Nestin expression is upregulated in the fibrotic rat heart and is localized in collagen-expressing mesenchymal cells and interstitial CD31(+)- cells.

Renal and lung fibrosis was characterized by the accumulation of collagen-immunoreactive mesenchymal cells expressing the intermediate filament protein nestin. The present study tested the hypothesis that nestin expression was increased in the hypertrophied/fibrotic left ventricle of suprarenal abdominal aorta constricted adult male Sprague-Dawley rats and induced in ventricular fibroblasts by pro-fibrotic peptide growth factors. Nestin protein levels were upregulated in the pressure-overloaded left ventricle and expression positively correlated with the rise of mean arterial pressure. In sham and pressure-overloaded hearts, nestin immunoreactivity was detected in collagen type I(+)-and CD31(+)-cells identified in the interstitium and perivascular region whereas staining was absent in smooth muscle α-actin(+)-cells. A significantly greater number of collagen type I(+)-cells co-expressing nestin was identified in the left ventricle of pressure-overloaded rats. Moreover, an accumulation of nestin(+)-cells lacking collagen, CD31 and smooth muscle α-actin staining was selectively observed at the adventitial region of predominantly large calibre blood vessels in the hypertrophied/fibrotic left ventricle. Angiotensin II and TGF-ß1 stimulation of ventricular fibroblasts increased nestin protein levels via phosphatidylinositol 3-kinase- and protein kinase C/SMAD3-dependent pathways, respectively. CD31/eNOS(+)-rat cardiac microvascular endothelial cells synthesized/secreted collagen type I, expressed prolyl 4-hydroxylase and TGF-ß1 induced nestin expression. The selective accumulation of adventitial nestin(+)-cells highlighted a novel feature of large vessel remodelling in the pressure-overloaded heart and increased appearance of collagen type I/nestin(+)-cells may reflect an activated phenotype of ventricular fibroblasts. CD31/collagen/nestin(+)-interstitial cells could represent displaced endothelial cells displaying an unmasked mesenchymal phenotype, albeit contribution to the reactive fibrotic response of the pressure-overloaded heart remains unknown.

Nestin upregulation characterizes vascular remodeling secondary to hypertension in the rat.

Proliferation and hypertrophy of vascular smooth muscle cells represent hallmark features of vessel remodeling secondary to hypertension. The intermediate filament protein nestin was recently identified in vascular smooth muscle cells and in other cell types directly participated in proliferation. The present study tested the hypothesis that vessel remodeling secondary to hypertension was characterized by nestin upregulation in vascular smooth muscle cells. Two weeks after suprarenal abdominal aorta constriction of adult male Sprague-Dawley rats, elevated mean arterial pressure increased the media area and thickness of the carotid artery and aorta and concomitantly upregulated nestin protein levels. In the normal adult rat carotid artery, nestin immunoreactivity was observed in a subpopulation of vascular smooth muscle cells, and the density significantly increased following suprarenal abdominal aorta constriction. Filamentous nestin was detected in cultured rat carotid artery- and aorta-derived vascular smooth muscle cells and an analogous paradigm observed in human aorta-derived vascular smooth muscle cells. ANG II and EGF treatment of vascular smooth muscle cells stimulated DNA and protein synthesis and increased nestin protein levels. Lentiviral short-hairpin RNA-mediated nestin depletion of carotid artery-derived vascular smooth muscle cells inhibited peptide growth factor-stimulated DNA synthesis, whereas protein synthesis remained intact. These data have demonstrated that vessel remodeling secondary to hypertension was characterized in part by nestin upregulation in vascular smooth muscle cells. The selective role of nestin in peptide growth factor-stimulated DNA synthesis has revealed that the proliferative and hypertrophic responses of vascular smooth muscle cells were mediated by divergent signaling events.

Nestin downregulation in rat vascular smooth muscle cells represents an early marker of vascular disease in experimental type I diabetes.

BACKGROUND: Nestin was reported to directly contribute to cell proliferation and the intermediate filament protein was detected in vascular smooth muscle cells. In experimental type I diabetes, nestin downregulation in the heart was identified as an incipient pathophysiological event. The following study tested the hypothesis that dysregulation of nestin expression in vascular smooth muscle cells represented an early event of vascular disease in experimental type I diabetes. METHODS/RESULTS: In the carotid artery and aorta of adult male Sprague-Dawley rats, a subpopulation of vascular smooth muscle cells co-expressed nestin and was actively involved in the cell cycle as reflected by the co-staining of nuclear phosphohistone-3. The infection of aortic vascular smooth muscle cells with a lentivirus containing a shRNAmir directed against nestin significantly reduced protein expression and concomitantly attenuated basal DNA synthesis. Two weeks following injection of adult male Sprague-Dawley rats with streptozotocin, the endothelial response of aortic rings to acetylcholine, vascular morphology and the total density of vascular smooth muscle cells in the vasculature of type I diabetic rats were similar to normal rats. By contrast, nestin protein levels and the density of nestin(+)/phosphohistone-3(+)-vascular smooth muscle cells were significantly reduced in type I diabetic rats. The in vivo observations were recapitulated in vitro as exposure of vascular smooth muscle cells to 30 mM D-glucose inhibited DNA synthesis and concomitantly reduced nestin protein expression. CONCLUSIONS: Hyperglycaemia-mediated nestin downregulation and the concomitant reduction of cycling vascular smooth muscle cells represent early markers of vascular disease in experimental type I diabetes.

Estradiol inhibits vascular endothelial cells pro-inflammatory activation induced by C-reactive protein.

In addition of being an important inflammatory biomarker and a risk factor for cardiovascular disease, much evidence indicates that the C-reactive protein (CRP) contributes to the atherosclerosis development process. This plasmatic protein synthesized by hepatocytes in response to inflammation and tissue injury induces pro-inflammatory molecules' expression by endothelial cells (ECs). Previous studies showed that the 17ß-estradiol (E2) has beneficial effects on vascular cells by reducing in vitro pro-inflammatory molecules expressions in EC. Therefore, we hypothesize that E2 blocks or reduces CRP-mediated inflammatory responses by modulating endogenous production of CRP in EC and/or activation mechanisms. Using human aortic ECs (HAECs), we first evaluated CRP production by vascular EC and second demonstrated its self-induction. Indeed, recombinant human CRP stimulation induces a fivefold increase of CRP expression. A 1-h pre-treatment of E2 at a physiologic dose (10(-9 )M) leads to an important decrease of CRP production suggesting a partial blockage of its amplification loop mechanism. Furthermore, in HAEC, E2 reduces the secretion of the most potent agonist of CRP induction, the IL-6, by 21 %. E2 pre-treatment also decreased the expression of pro-inflammatory molecules IL-8, VCAM-1, and ICAM-1 induced by CRP and involved in leukocytes recruitment. In addition, we demonstrated that E2 could restore vascular endothelial growth factor-mediated EC migration response impaired by CRP suggesting another pro-angiogenic property of this hormone. These findings suggest that E2 can interfere with CRP pro-inflammatory effects via activation signals using its rapid, non-genomic pathway that may provide a new mechanism to improve vascular repair.

A phosphorylcholine-modified chitosan polymer as an endothelial progenitor cell supporting matrix.

The aim of the present study was to develop a new biopolymer to increase endothelial progenitor cells (EPC) survival and amplification. As a cell culture platform, bone marrow-derived cells (BMDC) were used to investigate the biocompatibility of chitosan-phosphorylcholine (CH-PC). On CH-PC, BMDC were found in colonies with a mortality rate similar to that of fibronectin (FN), the control matrix. Adhesion/proliferation assays demonstrated a greater number of BMDC on CH-PC after 7 days with an amplification phase occurring during the second week. Difference in adhesion mechanisms between (CH-PC) and the control FN matrix suggest distinctive cell retention ability. Confocal microscopy analyses confirmed that (CH-PC) supported the survival/differentiation of endothelial cells. Moreover, flow cytometry analyses demonstrated that, (CH-PC) increased the percentage of progenitor cells (CD117(+)CD34(+)) (7.1 ± 0.8%, FN: 4.1 ± 0.8%) and EPC (CD117(+)CD34(+)VEGFR-2(+)CD31(+)) (2.33 ± 0.6%, FN: 0.25 ± 0.1%), while the mesenchymal stem cell fraction (CD44(+)CD106(+)CD90(+)) was decreased (0.07 ± 0.01%, FN: 0.55 ± 0.22%). Polymeric substrate CH-PC might provide a suitable surface to promote the amplification of EPC for future vascular therapeutic applications.