Published online before print June 14, 2007, doi: 10.1161/ATVBAHA.107.141713
© 2007 American Heart Association, Inc.
Brief Reviews |
Thrombospondins, Their Polymorphisms, and Cardiovascular Disease
From the Joseph J. Jacobs Center for Thrombosis and Vascular Biology and Department of Molecular Cardiology (O.I.S., E.F.P.), Cleveland Clinic, Ohio; and the Scripps Translational Science Institute and Division of Cardiovascular Diseases (E.J.T.), The Scripps Research Institute, La Jolla, Calif.
Correspondence to Edward F. Plow, PhD, Department of Molecular Cardiology/NB50, Cleveland Clinic Foundation/Lerner Research Institute, 9500 Euclid Avenue, Cleveland, OH 44195. E-mail plowe{at}ccf.org
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Abstract |
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 Top Abstract IntroductionThe ThrombospondinsThe Thrombospondins and...The TSP VariantsEffects of the TSP...Replication of the Disease...Concluding RemarksReferences |
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The thrombospondins are a 5-member gene family that mediate cell-cell and cell-matrix interactions. The thrombospondins are either trimers or pentamers, and their functions depend on their abilities to interact with numerous extracellular ligands and cell surface receptors through the multiple domains that compose each subunit. Recent genetic studies have indicated associations of particular single nucleotide polymorphisms in 3 of the 5 thrombospondins with cardiovascular disease. This observation has stimulated efforts to understand how the thrombospondins influence cardiovascular pathology, to dissect how the individual polymorphisms alter the structure and function of the parent thrombospondin molecules, and to replicate the genetic data in different patient populations. This review seeks to summarize current information that has emerged on each of these fronts.
Thrombospondins are large, extracellular matrix glycoproteins which mediate cell-cell and cell-matrix interactions by binding numerous ligands and cell-surface receptors. Particular single nucleotide polymorphisms in 3 of the 5 thrombospondins have been associated with myocardial infarction. This review summarizes current information linking the thrombospondins and their polymorphisms to cardiovascular pathophysiology.
Key Words: single nucleotide polymorphisms • thrombospondin • myocardial infarction • endothelial cells
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Introduction |
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TopAbstractIntroductionThe ThrombospondinsThe Thrombospondins and...The TSP VariantsEffects of the TSP...Replication of the Disease...Concluding RemarksReferences |
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The thrombospondins are members of a family of proteins that regulate cell-matrix and cell-cell-interactions. The diversity of their functions arises from their ability to bind to numerous ligands, including adhesive proteins and cellular receptors. The initial clue that thrombospondins might be implicated in coronary artery disease and myocardial infarction came from the first large-scale application of new, high throughput strategies for gene analysis to cardiology patients. This study, called GeneQuest, was designed to test relationships between a large number of single nucleotide polymorphisms (SNPs) in multiple candidate genes and coronary artery disease or myocardial infarction.1 To identify high frequency SNPs, 62 candidate genes, selected based on their involvement in vascular biology, thrombosis, and lipid metabolism, were sequenced in 114 DNA samples from a diverse population, and 72 high-frequency SNPs were identified in these genes. Association of these SNPs with coronary artery disease was analyzed in 352 families with members who had rigorously documented premature coronary artery disease (men <45 and women <50 years of age), a cohort in which genetic predisposition to disease is likely to be evident. The 3 SNPs that had statistically significant associations with myocardial infarction resided in genes of 3 different members of the thrombospondin (TSP) family. When analyzing 62 genes, the likelihood that 3 members, TSP-1, TSP-2, and TSP-4, of the 5-member TSP family would have a disease association by chance is extremely remote (P<10–5). Thus, these data suggested that there was a high likelihood that the implicated TSP family members play a role in cardiovascular pathology.
Despite the previous reports clearly showing the role for TSP-1 in vascular biology, this conclusion was unanticipated, because there was no genetic evidence that TSP family members had roles in coronary artery disease or myocardial infarction. Indeed, at the time there was no information to suggest that the variant forms of thrombospondins had unique biological activities let alone pathological functions. Thus, the leads revealed by GeneQuest have kindled renewed interest in the thrombospondin family and stimulated a flurry of basic and clinical research on thrombospondin SNPs. The purpose of this article is to provide background information on the thrombospondins and their roles in vascular biology, to consider how the implicated thrombospondin SNPs might influence the structure and function of the thrombospondins, to present experimental evidence of the effects of SNPs on thrombospondin functions, and to summarize recent genetic association studies that have tested further the associations of the thrombospondin SNPs with coronary artery disease and myocardial infarction.
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The Thrombospondins |
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TopAbstractIntroductionThe ThrombospondinsThe Thrombospondins and...The TSP VariantsEffects of the TSP...Replication of the Disease...Concluding RemarksReferences |
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The TSPs and Their Expression
The vertebrate thrombospondin family has 5 members. Each is a product of a distinct gene, and the 3 myocardial infarction-associated members, TSP-1, TSP-2, and TSP-4, reside on different chromosomes (15, 6, and 5, respectively). The 5 thrombospondins can be subdivided into 2 subgroups: those that form trimers (group A) and those that form pentamers (group B; Figure 1). Although the thrombospondin proteins have a high degree of similarity, the promoters of the genes and the untranslated mRNA regions are quite distinct. Hence, the levels of mRNA in different tissues of the thrombospondins are different, suggesting that they have distinct functions. Disruption of any one of the thrombospondin genes (2–5; Frolova E, Stenina OI, and Plow EF 2007, unpublished data) in mice is not embryonically lethal. Disruption of TSP-1, which is highly expressed in platelets and released on platelet activation, does not cause a platelet defect.2 However, the disruption of TSP-2, a protein not found in platelets, does cause a bleeding abnormality, apparently by affecting platelet formation by megakaryocytes.6 The roles of the individual thrombospondins in mice become overt on challenge, such as in wound or cancer models. However, point mutations affecting thrombospondin functions may cause severe abnormalities: mutations in THBS-5 in humans result in pseudoachondroplasia, a skeletal abnormality associated with dwarfism.7,8 Hence, mutations that alter thrombospondin function may be more pathogenic than a lack of expression.
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TSP-1 is expressed by all 3 major cell types of the vessel wall—endothelial cells, smooth muscle cells, and fibroblasts—and is abundant in platelets.9–11 The expression of recombinant TSP-2 and the development of TSP-2–deficient mice have allowed considerable progress in understanding of its functions.6,11–16 In contrast to TSP-1 and TSP-2, which belong to thrombospondin subgroup A, members of thrombospondin subgroup B (TSP-3, TSP-4, and TSP-5) have been discovered only recently and are less well-characterized. At the time of the GeneQuest report, there were very few publications on TSP-4, which harbors the most frequent myocardial infarction susceptibility SNP. Its expression has been demonstrated in heart, brain, cartilage, and tendon.
Molecular and Cellular Interactions of the TSPs
Thrombospondins are classified as "matricellular" proteins; they regulate cell-matrix and cell-cell interactions but do not maintain the structure of the extracellular matrix per se.9–11 Instead, thrombospondins act by binding to many different ligands, including adhesive proteins and cellular receptors. Matrix proteins that interact with TSP-1 include fibronectin, laminin, and collagen (reviewed in10). Cellular receptors for TSP-1 include CD36, a receptor for modified lipoproteins as well as many other ligands on monocytes, endothelial cells, and platelets17; integrin-associated protein (IAP, CD47), a component of a molecular complex of membrane proteins that modulates integrin function18,19; proteoglycans,20 and several integrins.21–24 The integrins that recognize TSP-1 on vascular cells include 
IIbβ3 on platelets,22 Vβ321, 3β1,25 and 6β126. The binding of the 2 β3 integrins to TSP-1 is inhibited by peptides that contain the amino acid sequence arginine-glycine-asparagine, so-called RGD-dependent binding. The binding of TSP-1 to other integrins is RGD-independent.
These various cellular receptors can mediate distinct and sometimes opposing responses to TSP-1. TSP-1 promotes chemotaxis of smooth muscle cells via CD4727 and Vβ3,28 and proliferation of smooth muscle cells via Vβ3,29 but induces apoptosis of T-cells and fibroblasts via CD47,30 and apoptosis of endothelial cells via CD36.31 TSP-2 has many of the properties of TSP-1. The leukocyte integrin Mβ2 recognizes TSP-4,32 the first identified receptor for this thrombospondin, and at least 1 receptor for TSP-4 is present on endothelial and smooth muscle cells.33 Altogether, the engagement of multiple receptors by these multidomainal proteins leads to complex, cell-specific responses, a challenge in assigning specific pathogenic mechanisms to the thrombospondin SNPs.
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The Thrombospondins and Cardiovascular Pathology |
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TopAbstractIntroductionThe ThrombospondinsThe Thrombospondins and...The TSP VariantsEffects of the TSP...Replication of the Disease...Concluding RemarksReferences |
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TSP-1
Although the relationship between the TSP-1 SNP and myocardial infarction was unexpected, a number of observations do link TSP-1 with atherosclerosis and restenosis (Figure 2). TSP-1 is present in early-stage atherosclerotic lesions34 and increases in large arteries of rabbits with hypercholesterolemia.35 Persistent elevation of TSP-1 in cardiac allografts correlates with vasculopathy.36 Increased TSP-1 levels in arterial wall could accelerate atherosclerosis and restenosis by affecting either smooth muscle cells or endothelial cells or both. In cultured endothelial cells, TSP-1 induces surface expression of vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule-1 (ICAM-1), and E-selectin, all of which promote leukocyte adhesion to endothelial cells.37 TSP-1 is a potent mitogen and chemoattractant for smooth muscle cells, with an activity equivalent to that of platelet-derived growth factor. Migration and proliferation of smooth muscle cells are key events in the formation of atherosclerotic lesions. In cell culture, TSP-1 stimulates smooth muscle cell growth and modulates the proliferative responses of these cells to growth factors.28,38–40 TSP-1 antagonizes cyclic GMP-mediated relaxation of SMC in response to nitric oxide.41,42 CD47 appears to be necessary for this inhibition.43,44 After aortic balloon catheter injury in rats, TSP-1 mRNA increases in the vessel wall within 2 hours after injury,45 and intraarterial delivery of an antibody against TSP-1 facilitates reendothelialization and reduces neointimal formation.29,35,46–48 TSP-1 is increased in the arteries of diabetic rats after vascular injury as compared with control animals,49 suggesting a role for TSP-1 in the increased susceptibility of diabetics to restenosis. Stimulation of smooth muscle cell proliferation by high glucose appears to be dependent on increased production of TSP-1.50
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TSP-1 is one of the most potent naturally occurring antiangiogenic proteins. There are inverse correlations between TSP-1 expression by cancer cells and their ability to promote angiogenesis (reviewed in51,52). The number of vasa vasorum in diabetic rat aortas is inversely related to the levels of TSP-1 in the vessel.49 Impaired angiogenesis in the inner layers of large vessels could result in ischemia, which may promote the progression of atherosclerotic lesions.
TSP-1 is selectively expressed in the infarct border zone in experimental infarct models and appears to serve as regulator of granulation, limiting fibrotic remodeling to the infarcted myocardium53: TSP-1 knockout mice had more extensive postinfarction remodeling than wild-type mice.
TSP-2 and TSP-4
Although neither TSP-2 nor TSP-4 had been implicated in cardiac or vascular pathology before GeneQuest, the abundant expression of these thrombospondins in heart and of TSP-2 in aortic tissue was known.54,55 Recent reports indicate that these proteins may be involved in remodeling of stressed hearts. The expression of TSP-2 increases in hearts of animal models of heart failure.56 A 3.5-fold increase in TSP-4 expression was found in failing human heart tissue.57 The expression of TSP-4 was dramatically increased in hearts of hypertensive SHR rats during the transition to diastolic hypertensive heart failure.58 TSP-2–deficient mice cannot resist the increased heart loading caused by angiotensin II, and as a consequence heart rupture or failure develops.56 Both TSP-259 and TSP-433 have chemotactic and mitogenic activities for vascular smooth muscle cells, activities also displayed by TSP-1. These common functions may be central to the roles of the thrombospondins in coronary artery disease and myocardial infarction.
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The TSP Variants |
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TopAbstractIntroductionThe ThrombospondinsThe Thrombospondins and...The TSP VariantsEffects of the TSP...Replication of the Disease...Concluding RemarksReferences |
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Of the 3 thrombospondin SNPs that are associated with myocardial infarction, 2 occur in coding regions (in TSP-1 and TSP-4) and 1 in the untranslated region of the mRNA (TSP-2). The TSP-1 SNP leads to a serine rather than the usual asparagine at position 700 (N700S). The S700 allele is recessive, associated with a high risk of myocardial infarction (adjusted OR=8.66), and is rare in Whites (Table
). The disease-associated SNP in TSP-4 substitutes a proline for the usual alanine at position 387 (A387P). The A387P variant gene is dominant and occurs with high frequency (34%) in Whites. The adjusted odds ratio for the risk of a myocardial infarction in carriers of the A387P polymorphism is 1.89 in the GeneQuest Population. The SNP in TSP-2, t3949g, resides in the 3'-untranslated region (3'-UTR) of its mRNA. Its effect is recessive and is protective against myocardial infarction (adjusted odds ratio=0.39). The frequency of this SNP is 10%. The Table summarizes the major features of the disease-associated SNPs in thrombospondins as analyzed in ethnically and etiologically different populations of patients with coronary artery disease.1,60–73 It is difficult to prove conclusively that a SNP associated with a disease plays a direct causative role, but experimental information on structural and functional consequences of TSP SNPs, which we now review, is consistent with this possibility.
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Effects of the TSP SNPs on Structure and Function |
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TopAbstractIntroductionThe ThrombospondinsThe Thrombospondins and...The TSP VariantsEffects of the TSP...Replication of the Disease...Concluding RemarksReferences |
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The TSP-1 SNP
The Type 2 (EGF-like) and Type 3 (Ca2+ binding) repeats in thrombospondin genes, in which the variant SNPs in TSP-1 and TSP-4 reside (see Figure 1), are similar to known coding regions of functionally important Ca2+-binding sites in other proteins. Moreover, Ca2+ binding affects the structure and function of thrombospondins.21,40,74–76 This is important because experimental data and predictive analyses demonstrate that the N700S SNP of TSP-1 perturbs the calcium binding properties of TSP-1. Particularly relevant is that the spontaneous mutations in TSP-5 that lead to pseudoachondroplasia alter the Ca2+ binding properties of TSP-5.7,77,78 The asparagine at position 700 in TSP-1, which is replaced by serine in S700, may, in fact, function as a coordinating residue in a calcium-binding site.79 A metal-binding site was indeed identified within this sequence surrounding SNP, and a difference in the calcium binding properties of TSP-1 variant compared with wild-type has been demonstrated in studies with synthetic peptides.79 Biophysical analyses indicate differences in the influence of calcium on the conformations of N700 and S700 TSP-1.37,80,81
With the abundance of TSP-1 in platelets, a differential effect of TSP-1 variants on platelet function is a prime candidate for a pathogenic mechanism in myocardial infarction. The prevailing view is that TSP-1 enhances platelet aggregation82 by providing additional bridges between aggregating platelets through binding to fibrinogen, for which TSP-1 has a high affinity (fibrinogen also binds to integrin IIbβ3 on the platelet surface). In vitro, the S700 variant of TSP-1 enhances platelet aggregation in comparison to N700 TSP-1. Also, expression of TSP-1 on the surface of platelets from carriers of S700 TSP-1 is increased.37 With the presence of more S700 TSP-1 on platelets and its greater capacity to support platelet aggregation, thrombus formation, the event underlying myocardial infarction, may be augmented, providing a mechanism for the increased risk in individuals having TSP-1 with the S700 SNP.
In addition to platelets, most vascular and blood cells also produce TSP-1 on stimulation, and this TSP-1 binds to receptors on endothelial cells. As noted above, TSP-1 induces apoptosis in endothelial cells and increases proliferation in smooth muscle cells, but whether the 2 forms of TSP-1 have different effects on vascular cells is unknown.
The TSP-4 SNP
TSP-4 P387 has dominant effect, and heterozygotes are as susceptible to myocardial infarction as homozygotes. Structural differences may underlie differences in the calcium binding properties of the A387 and P387 variants of TSP-4. Whereas the S700 substitution in TSP-1 leads to a decrease in calcium binding, the P387 substitution in TSP-4 increases its calcium binding by creating an additional binding site for the divalent cation.79,83 TSP-4 mRNA can be found in endothelial and smooth muscle cells from brain and coronary artery, and both forms of the TSP-4 protein are produced and secreted at similar levels by the brain endothelial cells, suggesting that function, rather than expression, is altered by the TSP-4 SNPs to account for pathology.33 Processing of TSP-4 protein by cultured endothelial cells is not affected by the substitution, suggesting that the molecular mechanism of P387 TSP-4 effects is not its retention in the endoplasmic reticulum as is found with the mutations of TSP-5 that cause pseudoachondroplasia.7
Against the background of known effects of TSP-1 on vascular cell functions and the role of these cells in atherogenesis, we examined the effects of the TSP-4 variants on the adhesive and proliferative responses of endothelial and smooth muscle cells. We found that TSP-4 regulates the functions of these cells and that P387 can affect the function of TSP-4.33 The P387 TSP-4 exerts a "gain-of-function" activity, interfering with endothelial cell adhesion and proliferation. The inhibitory effects of the P387 TSP-4 on endothelial cell repair functions, critical to maintain vessel wall integrity and function, coupled with the stimulatory effects of TSP-4 on smooth muscle cell proliferation,33 a key event in development of atherosclerotic lesion, can account for the association with coronary artery disease. Moreover, although the adhesion of neutrophils is equally supported by both TSP-4 variants, neutrophil activation is more robust in presence of P387 (TSP-4).32 This observation suggests that P387 (TSP-4) could play a role in inflammatory processes in the vascular wall.
The TSP-2 SNP
The t3949g SNP is located in the 3' untranslated region (3'UTR) of the TSP-2 mRNA, a region commonly implicated in the posttranscriptional regulation of protein expression (Figure 1B). The 3'-UTR of TSP-2 is large (
2kb), suggesting posttranscriptional regulation of expression. The rapid upregulation of TSP-2 levels in cell types present in vascular wall in response to stimuli45,84,85 also indicates posttranscriptional regulation of expression that is usually dependent on untranslated regions of mRNA. Presently, the effect of the t3949g substitution on the structure of the TSP-2 mRNA is based entirely on predictive analysis. Using the M-fold program86 to model mRNA structure, a notable difference in the secondary structure of t3949 and g3949 TSP-2 was predicted (Figure 3A). Such differences in secondary structure might lead to differential binding of RNA-binding proteins, which may, in turn, affect protein expression.
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Although TSP-2 had not been implicated in atherogenesis before GeneQuest, it is highly homologous to TSP-1, including domains implicated in angiogenesis, modulation of matrix metalloproteinase (MMP) activity, binding of growth factors, and recognition by CD36.11,15,87,88 Although certain functions of TSP-1 are shared by TSP-2 (eg, antiangiogenic properties), others have yet to be directly examined. In our reporter activity experiments, the g3949 variant 3'UTR of TSP-2, which was associated with the protective activity of TSP-2 against myocardial infarction in GeneQuest, regulated expression of the luciferase reporter in a cell type–dependent manner: in ECs, the expression of the reporter gene with the g3949 3'UTR was several-fold lower than the expression of the reporter gene with the t3949 3'UTR (Figure 3B). This result suggests that a decrease in TSP-2 expression in vascular wall caused by the g3949 SNP may account for the protective activity of TSP-2. However, the in vitro observation that the 3' UTR SNP has an effect on expression is far removed from the in vivo situation. Studies of the development of atherosclerotic lesions in TSP-2 knockout mice and the effect of SNP on TSP-2 expression in humans would clarify the mechanism of protection in individuals with g3949 SNP.
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Replication of the Disease Associations of the TSP SNPs |
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TopAbstractIntroductionThe ThrombospondinsThe Thrombospondins and...The TSP VariantsEffects of the TSP...Replication of the Disease...Concluding RemarksReferences |
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SNP association studies have inherent limitations, and even promising results are often not replicated.89,90 However, it is replication, not the failure to replicate, that is regarded as a major and necessary stamp of legitimacy on a SNP-association study. The association of each of the 3 thrombospondin SNPs with myocardial infarction has been replicated in at least 1 study (see Table
). It would be premature to conclude that a consensus has emerged. In some of these studies, it is clear that the populations analyzed were distinct based on the differences in the frequencies of the TSP variant alleles. Also, some of the studies differ in design with respect to the criteria used for patient and control selection. Association of P387 TSP-4 with the disease was established in 8 of 12 studies (Table).1,60–73
When the GeneQuest study was broadened to extend the initial analysis to 210 SNPs in 111 candidate genes, significant associations were found with polymorphisms in TSP-4, TSP-2, and plasminogen activator inhibitor (PAI)-2 genes, the strongest being with the A387P variant in TSP-4 (P=0.002).66 The coronary artery disease–associated genotype of TSP-4, with its odds ratio 1.85 for myocardial infarction and its high frequency, rivals the 
4 SNP in apolipoprotein E4 as the most prevalent and relevant SNP for susceptibility to the coronary artery disease in the population (frequency of the disease-associated apolipoprotein E4 genotype is approximately 20% with an odds ratio of 1.4).91 The protective effect of the TSP-2 SNP now has been reported in 2 independent population studies. With the rarity of the disease-associated S700 TSP-1 genotype, the replication of its association with myocardial infarction in an Italian population is notable.
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Concluding Remarks |
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TopAbstractIntroductionThe ThrombospondinsThe Thrombospondins and...The TSP VariantsEffects of the TSP...Replication of the Disease...Concluding RemarksReferences |
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Together with lifestyle and established risk factors, genetics plays a critical role in susceptibility for coronary artery disease and myocardial infarction. Indeed, a positive family history increases the risk of premature coronary artery disease that occurs in men under the age of 50 and women under the age of 45 by as much as 12-fold.92 GeneQuest was undertaken with this rationale in mind, and the 3 thrombospondin SNPs were implicated in coronary artery disease or protection from it. As summarized above, evidence is emerging that each of the 3 thrombospondin SNPs influences key molecular properties of the thrombospondins. Ultimately, a detailed understanding of how each SNP influences the structure of its parent TSP protein or its mRNA will be of fundamental importance. For TSP-1 and TSP-4, the variant molecules do induce distinct responses in vascular cells; and, it is likely that additional differential responses to the variant TSPs will be identified. The challenge will lie in determining which one(s) of these cellular responses drives cardiovascular pathology. Transgenic animal models and gene array profiling will be useful tools in this endeavor, but no single approach will necessarily provide a definitive answer. For TSP-2, answers may be easier to come by if our preliminary result that the TSP-2 SNP reduces expression holds up. In this case, the pathology in the TSP-2 knock-out will be quite informative. Now that whole genome SNP association studies have become feasible, the functional genomic issues and challenges to determine cause-and-effect for the thrombospondins may well be prototypic. Ultimately, the hope is that the TSP variants and many yet to be described SNPs and haplotypes in other genes will serve as new molecular targets for the treatment and prevention of cardiovascular disease.
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Acknowledgments |
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Sources of Funding
This study was supported by NIH P50 HL077107 (E.J.T./E.F.P.), NIH R01 DK067532 (O.I.S.), NIH K01 DK62128 (O.I.S.), 0565284B (American Heart Association) (O.I.S.), and funds from the Lerner Research Institute, Cleveland Clinic (O.I.S.).
Disclosures
None.
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Footnotes |
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Original received February 8, 2007; final version accepted May 16, 2007.
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