The Effects And Mechanisms Of Apelin-13Reverse The Impairment Induced Aldosterone

Background and Objective:Primary aldosteronism (PA) is defined by an excessive production of aldosterone, which leads to retention of sodium and loss of potassium, partially suppression of the renin-angiotensin system. PA is a disease characterized by low plasma renin levels, hypertension with or without hypokalemia, alkalosis, and nocturia increased. The excess of aldosterone secretion exerts a progressive damage on the cardiovascular system. Apelin, the endogenous ligand of the APJ receptor, is a recently described circulating peptide. Apelin has many active isoforms and Apelin-13is relatively effective. Apelin has the function of lowering blood pressure, increasing myocardial contraction force, regulating fluid homeostasis and glucose metabolism, promoting proliferation, migration and angiogenesis. Thus, Apelin/APJ system is expected to become a new target for the treatment of primary aldosteronism with cardiovascular and metabolic complications. This study observed the effects and mechanism of Apelin-13reverse the impairment induced by aldosterone in human umbilical vein endothelial cells (HUVECs).Part1:To investigate the effects of Apelin-13reverse the impairment of proliferation and tube formation induced by aldosterone in HUVECs, and then to find out the mRNA which has a significant difference between HUVECs treated with aldosterone and aldosterone plus Apelin-13. Part2:To investigate the effect of Apelin-13reverse the senescence induced by aldosterone in HUVECs and to explore the pathway of this effect by testing the expression of the proteins associated with senescence.Methods:Part1:HUVECs were cultured with RPMI-1640medium, supplemented with10%(v/v) fetal bovine serum. All cells in this experiment were used within10to15passages. Cells were pretreated with aldosterone for24hours, and then were incubated with or without apelin-13for another24hours. Afterwards, the proliferation ability of HUVECs was detected by MTS test and matrigel was used for testing the tube formation. Through the literature retrieval,10vascular endothelial function related mRNA (Angiopoietin-1, Angiopoietin-2, VEGFA, VEGFR-1, VEGFR-2, Tie-2, EGF, FGF-2, eNOS, HO-1, HIF-1α, Sirt-3) were selected and the expression levels of these mRNA were evaluated by qRT-PCR. Furthermore, we downregulate the level of APJ and inhibit the combination of Apelin-13and its receptor APJ in HUVECs by using APJ siRNA. Then MTS test and matrigel were used as above.Part2:Cultured HUVECs in vitro, drug intervention and APJ siRNA was transfected in HUVECs to downregulate the level of APJ were all the same as part1. SA-β-gal staining assay was used for measuring the cell senescence, and we further made use of Western-Blot to detect the expression levels of the proteins (Sirt-l,eNOS)which are related to vascular endothelial senescence function.Results:Part1:The abilities of proliferation and tube formation in HUVECs were inhibited by treating with aldosterone (10nmol/L) for48hours, while cells pretreated with aldosterone for24hours and then incubated with aldosterone and Apelin-13(1umol/l) for another24hours, the situation of proliferation and tube formation improved obviously. qRT-PCR array showed that compared to the aldosterone group, the levels of Angiogenin-1(Ang-1), Angiogenin-2(Ang-2), VEGFR-1, VEGFR-2and Tie-2were significantly upregulated in the group of aldosterone with Apelin-13. And furthermore, the effects of Apelin-13reverse the inhibition of proliferation and tube formation induced by aldosterone could be eliminated by using APJ siRNA to downregulate the Apelin receptor in HUVECs.Part2:The same as part1, aldosterone treatment alone could induce the characteristic of senescence in HUVECs, and Apelin-13as well could decrease the percentage of senescence cells significantly. Meanwhile, the group of APJ siRNA did not exhibit this reverse effect. Western-Blot showed that the expression of the two vascular endothelial senescence related proteins including Sirt-1and eNOS both decreased by aldosterone, then aldosterone with Apelin-13totally abolished the effects of aldosterone on HUVECs senescence related proteins.Conclusion:Part1:Aldosterone treatment could inhibit the abilities of proliferation and tube formation, and this inhibition could be reversed by Apelin-13. Then, the expression levels of Ang-1、Ang-2、VEGFR-1、VEGFR-2、Tie-2were significantly increased in the process of reverse. These mRNA were more likely to play a vital role in the effects of Apelin-13reverse the impairment induced by aldosterone.Part2:HUVECs exhibited the characteristic of senescence apparently by aldosterone, and Apelin-13could decrease the percentage of senescence cells. Then, the pathway of Sirt-1/eNOS may play an important role in the effect of Apelin-13reverse senescence induced by aldosterone.