Loss Of Jak2Impairs Endothelial Function By Attenuating The Axis Of Raf-1/MEK1/Sp-1Signaling Along With Altered ENOS And STAT Activities

ObjectiveThis study aims to explore the effects of Jak2in endothelial homeostasis and the consequences of Jak2deficiency. Given that Jak2deficiency leads to embryonic lethality, the exact role for Jak2in the regulation of postnatal endothelial function is yet to be fully elucidated. Here we generated a model in which Jak2deficiency can be induced by tamoxifen in adult mice. Using this Jak2gene knockout mouse model, we did thoracic aortic constriction and relaxation assay, explored the effects of Jak2on angiogenesis using Matrigel plug assay. In addition, we created the hindlimb ischemic model and explored the blood flow recovery after ischemic attack. On account of Jak2played important role in endothelial homeostasis and angiogenesis, we explored the mechanisms underlined these phenotypes.Method1. Explore endothelial function and angiogenesis in Jak2deficient mice(1) Jak2fl/fl mice were backcrossed with Cre-ERT2transgenic mice to generate Cre+-Jak2fl/fl mice. Male Cre+/+-Jak2fl/fl mice were intraperitoneally (i.p.) injected with tamoxifen (25mg/kg body weight) for5consecutive days at8weeks of age.(2) Thoracic aortic constriction and relaxation assay, Matrigel plug assay were performed in this Jak2knockout mouse model. The content of hemoglobin was measured in Matrigel plug and CD31staining was performed also. In addition, hindlimb ischemic surgery was conducted in Jak2deficient and control mice. The blood flow recovery was measured and CD31immunofluorescence was performed.2. Explore the mechanism underlying the compromised endothelial function and angiogenesis in Jak2deficient mouse(1) Extract protein from aortic lysate from Jak2deficient and control mice. Examine the expression level of eNOS, p-eNOS, MEK1, p-MEK1, AKT, p-AKT, ERK1/2, p-ERK1/2, p38and p-p38by Western Blot. Examine transcription factor Sp-1level by immunoprecipitation.(2) Treat cultured porcine aortic endothelial cells (PAECs) with TNFa and PD89059 (MEK1inhibitor) or AZD1480(Jak2inhibitor), detect the expression of Raf-1, pRaf-1, MEK1, pMEK1, Sp-1, pSp-1. Electrophoretic mobility shift assay (EMSA) was performed to measure the binding capacity of Sp-1to the promoter of eNOS.Results1. The effects of Jak2on endothelial function and angiogenesis(1) Loss of Jak2attenuates endothelial dependent responsive capacity to vasodilators: Compared to control mice, Jak2deficient mice exhibited diminished relaxation capacity induced by vasodilators.(2) Jak2is essential for endothelial angiogenesis:In Matrigel plug, hemoglobin assay confirmed that the content of hemoglobin in the gel plugs originated from littermate controls was3.3-fold higher than that of Jak2deficient mice (0.99±0.2μg/mg gel vs.0.23±0.1μg/mg gel, p<0.001). Immunostaining revealed that gel plugs derived from control mice showed significantly higher number of CD31-positive cells (ECs) along with more tubular like structures as compared with that of Jak2deficient mice.(3) Jak2deficiency attenuates perfusion recovery after hindlimb ischemic injury: After hindlimb surgery, the blood flow recovery was slower in Jak2deficient mice compared to control mice. Immunostaining data from ischemic muscle tissue showed the number of CD31positive and a-actin positive cells were significantly higher in control mice than that of Jak2deficient mice.2. Mechanisms underline the compromised endothelial function and angiogenesis in Jak2deficient mice(1) Jak2deficiency reduces eNOS and AKT expression:Western Blot data showed the expression of eNOS, p-eNOS, AKT and p-AKT was decreased in Jak2deficient mice aorta.(2) Loss of Jak2affects RAF1/MEK1signaling along with attenuated Sp-1activation: Data collected from Western Blot and immunoprecipitation showed the proteins involved in RAF1/MEK1signaling pathway decreased in Jak2deficient mice. EMSA showed the binding capacity of Sp-1to eNOS promoter was decreased in Jak2deficient mice.(3) Loss of Jak2leads to altered STAT signaling pathway:loss of Jak2also affects STAT3, STAT5and STAT6activation, which could synergize with the axis of Raf-1/MEKl/Sp-1/eNOS signaling to attenuate the capability of ECs to initiate angiogenesis.ConclusionOur study have demonstrated evidence supporting that loss of Jak2leads to endothelial dysfunction as manifested by the altered responsive capability to vasodilators, and impaired angiogenic response capacity along with delayed perfusion recovery after hindlimb ischemic surgery. Mechanistic studies revealed that loss of Jak2attenuates tyrosine phosphorylation of Raf-1, leading to a reduced MEK1activity, which then hampers Sp-1activity to impair eNOS expression. Jak2deficiency also attenuates STAT3, STAT5and STAT6activation, which could synergize with the above Rafl/MEKl/Sp-1/eNOS axis to impair angiogenic response. Our data not only provide novel information for fully understanding of the role of Jak2in the regulation of postnatal endothelial function, but also are an additional asset to evaluate Jak2-based therapeutic strategies against endothelial dysfunction in the settings of cardiovascular disorders.