Endothelin converting enzyme is located on alpha-actin filaments in smooth muscle cells.

OBJECTIVE: Endothelin converting enzyme is the key enzyme in the generation of endothelin-1 from big-endothelin-1. The mature endothelin-1 is a potent vasoconstrictor which also promotes mitogenesis and proliferation of smooth muscle cells. The objectives were to demonstrate in smooth muscle cells the presence of a phosphoramidon-sensitive endothelin converting enzyme activity, reveal the subcellular localization of the enzyme protein and determine the effects of the metalloproteinase inhibitor, phosphoramidon, the lysosomotrophic drug, chloroquine, and colchicine on the cycling pathway of the enzyme. METHODS: Subcellular localization of endothelin converting enzyme on human smooth muscle cells and the rat cell line, A7r5, was by immunofluorescence and confocal microscopy or by biotinylation of cell cultures and immunoblotting, after treatment of cell cultures with cytochalasin D, colchicine, chloroquine and phosphoramidon. Converting enzyme activity was determined by high performance liquid chromatographic assay. RESULTS: We detected phosphoramidon-sensitive endothelin converting enzyme activity in smooth muscle cells. In addition to its plasma membrane location, for the first time we revealed a striking co-localization of endothelin converting enzyme and alpha-actin filaments in smooth muscle cells. Colchicine treatment results in a perinuclear accumulation of endothelin converting enzyme. An increased level of endothelin converting enzyme protein was shown to be present in smooth muscle cells which had been grown in the presence of phosphoramidon or chloroquine. CONCLUSION: The 120 kDa endothelin converting enzyme co-localizes with alpha-actin in smooth muscle cells and resembles that found in endothelial cells in that it is present on both the plasma membrane and intracellularly.     

Tyrosine-kinase dependent TGF-beta and extracellular matrix expression by mechanical stretch in vascular smooth muscle cells.

Vascular hypertrophy, which is characterized by proliferation of vascular smooth muscle cells (VSMC) and accumulation of extracellular matrix (ECM), is a major pathological change in blood vessels after chronic exposure to hypertension. Blood pressure is transmitted to the arterial walls and counterbalanced by mechanical stress, leading to stretching of circumferentially oriented VSMC, which may play some role in the pathogenesis of vascular hypertrophy. The present study was designed, therefore, to investigate the effect of mechanical stretch on the expression of ECM components and transforming growth factor-beta (TGF-beta), a potent stimulator for ECM production, and to examine the signal transduction mechanisms of the induction of TGF-beta in cultured rat VSMC. VSMC were subjected to cyclic stretch to provide a maximal elongation of 20% at a rate of 60 cycles per minute for up to 24 h. Mechanical stretch stimulated TGF-beta1 mRNA expression in a time- and elongation-dependent manner. Indeed, the secretion of TGF-beta proteins into the culture media was increased after stretch. Stretch also stimulated mRNA expression of the ECM components, type I and type IV collagen, and fibronectin, which was largely inhibited by addition of neutralizing antibody against TGF-beta. The tyrosine kinase inhibitors genistein and herbimycin A blocked the induction of TGF-beta1 and type I collagen by stretch, while protein kinase C inhibitors, the calcium channel blockers nitrendipine and gadolinium, or Ca removal from the media had no effect. These results suggest that stretch-induced, tyrosine kinase-mediated autocrine/paracrine production of TGF-8 may play a critical role in the progression of vascular remodeling associated with high blood pressure.     

Glucose-induced TGF-beta1 and TGF-beta receptor-1 expression in vascular smooth muscle cells is mediated by protein kinase C-alpha

Sclerosis and increased matrix expression in diabetes are mediated by glucose-induced transforming growth factor (TGF)-beta1 expression. The intracellular effects of high glucose occur at least in part by way of protein kinase C (PKC). We previously described a role for PKC-alpha in glucose-induced permeability. We now investigated the hypothesis that glucose-induced expression of TGF-beta1 and its receptors (TGF-beta-R1 and -R2) are mediated by activation of this PKC isoform. TGF-beta1 and TGF-beta-R expressions were determined in vascular smooth muscle cells (VSMCs) by immunocytochemistry and Western blotting. PKC isoforms were assessed by confocal microscopy. PKC isoforms were inhibited with antisense oligodeoxynucleotides. PKC-alpha was upregulated by overexpression or microinjection. High glucose (20 mmol/L) increased VSMC TGF-beta1 and TGF-beta-R1 expression but not TGF-beta-R2 expression. PKC inhibitors and specific PKC-alpha downregulation by antisense treatment prevented this effect, whereas antisense treatment against PKC-beta, -epsilon, and -zeta had no influence. PKC-alpha overexpression increased TGF-beta1 and TGF-beta-R1 expression but not TGF-beta-R2 expression. PKC-alpha microinjection into individual VSMCs also increased TGF-beta1 and TGF-beta-R immunofluorescence. Last, VSMCs from PKC-alpha-deficient mice did not respond to high glucose compared with VSMCs from wild-type mice. We propose that high glucose-induced TGF-beta1 and TGF-beta-R1 expression is mediated by PKC-alpha. Our findings suggest an autocrine feedback mechanism and a possible role for PKC-alpha in diabetic vascular disease.     

Endoglin, a TGF-beta receptor-associated protein, is expressed by smooth muscle cells in human atherosclerotic plaques

Endoglin is a transmembrane protein that is found in association with transforming growth factor-beta (TGF-beta) superfamily receptor complexes and has an expression pattern that appears to be restricted primarily to endothelial cells, activated macrophages, trophoblasts, and fibroblasts. Since mutations in endoglin have been shown to be linked to hereditary hemorrhagic telangiectasia type 1, a disease manifested as vascular malformations characterized by excessive layers of vascular smooth muscle cells (VSMC), the expression of endoglin was investigated in VSMC. In vivo, the majority of SMC in human atherosclerotic plaques expressed high levels of endoglin, while endoglin was not detected in SMC from samples of the normal arterial wall. In vitro studies demonstrate that human aortic smooth muscle cells (HASMC) express the L-isoform of endoglin. Like endothelial cells, HASMC express endoglin protein as a dimer on the cell surface that binds TGF-beta1. In vitro, endoglin expression by HASMC is upregulated in response to TGF-beta1, suggesting that the presence of this factor in the atherosclerotic plaque might be responsible for the increased expression of endoglin. The demonstration of increased levels of endoglin in VSMC in human atherosclerotic plaques suggests a role for SMC endoglin in the maintenance of vascular integrity and in the response of the vessel wall to injury.     

Metabolism of glyceryl trinitrate to nitric oxide by endothelial cells and smooth muscle cells and its induction by Escherichia coli lipopolysaccharide.

Here, we demonstrate that the metabolism of glyceryl trinitrate (GTN) to nitric oxide (NO) occurs not only in bovine aortic smooth muscle cells (SMCs) but also in endothelial cells (ECs) and that this biotransformation is enhanced by pretreatment with Escherichia coli lipopolysaccharide (LPS). Two bioassay systems were used: inhibition of platelet aggregation and measurement of cGMP after stimulation by NO of guanylate cyclase in SMCs or ECs. In addition, NO produced from GTN by cells was measured as nitrite (NO2-), one of its breakdown products. Indomethacin (10 microM)-treated SMCs or ECs enhanced the platelet inhibitory activity of GTN. This effect was abrogated by coincubation with oxyhemoglobin (oxyHb; 10 microM), indicating release of NO from GTN. LPS (0.5 microgram/ml; 18 h) enhanced at least 2- to 3-fold the capacity of SMCs or ECs to form NO from GTN, and this enhancement was attenuated when cycloheximide (10 micrograms/ml) was incubated together with LPS. Furthermore, when incubated with GTN (200 microM) SMCs or ECs treated with LPS (0.5 microgram/ml; 18 h) released more NO from GTN than nontreated cells as indicated by a much higher (8- to 9-fold) increase in the levels of cGMP. Exposure of SMCs to GTN (600 microM) for 30 min led to an increase in the levels of NO2- dependent on cell numbers, which was enhanced when SMCs were treated with LPS. Incubation of nontreated or LPS-treated cells with NG-monomethyl-L-arginine (300 microM; 60 min) did not influence the metabolism of GTN to NO. SMCs failed to enhance the antiplatelet activity of sodium nitroprusside. Anesthetized rats treated with an intraperitoneal injection of LPS (20 mg/kg) 18 h beforehand showed enhanced hypotensive responses to GTN (0.25-1 mg/kg). These effects were blocked by methylene blue (10 mg/kg) but not by indomethacin (3 mg/kg). LPS did not alter the hypotensive responses induced by phentolamine, verapamil, or SIN-1. Thus, both in vitro and in vivo, LPS induces the enzyme(s) metabolizing GTN to NO.     

Nitric oxide synthase induction by ouabain in vascular smooth muscle cells from normotensive and hypertensive rats

OBJECTIVE: To investigate the effect of ouabain on inducible nitric oxide synthase (iNOS) activity and expression in cytokine-stimulated vascular smooth muscle cells (VSMC) from normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). METHODS: VSMC were treated for 24 h and afterwards, nitric oxide (NO) release was determined by the production of nitrite, a stable metabolite of NO. Activity of iNOS was measured by the conversion of [3H]-L-arginine to [3H]-L-citrulline and iNOS protein expression by Western blotting. RESULTS: Ouabain (0.01-1 mmol/l) further enhanced interleukin-1beta (II-1beta)-induced nitrite production by WKY and SHR VSMC, although a more pronounced effect was observed in SHR cells (maximum response 52.1 +/- 5.2 and 71.2 +/- 6.4% of 11-1beta effect in WKY and SHR cells, respectively). Such response on NO release was mimicked by the calcium ionophore A 23187 (0.01-1 micromol/l) and abolished by the voltage-operated calcium channels (VOCC) nifedipine (0.1 micromol/l). Expression of iNOS showed that ouabain increased the synthesis of the enzyme in WKY and SHR VSMC stimulated with II-1beta, and this effect was higher in SHR cells. The increased iNOS expression was significantly reduced by nifedipine. CONCLUSIONS: Ouabain stimulation of iNOS expression and activity in II-1beta-stimulated VSMCs from WKY rats and SHR seems to be related to increased intracellular calcium influx through VOCC. The more pronounced effect observed in SHR VSMC could be explained by an altered calcium entry in the hypertensive strain.     

HA-1077 suppress both proliferation of vascular smooth muscle cells and c-fos mRNA induction.

HA1077 is a newly synthesized vasodilator with unique intracellular calcium antagonistic action. In this study, its effect on the growth of vascular smooth muscle cells (VSMC) stimulated by fetal calf serum was examined. Both the proliferation and [3H]thymidine incorporation into DNA of the growth-arrested VSMC was dose-dependently inhibited by HA1077. The expression of a proto-oncogene, c-fos, which reached the maximum 30 min after addition of serum, was similarly inhibited by this agent in a dose-dependent manner. Thus, HA1077 is expected to be a useful vasodilator agent capable of suppressing the growth of VSMC which is thought to be an important underlying mechanism of atherosclerosis or restenosis after angioplasty.     

C/EBP-beta mediates iNOS induction by hypoxia in rat pulmonary microvascular smooth muscle cells

Exposure of rats to 10% O(2) for 4 days caused pulmonary hypertension and induced expression of both inducible nitric oxide synthase (iNOS) and CCAAT box enhancer binding protein-beta (C/EBP-beta) in rat lung. Electrophoretic mobility shift assays (EMSAs) showed that exposure to 1% O(2) increased the C/EBP-beta binding in rat pulmonary microvascular smooth muscle cells (rPSMs). To test the hypothesis that C/EBP-beta participates in hypoxia-induced iNOS expression in rPSMs, a C/EBP motif at -910 bp of rat iNOS promoter was mutated. rPSMs transfected with the rat iNOS promoter and exposed to 1% O(2) for 24 hours had significantly increased wild-type iNOS promoter activity. The hypoxia-induced promoter activity was abolished by the C/EBP motif mutation. Thus, C/EBP-beta mediates, at least in part, hypoxia-induced iNOS expression in rPSMs.     

Genetic differences in airway smooth muscle function

The genetic basis for airway smooth muscle properties is poorly explored. Contraction and relaxation are altered in asthmatic airway smooth muscle, but the basis for the alterations and the role that muscle-specific susceptibility genes may play is largely unexplored. Alterations in the beta-adrenergic receptor, signaling pathways affecting inositol phosphate metabolism, adenylyl and guanylyl cyclase activity, and contractile proteins such as the myosin heavy chain are all suggested by experimental model systems. Significant changes in proliferative and secretory capacities of asthmatic smooth muscle are also demonstrated, but their genetic basis also requires elucidation. Certain asthma-related genes such as ADAM33, although potentially important for smooth muscle function, have been incompletely explored.     

Mechanical strain-induced extracellular matrix production by human vascular smooth muscle cells: role of TGF-beta(1)

Elevated blood pressure imposes increased mechanical stress on the vascular wall, and mechanical strain is a mitogenic stimulus for vascular smooth muscle (VSM) cells. The role of mechanical forces in regulating the production of noncellular material by VSM cells for VSM cells of human origin remains undefined. We thus investigated the effects of chronic cyclical mechanical strain on extracellular matrix (ECM) protein production by cultured human VSM cells. To simulate a blood pressure of 120/80 mm Hg, human VSM cells were repetitively stretched and relaxed by 10% to 16% of their original length with the Flexercell apparatus. Fibronectin and collagen protein concentrations, matrix metalloproteinase (MMP) activity, and transforming growth factor-beta(1) (TGF-beta(1)) mRNA expression by human VSM cells were measured in response to mechanical strain. Exposing human VSM cells to 5 days of chronic cyclical mechanical strain increased fibronectin (+48%, P:<0.01) and collagen (+50%, P:<0.001) concentrations when compared with cells grown in static conditions. Mechanical strain also increased MMP-2 activity, the predominant matrix-degrading isoform (+11%, P:<0.05) in human VSM cells, thus strain-induced ECM accumulation was not due to inhibition of ECM protein degradation. Strain also increased TGF-beta(1) mRNA expression and the production of a soluble factor that increased ECM protein production. Moreover, a TGF-beta-blocking antibody inhibited the effect of strain-conditioned media on matrix production by human VSM cells. These results suggest that chronic cyclical mechanical strain can directly modulate the fibrogenic activity of human VSM cells by inducing ECM protein synthesis and MMP activity. This occurs, at least in part, through mechanical strain-induced TGF-beta(1) production, a mechanism that could explain the increased vascular ECM deposition in hypertension.     

Sodium cotransport in vascular smooth muscle cells.

Vascular smooth muscle cells possess a number of Na cotransport systems. Three of these cotransport systems, Na/Ca exchange, Na/H exchange and Na-K-Cl cotransport, have been the subject of an increasing number of investigations to determine the respective roles of these transporters in vascular smooth muscle cell function. Evidence has been obtained that the Na/Ca exchange system participates in regulation of intracellular Ca in vascular smooth muscle cells. The Na/H exchange system appears to function in concert with a Cl/HCO3 exchange system to regulate intracellular pH. The Na-K-Cl cotransport system is a major contributor to K flux across the plasma membrane of vascular smooth muscle cells and is regulated by a number of vasoactive agents, suggesting that this Na cotransport system is also an important component of vascular smooth muscle cell function. Cultured vascular smooth muscle cells derived from spontaneously hypertensive rats have been found to exhibit reduced Na-K-Cl cotransport activity compared to smooth muscle cells from normotensive controls. Thus, alteration of vascular smooth muscle Na-K-Cl cotransport activity may be related to changes in vascular tone. However, the precise function of Na-K-Cl cotransport in vascular smooth muscle cells remains to be clarified. Recent studies of Na-K-Cl cotransport in vascular endothelial cells provide evidence that the co-transporter is important for regulation of endothelial cell volume and suggest that this Na cotransport system may be vitally important for normal function of the vasculature.