Matthew J. Durand

Diversity in Mechanisms of Endothelium-Dependent Vasodilation in Health and Disease

Small arterioles (40–150 μm) contribute to the majority of vascular resistance within organs and tissues. Under resting conditions, the basal tone of these vessels is determined by a delicate balance between vasodilator and vasoconstrictor influences. Cardiovascular homeostasis and regional tissue perfusion is largely a function of the ability of these small blood vessels to constrict or dilate in response to the changing metabolic demands of specific tissues. The endothelial cell layer of these microvessels is a key modulator of vasodilation through the synthesis and release of vasoactive substances. Beyond their vasomotor properties, these compounds importantly modulate vascular cell proliferation, inflammation, and thrombosis. Thus, the balance between local regulation of vascular tone and vascular pathophysiology can vary depending upon which factors are released from the endothelium. This review will focus on the dynamic nature of the endothelial released dilator factors depending on species, anatomic site, and presence of disease, with a focus on the human coronary microcirculation. Knowledge how endothelial signaling changes with disease may provide insights into the early stages of developing vascular inflammation and atherosclerosis, or related vascular pathologies.

The Human Microcirculation: Regulation of Flow and Beyond

The microcirculation is responsible for orchestrating adjustments in vascular tone to match local tissue perfusion with oxygen demand. Beyond this metabolic dilation, the microvasculature plays a critical role in modulating vascular tone by endothelial release of an unusually diverse family of compounds including nitric oxide, other reactive oxygen species, and arachidonic acid metabolites. Animal models have provided excellent insight into mechanisms of vasoregulation in health and disease. However, there are unique aspects of the human microcirculation that serve as the focus of this review. The concept is put forth that vasculoparenchymal communication is multimodal, with vascular release of nitric oxide eliciting dilation and preserving normal parenchymal function by inhibiting inflammation and proliferation. Likewise, in disease or stress, endothelial release of reactive oxygen species mediates both dilation and parenchymal inflammation leading to cellular dysfunction, thrombosis, and fibrosis. Some pathways responsible for this stress-induced shift in mediator of vasodilation are proposed. This paradigm may help explain why microvascular dysfunction is such a powerful predictor of cardiovascular events and help identify new approaches to treatment and prevention.

Acute and chronic angiotensin-(1–7) restores vasodilation and reduces oxidative stress in mesenteric arteries of salt-fed rats

This study determined the effect of ANG-(1-7) on salt-induced suppression of endothelium-dependent vasodilatation in the mesenteric arteries of male Sprague-Dawley rats. Chronic intravenous infusion of ANG-(1-7), oral administration of the nonpeptide mas receptor agonist AVE-0991, and acute preincubation of the arteries with ANG-(1-7) and AVE-0991 all restored vasodilator responses to both ACh and histamine that were absent in the arteries of rats fed a high-salt (4% NaCl) diet. The protective effects of ANG-(1-7) and AVE-0991 were inhibited by acute or chronic administration of the mas receptor antagonist A-779, the ANG II type 2 (AT(2)) receptor blocker PD-123319, or N-nitro-l-arginine methyl ester, but not the ANG II type 1 receptor antagonist losartan. Preincubation with the antioxidant tempol or the nitric oxide (NO) donor diethylenetriamine NONOate and acute and chronic administration of the AT(2) receptor agonist CGP-42112 mimicked the protective effect of ANG-(1-7) to restore vascular relaxation. Acute preincubation with ANG-(1-7) and chronic infusion of ANG-(1-7) ameliorated the elevated superoxide levels in rats fed a high-salt diet, but the expression of Cu/Zn SOD and Mn SOD enzyme proteins in the vessel wall was unaffected by ANG-(1-7) infusion. These results indicate that both acute and chronic systemic administration of ANG-(1-7) or AVE-0991 restore endothelium-dependent vascular relaxation in salt-fed Sprague-Dawley rats by reducing vascular oxidant stress and enhancing NO availability via mas and AT(2) receptors. These findings suggest a therapeutic potential for mas/AT(2) receptor activation in preventing the vascular oxidant stress and endothelial dysfunction associated with elevated dietary salt intake.

Angiotensin-(1-7) and low-dose angiotensin II infusion reverse salt-induced endothelial dysfunction via different mechanisms in rat middle cerebral arteries

The goals of this study were to 1) determine the acute effect of ANG-(1-7) on vascular tone in isolated middle cerebral arteries (MCAs) from Sprague-Dawley rats fed a normal salt (NS; 0.4% NaCl) diet, 2) evaluate the ability of chronic intravenous infusion of ANG-(1-7) (4 ng·kg(-1)·min(-1)) for 3 days to restore endothelium-dependent dilation to acetylcholine (ACh) in rats fed a high-salt (HS; 4% NaCl) diet, and 3) determine whether the amelioration of endothelial dysfunction by ANG-(1-7) infusion in rats fed a HS diet is different from the protective effect of low-dose ANG II infusion in salt-fed rats. MCAs from rats fed a NS diet dilated in response to exogenous ANG-(1-7) (10(-10)-10(-5) M). Chronic ANG-(1-7) infusion significantly reduced vascular superoxide levels and restored the nitric oxide-dependent dilation to ACh (10(-10)-10(-5) M) that was lost in MCAs of rats fed a HS diet. Acute vasodilation to ANG-(1-7) and the restoration of ACh-induced dilation by chronic ANG-(1-7) infusion in rats fed a HS diet were blocked by the Mas receptor antagonist [D-ALA(7)]-ANG-(1-7) or the ANG II type 2 receptor antagonist PD-123319 and unaffected by ANG II type 1 receptor blockade with losartan. The restoration of ACh-induced dilation in MCAs of HS-fed rats by chronic intravenous infusion of ANG II (5 ng·kg(-1)·min(-1)) was blocked by losartan and unaffected by d-ALA. These findings demonstrate that circulating ANG-(1-7), working via the Mas receptor, restores endothelium-dependent vasodilation in cerebral resistance arteries of animals fed a HS diet via mechanisms distinct from those activated by low-dose ANG II infusion.

An acute rise in intraluminal pressure shifts the mediator of flow-mediated dilation from nitric oxide to hydrogen peroxide in human arterioles.

Endothelial nitric oxide (NO) is the primary mediator of flow-mediated dilation (FMD) in human adipose microvessels. Impaired NO-mediated vasodilation occurs after acute and chronic hypertension, possibly due to excess generation of reactive oxygen species (ROS). The direct role of pressure elevation in this impairment of human arteriolar dilation is not known. We tested the hypothesis that elevation in pressure is sufficient to impair FMD. Arterioles were isolated from human adipose tissue and cannulated, and vasodilation to graded flow gradients was measured before and after exposure to increased intraluminal pressure (IILP; 150 mmHg, 30 min). The mediator of FMD was determined using pharmacological agents to reduce NO [N(G)-nitro-l-arginine methyl ester (l-NAME), 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO)], or H2O2 [polyethylene glycol (PEG)-catalase], and mitochondrial (mt) ROS was quantified using fluorescence microscopy. Exposure to IILP decreased overall FMD (max %dilation: 82.7 ± 4.9 vs. 62 ± 5.6; P < 0.05). This dilation was abolished by treatment with l-NAME prepressure and PEG-catalase after IILP (max %dilation: l-NAME: 23.8 ± 6.1 vs. 74.8 ± 8.6; PEG-catalase: 71.8 ± 5.9 vs. 24.6 ± 10.6). To examine if this change was mediated by mtROS, FMD responses were measured in the presence of the complex I inhibitor rotenone or the mitochondrial antioxidant mitoTempol. Before IILP, FMD was unaffected by either compound; however, both inhibited dilation after IILP. The fluorescence intensity of mitochondria peroxy yellow 1 (MitoPY1), a mitochondria-specific fluorescent probe for H2O2, increased during flow after IILP (%change from static: 12.3 ± 14.5 vs. 127.9 ± 57.7). These results demonstrate a novel compensatory dilator mechanism in humans that is triggered by IILP, inducing a change in the mediator of FMD from NO to mitochondria-derived H2O2.

Critical Role for Telomerase in the Mechanism of Flow Mediated Dilation in the Human Microcirculation

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Acute Exertion Elicits a H2O2-Dependent Vasodilator Mechanism in the Microvasculature of Exercise-Trained but not Sedentary Adults

Brachial artery flow–mediated vasodilation in exercise-trained (ET) individuals is maintained after a single bout of heavy resistance exercise compared with sedentary individuals. The purpose of this study was to determine whether vasodilation is also maintained in the microcirculation of ET individuals. A total of 51 sedentary and ET individuals underwent gluteal subcutaneous fat biopsy before and after performing a single bout of leg press exercise. Adipose arterioles were cannulated in an organ bath, and vasodilation to acetylcholine was assessed±the endothelial nitric oxide inhibitorL-NG-nitroarginine methyl ester, the cyclooxygenase inhibitor indomethacin, or the hydrogen peroxide scavenger polyethylene glycol catalase. Separate vessels (isolated from the same groups) were exposed to an intraluminal pressure of 150 mm Hg for 30 minutes to mimic the pressor response, which occurs with isometric exercise. Vasodilation to acetylcholine was reduced in microvessels from sedentary subjects after either a single weight lifting session or exposure to increased intraluminal pressure, whereas microvessels from ET individuals maintained acetylcholine-mediated vasodilation. Before weight lifting, vasodilation of microvessels from ET individuals was reduced in the presence of L-NG-nitroarginine methyl ester and indomethacin. After weight lifting or exposure to increased intraluminal pressure, polyethylene glycol catalase significantly reduced vasodilation, whereas L-NG-nitroarginine methyl ester and indomethacin had no effect. These results indicate that (1) endothelium-dependent vasodilation in the microvasculature is maintained after heavy resistance exercise in ET individuals but not in sedentary subjects and that (2) high pressure alone or during weight lifting may induce a mechanistic switch in the microvasculature to favor hydrogen peroxide as the vasoactive mediator of dilation.

Low-Dose Angiotensin II Infusion Restores Vascular Function in Cerebral Arteries of High Salt–Fed Rats by Increasing Copper/Zinc Superoxide Dimutase Expression

BACKGROUND This study examined the vasoprotective role of circulating angiotensin II (ANG II) levels in the cerebral circulation of high salt (HS)-fed (SS.BN-(D13hmgc41-13hmgc23)/Mcwi) (Ren1-BN) congenic rats, which carry a normally functioning renin allele from the Brown Norway (BN) rat on the Dahl salt-sensitive genetic background. METHODS Ren1-BN rats were placed on an HS (4.0% NaCl) diet for 3 days. The vasodilator response to acetylcholine (ACh; 10(-10) - 10(-6) mol/L) was assessed in isolated middle cerebral arteries (MCAs), and Western blots were performed to assess the expression of the antioxidant enzymes copper (Cu)/zinc (Zn) superoxide dismutase (SOD) and manganese (Mn) SOD in cerebral resistance vessels. A separate group of HS-fed animals were infused with either a subpressor dose of ANG II (100ng/kg/min) or saline vehicle via osmotic minipump for 3 days. RESULTS HS diet eliminated acetylcholine (ACh)-induced dilation in the MCAs of the congenic rats. Western blot analysis of antioxidant enzymes showed that Cu/Zn SOD and Mn SOD expression were significantly reduced in the cerebral resistance arteries of the HS-fed rats compared with control animals fed a normal salt diet. Infusion of ANG II restored the vasodilator response to ACh in the MCAs and increased Cu/Zn SOD (but not Mn SOD) expression compared with saline-infused animals. CONCLUSIONS These results indicate that prevention of salt-induced ANG II suppression prevents vascular dysfunction in the cerebral circulation by preventing the downregulation of Cu/Zn SOD and vascular oxidant stress that normally occurs with HS diet.

Impaired relaxation of cerebral arteries in the absence of elevated salt intake in normotensive congenic rats carrying the Dahl salt-sensitive renin gene

This study evaluated endothelium-dependent vascular relaxation in response to acetylcholine (ACh) in isolated middle cerebral arteries (MCA) from Dahl salt-sensitive (Dahl SS) rats and three different congenic strains that contain a portion of Brown Norway (BN) chromosome 13 introgressed onto the Dahl SS genetic background through marker-assisted breeding. Two of the congenic strains carry a 3.5-Mbp portion and a 2.6-Mbp portion of chromosome 13 that lie on opposite sides of the renin locus, while the third contains a 2.0-Mbp overlapping region that includes the BN renin allele. While maintained on a normal salt (0.4% NaCl) diet, MCAs from Dahl SS rats and the congenic strains retaining the Dahl SS renin allele failed to dilate in response to ACh, whereas MCAs from the congenic strain carrying the BN renin allele exhibited normal vascular relaxation. In congenic rats receiving the BN renin allele, vasodilator responses to ACh were eliminated by nitric oxide synthase inhibition with N(G)-nitro-l-arginine methyl ester, angiotensin-converting enzyme inhibition with captopril, and AT(1) receptor blockade with losartan. N(G)-nitro-l-arginine methyl ester-sensitive vasodilation in response to ACh was restored in MCAs of Dahl SS rats that received either a 3-day infusion of a subpressor dose of angiotensin II (3 ng·kg(-1)·min(-1) iv), or chronic treatment with the superoxide dismutase mimetic tempol (15 mg·kg(-1)·day(-1)). These findings indicate that the presence of the Dahl SS renin allele plays a crucial role in endothelial dysfunction present in the cerebral circulation of the Dahl SS rat, even in the absence of elevated dietary salt intake, and that introgression of the BN renin allele rescues endothelium-dependent vasodilator responses by restoring normal activation of the renin-angiotensin system.

Introgression of the Brown Norway Renin Allele Onto the Dahl Salt-Sensitive Genetic Background Increases Cu/Zn SOD Expression in Cerebral Arteries

BACKGROUND Nitric oxide (NO)-dependent vasodilation is impaired in middle cerebral arteries (MCAs) from Dahl salt-sensitive (SS) rats that are fed normal salt (NS) diet, due to low plasma renin activity and chronic exposure to low plasma angiotensin II (ANG II) levels. NO-dependent vasodilator responses are rescued in MCAs from Ren1-BN congenic rats, which have a 2.0 Mbp portion of Brown Norway (BN) chromosome 13 containing the renin gene introgressed onto the Dahl SS genetic background. METHODS Vascular superoxide levels were measured with dihydroethidium (DHE) fluorescence in basilar arteries from 10- to 14-week-old, male Dahl SS and Ren1-BN congenic rats that fed NS diet. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and xanthine oxidase (XO) activity were also measured in cerebral artery tissue homogenates. Expression of the superoxide dismutase (SOD) enzymes was evaluated via western blotting in cerebral arteries from the two rat strains. RESULTS Superoxide levels were significantly higher in basilar arteries from Dahl SS rats compared to Ren1-BN congenic rats. NADPH oxidase and XO activity were similar between the two rat strains. Cu/Zn SOD expression was significantly higher in cerebral arteries from Ren1-BN congenic rats vs. those from Dahl SS rats. The expression of Mn-SOD was similar in cerebral arteries from both strains. CONCLUSIONS These findings suggest that introgressing the BN renin allele onto the Dahl SS genetic background to restore normal activity of the renin-angiotensin system (RAS) protects NO-dependent vascular relaxation in cerebral arteries by increasing the expression of Cu/Zn SOD and lowering vascular superoxide levels.