Effect and mechanism of clazosentan on phenotypic transformation of cerebrovascular smooth muscle cells after subarachnoid hemorrhage in rats

Abstract

Objective To explore the effect and mechanism of clazosentan (Cla) on phenotype transformation of vascular smooth muscle cells (VSMC) after subarachnoid hemorrhage (SAH) in rats. Methods Cell model of SAH was established by VSMC derived from basilar artery and middle artery of SD rats and cultured in the medium containing 10-6mol/L oxyhemoglobin from autologous whole blood. Then 1, 5 and 10 mg Cla were used to treat the cell model. Western blotting was adopted to detect the expression of phenotype transformation marker proteins of VSMC in control group, model group, l-Cla group, m-Cla group and h-Cla group. The content of endothelin-1 (ET-1) was detected by ELISA. CCK-8 assay and cell scratch test were respectively employed to detect cell proliferation and cell migration. The SAH model was established in SD rats, and Cla was injected intrathecally. The neurological function of rats in each group was evaluated 72 h after SAH modeling by modified Garcia neurological deficit scores. The diameter and thickness of basilar artery were detected by HE staining. Western blotting was used to detect the expression of VSMC phenotype transformation marker proteins. The content of ET-1 protein was measured by ELISA. Results When compared with the VSMC cells of the control group, the cells in the model group and Cla intervention groups had significantly lower expression levels of α-SMA and SM-22α, increased levels of MGP and OPN, and elevated ET-1 content and stronger proliferation activity and migration ability at different time points (P < 0.05). Opposite phenomena were observed when the Cla intervention groups compared with the model group (P < 0.05), and the differences were more significant with the increment of Cla dose. The Garcia scores were higher, diameter of basilar artery was smaller, and thickness of vascular wall was thicker in the model group and the Cla intervention groups of SD rats than the control rats, and the expression trends of α-SMA, SM-22α, MGP, OPN and ET-1 were similar to those in the in vitro study (all P < 0.05). In the rats of the Cla intervention groups, the above indicators showed similar trends as the cell model when compared with the rats of the model group (all P < 0.05). Conclusion Cla can inhibit the phenotypic transformation of brain VSMC induced by hemoglobin by promoting the expression of contractile marker proteins and inhibiting the expression of synthetic secretory marker proteins. It can also inhibit the phenotypic transformation of VSMC in SAH model rats and promote the repair of neural function injury by promoting pathological vascular remodeling of basilar artery.

Keywords

• clazosentan
• subarachnoid hemorrhage
• vascular smooth muscle cells
• phenotypic transformation