Endothelium-dependent Vasodilator Acetylcholine Research Articles

Endogenous Acetylcholine Contributes to Flow-Mediated Dilation in Humans

Background & Objective. Flow-mediated dilation (FMD) is a crucial function of the vascular endothelium wherein intraluminal shear stress stimulates endothelium-dependent vasodilation mediated primarily by nitric oxide. Brachial artery FMD (baFMD) is widely evaluated in clinical research as an index of endothelial function, and impaired baFMD is an independent predictor of cardiovascular risk. Despite the physiological and clinical relevance of FMD, the mechanisms by which shear stress is transduced to endothelium-dependent vasodilation (i.e., upstream of nitric oxide production) remain unclear. baFMD corresponds closely with sensitivity to the endothelium-dependent vasodilator acetylcholine (ACh), and preclinical models suggest ACh is released from endothelial cells in response to shear stress. Therefore, the present study tested the hypothesis that activation of muscarinic ACh receptors facilitates baFMD in humans. Methods. baFMD was evaluated using Doppler ultrasound in healthy participants (n=11; 5F/6M; 23±2 y; means ± SE) under control conditions and following local inhibition of muscarinic ACh receptors via intra-arterial atropine infused proximal to the measurement site. baFMD was assessed in response to transient and sustained shear stimuli in separate trials: 1) reactive hyperemia following 5 min of arterial cuff occlusion (RH-FMD) and 2) steady-state rhythmic handgrip exercise at graded intensities (10, 15, 20, & 25% maximal voluntary contraction, MVC) to produce step increases in shear rate (EX-FMD). Blockade effcacy was confirmed via a dose-response to intra-arterial ACh infusion pre and post atropine. Results. Baseline brachial artery diameter and forearm blood flow were similar in all trials (all P>0.05). Atropine reduced peak brachial artery dilation following cuff release (RH-FMD, Δ diameter, atropine: 4.6±0.8 vs. control: 6.5±0.8%, P=0.04) and increased the shear stimulus (shear rate area under the curve, SRAUC: atropine, 29±3 vs. control: 21±3 s−1×103, P=0.01) such that the percent change in brachial diameter was diminished by 35±16% when normalized to SRAUC (baFMD:SRAUC ratio, atropine: 1.7±0.3 vs. control: 3.9±1.0, P=0.02). Similarly, in the EX-FMD trial, atropine reduced brachial artery diameter at all exercise intensities (25% MVC, percent change in diameter: atropine, 10.8±1.4 vs. control, 15.5±1.7%, P=0.04) despite a greater shear stimulus (shear rate: atropine, 10.9±1.1 vs. control, 9.3±0.9 s−1×102, P=0.01). Thus, atropine reduced the slope describing the relationship between brachial artery vasodilation (percent change) and the change in shear rate by 38±8% across all exercise intensities (slope, atropine: 1.3±0.1 vs. control: 2.4±0.4% Δ×s×10−2, P=0.007). As expected, atropine reduced brachial artery vasodilation in response to upstream infusion of ACh (1.5 μg ACh/dl forearm volume/min, Δ diameter, atropine: 2±1 vs. control: 23±4%, P=0.001). Conclusions. The results suggest endogenous ACh facilitates baFMD in response to both transient and sustained shear stimuli, which highlights a novel physiological role of endogenous ACh in vascular regulation in humans and provides insight regarding the basic mechanisms of flow-mediated vasodilation. NIH R01 HL119337 and F31 HL142240. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Influence of a Decrease in Blood Viscosity on Arterial Pressure in Normotensive and Spontaneously Hypertensive Rats.

We investigated the effect of a decrease in blood viscosity on the mean BP during isovolumic hemodilution and vasodilating activity of the endothelium in normotensive Wistar rats and spontaneously hypertensive rats (SHR). Blood viscosity was reduced by isovolumic hemodilution (replacement of 10% of circulating blood with an equal volume of plasma). Hemodilution caused the same reduction in blood viscosity by 16% in both groups of rats. In Wistar rats, a decrease in blood viscosity did not significantly change in the mean BP; no significant correlations between blood viscosity and mean BP were observed before and after hemodilution. In SHR, a decrease in blood viscosity led to a significant decrease in the mean BP by 18%. Correlations were found between the mean BP and blood viscosity in SHR before (r=0.63; p=0.028) and after (r=0.71; p=0.009) isovolumic hemodilution. In SHR, a decrease in the index of vasodilating activity of the endothelium due to a decrease in the vasodilatory response to intravenous administration of the endothelium-dependent vasodilator acetylcholine was revealed. In SHR, BP passively follows the change, in this case, the decrease in blood viscosity, which attests to impaired BP regulation in response to changes in shear stress on the vascular endothelium caused by the development of endothelial dysfunction in hypertensive animals.

High-fat diet causes endothelial dysfunction in the mouse ophthalmic artery

Obesity is a significant health concern that leads to impaired vascular function and subsequent abnormalities in various organs. The impact of obesity on ocular blood vessels, however, remains largely unclear. In this study, we examined the hypothesis that obesity induced by high-fat diet produces vascular endothelial dysfunction in the ophthalmic artery. Mice were subjected to a high-fat diet for 20 weeks, while age-matched controls were maintained on a standard diet. Reactivity of isolated ophthalmic artery segments was assessed in vitro. Reactive oxygen species (ROS) were quantified in cryosections by dihydroethidium (DHE) staining. Redox gene expression was determined in ophthalmic artery explants by real-time PCR. Furthermore, the expression of nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2), the receptor for advanced glycation end products (RAGE), and of the lectin-like oxidized low-density-lipoprotein receptor-1 (LOX-1) was determined in cryosections using immunofluorescence microscopy. Ophthalmic artery segments from mice on a high-fat diet exhibited impaired vasodilation responses to the endothelium-dependent vasodilator acetylcholine, while endothelium-independent responses to nitroprusside remained preserved. DHE staining intensity in the vascular wall was notably stronger in mice on a high-fat diet. Messenger RNA expression for NOX2 was elevated in the ophthalmic artery of mice subjected to high fat diet. Likewise, immunostainings revealed increased expression of NOX2 and of RAGE, but not of LOX-1. These findings suggest that a high-fat diet triggers endothelial dysfunction by inducing oxidative stress in the ophthalmic artery via involvement of RAGE and NOX2.

Genetic background influences arterial vasomotor function in male and female mice.

The purpose of this study was to assess the influence of genetic background and sex on nitric oxide (NO)-mediated vasomotor function in arteries from different vascular territories. Vasomotor function was assessed in thoracic aorta, abdominal aorta, carotid arteries, and femoral arteries from the following mouse strains: SJL/J, DBA/2J, NZW/LacJ, and C57BL/6J. Contractile responses were assessed using the α1-adrenergic receptor agonist phenylephrine (PE, 10-9 -10-5 M). Vasorelaxation responses were assessed by examining relaxation to an endothelium-dependent vasodilator acetylcholine (ACh, 10-9 -10-5 M) and an endothelium-independent vasodilator sodium nitroprusside (SNP, 10-9 -10-5 M). To evaluate the role of NO, relaxation responses to ACh and SNP were assessed in the absence or presence of a nitric oxide synthase inhibitor (N omega-nitro-l-arginine methyl ester hydrochloride: 10-4 M). Vasomotor responses to ACh and PE varied across strains and among the arteries tested with some strains exhibiting artery-specific impairment. Results indicated some concentration-response heterogeneity in response to ACh and SNP between vessels from females and males, but no significant differences in responses to PE. Collectively, these findings indicate that vasomotor responses vary by genetic background, sex, and artery type.

Nitric oxide contribution to endothelium-mediated vasodilation is attenuated in postmenopausal compared to premenopausal females

Background: Hypertension prevalence increases in females following menopause. Poor endothelial microvascular function increases risk of hypertension. Endothelial-mediated vasodilation is dependent on nitric oxide (NO). Estrogen enhances NO production; therefore, reduced estrogen levels in postmenopausal females may result in a reduced contribution of NO to endothelial-mediated vasodilation, although studies are limited. Herein, we aimed to investigate the contribution of NO to endothelial-mediated vasodilation in premenopausal and postmenopausal females. We hypothesized that postmenopausal females would have attenuated endothelium-mediated vasodilation and reduced contribution of NO compared to premenopausal females. Methods: Ten premenopausal (27±3 yrs) and eleven postmenopausal (59±4 yrs) normotensive females were studied. Resting brachial blood pressures were measured using a standard arm cuff. Brachial arterial catheters were placed for continuous blood pressure measurement and pharmacologic infusions. Forearm blood flow (FBF) was measured using venous occlusion plethysmography at baseline (saline) and during infusion of an endothelium-dependent vasodilator (acetylcholine; 1.0, 2.0, 4.0, and 8.0 μg/100mL tissue/min) with and without a NO synthase inhibitor (L-NG-monomethylarginine [L-NMMA]; 1.0 mg/min) co-infusion. Forearm vascular conductance (FVC) was calculated as FBF/mean arterial pressure✕100. Data were analyzed using a two-way repeated-measures ANOVA. Results: Resting systolic blood pressure (SBP) but not diastolic blood pressure (DBP) was greater in postmenopausal compared to premenopausal females (SBP: 121±15 vs. 103±9 mmHg, respectively; P<0.01; DBP: 72±11 vs. 65±5 mmHg, respectively; P=0.07). FBF responses to acetylcholine did not differ between premenopausal and postmenopausal females (Group Main Effect: P=0.11). When correcting for blood pressure, premenopausal females exhibited greater FVC responses to acetylcholine compared to postmenopausal females (Group Main Effect: P=0.04). In premenopausal females, FBF and FVC responses to acetylcholine were reduced with co-infusion of L-NMMA compared to acetylcholine infusion only (L-NMMA Main Effect: P=0.02 and P=0.01, respectively). In postmenopausal females, the FBF and FVC responses to acetylcholine were unaffected by L-NMMA (L-NMMA Main Effect: P=0.79 and P=0.76, respectively). Conclusion: Compared to premenopausal females, postmenopausal females have impaired endothelial microvascular function. Attenuated NO contribution during endothelium-mediated vasodilation in postmenopausal women may partially contribute to the increased hypertension risk in this population. AHA 14PRE18040000, AHA 898649, NIH HL083947, NIH HL148144, NCATS UL1 TR000135 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The Role of Angiotensin 1‐7 in Isolated Human Arterioles with SARS‐CoV‐2

IntroductionThe vascular endothelium plays a crucial role to regulate vascular tone. Recently, our laboratory reported severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) infection significantly reduced endothelial‐dependent vasodilation months after exposed to SARS‐CoV‐2 in human microvessels. SARS‐CoV‐2 enters host cells through angiotensin‐converting enzyme (ACE)2 as a cell surface receptor. Downregulation of ACE2 protein expression by the virus leads to imbalance between angiotensin (ANG) II and ANG 1‐7, causing vasoconstriction, inflammation and increase oxidative stress. With this background, we intend to test our new hypothesis that administration of ANG 1‐7 will improve vasodilation in human arterioles from previously SARS‐CoV‐2 positive subjects.MethodsFresh human tissues were obtained as de‐identified surgical discarded specimens. Control subjects (n=9) were SARS‐CoV‐2 negative and without known virus exposure. We had 11 previously SARS‐CoV‐2 positive subjects or post‐COVID. For all post‐COVID patients, a negative COVID test was obtained prior to collection of surgical specimens. We isolated arterioles (100‐200 µm) and cannulated onto glass micropipettes under 60 mmHg and examined for diameter changes to the endothelial‐dependent vasodilator acetylcholine (ACh; 10−9 to 10−5 M) using videomicroscopy. Flow‐mediated dilation (FMD) was recorded at steady‐state during varying intraluminal pressure gradients (5‐100 cm H2O). All post‐COVID vessels were incubated in a culture media at 37°C for 12–15 hours with or without 1 nM of ANG 1‐7.ResultsDilation to ACh and FMD in arterioles from post‐COVID was significantly reduced (ACh max. dilation at 10−5 M: 74±6% vs. 93±4% in control, n=7‐9, P<0.001; FMD max. dilation at 100 cm H2O: 49±3% vs. 86±2% in control, n=6‐11, P<0.001) while overnight incubation of 1 nM ANG 1‐7 significantly improved the dilation (ACh max. dilation at 10−5 M: 74±6% in post‐COVID vs. 88±5% in post‐COVID + AND 1‐7, n=11, P<0.001; FMD max. dilation at 100 cm H2O: 49±3% in post‐COVID vs. 76±3% in post‐COVID + ANG 1‐7, n=11, P<0.001).ConclusionAdministration of ANG 1‐7 significantly improved vasodilation in isolated arterioles from post‐COVID subjects.

Genetic Background Influences Endothelium‐dependent Vasomotor Function in Large Arteries

Introduction Endothelial dysfunction is associated with a host of disease states including cardiovascular disease, diabetes, chronic kidney disease, and atherosclerosis. Previous research by our laboratory has shown that vasomotor function, and more specifically endothelium-dependent vasorelaxation responses, differ across large arteries from mice with distinct genetic backgrounds. However, it is unclear how much of the observed variation in endothelium-dependent vasorelaxation is due to nitric oxide (NO) mediated pathways. Objective The purpose of this study was to test the hypothesis that strain-dependent vasorelaxation differences were primarily the result of differences in reliance on NO-mediated pathways. Methods Vasomotor function was assessed in thoracic aorta, abdominal aorta, carotid arteries, and femoral arteries from four inbred strains (SJL/J (SJL), DBA/2J (DBA), NZW/LacJ (NZW) and C57BL/6J (B6)) of mice. Strains were chosen based on previously documented vasomotor variation and susceptibility to clinically significant pathologies. Arteries were dissected, cut into 2mm segments, and mounted in a wire myograph system. Arterial segments were stretched to a resting tension equivalent to a pressure of 90mmHg for all strains based on a passive stretch and relaxation tension-force assessment. Increasing concentrations of the endothelium-dependent vasodilator acetylcholine (ACh, 1x10-9 – 1x10-5M) and endothelium-independent vasodilator sodium nitroprusside (SNP, 1x10-9 – 1x10-5M) were used to assess vasorelaxation responses. To assess reliance on NO, vasorelaxation responses to Ach and SNP were assessed in the presence of a nitric oxide synthase inhibitor (NOS). One arterial segment from each group was treated with the NOS inhibitor N omega-Nitro-L-arginine methyl ester hydrochloride (L-NAME: 10x4 M) for thirty minutes. Results Two way ANOVA revealed a significant main effect of strain in vasorelaxation responses to ACh for all arteries tested (P <.0001) and no significant differences in response to SNP. Maximal relaxation responses to ACh were significantly inhibited in all arterial segments treated with L-NAME, while no differences were detected for L-NAME treated vessels in response to SNP. These findings indicate that large arteries have a strong dependence on NO-mediated, endothelium-dependent pathways to maintain vascular regulation. Furthermore, strain-dependent vasorelaxation differences were abolished in the presence of L-NAME indicating that strain differences were NO-dependent. Conclusion These findings indicate a strong influence of genetic background on endothelial function and suggest that these differences are primarily due to strain related reliance on NO for endothelium-dependent vasodilation.

TPA Deficiency Underlies Neurovascular Coupling Dysfunction by Amyloid-β.

The amyloid-β (Aβ) peptide, a key pathogenic factor in Alzheimer's disease, attenuates the increase in cerebral blood flow (CBF) evoked by neural activity (functional hyperemia), a vital homeostatic response in which NMDA receptors (NMDARs) play a role through nitric oxide, and the CBF increase produced by endothelial factors. Tissue plasminogen activator (tPA), which is reduced in Alzheimer's disease and in mouse models of Aβ accumulation, is required for the full expression of the NMDAR-dependent component of functional hyperemia. Therefore, we investigated whether tPA is involved in the neurovascular dysfunction of Aβ. tPA activity was reduced, and the tPA inhibitor plasminogen inhibitor-1 (PAI-1) was increased in male mice expressing the Swedish mutation of the amyloid precursor protein (tg2576). Counteracting the tPA reduction with exogenous tPA or with pharmacological inhibition or genetic deletion of PAI-1 completely reversed the attenuation of the CBF increase evoked by whisker stimulation but did not ameliorate the response to the endothelium-dependent vasodilator acetylcholine. The tPA deficit attenuated functional hyperemia by suppressing NMDAR-dependent nitric oxide production during neural activity. Pharmacological inhibition of PAI-1 increased tPA activity, prevented neurovascular uncoupling, and ameliorated cognition in 11- to 12-month-old tg2576 mice, effects associated with a reduction of cerebral amyloid angiopathy but not amyloid plaques. The data unveil a selective role of the tPA in the suppression of functional hyperemia induced by Aβ and in the mechanisms of cerebral amyloid angiopathy, and support the possibility that modulation of the PAI-1-tPA pathway may be beneficial in diseases associated with amyloid accumulation.SIGNIFICANCE STATEMENT Amyloid-β (Aβ) peptides have profound neurovascular effects that may contribute to cognitive impairment in Alzheimer's disease. We found that Aβ attenuates the increases in blood flow evoked by neural activation through a reduction in tissue plasminogen activator (tPA) caused by upregulation of its endogenous inhibitor plasminogen inhibitor-1 (PAI-1). tPA deficiency prevents NMDA receptors from triggering nitric oxide production, thereby attenuating the flow increase evoked by neural activity. PAI-1 inhibition restores tPA activity, rescues neurovascular coupling, reduces amyloid deposition around blood vessels, and improves cognition in a mouse model of Aβ accumulation. The findings demonstrate a previously unappreciated role of tPA in Aβ-related neurovascular dysfunction and in vascular amyloid deposition. Restoration of tPA activity could be of therapeutic value in diseases associated with amyloid accumulation.

Genetic Background influences Endothelial Function along the Mouse Vascular Tree

The endothelium is crucial for its role in maintaining vascular integrity and protecting against cardiovascular disease. Endothelial dysfunction is associated with a host of disease states including cardiovascular disease, diabetes, chronic kidney disease, and atherosclerosis. However, it is unclear whether endothelial function or dysfunction varies throughout the vasculature. In addition, accumulating evidence suggests that endothelial function, and more generally vasomotor function, is genetically regulated. The purpose of this study was to determine the influence of genetic background on vasomotor function in blood vessels of differing size. Vasomotor performance was measured in aorta (thoracic (TA), and abdominal (AA)) and conduit arteries (carotid artery (CA) and femoral artery (FA)) from four inbred strains (SJL/J (SJL), DBA/2J (DBA), NZW/LacJ (NZW) and C57BL/6J (B6)) of mice. Arteries were dissected, cut into 2 mm segments, and mounted in a wire myograph system. Increasing concentrations of phenylephrine (PE, 1×10−9 – 1×10−5M) were used to measure contractile responses, while increasing concentrations of the endothelium‐dependent vasodilator acetylcholine (ACh, 1×10−9 – 1×10−5M) and endothelium‐independent vasodilator sodium nitroprusside (SNP, 1×10−9 – 1×10−5M) were used to assess relaxation responses. Two way ANOVA revealed a significant main effect of strain in response to PE for AA (P = .0032) and CA (P = .0098). Post hoc analysis for max contractile response showed impaired vasoconstriction in NZW AA compared to B6 (P = .0004). In response to ACh, a significant main effect of strain was found for all vessels (TA: P = &lt;.0001, AA: P = .0156, CA: P = .0069, FA: P = &lt;.0001). Post hoc analysis showed that TA max vasorelaxation was impaired for all strains (NZW: P = &lt; .0001, DBA: P = .0140, SJL: P = &lt; .0001) and that NZW FA were impaired when compared to B6 ( P = .0015). A significant main effect of strain in response to SNP was found in TA (P = .0011), AA (P = .0086), and CA (P = &lt; .0001). Relaxation responses to SNP were not significantly different between strains. These findings indicate a strong influence of genetic background on endothelial function and could be important in understanding the implications that genetics have on vascular response to vasoactive agents.Support or Funding InformationThis work was supported by a Texas A&amp;M Triads for Transformation grant (MPM), a Texas A&amp;M Merit Fellowship (DH), a Texas A&amp;M College of Education Strategic Research Award (SYS), and J.L. Huffines Institute of Sports Medicine and Human Performance (SYS).

Heterogeneous effect of aging on vasorelaxation responses in large and small arteries.

Aging is associated with impaired vascular function characterized in part by attenuated vasorelaxation to acetylcholine (ACh) and sodium nitroprusside (SNP). Due to structural and functional differences between conduit and resistance arteries, the effect of aging on vasorelaxation responses may vary along the arterial tree. Our purpose was to determine age‐related differences in vasorelaxation responses in large and small arteries. Responses to the endothelium‐dependent vasodilator acetylcholine (ACh) and the endothelium‐independent vasodilator sodium nitroprusside (SNP) were assessed in abdominal aorta (AA), iliac arteries (IA), femoral arteries (FA), and gastrocnemius feed arteries (GFA) from young and old male rats. ACh‐mediated vasorelaxation was significantly impaired in old AA and IA. SNP‐mediated vasorelaxation was impaired in old AA. To investigate a potential mechanism for impaired relaxation responses in AA and IA, we assessed eNOS protein content and interactions with caveolin‐1 (Cav‐1), and calmodulin (CaM) via immunoprecipitation and immunoblot analysis. We found no age differences in eNOS content or interactions with Cav1 and CaM. Combined data from all rats revealed that eNOS content was higher in IA compared to AA and FA (p < .001), and was higher in GFA than AA (p < .05). Cav1:eNOS interaction was greater in FA than in AA and IA (p < .01), and in GFA compared to IA (p < .05). No differences in CaM:eNOS were detected. In conclusion, age‐related impairment of vasorelaxation responses occurred in the large conduit, but not small conduit or resistance arteries. These detrimental effects of age were not associated with changes in eNOS or its interactions with Cav‐1 or CaM.

Effect of Nearby Construction Activity on Endothelial Function, Sensitivity to Nitric Oxide, and Potassium Channel Activity in the Middle Cerebral Arteries of Rats.

The present study assessed the effect of nearby construction activity on the responses of rat middle cerebral arteries (MCA) to the endothelium-dependent vasodilator acetylcholine and the NO donor sodium nitroprusside (SNP) and the activity of MaxiK potassium channels in MCA smooth muscle cells from male Sprague-Dawley rats. Two monitoring systems were used to assess vibrations in the animal rooms during and immediately after construction activities near the research building where the animal facility is located. One was a commercially available system; the other was a Raspberry-Pi (RPi)-based vibration monitoring system designed in our laboratory that included a small computing unit attached to a rolling sensor (low sensitivity) and a piezoelectric film sensor (high sensitivity). Both systems recorded increased levels of vibration during construction activity outside the building. During the construction period, vasodilator responses to acetylcholine and SNP were abolished, and MaxiK single-channel current opening frequency and open-state probability in cell-attached patches of isolated MCA myocytes were dramatically decreased. Recovery of acetylcholine- and SNP-induced dilation was minimal in MCA from rats studied after completion of construction but housed in the animal facility during construction, whereas responses to acetylcholine and SNP were intact in rats purchased, housed, and studied after construction. Baseline levels of vibration returned after the completion of construction, concomitant with the recovery of normal endothelium-dependent vasodilation to acetylcholine and of NO sensitivity assessed by using SNP in MCA from animals obtained after construction. The results of this study indicate that the vibration associated with nearby construction can have highly disruptive effects on crucial physiologic phenotypes.

Aldosterone impairs coronary adenosine-mediated vasodilation via reduced functional expression of Ca2+-activated K+ channels

Elevated plasma aldosterone (Aldo) levels are associated with greater risk of cardiac ischemic events and cardiovascular mortality. Adenosine-mediated coronary vasodilation is a critical cardioprotective mechanism during ischemia; however, whether this response is impaired by increased Aldo is unclear. We hypothesized that chronic Aldo impairs coronary adenosine-mediated vasodilation via downregulation of vascular K+ channels. Male C57BL/6J mice were treated with vehicle (Con) or subpressor Aldo for 4 wk. Coronary artery function, assessed by wire myography, revealed Aldo-induced reductions in vasodilation to adenosine and the endothelium-dependent vasodilator acetylcholine but not to the nitric oxide donor sodium nitroprusside. Coronary vasoconstriction to endothelin-1 and the thromboxane A2 mimetic U-46619 was unchanged by Aldo. Additional mechanistic studies revealed impaired adenosine A2A, not A2B, receptor-dependent vasodilation by Aldo with a tendency for Aldo-induced reduction of coronary A2A gene expression. Adenylate cyclase inhibition attenuated coronary adenosine dilation but did not eliminate group differences, and adenosine-stimulated vascular cAMP production was similar between Con and Aldo mice. Similarly, blockade of inward rectifier K+ channels reduced but did not eliminate group differences in adenosine dilation whereas group differences were eliminated by blockade of Ca2+-activated K+ (KCa) channels that blunted and abrogated adenosine and A2A-dependent dilation, respectively. Gene expression of several coronary KCa channels was reduced by Aldo. Together, these data demonstrate Aldo-induced impairment of adenosine-mediated coronary vasodilation involving blunted A2A-KCa-dependent vasodilation, independent of blood pressure, providing important insights into the link between plasma Aldo and cardiac mortality and rationale for aldosterone antagonist use to preserve coronary microvascular function.NEW & NOTEWORTHY Increased plasma aldosterone levels are associated with worsened cardiac outcomes in diverse patient groups by unclear mechanisms. We identified that, in male mice, elevated aldosterone impairs coronary adenosine-mediated vasodilation, an important cardioprotective mechanism. This aldosterone-induced impairment involves reduced adenosine A2A, not A2B, receptor-dependent vasodilation associated with downregulation of coronary KCa channels and does not involve altered adenylate cyclase/cAMP signaling. Importantly, this effect of aldosterone occurred independent of changes in coronary vasoconstrictor responsiveness and blood pressure.

β‐Hydroxybutyrate (βHOB) Increases Nitric Oxide Synthase Activity in Resistance Arteries from Dahl Salt‐sensitive Rats

The deleterious link between overconsumption of dietary salt and hypertension is unequivocal. This dogma has recently expanded with the report that a high salt diet lowers the circulating levels of the ketone metabolite, beta‐hydroxybutyrate (βHOB), in Dahl salt‐sensitive rats (Dahl S‐HS). While stimulators of ketogenesis are well known for improving cardiovascular health (e.g., exercise and caloric restriction), less is known about negative‐regulation of ketone body metabolism and how it may impact hypertension and vascular aging. Therefore, we hypothesized that raising the circulating levels of βHOB would improve endothelium‐dependent relaxation in mesenteric resistance arteries (MRA) of Dahl S‐HS. To test this hypothesis, 1,3‐butanediol (20% v/v), which is a precursor of βHOB, was supplemented in the drinking water of 5 week old, male Dahl S‐HS for 8 weeks. Subsequently MRA function was analyzed using wire myographs. Although relaxation to endothelium‐dependent vasodilator acetylcholine (ACh) was not different [%Relaxation, Dahl S‐low salt (LS): 40±11 vs. Dahl S‐HS: 52±11 vs. Dahl S‐HS+βHOB: 44±11, p&gt;0.05], the mechanism of relaxation was different between the groups. Specifically, while nitric oxide synthase inhibitor (L‐NAME; 100 μM) completely prevented relaxation in Dahl S‐LS and Dahl S‐HS+βHOB groups, MRA from Dahl S‐HS still partially relaxed in the presence of L‐NAME (%Relaxation, Dahl S‐LS: 1±4 vs. Dahl S‐HS: 33±5 vs. Dahl S‐HS+βHOB: 6±4, p&lt;0.05; Figure 1A–C). These data suggest that MRA from Dahl S‐HS relax through nitric oxide‐independent mechanisms and that βHOB treatment restores nitric oxide synthase activity, despite consumption of a high salt diet. To test whether 1,3‐butanediol treatment had direct effects on arterial function, concentrations‐response curves were performed to βHOB and 1,3‐butanediol in MRA from naïve 5 week old, male Dahl S‐LS rats. We observed that βHOB and 1,3‐butanediol caused triphasic‐quadriphasic responses after pre‐contraction to phenylephrine (PE; Figure 2A–B). Specifically, we observed that low concentrations (&lt;3 nM) of βHOB caused pronounced vasodilation and this was inhibited by both L‐NAME and tetraethylammonium (TEA; potassium channel inhibitor; both, p&lt;0.05; Figure 3A–B). Intermediate concentrations of βHOB (3 nM–100 nM) caused contraction and this tended to be inhibited by indomethacin (cyclooxygenase inhibitor; p=0.08; Figure 3C). Finally, high concentrations (100 nM–30 μM) of βHOB caused relaxation again and this was predominately mediated by nitric oxide (p&lt;0.05; Figure 3A). Overall, these data suggest that βHOB stimulates the production of endothelium‐derived factors and low concentrations of βHOB could be a novel therapy against hypertension‐associated vascular aging by increasing nitric oxide synthesis.Support or Funding InformationAmerican Heart Association (18POST34060003) and National Institutes of Health (K99GM118885 and R01HL143082).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Elevated pulmonary arterial pressure in Zucker diabetic fatty rats.

Diabetes is a very strong predictor of chronic systemic vascular diseases and acute cardiovascular events. Recently, associations between metabolic disorders and pulmonary hypertension have also been reported in both humans and animal models. In order to get some further insight into the relationship of pulmonary hypertension with obesity, insulin resistance and hyperglycemia, herein we have used the Zucker diabetic fatty rats (ZDF/clr-lepr fa) at 20 weeks fed a standard diet and compared to their lean Zucker littermates (ZL). ZDF rats were obese, had elevated plasma glucose levels and insulin resistance, i.e. a clinically relevant model of type 2 diabetes. They presented elevated systolic, diastolic and mean pulmonary arterial pressures and a parallel increase in the Fulton index. Systemic arterial pressures were also increased but the left ventricle plus septum weight was similar in both groups and the heart rate was reduced. Wall media thickening was observed in the small pulmonary arteries from the ZDF rats. Isolated pulmonary arteries mounted in a wire myograph showed similar vasoconstrictor responses to phenylephrine and 5-HT and similar responses to the endothelium-dependent vasodilator acetylcholine. However, the iNOS inhibitor 1400W enhanced the vasoconstrictor responses in ZDF but not in ZL rats. The protein expression of eNOS and iNOS was not significantly different in the lungs of the two groups. The lung expression of Bmpr2 mRNA was downregulated. However, the mRNA expression of Kcna5, Kcnk3, Kcnq1, Kcnq4 or Kcnq5, which encode for the potassium channels Kv1.5, TASK-1, Kv7.1, Kv7.4 and Kv7.5, respectively, was similar in ZL and ZDF rats. In conclusion, ZDF rats show increased pulmonary arterial pressure, right ventricular hypertrophy, pulmonary arterial medial thickening and downregulated lung Bmpr2 despite leptin resistance. These changes were mild but are consistent with the view that diabetes is a risk factor for pulmonary hypertension.

Low but not high frequency of intermittent hypoxia suppresses endothelium-dependent, oxidative stress-mediated contractions in carotid arteries of obese mice.

Obstructive sleep apnea is characterized by intermittent hypoxia (IH) during sleep and predisposes to endothelial dysfunction. Obesity is a major risk factor for the occurrence of sleep apnea. The present study compared the functional impact of low- (IH10; 10 hypoxic events/h) and high-frequency (IH60; 60 hypoxic events/h) IH for 4 wk on endothelial function in male C57BL/6 mice with or without high-fat (HF) diet-induced obesity. Mean arterial blood pressure (tail cuff method) was increased in obese mice after IH60 exposure, i.e., HF + IH60 group. The serum levels of the oxidative stress marker malondialdehyde were augmented in lean IH60 and HF groups, with a further increase in HF + IH60 but a reduction in HF + IH10 mice compared with the HF group. Vascular responsiveness was assessed as changes in isometric tension in isolated arteries. Relaxations to the endothelium-dependent vasodilator acetylcholine were impaired in HF + IH60 aortae. Endothelium-dependent contractions (EDC; response to acetylcholine in the presence of the nitric oxide synthase inhibitor l-NAME) in carotid arteries were augmented in the HF group, but this HF-induced augmentation was suppressed by low-frequency IH exposure. The addition of apocynin (antioxidant) reduced EDC in HF and HF + IH60 groups but not in HF + IH10 group. In conclusion, these findings suggest that exposure of obese mice to mild IH exerts preconditioning-like suppression of endothelium-dependent and oxidative stress-mediated contractions. When IH severity increases, this suppression diminishes and endothelial dysfunction accelerates. NEW & NOTEWORTHY The present study demonstrates, for the first time, that low-frequency intermittent hypoxia may exert a preconditioning-like suppression of oxidative stress-induced endothelium-dependent contractions in mice with diet-induced obesity. This relative suppression was diminished as intermittent hypoxia became more severe, and a deleterious effect on endothelial function emerged.

Abstract 149: CD36 in Perivascular Macrophages Contributes to Neurovascular and Cognitive Dysfunction and Amyloid Angiopathy in Mice Overexpressing the Alzheimer Aβ Peptide

Amyloid-β (Aβ) exerts deleterious effects on the cerebral microcirculation, which may play a role in Alzheimer’s disease and mixed dementias. In mouse models of amyloid precursor protein (APP) overexpression, the vascular effects of Aβ require the innate immunity receptor CD36 in perivascular macrophages (PVM), immune cells located in the Virchow-Robin space surrounding penetrating cerebral vessels. PVM, in turn, induce vascular oxidative stress and dysfunction via NADPH oxidase (Park et al., Circ Res 2017). However, whether CD36 in PVM is involved in the cognitive impairment induced by Aβ remains unknown. We transplanted CD36 -/- bone marrow (BM) into irradiated male APP-overexpressing mice (Tg2576) (age 12 months) to repopulate their perivascular space with CD36 -/- PVM. Three months later, cerebral blood flow (CBF) was measured by laser-Doppler flowmetry in the somatosensory cortex of anesthetized mice with a cranial window (n=5/group). Cognition was also tested (n=10/group). In Tg2576 mice transplanted with WT BM (Wt→Tg), CBF responses induced by whisker stimulation (WS) or neocortical superfusion of the endothelium-dependent vasodilator acetylcholine (ACh) were attenuated compared to WT mice receiving WT BM (Wt→Wt) (WS, -35%; ACh, -36%; p&lt;0.05). CD36 -/- BM transplantation in Tg2576 mice (CD36 -/- →Tg) prevented the cerebrovascular dysfunction (CBF increases: WS: Wt→Tg, 9±1% vs. CD36 -/- →Tg, 21±2%; ACh: Wt→Tg, 9±1% vs CD36 -/- →Tg, 17±2%; p&lt;0.05), and reduced cerebrovascular amyloid deposition (thioflavin-S-based vascular amyloid load: -58%; p&lt;0.05 from Wt→Tg). Wt→Tg mice spent more time finding the escape hole at the Barnes maze test, indicating impairment in spatial memory (Wt→Wt, 965±166 sec; Wt→Tg, 1807±166 sec; p&lt;0.05), and showed impaired nest building ability (nesting score: Wt→Wt, 4.6±0.2; Wt→Tg, 1.8±0.2; p&lt;0.05). These cognitive deficits were ameliorated in CD36 -/- →Tg mice (Barnes maze: 1217±136 sec; nesting score: 2.7±0.3; p&lt;0.05 from Wt→Tg). We conclude that CD36 in PVM is critically involved in the cerebrovascular and cognitive dysfunctions induced by Aβ, and in cerebral amyloid angiopathy. CD36 in PVM may be a new therapeutic target to counteract the detrimental neurovascular and cognitive effects of amyloid pathology.

Paeonol Attenuates LPS-Induced Endothelial Dysfunction and Apoptosis by Inhibiting BMP4 and TLR4 Signaling Simultaneously but Independently.

Inflammatory injury of the endothelium leads to apoptosis and endothelial dysfunction. The current study explored the effect and mechanisms of paeonol in inflammation-induced apoptosis and endothelial dysfunction induced by lipopolysaccharides (LPSs). The effects of paeonol on LPS-induced inflammatory injury were assessed by Western blotting, flow cytometry and reactive oxygen species (ROS) measurement in human umbilical vein endothelial cells (HUVECs) and C57BL/6J mice. Vascular reactivity of isolated mouse aortae was examined using wire myographs. The exposure of HUVECs to LPS increased the protein presence of Toll-like receptor 4 (TLR4), bone morphogenic protein 4 (BMP4), BMP receptor type 1A, nicotinamide adenine dinucleotide phosphate oxidase subunit 2, mitogen-activated protein kinase (MAPK), inducible nitric oxide synthase (iNOS), and cleaved caspase 3, as well as decreased it in phosphorylated endothelial nitric oxide synthase; these effects were prevented by treatment with paeonol. Similarly, cotreatment with paeonol reversed BMP4-induced apoptosis in HUVECs. Relaxation in response to the endothelium-dependent vasodilator acetylcholine were impaired in mouse aortae after exposure to LPSs; this endothelial dysfunction was reversed by cotreatment with paeonol, noggin (a BMP4 inhibitor), TAK242 (TLR4 antagonist), apocynin (an ROS scavenger), MAPK inhibitors, and AG (an iNOS inhibitor). BMP4 small interfering RNAs (siRNAs) abolished LPS-induced upregulation of BMP4 and cleaved caspase 3 protein, but not in cells treated with TLR4 siRNA and vice versa. The silencing of TLR4 and BMP4 abolished the inhibitory effects of paeonol on LPS-induced activation of cleaved caspase 3. The present results demonstrate that paeonol reduces LPS-induced endothelial dysfunction and apoptosis by inhibiting TLR4 and BMP4 signaling independently.

Endothelium-dependent vasodilatory signalling modulates α1 -adrenergic vasoconstriction in contracting skeletal muscle of humans.

'Functional sympatholysis' describes the ability of contracting skeletal muscle to attenuate sympathetic vasoconstriction, and is critical to ensure proper blood flow and oxygen delivery to metabolically active skeletal muscle. The signalling mechanism responsible for sympatholysis in healthy humans is unknown. Evidence from animal models has identified endothelium-derived hyperpolarization (EDH) as a potential mechanism capable of attenuating sympathetic vasoconstriction. In this study, increasing endothelium-dependent signalling during exercise significantly enhanced the ability of contracting skeletal muscle to attenuate sympathetic vasoconstriction in humans. This is the first study in humans to identify endothelium-dependent regulation of sympathetic vasoconstriction in contracting skeletal muscle, and specifically supports a role for EDH-like vasodilatory signalling. Impaired functional sympatholysis is a common feature of cardiovascular ageing, hypertension and heart failure, and thus identifying fundamental mechanisms responsible for sympatholysis is clinically relevant. Stimulation of α-adrenoceptors elicits vasoconstriction in resting skeletal muscle that is blunted during exercise in an intensity-dependent manner. In humans, the underlying mechanisms remain unclear. We tested the hypothesis that stimulating endothelium-dependent vasodilatory signalling will enhance the ability of contracting skeletal muscle to blunt α1 -adrenergic vasoconstriction. Changes in forearm vascular conductance (FVC; Doppler ultrasound, brachial intra-arterial pressure via catheter) to local intra-arterial infusion of phenylephrine (PE; α1 -adrenoceptor agonist) were calculated during (1) infusion of the endothelium-dependent vasodilators acetylcholine (ACh) and adenosine triphosphate (ATP), the endothelium-independent vasodilator (sodium nitroprusside, SNP), or potassium chloride (KCl) at rest; (2) mild or moderate intensity handgrip exercise; and (3) combined mild exercise + ACh, ATP, SNP, or KCl infusions in healthy adults. Robust vasoconstriction to PE was observed during vasodilator infusion alone and mild exercise, and this was blunted during moderate intensity exercise (ΔFVC: -34±4 and -34±3 vs. -13±2%, respectively, P<0.05). Infusion of ACh or ATP during mild exercise significantly attenuated PE vasoconstriction similar to levels observed during moderate exercise (ACh: -3±4; ATP: -18±4%). In contrast, infusion of SNP or KCl during mild exercise did not attenuate PE-mediated vasoconstriction (-32±5 and -46±3%). To further study the role of endothelium-dependent hyperpolarization (EDH), ACh trials were repeated with combined nitric oxide synthase and cyclooxygenase inhibition. Here, PE-mediated vasoconstriction was blunted at rest (blockade: -20±5 vs. -31±3% vs.; P<0.05) and remained blunted during exercise (blockade: -15±5 vs. -14±5%). We conclude that stimulation of EDH-like vasodilatation can blunt α1 -adrenergic vasoconstriction in contracting skeletal muscle of humans.

Adverse vascular remodelling is more sensitive than endothelial dysfunction to hyperglycaemia in diabetic rat mesenteric arteries

Increased vascular stiffness and reduced endothelial nitric oxide (NO) bioavailability are characteristic of diabetes. Whether these are evident at a more moderate levels of hyperglycaemia has not been investigated. The objectives of this study were to examine the association between the level of glycaemia and resistance vasculature phenotype, incorporating both arterial stiffness and endothelial function. Diabetes was induced in male Sprague Dawley rats with streptozotocin (STZ; 55mg/kg i.v.) and followed for 8 weeks. One week post STZ, diabetic rats were allocated to either moderate (⿼20mM blood glucose, 6⿿7U/insulins.c. daily) or severe hyperglycaemia (⿼30mM blood glucose, 1⿿2U/insulins.c. daily as required). At study end, rats were anesthetized, and the mesenteric arcade was collected. Passive mechanical wall properties were assessed by pressure myography. Responses to the endothelium-dependent vasodilator acetylcholine (ACh) were assessed using wire myography. Our results demonstrated for the first time that mesenteric arteries from both moderate and severely hyperglycaemic diabetic rats exhibited outward hypertrophic remodelling and increased axial stiffness compared to arteries from non-diabetic rats. Secondly, mesenteric arteries from severely (⿼30mM blood glucose), but not moderately hyperglycaemic (⿼20mM blood glucose) rats exhibit a significant reduction to ACh sensitivity compared to their non-diabetic counterparts. This endothelial dysfunction was associated with significant reduction in endothelium-derived hyperpolarisation and endothelium-dependent NO-mediated relaxation. Interestingly, endothelium-derived nitroxyl (HNO)-mediated relaxation was intact. Therefore, moderate hyperglycaemia is sufficient to induce adverse structural changes in the mesenteric vasculature, but more severe hyperglycaemia is essential to cause endothelial dysfunction.

Intracerebroventricular application of S100B selectively impairs pial arteriolar dilating function in rats

S100B is an astrocyte-derived protein that can act through the receptor for advanced glycation endproducts (RAGE) to mediate either “trophic” or “toxic” responses. Its levels increase in many neurological conditions with associated microvascular dysregulation, such as subarachnoid hemorrhage (SAH) and traumatic brain injury. The role of S100B in the pathogenesis of microvasculopathy has not been addressed. This study was designed to examine whether S100B alters pial arteriolar vasodilating function. Rats were randomized to receive (1) artificial cerebrospinal fluid (aCSF), (2) exogenous S100B, and (3) exogenous S100B+the decoy soluble RAGE (sRAGE). S100B was infused intracerebroventricularly (icv) using an osmotic pump and its levels in the CSF were adjusted to achieve a concentration similar to what we observed in SAH. After 48h of continuous icv infusion, a cranial window/intravital microscopy was applied to animals for evaluation of pial arteriolar dilating responses to sciatic nerve stimulation (SNS), hypercapnia, and topical suffusion of vasodilators including acetylcholine (ACh), s-nitroso-N-acetyl penicillamine (SNAP), or adenosine (ADO). Pial arteriolar dilating responses were calculated as the percentage change of arteriolar diameter in relation to baseline. The continuous S100B infusion for 48h was associated with reduced responses to the neuronal-dependent vasodilator SNS (p<0.05) and the endothelial-dependent vasodilator ACh (p<0.05), compared to controls. The inhibitory effects of S100B were prevented by sRAGE. On the other hand, S100B did not alter the responses elicited by vascular smooth muscle cell-dependent vasodilators, namely hypercapnia, SNAP, or ADO. These findings indicate that S100B regulates neuronal and endothelial dependent cerebral arteriolar dilation and suggest that this phenomenon is mediated through RAGE-associated pathways.