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Insulin resistance associated with Type 2 diabetes contributes to impaired vasorelaxation. Previously, we showed the phosphorylation of myosin-bound phosphatase substrate MYPT1, a marker of the vascular smooth muscle cell (VSMC) contraction, was negatively regulated by Akt (protein kinase B) phosphorylation in response to insulin stimulation. In this study we examined the role of Akt phosphorylation on impaired insulin-induced vasodilation in the Goto-Kakizaki (GK) rat model of Type 2 diabetes. GK VSMCs had impaired basal and insulin-induced Akt phosphorylation as well as increases in basal MYPT1 phosphorylation, inducible nitric oxide synthase (iNOS) expression, and nitrite/nitrate production compared with Wistar-Kyoto controls. Both iNOS expression and the inhibition of angiotensin (ANG) II-induced MYPT1 phosphorylation were resistant to the effects of insulin in diabetic GK VSMC. We also measured the isometric tension of intact and denuded GK aorta using a myograph and observed significantly impaired insulin-induced vasodilation. Adenovirus-mediated overexpression of constitutively active Akt in GK VSMC led to significantly improved insulin sensitivity in terms of counteracting ANG II-induced contractile signaling via MYPT1, myosin light chain dephosphorylation, and reduced iNOS expression, S-nitrosylation and survivin expression. We demonstrated for the first time the presence of Akt-independent iNOS expression in the GK diabetic model and that the defective insulin-induced vasodilation observed in the diabetic vasculature can be restored by the overexpression of active Akt, which advocates a novel therapeutic strategy for treating diabetes. diabetes; insulin; inducible nitric oxide synthase; Akt; vasodilation

Carbon monoxide (CO) is endogenously produced by heme oxygenase (HO) and is involved in vascular, neural, and inflammatory responses in mammals. However, the biological activities of CO in nonmammalian vertebrates is unknown. To this extent, we used smooth muscle myography to investigate the effects of exogenously applied CO (delivered via a water-soluble CO-releasing molecule, CORM-3) on isolated lamprey (Petromyzon marinus) dorsal aortas and examined its mechanisms of action on trout (Oncorhynchus mykiss) efferent branchial (EBA) and celiacomesenteric (CMA) arteries. CORM-3 dose-dependently relaxed all vessels examined. Trout EBA were twofold more sensitive to CORM-3 when precontracted with norepinephrine (NE) than KC1 and CORM-3 relaxed five-fold more of the NE--than KC1-induced tension. Glybenclamide (10 [micro]M), an ATP-sensitive potassium channel inhibitor, inhibited NE-induced contraction, but did not affect CORM-3-induced relaxation. NS-2028 (10 [micro]M), a soluble guanylyl cyclase inhibitor, had no effect on a NE-contraction, but inhibited a subsequent CORM-3-induced relaxation. Zinc protopophyrin-IX (ZnPP-IX, 0.3-30 [micro]M), a HO inhibitor, elicited a small, yet dose-dependent and significant, increase in baseline tension but did not have any effect on subsequent NE-induced contractions or a nitric oxide-induced relaxation (via sodium nitroprusside). [ZnPP-IX] greater than 3 [micro]M, however, significantly reduced the predominant vasodilatory response of trout EBA to hydrogen sulfide. These results implicate an active HO/CO pathway in trout vessels having an impact on resting vessel tone and CO-induced vasoactivity that is at least partially mediated by soluble guanylyl cyclase. gasotransmitters; guanylyl cyclase; hydrogen sulfide; vasodilator

Endothelium-dependent vasorelaxation in large vessels is mainly attributed to [N.sup.[omega]]-nitro-L-arginine methyl ester (L-NAME)-sensitive endothelial nitric oxide (NO) synthase (eNOS)-derived NO production. Endothelium-derived hyperpolarizing factor (EDHF) is the component of endothelium-dependent relaxations that resists full blockade of NO synthases (NOS) and cyclooxygenases. [H.sub.2][O.sub.2] has been proposed as an EDHF in resistance vessels. In this work we propose that in mice aorta neuronal (n)NOS-derived [H.sub.2][O.sub.2] accounts for a large proportion of endothelium-dependent ACh-induced relaxation. In mice aorta rings, ACh-induced relaxation was inhibited by L-NAME and [N.sup.[omega]]nitro-L-arginine (L-NNA), two nonselective inhibitors of NOS, and attenuated by selective inhibition of nNOS with L-[Arg.sup.NO2]-L-Dbu-N[H.sub.2] 2TFA (L-[Arg.sup.NO2]-L-Dbu) and 1-(2-trifluoromethylphehyl)imidazole (TRIM). The relaxation induced by ACh was associated with enhanced [H.sub.2][O.sub.2] production in endothelial cells that was prevented by the addition of L-NAME, L-NNA, L-[Arg.sup.NO2]-L-Dbu, TRIM, and removal of the endothelium. The addition of catalase, an enzyme that degrades [H.sub.2][O.sub.2], reduced ACh-dependent relaxation and abolished ACh-induced [H.sub.2][O.sub.2] production. RT-PCR experiments showed the presence of mRNA for eNOS and nNOS but not inducible NOS in mice aorta. The constitutive expression of nNOS was confirmed by Western blot analysis in endothelium-containing vessels but not in endothelium-denuded vessels. Immunohistochemistry data confirmed the localization of nNOS in the vascular endothelium. Antisense knockdown of nNOS decreased both ACh-dependent relaxation and ACh-induced [H.sub.2][O.sub.2] production. Antisense knockdown of eNOS decreased ACh-induced relaxation but not [H.sub.2][O.sub.2] production. Residual relaxation in eNOS knockdown mouse aorta was further inhibited by the selective inhibition of nNOS with L-[Arg.sup.NO2]-L-Dbu. In conclusion, these results show that nNOS is constitutively expressed in the endothelium of mouse aorta and that nNOS-derived [H.sub.2][O.sub.2] is a major endothelium-dependent relaxing factor. Hence, in the mouse aorta, the effects of nonselective NOS inhibitors cannot be solely ascribed to NO release and action without considering the coparticipation of [H.sub.2][O.sub.2] in mediating vasodilatation. vasorelaxation; acetylcholine; antisense; knockdown

Impairment of flow-induced vasodilation in coronary resistance arterioles may contribute to the decline in coronary vasodilatory reserve that occurs with advancing age. This study investigated the effects of age on flow-induced signaling and activation of nitric oxide (NO)-mediated vasodilation in coronary resistance arterioles. Coronary arterioles were isolated from young (~6 mo) and old (~24 mo) male Fischer-344 rats to assess vasodilation to flow, vascular endothelial growth factor (VEGF), and ACh. Flow- and VEGF-induced vasodilation of coronary arterioles was impaired with age (P [less than or equal to] 0.05); however, ACh-induced vasodilation was preserved with age. [N.sup.G]-nitro-L-arginine methyl ester (L-NAME) (1 x [10.sup.-5] M) eliminated vasodilation to flow, VEGF, and ACh, indicating dependence of these responses on NO. SU-1498, an inhibitor of vascular endothelial growth factor receptor 2 (VEGFR, also known as Flk-1), abolished age-related differences in flow-induced vasodilation. Flow-stimulated phosphorylation of Flk-1 in coronary arterioles from young but not old rats and Flk-1 protein was reduced in coronary arterioles from old rats compared with those from young rats. Flow stimulated phosphorylation of endothelial nitric oxide synthase (eNOS) in coronary arterioles from both young and old rats. VEGF induced phosphorylation of both protein kinase B (Akt) and eNOS in coronary arterioles. VEGF-induced phosphorylation of Akt, but not eNOS, was significantly reduced in arterioles from old rats compared with arterioles from young rats. Wortmannin, an inhibitor of phosphatidylinositol (PI) 3-kinase, eliminated age-related differences in both flow- and VEGF-induced vasodilation. These results indicate that impairment of Flk-1/PI3-kinase signaling contributes to the reduction of flow-induced vasodilation in coronary arterioles with advancing age. phosphatidylinositol 3-kinase; endothelial nitric oxide synthase; acetylcholine; vascular endothelial growth factor; phosphorylated endothelial nitric oxide synthase; phophorylated protein kinase B; vasodilation

Diabetes and insulin resistance are associated with an increased risk of hypertension and cardiovascular disease. Recent evidence demonstrates that A[T.sub.2] receptors (AT2R) play an important role in the hemodynamic control of hypertension by vasodilation. The quantitative significance of AT2R in the establishment of diabetic vascular dysfunction, however, is not well defined and needs further investigation. Goto-Kakizaki (GK) rats, a polygenic model of spontaneous normotensive type 2 diabetes, were used to examine any abnormalities in cardiovascular function associated with AT2R at the early stage of the disease without endothelium influence. Using a myograph to measure the isometric force, we observed that ANG II-induced contraction was impaired in denuded GK aorta compared with control Wistar-Kyoto (WKY) aorta and exhibited a retarded AT1R antagonist response and enhanced Rho kinase signaling. When AT1R were blocked, ANG II induced a significant vasodilation of precontracted GK aorta via AT2R. The protein and mRNA of AT2R were increased in diabetic GK denuded aorta. Blocking AT2R restored the ANG II-induced contraction in the GK vasculature to control levels, demonstrating a counteractive role for AT2R in AT1R-induced contraction. Inhibition of inducible nitric oxide synthase (iNOS) by [N.sup.G]-monomethyl-L-arginine mimicked AT2R inhibition in denuded GK aorta, suggesting that AT2R-induced vasodilation was dependent on iNOS/NO generation. The protein and mRNA of iNOS were also increased in GK aorta. In conclusion, these results clearly demonstrate that enhanced AT2R and iNOS-induced, NO-mediated vasodilation impair ANG II-induced contraction in an endothelium-independent manner at the early stage of type 2 diabetes. angiotensin type 2 receptor; inducible nitric oxide synthase; vasodilation

Hyperhomocysteinemia (HHcy) impairs endothelium-dependent vasodilation by increasing reactive oxygen species, thereby reducing nitric oxide (NO*) bioavailability. It is unclear whether reduced expression or function of the enzyme that produces NO., endothelial nitric oxide synthase (eNOS), also contributes. It is also unclear whether resistance vessels that utilize both NO* and non-NO* vasodilatory mechanisms, undergo alteration of non-NO*mechanisms in this condition. We tested these hypotheses in male C57BL/6 mice with chronic HHcy induced by 6-wk high methionine/low-B vitamin feeding (Hey: 89.2 [+ or -] 49.0 [micro]M) compared with age-matched controls (Hey: 6.6 [+ or -] 1.9 [micro]M), using first-order mesenteric arteries. Dilation to ACh ([10.sup.-9]-[10.sup.-4] M) was measured in isolated, cannulated, and pressurized (75 mmHg) arteries with and without [N.sup.G]-nitro-L-arginine methyl ester (L-NAME) ([10.sup.-4] M) and/or indomethacin ([10.sup.-5] M) to test endothelium-dependent dilation and non-NO*-dependent dilation, respectively. The time course of dilation to ACh ([10.sup.-4] M) was examined to compare the initial transient dilation due to non-NO*, non-prostacyclin mechanism and the sustained dilation due to NO'. These experiments indicated that endothelium-dependent dilation was attenuated (P < 0.05) in HHcy arteries due to downregulation of only NO*-dependent dilation. Western blot analysis indicated significantly less (P < 0.05) basal eNOS and phospho-S1179-eNOS/eNOS in mesenteric arteries from HHcy mice but no difference in phospho-T495-eNOS/eNOS. S 1179 eNOS phosphorylation was also significantly less in these arteries when stimulated with ACh ex vivo or in situ. Real-time PCR indicated no difference in eNOS mRNA levels. In conclusion, chronic dietinduced HHcy in mice impairs eNOS protein expression and phos- phorylation at S 1179, coincident with impaired NO*-dependent dilation, which implicates dysfunction in eNOS post-transcriptional regulation in the impaired endothelium-dependent vasodilation and microvascular disease that is common with HHcy. homocysteine; endothelial nitric oxide synthase; S1179; vasodilation

Evidence from infusion studies suggests that soy isoflavones influence nitric oxide-dependent vasorelaxation. It is uncertain whether orally consumed isoflavones have similar effects. Healthy postmenopausal women (n = 22) consumed 2 low-fat test meals in random order 1 wk apart, with 80 mg isoflavones (ISO) or without isoflavones (CON). Endothelium-dependent vasodilation, assessed by brachial artery flow-mediated dilatation (FMD), was measured in fasting subjects, and 4 and 6 h following the test meal, in addition to blood pressure and pulse wave analysis to derive the peripheral augmentation index (pAIx). Blood samples were taken after fasting, and 5 and 7 h following the test meal for serum isoflavone, plasma 8-isoprostane [F.sub.2[alpha]], nitric oxide metabolites (NOx), glucose, and triacylglycerol analysis. Serum genistein and daidzein concentrations (geometric mean, 95% CI) reached 1.49 (1.20-1.84) [micro]mol/L and 0.95 (0.70-1.30)/[micro]mol/L, respectively, following ISO (7 h). FMD and plasma NOx concentrations were greater following ISO compared with CON, indicating better postprandial endothelial function. FMD values (%, mean [+ or -] SD) were: CON, 5.49 [+ or -] 2.32, 4.35 [+ or -] 2.32, 4.40 [+ or -] 2.26; ISO, 5.38 [+ or -] 1.91, 5.08 [+ or -] 1.74, 6.11 [+ or -] 2.60, at baseline, 4 h, and 6 h, respectively (P< 0.01). Plasma NQx concentrations ([micro]mol/L, mean [+ or -] SD) were: CON, 20.0 [+ or -] 5.1, 16.8 [+ or -] 5.1, 23.1 [+ or -] 6.0; ISO, 18.6 [+ or -] 6.3, 19.5 [+ or -] 5.1, 21.3 [+ or -] 10.1, at baseline, 5 h, and 7 h, respectively (P < 0.005). Treatment did not affect pAIx, blood pressure, or plasma 8-isoprostane [F.sub.2[alpha]] concentrations. In conclusion, consuming an isoflavone-enriched low- fat meal acutely increases endothelium-dependent vasodilation in postmenopausal women. Regular consumption of soy isoflavones may protect against endothelial dysfunction.

The coupling of hemoglobin sensing of physiological oxygen gradients to stimulation of nitric oxide (NO) bioactivity is an established principle of hypoxic blood flow. One mechanism proposed to explain this oxygen-sensing-NO bioactivity linkage postulates an essential role for the conserved Cys93 residue of the hemoglobin [beta]-chain ([beta]Cys93) and, specifically, for S-nitrosation of [beta]Cys93 to form S-nitrosohemoglobin (SNO-Hb) (1). The SNO-Hb hypothesis, which conceptually links hemoglobin and NO biology, has been debated intensely in recent years (2-3). This debate has precluded a consensus on physiological mechanisms and on assessment of the potential role of SNO-Hb in pathology. Here we describe new mouse models that exclusively express either human wild-type hemoglobin or human hemoglobin in which the [beta]Cys93 residue is replaced with alanine to assess the role of SNO-Hb in red blood cell-mediated hypoxic vasodilation. Substitution of this residue, precluding hemoglobin S-nitrosation, did not change total red blood cell S-nitrosothiol abundance but did shift S-nitrosothiol distribution to lower molecular weight species, consistent with the loss of SNO-Hb. Loss of [beta]Cys93 resulted in no deficits in systemic or pulmonary hemodynamics under basal conditions and, notably, did not affect isolated red blood cell-dependent hypoxic vasodilation. These results demonstrate that SNO-Hb is not essential for the physiologic coupling of erythrocyte deoxygenation with increased NO bioactivity in vivo., In addition to hemoglobin oxygen affinity, blood flow is a key component of the processes that match oxygen delivery to demand. Increased blood flow in response to hypoxia is an [...]

We tested the hypothesis that aging is associated with an impaired contraction-induced rapid vasodilation in healthy adults. We reasoned that employing single contractions of a small muscle mass would allow us to isolate the local rapid vasodilatory responses independent of systemic hemodynamic and sympathetic neural influences on forearm hemodynamics. We measured forearm blood flow (Doppler ultrasound) and arterial blood pressure (Finapres) on a beat-by-beat basis and calculated the changes in forearm vascular conductance ([DELTA]FVC) in response to forearm contractions in 18 young (24 [+ or -] 1 yr) and 13 older (62 [+ or -] 2 yr) healthy subjects. Single, 1-s dynamic forearm contractions were performed with the experimental arm slightly above heart level at 5, 10, 20, and 40% of the subjects' maximal voluntary contraction (MVC) in random order. In general, muscle contractions evoked a rapid increase in FVC that reached a peak within approximately four to five cardiac cycles postcontraction in both age groups. At 5% MVC, there were no significant age-related differences in contraction-induced forearm vasodilation. However, the peak vasodilatory responses were impaired ~40-45% in older adults at 10, 20, and 40% MVC, as were the total vasodilatory responses (area under curve ~40-50%; all P < 0.05). Additionally, the immediate vasodilation (first cardiac cycle postcontraction) for the 20% and 40% MVC trials was also impaired ~50% with age (P < 0.05). There were no significant age-group differences in MVC or forearm fat-free mass, and these variables were not correlated with local vasodilation within a given exercise intensity. Under the experimental conditions employed, the blunted responses with age reflect impaired local contraction-induced rapid vasodilation. vascular control; exercise; blood flow

In septic shock, systemic vasodilation and myocardial depression contribute to the systemic hypotension observed. Both components can be attributed to the effects of mediators that are released as part of the inflammatory response. We previously found that lysozyme (Lzm-S), released from leukocytes, contributed to the myocardial depression that develops in a canine model of septic shock. Lzm-S binds to the endocardial endothelium, resulting in the production of nitric oxide (NO), which, in turn, activates the myocardial soluble guanylate cyclase (sGC) pathway. In the present study, we determined whether Lzm-S might also play a role in the systemic vasodilation that occurs in septic shock. In a phenylephrine-contracted canine carotid artery ring preparation, we found that both canine and human Lzm-S, at concentrations similar to those found in sepsis, produced vasorelaxation. This decrease in force could not be prevented by inhibitors of NO synthase, prostaglandin synthesis, or potassium channel inhibitors and was not dependent on the presence of the vascular endothelium. However, inhibitors of the sGC pathway prevented the vasodilatory activity of Lzm-S. In addition, Aspergillus niger catalase, which breaks down [H.sub.2][O.sub.2], as well as hydroxyl radical scavengers, which included hydroquinone and mannitol, prevented the effect of Lzm-S. Electrochemical sensors corroborated that Lzm-S caused [H.sub.2][O.sub.2] release from the carotid artery preparation. In conclusion, these results support the notion that when Lzm-S interacts with the arterial vasculature, this interaction results in the formation of [H.sub.2][O.sub.2], which, in turn, activates the sGC pathway to cause relaxation. Lzm-S may contribute to the vasodilation that occurs in septic shock. reactive oxygen species; catalase; compound I; hydroxyl radical; septic shock; hypotension

Muscarinic receptor agonists have primarily been used to characterize endothelium-dependent vasodilator dysfunction with overweight/obesity. Reliance on a single class of agonist, however, yields limited, and potentially misleading, information regarding endothelial vasodilator capacity. The aims of this study were to determine 1) whether the overweight/obesity-related reduction in endothelium-dependent vasodilation extends beyond muscarinic receptor agonists and 2) whether the contribution of nitric oxide (NO) to endothelium-dependent vasodilation is reduced in overweight/obese adults. Eighty-six middle-aged and older adults were studied: 42 normal-weight (54 [+ or -] 1 yr, 21 men and 21 women, body mass index = 23.4 [+ or -] 0.3 kg/[m.sup.2]) and 44 overweight/obese (54 [+ or -] 1 yr, 28 men and 16 women, body mass index = 30.3 [+ or -] 0.6 kg/[m.sup.2]) subjects. Forearm blood flow (FBF) responses to intra-arterial infusions of acetylcholine in the absence and presence of the endothelial NO synthase inhibitor [N.sup.G]-monomethyl-L-arginine, methacholine, bradykinin, substance P, isoproterenol, and sodium nitropmsside were measured by strain-gauge plethysmograplay. FBF responses to each endothelial agonist were significantly blunted in the overweight/obese adults. Total FBF (area under the curve) to acetylcholine (50 [+ or -] 5 vs. 79 [+ or -] 4 ml/100 ml tissue), methacholine (55 [+ or -] 4 vs. 86 [+ or -] 5 ml/100 ml tissue), bradykinin (62 [+ or -] 5 vs. 85 [+ or -] 4 ml/100 ml tissue), substance P (37 [+ or -] 4 vs. 57 [+ or -] 5 ml/100 ml tissue), and isoproterenol (62 [+ or -] 4 vs. 82 [+ or -] 6 ml/100 ml tissue) were 30%-40% lower in the overweight/obese than normal-weight adults. [N.sup.G]-monomethyl-L-arginine significantly reduced the FBF response to acetylcholine to the same extent in both groups. There were no differences between the groups in the FBF responses to sodium nitroprusside. These results indicate that agonist-stimulated endothelium-dependent vasodilation is universally impaired with overweight/obesity. Moreover, this impairment appears to be independent of NO. cell surface receptors; intracellular signaling pathways; nitric oxide

The activation of initiator protein tissue factor (TF) is likely to be a crucial step in the blood coagulation process, which leads to fibrin formation. The stimuli responsible for inducing TF activation are largely undefined. Here we show that the oxidoreductase protein disulfide isomerase (PDI) directly promotes TF-dependent fibrin production during thrombus formation in vivo. After endothelial denudation of mouse carotid arteries, PDI was released at the injury site from adherent platelets and disrupted vessel wall cells. Inhibition of PDI decreased TF-triggered fibrin formation in different in vivo murine models of thrombus formation, as determined by intravital fluorescence microscopy. PDI infusion increased--and, under conditions of decreased platelet adhesion, PDI inhibition reduced--fibrin generation at the injury site, indicating that PDI can directly initiate blood coagulation. In vitro, human platelet--secreted PDI contributed to the activation of cryptic TF on microvesicles (microparticles). Mass spectrometry analyses indicated that part of the extracellular cysteine 209 of TF was constitutively glutathionylated. Mixed disulfide formation contributed to maintaining TF in a state of low functionality. We propose that reduced PDI activates TF by isomerization of a mixed disulfide and a free thiol to an intramolecular disulfide. Our findings suggest that disulfide isomerases can act as injury response signals that trigger the activation of fibrin formation following vessel injury., Introduction Fibrin formation represents a major defense and repair mechanism, which ensures the integrity of the vascular system (1-3). Together with platelets, which adhere and aggregate at sites of vessel [...]

In humans, endothelial vasodilator function serves as a surrogate marker for cardiovascular health and is measured as changes in conduit artery diameter after temporary ischemia [flow-mediated dilation (FMD)]. Here we present an FMD-related approach to study femoral artery (FA) vasodilation in anesthetized rats. Diameter and Doppler flow were monitored in the FA. Using high-resolution ultrasound (35 MHz) and automated analysis software, we detected dose-dependent vasodilation using established endothelium-independent [intravenous nitroglycerin E[C.sub.50] = 3.3 x [10.sup.-6] mol/l, peak 21[DELTA]% (SD 4)] and endothelium-dependent [intra-arterial acetylcholine E[C.sub.50] = 1.3 x [10.sup.-6] mol/l, peak 27[DELTA]% (SD 4)] pharmacological vasodilators. Wall shear stress induced by intra-aortic injection of adenosine and infusion of saline at increasing rates (1.5-4.5 ml/min) led to vasodilation at 1 to 2 min. Transient hindlimb ischemia by common iliac occlusion (5 min) led to reactive hyperemia with flow velocity and wall shear stress increase and was followed by FA dilation [16A% (SD 2)], the latter of which was completely abolished by nitric oxide synthase (NOS) inhibition with [N.sup.G]-monomethyl-L-arginine [1[DELTA]% (SD 2)]. FMD was significantly reduced in adult 20-24-wk-old animals compared with 9- to 10-wk-old animals, consistent with age-dependent endothelial dysfunction [16[DELTA]% (SD 3) vs. 10[DELTA]% (SD 3), P < 0.05]. Whereas FMD was completely NOS dependent in 9- to 10-wk-old animals, NOS-dependent mechanisms accounted for only half of the FMD in 20-24-wk-old animals, with the remainder being blocked by charybdotoxin and apamin, suggesting a contribution of endothelium-derived hyperpolarizing factor. To our knowledge, this is the first integrative physiological model to reproducibly study FMD of conduit arteries in living rats. nitric oxide synthase; endothelium-derived hyperpolarizing factor; rodents; age-dependent endothelial dysfunction

Nitric oxide (NO), generated from L-arginine by endothelial nitric oxide synthase (eNOS), is a key endothelial-derived factor whose bioavailability is essential to the normal function of the endothelium. Endothelium dysfunction is characterized by loss of NO bioavailability because of either reduced formation or accelerated degradation of NO. We have recently reported that overexpression of vascular cytochrome P-450 (CYP) 4A in rats caused hypertension and endothelial dysfunction driven by increased production of 20-hydroxyeicosatetraenoic acid (20-HETE), a major vasoconstrictor eicosanoid in the microcirculation. To further explore cellular mechanisms underlying CYP4A-20HETE-driven endothelial dysfunction, the interactions between 20HETE and the eNOS-NO system were examined in vitro. Addition of 20-HETE to endothelial cells at concentrations as low as 1 nM reduced calcium ionophore-stimulated NO release by 50%. This reduction was associated with a significant increase in superoxide production. The increase in superoxide in response to 20-HETE was prevented by [N.sup.G]-nitro-L-arginine methyl ester, suggesting that uncoupled eNOS is a source of this superoxide. The response to 20-HETE was specific in that 19-HETE did not affect NO or superoxide production, and, in fact, the response to 20-HETE could be competitively antagonized by 19(R)-HETE. 20-HETE had no effect on phosphorylation of eNOS protein at serine-1179 or threonine-497 following addition of calcium ionophore; however, 20-HETE inhibited association of eNOS with 90-kDa heat shock protein (HSP90). In vivo, impaired acetylcholine-induced relaxation in arteries overexpressing CYP4A was associated with a marked reduction in the levels of phosphorylated vasodilator-stimulated phosphoprotein, an indicator of bioactive NO, that was reversed by inhibition of 20-HETE synthesis or action. Because association of HSP90 with eNOS is critical for eNOS activation and coupled enzyme activity, inhibition of this association by 20-HETE may underlie the mechanism, at least in part, by which increased CYP4A expression and activity cause endothelial dysfunction. nitric oxide; vascular endothelium; vasodilator-stimulated phosphoprotein; cytochrome P-450; superoxide; endothelial nitric oxide synthase

The migration of vascular endothelial cells under flow can be modulated by the addition of chemical or mechanical stimuli. The aim of this study was to investigate how topographic cues derived from a substrate containing three-dimensional microtopography interact with fluid shear stress in directing endothelial cell migration. Subconfluent bovine aortic endothelial cells were seeded on fibronectin-coated poly(dimethylsiloxane) substrates patterned with a combinatorial array of parallel and orthogonal microgrooves ranging from 2 to 5 [micro]m in width at a constant depth of 1 [micro]m. During a 4-h time-lapse observation in the absence of flow, the majority of the prealigned cells migrated parallel to the grooves with the distribution of their focal adhesions (FAs) depending on the groove width. No change in this migratory pattern was observed after the cells were exposed to moderate shear stress (13.5 dyn/[cm.sup.2]), irrespective of groove direction with respect to flow. After 4-h exposure to high shear stress (58 dyn/ [cm.sup.2]) parallel to the grooves, the cells continued to migrate in the direction of both grooves and flow. By contrast, when microgrooves were oriented perpendicular to flow, most cells migrated orthogonal to the grooves and downstream with flow. Despite the change in the migration direction of the cells under high shear stress, most FAs and actin microfilaments maintained their original alignment parallel to the grooves, suggesting that topographic cues were more effective than those derived from shear stress in guiding the orientation of cytoskeletal and adhesion proteins during the initial exposure to flow. endothelial cell alignment; shear stress; focal adhesion

Rabbit 15-1ipoxygenase- 1 (15-LO-1) oxygenates arachidonic acid (AA) into 15-hydroperoxyeicosatetraenoic acid, which is then converted to the vasodilatory 15-hydroxy-11,12-epoxyeicosatrienoic acid (HEETA) and 11,12,15-trihydroxyeicosatrienoic acid (THETA). We studied the age-dependent expression of the 15-LO-1 in rabbit aorta and its effects on the synthesis of THETA, HEETA, and vasoactivity. Aortas of 1-wk-old rabbits express greater amounts of 15-LO-1 mRNA and protein compared with aortas of 4-, 8-, or 16-wk-old rabbits. The synthesis of THETA and HEETA in the rabbit aorta was also reduced with age. THETA synthesis was maximal in 1-wk-old aortas but decreased in aortas of 4- (42%), 8- (4%), and 16-wk-old (1%) rabbits. Similarly, THETA and HEETA synthesis decreased with age in mesenteric arteries from 1-, 4-, 8-, and 16-wk-old rabbits. The maximum vasorelaxation response to acetylcholine ([10.sup.-6] M) in the presence of indomethacin and nitro-L-arginine decreased in the order of 1 wk (64.5 [+ or -] 6.9%), 4 wk (52.6 [+ or -] 8.9%), 8 wk (53.0 [+ or -] 9.4%), and 16 wk (33.3 [+ or -] 6.6%). Similarly, the maximum relaxation to AA (3 x [10.sup.-4] M) decreased with age in the order of 1 wk (60.4 [+ or -] 8.9%), 4 wk (56.3 [+ or -] 5.8%), 8 wk (41.8 [+ or -] 12.3%), and 16 wk (28.9 [+ or -] 1.6%). In contrast, the vasorelaxation to sodium nitroprusside was not significantly altered by age. These data indicate that aortic 15-LO-1 expression and activity are downregulated with aging in rabbits. This decrease is paralleled by the reduced synthesis of vasoactive THETA and HEETA and aortic relaxations to acetylcholine and AA. endothelium-derived hyperpolarizing factor; arachidonic acid; acetylcholine; vasodilation

The mechanism by which acetylcholine (ACh) decreases systemic arterial pressure and hindlimb vascular resistance was investigated in the anesthetized rat. ACh injections caused dose-dependent decreases in systemic arterial pressure and hindlimb vascular resistance. [N.sup.[omega]]-nitro-L-arginine methyl ester (L-NAME) had little effect on the magnitude of depressor and vasodilator responses but decreased response duration when baseline parameters were corrected by a nitric oxide (NO) donor infusion. The decrease in the duration of the ACh depressor response was prevented by the administration of excess L-arginine. The L-NAME-resistant component of the depressor response to ACh was attenuated by ebselen, a glutathione peroxidase mimic. The calcium-activated potassium ([K.sub.Ca]) antagonists charybdotoxin (ChTX) and apamin decreased the magnitude but not the duration of the hindlimb vasodilator response to ACh. The combination of L-NAME, ChTX, and apamin reduced the magnitude and duration of the vasodilator response to ACh but not to sodium nitroprusside. Vasodepressor and hindlimb vasodilator responses to ACh were not modified by cytochrome P-450 and cyclooxygenase pathway inhibitors. These results suggest that the hindlimb vasodilator response to ACh has an initial L-NAME-resistant component mediated by the activation of [K.sub.Ca] channels and a sustained L-NAME-dependent component. The results with ebselen suggest that the L-NAME-resistant component of the depressor response involves a peroxide-sensitive mechanism. The present study suggests that vasodilator responses to ACh are not mediated by cytochrome P-450 products, since miconazole and 1-aminobentriazole alone or in combination did not affect either component of the response. The present data suggest that the hindlimb vasodilator response to ACh in the rat is mediated by two mechanisms with an initial ChTX- and apaminsensitive, L-NAME-resistant phase not mediated by cytochrome P-450 products and a secondary sustained phase mediated by NO. endothelium-dependent response; decrease in arterial pressure; hindlimb vasodilator response; calcium-activated potassium channels; cytochrome P-450

Astrocytes play an important role in the coupling between neuronal activity and brain blood flow via their capacity to 'sense' neuronal activity and transmit that information to parenchymal arterioles. Here we show another role for astrocytes in neurovascular coupling: the ability to act as a signaling conduit for the vitally important process of upstream vasodilation (represented by pial arterioles) during both excessive (seizure) and physiological (sciatic nerve stimulation) increases in cerebral cortical neuronal activity. The predominance of an astrocytic rather than a vascular route was indicated by data showing that pial arteriolar-dilating responses to neuronal activation were completely blocked following selective disruption of the superficial glia limitans, whereas interference with interendothelial signaling was without effect. Results also revealed contributions from connexin 43, implying a role for gap junctions and/or hemichannels in the signaling process and that signaling from the glia limitans to pial arterioles may involve a diffusible mediator. bicuculline; connexin; gap junction; glia limitans; L-[alpha]-aminoadipic acid; sciatic nerve stimulation

We have recently demonstrated that endothelium-derived hydrogen peroxide ([H.sub.2][O.sub.2]) is an endothelium-derived hyperpolarizing factor and that endothelial Cu/Zn-superoxide dismutase (SOD) plays an important role in the synthesis of endogenous [H.sub.2][O.sub.2] in both animals and humans. We examined whether SOD plays a role in the synthesis of endogenous [H.sub.2][O.sub.2] during in vivo reactive hyperemia (RH), an important regulatory mechanism. Mesenteric arterioles from wild-type and Cu,Zn-[SOD.sup.-/-]/mice were continuously observed by a pencil-type charge-coupled device (CCD) intravital microscope during RH (reperfusion after 20 and 60 s of mesenteric artery occlusion) in the cyclooxygenase blockade under the following four conditions: control, catalase alone, [N.sup.G]-monomethyl-L-arginine (L-NMMA) alone, and L-NMMA + catalase. Vasodilatation during RH was significantly decreased by catalase or L-NMMA alone and was almost completely inhibited by L-NMMA + catalase in wild-type mice, whereas it was inhibited by L-NMMA and L-NMMA + catalase in the Cu,Zn-[SOD.sup.-/-] mice. RH-induced increase in blood flow after L-NMMA was significantly increased in the wild-type mice, whereas it was significantly reduced in the Cu,Zn-[SOD.sup.-/-] mice. In mesenteric arterioles of the Cu,Zn-[SOD.sup.-/-] mice, Tempol, an SOD mimetic, significantly increased the ACh-induced vasodilatation, and the enhancing effect of Tempol was decreased by catalase. Vascular [H.sub.2][O.sub.2] production by fluorescent microscopy in mesenteric arterioles after RH was significantly increased in response to ACh in wild-type mice but markedly impaired in Cu,Zn-[SOD.sup.-/-] mice. Endothelial Cu,Zn-SOD plays an important role in the synthesis of endogenous [H.sub.2][O.sub.2] that contributes to RH in mouse mesenteric smaller arterioles. nitric oxide; endothelium-derived hyperpolarizing factor; arteriole; vasodilatation

Cholesterol is a primary constituent of the plasmalemma, including the lipid rafts/caveolae, where various G protein-coupled receptors colocalize with signaling proteins and channels. By manipulating cholesterol in rabbit and rat ventricular myocytes using methyl-[beta]-cyclodextrin (M[beta]CD), we studied the role of cholesterol in the modulation of L-type [Ca.sup.2+] currents ([I.sub.Ca,L]). M[beta]CD was mainly dialyzed from BAPTA-containing pipette solution during whole cell clamp. In rabbit myocytes dialyzed with 30 mM M[beta]CD for 10 min, a positive shift in membrane potential at half-maximal activation ([V.sub.0.5]) from -8 to -2 mV developed and was associated with an increase in current density at positive potentials (42% at +20 mV vs. time-matched controls). Isoproterenol (ISO) increased [I.sub.Ca,L] approximately threefold and caused a negative shift in [V.sub.0.5] in control cells, but it did not increase [I.sub.Ca,L]. in M[beta]CD-treated myocytes, nor did it shift [V.sub.0.5]. The effect of M[beta]CD (10 or 30 mM) was concentration dependent: 30 mM M[beta]CD suppressed the ISO-induced increase in [I.sub.Ca,L] more effectively than 10 mM M[beta]CD. M[beta]CD dialysis also abolished the increase in [I.sub.Ca,L] elicited by forskolin or dibutyryl cAMP, but not that elicited by (-)BAY K 8644. External application of M[beta]CD-cholesterol complex to rat myocytes attenuated the M[beta]CD-mediated inhibition of the ISO-induced increase of [I.sub.Ca,L]. Biochemical analysis confirmed that the myocytes' cholesterol content was diminished by M[beta]CD and increased by M[beta]CD-cholesterol complex. Cholesterol thus appears to contribute to the regulation of basal [I.sub.Ca,L] and [beta]-adrenergic cAMP/ PKA-mediated increases in [I.sub.Ca,L]. We suggest that cholesterol affects the structural coupling between L-type [Ca.sup.2+] channels and adjacent regulatory proteins. lipid raft; adenosine 3',5'-cyclic monophosphate; protein kinase A; phosphorylation

The mechanism of sensing hypoxia and hypoxia-induced activation of cerebral arterial [Ca.sup.2+]-activated [K.sup.+] (Kca) channel currents and vasodilation is not known. We investigated the roles of the cytochrome P-450 4A (CYP 4A) [omega]-hydroxylase metabolite of arachidonic acid, 20-hydroxyeicosatetraenoic acid (20-HETE), and generation of superoxide in the hypoxia-evoked activation of the [K.sub.Ca] channel current in rat cerebral arterial muscle cells (CAMCs) and cerebral vasodilation. Patch-clamp analysis of [K.sup.+] channel current identified a voltage- and [Ca.sup.2+]-dependent 238 [+ or -] 21-pS unitary [K.sup.+] currents that are inhibitable by tetraethylammonium (TEA, 1 mM) or iberiotoxin (100 nM). Hypoxia ( cytochrome P-450 4A [omega]-hydroxylase; superoxide; vasodilation; patch-clamp recording

Levosimendan enhances cardiac contractility via [Ca.sup.2+] sensitization and induces vasodilation through the activation of ATP-dependent [K.sup.+] and large-conductance [Ca.sup.2+]-dependent [K.sup.+] channels. However, the hemodynamic effects of levosimendan, as well as its metabolites, OR-1896 and OR-1855, relative to plasma concentrations achieved, are not well defined. Thus levosimendan, OR-1896, OR-1855, or vehicle was infused at 0.01, 0.03, 0.1, and 0.3 [micro]mol x [kg.sup.-1] x 30 [min.sup.-1], targeting therapeutic to supratherapeutic concentrations of total levosimendan (62.6 ng/ml). Results were compared with those of the [[beta].sub.1]-agonist dobutamine and the phosphodiesterase 3 inhibitor milrinone. Peak concentrations of levosimendan, OR-1896, and OR-1855 were 455 [+ or -] 21, 126 [+ or -] 6, and 136 [+ or -] 6 ng/ml, respectively. Levosimendan and OR-1896 produced dose-dependent reductions in mean arterial pressure (-31 [+ or -] 2 and -42 [+ or -] 3 mmHg, respectively) and systemic resistance without affecting pulse pressure, effects paralleled by increases in heart rate; OR-1855 produced no effect at any dose tested. Dobutamine, but not milrinone, increased mean arterial pressure and pulse pressure (17 [+ or -] 2 and 23 [+ or -] 2 mmHg, respectively). Regarding potency to elicit reductions in time to peak pressure and time to systolic pressure recovery: OR-1896 > levosimendan > milrinone > dobutamine. Levosimendan and OR-1896 elicited dose-dependent increases in change in pressure over time (118 [+ or -] 10 and 133 [+ or -] 13%, respectively), concomitant with reductions in left ventricular end-diastolic pressure and ejection time. However, neither levosimendan nor OR-1896 produced increases in myocardial oxygen consumption at inotropic and vasodilatory concentrations, whereas dobutamine increased myocardial oxygen consumption (79% above baseline). Effects of the levosimendan and OR-1896 were limited to the systemic circulation; neither compound produced changes in pulmonary pressure, whereas dobutamine produced profound increases (74 [+ or -] 13%). Thus levosimendan and OR-1896 are hemodynamically active in the anesthetized dog at concentrations observed clinically and elicit cardiovascular effects consistent with activation of both [K.sup.+] channels and [Ca.sup.2+] sensitization, whereas OR-1855 is inactive on endpoints measured in this study.

Intact female rats fed a high-phytoestrogen diet are protected against adverse left ventricular (LV) remodeling induced by chronic volume overload. We hypothesized that both phytoestrogens and ovarian hormones, particularly estrogen, are necessary for this dietary-induced cardioprotection. To test this hypothesis, eight groups of female rats were studied; rats were fed either a high-phytoestrogen (+phyto) or phytoestrogen-free diet. Groups included sham-operated rats, intact rats with fistula (Fist), ovariectomized rats with fistula (Fist-OX), and Fist-OX rats treated with estrogen (EST). Myocardial function and remodeling were assessed after 8 wk of volume overload using a blood-perfused isolated heart apparatus. Fist-OX rats developed significant ventricular dilatation and increased compliance vs. intact Fist rats, which were associated with a significant decrease in contractility. Estrogen treatment prevented pulmonary edema and attenuated LV hypertrophy and dilatation but did not maintain contractility. However, dietary phytoestrogens completely prevented LV dilatation in both the Fist + phyto and Fist-OX + EST + phyto groups but had no effect on LV remodeling in the Fist-OX + phyto group. Contractility was significantly greater in the estrogen-treated rats fed the phytoestrogen diet than in those treated with estrogen alone. Dietary phytoestrogens did not affect LV or uterine mass, serum estrogen, LV estrogen receptor expression, or cardiac function in sham animals. These data indicate that estrogen is not solely responsible for the cardioprotection exhibited by intact females and that phytoestrogens can work synergistically with ovarian hormones to attenuate ventricular remodeling induced by chronic volume overload in female rats. hormones; heart failure; remodeling; diet; physiology; hypertrophy; fistula

Previously we showed that pressor and differential regional sympathoexcitatory responses (adrenal > renal [greater than or equal to] lumbar) evoked by stimulation of [A.sub.1] adenosine receptors located in the nucleus of the solitary tract (NTS) were attenuated/abolished by baroreceptor denervation or blockade of glutamatergic transmission in the NTS, suggesting [A.sub.1] receptor-elicited inhibition of glutamatergic transmission in baroreflex pathways. Therefore we tested the hypothesis that stimulation of NTS [A.sub.1] adenosine receptors differentially inhibits/resets baroreflex responses of preganglionic adrenal (pre-ASNA), renal (RSNA), and lumbar (LSNA) sympathetic nerve activity. In urethane-chloralose-anesthetized male Sprague-Dawley rats (n = 65) we compared baroreflex-response curves (iv nitroprusside and phenylephrine) evoked before and after bilateral microinjections into the NTS of [A.sub.1] adenosine receptor agonist ([N.sup.6]-cyclopentyladenosine, CPA; 0.033-330 pmol/50 nl). CPA evoked typical dose-dependent pressor and differential sympathoexcitatory responses and similarly shifted baroreflex curves for pre-ASNA, RSNA, and LSNA toward higher mean arterial pressure (MAP) in a dose-dependent manner; the maximal shifts were 52.6 [+ or -] 2.8, 48.0 [+ or -] 3.6, and 56.8 [+ or -] 6.7 mmHg for pre-ASNA, RSNA, and LSNA, respectively. These shifts were not a result of simple baroreceptor resetting because they were two to three times greater than respective increases in baseline MAP evoked by CPA. Baroreflex curves for pre-ASNA were additionally shifted upward: the maximal increases of upper and lower plateaus were 41.8 [+ or -] 16.4% and 45.3 [+ or -] 8.7%, respectively. Maximal gain (%/mmHg) measured before vs. after CPA increased for pre-ASNA (3.0 [+ or -] 0.6 vs. 4.9 [+ or -] 1.3), decreased for RSNA (4.1 [+ or -] 0.6 vs. 2.3 [+ or -] 0.3), and remained unaltered for LSNA (2.1 [+ or -] 0.2 vs. 2.0 [+ or -] 0.1). Vehicle control did not alter the baroreflex curves. We conclude that the activation of NTS [A.sub.1] adenosine receptors differentially inhibits/resets baroreflex control of regional sympathetic outputs. nucleus of the solitary tract; purinergic receptors; adrenal nerve; renal nerve; lumbar nerve; pressor reflex

Byline: Olympia. Rudra, Arghyaprasun. Ghosh, Sudip. Ghosh, Deblina. Bhunia, Prabhakar. Mandal Sir, An 8-year-old boy presented to us with a raised asymptomatic cutaneous lesion over his left leg for the [...]

Various studies provide insights into developmental processes and molecular mechanisms controlling the formation and remodelling of the lymphatic vessels. The current understanding of the mechanisms of lymphatic development and contribution of lymphatic vessels to pathological conditions are reviewed and the essential and active role of lymphatic vessels in various pathological conditions is demonstrated.

We tested the hypotheses that the NO-cGMP-PKG pathway mediates inhibition of the store-operated cation channel (SOC) in human glomerular mesangial cells (HMC) and that TRPC4, a molecular component of SOC in HMC, is associated with PKG-phosphorylated vasodilator-stimulated phosphoprotein (VASP). Using fura 2 ratiometry, we measured intracellular [Ca.sup.2+] concentration [[[Ca.sup.2+]].sub.i] to determine whether sodium nitroprusside (SNP), an NO donor, and 8-Br-cGMP affected SOC-TRPC4 via PKG. We found that the SOC response in HMC was attenuated in the presence of 100 [micro]M SNP, an NO donor, or 100 [micro]M 8-Br-cGMP. Addition of DT-3 (2.5 [micro]M), a specific PKG-1[alpha] inhibitor, reversed the effects of 8-Br-cGMP on the SOC response. Application of 100 [micro]M cAMP did not significantly inhibit the SOC response. RT-PCR and Western blotting revealed PKG-1[alpha] transcript and protein in HMC. Immunocytochemical analysis localized PKG-1[alpha] to the cytoplasm and plasma membrane of HMC. Previous studies have shown that PKG-mediated phosphorylation of VASP attenuates cellular [Ca.sup.2+] entry, resulting in altered growth and proliferation. Therefore, we used Western blotting and immunocytochemistry to determine whether PKG-phosphorylated VASP associates with TRPC4. Western blot analysis revealed that 8-Br-cGMP enhanced the phosphorylation of VASP at serine 239 (Ser239), a known PKG phosphorylation site, in HMC within 5 min. Coimmunoprecipitation and coimmunostaining showed that P-Ser239-VASP associated with TRPC4. However, VASP that was unphosphorylated at Ser239 was not associated with TRPC4. These results indicate that VASP has a role in the NO/PKG1[alpha]-mediated inhibition of the TRPC4-SOC response in HMC. cGMP; SOC; nitric oxide; SNP

The extracellular matrix (ECM) plays a critical role in angiogenesis by providing biochemical and positional cues, as well as by mechanically influencing microvessel cell behavior. Considerable information is known concerning the biochemical cues relevant to angiogenesis, but less is known about the mechanical dynamics during active angiogenesis. The objective of this study was to characterize changes in the material properties of a simple angiogenic tissue before and during angiogenesis, During sprouting, there was an overall decrease in tissue stiffness followed by an increase during neovessel elongation. The fall in matrix stiffness coincided with peak matrix metalloproteinase mRNA expression and elevated proteolytic activity. An elevated expression of genes for ECM components and cell-ECM interaction molecules and a subsequent drop in proteolytic activity (although enzyme levels remained elevated) coincided with the subsequent stiffening. The results of this study show that the mechanical properties of a scaffold tissue may be actively modified during angiogenesis by the growing microvasculature, biomechanics; matrix metalloprotease

Vascular oscillation (vasomotion) occurs in the microcirculation and is thought to be a significant contributor to tissue perfusion. Our aims were to assess the relationship of vasomotion to perfusion in the cutaneous microcirculation of diabetic patients, to determine the influence on it of endothelium-dependent and nonendothelium-dependent vasodilatory stimuli, and to assess the relationship to perfusion and vasomotion of various biochemical markers of vascular function (HbA1c, LDL- and HDL-cholesterol, triglycerides, insulin resistance, high sensitive C-reactive protein, L- and E-selectin, soluble ICAM, von Willebrand factor) and microalbuminuria. Perfusion and vasomotion (spectral density at low and very low frequencies) were measured by laser-Doppler flowmetry after local heat and iontophoresis of ACh and sodium nitroprusside. Perfusion responses to all stimuli were impaired in patients with Type 2 diabetes (heat: F = 28.0, P < 0.001; ACh: F = 7.11, P = 0.003; sodium nitroprusside: F = 4.0, P = 0.028). Responses to endothelium-dependent stimuli were further impaired in microalbuminuric patients (heat: P = 0.035; ACh: P = 0.034). Vasomotion responses at low frequencies after endothelium-dependent stimuli were impaired in diabetic patients compared with that shown in controls (heat: F = 5.62, P = 0.002; ACh: F = 4.32, P = 0.015). Multivariate modeling showed microalbuminuria to be the only consistent predictor of perfusion and vasomotion responses. The results suggest that microalbuminuria in Type 2 diabetes reflects a generalized disturbance of microvascular function related to endothelium-dependent mechanisms. diabetes mellitus; laser-Doppler flowmetry; microcirculation; physiopathology

The stress and strain in the vessel wall are important determinants of vascular physiology and pathophysiology. Vessels are constrained radially by the surrounding tissue. The hypothesis in this work is that the surrounding tissue takes up a considerable portion of the intravascular pressure and significantly reduces the wall strain and stress. Ten swine of either sex were used to test this hypothesis. An impedance catheter was inserted into the carotid or femoral artery, and after mechanical preconditioning pressure-cross-sectional area relations were obtained with the surrounding tissue intact and dissected away (untethered), respectively. The radial constraint of the surrounding tissue was quantified as an effective perivascular pressure on the outer surface of the vessel, which was estimated as 50% or more of the intravascular pressure. For carotid arteries at pressure of 100 mmHg, the circumferential wall stretch ratio in the intact state was ~20% lower than in the untethered state and the average circumferential stress was reduced by ~70%. For femoral arteries, the reductions were ~15% and 70%, respectively. These experimental data support the proposed hypothesis and suggest that in vitro and in vivo measurements of the mechanical properties of vessels must be interpreted with consideration of the constraint of the surrounding tissue. stress analysis; strain; impedance planimetry; carotid artery; femoral artery

The consumption of garlic is inversely correlated with the progression of cardiovascular disease, although the responsible mechanisms remain unclear. Here we show that human RBCs convert garlic-derived organic polysulfides into hydrogen sulfide ([H.sub.2]S), an endogenous cardioprotective vascular cell signaling molecule. This [H.sub.2]S production, measured in real time by a novel polarographic [H.sub.2]S sensor, is supported by glucose-Maintained cytosolic glutathione levels and is to a large extent reliant on reduced thiols in or on the RBC membrane. [H.sub.2]S production from organic polysulfides is facilitated by allyl substituents and by increasing numbers of tethering sulfur atoms. Ailyl-substituted polysulfides undergo nucleophilic substitution at the [alpha] carbon of the allyl substituent, thereby forming a hydropolysulfide (R[S.sub.n]H), a key intermediate during the formation of [H.sub.2]S. Organic polysulfides (R-[S.sub.n]-R'; n > 2) also undergo nucleophilic substitution at a sulfur atom, yielding R[S.sub.n]H and [H.sub.2]S. Intact aorta rings, under physiologically relevant oxygen levels, also metabolize garlic-derived organic polysulfides to liberate [H.sub.2]S. The vasoactivity of garlic compounds is synchronous with [H.sub.2]S production, and their potency to mediate relaxation increases with [H.sub.2]S yield, strongly supporting our hypothesis that [H.sub.2]S mediates the vasoactivity of garlic. Our results also suggest that the capacity to produce [H.sub.2]S can be used to standardize garlic dietary supplements. Allium | aorta | polysulfides | red blood cells | vasorelaxation

Conducted vasodilation may coordinate blood flow in microvascular networks during skeletal muscle contraction. We tested the hypotheses that 1) exercise training enhances conducted vasodilation and 2) age-related changes in the capacity for conduction affect muscle perfusion during contractions. To address hypothesis 1, young (4-5 mo), adult (12-14 mo), and old (19-21 mo) C57BL6 male mice were sedentary or given access to running wheels for 8 wk. Voluntary running distances were significantly different (in km/day): young = 5.8 [+ or -] 0.1, adult = 3.9 [+ or -] 0.1, old = 2.2 [+ or -] 0.1 (P < 0.05). In gluteus maximus muscles, conducted vasodilation was greater in adult than in young or old mice (P < 0.05) and greater in young sedentary than in old sedentary mice but was not affected by exercise training. Citrate synthase activity was greater with exercise training at all ages (P < 0.05). mRNA for endothelial nitric oxide synthase did not differ among ages, but endothelial nitric oxide synthase protein expression was greater in adult and old mice with exercise training (P < 0.05). Connexin 37, connexin 40, and connexin 43 mRNA were not affected by exercise training and did not differ by age. To address hypothesis 2, perfusion of the gluteus maximus muscle during light to severe workloads was assessed by Doppler microprobe at 3-26 mo of age. Maximum perfusion decreased linearly across the lifespan. Perfusion at the highest workload, absolute and relative to maximum, decreased across the lifespan, with a steeper decline beyond ~20 mo of age. In this model, 1) exercise training does not alter conducted vasodilation and 2) muscle perfusion is maintained up to near maximum workloads despite age-related changes in conducted vasodilation. endothelial; smooth muscle; gap junction; microcirculation; aging

The extent to which sympathetic nerve activity restrains metabolic vasodilation in skeletal muscle remains unclear. We determined forearm blood flow (FBF; ultrasound/Doppler) and vascular conductance (FVC) responses to 10 min of ischemia [reactive hyperemic blood flow (RHBF)] and 10 min of systemic hypoxia (inspired [O.sub.2] fraction = 0.1) before and after regional sympathetic blockade with the [alpha]-receptor antagonist phentolamine via Bier block in healthy humans. In a control group, we performed sham Bier block with saline. Consistent with [alpha]receptor inhibition, post-phentolamine, basal FVC (FBF/mean arterial pressure) increased (pre vs. post: 0.42 [+ or -] 0.05 vs. 1.03 [+ or -] 0.21 units; P < 0.01; n = 12) but did not change in the saline controls (pre vs. post: 0.56 [+ or -] 0.14 vs. 0.53 [+ or -] 0.08 units; P = not significant; n = 5). Post-phentolamine, total RHBF (over 3 min) increased substantially (pre vs. post: 628 [+ or -] 75 vs. 826 [+ or -] 92 ml/min; P < 0.01) but did not change in the controls (pre vs. post: 618 [+ or -] 66 vs. 661 [+ or -] 35 ml/min; P = not significant). In all conditions, compared with peak RHBF, peak skin reactive hyperemia was markedly delayed. Furthermore, post-phentolamine (pre vs. post: 0.43 [+ or -] 0.06 vs. 1.16 [+ or -] 0.17 units; P < 0.01; n = 8) but not post-saline (pre vs. post: 0.93 [+ or -] 0.16 vs. 0.87 [+ or -] 0.19 ml/min; P = not significant; n = 5), the FVC response to hypoxia (arterial O2 saturation = 77 [+ or -] 1%) was markedly enhanced. These data suggest that sympathetic vasoconstrictor nerve activity markedly restrains skeletal muscle vasodilation induced by local (forearm ischemia) and systemic (hypoxia) vasodilator stimuli. vasodilation; sympathetic nervous system; reactive hyperemia; hypoxia; phentolamine

Caveolin-1 (Car-1) is essential for the morphology of membrane caveolae and exerts a negative influence on a number of signaling systems, including nitric oxide (NO) production and activity of the MAP kinase cascade. In the vascular system, ablation of caveolin-1 may thus be expected to cause arterial dilatation and increased vessel wall mass (remodeling). This was tested in Car-1 knockout (KO) mice by a detailed morphometric and functional analysis of mesenteric resistance arteries, shown to lack caveolae. Quantitative morphometry revealed increased media thickness and media-to-lumen ratio in KO. Pressure-induced myogenic tone and flow-induced dilatation were decreased in KO arteries, but both were increased toward wild-type (WT) levels following NO synthase (NOS) inhibition. Isometric force recordings following NOS inhibition showed rightward shifts of passive and active length-force relationships in KO, and the force response to [[alpha].sub.1]-adrenergic stimulation was increased. In contrast, media thickness and force response of the aorta were unaltered in KO vs. WT, whereas lumen diameter was increased. Mean arterial blood pressure during isoflurane anesthesia was not different in KO vs. WT, but greater fluctuation in blood pressure over time was noted. Following NOS inhibition, fluctuations disappeared and pressure increased twice as much in KO (38 [+ or -] 6%) compared with WT (17 [+ or -] 3%). Tracer-dilution experiments showed increased plasma volume in KO. We conclude that NO affects blood pressure more in Cav-1 KO than in WT mice and that restructuring of resistance vessels and an increased responsiveness to adrenergic stimulation compensate for a decreased tone in Cav-1 KO mice.

We studied the roles of estrogen receptors (ER) and aromatase in the mediation of flow-induced dilation (FID) in isolated arteries of male ER[alpha]-knockout (ER[alpha]-KO) and wild-type (WT) mice. FID was comparable between gracilis arteries of WT and ER[alpha]-KO mice. In WT arteries, inhibition of NO and prostaglandins eliminated FID. In ER[alpha]-KO arteries, [N.sup.[omega]]-nitro-L-arginine methyl ester (L-NAME) inhibited FID by ~26%, whereas indomethacin inhibited dilations by ~50%. The remaining portion of the dilation was abolished by additional administration of 6-(2-proparglyoxyphenyl)hexanoic acid (PPOH) or iberiotoxin, inhibitors of epoxyeicosatrienoic acid (EET) synthesis and large-conductance potassium channels, respectively. By using an electrophysiological technique, we found that, in the presence of 10 dyne/[cm.sup.2] shear stress, perfusate passing through donor vessels isolated from gracilis muscle of ER[alpha]-KO mice subjected to L-NAME and indomethacin elicited smooth muscle hyperpolarization and a dilator response of endothelium-denuded detector vessels. These responses were prevented by the presence of iberiotoxin in detector or PPOH in donor vessels. Gas chromatography-mass spectrometry (GC-MS) analysis indicated a significant increase in arterial production of EETs in ER[alpha]-KO compared with WT mice. Western blot analysis showed a significantly reduced endothelial nitric oxide synthase expression but enhanced expressions of aromatase and ER[beta] in ER[alpha]-KO arteries. Treatment of ER[alpha]-KO arteries with specific aromatase short-interfering RNA for 72 h, knocked down the aromatase mRNA and protein associated with elimination of EET-mediation of FID. Thus, FID in male ER[alpha]-KO arteries is maintained via an endothelium-derived hyperpolarizing factor/EET-mediated mechanism compensating for reduced NO mediation due, at least in part, to estrogen aromatized from testosterone. estradiol; testosterone; receptors; nitric oxide; endothelium-derived hyperpolarizing factor

With most cardiovascular disease risk factors, endothelium-dependent dilation of skeletal muscle resistance arterioles is compromised, although with hypercholesterolemia, impairments to reactivity are not consistently observed. Using apolipoprotein E (ApoE) and low-density lipoprotein receptor (LDLR) gene deletion male mouse models of hypercholesterolemia at 20 wk of age, we tested the hypothesis that arteriolar dilation would be maintained due to an increased stimulus-induced production of dilator metabolites via cyclooxygenase and cytochrome P-450 epoxygenase pathways. Arterioles from both strains demonstrated mild reductions in dilation to hypoxia and acetylcholine versus responses in C57/B1/6J (C57) controls. However, although inhibition of nitric oxide synthase (NOS) attenuated dilation in arterioles from C57 controls, this effect was absent in ApoE or LDLR strains. In contrast, cyclooxygenase-dependent portions of dilator reactivity were maintained across the three strains. Notably, although combined NOS and cyclooxygenase inhibition abolished arteriolar responses to hypoxia and acetylcholine in C57 controls, significant reactivity remained in ApoE and LDLR strains. Whereas inhibition of cytochrome P-450 [omega]-hydroxylase and epoxygenases had no effect on this residual reactivity in ApoE and LDLR strains, inhibition of 12/15-lipoxygenase with nordihydroguaiaretic acid abolished the residual reactivity. With both hypoxic and methacholine challenges, arteries from ApoE and LDLR strains demonstrated an increased production of both 12(S)- and 15(S)-hydroxyeicosatetraenoic acid, end products of arachidonic acid metabolism via 12/15-lipoxygenase, a response that was not present in C57 controls. These results suggest that with development of hypercholesterolemia, mechanisms contributing to dilator reactivity in skeletal muscle arterioles are modified such that net reactivity to endothelium-dependent stimuli is largely intact. skeletal muscle microcirculation; mouse models of cardiovascular disease

In a vulnerable plaque (VP), rupture often occurs at a site of high stress within the cap. It is also known that vessels do not become free of stress when all external loads are removed. Previous studies have shown that such residual stress/strain (RS/S) tends to make the stress distribution more uniform throughout the media of a normal artery. However, the influence of RS/S on the wall stress distribution in pathological coronaries remains unclear. The aim of this study was to investigate the effects of RS/S on the biomechanical stability of VPs. RS/S patterns were studied ex vivo in six human vulnerable coronary plaque samples. Because the existence of RS/S can only be assessed by releasing it, the opening angle technique was the experimental approach used to study the geometrical opening configurations of the diseased arteries, producing an arterial wall in a near-zero stress state. Reciprocally, these opening geometries were used in finite element simulations to reconstruct the RS/S distributions in closed arteries. It was found that the RS/S 1) is not negligible, 2) dramatically affects the physiological peak stress amplitude in the thin fibrous cap, 3) spotlights some new high stress areas, and 4) could be a landmark of the lipid core's developmental process within a VP. This study demonstrates that plaque rupture is not to be viewed as a consequence of intravascular pressure alone, but rather of a subtle combination of external loading and intraplaque RS/S. atherosclerosis; zero-stress state; lipid core; plaque growth; finite element analysis

Efforts to develop functional tissue-engineered blood vessels have focused on improving the strength and mechanical properties of the vessel wall, while the functional status of the endothelium within these vessels has received less attention. Endothelial cell (EC) function is influenced by interactions between its basal surface and the underlying extracellular matrix. In this study, we utilized a coculture model of a tissueengineered blood vessel to evaluate EC attachment, spreading, and adhesion formation to the extracellular matrix on the surface of quiescent smooth muscle cells (SMCs). ECs attached to and spread on SMCs primarily through the [[alpha].sub.5][[beta].sub.1]--integrin complex, whereas ECs used either [[alpha].sub.5][[beta].sub.1]- or [[alpha].sub.v][[beta].sub.3]-integrin to spread on fibronectin (FN) adsorbed to plastic. ECs in coculture lacked focal adhesions, but EC [[alpha].sub.5][[beta].sub.1]-integrin bound to fibrillar FN on the SMC surface, promoting rapid fibrillar adhesion formation. As assessed by both Western blot analysis and quantitative real-time RT-PCR, coculture suppressed the expression of focal adhesion proteins and mRNA, whereas tensin protein and mRNA expression were elevated. When attached to polyacrylamide gels with similar elastic moduli as SMCs, focal adhesion formation and the rate of cell spreading increased relative to ECs in coculture. Thus, the elastic properties are only one factor contributing to EC spreading and focal adhesion formation in coculture. The results suggest that the softness of the SMCs and the fibrillar organization of FN inhibit focal adhesions and reduce cell spreading while promoting fibrillar adhesion formation. These changes in the type of adhesions may alter EC signaling pathways in tissue-engineered blood vessels. focal adhesions; fibrillar adhesions; tissue engineering; integrin; elastic modulus

Advanced liver cirrhosis is associated with hyperdynamic circulation consisting of systemic hypotension, decreased peripheral resistance, and cardiac dysfunction, termed cirrhotic cardiomyopathy. Previous studies have revealed the role of endocannabinoids and vascular [CB.sub.1] receptors in the development of generalized hypotension and mesenteric vasodilation in animal models of liver cirrhosis, and [CB.sub.1] receptors have also been implicated in the decreased [beta]-adrenergic responsiveness of isolated heart tissue from cirrhotic rats. Here we document the cardiac contractile dysfunction in vivo in liver cirrhosis and explore the role of the endocannabinoid system in its development. Rats with C[Cl.sub.4]-induced cirrhosis developed decreased cardiac contractility, as documented through the use of the Millar pressure-volume microcatheter system, low blood pressure, and tachycardia. Bolus intravenous injection of the [CB.sub.1] antagonist AM251 (3 mg/kg) acutely increased mean blood pressure, as well as both load-dependent and -independent indexes of systolic function, whereas no such changes were elicited by AM251 in control rats. Furthermore, tissue levels of the endocannabinoid anandamide increased 2.7-fold in the heart of cirrhotic compared with control rats, without any change in 2-arachidonoylglycerol levels, whereas, in the cirrhotic liver, both 2-arachidonoylglycerol (6-fold) and anandamide (3.5-fold) were markedly increased. [CB.sub.1]-receptor expression in the heart was unaffected by cirrhosis, as verified by Western blotting. Activation of cardiac [CB.sub.1] receptors by endogenous anandamide contributes to the reduced cardiac contractility in liver cirrhosis, and [CB.sub.1]-receptor antagonists may be used to improve contractile function in cirrhotic cardiomyopathy and, possibly, in other forms of heart failure. cirrhosis; cannabinoid [CB.sub.1] receptors; endocannabinoids; cardiac contractility; pressure-volume loops

Although the endothelium co-generates both nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF), the relative contribution from each vasodilator is not clear. In studies where the endothelium is stimulated acutely, EDHF responses predominate in small arteries. However, the temporal relationship between endothelial-derived NO and EDHF over more prolonged periods is unclear but of major physiological importance. Here we have used a classical pharmacological approach to show that EDHF is released transiently compared with NO. Acetylcholine (3 x [10.sup.-6] mol/l) dilated second- and/or third-order mesenteric arteries for prolonged periods of up to 1 h, an effect that was reversed fully and immediately by the subsequent addition of L-NAME ([10.sup.-3] mol/l) but not TRAM-34 ([10.sup.-6] mol/l) plus apamin (5 x [10.sup.-7] mol/l). When vessels were pretreated with L-NAME, acetylcholine induced relatively transient dilator responses (declining over ~5 min), and vessels were sensitive to TRAM-34 plus apamin. When measured in parallel, the dilator effects of acetylcholine outlasted the smooth muscle hyperpolarization. However, in the presence of L-NAME, vasodilatation and hyperpolarization followed an identical time course. In vessels from [NOSIII.sup.-/-] mice, acetylcholine induced small but detectable dilator responses that were transient in duration and blocked by TRAM-34 plus apamin. EDHF responses in these mouse arteries were inhibited by an intracellular calcium blocker, TMB-8, and the phospholipase [A.sub.2] inhibitor [AACOCF.sub.3], suggesting a role for lipid metabolites. These data show for the first time that EDHF is released transiently, whereas endothelial-derived NO is released in a sustained manner. arteries; vasodilation; mediators; hyperpolarization; nitric oxide; endothelium-derived hyperpolarizing factor

Systemic sclerosis (scleroderma, SSc) is an autoimmune, connective tissue disorder that is characterized by impaired vascular function, increased oxidative stress, inflammation of internal organs, and impaired angiogenesis. Tight skin mice ([Tsk.sup.-/+]) have a defect in fibrillin-1, resulting in replication of many of the myocardial and vascular features seen in humans with SSc. D-4F is an apolipoprotein A-I (apoA-I) mimetic that improves vascular function in diverse diseases such as hypercholesterolemia, influenza, and sickle cell disease. [Tsk.sup.-/+] mice were treated with either phosphate-buffered saline (PBS) or D-4F (1 mg * [kg.sup.-1] * [day.sup.-1] for 6-8 wk). Acetylcholine and flow-induced vasodilation were examined in facialis arteries. Proinflammatory HDL (p-HDL) in murine and human plasma samples was determined by the cell-free assay. Angiostatin levels in murine and human plasma samples were determined by Western blot analysis. Hearts were examined for changes in angiostatin and autoantibodies against oxidized phosphotidylcholine (ox-PC). Angiogenic potential in thin sections of murine hearts was assessed by an in vitro vascular endothelial growth factor (VEGF)-induced endothelial cell (EC) tube formation assay. D-4F improved endothelium-, endothelial nitric oxide synthase-dependent, and flow-mediated vasodilation in [Tsk.sup.-/+] mice. [Tsk.sup.-/+] mice had higher plasma p-HDL and angiostatin levels than C57BL/6 mice, as did SSc patients compared with healthy control subjects. [Tsk.sup.-/+] mice also had higher triglycerides than C57BL/6 mice. D-4F reduced p-HDL, angiostatin, and triglycerides in the plasma of [Tsk.sup.-/+] mice. [Tsk.sup.-/+] hearts contained notably higher levels of angiostatin and autoantibodies against ox-PC than those of control hearts. D-4F ablated angiostatin in [Tsk.sup.-/+] hearts and reduced autoantibodies against ox-PC by >50% when compared with hearts from untreated [Tsk.sup.-/+] mice. Angiogenic potential in [Tsk.sup.-/+] hearts was increased only when the [Tsk.sup.-/+] mice were treated with D-4F (1 mg * [kg-.sup.-1] * [day.sup.-1], 6-8 wk), and cultured sections of hearts from the D-4F-treated [Tsk.sup.-/+] mice were incubated with D-4F (10 [micro]g/ml, 5-7 days). Failure to treat the thin sections of hearts and [Tsk.sup.-/+] mice with D-4F resulted in loss of VEGF-induced EC tube formation. D-4F improves vascular function, decreases myocardial inflammation, and restores angiogenic potential in the hearts of [Tsk.sup.-/+] mice. As SSc patients have increased plasma p-HDL and angiostatin levels similar to the [Tsk.sup.-/+] mice, D-4F may be effective at treating vascular complications in patients with SSc. systemic sclerosis; angiogenesis

In the microcirculation, longitudinal conduction of vasomotor responses provides an essential means of coordinating flow distribution among vessels in a complex network. Spread of current along the vessel axis can display a regenerative component, which leads to propagation of vasomotor signals over many millimeters; the ionic basis for the regenerative response is unknown. We examined the responses to 10 s of focal electrical stimulation (30 Hz, 2 ms, 30 V) of mouse cremaster arterioles to test the hypothesis that voltage-dependent [Na.sup.+] ([Na.sub.v]) and [Ca.sup.2+] channels might be activated in long-distance signaling in microvessels. Electrical stimulation evoked a vasoconstriction at the site of stimulation and a spreading, nondecremental conducted dilation. Endothelial damage (air bubble) blocked conduction of the vasodilation, indicating an involvement of the endothelium. The [Na.sub.v] channel blocker bupivacaine also blocked conduction, and TTX attenuated it. The [Na.sub.v] channel activator veratridine induced an endothelium-dependent dilation. The [Na.sub.v] channel isoforms [Na.sub.v]1.2, [Na.sub.v]1.6, and [Na.sub.v]1.9 were detected in the endothelial cells of cremaster arterioles by immunocytochemistry. These findings are consistent with the involvement of [Na.sub.v] channels in the conducted response. BAPTA buffering of endothelial cell [Ca.sup.2+] delayed and reduced the conducted dilation, which was almost eliminated by [Ni.sup.2+], amiloride, or deletion of [alpha].sub.1H] T-type [Ca.sup.2+] ([Ca.sub.v]3.2) channels. Blockade of endothelial nitric oxid synthase or [Ca.sup.2+]-activated [K.sup.+] channels also inhibited the conducted vasodilation. Our findings indicate that an electrically induced signal can propagate along the vessel axis via the endothelium and can induce sequential activation of [Na.sub.v] and [Ca.sub.v]3.2 channels. The resultant [Ca.sup.2+] influx activates endothelial nitric oxide synthase and [Ca.sup.2+]activated [K.sup.+] channels, triggering vasodilation. conducted vasodilation; T-type calcium channels; hypertension; gap junction; voltage-gated sodium channels

Sex differences exist in a variety of cardiovascular disorders. Sex hormones have been shown to mediate pulmonary artery (PA) vasodilation. However, the effects of fluctuations in physiological sex hormone levels due to sex and menstrual cycle on PA vasoreactivity have not been clearly established yet. We hypothesized that sex and menstrual cycle affect PA vasoconstriction under both normoxic and hypoxic conditions. Isometric force displacement was measured in isolated PA rings from proestrus females (PF), estrus and diestrus females (E/DF), and male (M) Sprague-Dawley rats. The vasoconstrictor response under normoxic conditions (organ bath bubbled with 95% [O.sub.2]-5% C[O.sub.2]) was measured after stimulation with 80 mmol/1 KCI and 1 [micro]mol/l phenylephrine. Hypoxia was generated by changing the gas to 95% N2-5% C[O.sub.2]. PA rings from PF demonstrated an attenuated vasoconstrictor response to KCl compared with rings from E/DF (75.58 [+ or -] 3.2% vs. 92.43 [+ or -] 4.24%, P < 0.01). Rings from M also exhibited attenuated KCl-induced vasoconstriction compared with E/DF (79.34 [+ or -] 3.2% vs. 92.43 [+ or -] 4.24%, P < 0.05). PA rings from PF exhibited an attenuated vasoconstrictor response to phenylephrine compared with E/DF (59.61 [+ or -] 2.98% vs. 70.03 [+ or -] 4.61%, P < 0.05). While the maximum PA vasodilation during hypoxia did not differ between PF, E/DF, and M, phase II of hypoxic pulmonary vasoconstriction was markedly diminished in the PA from PF (64.10 [+ or -] 7.10% vs. 83.91 [+ or -] 5.97% in M, P < 0.05). We conclude that sex and menstrual cycle affect PA vasoconstriction in isolated PA rings. Even physiological increases in circulating estrogen levels attenuate PA vasoconstriction under both normoxic and hypoxic conditions. sex hormones; genomic and nongenomic effects; phenylephrine; potassium chloride

Endothelial cell migration is critical for proper blood vessel development. Signals from growth factors and matrix proteins are integrated through focal adhesion proteins to alter cell migration. Hydrogen peroxide-inducible clone 5 (Hic-5), a paxillin family member, is enriched in the focal adhesions in bovine pulmonary artery endothelial (BPAE) cells, which migrate to lysophosphatidic acid (LPA) on denatured collagen. In this study, we investigate the role of Hic-5 in LPA-stimulated endothelial cell migration. LPA recruits Hic-5 to the local adhesions and to the pseudopodia in BPAE cells plated on collagen, suggesting that recruitment of Hic-5 to focal adhesions is associated with endothelial cell migration. Knockdown of endogenous Hic-5 significantly decreases migration toward LPA, confirming involvement of Hic-5 in migration. To address the role of Hic-5 in endothelial cell migration, we exogenously expressed wild-type (WT) Hic-5 and green fluorescent protein Hic-5 C369A/C372A (LIM3 mutant) constructs in BPAE cells. WT Hic-5 expression increases chemotaxis of BPAE cells to LPA, whereas migration toward LPA of the green fluorescent protein Hic-5 C369A/C372A-expressing cells is similar to that shown in vector control cells. Additionally, ERK phosphorylation is enhanced in the presence of LPA in WT Hic-5 cells. A pharmacological inhibitor of MEK activity inhibits LPA-stimulated WT Hic-5 cell migration and ERK phosphorylation, suggesting Hic-5 enhances migration via MEK activation of ERK. Together, these studies indicate that Hic-5, a focal adhesion protein in endothelial cells, is recruited to the pseudopodia in the presence of LPA and enhances migration. pseudopodia; focal adhesion proteins; angiogenesis doi:10.1152/ajpheart.00728.2006

Pulmonary hypertension (PH) causes right ventricular (RV) hypertrophy and, according to the extent of pressure overload, eventual heart failure. We tested the hypothesis that the mechanical stress in PH-RV impairs the vasoreactivity of the RV coronary microvessels of different sizes with increased superoxide levels. Five-week-old male Sprague-Dawley rats were injected with monocrotaline (n = 126) to induce PH or with saline as controls (n = 114). After 3 wk, coronary arterioles (diameter = 30-100 [micro]m) and small arteries (diameter = 100-200 [micro]m) in the RV were visualized using intravital videomicroscopy. We evaluated ACh-induced vasodilation alone, in the presence of [N.sup.[omega]]-nitro-L-arginine methyl ester (L-NAME), in the presence of tetraethylammonium (TEA) or catalase with or without L-NAME, and in the presence of SOD. The degree of suppression in vasodilation by L-NAME and TEA was used as indexes of the contributions of endothelial nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF), respectively. In PH rats, ACh-induced vasodilation was significantly attenuated in both arterioles and small aretries, especially in arterioles. This decreased vasodilation was largely attributable to reduced NO-mediated vasoreactivity, whereas the EDHF-mediated vasodilation was relatively robust. The suppressive effect on arteriolar vasodilation by catalase was similar to TEA in both groups. Superoxide, as measured by lucigenin chemiluminescence, was significantly elevated in the RV tissues in PH. SOD significantly ameliorated the impairment of ACh-induced vasodilation in PH. Robust EDHF function will play a protective role in preserving coronary microvascular homeostasis in the event of NO dysfunction with increased superoxide levels. acetylcholine; [N.sup.[omega]]-nitro-L-arginine methyl ester; tetraethylanmonium; catalase; superoxide dimutase; endothelium-derived hyperpolarizing factor doi:10.1152/ajpheart.00548.2006

To further explore the limitations to maximal [O.sub.2] consumption (V[O.sub.2 max]) in exercise-trained skeletal muscle, six cyclists performed graded knee-extensor exercise to maximum work rate (W[R.sub.max]) in hypoxia (12% [O.sub.2]), hyperoxia (100% [O.sub.2]), and hyperoxia + femoral arterial infusion of adenosine (ADO) at 80% W[R.sub.max]. Arterial and venous blood sampling and thermodilution blood flow measurements allowed the determination of muscle [O.sub.2] delivery and [O.sub.2] consumption. At W[R.sub.max], [O.sub.2] delivery rose progressively from hypoxia (1.0 [+ or -] 0.04 l/min) to hyperoxia (1.20 [+ or -] 0.09 l/min) and hyperoxia + ADO (1.33 [+ or -] 0.05 l/min). Leg V[O.sub.2] max varied with [O.sub.2] availability (0.81 [+ or -] 0.05 and 0.97 [+ or -] 0.07 l/min in hypoxia and hyperoxia, respectively) but did not improve with ADO-mediated vasodilation (0.80 [+ or -] 0.09 l/min in hyperoxia + ADO). Although a vasodilatory reserve in the maximally working quadriceps muscle group may have been evidenced by increased leg vascular conductance after ADO infusion beyond that observed in hyperoxia (increased blood flow but no change in blood pressure), we recognize the possibility that the ADO infusion may have provoked vasodilation in nonexercising tissue of this limb. Together, these findings imply that maximally exercising skeletal muscle may maintain some vasodilatory capacity, but the lack of improvement in leg V[O.sub.2max] with significantly increased [O.sub.2] delivery (hyperoxia + ADO), with a degree of uncertainty as to the site of this dilation, suggests an ADO-induced mismatch between [O.sub.2] consumption and blood flow in the exercising limb. exercise; blood flow; vasodilatory reserve doi: 10.1152/ajpheart.01396.2006.