Current Drug Targets - Volume 9, Issue 10, 2008

During tissue development and remodeling, cells obtain informational cues from the proteins that comprise the extracellular matrix. The thrombospondins are a family of extracellular, calcium-binding proteins that modulate cellular proliferation, migration and differentiation. At least one of the five members of this gene family is expressed in virtually every tissue of the body. The thrombospondins participate in embryonic development, axon guidance, synaptogenesis, wound healing, and angiogenesis. They are up-regulated and function in various disease states including, diabetes, atherosclerosis, scleroderma and tumor progression. The importance of the thrombospondins in tissue remodeling is underscored by the observations that mice that lack thrombospondin-1 (TSP-1) or -2 have significant abnormalities in response to a wide range of challenges. In addition, mutations in cartilage oligomeric matrix protein (COMP or TSP-5) cause the human dwarfing conditions pseudoachondroplasia and multiple epiphyseal dysplasia. The reviews in this series focus on the importance of thrombospondins in various disease states and discuss potential ways that the knowledge that has been gained to date may be used to diagnose and treat disease. Recently, thrombospondin-1 has been shown to regulate thrombosis by inhibiting the degradation of high molecular weight von Willebrand factor. The review by Drs. Bonnefoy and Hoelerts in this series describes the effect of TSP-1 on von Willebrand factor function. TSP-1 binds to von Willebrand factor and protects it from degradation by ADAMTS13 (A Disintegrin And Metalloprotease with ThromboSpondin-1 repeat 13). In the absence of TSP-1, there is a decrease in the formation of the long strands of von Willebrand factor that support thombosis. TSP-1 also profoundly affects the cardiovascular system through the suppression of nitric oxide (NO). The review by Dr. Isenberg and colleagues describes the molecular basis for the inhibition of NO signaling by TSP-1, CD47 and CD36. TSP-1 limits NO function in endothelial cells, smooth muscle cells and platelets. The authors point out that therapeutic strategies that target TSP-1 and/or CD47 would enhance the beneficial effects of NO on blood flow and vascular remodeling. The TSP-1/CD47 axis has also been shown to be involved in the regulation of the immune response. The review by Dr. Sarfati and colleagues explains how the interaction of TSP-1 with CD47 suppresses dendritic and T cell function. This interaction contributes to the maintenance of peripheral tolerance and the cross talk between antigen presenting cells and T cells. In adult tissue, the inhibition of angiogenesis in the tumor microenvironment has received significant attention. The review by Dr. Volpert and coworkers describes the molecular mechanisms that underlie the inhibition of angiogenesis by TSP-1, with particular focus on the induction of apoptosis. Like most anti-angiogenic therapies, TSP-1-based therapies are well tolerated and have limited activity as a monotherapy. The review by Dr. Volpert and colleagues discusses the importance of developing strategies for the use of TSP-1-based combination therapies. The thrombospondins regulate tissue genesis and remodeling by modulating the structure of the extracellular matrix and cellular behavior. They exert their effect on the extracellular matrix in several ways. Through the activation of transforming growth factor (TGF) β, TSP-1 can affect the composition of the extracellular matrix. TSPs bind directly to other matrix proteins including laminin, collagens, fibronectin and fibrinogen. The inclusion of TSP-1 into fibrin preparations decreases the fibril thickness. The absence of TSP-2 results in abnormal collagen fibril assembly in the skin. TSPs also modulate extracellular matrix structure by inhibiting proteinases, including elastase, cathepsin G, plasmin and urokinase. Proteases, in turn, may regulate TSP-1 function. The review by Dr. Iruela-Arispe describes the characterization of proteases that cleave TSP-1. The members of the ADAMTS family of extracellular proteases contain multiple copies of the type 1 repeats (TSRs). Proteolytic cleavage of the TSPs may have dramatic effects on the activity and bioavailability of specific regions of the proteins during tissue remodeling.

Thrombospondin-1 (TSP1), expressed in many cells and tissues is abundantly present in platelet α-granules, from where it is released upon platelet activation. Murine Tsp1-/- platelet studies have revealed that TSP1 is redundant for platelet aggregation, but that it reinforces platelet aggregate stabilization, especially in a shear field. von Willebrand factor (VWF), synthesized by megakaryocytes and endothelial cells is stored both in platelet α-granules and in endothelial Weibel- Palade bodies as ultralarge multimers. When released from endothelial cells, these multimers are temporarily retained on the endothelium, to be cleaved by the plasma protease ADAMTS13 into smaller and hemostatically less reactive multimers, released in plasma. This protease shows partial sequence identity with the type 1 (TSR1) and type 2 (TSR2) repeats of TSP1 and contains 1 TSR1 and 6 TSR2 repeats. TSP1, locally released by platelets, competes with ADAMTS13 during VWF proteolysis and controls the degree of VWF multimer processing. In addition, TSP1 and VWF both interact with the platelet GPIb/V/IX membrane complex, primarily in flow. These interactions control the recruitment of platelets to (sub) endothelial VWF and TSP1, exposed to the circulation, as a consequence of vascular inflammation and endothelial injury. TSP1-VWF interactions do not strictly enhance platelet recruitment and secreted TSP1 even weakly competes with the dynamic platelet rolling and adhesion onto VWF. Hence, TSP1 and VWF show partially related hemostatic functions, the most important one being the TSP1 role in the ADAMTS13 operated VWF multimer processing, in proinflammatory and thrombogenic conditions.

Activation of soluble guanylate cyclase by nitric oxide (NO) controls signaling pathways that play critical roles in normal vascular physiology and in the pathogenesis of cardiovascular disease. We have identified the secreted protein thrombospondin-1 as a key regulator of NO signaling. Thrombospondin-1 limits the angiogenic activity of NO in endothelial cells, its vasodilator activity in vascular smooth muscle, and its antithrombotic activity in platelets. Loss of either thrombospondin-1 or its receptor CD47 in transgenic mice results in hyperdynamic responses to NO and reveals the importance of this pathway in normal physiology. Thrombospondin-1 and CD47 null mice show improved abilities to respond to ischemic stress, suggesting that therapeutic targeting of this pathway could benefit patients with a variety of ischemic conditions. We review the preclinical development of therapeutics targeting thrombospondin-1 or CD47 for improving survival of fixed ischemia, ischemia due to aging and peripheral vascular disease, and skin grafting.

The past decades have been marked by spectacular progress towards understanding how dendritic cells (DCs) interact with T cells to elicit protective immune responses to fight infectious diseases and cancer. DCs that are lying at the interface between innate and adaptive immunity, are educated in peripheral tissues prior to their journey to the secondary lymphoid organs (SLO) whereby they dictate different classes of T cell responses. Uncontrolled or unwanted inflammatory responses are the price to pay to eliminate pathogens. However, if not self-limited, they may induce collateral damages that result in chronic inflammation often associated with autoimmune disorders. CD47 and its two ligands, i.e. thrombospondin 1 (TSP-1) and SIRP-α, were identified as a previously unappreciated inhibitory axis of DC and T cell functions. TSP-1 is predominantly a negative regulator of DC and T cell function while basal SIRP-α ligation on APC by CD47 enforces tolerance. Yet, CD47/SIRP-α interaction positively controls DC and innate cell transendothelial migration. Due to the promiscuity of the protein interactions for CD47 and its ligands, it is quite interesting to note that deletion of the CD47 gene in mice largely agrees with the in vitro data with human cells. In fact, the well-conserved tissue distribution of CD47 and SIRP-α across species may facilitate the transition from bench to bedside. We thus propose CD47/TSP- 1/SIRP-α axis as an important sensor to maintain homeostasis and regulate innate and adaptive immune responses.

Thrombospondin-1 is the first and most studied naturally occurring protein inhibitor of angiogenesis. Its characteristic multi-domain structure determines thrombospondin-1 divergent functions, which include but are not limited to the regulation of angiogenesis. Below we overview the structural determinants and receptors expressed on the endothelial and other cell types, that are at the root of thrombospondin-1 striking ability to block neovascularization. We specifically emphasize thrombospondin-1 direct apoptotic action on the remodeling vascular endothelium and summarize current knowledge of its pro-apoptotic signaling and transcriptional networks. Further, we provide comprehensive survey of the thrombospondin-based anti-angiogenic strategies with special focus on the combination treatments. We convincingly illustrate how precise knowledge of the pro-apoptotic events and intermediates elicited by thrombospondin in the vascular endothelial cells facilitates the design of the most effective treatment combinations, where the efficacy of thrombospondin- derived compounds is maximized by the partner drug(s) (“complementation” strategies) and provide examples of such fine-tuning of the thrombospondin-based anti-angiogenic treatments.

The contribution of proteases to developmental, physiological and pathological processes has been well accepted. Cleavage of matrix proteins is a key requirement for cell migration and remodeling of the extracellular environment. The constant process of matrix turnover is dependent on the delicate balance between degradation and synthesis. In addition, regulated proteolysis also allows for the release and activation of growth factors and cytokines. Similarly to other extracellular matrix proteins, thrombospondins are also targets of proteolysis. While in some cases enzymatic activity is associated with degradation of the protein; in other situations, targeted and selective cleavage offers the means to release polypeptides with either alternative or enhanced function. Here, we provide a summary of the published information related to thrombospondin proteolysis within the context of how proteolysis of extracellular matrix proteins impacts diversification of protein function. We also discuss its biological relevance and potential therapeutic value of thrombospondin proteolysis with particular emphasis on angiogenesis.

Cartilage oligomeric matrix protein is a non-collagenous extracellular matrix protein expressed primarily in cartilage, ligament, and tendon. Cartilage oligomeric matrix protein has been studied extensively because mutations in the gene cause two skeletal dysplasias, pseudoachondroplasia and multiple epiphyseal dysplasia. Pseudoachondroplasia is a disproportionate dwarfing condition associated with joint abnormalities, while multiple epiphyseal dysplasia is less severe. Both of these skeletal dysplasias have a characteristic chondrocyte pathology that consists of intracellular retention of cartilage oligomeric matrix protein and other extracellular matrix proteins in an enlarged rough endoplasmic reticulum. This toxic intracellular retention of extracellular matrix proteins causes chondrocyte cell death thereby decreasing linear bone growth. Additionally, when cartilage oligomeric matrix protein and the other co-retained proteins are not exported to the extracellular matrix, the resulting matrix is abnormal and easily erodes with normal physical activity. Cartilage oligomeric matrix protein is also a marker for joint destruction associated osteoarthritis, rheumatoid arthritis, joint trauma, and intense activity. Serum cartilage oligomeric matrix protein levels are higher in aggressive cases of arthritis and levels are used to predict future disease progression. Recent studies have identified molecular functions of cartilage oligomeric matrix protein that may contribute to its role in skeletal disease. These molecular functions include: binding other ECM proteins, catalyzing polymerization of type II collagen fibrils, and regulation of chondrocyte proliferation. Here, we review cartilage oligomeric matrix protein's role in skeletal disease and potential molecular mechanisms.

Laryngeal cancer is a common head and neck cancer. Despite advances in treatment, improvement in survival and quality of life of patients still remains a challenge. Chemotherapy has become more and more important since this treatment is able to preserve laryngeal function. There is increasing evidence showing that many chemotherapeutic agents kill laryngeal tumor cells via apoptotic mechanisms. The balance between anti-apoptotic molecules and pro-apoptotic ones plays a critical role in determining the sensitivity of the tumor cells to chemotherapy. The commonly used chemotherapeutic agents for laryngeal cancer include cisplatin, 5-fluorouracil and paclitaxel. These three agents may target common apoptotic molecules, but more importantly they have their own particular targets. Systemically analysis of these targets will not only help to optimize the treatment but also provide a rationale for the best combination of two or more agents for the chemotherapy of laryngeal cancer.

Management of acute and chronic pain has always been a key area of clinical research. Enkephalinase inhibitors (EIs) seem to be promising as therapeutic agents having antinociceptive action. They additionally possess anticraving, antidiarrhoeal and antidepressant actions. The antinociceptive action of EIs has been reported for over a decade however, their therapeutic potential is yet to be effectively explored. EIs may be broadly classified as endogenous and those that are obtained synthetically. Endogenous EIs include peptides like spinorphin and opiorphin. And compounds like RB 101, RB 120, RB 3007 constitute the synthetically obtained EIs. Endogenous and synthetic inhibitors enkephalin degrading enzymes have been studied in vivo using standard animal models. The potential EI targets appear to be APN (Aminopeptidase N), NEP (Neutral endopeptidase), DPP-III (Dipeptidyl peptidase). EIs possess the advantage that they lack the opioid side effects. This article reviews the mechanisms by which EIs act and elucidates the pathways involved.

In the context of drug delivery it is crucial to gain knowledge of nature of the cell's internal barriers, as well as one needs to be aware of requirements for the study of spatial and temporal interactions of drug delivery vehicles with the cell. Fluorescent imaging technology can be a great innovation in the field of science as far as study of live cell imaging and dynamic events are concerned. The technique has also demonstrated the ability to integrate the anatomic, functional, and statistical data. The current review article discusses various fluorescent techniques and also elaborates the scope of fluorescent imaging in the field of drug delivery.

Type-2 diabetes is strongly linked to visceral obesity and elevated levels of circulating free fatty acids. For years this correlation of obesity to diabetes has intrigued the minds of researchers and research in this direction has led to a possible solution to this question. Human Genome project has identified nearly 150 orphan GPCRs. The reverse pharmacology approaches have identified free fatty acids as ligands for the GPR40 family of orphan receptors. This review mainly emphasizes on the role of GPR40 carboxylic acid receptor family in the development of diabetes alongwith detailed coverage of each receptor of the family. GPR40 family has provided an insight into regulation of carbohydrate and lipid metabolism in vertebrates and has further provided targets for the development of therapeutic agents useful for treating or preventing disorders such as Type-2 diabetes. This review also suggests where further research and development could be beneficial.

Diabetes mellitus (DM) has been recognized as a growing world-wide epidemic by many health advocacy groups including the World Health Organization (WHO). DM affects about 6% of the North American population. A recent report estimated that 8.2% of adult population worldwide has impaired glucose tolerance. Current treatment approaches include diet, exercise, and a variety of pharmacological agents including insulin, biguanides, sulfonylureas and thiazolidinediones. New therapies are still needed to control metabolic abnormalities, and also to preserve β-cell mass and to prevent loss of β-cell function. In many cases monotherapy gradually fails to improve blood glucose control and combination therapy is employed. The long-term success of these treatments varies substantially. Thus, there is an imperative need for novel therapeutic approaches for glycemic control that can complement existing therapies and possibly attempt to preserve normal physiological response to meal intake. Glucagon-like peptide 1 (GLP-1) is a drug candidate which potentially fulfils these conditions. Glucoregulatory actions of GLP-1 include glucose-dependent enhancement of insulin secretion, inhibition of glucagon secretion, slowing of gastric emptying and reduction of food intake. GLP-1 is rapidly inactivated by amino peptidase, Dipeptidyl Peptidase-IV (DPP-IV) and the utility of DPP-IV inhibitors are also under investigation. There is a recent upsurge in the development of GLP-1 mimetics and DPP-IV inhibitors as potential antidiabetic agents. The present review summarizes the concepts of GLP-1 based therapy for type 2 diabetes and the current preclinical and clinical development in GLP-1 modulators.