Sophocles Chrissobolis

Nitroxyl (HNO) suppresses vascular Nox2 oxidase activity.

Nox2 oxidase activity underlies the oxidative stress and vascular dysfunction associated with several vascular-related diseases. We have reported that nitric oxide (NO) decreases reactive oxygen species production by endothelial Nox2. This study tested the hypothesis that nitroxyl (HNO), the redox sibling of NO, also suppresses vascular Nox2 oxidase activity. Specifically, we examined the influence of two well-characterized HNO donors, Angelis salt and isopropylamine NONOate (IPA/NO), on Nox2-dependent responses to angiotensin II (reactive oxygen species production and vasoconstriction) in mouse cerebral arteries. Angiotensin II (0.1μmol/L)-stimulated superoxide (measured by lucigenin-enhanced chemiluminescence) and hydrogen peroxide (Amplex red fluorescence) levels in cerebral arteries (pooled basilar and middle cerebral (MCA)) from wild-type (WT) mice were ~60% lower (P<0.05) in the presence of either Angelis salt (1μmol/L) or IPA/NO (1μmol/L). Similarly, phorbyl 12,13-dibutyrate (10μmol/L; Nox2 activator)-stimulated hydrogen peroxide levels were ~40% lower in the presence of IPA/NO (1μmol/L; P<0.05). The ability of IPA/NO to decrease superoxide levels was reversible and abolished by the HNO scavenger l-cysteine (3mmol/L; P<0.05), but was unaffected by hydroxocobalamin (100μmol/L; NO scavenger), ODQ (10μmol/L; soluble guanylyl cyclase (sGC) inhibitor), or Rp-8-pCPT-cGMPS (10μmol/L; cyclic guanosine monophosphate (cGMP)-dependent protein kinase inhibitor). Angiotensin II-stimulated superoxide was substantially less in arteries from Nox2-deficient (Nox2(-/y)) versus WT mice (P<0.05). In contrast to WT, IPA/NO (1μmol/L) had no effect on superoxide levels in arteries from Nox2(-/y) mice. Finally, angiotensin II (1-1000μmol/L)-induced constriction of WT MCA was virtually abolished by IPA/NO (1μmol/L), whereas constrictor responses to either the thromboxane A2 mimetic U46619 (1-100 nmol/L) or high potassium (122.7mmol/L) were unaffected. In conclusion, HNO suppresses vascular Nox2 oxidase activity via a sGC-cGMP-independent pathway. Thus, HNO donors might be useful therapeutic agents to limit and/or prevent Nox2-dependent vascular dysfunction.

Aldosterone-induced oxidative stress and inflammation in the brain are mediated by the endothelial cell mineralocorticoid receptor

Elevated aldosterone levels, which promote cerebral vascular oxidative stress, inflammation, and endothelial dysfunction, may increase stroke risk, independent of blood pressure and other risk factors. The main target receptor of aldosterone, the mineralocorticoid receptor (MR), is expressed in many cell types, including endothelial cells. Endothelial cell dysfunction is thought to be an initiating step contributing to cardiovascular disease and stroke; however the importance of MR expressed on endothelial cells in the brain is unknown. Here we have examined whether endothelial cell MR mediates cerebral vascular oxidative stress and brain inflammation during aldosterone excess. In male mice, aldosterone (0.72 mg/kg/day, 14 days) caused a small increase (~14 mmHg) in blood pressure. The MR blocker spironolactone (25 mg/kg/d, ip) abolished this increase, whereas endothelial cell MR-deficiency had no effect. Aldosterone increased superoxide production capacity in cerebral arteries, and also mRNA expression of the pro-inflammatory cytokines chemokine (C-C motif) ligand 7 (CCL7), CCL8 and interleukin (IL)-1β in the brain. These increases were prevented by both spironolactone treatment and endothelial cell MR-deficiency; whereas IL-1β expression was blocked by spironolactone only. Endothelial cell MR mediates aldosterone-induced increases in cerebrovascular superoxide levels and chemokine expression in the brain, but not blood pressure or brain IL-1β. Endothelial cell-targeted MR antagonism may represent a novel approach to treat cerebrovascular disease and stroke, particularly during conditions of aldosterone excess.

Aldosterone and the mineralocorticoid receptor in the cerebral circulation and stroke

Ischemic stroke is a leading cause of morbidity and mortality worldwide. Elevated plasma aldosterone levels are an independent cardiovascular risk factor and are thought to contribute to hypertension, a major risk factor for stroke. Evidence from both experimental and human studies supports a role for aldosterone and/or the mineralocorticoid receptor (MR) in contributing to detrimental effects in the cerebral vasculature and to the incidence and outcome of ischemic stroke. This article reviews the evidence, including the protective effects of MR antagonism. Specifically, the effects of aldosterone and/or MR activation on cerebral vascular structure and on immune cells will be reviewed. The existing evidence suggests that aldosterone and the MR contribute to cerebral vascular pathology and to the incidence and outcome of stroke. We suggest that further research into the signaling mechanisms underlying the effects of aldosterone and MR activation in the brain and its vasculature, especially with regard to cell-specific actions, will provide important insight into causes and potential treatments for cerebrovascular disease and stroke.

Pressor response to angiotensin II is enhanced in aged mice and associated with inflammation, vasoconstriction and oxidative stress

Aging is commonly associated with chronic low-grade inflammation and hypertension but it is unknown whether a cause-effect relationship exists between them. We compared the sensitivity of young adult (8-12 w) and aged (23-31 mo) male C57Bl6J mice to develop hypertension in response to a slow-pressor dose of angiotensin II (Ang II; 0.28 mg/kg/d; 28 d). In young mice, the pressor response to Ang II was gradual and increased to 142±8 mmHg over 28 d. However, in aged mice, Ang II promptly increased SBP and reached 155±12 mmHg by 28 d. Aging increased renal but not brain expression of Ang II receptors (At1ar and At2r) and elevated AT1R:AT2R expression ratio in mesenteric artery. Maximal contractile responses of mesenteric arteries to Ang II were enhanced in aged mice and were not affected by L-NAME, indomethacin or tempol. Mesenteric arteries and thoracic aortae from aged mice exhibited higher Nox2-dependent superoxide production. Despite having higher renal expression of Nlrp3, Casp-1 and Il-1β, Ang II-induced hypertension (SBP: 139±7 mmHg) was unaffected by co-infusion of the NLRP3 inflammasome inhibitor, MCC950 (10 mg/kg/d; SBP: 145±10 mmHg). Thus, increased vascular AT1R:AT2R expression, rather than NLRP3 inflammasome activation, may contribute to enhanced responses to Ang II in aging.

Advanced atherosclerosis is associated with inflammation, vascular dysfunction and oxidative stress, but not hypertension

ABSTRACT Although hypertension may involve underlying inflammation, it is unknown whether advanced atherosclerosis − a chronic inflammatory condition − can by itself promote hypertension. We thus tested if advanced atherosclerosis in chronically hypercholesterolemic mice is associated with systemic and end‐organ inflammation, vascular dysfunction and oxidative stress, and whether blood pressure is higher than in control mice. Male ApoE−/− and wild‐type (C57Bl6J) mice were placed on a high fat or chow diet, respectively, from 5 to 61 weeks of age. Expression of several cytokines (including IL‐6, TNF‐&agr;, IFN‐&ggr; and/or IL‐1&bgr;) was elevated in plasma, brain, and aorta of ApoE−/− mice. Aortic superoxide production was ˜3.5‐fold greater, and endothelium‐dependent relaxation was markedly reduced in aorta and mesenteric artery of ApoE−/− versus wild‐type mice. There was no difference in blood pressure of aged ApoE−/− (104 ± 3 mmHg, n = 13) and wild‐type mice (113 ± 1 mmHg, n = 18). To clarify any effects of aging alone, findings from 61 week‐old wild‐type mice were compared with those from young (8–12 weeks old) chow‐fed wild‐type mice. The data indicate that aging alone increased renal and aortic expression of numerous cytokines (including CCL2, CCL7 and IL‐1&bgr;). Aging had no effect on blood pressure, systemic inflammation, oxidative stress or endothelial function. Despite systemic and end‐organ inflammation, oxidative stress and endothelial dysfunction, advanced atherosclerosis does not necessarily result in elevated blood pressure.

Role of Oxidative Stress in Hypertension

Oxidative stress, defined as an increase in steady-state levels of superoxide, is involved in the pathogenesis of several cardiovascular diseases. Hypertension is a major risk factor for cardiovascular diseases, and there exists much experimental support for a role of oxidative stress in hypertension, often in association with vascular abnormalities including endothelial dysfunction. Vascular NADPH oxidases (Nox) are by far the most researched topic amongst the sources of reactive oxygen species (ROS) in hypertension, and are thought to be a predominant underlying cause of oxidative stress in hypertension. The purpose of this chapter is to discuss the involvement of oxidative stress in association with vascular abnormalities in animal models of hypertension, with a particular emphasis on evidence for involvement of Nox in three commonly studied models: angiotensin II (Ang II)-induced hypertension, mineralocorticoid-dependent hypertension and the spontaneously hypertensive rat (SHR). Antioxidant defence mechanisms (i.e. superoxide dismutases [SOD]’s and glutathione peroxidases [GPx’s]) may limit vascular oxidative stress, thus experimental evidence discussing their likely protection against vascular oxidative stress and hypertension will be discussed. Recent concepts regarding the link between oxidative stress, the immune system and hypertension will also be covered, and finally we will briefly address clinical data providing an association between oxidative stress and hypertension, in particular the link between genetic abnormalities and oxidative stress in hypertension.

Cell-specific mineralocorticoid receptors: future therapeutic targets for stroke?

The mineralocorticoid receptor (MR), well known to be expressed in renal epithelial cells where it is important in fluid and electrolyte homeostasis, has aldosterone as one of its main agonists. Much research in the last 10–15 years indicates that MRs are also expressed outside of the kidney, including in the brain, vasculature and heart, where they contribute to the pathophysiology of disease (Dinh et al., 2012; Jaisser and Farman, 2016). Excess aldosterone is a cardiovascular risk factor, and MR antagonism is beneficial in the setting of cardiovascular disease (both clinically and experimentally), including in experimental stroke, whereby MR antagonism is beneficial in reducing both cerebral infarct size (Iwanami et al., 2007; Oyamada et al., 2008) and cerebral vascular remodeling (reviewed in Dinh et al., 2012) following cerebral ischemia. MR antagonism also reverses remodeling, both during aldosterone/mineralocorticoid excess and following cerebral ischemia. The advent of technology to generate mice which lack specific genes in specific cell types has allowed investigation into the contribution of MR in cell types such as vascular endothelial and myeloid cells to the pathophysiology of cerebrovascular disease and stroke (Frieler et al., 2011, 2012; Dinh et al., 2016). Endothelial cell MRs mediate cerebrovascular oxidative stress and brain inflammation in response to excess aldosterone (Dinh et al., 2016), and myeloid MR contribute to the ischemic damage, inflammation and neurological impairment following cerebral ischemia/reperfusion (Frieler et al., 2011, 2012). These and further investigations into the contribution of cell-specific MRs to cerebrovascular disease and stroke can help guide the future design of therapeutic strategies for stroke treatment.

Obstructive sleep apnoea is common in patients with primary aldosteronism and improves with adrenalectomy or mineralocorticoid receptor antagonists

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