| Objective:Extracellular matrix plays a momentous role in the development and repair of various organs,including post cardiac insult.We identified that nephronectin(NPNT),an extracellular protein,is required in cardiac development and has been implicated in cardiac repair post MI via bioimformation analysis from open access-RNA-seq database.However,the impact of nephronectin on cardiac repair in myocardial infarction(MI)mouse model remains elusive.This study aimed to determine whether NPNT is involved in cardiac repair and cardiac remodelling after MI and,if so,to elucidate the underlying mechanisms.Methods:1.Animals were random Ly allocated into Sham group,MI-PBS group,MI-EV group and MI-NPNT group.AAV2/9-NPNT-c Tn T-GFP virus and a negative control were delivered into mouse hearts 28 days before MI surgery to overexpress the NPNT gene.(1)GFP fluorescence,WB and q PCR techniques were used to determine the NPNT overexpression in hearts.HE stating,CD31 staining and echocardiography test were performed to observe effect of NPNT exert on morphology,capillary density and cardiac function;(2)cardiac ultrasonography were performed on 1day and 28 days.Left ventricular end-diastolic diameter(LVID-d),left ventricular end-systolic diameter(LVID-s),ejection fraction(EF)and left ventricular shortening fraction(FS)values were documented.(3)Masson staining was performed to determine the infarct size in each mouse group and survival analysis were to observe the impact of NPNT on survival post MI.(4)Masson were stained to observe the pathological changes and cardiac fibrosis area in the infarct zone,border zone and remote zone.(5)Angiogenesis and arteriagenesis after myocardial infarction were analyzed by immunostaining.2.(1)In vitro,migration and tube formation experiments were performed in HUVEC stimulated with recombinant NPNT protein;(2)HUVEC were stimulated with recombinant NPNT protein in vitro and collected for immunoblots to determine protein levels and phosphorylation protein levels of EGFR and its downstream target molecules such as JAK2/STAT3.(3)Pretreat HUVEC with an EGFR inhibitor or STAT3 inhibitor and then repeated the above-mentioned migration assay and tube formation assay,along with the EGFR/JAK2/STAT3 signaling pathway.(4)WB was used to detect the changes of EGFR/JAK2/STAT3 and their phosphorylation levels in the infarct border zone in four groups of mice.3.(1)EdU stain and CCK-8 kit were tested in H9C2 overexpressing NPNT;(2)Expression of Cx43 was detected by immunofluorescence in NRCM seeded on plates coated with recombinant NPNT protein in vitro.Results:1.(1)GFP fluorescence,WB and q PCR techniques indicated successful NPNT overexpression,HE staining,CD31 staining and echocardiography showed no impact on morphology,capillary density and cardiac function;(2)The results of echocardiography at 1 day and 28 days after myocardial infarction showed significantly reduced LVID-d and LVID-s,accompanying by increased EF and FS in MI-NPNT group compared with MI-PBS group,suggesting that cardiac function after myocardial infarction in the MI-NPNT group was significantly improved compared with the control group;(3)The infarct size in the NPNT group was significantly reduced compared with the PBS group.Survival analysis of the mice in each group also showed myocardial-specific overexpression of NPNT group improved survival after myocardial infarction;(4)The results of Masson staining suggested that myocardial fibrosis area in the infarct border zone in mice in the MI-NPNT group is significantly smaller than that in the control group;(5)It was found that the angiogenesis and arteriagenesis in the NPNT group were significant increased compared with MI-PBS group.2.In vitro,treatment with NPNT enhanced human umbilical vascular endothelial cell(HUVEC)migration and tube formation;Treatment with NPNT increased the phosphorylation of EGFR and the phosphorylation of downstream genes such as JAK2/STAT3 in endothelial cells.Notably,all these results could be revoked by an EGFR inhibitor or STAT3 inhibitor;WB results showed that myocardial-specific overexpression of NPNT activated the EGFR/JAK2/STAT3 signaling pathway in border zone of infarcted myocardial tissue.3.EdU stain and CCK-8 kit results showed NPNT did not enhance cell viability or proliferation.While NPNT promotes cell intercellular communication in NRCM.Conclusions:The present study found that NPNT enhanced migration and tube formation in endothelial cells in vitro and promoted angiogenesis and improved cardiac function in infarcted mouse hearts in vivo partly through the EGFR/JAK2/STAT3 signalling pathway.Additionally,NPNT did not promote cardiomyocytes proliferation,it can promote the expression of Cx43,indicating a closer information communication.Thuw,our study could pave the way for future research on NPNT and cardiac repair. |
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