Peptidase Inhibitor 16 Attenuates Hypertension Related Aortic Remodeling By Regulation Of Angiotensin Ⅱ Type1 Receptor-Dot1L Signaling

Part I:Peptidase Inhibitor 16 Attenuates Aortic Remodeling by Interacting with Angiotensin II Type 1 ReceptorObjective:Hypertension has become the chronic noncommunicable disease with the highest prevalence in the world,and is a common risk factor for many clinical cardiovascular diseases with high incidence.The aortic stiffness caused by hypertension is complementary to hypertension itself,but its mechanism is still unclear.The role of peptidase inhibitor 16（PI16）in hypertension and aortic remodeling is not fully understood.This study aims to explore the role of PI16 in hypertension and aortic remodeling,as well as the regulation of angiotensin II type 1 receptor（AT1R）by PI16.Materials and methods:The plasma levels of PI16 of hypertensive and non-hypertensive patients were measured by ELISA.PI16 transgenic（Tg）mice and PI16-knockout(PI16^-/-)mice were generated,and an Ang II infusion mouse model was used for in vivo study.The primary rat aortic vascular smooth muscle cells（RAVSMCs）were extracted for in vitro study.The interaction between PI16 and AT1R was detected by co-immunoprecipitation（CO-IP）,and expression vectors containing fragments of PI16 amino acids was constructed to explore the binding domain between PI16 and AT1R.The effects of PI16 on AT1R ubiquitination were investigated using tandem ubiquitin binding entities（TUBES）.The transformation study of small peptide in binding domain of PI16 and AT1R was carried out using spontaneously hypertensive rats（SHR）and Ang II infusion mouse model.Results:Plasma PI16 level was decreased in hypertensive patients and negatively correlated to pulse wave velocity（PWV）,a key index for arterial elasticity.Aortic thickness,collagen deposition and the levels of fibrosis-associated markers,including connective tissue growth factor（CTGF）and collagens,were significantly reduced in PI16 transgenic（Tg）mice compared with WT mice,but aggravated in PI16-knockout(PI16^-/-)mice after Ang II treatment.Mechanistically,PI16 directly binds to AT1R via amino acids 41–50.Overexpression of PI16can promote the interaction of AT1R andβ-arrestin.The increased recruitment ofβ-arrestin thus promotes the internalization of AT1R and inhibits the ubiquitination of AT1R,finally down-regulates the expression of AT1R.Further a synthesized peptide of PI16 in binding domain（41-50 aa）with AT1R inhibited hypertension and aortic remodeling of both SHR and Ang II infusion mouse model.Conclusion:PI16 interacts with AT1R through amino acid 41-50 to down-regulate the expression level of AT1R,thereby reducing hypertension and vascular remodeling.Pi16-AT1R-β-arrestin axis may provide a potential therapeutic target for hypertension and aortic remodeling,and the short peptide（41-50 aa）of PI16 and AT1R binding domain may become a potential drug for the clinical treatment of hypertension and large artery stiffening.Part Ⅱ: Epigenetic Regulation of Aortic Remodeling by Interaction of Peptidase Inhibitor 16 with Disruptor of Telomeric Silencing 1-likeObjective: Aortic remodeling contributes to aortic disease and high prevalence of cardiovascular events.However,mechanisms of epigenetic regulation for aortic remodeling are not fully understood.Here,we aimed to explore the epigenetic regulation of histone H3 Lys79（H3K79）methylation by peptidase inhibitor 16（PI16）in aortic remodeling.Materials and methods: Coimmunoprecipitation（CO-IP）and mass spectrometry were conducted to examine the interaction between PI16 and disruptor of telomeric silencing 1-like（Dot1L）.RNA-sequencing was used to address the mechanism by which PI16 affected vascular smooth muscle cells（VSMCs）phenotypic transition.H3K79 methylation downstream target genes were confirmed by chromatin immunoprecipitation（Ch-IP）assays.Results: PI16 directly bound to methyltransferase Dot1 L via amino acids 116–128.Overexpression of PI16 prevented the decline of Dot1 L after Ang II treatment through inhibiting Dot1 L ubiquitination and resulted in preserved H3K79 methylation.RNA-seq revealed that antifibrotic factor Krüppel-like factor 4（KLF4）was a key downstream gene regulated by overexpression of PI16.Preserved H3K79 methylation by PI16 after Ang II treatment maintained the level of KLF4 preventing the fibroblastic switching of VSMCs.We further synthesized a small peptide consisting of 116-128 amino acids of PI16,which resembled the effects of full-length PI16 on blood pressure and aortic fibrosis in spontaneous hypertensive rats.Conclusion: The preserved H3K79 methylation by 116-128 amino acids of PI16 binding with Dot1 L attenuates aortic thickening and fibrosis.The PI16-Dot1L-H3K79 axis may therefore provide a novel therapeutic target for hypertension and vascular fibrosis.