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

TGF-β is a multifunctional cytokine affecting many cell types and implicated in tissue remodeling processes. Due to its many functions and cell-specific effects, the consequences of TGF-β signaling are process-and stage-dependent, and it is not uncommon that TGF-β exerts distinct and sometimes opposing effects on a disease progression depending on the stage and on the pathological changes associated with the stage. The mechanisms underlying cell- and process-specific effects of TGF-β are poorly understood. We are describing a novel pathway that mediates induction of angiogenesis in response to TGF-β1. We found that in endothelial cells (EC) TSP-4, a secreted extracellular matrix (ECM) protein is upregulated in response to TGF-β1 and mediates the effects of TGF-β1 on angiogenesis. Upregulation of TSP-4 does not require the synthesis of new protein, is not caused by decreased secretion of TSP-4, and is mediated by activation of SMAD3. Using Thbs4−/− mice and TSP-4 shRNA, we found that TSP-4 mediated pro-angiogenic functions on cultured EC and angiogenesis in vivo in response to TGF-β1. We observed ~ 3-fold increases in tumor mass and levels of angiogenesis markers in animals injected with TGF-β1, and these effects did not occur in Thbs4−/− animals. Injections of an inhibitor of TGF-β1 signaling SB431542 also decreased the weights of tumors and cancer angiogenesis. Our results from in vivo angiogenesis models and cultured EC document that TSP-4 mediates upregulation of angiogenesis by TGF-β1. Upregulation of pro-angiogenic TSP-4 and selective effects of TSP-4 on EC may contribute to stimulation of tumor growth by TGF-β despite the inhibition of cancer cell proliferation.