The Effect And The Molecular Mechanism Of PI16 On Angiotensin Ⅱ Induced Hypertension And Vascular Remodeling

Background:Hypertension is the most common chronic cardiovascular disease in clinical practice.Vascular remodeling induced by long-term hypertension is an important cause of the progression of hypertension and cardiovascular complications.The vascular remodeling caused by hypertension is mainly manifested in hypertrophy of vascular wall,increased extracellular matrix and fibrosis,and the process involves the proliferation of vascular smooth muscle,inflammatory response,apoptosis and the proliferation of extracellular matrix and other cellular biological processes.Our previous research found a new protein with uncertain function called Peptidase inhibitor 16(PI16),which is highly expressed in vascular tissues.It is not clear whether PI 16 plays a role in vascular remodelingObjective:To investigate the effect and the molecular mechanism of PI16 on angiotensin Ⅱ induced hypertension and aortic vascular remodeling through overexpression of PI16 in angiotensin Ⅱ induced models both in vitro and in vivo,and to find potential new targets for the prevention and treatment of clinical hypertension and vascular remodelingMethods:1.By way of submerged pump,PI16 transgenic mice were infused with angiotensin Ⅱ to set up a model of high blood pressure and vascular remodeling.Blood pressure was measured by caudal arterial method,and thoracic aorta mason staining and Western Blot,PCR techniques were used to detect the level of aortic fibrosis in mice and fibrosis related molecular expression;2.Giving PI16 adenovirus infection to rat aortic smooth muscle cells in vitro for overexpression of PI16,Western Blot and PCR techniques were used to detect the fibrosis related molecular expression in rat aortic smooth muscle cells and to evaluate the role of PI16 in the fibrosis of smooth muscle cells;3.Co-immunoprecipitation,immunofluorescence and Chip techniques were used to explore the potential molecular mechanism of P116’s effect on hypertension and vascular remodeling induced by angiotensin Ⅱ.Results:1.Blood pressure of wild-type mice increased significantly after AngⅡstimulation,while blood pressure and vascular fibrosis of PI16 transgenic mice were significantly lower than those of wild-type mice;2.Compared with cells in control group,overexpression of PI16 significantly inhibited the expression of fibrosis related molecules such as TGF-β and CTGF in rat aortic smooth muscle cells treated with AngⅡ;3.The co-immunoprecipitation results showed that PI16 could directly bind with the specific histone H3K79 methylation transferase DOT1L,increasing H3K79 methylation(including mono-methylation,di-methylation and tri-methylation);4.Further chip analysis showed that PI16 could increase the binding of methylated H3K79 to the promoter regions of ATF4,KLF4 and other transcription factors,activating their expression in nucleic acid and protein levels and inhibiting the downstream AT1 receptor of KLF4;5.After inhibiting the function of DOT1L in vivo and in vivo through small molecular compounds,the protective effect of PI16 on angiotensin Ⅱ induced hypertension and vascular fibrosis was significantly weakened.Conclusion:Overexpression of PI16 can significantly improve the high blood pressure and vascular remodeling in mice induced by AngⅡ.The main mechanism is:PI16 binds with histone H3K79 methylation transferase DOT1L to enhance H3K79 methylation level,inhibiting the expression of AT1 receptor by promoting ATF4,KLF4 gene transcription and expression,and eventually improving the angiotensin Ⅱinduced fibrosis of smooth muscle.