Thrombospondin-4 regulates fibrosis and remodeling of the myocardium in response to pressure overload.

Thrombospondin-4 (TSP-4) expression increases dramatically in hypertrophic and failing hearts in rodent models and in humans. The aim of this study was to address the function of TSP-4 in the heart. TSP-4-knockout (Thbs4(-/-)) and wild-type (WT) mice were subjected to transverse aortic constriction (TAC) to increase left ventricle load. After 2 wk, Thbs4(-/-) mice had a significantly higher heart weight/body weight ratio than WT mice. The additional increase in the heart weight in TAC Thbs4(-/-) mice was due to increased deposition of extracellular matrix (ECM). The levels of interstitial collagens were higher in the knockout mice, but the size of cardiomyocytes and apoptosis in the myocardium was unaffected by TSP-4 deficiency, suggesting that increased reactive fibrosis was the primary cause of the higher heart weight. The increased ECM deposition in Thbs4(-/-) mice was accompanied by changes in functional parameters of the heart and decreased vessel density. The expression of inflammatory and fibrotic genes known to be influential in myocardial remodeling changed as a result of TSP-4 deficiency in vivo and as a result of incubation of cells with recombinant TSP-4 in vitro. Thus, TSP-4 is involved in regulating the adaptive responses of the heart to pressure overload, suggesting its important role in myocardial remodeling. Our study showed a direct influence of TSP-4 on heart function and to identify the mechanism of its effects on heart remodeling.

Thrombospondin-4 regulates vascular inflammation and atherogenesis.

RATIONALE: Thrombospondin (TSP)-4 is an extracellular protein that has been linked to several cardiovascular pathologies. However, a role for TSP-4 in vascular wall biology remains unknown. OBJECTIVE: We have examined the effects of TSP-4 gene (Thbs4) knockout on the development of atherosclerotic lesions in ApoE(-/-) mice. METHODS AND RESULTS: Deficiency in TSP-4 reduced atherosclerotic lesions: at 20 weeks of age, the size of the aortic root lesions in Thbs4(-/-)/ApoE(-/-) mice was decreased by 48% in females and by 39% in males on chow diets; in mice on Western diets, lesions in the descending aorta were reduced by 30% in females and 33% in males. In ApoE(-/-) mice, TSP-4 was abundant in vessel areas prone to lesion development and in the matrix of the lesions themselves. TSP-4 deficiency reduced the number of macrophages in lesions in all groups by ≥ 2-fold. In addition, TSP-4 deficiency reduced endothelial cell activation (expression of surface adhesion molecules) and other markers of inflammation in the vascular wall (decreased production of monocyte chemoattractant protein-1 and activation of p38). In vitro, both the adhesion and migration of wild-type macrophages increased in the presence of purified recombinant TSP-4 in a dose-dependent manner (up to 7- and 4.7-fold, respectively). These responses led to p38-MAPkinase activation and were dependent on ß(2) and ß(3) integrins, which recognize TSP-4 as a ligand. CONCLUSIONS: TSP-4 is abundant in atherosclerotic lesions and in areas prone to development of lesions and may influence the recruitment of macrophages by activating endothelial cells and directly interacting with macrophages to increase their adhesion and migration. Our observations suggest an important role for this matricellular protein in the local regulation of inflammation associated with atherogenesis.

A novel transcriptional mechanism of cell type-specific regulation of vascular gene expression by glucose.

OBJECTIVE: Vascular diabetic complications are associated with abnormal extracellular matrix and dysfunction of vascular cells, which later result in aberrant angiogenesis and development of atherosclerotic lesions. The tissue and cell specificity of the effects of high glucose are well recognized, but the underlying cell type-specific molecular mechanisms controlled by glucose are still unclear. We sought to identify cell type-specific mechanisms by which high glucose regulates transcription of genes in vascular cells. METHODS AND RESULTS: Thrombospondin-1 is a potent antiangiogenic protein associated with development of several diabetic complications and regulated by high glucose in multiple cell types. We report that distinct cell type-specific mechanisms regulate thrombospondin-1 gene (THBS1) transcription in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) in response to high glucose: although a proximal fragment of 280 nucleotides is sufficient to drive transcription in ECs, THBS1 was regulated cooperatively by interaction between proximal (-272 to -275) and distal (-1016 to -1019) promoter elements in VSMCs. Transcription factors activated by high glucose in VSMCs were cell type-specific. The formation of a single complex interacting with both distal and proximal glucose-responsive elements of THBS1 promoter in VSMCs was confirmed using gel-shift assays, binding sequence decoy oligomers, and specific mutant promoter fragments. CONCLUSIONS: Transcriptional response of vascular cells to high glucose is cell type-specific and involves activation of distinct transcription factors, providing a basis for tissue-specific changes of vasculature in diabetics.

Aryl hydrocarbon receptor is activated by glucose and regulates the thrombospondin-1 gene promoter in endothelial cells.

Hyperglycemia is an independent risk factor for development of diabetic vascular complications. The molecular mechanisms that are activated by glucose in vascular cells and could explain the development of vascular complications are still poorly understood. A putative binding site for the transcription factor aryl hydrocarbon receptor (AhR) was identified in the glucose-responsive fragment of the promoter of thrombospondin-1, a potent antiangiogenic and proatherogenic protein involved in development of diabetic vascular complications. AhR was expressed in aortic endothelial cells (ECs), activated, and bound to the promoter in response to high glucose stimulation of ECs. The constitutively active form of AhR induced activation of the thrombospondin-1 gene promoter. In response to high glucose stimulation, AhR was found in complex with Egr-1 and activator protein-2, which are 2 other nuclear transcription factors activated by glucose in ECs that have not been previously detected in complex with AhR. The activity of the DNA-binding complex was regulated by glucose through the activation of hexosamine pathway and intracellular glycosylation. This is the first report of activation of AhR (a receptor for xenobiotic compounds) by a physiological stimulus. This report links the activation of AhR to the pathological effects of hyperglycemia in the vasculature.

Thrombospondins, their polymorphisms, and cardiovascular disease.

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.

Polymorphisms A387P in thrombospondin-4 and N700S in thrombospondin-1 perturb calcium binding sites.

Recent genetic studies have associated members of the thrombospondin (TSP) gene family with premature cardiovascular disease. The disease-associated polymorphisms lead to single amino acid changes in TSP-4 (A387P) and TSP-1 (N700S). These substitutions reside in adjacent domains of these highly homologous proteins. Secondary structural predictive programs and the homology of the domains harboring these amino acid substitutions to those in other proteins pointed to potential alterations of putative Ca2+ binding sites that reside in close proximity to the polymorphic amino acids. Since Ca2+ binding is critical for the structure and function of TSP family members, direct evidence for differences in Ca2+ binding by the polymorphic forms was sought. Using synthetic peptides and purified recombinant variant fragments bearing the amino acid substitutions, we measured differences in Tb3+ luminescence as an index of Ca2+ binding. The Tb3+ binding constants placed the TSP-1 region affected by N700S polymorphism among other high-affinity Ca2+ binding sites. The affinity of Ca2+ binding was lower for peptides (3.5-fold) and recombinant fragments (10-fold) containing the S700 vs. the N700 form. In TSP-4, the P387 form acquired an additional Ca2+ binding site absent in the A387 form. The results of our study suggest that both substitutions (A387P in TSP-4 and N700S in TSP-1) alter Ca2+ binding properties. Since these substitutions exert the opposite effects on Ca2+ binding, a decrease in TSP-1 and an increase in TSP-4, the two TSP variants are likely to influence cardiovascular functions in distinct but yet pathogenic ways.

Increased expression of thrombospondin-1 in vessel wall of diabetic Zucker rat.

BACKGROUND: Thrombospondin-1 (TSP-1) expression in the vascular wall has been related to the development of atherosclerotic lesions and restenosis. TSP-1 promotes the development of neointima and has recently been associated with atherogenesis at a genetic level. Because TSP-1 expression is responsive to glucose stimulation in mesangial cells, we hypothesized that glucose may stimulate its production by vascular cells. Thus, TSP-1 expression in the blood vessel wall may increase, providing a molecular link between diabetes and accelerated vascular lesion development. METHODS AND RESULTS: To determine whether the expression level of TSP-1 in vessel wall is increased in diabetes, aorta and carotid arteries of Zucker rats were used for immunostaining, Western blotting, and in situ RNA hybridization. A significant increase in TSP-1 expression was found in the adventitia of blood vessels from diabetic rats. Consistent with the well-known antiangiogenic effect of TSP-1, the number of vasa vasorum was reduced in aortas from diabetic rats. In cultured endothelial cells, vascular smooth muscle cells, and fibroblasts, TSP-1 expression increased in response to glucose stimulation (>30-fold). After balloon catheter injury to carotid arteries, expression of TSP-1 protein and mRNA was higher at all time points in the vessels of diabetic rats. CONCLUSIONS: Increased expression of TSP-1 in blood vessels in diabetes may represent a new link between diabetes, atherogenesis, and accelerated restenosis. This increase in TSP-1 production may be a direct response of vascular cells to glucose.

Thrombospondin-4 and its variants: expression and differential effects on endothelial cells.

BACKGROUND: In a recent large-scale genetic association study, a single nucleotide polymorphism in the thrombospondin-4 (TSP-4) gene, resulting in a proline-for-alanine substitution at position 387, was associated with a significantly increased risk for premature atherosclerosis. TSP-4 had not previously been implicated in vascular pathology, and very little information is available on its expression and functions. METHODS AND RESULTS: The goal of this study was to assess TSP-4 expression in vessel wall and to identify differences in functions of TSP-4 variants that could account for the proatherogenic effects of the (P387)TSP-4 variant. TSP-4 expression was demonstrated in human endothelial cells (ECs) and vascular smooth muscle cells from brain blood vessels and coronary arteries. (P387)TSP-4 and its fragment (residues 326 to 722), but not the A(387) forms, suppressed EC adhesion and proliferation. The (P387)TSP-4 was more active in inducing the phosphorylation of focal adhesion kinase, consistent with inhibition of proliferation. Both variant fragments increased the proliferation of human aortic smooth muscle cells. CONCLUSIONS: TSP-4 is expressed by vascular cells and influences the vessel wall by modulating the proliferation of ECs and smooth muscle cells. The A387P substitution is a "gain-of-function" mutation, favoring a form of TSP-4 that interferes with EC adhesion and proliferation and may thereby be proatherogenic.

Coronary artery disease and the thrombospondin single nucleotide polymorphisms.

GeneQuest was a high throughput, large-scale analysis of single nucleotide polymorphisms (SNPs) to identify gene associated with familial, premature coronary artery disease and myocardial infarction. The three SNPs showing the highest and most significant associations with disease were all members of the thrombospondin gene family, thrombospondin-1, thrombospondin-2 and thrombospondin-4. These unanticipated associations have kindled efforts to understand how the three SNPs influence the structures and functions of the thrombospondins. The SNP in thrombospondin-1 and thrombospondin-4 reside in their coding regions and result in single amino acid changes: in thrombospondin-1, the predominant asparagine at position 700 is changed to a serine while, in thrombospondin-4, it is a change of an alanine to a proline at position 387. The SNP in thrombospondin-2 is a base change in the 3'-untranslated region of the mRNA. At this early stage of investigation, predictive analyses suggest that the substitutions in thrombospondin-2 and thrombospondin-4 should alter structure, and there is direct evidence to indicate that the thrombospondin-1 SNP alters conformational stability. In addition, profound differences in the function of the thrombospondin-4 SNP variants have been identified with respect to their capacity to support endothelial cell adhesion and proliferation. While substantial additional information is needed to understand if and how the polymorphic forms of the thrombospondins affect coronary artery disease, the data assembled to date suggest marked effects of these SNPs on the structures and functions of the thrombospondins, which are consistent with induction of a proatherogenic and prothrombotic phenotype.

Regulation of gene expression in vascular cells by coagulation proteins.

Receptors of vascular cells and coagulation proteins form a tightly integrated and balanced system, providing regulation to coagulation and mediating a response to coagulation by the vascular cells. Endothelial and smooth muscle cells express a variety of proteins directly participating in hemostasis. Engagement of activated coagulation proteins by their specific receptors on the vascular cell surface, in turn, activates these cells and leads to expression of genes involved in coagulation, angiogenesis, leukocyte adhesion, regulation of the vascular wall tone, etc. The signals inducing the expression of target genes are mediated by protease-activated receptors, which are shared among coagulation proteases. However, differences in mechanisms of activation of these receptors, as well as the presence of specific receptors for each coagulation protein and structures of promoters of target genes may provide specificity in the responses of vascular cell types to different coagulation factors. Activation of gene expression in vascular cells by coagulation proteases accounts for the long-term consequences of coagulation in disorders such as atherosclerotic lesion development, cancer growth, and inflammation. Multiple intracellular pathways and specific transcriptional mechanisms activated by coagulation proteins represent an attractive target for drug design, providing the possibility of controlling the adverse effects of coagulation activation without interfering with the hemostatic requirements of coagulation. This review discusses regulation of gene expression in vascular cells by thrombin, tissue factor, factor VIIa, factor Xa and protein C. Differences and similarities in mechanisms of receptor activation, the pathological profiles of genes activated by these coagulation factors, and recently described transcriptional mechanisms that they induce are discussed.

Cell type-specific post-transcriptional regulation of production of the potent antiangiogenic and proatherogenic protein thrombospondin-1 by high glucose.

Hyperglycemia is an independent risk factor for development of vascular diabetic complications. Vascular dysfunction in diabetics manifests in a tissue-specific manner; macrovasculature is affected by atherosclerotic lesions, and microvascular complications are described as "aberrant angiogenesis": in the same patient angiogenesis is increased in some tissues (e.g. retinal neovascularization) and decreased in others (e.g. in skin). Molecular cell- and tissue-specific mechanisms regulating the response of vasculature to hyperglycemia remain unclear. Thrombospondin-1 (TSP-1), a potent antiangiogenic and proatherogenic protein, has been implicated in the development of several vascular diabetic complications (atherosclerosis, nephropathy, and cardiomyopathy). This study examines cell type-specific regulation of production of thrombospondin-1 by high glucose. We previously reported the increased expression of TSP-1 in the large arteries of diabetic animals. mRNA and protein levels were up-regulated in response to high glucose. Unlike in macrovascular cells, TSP-1 protein levels are dramatically decreased in response to high glucose in microvascular endothelial cells and retinal pigment epithelial cells (RPE). This down-regulation is post-transcriptional; mRNA levels are increased. In situ mRNA hybridization and immunohistochemistry revealed that the level of mRNA is up-regulated in RPE of diabetic rats, whereas the protein level is decreased. This cell type-specific posttranscriptional suppression of TSP-1 production in response to high glucose in microvascular endothelial cells and RPE is controlled by untranslated regions of TSP-1 mRNA that regulate coupling of TSP-1 mRNA to polysomes and its translation. The cell-specific regulation of TSP-1 suggests a potential mechanism for the aberrant angiogenesis in diabetics and TSP-1 involvement in development of various vascular diabetic complications.

Glycosylation mediates up-regulation of a potent antiangiogenic and proatherogenic protein, thrombospondin-1, by glucose in vascular smooth muscle cells.

Accelerated development of atherosclerotic lesions remains the most frequent and dangerous complication of diabetes, accounting for 80% of deaths among diabetics. However, our understanding of the pathways mediating glucose-induced gene expression in vascular cells remains controversial and incomplete. We have identified an intracellular metabolic pathway activated by high glucose in human aortic smooth muscle cells that mediates up-regulation of thrombospondin-1 (TSP-1). TSP-1 is a potent antiangiogenic and proatherogenic protein that may represent an important link between diabetes and vascular complications. Using different glucose analogs and metabolites sharing distinct, limited metabolic steps with glucose, we demonstrated that activation of TSP-1 transcription is mediated by the hexosamine pathway of glucose catabolism, possibly resulting in modulation of the activity of nuclear proteins activity through their glycosylation. Specific inhibitors of glutamine: fructose 6-phosphate amidotransferase (GFAT), an enzyme controlling the hexosamine pathway, as well as direct inhibitors of protein glycosylation efficiently inhibited TSP-1 transcription and the activity of a TSP-1 promoter-reporter construct stimulated by high glucose. Overexpression of recombinant GFAT resulted in increased TSP-1 levels. Pharmacological inhibition of GFAT or protein glycosylation inhibited increased proliferation of human aortic smooth muscle cells caused by glucose. We have demonstrated that the hexosamine metabolic pathway mediates up-regulation of TSP-1 by high glucose. Our results suggest that the hexosamine pathway and intracellular glycosylation may control important steps in initiation and development of atherosclerotic lesions.

Mechanism and effect of thrombospondin-4 polymorphisms on neutrophil function.

High-throughput genomic technology identified an association between a single nucleotide polymorphism (SNP), a proline (P387) rather than the predominant alanine (A387) at position 387 in thrombospondin-4 (TSP-4) and premature myocardial infarction. The inflammatory hypothesis of atherosclerosis invokes a prominent role of leukocytes and cytokines in pathogenesis. As the expression of TSP-4 by vascular cells permits its exposure to circulating leukocytes, the interactions of human neutrophils (polymorphonuclear leukocytes [PMNs]) with both TSP-4 variants were investigated. Phorbol 12-myristate 13-acetate (PMA)-stimulated PMNs adhered and migrated well and equally on the TSP-4 variants. Integrin alpha(M)beta2 was identified as the TSP-4 receptor mediating these responses, and the 3 epidermal growth factor (EGF)-like domains of TSP-4 harboring the SNPs interacted with the alpha(M)I-domain. Despite the similarity in these responses, the P387 variant induced more robust tyrosine phosphorylation of the stress-related mitogen-activated protein kinases (MAPKs): p38MAPK and c-Jun NH2-terminal kinase (JNK), as well as signal transducer and activator of transcription-1 (STAT1) and heat shock protein 27 (HSP27) than the A387 variant. Additionally, cells adherent to P387 TSP-4 variant released 4-fold more H2O2 and secreted 2-fold more interleukin 8 (IL-8) as compared with the A387. H2O2 release and p38MAPK activation were totally inhibited by blockade of alpha(M)beta2. Thus, alpha(M)beta2 plays a central role in proinflammatory activities of TSP-4 (P387) and may contribute to the prothrombotic phenotype associated with this variant.