The Role Of Lymphangiogenesis And Permeability Changes In Regulating Ang Ⅱ-induced Cardiac Remodeling And The Underlying Mechanism

Background:Cardiac lymphangiogenesis and integrity play an important role in the maintenance of myocardial tissue fluid balance.The disturbance of lymphangiogenesis is closely related to cardiac edema and remodeling induced by myocardial ischemia/reperfusion（I/R）injury and pressure overload.However,the role of lymphangiogenesis and integrity in the regulation of Ang Ⅱ-induced cardiac remodeling and the underlying mechanism remain unclear.Objective:To clarify the changes of lymphangiogenesis and permeability during Ang Ⅱ-induced cardiac remodeling in mice,and to elucidate the molecular mechanism for cardiac lymphangiogenesis and permeability changes in regulating cardiac remodeling.This study will provide new therapeutic target and strategy for the prevention and treatment of cardiac remodeling.Methods:1.Animal model and treatmentWild-type（WT）C57BL/6J mice,VEGFR-3^f/-mice and VEGFR-3^f/fmice were used to establish cardiac remodeling with infusion of Ang Ⅱ（1000 ng/kg/min）for 3,7 and 14 days.Epoxomicin（0.58mg/kg/day）was injected intraperitoneally for 14 days to investigate the role of proteasome activity in Ang Ⅱ-induced cardiac remodeling.2.Measurement of blood pressure and cardiac functionThe blood pressure at basal condition and at different time points of Ang Ⅱ infusion were monitored by Tail-cuff method.Cardiac function was evaluated by M-type echocardiography,Cardiac parameters include ejection fraction（EF%）,shortening of short axis（FS%）,thickness of anterior and posterior wall of left ventricle（LVAW,LVPW）,and left ventricular internal diameter（LVID）.3.GravimetryThe water content in the hearts of mice was measured by the dry and wet weight of hearts.The formula was:the water content in the hearts of mice（%）=（wet weight-dry weight）/wet weight of mice heart x 100%4.Histological examinationsHeart tissue sections were stained with hematoxylin-eosin（H&E）,Masson trichrome（Masson）,immunohistochemistry（IHC）,wheat germ agglutinin（WGA）,dihydroethidium（DHE）and immunofluorescence（IF）,respectively.5.Lymphatic permeability assays in vivoAnimals:20μl Evans Blue was injected into the foot pad of mice.The popliteal lymph nodes were removed after 16 hours.After grinding,the absorbance（OD）of the lymph nodes was measured at620 nm using a microplate reader.Lymphatic endothelial cells（LECs）in vitro:The permeability of LECs was measured with Evans blue and FITC-dextran.6.Measurement of proteasome activityThe proteins were extracted from mouse heart or LECs,added with fluorescent substrate.The activities of caspase-like,trypsin-like and chymotrypsin-like were measured using a microplate reader.7.Analysis of gene and protein expressionThe m RNA level of each gene was detected by real-time quantitative PCR.The protein levels were measured using Western blot analysis.8.StatisticsAll data in this study were expressed as mean±standard deviation.Statistical analysis using Graph Pad Prism 9 software.Firstly,the normal distribution of the detected data is analyzed.Independent t-test was used to determine the statistical differences between the two groups if the experimental data were in normal distribution,and Mann-Whitney test was used to analyze the data if they were not in normal distribution.One-way ANOVA and two-way ANOVA were used to analyze the differences between the two groups.P<0.05 showed that there was a statistical difference.Results:1.Ang Ⅱ infusion induces increase of systolic pressure,cardiac remodeling,and lymphangiogenesis.After 3,7,14 days of Ang Ⅱ infusion in WT mice,systolic blood pressure,myocardial hypertrophy,fibrosis,inflammatory response,superoxide production,Lyve1⁺/VEGFR3⁺lymphatics,the ratio of Lyve-1⁺lymphatics to cardiomyocytes as well as levels of VEGF-C and VEGFR3 increased in a time-dependent manner.2.Ang Ⅱ infusion enhances lymphatic permeability and cardiac edema.Compared with saline group,the lymphatic permeability（expressed by the OD value of popliteal lymph nodes）and its related mediator p38 MAPK activity were time-dependenly increased in mice.But the protein levels of MKP5 and VE-cadherin were decreased in a time-dependent manner.In addition,myocardial water content（%）was also time-dependently increased in mice.3.VEGFR-3 knockout reduces cardiac lymphangiogenesis,and aggravates Ang Ⅱ-induced increase of lymphatic permeability,cardiac edema,remodeling,and dysfunction.After 14 days of Ang Ⅱ infusion,compared with VEGFR3^f/fcontrol mice,the systolic blood pressure was significantly higher,but the number of lymphangiogenesis（expressed by the Lyve1⁺/VEGFR3⁺lymphatics,the ratio of Lyve-1⁺lymphangiogenesis to cardiomyocytes,and the expression of VEGFR3,p-ERK1/2 and p-AKT）was markedly lower in VEGFR-3 knockout mice.Accordingly,the degree of cardiac remodeling（including myocardial hypertrophy,fibrosis,superoxide production and the number of CD68⁺macrophages）and the related signal pathways and cardiac dysfunction were further aggravated in VEGFR-3 knockout mice.4.Ang Ⅱ upregulates proteasome activity and lymphatic endothelial cell permeability.Compared with saline group,Ang Ⅱ infusion for 14 days significantly increased both trypsin-like and chymotrypsin-like activities as well as the expression of catalytic subunits（β2i andβ5i）in cardiac tissues.Similarly,Ang Ⅱ treatment also had the same effect on proteasome activities and the expressions ofβ2i andβ5i in LECs.In addition,Ang Ⅱ activates p38 MAPK in LECs,and decreased MKP5 and VE-cadherin protein levels.5.Proteasome inhibitor epoxomicin reduced the permeability of lymphatic endothelial cells In vitro.Compared with saline control group,Ang Ⅱ treatment-induced hyperpermeability in LECs（detected by Evans blue and FITC-dextran）and downregulation of VE-cadherin fluorescence intensity were completely reversed by treatment of AT1R inhibitor or epoxomicin.Meanwhile,losartan or epoxomicin could reverse Ang Ⅱ-induced activation of p38 MAPK and decrease of MKP5 and VE-cadherin protein levels.6.Administration of epoxomicin reduces cardiac edema,remodeling,and dysfunction in mice.Compared with the control group,administration of epoxomicin in mice significantly reduced Ang Ⅱ-induced elevation of proteasome activity and blood pressure,attenuated the degree of cardiac edema,lymphatic permeability（as reflected by decrease of p-p38 MAPK,and increase of MKP5 and VE-cadherin protein levels）and cardiac remodeling（as indicated by hypertrophy,fibrosis,superoxide production and number of CD68⁺macrophages）,and improved cardiac dysfunction.Conclusions:Here,our results reveal that both cardiac lymphangiogenesis and lymphatic hyperpermeability are involved in Ang Ⅱ-induced adaptive hypertrophy.Ang Ⅱ induce LEC hyperpermeability leading to cardiac edema,hypertrophic remodeling,and dysfunction likely through MKP5/p38 MAPK/VE-cadherin signaling pathway.Thus,selective stimulation of lymphangiogenesis and/or inhibition of proteasome activity may be novel therapeutic strategies for treating hypertension-induced cardiac remodeling.Further researches are needed to assess the role of MKP5/p38 MAPK/VE-cadherin signaling in other types of hypertrophic remodeling caused by high-salt or pressure overload.