| Background:Pulmonary arterial hypertension (PAH) is a disease of the pulmonary vasculature, which is characterized by proliferation in the vascular wall. Chronic hypoxia is the most important factor contributing to PAH. Hypoxic pulmonary hypertension (HPH) is characterized by a process of pulmonary vasoconstriction, pulmonary hypertrophy and proliferation of pulmonary smooth muscle cells (PASMCs) and pulmonary vascular remodeling, and it leads to high mortality and accounts for most deaths of chronic obstructive pulmonary diseases (COPD). HPH is involved in endothelial dysfunction and proliferative remodeling in intima and media. In pathological detection, the pulmonary arteries manifest pathological proliferative vascular remodeling that includes cellular proliferation in both the intima and the media and muscularization of the normally thin-walled distal pulmonary arteries.The imbalance of vasoconstrictors and vasodilators is the main cause and the basis of therapies of PAH. Chronic hypoxia causes pulmonary endothelial cell dysfunction and over expression of ET-1 and in HPH models, VEGF and its receptors expressions are increased. Voltage-gated K+ channels (KV channels) are found on many kinds of cells, including PASMCs. They are critical to hypoxic vasoconstriction in HPH. By controlling the membrane potential in PASMCs, KV channels regulate the opening of the voltage-gated L-type Ca2+ channels. When KV channels are inhibited, the influx of Ca2+ causes both PASMCs contraction and increased proliferation rates. On PASMCs, KV2.1 channel is very important and being most extensively studied.Sodium tanshinone IIA sulphonate (STS), a derivative of tanshinone II A, is isolated from the root of Salvia miltiorrhiza known as Danshen in China. Injection of Salvia miltiorrhiza extract was successfully used for cardiac diseases but the exact mechanisms are poorly understood. To date, researches have indicated that Salvia miltiorrhiza has showed properties such as anti-tumor, anti-inflammation, anti-proliferation, and anti- reduced-oxidation etc. Clinical statistics data also suggest that Salvia miltiorrhiza extract imposes a helpful effect on the PAH patients by diminishing the increased pulmonary arterial pressure. However, the underlying mechanisms by which it exhibits its beneficial effect in the treatment of PAH are not well defined. And limited information is available on the effect of STS on HPH in rat models.Objective:The aims of this study were to study the roles of ET-1 and VEGF in the different reaction of pulmonary arteries and systemic arteries to hypoxia, namely the former leads to increased blood pressure whereas the latter not.; to detect their concentrations in pulmonary artery and carotid artery of HPH rats respectively; to study KV2.1 channels expression in lungs and pulmonary arteries from HPH rat; to estimate the effect of STS on cultured PASMCs proliferation induced by hypoxia and to study if STS has effects on KV2.1 channels regulation and ET-1 secretion activity of PASMCs.Methods:HPH rat model was established. And the contents of ET-1 in plasma of pulmonary artery and carotid artery and in homogenates of lung and systemic arteries were determined by radioimmunoassay, and the contents of VEGF in serum of pulmonary artery and carotid artery were determined by ABC-ELISA. PASMCs, isolated from rat pulmonary arteries, were cultured under normal and hypoxic circumstances, respectively. Simultaneously, they were incubated with STS of various concentrations. KV2.1 mRNA and protein in lungs, pulmonary arteries from HPH rats and PASMCs were detected by RT-PCR and western blot.Results:32 rats were divided into 4 groups, i.e. N2w, N3w, H2w and H3w group. HPH rat model was successfully established. The levels of mPAP and the values of [RV/ (LV + S)] were significantly higher in the hypoxic models than those of the control groups. Compared with control groups, the values of ET-1 were both enhanced in carotid artery and pulmonary artery plasma in model groups. In the HPH groups, the level of pulmonary artery plasma ET-1 was significantly lower than that of carotid artery plasma, but just the reverse was ET-1 in control rats. The levels of ET-1 in homogenates of lungs in HPH models were significantly higher than those in homogenates of lungs from control groups, and markedly higher than those in homogenates of systemic arteries from HPH rats. Those results indicated that concentration of ET-1 around pulmonary arteries is significantly higher than that around systemic arteries. In H2w group, there was a significant positive arteriolar-venular (AV) gradient of VEGF levels across the pulmonary circulation. The values of VEGF in serum of pulmonary artery in H3w group were significantly higher than in control groups and H2w group. In serum of carotid artery, the values of VEGF in the HPH models were higher than those in the control groups. KV2.1 expressions in lungs and pulmonary arteries were decreased in HPH models.It was shown that hypoxia induced proliferation, protein synthesis and ET-1 secretion and inhibited KV2.1 channels expression of PASMCs. STS (2, 10, 25 and 50μg/ml) inhibited PASMCs proliferation induced by hypoxia in a dose-dependent manner. 10, 25 and 50μg/ml of STS also inhibited the role of inhibiting Kv2.1 expression by hypoxia in PASMCs. In addition, STS (10, 25 and 50μg/ml) decreased the secretion of ET-1 in cultured PASMCs exposed to hypoxia.Conclusion:1. There is a significant positive arteriolar-venular (AV) gradient of ET-1 levels, which has been interpreted as evidence that lung is an important place to modulate ET-1 levels in HPH rat models; the levels of ET-1 in homogenates of lung were markedly higher than those in systemic arteries from HPH rats. The result that concentrations of ET-1 around pulmonary arteries and systemic arteries are significantly different may be one mechanism accounting for the different reaction of them to hypoxia; ET-1 and VEGF plays important roles in the pathogenesis of HPH. Hypoxia induced proliferation, protein synthesis and ET-1 secretion in cultured PASMCs. 2. KV2.1 expressions in lungs and pulmonary arteries were decreased in HPH models. It indicated that KV2.1 is closely related to the pathogenesis of HPH. Hypoxia inhibited KV2.1 channels expression in cultured PASMCs.3. The beneficial regulatory effects of STS on PASMCs hypoxia-induced proliferation may be through decreasing ET-1 secretion and up regulating KV2.1 channels in PASMCs under hypoxic circumstances. And it makes STS have potential to develop a new method for treatment of HPH.
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