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Focal adhesion protein zyxin is a mechanosensitive modulator of gene expression in vascular smooth muscle cells
No full textExcessive deformation of vascular smooth muscle cells (SMCs) caused by a prolonged increase in blood pressure (eg, in hypertension) results in an adaptive remodeling of the vessel wall that is characterized by SMC hypertrophy or hyperplasia and contributes to fixation of the increase in blood pressure. The onset of this process is characterized by a unique change in gene expression in the SMC. However, thus far, no transcription factor has been identified that specifically mediates mechanosensitive gene expression in these cells. Therefore, the role of a putative mechanotransducer, the cytoskeletal protein zyxin, was investigated in rat aortic cultured SMCs. Immunofluorescence and Western blot analysis revealed that on exposure to cyclic stretch, but not to osmotic stress or treatment with proinflammatory cytokines, zyxin dissociates from focal adhesions and accumulates in the nucleus. Unlike zyxin, vinculin, another focal adhesion-associated protein, did not translocate. Moreover, antisense oligonucleotide downregulation of zyxin protein abundance suggested that zyxin accumulation in the nucleus is a prerequisite for mechanosensitive gene expression in these cells. Thus, stretch-induced endothelin B receptor expression, for example, was attenuated, whereas that of tenascin-C was augmented after zyxin suppression. The data are consistent with a role for zyxin in transducing mechanical stimuli from the cell membrane to the nucleus in vascular SMCs and in controlling the expression of mechanosensitive genes that have been implicated in hypertension-induced arterial remodeling
Cytokine-induced down-regulation of zfm1/splicing factor-1 promotes smooth muscle cell proliferation
No full textOne hallmark of inflammation is the proliferation of bystander cells such as vascular smooth muscle cells (SMC), a process governed by growth factors and cytokines. Whereas cytokine induction of gene products promoting inflammation and proliferation is well characterized, little is known about the concomitant down-regulation of potentially counter-regulatory gene products in these cells. By employing the suppression subtractive hybridization-PCR technique, RNA isolated from rat aortic SMC treated with the cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) was subtracted from RNA of control cells. Eleven genes were identified, the expression of which fell by 44-77%. One, the transcriptional repressor splicing factor-1 or zfm1, was characterized further. Antisense oligonucleotide suppression of zfm1 protein synthesis mimicked the stimulatory effects of IL-1beta and TNFalpha on SMC proliferation and expression of the chemokine MCP-1 and the vascular cell adhesion molecule-1. Moreover, in an in vivo mouse model of atherosclerosis, zfm1 abundance was decreased in proliferating arterial SMC. These findings suggest a role for zfm1 in controlling both proliferation and expression of pro-inflammatory gene products in SMC. Therefore, cytokine-induced down-regulation of zfm1 expression may contribute to the pathogenesis of hyperproliferative inflammatory diseases
ACE inhibitor and AT(1) antagonist blockade of deformation-induced gene expression in the rabbit jugular vein through B-2 receptor activation
No full textDeformation-induced endothelin-1 synthesis in endothelial cells may contribute to the intimal hyperplasia of venous bypass grafts. ACE inhibitors and angiotensin II type 1 (AT(1)) receptor antagonists are capable of reducing vein graft disease. Therefore, the effects of these drugs on endothelial preproendothelin-1 (ppET-1) and smooth muscle endothelin B receptor (ETB-R) expression were investigated in isolated perfused segments of the rabbit jugular vein. Pretreatment with ramiprilat (0.3 mu mol/L) or irbesartan (0.01 to 1 mu mol/L) had no effect on basal ppET-1 or ETB-R expression but markedly attenuated the deformation-induced expression of these gene products, and these effects were reversed by the B-2 receptor antagonist icatibant (Hoe 140) and by the NO synthase inhibitor N-G-nitro-L-arginine. Candesartan (1 mu mol/L) mimicked the inhibitory effect of irbesartan, Moreover, reporter gene analysis with a rat ppET-1 promoter-luciferase construct transiently transfected into porcine aortic cultured endothelial cells revealed that the inhibitory effect of both ramiprilat and irbesartan on deformation-induced ppET-1 expression is species independent and mediated at the level of transcription. In addition, RT-PCR analysis detected only AT(1) receptor expression in the endothelium-intact rabbit jugular vein, and neither irbesartan nor ramiprilat affected endothelial NO synthase expression. Thus, ACE inhibitors and AT(1) receptor antagonists are capable of suppressing deformation-induced gene expression in the vessel wall in both an autocrine (ppET-1) and a paracrine (ETB-R) manner via a common mechanism of action that constitutes a B-2 receptor-mediated increase in endothelial NO release
Disinhibition of SOD-2 Expression to Compensate for a Genetically Determined NO Deficit in Endothelial Cells-Brief Report
No full textObjective-Homozygosity for the -786C-variant of the human nos-3 gene is a risk factor for coronary artery disease (CAD). Interestingly, affected individuals develop CAD more frequently but not earlier than the general population. Methods and Results-Genotyped primary human umbilical vein endothelial cells (ECs) were exposed to fluid shear stress (FSS) and analyzed for nitric oxide (NO) and superoxide anion (O-2(-)) formation as well as mRNA and protein expression of different antioxidant enzymes. Dysfunctional CC-genotype ECs failed to upregulate NO synthase expression in response to FSS and exhibited a reduced NO synthesis capacity when compared to functionally intact TT-genotype ECs. However, only CC-genotype ECs responded to FSS with an Egr-1-mediated increase in manganese-containing superoxide dismutase (SOD-2) expression, shielding them from endothelin-1-induced oxidative stress in a NO-independent manner. Conclusions-This FSS-induced rise in SOD-2 expression in CC-genotype ECs effectively stabilizes their antiatherosclerotic phenotype and may explain not only the comparatively slow onset of CAD in homozygous carriers of the C-allele of the nos-3 gene but also define a general strategy for preventing endothelial dysfunction at the outset of atherosclerosis. (Arterioscler Thromb Vasc Biol. 2009; 29: 1890-1893.)Deutsche Forschungsgemeinschaft [HE 1587/9-1
ACE inhibitor and AT(1) antagonist blockade of deformation-induced gene expression in the rabbit jugular vein through B-2 receptor activation
No full textDeformation-induced endothelin-1 synthesis in endothelial cells may contribute to the intimal hyperplasia of venous bypass grafts. ACE inhibitors and angiotensin II type 1 (AT(1)) receptor antagonists are capable of reducing vein graft disease. Therefore, the effects of these drugs on endothelial preproendothelin-1 (ppET-1) and smooth muscle endothelin B receptor (ETB-R) expression were investigated in isolated perfused segments of the rabbit jugular vein. Pretreatment with ramiprilat (0.3 mu mol/L) or irbesartan (0.01 to 1 mu mol/L) had no effect on basal ppET-1 or ETB-R expression but markedly attenuated the deformation-induced expression of these gene products, and these effects were reversed by the B-2 receptor antagonist icatibant (Hoe 140) and by the NO synthase inhibitor N-G-nitro-L-arginine. Candesartan (1 mu mol/L) mimicked the inhibitory effect of irbesartan, Moreover, reporter gene analysis with a rat ppET-1 promoter-luciferase construct transiently transfected into porcine aortic cultured endothelial cells revealed that the inhibitory effect of both ramiprilat and irbesartan on deformation-induced ppET-1 expression is species independent and mediated at the level of transcription. In addition, RT-PCR analysis detected only AT(1) receptor expression in the endothelium-intact rabbit jugular vein, and neither irbesartan nor ramiprilat affected endothelial NO synthase expression. Thus, ACE inhibitors and AT(1) receptor antagonists are capable of suppressing deformation-induced gene expression in the vessel wall in both an autocrine (ppET-1) and a paracrine (ETB-R) manner via a common mechanism of action that constitutes a B-2 receptor-mediated increase in endothelial NO release
Interleukin-10 induction of nitric-oxide synthase expression attenuates CD40-mediated interleukin-12 synthesis in human endothelial cells
No full textInterleukin-10 (IL-10) is a potent anti-inflammatory cytokine in Th1 cell-mediated chronic inflammatory diseases such as, e. g. Crohn's disease. Moreover, IL-10 has been shown to limit the progression of atherosclerosis, presumably by influencing endothelial cell function. Here we demonstrate that under pro-inflammatory conditions expression of the human IL-10 receptor gene is enhanced in endothelial cells in vitro and in vivo. Subsequent exposure to IL-10 results in an up-regulation of both endothelial nitric-oxide synthase (NOS-3) expression and activity. Gel mobility shift analyses and decoy oligonucleotide experiments suggest that this effect of IL-10 is mediated through activation of the transcription factor STAT-3 ( signal transducer and activator of transcription-3). One functional consequence of IL-10 up-regulation of NOS-3 abundance in cultured endothelial cells is the attenuation of CD154-induced IL-12 p40 expression. Moreover, CD154-induced IL-12 p40 expression is enhanced after blockade of NOS-3 activity but attenuated in the presence of exogenous nitric oxide. Increased NOS-3 expression may, thus, be one mechanism by which IL-10 exerts its anti-inflammatory effects in Th1 cell-mediated chronic inflammatory diseases
Pressure-induced upregulation of preproendothelin-l and endothelin B receptor expression in rabbit jugular vein in situ - Implications for vein graft failure?
No full textUpregulation of endothelin-1 (ET-1) synthesis in venous bypass grafts in response to arterial levels of blood pressure may play a major role in graft failure. To investigate this hypothesis, isolated segments of the rabbit jugular vein were perfused at physiological (0 to 5 mm Hg) and nonphysiological (20 mm Hg) levels of intraluminal pressure. As judged by reverse transcription-polymerase chain reaction analysis (mRNA level), neither endothelin-converting enzyme nor endothelin A receptor expression appeared to be pressure sensitive. In contrast, there was a profound and time-dependent increase in endothelial prepro-ET-1 mRNA and intravascular ET-1 abundance (by ELISA) as well as in smooth muscle endothelin B receptor mRNA and functional protein (by superfusion bioassay) on raising the perfusion pressure from 5 to 20 mm HE, but not from 0 to 5 mm Hg, for up to 12 hours. Video microscopy analysis revealed that the segments were distended by 75% at 5 mm Hg and near maximally at 20 mm Hg compared with the resting diameter at 0 to 1 mm Hg. Treatment of the segments with actinomycin D (1 mu mol/L), the specific protein kinase C inhibitor, Ro 31-8220 (0.1 mu mol/L), or the c-Src family-specific tyrosine kinase inhibitor, herbimycin A (0.1 mu mol/L), demonstrated that the pressure-induced expression of these gene products occurs at the level of transcription and requires activation of protein kinase C, but not c-Src. In venous bypass grafts such deformation-induced changes in gene expression may contribute not only to acute graft failure through ET-1-induced vasospasm but also to endothelin A receptor- and/or endothelin B receptor-mediated smooth muscle cell hyperplasia and graft occlusion
Elevated Perfusion Pressure Upregulates Endothelin-1 and Endothelin B Receptor Expression in the Rabbit Carotid Artery
No full textTo investigate the hypothesis that high blood pressure activates the endothelin system in the vessel wall, isolated segments of the rabbit carotid artery were subjected to different levels of perfusion pressure. Both preproendothelin-l (ppET-1) mRNA abundance and intravascular ET-1 peptide content were strongly upregulated on raising the intraluminal pressure from 90 to 160 mm Hg for 3 to 12 hours, and this increase in ppET-1 mRNA occurred predominantly in the endothelial cells. Endothelin-converting enzyme-1 and endothelin A receptor (ETA-R) expression were pressure-insensitive, whereas that of the ETB-R in the smooth muscle cells was also significantly enhanced. Both the pressure-induced increase in ppET-1 and ETB-R expression required RNA synthesis because they were abolished by actinomycin D. The nuclear signaling mechanisms involved therein, however, appeared to be different. Thus, the pressure-induced expression of ppET-1 and activation of CCAAT-enhancer binding proteins beta and delta were blocked by the tyrosine kinase inhibitor herbimycin A, whereas ETB-R expression and the nuclear translocation of activator protein-1 were abolished by the protein kinase C inhibitor Ro 31-8220. One consequence of these presumably deformation-induced changes in gene expression was an increased rate of apoptosis of the smooth muscle cells in the media that if transferable to the situation in human blood vessels may contribute to hypertension-induced arterial remodeling
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