Current Pharmaceutical Design - Volume 13, Issue 26, 2007

Atherosclerosis is the leading cause of death in the Western world and anti-thrombotic therapy plays a key role in the management of this disease. However, despite the importance of platelets in the disease process there has been little progress in developing novel anti-thrombotic agents. Current anti-thrombotic therapy revolves around aspirin, a drug that has been around for thousands of years. More recently GPIIb/IIIa antagonists were hailed as the future of anti-thrombotic therapy. However, with the failure of the oral GPIIb/IIIa antagonists [1] these drugs have been restricted to high-risk patients under-going interventions [2]. Clopidogrel has proven to be a significant breakthrough as it is orally active and is very effective at inhibiting platelet aggregation [3]. So do we need another anti-platelet agent? There is convincing evidence for aspirin resistance and this has been shown to be associated with poor outcome in cardiovascular patients [4]. The requirement for intravenous administration has restricted the use of GPIIb/IIIa antagonists. Clopidogrel is the most effective anti-platelet agent available today however, there are problems associated with its use. Patients who have had a stent implanted can develop in-stent thrombosis even when on clopidogrel [5]. Since this can be fatal there is a definite need for more effective anti-thrombotic agents. There are three potential reasons for the failure of aspirin and clopidogrel to prevent thrombosis. The first is due to underdosing. The recommendations for the use of clopidogrel have all moved towards increased loading dose and to extending the treatment period [6]. A second potential problem is inherent resistance to the effect of anti-platelet agents. There is evidence that some people do not respond to aspirin and continue to have normal platelet function despite aspirin therapy [7, 8]. A similar phenomenon appears to exist with clopidogrel although not as well characterized. Some patients appear to be resistant to both anti-platelet agents [9]. A third limitation with aspirin and clopidogrel is their mechanism of action. Aspirin acts to inhibit cyclooxygenase-mediated events while clopidogrel inhibits ADP-mediated events. However, non-COX and non-ADPmediated events can occur as well. Thrombin will activate platelets in a COX-independent manner and does not require ADP for its action although secreted ADP may enhance its actions. Thus, existing anti-platelet agents may be effective in situations where there is collagen exposure but would be ineffective if thrombin generation is occurring. While there is a case to be made for the development of new anti-platelet agents improved versions of aspirin or clopidogrel are unlikely to be effective as they will still be limited by their mechanism of action. Thus it is necessary to identify novel drug targets. There are two approaches that can be used. The first is to target proteins that are known to play a role in thrombus formation. The second approach is to identify novel proteins that may be involved. In this issue we look at both approaches to developing new platelet drug targets. One key receptor in platelet function is GPIb. It is well known to mediate platelet adhesion to von Willebrand factor under high shear. Since thrombosis in the coronary vessels usually occurs in a high shear environment the GPIb-vWf interaction is an ideal target. It also promises to be free of the major adverse effect of other anti-thrombotics as it should not prolong bleeding as this usually occurs in a low shear environment. Many companies have tried to develop inhibitors of this interaction with little success. Hans Deckmyn's group in Leuven, Belgium has many years of experience working with GPIb. Karen Vanhoorelbeke from this group writes about the progress in developing inhibitors of GPIb. In most cases these are antibodies or proteins which will restrict them to acute use. However, this is not necessarily a problem since these agents will only inhibit the initial step of platelet adhesion and will have no effect on thrombus growth. Thus they are likely to be most effective when given early such as prior to angioplasty. Another promising target is the collagen receptors. The interaction between platelets and collagen is central to thrombus formation and thus is an ideal drug target. However, there are two different collagen receptors and we are only beginning to understand the interplay between the two and their role in thrombosis.........

The platelet releasate comprises of a multitude of inflammatory and vasoactive substances, which can attract atherogenic leukocytes from the circulation, activate endothelial cells and stimulate vessel growth and repair by triggering vascular cell proliferation, migration, and inflammation. Thus, platelets are believed central in the development and progression of atherosclerotic lesions and recent progress in uncovering more than 300 proteins in the thrombin-activated platelet releasate may advance our ability to understand the events involved and responses triggered in the progression of atherosclerosis. Furthermore, neutralisation of these platelet-derived proinflammatory factors may become an interesting means for therapeutic or preventative intervention in atherosclerosis.

Platelets are the principle effectors of cellular haemostasis and key mediators in the pathogenesis of thrombosis. A variety of membrane receptors determine platelet reactivity with numerous agonists and adhesive proteins, and therefore represent key targets for the development of antiplatelet drug therapy. Here, we summarise recent advances in the analysis of the complex platelet membrane system achieved through the integration of platelet biology and proteomics.

GAS6, the product of growth arrest specific (GAS) gene 6 is a ligand for the tyrosine protein kinase receptors Axl, Tyro3 and Mer whose signaling has been implicated in cell growth, survival, adhesion and migration. Although a secreted human vitamin Kdependent protein with close structural similarity with protein S, GAS6 does not exhibit anticoagulant properties but rather may be an important regulator of vascular homeostasis and platelet signaling. GAS6 signals via its receptor tyrosine kinases and appears to modulate platelet outside-in signaling via GP αIIbβIII, playing a key role in the perpetuation of platelet aggregates and clot retraction. GAS6 is also implicated in foam cell formation and neointimal proliferation in response to vascular injury. Thus GAS6 acts at key points in the pathophysiology of atherosclerosis and thrombosis; two processes implicated in most acute cardiovascular pathology. Inhibition of GAS6 or its receptors may provide antithrombotic activity in the absence of increased bleeding and thus presents an attractive drug target. GAS6 signaling may be modulated through direct antibody inhibition, blockade of its receptors or GAS6 trapping. However, ubiquitous expression of GAS6 and its receptors and the diverse biological effects of the pathway may make selective drug targeting difficult.

Recent studies of the platelet transcriptome have shown it to be complex and readily analysed by modern techniques. Among the thousands of distinct transcripts are many not previously described in platelets. Differences in message abundance between groups are apparent, and these are reflected at the protein level. Platelets are enriched in messages for receptors, signal transduction proteins and cytokines. Categories of potential drug targets include novel receptors mediating platelet activation and proteins involved in signal transduction. In addition, proteins released or secreted by activated platelets, or specifically translated from mRNA following platelet activation represent a new category of potential drug target for the treatment and prevention of thrombosis and atherogenesis. Transcriptional studies provides a means for the identification and characterisation of novel platelet drug targets in all these categories.

Although somewhat controversial, there is good evidence that long-distance travel in general is a risk factor for venous thromboembolism, even in the absence of other risk factors. This is probably due to effects consequent to prolonged sitting but air travel in particular may be associated with risk factors other than this. One likely factor is hypoxia caused by the low ambient pressure of aircraft cabins. There is an association between venous thromboembolism and the hypoxia of altitude, chronic respiratory disease, neonatal hypoxia, sleep apnoea and experimentally-induced hypoxia. Platelet number and/or function are altered in all of these circumstances. Platelet aggregation is pivotal to venous thromboembolism and hypoxia alters platelet number and function. The early-onset thrombocytosis caused by hypoxia may be due to increased release of platelets from megakaryocytes and the late-onset thrombocytopaenia may be due to decreased platelet production and/or stem cell competition between erythrocytes and megakaryocytes. Hypoxia-induced platelet activation and aggregation may be due to increased circulating catecholamine levels but it is not known whether hypoxia can affect platelets directly. There is a need for further studies on the possible involvement of hypoxia-induced changes in platelet number and function in air travel-related venous thromboembolism.

Platelets have important roles in atherosclerosis and thrombosis and their inhibition reduces the risk of these disorders. There is still a need for platelet inhibitors affecting pathways that reduce thrombosis and atherosclerosis while leaving normal hemostasis relatively unaffected, thus reducing possible bleeding complications. Although combinations show progress in achieving these goals none of the present inhibitors completely fulfill these requirements. Collagen receptors offer attractive possibilities as alternative targets at early stages in platelet activation. Three major collagen receptors are assessed in this review; the α2β1 integrin, responsible primarily for platelet adhesion to collagen; GPVI, the major signaling receptor for collagen; and GPIb-V-IX, which is indirectly a collagen receptor via von Willebrand factor. Several thrombosis models and experimental approaches suggest that all three are interesting targets and merit further investigation.

The platelet receptor glycoprotein (GP)Ib-IX-V complex plays a dominant role in the first steps of platelet adhesion and arterial thrombus formation. Through its interaction with the multimeric plasma protein von Willebrand factor (VWF), which is bound to the damaged subendothelial structures, GPIb-IX-V tethers the platelets from the flowing blood thereby slowing them down. This step is a prerequisite for the collagen receptors to participate in firm adhesion resulting in the formation of a first platelet layer which is the basis for further thrombus formation. Recently, other ligands for GPIb-IX-V besides the extensively studied VWF have been identified, such as : α-thrombin, coagulation factor XII (FXII), high molecular weight kininogen (HMWK), factor XI (FXI), integrin Mac-1 and P-selectin. In this review, the interaction of GPIb-IX-V with its different ligands is described and the anticipated or demonstrated in vivo effects are discussed.

This issue of Current Pharmaceutical Design, for which I have the great pleasure to be Executive Guest Editor, addresses topical issues relating to the pathogenesis and treatment of diabetic complications, retinopathy, nephropathy and cardiomyopathy. Fletcher et al. [1] describes neuronal and glial cell dysfunction in diabetic retinopathy, and how these changes relate to vascular compromise. Marshall [2] discusses the importance of the podocyte in the development of diabetic nephropathy, and how a variety of factors including metabolic and hemodynamic abnormalities affect podocyte integrity. Connelly et al. [3] examines the prevalence of coronary artery disease and cardiac failure in the diabetic population, and how factors such as angiotensin II are crucial for the development of diabetic cardiac disease Dean and Burrell [4] examines the role of the recently identified enzyme, ACE2, in microvascular and macrovascular disease in diabetes, and how compounds that target ACE2 may potentially be of clinical value for the treatment of diabetic complications. Calkin et al. [5] reviews evidence that PPARα agonists have potential benefits for the treatment of diabetes-associated atherosclerosis. Given the excellence of the reviews in this issue, I hope the readers of Current Pharmaceutical Design will find this issue informative with regard to updating their knowledge about the variety of factors implicated in the development and progression of diabetic complications. The reviews identify the potential for the development of new and improved treatment strategies for the better management of diabetic micro- and macro-vascular disease. References [1] Fletcher EL, Phipps JA, Ward MM, Puthussery T, Wilkinson-Berka JL. Neuronal and Glial Cell Abnormality as Predictors of Progression of Diabetic Retinopathy. Curr Pharm Des 2007; 13(26): 2699-2712. [2] Marshall SM. The Podocyte: a Potential Therapeutic Target in Diabetic Nephropathy. Curr Pharm Des 2007; 13(26): 2713-2720. [3] Connelly KA, Boyle AJ, Kelly DJ. Angiotensin II and the Cardiac Complications of Diabetes Mellitus. Curr Pharm Des 2007; 13(26): 2721-2729. [4] Dean RG, Burrell LM. ACE2 and Diabetic Complications. Curr Pharm Des 2007; 13(26): 2730-2735. [5] Calkin AC, Jandeleit-Dahm KA, Sebekova E, Allen TJ, Mizrahi J, Cooper ME, Tikellis C. PPARs and Diabetes- Associated Atherosclerosis. Curr Pharm Des 2007; 13(26): 2736-2741.

Diabetes is known to cause significant alterations in the retinal vasculature. Indeed, diabetic retinopathy is the leading cause of blindness in those of working age. Considerable evidence is emerging that indicates that retinal neurons are also altered during diabetes. Moreover, many types of neuronal deficits have been observed in animal models and patients prior to the onset of vascular compromise. Such clinical tools as the flash ERG, multifocal ERG, colour vision, contrast sensitivity and short-wavelength automated perimetry, all provide novel means whereby neuronal dysfunction can be detected at early stages of diabetes. The underlying mechanisms that lead to neuronal deficits are likely to be broad. Retinal glial cells play an essential role in maintaining the normal function of the retina. There is accumulating evidence that Muller cells are abnormal during diabetes. They are known to become gliotic, display altered potassium siphoning, glutamate and GABA uptake and are also known to express several modulators of angiogenesis. This review will examine the evidence that neurons and glia are altered during diabetes and the relationship these changes have with vascular compromise.

Over the last five years, much work has underlined the important role of the podocyte in the development of diabetic nephropathy. The metabolic and haemodynamic abnormalities of the diabetic milieu act in concert, perhaps via the common effector path of oxidative stress and development of reactive oxygen species, to promote podocyte damage. There is loss of nephrin from the slit diaphragm, increased synthesis of some of the components of the glomerular basement membrane, activation of pro-apoptotic and hypertrophic pathways, loss of the α3β1integrin and increased secretion of VEGF. These changes interact to lead to increased permeability, accumulation of abnormal extracellular matrix, apoptosis, foot process detachment and podocyte loss. The foot processes of the remaining podocytes hypertrophy and widen, with reduced filtration slit width. The end result is increasing proteinuria, basement membrane thickening and accumulation of mesangial matrix and declining renal function. Some currently used therapies, such as tight glucose control and inhibition of the renin angiotensin system, ameliorate these changes and prevent podocyte loss. Statins may also have a specific podocyte protective role. Other potential therapies include inhibitors of glycation, antioxidants, and inhibitors of growth factor and signalling pathways.

The prevalence of diabetes has reached epidemic proportions in the developed world and is expect to increase to 5.4% by 2025. This has resulted in an unprecedented number of patients experiencing the macro- and micro-vascular complications of diabetes, such as renal, retinal, neurological and cardiac dysfunction. Premature coronary artery disease and cardiac failure are vastly overrepresented in the diabetic population, with significant morbidity and mortality. In fact, the rate of cardiac events in patients with diabetes is equivalent to non-diabetic patients with a previous myocardial infarction. Epidemiological evidence, combined with the results of large scale trials blocking the renin-angiotensin system (RAS) have provided data to support the hypothesis that angiotensin II and its interaction with the metabolic changes associated with diabetes mellitus is responsible for the pathogenesis of many of these complications. This review focuses on the role of the RAS and the development of diabetic cardiac disease.

Angiotensin converting enzyme (ACE) is a key enzyme in the renin angiotensin system (RAS) and converts angiotensin (Ang) I to the vasoconstrictor Ang II, which is thought to be responsible for most of the physiological and pathophysiological effects of the RAS. This classical view of the RAS was challenged with the discovery of the enzyme, ACE2 which both degrades Ang II and leads to formation of the vasodilatory and anti-proliferative peptide, Ang 1-7. Activation of the RAS is a major contributor to diabetic complications, and blockade of the vasoconstrictor and hypertrophic actions of Ang II, slows but does not prevent the progression of such complications. The identification of ACE2 in the heart and kidney adds further complexity to the RAS, provides the rationale to explore the role of this enzyme in pathophysiological states, including the microvascular and macrovascular complications of diabetes. It is believed that ACE2 acts in a counter-regulatory manner to ACE to modulate the balance between vasoconstrictors and vasodilators within the heart and kidney, and may thus play a significant role in the pathophysiology of cardiac and renal disease. Relatively little is known about ACE2 in diabetes, and this review will explore and discuss the data that is currently available. The discovery of ACE2 presents a novel opportunity to develop drugs that specifically influence ACE2 activity and/or expression, and it is possible that such compounds may have considerable clinical value in the prevention and treatment of the complications of diabetes.

Peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors affecting the regulation of various genes relevant to the pathogenesis of diabetic complications. A number of drugs have been developed to act as agonists of the three PPARs. To date, PPAR isoforms that have been identified are the α, β/δ, and γ isosforms. Fenofibrate and gemfibrozil are two drugs that act as PPARα agonists and are currently in use in the clinical setting. Rosiglitazone is a PPARγ agonist also in clinical use. These drugs have proved very useful in regulation of either glucose or lipid metabolism and consequently are used in patients with type 2 diabetes. Here, we will review the anti-atherosclerotic potential of PPAR agonists with particular emphasis on recent studies in an animal model of diabetes-associated atherosclerosis, the streptozotocin diabetic apolipoprotein E deficient mouse. These studies have shown both PPARα agonists, gemfibrozil and fenofibrate, confer anti-atherosclerotic effects, partly independent of their metabolic effects. Similar positive findings have also been detected in a dose-dependent manner with the PPARγ agonist, rosiglitazone. The potential clinical implications of these findings are also discussed in view of the recently reported results of the PROACTIVE and FIELD clinical trials with the PPAR agonists rosiglitazone and fenofibrate respectively..