Dilation Of Resistance Arteries Research Articles

New insights into mechanisms of endothelial insulin resistance in type 2 diabetes.

Insulin resistance in the vasculature is a hallmark of type 2 diabetes (T2D), and blunting of insulin-induced vasodilation is its primary consequence. Individuals with T2D exhibit a marked impairment in insulin-induced dilation in resistance arteries across vascular beds. Importantly, reduced insulin-stimulated vasodilation and blood flow to skeletal muscle limits glucose uptake and contributes to impaired glucose control in T2D. The study of mechanisms responsible for the suppressed vasodilatory effects of insulin has been a growing topic of interest for not only its association with glucose control and extension to T2D but also its relationship with cardiovascular disease development and progression. In this mini-review, we integrate findings from recent studies by our group with the existing literature focused on the mechanisms underlying endothelial insulin resistance in T2D.

Endothelium-Specific Deficiency of Polycystin-1 Promotes Hypertension and Cardiovascular Disorders.

Autosomal dominant polycystic kidney disease is the most frequent hereditary kidney disease and is generally due to mutations in PKD1 and PKD2, encoding polycystins 1 and 2. In autosomal dominant polycystic kidney disease, hypertension and cardiovascular disorders are highly prevalent, but their mechanisms are partially understood. Since endothelial cells express the polycystin complex, where it plays a central role in the mechanotransduction of blood flow, we generated a murine model with inducible deletion of Pkd1 in endothelial cells (Cdh5-CreERT2;Pkd1fl/fl) to specifically determine the role of endothelial polycystin-1 in autosomal dominant polycystic kidney disease. Endothelial deletion of Pkd1 induced endothelial dysfunction, as demonstrated by impaired flow-mediated dilatation of resistance arteries and impaired relaxation to acetylcholine, increased blood pressure and prevented the normal development of arteriovenous fistula. In experimental chronic kidney disease induced by subtotal nephrectomy, endothelial deletion of Pkd1 further aggravated endothelial dysfunction, vascular remodeling, and heart hypertrophy. Altogether, this study provides the first in vivo demonstration that specific deletion of Pkd1 in endothelial cells promotes endothelial dysfunction and hypertension, impairs arteriovenous fistula development, and potentiates the cardiovascular alterations associated with chronic kidney disease.

Endotheilum-specific deficiency of polycystin-1 promotes hypertension and cardiovascular disorders, and impairs arterio-venous fistula maturation

Autosomal dominant polycystic kidney disease (ADPKD) is the most frequent hereditary kidney disease and is generally due to mutations in PKD1 and PKD2, encoding polycystins 1 and 2. In ADPKD, hypertension and cardiovascular (CV) disorders are highly prevalent but their mechanisms are partially understood. The objective of this study was to determine whether hypertension and the CV disorders of ADPKD are a consequence of the endothelial deficiency in the polycystin complex, rather than to the kidney disease, as is currently believed. Since endothelial cells express the polycystin complex, where it plays a central role in the mechanotransduction of blood flow, we generated a murine model with inducible deletion of Pkd1 in endothelial cells (Cdh5-CreERT2; Pkd1fl/fl) to specifically determine the role of endothelial polycystin-1 in ADPKD. We analyzed the endothelial phenotype associated with Pkd1 deficiency, invasive and noninvasive blood pressure, vascular function on isolated vessels, and echocardiography. The impact on the development of a model of aorto-cava arterio-venous fistula and on the CV consequences of superimposed chronic kidney disease were analyzed to further establish the clinical relevance of our results. As expected, transgenic mice presented normal kidney structure and function. Endothelial deletion of Pkd1 induced endothelial dysfunction, as demonstrated by impaired flow-mediated dilatation of resistance arteries and impaired relaxation to acetylcholine, increased blood pressure and prevented the normal development of arterio-venous fistula. In experimental chronic kidney disease induced by subtotal nephrectomy, endothelial deletion of Pkd1 further aggravated endothelial dysfunction, vascular remodelling and heart hypertrophy ( Fig. 1 ). Altogether, this study provides the first in vivo demonstration that specific deletion of Pkd1 in endothelial cells promotes endothelial dysfunction and hypertension independently of kidney cysts and of impaired kidney function, impairs arterio-venous fistula development and potentiates the cardiovascular alterations associated with chronic kidney disease. It establishes the endothelium as a significant contributor to cardiovascular disease in ADPKD.

ENDOTHELIUM-SPECIFIC DEFICIENCY OF POLYCYSTIN-1 PROMOTES HYPERTENSION AND CARDIOVASCULAR DISORDERS

Objective: Autosomal dominant polycystic kidney disease (ADPKD) is the most frequent hereditary kidney disease and is generally due to mutations in PKD1 and PKD2, encoding polycystins 1 and 2. In ADPKD, hypertension and cardiovascular (CV) disorders are highly prevalent but their mechanisms are partially understood. The objective of this study was to determine whether hypertension and the CV disorders of ADPKD are a consequence of the endothelial deficiency in the polycystin complex, rather than to the kidney disease, as is currently believed. Design and method: Since endothelial cells express the polycystin complex, where it plays a central role in the mechanotransduction of blood flow, we generated a murine model with inducible deletion of Pkd1 in endothelial cells (Cdh5-CreERT2;Pkd1fl/fl) to specifically determine the role of endothelial polycystin-1 in ADPKD. We nalysed the endothelial phenotype associated with Pkd1 deficiency, invasive and non-invasive blood pressure, vascular function on isolated vessels, and echocardiography. The impact on the development of a model of aorto-cava arterio-venous fistula and on the CV consequences of superimposed chronic kidney disease were nalysed to further establish the clinical relevance of our results. Results: As expected, transgenic mice presented normal kidney structure and function. Endothelial deletion of Pkd1 induced endothelial dysfunction, as demonstrated by impaired flow-mediated dilatation of resistance arteries and impaired relaxation to acetylcholine, increased blood pressure and prevented the normal development of arterio-venous fistula. In experimental chronic kidney disease induced by subtotal nephrectomy, endothelial deletion of Pkd1 further aggravated endothelial dysfunction, vascular remodelling and heart hypertrophy. Conclusions: Altogether, this study provides the first in vivo demonstration that specific deletion of Pkd1 in endothelial cells promotes endothelial dysfunction and hypertension independently of kidney cysts and of impaired kidney function, impairs arterio-venous fistula development and potentiates the cardiovascular alterations associated with chronic kidney disease. It establishes the endothelium as a significant contributor to cardiovascular disease in ADPKD.

Small and intermediate Ca2+-sensitive K+ channels do not play a role in vascular conductance during resting blood flow in the anaesthetised pig.

Flow-induced dilation in resistance arteries is mediated by endothelium-dependent hyperpolarisation via small and intermediate conducting Ca2+ sensitive K+ channels. The aim of the current study was to assess the effect of blocking both channels, using the toxins apamin and charybdotoxin, on flow-induced dilation in a conduit artery and vascular conductance. Experiments were carried out on the iliac and its vascular bed in anaesthetised pigs (n=4). Flow-induced dilation and vascular conductance (∆F/∆P) were assessed before and after administration of toxins intra-arterially (i.a.) at 50µgkg-1. Iliac diameter increased from baseline to 2.39±0.4mm before and 2.09±0.46mm after toxin administration, which was not significantly different (P = 0.63, Student's paired t test). Control conductance was 1.49 ± 0.27mlmin-1mmHg-1 (P < 0.00001, ANOVA), and 1.53 ± 0.18mlmin-1mmHg-1 (P < 0.00001, ANOVA) in the presence of the toxins which was not significantly different (P = 0.93 homogeneity of regression analysis). There was a small but significant increase in mean arterial pressure after the toxins were administered, from 74 ± 5 to 80 ± 9mmHg (P = 0.03, Student's paired t test); but all other measured parameters were not significantly affected. Small- and intermediate-conducting Ca2+-sensitive K+ channels are not involved in flow-mediated dilation in conduit arteries and do not play a role in resistance vessel diameter maintenance at resting blood flow.

VEGF-A inhibits agonist-mediated Ca2+ responses and activation of IKCa channels in mouse resistance artery endothelial cells.

Prolonged exposure to vascular endothelial growth factor A (VEGF-A) inhibits agonist-mediated endothelial cell Ca2+ release and subsequent activation of intermediate conductance Ca2+ -activated K+ (IKCa ) channels, which underpins vasodilatation as a result of endothelium-dependent hyperpolarization (EDH) in mouse resistance arteries. Signalling via mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) downstream of VEGF-A was required to attenuate endothelial cell Ca2+ responses and the EDH-vasodilatation mediated by IKCa activation. VEGF-A exposure did not modify vasodilatation as a result of the direct activation of IKCa channels, nor the pattern of expression of inositol 1,4,5-trisphosphate receptor 1 within endothelial cells of resistance arteries. These results indicate a novel role for VEGF-A in resistance arteries and suggest a new avenue for investigation into the role of VEGF-A in cardiovascular diseases. Vascular endothelial growth factor A (VEGF-A) is a potent permeability and angiogenic factor that is also associated with the remodelling of the microvasculature. Elevated VEGF-A levels are linked to a significant increase in the risk of cardiovascular dysfunction, although it is unclear how VEGF-A has a detrimental, disease-related effect. Small resistance arteries are central determinants of peripheral resistance and endothelium-dependent hyperpolarization (EDH) is the predominant mechanism by which these arteries vasodilate. Using isolated, pressurized resistance arteries, we demonstrate that VEGF-A acts via VEGF receptor-2 (R2) to inhibit both endothelial cell (EC) Ca2+ release and the associated EDH vasodilatation mediated by intermediate conductance Ca2+ -activated K+ (IKCa ) channels. Importantly, VEGF-A had no direct effect against IKCa channels. Instead, the inhibition was crucially reliant on the downstream activation of the mitogen-activated protein/extracellular signal-regulated kinase kinase 1/2 (MEK1/2). The distribution of EC inositol 1,4,5-trisphosphate (IP3 ) receptor-1 (R1) was not affected by exposure to VEGF-A and we propose an inhibition of IP3 R1 through the MEK pathway, probably via ERK1/2. Inhibition of EC Ca2+ via VEGFR2 has profound implications for EDH-mediated dilatation of resistance arteries and could provide a mechanism by which elevated VEGF-A contributes towards cardiovascular dysfunction.

SKA-31, an activator of endothelial Ca2+-activated K+ channels evokes robust vasodilation in rat mesenteric arteries

It is now well recognized that endothelial KCa2.3 and KCa3.1 channel activities contribute to dilation of resistance arteries via endothelium-mediated hyperpolarization and vascular smooth muscle relaxation. In this study, we have investigated the functional effect of the KCa channel activator SKA-31 in third order rat mesenteric arteries using arterial pressure myography. Isolated arteries were cannulated, pressurized intraluminally to 70 mmHg at 36 °C and then constricted with 1 μM phenylephrine. Acute bath exposure to SKA-31 evoked a robust and reversible inhibition of developed tone (IC50 = 0.22 μM). The vasodilatory effects of SKA-31 and acetylcholine were blunted in the presence of KCa2.3 and KCa3.1 channel antagonists, and were largely prevented following endothelial denudation. Western blot and q-PCR analyses of isolated mesenteric arteries revealed KCa2.3 and KCa3.1 channel expression at the protein and mRNA levels, respectively. Penitrem-A, an inhibitor of KCa1.1 channels, decreased vasodilatory responses to acetylcholine, sodium nitroprusside and NS-1619, but had little effect on SKA-31. Similarly, bath exposure to the eNOS inhibitor L-NAME did not alter SKA-31 and acetylcholine-mediated vasodilation. Collectively, these data highlight the major cellular mechanisms by which the endothelial KCa channel activator SKA-31 inhibits agonist-evoked vasoconstriction in rat small mesenteric arteries.

Klf4 has an unexpected protective role in perivascular cells within the microvasculature.

Recent smooth muscle cell (SMC) lineage-tracing studies have revealed that SMCs undergo remarkable changes in phenotype during development of atherosclerosis. Of major interest, we demonstrated that Kruppel-like factor 4 (KLF4) in SMCs is detrimental for overall lesion pathogenesis, in that SMC-specific conditional knockout of the KLF4 gene ( Klf4) resulted in smaller, more-stable lesions that exhibited marked reductions in the numbers of SMC-derived macrophage- and mesenchymal stem cell-like cells. However, since the clinical consequences of atherosclerosis typically occur well after our reproductive years, we sought to identify beneficial KLF4-dependent SMC functions that were likely to be evolutionarily conserved. We tested the hypothesis that KLF4-dependent SMC transitions play an important role in the tissue injury-repair process. Using SMC-specific lineage-tracing mice positive and negative for simultaneous SMC-specific conditional knockout of Klf4, we demonstrate that SMCs in the remodeling heart after ischemia-reperfusion injury (IRI) express KLF4 and transition to a KLF4-dependent macrophage-like state and a KLF4-independent myofibroblast-like state. Moreover, heart failure after IRI was exacerbated in SMC Klf4 knockout mice. Surprisingly, we observed a significant cardiac dilation in SMC Klf4 knockout mice before IRI as well as a reduction in peripheral resistance. KLF4 chromatin immunoprecipitation-sequencing analysis on mesenteric vascular beds identified potential baseline SMC KLF4 target genes in numerous pathways, including PDGF and FGF. Moreover, microvascular tissue beds in SMC Klf4 knockout mice had gaps in lineage-traced SMC coverage along the resistance arteries and exhibited increased permeability. Together, these results provide novel evidence that Klf4 has a critical maintenance role within microvascular SMCs: it is required for normal SMC function and coverage of resistance arteries. NEW & NOTEWORTHY We report novel evidence that the Kruppel-like factor 4 gene ( Klf4) has a critical maintenance role within microvascular smooth muscle cells (SMCs). SMC-specific Klf4 knockout at baseline resulted in a loss of lineage-traced SMC coverage of resistance arteries, dilation of resistance arteries, increased blood flow, and cardiac dilation.

The AMP-Related Kinase (AMPK) Induces Ca 2+ -Independent Dilation of Resistance Arteries by Interfering With Actin Filament Formation

Decreasing Ca2+ sensitivity of vascular smooth muscle (VSM) allows for vasodilation without lowering of cytosolic Ca2+. This may be particularly important in states requiring maintained dilation, such as hypoxia. AMP-related kinase (AMPK) is an important cellular energy sensor in VSM. Regulation of Ca2+ sensitivity usually is attributed to myosin light chain phosphatase activity, but findings in non-VSM identified changes in the actin cytoskeleton. The potential role of AMPK in this setting is widely unknown. To assess the influence of AMPK on the actin cytoskeleton in VSM of resistance arteries with regard to potential Ca2+ desensitization of VSM contractile apparatus. AMPK induced a slowly developing dilation at unchanged cytosolic Ca2+ levels in potassium chloride-constricted intact arteries isolated from mouse mesenteric tissue. This dilation was not associated with changes in phosphorylation of myosin light chain or of myosin light chain phosphatase regulatory subunit. Using ultracentrifugation and confocal microscopy, we found that AMPK induced depolymerization of F-actin (filamentous actin). Imaging of arteries from LifeAct mice showed F-actin rarefaction in the midcellular portion of VSM. Immunoblotting revealed that this was associated with activation of the actin severing factor cofilin. Coimmunoprecipitation experiments indicated that AMPK leads to the liberation of cofilin from 14-3-3 protein. AMPK induces actin depolymerization, which reduces vascular tone and the response to vasoconstrictors. Our findings demonstrate a new role of AMPK in the control of actin cytoskeletal dynamics, potentially allowing for long-term dilation of microvessels without substantial changes in cytosolic Ca2+.

Short-term high salt intake reduces brachial artery and microvascular function in the absence of changes in blood pressure.

The aims of this study were to test the hypothesis that short-term high salt intake reduces macrovascular and microvascular endothelial function in the absence of changes in blood pressure and to determine whether acute exercise restores endothelial function after high salt in women. Twelve women were administered high salt (11 g of sodium chloride for 7 days) and then underwent a weightlifting session. Brachial artery flow-mediated dilation and nitroglycerin dilation were measured with ultrasound at baseline, after high salt, and after weightlifting. Subcutaneous fat tissue biopsies were obtained at baseline, after high salt, and after weightlifting. Resistance arteries from biopsies were cannulated for vascular reactivity measurements in response to flow [flow-induced dilation (FID)] and acetylcholine. Blood pressure was similar before and after high salt diet. Brachial flow-mediated dilation was reduced after high salt diet but was not affected by acute weightlifting. Brachial nitroglycerin dilations were similar before and after high salt. FID and acetylcholine-induced dilation of resistance arteries were similar to that of before and after high salt diet. FID and acetylcholine-induced dilation was not altered by weightlifting after high salt diet. However, N-nitro-L-arginine methyl ester significantly reduced FID at baseline and after exercise but had no effect dilator reactivity after high salt diet alone. These data suggest that high salt intake reduces brachial artery endothelial function and switches the mediator of vasodilation in the microcirculation to a non-nitric oxide-dependent mechanism in healthy adults and acute exercise may switch the dilator mechanism back to nitric oxide during high salt diet.

AKAP150-dependent cooperative TRPV4 channel gating is central to endothelium-dependent vasodilation and is disrupted in hypertension.

Endothelial cell dysfunction, characterized by a diminished response to endothelial cell-dependent vasodilators, is a hallmark of hypertension. TRPV4 channels play a major role in endothelial-dependent vasodilation, a function mediated by local Ca(2+) influx through clusters of functionally coupled TRPV4 channels rather than by a global increase in endothelial cell Ca(2+). We showed that stimulation of muscarinic acetylcholine receptors on endothelial cells of mouse arteries exclusively activated TRPV4 channels that were localized at myoendothelial projections (MEPs), specialized regions of endothelial cells that contact smooth muscle cells. Muscarinic receptor-mediated activation of TRPV4 depended on protein kinase C (PKC) and the PKC-anchoring protein AKAP150, which was concentrated at MEPs. Cooperative opening of clustered TRPV4 channels specifically amplified Ca(2+) influx at MEPs. Cooperativity of TRPV4 channels at non-MEP sites was much lower, and cooperativity at MEPs was greatly reduced by chelation of intracellular Ca(2+) or AKAP150 knockout, suggesting that Ca(2+) entering through adjacent channels underlies the AKAP150-dependent potentiation of TRPV4 activity. In a mouse model of angiotensin II-induced hypertension, MEP localization of AKAP150 was disrupted, muscarinic receptor stimulation did not activate TRPV4 channels, cooperativity among TRPV4 channels at MEPs was weaker, and vasodilation in response to muscarinic receptor stimulation was reduced. Thus, endothelial-dependent dilation of resistance arteries is enabled by MEP-localized AKAP150, which ensures the proximity of PKC to TRPV4 channels and the coupled channel gating necessary for efficient communication from endothelial to smooth muscle cells in arteries. Disruption of this molecular assembly may contribute to altered blood flow in hypertension.

Aerobic exercise training enhances hydrogen peroxide mediated flow induced dilation of resistance arteries following exertion in overweight adults (678.8)

Exercise training protects against exertion‐induced microvasculature dysfunction. The purpose of this study was to determine if aerobic exercise training (AT) in overweight adults confers a protective effect. We hypothesized 1) that AT is protective and 2) H2O2 mediates this effect against microvasculature dysfunction. Eleven normotensive, overweight and obese adults (BMI 蠅25) enrolled in AT (30‐40 min. at 75% HRmax; 3 x per week) for 8 weeks, with a matched control group. Gluteal biopsies were performed at rest and following exertion at wk. 8 (6‐8 sets leg press at 10RM). Resistance arteries (RAs) were dissected from biopsies and cannulated for measures of reactivity to flow and acetylcholine induced dilation (FID; ACh) with and without the H2O2 scavenger, PEG‐CAT (500 U/ml) or the NOS inhibitor L‐NAME (10‐4 M). Nitric oxide (NO) and H2O2 production in RAs was assessed via fluorescent microscopy. FID and ACh dilations were impaired following exertion in control subjects. In contrast, FID and ACh were preserved following exertion in AT. PEG‐CAT significantly reduced FID (59 ± 8%; P&lt; 0.01) and AChD (66 ± 6%; P&lt; 0.01 post exertion in the AT group. Flow‐ induced NO was reduced and H2O2 production was increased in RAs of the AT subjects following exertion (H2O2:NO ↑ 2.4fold P&lt; 0.05). We conclude that 1) AT is protective against exertion induced microvasculature dysfunction and 2) H2O2 contributes to this protective effect.Grant Funding Source: Supported by NHLBI K23HL85614, RO1HL095701, and HL095701‐01A2S

Levosimendan and its metabolite OR-1896 elicit KATP channel-dependent dilation in resistance arteries in vivo

BackgroundLevosimendan and its long-lived metabolite OR-1896 produce vasodilation in different types of vessels by activating ATP-sensitive (KATP) and other potassium channels. MethodsIn the present study we applied intravital videomicroscopy to investigate the in situ effects of levosimendan and OR-1896 on the diameters of real resistance arterioles (rat cremaster muscle arterioles with diameters of ~ 20μm). ResultsLevosimendan and OR-1896 induced concentration-dependent (1nM – 100μM) dilations to similar extents in these arterioles (maximal dilation from 23±2 to 33±2μm and from 22±1 to 32±1μm, respectively). The arteriolar dilations induced by the selective KATP channel opener pinacidil (1nM – 10μM) (maximal dilation from 22±4μm to 35±3μm) were diminished in the presence of the selective KATP channel blocker – glibenclamide (5μM) (maximal diameter attained: 22±1μm). Glibenclamide also counteracted the maximal dilations in response to levosimendan or OR-1896 (to 23±3μm or 22±5μm, respectively). ConclusionsIn conclusion, this is the first demonstration that levosimendan and OR-1896 elicit arteriolar dilation in vivo, via activation of KATP channels in real resistance vessels in the rat.

Elementary Ca2+signals through endothelial TRPV4 channels regulate vascular function

The endothelial cells (ECs) lining blood vessels are pivotal regulators of vascular tone. Endothelial-dependent vasodilation by classic agents such as acetylcholine (ACh) depends on an elevation of EC Ca2+, and can act through the generation of nitric oxide (NO). An elevation of EC Ca2+ also activates small- and intermediate-conductance (SK and IK) potassium channels. This leads to endothelium-dependent hyperpolarization (“EDH”), which is spread through gap junctions in specialized EC projections to adjacent smooth muscle cells (SMCs) to cause vasodilation of small resistance arteries and arterioles, and this pathway is responsible for the majority of the ACh-induced dilation in resistance arteries [1]. We have recently reported the first measurements of elementary Ca2+ influx events (“sparklets”) through single TRPV4 (transient receptor potential vanilloid 4) channels in ECs of intact, small mesenteric arteries. Cooperative opening of as few as 3 TRPV4 channels per EC caused maximum vasodilation primarily through activation of EC IK channels [2]. This raises the fundamental question about how these sparse channels maintain the functional linkages necessary for efficient signaling. An architectural feature of ECs that is likely centrally important in this regard is the myoendothelial projection (MEP). These specialized projections through the internal elastic lamina (IEL) connect ECs with adjacent SMCs through gap junctions. The objective of this study was to elucidate the signaling network that enables efficient and effective endothelial-dependent vasodilation through the EDH pathway. Our results indicate that muscarinic receptor agonists activate TRPV4 sparklets exclusively at MEPs in a protein kinase Cα (PKCα) and A-kinase anchoring protein (AKAP150)-dependent manner. We also found that elevation of extracellular K+ activates EC inward rectifier K+ (Kir) channels to cause vasodilation, and that TRPV4-mediated vasodilation is attenuated by block of Kir channels. Our results support the concept that AKAP150 localization to MEPs ensures the proximity of PKC to TRPV4 channels and enhances channel cooperativity. The resultant activation of MEP IK channels by TRPV4 sparklets causes a hyperpolarizing current as well as local accumulation of K+ ions in the “nanospace” between the MEP and SM membranes. This in turn could activate EC Kir channels to augment EDH. Compartmentalization of signaling elements by anchoring proteins at MEPs, along with a high-amplification network of ion channels at MEPs makes it possible for a small number of ion channels to exert a profound influence on vascular function independent of NO.

Binge Drinking Impairs Vascular Function in Young Adults

The aim of this study was to assess whether young binge drinkers (BD) have impaired macrovascular and microvascular function and cardiovascular disease risk factors compared with age-matched alcohol abstainers (A). Binge drinking rates are highest on college campuses and among those age 18 to 25 years; however, macrovascular and microvascular endothelial function in young adults with histories of repeated binge drinking (≥ 5 standard drinks in 2 h in men, ≥ 4 standard drinks in 2 h in women) has not been investigated. Cardiovascular profiles, brachial artery endothelial-dependent flow-mediated dilation (FMD), and flow-independent nitroglycerin (NTG)-mediated dilation and vasoreactivity of resistance arteries (isolated from gluteal fat biopsies) were evaluated in A and BD. Men and women (18 to 25 years of age; A, n = 17; BD, n = 19) were enrolled. In the BD group, past-month mean number of binge episodes was 6 ± 1, and the mean duration of binge drinking behavior was 4 ± 0.6 years. FMD and NTG-mediated dilation were significantly lower in the BD group (FMD: 8.4 ± 0.7%, p = 0.022; NTG-mediated dilation: 19.6 ± 2%, p = 0.009) than in the A group (FMD: 11 ± 0.7%; NTG-mediated dilation: 28.6 ± 2%). Acetylcholine-induced and sodium nitroprusside-induced dilation in resistance arteries was not significantly different between the A and BD groups. However, endothelin-1-induced constriction was significantly enhanced in the BD group compared with the A group (p = 0.032). No differences between groups were found in blood pressure, lipoproteins, and C-reactive protein. Alterations in the macrocirculation and microcirculation may represent early clinical manifestations of cardiovascular risk in otherwise healthy young BD. This study has important clinical implications for screening young adults for a repeated history of binge drinking.

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.

Desensitization of endothelial P2Y1 receptors by PKC‐dependent mechanisms in pressurized rat small mesenteric arteries

Extracellular nucleotides play a crucial role in the regulation of vascular tone and blood flow. Stimulation of endothelial cell P2Y1 receptors evokes concentration-dependent full dilatation of resistance arteries. However, this GPCR can desensitize upon prolonged exposure to the agonist. Our aim was to determine the extent and nature of P2Y1 desensitization in isolated and pressurized rat small mesenteric arteries. The non-hydrolyzable selective P2Y1 agonist ADPbetaS (3 microM) was perfused through the lumen of arteries pressurized to 70 mmHg. Changes in arterial diameter and endothelial cell [Ca(2+)](i) were obtained in the presence and absence of inhibitors of protein kinase C (PKC). ADPbetaS evoked rapid dilatation to the maximum arterial diameter but faded over time to a much-reduced plateau closer to 35% dilatation. This appeared to be due to desensitization of the P2Y1 receptor, as subsequent endothelium-dependent dilatation to acetylcholine (1 microM) remained unaffected. Luminal treatment with the PKC inhibitors BIS-I (1 microM) or BIS-VIII (1 microM) tended to augment concentration-dependent dilatation to ADPbetaS (0.1-3 microM) and prevented desensitization. Another PKC inhibitor, Gö 6976 (1 microM), was less effective in preventing desensitization. Measurements of endothelial cell [Ca(2+)](i) in pressurized arteries confirmed the P2Y1 receptor but not M(3) muscarinic receptor desensitization. These data demonstrate for the first time the involvement of PKC in the desensitization of endothelial P2Y1 receptors in pressurized rat mesenteric arteries, which may have important implications in the control of blood flow by circulating nucleotides.

Flow-Induced Remodeling in Resistance Arteries From Obese Zucker Rats Is Associated With Endothelial Dysfunction

Chronic increases in blood flow increase arterial diameter and NO-dependent dilation in resistance arteries. Because endothelial dysfunction accompanies metabolic syndrome, we hypothesized that flow-mediated remodeling might be impaired in obese rat resistance arteries. Obese and lean Zucker rat mesenteric resistance arteries were exposed to chronic flow increases through arterial ligation in vivo: arteries exposed to high flow were compared with normal flow arteries. Diameter was measured in vitro in cannulated arteries using pressure arteriography. After 7 days, outward remodeling (diameter increased from 346+/-9 to 412+/-11 mum at 100 mm Hg) occurred in lean high-flow arteries. Endothelium-dependent tone was reduced in high-flow arteries from obese rats by contrast with lean animals. On the other hand, diameter enlargement occurred similarly in the 2 strains. The involvement of NO in endothelium-dependent dilation (evidenced by NO blockade) and endothelial NO synthase phosphorylation was smaller in obese than in lean rats. Superoxide anion and reduced nicotinamide-adenine dinucleotide phosphate oxidase subunit expression (p67phox and gp91phox) increased in obese rats and were higher in high-flow than in control arteries. Acute Tempol (a catalase mimetic), catalase plus superoxide dismutase, and l-arginine plus tetrahydrobiopterin restored endothelium-dependent dilation in obese rat normal and high-flow arteries to the level found in lean control arteries. Thus, flow-induced remodeling in obese resistance arteries was associated with a reduced endothelium-mediated dilation because of a decreased NO bioavailability and an excessive superoxide production. This dysfunction might have negative consequences in ischemic diseases in patients with obesity or metabolic syndrome.

Age-associated impairment in vasorelaxation to fluid shear stress in the female vasculature is improved by TNF-α antagonism

Aging is associated with alterations in vascular homeostasis, including a reduction in flow-mediated vasodilation, which in women is related to the onset of menopause. We previously found that in female animals, aging is associated with an increase in TNF-alpha. Thus we investigated the role of in vivo TNF-alpha inhibition on vascular responses to shear stress in aging female rats. Mesenteric arteries (approximately 150 microm) were isolated from young (3 mo) and ovariectomized Sprague-Dawley female rats approaching reproductive senescence (12 mo) treated with either placebo or a TNF-alpha inhibitor (etanercept; 0.3 mg/kg) and were mounted on a pressure myograph system. Vessels were equilibrated at an intraluminal pressure of 60 mmHg and then preconstricted with phenylephrine at approximately 70% of their initial diameter. Perfusate flow was increased in steps from 0 to 150 microl/min. Compared with young vessels, aged vessels have a decrease in flow-mediated dilation [maximal dilation (means +/- SE): 52 +/- 4 vs. 24 +/- 15%; P < 0.05], which was improved by TNF-alpha inhibition. Moreover, in aged vessels maximal dilation to flow was achieved at higher levels of shear stress compared with young vessels. In all groups, flow-mediated dilation was abolished by either endothelial removal or nitric oxide synthase inhibition with N(G)-nitro-L-arginine methyl ester. However, the modulation by N(G)-nitro-L-arginine methyl ester was reduced in vessels from aged animals compared with young animals but was improved in the etanercept-treated aged animals. In vivo chronic TNF-alpha inhibition improves flow-mediated arterial dilation in resistance arteries of aged female animals.

Increased superoxide leads to decreased flow-induced dilation in resistance arteries of Mn-SOD-deficient mice

The role of mitochondrial manganese-superoxide dismutase (Mn-SOD) in the maintenance of vascular function has not yet been studied. Thus we examined flow- and agonist-induced dilations in isolated mesenteric arteries (approximately 90 microm in diameter) of Mn-SOD heterozygous (Mn-SOD+/-) and wild-type (WT) mice. Increases in flow elicited dilations in all vessels, but the magnitude of the dilation was significantly less in vessels of Mn-SOD+/- mice than in those of WT mice (64 vs. 74% of passive diameter). N(omega)-nitro-L-arginine methyl ester inhibited the dilation in vessels of WT mice but had no effect on vessels of Mn-SOD+/- mice. Tempol or tiron (superoxide scavengers) increased flow-induced dilation in vessels of Mn-SOD+/- mice. Acetylcholine- and sodium nitroprusside-induced, but not adenosine-induced, dilations were also decreased in arteries of Mn-SOD+/- mice. Superoxide levels in the arteries of Mn-SOD+/- mice were significantly increased. Western blot analysis confirmed a 50% reduction of Mn-SOD protein in the vessels of Mn-SOD+/- mice. A 41% reduction in endothelial nitric oxide synthase (eNOS) protein and a 37% reduction in eNOS activity were also found in the vessels of Mn-SOD+/- mice. Whereas there was no difference in eNOS protein in kidney homogenates of WT and Mn-SOD+/- mice, a significant reduction of nitric oxide synthase activity was found in Mn-SOD+/- mice, which could be restored by the administration of tiron. We conclude that an increased concentration of superoxide due to reduced activity of Mn-SOD, which inactivates nitric oxide and inhibits eNOS activity, contributes to the impaired vasodilator function of isolated mesenteric arteries of Mn-SOD+/- mice. These results suggest that Mn-SOD contributes significantly to the regulation of vascular function.