glucose metabolism. Synthetic compounds that activate PPARa (GenBank accession no. NM_001001928) (e.g. fibrates) and PPARg (GenBank accession no. NM_138712) (e.g. thiazolidinediones, TZDs) are used clinically to decrease lipid abnormalities and as insulin sensitizers, respectively. These receptors are also expressed in vascular cells and macrophages where they have been shown to exhibit potent anti-inflammatory effects – at least in part through inhibition of NF-kB and AP-1 pathways. In models of experimental diabetes, PPARa and PPARg levels are reduced [38]. Activation of PPARs can inhibit adhesion molecule expression, cytokine production, PKC activation, RAGE expression, elaboration of matrix metalloproteinases and cellular proliferation. A recent study comprehensively evaluated the effect of various PPAR agonists in experimental atherosclerosis and demonstrated that PPARa and PPARg ligands can reduce atherogenesis in mice [38]. Finally, clinical observations support an important role for
Drug Discovery Today: Disease Mechanisms | Cardiovascular diseases
PPARa and PPARg ligands in the treatment of vasculopathy. For example, treatment of patients with TZDs has been shown to reduce intimal hyperplasia after coronary stenting [39]. Pilot studies assessing effects of TZDs on carotid artery intimal-medial thickness in patients with type II diabetes are also encouraging and demonstrate a reduction in plaque thickness [40]. Furthermore, treatment of patients with the PPARa agonist gemfibrozil lowered vascular events in patients with type II diabetes [41].
Others KLF2
KLF2 is a zinc-finger protein that is expressed in the endothelial layer of the blood vessel wall. Recent studies from our group indicate that this factor can confer anti-adhesive, antithrombotic and anti-inflammatory properties in endothelial cells ([42], and references therein). The reduction in KLF2 expression by inflammatory stimuli could be a key mechanism by which the endothelium is rendered dysfunctional in disease states such as diabetes. Identification of mechanisms to induce KLF2 in endothelial cells can provide novel approaches in the treatment of diabetic vasculopathy. In fact, multiple statins significantly induce endothelial KLF2 expression, and the statin-dependent induction of eNOS and thrombomodulin requires KLF2 [43]. CREB
The role of this transcription factor in diabetic vasculopathy remains controversial. Studies in monocytes suggest that this factor might cooperate with NF-kB and induce genes such as COX-2 in hyperglycemic conditions. However, in VSMCs, hyperglycemia led to decreased CREB protein and increased enhanced chemokinesis (a sign of phenotypic modulation) [44]. Clearly, additional studies are needed to elucidate the role of CREB in diabetic vasculopathy.
Conclusions The burden of DV on the global population is enormous and growing ever greater. In addition to lifestyle modification, novel therapies are clearly needed in the treatment of diabetes. Over the past two decades, considerable progress has been made in our understanding of the key cellular signaling pathways involved in the pathogenesis of DV. As noted in this review, these studies have provided novel approaches to the treatment of DV. However, given the complexity of this disease process, it is probable that additional therapeutic approaches will be required. Apropos unique approaches, an emerging field that shows great promise – but is beyond the scope of this review – is that concerning the anti-inflammatory activities of adipocytokines such as adiponectin (GenBank accession no. NP_004788). Ultimately, many of these pathways will converge in nuclear events that regulate gene expression and, as a consequence, www.drugdiscoverytoday.com
15
Drug Discovery Today: Disease Mechanisms | Cardiovascular diseases
Figure 2. Transcription factors implicated in regulation of the development of diabetic vasculopathy. NF-kB and PPARa,g have been demonstrated to induce and inhibit, respectively, development of diabetic vasculopathy. Emerging data implicate the additional factors listed under Section ‘Others’; however, current data are most compelling for NF-kB and PPARs. See text for details.
cellular function. As such, an understanding of the nuclear mechanism(s) involved in the development of DV is a crucial but poorly developed area of research, deserving of greater investigation. Integral involvement in DV of the transcriptional regulators NF-kB and the PPARs has been demonstrated experimentally and clinically, respectively (summarized in Fig. 2). However, few studies have been performed to date that take advantage of contemporary molecular techniques (chromatin immunoprecipitation, cofactor identification, analysis of modification of transcription factors and chromatin by acylation and methylation, confirmatory experiments in transgenic mice). It is probable that through such studies we will identify novel therapeutic strategies that will ultimately allow us to curtail the increasing burden of DV and its attendant consequences.
References 1 Sheetz, M.J. and King, G.L. (2002) Molecular understanding of hyperglycemia’s adverse effects for diabetic complications. J. Am. Med. Assoc. 288, 2579–2588 2 Beckman, J.A. et al. (2002) Diabetes and atherosclerosis: epidemiology, pathophysiology, and management. J. Am. Med. Assoc. 287, 2570–2581 3 Nishikawa, T. et al. (2000) Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature 404, 787–790 4 Ceriello, A. and Motz, E. (2004) 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 5 Wautier, J.L. and Schmidt, A.M. (2004) Protein glycation: a firm link to endothelial cell dysfunction. Circ. Res. 95, 233–238 6 Yan, S.F. et al. (2003) Glycation, inflammation, and RAGE: a scaffold for the macrovascular complications of diabetes and beyond. Circ. Res. 93, 1159–1169 7 Bakris, G.L. et al. (2004) Advanced glycation end-product cross-link breakers: a novel approach to cardiovascular pathologies related to the aging process. Am. J. Hypertens. 17 (12 Pt 2), 23S–30S
16
www.drugdiscoverytoday.com
Vol. 2, No. 1 2005
8 Forbes, J.M. et al. (2004) Advanced glycation end product interventions reduce diabetes-accelerated atherosclerosis. Diabetes 53, 1813–1823 9 Thornalley, P.J. (2003) Use of aminoguanidine (Pimagedine) to prevent the formation of advanced glycation endproducts. Arch. Biochem. Biophys. 419, 31–40 10 Hammes, H.P. et al. (2003) Benfotiamine blocks three major pathways of hyperglycemic damage and prevents experimental diabetic retinopathy. Nat. Med. 9, 294–299 11 Naka, Y. et al. (2004) RAGE axis: animal models and novel insights into the vascular complications of diabetes. Arterioscler. Thromb. Vasc. Biol. 24, 1342–1349 12 Falcone, C. et al. (2005) Plasma levels of soluble receptor for advanced glycation end products and coronary artery disease in nondiabetic men. Arterioscler. Thromb. Vasc. Biol. 25, 1032–1037 13 Rask-Madsen, C. and King, G.L. (2005) Proatherosclerotic mechanisms involving protein kinase C in diabetes and insulin resistance. Arterioscler. Thromb. Vasc. Biol. 25, 487–496 14 Brownlee, M. (2001) Biochemistry and molecular cell biology of diabetic complications. Nature 414, 813–820 15 Way, K.J. et al. (2001) Protein kinase C and the development of diabetic vascular complications. Diabet. Med. 18, 945–959 16 Kouroedov, A. et al. (2004) Selective inhibition of protein kinase Cbeta2 prevents acute effects of high glucose on vascular cell adhesion molecule-1 expression in human endothelial cells. Circulation 110, 91–96 17 El-Kabbani, O. et al. (2004) Aldose reductase structures: implications for mechanism and inhibition. Cell. Mol. Life Sci. 61, 750–762 18 Ramana, K.V. et al. (2004) Activation of nuclear factor-kappaB by hyperglycemia in vascular smooth muscle cells is regulated by aldose reductase. Diabetes 53, 2910–2920 19 Mabley, J.G. et al. (2002) NFkappaB1 (p50)-deficient mice are not susceptible to multiple low-dose streptozotocin-induced diabetes. J. Endocrinol. 173, 457–464 20 Yuan, M. et al. (2001) Reversal of obesity- and diet-induced insulin resistance with salicylates or targeted disruption of Ikkbeta. Science 293, 1673–1677 21 Hofmann, M.A. et al. (1998) Insufficient glycemic control increases nuclear factor-kappa B binding activity in peripheral blood mononuclear cells isolated from patients with type 1 diabetes. Diabetes Care 21, 1310–1316 22 Schmidt, A.M. et al. (1995) Advanced glycation endproducts interacting with their endothelial receptor induce expression of vascular cell adhesion molecule-1 (VCAM-1) in cultured human endothelial cells and in mice. A potential mechanism for the accelerated vasculopathy of diabetes. J. Clin. Invest. 96, 1395–1403 23 Morigi, M. et al. (1998) Leukocyte-endothelial interaction is augmented by high glucose concentrations and hyperglycemia in a NF-kB-dependent fashion. J. Clin. Invest. 101, 1905–1915 24 Tanaka, N. et al. (2000) The receptor for advanced glycation end products is induced by the glycation products themselves and tumor necrosis factor-alpha through nuclear factor-kappa B, and by 17 beta-estradiol through Sp-1 in human vascular endothelial cells. J. Biol. Chem. 275, 25781–25790 25 Yerneni, K.K. et al. (1999) Hyperglycemia-induced activation of nuclear transcription factor kappaB in vascular smooth muscle cells. Diabetes 48, 855–864 26 Peiro, C. et al. (2003) Glycosylated human oxyhaemoglobin activates nuclear factor-kappaB and activator protein-1 in cultured human aortic smooth muscle. Br. J. Pharmacol. 140, 681–690 27 Guha, M. et al. (2000) Molecular mechanisms of tumor necrosis factor alpha gene expression in monocytic cells via hyperglycemia-induced oxidant stress-dependent and -independent pathways. J. Biol. Chem. 275, 17728–17739 28 Miao, F. et al. (2004) In vivo chromatin remodeling events leading to inflammatory gene transcription under diabetic conditions. J. Biol. Chem. 279, 18091–18097 29 Nakamura, N. et al. (2000) Induction of aldose reductase in cultured human microvascular endothelial cells by advanced glycation end products. Free Radic. Biol. Med. 29, 17–25
Vol. 2, No. 1 2005
30
31
32
33
34
35
36
Srinivasan, S. et al. (2004) Hyperglycaemia-induced superoxide production decreases eNOS expression via AP-1 activation in aortic endothelial cells. Diabetologia 47, 1727–1734 Chen, Y.Q. et al. (1998) Sp1 sites mediate activation of the plasminogen activator inhibitor-1 promoter by glucose in vascular smooth muscle cells. J. Biol. Chem. 273, 8225–8231 Du, X.L. et al. (2000) Hyperglycemia-induced mitochondrial superoxide overproduction activates the hexosamine pathway and induces plasminogen activator inhibitor-1 expression by increasing Sp1 glycosylation. Proc. Natl. Acad. Sci. USA 97, 12222– 12226 Hasan, R.N. et al. (2003) Differential regulation of early growth response gene-1 expression by insulin and glucose in vascular endothelial cells. Arterioscler. Thromb. Vasc. Biol. 23, 988–993 Hassa, P.O. and Hottiger, M.O. (2002) The functional role of poly(ADPribose)polymerase 1 as novel coactivator of NF-kappaB in inflammatory disorders. Cell. Mol. Life Sci. 59, 1534–1553 Soriano, F.G. et al. (2001) Diabetic endothelial dysfunction: role of reactive oxygen and nitrogen species production and poly(ADP-ribose) polymerase activation. J. Mol. Med. 79, 437–448 Szabo, C. et al. (2002) Poly(ADP-ribose) polymerase is activated in subjects at risk of developing type 2 diabetes and is associated with impaired vascular reactivity. Circulation 106, 2680–2686
Drug Discovery Today: Disease Mechanisms | Cardiovascular diseases
37
38
39
40
41
42 43 44
Li, J.H. et al. (2004) Advanced glycation end products activate Smad signaling via TGF-beta-dependent and independent mechanisms: implications for diabetic renal and vascular disease. FASEB J. 18, 176–178 Kanie, N. et al. (2003) Relationship between peroxisome proliferatoractivated receptors (PPAR alpha and PPAR gamma) and endotheliumdependent relaxation in streptozotocin-induced diabetic rats. Br. J. Pharmacol. 140, 23–32 Takagi, T. et al. (2003) Pioglitazone reduces neointimal tissue proliferation after coronary stent implantation in patients with type 2 diabetes mellitus: an intravascular ultrasound scanning study. Am. Heart J. 146, E5 Koshiyama, H. et al. (2001) Rapid communication: inhibitory effect of pioglitazone on carotid arterial wall thickness in type 2 diabetes. J. Clin. Endocrinol. Metab. 86, 3452–3456 Tonelli, M. et al. (2004) Gemfibrozil for secondary prevention of cardiovascular events in mild to moderate chronic renal insufficiency. Kidney Int. 66, 1123–1130 Lin, Z. et al. (2005) Kruppel-like factor 2 (KLF2) regulates endothelial thrombotic function. Circ. Res. 96, e48–e57 Sen-Banerjee, S. et al. KLF2 as a novel mediator of statin effects in endothelial cells. Circulation (in press) Watson, P.A. et al. (2001) Diabetes-related changes in cAMP response element-binding protein content enhance smooth muscle cell proliferation and migration. J. Biol. Chem. 276, 46142–46150
www.drugdiscoverytoday.com
17