The Role And Molecular Mechanism Of Cardiac Lymphangiogenesis In Pressure Overload Triggers Mice Cardiac Remodeling

Background:Prolonged pressure overload triggers cardiac hypertrophy and frequently leads to heart failure.Vascular endothelial growth factor-C(VEGF-C)and its receptor VEGFR-3 are components of the central pathway for lymphatic vessel growth(also known as lymphangiogenesis),which has crucial functions in the maintenance of tissue fluid balance and myocardial function after ischemic injury.However,its roles in the development of cardiac hypertrophy and dysfunction during pressure overload remain largely unknown.Aims:Cardiac remodeling models were induced by transverse aortic constriction(TAC)using wild-type,VEGFR-3 knockdown or recombinant human VEGF-C_156S mutant mice.We aim to illuminate molecular mechanism of VEGF-C-VEGFR-3 pathway in regulating cardiac lymphangiogenesis,and elucidate the potential role of lymphangiogenesis in cardiac hypertrophy and dysfunction induced by pressure overload,and finally provide new pharmacological targets for prevention and treatment of clinical heart failure.Methods:1.Animal model and treatmentWT or VEGFR-3^f/-mice were subjected to TAC surgery for 1-6 weeks to construct cardiac hypertrophy and heart failure models.Delivering VEGF-C_156Sdose-dependently to WT mice with low-dose(33 ng/g,VEGF-C-L)and high-dose(100 ng/g,VEGF-C-H)by intraperitoneal injection initiated 2 days before TAC and sustained for 6 weeks for the protection of heart failure.VEGF-C_156Sat high-dose(100 ng/g)was intraperitoneally injected after 4 weeks of TAC for the reversion of heart failure.The controls were injected with saline.2.Cardiac function detectionThe mice echocardiograph parameter of fractional shortening(FS%),ejection fraction(EF%),left ventricular anterior wall thickness(LVAW),left ventricular posterior wall thickness(LVPW)and left ventricular inner diameter(LVID)were obtained by the M-mode tracings using a 30 MHz probe after anesthetized with ketamine(0.2 g/kg)and xylazine(0.01 g/kg)by intraperitoneal injection.The mice pressure-volume(PV)relations were performed with a catheter to evaluate in vivo LV function after anesthetized with isoflurane(3%).3.Cardiac water content evaluationThe mice cardiac water content was evaluated by magnetic resonance imaging(MRI)after intraperitoneally anesthetized with ketamine(0.2 g/kg)and xylazine(0.01 g/kg)post 6 weeks TAC,collecting and analyzing the T2 map images by Para Vision 5.1 software.Cardiac wet weight-dry weight method was performed for further analysis of cardiac water content following the equation:Heart water content(%)=(wet weight-dry weight)/wet weight x 100(%).4.Histopathological analysesMice hearts were removed and fixed in 4%paraformaldehyde for 24 h then dehydrated overnight and embedded in paraffin.The 5?m slides were performed pathological staining of Hematoxylin and eosin(H&E),Masson Trichrome Stain,Wheat Germ Agglutinin(WGA)and immunohistochemistry staining.For the immunofluorescence and cardiomyocyte apoptosis staining,cardiac frozen sections were obtained and fixed with 4%paraformaldehyde,dried naturally for further experiments.5.Gene and protein expression analysesTotal m RNA from fresh mice heart was extracted by TRIzol method and was performed reverse-transcribed with 1?g to obtain the c DNA strain.The m RNA expression levels of ANF,collagen I,collagen III,?-SMA and CD31 were analyzed by qPCR.Total protein was extracted from heart using Tissue Protein Extraction Reagent and performed western blot analysis.6.Statistical analysisAll data were presented as mean±standard deviation(SD).Statistical analyses were performed with Graph Pad Prism 8.0.Student's t test was performed to evaluate statistical difference between two groups when data were normally distributed,and Mann-Whitney test was used when data were not normally distributed.The statistical difference between multiple groups were analyzed by One-way ANOVA.p<0.05 was considered statistically significant.Results:1.Relationships between cardiac lymphangiogenesis and cardiac function after TAC surgeryMice cardiac function reached a peak at 2 weeks and then significantly descend at 4 to 6 weeks after TAC.The lung weight to tibia length ratio was higher after TAC indicating pulmonary edema.Cardiac hypertrophy,myocardial fibrosis and the cardiomyocytes apoptosis were time-dependently increase and reached a peak on 6 weeks TAC.The number of microvessels and lymphatic vessels were markedly increased in week 1,and peaked in week 2 then remarkably decreased in weeks 4 to 6.2.VEGFR-3 knockdown destroys cardiac lymphangiogenesisThe number of lymphatic capillaries and ratio of lymphatic-to-cardiomyocyte were significantly reduced in VEGFR-3 knockdown mice after sham or TAC surgery.Cardiac water content was substantially higher in TAC-treated VEGFR-3 knockdown mice.Also,the protein levels of VEGFR-3,p-AKT and p-ERK1/2 were significantly decreased in VEGFR-3 knockdown mice.3.VEGFR-3 knockdown aggravates cardiac hypertrophy and dysfunctionVEGFR-3 knockdown mice aggravated HF after 6 weeks of TAC.Furthermore,cardiac hypertrophy,LV fibrosis and cardiomyocytes apoptosis were significantly exacerbated after VEGFR-3 knockdown.Moreover,VEGRFR-3knockdown promotes recruitment of total macrophage and M1 polarization,which promoted cardiac hypertrophy and dysfunction post TAC.4.VEGF-C_156S promotes cardiac lymphangiogenesis and attenuates cardiac edemaThe VEGF-C_156S treated mice showed a dose-dependent increase in cardiac lymphatic vessels and reduction of cardiac water content(%)and the protein levels of VEGFR-3,p-AKT and p-ERK1/2 after 6 weeks the TAC surgery.In addition,the VEGF-C_156S had no effect on cardiac angiogenesis.5.VEGF-C_156S ameliorates cardiac hypertrophy and dysfunctionAdministration of VEGF-C_156S dose-dependently improved HF,attenuated cardiac hypertrophy and fibrotic response and abrogated cardiomyocyte apoptosis after 6 weeks TAC.Moreover,the VEGF-C_156S has no direct effect on cardiomyocytes hypertrophy in vitro.6.VEGF-C_156S reverses established cardiac dysfunctionVEGF-C_156Sreverses TAC induced cardiac lymphangiogenesis,severe cardiac edema,cardiac hypertrophy,fibrosis and improved cardiac contractile function.Conclusions:The current results indicate that stimulating cardiac lymphangiogenesis through VEGF-C-VEGFR-3 axis activation plays a protective role in the transition from cardiac hypertrophy to heart failure.Highlighting the potential and new approach for prevention and treatment of cardiac hypertrophy and heart failure by therapeutic lymphangiogenesis.