Effect of pressure on cultured smooth muscle cells.

Recent studies indicate that smooth muscle cell (SMC) growth and morphology can be modulated by repetitive strain. However, there is very little known about the influence of pressure, without an associated cell stretch, on SMC phenotype. To study this, cultured bovine aortic SMC were grown on a rigid surface and placed in a custom-designed plexiglass pressure chamber with a carefully regulated 5% CO2/air environment. SMC were exposed to either atmospheric, 105 mm Hg or 120/90 mm Hg pressure at a frequency of 60 cycles/min (0.5 s systole, 0.5 s diastole). SMC number was determined on days 1, 3, 5, 7 and 9. SMC exposed to pressure were more elongated and displayed a significant increase in cell number by day 5 which persisted until day 9. Lactate dehydrogenase (LDH) in the conditioned media, an index of cytotoxicity, was not different between the groups at each time point. There was also no difference in pH or pCO2 of the media of SMC in any group. This is the first report of the effects of increased static and pulsatile pressure on SMC in vitro and indicates an increased proliferative rate. We hypothesize that the systemic pressure that the blood vessel is exposed to in vivo may have a significant regulatory influence on the phenotype of the smooth muscle cells which may affect the SMC response to injury or stimuli.

Cyclic strain upregulates nitric oxide synthase in cultured bovine aortic endothelial cells.

In vivo, endothelial cells (EC) are subjected to hemodynamic forces which may influence the production of nitric oxide. This study was designed to examine the effect of cyclic strain on the expression of endothelial nitric oxide synthase (eNOS) in cultured bovine aortic EC. EC were grown on flexible membranes which were subjected to deformation at 60 cycles/min with -5 or -20 kPa of vacuum. This results in an average strain of 6 and 10%, respectively, which is transmitted to the attached cells. Northern blot analysis of total cytosolic RNA demonstrated an increase in eNOS gene expression with both strain regimens but the increase with 10% average strain was greater than that at 6%. Nuclear runoff transcription assays confirmed the induction of eNOS transcripts. Western blot analysis showed an increase in eNOS level after 24 h of cyclic 10% average strain compared with controls or 6% average strain. Immunohistochemical staining of EC for eNOS was increased in the high strain periphery (7-24% strain) of membranes deformed with -20 kPa vacuum. These results demonstrate that cyclic strain upregulates the expression of eNOS transcripts and protein levels in bovine aortic EC thus emphasizing the importance of hemodynamic forces in the regulation of eNOS in vivo.

Cyclic strain increases endothelial nitric oxide synthase activity.

BACKGROUND: Endothelial nitric oxide synthase (eNOS) is an important enzyme that controls the production of a potent vascular smooth muscle relaxing factor, nitric oxide. However, the role of hemodynamic forces (blood pressure, cyclic strain, and shear stress) on the regulation of eNOS has not been fully elucidated. Recently, we showed that cyclic strain increases eNOS gene and protein in cultured bovine aortic endothelial cells (EC). Because an increase in gene transcription and protein synthesis may not necessarily translate into an increase in functional activity, the aim of this study was to determine the effects of cyclic strain on eNOS activity. METHODS: EC were seeded onto plates with flexible bottoms that can be deformed by vacuum and were then exposed to 60 cycles/minute of either 24% maximum strain (-20 kPa vacuum) or 10% maximum strain (-5 kPa vacuum) for 24 hours. eNOS activity was assessed, and nitric oxide production was determined (as nitrite) by the Greiss reaction. RESULTS: Twenty-four percent strain, at 60 cycles/min, but not 10% strain significantly increases eNOS activity compared with stationary controls. Both strain regimens increased nitric oxide (as nitrite) in culture media compared with stationary controls, although nitrite in media of EC exposed to high strain were significantly increased compared with the lower strain. CONCLUSIONS: Cyclic strain increases eNOS activity in cultured bovine aortic EC. These results may indicate the importance of hemodynamic forces in the regulation of eNOS in vivo.

Increased ambient pressure stimulates proliferation and morphologic changes in cultured endothelial cells.

Very little is known about the effects of pressure within the vascular system on EC phenotype. To study this, bovine aortic EC were seeded on rat type I collagen plates (2,000/cm2) and allowed to attach for 24 hours. The cells were exposed to either atmospheric, 40, 80, or 120 mm Hg pressure by placing them in a plexiglass pressure chamber loaded with 5% CO2/air and maintained at 37 degrees C inside an incubator. Chamber pressure was continuously monitored with an amplified voltage transducer connected to a digital monitor. EC were maintained in DMEM supplemented with 10% calf serum and substrates for up to 9 days. The results indicate that EC proliferation is influenced by their ambient pressure. EC subjected in vitro to pressures comparable to mean systemic blood pressures had a significant increase in cell number compared to EC exposed to atmospheric pressures. EC were elongated and appeared to align randomly. We hypothesize that the systemic pressure which the endothelium is exposed to in vivo may have a significant regulatory influence on the ability of the endothelium to proliferate which may affect the endothelial cell response to injury.