Role And Mechanism Of Endothelial Cell-specific Deletion Of CD38 Gene In AngⅡ-induced Hypertension And Vascular Remodeling In Mice

Background and aims:Hypertension is a vascular injury disease characterized by elevated arterial blood pressure including systolic and diastolic blood pressures and accompanied with the lesions of multiple organs such as heart,brain,kidney,retina and so on,and is the leading cause of premature death of cardiovascular disease worldwide.Therefore,it is important to find some effective antihypertensive interventions to reduce hypertension-related morbidity and mortality.Structural and functional abnormalities of blood vessels are the common pathological basis of various cardiovascular diseases,in which endothelium is an important barrier to maintain vascular homeostasis.Adhesion of inflammatory cells,oxidative stress damage and thrombosis caused by endothelial dysfunction play an important role in the occurrence and development of cardiovascular diseases.CD38 is a membrane protein with dual enzymatic activities of ADPR cyclase and NAD⁺hydrolase,and the previous studies from our laboratory showed that CD38 deficiency inhibited angiotensin Ⅱ（Ang Ⅱ）-induced hypertension,vascular remodeling and cardial hypertrophy.However,role and mechanism of endothelial cells in Ang Ⅱ-induced hypertension and vascular remodeling remains to be explored.Therefore,effect of endothelial cell-specific CD38 deficient mice on Ang Ⅱ-induced hypertension and vascular remodeling was evaluated in vivo.In addition,in vitro,effect of knockdown CD38 in primary human umbilical vein endothelial cell（HUVECs）on endothelial cell injury model was investigated.This project aims to elucidate the role and mechanism of CD38 in Ang Ⅱ-induced hypertension and vascular injury in vivo and in vitro,to provide an insight in prevention and treatment of hypertension.Methods:1.Preparation and genotypic identification of endothelial cell-specific CD38deficient mice:CD38^flox/floxmice were mated with vascular endothelium-specific expression of Cdh5^cremice to obtain endothelial cell-specific CD38 deficient(CD38^fl/flCdh5^cre)mice,and the genotyped was identified by PCR with mouse tail DNA.2.Preparation and analysis of Ang Ⅱ-induced mouse hypertension model:1)Twelve CD38^flox/floxand twelve CD38^fl/flCdh5^cremale mice with age of 8-12 weeks were used to prepare a hypertension model by subcutaneous implantation of micro-osmotic pumps at the scapula of the mice and administered with Ang Ⅱ（1500ng/kg/min）for 4 weeks.The blood pressure was monitored weekly by tail-cuff method and the cardiac function was detected by echocardiography at weeks 0 and 4;2)At the end of modeling,the thoracic aorta tissue was collected.Paraffin sections were prepared for HE&EVG staining to detect the thickness of membranes in blood vessels and frozen sections were prepared for DHE staining to examine the amount of ROS in blood vessels;3)HE&Masson&Sirius Red staining of heart tissue was used to detect the cardiac fibrosis;4)Eyeball blood was obtained to detect the liver and kidney functions;5)PAS staining of kidney tissue was used to detect the pathological damage of glomerular basement membrane.The expressions of inflammation-related proteins such as ICAM1,caspase-1,NLRP3 in kidney tissues were detected by Western Blot assay and the m RNA expressions of TNFα,MCP-1 in kidney tissues were detected by Q-PCR assay.3.Preparation of CD38 interfered endothelial cell lines:1)HUVECs were isolated by 1 mg/m L collagenase I digestion.Then CD38 knockdown HUVECs was prepared by lentivirus infection;2)The optimal MOI was determined by infecting HUVEC cells with different titers;3)Western Blot and Q-PCR assays were used to verify the interference efficiency of CD38 in HUVEC after lentivirus（lentiviruses sh RNA1,2,3 and scramble）infection.The lentivirus with the highest interference efficiency was selected for follow-up experiments.4.Preparation and analysis of Ang Ⅱ-induced inflammatory model in endothelial cell:1)Q-PCR assay was used to detect the expression of TNFαin HUVECs after Ang Ⅱ（1μM）stimulation for 48,72 h;2)Western Blot and Q-PCR assays were used to detect the protein and m RNA expressions of CD38 in HUVECs after TNFα（5 ng/m L）stimulation for 0,4,6,8,12 and 24 h;3)Adhesion assay was used to observe the adhesion of monocytes THP-1 to HUVECs in the scramble and lenti-sh RNA-CD38 HUVECs after TNFα（5 ng/m L）stimulation for 6 h;4)Western Blot assay was used to detect the expressions of adhesion molecules such as ICAM1,VCAM1 and inflammatory factor such as IL-1βin the scramble and lenti-sh RNA-CD38 HUVECs after TNFα（5 ng/m L）stimulation for 0,4,6,8,12,24h;5)Western Blot assays was used to detect the expressions of phosphorylated protein of inflammation-related signaling pathways in the scramble and lenti-sh RNA-CD38 HUVECs after TNFα（5 ng/m L）stimulation for 0,15,30,60 min;6)Western Blot assay was used to detect the expressions of ICAM1,VCAM1 and IL-1βin the control and CD38 inhibitor 78C-treated HUVECs after TNFα（5 ng/m L）stimulation for 0,4,6,8,12 and 24 h.5.Preparation and analysis of Ang Ⅱ-induced senescent model in endothelial cell:1)SA-β-gal staining was used to detect the senescent of scramble and lenti-sh RNA-CD38 HUVECs after Ang Ⅱ（1μM）stimulation for 72 h;2)Western Blot and Q-PCR assays were used to detect the protein and m RNA expressions of senescent marker protein P21 followed Ang Ⅱ treatment.Results:1.Endothelial cell-specific CD38 deficiency improves Ang Ⅱ-induced hypertension and vascular remodeling in mice:Blood pressure,vascular media thickness and vascular ROS accumulation were significantly elevated by Ang Ⅱ stimulation in mice,whereas endothelial cell-specific CD38 deficiency ameliorated Ang Ⅱ-induced elevation of blood pressure including SBP and DBP,vascular media thickness and vascular ROS accumulation.2.Endothelial cell-specific CD38 deficiency improves Ang Ⅱ-induced cardiac remodeling in mice:The results showed that Ang Ⅱ remarkably induced cardial hypertrophy and fibrosis in mice.In hypertensive mouse model,compared with the control group,CD38^fl/flCdh5^cremice significantly ameliorated Ang Ⅱ-induced cardial hypertrophy and fibrosis.3.Endothelial cell-specific CD38 deficiency inhibits hypertension-induced injury of renal function in mice:CD38 deficiency significantly inhibited Ang Ⅱ-induced injury of the glomerular basement membrane and the elevation of protein expressions of renal inflammation-associated proteins such as ICAM1,caspase-1,NLRP3 and m RNA expression of TNF-αand MCP-1 in CD38^fl/flCdh5^cremice compared with control mice.4.Lentiviral interference with CD38 inhibits Ang Ⅱ-induced endothelial cell inflammation:The results showed that Ang Ⅱ increased TNFαand CD38 expressions in HUVECs.CD38 knockdown significantly reduced the adherence of TNFα-induced monocytes to HUVECs in lenti-sh RNA-CD38 HUVECs compared with scramble HUVECs.In addition,CD38 knockdown or inhibition of its activity with 78C significantly inhibited TNFα-induced expressions of adhesion molecules such as ICAM1,VCAM1 and inflammatory factors such as IL-1β.Moreover,CD38knockdown markedly inhibited TNFα-induced phosphorylated protein expressions of NF-κB,MAPK,and AKT signaling pathways in lenti-sh RNA-CD38 HUVECs compared with scramble HUVECs.5.Lentivirus interference with CD38 inhibits Ang Ⅱ-induced endothelial cell senescent:Our results showed that CD38 knockdown significantly reduced Ang Ⅱ-induced SA-β-gal formation and the expression of P21 in lenti-sh RNA-CD38HUVECs compared with scramble HUVECs,suggesting that CD38 deficiency will be able to inhibit Ang Ⅱ-induced senescence of HUVECs.Conclusion:Endothelial cell-specific CD38 deficiency ameliorated Ang Ⅱ-induced elevation of blood pressure,vascular remodeling and hypertension-induced cardiac and renal impairment in mice.The mechanism may be related to that CD38 deficiency or inhibition of CD38 function reduced endothelial cell inflammation,inflammatory cell adhesion and migration through inhibiting the NF-κB,MAPK,and AKT signaling pathways,and inhibited endothelial cell senescence via reducing P21 expression.