Activation of AMP-activated protein kinase by 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside in the muscle microcirculation increases nitric oxide synthesis and microvascular perfusion.

OBJECTIVE: To investigate the effects of activation of the AMP-activated protein kinase (AMPK) on muscle perfusion and to elucidate the mechanisms involved. METHODS AND RESULTS: In a combined approach, we studied the vasoactive actions of AMPK activator by 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) on rat cremaster muscle resistance arteries ( approximately 100 mum) ex vivo and on microvascular perfusion in the rat hindlimb in vivo. In isolated resistance arteries, AICAR increased Thr172 phosphorylation of AMPK in arteriolar endothelium, which was predominantly located in microvascular endothelium. AICAR induced vasodilation (19+/-4% at 2 mmol/L, P<0.01), which was abolished by endothelium removal, inhibition of NO synthase (with N-nitro-L-arginine), or AMPK (with compound C). Smooth muscle sensitivity to NO, determined by studying the effects of the NO donor S-nitroso-N-acetylpenicillamine (SNAP), was not affected by AICAR except at the highest dose. AICAR increased endothelial nitric oxide synthase activity, as indicated by Ser1177 phosphorylation. In vivo, infusion of AICAR markedly increased muscle microvascular blood volume (approximately 60%, P<0.05), as was evidenced by contrast-enhanced ultrasound, without effects on blood pressure, femoral blood flow, or hind leg glucose uptake. CONCLUSIONS: Activation of AMPK by AICAR activates endothelial nitric oxide synthase in arteriolar endothelium by increasing its Ser1177 phosphorylation, which leads to vasodilation of resistance arteries and recruitment of microvascular perfusion in muscle.

ρ-Kinase inhibition reduces early microvascular leukocyte accumulation in the rat kidney following ischemia-reperfusion injury: roles of nitric oxide and blood flow.

AIM: To study whether microvascular leukocyte accumulation after rat renal ischemia and reperfusion (IR) is decreased by Rho kinase inhibition, independently of effects upon nitric oxide (NO) and renal blood flow. METHODS: Male Wistar rats were subjected to 60 min of ischemia by bilateral clamping and 60 min of reperfusion of the renal arteries, or a sham procedure. Haemodynamics were monitored and microsphere blood flow to the kidneys was measured. The infusion of the Rho kinase inhibitor (Y27632) was commenced before clamping and IR. The NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), was administered after the start of reperfusion whilst the dopamine-1 receptor agonist fenoldopam, a renal vasodilator, was infused during the reperfusion period. Digital imaging microscopy analysis of cryosections was done to determine leukocyte accumulation and vasodilator-stimulated phosphoprotein serine 239 phosphorylation (p-VASP ser 239), a marker of endothelial NO. RESULTS: Leukocytes (60-70% neutrophils) accumulated within blood vessels in the corticomedullary junction and medulla of the kidney. Leukocyte accumulation was markedly reduced by the Rho kinase inhibitor but not by fenoldopam. However, both drugs improved renal blood flow and microvascular expression of p-VASP ser 239 in the corticomedullary junction and medulla, which were decreased following IR. L-NAME treatment of IR animals pretreated with the Rho kinase inhibitor reduced blood flow and p-VASP ser 239 expression and increased leukocyte accumulation. CONCLUSION: Early microvascular leukocyte accumulation in the corticomedullary junction and medulla of the rat kidney after IR is ameliorated by Rho kinase inhibition. This effect is partly independent upon attenuation of decreased NO and renal blood flow.

Role of cyclooxygenase and derived reactive oxygen species in rho-kinase-mediated impairment of endothelium-dependent vasodilation and blood flow after ischemia-reperfusion of the rat kidney.

BACKGROUND/AIMS: Decreased endothelium-dependent vasodilation and blood flow in renal ischemia-reperfusion (IR) may result in part from rho-kinase activation, and cyclooxygenase (COX) activation, and resultant reactive oxygen species (ROS) may be involved. METHODS: We tested this hypothesis in male Wistar rats, subjected to 60 min of bilateral clamping of the renal arteries and 60 min of reperfusion or a sham procedure, and treated by the rho-kinase inhibitor Y27632 (1 mg/kg) and/or the nonspecific COX inhibitor diclofenac (10 mg/kg). Renal blood flow was measured by fluorescent microspheres, and ROS in the arterial endothelium was quantified by dihydroethidium staining. Endothelium-dependent vasodilation was determined by an acetylcholine concentration-response curve in the presence or absence of diclofenac (10 microM). RESULTS: Y27632 increased renal blood flow and reduced ROS in vivo, and improved endothelium-dependent vasodilation in vitro, following IR with or without diclofenac. Following IR, diclofenac had no effect on renal blood flow and ROS in vivo, but improved endothelium-dependent vasodilation in vitro. CONCLUSION: Activation of rho-kinase impairs endothelium-dependent vasodilation and perfusion following renal IR, independently of COX and resultant ROS. In contrast, the vasodilatory effect of rho-kinase inhibition may be partly mediated by decreasing ROS, unrelated to COX and resultant vasoconstricting prostanoids.

Endothelial dysfunction and diabetes: roles of hyperglycemia, impaired insulin signaling and obesity.

Endothelial dysfunction comprises a number of functional alterations in the vascular endothelium that are associated with diabetes and cardiovascular disease, including changes in vasoregulation, enhanced generation of reactive oxygen intermediates, inflammatory activation, and altered barrier function. Hyperglycemia is a characteristic feature of type 1 and type 2 diabetes and plays a pivotal role in diabetes-associated microvascular complications. Although hyperglycemia also contributes to the occurrence and progression of macrovascular disease (the major cause of death in type 2 diabetes), other factors such as dyslipidemia, hyperinsulinemia, and adipose-tissue-derived factors play a more dominant role. A mutual interaction between these factors and endothelial dysfunction occurs during the progression of the disease. We pay special attention to the possible involvement of endoplasmic reticulum stress (ER stress) and the role of obesity and adipose-derived adipokines as contributors to endothelial dysfunction in type 2 diabetes. The close interaction of adipocytes of perivascular adipose tissue with arteries and arterioles facilitates the exposure of their endothelial cells to adipokines, particularly if inflammation activates the adipose tissue and thus affects vasoregulation and capillary recruitment in skeletal muscle. Hence, an initial dysfunction of endothelial cells underlies metabolic and vascular alterations that contribute to the development of type 2 diabetes.

Rho-kinase-dependent F-actin rearrangement is involved in the inhibition of PI3-kinase/Akt during ischemia-reperfusion-induced endothelial cell apoptosis.

Activation of cytoskeleton regulator Rho-kinase during ischemia-reperfusion (I/R) plays a major role in I/R injury and apoptosis. Since Rho-kinase is a negative regulator of the pro-survival phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway, we hypothesized that inhibition of Rho-kinase can prevent I/R-induced endothelial cell apoptosis by maintaining PI3-kinase/Akt activity and that protective effects of Rho-kinase inhibition are facilitated by prevention of F-actin rearrangement. Human umbilical vein endothelial cells were subjected to 1 h of simulated ischemia and 1 or 24 h of simulated reperfusion after treatment with Rho-kinase inhibitor Y-27632, PI3-kinase inhibitor wortmannin, F-actin depolymerizers cytochalasinD and latrunculinA and F-actin stabilizer jasplakinolide. Intracellular ATP levels decreased following I/R. Y-27632 treatment reduced I/R-induced apoptosis by 31% (P < 0.01) and maintained Akt activity. Both effects were blocked by co-treatment with wortmannin. Y-27632 treatment prevented the formation of F-actin bundles during I/R. Similar results were observed with cytochalasinD treatment. In contrast, latrunculinA and jasplakinolide treatment did not prevent the formation of F-actin bundles during I/R and had no effect on I/R-induced apoptosis. Apoptosis and Akt activity were inversely correlated (R (2) = 0.68, P < 0.05). In conclusion, prevention of F-actin rearrangement by Rho-kinase inhibition or by cytochalasinD treatment attenuated I/R-induced endothelial cell apoptosis by maintaining PI3-kinase and Akt activity.

3beta-OH-tibolone acutely dilates rat muscle arterioles similar to 3alpha-OH-tibolone.

In an earlier study, we focused on the vasoactive effect of 3alpha-OH-tibolone on spontaneously constricted isolatedfemale rat gracilis muscle arterioles. Vasodilator effects (from 10 to 10 M) of 3alpha-OH-tibolone were similar to those of 17beta-estradiol. It was reported that 3beta-OH-tibolone like estradiol altered GABAB activation in neurons through a membrane estrogen receptor, whereas the 3alpha-OH metabolite did not. We therefore hypothesized that the 3beta-OH metabolite may also have a vasodilating effect in our isolated arteriole model. The results indicate that 3beta-OH-tibolone induces a vasodilator effect in small arterioles that is comparable with that of 3alpha-OH-tibolone at the same concentration. This is intriguing because the binding affinity of 3alpha-OH-tibolone to the estrogen receptor is almost twice that of 3beta-OH-tibolone. Other mechanisms may play a role.

Production of endothelin-1 and reduced blood flow in the rat kidney during lung-injurious mechanical ventilation.

INTRODUCTION: The mechanisms by which mechanical ventilation (MV) can injure remote organs, such as the kidney, remain poorly understood. We hypothesized that upregulation of systemic inflammation, as reflected by plasma interleukin-6 (IL-6) levels, in response to a lung-injurious ventilatory strategy, ultimately results in kidney dysfunction mediated by local endothelin-1 (ET-1) production and renal vasoconstriction. METHODS: Healthy, male Wistar rats were randomized to 1 of 2 MV settings (n=9 per group) and ventilated for 4 h. One group had a lung-protective setting using peak inspiratory pressure of 14 cm H2O and a positive end-expiratory pressure of 5 cm H2O; the other had a lung-injurious strategy using a peak inspiratory pressure of 20 cm H2O and positive end-expiratory pressure of 2 cm H2O. Nine randomly assigned rats served as nonventilated controls. We measured venous and arterial blood pressure and cardiac output (thermodilution method), renal blood flow (RBF) using fluorescent microspheres, and calculated creatinine clearance, urine flow, and fractional sodium excretion. Histological lung damage was assessed using hematoxylin-eosin staining. Renal ET-1 and plasma ET-1 and IL-6 concentrations were measured using enzyme-linked immunosorbent assays. RESULTS: Lung injury scores were higher after lung-injurious MV than after lung-protective ventilation or in sham controls. Lung-injurious MV resulted in significant production of renal ET-1 compared with the lung-protective and control groups. Simultaneously, RBF in the lung-injurious MV group was approximately 40% lower (P<0.05) than in the control group and 28% lower (P<0.05) than in the lung-protective group. Plasma ET-1 and IL-6 levels did not differ among the groups and systemic hemodynamics, such as cardiac output, were comparable. There was no effect on creatinine clearance, fractional sodium excretion, urine output, or kidney histology. CONCLUSIONS: Lung-injurious MV for 4 h in healthy rats results in significant production of renal ET-1 and in decreased RBF, independent of IL-6. Decreased RBF has no observable effect on kidney function or histology.

Physiological concentrations of insulin induce endothelin-dependent vasoconstriction of skeletal muscle resistance arteries in the presence of tumor necrosis factor-alpha dependence on c-Jun N-terminal kinase.

OBJECTIVE: Tumor necrosis factor-alpha (TNF-alpha) has been linked to obesity-related insulin resistance and impaired endothelium-dependent vasodilatation, but the mechanisms have not been elucidated. To investigate whether TNF-alpha directly impairs insulin-mediated vasoreactivity in skeletal muscle resistance arteries and the role of c-Jun N-terminal kinase (JNK) in this interference. METHODS AND RESULTS: Insulin-mediated vasoreactivity of isolated resistance arteries of the rat cremaster muscle to insulin (4 to 3400 microU/mL) was studied in the absence and presence of TNF-alpha (10 ng/mL). Although insulin or TNF-alpha alone did not affect arterial diameter, insulin induced dose-dependent vasoconstriction of cremaster resistance arteries in the presence of TNF-alpha, (-12+/-1% at 272 microU/mL). Blocking endothelin receptors in the absence of TNF-alpha uncovered insulin-mediated vasodilatation (18+/-6% at 272 microU/mL) but not in the presence of TNF-alpha (2+/-2% at 272 microU/mL), showing that TNF-alpha inhibits vasodilator effects of insulin. Using digital imaging microscopy, we discovered that TNF-alpha activates JNK in arterial endothelium, visible as an increase in phosphorylated JNK. Moreover, inhibition of JNK with the cell-permeable peptide inhibitor L-JNKI abolished insulin-mediated vasoconstriction in the presence of TNF-alpha, showing that JNK is required for interaction between TNF-alpha and insulin. CONCLUSIONS: TNF-alpha inhibits vasodilator but not vasoconstrictor effects of insulin in skeletal muscle resistance arteries, resulting in insulin-mediated vasoconstriction in the presence of TNF-alpha. This effect of TNF-alpha is critically dependent on TNF-alpha-mediated activation of JNK.

Globular adiponectin controls insulin-mediated vasoreactivity in muscle through AMPKα2.

Decreased tissue perfusion increases the risk of developing insulin resistance and cardiovascular disease in obesity, and decreased levels of globular adiponectin (gAdn) have been proposed to contribute to this risk. We hypothesized that gAdn controls insulin's vasoactive effects through AMP-activated protein kinase (AMPK), specifically its α2 subunit, and studied the mechanisms involved. In healthy volunteers, we found that decreased plasma gAdn levels in obese subjects associate with insulin resistance and reduced capillary perfusion during hyperinsulinemia. In cultured human microvascular endothelial cells (HMEC), gAdn increased AMPK activity. In isolated muscle resistance arteries gAdn uncovered insulin-induced vasodilation by selectively inhibiting insulin-induced activation of ERK1/2, and the AMPK inhibitor compound C as well as genetic deletion of AMPKα2 blunted insulin-induced vasodilation. In HMEC deletion of AMPKα2 abolished insulin-induced Ser(1177) phosphorylation of eNOS. In mice we confirmed that AMPKα2 deficiency decreases insulin sensitivity, and this was accompanied by decreased muscle microvascular blood volume during hyperinsulinemia in vivo. This impairment was accompanied by a decrease in arterial Ser(1177) phosphorylation of eNOS, which closely related to AMPK activity. In conclusion, globular adiponectin controls muscle perfusion during hyperinsulinemia through AMPKα2, which determines the balance between NO and ET-1 activity in muscle resistance arteries. Our findings provide a novel mechanism linking reduced gAdn-AMPK signaling to insulin resistance and impaired organ perfusion.

Endothelium-dependent dilatory effects of 3alpha-OH-tibolone in gracilis muscle arterioles of the female Wistar rat.

OBJECTIVE: Tibolone is a synthetic steroid used for the treatment of the symptoms of menopause that, once metabolized, has estrogenic, progestogenic, and androgenic properties. We investigated the direct vasodilatory effects of the major active tibolone metabolite 3alpha-OH-tibolone and its sulfated form on female rat skeletal muscle arterioles, which play an important role in the control of blood pressure. DESIGN: In isolated, pressurized spontaneously constricted arterioles (mean passive diameter 83 +/- 3 microm), we investigated the vasodilatory effect of 3alpha-OH-tibolone and its sulfated form. To study the role of the endothelium and in particular that of nitric oxide, we repeated the experiments with 3alpha-OH-tibolone after removal of the endothelium and on vessels pretreated with the nitric oxide synthesis inhibitor, Nomega-nitro-L-arginine (L-Na). Finally we compared the vasodilatory effect of 3alpha-OH-tibolone with 17beta-estradiol. RESULTS: A dose-dependent dilatation to 3alpha-OH-tibolone was observed starting at a concentration of 10 M. With the sulfated form of 3alpha-OH-tibolone, dilatation was only present at the highest concentration (10 M). In the denuded vessels, the vasodilatory effect was absent at concentrations from 10 to 10 M. The dilatation induced by 3alpha-OH-tibolone was not significantly reduced by L-Na. The vasodilatory effect of 3alpha-OH-tibolone did not differ from that of 17beta-estradiol. CONCLUSIONS: 3alpha-OH-tibolone has a dose-dependent vasodilatory effect on isolated skeletal muscle arterioles from the rat. The sulfated form has no vasodilatory effect in this setup. This finding suggests that during this short incubation time there was no conversion of the sulfated metabolite into its active form by the vascular endothelium. The vasodilatory effect of 3alpha-OH-tibolone is endothelium dependent at physiologic concentrations and comparable to that of 17beta-estradiol.

Physiological concentrations of insulin induce endothelin-mediated vasoconstriction during inhibition of NOS or PI3-kinase in skeletal muscle arterioles.

OBJECTIVE: To determine the roles of nitric oxide, endothelin-1 and phosphatidylinositol 3-kinase (PI3-kinase) in acute responses of isolated rat skeletal muscle arterioles to insulin. METHODS: Rat cremaster first order arterioles were separated from surrounding tissue, cannulated in a pressure myograph and responses to insulin (4 microU/ml-3.4 mU/ml) were studied without intraluminal blood or flow. RESULTS: Insulin alone did not significantly affect arteriolar diameter. Non-selective antagonism of endothelin receptors, with PD-142893, uncovered insulin-induced vasodilatation (25+/-8% from baseline at 3.4 mU/ml), which was abolished by inhibition of NO synthesis with N(G)-nitro-L-arginine (L-NA). Inhibition of NO synthesis alone uncovered insulin-induced vasoconstriction at physiological concentrations (21+/-5% from baseline diameter at 34 microU/ml), which was abolished by PD-142893. The NO donor, S-nitroso-N-acetyl-penicillamine (SNAP) inhibited insulin-induced vasoconstriction during NOS inhibition, even at a concentration that did not elicit vasodilatation itself. Inhibition of PI3-kinase, an intracellular mediator of insulin-induced NO production, with wortmannin, also uncovered insulin-induced vasoconstriction (13+/-3% from baseline at 34 microU/ml) that was abolished by PD-142893. CONCLUSIONS: Insulin induces both nitric oxide and endothelin-1 activity in rat cremaster first-order arterioles. This study demonstrates for the first time that vasoconstrictive effects of physiological concentrations of insulin during inhibition of NOS activity are mediated by endothelin and that insulin induces endothelin-1-mediated vasoconstriction in isolated skeletal muscle arterioles during inhibition of PI3-kinase. These findings support the hypothesis of altered microvascular reactivity to insulin in conditions of diminished PI3-kinase activity, a prominent feature of insulin resistance.

Tumor necrosis factor-alpha impairs endothelium-dependent relaxation of rat renal arteries, independent of tyrosine kinase.

We hypothesized that tumor necrosis factor-alpha (TNF-alpha) mimics endotoxin in attenuating endothelium-dependent vasodilation and smooth muscle constriction of rat renal arteries, and that tyrosine kinase is involved. Isolated rat renal arteries (n =6 per group), pretreated for 2 h by genistein (4',5,7-trihydroxyisoflavone, 10 microg/mL, a tyrosine kinase inhibitor) or vehicle, were exposed for 2 h to recombinant human (rh) TNF-alpha (100 ng/mL) or vehicle. rhTNF-alpha attenuated (P < 0.05) the constriction response to depolarizing 125 mM KCl (952.6+/-125.3 mg/mm vs. 1191.4+/-136.8 mg/mm in rhTNF-alpha-exposed and control segments, respectively), but did not affect the constriction response to norepinephrine (NE, 0.01-10 microM). Genistein did not affect the constriction response to KCl. The concentration-response relation to NE in genistein-pretreated control segments showed (P < 0.05) a rightward shift, while the maximum constriction was not affected. Genistein did not prevent a reduction (P < 0.05) by rhTNF-alpha in the maximum response to NE (721.7+/-42.4 mg/mm vs. 999.8+/-84.4 mg/mm in controls). The endothelium-dependent relaxation induced by (acetyl choline) ACh (0.001-1.0 microM) was attenuated (P < 0.05) by rhTNF-alpha (39.4%+/-6.7% and 77.4%+/-10.0% in rhTNF-alpha-exposed and control segments, respectively). The reduction (P < 0.05) in maximum ACh-induced relaxation after exposure to rhTNF-alpha was not affected by genistein (44.6%+/-3.4% and 70.8% x 2.2% in genistein-pretreated rhTNF-alpha-exposed and control segments, respectively). Hence, the attenuated endothelium-dependent relaxation and smooth muscle constriction of rat renal arteries following short-term rhTNF-alpha exposure, mimicking the effect of endotoxin, does not involve the activity of tyrosine kinase. The latter may be involved in pharmacomechanical coupling, by increasing Ca2+ sensitivity, but less in the electromechanical coupling of smooth muscle constriction.

Homocysteine impairs estrogen-induced vasodilation in isolated rat arterioles.

OBJECTIVE: Clinical and basic studies have provided evidence that the cardiovascular protective effects of estrogens are partly due to effects on vasoreactivity and changes in homocysteine metabolism. Moreover, homocysteine has also been shown to influence vasoreactivity. We investigated the influence of homocysteine on the rapid vasodilatory effects of estradiol in an isolated vessel setup. DESIGN: Isolated, spontaneously constricted, gracilis muscle arterioles (diameter approximately 50 micromol/L) from female Wistar rats were cumulatively exposed to 10-10 to 10-4 mol/L 17beta-estradiol in the presence of 50 micromol/L homocysteine or N-nitro-L-arginine (L-NA) (a blocker of nitric oxide synthesis), or both. Control experiments were done without L-NA or homocysteine (n = 6 for each series). RESULTS: The dose-dependent dilation during short-term exposure to 17beta-estradiol was significantly less or absent in arterioles where L-NA, homocysteine, or both were present. The addition of 50 micromol/L homocysteine significantly increased the spontaneous constriction by 6% to 10%. CONCLUSIONS: We showed that a pathophysiological concentration of homocysteine increases the spontaneous arteriolar constriction and inhibits the 17beta-estradiol-induced, endothelium-mediated, rapid vasodilatory effect on muscle arterioles from the female rat. The endothelium-independent vasodilation remained unchanged.

Pressure-diameter relationship in the human greater saphenous vein.

BACKGROUND: Compliance of artificial and autologous vascular grafts is related to future patency. We investigated whether differences in compliance exist between saphenous vein grafts derived from the upper or lower leg, which might indicate upper or lower leg saphenous vein preference in coronary artery bypass surgery. Furthermore, the effect of perivenous application of fibrin glue on mechanical vein wall properties was studied to evaluate its possible use as perivenous graft support. METHODS: Vein segments (N = 10) from upper or lower leg saphenous vein grafts were collected for histopathologic examination and smooth muscle cell/extracellular matrix (SMC/ECM) ratio was calculated. This ratio is suggested to be related with vascular elastic compliance. In a second group vein graft segments (N = 6) from upper and lower leg were placed in an in vitro model generating stepwise increasing static pressure up to 150 cm H(2)O. Outer diameter was measured continuously with a video micrometer system. Distensibility was calculated from the pressure-diameter curves. A third group of vein graft segments (N = 7) was pressurized after fibrin glue application to prevent overdistension, and studied in the same setup. RESULTS: Vein segments from the lower leg demonstrated a consistent higher relative response compared with the upper leg saphenous vein graft (0.9176 +/- 0.03993 vs 0.5245 +/- 0.02512). Both reach a plateau in the high-pressure range (> 100 cm H(2)O). A significant difference in in vitro distensibility between upper and lower leg saphenous vein was only found at a pressure of 50 cm H(2)O (p < 0.05). With fibrin glue, support overdistension is prevented as revealed by the maximum relative response between fibrin glue supported upper and lower leg saphenous vein segments (0.4080 +/- 0.02464 vs 0.582 +/- 0.051), and no plateau is reached in the pressure range up to 150 cm H(2)O. CONCLUSIONS: No upper or lower leg saphenous vein preference could be deduced from the differences in pressure-diameter response due to loss of distensibility (and thus of compliance) in the high-pressure range. Fibrin glue effectively prevents overdistension and preserves some distensibility in the high-pressure range in both the upper and lower leg saphenous vein. This might provide a basis for clinical application of perivenous support.

Effect of cyclic axial stretch of rat arteries on endothelial cytoskeletal morphology and vascular reactivity.

Pulsatile fluid shear stress and circumferential stretch are responsible for the axial alignment of vascular endothelial cells and their actin stress fibers in vivo. We studied the effect of cyclic alterations in axial stretch independent of flow on endothelial cytoskeletal organization in intact arteries and determined if functional alterations accompanied morphologic alterations. Rat renal arteries were axially stretched (20%, 0.5 Hz) around their in vivo lengths, for up to 4h. Actin stress fibers were examined by immunofluorescent staining. We found that cyclic axial stretching of intact vessels under normal transmural pressure in the absence of shear stress induces within a few hours realignment of endothelial actin stress fibers toward the circumferential direction. Concomitant with this morphologic alteration, the sensitivity (log(EC(50))) to the endothelium-dependent vasodilator (acetylcholine) was significantly decreased in the stretched vessels (after stretching -5.15+/-0.79 and before stretching -6.71+/-0.78, resp.), while there was no difference in sodium nitroprusside (SNP) sensitivity. There was no difference in sensitivity to both acetylcholine and SNP in time control vessels. Similar to cultured cells, endothelial cells in intact vessels subjected to cyclic stretching reorganize their actin filaments almost perpendicular to the stretching direction. Accompanying this morphological alteration is a loss of endothelium-dependent vasodilation but not of smooth muscle responsiveness.

Subendocardial and subepicardial pressure-flow relations in the rat heart in diastolic and systolic arrest.

Ischemic heart disease is more apparent in the subendocardial than in subepicardial layers. We investigated coronary pressure-flow relations in layers of the isolated rat left ventricle, using 15 microm microspheres during diastolic and systolic arrest in the vasodilated coronary circulation. A special cannula allowed for selective determination of left main stem pressure-flow relations. Arterio-venous shunt flow was derived from microspheres in the venous effluent. We quantitatively investigated the pressure-flow relations in diastolic arrest (n=8), systolic arrest at normal contractility (n=8) and low contractility (n=6). In all three groups normal and large ventricular volume was studied. In diastolic arrest, at a perfusion pressure of 90 mmHg, subendocardial flow is larger than subepicardial flow, i.e., the endo/epi ratio is approximately 1.2. In systolic arrest the endo/epi ratio is approximately 0.3, and subendocardial flow and subepicardial flow are approximately 12% and approximately 55% of their values during diastolic arrest. The endo/epi ratio in diastolic arrest decreases with increasing perfusion pressure, while in systole the ratio increases. The slope of the pressure-flow relations, i.e., inverse of resistance, changes by a factor of approximately 5.3 in the subendocardium and by a factor approximately 2.2 in the subepicardium from diastole to systole. Lowering contractility affects subendocardial flow more than subepicardial flow, but both contractility and ventricular volume changes have only a limited effect on both subendocardial and subepicardial flow. The resistance (inverse of slope) of the total left main stem pressure-flow relation changes by a factor of approximately 3.4 from diastolic to systolic arrest. The zero-flow pressure increases from diastole to systole. Thus, coronary perfusion flow in diastolic arrest is larger than systolic arrest, with the largest difference in the subendocardium, as a result of layer dependent increases in vascular resistance and intercept pressure. Shunt flow is larger in diastolic than in systolic arrest, and increases with perfusion pressure. We conclude that changes in contractility and ventricular volume have a smaller effect on pressure-flow relations than diastolic-systolic differences. A synthesis of models accounting for the effect of cardiac contraction on perfusion is suggested.

Perivascular adipose tissue control of insulin-induced vasoreactivity in muscle is impaired in db/db mice.

Microvascular recruitment in muscle is a determinant of insulin sensitivity. Whether perivascular adipose tissue (PVAT) is involved in disturbed insulin-induced vasoreactivity is unknown, as are the underlying mechanisms. This study investigates whether PVAT regulates insulin-induced vasodilation in muscle, the underlying mechanisms, and how obesity disturbs this vasodilation. Insulin-induced vasoreactivity of resistance arteries was studied with PVAT from C57BL/6 or db/db mice. PVAT weight in muscle was higher in db/db mice compared with C57BL/6 mice. PVAT from C57BL/6 mice uncovered insulin-induced vasodilation; this vasodilation was abrogated with PVAT from db/db mice. Blocking adiponectin abolished the vasodilator effect of insulin in the presence of C57BL/6 PVAT, and adiponectin secretion was lower in db/db PVAT. To investigate this interaction further, resistance arteries of AMPKα2(+/+) and AMPKα2(-/-) were studied. In AMPKα2(-/-) resistance arteries, insulin caused vasoconstriction in the presence of PVAT, and AMPKα2(+/+) resistance arteries showed a neutral response. On the other hand, inhibition of the inflammatory kinase Jun NH(2)-terminal kinase (JNK) in db/db PVAT restored insulin-induced vasodilation in an adiponectin-dependent manner. In conclusion, PVAT controls insulin-induced vasoreactivity in the muscle microcirculation through secretion of adiponectin and subsequent AMPKα2 signaling. PVAT from obese mice inhibits insulin-induced vasodilation, which can be restored by inhibition of JNK.

Protein kinase C theta activation induces insulin-mediated constriction of muscle resistance arteries.

OBJECTIVE: Protein kinase C (PKC) theta activation is associated with insulin resistance and obesity, but the underlying mechanisms have not been fully elucidated. Impairment of insulin-mediated vasoreactivity in muscle contributes to insulin resistance, but it is unknown whether PKC theta is involved. In this study, we investigated whether PKC theta activation impairs insulin-mediated vasoreactivity and insulin signaling in muscle resistance arteries. RESEARCH DESIGN AND METHODS: Vasoreactivity of isolated resistance arteries of mouse gracilis muscles to insulin (0.02-20 nmol/l) was studied in a pressure myograph with or without PKC theta activation by palmitic acid (PA) (100 micromol/l). RESULTS: In the absence of PKC theta activation, insulin did not alter arterial diameter, which was caused by a balance of nitric oxide-dependent vasodilator and endothelin-dependent vasoconstrictor effects. Using three-dimensional microscopy and Western blotting of muscle resistance arteries, we found that PKC theta is abundantly expressed in endothelium of muscle resistance arteries of both mice and humans and is activated by pathophysiological levels of PA, as indicated by phosphorylation at Thr(538) in mouse resistance arteries. In the presence of PA, insulin induced vasoconstriction (21 +/- 6% at 2 nmol/l insulin), which was abolished by pharmacological or genetic inactivation of PKC theta. Analysis of intracellular signaling in muscle resistance arteries showed that PKC theta activation reduced insulin-mediated Akt phosphorylation (Ser(473)) and increased extracellular signal-related kinase (ERK) 1/2 phosphorylation. Inhibition of PKC theta restored insulin-mediated vasoreactivity and insulin-mediated activation of Akt and ERK1/2 in the presence of PA. CONCLUSIONS: PKC theta activation induces insulin-mediated vasoconstriction by inhibition of Akt and stimulation of ERK1/2 in muscle resistance arteries. This provides a new mechanism linking PKC theta activation to insulin resistance.

Selective resistance to vasoactive effects of insulin in muscle resistance arteries of obese Zucker (fa/fa) rats.

UNLABELLED: Obesity is related to insulin resistance and hypertension, but the underlying mechanisms are unclear. Insulin exerts both vasodilator and vasoconstrictor effects on muscle resistance arteries, which may be differentially impaired in obesity. OBJECTIVES: To investigate whether vasodilator and vasoconstrictor effects of insulin are impaired in muscle resistance arteries of obese rats and the roles of Akt and endothelial NO synthase (eNOS). METHODS/RESULTS: Effects of insulin were studied in resistance arteries isolated from cremaster muscles of lean and obese Zucker rats. In arteries of lean rats, insulin increased activity of both NO and endothelin (ET-1), resulting in a neutral effect under basal conditions. In arteries of obese rats, insulin induced endothelin-mediated vasoconstriction (-15 +/- 5% at 1 nM, P < 0.05 vs. lean). Insulin induced vasodilatation during endothelin receptor blockade in arteries of lean rats (20 +/- 5% at 1 nM) but not in those of obese rats. Inhibition of NO synthesis increased vascular tone (by 12 +/- 2%) and shifted insulin-mediated vasoreactivity to vasoconstriction (-25 +/- 1% at 1 nM) in lean rats but had no effect in arteries of obese rats, indicating reduced NO activity. Protein analysis of resistance arteries revealed that insulin-mediated activation of Akt was preserved in obese rats, whereas expression of eNOS was markedly decreased. CONCLUSIONS: Vasodilator but not vasoconstrictor effects of insulin are impaired in muscle resistance arteries of obese rats, and this selective impairment is associated with decreased protein levels of eNOS. These findings provide a new mechanism linking obesity to insulin resistance and hypertension.

Cross-talk between cardiac muscle and coronary vasculature.

The cardiac muscle and the coronary vasculature are in close proximity to each other, and a two-way interaction, called cross-talk, exists. Here we focus on the mechanical aspects of cross-talk including the role of the extracellular matrix. Cardiac muscle affects the coronary vasculature. In diastole, the effect of the cardiac muscle on the coronary vasculature depends on the (changes in) muscle length but appears to be small. In systole, coronary artery inflow is impeded, or even reversed, and venous outflow is augmented. These systolic effects are explained by two mechanisms. The waterfall model and the intramyocardial pump model are based on an intramyocardial pressure, assumed to be proportional to ventricular pressure. They explain the global effects of contraction on coronary flow and the effects of contraction in the layers of the heart wall. The varying elastance model, the muscle shortening and thickening model, and the vascular deformation model are based on direct contact between muscles and vessels. They predict global effects as well as differences on flow in layers and flow heterogeneity due to contraction. The relative contributions of these two mechanisms depend on the wall layer (epi- or endocardial) and type of contraction (isovolumic or shortening). Intramyocardial pressure results from (local) muscle contraction and to what extent the interstitial cavity contracts isovolumically. This explains why small arterioles and venules do not collapse in systole. Coronary vasculature affects the cardiac muscle. In diastole, at physiological ventricular volumes, an increase in coronary perfusion pressure increases ventricular stiffness, but the effect is small. In systole, there are two mechanisms by which coronary perfusion affects cardiac contractility. Increased perfusion pressure increases microvascular volume, thereby opening stretch-activated ion channels, resulting in an increased intracellular Ca2+ transient, which is followed by an increase in Ca2+ sensitivity and higher muscle contractility (Gregg effect). Thickening of the shortening cardiac muscle takes place at the expense of the vascular volume, which causes build-up of intracellular pressure. The intracellular pressure counteracts the tension generated by the contractile apparatus, leading to lower net force. Therefore, cardiac muscle contraction is augmented when vascular emptying is facilitated. During autoregulation, the microvasculature is protected against volume changes, and the Gregg effect is negligible. However, the effect is present in the right ventricle, as well as in pathological conditions with ineffective autoregulation. The beneficial effect of vascular emptying may be reduced in the presence of a stenosis. Thus cardiac contraction affects vascular diameters thereby reducing coronary inflow and enhancing venous outflow. Emptying of the vasculature, however, enhances muscle contraction. The extracellular matrix exerts its effect mainly on cardiac properties rather than on the cross-talk between cardiac muscle and coronary circulation.