Reactive oxygen species (ROS) contribute to many aspects of physiological and pathological cardiovascular processes. However, the underlying mechanism of ROS induction by low shear stress (LSS) remains unclear. Accumulating evidence has shown that the angiotensin II type 1 receptor (AT1R) is involved in inflammation, apoptosis, and ROS production. Our aim was to explore the role of AT1R in LSS-mediated ROS induction. We exposed human umbilical vein endothelial cells (HUVECs) to LSS (3 dyn/cm ) for different periods of time. Western blotting and immunofluorescence showed that LSS significantly induced AT1R expression in a time-dependent manner. Using immunohistochemistry, we also noted a similar increase in AT1R expression in the inner curvature of the aortic arch compared to the descending aorta in C57BL/6 mice. Additionally, HUVECs were cultured with a fluorescent probe, either DCFH, DHE or DAF, after being subjected to LSS. Cell chemiluminescence and flow cytometry results revealed that LSS stimulated ROS levels and suppressed nitric oxide (NO) generation in a time-dependent manner, which was reversed by the AT1R antagonist Losartan. We also found that Losartan markedly increased endothelial NO synthase (eNOS) phosphorylation at Ser(633,1177) and dephosphorylation at Thr(495), which involved AKT and ERK. Moreover, the ROS level was significantly reduced by endogenous and exogenous NO donors (L-arginine, SNP) and increased by the eNOS inhibitor L-NAME. Overall, we conclude that LSS induces ROS via AT1R/eNOS/NO.
Excessive proliferation, migration, and antiapoptosis of pulmonary artery (PA) smooth muscle cells (PASMCs) underlies the development of pulmonary vascular remodeling. The innervation of the PA is predominantly sympathetic, and increased levels of circulating catecholamines have been detected in pulmonary arterial hypertension (PAH), suggesting that neurotransmitters released by sympathetic overactivation may play an essential role in PAH. However, the responsible mechanism remains unclear. Here, to investigate the effects of norepinephrine (NE) on PASMCs and the related mechanism, we used 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, the proliferating cell nuclear antigen and the cell counting kit-8 assay to evaluate the proliferation of PASMCs, Boyden chamber migration, and wound-healing assays to assess migration and western blot analysis to investigate protein expression. We demonstrated that the phosphorylation level of the protein phosphatase 2A (PP2A) catalytic subunit (Y307) was higher in PAH patients and PAH models than in controls, both in vivo and in vitro. In addition, NE induced the proliferation and migration of PASMCs, which was attenuated by berberine (BBR), a Chinese herbal medicine, and/or PP2A overexpression. PP2A inhibition worsened NE-induced PAH and could not be reversed by BBR. Thus, PP2A is critical in driving PAH, and BBR may alleviate PAH via PP2A signaling pathways, thereby offering a potential therapeutic option for PAH.
Uncoupled endothelial nitric oxide synthase (eNOS) produces O instead of nitric oxide (NO). Earlier, we reported rapamycin, an autophagy inducer and inhibitor of cellular proliferation, attenuated low shear stress (SS) induced O production. Nevertheless, it is unclear whether autophagy plays a critical role in the regulation of eNOS uncoupling. Therefore, this study aimed to investigate the modulation of autophagy on eNOS uncoupling induced by low SS exposure. We found that low SS induced endothelial O burst, which was accompanied by reduced NO release. Furthermore, inhibition of eNOS by L-NAME conspicuously attenuated low SS-induced O releasing, indicating eNOS uncoupling. Autophagy markers such as LC3 II/I ratio, amount of Beclin1, as well as ULK1/Atg1 were increased during low SS exposure, whereas autophagic degradation of p62/SQSTM1 was markedly reduced, implying impaired autophagic flux. Interestingly, low SS-induced NO reduction could be reversed by rapamycin, WYE-354 or ATG5 overexpression vector via restoration of autophagic flux, but not by N-acetylcysteine or apocynin. eNOS uncoupling might be ascribed to autophagic flux blockade because phosphorylation of eNOS Thr495 by low SS or PMA stimulation was also regulated by autophagy. In contrast, eNOS acetylation was not found to be regulated by low SS and autophagy. Notably, although low SS had no influence on eNOS Ser1177 phosphorylation, whereas boosted eNOS Ser1177 phosphorylation by rapamycin were in favor of the eNOS recoupling through restoration of autophagic flux. Taken together, we reported a novel mechanism for regulation of eNOS uncoupling by low SS via autophagy-mediated eNOS phosphorylation, which is implicated in geometrical nature of atherogenesis.
Our data indicate that H S is a novel regulator of FoxO1 in cardiac cells and provide evidence supporting the potential of H S in inhibiting the progression of DCM.
Low shear stress (LSS) increases degradation of the endothelial glycocalyx, leading to production of endothelial inflammation and atherosclerosis. However, the underlying mechanisms of how LSS diminishes the endothelial glycocalyx remain unclear. We showed that LSS inactivated AMPK, enhanced Na+‐H+ exchanger (NHE)1 activity, and induced glycocalyx degradation. Activation of AMPK prevented LSS‐induced NHE1 activity and endothelial glycocalyx impairment. We further identified hyaluronidase 2 (HYAL2) as a mediator of endothelial glycocalyx impairment in HUVECs exposed to LSS. Inactivation of AMPK by LSS up‐regulates the activity of HYAL2, which acts downstream of NHE1. We characterized a left common carotid artery partial ligation (PL) model of LSS in C57BL/6 mice. The results showed decreased expression of hyaluronan (HA) in the endothelial glycocalyx and decreased thickness of the endothelial glycocalyx in PL mice. Pharmacological activation of AMPK by ampkinone not only attenuated glycocalyx impairment due to HA degradation but also blocked vascular cell adhesion molecule 1 and intercellular adhesion molecule 1 expression increase and macrophage recruitment in the endothelia of PL mice. Our results revealed that AMPK dephosphorylation induced by LSS activates NHE1 and HYAL2 to promote HA degradation and glycocalyx injury, which may contribute to endothelial inflammatory reaction and macrophage recruitment.—Zhang, J., Kong, X., Wang, Z., Gao, X., Ge, Z., Gu, Y., Ye, P., Chao, Y., Zhu, L., Li, X., Chen, S. AMP‐activated protein kinase regulates glycocalyx impairment and macrophage recruitment in response to low shear stress. FASEB J. 33, 7202–7212 (2019). http://www.fasebj.org
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