Vascular Nox (NADPH Oxidase) Compartmentalization, Protein Hyperoxidation, and Endoplasmic Reticulum Stress Response in Hypertension

Abstract

Vascular Nox (NADPH oxidase)-derived reactive oxygen species and endoplasmic reticulum (ER) stress have been implicated in hypertension. However, relationships between these processes are unclear. We hypothesized that Nox isoforms localize in a subcellular compartment-specific manner, contributing to oxidative and ER stress, which influence the oxidative proteome and vascular function in hypertension. Nox compartmentalization (cell fractionation), O (lucigenin), HO (amplex red), reversible protein oxidation (sulfenylation), irreversible protein oxidation (protein tyrosine phosphatase, peroxiredoxin oxidation), and ER stress (PERK [protein kinase RNA-like endoplasmic reticulum kinase], IRE1α [inositol-requiring enzyme 1], and phosphorylation/oxidation) were studied in spontaneously hypertensive rat (SHR) vascular smooth muscle cells (VSMCs). VSMC proliferation was measured by fluorescence-activated cell sorting, and vascular reactivity assessed in stroke-prone SHR arteries by myography. Noxs were downregulated by short interfering RNA and pharmacologically. In SHR, Noxs were localized in specific subcellular regions: Nox1 in plasma membrane and Nox4 in ER. In SHR, oxidative stress was associated with increased protein sulfenylation and hyperoxidation of protein tyrosine phosphatases and peroxiredoxins. Inhibition of Nox1 (NoxA1ds), Nox1/4 (GKT137831), and ER stress (4-phenylbutyric acid/tauroursodeoxycholic acid) normalized SHR vascular reactive oxygen species generation. GKT137831 reduced IRE1α sulfenylation and XBP1 (X-box binding protein 1) splicing in SHR. Increased VSMC proliferation in SHR was normalized by GKT137831, 4-phenylbutyric acid, and STF083010 (IRE1-XBP1 disruptor). Hypercontractility in the stroke-prone SHR was attenuated by 4-phenylbutyric acid. We demonstrate that protein hyperoxidation in hypertension is associated with oxidative and ER stress through upregulation of plasmalemmal-Nox1 and ER-Nox4. The IRE1-XBP1 pathway of the ER stress response is regulated by Nox4/reactive oxygen species and plays a role in the hyperproliferative VSMC phenotype in SHR. Our study highlights the importance of Nox subcellular compartmentalization and interplay between cytoplasmic reactive oxygen species and ER stress response, which contribute to the VSMC oxidative proteome and vascular dysfunction in hypertension.

Oxidative Stress: A Unifying Paradigm in Hypertension

“…We demonstrated that vascular hypercontractility in stroke-prone spontaneously hypertensive rats (SHR-SPs) involves oxidative and ER stress through Nox4-dependent processes. 18 Inhibition of ER stress with the use of 4-phenylbutyric acid (4-PBA) and STF083010 (an IRE1-XBP1 disruptor) ameliorated vascular dysfunction in SHR-SPs. 18 Treatment of SHRs with 4-PBA reduced blood pressure and improved vascular function and structure by ameliorating ER stress.…”

“…18 Inhibition of ER stress with the use of 4-phenylbutyric acid (4-PBA) and STF083010 (an IRE1-XBP1 disruptor) ameliorated vascular dysfunction in SHR-SPs. 18 Treatment of SHRs with 4-PBA reduced blood pressure and improved vascular function and structure by ameliorating ER stress. 12 Although ERand mitochondria-derived ROS may contribute in part to oxidative stress in hypertension, the upstream driving factor appears to be Nox activation.…”

“…97 Vascular oxidative stress is also associated with altered phosphatase activity that further amplifies kinase signalling and thus contributes to vascular damage in hypertension. 18 In Ang IIeinduced hypertension in mice and rats, expression of Nox subunits, activity of Noxs, ROS generation, and oxidation of signalling molecules are increased. 18 These processes cause oxidative damage of cells and tissues with consequent endothelial dysfunction, renal injury, and cardiovascular remodelling, processes that are attenuated by angiotensin-converting enzyme inhibitors, ROS scavengers, and Nox inhibitors.…”

“…18 In Ang IIeinduced hypertension in mice and rats, expression of Nox subunits, activity of Noxs, ROS generation, and oxidation of signalling molecules are increased. 18 These processes cause oxidative damage of cells and tissues with consequent endothelial dysfunction, renal injury, and cardiovascular remodelling, processes that are attenuated by angiotensin-converting enzyme inhibitors, ROS scavengers, and Nox inhibitors. 98 Moreover, in mice deficient in Nox2 and p47phox, the blood pressureeelevating effect of Ang II is blunted.…”

Endoplasmic Reticulum Stress as Therapeutic Target against Hypertension

“…chaperones 4-phenyl butyric acid (4-PBA) and taurineconjugated ursodeoxycholic acid (TUDCA) are approved by US Food and Drug Administration (FDA) for treating urea cycle disorders and biliary cirrhosis respectively. Nox (NADPH-oxidase) subcellular compartmentalization contributes to oxidative and ER stress, resulting in increased proliferation of vascular smooth muscle cells (VSMCs), protein hyperoxidation and vascular dysfunction in hypertension; and 4-PBA attenuates hypercontractility and vascular reactive oxygen species (ROS) formation in the stroke-prone spontaneous hypertensive rats (SHRs) [9]. Oral administration of 4-PBA lowers blood pressure, reduces vasoconstriction and enhances vasodilation in small mesenteric arteries from SHRs through inhibition of ER stress and oxidative stress [10].…”