Effects Of β₁-adrenoceptor On The Growth Of Cultured Vascular Smooth Muscle Cells

OBJECTIVE:To study whether β-adrenoceptor and which β-adrenoceptor subtype involved in the growth of vascular smooth muscle cells (VSMCs) and the relationship between VSMCs growth and the expression of matrix metalloproteinases-2 and its tissue inhibitor of metalloproteinase-2 (MMP-2/TIPM-2).To explore the underlying mechanisms of Pi-adrenoceptor blocker's applications in clinic.METHODS: 1. The New Zealand rabbit aortic VSMCs were cultured and identified with light-microscopy and a-actin and Vimentin immunocytochemistrical staining. 2. First, cultured cells were incubated with isoproterenol(ISO) at different concentrations for different hours to characterize the growth effects of ISO on VSMCs and to determine the optimal experimental conditions. Second, cells were divided into the following four groups. ISO, ISO plus Atenolol(a selectiveB1-adrenoceptor antagonist) ,ISO plus ICI118551(a selective B2-adrenoceptor antagonist) and IMDM group(the control) to characterize β-AR subtype involved in the above effect. 3. Cell proliferation was observed by cell number count, WST-1 and BrdU ELISA simultaneously measurement and Ki67 labelling index. Cell hypertrophy was measured by the cell cross-sectional area and nucleus/plasma ratio through the automatic photomicrographic system. 4. The expression of the matrix metalloproteinase-2(MMP-2) and its tissue inhibitors (TIMP-2) were assessed by immunocytochemistry, using monoclonal antibody to MMP-2 and TIMP-2 respectively.RESULTS: 1 VSMCs cultured from the rabbit thoracic aorta showed the characterizatic of VSMCs including spindle shaped cells arranged in an interwoven and a peak and valley pattern and immunocytochemistry staining positively for a-actin and vimentin.2 After treated with ISO, VSMCs number, DNA synthesis ,metabolic activity and Ki67 labeling index increased significantly in amanner of concentration-and time dependence(P<0.01). And the maximum effect was obtained when the cells treated with ISO at the concentration of 10-5mol/L for 24 hours (P<0.01) . 3 Incubated with ISO at the concentration of 10-5mol/L for 24h, VSMCs cross-sectional area and nucleus/plasma ratio increased markedly (28.0% P<0.05,29.7% P<0.01 respectively). The cellular DNA synthesis, metabolic activity and Ki67 labeling index decreased significantly (48.2% 40.2% and 54.9% respectively) in ISO plus Atenolol group when compared with ISO group(P<0.01); however, there were no change in ISO plus ICI118551 group(P>0.05). 5 The expression of MMP-2 on VSMCs increased significantly in cells at the presence of 1x10-5mol/L ISO for 24 hours(P<0.01). Atenolol not ICI118551 attenuated markedly MMP-2 expression on VSMCs treated with ISO(P<0.01); while there was no significant change in the expression of TIMP-2 on VSMCs in the experimental groups (p>0.05).CONCLUSIONS: The study demonstrates that ISO not only promotes VSMCs proliferation but also induces VSMCs hypertrophy as well as increases expression of MMP-2 on VSMCs in vitro. Theβ1 -adrenoceptor subtype mediates the above effects of ISO in VSMCs.