Effects Of S-Propargyl-Cysteine On Angiogenesis And The Underlying Molecular Mechanisms

Objective:Conventional revascularization strategies or drug therapies for ischemic heart disease (IHD) are designed for reperfusion of coronary arteries to salvage cardiomyocytes, but occasionally myocardial reperfusion injury can occur because of microcirculatory dysfunction. Therefore, a more microcirculation-friendly strategy should be explored to overcome and compensate the shortcomings of conventional strategies. Angiogenesis is a physiological or pathological process characterized by the sprouting of new blood vessels from existing vessels. Vascular endothelial growth factor (VEGF) is the crucial regulator of angiogenesis that primarily binds to VEGF receptor 2 (VEGFR2). Signal transducer and activator of transcription 3 (STAT3) belongs to an important family of nuclear factors. It plays an important role in cell survival, and is required for myocardial capillary growth after ischemic injury. Hydrogen sulfide is a new gaseous transmitter, and its proangiogenic effect has been disclosed these years. However, hydrogen sulfide cannot be used for clinical therapy because of its gaseous nature. In this work, we investigated the proangiogenic effect of S-propargyl-cysteine (SPRC), a novel water-soluble modulator of endogenous hydrogen sulfide, and elucidated the possible involved mechanisms to provide an experimental basis for angiogenesis-mediated drug therapy for IHD.Methods:MTT assay and EdU incorporation assay were used to determine cell proliferation. Wound healing assay and transwell assay were used to determine cell migration. Matrigel was used to culture cells to form tube. These in vitro assays were performed to investigate possible pro-angiogenic effects of SPRC on primary human umbilical vein endothelial cells (HUVEC). In addition, rat aortic ring model, Matrigel plug model, sponge implantation model, burn wound model and chorioallantoic membranes (CAM) model were performed to investigate possible pro-angiogenic effects of SPRC in vivo under normal condition. Hindlimb ischemia model and myocardial ischemia model were performed to investigate possible pro-angiogenic effects of SPRC in vivo under ischemic condition. In hindlimb ischemia model, laser Doppler perfusion imaging was used to determine blood flow, microangiography and CD31 staining were used to determine vascular density in hindlimb muscles. In myocardial ischemia model, echocardiography was used to determine heart function, TTC and Masson’s trichrome staining were used to determine myocardial infarct and fibrosis size, respectively, and microangiography was used to determine vascular density in heart.We determined the phosphorylated protein levels of STAT3, MAPK family and Akt by Western blot, and then transfected primary HUVEC with STAT3 siRNA to disclose the crucial role of STAT3 in SPRC-induced angiogenesis. We first performed co-crystallization between STAT3 and SPRC to test whether STAT3 could be directly activated by SPRC. Co-immunoprecipitation was used to determine possible interaction between VEGFR2 and STAT3. We also pretreated the cells with SU5416, a selective tyrosine-kinase inhibitor of VEGFR2, and VEGFR2 siRNA to confirm the signal transduction from VEGFR2 to STAT3. STAT3 translocation was observed by Western blot or confocal laser scanning microscopy. Electrophoretic mobility shift assay (EMSA) was performed to determine the STAT3 activity in nuclear extract, and chromatin immunoprecipitation (ChIP) was performed to analyze interaction between STAT3 and promoters of Vegf, Akt, Erk, and Cth.Results:SPRC promoted cell proliferation, adhesion, migration, and tube formation of primary HUVEC and increased angiogenesis in the rat aortic ring model, Matrigel plug model, sponge implantation model, burn wound model and CAM model under normal condition. In hindlimb ischemia model, SPRC promoted angiogenesis after ligation of the left femoral artery to ameliorate ischemic condition, with increased blood flow, collateral vessel density and capillary density. In myocardial ischemia model, SPRC also promoted angiogenesis after ligation of coronary artery, with improved cardiac function, reduced myocardial infarct and fibrosis size, and increased collateral vessel density.In primary HUVEC, STAT3 phosphorylation was significantly induced after SPRC treatment. In addition, SPRC also induced phosphorylation of Erk, JNK and Akt, while reduced that of p38. The critical roles of STAT3 in mediating the proangiogenic effect of SPRC were confirmed by RNA interference. Co-crystallization excluded the possible direct interaction between SPRC and STAT3, whereas co-immunoprecipitation revealed an enhanced interaction between VEGFR2 and STAT3 after SPRC treatment. With SU5416 treatment and VEGFR2 siRNA interference, we also demonstrated possible signal transduction from VEGFR2 to STAT3. Meanwhile, immunofluorescence, EMSA and ChIP showed that SPRC induced the nuclear translocation of STAT3, followed by transcriptional activation of downstream promoters, particularly the Vegf and Cth promoters. We observed a time-dependent increase in protein synthesis of VEGF and CTH several hours after SPRC treatment.Conclusion:We present a novel STAT3-mediated angiogenic mechanism induced by SPRC, a novel modulator of endogenous hydrogen sulfide, and demonstrate the therapeutic potential of SPRC in ischemic disease through the promotion of angiogenesis, which sheds new light on hydrogen sulfide-mediated drug therapy.