Mechanisms Of Augmented Vasoconstriction Induced By The Injury Of The Adventitia In The Rat Carotid And Effect Of Drug Intervention

IntroductionBlood Vessel wall is composed of three layers:adventitia,media,and intima. Accumulating evidence now supports the idea that the adventitia plays more than a structural role in vascular function.The role of adventitial in the regulation of vascular smooth muscle function is largely unexplored.Perivascular injury caused by the positioning of a silicone collar around the rabbit carotid artery resulted in the formation of a neointima under a morphologically continuous endothelium.Collar-induced lesions also display functional changes similar to those that occur in early human atherosclerosis including augmented vasoconstriction and hypersensitivity to the vasoconstrictor action of 5-hydroxytryptamine(5-HT).The mechanisms of augmented vasoconstriction induced by the injury of the adventitia remains unclear.Angiotensinâ…¡(Angâ…¡) is a potent vasoconstrictor agent.Although Angâ…¡has been thought to mediate its effects on the vasculature by way of its direct contractile effects mediated by Angâ…¡type 1(AT₁) receptors,it has recently become apparent that vascular superoxide anion production may have a role in this mediation.Previous studies have confirmed a high expression of AT₁ receptor in the vascular tissue of the animal model with the adventitia injury.Moreover,it has been proven in numerous studies that the enhancement of oxidative stress plays a significant role in the pathogenesis of vascular injury.It remained to be determined whether Angâ…¡and its receptor participate in the regulation of vascular tone by the adventitia through oxidative stress mediation.The vascular smooth muscle contraction is regulated by the Ca²⁺ concentration and the Ca²⁺ sensitization of vascular smooth muscle cell(VSMC).RhoA/Rho kinase signaling pathway is the major regulation mechanism for the Ca²⁺ sensitization of VSMC.Previous studies have confirmed that the chronic inflammation stimulation of the adventitia increases RhoA/Rho kinase activity.However,the activation of Rho kinase contributes to adventitia injury and the regulation of the RhoA/Rho kinase signal pathway is still not clear.Tongxinluo is a TCM that is developed on the basis of meridian pathology of TCM,with the function of alleviating the coronaryospasm.Previous studies have confirmed that tongxinluo is able to improve the endiotelum function,regulate the RhoA/Rho kinase signal pathway of VSMCs and restrain vasospasm.It needs further investigation on the effectiveness of tongxinluo to the augmented vasoconstriction induced by the injury of the adventitia and the possible mechanism.ObjectiveThe present study was undertaken to investigate the mechanisms of altering vasoconstriction and vascular reactivity induced by adventitia injury in the rat carotid and the effects of the treatment of tongxinluo on this animal model.MethodsA non-occlusive silicone collar was positioned around the right carotid artery of rats to establish an animal model with adventitia injury in one side of the carotid.The contralateral carotid artery was served as a control(non-operated).Both carotids were harvested for morphometry and analysis of AT₂R and p22^phox receptor expression by immunohistochemistry at days 3(n=4),7(n=4),and 14(n=4).Blood flow of both carotids were determined at days 7(n=10) by using transit-time perivascular flowmeter (Transonic systems,TS420,USA) and vascular responses were examined in response to topical application of 5-HT.Then another 84 Wistar Kyoto rats were randomly assigned to 7 groups of 12 rats each:vehicle,valsartan(30 mg kg^-1 day^-1),fasudil(15 mg kg^-1 day^-1),valsartan plus fasudil,and tongxinluo of different dosage(200 mg kg^-1 day^-1,400 mg kg^-1 day^-1 and 800 mg kg^-1 day^-1 respectively).All treatments were administered by intragastric administration and from 7 days before collar placement until the end of the experiment except fasudil which was administered by intraperitoneal injecting and initiated 3 days before placing the collar.The control group rats were fed with water of equivalent amount.Blood flow and vascular reactivity of both carotids were determined 7 days after placing the collar.The blood from the rat's heart was taken to measure the concentration of Angâ…¡in the serum with the ELISA.Both carotids were harvested for morphometry by light microscopy,for detecting of AT₁ receptor,AT₂ receptor,p22^phox and p-MYPT1^Thr696 protein expression by Western blotting analysis and for determining of AT₁ receptor,AT₂ receptor,p22^phox and RhoK mRNA expression by RT-PCR.ResultsPositioning of the silicone collar around the rat carotid artery for three days and one week leaded to the characteristic histological changes of chronic vasospasm in the form of corrugation of the internal elastic lamina,medial thickening,and significantly reduction in luminal area without intimal proliferation.Two weeks after collar placement,injured artery appeared a diffuse intimal hyperplasia.As compared with LCA,MD of RCA increased by 23.04±5.96%(p=0.009),IELA reduced by 12.15±2.29%(p=0.003) three days after collar placement,while MD of RCA increased by 61.65±10.32%(p＜0.001) and IELA reduced by 45.17±3.84% (p＜0.001) one week after collar placement.Two weeks after collar placement,MD thickened by 31.52±4.56%(p=0.012),LA reduced for by 37.17±4.57%(p＜0.001) and the average NA was 0.2±0.05mm² in RCA.As compared with the control group,the small dosage of tongxinluo had no significant impact on the vascular morphological changes(p＞0.05) while all other intervention inclouding valsartan,fasudil,valsartan +fasudil,medium dosage and large tongxinluo can effectively prevent the chronic vasoconstriction induced by adventitia injure(all with p＜0.05). The collar placement significantly reduced the carotid blood flow (2.79±0.22ml/min vs.3.67±0.26 mi/min compare to non-collared artery,p=0.0002) and raised the sensitivity to topical application of 5-HT.As compared with the control group,the small dosage of tongxinluo has no significant impact on blood flow of RCA and the vessel's reactivity to 5-HT(p＞0.05),while all other intervention inclouding valsartan,fasudil,valsartan +fasudil,medium and large dosage of tongxinluo abolished collar-induced the reduction of blood flow(all with p＜0.05) and normalized the hypersensitivity to 5-HT in collared arteries(all with p＜0.05).Adventitia injure of the rat carotid induced remarkably increasing of serum Angâ…¡concentration(45.21±4.52 pg/ml vs.19.83±0.5 pg/ml to normal rat,p=0.0148). valsartan further increased serum Angâ…¡concentration(89.73±20.44 pg/ml vs. 45.21±4.52 pg/ml.p=0.001),while fasudil inhibited the increasing of serum Angâ…¡concentration(24.73±2.4pg/ml vs.45.21±4.52 pg/ml,p=0.0403).The combination of valsartan and fasudil also further increased serum Angâ…¡concentration (72.81±5.51pg/ml vs.45.21±4.52pg/ml,p=0.0095).Medium or large dosage of tongxinluo reduced serum Angâ…¡level(p＜0.05 as compared with the control group), while small dose of tongxinluo did not affect(p=0.2187) on serum Angâ…¡level.Adventitia injury increased AT₁ receptor,AT₂ receptor,p22^Phox,and Rho kinase mRNA expression,also increased AT₁ receptor,AT₂ receptor,p22^Phox,and p-MYPT1^Thr696 protein expression in collared arteries.The results of immunolhistochemistry had shown that the p22^phox expression of vascular wall increased in all layers three days after collar placement and reached its peak at one week after collar placement.Two weeks after the collar placement,the p22^phox expression in the media and the adventitia reduced to some extent and there were some p22^phox protein expression in the neo-intima.There was almost no AT₂ receptor protein expression in the vascular wall of LCA.Three days and one week after the collar placement,AT₂ receptor was highly expressed in intima,with no expression in media or adventitia.Two weeks after the surgery,there was a little AT₂ receptor expression in the neo-intima.The block of AT₁ receptor with valsartan did not affect the AT₁ receptor expression of RCA,further increased AT₂ receptor expression,restrained the increase of p22^Phox expressions,lowered the Rho kinase expression and reduced the p-MYPT1^Thr696 protein expression.The block of Rho kinase with fasudil did not affect AT₁ receptor and AT₂ receptor expression,decreased the p22^phox expression and significantly restrained the p-MYPT1^Thr696 protein expression without affecting the Rho kinase expression in collared arteries.Tongxinluo did not affect AT₁ receptor expression of RCA,and the large dosage of tongxinluo slightly increased AT₂ receptor mRNA expression,drastically reduced the p22^phox expression,and restrained the synthesis and activity of Rho kinase.This indicates that tongxinluo can alleviate the vasospasm induced by adventitia injury via decreasing the serum Angâ…¡level,mitigating the oxidative stress of the vascular wall, restraining the activity of the RhoA/RhoK signal pathway.Conclusions1,Adventitia injury induced by silicone collar placement resulted in chronic vasospasm and hypersensitivity to 5-HT in the rat carotid artery.Two weeks after collar placement,injured artery appeared a diffuse intimal thickening which resembled early lesions of atherosclerosis.2,Cuff-treatment of the rat carotid artery increased serum Angâ…¡level, upregulated Angâ…¡receptor expression in the vascular well,activated the oxidative stress of the vascular wall,and increased the activity of the RhoA/Rho kinase pathway. Valsartan and fasudil can prevent the collar-induced chronic vasoconstriction and vascular hypersensitivity to 5-HT.3,Tongxinluo can effectively prevented the chronic vasoconstriction and the vascular hypersensitivity to 5-HT induced by the adventitia injury.The tongxinluo played its role in alleviating vasospasm mainly through lowering serum Angâ…¡level, restraining the significantly enhanced oxidative stress induced by the adventitia injury, restrained the synthesis and activity of Rho kinase.