The Novel Mechanisms Of Frequently Used Antihypertensive Drugs On The MAPKs Phosphorylation, Proliferation/Apoptosis And DNA Methylation Of Vascular Smooth Muscle Cells Induced By Mechanical Stretch Stress

AIMS: Mechanical stretch induced by hypertension on vascular wall can cause vascular remodeling. Traditionally the first-line antihypertensive drugs are regarded as a therapeutic role of treating hypertension through reducing blood pressure. Whether antihypertensive drugs have a direct block effect on vascular remodeling induced by stretch stress or not is still unknown. Diuretic hydrochlorothiazide, calcium blockers nifedipine, angiotensin receptor blockers valsartan and angiotensin converting enzyme inhibitor enalapril which are commonly used in clinical application are selected as experimental drugs. Our revious studies have proved that mechanical stretch can nonspecifically activate almost all of the ion channels, transmembrane proteins and receptors in cell membrane, induce activation of MAPK signaling pathways, increase gene transcription and protein expression, change the state of VSMCs proliferation, apoptosis, migration and inflammation and ultimately speed up the process of vascular remodeling. Thus, this study was designed to investigate the effects of the four drugs(hydrochlorothiazide, nifedipine, valsartan and enalapril) on mechanical stretch stress mediated MAPK signaling pathway,Ki67/Tunel positive ratio and DNA methylation in VSMCs to discover new pharmacological mechanisms of traditional medication and provide new experimental evidence for the clinical application of antihypertensive drugs.Methods:(1)VSMCs were separated from C57BL/6J mice aorta and cultured in vitro. Morphological features of cultured VSMCs were observed under inverted phase contrast microscope and photographed and subsequent morphological analysis and identification, followed by immunofluorescence fluorescence method on smooth muscle cell-specific biological markers of smooth muscle α-actin(SM-α-actin) were identified, recognized as a high purity VSMCs continue after subculture expanded cells, follow-up experiments for research.(2)Cultured quiescent VSMCs were pretreated with hydrochlorothiazide, nifedipine,valsartan and enalapril respectively and subjected to treatment with mechanical stretch stress. Level of p ERK1/2, p JNK1/2 and p-p38 in the treated cells was detected by Western blotting.(3)Cultured VSMC cycle was synchronized by serum starvation,pretreated with hydrochlorothiazide, nifedipine, valsartan and enalapril respectively and subjected to mechanical stretch stress. Ki67/Tunel positive cells were detected by immunofluorescent staining.(4)Cultured VSMC cycle was synchronized by serum starvation,pretreated with hydrochlorothiazide and nifedipine respectively and subjected to mechanical stretch stress. Methylation(5m C positive) was detected by immunofluorescent staining.Results:(1)VSMCs were successfully separated from mouse aorta after primary culture of aortic intima for 7 days. All separated cells presented a long spindle shape, showed a peak-valley shape growth and positively expressed the smooth muscle cells-specific antigen SM-α-actin.(2)Compared with the negative control group, hydrochlorothiazide or nifedipine had no effects on MAPK signaling pathway in quiescent VSMCs. Mechanical stretch stress stimulation significantly increased levels of p ERK1/2, p JNK1/2, p-p38. Mechanical stretch stress-initiated increased levels of p ERK1/2, p JNK1/2, p-p38 were synergistically enhanced by hydrochlorothiazide, and inhibited by nifedipine in a concentration-dependent manner.(3)Compared with the negative control group, hydrochlorothiazide or nifedipine had no effects on Ki67/Tunel positive ratio in quiescent VSMCs. Mechanical stretch stress stimulation significantly increased levels of Ki67/Tunel positive ratio. Mechanical stretch stress-initiated increased levels of Ki67/Tunel positive ratio were synergistically enhanced by hydrochlorothiazide, and inhibited by nifedipine in a concentration-dependent manner.(4)The methylation level of the negative control group was high, hydrochlorothiazide had no effects on methylation level in quiescent VSMCs. But nifedipine decreased methylation level in quiescent VSMCs. Mechanical stretch stress-initiated decreases of methylation levels were inhibited by nifedipine and hydrochlorothiazide.(5)Compared with the negative control group, valsartan or enalapril had no effects on MAPK signaling pathway in quiescent VSMCs. Mechanical stretch stress stimulation significantly increased levels of p ERK1/2 and p-p38. Mechanical stretch stress-initiated increased levels of p ERK1/2 and p-p38 were synergistically inhibited by valsartan and enalapril in a concentration-dependent manner.Conclusions:(1)Hydrochlorothiazide synergistically promotes increased p ERK1/2, p JNK1/2, p-p38 in VSMCs induced by mechanical stretch stress, which can be inhibited by nifedipine in a concentration-dependent manner. Valsartan and enalapril are as same as nifedipine.(2)Hydrochlorothiazide synergistically promotes increased Ki67/Tunel positive ratio in VSMCs induced by mechanical stretch stress, which can be inhibited by nifedipine in a concentration-dependent manner.(3)Mechanical stretch stress stimulation significantly decreased the methylation level, which can be inhibited by nifedipine and hydrochlorothiazide.