Confluent Vascular Smooth Muscle Cells Research Articles

The Methanol Seed Extract of Garcinia kola Attenuated Angiotensin II- and Lipopolyssacharide-Induced Vascular Smooth Muscle Cell Proliferation and Nitric Oxide Production

Abstract All over the world, cardiovascular diseases are a risk factor for poor health and early death with predisposing factors to include age, gender, tobacco use, physical inactivity, excessive alcohol consumption, unhealthy diet, obesity, family history of cardiovascular disease, hypertension, diabetes mellitus, hyperlipidemia, psychosocial factors, poverty and low educational status, and air pollution. It is envisaged that herbal products that can stem this trend would be of great benefit. Garcinia kola (GK), also known as bitter kola is one of such plants. Generally used as a social snack and offered to guests in some cultural settings, bitter kola has been indicated in the treatment of laryngitis, general inflammation, bronchitis, viral infections and diabetes. In this study, the effects of methanol seed extract of Garcinia kola on the proliferation of Vascular Smooth Muscle Cells (VSMCs) in cell culture by Angiotensin II (Ang II) and LPS-induced NO production were carried out. Confluent VSMCs were exposed to GK (25, 50 and 100 μg/ml) before or after treatment with lipopolyssacharide (100μg/ml), and Angiotensin II (10−8-10−6M). Cellular proliferation was determined by MTT assay and NO production by Griess assay. Treatment with Angiotensin II (10−8, 10−6) or LPS significantly enhanced proliferation of VSM cells while LPS significantly increased nitric oxide (NO) production. Treatment with GK (25, 50 & 100 μg/ml) attenuated VSM cell proliferation. The results indicate that GK has potential to inhibit mitogen activated vascular cell growth and possibly inhibit inflammatory responses to LPS. Thus GK may be useful in condition that is characterized by cellular proliferation and inflammatory responses.

Apposite Insulin-like Growth Factor (IGF) Receptor Glycosylation Is Critical to the Maintenance of Vascular Smooth Muscle Phenotype in the Presence of Factors Promoting Osteogenic Differentiation and Mineralization

Vascular calcification is strongly linked with increased morbidity and mortality from cardiovascular disease. Vascular calcification is an active cell-mediated process that involves the differentiation of vascular smooth muscle cells (VSMCs) to an osteoblast-like phenotype. Several inhibitors of this process have been identified, including insulin-like growth factor-I (IGF-I). In this study, we examined the role of the IGF receptor (IGFR) and the importance of IGFR glycosylation in the maintenance of the VSMC phenotype in the face of factors known to promote osteogenic conversion. IGF-I (25 ng/ml) significantly protected VSMCs from β-glycerophosphate-induced osteogenic differentiation (p < 0.005) and mineral deposition (p < 0.01). Mevalonic acid depletion (induced by 100 nm cerivastatin) significantly inhibited these IGF protective effects (p < 0.01). Mevalonic acid depletion impaired IGFR processing, decreased the expression of mature IGFRs at the cell surface, and inhibited the downstream activation of Akt and MAPK. Inhibitors of N-linked glycosylation (tunicamycin, deoxymannojirimycin, and deoxynojirimycin) also markedly attenuated the inhibitory effect of IGF-I on β-glycerophosphate-induced mineralization (p < 0.05) and activation of Akt and MAPK. These results demonstrate that alterations in the glycosylation of the IGFR disrupt the ability of IGF-I to protect against the osteogenic differentiation and mineralization of VSMCs by several interrelated mechanisms: decreased IGFR processing, reduced IGFR cell-surface expression, and reduced downstream signaling via the Akt and MAPK pathways. IGF-I thus occupies a critical position in the maintenance of normal VSMC phenotype and protection from factors known to stimulate vascular calcification.

Axl/Phosphatidylinositol 3-Kinase Signaling Inhibits Mineral Deposition by Vascular Smooth Muscle Cells

The calcification of blood vessels correlates with increased morbidity and mortality in patients with atherosclerosis, diabetes, and end-stage kidney disease. The receptor tyrosine kinase Axl is emerging as an important regulator of adult mammalian physiology and pathology. This study tests the hypothesis that Axl prevents the deposition of a calcified matrix by vascular smooth muscle cells (VSMCs) and that this occurs via the phosphatidylinositol 3-kinase (PI3K) signaling pathway. First, we demonstrate that Axl is expressed and phosphorylated in confluent VSMCs and that its expression is markedly downregulated as these cells calcify their matrix. Second, we demonstrate that overexpression of wild-type Axl, using recombinant adenoviruses, enhances Axl phosphorylation and downstream signaling via PI3K and Akt. Furthermore, overexpression of Axl significantly inhibits mineral deposition by VSMCs, as assessed by alizarin red staining and (45)Ca accumulation. Third, the addition of a PI3K inhibitor, wortmannin, negates the inhibition of mineralization by overexpression of wild-type Axl, suggesting that activation of downstream signaling via PI3K is crucial for its inhibitory activity. In contrast, Axl-mediated signaling is not enhanced by overexpression of kinase-dead Axl and mineralization is accelerated, although beta-glycerophosphate is still required for this effect. Finally, the caspase inhibitor zVAD.fmk attenuates the increased mineralization induced by kinase-dead Axl, suggesting that kinase-dead Axl stimulates mineralization by inhibiting the antiapoptotic effect of endogenous Axl. Together, these results demonstrate that signaling through Axl inhibits vascular calcification in vitro and suggest that therapeutics targeting this receptor may open up new avenues for the prevention of vascular calcification in vivo.

Hyperthermia at 43°C for 2 h Inhibits the Proliferation of Vascular Smooth Muscle Cells, but not Endothelial Cells

K. Orihara, S. Biro, S. Hamasaki, H. Eto, M. Miyata, Y. Ikeda and C. Tei. Hyperthermia at 43°C for 2h Inhibits the Proliferation of Vascular Smooth Muscle Cells, but not Endothelial Cells. Journal of Molecular and Cellular Cardiology (2002) 34, 1205–1215. Restenosis after angioplasty is one of the most critical problems of the various interventional therapies for myocardial ischemia. It has been difficult to prevent the vascular smooth muscle cells (VSMCs) proliferation resulting in restenosis. The goal of this study was to prove the treatment by hyperthermia to be effective in suppressing VSMC's proliferation in vitro. When just-stimulated VSMCs, which were incubated for 2h after 5% FBS stimulation to quiescent VSMCs, were exposed to hyperthermia (43°C, 2h), the cell cycle progression to S and G2/M phase was significantly delayed 24h after 5% FBS stimulation. And another 24h later, cell death was observed partly (19%) of heat-treated VSMCs. Nonetheless, hyperthermia under the same conditions did not result in the death of quiescent VSMCs, and did not inhibit the proliferation of cultured bovine aortic endothelial cells (BAECs). In addition, we found that hyperthermia (43°C, 2h) elevated p27Kip1 over the amount induced in confluent VSMCs. Much elevation of p27Kip1, which is a negative regulator of G1/S progression, may play a role in heat-induced G1 arrest of VSMCs. In conclusion, we have found that hyperthermia (43°C, 2h) inhibited the proliferation of the dividing VSMCs mainly due to G1 arrest with neither inhibiting the generation of BAECs nor damaging quiescent VSMCs. Hence, our data suggest that hyperthermia may be clinically applicable for the prevention of restenosis.

Hyperthermia at 43°C for 2h Inhibits the Proliferation of Vascular Smooth Muscle Cells, but not Endothelial Cells

<h2>Abstract</h2> K. Orihara, S. Biro, S. Hamasaki, H. Eto, M. Miyata, Y. Ikeda and C. Tei. Hyperthermia at 43°C for 2h Inhibits the Proliferation of Vascular Smooth Muscle Cells, but not Endothelial Cells. <i>Journal of Molecular and Cellular Cardiology</i> (2002) <b>34</b>, 1205–1215. Restenosis after angioplasty is one of the most critical problems of the various interventional therapies for myocardial ischemia. It has been difficult to prevent the vascular smooth muscle cells (VSMCs) proliferation resulting in restenosis. The goal of this study was to prove the treatment by hyperthermia to be effective in suppressing VSMC's proliferation <i>in vitro</i>. When just-stimulated VSMCs, which were incubated for 2h after 5% FBS stimulation to quiescent VSMCs, were exposed to hyperthermia (43°C, 2h), the cell cycle progression to S and G2/M phase was significantly delayed 24h after 5% FBS stimulation. And another 24h later, cell death was observed partly (19%) of heat-treated VSMCs. Nonetheless, hyperthermia under the same conditions did not result in the death of quiescent VSMCs, and did not inhibit the proliferation of cultured bovine aortic endothelial cells (BAECs). In addition, we found that hyperthermia (43°C, 2h) elevated p27^Kip1 over the amount induced in confluent VSMCs. Much elevation of p27^Kip1, which is a negative regulator of G1/S progression, may play a role in heat-induced G1 arrest of VSMCs. In conclusion, we have found that hyperthermia (43°C, 2h) inhibited the proliferation of the dividing VSMCs mainly due to G1 arrest with neither inhibiting the generation of BAECs nor damaging quiescent VSMCs. Hence, our data suggest that hyperthermia may be clinically applicable for the prevention of restenosis.

Differential regulation of transforming growth factor receptors by angiotensin II and transforming growth factor-beta1 in vascular smooth muscle.

Angiotensin II (Ang II) and transforming growth factor (TGF) beta1 play a role in vascular remodeling in hypertension. In this process they may interact on various levels, including that of receptor regulation. This consideration prompted the present study on transcriptional regulation of TGF-beta receptors by Ang II and TGF-beta in vascular smooth muscle cells. Transcriptional expression of the components of the TGF-beta system was demonstrated for TGF-beta and for TGF-beta receptors I, II, and III. As measured by quantitative reverse transcriptase polymerase chain reaction, TGF-beta mRNA increased about 2.4-fold in the presence of 40 pM exogenous TGF-beta. Ang II at 10(-6) M increased TGF-beta mRNA 2.5-fold compared to control cells (P<0.05). Ang II also significantly increased TGF-beta protein concentration in the supernatant of confluent vascular smooth muscle cells. Ang II caused the induction of TGF-beta, but short-term experiments showed TGF-beta receptor II mRNA to be differentially regulated by Ang II and TGF-beta; while TGF-beta caused a 40% decrease in TGF-beta receptor II mRNA after 4 h (P<0.05), Ang II caused an increase by about 70%. In contrast, both TGF-beta and Ang II increased TGF-beta receptor I mRNA to about 260% or 180% of controls (P<0.05). TGF-beta effects were abrogated by coincubation with a TGF-beta neutralizing antibody, and Ang II effects were abrogated by losartan, an AT-1 receptor antagonist. Coincubation of Ang II with the TGF-beta neutralizing antibody did not inhibit the effect of Ang II, indicating that the short-term effects of Ang II on the expression of the TGF-beta receptors are not mediated via TGF-beta. Furthermore, Ang II stimulated DNA synthesis even in the presence of the TGF-beta neutralizing antibody. In conclusion, this study indicates (a) that in vascular smooth muscle TGF-beta receptors are regulated on the RNA level by TGF-beta and Ang II, and (b) that Ang II dependent regulation of TGF-beta receptors is at least partially independent of endogenous TGF-beta. Stimulation of the transcriptional expression of TGF-beta receptors by Ang II may increase sensitivity of vascular smooth muscle cells to TGF-beta.

Adenosine inhibits collagen and total protein synthesis in vascular smooth muscle cells.

-The objective of this study was to characterize the effects of exogenous, drug-induced and cAMP-adenosine pathway-derived adenosine on collagen synthesis by and hypertrophy of vascular smooth muscle cells (SMCs). Confluent vascular SMCs were stimulated with 2.5% fetal calf serum in the presence and absence of adenosine receptor agonists [adenosine, 2-chloroadenosine, N6-cyclopentyladenosine, 5'-N-ethylcarboxamidoadenosine, 5'-N-methylcarboxamidoadenosine, and 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamino adenosine], drugs that increase levels of endogenous adenosine [erythro-9-(2-hydroxy-3-nonyl) adenine, dipyridamole, and iodotubericidin], and cAMP (increases adenosine by conversion to AMP and hence to adenosine via the cAMP-adenosine pathway). Adenosine receptor agonists inhibited fetal calf serum-induced collagen and total protein synthesis (as assessed by [3H]proline and [3H]leucine incorporation, respectively) with a relative potency profile consistent with the effects being mediated by adenosine A2B receptors. Erythro-9-(2-hydroxy-3-nonyl) adenine, dipyridamole, iodotubericidin, and cAMP also inhibited collagen and total protein synthesis. The effects of 2-chloroadenosine, erythro-9-(2-hydroxy-3-nonyl) adenine, iodotubericidin, and cAMP on collagen and total protein synthesis were attenuated by KF17837 and 1,3-dipropyl-8-p-sulfophenylxanthine (selective and nonselective A2 receptor antagonists, respectively) but not by 8-cyclopentyl-1, 3-dipropylxanthine (selective A1 receptor antagonist). These studies indicate that exogenous, drug-induced and cAMP-adenosine pathway-derived adenosine inhibit vascular SMC collagen synthesis and hypertrophy via A2B receptors. Thus, exogenous A2B receptor agonists and drugs that modulate endogenous adenosine levels may protect against vasoocclusive disorders by attenuating extracellular matrix synthesis by and cellular hypertrophy of vascular SMCs. Moreover, the cAMP-adenosine pathway may protect against vascular hypertrophy.

Cigarette smoke extract is a modulator of mitogenic action in vascular smooth muscle cells

Cigarette smoking is associated with an increased incidence of atherosclerotic disease. In this study, we examined the mechanism underlying the growthmodulating effects of cigarette smoke extract (CSE) in confluent vascular smooth muscle cells (VSMCs). The treatment of VSMC by CSE decreased the activities of Superoxide dismutase (SOD), catalase and glutathione peroxidase of VSMC in a time-dependent manner. In mitogenesis assays using the confluent cells, CSE was not a direct mitogen for VSMC, but potentiated the stimulatory effect of hydrogen peroxides. The reduction of activities of catalase and glutathione peroxidase was partially prevented by SH-containing compounds. In summary, CSE enhanced the mitogenic effect response of hydrogen peroxides, largely depending on the dysregulation of the activities of SOD, catalase and glutathione peroxidase by CSE.

Alterations in Calcium Stores in Aortic Myocytes From Spontaneously Hypertensive Rats

The aim of the present work was to further characterize intracellular calcium stores released by angiotensin II (Ang II) in spontaneously hypertensive rat (SHR) and Wistar-Kyoto rat (WKY) vascular smooth muscle cells (VSMCs) and to study their alterations associated with proliferation. Intracellular Ca2+ concentration was monitored by image analysis in aortic myocytes loaded with fura 2. In the presence of extracellular Ca2+, sensitivity to Ang II in proliferating VSMCs was not different in the two strains, but it increased 10-fold in confluent VSMCs from SHR-compared with those from WKY. In Ca(2)+-free medium, Ca2+ release induced by thapsigargin (10 mumol/L) was significantly greater (about twofold) in SHR than WKY, in both proliferating and confluent cultures, with responses during proliferation being 0.7-fold smaller. Responses to Ang II were abolished after exposure of the cells to thapsigargin. In proliferating cultures, ryanodine (10 mumol/L) did not modify the rises in intracellular Ca2+ concentration induced by Ang II in VSMCs from both strains. Conversely, in confluent cultures, ryanodine reduced Ang II (100 nmol/L)-induced Ca2+ release to the same level as in proliferating cultures, and it suppressed the difference between SHR and WKY. These results show that the ryanodine-sensitive Ca2+ release induced by Ang II is enhanced in VSMCs from SHR at confluence and is impaired during proliferation. Thus, they suggest that differences in Ca2+(-)induced Ca2+ release from the sarcoplasmic reticulum may participate in increased responsiveness of VSMCs to Ang II in SHR and in phenotypic modulation of vascular myocytes during proliferation.

Transforming Growth Factor β Is a Modulator of Platelet-Derived Growth Factor Action in Vascular Smooth Muscle Cells: A Possible Role for Catalase Activity and Glutathione Peroxidase Activity

Transforming growth factor-β (TGF-β) has been implicated in mediating the growth of vascular smooth muscle cells (VSMCs) after vascular injury. In this study, we examined the mechanism underlying the growth-modulating effects of TGF-β in confluent VSMCs. Stimulation of rat VSMC by TGF-β decreased both their catalase activity and glutathione peroxidase activity in a dose-dependent manner. In mitogenesis assays using the confluent cells, TGF-β was not a direct mitogen for VSMC, but potentiated the stimulatory effect of platelet-derived growth factor (PDGF)-BB. This enhancement of mitogenesis was blunted by the addition of the scavenging enzyme catalase or the chemical antioxidant N-acetyl-L-cysteine. In summary, TGF-β enhances the mitogenic effect response of PDGF-BB, largely depending on the dysregulation of catalase activity and glutathione peroxidase activity by TGF-β.

Plasma membrane calcium pump-mediated calcium efflux and bulk cytosolic free calcium in cultured aortic smooth muscle cells from spontaneously hypertensive and Wistar-Kyoto normotensives rats

To compare the efficacy of the calcium pump-mediated calcium efflux pathway in spontaneously hypertensive rats (SHR) with that in Wistar-Kyoto normotensive rats (WKY), at rest and after angiotensin II stimulation. The intracellular free calcium concentration and calcium-45 efflux were measured in parallel, in cultured aortic smooth muscle cells isolated from 10-week-old male SHR and WKY rats. The intracellular free calcium concentration and calcium-45 efflux were studied in confluent vascular smooth muscle cells in culture. Experiments were performed in the absence of added extracellular calcium and sodium. Fura-2 was used to measure basal and angiotensin II-stimulated intracellular free calcium concentration. Effluxed calcium-45 was measured over 5s intervals to determine basal and angiotensin II-stimulated calcium efflux rates in SHR and in WKY rats. No significant difference between SHR and WKY rats was observed in basal intracellular free calcium concentration or 100nmol/l angiotensin II-stimulated peak intracellular free calcium concentration. However, significantly elevated basal and 100 nmol/l angiotensin II-stimulated calcium-45 efflux rates were found in SHR. The calcium-45 efflux rates in SHR were elevated when the efflux was normalized with respect to the bulk intracellular free calcium concentration. The time taken to reach the maximum calcium-45 efflux rate after angiotensin II stimulation was reduced in SHR compared with that in WKY rats and was dose-dependent in both rat strains. The calcium-pump mediated calcium efflux pathway appears to be more efficient in SHR. This may be the result of post-translational modification, enhanced calcium pump sites in a critical region of the membrane, or the presence of a pool of calcium near the plasma membrane that is not readily detected by cytosolic Fura-2 but is higher in SHR both before and after angiotensin II stimulation.

Vascular cell-derived heparan sulfate shows coupled inhibition of basic fibroblast growth factor binding and mitogenesis in vascular smooth muscle cells.

Basic fibroblast growth factor (bFGF) has been previously shown to be mitogenic for vascular smooth muscle cells (VSMCs) in vivo, but only after vascular injury. We show in the present study that the regulation of bFGF-stimulated VSMC proliferation, by vascular cell-secreted heparin-like compounds, correlates with inhibition of bFGF binding to cell-associated heparin sulfate proteoglycans. The stimulation of cultured VSMC proliferation by bFGF was markedly reduced when these cells were cocultured with confluent endothelial cells or confluent VSMCs (100.8 +/- 8.4% and 55.6 +/- 2.3% inhibition, respectively) or with conditioned media from these two cell types. Balb/c3T3 fibroblasts had no statistically significant effect on bFGF-stimulated VSMC proliferation. Vascular cell-conditioned media also inhibited bFGF binding to heparan sulfate proteoglycans on VSMCs, and the inhibition of binding correlated linearly with the inhibition of proliferation after a critical amount of binding was inhibited (44%) (r = .952, P < .0001). Heparinase or heparitinase treatment of conditioned media removed the bFGF-inhibitory effects, presumably by degrading heparin-like compounds. Indeed, heparin itself mimicked the inhibitory effects of conditioned media on bFGF-mediated proliferation and binding to heparin sulfate proteoglycans. These results suggest a bFGF regulatory role for vascular cell-produced heparin-like compounds, linking the mitogenic effects with binding to heparan sulfate proteoglycans for this heparin-binding growth factor.

Osteopontin is elevated during neointima formation in rat arteries and is a novel component of human atherosclerotic plaques.

In an earlier report, we used differential cloning to identify genes that might be critical in controlling arterial neointima formation (Giachelli, C., N. Bae, D. Lombardi, M. Majesky, and S. Schwartz. 1991. Biochem. Biophys. Res. Commun. 177:867-873). In this study, we sequenced the complete cDNA and conclusively identified one of these genes, 2B7, as rat osteopontin. Using immunochemistry and in situ hybridization, we found that medial smooth muscle cells (SMC) in uninjured arteries contained very low levels of osteopontin protein and mRNA. Injury to either the adult rat aorta or carotid artery using a balloon catheter initiated a qualitatively similar time-dependent increase in both osteopontin protein and mRNA in arterial SMC. Expression was transient and highly localized to neointimal SMC during the proliferative and migratory phases of arterial injury, suggesting a possible role for osteopontin in these processes. In vitro, basic fibroblast growth factor (bFGF), transforming growth factor-beta (TGF-beta), and angiotensin II (AII), all proteins implicated in the rat arterial injury response, elevated osteopontin expression in confluent vascular SMC. Finally, we found that osteopontin was a novel component of the human atherosclerotic plaque found most strikingly associated with calcified deposits. These data implicate osteopontin as a potentially important mediator of arterial neointima formation as well as dystrophic calcification that often accompanies this process.

Primary Confluent Vascular Smooth Muscle Cells (VSMCs) are Inhomogeneous with Respect to Smooth Muscle Specific Myosin Heavy Chain (SM-MHC) Content

Primary Confluent Vascular Smooth Muscle Cells (VSMCs) are Inhomogeneous with Respect to Smooth Muscle Specific Myosin Heavy Chain (SM-MHC) Content