Cardioprotective prostacyclin signaling in vascular smooth muscle

Prostacyclin plays an important cardioprotective role, which has been increasingly appreciated in recent years in light of adverse effects of COX-2 inhibitors in clinical trials. This cardioprotection is thought to be mediated, in part, by prostacyclin inhibition of platelet aggregation. Multiple lines of evidence suggest that prostacyclin additionally protects from cardiovascular disease by pleiotropic effects on vascular smooth muscle. Genetic deletion of the prostacyclin receptor in mice revealed an important role for prostacyclin in preventing the development of atherosclerosis, intimal hyperplasia, and restenosis. In vitro studies have shown these effects may be due to prostacyclin inhibition of vascular smooth muscle cell proliferation and migration. Prostacyclin has also been shown to promote vascular smooth muscle cell differentiation at the level of gene expression through the Gs/cAMP/PKA pathway. Recently identified single nucleotide polymorphisms in the prostacyclin receptor that compromise receptor function suggest that some genetic variations may predispose individuals to increased cardiovascular disease. Herein, we review the literature on the cardioprotective effects of prostacyclin on vascular smooth muscle, and the underlying molecular signaling mechanisms. Understanding the role of prostacyclin and other eicosanoid mediators in the vasculature may lead to improved therapeutic and preventative options for cardiovascular disease.     

Contraction of vascular smooth muscle in cell culture

The use of cultured vascular smooth muscle cells for the study of events related to excitation and contraction of smooth muscle has been limited by the inability to reliably induce contractile responses after subculturing of the cells. This limitation has been overcome by the cell culture preparation described herein. We demonstrate that appropriate responses to both smooth muscle agonists and vasodilators were preserved in cells that were serially subcultured. Fetal bovine pulmonary artery and aortic cell cultures were established following enzymatic dispersion of the medial portion of freshly harvested vessels. At various times after isolation, cells were transferred to microscope coverslips coated with a polymerized silicone preparation (polydimethyl siloxane). Tension forces generated by the cells were manifested as wrinkles and distortions of this flexible growth surface. Visual evidence of cell contraction in the form of increased wrinkling was documented for cells exposed to angiotensin II, carbachol, and KCl. Decreases in cell tension occurred following treatment with isoproterenol, and those relaxing effects were overcome by subsequent treatment with the agonist carbachol. The contractile responses did not diminish with prolonged maintenance in culture or repeated subculturing. Phosphorylation of the light chains on the contractile protein myosin was also measured as a biochemical index of agonist-induced contraction. Cells depolarized with KCl or exposed to carbachol showed increased myosin phosphorylation when analyzed by 2-dimensional gel electrophoresis. The responses remained intact through 7 passages and 9 weeks in culture. These results show that cultured vascular smooth muscle cells do not necessarily undergo a phenotypic modulation with loss of contractility under prolonged maintenance in culture.     

Endotoxin elicits nitric oxide release in rat but prostacyclin synthesis in human and bovine vascular smooth muscle cells

Lipopolysaccharide (LPS) exposure to cells and tissues can mimic the biochemical events leading to septic shock. Previous data demonstrated a massive upregulation of prostaglandin endoperoxide H2 synthase (PGHS-2), but not NO synthase-2 (NOS-2) in bovine smooth muscle cells (SMC) between 2 and 12 h of LPS exposure. This caused an abundant release of prostacyclin (PGI2) by constitutive PGI2-synthase as a counterregulation to a dysfunctional endothelium. We here report that human as well as bovine SMC mainly respond by the induction of PGHS-2 and the subsequent release of PGI2, whereas rat SMC exhibited a distinct induction of NOS-2 and released significantly higher amounts of *NO compared with cattle and human. The induction of either PGHS-2 or NOS-2 in the three different species investigated seems to be mutually exclusive in the time window of 2-24 h. This finding should be considered in the setup of experimental models for the investigation of septic shock.     

Nicotine-induced vascular endothelial growth factor release via the EGFR-ERK pathway in rat vascular smooth muscle cells

Cigarette smoke has been firmly established as an independent risk factor for atherosclerosis and other vascular diseases. The proliferation and migration of vascular smooth muscle cells (VSMC) induced by growth factors have been proposed to play an important role in the progression of atherosclerosis. In the present study, we investigated the effects of nicotine, which is one of the important constituents of cigarette smoke, on vascular endothelial growth factor (VEGF) release, in rat VSMC. The stimulation of cells with nicotine resulted in a time- and concentration-dependent release of VEGF. Hexamethonium, an antagonist of nicotinic acetylcholine receptor (nAChR), inhibited nicotine-induced VEGF release. We next investigated the mechanisms by which nicotine induces VEGF release in the cells. The nicotine-induced VEGF release was inhibited by treatment with U0126, a selective inhibitor of MEK, which attenuated the nicotine-induced ERK phosphorylation. Nicotine induced a transient phosphorylation of ERK. Furthermore, AG1478, a selective inhibitor of epidermal growth factor receptor (EGFR) kinase, inhibited nicotine-induced ERK phosphorylation and VEGF release. These data suggest that nicotine releases VEGF through nAChR in VSMC. Moreover, VEGF release induced by nicotine is mediated by an EGFR-ERK pathway in VSMC. VEGF may contribute to the risk of cardiovascular diseases in cigarette smokers.     

Characterization of vascular smooth muscle cell phenotype in long-term culture

Summary Studies of bovine carotid artery smooth muscle cells, during long-term in vitro subcultivation (up to 100 population doublings), have revealed phenotypic heterogeneity among cells, as characterized by differences in proliferative behavoir, cell morphology, and contractile-cytoskeletal protein profiles. In vivo, smooth muscle cells were spindle-shaped and expressed desmin and alpha-smooth muscle actin (50% of total actin) as their predominant cytoskeletal and contractile proteins. Within 24 h of culture, vimentin rather than desmin was the predominant intermediate filament protein, with little change in alpha-actin content. Upon initial subcultivation, all cells were flattened and fibroblastic in appearance with a concommitant fivefold reduction in alpha-actin content, whereas the beta and gamma nonmuscle actins predominated. In three out of four cell lines studied, fluctuations in proliferative activity were observed during the life span of the culture. These spontaneous fluctuations in proliferation were accompanied by coordinated changes in morphology and contractile-cytoskeletal protein profiles. During periods of enhanced proliferation a significant proportion of cells reverted to their original spindle-shaped morphology with a simultaneous increase in alpha-actin content (20 to 30% of total actin). These results suggest that in long-term culture smooth muscle cells undergo spontaneous modulations in cell phenotype and may serve as a useful model for studying the regulation of intracellular protein expression. This work was supported by grants from from National Institutes of Health, Bethesda, MD, to DMW (HL35684), JW (HL36412), and JM and RL (SCOR HL 14212).     

Isolation and culture of vascular smooth muscle cells from rat placenta

We developed a new separation method for isolating placental vascular smooth muscle cells (PVSMCs) from a rat in this study. Our method used the magnetic force between a magnet and ferrous ferric oxide (Fe₃O ₄) to make the separation and extraction processes easier and more efficient. From the first to sixth generation, the cells isolated using this protocol were identified as smooth muscle cells (SMCs) by their immunoreactivity to the SMC markers and by the “hill and valley” morphology. PVSMCs were exposed to angiotensin II (1 μmol/L) and resulted in sharply increased intracellular Ca ²⁺ concentration. Furthermore, activation of protein kinase C (PKC) increased concomitantly with a decrease in calponin expression. These results indicate that the isolated cells had biological activity. Our method of isolating PVSMCs from rat leads to isolation of cultured cells with activity and high purity. The approach will be useful in research studies on placental vascular diseases.     

Angiotensin II-induced growth effects in vascular smooth muscle in cell culture and in the aortic tunica media in organ culture

Several different studies have investigated the growth effects of angiotensin II on vascular smooth muscle cells in culture. However, smooth muscle cells change their phenotype when placed in culture. The objective of the present study was to investigate the effects of angiotensin II on (3)H-thymidine and (3)H-proline incorporation in vascular smooth muscle cells in culture and in the tunica media of blood vessels perfused at normal physiological pressures in organ culture, thus avoiding the phenotypic changes observed in cell culture. The perfusion system consisted of a peristaltic pump and a closed circuit of plastic tubing connected to a culture media bottle where thoracic rat aortae were placed. Angiotensin II induced an increase in (3)H-thymidine and (3)H-proline incorporation in both culture systems. The results suggest that angiotensin II may play a role in mediating cell growth in vascular smooth muscle cells in their 'contractile' as well as in their 'synthetic' phenotype.     

Permanently proliferating rat vascular smooth muscle cell with maintained expression of smooth muscle characteristics, including actin of the vascular smooth muscle type

Cells of an established clonal line (RVF-SMC) derived from rat vena cava are described by light and electron microscope methods and biochemical analysis of the major proteins. The cells are flat, and they moderately elongate and form monolayers. They are characterized by prominent cables of microfilaments bundles decoratable with antibodies to actin and alpha-actinin. These bundles contain numerous densely stained bodies and are often flanked by typical rows of surface caveolae and vesicles. The cells are rich in intermediate-sized filaments of the vimentin type but do not show detectable amounts of desmin and cytokeratin filaments. Isoelectric focusing and protein chemical studies have revealed actin heterogeneity. In addition to the two cytoplasmic actins, beta and gamma, common to proliferating cells, two smooth muscle-type actins (an acidic alpha-like and a gamma-like) are found. The major (alpha-type) vascular smooth muscle actin accounts for 28% of the total cellular actin. No skeletal muscle or cardiac muscle actin has been detected. The synthesis of large amounts of actin and vimentin and the presence of at least three actins, including alpha- like actin, have also been demonstrated by in vitro translation of isolated poly(A)+ mRNAs. This is, to our knowledge, the first case of expression of smooth muscle-type actin in a permanently growing cell. We conclude that permanent cell growth and proliferation is compatible with the maintained expression of several characteristic cell features of the differentiated vascular smooth muscle cell including the formation of smooth muscle-type actin.     

Regulation of vascular smooth muscle cell growth by aldose reductase

Aldose reductase (AR) is a broad-specificity aldo-keto reductase with wide species and tissue distribution. The enzyme has been implicated in the development of pleiotropic complications of long-term diabetes. However, the euglycemic function of the enzyme remains unclear. To examine its potential role in cell growth, changes in AR mRNA and protein were measured in human aortic smooth muscle cells exposed in culture to serum or thrombin. Stimulation by these mitogens led to an increase in the abundance of AR mRNA and protein. Furthermore, inhibition of the AR by tolrestat and sorbinil diminished DNA synthesis and cell proliferation in response to serum. Immunohistochemical staining with anti-AR antibodies revealed no significant expression of AR in the smooth muscle cells of rat carotid arteries. However, 10 and 21 days after balloon injury, intense staining was associated with the proliferating cells of the neointima. Treatment of these animals with 40 mg/kg/day sorbinil diminished the ratio of neointima to the media. Together, these observations suggest that, in vascular smooth muscle cells (VSMC), AR is a growth-responsive gene product and that inhibition of AR prevents VSMC growth and decreases intimal hyperplasia and restenosis.     

Regulation of vascular smooth muscle cell growth by aldose reductase

Aldose reductase (AR) is a broad-specificity aldo-keto reductase with wide species and tissue distribution. The enzyme has been implicated in the development of pleiotropic complications of long-term diabetes. However, the euglycemic function of the enzyme remains unclear. To examine its potential role in cell growth, changes in AR mRNA and protein were measured in human aortic smooth muscle cells exposed in culture to serum or thrombin. Stimulation by these mitogens led to an increase in the abundance of AR mRNA and protein. Furthermore, inhibition of the AR by tolrestat and sorbinil diminished DNA synthesis and cell proliferation in response to serum. Immunohistochemical staining with anti-AR antibodies revealed no significant expression of AR in the smooth muscle cells of rat carotid arteries. However, 10 and 21 days after balloon injury, intense staining was associated with the proliferating cells of the neointima. Treatment of these animals with 40 mg/kg/day sorbinil diminished the ratio of neointima to the media. Together, these observations suggest that, in vascular smooth muscle cells (VSMC), AR is a growth-responsive gene product and that inhibition of AR prevents VSMC growth and decreases intimal hyperplasia and restenosis.     

Platelet-derived growth factor and transforming growth factor-beta regulate plasminogen activator inhibitor-1 synthesis in vascular smooth muscle cells

Bovine vascular smooth muscle cells (SMC) were examined for production of plasminogen activator inhibitor-1 (PAI-1) which may play a key role in regulating the fibrinolytic system. Growth-arrested SMC released active PAI (101 arbitrary units (AU)/10(6) cells/24 h) and a latent form of PAI (880 AU/10(6) cells/24 h) into the conditioned medium (CM). The levels of PAI were significant since 880 AU of PAI could inhibit approximately 1 microgram of tissue plasminogen activator. The extracellular matrix of SMC also contained PAI activity; however, the level was 17-fold less than that observed in the CM. SMC-PAI was a rapid inhibitor of tissue plasminogen activator (kass greater than 10(7) M-1 S-1) and was identified as a 45-kDa protein immunologically related to endothelial cell PAI-1. PAI-1 comprised 20 and 30%, respectively, of the newly synthesized protein detected in the CM and extracellular matrix of SMC. The SMC growth modulators, platelet-derived growth factor and transforming growth factor-beta, induced PAI-1 activity and protein synthesis by 2- and 3-fold, respectively, in a dose- and time-dependent manner. The increases in PAI-1 activity and protein synthesis were ascribed to elevated levels of PAI-1 mRNA as judged by Northern blot analysis of total RNA prepared from control and platelet-derived growth factor- and transforming growth factor-beta-treated cells. Increases in PAI-1 mRNA levels were evident 1 h after growth factor treatment and were maximal after 4 h. PAI-1 mRNA levels were unaffected by cycloheximide treatment. The results indicate that SMC synthesize and release PAI-1 which could regulate the normal fibrinolytic environment of the arterial wall. During atherosclerosis or after vascular injury increases in platelet-derived or locally produced mitogens may stimulate further PAI-1 synthesis and generate a prothrombotic state.     

Evidence for prostacyclin and cAMP upregulation by bradykinin and insulin-like growth factor 1 in vascular smooth muscle cells

Although bradykinin (BK) and insulin like growth factor-1 (IGF-1) have been shown to modulate the functional and structural integrity of the arterial wall, the cellular mechanisms through which this regulation occurs is still undefined. The present study examined the role of second messenger molecules generated by BK and IGF-1 that could ultimately result in proliferative or antiproliferative signals in vascular smooth muscle cells (VSMC).

Activation of BK or IGF-1 receptors stimulated the synthesis and release of prostacyclin (PGI₂) leading to increased production of cAMP in VSMC. Inhibition of p42/p44^mapk or src kinases prevented the increase in PGI₂ and cAMP observed in response to BK or IGF-1, indicating a role for these kinases in the regulation of cPLA₂ activity in the VSMC. Inhibition of PKC failed to alter production of PGI₂ in response to BK, but further increased both p42/p44^mapk activation and the synthesis of PGI₂ produced in response to IGF-1. In addition, both BK and IGF-1 significantly induced the expression of c-fos mRNA levels in VSMC, and this effect of BK was accentuated in the presence a cPLA₂ inhibitor. Finally, inhibition of cPLA₂ activity and/or cyclooxygenase activity enhanced the expression of collagen I mRNA levels in response to BK and IGF-1 stimulation.

These findings indicate that the effect of BK or IGF-1 to stimulate VSMC growth is an integrated response to the activation of multiple signaling pathways. Thus, the excessive cell growth that occurs in certain forms of vascular disease could reflect dysfunction in one or more of these pathways.     

Regulation of prostacyclin synthesis by angiotensin II and TNF-alpha in vascular smooth muscle

We had previously established that in a model of Ang II-induced hypertension, administration of an anti-TNF-alpha antibody caused additional increases in mean arterial pressure. Production of vasodilator prostanoids (i.e. PGI2 and PGE2) is increased by Ang II in vascular smooth muscle and is part of a counter-regulatory mechanism that opposes increases in vascular tone. We, therefore, examined the effects of TNF-alpha on Ang II-induced increases in PGI2 production in vascular smooth muscle cells (VSMC). Addition of Ang II caused an increase in the production of PGI2, while addition of TNF-alpha had no effect. However, pretreatment with TNF-alpha potentiated the stimulatory effects of Ang II. The potentiating effect of TNF-alpha was neither at the level of prostacyclin synthetase nor at the level of acyl hydrolase activity. This potentiation was dependent on tyrosine kinase activity, as preincubation with genistein completely abolished the effect of TNF-alpha. TNF-alpha upregulated AA-induced PGI2 synthesis, indicating that the effect of TNF-alpha is at the level of cyclooxygenase (COX). These data suggest that TNF-alpha potentiates Ang II-induced synthesis of PGI2 and PGE2 in a tyrosine kinase-dependent manner, an effect that may contribute to the counter-regulatory influence of prostaglandins on the pressor effects of Ang II in the vasculature.     

血管平滑肌细胞的钙动力学

血管平滑肌细胞外的Ca~(2+)通过多种通道进入细胞内。Ca~(2+)通道的本质是镶嵌在膜脂质双分子层中的糖蛋白,神经介质和药物可影响Ca~(2+)通道的功能。靠近胞膜的肌质网和胞膜内侧面的高亲和性Ca~(2+)结合位点是血管平滑肌细胞内储存和释放Ca~(2+)的主要部位。胞浆[Ca~(2+)]增高后在钙调蛋白的介导下引起血管收缩。高血压等血管性疾病的发生与其平滑肌细胞的钙动力学异常有关。