To be, or nox to be, endoplasmic reticulum stress in hypertension

Precise Nox localization for specific reactive oxygen species signaling in the vasculature

In the vasculature, reactive oxygen species (ROS) play key roles in signal transduction related to contraction, relaxation, hypertrophy, proliferation, migration, and cell death and the NADPH oxidase (Nox) isozymes are a major source of these ROS¹. Over the past 20 years, it has been established that Nox enzymes have specific subcellular localizations². In fact, several studies have identified and refined the specific locations of Nox redox signaling pathways that are involved in vascular physiology and pathophysiology. For example, in cultured vascular smooth muscle cells (VSMC), Nox-1 is co-localized with caveolin in caveolin-enriched fractions on the cell surface³, whereas Nox-4 is co-localized with vinculin at focal adhesions³.

Given that ROS production is a precisely controlled process during homeostatic conditions, disturbance of this mechanism could lead to excessive ROS production, oxidative stress, and irreversible injury to the vasculature. However, the connection between subcellular localization of Nox enzymes, and organelle dysfunction, especially in disease conditions, still needs clarification. In this issue of Hypertension, Camargo and colleagues⁴ provide important evidence of Nox-4 upregulation on the endoplasmic reticulum (ER), contributing to a state of ER stress in VSMC, a well-established pathogenic mechanism in the vasculature of spontaneously hypertensive rats (SHR) ⁵ and angiotensin (Ang) II-induced hypertensive rats⁶. These observations have important implications for the understanding of the mechanisms associated with the genesis and/or maintenance of vascular dysfunction during hypertension.

Endoplasmic reticulum stress and hypertension

The ER is a multifunctional intracellular organelle and disturbances in its normal functions lead to the misfolding and aggregation of proteins, causing ER stress⁷. During ER stress there is an increase in the unfolded protein response (UPR), which acts through three transmembrane stress sensors, pancreatic ER kinase (PKR)-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1), and activating transcription factor 6 (ATF6) ⁷. Recently, it was reported by our group that chronic ER stress contributes to the increase of blood pressure and vascular injury in hypertension^{5, 6}. Specifically, we reported that SHR chronically treated with two different ER stress inhibitors, tauroursodeoxycholic acid (TUDCA) or 4-phenlybutyric acid (PBA) for 14 days, had decreased systolic blood pressure compared with SHR that received vehicle⁵. Also, it was observed in SHR, that treatment with TUDCA or PBA abolished the contraction to acetylcholine in aorta via cyclooxygenase-derived prostanoids⁵. In a subsequent study, we found that the induction of ER stress with tunicamycin in normotensive Sprague-Dawley rats caused an increase in systolic blood pressure compared with vehicle-treated or tunicamycin-treated rats that were co-treated with ER stress inhibitor PBA. On the other hand, inhibition of ER stress in Ang II-induced hypertension, led to a 20 mm Hg decrease in blood pressure⁶. Indices of vascular injury including, aortic apoptosis, increased collagen content, and fibrosis in Ang II-treated rats were attenuated with ER stress inhibition⁶. Collectively, these studies revealed that ER stress is an organelle dysfunction that contributes to vascular injury and hypertension. Supporting our findings, Camargo et al. demonstrated that the hyperproliferative and hypercontractile vascular response in SHR and SHR stroke prone (SHRSP), respectively, were attenuated by PBA⁴. Camargo et al. also revealed the cyclical nature of ROS generation and ER stress (Figure 1), with inhibition of ER stress preventing ROS generation (particularly H₂O₂) and Nox-4 upregulation, as well as Nox inhibition preventing the UPR response⁴.

Figure 1.

Nox-1 and Nox-4 exhibit distinct subcellular locations in vascular smooth muscle cells from SHR. Of note, Nox-4 activity is able to mediate ER stress and ER stress is able to cause increases in Nox-4 expression and activity.

Endoplasmic reticulum-dependent Nox-4 signaling in the vasculature

One of the major observations by Camargo and colleagues was that Nox-4 subcellular localization in ER was upregulated in VSMC from SHR and this was associated with ER stress⁴. While they also observed expression of Nox-4 on the plasma membrane and nuclear fractions, there was not a difference between SHR and Wistar Kyoto rats (WKY) ⁴.

The notable concept that Nox-4 is localized on the ER and contributes to ER stress has been previously investigated in VSMC. Pedruzzi and colleagues first reported in 2004 that Nox4 was localized in a paranuclear localization in VSMC and that 7-ketocholesterol (an oxysterol that is the one most frequently detected at high levels in atherosclerotic plaques) triggered the overexpression of Nox-4 and induced indices of ER stress⁸. When Nox-4 mRNA was silenced by using RNA interference, ER stress caused by 7-Kchol was decreased⁸. This publication was closely followed by further reports that revealed a spatial and/or mechanistic association between Nox-4 and the endoplasmic reticulum in VSMC^{9, 10}. Nonetheless, the results presented by Camargo and colleagues were the first to extend these findings into hypertension⁴, in which ER stress is a known to be a significant contributor to its pathogenesis^{5, 6}

Potential implications of endoplasmic reticulum-dependent Nox4 signaling in hypertension

Given that the upregulation of Nox-4 on the ER of SHR VSMC, it begs the question, could Nox-4 be a new target for preventing ER stress in the vasculature of hypertensive animals and patients? The findings from the investigation by Camargo et al. ⁴ extend the therapeutic potential of ER stress inhibition in hypertension. Not only could the proteotoxicity associated with the misfolded and aggregated proteins be prevented by treatment with ER stress inhibitors, but also the generation of excessive ROS, which could serve as a cause or exacerbate disturbed proteostasis. Additionally, if a pharmacologic drug could specifically target the ER bound Nox-4 isoform, this agent would have the therapeutic potential to prevent proteotoxicity-associated with ER stress in hypertension, as well as counter-act the other damaging effects of oxidative stress.