Kallikrein-1 Overexpression Alleviates Age-Associated Cardiac Fibrosis Through Alternatively Regulating Activated Macrophage Polarization

Object: Experimental evidence indicates that the KLK1 protects against fibrosis in various pathogenic states and recruitment and polarization of macrophages contribute to the development of cardiac fibrosis.The present study was designed to determine the effect of KLK1 on cardiac aging and to explore the mechanism of KLK1 regulating the aging related fibrosis.Methods: In the in vivo study,animal experiments are carried out using 3 and 24-month-old SD and h KLK1 overexpression rats fed up at lab animal environment of SPF.All groups were performed for haemodynamics and echocardiography to determine the cardiac function.Then the animals were sacrificed and their hearts were harvested for HE staining,Picrosirius red staining,Masson staining to evaluate cardiac fibrosis.The specimen were also used to detect mitochondria ultrastructure by electron microscope,expression of NAPDH subunits p47-phox,p67-phox,gp91-phox,expression of anti-oxidative stress enzymes SOD-1,SOD-2,ROS level by DHE staining.Finally immunohistochemistry of CD68,immunofluorescence co-localization of CD68 and CD163,Th1 and Th2 cytokines examinations were used to measure macrophage polarization level.In the in vitro research,IL-4 induced Raw264.7,administrated by H2O2 or not,were utilized to evaluate differential capacity of macrophage.In addition,the cell line were cultivated to investigate migration ability administrated by hydrogen peroxide,bradykinin and bradykinin receptor antagonist HOE-140.In the in vitro study bradykinin was used to determine the migration capacity of Raw264.7 induced by different dose of H2O2 administration with or without HOE-140 treatment.Flow cytometry was used to examine the differentiation capacity of M0 towards M2 macrophage under the induction of H2O2 before IL-4 induction.Results: Echocardiographic measurements showed that aging caused significant alternations in cardiac morphology,hypertrophy,and fibrosis in rats,and h KLK1 overexpression protected against aging-induced cardiac dysfunction.Compared with wild-type hearts,the h KLK1 transgene decreased the expression of monocyte chemoattractant protein(MCP-1)and suppressed mitochondrial dysfunction and excess oxidative stress,leading to decreased recruitment and retention of alternatively activated(M2)macrophages and reduced secretion of pro-fibrotic cytokines mediated by the transforming growth factor(TGF)-?1/Smad3 signaling pathway in hearts of aging rats.Furthermore,these cardio-protective effects of h KLK1 overexpression were associated with the Janus kinase(JAK)/signal transducer and activator of transcription 3(STAT3)signaling pathway.H2O2-induced senescence caused differentiation of RAW264.7 cells into M2-type cells and increased the secretion of Th2-type cytokines.Bradykinin treatment relieved the migratory capacity of macrophages induced by H2O2.Thus,MCP-1 secretion and TGF?1 signaling shifted macrophage polarity,and h KLK1 overexpression reduced cardiac fibrosis and improved aging related heart dysfunction through JAK/STAT3 activation,indicating that h KLK1 may alleviate aging-induced cardiac dysfunction.Conclusion: h KLK1 overexpression decreased M2 recruitment and retention in heart tissues by inhibiting p53-PGC-1? mitochondria axis,reducing oxidative stress level and pro-fibrotic MCP-1 expression,subsequently blocking activation of the TGF-?1/Smad3 pathway,decreasing collagen secretion and fibrosis,and thus ameliorating cardiac dysfunction in aging rats.In the in vitro study,H2O2 treatments together with IL-4 assistant with differentiation of M0 towards M2.In addition,bradykinin treatments reverse the enhanced migration capacity of Raw264.7induced by H2O2 treatments while HOE-140 administration recover the migration ability of macrophage.