Pathological Cyclic Strain Promotes Proliferation Of Vascular Smooth Muscle Cells Via The ACTH/ERK/STAT3 Pathway

Hypertension disease is one of the most common cardiovascular diseases.It is mainly defined by clinical characterizations with persistent high blood pressure.In the course of high blood pressure disease,an abnormal increase in blood pressure leads to significant changes in the hemodynamic environment,with an increase in periodic tensile strain and local disturbances in shear stress as the main features.At the same time,vascular kinetic studies have shown that the main bearing cells of the forces generated during blood flow have a certain bias,in which the periodic tensile strain due to pulse pressure is mainly caused by the vascular smooth muscle cells(VSMCs)in the middle layer of blood vessels.Related studies have shown that changes and disturbances in this local mechanical environment can directly or indirectly induce vascular remodeling.In the course of vascular remodeling,abnormal proliferation of smooth muscle cells plays an important role.A previous study in the laboratory found that the expression of Adrenocorticotropic hormone(ACTH)in thoracic aorta in Pregnancy hypertension rats was significantly higher than that in wild-type rats.The results suggest that ACTH may play an important role in the pathogenesis of vascular remodeling as animportant molecule in the course of hypertension.In the first part of this paper,we used cell-based cyclic strain loading experiments to investigate whether pathological cyclic high strain regulates the expression of ACTH and its receptor MC2R(Melanocortin 2 receptor)in VSMCs.First of all,in order to further verify the relationship between pathological high cyclic strain and abnormal proliferation of VSMCs,we applied FX-5000 T Strain Unit to apply 5% physiological strain and 15%pathological high strain to VSMCs cultured in vitro.Brud Elisa detect the cell proliferation after cyclic strain loading 12 h and 24 h.At the same time,in order to investigate whether ACTH and its receptor MC2 R act as mechanically sensitive factors,radioimmunity assay and Western blotting be used to detect the protein level of intracellular and culture supernatants of ACTH and the intracellular protein expression of receptor MC2 R after12 h and 24 h of cyclic strain loading.Finally,under the conditions of 15%pathological strain in vitro cultured VSMCs,the RNAi(RNA interference)technology was used to down-regulate the intracellular expression of MC2 R.the interference effect was detected by Western blotting technique.meanwhile,the proliferation detection was performed by Brdu Elisa.Cell proliferation level.Analysis of the results revealed that:(1)Compared to the 5% physiological strain-loaded group,15% of pathological high strain-loaded groups significantly increased the level of cell proliferation after 12 hours and 24 hours of cyclic strain loading.(2)Correspondingly,compared with the physiological group,the intracellular protein level of ACTH in the pathological group was significantly up-regulated after 12 hours of cyclic tensile strain loading,and the protein level in the supernatant was significantly up-regulated after 24 hours.At the same time,the protein levels of receptor MC2 R were significantly up-regulated after12 h and 24 h of high cyclic strain loading,respectively.(3)Under the condition of 15% pathological high cyclic strain loading,the level of cell proliferation was significantly decreased after down-regulating the expression level of receptor MC2 R.The above results suggest that ACTH and its receptor MC2 R as a mechanical sensitivity factor in response to pathological hypertensive strain under hypertensive conditions and regulate the proliferation of VSMC.The second part investigates the specific molecular mechanism of action of ACTH static stimulation on the proliferation of VSMC.First,in order to reveal the regulation effect of ACTH stimulation on the proliferation of VSMC,we used different concentrations of ACTH(10 nM,100 nM,1000 nM)to stimulate VSMCs cultured.Brdu Elisa was used to detect the cell proliferation.Later,in order to investigate the specific mechanism of ACTH-mediated VSMC proliferation,we selected the ACTH(1000 nM)concentration with the most pro-proliferative effect to stimulate VSMCs.Western blotting was used to detect different time points(0 min,15 min,30 min,1 h,3 h,6 h)Phosphorylated protein levels of extracellular signal-regulated kinase(ERK),signal transduction and activators of transcription 3(727 and 705 Site)Expression changes and Brdu Elisa was used to detect cell proliferation.Next,in order to investigate the relationship between ERK and STAT3 727 of phosphorylation and their relationship with cell proliferation,we used different concentrations of PD98059(5 μM,20 μM,50 μM)to antagonize ERK phosphorylation,while the protein expression of p-ERK and p-STAT3-727 was detected by Western blotting.Finally,to investigate therole of ERK and STAT3 phosphorylation in ACTH-dependent cell proliferation,we used PD98059(50 μM)to antagonize ERK with ACTH(1000 nM)stimulating.At the same time,Brdu Elisa was used to detect the cell proliferation.The experimental results showed that:(1)compared with the blank group,the proliferative effect of ACTH with 10 nM,100 nM,and 1000 nM on VSMCs increased with increasing concentration,and ACTH with 1000 nM had the most significant proliferative effect.(2)After stimulation of VSMCs with 1000 nM ACTH,the phosphorylation levels of ERK and STAT3 in the downstream molecules were significantly upregulated at 15 min and 30 min,respectively.(3)Phosphorylation at site727 of STAT3 was found to be dependent on PD98059 antagonistic experiments.Phosphorylation of ERK,and when the concentration of PD98059 was 50 μM,could basically antagonize the phosphorylation of ERK.(4)ACTH stimulated VSMCs and antagonized with PD98059(50μM).After antagonizing the phosphorylation of ERK,it can be partially Eliminating the phosphorylation of 727 sites of ERK and STAT3 downstream of ACTH can reverse the pro-proliferation effect of ACTH.In summary,abnormally high cyclic strain can stimulate the ACTH and its receptor MC2 R to the up-regulation in VSMCs,and regulate the proliferation of VSMCs through p-ERK and p-STAT3 signaling pathway.This study revealed the regulation of cell proliferation by ACTH and its receptor MC2 R in VSMCs,which providing new possible targets for the treatment of hypertension.