The role of thrombospondins in wound healing, ischemia, and the foreign body reaction

Thrombospondin (TSP) 1 and TSP2 have been implicated in the regulation of several processes during tissue repair. Due to their matricellular nature, these proteins are thought to modulate cell-matrix interactions through a variety of mechanisms specific to the spatio-temporal context of their expression. Most notably, TSP1 and TSP2 appear to play distinct, non-overlapping roles in the healing of skin wounds. In contrast, both proteins have been implicated as regulators of ischemia-induced angiogenesis. Moreover, TSP2 has been shown to be a critical regulator of angiogenesis in the foreign body response (FBR). In this review, we discuss the role of TSPs in tissue repair and examine the mechanistic data regarding the ability of the thrombospondins to modulate cell-matrix interactions in this context.     

Modulation of the extracellular matrix patterning of thrombospondins by actin dynamics and thrombospondin oligomer state

Thrombospondins (TSPs) are evolutionarily-conserved, secreted glycoproteins that interact with cell surfaces and extracellular matrix (ECM) and have complex roles in cell interactions. Unlike the structural components of the ECM that form networks or fibrils, TSPs are deposited into ECM as arrays of nanoscale puncta. The cellular and molecular mechanisms for the patterning of TSPs in ECM are poorly understood. In the present study, we investigated whether the mechanisms of TSP patterning in cell-derived ECM involves actin cytoskeletal pathways or TSP oligomer state. From tests of a suite of pharmacological inhibitors of small GTPases, actomyosin-based contractility, or actin microfilament integrity and dynamics, cytochalasin D and jasplakinolide treatment of cells were identified to result in altered ECM patterning of a model TSP1 trimer. The strong effect of cytochalasin D indicated that mechanisms controlling puncta patterning depend on global F-actin dynamics. Similar spatial changes were obtained with endogenous TSPs after cytochalasin D treatment, implicating physiological relevance. Under matched experimental conditions with ectopically-expressed TSPs, the magnitude of the effect was markedly lower for pentameric TSP5 and Drosophila TSP, than for trimeric TSP1 or dimeric Ciona TSPA. To distinguish between the variables of protein sequence or oligomer state, we generated novel, chimeric pentamers of TSP1. These proteins accumulated within ECM at higher levels than TSP1 trimers, yet the effect of cytochalasin D on the spatial distribution of puncta was reduced. These findings introduce a novel concept that F-actin dynamics modulate the patterning of TSPs in ECM and that TSP oligomer state is a key determinant of this process.     

The role of thrombospondins 1 and 2 in the regulation of cell-matrix interactions, collagen fibril formation, and the response to injury

Thrombospondins (TSPs) 1 and 2 are extracellular modular glycoproteins that are best known for their anti-angiogenic properties and their ability to modulate cell-matrix interactions. However, these proteins, and in particular TSP2, are pleiotropic in function and affect processes as disparate as bone growth and hemostasis. In recognition of their ability to influence a wide variety of cell functions, and in the absence of convincing evidence for their participation as integral components of extracellular structures, the term 'matricellular' has been applied to these and a small group of functionally related proteins. In this review, we focus on the role of TSP1 and 2 in two forms of injury in mice, excisional skin wounds and subcutaneously implanted biomaterials, and take advantage of mice with targeted disruptions of one or both genes to identify likely biochemical mechanisms that could account for the characteristics of the injury response in these knockout mice. In work that stems largely from our own laboratory, we show that pericellular levels of the matrix metalloproteinase, MMP2, are controlled to a large extent by TSP2 (and potentially also by TSP1), and that elevated levels of MMP2 are likely to account in part for defects as diverse as reduced cellular adhesion, abnormal collagen fibril structure, and increased endothelial cell and vascular proliferation.     

The thrombospondins

Thrombospondin-1 (TSP-1) was studied in the 1980s as a major component of platelet alpha-granules released upon platelet activation and also as a cell adhesion molecule. In 1993, we published a short review that discussed the exciting identification by molecular cloning of four additional vertebrate gene products related to TSP-1 [Current Biology 3 (1993) 188]. We put forward a structurally based classification for the newly identified proteins and discussed the functional and evolutionary implications of the new gene family. Since that time, the depth and breadth of knowledge on vertebrate TSPs and their functions in cells and tissues in health and disease has expanded into important new areas. Of particular interest is the new knowledge on the complex, domain and cell-type specific effects of TSPs on cell-signaling and cell-adhesion behaviour, the roles of TSP-1 and TSP-2 as anti-angiogenic agents, the roles of TSP-1 and TSP-2 in wound-healing, and associations of point mutations and polymorphisms in TSP-1, TSP-4 and TSP-5/COMP with human genetic diseases. The TSP family also now includes invertebrate members. In this article, we give the 2004 view on TSPs and our perspectives on the significant challenges that remain. Other articles in this issue discuss the functions of vertebrate TSPs in depth.     

Thrombospondins in the heart: potential functions in cardiac remodeling

Cardiac remodeling after myocardial injury involves inflammation, angiogenesis, left ventricular hypertrophy and matrix remodeling. Thrombospondins (TSPs) belong to the group of matricellular proteins, which are non-structural extracellular matrix proteins that modulate cell–matrix interactions and cell function in injured tissues or tumors. They interact with different matrix and membrane-bound proteins due to their diverse functional domains. That the expression of TSPs strongly increases during cardiac stress or injury indicates an important role for them during cardiac remodeling. Recently, the protective properties of TSP expression against heart failure have been acknowledged. The current review will focus on the biological role of TSPs in the ischemic and hypertensive heart, and will describe the functional consequences of TSP polymorphisms in cardiac disease.     

Interactions of thrombospondins with alpha4beta1 integrin and CD47 differentially modulate T cell behavior

Thrombospondin (TSP)-1 has been reported to modulate T cell behavior both positively and negatively. We found that these opposing responses arise from interactions of TSP1 with two different T cell receptors. The integrin alpha4beta1 recognizes an LDVP sequence in the NH2-terminal domain of TSP1 and was required for stimulation of T cell adhesion, chemotaxis, and matrix metalloproteinase gene expression by TSP1. Recognition of TSP1 by T cells depended on the activation state of alpha4beta1 integrin, and TSP1 inhibited interaction of activated alpha4beta1 integrin on T cells with its counter receptor vascular cell adhesion molecule-1. The alpha4beta1 integrin recognition site is conserved in TSP2. A recombinant piece of TSP2 containing this sequence replicated the alpha4beta1 integrin-dependent activities of TSP1. The beta1 integrin recognition sites in TSP1, however, were neither necessary nor sufficient for inhibition of T cell proliferation and T cell antigen receptor signaling by TSP1. A second TSP1 receptor, CD47, was not required for some stimulatory responses to TSP1 but played a significant role in its T cell antigen receptor antagonist and antiproliferative activities. Modulating the relative expression or function of these two TSP receptors could therefore alter the direction or magnitude of T cell responses to TSPs.     

Thrombospondins: structure and regulation of expression.

Thrombospondin (TSP) is a large, trimeric, modular glycoprotein that is a major constituent of platelet alpha granules. TSP is also secreted by a wide variety of epithelial and mesenchymal cells in patterns that reflect developmental changes in the embryo and response to injury in the adult. In addition to its role in blood coagulation, TSP has been reported to serve both adhesive and anti-adhesive functions, to foster neurite outgrowth, stimulate and inhibit cell growth and migration, and inhibit angiogenesis. Although this diversity in apparent function can be attributed, in part, to the ability of a single TSP to interact with several different cell-surface receptors, it is now known that the TSPs are encoded by at least three homologous genes in both human and mouse. TSP1, the commonly recognized protein isolated from platelets, is similar to TSP2 in structure. Both proteins contain NH2-terminal, COOH-terminal, and procollagen homology domains, and type I (TSP or properdin), type II (EGF-like), and type III (Ca(2+)-binding) repeats. However, the two TSPs differ in amino acid sequence and in the regulation of their expression. TSP1 is rapidly induced by serum and growth factors. An SRE and a binding site for NF-Y have been shown to mediate the serum response of the human TSP1 gene. On the other hand, TSP2 is far less responsive to serum than TSP1 and lacks the promoter elements that mediate the serum responsiveness of TSP1. TSP3 resembles TSP1 and TSP2 in its COOH-terminal domain and type III repeats, but contains four rather than three type II repeats and lacks type I repeats and a procollagen homology. The NH2-terminal domain of TSP3 also differs from that of either TSP1 or TSP2. All three TSPs demonstrate characteristic patterns of expression in the developing and adult mouse. It is therefore likely that each protein subserves a discrete function. In the future it will be necessary to distinguish among the three TSPs in addressing the function of these proteins.     

Thrombospondins 1 and 2 function as inhibitors of angiogenesis.

Thrombospondins (TSPs) 1 and 2 are matricellular proteins with the well-characterized ability to inhibit angiogenesis in vivo, and the migration and proliferation of cultured microvascular endothelial cells (ECs). Angiogenesis in developing tumors and in various models of wound healing is diminished or delayed by the presence of TSP1 or 2. Sequences within the type I repeats of TSP1 and 2 have been demonstrated to mediate the anti-migratory effects of TSPs on microvascular EC, although, paradoxically, sequences in the N- and C-terminal domains have pro-angiogenic effects. A scavenger receptor, CD36, recognizes the active sequences in the type I repeats, and is required for the anti-angiogenic effects of TSP1 in the corneal neovascularization assay. However, interactions of TSPs with growth factors, proteases, histidine-rich glycoprotein, and other cell-surface receptors on EC have the potential to modulate CD36-mediated effects. Binding of TSP1 to CD36 has been shown to activate apoptosis by inducing p38 and Jun N-terminal kinase, members of the mitogen-activated protein kinase superfamily, and subsequently the cell-surface expression of FasL. Ligation of Fas by FasL then induces a caspase cascade and apoptotic cell death. However, we have recently shown that inhibition of proliferation of microvascular EC by TSPs can occur in the absence of cell death. This finding raises the possibility that TSPs can activate separate cell death and anti-proliferative pathways.     

Functions of the conserved thrombospondin carboxy-terminal cassette in cell-extracellular matrix interactions and signaling

Thrombospondins (TSPs) are extracellular, multidomain, calcium-binding glycoproteins that function at cell surfaces, in extracellular matrix (ECM) and as bridging molecules in cell-cell interactions. TSPs are multifunctional and modulate cell behavior during development, wound-healing, immune response, tumor growth and in the homeostasis of adult tissues. TSPs are assembled as oligomers that are composed of homologous polypeptides. In all the TSP polypeptides, the most highly-conserved region is the carboxyl-region, which contains a characteristic set of domains comprising EGF domains, TSP type 3 repeats and a globular carboxy-terminal domain. This large region is termed here the thrombospondin carboxy-terminal cassette (TSP-CTC). The strong conservation of the TSP-CTC suggests that it may mediate ancestral functions that are shared by all TSPs. This review summarizes the current knowledge of the TSP-CTC and areas of future interest.     

Thrombospondins and tumor angiogenesis

The thrombospondins (TSPs) are a family of five secreted proteins that are widely distributed in the extracellular matrix of numerous tissues. TSPs are multimodular and each domain specifies a distinct biological function through interaction with a specific receptor. TSP1 and TSP2 have anti-angiogenic activity, which, at least for TSP1, involves interaction with the microvascular endothelial cell receptor CD36. Expression of TSP1 and TSP2 is modulated by hypoxia and by oncogenes. In several tumors (thyroid, colon, bladder carcinomas), TSP1 expression is inversely correlated with tumor grade and survival rate, whereas in others (e.g. breast carcinomas), it is correlated with the stromal response and is of little prognostic value. Recent studies suggest that TSPs or TSP-derived peptides retaining biological activity could be developed into promising new therapeutic strategies for the anti-angiogenic treatment of solid tumors.     

Thrombospondins function as regulators of angiogenesis

Thrombospondins (TSPs) -1 and -2 were among the first protein inhibitors of angiogenesis to be identified, a property that was subsequently attributed to the interactions of sequences in their type I repeats with endothelial cell-surface receptors. The interactions of TSPs-1 and -2 with cell-surface receptors, proteases, growth factors, and other bioactive molecules, coupled with the absence of direct structural functions that can be attributed to these matrix proteins, qualify them for inclusion in the category of ‘matricellular proteins’. The phenotypes of TSP-1, TSP-2, and double TSP-1/2-null mice confirm the roles that these proteins play in the regulation of angiogenesis, and provide clues to some of the other important functions of these multi-domain proteins. One of these functions is the ability of TSP-1 to activate the latent TGFβ1 complex, a property that is not shared by TSP-2. A major pathway by which TSP1 or TSP2 inhibits angiogenesis involves an interaction with CD 36 on endothelial cells, which leads to apoptosis of both the liganded and adjacent cells. However a homeostatic mechanism, which inhibits endothelial cell proliferation, and may be physiologically preferable under some circumstances, has also been elucidated, and involves interaction with the very low density lipoprotein receptor (VLDLR). The interaction of TSP1with its receptor, CD47, further inhibits angiogenesis by antagonizing nitric oxide signaling in endothelial and vascular smooth muscle cells. Paradoxically, there is also evidence that TSP-1 can function to promote angiogenesis. This apparent contradiction can be explained by the presence of sequences in different domains of the protein that interact with different receptors on endothelial cells. The anti-angiogenic function of TSPs has spurred interest in their use as anti-tumor agents. Currently, peptide mimetics, based on sequences in the type I repeats of TSPs that have been shown to have anti-angiogenic properties, are undergoing clinical testing.     

Thrombospondins as key regulators of synaptogenesis in the central nervous system

Thrombospondins (TSPs) are a family of large, oligomeric multidomain glycoproteins that participate in a variety of biological functions as part of the extracellular matrix (ECM). Through their associations with a number of binding partners, TSPs mediate complex cell-cell and cell-matrix interactions in such diverse processes as angiogenesis, inflammation, osteogenesis, cell proliferation, and apoptosis. It was recently shown in the developing central nervous system (CNS) that TSPs promote the formation of new synapses, which are the unique cell-cell adhesions between neurons in the brain. This increase in synaptogenesis is mediated by the interaction between astrocyte-secreted TSPs and their neuronal receptor, calcium channel subunit α2δ-1. The cellular and molecular mechanisms that underlie induction of synaptogenesis via this interaction are yet to be fully elucidated. This review will focus on what is known about TSP and synapse formation during development, possible roles for TSP following brain injury, and what the previously established actions of TSP in other biological tissues may tell us about the mechanisms underlying TSP's functions in CNS synaptogenesis.     

Members of the thrombospondin gene family bind stromal interaction molecule 1 and regulate calcium channel activity

The thrombospondins (TSPs) are a family of matricellular proteins that regulate cellular phenotype through interactions with a myriad of other proteins and proteoglycans. We have identified a novel interaction of the members of the TSP gene family with stromal interaction molecule 1 (STIM1). This association is robust since it is preserved in Triton X-100, can be detected with multiple anti-TSP-1 and anti-STIM1 antibodies, and is detected in a wide range of cell types. We have also found that STIM1 co-immunoprecipitates with TSP-4 and cartilage oligomeric matrix protein (COMP), and that a recombinant version of the N-terminal domain of STIM1 binds to the signature domain of TSP-1 and COMP. The association of the TSPs with STIM1 is observed in both the presence and absence of calcium indicating that the calcium-dependent conformation of the signature domain of TSPs is not required for binding. Thus, this interaction could occur in the ER under conditions of normal or low calcium concentration. Furthermore, we observed that the expression of COMP in HEK 293 cells decreases STIM1-mediated calcium release activated calcium (CRAC) channel currents and increases arachidonic acid calcium (ARC) channel currents. These data indicate that the TSPs regulate STIM1 function and participate in the reciprocal regulation of two channels that mediate calcium entry into the cell.     

The thrombospondins

Thrombospondins are evolutionarily conserved, calcium-binding glycoproteins that undergo transient or longer-term interactions with other extracellular matrix components. They share properties with other matrix molecules, cytokines, adaptor proteins, and chaperones, modulate the organization of collagen fibrils, and bind and localize an array of growth factors or proteases. At cell surfaces, interactions with an array of receptors activate cell-dependent signaling and phenotypic outcomes. Through these dynamic, pleiotropic, and context-dependent pathways, mammalian thrombospondins contribute to wound healing and angiogenesis, vessel wall biology, connective tissue organization, and synaptogenesis. We overview the domain organization and structure of thrombospondins, key features of their evolution, and their cell biology. We discuss their roles in vivo, associations with human disease, and ongoing translational applications. In many respects, we are only beginning to appreciate the important roles of these proteins in physiology and pathology.     

Thrombospondins use the VLDL receptor and a nonapoptotic pathway to inhibit cell division in microvascular endothelial cells

TSPs 1 and 2 function as endogenous inhibitors of angiogenesis. Although thrombospondins (TSPs) have been shown to induce apoptosis in HMVECs, we reasoned that a homeostatic mechanism would also be needed to inhibit EC growth without causing cell death, e.g., in the maintenance of a normal vascular endothelium. HMVECs, cultured in low serum, responded to VEGF with an increase in [(3)H]thymidine incorporation that was inhibited by TSPs and was accompanied by decreases in the phosphorylation of Akt and MAPK, without an increase in apoptosis. RAP, an inhibitor of the low-density lipoprotein (LDL) family of endocytic receptors, and blocking antibodies to VLDLR were as effective as TSPs in the inhibition of thymidine uptake in response to VEGF, and the effects of these agents were not additive. Supportive evidence for the role of the VLDLR in mediating this inhibition was provided by the demonstration of a high-affinity interaction between TSPs and the VLDLR. We propose that TSP1 and TSP2, together with the VLDLR, initiate a nonapoptotic pathway for maintenance of the normal adult vascular endothelium in a quiescent state, similar to that invoked for the regulation of mitogenesis by PDGF, but involving signaling via the VLDLR rather than LRP1.