Bovine Aortic Smooth Muscle Cells Research Articles

PH and nitric oxide synthase activity and expression in bovine aortic endothelial cells

Abstract Aim: Nitric oxide (NO) plays an important role in the transition from intrauterine to extrauterine life. If this transition fails, a condition called persistent pulmonary hypertension of the neonate (PPHN) may develop. The current treatment modalities for this disease include induction of alkalosis by hyperventilation or alkali infusion, inhaled nitric oxide (iNO) and extracorporeal membrane oxygenation. There is evidence from animal studies that the elevated pH, not the low pCO2 is responsible for the resultant pulmonary vasodilatation. In this study, we examined the effect of pH on the activity and expression of endothelial nitric oxide synthase (eNOS) in cultured bovine aortic endothelial cells (BAEC) as a possible explanation for the pH dependent drop in pulmonary vascular resistance. Methods: BAEC were exposed to a pH gradient of 7.1–7.6 for 4 h (short‐term) and 16 h (long‐term). Standard Western blotting technique was used to detect expression of eNOS. Activity was measured by an indirect assay using bovine aortic smooth muscle cells (BASM) as reporter cells and measuring cGMP levels as a marker of NO production. The cells were exposed to the pH gradient for a total of 4 h and measurement were made at 30, 60 and 90 min, and 2, 3 and 4 hours. Results: eNOS activity and expression remained unchanged during the four and sixteen hours of exposure. Conclusion: In this in vitro experiment, we could not demonstrate an alkalosis‐induced increase in eNOS activity and expression. The clinically observed pH dependent vasodilatation does not appear to be directly mediated through the induction of eNOS.

PH and nitric oxide synthase activity and expression in bovine aortic endothelial cells

Nitric oxide (NO) plays an important role in the transition from intrauterine to extrauterine life. If this transition fails, a condition called persistent pulmonary hypertension of the neonate (PPHN) may develop. The current treatment modalities for this disease include induction of alkalosis by hyperventilation or alkali infusion, inhaled nitric oxide (iNO) and extracorporeal membrane oxygenation. There is evidence from animal studies that the elevated pH, not the low pCO2 is responsible for the resultant pulmonary vasodilatation. In this study, we examined the effect of pH on the activity and expression of endothelial nitric oxide synthase (eNOS) in cultured bovine aortic endothelial cells (BAEC) as a possible explanation for the pH dependent drop in pulmonary vascular resistance. BAEC were exposed to a pH gradient of 7.1-7.6 for 4 h (short-term) and 16 h (long-term). Standard Western blotting technique was used to detect expression of eNOS. Activity was measured by an indirect assay using bovine aortic smooth muscle cells (BASM) as reporter cells and measuring cGMP levels as a marker of NO production. The cells were exposed to the pH gradient for a total of 4 h and measurement were made at 30, 60 and 90 min, and 2, 3 and 4 hours. eNOS activity and expression remained unchanged during the four and sixteen hours of exposure. In this in vitro experiment, we could not demonstrate an alkalosis-induced increase in eNOS activity and expression. The clinically observed pH dependent vasodilatation does not appear to be directly mediated through the induction of eNOS.

Regulation of cGMP-dependent Protein Kinase Expression by Rho and Krüppel-like Transcription Factor-4

Type I cGMP-dependent protein kinase (PKG I) plays a major role in vascular homeostasis by mediating smooth muscle relaxation in response to nitric oxide, but little is known about the regulation of PKG I expression in smooth muscle cells. We found opposing effects of RhoA and Rac1 on cellular PKG I expression: (i) cell density-dependent changes in PKG I expression varied directly with Rac1 activity and inversely with RhoA activity; (ii) RhoA activation by calpeptin suppressed PKG I, whereas RhoA down-regulation by small interfering RNA increased PKG I expression; and (iii) PKG I promoter activity was suppressed in cells expressing active RhoA or Rho-kinase but was enhanced in cells expressing active Rac1 or a dominant negative RhoA. Sp1 consensus sequences in the PKG I promoter were required for Rho regulation and bound nuclear proteins in a cell density-dependent manner, including the Krüppel-like factor 4 (KLF4). KLF4 was identified as a major trans-acting factor at two proximal Sp1 sites; active RhoA suppressed KLF4 DNA binding and trans-activation potential on the PKG I promoter. Experiments with actin-binding agents suggested that RhoA could regulate KLF4 via its ability to induce actin polymerization. Regulation of PKG I expression by RhoA may explain decreased PKG I levels in vascular smooth muscle cells found in some models of hypertension and vascular injury.

Spatial variations regulation of expression of CuZn‐SOD and Mn‐SOD by shear stress in vascular tissue

Introduction There are important spatial variations between shear stress and focal and eccentric atherosclerotic lesions. We tested whether pulsatile shear stress (PSS) vs. oscillatory shear stress (OSS) regulates SOD isoforms; namely, SOD1 (CuZn-SOD), SOD2 (Mn-SOD), and SOD3 (extracellular SOD), in bovine aortic endothelial cells (BAEC) and vascular smooth muscle cells (VSMC). Methods Confluent BAEC and VSMC monolayers were exposed to PSS at a mean shear stress (τave) of 23 dyn.cm−2 and a temporal gradient (∂τ/∂ t) at 71 dyn.cm. −2.sec−2; and OSS at τave= 0.02 ± 3 dyn.cm−2 in a dynamic parallel plate flow system for 4 hours. SOD-mRNA isoforms were measured with real-time RT-PCR. Results a)BAEC (Fig. 1): SOD2 was significantly up-regulated in BAEC in response to shear stress (P <0.05, n=3). PSS increased SOD2 expression by 10-fold and OSS by 5-fold. SOD1 was increased by 2-fold in response to PSS, but remained unchanged in response to OSS. SOD3 did not respond to shear stress. Figure 1Open in figure viewerPowerPoint Endothelial SOD mRNA expression b)VSMC (Fig. 2): SOD2 was up-regulated in SMC in response to PSS by 2-fold, but was down-regulated in response to OSS (P < 0.05, n=3). SOD1 expressed the similar trend, but was statistically insignificant. SOD3 expression was statistically insignificant. Figure 2Open in figure viewerPowerPoint Smooth muscle SOD mRNA expression Conclusion Shear stress increased expression of SOD1 and -2 suggesting that spatial variations in shear stress may contribute to spatial differences in vascular oxidative stress.

Induction of BMP4 in Vascular Smooth Muscle Cells by Shear Stress

Introduction Bone morphogenic protein-4 (BMP4) promotes inflammatory responses and vascular calcification. Smooth muscle cells proliferation and migration occur in the denuded arteries post angioplasty. We assessed whether pulsatile shear stress (PSS) vs. oscillatory shear stress (OSS) regulated BMP2 and BMP4 expression in bovine aortic endothelial cell (BAEC) and vascular smooth muscle cell (VSMC). Methods Confluent BAEC and VSMC monolayers were exposed to PSS at a mean shear stress (τave) of 23 dyn.cm−2 and a temporal gradient (∂τ/∂t) at 71 dyn.cm.−2.sec−2; and OSS at τave= 0.02 ± 3 dyn.cm−2 in a dynamic parallel plate flow system for 4 hours. BMP-mRNA was measured with real-time RT-PCR Results a) VSMC (Fig. 1): OSS significantly up-regulated BMP4 by 2.2-fold and PSS by 1.64-fold (P<0.05, n=3) in VSMC. OSS significantly down-regulated BMP2 by 0.32-fold (P < 0.05), but PSS-induced down-regulation was statistically insignificant. Control samples were under static condition. Figure 1Open in figure viewerPowerPoint Smooth Muscle cell BMP mRNA expression b) BAEC (Fig. 2): OSS up-regulated BMP2 by 2-fold, and BMP4 by 1.5-fold in BAEC. Similarly, PSS induced BMP2 and BMP4 expression by 1.6- and 1.4-fold, respectively. However, these differences were statistically insignificant. Figure 2Open in figure viewerPowerPoint Endothelial BMP mRNA expression Discussion OSS was a stronger inducer of BMP4 expression in VSMC than PSS. The findings suggest that shear stress mediated increases in BMP4 expression may contribute to inflammation in the denuded regions of stented arteries.

High density lipoproteins downregulate basic fibroblast growth factor production and release in minimally oxidated-LDL treated smooth muscle cells

Increase in plasma low density lipoprotein (LDL) levels and/or decrease in high density lipoprotein (HDL) levels are major risk factors for the development of atherosclerosis. An oxidative modification of LDL represents a key process in atherogenesis. It is well known that the LDL/HDL ratio is more important than the individual LDL and HDL levels to predict atherosclerosis. The purpose of our study was to investigate the effects of mildly oxidized LDL (minimally modified LDL: MM-LDL) and HDL, administrated alone or in combination, on the production and release of basic fibroblast growth factor (bFGF) by bovine aortic smooth muscle cells (SMCs) in culture. MM-LDL and HDL have opposite effects on aortic SMCs: MM-LDL increases both bFGF production and release and SMC proliferation, while HDL decreases both bFGF production and release and SMC proliferation. The effects of either MM-LDL or HDL on SMCs are mediated through a Gi-protein-coupled receptor. The simultaneous treatment of SMCs with MM-LDL and HDL (MM-LDL/HDL ratio = 4.0) produced the inhibition of MM-LDL effects. Our data suggest that the protective role of HDL could also be exerted through the inhibition of the pro-atherosclerotic effects of MM-LDL on SMCs.

Resveratrol inhibits vascular smooth muscle cell proliferation and induces apoptosis

In France, despite a high intake of dietary cholesterol and saturated fat, the cardiovascular death rate is one of the lowest among developed countries. This "French paradox" has been postulated to be related to the high red wine intake in France. The aim of this study was to determine the effects of resveratrol, a major polyphenol component of red wine, on vascular smooth muscle cell (SMC) proliferation in vitro. SMCs were exposed to 10(-6) to 10(-4) M resveratrol and cell proliferation was assessed by cell counting. Cell cycle analysis was done by treating cells with propidium iodide followed by flow-activated cell sorting. Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling staining. We demonstrate that resveratrol inhibited bovine aortic SMC proliferation in a dose-dependent manner. The lowest concentration of resveratrol resulting in a significant decrease in SMC proliferation compared with control was 10(-5) M. By flow cytometry, we observed a block in the G1-S phase of the SMC cycle. Resveratrol treatment also resulted in a dose-dependent apoptosis of SMCs but had no effects on SMC morphology. The results indicated that vascular SMC proliferation could be inhibited by resveratrol through a block on G1-S phase and by an increase in apoptosis. It supports the conjecture that red wine consumption may have a beneficial effect on cardiovascular mortality. Our results suggest that resveratrol inhibits, in a dose-dependent manner, smooth muscle cell proliferation, which may help to partially explain a beneficial effect of wine drinking. This inhibition is related to an early block in the cell cycle and also to a dose-dependent apoptotic effect. The present study demonstrates that resveratrol not only is an indirect marker of a healthy life style and alimentation but may also be directly responsible for the French paradox.

Bovine Retinal Pericytes Are Resistant to Glucose-Induced Oxidative StressIn Vitro

Diabetic retinopathy is a sight-threatening complication of diabetes, and loss of pericytes represents early signs of its development. We tested the hypothesis that high glucose levels may induce signs of oxidative stress in cultured bovine retinal pericytes. Pericytes were exposed to either normal (5.5 mM) or high (22 mM) glucose levels for 1, 3, and 5 days. Signs of oxidative stress were measured by expression of copper/zinc superoxide dismutase, manganese superoxide dismutase, catalase, and glutathione peroxidase using real-time RTPCR. To elucidate the role of oxidative stress, we also measured glutathione (GSH) concentration in the cells and investigated the impact of thiol-reactive metal ions and hydrogen peroxide (H(2)O(2)) on intracellular GSH. Despite the stimulation with high glucose, thiol-reactive metal ions, or H(2)O(2), there was no clear increased expression of antioxidant enzymes or influence of GSH levels. Lipid peroxidation (malondialdehyde level) was increased in bovine aortic smooth muscle cells, but not in bovine retinal pericytes. The data indicate that pericytes do not develop oxidative stress in response to hyperglycemia. However, it is not definitively excluded that oxidative stress may occur after longer time periods of glucose stimulation.

Calreticulin Destabilizes Glucose Transporter-1 mRNA in Vascular Endothelial and Smooth Muscle Cells Under High-Glucose Conditions

Substrate autoregulation of glucose transporter-1 (GLUT-1) mRNA and protein expression provides vascular endothelial and smooth muscle cells a sensitive mechanism to adapt their rate of glucose transport in response to changing glycemic conditions. Hyperglycemia-induced downregulation of glucose transport is particularly important in protecting these cells against an excessive influx of glucose and consequently increased intracellular protein glycation and generation of free radicals; both are detrimental in the development of vascular disease in diabetes. We aimed to investigate the molecular mechanism of high glucose-induced downregulation of GLUT-1 mRNA expression in primary bovine aortic vascular endothelial (VEC) and smooth muscle (VSMC) cell cultures. Using RNA mobility shift, UV cross-linking, and in vitro degradation assays, followed by mass-spectrometric analysis, we identified calreticulin as a specific destabilizing trans-acting factor that binds to a 10-nucleotide cis-acting element (CAE(2181-2190)) in the 3'-untranslated region of GLUT-1 mRNA. Pure calreticulin accelerated the rate of GLUT-1 mRNA-probe degradation in vitro, whereas overexpression of calreticulin in vascular cells decreased significantly the total cell content of GLUT-1 mRNA and protein. The expression of calreticulin was augmented in vascular cells exposed to high glucose in comparison with low-glucose conditions. Similarly, increased expression of calreticulin was observed in aortae of diabetic Psammomys obesus in comparison with normoglycemic controls. These data suggest that CAE(2181-2190)-calreticulin complex, which is formed in VSMC and VEC exposed to hyperglycemic conditions, renders GLUT-1 mRNA susceptible to degradation. This interaction underlies the process of downregulation of glucose transport in vascular cells under high-glucose conditions.

Identification of Common and Specific Growth Factor Binding Sites in Heparan Sulfate Proteoglycans

The structural complexity within heparan sulfate has suggested that it contains multiple protein-specific binding sites. To evaluate the selectivity of growth factor binding to heparan sulfate, we conducted a detailed study of the intercompetition of fibroblast growth factor-2 (FGF-2) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) binding to heparan sulfate (HS) on bovine aortic smooth muscle cells. Radioligand binding assays were conducted, and an analytical method was developed for determining the apparent binding constants and numbers of specific and shared binding sites within HS. These studies revealed the presence of two general classes of HS-binding sites for FGF-2 and HB-EGF. The major class (approximately 10(6) sites per cell) was able to bind to either growth factor with relatively low affinity (K(d) = 12 and 44 nM for FGF-2 and HB-EGF, respectively) and was termed "common" binding sites. However, both FGF-2 and HB-EGF also showed specific high affinity (0.6 and 6.1 nM for FGF-2 and HB-EGF, respectively) binding to a minor subset (118,000 and 28,000 sites per cell for FGF-2 and HB-EGF, respectively) of "unique" binding sites, which were unable to bind the other growth factor. These studies indicate that growth factor binding to HS involves multiple binding sites of variable affinity, density, and selectivity. The approach outlined in this study could be applied to aid in the evaluation of the relative biological roles of these selective and nonselective growth factor binding domains within HS.

Regulation of RANKL-induced osteoclastic differentiation by vascular cells

Vascular calcification is a regulated process of biomineralization resembling osteogenesis. Many bone-related factors, including resorptive osteoclast-like cells, although in low abundance, have been found in calcified atherosclerotic lesions. The regulatory mechanisms governing them in the vasculature, however, are not clear. Previously, we found that calcifying vascular cells (CVC), a subpopulation of bovine aortic smooth muscle cells (BASMC), undergo osteoblastic differentiation and form mineralized nodules. Since osteoblasts and marrow stromal preosteoblasts regulate osteoclastic differentiation in bone, we hypothesized that vascular cells also regulate differentiation of osteoclastic precursors in the artery wall. Peripheral blood monocytes, which are osteoclast precursors, were co-cultured with CVC or BASMC. Results showed that monocytes co-cultured with both of the vascular cells yielded fewer resorption pits than monocytes cultured alone. Furthermore, monocytes co-cultured with CVC had fewer resorption pits than those co-cultured with BASMC. Conditioned media from the vascular cells also inhibited resorptive activity of monocytes suggesting that the inhibitory effect was mediated in part by soluble factors. Compared with BASMC, CVC had lower mRNA expression for osteopontin, which promotes osteoclast attachment, but greater mRNA expression for the soluble inhibitory cytokine, IL-18. Increased osteoclastic differentiation was observed when neutralizing antibody to IL-18 receptor was added to the cultures of preosteoclasts with CVC conditioned media. Osteoprotegerin, another osteoclast inhibitory cytokine, was expressed at similar levels in both cultures. These results suggest that vascular cells inhibit osteoclastic differentiation, and that CVC have greater inhibitory effects than BASMC.

Effect of crocin on experimental atherosclerosis in quails and its mechanisms

In the present study, we examined the prophylaxis effect of crocin on experimental atherosclerosis and its possible mechanisms. The atherosclerosis formation was induced by hyperlipidamic diet in quails. At the 9th week, serum lipid, MDA and NO were measured, and HE staining was used to investigate the histopathological changes of aorta. Bovine aortic endothelial cells (EC) were obtained from the thoracic aorta of newborn calves. After incubation of the cells with Ox-LDL (50 mg·L − 1) for 24 h, the activities of LDH, NO in culture media and activity of NOS in endothelial cells were measured, flow cytometer was used to determine the rate of endothelial cells apoptosis. Peritoneal macrophages were obtained from thioglycolate-injected mice. Cholesterol and free cholesterol in cells were assayed after incubation of the cells with Ox-LDL. Bovine aortic smooth muscle cells (SMC) were obtained from the thoracic aorta of newborn calf. Proliferation was induced by 100 μg·L − 1 Ox-LDL and antiproliferative effect of crocin on SMCs were observed. SMCs cycle phases were measured by flow cytometry. SMCs were loaded with Fluo-3/AM and [Ca 2+] i was measured by Laser Scanning Confocal Microscope (LSCM). Crocin could reduce the level of serum TC, TG, LDL-C and inhibit the formation of aortic plaque. Crocin could reduce MDA and inhibit the descending of NO in serum. Compared with control, Ox-LDL group could increase the activity of LDH and decrease activity of NO in culture media and activity of NOS in endothelial cells, preincubated with crocin, the effects of Ox-LDL were inhibited. Crocin could decrease the EC apoptosis induced by Ox-LDL. Crocin concentration-dependently inhibited the TC and CE elevation induced by Ox-LDL in macrophages. Crocin could inhibit the proliferation of SMCs induced by Ox-LDL. In the presence or absence of extracellular Ca 2+, crocin concentration-dependently inhibited the [Ca 2+] i elevation induced by 120mg·L − 1Ox-LDL, In the absence of extracellular Ca 2+, crocin could inhibit the [Ca 2+] i elevation induced by CHCl 3 in a concentration-dependent manner. The results indicated that crocin could inhibit the formation of atherosclerosis in quails. Crocin had protective effects on endothelial cells. Crocin could decrease CE in macrophages and uptake of Ox-LDL, inhibiting the formation of foam cell, which would promote the initiation and progression of atherosclerosis. Crocin could inhibit the [Ca 2+] i elevation in smooth muscle cell, Ca 2+ is an important second messenger that regulates a variety of cellular processes, including smooth muscle cell proliferation and gene expression . Crocin exerted antiatherosclerotic effects through decreasing the level of Ox-LDL that plays an important role in the initiation and progression of atherosclerosis.

Homocysteine promotes p38-dependent chemotaxis in bovine aortic smooth muscle cells

Increased levels of homocysteine in the blood are a risk factor for atherosclerosis. The purpose of this study was to examine the effects of homocysteine on smooth muscle cell (SMC) migration and to determine whether p38 was involved in this process. The effect of 0.5 to 2.0 mmol/L d , l -homocysteine as a chemoattractant for SMCs was assayed with a modified Boyden chamber. To determine the functional role of p38 in SMC chemotaxis induced by d , l -homocysteine, we treated SMCs with a p38 inhibitor, SB203580, before the assay. The number of migrated cells was increased 7.0 +/- 1.2-fold (n = 15; P < .001) by 2.0 mmol/L d , l -homocysteine. SB203580 partially prevented the migration of SMCs toward homocysteine. Preconditioning SMCs with 2.0 mmol/L d , l -homocysteine significantly enhanced chemotaxis toward 10% fetal bovine serum compared with nonconditioned control SMCs (28.9 +/- 3.3-fold vs 15.6 +/- 2.8-fold; P < .05). There was a fourfold p38 activation after exposure of SMCs to 2.0 mmol/L d , l -homocysteine by immunoblot. These results suggest that homocysteine not only is a chemoattractant for SMC but can also enhance SMC chemotactic potential. The mechanism of these effects may involve p38 activation. This study demonstrates that homocysteine can promote chemotaxis of SMCs through a p38-dependent pathway. To our knowledge, this is the first report that homocysteine may influence SMC chemotaxis. It may be an important mechanism for homocysteine-induced atherogenesis, because the migration of SMCs from the media is believed to play a critical role in progressive intimal thickening. Although homocysteine promotes atherogenesis and thrombosis by a variety of mechanisms, the effects of homocysteine on SMC proliferation and migration might be critical elements that may have potential therapeutic implications, because selective blockade of the p38 pathway is feasible.

Cellular growth under hydrostatic pressure using bovine aortic EC-SMC co-cultured ePTFE vascular graft

High blood pressure (hypertension) is implicated in the development of atherosclerosis. Blood vessels are constantly subjected to stretch due to blood pressure and changes in stretch usually instigate adaptive vascular remodeling, including abnormal growth and proliferation of vascular smooth muscle cells (VSMCs) as well as extracellular matrix (ECM). In this experiment, we used bovine aortic endothelial cells and smooth muscle cells (EC-SMC) co-cultured ePTFE vascular grafts subjected to normal atmospheric pressure (as a control), and 100 mmHg hydrostatic pressure for 7 d. The increase of cell layer thickness was observed. When measured, the cell layer thickness increased by 116.2%. The increase of collagen (Type IV) synthesis was also observed in the immunohistochemistry assay. When stained with toluidine blue, the cells showed metachromatic phenomenon.

Inhibition of Cultured Bovine Aortic Smooth Muscle Cell Proliferation by Colominic Acid

Colominic acid (CA) is an alpha2,8-linked polymer of sialic acid, originally isolated from capsular Escherichia coli K1. Since inhibition of arterial smooth muscle cell hyperplasia is one of the effective strategies to prevent atherosclerosis, we investigated the effect of CA, purified as an alpha2,8-linked homopolymer of N-acetylneuraminic acid, on the proliferation of bovine aortic smooth muscle cells in culture. The results demonstrate that CA inhibits the proliferation of the cells without nonspecific cell damage. Sulfation did not modify the inhibitory effect of CA. Specifically, the inhibitory effect of sulfated CA was almost equal to that of CA in vascular smooth muscle cell proliferation. On the other hand, it was suggested that the inhibition of the proliferation by CA is in a degree similar to that by heparin but weaker than that by sodium/calcium-spirulans, known sulfated polysaccharides as the potent inhibitor of vascular smooth muscle cells. The present data suggest that CA with or without sulfate groups can be an origin of beneficial agents that prevents atherosclerosis through a moderate inhibition of arterial smooth muscle cell proliferation.

An Organobismuth Compound that Exhibits Selective Cytotoxicity to Vascular Endothelial Cells in Vitro

Selective cytotoxicity of chemical compounds to vascular endothelial cells is often beneficial for clinical applications including antivascular cancer therapy. In this study, the cytotoxicity of 35 organic compounds containing bismuth or antimony was evaluated based on the leakage of lactate dehydrogenase and morphologic observations in cultured bovine aortic endothelial cells and other cell types. The results indicate that only tris[2-(N,N-dimethylaminomethyl)phenyl]-bismuthane (TDPBi) had potent cytotoxicity to bovine aortic endothelial cells but not to bovine aortic smooth muscle cells and porcine kidney epithelial LLC-PK1 cells; the compound exhibited moderate cytotoxicity to human fetal lung fibroblastic IMR-90 cells. Neither inorganic bismuth nor tris[2-(N,N-dimethylaminomethyl)phenyl]stibane, in which antimony substitutes for bismuth with the same organic structure as TDPBi, exhibited cytotoxicity to vascular endothelial cells, suggesting that the entire structure of TDPBi is required for selective cytotoxicity. The present data revealed that TDPBi has selective cytotoxicity to vascular endothelial cells, which may be applicable in antivascular cancer therapy.

The effect of heparin on osteoblast differentiation and activity in primary cultures of bovine aortic smooth muscle cells

Recent studies have suggested that aortic smooth muscle cells undergo a phenotypic transition into osteoblast-like cells and mineralize when cultured in the presence of β-glycerophosphate. Since we had previously demonstrated that heparin could inhibit osteoblast differentiation and mineralization in primary cultures of murine calvaria cells, we were interested in determining if heparin would have a similar effect when primary aortic smooth muscle cells were cultured in the presence of β-glycerophosphate. The effect of heparin and low molecular weight heparin (LMWH) on osteoblast differentiation and activity was therefore examined in primary cultures of bovine aortic smooth muscle cells (BASMC) over a 14-day period. Here, we report that BASMC differentiate into osteoblast-like cells when cultured in the presence of β-glycerophosphate. Moreover, we report that heparin not only inhibits this process but that it also inhibits the ability of BASMC to mineralize as well. Importantly, these effects were found not to be dependent upon heparins’ anticoagulant activity since unfractionated heparin and heparins with low anti-thrombin III affinities inhibited the mineralization process equally well. Sulfation, however, was found to be a major determinant of heparins ability to inhibit BASMC mineralization since neither dermatan sulfate nor N-desulfated heparin were able to demonstrate an effect. We conclude that BASMC cultures can undergo a phenotypic transition into mature osteoblasts and that both the differentiation process and their ability to mineralize are inhibited by heparin.

Decorin promotes aortic smooth muscle cell calcification and colocalizes to calcified regions in human atherosclerotic lesions.

Ectopic calcification localized to the intima of atherosclerotic plaque is a risk marker for cardiovascular events and increases the risk of aortic dissection during angioplasty. A variety of extracellular matrix molecules such as collagen type 1, bone sialoprotein, and osteopontin are known to regulate the biomineralization of bone and ectopic vascular calcification. In the present study, it was investigated whether decorin, a small leucine-rich proteoglycan expressed in bone and atherosclerotic plaque, is involved in arterial calcification. Calcification was induced in cultured bovine aortic smooth muscle cell (BASMC) by the addition of beta-glycerophosphate or inorganic phosphate. Northern and Western analysis revealed that decorin expression was strongly upregulated in mineralizing BASMC. Furthermore, overexpression of decorin using a retroviral expression vector resulted in a 3- to 4-fold elevation of calcium deposited on the BASMC monolayer. Increased calcification in response to decorin could also be mimicked by adding exogenous decorin to the cultures. In addition, human coronary atherosclerotic lesions taken from sudden-death patients showed marked colocalization of calcium deposits with decorin. Decorin induces calcification of arterial smooth muscle cell cultures and colocalizes to mineral deposition in human atherosclerotic plaque, suggesting that decorin functions as promoter of intimal calcification.

Pulse Pressure-Induced Transmural Fluid Flux Increases Bovine Aortic Smooth Muscle Cell Apoptosis in a Mitogen Activated Protein Kinase Dependent Manner

Background: Mechanical forces associated with blood flow are critical in the regulation of vascular smooth muscle cell (VSMC) growth, migration, differentiation and apoptosis as fundamental features in the pathogenesis of vascular disease. We investigated the effect of pulse pressure on VSMC apoptosis. Methods: Using a perfused transcapillary co-culture system, bovine thoracic aortic SMC (BASMC) were exposed to increases in pulsatile flow (0.3–17 ml/min) and hence pulse pressure (amplitude of pulse 6–50 mm Hg) in the absence or presence of bovine aortic endothelial cells (BAEC). The extent of apoptosis was determined by measuring caspase-3 activity, the levels of pro- and anti-apoptotic Bcl-2 family proteins, FasL and cellular apoptosis susceptibility (CAS) protein expression and the extent of DNA fragmentation. Results: Changes in pulse pressure resulted in a significant force- and time-dependent increase in caspase-3 activity in BASMC. This effect was maximal after 6 h, independent of BAEC presence, and attenuated following inhibition of mitogen-activated protein kinase (MAPK) activity with PD98059. In parallel cultures, there was a significant increase in Bad and Bax expression, concomitant with an increase in DNA fragmentation, and a significant decrease in Bcl-2 and Bcl-X_L expression. The pro-apoptotic effects of pulse pressure were specific for differentiated cells but independent of p53, inasmuch as FasL and CAS expression were enhanced in differentiated adult cells but decreased in de-differentiated embryonic cells in response to flow. Conclusions: These results suggest that pulse pressure promotes phenotypically distinct VSMC apoptosis in vitro in an endothelial-independent, MAPK-dependent, manner.

Heparin-binding EGF-like growth factor induces expression of lectin-like oxidized LDL receptor-1 in vascular smooth muscle cells

Receptor-mediated endocytosis of oxidized LDL (Ox-LDL) has been implicated in lipid accumulation and vascular cell dysfunction. Lectin-like Ox-LDL receptor-1 (LOX-1) is highly inducible by proinflammatory cytokines, as well as angiotensin II and Ox-LDL in vitro. LOX-1 is expressed in macrophages and smooth muscle cells accumulated in the intima of advanced atherosclerotic plaques in vivo. Here we show that heparin-binding epidermal growth factor-like growth factor (HB-EGF), a potent mitogen for vascular smooth muscle cells, induces LOX-1 expression in cultured bovine aortic smooth muscle cells. HB-EGF (1–100 ng/ml) induced LOX-1 expression, which was peaked between 8 and 16 h after HB-EGF stimulation. HB-EGF-induced expression of LOX-1 was suppressed by ZD1839, an inhibitor of EGF receptor phosphorylation. Both MEK and p38 mitogen-activated protein kinase (MAPK) inhibitors significantly blocked LOX-1 upregulation induced by HB-EGF. Phosphatidylinositol 3-kinase (PI3K) inhibitors also blocked HB-EGF-induced LOX-1 expression. HB-EGF induced phosphorylation of ERK, p38 MAPK and Akt, which were suppressed by ZD1839. Upregulated expression of LOX-1 was associated with enhanced uptake of DiI-labeled Ox-LDL in smooth muscle cells. Taken together, HB-EGF can also act as an inducer of LOX-1 expression and play an integral role in foam cell transformation, cellular dysfunction, and proliferation of smooth muscle cells in atherogenesis.