Bovine Smooth Muscle Cells Research Articles

Textured soy protein scaffolds enable the generation of three-dimensional bovine skeletal muscle tissue for cell-based meat

Cell-based meat (CBM) production is a promising technology that could generate meat without the need of animal agriculture. The generation of tissue requires a three-dimensional (3D) scaffold to provide support to the cells and mimic the extracellular matrix (ECM). For CBM, the scaffold needs to be edible and have suitable nutritional value and texture. Here, we demonstrate the use of textured soy protein—an edible porous protein-based biomaterial—as a novel CBM scaffold that can support cell attachment and proliferation to create a 3D engineered bovine muscle tissue. The media composition was optimized for 3D bovine satellite cell (BSC) proliferation and differentiation by adding myogenic-related growth factors. Myogenesis of several cell combinations was compared, and elevated myogenesis and ECM deposition were shown in co-culture of BSCs with bovine smooth muscle cells and tri-cultures of BSCs, bovine smooth muscle cells and bovine endothelial cells. The expression of proteins associated with ECM gene sets was increased in the co-culture compared with BSC monoculture. Volunteers tasted the product after cooking and noted its meaty flavour and sensorial attributes, achieving the goal of replicating the sensation and texture of a meat bite. This approach represents a step forward for the applied production of CBM as a food product. Animal skeletal muscle cell culture has the potential to provide new protein sources without the need for conventional animal agriculture. Ben-Ayre et. al. address one of the challenges of growing cell-based meat in vitro by using textured soy protein as an extracellular matrix scaffold. This approach allowed three-dimensional skeletal muscle cell development to produce a meat-like product for human consumption.

Role of PKCζ-NADPH oxidase signaling axis in PKCα-mediated Giα2 phosphorylation for inhibition of adenylate cyclase activity by angiotensin II in pulmonary artery smooth muscle cells.

We sought to determine the mechanism by which angiotensin II (AngII) inhibits isoproterenol induced increase in adenylate cyclase (AC) activity and cyclic adenosine monophosphate (cAMP) production in bovine pulmonary artery smooth muscle cells (BPASMCs). Treatment with AngII stimulates protein kinase C-ζ (PKC-ζ), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and PKC-α activities, and also inhibits isoproterenol induced increase in AC activity and cAMP production in the cells. Pertussis toxin pretreatment eliminates AngII caused inhibition of isoproterenol induced increase in AC activity without a discernible change in PKC-ζ, NADPH oxidase, and PKC-α activities. Treatment of the cells with AngII increases α2 isoform of Gi (Giα2) phosphorylation; while pretreatment with chemical and genetic inhibitors of PKC-ζ and NADPH oxidase attenuate AngII induced increase in PKC-α activity and Giα2 phosphorylation, and also reverse AngII caused inhibition of isoproterenol induced increase in AC activity. Pretreatment of the cells with chemical and genetic inhibitors of PKC-α attenuate AngII induced increase in Giα2 phosphorylation and inhibits isoproterenol induced increase in AC activity without a discernible change in PKC-ζ and NADPH oxidase activities. Overall, PKCζ-NADPH oxidase-PKCα signaling axis plays a crucial role in Giα2 phosphorylation resulting in AngII-mediated inhibition of isoproterenol induced increase in AC activity in BPASMCs.

Primary Culture 한 소 모양체근 세포에서의 PLC/IP₃ 및 cAMP/PKA 경로를 통한 차즈기 열수 추출물의 수축 조절 효과

Primary Culture 한 소 모양체근 세포에서의 PLC/IP₃ 및 cAMP/PKA 경로를 통한 차즈기 열수 추출물의 수축 조절 효과

Eukaryotic elongation factor 2 kinase controls proliferation and migration of vascular smooth muscle cells.

Eukaryotic elongation factor 2 kinase (eEF2K), also known as calmodulin (CaM)-dependent protein kinase (CaMK) III, is a unique member of CaMK family protein. We have recently found that expression of eEF2K protein increased in mesenteric artery from spontaneously hypertensive rats. As pathogenesis of hypertension is in part regulated by vascular structural remodelling via proliferation and migration of vascular smooth muscle cells (SMCs), we tested the hypothesis that eEF2K controls SMCs proliferation and migration. In rat mesenteric arterial SMCs, an eEF2K inhibitor, A-484954 (10 μm), significantly inhibited platelet-derived growth factor (PDGF)-BB (10 ng mL(-1) )-induced SMCs proliferation as determined by a cell counting and bromodeoxyuridine incorporation assay. PDGF-BB (10 ng mL(-1) )-induced SMCs migration was significantly inhibited by A-484954 (10 μm) as determined by a Boyden chamber assay. A-484954 (10 μm) significantly inhibited PDGF-BB (10 ng mL(-1) )-induced phosphorylation of eEF2K, extracellular signal-regulated kinase (ERK), Akt, p38 and heat-shock protein (HSP) 27 as determined by Western blotting. It was confirmed that a CaM inhibitor, W-7 (50 μm), inhibited PDGF-BB (10 ng mL(-1) )-induced phosphorylation of eEF2K. In an ex vivo mesenteric arterial ring assay, 10% foetal bovine serum-induced SMCs outgrowth was significantly inhibited by A-484954 (10 μm). We for the first time revealed that eEF2K mediates PDGF-BB-induced SMCs proliferation and migration through activating ERK, Akt, p38 and HSP27 signals in a CaM-dependent manner. Our results suggest eEF2K as a novel pharmaceutical target for the prevention of hypertensive cardiovascular diseases.

Identification, purification and partial characterization of low molecular weight protein inhibitor of Na⁺/K⁺-ATPase from pulmonary artery smooth muscle cells.

We have identified a novel endogenous low mol wt. (15.6 kDa) protein inhibitor of Na(+)/K(+)-ATPase in cytosolic fraction of bovine pulmonary artery smooth muscle cells. The inhibitor showed different affinities toward the α₂β₁ and α₁β₁ isozymes of Na(+)/K(+)-ATPase, where α₂ is more sensitive than α₁. The inhibitor interacted reversibly to the E1 site of the enzyme and blocked the phosphorylated intermediate formation. Circular dichroism study suggests that the inhibitor causes an alteration in the confirmation of the enzyme.

Construction of Tissue-Engineered Small-Diameter Vascular Grafts in Fibrin Scaffolds in 30 Days

Tissue-engineered small-diameter vascular grafts have been developed as a promising alternative to native veins or arteries for replacement therapy. However, there is still a crucial need to improve the current approaches to render the tissue-engineered blood vessels more favorable for clinical applications. A completely biological blood vessel (3-mm inner diameter) was constructed by culturing a 50:50 mixture of bovine smooth muscle cells (SMCs) with neonatal human dermal fibroblasts in fibrin gels. After 30 days of culture under pulsatile stretching, the engineered blood vessels demonstrated an average burst pressure of 913.3±150.1 mmHg (n=6), a suture retention (53.3±15.4 g) that is suitable for implantation, and a compliance (3.1%±2.5% per 100 mmHg) that is comparable to native vessels. These engineered grafts contained circumferentially aligned collagen fibers, microfibrils and elastic fibers, and differentiated SMCs, mimicking a native artery. These promising mechanical and biochemical properties were achieved in a very short culture time of 30 days, suggesting the potential of co-culturing SMCs with fibroblasts in fibrin gels to generate functional small-diameter vascular grafts for vascular reconstruction surgery.

Plasma surface chemical treatment of electrospun poly(L-lactide) microfibrous scaffolds for enhanced cell adhesion, growth, and infiltration.

Poly(l-lactide) (PLLA) microfibrous scaffolds produced by electrospinning were treated with mild Ar or Ar-NH3/H2 plasmas to enhance cell attachment, growth, and infiltration. Goniometry, atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS) measurements were used to evaluate the modification of the scaffold surface chemistry by plasma treatment. AFM and XPS measurements showed that both plasma treatments increased the hydrophilicity without affecting the integrity of the fibrous structure and the fiber roughness, whereas Ar-NH3/H2 plasma treatment also resulted in surface functionalization with amine groups. Culture studies of bovine aorta endothelial cells and bovine smooth muscle cells on the plasma-treated PLLA scaffolds revealed that both Ar and Ar-NH3/H2 plasma treatments promoted cell spreading during the initial stage of cell attachment and, more importantly, increased the cell growth rate, especially for Ar plasma treatment. In vitro cell infiltration studies showed that both plasma treatments effectively enhanced cell migration into the microfibrous scaffolds. In vivo experiments involving the subcutaneous implantation of plasma-treated PLLA scaffolds under the skin of Sprague-Dawley rats also showed increased cell infiltration. The results of this study indicate that surface treatment of PLLA microfibrous scaffolds with mild Ar or Ar-NH3/H2 plasmas may have important implications in tissue engineering. Further modifications with bioactive factors should improve the functions of the scaffolds for specific applications.

Olive Oil Polyphenols Differentially Inhibit Smooth Muscle Cell Proliferation through a G1/S Cell Cycle Block Regulated by ERK1/2

We hypothesized that polyphenols contained in olive oil play a role in reducing the risk of atherosclerosis. The aim of this study was to determine if the polyphenols in olive oil, oleuropein (Ole), hydroxytyrosol (HT), and tyrosol (Tyr) could inhibit smooth muscle cell (SMC) proliferation through its influence on cell cycle regulation. Bovine vascular SMC were cultured in the presence of Ole, HT, or Tyr at concentration of 1, 10, or 100 μmol/L. On days 1, 3, and 5, numbers of cells were counted. Cell cycle analysis was performed by flow cytometry on day 1 after SMC were stained with propidium iodide. Cell populations grown in the presence of Ole or HT at 100 μmol/L concentration were significantly inhibited after 5 days of exposure. Tyr had a similar tendency but it did not attain significance. Cell cycle analysis revealed that 66% of cells were in G1 phase in Ole group, compared with 48% in control group. To examine the cell cycle block between G1 and S phases, we performed Western blotting and found that ERK1/2 activation was inhibited by Ole or HT. We conclude that olive oil polyphenols could inhibit SMC proliferation through a cell cycle block between G1 and S phases which may be regulated by ERK1/2. These results demonstrate a mechanism by which olive oil consumption may be atheroprotective by inhibiting SMC proliferation.

Non‐linear microscopy of smooth muscle cells in artificial extracellular matrices made of cellulose

Non-linear microscopy has been used to characterize bovine smooth muscle cells and their proliferation, migration, and differentiation in hydrogel cellulose scaffolds, toward the development of fully functional blood vessel implants. The extracellular matrix (ECM) composed of cellulose and endogenous collagen fibers was imaged using Second Harmonic Generation (SHG) microscopy and the cell morphology by Coherent Anti-Stokes Raman Scattering (CARS) microscopy. Images prove that cells adhere on the cellulose scaffold without additional surface modification and that both contractile and proliferating phenotypes are developed. This work shows that non-linear microscopy contributes with unique insights in cell interactions with (artificial) ECM components and has the potential to become an established characterization method in tissue engineering.

Olive Oil Polyphenol Oleuropein Inhibits Smooth Muscle Cell Proliferation

Objectives The Mediterranean diet, with a high content of olive oil, is associated with a reduced risk of coronary artery disease. The aim of this study was to determine the effect of oleuropein, one of the polyphenols in olive oil, on vascular smooth muscle cell (SMC) proliferation in vitro. Design This was an experimental study. Materials and methods Bovine vascular SMCs were cultured in the presence of 100 μM of oleuropein. On days 1, 3 and 5, cell number was counted. Cell cycle analysis was performed by flow cytometry. Cell cycle regulators were assessed by immunoblotting. Results Cell proliferation in the presence of oleuropein was significantly inhibited by 92%. Cell cycle analysis revealed that oleuropein treatment blocked cells in the G1–S phase compared with the non-treated group. Among G1 phase regulators, retinoblastoma protein, cyclinD, p21 and p27 were not affected by oleuropein, but extracellular signal-regulated kinase 1/2 (ERK1/2) activation was inhibited. Growth of SMC treated with 100 μM of the mitogen-activated protein (MAP) kinase/ERK kinase 1 (MEK1) inhibitor PD98059 was also significantly inhibited by 70%. Conclusions Oleuropein inhibited SMC proliferation through a cell cycle block between the G1 and the S phases, which may be regulated by ERK1/2. These results suggest a mechanism by which olive oil consumption may have beneficial effects on cardiovascular mortality by inhibiting SMC proliferation.

Bacterial cellulose as a potential vascular graft: Mechanical characterization and constitutive model development

Bacterial cellulose (BC) is a polysaccharide produced by Acetobacter Xylinum bacteria with interesting properties for arterial grafting and vascular tissue engineering including high-burst pressure, high-water content, high crystallinity, and an ultrafine highly pure fibrous structure similar to that of collagen. Given that compliance mismatch is one of the main factors contributing to the development of intimal hyperplasia in vascular replacement conduits, an in depth investigation of support mechanical properties of BC is required to further supporting its use in cardiovascular-grafting applications. The aim of this study was to mechanically characterize BC and also study its potential to accommodate vascular cells. To achieve these aims, inflation tests and uniaxial tensile tests were carried out on BC samples. In addition, dynamic compliance tests were conducted on BC tubes, and the results were compared to that of arteries, saphenous vein, expanded polytetrafluoroethylene, and Dacron grafts. BC tubes exhibited a compliance response similar to human saphenous vein with a mean compliance value of 4.27 × 10(-2) % per millimeter of mercury over the pressure range of 30-120 mmHg. In addition, bovine smooth muscle cells and endothelial cells were cultured on BC samples, and histology and fluorescent imaging analysis were carried out showing good adherence and biocompatibility. Finally, a method to predict the mechanical behavior of BC grafts in situ was established, whereby a constitutive model for BC was determined and used to model the BC tubes under inflation using finite element analysis.

Role of Protein Transamidation in Serotonin-Induced Proliferation and Migration of Pulmonary Artery Smooth Muscle Cells

Pulmonary hypertension is characterized by elevated pulmonary artery pressure and pulmonary artery smooth muscle cell (SMC) proliferation and migration. Clinical and experimental evidence suggests that serotonin (5-HT) is important in these responses. We previously demonstrated the participation of the 5-HT transporter and intracellular 5-HT (5-HTi) in the pulmonary vascular SMC-proliferative response to 5-HT. However, the mechanism underlying the intracellular actions of 5-HT is unknown. We speculated that 5-HTi activates SMC growth by post-translational transamidation of proteins via transglutaminase (TGase) activity, a process referred to as serotonylation. To test this hypothesis, serotonylation of pulmonary artery SMC proteins, and their role in 5-HT-induced proliferative and migratory responses, were assessed. 5-HT caused dose- and time-dependent increase in serotonylation of multiple proteins in both bovine and rat pulmonary artery SMCs. Inhibition of TGase with dansylcadaverin blocked this activity, as well as SMC-proliferative and migratory responses to 5-HT. Serotonylation of proteins also was blocked by 5-HT transporter inhibitors, and was enhanced by inhibition of monoamine oxidase, an enzyme known to degrade 5-HTi, indicating that 5-HTi levels regulate serotonylation. Immunoprecipitation assays and HPLC-mass spectral peptide sequencing revealed that a major protein serotonylated by TGase was fibronectin (FN). 5-HT-stimulated SMC serotonylation and proliferation were blocked by FN small interfering (si) RNA. These findings, together with previous observations that FN expression in the lung strongly correlates with the progression of pulmonary hypertension in both experimental animals and humans, suggest an important role of FN serotonylation in the pathogenesis of this disease.

Self‐organization and pattern formation in calcifying vascular stem cells

We previously observed patterns of evenly spaced stripes of calcified cellular aggregates in cultures of calcifying vascular cells (CVC), bovine smooth muscle cells purified by dilutional cloning. We hypothesized that the patterns were formed by a reaction‐diffusion process driven by interaction of a known morphogen, bone morphogenetic protein‐2 (BMP‐2), with its inhibitor, matrix GLA protein (MGP). We used mathematical models based on the biochemical and physical properties of BMP‐2 (autocatalytic, chemoattractant, and osteogenic) and MGP (inhibitory of BMP‐2 and rapidly diffusing).Computer simulations based on the math models predicted 2 experimental findings. Over 10–14 days, CVC cultures treated with exogenous MGP produced evenly spaced spots (nodules) rather than stripes, and CVC treated with warfarin (an inhibitor of MGP) produced stripes at double the density of untreated cultures. In addition, the models predicted conversion of diffuse cell density to focal nodules. In vivo, when CVC were implanted for 8 wks in subcutaneous diffusion chambers in hyperlipidemic mice, they produced tissue nodules resembling calcified atherosclerotic plaque.Thus, calcified atherosclerotic lesions may arise from cellular aggregates formed by reaction‐diffusion processes. Harnessing this mechanism may ultimately allow clinical control of focal vs. diffuse vascular disease.

Low-Energy Electromagnetic Fields Promote Proliferation of Vascular Smooth Muscle Cells

The rationale was to investigate the effects of low-energy electromagnetic fields (EMF) on the proliferation of bovine coronary and murine aortic smooth muscle cells (SMC). EMF were applied to SMC at field frequencies of 25, 50, or 100 Hz, and exposure time was set to 5, 15, or 30 minutes. Significant increases in SMC-counts compared with sham exposed controls were found for all EMF-frequencies tested. The effect was most pronounced for 50 Hz fields with maximum increases of 1.2-fold over controls. Sequential double exposure of mouse aortic SMC to 50 Hz fields revealed significantly enhanced cell proliferation by 1.2 fold compared with single exposure (p < 0.05). Experiments performed on bovine SMC also revealed significant increases in cell proliferation. The results demonstrate that EMF are capable of significantly enhancing the proliferation of vascular SMC. These results rise the question whether EMF would qualify as supportive means to angio-/arteriogenic approaches.

Study of vascular smooth muscle cell calcification induced by hyperphosphate and intervented by phosphonoformic acid

Objective To evaluate the effects of different concentrations of phosphate on calcium deposition and osteocalcin level in cultured bovine aortic smooth muscle cell, investigate the mechanism of hyperphosphatemia to evoke calcification of vascular smooth muscle cell and observe the effects of phosphonoformic acid(PFA) in different concentrations on vascular calcification. Methods The bovine aortic smooth muscle cells (BASMC) were cultured. Calcium deposition and the expression of osteocalcin of BASMC in different concentrations of phosphate (1.5 mmol/L and 2.0 mmol/L) and PFA were determined by o-cresolphthalein complexone and radioimmunity methods, respectively. Osteocalcin mRNA expressions were determined by RT-PCR. Results After six or nine days of BASMC cultured, the calcium deposition in Pi 2.0 mmol/L group was more than that in Pi 1.5 mmol/L group[(77.187 ±11.692)μg/(mg•protein) vs(25.768 ±1.750)μg/(mg•protein), P < 0.01 and(125.399 ±16.677)μg/(mg•protein) vs(29.046 ±2.635)μg/(mg•protein), P < 0.01 respectively]. The calcium deposition was dependent on time and dosage of phosphate treatment. After 72 h culture the osteocalcin in Pi 2.0 mmol/L group was more than that in Pi 1.5 mmol/L group[in supernatant,(1.503 ±10–2 ± 2.601 × 10–3)ng/(μg•protein) vs(2.981 ×10–3 ± 8.382 × 10–4)ng/(μg•protein), P < 0.001], the same was found in osteocalcin mRNA expression[OC/GAPDH, (1.906 ±0.132) vs(0.748 ±0.037), P < 0.001]. Compared to Pi 1.5mmol/L group, bovine smooth muscle cells(BSMC) cultured in media containing Pi 2.0 mmol/L phosphate levels increased calcium deposition[On day 6,(77.187 ±11.692)μg/(mg•protein) vs (25.768 ±1.750)μg/(mg•protein), P < 0.001]. Elevated phosphate treatment of BSMCs also enhanced the expression of the osteoblastic differentiation marker osteocalcin[On day 3, Pi 2.0 mmol/L group vs Pi 1.5mmol/L group,(1.503 ×10–2 ± 2.601 × 10–3)ng/(μg•protein) vs(2.981 ×10–3 ± 8.382 × 10–4)ng/(μg•protein), P < 0.001]. PFA decreased ciacium deposition and osteocalcin expression statistically[Pi 2.0 mmol/L+PFA1.0 mmol/L group vs Pi 2.0mmol/L group, ciacium deposition, (37.729 ±5.899)μg/(mg•protein) vs (77.187 ±11.692)μg/(mg•protein), P < 0.001]; Osteocalcin in supernatant, (4.529 ±10–3 ± 1.250 × 10–3)ng/(μg•protein) vs(1.503 ×10–2 ± 2.601 × 10–3) ng/(μg•protein), P < 0.001; osteocalcin mRNA expression, OC/GAPDH, (0.642 ±0.092) vs (1.89±0.165), P < 0.01]. Conclusion Hyperphosphate may directly promote calcium deposition and the osteocalcin expression of BASMCs. It may be a new explanation for the phenomenon of vascular calcification in hyperphosphatemic conditions. Hyperphosphatemia is an independent factor to stimulate vascular calcification. PFA can inhibit calcium deposition and osteocalcin expression induced by elevated phosphate. PFA may be a new medicine to treat vascular calcification induced by elevated phosphate.

Latent TGF-β-binding protein 2 binds to DANCE/fibulin-5 and regulates elastic fiber assembly

Elastic fibers play the principal roles in providing elasticity and integrity to various types of human organs, such as the arteries, lung, and skin. However, the molecular mechanism of elastic fiber assembly that leads to deposition and crosslinking of elastin along microfibrils remains largely unknown. We have previously shown that developing arteries and neural crest EGF-like protein (DANCE) (also designated fibulin-5) is essential for elastogenesis by studying DANCE-deficient mice. Here, we report the identification of latent transforming growth factor-beta-binding protein 2 (LTBP-2), an elastic fiber-associating protein whose function in elastogenesis is not clear, as a DANCE-binding protein. Elastogenesis assays using human skin fibroblasts reveal that fibrillar deposition of DANCE and elastin is largely dependent on fibrillin-1 microfibrils. However, downregulation of LTBP-2 induces fibrillin-1-independent fibrillar deposition of DANCE and elastin. Moreover, recombinant LTBP-2 promotes deposition of DANCE onto fibrillin-1 microfibrils. These results suggest a novel regulatory mechanism of elastic fiber assembly in which LTBP-2 regulates targeting of DANCE on suitable microfibrils to form elastic fibers.

Anti-Atherogenic Effects of a Mixture of Ascorbic Acid, Lysine, Proline, Arginine, Cysteine, and Green Tea Phenolics in Human Aortic Smooth Muscle Cells

Certain drastic behavioral modifications by arterial wall smooth muscle cells (SMC) have been considered key steps in the formation of atherosclerotic lesions: massive migration of SMC from the media to the intima layer of the vessel, dedifferentiation of SMC to proliferating phenotype, and increased secretion of inflammatory cytokines as a response to inflammatory stimuli. We investigated the anti-atherogenic effects of naturally occurring compounds (ascorbic acid, green tea extract, lysine, proline, arginine, and N-acetyl cysteine) using the model of cultured aortic SMC. Cell growth was measured by DNA synthesis, cell invasiveness was measured through Matrigel, matrix metalloproteinase-2 (MMP-2) secretion was measured by zymography, and SMC secretion of monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) was measured by immunochemistry. Fetal bovine serum-stimulated SMC growth was inhibited by the nutrient mixture (NM) with 85% inhibition at 100 microg/mL. A corresponding concentration of epigallocatechin gallate (EGCG; 15 microM), the most active tea phenolic, produced a significant effect but one lower than NM. NM inhibited aortic SMC Matrigel invasion in a dose-dependent manner and significantly decreased MMP-2 expression. Stimulation of SMC with tumor necrosis factor-alpha significantly increased production and secretion of such mediators of inflammation as IL-6 and MCP-1; addition of 100 microg/mL NM inhibited secretion of MCP-1 and IL-6 by 65% and 47%, respectively. These data suggest that the NM of ascorbic acid, tea phenolics, and selected amino acids has potential in blocking the development of atherosclerotic lesions by inhibiting atherogenic responses of vascular SMC to pathologic stimuli and warrants in vivo studies.

Subcellular Changes in NAD(P)H Redox Caused by Hypoxia in Bovine Coronary Artery Smooth Muscle Cells.

Subcellular Changes in NAD(P)H Redox Caused by Hypoxia in Bovine Coronary Artery Smooth Muscle Cells.

Expression and Functions of Nox Isoforms in Bovine Coronary Arterial Smooth Muscle Cells ‐ Contribution to Extracellular O 2 ·− sLevels

Expression and Functions of Nox Isoforms in Bovine Coronary Arterial Smooth Muscle Cells ‐ Contribution to Extracellular O ₂ ^·− sLevels

Effect of Fluid Shear Stress on Migration of Vascular Smooth Muscle Cells in Cocultured Model

Migration of smooth muscle cells (SMCs) in hyperplasia is thought to have a correlation with blood flow conditions. In this study, the effect of shear stress applied to endothelial cells (ECs) on SMC migration was examined using a newly designed EC-SMC coculture model (CM), in which bovine SMCs and ECs were separated by a collagen layer and a membrane filter. After exposing the CM to shear stresses of 0.5, 1.0, or 1.5 Pa for 48 h, the number of SMCs migrating into the collagen layer was counted. Under static conditions, the migration of SMCs in the CM increased compared with SMCs cultured alone. Shear stress of 1.5 Pa significantly suppressed the SMC migration (p < 0.05) compared with the static CM. Media conditioned with the CM exposed to shear stress of 1.0 Pa (p < 0.05) and 1.5 Pa (p < 0.005) exhibited reduction in activated matrix metalloproteinase-2 (MMP-2) compared with the static CM, as analyzed by zymography. Addition of an inhibitor of nitric oxide (NO) synthase, N (omega)-nitro-L: -arginine methyl ester, to the media inhibited the effect of 1.5 Pa shear stress on SMC migration but MMP-2 activity was unaffected. These results suggest that physiological shear stress has protective roles in atherosclerogenesis.