Vitamin D metabolites influence the expression of various genes involved in calcium homeostasis, cell differentiation, and regulation of the immune system. Expression of these genes is mediated by the activation of the nuclear vitamin D receptor (VDR). Previous studies have shown that a hormonally active form of vitamin D, 1␣,25-dihydroxyvitamin D3, exerts anticoagulant effects in cultured monocytic cells. To clarify whether activation of VDR plays any antithrombotic actions in vivo, hemostatic/thrombogenic systems were examined in normocalcemic VDR knock-out (KO) mice on a high calcium diet and compared with wild type and hypocalcemic VDRKO mice that were fed a regular diet. Platelet aggregation was enhanced significantly in normocalcemic VDRKO mice compared with wild type and hypocalcemic VDRKO mice. Aortic endothelial nitric-oxide (NO) synthase expression and urinary NOx excretions were reduced in hypocalcemic VDRKO mice, but not in normocalcemic VDRKO mice. Northern blot and RT-PCR analyses revealed that the gene expression of antithrombin in the liver as well as that of thrombomodulin in the aorta, liver and kidney was down-regulated in hypo-and normocalcemic VDRKO mice. Whereas tissue factor mRNA expression in the liver and kidney was up-regulated in VDRKO mice regardless of plasma calcium level. Furthermore, VDRKO mice manifested an exacerbated multi-organ thrombus formation after exogenous lipopolysaccharide injection regardless of the calcemic conditions. These results demonstrate that activation of nuclear VDR elicits antithrombotic effects in vivo, and suggest that the VDR system may play a physiological role in the maintenance of antithrombotic homeostasis.

Neointimal SMCs show features of an undifferentiated state, indicated by altered expression of SM MHC, and undergo redifferentiation in a time-dependent manner. The expression of SM MHC isoforms provides insight into the biology of healing after angioplasty and furnishes useful tools for the understanding of the roles of differentiation and phenotypic modulation of SMCs in human vascular lesions.

digested by collagenase/trypsin, and the digested cardiac cells were allowed to attach to the plates overnight. The attached cells included macrophages and myofibroblasts (positive for α smooth muscle actin [αSMA]) as well as other cardiac cells (Supplemental Figure 1B). Notably, cardiac myofibroblasts seemed to be more difficult than cardiac macrophages to collect using our isolation method from infarcted hearts because, as revealed by our immunohistochemical analysis, the number of cardiac myofibroblasts was the same as that of cardiac macrophages in the infarcted area (Supplemental Figure 1C). When the overnight-attached cells were cultured in 10% FBS/DMEM for more than 6 days, almost all of the cells on the plates were positive for αSMA and SM22α, 2 myofibroblast marker proteins (18, 19) (>97.9% and >93.8%, respectively) (Supplemental Figure 1, D and E), indicating that the cells were primarily composed of cardiac myofibroblasts. This is probably because only myofibroblasts were able to grow rapidly in the culture medium.Isolated cardiac macrophages and myofibroblasts were allowed to engulf fluorescently labeled apoptotic cells, and we assessed the fluorescence taken up by cardiac macrophages and administration promoted the restoration of cardiac function and morphology after MI, suggesting that MFG-E8 is a new therapeutic target for the treatment of MI.

Mouse milk fat globule epidermal growth factor 8 (MFG-E8), which is secreted by a subset of activated macrophages, binds to apoptotic cells by recognizing phosphatidylserine and promotes their engulfment. Many apoptotic cells are left unengulfed in the germinal centers of the spleen in MFG-E8(-/-) mice, and these mice develop an autoimmune disease resembling human systemic lupus erythematosus (hSLE). Here, we report that hMFG-E8 bound to phosphatidylserine and an integrin alpha(v)beta(3) complex. Increasing concentrations of MFG-E8 generated a bell-shaped response curve for the efficiency of phagocytosis. That is, in NIH3T3 and MFG-E8(-/-) thioglycollate-elicited peritoneal macrophages that do not express MFG-E8, hMFG-E8 enhanced engulfment at low concentrations but inhibited it at high concentrations. On the other hand, hMFG-E8 dose-dependently inhibited the engulfment of apoptotic cells by MFG-E8(+/+) thioglycollate-elicited peritoneal macrophages, indicating that an excess of MFG-E8 has an inverse effect on the engulfment of apoptotic cells. To investigate the role of MFG-E8 in human disease, we generated two mAb against MFG-E8 and screened human blood samples for MFG-E8 using an ELISA. We found that some childhood-onset and adult SLE patients carried a significant level of MFG-E8 in their blood samples. These results suggested that the aberrant expression of MFG-E8 is involved in the pathoetiology of some cases of hSLE.