Background-Recent advances in high-throughput genomics technology have expanded our ability to catalogue allelic variants in large sets of candidate genes related to premature coronary artery disease. Methods and Results-A total of 398 families were identified in 15 participating medical centers; they fulfilled the criteria of myocardial infarction, revascularization, or a significant coronary artery lesion diagnosed before 45 years in men or 50 years in women. A total of 62 vascular biology genes and 72 single-nucleotide polymorphisms were assessed. Previously undescribed variants in 3 related members of the thrombospondin protein family were prominent among a small set of single-nucleotide polymorphisms that showed a statistical association with premature coronary artery disease. A missense variant of thrombospondin 4 (A387P) showed the strongest association, with an adjusted odds ratio for myocardial infarction of 1.89 (Pϭ0.002 adjusted for covariates) for individuals carrying the P allele. A variant in the 3Ј untranslated region of thrombospondin-2 (change of thymidine to guanine) seemed to have a protective effect against myocardial in individuals homozygous for the variant (adjusted odds ratio of 0.31; Pϭ0.0018). A missense variant in thrombospondin-1 (N700S) was associated with an adjusted odds ratio for coronary artery disease of 11.90 (Pϭ0.041) in homozygous individuals, who also had the lowest level of thrombospondin-1 by plasma assay (Pϭ0.0019). Conclusions-This large-scale genetic study has identified the potential of multiple novel variants in the thrombospondin gene family to be associated with familial premature myocardial infarction. Notwithstanding multiple caveats, thrombospondins specifically and high-throughput genomic technology in general deserve further study in familial ischemic heart disease. (Circulation. 2001;104:2641-2644.)

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.

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 ␤ 2 and ␤ 3 integrins, which recognize TSP-4 as a ligand. 7 In vivo, expression of TSP-4 increases dramatically in response to pressure overload, 9 in failing hearts and heart hypertrophy, 10,11 and in response to ischemia. 12 Furthermore, multiple reports of various populations have documented a genetic association between TSP-4 and accelerated atherogenesis. [13][14][15][16][17][18] The effect of TSP-1 deficiency on the development of atherosclerotic lesions in a mouse model has been recently reported, 19 namely promoting development of atherosclerotic lesions at the initial stages, but exerting a beneficial effect in Original Conclusions: TSP-4 is abundant in atherosclerotic lesions

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 (Ͼ30-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

Thrombospondins are matricellular proteins that regulate cell-cell and cell-matrix interactions (1, 2). Recent genetic association studies link the thrombospondin (TSP) 2 protein family to the development of atherosclerotic lesions (3-10). TSP-1 was found in early atherosclerotic lesions (11), in injured vascular walls (12,13), and in cardiac allografts where its expression correlated with the degree of vasculopathy (14). The genetic disruption of TSP-1 reduced the atherosclerotic lesion area in the mouse model of atherosclerosis and suggested an important role for TSP-1 in the evolution of plaque and its composition (15). In both in vivo and in vitro studies TSP-1 induced proliferation of vascular smooth muscle cells (SMC) (16,17), and both TSP-1 and TSP-2 inhibited growth of endothelial cells (18 -21); both effects are considered proatherogenic.Previous studies have documented increased TSP-1 levels in the plasma and kidneys of diabetic patients and diabetic animal models (22-25). In mesangial cells, the level of TSP-1 was upregulated by glucose by a transcriptional mechanism (26 -28). We have recently reported increased levels of TSP-1 in the blood vessels of diabetic animals (29). Moreover, TSP-1 was up-regulated by high glucose in vitro in major cell types from large blood vessels. These observations suggest that TSP-1 represents an important link between diabetes, hyperglycemia, and accelerated atherogenesis.A large number of clinical studies and trials have conclusively identified hyperglycemia as an independent risk factor for development of both micro-and macrovascular complications (30 -33). Recently, the Epidemiology of Diabetes Intervention and Complications study reported that, as compared with conventional therapy, intensive glycemic control reduced the risk of the most serious cardiovascular events such as heart attacks, stroke, and death by nearly 60%. These findings clearly underscore the importance of hyperglycemia as a critical player in the development of pathogenic complications associated with diabetes. Glucose regulates the expression of a number of vascular * This work was supported by National Institutes of Health Grants R01 DK067532, K01 DK62128, and P50 HL077107, American Heart Association Grant 0565284B, and funds from the Lerner Research Institute (Cleveland Clinic) (to O. I. S.) and by National Institutes of Health Grant R01 45418 (to P. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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 downregulation 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 cellspecific 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.Despite the significant advances in the therapeutic methods to control blood glucose and insulin levels in diabetic patients, the precise regulation of these levels remains a problem. Vascular diabetic complications remain most prevalent and dangerous and account for the greatest numbers of deaths and hospitalizations in diabetic patients. The molecular basis for the vascular complications of diabetes is not well understood. Recent reports indicate that both microvascular and macrovascular complications of diabetes correlate directly with glucose levels in both patients and animal models (1-5). Some of these reports revealed the pathogenic role of impaired glucose tolerance and post-prandial hyperglycemia even in the absence of diabetes, e.g. (6). In vascular cells, glucose regulates expression of many genes that have been linked to the development of atherosclerosis or abnormal angiogenesis (reviewed in Ref. 7). One of them is thrombospondin-1 (TSP-1), 3 a cell matrix protein implicated in both atherogenesis (8 -12) and angiogenesis (13)(14)(15)(16)(17)(18)(19). Several lines of evidence indicate that TSP-1 may represent a lin...

Abstract-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.

The vitamin K-dependent (VKD) carboxylase binds VKD proteins via their propeptide and converts Glu's to gamma-carboxylated Glu's, or Gla's, in the Gla domain. Multiple carboxylation is required for activity, which could be achieved if the carboxylase is processive. In the only previous study to test for this capability, an indirect assay was used which suggested processivity; however, the efficiency was poor and raised questions regarding how full carboxylation is accomplished. To unequivocally determine if the carboxylase is processive and if it can account for comprehensive carboxylation in vivo, as well as to elucidate the enzyme mechanism, we developed a direct test for processivity. The in vitro carboxylation of a complex containing carboxylase and full-length factor IX (fIX) was challenged with an excess amount of a distinguishable fIX variant. Remarkably, carboxylation of fIX in the complex was completely unaffected by the challenge protein, and comprehensive carboxylation was achieved, showing conclusively that the carboxylase is processive and highly efficient. These studies also showed that carboxylation of individual fIX/carboxylase complexes was nonsynchronous and implicated a driving force for the reaction which requires the carboxylase to distinguish Glu's from Gla's. We found that the Gla domain is tightly associated with the carboxylase during carboxylation, blocking the access of a small peptide substrate (EEL). The studies describe the first analysis of preformed complexes, and the rate for full-length, native fIX in the complex was equivalent to that of the substrate EEL. Thus, intramolecular movement within the Gla domain to reposition new Glu's for catalysis is as rapid as diffusion-limited positioning of a small substrate, and the Gla domain is not sterically constrained by the rest of the fIX molecule during carboxylation. The rate of carboxylation of fIX in the preformed complex was 24-fold higher than for fIX modified by free carboxylase, which supports carboxylase processivity and which indicates that binding and/or release is the rate-limiting step in protein carboxylation. These data indicate a model of tethered processivity, in which the VKD proteins remain bound to the carboxylase throughout the reaction via their propeptide, while the Gla domain undergoes intramolecular movement to reposition new Glu's for catalysis to ultimately achieve comprehensive carboxylation.