The Proangiogenic Effects Of Hydrogen Sulfide In Different Ischemia Models

Hydrogen sulfide （H₂S）, a colorless gas with the characteristic smell of rotten eggs, is considered to be toxic by inhibiting the activity of cytochrome oxidase of the oxidation chain in mitochondrion, and then affects the activities of central nervous system and respiratory system. By the year of 1989, the discoveries of endogenous H₂S in the brains of rats and human beings as well as three enzymes involving H₂S production suggest that H₂S may have some important physiological functions. In the nervous system, H₂S can function as a neuromodulator and a neurotrophin. In a neuron, physiological concentrations of H₂S specifically enhances the activity of N-methyl-d-aspartate （NMDA） receptors and facilitates the induction of hippocampal long-term potentiation, affecting the physiological process of memory. In cardiovascular system, H₂S can relax smooth muscle cells by activating ATP-dependent K+channels （KATP） and decrease the blood pressure. In a vivo model of myocardial ischemia-reperfusion, H₂S can decrease infarct size and preserve left ventricular function. In digestive system, H₂S accelates the uptakes of blood glucose by fat cells and skeletal muscle cells and improves glucose tolerance and insulin sensitivity. As a small molecule of gas, H₂S freely permeables to membrane, endogenously and enzymatically generates, effect physiological functions. Together with nitric oxide （NO） and carbon monoxide （CO）, it has been regarded as the third gasotransmitter.Angiogenesis, the formation of new capillaries from pre-existing blood vessels, is a process of the degradation of extracellular matrix, endothelial cell proliferation, migration, and the formation of mature vessels. It occurs in many physiological and pathological processes, such as embryonic development, wound healing and tumor growth. Our lab has provided the first evidence on the proangiogenic effect of exogenous H₂S by showing that H₂S promoted angiogenesis in vitro by promoting endothelial cells （EC） growth, migration and tube-like structure formation, as well as in an in vivo mouse model of a Matrigel plug assay. The proangiogenic effect of H₂S was also confirmed by Szabo C on a chicken chorioallantoid membranes （CAM） model.Ischemic diseases with its high morbidity and high mortality, are caused by the pathological enviromental blood flow decrease or interruption, resulting in the loss of supplement of oxygen and nutrients to the functional areas of the vessels. Conventional revascularization strategies are designed for reperfusion, neglecting the fact that occasionally reperfusion injury can occur because of microcirculatory dysfunction. So comes the therapeutic angiogenesis, which transfer the angiogenic factors （eg. VEGF, bFGF） or their genes or stem cells into the ischemic areas to promote collateral vessel formation and regional blood flow.It’s known that under physiological conditions, H₂S can promote angiogenesis, but it is more important to investigate whether H₂S can exhibit its effect of proangiogenesis in pathological states, such as tissue ischemia. If true, what is the underneath mechanism. In the present research, we mainly focused on the proangiogenic role of H₂S in two kinds of tissue ischemia models, providing a more microcirculation-friendly strategy to therapeutic angiogenesis.Firstly, the proangiogenesis of H₂S in a rat model of hind limb ischemia was investigated. NaHS 20 and 50 μmol/kg/day treatments for 4 weeks significantly promoted regional blood flow by fluorescent microsphere detect; As showed in the microangiography assay, there were more angiographically visible collateral vessels with typical "corkscrew" appearance in NaHS 50 and 100 μmol/kg/day groups; Capillary density visualized by immunohistochemical staining showed that NaHS treatment was associated with capillary density, while a dose of 50 mmol/kg/d had the most apparent effect. NaHS 50 μmol/kg/d treatment promoted PCNA expression in ischemic area, suggesting the proliferation of vascular endothelial cells and skeletal muscle cells. Furthermore immunohistochemical staining demonstrated that in ischemic hind limb muscles, VEGF expression was mainly localized in the skeletal muscle cells, while VEGFR2 and phospho-VEGFR2 expression were localized in the neighboring vascular endothelial cells; NaHS treatment could increase the expression of VEGF and phosphorylation of VEGFR2, but had no effect on the expression of VEGFR2 detected by immunohistochemical staining and western blot. At the same time, NaHS treatment could enhance the phosphorylation of Akt in ischemic hind limb muscles. Above all, NaHS treatment could promote angiogenesis with improvement of regional blood flow in ischemic hind limb, and this promotion may be mediated by increasing VEGF expression in skeletal muscle cells and enhancing the phos phorylation of VEGFR2 in the neighboring vascular endothelial cells.Secondly, a myocardial infarction model in mice was successfully setted up. Many methods were applied including ECG, echocardiography, myocardial enzyme spectrum assay, Evans’Blue-TTC staining and HE staining to detect whether the operation was successful. When the Left Anterior Descending （LAD） was occlused, the anterior wall of the heart turned pale with an elavation of ST; In the echocardiography, the values of EF and FS were decreased; In myocardial enzyme spectrum assay, the concentrations of LDH, CK and CK-MB were increased. And an infarcted area was easy to be identified by Evans’Blue-TTC staining. Furthermore, HE staining showed that there were many fibrous tissues in the infarcted areas after myocardial infarction for 2 weeks. In conculsion, the myocardial infarction model on mice was successfully built up.Thirdly, the proangiogenic effect of H₂S in a mouse model of myocardial infarction was studied. NaHS 50 mmol/kg/d treatment significantly increased the capillary density in the area of risk detected by immunohistochemical staining. And different concentrations of NaHS treatment could decrease the mortality of mice after myocardial infarction and ameliorate the cardic functions examed by echocardiography and myocardial enzyme spectrum assay. But all improvements by NaHS treatment was not mainly mediated by VEGF, considering that the expression of tissue VEGF was not significantly changed after NaHS treatment detected by ELISA. While in western blot, NaHS treatment significantly increased the expressions of three enzymes that catalyzing the production of H₂S, and increased the concentration of H₂S in plasm suggesting that the proangiogenic effect of H₂S in a mouse model of myocardial infarction may be regulated by the expressions of the three enzymes and maintain the concentration of H₂S.Fourthly, the research of the endogenous H₂S on the proangiogenic effect was carried out in CSE knock-out mice. The results revealed that there were less capillaries in the CSE knock-out mice after myocardial infarction by immunohistochemical staining. Although the expressions of CBS and 3-MST were increased in CSE knock-out mice after myocardial infarction, the H₂S concentration in the plasm was lower in CSE knock-out mice before or after myocardial infarction compared to wild type mice. ELISA results showed that the VEGF expression in CSE knock-out mice was decreased after MI. All these results suggested that endogenous H₂S produced by CSE took important part in the proangiogenic effect in the mice myocardial infarction model.Lastly, the protection of H₂S on the primary cardiomyocytes in hypoxia was investigated. After 6 hours’hypoxia, the cardiomyocytes showed low viability and great damage with the detection of LDH and CCK8 kits. While NaHS treatment could change this situation. Both Western blotting and ELISA assay displayed the increase of VEGF expression in the cardiomyocytes and the secretion in the medium after in hypoxia for 6 hours, and H₂S didn’t affect the VEGF expression after hypoxia. The expressions of CSE and 3-MST didn’t change significantly while CBS increased after hypoxia. H₂S didn’t changed the expressions of CBS and 3-MST, but it could significantly increase the expression of CBS. Meanwhile, HPLC-MS revealed that the concentration of H₂S in the medium dropped after hypoxia for 6 hours and increased by H₂S treatments. CSE SiRNA could significantly knock down the expression of CSE. After CSE was knocked down, the expressions of CBS and 3-MST increased. At the same time, H₂S concentration in the medium dropped significantly. Cardiomyocytes after CSE SiRNA treatment displayed worse damage by LDH and CCK8 detects, suggesting the protection of endogenous H₂S on the cardiomyocytes in hypoxia.In conclusion, NaHS treatment could promote angiogenesis in ischemic hind limb mediated by VEGF paracrine; NaHS treatment could promote angiogenesis and improve heart function in a mouse model of myocardial infarction partially mediated by enhancing expressions of the three enzymes; Endogenous H₂S also took part in the promotion; NaHS treatment could protect the cardiomyocytes from hypoxic damage, and endogenous H₂S may take an important role this protection.