The Effect And Mechanism Of SIRT3 In Hypertensive Heart Injury And Lymphangiogenesis

BackgroundsHypertension is an crucial risk factor for various cardiovascular diseases,among which cardiac remodeling caused by hypertension is the main target organ injury and one of the common diseases in cardiovascular medicine,but the pathogenesis of hypertensive heart injury is not completely clear.Recently,the study of cardiac microcirculation has attracted people's attention.The function of the lymphatic system begins with capillary lymphatic vessels,which consist of a special layer of lymphatic endothelial cells.The lymphatic vessels are vital in the regulation of myocardial interstitial fluid and immune cell homeostasis.The lymphatic system is closely associated with the development of hypertensive heart injury.Cardiac lymphatic dysfunction may delay the regression of inflammation and immune response and activate immune cells to trigger pro-fibrotic mechanisms.Lymphangiogenesis has been found to improve edema and fibrosis of overloaded heart.Lymphangiogenesis is regulated mainly by VEGFC-VEGFR3 axis.The intracellular pathways of lymphatic angiogenesis are mediated by ERK and AKT.However,in the hypertensive mouse models under the background of RAAS system activation,the changes of lymphangiogenesis in the occurrence and development of cardiac remodeling remain unclear.SIRT3 is an important regulator of hypertensive cardiac dysfunction,hypertrophy and fibrosis.SIRT3 plays an important protective role in maintaining endothelial cells from functional damage induced by Angll,regulating endothelial cell metabolism,and alleviating oxidative stress and senescence of endothelial cells.Lymphatic endothelial cells and vascular endothelial cells have a high degree of homology,so SIRT3 is likely to play an important role in lymphangiogenesis.We propose the hypothesis:SIRT3 can improve cardiac function in Ang? induced hypertensive heart injury mouse models by regulate lymphangiogenesis;SIRT3 protects Ang? stimulated lymphatic endothelial cells through VEGFC-VEGFR3 and ERK pathways.Objectives1.To investigate the effect of SIRT3 modulation of lymphangial regeneration on hypertensive heart injury.2.To explore whether SIRT3 regulates lymphangiogenesis.3.To explore the mechanism of SIRT3 regulating lymphangiogenesis.Methods1.Animal ModelAll experiments were permitted by the ethics boards of Cheelo Hospital of Shandong University.Eight-week-old wild-type(WT)mice were purchased.SIRT3 knockout(SIRT3-KO)mice were purchased from Jackson Laboratory.SIRT3 overexpression(SIRT3-LV)mice were generated by caudal vein injection of lentivirus,and control group mice were generated by injection of negative control lentivirus(NC-LV).Mice were randomized,and ten mice were used in each group.All experimental mice anesthetized were implanted subcutaneously corresponding osmotic pumps beforehand filled with Ang?(1000 ng/kg per min)or saline for 28 days.2.Blood pressure and Echocardiography measurementBlood pressures were measured by a non-invasive tail-cuff system(Softron).Echocardiography was performed with a Visual Sonics Vevo 770 machine,M-mode ultrasound,blood flow Doppler,and tissue Doppler echocardiography were collected from each mouse.3.Histopathology and ImmunohistochemistryThe mice were euthanized,and the tissue was fixed using 4%paraformaldehyde.The heart tissues were cut into 5 ?m paraffin sections.The sections were stained with WGA to measure cardiomyocyte size.Masson's staining and Sirius red staining were used to measure interstitial and perivascular fibrosis.Immunohistochemical LYVE1 staining marks the lymphatic vessels of the heart.4.Cardiac gravimetryWeigh the tissue wet weight of mice ventricle,dry at 65 ? for 5 days,weigh the tissue dry weight.Gravimetry is calculated by the difference between dry and wet weight.5.Cell CultureUsing Ang? treatment at appropriate dose(10-6mol/L)in culture medium for 48 hours.To inhibit expression of SIRT3 in vitro,the LECs were transfected with 50 nM of small interfering RNA of SIRT3.To overexpression SIRT3 in vitro,lentivirus was infected.6.Endothelial cell function testLymphatic endothelial cells were grouped and treated with CCK-8 reagent,and cell activity was detected.Cell proliferation was revealed by EDU staining.Transwell test and wound healing test were used to detect endothelial cell migration.7.Western Blot analysisAppropriate protein samples from mouse ventricular tissue and LECs were run on SDS-PAGE gel and immunoblotted with the primary antibodies overnight at 4?,and incubated with secondary antibodies to exposure in the next day.8.RT-qPCRRNA samples are extracted and the corresponding primer amplification assay is used after reverse transcription.9.Data and statistical analysisWe use GraphPad prism 8 to statistically analyze data and plot charts.The data were expressed as the mean±standard error of mean.Comparisons between the two groups were performed using a t-test of independent samples,and comparisons between multiple groups were tested by one-way ANOVA.p<0.05 was considered statistically significant.Results1.Model of hypertensive heart injuryHomozygous SIRT3 knockout mice and lentivirus overexpression mice were successfully constructed.Mice were all infused with Ang? or saline for 28 days.As demonstrated,Ang? induced systolic blood pressure over 140 mmHg.SIRT3 did not affect the baseline level nor the changes in blood pressure induced by Angll.Compared with the control group,SIRT3 expression level in experimental group was significantly downregulated.2.SIRT3 deficiency aggravated Ang? induced hypertensive heart injuryCompared with control,the mice in the Ang? group had significantly reduced cardiac function,elevated levels of myocardial injury markers,increased myocardial hypertrophy and fibrosis levels,and SIRT3-KO aggravated the above changes.3.SIRT3 deficiency aggravated lymphangiogenesis insufficiency and cardiac edema in Ang? induced hypertensive heart injuryCompared with control,the mice in the Ang? group had elevated water content and lower lymphatic vessel density,and SIRT3-KO further aggravated the above changes.4.SIRT3 regulated cardiac lymphangiogenesis through VEGFC-VEGFR3 axisCompared with control,the mRNA and protein expressions of VEGFR3 and LYVE1 in the Ang? group were reduced,the serum VEGFC level was reduced,and the expression of the VEGFC-VEGFR3 axis and LYVE1 was further downregulated by SIRT3-KO.5.SIRT3 alleviated functional injury of lymphatic endothelial cellsThe proliferation function,migration function and cell activity of lymphangial endothelial cells after Ang? treatment were reduced,SIRT3 knockdown further aggravated the above changes of lymphatic endothelial cells,and SIRT3 overexpression improved cell function.6.SIRT3 promote lymphatic marker expression in LECs and regulated ERK pathwayThe expression of VEGFR3 and the phosphorylation level of ERK after Ang? treatment were reduced,which was more pronounced in the SIRT3 knockout group,and sirt3 overexpression alleviated this change.After inhibition of the ERK pathway,the proliferation of lymphatic endothelial cells is reduced.7.SIRT3 overexpression upregulated cardiac lymphangiogenesisCompared with the control group,the Ang? group of mice had insufficient lymphangiogenesis in cardiac tissue and aggravated edema,and compared with control group,the SIRT3 overexpression group promoted cardiac lymphatic vessel regeneration and alleviated cardiac edema.8.SIRT3 overexpression reversed hypertensive cardiac injurySIRT3 overexpression relieved Ang?-induced cardiac fibrosis,ventricular hypertrophy,and improved cardiac function compared with the negative control group.Conclusions1.SIRT3 alleviates hypertensive cardiac injury by regulating lymphangiogenesis.2.SIRT3 can regulate lymphatic endothelial cell activity,proliferation,and migration.3.SIRT3 promotes lymphangiogenesis via VEGFC-VEGFR3 axis and ERK pathway.