Marco Cattaruzza

Regulation of inducible nitric oxide synthase gene expression in vascular smooth muscle cells.

1. Formation of nitric oxide (NO) by the constitutive calcium-dependent NO synthase expressed in endothelial cells plays an important role in the control of local blood flow and vascular homeostasis. Expression of the inducible calcium-independent NO synthase (iNOS) in vascular smooth muscle cells (VSMC), on the other hand, is thought to play a potentially detrimental role in the pathogenesis of chronic inflammation or septic shock. In vascular injury, however, iNOS expression in VSMC may be beneficial as a compensatory mechanism for the lack of endothelial NO synthesis, e.g., by preventing restenosis following angioplasty or heart transplant vasculopathy. 2. Because iNOS activity does not seem to be controlled once the enzyme is expressed, regulation of NO release from iNOS-expressing cells predominantly occurs at the transcriptional and/or posttranscriptional level. 3. This review summarizes what is currently known about the regulation of expression of this enzyme in VSMC, details some of the transcription factors involved therein as well as their mode of activation, and highlights some pharmacological strategies based on these findings that may be employed for the control of iNOS expression in VSMC in the clinical arena.

Stretch-induced endothelin B receptor-mediated apoptosis in vascular smooth muscle cells

Growing evidence suggests that a pressure‐induced increase in the synthesis of endothelin (ET‐1) is involved in arterial remodeling and, as a consequence, in the manifestation of chronic hypertension. To study potential stretch‐induced changes in gene expression and their functional consequences, we have cultured rat aortic smooth muscle cells (raSMC) and porcine aortic endothelial cells (PAEC) on flexible elastomer membranes. The cells were periodically stretched (up to 20% elongation, 0.5 Hz, 6 h) and the expression of prepro‐ET‐1 and that of the endothelin A and B receptors (ETA‐R and ETB‐R) were analyzed by semi‐quantitative RT‐PCR analysis and ELISA (ET‐1). In contrast to PAEC where ET‐1 synthesis was up‐regulated up to eightfold on exposure to cyclic stretch, ET‐1 synthesis in raSMC was decreased by more than 80% under these conditions. ETA R‐mRNA expression in stretched raSMC declined to 50% whereas ETB R‐mRNA levels were increased up to 10‐fold. One functional consequence of this apparent shift in receptor abundance was an apoptosis‐promoting action of exogenous ET‐1 (10 nM), as judged by the appearance of subdiploid peaks during FACS analysis, caspase‐3 activation and chromatin condensation. This ET‐1‐induced apoptosis appeared to be ETB‐R mediated, as it was completely suppressed by the ETB‐R antagonist BQ 788 but not by the ETA‐R antagonist BQ 123. Moreover, raSMC derived from homozygous spotting lethal rats, which lack a functional ETB‐R, showed no signs of apoptosis after exposure to cyclic strain and exogenous ET‐1. These findings suggest a central role for the endothelin system in the onset of hypertension‐induced remodeling in conduit arteries, which may proceed via an initial stretch‐induced apoptosis of the smooth muscle cells.—Cattaruzza, M., Dimigen, C., Ehrenreich, H., Hecker, M. Stretch‐induced endothelin B receptor‐mediated apoptosis in vascular smooth muscle cells. FASEB J. 14, 991–998 (2000)

Shear Stress Insensitivity of Endothelial Nitric Oxide Synthase Expression as a Genetic Risk Factor for Coronary Heart Disease

Coronary heart disease (CHD) is based on the development of atherosclerosis in coronary arteries. Shear stress-induced endothelial nitric oxide (NO) release not only contributes to local blood pressure control but also effectively helps to retard atherosclerosis. Therefore, functionally relevant polymorphisms in the endothelial NO synthase (NOS-3) gene may contribute to the development of CHD. NOS-3 expression was analyzed in endothelial cells isolated from umbilical cords genotyped for the −786C/T single nucleotide polymorphism (SNP) of the human nos-3 gene. Moreover, NO-dependent relaxation was examined in segments of saphenous vein isolated from genotyped patients undergoing aortocoronary bypass surgery, and patients subjected to quantitative coronary angiography were genotyped to verify an association between this SNP and CHD. Shear stress-induced NOS-3 mRNA and protein expression was present in TT and CT genotype cells but absent in cells with CC genotype. Pretreatment of these cells with a decoy oligonucleotide comprising position −800 to −779 of the C-type nos-3 promoter reconstituted shear stress-induced NOS-3 expression. These results were confirmed by reporter gene analysis with the corresponding nos-3 promoter luciferase constructs. In addition, the NO-mediated relaxant response of vein grafts from CC genotype patients was significantly attenuated as compared with the CT or TT genotype, and in CHD-positive patients, the CC genotype was significantly more frequent (19.0%) than in CHD-negative patients (4.4%). The −786C/T SNP of the nos-3 gene thus constitutes a genetic risk factor for CHD, presumably due to binding of an inhibitory transcription factor to the C-type promoter blocking shear stress-dependent maintenance of NOS-3 expression.

Focal Adhesion Protein Zyxin Is a Mechanosensitive Modulator of Gene Expression in Vascular Smooth Muscle Cells

Abstract—Excessive 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-inducible CD40 gene expression in vascular smooth muscle cells is mediated by nuclear factor κB and signal transducer and activato of transcription-1

The interaction of T‐lymphocytes expressing the CD40 ligand (CD154) and cells of the vessel wall expressing the corresponding receptor protein (CD40) may play an important role in chronic inflammation including arteriosclerosis. One way of interfering with CD40‐CD154 signalling is to prevent CD40 expression, the regulation of which, however, has yet to be elucidated. Therefore, we studied CD40 expression in rat aortic cultured smooth muscle cells. Both CD40 mRNA and protein expression in these cells was markedly enhanced as early as 6 h after exposure to different pro‐inflammatory cytokines. Experiments with actinomycin D and subsequent run‐on analyses revealed that CD40 expression in response to these cytokines was regulated at the level of transcription. Moreover, electrophoretic mobility shift analyses along with the employment of transcription factor decoy oligodeoxynucleotides demonstrated that tumor necrosis factor α via nuclear κB and interferon‐γ via signal transducer and activator of transcription‐1 up‐regulate CD40 gene expression in rat aortic cultured smooth muscle cells.

Interleukin-10 Induction of Nitric-oxide Synthase Expression Attenuates CD40-mediated Interleukin-12 Synthesis in Human Endothelial Cells

Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine in Th1 cell-mediated chronic inflammatory diseases such as, e.g. Crohns 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.

Elevated Perfusion Pressure Upregulates Endothelin-1 and Endothelin B Receptor Expression in the Rabbit Carotid Artery

To 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-1 (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 (ET(A)-R) expression were pressure-insensitive, whereas that of the ET(B)-R in the smooth muscle cells was also significantly enhanced. Both the pressure-induced increase in ppET-1 and ET(B)-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 ET(B)-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.

Zyxin Mediation of Stretch-Induced Gene Expression in Human Endothelial Cells

Rationale: Prolonged exposure to enhanced stretch, such as in hypertension, triggers endothelial dysfunction, a hallmark of pathological vascular remodeling processes. Despite its clinical relevance, little is known about stretch-induced gene expression in endothelial cells. Objective: Here, we have characterized a new stretch-inducible signaling pathway and the subsequent changes in endothelial gene expression in response to stretch. Methods and Results: Using human primary endothelial cells, we observed that the protein zyxin translocates from focal adhesions to the nucleus solely in response to stretch. There, it orchestrates complex changes in gene expression by interacting with a novel cis-acting element found in all zyxin-regulated genes analyzed so far. By way of DNA microarray pathway analyses, stretch-induced changes in endothelial cell gene expression were systematically explored, revealing that zyxin mainly regulates proinflammatory pathways. Conclusions: Stretch appears to be an important factor in the development of endothelial dysfunction with zyxin as a potential therapeutic target to interfere with these early changes in endothelial cell phenotype.

T-786C polymorphism of the NOS-3 gene and the endothelial cell response to fluid shear stress-a proteome analysis.

Endothelial dysfunction is a common denominator of cardiovascular disease. Central to endothelial dysfunction is a decrease in the bioavailability of nitric oxide (NO) synthesized by endothelial NO synthase (NOS-3). In vivo, the level of fluid shear stress (FSS) exerted by the flowing blood determines NOS-3 expression. However, in contrast to the -786T variant of the nos-3 gene, the -786C variant is not sensitive to shear stress. Consequently, cells homozygous for this variant have an inadequate capacity to synthesize NO. Therefore, we have compared shear stress-induced protein expression in human primary cultured endothelial cells with TT or CC genotype. Cells with the CC genotype exhibited a greatly reduced FSS-induced NOS-3 expression as well as a diminished NO synthesis capacity when compared to TT genotype cells. Proteome changes in response to FSS (30 dyn/cm(2) for 24 h) were monitored by 2D-gel electrophoresis/densitometry/mass spectrometry. Of a total of 14 FSS-sensitive proteins, 8 were identically expressed in all cells. Four proteins, all of them part of the NO-dependent endoplasmic reticulum-stress response, were up-regulated by FSS only in cells with TT genotype. In contrast, CC genotype cells responded to FSS with a unique increase in manganese-containing superoxide dismutase expression. These differences in protein expression may (i) reflect the low bioavailability of NO in cells homozygous for the -786C variant of the nos-3 gene and (ii) point to a mechanism by which this deficit is counterbalanced by protecting the less abundant NO from rapid degradation.

Pressure-Induced Upregulation of Preproendothelin-1 and Endothelin B Receptor Expression in Rabbit Jugular Vein In Situ Implications for Vein Graft Failure?

Upregulation 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 Hg, 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 micromol/L), the specific protein kinase C inhibitor, Ro 31-8220 (0.1 micromol/L), or the c-Src family-specific tyrosine kinase inhibitor, herbimycin A (0.1 micromol/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.