cGMP-elevating agents suppress proliferation of vascular smooth muscle cells by inhibiting the activation of epidermal growth factor signaling pathway

BACKGROUND: Abnormal proliferation of vascular smooth muscle cells (VSMC) is a key event in the pathogenesis of atherosclerosis and many vascular diseases. It is known that nitric oxide released from the endothelium participates in the regulation of VSMC proliferation via a cyclic 3',5'-guanosine monophosphate (cGMP)-mediated mechanism. In a series of experiments, sodium nitroprusside (SNP) and A02131-1 were evaluated for their antiproliferative effect and the mechanism of their cGMP-elevating action. METHODS AND RESULTS: The effect of SNP and A02131-1 on epidermal growth factor (EGF)-stimulated proliferation of rat aortic smooth muscle cells (VSMC) was examined. Cell proliferation was measured in terms of [3H]thymidine incorporation, flow cytometry, and the cell number. Further, their effect on the EGF-activated signal transduction pathway was assessed by measuring mitogen-activated protein kinases (MAPK), MAPK kinase (MEK). Raf-1 activity, and the formation of active form of Ras. SNP and A02131-1 inhibited EGF-induced DNA synthesis and subsequent proliferation of VSMC. These two increased cGMP but only a little cAMP in VSMC. A similar antiproliferative effect was observed with 8-bromo-cGMP. The antiproliferative effect of the two was reversed by KT5823 but not by dideoxyadenosine nor Rp-cAMPS. SNP and A02131-1 blocked the EGF-inducible cell cycle progression at the G1/S phase. Further experiments indicated that the two cGMP-elevating agents primarily blocked the activation of Raf-1 by EGF-activated Ras. CONCLUSIONS: These results demonstrate that cGMP-elevating agents inhibit [3H]thymidine incorporation and thus the growth of VSMC, and this inhibition appears to attenuate EGF-activated signal transduction pathway by preventing Ras-dependent activation of Raf-1.     

Ascorbate affects proliferation of guinea-pig vascular smooth muscle cells by direct and extracellular matrix-mediated effects

Proliferation of smooth muscle cells and deposition of extracellular matrix proteins are important events in the formation of atherosclerotic plaques. We have investigated the direct and matrix-mediated effects of ascorbate on the proliferation rate of vascular smooth muscle cells (VSMC) isolated from the guinea-pig aorta. In the presence of ascorbate, cells showed a bi-phasic growth pattern. At 125 microM ascorbate, -3H--thymidine incorporation was stimulated 25%. However, higher concentrations of ascorbate gradually decreased cell-incorporated radioactivity up to 50% at 2 mM ascorbate. These effects of ascorbate on DNA synthesis in VSMC were paralleled by the changes in cell number and were not due to ascorbate cytotoxicity. Alpha-tocopherol (0.1 mM), individually and in combinations with 1 mm ascorbate, also inhibited DNA synthesis in VSMC. Ascorbate also influenced proliferation of smooth muscle cells through matrix-mediated effect. New VSMC culture plated on extracellular matrices deposited by smooth muscle cells in the presence of 0.1-1 mM ascorbate had up to 50% lower proliferation rate than on matrices from ascorbate-deficient cells, as assessed by [3H]-thymidine incorporation. This effect was independent from alpha-tocopherol and specific inhibitors of collagen synthesis: L-azetidine-2-carboxylic acid and pyridine-2,4-dicarboxylic acid. An ascorbate-dependent matrix effect was specific for smooth muscle cells grown on VSMC and human skin fibroblast-originated matrices, but not for human vascular endothelial cells. The possible involvement of ascorbate in the regulation of smooth muscle cells proliferation by its antioxidant/pro-oxidant effects and regulation of extracellular matrix composition are discussed.     

Differential effects of IFN-beta1b on the proliferation of human vascular smooth muscle and endothelial cells

The effect of human interferon (IFN)-beta1b (Betaseron) on the proliferation of cultured human vascular smooth muscle and endothelial cells was tested in vitro. IFN-beta1b inhibited thymidine incorporation and growth of primary cultures of human aortic and coronary artery smooth muscle in a concentration-dependent manner. The same concentrations of IFN-beta1b did not inhibit thymidine incorporation or growth of primary cultures of human aortic or coronary artery endothelial cells. IFN-beta1b induced the expression of MxA (an antiviral protein induced by type I IFNs) in both smooth muscle and endothelial cells, suggesting that both cell types express receptors for type I IFNs. The growth-inhibitory effect of IFN-beta1b could be mimicked by commercially available human IFN-beta, but not by IFN-alpha2 or IFN-alpha8. The effect of IFN-beta1b was species specific, as it did not inhibit thymidine incorporation in aortic smooth muscle cells derived from pig, rabbit, rat, or mouse. The action of IFN-beta1b on smooth muscle cells persisted for at least 4 days following a 24 h preincubation with IFN-beta1b. Human vascular smooth muscle cells treated with IFN-beta1b did not release lactate dehydrogenase, nor did they show any morphologic change, suggesting that IFN-beta1b was not toxic to the human vascular smooth muscle cells. IFN-beta1b inhibited vascular smooth muscle growth while having no growth-inhibitory effect on endothelial cells obtained from the same blood vessel, making it a potential candidate for treating pathologic conditions where abnormal vascular smooth muscle proliferation is implicated, such as restenosis following balloon angioplasty or smooth muscle proliferation following vascular stenting.     

Progesterone inhibits arterial smooth muscle cell proliferation

Mortality from atherosclerotic cardiovascular disease is lower in premenopausal women than in age-matched men. It is also lower in postmenopausal women who take estrogens and progestins together rather than estrogens alone. Progesterone receptors were detected in human and rat aortic smooth muscle cells in vivo and in vitro (in subculture). We examined the effect of progesterone on proliferation of smooth muscle cells, important constituents of atherosclerotic plaques. Progesterone at physiologic levels inhibited DNA synthesis and proliferation in these cells in a dose-dependent manner, and pretreatment with the progesterone receptor antagonist RU486 blocked inhibition. Cyclin A and E messenger RNA levels decreased after progesterone treatment but those of cyclin B and D1 did not change. This cell cycle-dependent inhibition of arterial smooth muscle cell proliferation by progesterone may represent a mechanism for the hormone's protective effect against atherosclerosis.     

Mechanism of catecholamine-induced proliferation of vascular smooth muscle cells

BACKGROUND: Catecholamines have been shown to aggravate atherosclerosis in animals and humans, and abnormal proliferation of vascular smooth muscle cells (VSMC) is a key event in the early stage of atherosclerosis. Catecholamines may be involved in such cell growth. Therefore, a series of experiments using cultured VSMC was performed to elucidate their possible mitogenic effect. METHODS AND RESULTS: We examined the mitogenic effect of catecholamines using rat aortic smooth muscle cells (VSMC) by measuring [3H]thymidine incorporation, checking with flow cytometry, and counting the cell number directly. Furthermore, the catecholamine-activated signal transduction pathway was assessed by measurement of the formation of inositol 1, 4, 5-triphosphate, intracellular Ca2+ concentration, mitogen-activated protein kinase (MAPK) activity, and mitogenic gene expression. Norepinephrine (NE) and phenylephrine stimulated [3H]thymidine incorporation and cell growth. Clonidine and isoproterenol showed little of such effects. Prazosin was more effective than either yohimbine or propranolol in suppressing the mitogenic effect of NE, indicating that catecholamine-induced VSMC proliferation is mediated by alpha 1-adrenoceptors. The alpha 1-adrenoceptor activation was coupled to pertussis toxin-insensitive Gq-protein and triggered phosphoinositide hydrolysis with subsequent activation of protein kinase C and MAPK in VSMC. In response to NE, both 42- and 44-kD MAPK were activated and tyrosine was phosphorylated. alpha 1-Adrenoceptor stimulation with NE also caused accumulation of c-fos, c-jun, and c-myc mRNA. Chloroethylclonidine completely blocked the alpha 1-adrenoceptor-mediated mitogenesis. CONCLUSIONS: The effect of catecholamines appears to be mediated via the activation of the chloroethylclonidine-sensitive alpha 1-adrenoceptors that triggers the phosphoinositide hydrolysis and activates the MAPK pathway, leading to DNA synthesis and cell proliferation.     

Nitric oxide modulates basal and endothelin-induced coronary artery vascular smooth muscle cell proliferation and collagen levels

The mechanisms governing the pathological accumulation of collagen in the extracellular matrix following angioplasty are complex, but may involve interactions between endothelium-derived paracrine agents and vascular cellular components. We tested the hypothesis that nitric oxide (NO) directly decreases collagen levels and decreases endothelin (ET-1)-stimulated increases in levels of specific collagen subtypes in coronary vascular smooth muscle cells (VSMC). Cultured VSMC were incubated for 48 h with the NO donor CAS 754 (10(-4) M), ET-1 (10(-8) M), or ET-1 plus CAS 754. In some experiments, angiotensin II (Ang II; 10(-8) M) was utilized in place of ET-1. Soluble collagen types I and III were quantitated with an ELISA method, and cell counts were performed. CAS 754 significantly inhibited cell proliferation (-17+/-2% v control), basal total protein synthesis (-65+/-7% v control), and basal collagen type I levels (-39+/-6% v control), but not collagen type III levels. ET-1 and Ang II both significantly stimulated cell proliferation (26+/-5% v control), total protein synthesis (169+/-6% v control), and collagen type I levels (200+/-11% v control). Ang II, but not ET-1, significantly increased collagen type III levels. Co-incubations of ET-1 and CAS 754 resulted in a significant decrease in cell proliferation, protein synthesis, and collagen levels (-23+/-2% v control, 90+/-5% v control, and 63+/-3% v control, respectively) compared to ET-1 alone. In contrast, co-incubation of Ang II and CAS 754 had no significant effect on cell proliferation, protein synthesis, and collagen levels seen with Ang II alone. These results demonstrate that NO inhibits basal collagen levels and cell division. Additionally, NO alters ET-1 stimulation of VSMC proliferation, protein synthesis, and production of extracellular matrix components. Thus, an imbalance in key endothelium-derived compounds could significantly impact upon extracellular matrix deposition following mechanical revascularization.     

Neonatal estrogen stimulates proliferation of periductal fibroblasts and alters the extracellular matrix composition in the rat prostate

The purpose of this study was to examine whether changes in extracellular matrix (ECM) molecules are associated with the growth inhibition and differentiation defects of the prostate gland following neonatal exposure to estradiol. Using immunocytochemistry (ICC), laminin and collagen IV were localized to the basement membrane (BM) as well to the basal lamina of the periductal smooth muscle of the control developing prostates. In contrast, fibronectin and collagen III were localized throughout the stromal ECM. Exposure to neonatal estrogen altered the staining profile for specific ECM molecules. In the estrogenized rats, a thick layer of cells negative for laminin and collagen IV was observed adjacent to the BM. Electron microscopy and ICC for alpha-actin, fibronectin, and vimentin identified this multicellular layer of periductal cells as differentiated fibroblasts. Peripheral to these fibroblasts, actin-positive smooth muscle formed a second layer of periductal stromal cells. PCNA labeling showed that estrogen exposure increased the fibroblast proliferation. Because many periductal fibroblasts were positive for estrogen receptor alpha (ER alpha) in estrogenized rats, a direct effect of estradiol on their proliferation is suggested. Gelatinolytic gels revealed that estrogen exposure did not alter the activity of matrix metalloproteinases associated with tissue remodeling during prostate morphogenesis. However, the periductal fibroblast layer in estrogenized prostates was devoid of urokinase- and tissue-plasminogen activator, which may potentially alter the localized proteolysis involved in matrix remodeling. It is proposed that proliferation of a multicellular layer of periductal fibroblasts in estrogenized prostates results in a physical barrier that constrains branching morphogenesis and blocks paracrine communications between smooth muscle and epithelial cells which normally regulate differentiation.     

Dipyridamole inhibits PDGF- and bFGF-induced vascular smooth muscle cell proliferation

Dipyridamole is the only pharmacologic agent demonstrated to reduce polytetrafluoroethylene (PTFE) graft occlusion in hemodialysis patients. However, the mechanism of action of dipyridamole in preventing graft occlusion is unknown. The purpose of this study was to examine the direct effects of dipyridamole on both platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF)-induced vascular smooth muscle cell (VSMC) proliferation. Human aortic smooth muscle cells were grown to confluence in 96 well plates. A total of 5 x 10(-6) molar dipyridamole, PDGF 10 ng/ml, or bFGF 10 ng/ml were added to appropriate wells at the start of each experiment. Cell proliferation at 48 hours was determined using tritiated thymidine uptake. Intracellular cyclic AMP (cAMP) was measured using a competitive enzyme immunoassay. Treatment of VSMC with 5 microM dipyridamole dramatically reduced basal proliferation rates compared to controls [5229 +/- 1131 counts per minute (CPM) versus 387 +/- 68 CPM, P < 0.001]. Treatment with dipyridamole also reduced PDGF-stimulated VSMC proliferation (7311 +/- 1655 CPM vs. 593 +/- 110 CPM, P < 0.001) as well as the response to bFGF (5632 +/- 1270 CPM vs. 310 +/- 31 CPM, P < 0.001). Treatment of VSMC with either 5 or 20 microM dipyridamole did not change intracellular cAMP levels. Furthermore, the addition of dibutyryl cAMP to VSMC demonstrated only a modest inhibitory effect on proliferation. We conclude that dipyridamole inhibits both PDGF- and bFGF-stimulated VSMC proliferation. The effects of dipyridamole on VSMC proliferation do not appear to be entirely mediated by changes in intracellular cAMP concentrations. The direct effect of dipyridamole on VSMC proliferation may account for its efficacy in reducing PTFE graft thrombosis in hemodialysis patients.     

Prolonged heparin after uncomplicated coronary interventions: a prospective, randomized trial

BACKGROUND: Both ischemic and direct vascular injury (angioplasty) result in the elaboration of proinflammatory substances, including tumor necrosis factor alpha (TNF), which may regulate vascular smooth muscle cell (VSMC) proliferation and promote vessel stenosis. Interleukin-10 (IL-10) is a pleiotropic cytokine with potent antiinflammatory effects in many cells lines. We hypothesized that IL-10 could be used therapeutically to influence vascular remodeling by inhibiting TNF-induced VSMC proliferation. The purposes of this study were (1) to determine whether human myocardium produces endogenous TNF in response to ischemia-reperfusion, (2) to examine the effect of TNF on human arterial smooth muscle proliferation, and (3) to explore the potential therapeutic effect of IL-10 on unstimulated and TNF-stimulated VSMC proliferation. MATERIALS AND METHODS: Right atrial muscle was obtained from patients undergoing elective cardiac surgery. Atrial muscle was subjected to simulated ischemia and reperfusion in vitro and TNF was measured by immunoassay. Human aortic VSMCs were isolated and cultured. Proliferation assays were performed to determine the effect of TNF and IL-10 on VSMC growth. RESULTS: Ischemia-reperfusion resulted in an increase in atrial myocellular TNF (94.5 +/- 15.8 pg/g wet tissue versus control 12.9 +/- 4.4 pg/g wet tissue, P < 0.002). Compared with control, TNF stimulated concentration-dependent VSMC proliferation (P < 0.005). IL-10 alone did not influence VSMC growth. However, following TNF stimulation, IL-10 inhibited VSMC growth at a dose as low as 0.1 pg/ml (P < 0.005). CONCLUSIONS: Ischemia-reperfusion insult results in increased endogenous myocardial TNF accumulation. TNF stimulates VSMC growth which is abrogated by physiologically relevant levels of IL-10. This antiinflammatory cytokine may prove to be an effective therapeutic agent in regulating vessel wall remodeling following both ischemic and direct cardiovascular injury.     

Clinical pharmacokinetics of irinotecan

Nitric oxide (NO) and cyclic guanosine 3',5'-monophosphate (cGMP) have been reported to prevent vascular smooth muscle cell (VSMC) proliferation and have beneficial effects to reduce intimal thickening in response to arterial injury. The purpose of this study was to determine whether the downstream effector molecule of NO-cGMP signaling, cyclic GMP-dependent protein kinase (PKG), regulates phenotypic modulation and proliferation in cultured rat aortic VSMC. PKG-expressing VSMC lines were created by transfection of PKG-deficient cell lines and characterized. All forms of PKG, i.e. PKG-I alpha and PKG-I beta, as well as the constitutively active catalytic domain of PKG-I, transformed dedifferentiated 'synthetic' VSMC to a more contractile-like morphology. PKG expression resulted in an increased production of the contractile phenotype marker proteins, smooth muscle myosin heavy chain-2, calponin and alpha-actin and restored the capacity of cAMP and cGMP analogues to inhibit platelet-derived growth factor (PDGF)-induced cell migration. On the other hand, PKG expression had no significant effects on PDGF-induced cell proliferation. These results suggest that PKG expression contributes to the regulation of a contractile-like phenotypic expression in cultured VSMC, and the suppression of PKG expression during cultured growth in vitro may permit the modulation of cells to a more synthetic, dedifferentiated phenotype.     

Low- and high-density lipoproteins as mitogenic factors for vascular smooth muscle cells: individual, additive and synergistic effects

The mitogenic activities of low (LDL)- and high (HDL)-density lipoproteins have been examined in cultures of human vascular smooth muscle cells (VSMC). LDL and HDL3 dose-dependently (EC50 values approximately 50 micrograms/ml) stimulated DNA and protein synthesis ([3H]-thymidine and [3H]-leucine incorporation, respectively) in the absence of exogenously added mitogens. The synthetic responses of VSMC to combinations of LDL and HDL3 were additive, indicating that each lipoprotein mediates discrete effects. LDL or HDL3 promoted VSMC proliferation under strict mitogen-free conditions, but this growth response was not sustained. VSMC exposed to combinations of lipoproteins (either LDL or HDL3) and growth factors (either PDGF-BB, EGF, bFGF or IGF) exhibited synergistic DNA synthesis responses. In the combined presence of PDGF-BB and either LDL or HDL3, VSMC proliferation was sustained. Anionized lipoprotein preparations (oxidized, acetylated, carbamylated or malonimylated) also stimulated DNA and protein synthesis. Since the antioxidant beta-hydroxylated toluene did not block the effect of native LDL on DNA synthesis, and fucoidin, a specific competitor for the 'scavenger' receptor, did not inhibit oxidized LDL-induced DNA synthesis, activation of mitogenic signals by lipoproteins does not depend on lipid peroxidation. Rather, the apparent intrinsic mitogenic potential of lipoproteins may depend upon their direct activation of replication-coupled signal transduction systems.     

The effects of glucose-induced oxidative stress on growth and extracellular matrix gene expression of vascular smooth muscle cells

Vascular smooth muscle cell (VSMC) dysfunction plays a role in diabetic macrovasculopathy and this may include abnormalities in growth characteristics and the extracellular matrix. As the actual mechanisms by which glucose induces VSMC dysfunction remain unclear, the aim of this study was to assess the potential role of glucose-induced oxidative stress. Porcine aortic VSMCs were cultured for 10 days in either 5 mmol/l normal glucose or 25 mmol/l D-glucose (high glucose). There was evidence of oxidative stress as indicated by a 50% increase in intracellular malondialdehyde (p < 0.05), increased mRNA expression of CuZn superoxide dismutase and Mn superoxide dismutase (by 51% and 37% respectively, p < 0.01) and a 50% decrease in glutathione in 25 mmol/l D-glucose (p < 0.001). Growth was increased by 25.0% (p < 0.01). mRNA expression of extracellular matrix proteins (collagens I, III, IV and fibronectin) was not altered by high glucose in these experimental conditions. Repletion of glutathione with N-acetyl L-cysteine (1 mmol/l) in VSMC grown in high glucose was associated with reduction in malondialdehyde and restored growth to that of normal glucose. The water soluble analogue of vitamin E, Trolox (200 mumol/l), reduced malondialdehyde concentrations, but had no effect on glutathione depletion or the increased growth rate seen with high glucose. The addition of buthionine sulphoximine (10 mumol/l) to VSMC cultured in normal glucose reduced glutathione, increased malondialdehyde and increased growth to a similar extent as that found in high glucose alone. These results suggest that thiol status, rather than lipid peroxides, is a key factor in modulating VSMC growth and that mRNA expression of extracellular matrix proteins is not increased in VSMC under conditions of glucose-induced oxidative stress.     

Proliferative effects of oxidized low-density lipoprotein on vascular smooth muscle cells: role of dietary habits

The effects were studied of native, partially-oxidized and totally-oxidized human low-density lipoprotein (LDL) on the proliferation of cultured rat aortic smooth muscle cells (VSMC), measured as an altered DNA synthesis. The LDL was obtained from three different human long-term diet groups (a control diet rich in saturated fats, a vegetarian diet, and a fish diet). The oxidized LDLs were prepared by oxidizing the LDL with copper sulfate. The DNA synthesis was measured by [3H]-thymidine incorporation into the DNA. The partially-oxidized LDL was the most potent promoter of DNA synthesis compared to the native or totally-oxidized LDL of the same diet group. The partially-oxidized LDL had a true mitogenic effect in the absence of exogenous growth factors. The native and totally-oxidized LDL induced a significant increase in DNA synthesis, if they were obtained from the fish diet group. This study suggests an enhanced proliferative effect of partially-oxidized LDL on VSMC growth.     

Effect of tumour necrosis factor-alpha on proliferation, activation and protein synthesis of rat hepatic stellate cells

BACKGROUND/AIMS: Hepatic stellate cells represent the principal matrix-synthesising cells of damaged liver and are targets of a number of cytokines currently under investigation. The study analyses the effects of tumour necrosis factor-alpha and interferon-gamma on proliferation, "activation" and protein synthesis of hepatic stellate cells. METHODS: Primary cultures of hepatic stellate cells were exposed to tumour necrosis factor-alpha and interferon-gamma. Cell proliferation was studied by 3H-thymidine and bromo-deoxy-uridine incorporation. Protein synthesis was analysed using immunoprecipitation, Western- and Northern blotting techniques. RESULTS: Proliferation of hepatic stellate cells was reduced by tumor necrosis factor-alpha and interferon-gamma, while "activation" of hepatic stellate cells as assessed by expression of smooth muscle alpha-actin and of TGF-beta/activin type I receptor was induced by tumour necrosis factor-alpha but downregulated by interferon-gamma. Tumour necrosis factor-alpha increased the synthesis of distinct extracellular matrix proteins, particularly of fibronectin and tenascin, but decreased collagen type III expression. In contrast, interferon-gamma reduced the synthesis of all connective tissue proteins tested. Among the protease inhibitors, interferon-gamma induced C1-esterase inhibitor synthesis, while tumour necrosis factor-alpha stimulated plasminogen activator inhibitor type 1 production. CONCLUSIONS: Tumour necrosis factor-alpha and interferon-gamma decrease proliferation of hepatic stellate cells, while "activation" of hepatic stellate cells and synthesis of proteins involved in matrix metabolism are regulated in a differential, cytokine-specific manner, suggesting that both cytokines play an important role in liver repair.     

Transitions in cell organization and in expression of contractile and extracellular matrix proteins during development of chicken aortic smooth muscle: evidence for a complex spatial and temporal differentiation program

Whereas the understanding of the mechanisms underlying skeletal and cardiac muscle development has been increased dramatically in recent years, the understanding of smooth muscle development is still in its infancy. This paper summarizes studies on the ontogeny of chicken smooth muscle cells in the wall of the aorta and aortic arch-derived arteries. Employing immunocytochemistry with antibodies against smooth muscle contractile and extracellular matrix proteins we trace smooth muscle cell patterning from early development throughout adulthood. Comparing late stage embryos to young and adult chickens we demonstrate, for all the stages analyzed, that the cells in the media of aortic arch-derived arteries and of the thoracic aorta are organized in alternating lamellae. The lamellar cells, but not the interlamellar cells, express smooth muscle specific contractile proteins and are surrounded by basement membrane proteins. This smooth muscle cell organization of lamellar and interlamellar cells is fully acquired by embryonic day 11 (ED 11). We further show that, during earlier stages of embryogenesis (ED3 through ED7), cells expressing smooth muscle proteins appear only in the peri-endothelial region of the aortic and aortic arch wall and are organized as a narrow band of cells that does not demonstrate the lamellar-interlamellar pattern. On ED9, infrequent cells organized in lamellar-interlamellar organization can be detected and their frequency increases by ED10. In addition to changes in cell organization, we show that there is a characteristic sequence of contractile and extracellular matrix protein expression during development of the aortic wall. At ED3 the peri-endothelial band of differentiated smooth muscle cells is already positive for smooth muscle alpha actin (alphaSM-actin) and fibronectin. By the next embryonic day the peri-endothelial cell layer is also positive for smooth muscle myosin light chain kinase (SM-MLCK). Subsequently, by ED5 this peri-endothelial band of differentiated smooth muscle cells is positive for alphaSM-actin, SM-MLCK, SM-calponin, fibronectin, and collagen type IV. However, laminin and desmin (characteristic basement membrane and contractile proteins of smooth muscle) are first seen only at the onset of the lamellar-interlamellar cell organization (ED9 to ED10). We conclude that the development of chicken aortic smooth muscle involves transitions in cell organization and in expression of smooth muscle proteins until the adult-like phenotype is achieved by mid-embryogenesis. This detailed analysis of the ontogeny of chick aortic smooth muscle should provide a sound basis for future studies on the regulatory mechanisms underlying vascular smooth muscle development.     

Mineralocorticoids and hypertension]

The degree of lumen narrowing in advanced lesions correlates poorly with the amount of intimal mass accumulated in the atherosclerotic plaque. As an alternate mechanism of stenosis, we propose that human smooth muscle cells bind to fibrin deposited in the matrix and exert contractile forces to cause a narrowing of the lumen. In the present study we demonstrated in vitro that human newborn aortic smooth muscle cell lines can contract and adhere to fibrin clots composed of either fibronectin-depleted plasma ("plasma") or recombinant fibrin. By using neutralizing antibodies and RGD peptides, we showed that members of the integrin family mediated the interaction between human newborn smooth muscle cells and fibrin. Neutralizing antibodies against the integrin alphavbeta3 (c7E3 Fab and LM609) did not inhibit either plasma clot contraction or recombinant fibrin clot contraction by human newborn smooth muscle cells. In contrast, antibodies against alpha5, beta1, and alpha5/beta1 inhibited contraction of clots composed of either plasma or recombinant fibrin. Anti-alphavbeta3, anti-alphav, anti-alpha5, anti-beta1, and anti-alpha5beta1 antibodies inhibited human newborn smooth muscle cell adhesion to plasma clots; however, only anti-alpha5, anti-beta1, and anti-alpha5beta1 antibodies significantly inhibited adhesion to recombinant fibrin. While the linear RGD peptides had no effect, the cyclic peptide penRGD inhibited adhesion to plasma clots and recombinant fibrin. However, it did not block contraction of recombinant fibrin clots. These results suggest that during the interaction of human newborn smooth muscle cell lines with fibrin, alpha5beta1 plays a significant role. This interaction is of potential interest as a target for efforts to block vascular contraction.     

Preeclampsia, lipid peroxidation, and calcium adenosine triphosphatase activity of red blood cell ghosts

1. The effects of oxatriazole-type (GEA 3162 and GEA 5624) nitric oxide (NO) donors on mitogenesis and proliferation were studied in vascular smooth muscle cell (VSMC) culture. The effects of the GEA-compounds were compared with well-known NO-donors 3-morpholinosydnonimine (SIN-1) and S-nitroso-N-acetylpenicillamine (SNAP). 2. All NO-donors released NO and increased the production of cyclic GMP concentration-dependently. The production of cyclic GMP was inhibited by the guanylate cyclase inhibitor, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). 3. The NO-donors inhibited basal and serum-induced DNA synthesis concentration-dependently. The GEA-compounds were needed in concentrations 10 times lower than SIN-1 and SNAP. GEA 3162, SIN-1 and SNAP were also able to inhibit serum-induced cell proliferation. GEA 5624 was ineffective. The antimitogenic effect of NO-donors was not reduced by inhibiting the guanylate cyclase. 4. These results suggest that NO inhibits serum-induced DNA synthesis and proliferation of VSMC by a cyclic GMP-independent mechanism. The oxatriazole-type NO-donor GEA 3162 was found to be a more potent inhibitor of mitogenesis and cell proliferation than SIN-1 and SNAP.     

Molecular anatomy of the perivascular sheath in human placental stem villi: the contractile apparatus and its association to the extracellular matrix

In previous studies, we have shown that smooth muscle cells and myofibroblast subpopulations of the perivascular stem villous sheath of the human placenta contain focal adhesion plaques and talin immunoreactivity. The close association of these cells to elastic and collagen fibres have led to the assumption of a functional myofibroelastic unit within the perivascular stem villous sheath. Interactions between the extracellular matrix and smooth muscle cells depend on a variety of structural protein assemblies. In the present study, we examined, by immunocytochemistry, whether the molecular assembly of extracellular matrix proteins and molecules of focal adhesions, known to be essential for signal transduction in smooth muscle cells, are also found in smooth muscle cells of the perivascular stem villous sheath of the human placenta. Vascular and extravascular smooth muscle cells were immunoreactive for alpha-actinin, vinculin, paxillin and tensin, the integrin chains alpha1 and beta1, and the basement membrane components laminin and heparan/-chondroitin sulfate proteoglycan perlecan. pp125(FAK) did not react. In the extracellular matrix of blood vessel walls and the perivascular stem villous sheath, we found immunoreactivity of fibronectin and collagen types I, VI and undulin (collagen type XIV). From our data we conclude that within the perivascular stem villous sheath, there exists a system of signal transduction molecules, indicating a cross talk between the smooth muscle cells of this sheath and their surrounding extracellular matrix.     

Effects of brief glucocorticoid exposure on growth of vascular smooth muscle cells in culture

While prolonged exposure of vascular smooth muscle cells (VSMC) to glucocorticoid has been shown to inhibit cell proliferation, the effect of a brief pulse exposure is not known. We studied the short-term effects of pulse exposure to dexamethasone (DEX) on DNA synthesis in cultured VSMC. VSMC were pulsed with DEx for varying time intervals and [3H]thymidine incorporation into cells after 24 h was measured. Exposure to DEX for 24 h decreased [3H]thymidine incorporation, while pulse treatments with DEX from 2 min to 6 h significantly increased [3H]thymidine incorporation. Maximal proliferative effect was observed with a 20-min exposure. The effect of a 20-min pulse was dose-dependent, with the half-maximal dose of DEX being approximately 10(-7) M. A selective glucocorticoid receptor antagonist, RU486, inhibited the proliferative effect of DEx. Concentrated conditioned medium from cells exposed to 10(-6) M DEX increased [3H]thymidine incorporation by other VSMC in a dose-dependent manner. These results suggest that short-term pulse DEX exposure is capable of producing one or more autocrine growth factors in VSMC via a glucocorticoid receptor action. This effect of glucocorticoid pulses may contribute to the pathogenesis of arteriosclerosis and hypertension.