Study The Mechanism Of A Small Molecule RhoA Inhibitor, Hl07, On Vasodilation

The compound HL07 is one of the first-in-class small molecule RhoA inhibitors that we have been synthesized through computer-aided virtual screening, chemical syntheses, molecular biology, structural biology, and etc.Objective: 1) To observe the effect of HL07 on agonist-induced vasoconstriction; 2) To explore the possible mechanism of HL07 on vasodilation ; 3) To investigate the effect of HL07 on cerebral vasospasm (CVS) following subarachnoid hemorrhage (SAH) and to discover a suitable disease model for HL07.Methods: 1) We used Isometric tension determination to monitor the changes of vascular tension caused by HL07; 2) We applied G-LISA method, quantitative competitive RT-PCR assay to measure RhoA activation and RhoA mRNA level in the human cerebrovascular smooth muscle cells (HBVSMCs); 3) We established the rat SAH-CVS model using the method of double-heorrhage by injecting autologous arterial blood into the prechiasmatic cistern, and observed the influence of HL07 on the morphology and the perimeter of inside diameter of basilar artery (BA). All SD rats were divided into 5 groups randomly: Normal group, SAH group, SAH+Fasudil group, SAH+HL07 group, SAH+DMSO group (n=9 in each group).Results: 1) In isolated endothelium-denuded rat thoracic aorta (TA) rings, HL07 (0~180μM) caused dose-dependent relaxation on PE-induced contraction (IC50=156.93μM), but far more weak relaxation on KCl-induced contraction; Furthermore, in the presence of nifedipine and thapsigargin (Nif/TSG), HL07 showed a greater relaxation with IC50=149.52μM (P<0.05); On the other hand, HL07 displayed better relaxation with IC50=134.97μM on PE-induced contraction in pulmonary artery (PA) rngs than in TA rings (P<0.05); 2) HL07 potently blocked PE-induced RhoA activation in dose-dependent manner in HBVSMCs (P<0.05) (at the concentration of 0.25, 2.0, 10.0μM,the inhibitory rate are 30.88±5.77%, 41.87±7.19%, 64.09±4.83%, respectively); But HL07 had no significant effect on RhoA mRNA level (P>0.05); 3) The comparison of the perimeter of inside diameter of BA in each group: there were significantly statistical difference between SAH group (215.14±19.33μm) and normal group (269.80±13.12μm) (P<0.05), and SAH+Fasudil group (264.78±14.90μm) (P<0.05), and SAH+HL07 group (269.68±33.61μm) (P<0.05); There were no significantly difference between SAH group and SAH+DMSO group (222.93±16.10μm) as well as between SAH+HL07 group and SAH+Fasudil group.Conclusion: 1) HL07 caused significantly relaxation on PE-induced contraction in artery rings. 2) The mechanism of the relaxation might be contributed to the inhibition of HL07 on RhoA/Rho-kinase pathway through blocking RhoA activation rather than regulating RhoA mRNA level. 3) HL07 could relieve the CVS of the BA after SAH. In summation, the results indicate that HL07 can cause inhibitory effect on bioactive substances induced vasoconstriction; its inhibition on RhoA activation could be one of the mechanisms of HL07 on vasodilation.