Arteriolar Dilation Research Articles

Stromal Vascular Fraction Restores Vasodilatory Function by Reducing Oxidative Stress in Aging-Induced Coronary Microvascular Disease.

Aims: The objective of this study is to identify mechanisms for adipose stromal vascular fraction's (SVF) restorative effects on vasodilation in aging-induced coronary microvascular disease (CMD). We hypothesize that reactive oxygen species (ROS) diminish β1-adrenergic receptor (β1ADR)- and flow-mediated dilation (FMD) in coronary arterioles, reversible by SVF and adipose-derived stem cells (ADSCs). Results: SVF attenuates aging-induced chronic accumulation of ROS and pro-oxidant gene and protein expression with enhancement of antioxidant gene and protein expression and glutathione, but not nitric oxide. ADSCs attenuate hydrogen peroxide while restoring nitric oxide and glutathione. Mass spectrometry of SVF- and ADSC-conditioned media reveals abundant antioxidant proteins suggesting a paracrine mechanism. FMD and β1ADR-mediated dilation diminished with aging, restored with SVF and ADSCs. FMD was restored by a switch in the acute signaling mediator from hydrogen peroxide in aging to peroxynitrite with SVF and ADSCs. Vasorelaxation to β1ADR-agonism was mechanistically linked with hydrogen peroxide, nitric oxide, and glutathione. Exogenous ROS eliminates isoproterenol-mediated dilation in youth that is blocked by inhibition of pro-desensitization and internalization proteins while nitric oxide enhances isoproterenol-mediated dilation in aging. Innovation: We introduce a novel mechanism by which ROS impacts β1ADR trafficking: the ROS/RNS-β1ADR desensitization and internalization axis. Aging-induced ROS shunts β1ADR from the plasma membrane into endosomes. SVF reduces oxidative burden, restoring functional β1ADR. Conclusions: SVF (and ADSCs to a lesser extent) reduce oxidative stress, and restore flow- and β1ADR-mediated vasodilation in aging. SVF represents a promising therapeutic strategy for CMD by addressing root cause of pathology; that is, oxidative stress-mediated hyperconstriction. Antioxid. Redox Signal. 38, 261-281.

Retinal microvascular function is associated with the cerebral microcirculation as determined by intravoxel incoherent motion MRI

Background and aimsThe easily accessible retinal vessels provide a unique opportunity to study a proxy for cerebral small vessels. Associations between retinal vessel diameters and macrostructural brain white matter changes have already been demonstrated. Alterations in microvascular function, likely precede these structural abnormalities. We examined whether retinal microvascular function is related to cerebral microvascular properties, assessed by the intravoxel incoherent motion (IVIM) effect in brain MRI. MethodsSeventy participants (age 60 ± 8 years, 41% women) from the population-based Maastricht Study underwent brain IVIM diffusion imaging (3 Tesla) to determine the microvascular measures f (perfusion volume fraction) and D* (pseudo-diffusion of circulating blood). The retinal arteriolar and venular dilation response to flicker light stimulation were measured by a dynamic vessel analyzer. Linear regression analysis was used to investigate associations between retinal vasoreactivity and IVIM measures in white matter hyperintensities (WMH), normal-appearing white matter (NAWM) and cortical gray matter (CGM). ResultsMore retinal arteriolar dilation was significantly associated with stronger pseudo-diffusion (D*) in the NAWM and CGM (β 0.280 [95% CI 0.084–0.475], and β 0.310 [95% CI 0.091–0.528], respectively), but not with the cerebral blood volume fraction (f). No associations were observed between retinal venular dilation response and cerebrovascular IVIM measures. ConclusionsVariations in retinal arteriolar microvascular function and microcirculatory properties in the brain are linked. The retina could serve as a proxy for early detection of brain microvascular dysfunction.

Persistent Coronary Vasomotor Tone During Myocardial Ischemia Occurs at the Capillary Level and May Involve Pericytes.

BackgroundThere is persistent coronary vasomotor tone during myocardial ischemia, despite ongoing coronary arteriolar dilatation. The mechanism underlying this vasodilatory tone, which can be unmasked by coronary vasodilators, is unclear. We hypothesized that persistent microvascular resistance during myocardial ischemia occurs at the level of capillaries and may be caused by pericytes.MethodsWe studied nine instrumented dogs where coronary blood flow and coronary driving pressure were reduced to half by placement of stenoses. Myocardial blood flow and myocardial blood volume were measured with myocardial contrast echocardiography before and during adenosine administration. In three animals, the heart was perfusion-fixed under these conditions for electron microscopic assessment of capillary and pericyte size.ResultsDuring ischemia, myocardial blood volume decreased and myocardial vascular resistance remained unchanged. Adenosine administration reversed the decline in myocardial blood volume and decreased myocardial vascular resistance. Electron microscopy showed larger capillaries in ischemic beds receiving adenosine than ischemic beds not receiving adenosine. Pericytes in beds receiving adenosine also tended to be larger.ConclusionCapillaries are the site of persistent vasomotor tone during myocardial ischemia; any other site of vascular regulation (arterioles or venules) cannot explain these myocardial contrast echocardiography findings, which are confirmed on post-mortem electron microscopic examination. The decrease in capillary size is likely caused by pericyte contraction in an attempt to maintain a constant capillary hydrostatic pressure. Adenosine relaxes pericytes, restores myocardial blood volume, reduces myocardial vascular resistance, and improves regional function during ischemia. These findings could have important therapeutic implications.

Ex vivo capillary-parenchymal arteriole approach to study brain pericyte physiology.

.SignificanceVascular mural cells, defined as smooth muscle cells (SMCs) and pericytes, influence brain microcirculation, but how they contribute is not fully understood. Most approaches used to investigate pericyte and capillary interactions include ex vivo retinal/slice preparations or in vivo two-photon microscopy. However, neither method adequately captures mural cell behavior without interfering neuronal tissue. Thus, there is a need to isolate vessels with their respective mural cells to study functional and pathological changes.AimThe aim of our work was to implement an ex vivo method that recapitulates vessel dynamics in the brain.ApproachExpanding upon our established ex vivo capillary-parenchymal arteriole (CaPA) preparation, we isolated and pressurized arteriole-capillary branches. Using Alexa Fluor™ 633 Hydrazide, we distinguished arterioles (containing elastin) versus capillaries (lacking elastin). In addition, our transgenic SMMHC-GCaMP6f mice allowed for us to visualize mural cell morphology and signals. Lastly, isolated microvasculature was cultured in DMEM media (up to 72 h), mounted, and pressurized using our CaPA preparation.ResultsU46619 induced a decrease in capillary lumen diameter using both a bath perfusion and local application. In addition, U46619 increased signaling both globally and locally in contractile pericytes. In our SMMHC-GCaMP6f mice, we saw that thin strand pericytes had sparse processes while contractile pericytes had long, thick processes that wrapped around the lumen of the capillary. Fresh and cultured pericytes constricted in response to U46619 to the same level, and upstream arteriolar dilation induced by capillary stimulation with 10 mM remained unchanged by culture conditions adding another application of longer treatment to our approach.ConclusionOur ex vivo CaPA methodology facilitates observation of arteriolar SMC and pericyte dynamic changes in real-time without environmental factors. This method will help to better understand how mural cells differ based on microvasculature location.

N-Methyl-D-Aspartic Acid Receptor-Mediated Vasodilation Is Attenuated in the Retinas of Diabetic Rats

Purpose Activation of N-methyl-d-aspartic acid (NMDA) receptors enhances nitric oxide (NO) production in retinal neuronal cells, and in turn, NO released from neuronal cells induces glial cell-mediated dilation of retinal arterioles in rats. The purpose of this study was to examine how neuronal cell-dependent, glial cell-mediated vasodilation is impacted in diabetic rat retinas. Methods Diabetes was induced in 6-week-old male Wistar rats by combining streptozotocin injection and D-glucose feeding. Two weeks later, the dilator function of retinal arterioles was assessed. Results Compared with non-diabetic rats, the dilator responses of retinal arterioles induced by intravitreal injection of NMDA and NOR3, an NO donor, were reduced in diabetic rats. Following the blockade of large-conductance Ca2+-activated K+ (BKCa) channels with iberiotoxin, no significant difference in the retinal vasodilator response to NOR3 was observed between non-diabetic and diabetic rats. Intravitreal injection of 14,15-epoxyeicosatrienoic acid, a vasodilatory factor released from glial cells, dilated retinal arterioles, and the response was diminished by diabetes. Conclusion These findings suggest that the impaired BKCa channel function in vascular cells is responsible for the diminished neuronal cell-dependent, glial cell-mediated dilation of retinal arterioles during the early stage of diabetes.

Association of cerebral microvascular dysfunction and white matter injury in Alzheimer's disease.

Patients with Alzheimer's disease (AD) often have cerebral white matter (WM) hyperintensities on MRI and microinfarcts of presumed microvascular origin pathologically. Here, we determined if vasodilator dysfunction of WM-penetrating arterioles is associated with pathologically defined WM injury and disturbances in quantitative MRI-defined WM integrity in patients with mixed microvascular and AD pathology. We analyzed tissues from 28 serially collected human brains from research donors diagnosed with varying degrees of AD neuropathologic change (ADNC) with or without cerebral microinfarcts (mVBI). WM-penetrating and pial surface arteriolar responses to the endothelium-dependent agonist bradykinin were quantified ex vivo with videomicroscopy. Vascular endothelial nitric oxide synthase (eNOS) and NAD(P)H-oxidase (Nox1,2 and 4 isoforms) expression were measured with quantitative PCR. Glial fibrillary acidic protein (GFAP)-labeled astrocytes were quantified by unbiased stereological approaches in regions adjacent to the sites of WM-penetrating vessel collection. Post-mortem diffusion tensor imaging (DTI) was used to measure mean apparent diffusion coefficient (ADC) and fractional anisotropy (FA), quantitative indices of WM integrity. In contrast to pial surface arterioles, white matter-penetrating arterioles from donors diagnosed with high ADNC and mVBI exhibited a significantly reduced dilation in response to bradykinin when compared to the other groups. Expression of eNOS was reduced, whereas Nox1 expression was increased in WM arterioles in AD and mVBI cases. WM astrocyte density was increased in AD and mVBI, which correlated with a reduced vasodilation in WM arterioles. Moreover, in cases with low ADNC, bradykinin-induced WM arteriole dilation correlated with lower ADC and higher FA values. Comorbid ADNC and mVBI appear to synergistically interact to selectively impair bradykinin-induced vasodilation in WM-penetrating arterioles, which may be related to reduced nitric oxide- and excess reactive oxygen species-mediated vascular endothelial dysfunction. WM arteriole vasodilator dysfunction is associated with WM injury, as supported by reactive astrogliosis and MRI-defined disrupted WM microstructural integrity.

Mortality prediction of retinal vessel diameters and function in a long-term follow-up of haemodialysis patients.

Retinal vessel diameters are candidate biomarkers of mortality prediction in large population-based studies. We aimed to investigate the predictive value of retinal vessel diameters and flicker-induced retinal arteriolar and venular dilation on all-cause mortality in long-term follow-up of haemodialysis patients. Retinal vessel diameters as well as maximum arteriolar (aMax) and venular dilation (vMax) were investigated in 275 and 214 haemodialysis patients, respectively. Patients were observed in a long-term follow-up for a median period of 73 months. About 36% (76/214) and 41% (113/275) of patients died. Arteriolar and venular diameters were 175 ± 19 and 208 ± 20 µm, respectively. Median aMax and vMax were 1.6 (0.3-3.3) and 3.2 (2.0-5.1)%. Patients within the lowest tertile of vMax showed lower 5-year survival rates compared with the highest tertile (50.6 vs. 82.1%) and also exhibited a higher incidence of infection-related deaths (21.7 vs. 4.0%). Univariate hazard ratio (HR) per standard deviation increase of vMax for all-cause mortality was 0.69 (0.54-0.88) and was even more pronounced for infection-related mortality [HR 0.53 (0.33-0.83)]. Regarding all-cause mortality, multivariate adjustment for eight non-retinal mortality predictors including interleukin-6 did not attenuate the HR relevantly [0.73 (0.54-0.98)]. Arteriolar and venular diameters did not predict all-cause nor cardiovascular and infection-related mortality. Long-term follow-up of patients on haemodialysis demonstrated the potential of retinal venular dilation capacity for mortality prediction, which was most pronounced for infection-related mortality. In the same cohort, retinal arteriolar and venular diameters showed no predictive value for hard endpoints. Retinal venular dilation but not arteriolar and venular diameters is a valuable diagnostic biomarker for risk prediction in patients with end-stage renal disease and should be considered for monitoring of critically ill patients.

Arterial vasodilation drives convective fluid flow in the brain: a poroelastic model

The movement of fluid into, through, and out of the brain plays an important role in clearing metabolic waste. However, there is controversy regarding the mechanisms driving fluid movement in the fluid-filled paravascular spaces (PVS), and whether the movement of metabolic waste in the brain extracellular space (ECS) is primarily driven by diffusion or convection. The dilation of penetrating arterioles in the brain in response to increases in neural activity (neurovascular coupling) is an attractive candidate for driving fluid circulation, as it drives deformation of the brain tissue and of the PVS around arteries, resulting in fluid movement. We simulated the effects of vasodilation on fluid movement into and out of the brain ECS using a novel poroelastic model of brain tissue. We found that arteriolar dilations could drive convective flow through the ECS radially outward from the arteriole, and that this flow is sensitive to the dynamics of the dilation. Simulations of sleep-like conditions, with larger vasodilations and increased extracellular volume in the brain showed enhanced movement of fluid from the PVS into the ECS. Our simulations suggest that both sensory-evoked and sleep-related arteriolar dilations can drive convective flow of cerebrospinal fluid not just in the PVS, but also into the ECS through the PVS around arterioles.

Acute Antioxidant and Cytoprotective Effects of Caffeine in the Model of Neurovascular Injury by Oxidative Stress

Caffeine is commonly used in the neonatal intensive care units for respiratory disorders, but the effects of this medication in the neonatal cerebral circulation remain largely unexplored. We used newborn pigs as a clinically relevant model of the neonatal human brain to investigate the effects of caffeine on cerebral arterioles and primary neurovascular cells. First, we examined dose‐dependent cerebrovascular and systemic effects of water‐soluble anhydrous caffeine in anesthetized newborn pigs equipped with closed cranial windows. Caffeine administered systemically at therapeutic concentrations (20‐40 mg/kg i.v.) had no significant effects on diameter of pial arterioles, mean arterial blood pressure (MABP), and heart rate within 10‐60 min after the injection. When used at a higher concentration (70 mg/kg i.v.), caffeine produced dilation of pial arterioles (15‐20%) accompanied by an increased heart rate, without any effect on MABP. Topical application of caffeine (10‐5‐10‐3M) under the cranial window produced a dose‐dependent dilation of pial cerebral arterioles (15‐30%) that was fully reversed by a brief flushing with artificial cerebrospinal fluid. These in vivo data indicate that cerebral vascular cells are potential targets for caffeine. Next, we tested the hypothesis that caffeine exhibits cytoprotective properties in cerebral circulation during oxidative stress conditions. We used an in vitro model of acute oxidative stress‐induced neurovascular injury caused by excitotoxic and inflammatory conditions concomitant with epileptic seizures, asphyxia, and cerebral ischemia. Endothelial cells and cortical astrocytes are the key components of the neurovascular unit that are most vulnerable to oxidative stress injury. Primary cultures of cerebral vascular endothelial cells (CVEC) and cortical astrocytes from newborn pigs responded to pro‐inflammatory cytokine TNF‐alpha (30 ng/ml) and excitotoxic glutamate (1‐2 mM) by a surge in reactive oxygen species (ROS) production leading to apoptosis. NADPH oxidase is the major ROS‐generating system in the neurovascular unit during inflammation and excitotoxicity. We examined the acute effects of caffeine on oxidative stress‐induced neurovascular cell death caused by TNF‐alpha and excitotoxic glutamate. Caffeine (0.1‐10 µM) inhibited NADPH oxidase activation, reduced overall ROS generation, and attenuated major key events of apoptosis (caspase‐3 activation, DNA fragmentation, and loss of cell‐cell contacts) in CVEC and astrocytes exposed to TNF‐a and glutamate. These data demonstrate, for the first time, that caffeine exhibits acute antioxidant and cytoprotective effects in cerebral vascular endothelial cells and astrocytes. Overall, endothelial and astrocytic components of the neurovascular unit represent target cells for caffeine cytoprotective therapy in the neonatal cerebral circulation during oxidative stress.

Sex Differences in Hydrogen Peroxide‐Induced K+ Channel Activation in Arterioles of Ischemic Myocardium after Exercise Training

The objective of this study was to determine sex differences in potassium (K+) channel contribution to H2O2‐mediated dilation after exercise training in arterioles distal to chronic coronary artery occlusion. We tested the hypothesis that females would have enhanced H2O2‐mediated vasodilation compared to males through enhanced coupling of H2O2 with large‐conductance, calcium‐dependent K+ (BKCa; iberiotoxin 100 nM) channels and 4‐aminopyridine (4‐AP; 1 mM)‐sensitive voltage‐gated K+ (Kv). Adult Yucatan miniature swine were surgically instrumented with an Ameroid constrictor around the proximal left circumflex coronary artery, which gradually induced occlusion and created a collateral‐dependent vascular bed. Arterioles from the left anterior descending artery myocardial region served as nonoccluded control vessels. Pigs of each sex were separated into two groups: one that underwent a progressive exercise training program (treadmill run; 5 days/week for 14 weeks) and another that remained sedentary and was limited to normal pen activity. Collateral‐dependent arterioles from female, sedentary pigs were significantly less sensitive to H2O2‐induced dilation compared with nonoccluded arterioles (EC50: ‐4.55 ± 0.15 vs. ‐4.91 ± 0.15 log M, respectively; P<0.05), whereas exercise training reversed the impaired dilation so that collateral‐dependent and nonoccluded arterioles responded similarly to H2O2 (EC50: ‐4.86 ± 0.16 vs. ‐4.95 ± 0.18 log M, respectively). Conversely, male pigs demonstrated enhanced vasodilation to H2O2 in collateral‐dependent compared with nonoccluded arterioles (‐4.92 ± 0.16 vs. –4.35 ± 0.16 log M, respectively) of sedentary pigs, which persisted after exercise training (‐4.94 ± 0.11 vs. ‐4.50 ± 0.21 log M, respectively). In female sedentary pigs, H2O2‐mediated dilation in nonoccluded or collateral‐dependent arterioles was not altered by inhibitors of BKCa or Kv channels. In contrast, after exercise training, Kv channels contributed significantly to dilation in nonoccluded arterioles and both BKCa and Kv channels played a significant role in dilation of collateral‐dependent arterioles in female pigs. Blockade of BKCa or Kv channels in nonoccluded or collateral‐dependent arterioles from male pigs had no effect on H2O2‐mediated vasodilation, independent of exercise‐training. Taken together, our studies suggest that coronary arterioles in females compensate for impaired H2O2‐mediated vasodilation by modulating BKCa and 4‐AP‐sensitive Kv channels after exercise‐training. Males do not appear to utilize the particular pathway involving H2O2 action on BKCa or 4‐AP‐sensitive Kv channels.

5‐HT7 Receptors Mediate Dilation of Rat Cremaster Muscle Arterioles in vivo

Serotonin (5‐HT) infusion in vivo causes hypotension and a fall in total peripheral resistance (TPR) that is mediated by 5‐HT7 receptors. 5‐HT dilates small arterioles in skeletal muscle, a microcirculation that importantly contributes to TPR. However, the receptors that mediate this dilation have not been established. Therefore, we tested the hypothesis that 5‐HT7 receptors mediate the arteriolar dilation in the skeletal muscle microcirculation. Cremaster muscles of isoflurane‐anesthetized male Sprague‐Dawley rats were prepared for digital in vivo microscopy and superfused with physiological salt solution (PSS) at 34 °C. Bolus (1‐10 nmols) application of 5‐HT into the superfusate caused substantial dilation of 3rd to 5th order arterioles from 10 ± 1 to 27 ± 1 μm (n = 10 arterioles from 4 rats, p <0.0001) that recovered within 2‐3 min. Dilation to bolus application of 5‐HT was inhibited by superfusion with 1 μM SB269970, a selective 5‐HT7 receptor antagonist (diameter in SB = 9 ± 1 μm; diameter in SB + 5‐HT = 10 ± 1 μm; n = 10 arterioles from 4 rats; p = 0.8763). Similarly, steady‐state superfusion with 10‐30 nM of the 5‐HT1/7 receptor agonist 5‐carboxamidotryptamine (5‐CT) dilated 3rd‐5th‐order arterioles from 16 ± 3 to 37 ± 5 μm (n = 7 arterioles from 3 rats, p = 0.0004). This dilation was reversed (diameter in 1 μM SB269970 = 13 ± 2 μm, n = 7, p = 0.1479 vs. baseline) or blocked (diameter in 1 μM SB269970 + 10‐30 nM 5‐CT = 10 ± 2 μm, n = 7, p = 0.0005 vs. 5‐CT alone) by 1 μM SB269970. Dilation of arterioles to the muscarinic agonist, methacholine (MCH) was not inhibited by SB269970 verifying the specificity of the antagonist; arterioles dilated from 13 ± 2 μm to 25 ± 2 μm to 100 nmols MCH and dilated from 9 ± 2 μm to 32 ± 2 μm in response to MCH in the presence of 1 μM SB269970; n = 10 arterioles from 4 rats; p = 0.7232 vs. dilation in the absence of SB269970. Consistent with these data, pooled samples (4‐5 rats) of 1st and 2nd – order cremaster arterioles expressed message for 5‐HT7 receptors by quantitative real‐time PCR (CT = 36.4 ± 0.74, n = 3 pooled samples, p = 0.039 vs CT = 40). These data support our hypothesis that 5‐HT7 receptors mediate dilation of small arterioles in skeletal muscle and likely contribute to the observed hypotension when these receptors are engaged in vivo.

Endothelial connexin hemichannels as a novel endothelial regulatory mechanism of vasodilation

The vascular endothelium regulates vasomotor tone in arterioles through intercellular conduction of electrical signals by gap junctions. Endothelial vasodilation is mediated by nitric oxide (NO) and by a hyperpolarizing current initiated in the endothelium. Both signals are transmitted to smooth muscle cells evoking relaxation. Endothelial cells express connexin (Cx) 37,40,43. Cx hemichannels allow the exchange of ions and molecules between intracellular and extracellular compartments that may be associated with endothelial electrical activity. Whether or not Cx hemichannels regulate vasomotor tone is unknown. We evaluated the role of Cx‐hemichannels in the vasodilation process by measuring dye uptake in primary culture of endothelial cells and in isolated mouse mesenteric arterioles. We detected that both acetylcholine (ACh) and bradykinin (BK) increase dye permeability, which was inhibited by connexin mimetic peptides that block Cx43 hemichannels (Gap19). We determined that NO mediates Cx43 hemichannels activity inasmuch as blocking NOS inhibits hemichannel activity. Using biotin switch assay, we observed that ACh induced S‐nitrosylation only in Cx43 at cysteine 271. ACh and BK open endothelial Cx43 hemichannels activity and promote endothelial Ca2+entry from the extracellular space and lead to endothelium hyperpolarization signaling in isolated mesenteric arteries. Blocking Cx43‐hemichannels reduces calcium entry and hyperpolarization. To further assess the physiological role of Cx43 endothelial hemichannels activity, we cannulated isolated mesenteric arterioles and studied ACh‐induced arteriolar relaxation in the presence and absence of connexin mimetic peptides that block Cx43 hemichannels (Gap19) and Cx40 (40Gap 27, a connexin mimetic peptide). Gap19 attenuates ACh‐induced relaxation, but 40Gap27 fails to reduce relaxation. These results indicate that endothelial Cx43 hemichannels participate in arteriolar dilatation. In addition, we replaced cysteine 271 by serine to create a Cx43C271S mouse that failed to cause S‐nitrosylation of Cx43. We detected that this knockin mouse displayed hypertension compared to wild type mice, indicating that Cx43 hemichannels and S‐nitrosylated Cx43 play a role in vascular resistance. Our results suggest that Cx43 hemichannels are a potential therapeutic target in hypertension and vascular disease.

Exercise Training Shifts Dependency from NADPH Oxidase 2 to NADPH Oxidase 4 in Endothelium‐Dependent Dilation in Coronary Arterioles Distal to Chronic Occlusion

Previously, we have reported that exercise training enhances endothelium‐dependent dilation in coronary arterioles distal to chronic occlusion, through increased contribution of the redox signaling molecule, hydrogen peroxide (H2O2). In the present study, we tested the hypothesis that enhanced endothelium‐dependent dilation in collateral‐dependent arterioles after exercise training would be attributable to an increased contribution of NADPH oxidase (NOX) 4‐derived reactive oxygen species. Yucatan miniature swine were surgically instrumented with an ameroid constrictor around the proximal left circumflex artery, which gradually induced occlusion of the artery and created a collateral‐dependent vascular bed. Eight weeks postoperatively, swine were assigned to either a sedentary protocol consisting of normal pen activity or a progressive exercise‐training regimen comprised of treadmill running 5 days a week, for 14 weeks. Coronary arterioles were isolated from the nonoccluded and collateral‐dependent myocardial regions and bradykinin‐mediated dilation via pressure myography was performed in the presence and absence of ML‐171, gp91ds‐tat and GKT136901, inhibiting NOX1, NOX2, and NOX1/4 isoforms, respectively. Additionally, using the superoxide probe, dihydroethidium, HPLC analysis was performed on isolated microvascular endothelial cells, pretreated with NOX inhibitors, to determine candidate sources of superoxide. Inhibition of NOX2 produced a significant rightward shift in EC50 values of arterioles from sedentary animals, independent of occlusion, as well as the nonoccluded arterioles of exercise‐trained pigs. EC50 values of collateral‐dependent arterioles from exercise‐trained swine were not significantly altered by NOX2 inhibition. Contrastingly, collateral‐dependent arterioles from exercise‐trained swine exhibited a significant shift in EC50 values in the presence NOX1/4 inhibition, which was absent in other arteriole treatment groups. Importantly, NOX1 inhibition did not significantly shift EC50 values in any treatment group, suggesting responses to the NOX1/4 inhibitor, GKT136901, reflect the contribution of NOX4. Interestingly, microvascular endothelial cell superoxide levels were not different across arteriole treatment groups. Our present findings suggest that exercise training produces adaptations in collateral‐dependent arterioles that appear to shift dependence from NOX2‐ to NOX4‐derived reactive oxygen species for endothelium‐dependent dilation. Taken together with our previous findings, our data support the assertion that enhanced dilation of collateral‐dependent arterioles after exercise training is driven by enhanced NOX4‐derived H2O2 levels.

Constrictor Responses of Cerebral Resistance Arterioles Preserved in Male and Female Rats Exposed to Prenatal Alcohol

Fetal alcohol spectrum disorder (FASD) is the result of exposure to alcohol in utero. This disorder causes lifelong intellectual and behavioral disabilities, as well as cardio and cerebrovascular dysfunction. However, the mechanisms by which these abnormalities arise are poorly understood, and no treatment exists. Past studies have shown that dilation of cerebral arterioles to nitric oxide synthase (NOS)‐dependent agonists is impaired in rats exposed to prenatal alcohol, yet no studies have examined the influence of prenatal alcohol on constrictor responses of cerebral arterioles. We hypothesize that in uteroalcohol exposure may impair the activity of the vascular smooth muscle, which could lead to an alteration in the ability of the cerebral arterioles to constrict in response to endogenous vasoconstrictor agents. Therefore, the goal of this study was to determine whether responses of cerebral arterioles to vasoconstrictors are altered in rats exposed to alcohol in utero. We fed Sprague‐Dawley dams a liquid diet with or without alcohol (3% ethanol) for the duration of their pregnancy (21‐23 days). We then examined the reactivity of the cerebral arterioles of the pups to arginine vasopressin (AVP; 0.1 and 1 nM), thromboxane (U‐46619; 10 and 100 nM), and angiotensin II (1 and 10 µM) in four groups: control male, control female, prenatal alcohol male, and prenatal alcohol female, at two different age ranges: 4‐6 weeks of age and 14‐16 weeks of age. We found that constrictor responses of cerebral arterioles to AVP and the thromboxane analog (U‐46619) were similar in control rats and rats exposed to prenatal alcohol at both age groups for both sexes. In addition, adolescent male and female rats showed no difference to angiotensin II following prenatal exposure to alcohol. However, alcohol‐exposed females exhibited an unexpected dilation to the high concentration of angiotensin II in adulthood, which was absent in males. We suggest that the findings from these studies may have implications regarding the susceptibility of the brain to cerebral ischemic damage. We speculate that impaired vasodilation, with preserved vasoconstriction, can lead to a scenario favoring a decrease in cerebral blood flow during times of increased metabolic demand. This is the first published study to examine the influence of in uteroexposure to alcohol on constrictor responses of cerebral arterioles. Further understanding of how cerebrovascular blood flow may be altered in rats with FASD can ultimately lead to an understanding of how the resulting ischemic damage may relate to intellectual and behavioral abnormalities.

Post‐menopausal Cerebral Microvascular Alterations in a Mouse Model of Alzheimer’s Disease

Cognitive decline, as seen in Alzheimer’s disease (AD), is a growing public health concern and is linked to decreased cerebral blood flow, particularly in women after menopause. Impaired cerebrovascular function precedes the onset of AD, possibly due to reduced endothelial function in parenchymal arterioles, the bottlenecks of the cerebral microcirculation, although exact mechanisms by menopause remain elusive. The goal of this study was to determine whether menopause impairs endothelial function in parenchymal arterioles of wild‐type (WT) and 5x‐FAD mouse model of AD. Menopause was induced by the 4‐vinylcyclohexene diepoxide (VCD) model, which induces gradual ovarian failure. Cerebral parenchymal arterioles were isolated and studied by pressure myography. Data are means ± SEM. In WT mice, menopause caused a significant increase in myogenic tone (myogenic tone: 24.73 ± 1.2 vs. 34 ± 2.6%, vehicle vs VCD, n = 5 / 8; p<0.05), as well as a small but significant reduction in resting lumen diameter (32.68 ± 1.3 vs. 28.40 ± 2.1 µm, vehicle vs VCD, n = 5 / 8; p<0.05). We then tested endothelial function, focusing on K+ channels, namely small and intermediate Ca2+‐activated K+ channels (SKCa / IKCa) and inwardly rectifying K+ channels (KIR2). Menopause did not affect arteriolar dilation to a cumulative concentration‐response curve of the SKCa/ IKCa activator NS‐309 (at 1 µM, vasodilation: 23.49 ± 11.5 vs. 17.80 ± 1.7%, vehicle vs VCD, n = 3 / 6). Similarly, KIR2 function was unchanged (vasodilation: 16.06 ± 6.6 vs. 16.21 ± 2.9%, vehicle vs VCD, n = 4 / 8). In the 5x‐FAD mice, menopause did not affect myogenic tone (myogenic tone: 23.49 ± 2.2 vs. 28 3.2%, vehicle vs VCD, n = 10 / 7), although there was a significant decrease in resting lumen diameter (39.16 ± 2.6 vs. 27.79 ± 2.0 µm, vehicle vs VCD, n = 10 / 7 p<0.05). Arterioles from menopausal 5x‐FAD mice showed a significantly blunted response to NS‐309 (at 1 µM, vasodilation: 14.96 ± 2.9 vs. 9.42 ± 1.4%, vehicle vs VCD, n = 7 / 4, p<0.05), without differences in KIR2‐induced dilation (vasodilation: 16.02 ± 1.3 vs. 13.69 ± 2.3%, vehicle vs VCD n = 9 / 6). In conclusion, we found that in WT mice, menopause leads to changes in myogenic tone without affecting K+‐dependent dilation. However, in 5x‐FAD mice, menopause impairs endothelial SKCa/IKCa channels independent of KIR2 function or myogenic tone. These findings identify new targets modulated by menopause in the cerebral microvasculature of WT and AD mice, which may contribute to cognitive dysfunction.

NADPH oxidase 4 contributes to TRPV4-mediated endothelium-dependent vasodilation in human arterioles by regulating protein phosphorylation of TRPV4 channels.

Impaired endothelium-dependent vasodilation has been suggested to be a key component of coronary microvascular dysfunction (CMD). A better understanding of endothelial pathways involved in vasodilation in human arterioles may provide new insight into the mechanisms of CMD. The goal of this study is to investigate the role of TRPV4, NOX4, and their interaction in human arterioles and examine the underlying mechanisms. Arterioles were freshly isolated from adipose and heart tissues obtained from 71 patients without coronary artery disease, and vascular reactivity was studied by videomicroscopy. In human adipose arterioles (HAA), ACh-induced dilation was significantly reduced by TRPV4 inhibitor HC067047 and by NOX 1/4 inhibitor GKT137831, but GKT137831 did not further affect the dilation in the presence of TRPV4 inhibitors. GKT137831 also inhibited TRPV4 agonist GSK1016790A-induced dilation in HAA and human coronary arterioles (HCA). NOX4 transcripts and proteins were detected in endothelial cells of HAA and HCA. Using fura-2 imaging, GKT137831 significantly reduced GSK1016790A-induced Ca2+ influx in the primary culture of endothelial cells and TRPV4-WT-overexpressing human coronary artery endothelial cells (HCAEC). However, GKT137831 did not affect TRPV4-mediated Ca2+ influx in non-phosphorylatable TRPV4-S823A/S824A-overexpressing HCAEC. In addition, treatment of HCAEC with GKT137831 decreased the phosphorylation level of Ser824 in TRPV4. Finally, proximity ligation assay (PLA) revealed co-localization of NOX4 and TRPV4 proteins. In conclusion, both TRPV4 and NOX4 contribute to ACh-induced dilation in human arterioles from patients without coronary artery disease. NOX4 increases TRPV4 phosphorylation in endothelial cells, which in turn enhances TRPV4-mediated Ca2+ entry and subsequent endothelium-dependent dilation in human arterioles.

Role of Adiponectin Receptor 1 in Promoting Nitric Oxide-Mediated Flow-Induced Dilation in the Human Microvasculature.

Chronic administration of exogenous adiponectin restores nitric oxide (NO) as the mediator of flow-induced dilation (FID) in arterioles collected from patients with coronary artery disease (CAD). Here we hypothesize that this effect as well as NO signaling during flow during health relies on activation of Adiponectin Receptor 1 (AdipoR1). We further posit that osmotin, a plant-derived protein and AdipoR1 activator, is capable of eliciting similar effects as adiponectin. Human arterioles (80–200 μm) collected from discarded surgical adipose specimens were cannulated, pressurized, and pre-constricted with endothelin-1 (ET-1). Changes in vessel internal diameters were measured during flow using videomicroscopy. Immunofluorescence was utilized to compare expression of AdipoR1 during both health and disease. Administration of exogenous adiponectin failed to restore NO-mediated FID in CAD arterioles treated with siRNA against AdipoR1 (siAdipoR1), compared to vessels treated with negative control siRNA. Osmotin treatment of arterioles from patients with CAD resulted in a partial restoration of NO as the mediator of FID, which was inhibited in arterioles with decreased expression of AdipoR1. Together these data highlight the critical role of AdipoR1 in adiponectin-induced NO signaling during shear. Further, osmotin may serve as a potential therapy to prevent microvascular endothelial dysfunction as well as restore endothelial homeostasis in patients with cardiovascular disease.

Astrocyte regulation of cerebral blood flow during hypoglycemia

Hypoglycemia triggers increases in cerebral blood flow (CBF), augmenting glucose supply to the brain. We have tested whether astrocytes, which can regulate vessel tone, contribute to this CBF increase. We hypothesized that hypoglycemia-induced adenosine signaling acts to increase astrocyte Ca2+ activity, which then causes the release of prostaglandins (PGs) and epoxyeicosatrienoic acids (EETs), leading to the dilation of brain arterioles and blood flow increases. We used an awake mouse model to investigate the effects of insulin-induced hypoglycemia on arterioles and astrocytes in the somatosensory cortex. During insulin-induced hypoglycemia, penetrating arterioles dilated and astrocyte Ca2+ signaling increased when blood glucose dropped below a threshold of ∼50 mg/dL. Application of the A2A adenosine receptor antagonist ZM-241385 eliminated hypoglycemia-evoked astrocyte Ca2+ increases and reduced arteriole dilations by 44% (p < 0.05). SC-560 and miconazole, which block the production of the astrocyte vasodilators PGs and EETs respectively, reduced arteriole dilations in response to hypoglycemia by 89% (p < 0.001) and 76% (p < 0.001). Hypoglycemia-induced arteriole dilations were decreased by 65% (p < 0.001) in IP3R2 knockout mice, which have reduced astrocyte Ca2+ signaling compared to wild-type. These results support the hypothesis that astrocytes contribute to hypoglycemia-induced increases in CBF by releasing vasodilators in a Ca2+-dependent manner.

Cognitive impairment and microvascular function in end-stage renal disease.

ObjectiveHemodialysis patients show an approximately threefold higher prevalence of cognitive impairment compared to the age‐matched general population. Impaired microcirculatory function is one of the assumed causes. Dynamic retinal vessel analysis is a quantitative method for measuring neurovascular coupling and microvascular endothelial function. We hypothesize that cognitive impairment is associated with altered microcirculation of retinal vessels.Methods152 chronic hemodialysis patients underwent cognitive testing using the Montreal Cognitive Assessment. Retinal microcirculation was assessed by Dynamic Retinal Vessel Analysis, which carries out an examination recording retinal vessels' reaction to a flicker light stimulus under standardized conditions.ResultsIn unadjusted as well as in adjusted linear regression analyses a significant association between the visuospatial executive function domain score of the Montreal Cognitive Assessment and the maximum arteriolar dilation as response of retinal arterioles to the flicker light stimulation was obtained.ConclusionThis is the first study determining retinal microvascular function as surrogate for cerebral microvascular function and cognition in hemodialysis patients. The relationship between impairment in executive function and reduced arteriolar reaction to flicker light stimulation supports the involvement of cerebral small vessel disease as contributing factor for the development of cognitive impairment in this patient population and might be a target for noninvasive disease monitoring and therapeutic intervention.

Role of Caveolae in the Development of Microvascular Dysfunction and Hyperglycemia in Type 2 Diabetes.

In type 2 diabetes (T2D) microvascular dysfunction can interfere with tissue glucose uptake thereby contributing to the development of hyperglycemia. The cell membrane caveolae orchestrate signaling pathways that include microvascular control of tissue perfusion. In this study, we examined the role of caveolae in the regulation of microvascular vasomotor function under the condition of hyperglycemia in T2D patients and rodent models. Human coronary arterioles were obtained during cardiac surgery from T2D patients, with higher perioperative glucose levels, and from normoglycemic, non-diabetic controls. The coronary arteriole responses to pharmacological agonists bradykinin and acetylcholine were similar in T2D and non-diabetic patients, however, exposure of the isolated arteries to methyl-β-cyclodextrin (mβCD), an agent known to disrupt caveolae, reduced vasodilation to bradykinin selectively in T2D subjects and converted acetylcholine-induced vasoconstriction to dilation similarly in the two groups. Dilation to the vascular smooth muscle acting nitric oxide donor, sodium nitroprusside, was not affected by mβCD in either group. Moreover, mβCD reduced endothelium-dependent arteriolar dilation to a greater extent in hyperglycemic and obese db/db mice than in the non-diabetic controls. Mechanistically, when fed a high-fat diet (HFD), caveolin-1 knockout mice, lacking caveolae, exhibited a significantly reduced endothelium-dependent arteriolar dilation, both ex vivo and in vivo, which was accompanied by significantly higher serum glucose levels, when compared to HFD fed wild type controls. Thus, in T2D arterioles the role of caveolae in regulating endothelium-dependent arteriole dilation is altered, which appears to maintain vasodilation and mitigate the extent of hyperglycemia. While caveolae play a unique role in microvascular vasomotor regulation, under the condition of hyperglycemia arterioles from T2D subjects appear to be more susceptible for caveolae disruption-associated vasomotor dysfunction and impaired glycemic control.