Vasodilation In Resistance Arteries Research Articles

Polarized localization of phosphatidylserine in the endothelium regulates Kir2.1

Lipid regulation of ion channels is largely explored using in silico modeling with minimal experimentation in intact tissue; thus, the functional consequences of these predicted lipid-channel interactions within native cellular environments remain elusive. The goal of this study is to investigate how lipid regulation of endothelial Kir2.1 - an inwardly rectifying potassium channel that regulates membrane hyperpolarization - contributes to vasodilation in resistance arteries. First, we show that phosphatidylserine (PS) localizes to a specific subpopulation of myoendothelial junctions (MEJs), crucial signaling microdomains that regulate vasodilation in resistance arteries, and in silico data have implied that PS may compete with phosphatidylinositol 4,5-bisphosphate (PIP2) binding on Kir2.1. We found that Kir2.1-MEJs also contained PS, possibly indicating an interaction where PS regulates Kir2.1. Electrophysiology experiments on HEK cells demonstrate that PS blocks PIP2 activation of Kir2.1 and that addition of exogenous PS blocks PIP2-mediated Kir2.1 vasodilation in resistance arteries. Using a mouse model lacking canonical MEJs in resistance arteries (Elnfl/fl/Cdh5-Cre), PS localization in endothelium was disrupted and PIP2 activation of Kir2.1 was significantly increased. Taken together, our data suggest that PS enrichment to MEJs inhibits PIP2-mediated activation of Kir2.1 to tightly regulate changes in arterial diameter, and they demonstrate that the intracellular lipid localization within the endothelium is an important determinant of vascular function.

The internal elastic lamina of resistance arteries facilitates vasodilation

In large conduit arteries, smooth muscle cell elastin produces elastic laminae, while in resistance arteries endothelial cell derived elastin is the major contributor to the internal elastic lamina (IEL). The IEL in resistance arteries separates smooth muscle and endothelium and contain distinct holes in the IEL (HIEL) where the two cell types communicate via a structure termed the myoendothelial junction (MEJ). The MEJ is an anatomical hallmark of resistance arteries that pierces through the IEL and facilitates vasodilation by movement of endothelial derived hyperpolarization (EDH) to smooth muscle. In contrast, large arteries dilate via nitric oxide (NO) and do not contain MEJs. To study the role of the IEL in resistance arteries in facilitating MEJ formation and EDH‐based vasodilation, we used a mouse with a Cdh5Cre to constitutively knockout elastin from endothelial cells (elastin ECKO). In en face preparations from third order mesenteric arteries, elastin ECKO mice had a 55.49% reduction in IEL surface area, and, importantly, completely lacked the distinct HIEL where MEJs could form. Regardless of this, en face claudin‐5 staining demonstrated a normal endothelial cell morphology. The lack of distinct HIEL, the canonical MEJ sites, led us to hypothesize vasodilatory mechanisms may be disrupted. To test this hypothesis, we first performed pressure myography on third order mesenteric arteries from elastin ECKO mice. Myogenic tone began to diverge from littermate controls starting at 80 mmHg, with preserved active, but not passive tone. For this reason, to test for changes in EDH between control and elastin ECKO, we used arteries pressurized to 60 mmHg and performed acetylcholine (Ach) dose‐response curves. We found elastin ECKO mice have decreased responses to Ach. Additionally, in the presence of 30 uM L‐NAME, the majority of dilation in elastin ECKO arteries was inhibited, compared to no inhibition with L‐NAME in the littermate controls. This indicated that NO‐mediated dilation was present in elastin ECKO mice and not littermate controls. En face immunohistochemistry demonstrated disruption of proteins localized to MEJs that regulate EDH in resistance arteries: both phosphatidylserine (See Abstract ID:R5515) and alpha globin, which is used to chelate NO. These results indicated that the disruption in the IEL, and thus canonical MEJ sites, affected the polarization of EDH‐associated proteins in the endothelium, supporting the observation of increased NO‐based signaling in elastin ECKO arteries. Overall, these data imply endothelial‐derived elastin is necessary for the functional formation of MEJs, the vasodilatory capacity of the endothelium, and the pathway of vasodilation in resistance arteries.

Exploring Endothelial KCa Channel Activation as a Novel Strategy to Restore Cardiac Function Following Acute Myocardial Infarction in Mice

Endothelial dysfunction (ED) is a common underlying feature of several premorbid conditions (e.g. obesity, Type 2 Diabetes (T2D), atherosclerosis, hypertension) associated with the development of myocardial infarction (MI). The ability of an artery to dilate properly and oppose plaque formation within the vascular wall is compromised by ED, which ultimately leads to reduced blood flow. In the coronary circulation, ED can decrease blood supply to the working myocardium and the resulting prolonged ischemia can promote MI. Recent studies from our group have demonstrated that a small molecule activator of endothelial KCa2.3 and KCa3.1 channel activity (i.e. SKA-31) improves agonist-evoked vasodilation in resistance arteries from multiple vascular beds and species, and this effect remains robust in arteries from T2D tissues. We have further observed that prolonged administration of SKA-31 to aged rats improves both their cardiac and vascular functions. Thus, the goal of this project is to investigate the potential cardio-protective effects of SKA-31 in male mice (12-15 weeks of age, C57B1/6 strain) following an acute MI induced by permanent surgical ligation of the left-anterior descending coronary artery. Within 48 h post-surgery, MI and sham surgery mice were treated orally with either vehicle or 10 mg/kg of SKA-31 daily for 6 weeks. Myocardial damage in MI mice was confirmed by histological staining. Echocardiographic assessment of cardiac function 4 weeks post-surgery revealed that Ejection Fraction (EF) and Fractional Shortening (FS) were similar in sham animals treated with either vehicle or SKA-31 (Table 1). Similar results were observed for measurements of end systolic volume (ESV) and end diastolic volume (EDV). In contrast, the MI animals treated with vehicle displayed ~50% decreases in EF and FS, and significantly larger values for ESV and EDV. These functional and structural parameters thus confirm the presence of sustained cardiac dysfunction in the MI mice. Treatment of MI mice with SKA-31 did not lead to improvements in EF, FS, ESV or EDV compared with vehicle treated animals. Our preliminary findings indicate that SKA-31 treatment did not affect the healthy cohort, but also did not improve gross cardiac function in the MI group. Recent data indicate that an acute MI is typically followed by a robust inflammatory response within the injured myocardium that peaks 3 days post-MI and declines to baseline by 7-9 days. We speculate that this local immune response is exacerbated by SKA-31 administration within 48 h post-injury, thereby negating any potential benefit of SKA-31 at the level of the vasculature. Delaying SKA-31 administration to coincide with the full decline of the local immune response may prove more effective in restoring cardiac function.

Endothelial Ca2+ signaling-dependent vasodilation through transient receptor potential channels.

Ca2+ signaling of endothelial cells plays a critical role in controlling blood flow and pressure in small arteries and arterioles. As the impairment of endothelial function is closely associated with cardiovascular diseases (e.g., atherosclerosis, stroke, and hypertension), endothelial Ca2+ signaling mechanisms have received substantial attention. Increases in endothelial intracellular Ca2+ concentrations promote the synthesis and release of endothelial-derived hyperpolarizing factors (EDHFs, e.g., nitric oxide, prostacyclin, or K+ efflux) or directly result in endothelial-dependent hyperpolarization (EDH). These physiological alterations modulate vascular contractility and cause marked vasodilation in resistance arteries. Transient receptor potential (TRP) channels are nonselective cation channels that are present in the endothelium, vascular smooth muscle cells, or perivascular/sensory nerves. TRP channels are activated by diverse stimuli and are considered key biological apparatuses for the Ca2+ influx-dependent regulation of vasomotor reactivity in resistance arteries. Ca2+-permeable TRP channels, which are primarily found at spatially restricted microdomains in endothelial cells (e.g., myoendothelial projections), have a large unitary or binary conductance and contribute to EDHFs or EDH-induced vasodilation in concert with the activation of intermediate/small conductance Ca2+-sensitive K+ channels. It is likely that endothelial TRP channel dysfunction is related to the dysregulation of endothelial Ca2+ signaling and in turn gives rise to vascular-related diseases such as hypertension. Thus, investigations on the role of Ca2+ dynamics via TRP channels in endothelial cells are required to further comprehend how vascular tone or perfusion pressure are regulated in normal and pathophysiological conditions.

Activation of the cation channel TRPM3 in perivascular nerves induces vasodilation of resistance arteries

The Transient Receptor Potential Melastatin 3 (TRPM3) is a Ca2+-permeable non-selective cation channel activated by the neurosteroid pregnenolone sulfate (PS). This compound was previously shown to contract mouse aorta by activating TRPM3 in vascular smooth muscle cells (VSMC), and proposed as therapeutic modulator of vascular functions. However, PS effects and the role of TRPM3 in resistance arteries remain unknown. Thus, we aimed at determining the localization and physiological role of TRPM3 in mouse mesenteric arteries. Real-time qPCR experiments, anatomical localization using immunofluorescence microscopy and patch-clamp recordings in isolated VSMC showed that TRPM3 expression in mesenteric arteries is restricted to perivascular nerves. Pressure myography experiments in wild type (WT) mouse arteries showed that PS vasodilates with a concentration-dependence that was best fit by two Hill components (effective concentrations, EC50, of 14 and 100 μM). The low EC50 component was absent in preparations from Trpm3 knockout (KO) mice and in WT arteries in the presence of the CGRP receptor antagonist BIBN 4096. TRPM3-dependent vasodilation was partially inhibited by a cocktail of K+ channel blockers, and not mediated by β-adrenergic signaling. We conclude that, contrary to what was found in aorta, PS dilates mesenteric arteries, partly via an activation of TRPM3 that triggers CGRP release from perivascular nerve endings and a subsequent activation of K+ channels in VSMC. We propose that TRPM3 is implicated in the regulation of the tone of resistance arteries and that its activation by yet unidentified endogenous damage-associated molecules lead to protective vasodilation responses in mesenteric arteries.

Placental protein 13 (PP13) stimulates rat uterine vessels after slow subcutaneous administration.

IntroductionReduced concentrations of placental protein 13 (PP13) during the first trimester of human pregnancy are associated with elevated risk for the subsequent development of preeclampsia, which is one of the deadliest obstetrical complications of pregnancy. Previous studies by our group have shown that PP13 lowers blood pressure in pregnant rats, increases the size and weight of pups and placentas, and induces vasodilation of resistance arteries through endothelial signaling pathways involving endothelial nitric oxid synthase and prostaglandin.MethodsIn the present study, the effect of PP13 was investigated in nonpregnant female Sprague Dawley rats (n=27). Osmotic pumps were surgically implanted subcutaneously that released a constant dose of PP13 or saline over 7 days. Most animals were sacrificed 6 days after the end of PP13 release (on day 13), while some were sacrificed immediately at the end of day 7 (the last PP13 release day), to compare the short- and long-term impact of PP13 on vessels’ growth and size.ResultsThe uterine vessels were significantly expanded in the group exposed to recombinant PP13 (rPP13) compared to the control (saline) group. Both veins and arteries were significantly expanded by rPP13 with a more pronounced effect after 13 days compared to the corresponding vessels after 7 days. Furthermore, the long-term effect of treatment by rPP13 was more pronounced in the veins compared to the corresponding arteries. The effect of a PP13 variant with a histidine-tag (His-PP13) remained the same between 7 and 13 days.ConclusionIn conclusion, PP13 might play a key role in the expansive remodeling of the uterine vessels, reflecting what would happen if the rat was pregnant, preparing the uterine vascu-lature for the increase in uteroplacental blood flow, which is necessary for normal pregnancy. We suggest that PP13 could act by NO signaling pathways, a hypothesis that requires future study.

Aspirin causes endothelium-dependent vasodilation of resistance arteries from non-gravid and gravid rats.

The objective of this study was to understand the effect of acetylsalicylic acid (aspirin) on resistance arteries from mesentery and uterus. During pregnancy, the uterine vasculature undergoes consistent growth to provide sufficient uteroplacental blood flow, a process whose failure is associated with pregnancy complications characterized by high uterine vascular resistance. Uterine arcuate (UA) and mesenteric arteries (MA; diameter <300 µm) isolated from non-gravid, mid-gravid (day 14), and late-gravid rats (day 20) were exposed to aspirin (10-12 to 10-5 M). Further, in UA from late-gravid rats, aspirin was evaluated in presence of inhibitors of nitric oxide synthases, cyclooxygenase, cyclic nucleotides (cAMP, cGMP) and BK channels, and also on endothelium-denuded vessels. Aspirin dilated both UA and MA in a dose dependent manner. Pregnancy increased aspirin vasodilation in MA and UA from mid-gravid rats, an effect that was reduced in vessels from late gravid animals at concentrations >10-7 M. Further, uterine vasodilation was significantly reduced when the endothelium was removed (p < 0.001), and by inhibitors of nitric oxide synthase (p < 0.001), cyclooxygenase synthase (p < 0.05), cyclic nucleotides cGMP/cAMP and BK channels. This is the first study to show a direct vasodilatory effect of aspirin on rat uterine artery that is mediated by a combination of cellular - primarily endothelial - mechanisms. Our results in UA suggest that the use of aspirin may be effective in enhancing uteroplacental blood flow, while its vasodilation effect on MA may lower peripheral resistance.

Placental protein 13 (PP13)-induced vasodilation of resistance arteries from pregnant and nonpregnant rats occurs via endothelial-signaling pathways

ABSTRACTPlacental protein 13 (PP13) induces hypotension in rats. This study aims to evaluate PP13 effects on isolated uterine arteries from nonpregnant and mid-pregnant rats. Vessels were isolated, cannulated, and pressurized to 50 mmHg within an arteriograph, preconstricted and exposed to increasing PP13 concentrations (10−13–10−8 M). PP13 elicited 38–50% arterial vasodilation with half-maximum response (EC50) = 1 pM. The relaxation was mediated by activating the endothelial-signaling pathways of prostaglandin and nitric oxide (NO). Accordingly, these results encourage evaluation of PP13 as a possible therapy for gestational diseases characterized by insufficient uteroplacental blood flow and/or maternal hypertension.

Nitric oxide and Kir6.1 potassium channel mediate isoquercitrin-induced endothelium-dependent and independent vasodilation in the mesenteric arterial bed of rats

The vascular effect of flavonoid isoquercitrin was investigated in the perfused mesenteric vascular bed of rats. In preparations with functional endothelium isoquercitrin (100, 300 and 1000nmol) dose-dependently reduced the perfusion pressure by 13±2.2, 33±3.9, and 58±3.7mm Hg, respectively. Endothelium removal or inhibition of the nitric oxide synthase enzymes by l-NAME did not change the effects of 100 and 300 nmol isoquercitrin, but reduced by 30–40% the vasodilation induced by 1000 nmol isoquercitrin. Perfusion with nutritive solution containing 40mM KCl abolished the vasodilatory effect of all isoquercitrin doses. Treatment with glibenclamide, a Kir6.1 (ATP-sensitive) potassium channel blocker, inhibited vasodilation induced by 100 and 300 nmol isoquercitrin, but only partially reduced the effect of 1000 nmol isoquercitrin. The non-selective KCa (calcium-activated) potassium channel blocker tetraethylammonium, but not the selective KCa1.1 channel blocker iberiotoxin, reduced by around 60% vasodilation induced by all isoquercitrin doses. In addition, association of tetraethylammonium and glibenclamide, or l-NAME and glibenclamide, fully inhibited isoquercitrin-induced vasodilation. Our study shows that isoquercitrin induces vasodilation in resistance arteries, an effect mediated by K+ channel opening and endothelial nitric oxide production.

Abstract 024: The Direct Renin Inhibitor Aliskiren Improves Vasodilation and Endothelial Function in Resistance Arteries from Diabetic and Hypertensive Patients

We previously demonstrated that the direct renin inhibitor aliskiren (ALK) significantly reduced the remodeling of subcutaneous resistance arteries of hypertensive patients as compared to the angiotensin-converting enzyme inhibitor ramipril (RAM). Here we questioned whether endothelial function of resistance arteries would improve after 1 year of blood pressure (BP) control with ALK or RAM. Sixteen diabetic patients with mild essential hypertension were randomized to ALK (150-300 mg once daily, n=9) or RAM (5-10 mg once daily, n=7). Subcutaneous resistance arteries were dissected from gluteal biopsy and mounted on a pressurized micromyograph. Endothelium-dependent and -independent relaxations were assessed by concentration-response curves to acetylcholine (1 nM to 100 μM) and sodium nitroprusside (10 nM to 1 mM) respectively, in arteries pre-contracted with norepinephrine (10 μM). Carotid-femoral pulse wave velocity (PWV) was assessed by applantion tonometry. Forearm flow mediated dilation (FMD) was assessed by ultrasounds. The expression of P-eNOS/e-NOS and the markers of oxidative stress nitrotyrosine and LOX-1 were assessed by immunohistochemistry in resistance arteries. Patients were similar for age, sex, BMI and glycemic and metabolic control. Systolic BP was significantly and equally reduced by both ALK and RAM (153±8.9 mmHg reduced to 128±7.3 mmHg and 151±10.6 mmHg reduced to 121±12.1, respectively, P&lt;0.01) whereas diastolic BP was significantly reduced only in ALK-treated but not in RAM-treated patients (94.2±7.17 mmHg reduced to 81.4±6.31 mmHg, P&lt;0.01; and 84.7±12.22 reduced to 78.6±7.48, NS, respectively). PWV and FMD were similar in both groups before and after treatment. Endothelium-dependent vasodilation was improved only by ALK (max dilation 92.5±3.8% vs 50.5±14%, P&lt;0.05) but not by RAM (max dilation 72.9±5.6% vs 61.8±14.5%, NS). Endothelium-independent vasodilation was similar in all the groups. Only ALK increased P-eNOS/eNOS expression (+44% vs before treatment, p&lt;0.05). The markers of oxidative stress were similar in both groups before and after treatment. In conclusion ALK improved endothelial function and induced vasodilation in resistance arteries from diabetic and hypertensive patients.

Abstract 18339: Weight Loss with Low Carbohydrate Diets Improves Flow Induced Vasodilation in Resistance Arteries

Introduction: Obesity is associated with microvascular dysfunction and reduced nitric oxide (NO). We tested the hypothesis that NO-dependent, flow induced vasodilation in microvessels is improved by low carbohydrate diets designed for weight loss. Methods: Obese female adults (BMI = 32.0 ± 0.6 kg/m 2 , pre &amp; post, n=22) were randomly assigned to a low carbohydrate (LC) diet for weight loss (LCWL, n=11, 500 calorie/day deficit). All meals were prepared for 6 weeks (10% carbohydrate, 30% protein, 60% fat). Resistance arteries (RAs, internal diameter of 16021 μm, n=22) were dissected from gluteal subcutaneous fat biopsies at weeks 0 (pre) and 6 (post). Vessels were cannulated and exposed to pressure gradients (Δ10-Δ100 cmH 2 O), with or without L-NAME (10 -4 M, inhibitor of eNOS), PEG-catalase (PEG-CAT, 500 Units/ml, scavenger of H 2 O 2 ), indomethacin (INDO, 10 -5 M, inhibitor of cyclooxygenase), or pioglitazone (PIOG, 10 -5 M, PPARγ agonist) and to papaverine (10 -4 M), pre and post LC. NO or H 2 O 2 in RAs was measured by fluorescence microscopy. Results: LC diets reduced BMI (post 32.7±0.8% vs. pre 31.4±1.0%, p&lt;0.01), and increased flow-induced vasodilation (FID, post 81 ± 3% vs. pre 63 ± 4%, at Δ100 cmH 2 O (p&lt;0.01) that was reduced by L-NAME and PEG-CAT (p&lt;0.05). Consistently, vascular NO (fluorescence ratio: 1.5 ± 0.3, post vs. pre, p&lt;0.05) and H 2 O 2 (2.6 ± 1.2, p&lt;0.05) were increased with flow, and inhibited by L-NAME and PEG-CAT, respectively. INDO reduced FID in post, less (p&lt;0.05) than in pre LC. There was no effect of pioglitazone on FID both in pre and in post (p&gt;0.05). Endothelium independent responses to papaverine were similar in pre and post LC. Conclusion: We found that after 6 weeks of WL on low carbohydrate diet, 1) BMI was reduced, and microvascular vasodilator reactivity was improved; 2) microvascular NO and H 2 O 2 generation were increased; and 3) there was no effect of pioglitazone on FID in resistance arteries. These data indicate that weight reduction on low carbohydrate diet is critical for improved and endothelium-dependent microvascular function. NIH ( R01HL095701 ).

AKAP150‐dependent cooperative TRPV4 channel gating is central to endothelium‐dependent vasodilation and is disrupted in hypertension (678.10)

Endothelial cell (EC) dysfunction is a hallmark of hypertension. We recently discovered that local Ca2+ influx through clusters of functionally coupled TRPV4 channels (detected optically as “TRPV4 sparklets”) drives physiological vasodilation. Here, we show that stimulation of EC muscarinic receptors activated TRPV4 sparklets exclusively at discrete sites at myoendothelial projections (MEPs)—specialized regions of ECs that contact adjacent smooth muscle cells. This activation was dependent on protein kinase C (PKC) and was absent in mice lacking the PKC‐anchoring protein AKAP150 (A‐kinase anchoring protein 150), which was localized predominantly to MEPs. Cooperative gating of TRPV4 channels within a cluster amplified Ca2+ influx at MEPs by more than 2‐fold. This cooperativity was largely absent at non‐MEP sites and was virtually eliminated by chelation of intracellular Ca2+ or AKAP150 knockout, suggesting AKAP150‐dependent potentiation of TRPV4 activity by Ca2+ influx via adjacent channels. Notably, MEP‐localization of AKAP150 was disrupted in angiotensin II‐induced hypertension, leading to a complete loss of muscarinic activation of TRPV4 channels, much weaker coupling among TRPV4 channels at MEPs, and approximately an 80% reduction in carbachol‐induced vasodilation. Our results support the concept that endothelial‐dependent vasodilation of resistance arteries is enabled by MEP‐localized AKAP150, which ensures the proximity of PKC to TRPV4 channels and coupled channel gating necessary for efficient communication of the endothelium to the smooth muscle cells in arteries—a molecular configuration that is disrupted in hypertension.Grant Funding Source: Supported by HL044455, 1P01HL095488, R37DK053832, R01HL098243 to MTN

Aging Impairs Electrical Conduction Along Endothelium of Resistance Arteries Through Enhanced Ca2+-Activated K + Channel Activation

Intercellular conduction of electrical signals underlies spreading vasodilation of resistance arteries. Small- and intermediate-conductance Ca(2+)-activated K(+) channels of endothelial cells serve a dual function by initiating hyperpolarization and modulating electrical conduction. We tested the hypothesis that regulation of electrical signaling by small- and intermediate-conductance Ca(2+)-activated K(+) channels is altered with advancing age. Intact endothelial tubes (60 µm wide; 1-3 mm long) were freshly isolated from male C57BL/6 mouse (Young: 4-6 months; Intermediate: 12-14 months; Old: 24-26 months) superior epigastric arteries. Using dual intracellular microelectrodes, current was injected (± 0.1-3 nA) at site 1 while recording membrane potential (Vm) at site 2 (separation distance: 50-2000 µm). Across age groups, greatest differences were observed between Young and Old. Resting Vm in Old (-38 ± 1 mV) was more negative (P<0.05) than Young (-30 ± 1 mV). Maximal hyperpolarization to both direct (NS309) and indirect (acetylcholine) activation of small- and intermediate-conductance Ca(2+)-activated K(+) channels was sustained (ΔVm ≈-40 mV) with age. The length constant (λ) for electrical conduction was reduced (P<0.05) from 1630 ± 80 µm (Young) to 1320 ± 80 µm (Old). Inhibiting small- and intermediate-conductance Ca(2+)-activated K(+) channels with apamin+charybdotoxin or scavenging hydrogen peroxide (H2O2) with catalase improved electrical conduction (P<0.05) in Old. Exogenous H2O2 (200 µmol/L) in Young evoked hyperpolarization and impaired electrical conduction; these effects were blocked by apamin+charybdotoxin. Enhanced current loss through Ca2+-activated K+ channel activation impairs electrical conduction along the endothelium of resistance arteries with aging. Attenuating the spatial domain of electrical signaling will restrict the spread of vasodilation and thereby contribute to blood flow limitations associated with advanced age.

The impact of outward remodeling on vasodilation in skeletal muscle resistance arteries

Collateral enlargement following arterial occlusion is thought to rescue downstream tissues from severe ischemic insult in patients with ischemic disease, such as peripheral arterial occlusive disease. However, in animal models, collateral‐dependent hyperemia is reduced after arterial occlusion, suggesting that vasodilation may be impaired. Impaired vasodilation would compromise the effectiveness of collaterals, as ischemia occurs during periods of increased metabolic demand. To assess the impact of collateral enlargement on vasodilation in resistance arteries, we ligated the femoral arteries of young, otherwise healthy mice to induce outward remodeling in the collateral circuit. Outward remodeling transiently impairs functional vasodilation, which is reduced at day‐7 (62 ± 3% vs. 93 ± 10%, p &lt; 0.05), but improves by day‐28 (101 ± 4% vs. 115 ± 10%). Vascular reactivity studies indicate the impaired vasodilation is due to impaired smooth muscle‐dependent responses during outward remodeling, as both endothelial‐ and smooth muscle‐dependent vasodilation is impaired at day‐7 (100 ± 16% vs. 149 ± 15%, p &lt; 0.05 and 103 ± 15% vs. 125 ± 15%, p &lt; 0.05, respectively), but not at day‐28 (136 ± 24% vs. 142 ± 20% and 146 ± 18% vs. 124 ± 10%). Studies examining the cellular mechanism of the day‐7 impairment and the impact of impaired vasodilation on hemodynamics are currently underway.

Circulation: Clinical Summaries

Circulation: Clinical Summaries

Elementary TRPV4 Ca 2+ events in intact vascular endothelium

Transient receptor potential vanilloid 4 (TRPV4) channel serves as a Ca2+ influx pathway in endothelial cells (ECs). The objective of this study was to identify elementary TRPV4 Ca2+ events in vascular ECs. We used third-order mesenteric arteries (MAs) from mice that express Ca2+ biosensor (GCaMP2) selectively in ECs. TRPV4 agonist GSK1016790A (GSK, 10 nM) produced EC-dependent dilation of pressurized MAs. High speed confocal imaging of slit-open MAs revealed that TRPV4 agonists (GSK, 4α-PDD, 11,12-EET) activate local Ca2+ events (“TRPV4 Ca2+ sparklets”) in ECs in absence of Ca2+ release from intracellular stores. Fractional fluorescence changes (ΔF/F0) for 19 event sites showed a quantal level of 0.22 ± 0.03. Mean open probability (NPs, N = number of quanta, Ps = probability of occurrence) of the sparklets at a site was 0.46 ± 0.06 (n = 10 sites), 0.19 ± 0.03 (n = 5 sites) and 0.57 ± 0.19 (n = 5 sites) for GSK (10 nM), 4α-PDD (5 μM) and 11, 12-EET (1 μM), respectively. TRPV4 inhibitor HC067047 (1 μM) significantly reduced NPs of GSK-induced sparklets to 0.16 ± 0.05 (n = 5 sites, p<0.05). In conclusion, this is the first detection of elementary TRPV4 Ca2+ sparklets that initiate EC-dependent vasodilation of resistance arteries. Supported by AHA-PHA 10POST3690006 (SKS) and NIH HL44455 (MTN).

Endothelial Function in Resistance and Conduit Arteries and 5-Year Risk of Cardiovascular Disease

Impaired endothelial function has been implicated as a cause of cardiovascular disease. Little is known of the relations of measures of endothelial function in resistance and conduit arteries to incident cardiovascular disease in the general population, and available techniques have not been compared. In 1016 participants (70 years of age) of the population-based Prospective Study of the Vasculature in Uppsala Seniors (PIVUS) study (52% women), we measured endothelium-dependent vasodilation using the invasive forearm technique with acetylcholine given in the brachial artery, the brachial artery ultrasound technique with measurement of flow-mediated dilatation, and the pulse-wave analysis-based method with β-2-agonist terbutaline provocation. During 5 years of follow-up, 101 participants experienced a composite end point of myocardial infarction, stroke, or death, excluding the 85 persons with a history of myocardial infarction or stroke at baseline. In logistic regression models adjusted for several established and novel cardiovascular disease risk factors and medications, endothelium-dependent vasodilation by the invasive forearm technique with acetylcholine was associated with risk of the end point (odds ratio, 0.72 per SD; 95% confidence interval, 0.56 to 0.93; P=0.01). Endothelial function by the other 2 methods was not related to risk of the end point. Addition of endothelium-dependent vasodilation to the Framingham risk score improved discrimination of risk of the end point. Endothelium-dependent vasodilation in resistance arteries, but not in the brachial conduit artery (flow-mediated dilatation), was associated with 5-year risk of a composite end point of death, myocardial infarction, or stroke independently of major cardiovascular disease risk factors. This vascular measurement improved risk discrimination when added to an established risk score in an elderly population.

Globular and Full-Length Adiponectin Induce NO-Dependent Vasodilation in Resistance Arteries of Zucker Lean but Not Zucker Diabetic Fatty Rats

Adiponectin increases nitric oxide (NO) production in endothelial cell cultures and is reduced in the circulation of obese and diabetic patients, but its functional effect on resistance arteries is not yet studied in detail. We assessed the direct vasodilatory response of isolated mesenteric resistance arteries of Zucker diabetic fatty (ZDF) rats and Zucker lean (ZL) rats to globular adiponectin (gAd) and full-length adiponectin (fAd) and tested the effect of additional reactive oxygen species (ROS) inhibitors in vitro. Serum adiponectin and insulin levels were measured by ELISA. The mRNA expressions of the adiponectin receptors and the downstream signaling molecules adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 1 (APPL1), adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 2 (APPL2), and endothelial NO synthase (eNOS) in mesenteric resistance arteries were quantified by real-time reverse transcriptase PCR. Both gAd and fAd induced a relevant dose-dependent vasodilation in ZL, but not in hypoadiponectinemic ZDF rats. This effect was totally blunted by L-nitroarginine-methyl-ester indicating NO dependency. The addition of ROS inhibitors could not improve the vasodilatory effect of adiponectin. Vasodilatory response to acetylcholine was reduced in ZDF rats, which could not be enhanced by low-dose adiponectin. Adiponectin receptor 1 (AdipoR1) was higher expressed than adiponectin receptor 2 (AdipoR2) with no significant differences between both animal groups, but APPL1 was significantly decreased in ZDF rats. The eNOS expression was not significantly different between ZL and ZDF rats. Adiponectin exerts a NO-dependent vasodilation in resistance arteries of normoglycemic ZL rats, but not diabetic ZDF rats. This may contribute to endothelial dysfunction in ZDF rats. Alterations in the expression of APPL1 may be involved in the observed insensitivity to adiponectin in ZDF rats.

GENETIC VARIATION IN THE DDAH-1 GENE IN RELATION TO ADMA LEVELS AND ENDOTHELIAL FUNCTION: PP.6.251

Background: Asymmetric dimethylarginine (ADMA), an endogenous methylated amino acid, has been identified as a competitive inhibitor of nitric oxide synthase (NOS). Elevated ADMA levels have been demonstrated in cardiovascular disorders and many studies have reported an inverse correlation between ADMA and endothelial function. The majority of ADMA is metabolized by dimethylarginine dimethylaminohydrolases (DDAH-1 and DDAH-2). Our aim was to study the genetic variation in the DDAH-1 gene in relation to ADMA levels and endothelial function. Methods: A total of 959 individuals, aged 70, from the community based Prospective Study of the Vasculature in Uppsala Seniors (PIVUS) study were included. Plasma concentrations of ADMA were measured by high performance liquid chromatography. Endothelial function was evaluated with both the invasive forearm technique (measuring endothelial dependent vasodilation in resistance arteries) and with brachial artery ultrasound (measuring flow mediated dilation in a conduit artery). Forty common single nucleotide polymorphisms (SNPs) in the DDAH-1 gene were analyzed. Results: There were significant associations between DDAH-1 genotype and ADMA levels in 20 of the 40 selected SNPs. However, no associations were seen between DDAH-1 genotype and endothelial function. Conclusion: These results indicate that genetic variation in the DDAH-1 gene has an impact on ADMA concentration in plasma. However, it does not seem to have a major influence on endothelial function.

High intravascular pressure decreases endothelial Ca2+ pulsars and impairs endothelium‐dependent vasodilation in mouse mesenteric arteries

Ca2+ pulsars are inositol 1,4,5‐triphosphate (IP3) receptor‐mediated Ca2+ events in endothelial cells. Increase in Ca2+ pulsars by acetylcholine (ACh) is associated with endothelium‐dependent vasodilation. The objective of this study was to evaluate the effect of intravascular pressure on Ca2+ pulsars and endothelium dependent vasodilation of resistance arteries. Third‐order mesenteric arteries (MAs) from C57BL6 mice were pressurized at 80 or 120 mmHg and were allowed to develop myogenic tone (26± 1% and 36 ± 1 % at 80 and 120 mmHg respectively, n=9). Endothelium dependent vasodilation to ACh (5 μM) was significantly higher at 80 mmHg compared to 120 mmHg (76.8 ± 7.8 % and 30.1 ± 4.6% respectively, n=4–6, p&lt;0.05). To evaluate the effect of intravascular pressure on Ca2+ pulsars, we used MAs from mice that express a calcium biosensor (GCaMP2) exclusively in endothelial cells. Frequency of Ca2+ pulsars (pulsars/second/site) was higher at 80 mmHg compared to 120 mmHg (0.11 ± 0.01 and 0.07 ± 0.01 respectively, n=15–31 sites). Following ACh treatment, the Ca2+ pulsar frequency increased to 0.19 ± 0.02 at 80 mmHg compared to 0.09 ± 0.01 at 120 mmHg (n=9–10 sites, p&lt;0.05). Taken together, these results indicate that impaired endothelium‐dependent dilation of resistance arteries at high intravascular pressure is associated with a decrease in Ca2+ pulsars in the endothelial cells. Supported by NIH HL44455.