Abstract
Background and Aims: The angiotensin-(1-7)/angiotensin-converting enzyme 2/Mas receptor axis counter-regulates the detrimental effects of angiotensin II. Beneficial effects of angiotensin-(1-7), including anti-inflammation, oxidative stress reduction, and anti-thrombosis, have been reported. Previous studies documented that ramipril decreased thrombin generation in human hypertension and that the anti-thrombotic effects of captopril and losartan were angiotensin-(1-7)-dependent, suggesting an interaction between thrombin and angiotensin-(1-7). However, it is not clear whether angiotensin-(1-7) can alleviate the endothelial phenotypic changes induced by thrombin. We have previously documented cytoskeleton remodeling, cell adhesion, and cell migration as dominant altered phenotypes in thrombin-stimulated human aortic endothelial cells (HAECs). In this study, we investigated whether angiotensin-(1-7) can modulate thrombin-induced phenotypic changes. Furthermore, we investigated whether NAPDH oxidase 5 (Nox5)-produced reactive oxygen species (ROS) play a significant role in angiotensin-(1-7)-mediated phenotypic changes.Methods: HAECs were pretreated with 100 nM angiotensin-(1-7) for 1 h, followed by stimulation with 2 units/mL thrombin for different times. Immunofluorescent assay, monocyte adhesion assay, wound-healing assay, ROS assay, real-time PCR, Western blotting, and Nox5 siRNA transfection were conducted. HAECs were pretreated with the ROS scavenger N-acetylcysteine (NAC) to determine whether thrombin-induced phenotypic changes depended on ROS production.Results: Angiotensin-(1-7) prevented thrombin-induced actin cytoskeleton derangements, monocyte adhesion, and migratory impairment. Nox5 siRNA transfection confirmed that thrombin-induced Nox5 expression stimulated ROS production and increased HO-1/NQO-1/ICAM-1/VCAM-1 gene expression, all of which were decreased by angiotensin-(1-7). Phenotypic changes induced by thrombin were prevented by NAC pretreatment.Conclusion: Angiotensin-(1-7) prevents actin cytoskeleton derangement, monocyte adhesion, and migration impairment induced by thrombin via downregulation of ROS production. In addition, thrombin-induced Nox5 expression is involved in the production of ROS, and angiotensin-(1-7) decreases ROS through its inhibitory effect on Nox5 expression.
Highlights
Atherosclerosis is a chronic inflammatory disease initiated through endothelial dysfunction caused by several factors, including low-density lipoproteins, hyperglycemia, oscillatory shear stress, oxidants, tumor necrosis factor-α, endotoxin, and thrombin (Steffel et al, 2006; Hess and Grant, 2011)
We identified that cytoskeleton remodeling, cell adhesion, and cell migration are the dominant altered phenotypes in thrombinstimulated Human aortic endothelial cells (HAECs) (Wang et al, 2015)
Thrombin induced a significant derangement of the actin cytoskeleton, with variable cell retraction and increased cortical ring formation
Summary
Atherosclerosis is a chronic inflammatory disease initiated through endothelial dysfunction caused by several factors, including low-density lipoproteins, hyperglycemia, oscillatory shear stress, oxidants, tumor necrosis factor-α, endotoxin, and thrombin (Steffel et al, 2006; Hess and Grant, 2011). Atherosclerotic plaques undergo spontaneous rupture with inflammatory substances such as tissue factor contacting with the circulating clotting factor VIIa. The tissue factor:VIIa complex may activate coagulation factors IX and Xa. Factor Xa further catalyzes the production of thrombin, resulting in the formation of fibrin (Steffel et al, 2006). Previous studies documented that ramipril decreased thrombin generation in human hypertension and that the anti-thrombotic effects of captopril and losartan were angiotensin-(1-7)-dependent, suggesting an interaction between thrombin and angiotensin-(1-7). It is not clear whether angiotensin-(1-7) can alleviate the endothelial phenotypic changes induced by thrombin. We investigated whether NAPDH oxidase 5 (Nox5)-produced reactive oxygen species (ROS) play a significant role in angiotensin-(1-7)-mediated phenotypic changes
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