Experimental Investigation Of The Relationship Between Endothelial Cell Dysfunction And The Mechanism Of Hypertension

Recent studies have revealed that vascular tone and endothelial cell function plays an important role in the cardiovascular events. The changes of the endothelial function and endothelial vasoactive substances may lead to hypertension. This study based on animal model and cell culture to explore the relationship between endothelial cell dysfunction and hypertension.Objective: (1)To study the role of angiotensin II (Ang II), nitric oxide(NO) and superoxide anion(AngII) in hypertension pathogenesis. (2)To explore the change of serum Ang II, NO and O2- in hypertensive rats induced by nitric oxide-deficiency. (3)To investigate the effects of insulin on human umbilical vein endothelial cells(HUVEC) secreting Ang II, NO and O2-. (4)To investigate the effects of acetylcholine on human umbilical vein endothelial cells(HUVEC) secreting Ang II, NO and O2-. (5)To determine the influences of local Ang II formation on HUVEC generating NO and O2-. (6)To investigate the effects of Ca 2+ on human umbilical vein endothelial cells(HUVEC) secreting Ang II, NO and O2-. (7)The impact of different provocations on IP3/ Ca~2+ signaling system in human umbilical artery smooth muscle cells. (8)The impact of different provocations on IP3/ Ca2+ signaling system in human umbilical artery smooth muscle cells of co-culture.Methods: In the chapter one, we set up 2K1C renal hypertensive rats model, blood pressure, heart rate, left ventricle weight index(LWI) were measured and serum Ang II, NO and O2- level were tested. In the second chapter, rats were induced hypertension by feeding L-NAME, to observe the changes of blood pressure, heart rate, LWI and serum Ang II, NO and O2- level. In the part of cell culture, we investigated the effects of insulin, acetylcholine, Ca~2+, and Ang II of local formation on HUVEC secreting Ang II, NO and O2- level. We also investigated the impact of different provocations on IP3/ Ca2+ signaling system inhuman umbilical artery smooth muscle cells and in human umbilical artery smooth muscle cells of co-culture as well. NO was tested by nitric acid reductase method, SOD activity used xanthine oxidase method, O2* was tested based on fenton reaction, Ang II, IP3 and CaM was used radio-immunity assay.Results: Systolic blood pressure and LVI were higher in comfort and Ins sitimulated group than in that control group (P<0.05). Captopril or irbesartan, and combination of these two drugs decreased significantly compared with comfort group. Heart rate did not change significantly after experiment(P > 0.05). Ang II was higher in comfort and Ins sitimulated group as compared with compared with control group. The concentration of NO was lower in comfort and Ins sitimulated group(P<0.05), NO was higher in captopril or irbesartan, and combination of these two drugs group than that in comfort group(P<0.05). O2" was higher in comfort and Ins sitimulated group than that in control group(P<0.05), O2 was lower in captopril or irbesartan, and combination of these two drugs(P<0.05). SOD activity was lower in comfort group than that in control group(P<0.05), higher in captopril or irbesartan, and combination of these two drugs than that in comfort group(P<0.05). The level of serum insulin was higher in Ins stimulating group, and blood glucose elevated than that in control group(P<0.05). It was suggested that insulin resistance may be existing.In the model of hypertension induced by L-NAME, systolic blood pressure and LVI were higher in L-N group and L-N+Ins group than in that control group (P<0.05) . Captopril or irbesartan, and combination of these two drugs decreased significantly compared with L-N group. Heart rate did not change significantly after experiment(P > 0.05). Ang II was not higher in L-N and L-N+Ins group as compared with compared with control group(P>0.05). The concentration of NO was lower in L-N and L-N+Ins group(P<0.05), NO was higher in captopril or irbesartan, and combination of these two drugs group thanthat in L-N group(P<0.05). O2" was higher in L-N and L-N+Ins group than that in control group(P<0.05), O2" was lower in captopril or irbesartan, and combination of these two drugs(P<0.05). SOD activity did not change in all group than that in control group(P>0.05). The level of serum insulin was higher in L-N+Ins group, and blood glucose elevated than that in control or in L-N group(P<0.05). It was suggested that insulin resistance may be existing.NOS activity, the concentration of NO and Ang II elevated in the HUVEC culture supernatant in the different concentration of insulin, O2" was lower while L-Arg was supplemented(P<0.05), SOD activity did not change compared with control group(P>0.05). NOS activity, the concentration of NO decreased while SOD activity, Ang II and O2" did not change in the HUVEC culture supernatant in the presence of L-NAME. However, in the present concentration of L-NAME, adding different concentration of insulin, Ang II and O2" increased while NOS activity, the concentration of NO and SOD activity did not changed than that in L-NAME. It is suggested that insulin did not improve function of HUVEC damaged by L-NAME. NOS activity, the concentration of NO and Ang II elevated in the HUVEC culture supernatant in the different concentration of acetylcholine, O2" was lower while L-Arg was supplemented(P<0.05), SOD activity did not change compared with control group(P>0.05). NOS activity, the concentration of NO decreased while SOD activity, Ang II and O2" did not change in the HUVEC culture supernatant in the presence of L-NAME. However, in the present concentration of L-NAME, adding different concentration of acetylcholine, Ang II and O2" increased while NOS activity, the concentration of NO and SOD activity did not changed than that in L-NAME. It is suggested that acetylcholine did not improve function of HUVEC damaged by L-NAME. NOS activity, the concentration of NO, Ang II, O2" and SOD activity did not change significantly in the present concentration of Ca2+ in cultured HUVEC supernatant. However, Ca2+adding acetylcholine, NOS activity, the concentration of NO elevated significantly compared with acetylcholine only(P<0.05), Ca2+ boosted up effects of acetylcholine on HUVEC. While Ca2+ adding insulin, NOS activity, the concentration of NO did not change significantly compared with insulin only(P> 0.05), Ca2+ did not boost up effects of insuoin on HUVEC. Cultured HUVEC could convert AngI to Ang II, NOS activity, the concentration of NO, SOD activity decreased, while O2" increased by Ang II. Irbesartan could not block AngI convert to Ang II, but NOS activity, the concentration of NO, SOD activity increased and O2" decreased by treating with irbesartan.NOS activity and the level of NO increased and O2" decreased while SOD did not change significantly in acetylcholine and acetylcholine adding L-Arg in cultured HUASMC supernatant. Atropine could reverse the effects of acetylcholine. NOS activity, the level of NO and Ang II increased while O2' and SOD did not change significantly in insulin and insuoine adding L-Arg in cultured HUASMC supernatant. Atropine could reverse the effects of acetylcholine. Cultured HUASMC could convert AngI to Ang II, NOS activity, the concentration of NO, SOD activity decreased, while O2" increased by Ang II .The level of NO increased and O2" decreased while NOS activity and SOD did not change significantly in SNP in cultured HUASMC supernatant. NOS activity, the concentration of NO, Ang II, O2" and SOD activity did not change significantly in the present concentration of Ca2+ in cultured HUASMC supernatant. NOS activity, the concentration of NO increased while O2" decreased in Ca2+ adding SNP and CCB. The drugs elevated the concentration of NO could decrease IP3 and CaM activity, whereas those decreased the the concentration of NO could increase IP3 and CaM activity.NOS activity and the level of NO increased and O2" decreased while SOD did not change significantly in acetylcholine and acetylcholine adding L-Arg inco-cultured cells supernatant. Atropine could reverse the effects of acetylcholine. NOS activity, the level of NO and Ang II increased while O2" and SOD did not change significantly in insulin and insulin adding L-Arg in co-cultured cells supernatant. Atropine could reverse the effects of acetylcholine. Cultured HUASMC could convert AngI to Ang II, NOS activity, the concentration of NO, SOD activity decreased, while O2* increased by Ang II .The level of NO increased and O2" decreased while NOS activity and SOD did not change significantly in SNP in co-cultured cells supernatant. NOS activity, the concentration of NO, Ang II, O2" and SOD activity did not change significantly in the present concentration of Ca2+ in co-cultured cells supernatant. NOS activity, the concentration of NO increased while O2" decreased in Ca2+adding SNP and CCB. The drugs elevated the concentration of NO could decrease IP3 and CaM activity, whereas those decreased the the concentration of NO could increase IP3 and CaM activity in co-cultured cells supernatant.Conclusions: (1)2K1C renal hypertensive rats, serum Ang II and O2 and LVI elevated, NO decreased and insulin resistance, the imbalance of Ang II, NO and O2" play an important role in hypertension pathogenesis. Captopril and irbesartan can prevent blood pressure and LWI elevated, increase NO and decrease O2". (2) In the model of hypertension induced by L-NAME, serum O2" and LVI elevated, NO decreased and insulin resistance. Captopril and irbesartan can prevent blood pressure and LWI elevated, increase NO and decrease O2'. (3)Insulin may increase Ang II and NO generation, when endothelial cell function was damaged by L-NAME, Ang II and O2" elevated, surprisingly. It is suggested that insulin did not improve endothelial dysfunction. (4) Acetylcholine may increase Ang II and NO generation, when endothelial cell function was damaged by L-NAME, Ang II and O2" elevated, surprisingly. It is suggested that Acetylcholine did not improve endothelial dysfunction. (5)Situ Ang II formation...