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The earliest blood vessels in mammalian embryos are formed when endothelial cells differentiate from angioblasts and coalesce into tubular networks. Thereafter, the endothelium is thought to expand solely by proliferation of pre-existing endothelial cells. Here we show that a complementary source of endothelial cells is recruited into pre-existing vasculature after differentiation from the earliest precursors of erythrocytes, megakaryocytes and macrophages, the erythro-myeloid progenitors (EMPs) that are born in the yolk sac. A first wave of EMPs contributes endothelial cells to the yolk sac endothelium, and a second wave of EMPs colonizes the embryo and contributes endothelial cells to intraembryonic endothelium in multiple organs, where they persist into adulthood. By demonstrating that EMPs constitute a hitherto unrecognized source of endothelial cells, we reveal that embryonic blood vascular endothelium expands in a dual mechanism that involves both the proliferation of pre-existing endothelial cells and the incorporation of endothelial cells derived from haematopoietic precursors.New blood vessel endothelial cells arise from differentiation of erythro-myeloid progenitor cells to complement the proliferation of pre-existing endothelial cells., Author(s): Alice Plein [sup.1] , Alessandro Fantin [sup.1] , Laura Denti [sup.1] , Jeffrey W. Pollard [sup.2] , Christiana Ruhrberg [sup.1] Author Affiliations:(1) UCL Institute of Ophthalmology, University College London, [...]

Primary open-angle glaucoma (POAG) is often caused by elevated intraocular pressure (IOP), which arises due to increased resistance to aqueous humor outflow (AHO). Aqueous humor flows through Schlemm's canal (SC), a lymphatic-like vessel encircling the cornea, and via intercellular spaces of ciliary muscle cells. However, the mechanisms underlying increased AHO resistance are poorly understood. Here, we demonstrate that signaling between angiopoietin (Angpt) and the Angpt receptor Tie2, which is critical for SC formation, is also indispensable for maintaining SC integrity during adulthood. Deletion of Angpt1/Angpt2 or Tie2 in adult mice severely impaired SC integrity and transcytosis, leading to elevated IOP, retinal neuron damage, and impairment of retinal ganglion cell function, all hallmarks of POAG in humans. We found that SC integrity is maintained by interconnected and coordinated functions of Angpt-Tie2 signaling, AHO, and Prox1 activity. These functions diminish in the SC during aging, leading to impaired integrity and transcytosis. Intriguingly, Tie2 reactivation using a Tie2 agonistic antibody rescued the POAG phenotype in Angpt1/Angpt2-deficient mice and rejuvenated the SC in aged mice. These results indicate that the Angpt-Tie2 system is essential for SC integrity. The impairment of this system underlies POAGassociated pathogenesis, supporting the possibility that Tie2 agonists could be a therapeutic option for glaucoma., Introduction Glaucoma is a leading cause of irreversible blindness, affecting 3.5% of the population aged 40 to 80 years worldwide (1). It involves cupping of the optic nerve head and [...]

Glaucoma is a leading cause of blindness, with an estimated world-wide prevalence of 3.5% in members of the population older than 40 years of age. Elevated intraocular pressure as the result of abnormal resistance to aqueous humor drainage is a major contributing, and the only preventable, factor in glaucoma development. Schlemm's canal (SC), a lymphatic-like vessel encircling the anterior portion of the eye, plays a key role in promoting aqueous humor outflow and maintenance of normal intraocular pressure. The risk of developing glaucoma increases with age; therefore, understanding mechanisms of SC maintenance and how aging affects SC function are of special importance, both for prevention and novel treatment approaches to glaucoma. Using a compelling array of genetic models, Kim et al. report in this issue of the JCI that continuous angiopoietin/TIE2 signaling is required for maintaining SC identity and integrity during adulthood and show that its age-related changes can be rescued by a TIE2 agonistic antibody., A 'classical' view of lymphatic vasculature The lymphatic vasculature serves as a one-way drainage system for extracellular fluid and immune cells from the periphery of the body into the bloodstream. [...]

Contrary to previous assumptions, it seems the cells that line blood vessels are derived from more than one source. In addition to their known developmental path, they can arise from progenitors of embryonic blood cells.Endothelial cells arise from erythro-myeloid progenitors., Author(s): M. Luisa Iruela-ArispeAuthor Affiliations:A dual origin for blood vessels Transient embryonic populations of red blood and immune cells arise early in development, before the emergence of HSCs, from precursor [...]

The increasing prevalence of diabetes has resulted in a global epidemic.sup.1. Diabetes is a major cause of blindness, kidney failure, heart attacks, stroke and amputation of lower limbs. These are often caused by changes in blood vessels, such as the expansion of the basement membrane and a loss of vascular cells.sup.2-4. Diabetes also impairs the functions of endothelial cells.sup.5 and disturbs the communication between endothelial cells and pericytes.sup.6. How dysfunction of endothelial cells and/or pericytes leads to diabetic vasculopathy remains largely unknown. Here we report the development of self-organizing three-dimensional human blood vessel organoids from pluripotent stem cells. These human blood vessel organoids contain endothelial cells and pericytes that self-assemble into capillary networks that are enveloped by a basement membrane. Human blood vessel organoids transplanted into mice form a stable, perfused vascular tree, including arteries, arterioles and venules. Exposure of blood vessel organoids to hyperglycaemia and inflammatory cytokines in vitro induces thickening of the vascular basement membrane. Human blood vessels, exposed in vivo to a diabetic milieu in mice, also mimic the microvascular changes found in patients with diabetes. DLL4 and NOTCH3 were identified as key drivers of diabetic vasculopathy in human blood vessels. Therefore, organoids derived from human stem cells faithfully recapitulate the structure and function of human blood vessels and are amenable systems for modelling and identifying the regulators of diabetic vasculopathy, a disease that affects hundreds of millions of patients worldwide.Organoids derived from human stem cells recapitulate the structure and functions of human blood vessels, and can be used to model and identify regulators of diabetic vasculopathy., Author(s): Reiner A. Wimmer [sup.1] , Alexandra Leopoldi [sup.1] , Martin Aichinger [sup.2] , Nikolaus Wick [sup.3] , Brigitte Hantusch [sup.3] , Maria Novatchkova [sup.1] , Jasmin Taubenschmid [sup.1] , [...]

This study evaluated endothelium-dependent vascular relaxation in response to acetylcholine (ACh) in isolated middle cerebral arteries (MCA) from Dahl salt-sensitive (Dahl SS) rats and three different congenic strains that contain a portion of Brown Norway (BN) chromosome 13 introgressed onto the Dahl SS genetic background through marker-assisted breeding. Two of the congenic strains carry a 3.5-Mbp portion and a 2.6-Mbp portion of chromosome 13 that lie on opposite sides of the renin locus, while the third contains a 2.0-Mbp overlapping region that includes the BN renin allele. While maintained on a normal salt (0.4% NaCl) diet, MCAs from Dahl SS rats and the congenic strains retaining the Dahl SS renin allele failed to dilate in response to ACh, whereas MCAs from the congenic strain carrying the BN renin allele exhibited normal vascular relaxation. In congenic rats receiving the BN renin allele, vasodilator responses to ACh were eliminated by nitric oxide synthase inhibition with [N.sup.G]-nitro-L-arginine methyl ester, angiotensin-converting enzyme inhibition with captopril, and [AT.sub.1] receptor blockade with losartan. [N.sup.G]-nitro-L-arginine methyl ester-sensitive vasodilation in response to ACh was restored in MCAs of Dahl SS rats that received either a 3-day infusion of a subpressor dose of angiotensin II (3 ng x [kg.sup.-1] x [min.sup.-1] iv), or chronic treatment with the superoxide dismutase mimetic tempol (15 mg x [kg.sup.-1] x [day.sup.-1]). These findings indicate that the presence of the Dahl SS renin allele plays a crucial role in endothelial dysfunction present in the cerebral circulation of the Dahl SS rat, even in the absence of elevated dietary salt intake, and that introgression of the BN renin allele rescues endothelium-dependent vasodilator responses by restoring normal activation of the renin-angiotensin system. renin-angiotensin system; vascular dysfunction; physiological genomics; vasodilation; angiotensin II; oxidant stress doi: 10.1152/ajpheart.00700.2010.

BAY 41-8543 is a nitric oxide (NO)-independent stimulator of soluble guanylyl cyclase (sGC). Responses to intravenous injections of BAY 41-8543 were investigated under baseline and elevated tone conditions and when NO synthase (NOS) was inhibited with [N.sup.[omega]]-nitro-L-arginine methyl ester (L-NAME). Under baseline conditions, intravenous injections of BAY 41-8543 caused small decreases in pulmonary arterial pressure, larger decreases in systemic arterial pressure, and increases in cardiac output. When pulmonary arterial pressure was increased to ~30 mmHg with an intravenous infusion of U-46619, intravenous injections of BAY 41-8543 produced larger dose-dependent decreases in pulmonary arterial pressure, and the relative decreases in pulmonary and systemic arterial pressure in response to the sGC stimulator were similar. Treatment with L-NAME markedly decreased responses to BAY 41-8543 when pulmonary arterial pressure was increased to similar values (~30 mmHg) in U-46619-infused and in U-46619-infused plus L-NAME-treated animals. The intravenous injection of a small dose of sodium nitroprusside (SNP) when combined with BAY 41-8543 enhanced pulmonary and systemic vasodilator responses to the sGC stimulator in L-NAME-treated animals. The present results indicate that BAY 41-8543 has similar vasodilator activity in the systemic and pulmonary vascular beds when pulmonary vasoconstrictor tone is increased with U-46619. These results demonstrate that pulmonary and systemic vasodilator responses to BAY 41-8543 are significantly attenuated when NOS is inhibited by L-NAME and show that vasodilator responses to BAY 41-8543 are enhanced when combined with a small dose of SNP in L-NAME-treated animals. The present results are consistent with the concept that pulmonary and systemic vasodilator responses to the sGC stimulator are NO-independent; however, the vasodilator activity of the compound is greatly diminished when endogenous NO production is inhibited with L-NAME. These data show that BAY 41-8543 has similar vasodilator activity in the pulmonary and systemic vascular beds in the rat. soluble guanylyl cyclase stimulator; pulmonary and systemic vascular beds; U-46619; [N.sup.[omega]]-nitro-L-arginine methyl ester; sodium nitroprusside doi: 10.1152/ajpheart.01101.2009.

Chronic cytomegalovirus (CMV) infections are implicated in vascular diseases. Recently, we showed that an active mouse CMV (mCMV) infection in nonpregnant mice increased endothelial-dependent vasodilation in isolated mesenteric and uterine arteries. In late pregnancy, while increased vasodilation was found in mesenteric arteries from infected mice, there was a dramatic decrease in uterine arteries. Understanding the mechanisms for these vascular changes during CMV infections is important for pregnancy outcomes and long-term consequences of this chronic infection. Increased nitric oxide (NO) is implicated in CMV-associated atherosclerosis, and CMV replication is dependent on prostaglandin H synthase (PGHS) activity. Alternatively, CMV infections decrease NO under inflammatory conditions. We therefore hypothesized that changes in the contribution by NO or PGHS-induced vasodilators would explain the increased or decreased endothelial-dependent vasodilation in arteries from nonpregnant and late pregnant mice, respectively. We found that the contribution by NO to methacholine-induced vasodilation was significantly increased in mesenteric, but not uterine, arteries isolated from nonpregnant and pregnant mCMV-infected mice. Prostaglandin inhibition did not affect endothelial-dependent vasodilation in any group. Vasodilation responses to sodium nitroprusside, an NO donor, were increased in mesenteric and uterine arteries isolated only from mCMV-infected nonpregnant mice. These results explain the increased vasodilation responses observed in mesenteric arteries from mCMV-infected mice; however, the decreased vasodilation in uterine arteries from pregnant mice could not be explained by these mechanisms. Thus CMV infection affects the contribution of NO differently in endothelial-dependent vasodilation in pregnant compared with nonpregnant mice and also in the mesenteric compared with the uterine vascular bed. pregnancy; endothelium; vascular smooth muscle; methacholine; sodium nitroprusside; vascular function doi: 10.1152/ajpheart.01113.2009.

The goals of this study were to 1) determine the acute effect of ANG-(1-7) on vascular tone in isolated middle cerebral arteries (MCAs) from Sprague-Dawley rats fed a normal salt (NS; 0.4% NaC1) diet, 2) evaluate the ability of chronic intravenous infusion of ANG-(1-7) (4 ng x [kg.sup.-1] x [min.sup.-l]) for 3 days to restore endothelium-dependent dilation to acetylcholine (ACh) in rats fed a high-salt (HS; 4% NaCl) diet, and 3) determine whether the amelioration of endothelial dysfunction by ANG-(1-7) infusion in rats fed a HS diet is different from the protective effect of low-dose ANG II infusion in salt-fed rats. MCAs from rats fed a NS diet dilated in response to exogenous ANG-(1-7) ([10.sup.-10] - [10.sup.-5] M). Chronic ANG-(1-7) infusion significantly reduced vascular superoxide levels and restored the nitric oxide-dependent dilation to ACh ([10.sup.-10]-[10.sup.-5] M) that was lost in MCAs of rats fed a HS diet. Acute vasodilation to ANG-(1-7) and the restoration of ACh-induced dilation by chronic ANG-(1-7) infusion in rats fed a HS diet were blocked by the Mas receptor antagonist [D-ALA(7)]-ANG-(1-7) or the ANG II type 2 receptor antagonist PD-123319 and unaffected by ANG II type 1 receptor blockade with losartan. The restoration of ACh-induced dilation in MCAs of HS-fed rats by chronic intravenous infusion of ANG II (5 ng x [kg.sup.-1] x [min.sup.-1]) was blocked by losartan and unaffected by D-ALA. These findings demonstrate that circulating ANG-(1-7), working via the Mas receptor, restores endothelium-dependent vasodilation in cerebral resistance arteries of animals fed a HS diet via mechanisms distinct from those activated by low-dose ANG II infusion. Mas receptor; nitric oxide; oxidative stress; angiotensin II doi: 10.1152/ajpheart.00328.2010.

Data suggest that polyphenol-rich products may improve endothelial function and other cardiovascular health risk factors. Grape and wine contain high amounts of polyphenols, but effects of these polyphenols have hardly been investigated in isolation in randomized controlled studies. Our objective in this study was to test the chronic effect of polyphenol-rich solids derived from either a wine grape mix or grape seed on flow-mediated dilation (FMD). Blood pressure and other vascular function measures, platelet function, and blood lipids were secondary outcomes. Thirty-five healthy males were randomized in a double-blind, placebo-controlled crossover study consisting of three 2-wk intervention periods separated by 1-wk washout periods. The test products, containing 800 mg of polyphenols, were consumed as capsules. At the end of each intervention period, effects were measured after consumption of a low-fat breakfast (~751 kJ, 25% fat) and a high-fat lunch (~3136 kJ, 78% fat). After the low-fat breakfast, the treatments did not significantly affect FMD. The absolute difference after the wine grape solid treatment was -0.4% (95% CI = -1.8 to 0.9; P = 0.77) and after grape seed solids, 0.2% (95% CI = -1.2 to 1.5; P = 0.94) compared with after the placebo treatment. FMD effects after the high-fat lunch and effects on secondary outcomes also showed no consistent differences between both of the grape solids and placebo treatment. In conclusion, consumption of grape polyphenols has no major impact on FMD in healthy men. Future studies should address whether grape polyphenols can improve FMD and other cardiovascular health risk factors in populations with increased cardiovascular risk. J. Nutr. 140: 1769-1773, 2010. doi: 10.3945/jn.110.125518.

It has been reported that mitochondrial aldehyde dehydrogenase (ALDH2) catalyzes the formation of glyceryl dinitrate and inorganic nitrite from glyceryl trinitrate (GTN), leading to an increase in cGMP and vasodilation in the coronary and systemic vascular beds. However, the role of nitric oxide (NO) formed from nitrite in mediating the response to GTN in the pulmonary vascular bed is uncertain. The purpose of the present study was to determine if nitrite plays a role in mediating vasodilator responses to GTN. In this study, intravenous injections of GTN and sodium nitrite decreased pulmonary and systemic arterial pressures and increased cardiac output. The decreases in pulmonary arterial pressure under baseline and elevated tone conditions and decreases in systemic arterial pressure in response to GTN and sodium nitrite were attenuated by cyanamide, an ALDH2 inhibitor, whereas responses to the NO donor, sodium nitroprusside (SNP), were not altered. The decreases in pulmonary and systemic arterial pressure in response to GTN and SNP were not altered by allopurinol, an inhibitor of xanthine oxidoreductase, whereas responses to sodium nitrite were attenuated. GTN was ~1,000-fold more potent than sodium nitrite in decreasing pulmonary and systemic arterial pressures. These results suggest that ALDH2 plays an important role in the bioactivation of GTN and nitrite in the pulmonary and systemic vascular beds and that the reduction of nitrite to vasoactive NO does not play an important role in mediating vasodilator responses to GTN in the intact chest rat. mitochondrial aldehyde dehydrogenase; xanthine oxidoreductase; nitric oxide; glyceryl trinitrate; sodium nitrite; sodium nitroprusside; allopurinol; cyanamide; U-46619; [N.sup.G]-nitro-L-arginine methyl ester doi: 10.1152/ajpheart.00959.2009.

Tissue engineering of small diameter ( Key words: biological scaffolds, blood vessel, decellularized matrices, endothelial cells, extracellular matrix, polymer scaffolds, tissue engineering, vascular smooth muscle cells. La reconstitui ion par ingenierie tissulaire de vaisseaux sanguins de petit diametre ( Mots-cles: supports biologiques, vaisseau sanguin, matrices decellularisees, cellules endotheliales, matrice extracellulaire, supports de polymeres, ingenierie tissulaire, cellules musculaires lisses vasculaires. [Traduit par la Redaction], Introduction Cardiovascular disease is a leading cause of morbidity and mortality worldwide. According to recent statistics by the American Heart Association, 34.3% of all deaths in the United States are [...]

Changes in neuronal activity are accompanied by the release of vasoactive mediators that cause microscopic dilation and constriction of the cerebral microvasculature and are manifested in macroscopic blood oxygenation level-dependent (BOLD) functional MRI (fMRI) signals. We used two-photon microscopy to measure the diameters of single arterioles and capillaries at different depths within the rat primary somatosensory cortex. These measurements were compared with cortical depth-resolved fMRI signal changes. Our microscopic results demonstrate a spatial gradient of dilation onset and peak times consistent with 'upstream' propagation of vasodilation toward the cortical surface along the diving arterioles and 'downstream' propagation into local capillary beds. The observed BOLD response exhibited the fastest onset in deep layers, and the 'initial dip' was most pronounced in layer I. The present results indicate that both the onset of the BOLD response and the initial dip depend on cortical depth and can be explained, at least in part, by the spatial gradient of delays in microvascular dilation, the fastest response being in the deep layers and the most delayed response in the capillary bed of layer I. blood flow | cortical layer | hemodynamic | imaging | somatosensory doi/ 10.1073/pnas.1006735107

Adenosine is an important paracrine agent regulating renal vascular tone via adenosine [A.sup.1] and [A.sup.2] receptors. While A2B receptor message and protein have been localized to preglomerular vessels, functional evidence on the role of [A.sup.2]B receptors in mediating the vasodilator action of adenosine on afferent arterioles is not available. The present study determined the role of [A.sup.2]B receptors in mediating the afferent arteriolar dilation and compared the effects of [A.sup.2]B and [A.sup.2]A receptor blockade on afferent arterioles. We used the rat in vitro blood-perfused juxtamedullary nephron technique combined with videomicroscopy. Single afferent arterioles of Sprague-Dawley rats were visualized and superfused with solutions containing adenosine or adenosine [A.sup.2] receptor agonist (CV-1808) along with adenosine [A.sup.2]B and [A.sup.2]A receptor blockers. Adenosine (10 [micro]mol/1) caused modest constriction and subsequent superfusion with SCH-58261 (SCH), an [A.sup.2A] receptor blocker, at concentrations up 10 [micro]mol/1 elicited only slight additional decreases in afferent arteriolar diameter with maximum effect at a concentration of 1 [micro]mol/l (- 11.0 [+ or -] 2.5%, n = 6, P < 0.05). However, superfusion of adenosine-treated vessels with MRS-1754 (MRS), an [A.sup.2]B receptor blocker, elicited greater decreases in afferent arteriolar diameter (-26.0 [+ or -] 4.7%, n = 5, P < 0.01). SCH did not significantly augment the adenosine-mediated afferent constriction elicited by MRS; however, adding MRS after SCH caused further significant vasoconstriction. Superfusion with CV-1808 dilated afferent arterioles (17.2 [+ or -] 2.4%, n = 6, P < 0.01). This effect was markedly attenuated by MRS (-22.6 [+ or -] 2.0%, n = 5, P < 0.01) but only slightly reduced by SCH (-9.0 [+ or -] 1.1%, n = 5, P < 0.05) and completely prevented by adding MRS after SCH (-24.7 [+ or -] 1.8%, n = 5, P < 0.01). These results indicate that, while both [A.sup.2A] and [A.sup.2B] receptors are functionally expressed in juxtamedullary afferent arterioles, the powerful vasodilating action of adenosine predominantly involves [A.sup.2B] receptor activation, which counteracts [A.sup.1] receptor-mediated vasoconstriction. adenosine receptor antagonists; renal microcirculation; vascular biology; afferent arterioles; kidney doi: 10.1152/ajprenal.00149.2010.

The sole FDA-approved treatment for acute stroke is recombinant tissue-type plasminogen activator (rtPA). However, rtPA aggravates the impairment of cerebrovasodilation induced by global hypoxia/ischemia; this impairment is attenuated by the preinjury treatment with the plasminogen activator inhibitor derivative EEIIMD. MAPK (a family of kinases, p38, and JNK) is upregulated after cerebral ischemia. In this study, we determined whether the novel plasminogen activator inhibitor-derived peptide, Ac-RMAPEEIIMDRPFLYVVR-amide, (PAI-1-DP) given 30 min before or 2 h after, focal central nervous system injury induced by photothrombosis would preserve responses to cerebrovasodilators and the role of p38 and JNK MAPK in such effects. Cerebrospinal fluid JNK and p38 levels were elevated by photothrombotic injury, an effect potentiated by rtPA. Cerebrovasodilation was blunted by photothrombosis and reversed to vasoconstriction by rtPA but restored to dilation by PAI-I-DP pre- and posttreatment. PAI-1-DP blocked JNK, but preserved p38 MAPK upregulation after photothrombosis. The JNK MAPK antagonist SP600125 prevented, and the p38 antagonist SB203580 potentiated, impaired cerebrovasodilation after photothrombosis. These data indicate that rtPA impairs cerebrovasodilation after injury by activating JNK, while p38 MAPK is protective, and that the novel peptide PAI-1-DP protects by inhibiting activation of JNK by rtPA. JNK MAPK inhibitors, including PAI-1-DP, may offer a novel approach to increase the benefit-to-risk ratio of thrombolytic therapy and enable its use in central nervous system ischemic disorders. cerebral circulation; newborn; plasminogen activators; signal transduction doi: 10.1152/ajpregu.00256.2010.

Carbon monoxide (CO) causes cerebral arteriolar dilation in newborn pigs by the activation of large-conductance [Ca.sup.2+]-activated [K.sup.+] channels. In adult rat cerebral and skeletal muscle arterioles, CO has been reported to produce constriction caused by the inhibition of nitric oxide (NO) synthase (NOS). We hypothesized that, in contrast to dilation to acute CO, more prolonged exposure of newborn cerebral arterioles to elevated CO produces constriction by reducing NO. In piglets with closed cranial windows, pial arteriolar responses to isoproterenol ([10.sup.-6] M), sodium nitroprusside (SNP; [10.sup.-7] and 3 x [10.sup.-7] M), and L-arginine ethyl ester (L-Arg; [10.sup.-5] and [10.sup.-4] M) were determined before and after 2 h of treatment with CO. CO ([10.sup.-7] M) caused transient dilation and had no further effects. CO (2 x [10.sup.-7] and [10.sup.-6] M) initially caused vasodilation, but over the 2-h exposure, pial arterioles constricted and removal of the CO caused dilation. Exposure to elevated CO (2 h) did not alter dilation to SNP or isoproterenol. Conversely, the NOS substrate L-Arg caused dilation before CO that was progressively lost over 90 min of elevated CO. If NO was held constant, CO caused dilation that was sustained for 2 h. We conclude that in neonates, cerebral arteriole responses to CO are biphasic: dilation to acute elevation with subsequent constriction from NOS inhibition after more prolonged exposure. As a result, short episodic production of CO allows function as a dilator gasotransmitter, whereas prolonged elevation can reduce NO to elevate cerebrovascular tone. The interaction between heine oxygenase/CO and NOS/NO could form a negative feedback system in the control of cerebral vascular tone. nitric oxide; neonate doi: 10.1152/ajpheart.00258.2010.

Myosin phosphatase target subunit 1 (MYPT1) is the regulatory subunit of myosin light chain phosphatase (MLCP). It plays a critical role in vasodilatation induced by cGMP-elevating agents such as nitric oxide (NO). The present study was performed to determine the role of MYPT1 in the development of tolerance of the pulmonary artery to NO. Incubation of isolated porcine pulmonary arteries for 24 or 48 h with DETA NONOate (DETA NO) significantly reduced protein levels of MYPT1 and the leucine zipper-positive (LZ+) isoform of MYPTI but not that of PPlcS. The extent of reduction in total MYPT1 protein level was comparable to that of MYPTI (LZ+). The decrease in MYPT1 protein caused by 48-h DETA NO incubation was prevented by ODQ, an inhibitor of guanylyl cyclase, and by inhibitors of proteasomes (MG-132 and lactacystin) but was not affected by the inhibitor of protein synthesis, cycloheximide. A reduction in MYPT1 protein was also obtained with 8-bromo-cGMP, but this was prevented by Rp-8bromo-PET-cGMP [inhibitor of cGMP-dependent protein kinase (PKG)]. Incubation for 48 h with DETA NO also reduced dephosphorylation of myosin light chain and relaxation of the artery in response to DETA NO, which was prevented by MG-132. These results suggest that the reduction in MYPT1 protein contributes to the development of tolerance of pulmonary arteries to NO. This may result from increased degradation of MYPT1 after prolonged PKG activation. myosin light chain phosphatase; guanosine 3',5'-cyclic monophosphate-dependent protein kinase; vasodilatation doi: 10.1152/ajplung.00340.2009.

Capsaicin-sensitive extrinsic sensory nerves and submucosal vasodilator neurons provide important vasodilator input to submucosal arterioles, but relatively little is known about the signaling between these populations and the sympathetic vasoconstrictor innervation. This study examined whether release of sympathetic purines can modulate dilator nerves. In vitro submucosal preparations from guinea pig ileum were modified to leave the parent mesenteric artery intact so that perivascular sympathetic and extrinsic afferent nerves could be activated by a bipolar stimulating electrode placed on the parent artery, and submucosal vasodilator neurons were activated using focal electrodes placed on submucosal ganglia. The outside diameter of submucosal arterioles was monitored using videomicroscopy, and dilator responses were examined after preconstricting vessels 80-95% with prostaglandin [F.sub.2[alpha]] (400 nM). Mesenteric nerve stimulation evoked a frequency-dependent dilation, with suramin (100 [micro]M) present throughout to inhibit P2x receptor-mediated vasoconstrictions. In the presence of guanethidine (10 [micro]M) to inhibit sympathetic purine release, superfusion of ATP (200 nM-6 [micro]M) caused a concentration-dependent inhibition of nerve-evoked dilations. Vasodilations to substance P (10 nM) were not inhibited by ATP in the presence of guanethidine, implicating a presynaptic effect of ATP on neurotransmitter release. The inhibitory effect of ATP was blocked by the adenosine receptor antagonist 8-phenyltheophylline (8-PT; 10 [micro]M). In addition, 8-PT increased the amplitude of nerve-evoked dilations, suggesting a tonic inhibitory effect of adenosine receptors on vasodilator release. Dilations evoked by electrical stimulation of submucosal ganglia were also inhibited almost 50% by ATP (2 [micro]M) and its nonhydrolyzable analog, [alpha],[beta]-methylene-ATP (10 [micro]M). These data suggest that sympathetic varicosities release ATP or a related purine that can act at presynaptic adenosine receptors on extrinsic sensory and submucosal vasodilator neurons to inhibit neurotransmitter release. purinergic neurotransmission; vasodilation; extrinsic afferent neuron; substance P doi: 10.1152/ajpgi.00291.2009.

This investigation tested the hypothesis that metabolic syndrome decreases the relative contribution of specific [K.sup.+] channels to coronary reactive hyperemia. [Ca.sup.2+]-activated ([BK.sub.ca]), voltage-activated ([K.sub.v]), and ATP-dependent ([K.sub.ATP]) [K.sup.+] channels were investigated. Studies were conducted in anesthetized miniature Ossabaw swine fed a normal maintenance diet (11% kcal from fat) or an excess calorie atherogenic diet (43% kcal from fat, 2% cholesterol, 20% kcal from fructose) for 20 wk. The latter diet induces metabolic syndrome, increasing body weight, fasting glucose, total cholesterol, and triglyceride levels. Ischemic vasodilation was determined by the coronary flow response to a 15-s occlusion before and after cumulative administration of antagonists for [BK.sub.ca] (penitrem A; 10 [micro]g/kg iv), [K.sub.v] (4-aminopyridine; 0.3 mg/kg iv) and [K.sub.ATP] (glibenclamide; 1 mg/kg iv) channels. Coronary reactive hyperemia was diminished by metabolic syndrome as the repayment of flow debt was reduced ~30% compared with lean swine. Inhibition of BKc, channels had no effect on reactive hyperemia in either lean or metabolic syndrome swine. Subsequent inhibition of Kv channels significantly reduced the repayment of flow debt (~25%) in both lean and metabolic syndrome swine. Additional blockade of [K.sub.ATP] channels further diminished (~45%) the repayment of flow debt in lean but not metabolic syndrome swine. These data indicate that the metabolic syndrome impairs coronary vasodilation in response to cardiac ischemia via reductions in the contribution of [K.sup.+] channels to reactive hyperemia. coronary reactive hyperemia; calcium-activated potassium channels; voltage-activated potassium channels; adenosine triphosphate-dependent potassium channels; Ossabaw miniature swine; type 2 diabetes doi: 10.1152/ajpheart.00888.2009.

This study determined the role of nitric oxide (NO) in neurogenic vasodilation in mesenteric resistance arteries of the toad Bufo marinus. NO synthase (NOS) was anatomically demonstrated in perivascular nerves, but not in the endothelium. ACh and nicotine caused TTX-sensitive neurogenic vasodilation of mesenteric arteries. The ACh-induced vasodilation was endothelium-independent and was mediated by the NO/ soluble guanylyl cyclase signaling pathway, inasmuch as the vasodilation was blocked by the soluble guanylyl cyclase inhibitor 1H[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one and the NOS inhibitors [N.sup.[omega]]-nitro-L-arginine methyl ester and [N.sup.[omega]]-nitro-L-arginine. Furthermore, the ACh-induced vasodilation was significantly decreased by the more selective neural NOS inhibitor [N.sup.5]-(1-imino-3-butenyl)-L-ornithine. The nicotine-induced vasodilation was endothelium-independent and mediated by NO and calcitonin gene-related peptide (CGRP), inasmuch as pretreatment of mesenteric arteries with a combination of [N.sup.[omega]]-nitro-L-arginine and the CGRP receptor antagonist CGRP-(8-37) blocked the vasodilation. Clotrimazole significantly decreased the ACh-induced response, providing evidence that a component of the NO vasodilation involved [Ca.sup.2+]-activated [K.sup.+] or voltage-gated [K.sup.+] channels. These data show that NO control of mesenteric resistance arteries of toad is provided by nitrergic nerves, rather than the endothelium, and implicate NO as a potentially important regulator of gut blood flow and peripheral blood pressure. amphibian; autonomic nervous system; nitric oxide synthase; endothelium doi:10.1152/ajpregu.00148.2009

Oxidative stress has been shown to suppress endothelial nitric oxide synthase expression through activation of the transcription factor fork-head box O 1 (FOXO1) in cultured endothelial cells. We previously reported that circulating kallistatin levels are markedly reduced in rats with chronic oxidative organ damage. In this study, we investigated the potential role of oxidative stress in suppression of kallistatin expression via FOXO1 activation. In Dahl salt-sensitive (DSS) rats, we found that high salt intake induced a time-dependent correlation of increased thiobarbituric acid reactive substances (TBARS, an indicator of lipid peroxidation) with reduced serum kallistatin levels. Moreover, salt loading provoked an elevation of in situ aortic superoxide formation in association with reduced kallistatin levels. Expression of kallistatin was identified in cultured endothelial cells by immunocytochemistry and flow cytometry; however, [H.sub.2][O.sub.2] dose-dependently lowered kallistatin mRNA and protein levels as determined by real-time PCR and Western blot, respectively. Downregulation of kallistatin synthesis by oxidative stress was restored by knockdown of FOXO1 expression with small-interfering RNA. [H.sub.2][O.sub.2] rapidly induced FOXO1 nuclear translocation, but the effect was blocked by c-Jun N[H.sub.2]-terminal kinase (JNK) inhibitor. Inhibition of JNK by pharmacological inhibitor or small-interfering RNA reversed [H.sub.2][O.sub.2]'s effect on kallistatin expression in endothelial cells. This study demonstrates that an inverse relationship exists between oxidative stress and kallistatin levels in the circulation and blood vessels and that kallistatin expression is negatively regulated by oxidative stress via JNK-dependent FOXO1 activation in cultured endothelial cells. kallistatin; Forkhead box O 1; oxidative stress; c-Jun N[H.sub.2]-terminal kinase; blood vessel doi:10.1152/ajpheart.00826.2009

The transient receptor potential vallinoid type 4 (TRPV4) channel has been implicated in the endothelial shear response and flow-mediated dilation, although the precise functions of this channel remain poorly understood. In the present study, we investigated the role of TRPV4 in shear stress-induced endothelial Ca2+ entry and the potential link between this signaling response and relaxation of small resistance arteries. Using immunohistochemical analysis and RT-PCR, we detected strong expression of TRPV4 protein and mRNA in the endothelium in situ and endothelial cells freshly isolated from mouse small mesenteric arteries. The selective TRPV4 agonist GSK1016790A increased endothelial Ca2+ and induced potent relaxation of small mesenteric arteries from wild-type (WT) but not [TRPV4.sup.-/-] mice. Luminal flow elicited endothelium-dependent relaxations that involved both nitric oxide and EDHFs. Both nitric oxide and EDHF components of flow-mediated relaxation were markedly reduced in TRPV4 /- mice compared with WT controls. Using a fura-2/[Mn.sup.2+] quenching assay, shear was observed to produce rapid [Ca.sup.2+] influx in endothelial cells, which was markedly inhibited by the TRPV4 channel blocker ruthenium red and TRPV4-specific short interfering RNA. Flow elicited a similar TRPV4-mediated [Ca2.sup.+] entry in HEK-293 cells transfected with TRPV4 channels but not in nontransfected cells. Collectively, these data indicate that TRPV4 may be a potential candidate of mechanosensitive channels in endothelial cells through which the shear stimulus is transduced into [Ca.sup.2+] signaling, leading to the release of endothelial relaxing factors and flow-mediated dilation of small resistance arteries. transient receptor potential; endothelium; endothelium-derived factors; calcium signaling doi: 10.1152/ajpheart.00854.2009

Inorganic nitrite has recently been recognized to possess vascular activity that is enhanced in hypoxia. This has been demonstrated in humans in the forearm vascular bed. In animal models nitrite reduces pulmonary vascular resistance, but its effects upon the pulmonary circulation of humans have not yet been demonstrated. This paradigm is of particular interest mechanistically since the pulmonary vasculature is known to behave differently to the systemic. To investigate, 18 healthy volunteers were studied in a hypoxic chamber (inspired oxygen, 12%) or while breathing room air. Each received an infusion of sodium nitrite (1 [micro]mol/min) or 0.9% saline. Three protocols were performed: nitrite/ hypoxia (n = 12), saline/hypoxia (n = 6), and nitrite/normoxia (n = 6). Venous blood was sampled for plasma nitrite, forearm blood flow was measured by strain-gauge plethysmography, and pulmonary arterial pressure was measured by transthoracic echocardiography. Plasma nitrite doubled and clearance kinetics were similar whether nitrite was infused in hypoxia or normoxia. During hypoxia, nitrite increased forearm blood flow (+36%, P < 0.001) and reduced three separate indirect indexes of pulmonary arterial pressure by 16%, 12%, and 17% (P < 0.01). Pulmonary, but not systemic, arterial effects persisted 1 h after stopping the infusion, at a time when plasma nitrite had returned to baseline. No effects were observed during normoxia. Therefore, in hypoxic but not normoxic subjects, sodium nitrite causes arterial and pulmonary vasodilatation. In addition, hypoxia-induced pulmonary vasoconstriction was attenuated for a prolonged period and not dependent on a simultaneous elevation of plasma nitrite. This finding is consistent with the direct extravascular metabolism of nitrite to nitric oxide to effect hypoxia-associated bioactivity. hypoxia; pulmonary hypertension; vasodilatation; nitric oxide doi: 10.1152/ajpheart.00583.2009

Am J Physiol Regul Integr Comp Physiol 298: R51-R60, 2010. First published November 4, 2009; doi: 10.1152/ajpregu.00576.2009.--Hypoxic pulmonary vasoconstriction (HVC), an intrinsic and assumed ubiquitous response of mammalian pulmonary blood vessels, matches regional ventilation to perfusion via an unknown O2-sensing mechanism. Global pulmonary hypoxia experienced by individuals suffering from chronic obstructive pulmonary disease or numerous hypoventilation syndromes, including sleep apnea, often produces maladaptive pulmonary hypertension, but pulmonary hypertension is not observed in diving mammals, where profound hypoxia is routine. Here we examined the response of cow and sea lion pulmonary arteries (PA) to hypoxia and observed the expected HVC in the former and a unique hypoxic vasodilation in resistance vessels in the latter. We then used this disparate response to examine the [O.sub.2]-sensing mechanism. In both animals, exogenous [H.sub.2]S mimicked the vasoactive effects of hypoxia in isolated PA. [H.sub.2]S-synthesizing enzymes, cystathionine [beta]-synthase, cystathionine [gamma]-lyase, and 3-mercaptopyruvate sulfur transferase, were identified in lung tissue from both animals by one-dimensional Western blot analysis and immunohistochemistry. The relationship between [H.sub.2]S production/consumption and [O.sub.2] was examined in real time by use of amperometric [H.sub.2]S and [O.sub.2] sensors. H2S was produced by sea lion and cow lung homogenate in the absence of [O.sub.2], but it was rapidly consumed when [O.sub.2] was present. Furthermore, consumption of exogenous [H.sub.2]S by cow lung homogenate, PA smooth muscle cells, and heart mitochondria was [O.sub.2] dependent and exhibited maximal sensitivity at physiologically relevant [Po.sub.2] levels. These studies show that HVC is not an intrinsic property of PA and provide further evidence for [O.sub.2]-dependent [H.sub.2]S metabolism in [O.sub.2] sensing. hypoxic vasoconstriction; pinniped; pulmonary hypertension

Shibasaki M, Aoki K, Morimoto K, Johnson JM, Takamata A. Plasma hyperosmolality elevates the internal temperature threshold for active thermoregulatory vasodilation during heat stress in humans. Am J Physiol Regul Integr Comp Physiol 297:R1706-R 1712, 2009. First published October 7, 2009; doi: 10.1152/ajpregu.00242.2009.-Plasma hyperosmolality delays the response in skin blood flow to heat stress by elevating the internal temperature threshold for cutaneous vasodilation. This elevation could be because of a delayed onset of cutaneous active vasodilation and/or to persistent cutaneous active vasoconstriction. Seven healthy men were infused with either hypertonic (3% NaCL) or isotonic (0.9% NaCl) saline and passively heated by immersing their lower legs in 42[degrees]C water for 60 rain (room temperature, 28[degrees]C; relative humidity, 40%). Skin blood flow was monitored via laser-Doppler flowmetry at sites pretreated with bretylium tosylate (BT) to block sympathetic vasoconstriction selectively and at adjacent control sites. Plasma osmolality was increased by ~13 mosmol/kg[H.sub.2]O following hypertonic saline infusion and was unchanged following isotonic saline infusion. The esophageal temperature ([T.sub.es]) threshold for cutaneous vasodilation at untreated sites was significantly elevated in the hyperosmotic state (37.73 [+ or -] 0.11[degrees]C) relative to the isosmotic state (36.63 [+ or -] 0.12[degrees]C, P < 0.001). A similar elevation of the [T.sub.es] threshold for cutaneous vasodilation was observed between osmotic conditions at the BT-treated sites (37.74 [+ or -] 0.18 vs. 36.67 [+ or -] 0.07[degrees]C, P < 0.001) as well as sweating. These results suggest that the hyperosmotically induced elevation of the internal temperature threshold for cutaneous vasodilation is due primarily to an elevation in the internal temperature threshold for the onset of active vasodilation, and not to an enhancement of vasoconstrictor activity. osmoregulation; body fluid; thermoregulation; skin blood flow; sweating doi: 10.1152/ajpregu.00242.2009

LeBianc A J, Reyes R, Kang LS, Dailey RA, Stallone JN, Moningka NC, Muller-Delp JM. Estrogen replacement restores fiow-induced vasodilation in coronary arterioles of aged and ovariectomized rats. Am J Physiol Regul Integr Comp Physiol 297: R1713-R1723, 2009. First published October 7, 2009; doi: 10.1152/ajpregu.00178.2009.--The risk for cardiovascular disease (CVD) increases with advancing age; however, the age at which CVD risk increases significantly is delayed by more than a decade in women compared with men. This cardioprotection, which women experience until menopause, is presumably due to the presence of ovarian hormones, in particular, estrogen. The purpose of this study was to determine how age and ovarian hormones affect flow-induced vasodilation in the coronary resistance vasculature. Coronary arterioles were isolated from young (6 mo), middle-aged (14 mo), and old (24 mo) intact, ovariectomized (OVX), and ovariectomized + estrogen replaced (OVE) female Fischer-344 rats to assess flow-induced vasodilation. Advancing age impaired flow-induced dilation of coronary arterioles (young: 50 [+ or -] 4 vs. old: 34 [+ or -] 6; % relaxation). Ovariectomy reduced flow-induced dilation in arterioles from young females, and estrogen replacement restored vasodilation to flow. In aged females, flow-induced vasodilation of arterioles was unaltered by OVX; however, estrogen replacement improved flow-induced dilation by ~160%. The contribution of nitric oxide (NO) to flow-induced dilation, assessed by nitric oxide synthase (NOS) inhibition with [N.sup.g]-nitro-L-arginine methyl ester (L-NAME), declined with age. L-NAME did not alter flow-induced vasodilation in arterioles from OVX rats, regardless of age. In contrast, L-NAME reduced flow-induced vasodilation of arterioles from estrogen-replaced rats at all ages. These findings indicate that the age-induced decline of flow-induced, NO-mediated dilation in coronary arterioles of female rats is related, in part, to a loss of ovarian estrogen, and estrogen supplementation can improve flow-induced dilation, even at an advanced age. endothelial nitric oxide synthase; Akt; nitric oxide; ovariectomy doi: 10.1152/ajpregu.00178.2009

Reglin B, Secomb TW, Pries AR. Structural adaptation of microvessel diameters in response to metabolic stimuli: where are the oxygen sensors? Am J Physiol Heart Circ Physiol 297: H2206-H2219, 2009. First published September 25, 2009; doi: 10.1152/ajpheart.00348.2009.-- Maintenance of functional vascular networks requires structural adaptation of vessel diameters in response to hemodynamic and metabolic conditions. The mechanisms by which diameters respond to the metabolic state are not known, but may involve the release of vasoactive substances in response to low oxygen by tissue ('tissue signaling', e.g., C[O.sub.2], adenosine), by vessel walls ('wall signaling', e.g., prostaglandins, adenosine), and/or by red blood cells (RBCs) ('RBC signaling', e.g., ATP and nitric oxide). Here, the goal was to test the potential of each of these locations of oxygen-dependent signaling to control steady-state vascular diameters and tissue oxygenation. A previously developed theoretical model of structural diameter adaptation based on experimental data on microvascular network morphology and hemodynamics was used. Resulting network characteristics were analyzed with regard to tissue oxygenation (Oxdef; percentage of tissue volume with [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] < 1 Torr) and the difference between estimated blood flow velocities and corresponding experimental data [velocity error ([V.sub.err]); root mean square deviation of estimated vs. measured velocity]. Wall signaling led to Oxdef < 1% and to the closest hemodynamic similarity ([V.sub.err]: 0.60). Tissue signaling also resulted in a low oxygen deficit, but a higher [V.sub.err] (0.73) and systematic diameter deviations. RBC signaling led to widespread hypoxia (Oxdef: 4.7%), unrealistic velocity distributions ([V.sub.err]: 0.81), and shrinkage of small vessels. The results suggest that wall signaling plays a central role in structural control of vessel diameters in microvascular networks of given angioarchitecture. Tissue-derived and RBC-derived signaling of oxygen levels may be more relevant for the regulation of angiogenesis and/or smooth muscle tone. oxygen sensing; vascular diameter; angioadaptation doi: 10.1152/ajpheart.00348.2009.

The blood and lymphatic vasculatures are structurally and functionally coupled in controlling tissue perfusion, extracellular interstitial fluids, and immune surveillance. Little is known, however, about the molecular mechanisms that underlie the regulation of bloodlymphatic vessel connections and lymphatic perfusion. Here we show in the adult zebrafish and glass catfish (Kryptopterus bicirrhis) that blood-lymphatic conduits directly connect arterial vessels to the lymphatic system. Under hypoxic conditions, arterial-lymphatic conduits (ALCs) became highly dilated and linearized by NO-induced vascular relaxation, which led to blood perfusion into the lymphatic system. NO blockage almost completely abrogated hypoxia-induced ALC relaxation and lymphatic perfusion. These findings uncover mechanisms underlying hypoxia-induced oxygen compensation by perfusion of existing lymphatics in fish. Our results might also imply that the hypoxia-induced NO pathway contributes to development of progression of pathologies, including promotion of lymphatic metastasis by modulating arterial-lymphatic conduits, in the mammalian system. blood vessel | lymphatic vessel doi/10.1073/pnas.0907608106

It has been suggested that nitric oxide (NO) stimulates the cyclooxygenase (COX)-dependent mechanisms in the ocular vasculature; however, the importance of the pathway in regulating retinal circulation in vivo remains to be elucidated. Therefore, we investigated the role of COX-dependent mechanisms in NO-induced vasodilation of retinal blood vessels in thiobutabarbital-anesthetized rats with and without neuronal blockade (tetrodotoxin treatment). Fundus images were captured with a digital camera that was equipped with a special objective lens. The retinal vascular response was assessed by measuring changes in diameter of the retinal blood vessel. The localization of COX and soluble guanylyl cyclase in rat retina was examined using immunohistochemistry. The NO donors (sodium nitroprusside and NOR3) increased the diameter of the retinal blood vessels but decreased systemic blood pressure in a dose-dependent manner. Treatment of rats with indomethacin, a nonselective COX inhibitor, or SC-560, a selective COX-1 inhibitor, markedly attenuated the vasodilation of retinal arterioles, but not the depressor response, to the NO donors. However, both the vascular responses to NO donors were unaffected by the selective COX-2 inhibitors NS-398 and nimesulide. Indomethacin did not change the retinal vascular and depressor responses to hydralazine, 8-(4-chlorophenylthio)-guanosine-3', 5'-cyclic monophosphate (a membrane-permeable cGMP analog) and 8-(4-chlorophenylthio)-adenosine-3', 5'-cyclic monophosphate (a membrane-permeable cAMP analog). Treatment with SQ 22536, an adenylyl cyclase inhibitor, but not ODQ, a soluble guanylyl cyclase inhibitor, significantly attenuated the NOR3-induced vasodilation of retinal arterioles. The COX-1 immunoreactivity was found in retinal blood vessels. The retinal blood vessel was faintly stained for soluble guanylyl cyclase, although the apparent immunoreactivities on mesenteric and choroidal blood vessels were observed. These results suggest that NO exerts a substantial part of its dilatory effect via a mechanism that involves COX-1-dependent pathway in rat retinal vasculature. cyclooxygenases; prostaglandins; retinal blood vessel doi: 10.1152/ajpregu.91005.2008.

A role for nitric oxide (NO) produced during the reduction of nitrite by deoxygenated red blood cells (RBCs) in regulating vascular dilation has been proposed. It has not, however, been satisfactorily explained how this NO is released from the RBC without first reacting with the large pools of oxyhemoglobin and deoxyhemoglobin in the cell. In this study, we have delineated a mechanism for nitrite-induced RBC vasodilation that does not require that NO be released from the cell. Instead, we show that nitrite enhances the ATP release from RBCs, which is known to produce vasodilation by several different methods including the interaction with purinergic receptors on the endothelium that stimulate the synthesis of NO by endothelial NO synthase. This mechanism was established in vivo by measuring the decrease in blood pressure when injecting nitrite-reacted RBCs into rats. The observed decrease in blood pressure was not observed if endothelial NO synthase was inhibited by [N.sup.[omega]]-nitro-L-arginine methyl ester (L-NAME) or when any released ATP was degraded by apyrase. The nitrite-enhanced ATP release was shown to involve an increased binding of nitrite-modified hemoglobin to the RBC membrane that displaces glycolytic enzymes from the membrane, resulting in the formation of a pool of ATP that is released from the RBC. These results thus provide a new mechanism to explain nitrite-induced vasodilation. nitrite reduction; nitric oxide; anerobic glycolysis; hypoxia; red blood cell; adenosine 5'-triphosphate doi: 10.1152/ajpheart.01233.2008

Flow-mediated dilatation (%FMD), an index of nitric oxide (NO)-mediated vasodilator function, is regarded as a surrogate marker of cardiovascular disease. Aging is associated with endothelial dysfunction, but underlying sex-related differences may exist and the effects of fitness and exercise on endothelial dysfunction in men (M) and women (W) are poorly understood. We compared %FMD of the brachial artery in 18 young [Y, 26 [+ or -] 1 yr; 9 M and 9 W], 12 older fit (OF, 57 [+ or -] 2 yr; 6 M and 6 W), and 16 older sedentary (OS, 59 [+ or -] 2 yr; 8 M and 8 W) subjects. Glyceryl trinitrate (GTN) administration was used to assess endothelium-independent vasodilatation, and the FMD-to-GTN ratio was calculated to characterize NO dilator function in the context of smooth muscle cell sensitivity. Brachial %FMD in Y (7.1 [+ or -] 0.8%) was significantly higher compared with OS (4.8 [+ or -] 0.7%, P < 0.05), but not OF (6.4 [+ or -] 0.7%). Differences between Y and OS subjects were due primarily to lower FMD in the OS women (4.3 [+ or -] 0.6%). OS women exhibited significantly lower FMD-to-GTN ratios compared with Y (P < 0.05) and OF women (P < 0.05), whereas these differences were not apparent in men. Exercise training improved brachial artery NO dilator function (FMD-to-GTN ratio) after 24 wk (P < 0.05) in OS women, but not men. These findings indicate that maintaining a high level of fitness, or undertaking exercise training, prevents the age-related decline in the brachial artery vasodilator function evident in women. In OS men, who had relatively preserved NO dilator function, no training adaptations were observed. This study has potential implications for the prevention of conduit artery endothelial dysfunction in men and women. fitness; exercise training; upper-limb nitric oxide dilator function

In animal models, administration of the adenosine analog AICA-riboside has shown beneficial effects on ischemia-reperfusion injury and glucose homeostasis. The vascular and/or metabolic effects of AICA-riboside administration in humans remain to be established. AICA-riboside was infused intra-arterially in four different dosages up to 8 mg x [min.sup.-1] x [dl.sup.-1] in 24 healthy subjects. Forearm blood flow (FBF) and glucose uptake and plasma glucose, free fatty acid, and AICA-riboside concentrations were assessed. We also combined AICA-fiboside infusion (2 mg x [min.sup.-1] x [dl.sup.-1]) with the intra-arterial administration of the adenosine receptor antagonist caffeine (90 [micaro]g x [min.sup.-1 x [dl.sup.-1]; n = 6) and with the endothelial NO synthase inhibitor L-NMMA (0.4 mg x [min.sup.-1] x [dl.sup.-1] = 6). Additional in vitro experiments were performed to explain our in vivo effects of AICA-riboside in humans. AICA-riboside increased FBF dose dependently from 2.0 [+ or -] 0.2 to 13.2 [+ or -] 1.9 ml x [min.sup.-1] x [dl.sup.-1] maximally (P < 0.05 for all dosages). The latter was not reduced by caffeine administration but was significantly attenuated by L-NMMA infusion. Despite high plasma AICA-riboside concentrations, forearm glucose uptake did not change. In vitro experiments showed rapid uptake of AICA-riboside by the equilibratire nucleoside transporter in erythrocytes and subsequent phosphorylation to AICA-ribotide. We conclude that AICA-riboside induces a potent vasodilator response in humans that is mediated by NO. Despite high local plasma concentrations, AICA-riboside does not increase skeletal muscle glucose uptake. 5-aminoimidazole-4-carboxamide; nitric oxide; forearm blood flow; forearm glucose uptake

Background/Objectives: Nitric oxide (NO) has been proposed as a mediator of vascular expansion during pregnancy. Inability to increase NO synthesis and/or production of its precursor, arginine, may be a contributor to pregnancy-induced hypertension or preeclampsia. Because maternal weight is associated with blood pressure and risk of preeclampsia during pregnancy, it may also influence arginine and/or NO production. The purpose of this study was to determine the in vivo arginine production and NO synthesis rate in pregnant women with normal (n=10) and low (n=10) body mass indices (BMIS). Subjects/Methods: Arginine flux and NO synthesis rate were measured in the postabsorptive state with constant infusions of [sup.15][N.sub.2]-arginine and [sup.13]C, [sup.2.H.sub.4]-citrulline. Plasma concentrations of arginine and NO metabolites were also measured. Kinetic parameters were correlated to maternal variables, gestational age, birth weight and blood pressure. Results: Endogenous arginine flux was significantly faster in the low-BMI compared with normal-BMI women in the first trimester (63.1 [+ or -] 3.4 vs 50.2 [+ or -] 2.0[[micro]mol/kg per h, P Conclusions: These findings suggest, but do not prove, that maternal BMI may be a factor in the ability to produce NO during pregnancy and may be one way by which BMI influences blood pressure during pregnancy. doi:10.1038/ejcn.2009.24; published online 13 May 2009 Keywords: arginine production; nitric oxide synthesis; maternal weight; maternal body mass index, Introduction A physiological adaptation of pregnancy is expansion of the maternal vascular compartment. It starts with vasodilatation as peripheral resistance falls by 6 weeks and reaches a minimum in mid-pregnancy [...]

The aims of this study were to study the influence of the duration of diabetes, the role of endothelial-derived vasodilators, and the role of phosphodiesterase (PDE) isoform activity in the early changes in vascular reactivity of aortic rings from diabetic rats. Diabetes mellitus was induced in female rats by intravenous streptozotocin (85 mg/kg). Two or 4 wk later, thoracic aortic rings from control and diabetic rats were isolated, and vascular responses to acetylcholine (ACh), S-nitrosoN-acetylpenicillamine (SNAP) [nitric oxide (NO) donor], DMPPO (PDE5 inhibitor), and phenylephrine (PE) were obtained in the presence and absence of endothelium or other drugs. PDE isoform activity was also measured. At 2 wk, responses to ACh and DMPPO were enhanced, whereas those to PE were attenuated in diabetic rats relative to controls. Indomethacin and SQ-29548 (a thromboxane [A.sub.2] receptor antagonist), but not [N.sup.G]-nitro-L-arginine methyl ester, corrected these differences. The responses to SNAP, and cAMP and cGMP hydrolytic activities, were similar in the two groups. In contrast, at 4 Wk, ACh, DMPPO, and PE produced similar responses in the two groups: [N.sup.G]-nitro-L-arginine methyl ester rendered the response to PE lower in the diabetic group, and this was corrected by indomethacin, but not SQ-29548, treatment. The response to SNAP was greater in the diabetic group, and this was corrected by DMPPO. Activity of all PDEs was decreased at 4 wk. We conclude that, at 2 wk, there is modulation of thromboxane [A.sub.2] production, but no change in the NO system or PDE isoform activities. At 4 wk, a reduction in NO activity is superimposed; at this stage, PDE activity is reduced, together with increased production of vasodilating prostaglandins, possibly as a compensatory mechanism to maintain normal vascular reactivity. endothelium; vasodilation; vasoconstriction

Impaired nitric oxide-cGMP signaling contributes to severe pulmonary hypertension after birth, which may in part be due to decreased soluble gnanylate cyclase (sGC) activity. Cinacignat (BAY 58-2667) is a novel sGC activator that causes vasodilation, even in the presence of oxidized heine or heine-free sGC, but its hemodynamic effects have not been studied in the perinatal lung. We performed surgery on eight fetal (126 [+ or -] 2 days gestation) lambs (full term = 147 days) and placed catheters in the main pulmonary artery, aorta, and left atrium to measure pressures. An ultrasonic flow transducer was placed on the left pulmonary artery to measure blood flow, and a catheter was placed in the left pulmonary artery for drug infusion. Cinaciguat (0.1-100 [micro]g over 10 rain) caused dose-related increases in pulmonary blood flow greater than fourfold above baseline and reduced pulmonary vascular resistance by 80%. Treatment with IH-[l,2,4]oxadiazolo[4,3-a]quinoxalin-l-one (ODQ), an sGC-oxidizing inhibitor, enhanced cinacignat-induced pulmonary vasodilation by > 120%. The pulmonary vasodilator effect of cinacignat was prolonged, decreasing pulmonary vascular resistance for > 1.5 h after brief infusion. In vitro stimulation of ovine fetal pulmonary artery smooth muscle cells with cinacignat after ODQ treatment resulted in a 14-fold increase in cGMP compared with non-ODQ-treated cells. We conclude that cinaciguat causes potent and sustained fetal pulmonary vasodilation that is augmented in the presence of oxidized sGC and speculate that cinacignat may have therapeutic potential for severe neonatal pulmonary hypertension. BAY 58-2667; cGMP; nitric oxide; persistent pulmonary hypertension of the newborn; pulmonary hypertension

Store-operated [Ca.sup.2+] entry (SOCE) has recently been proposed to contribute to [Ca.sup.2+] influx in vascular smooth muscle cells (VSMCs). Adenosine is known for its protective role against hypoxia and ischemia by increasing nutrient and oxygen supply through vasodilation. This study was designed to examine the hypothesis that SOCE have a functional role in adenosine-induced vasodilation. Small mesenteric resistance arteries and mesenteric VSMCs were obtained from rats. Isometric tensions of isolated artery tings were measured by a sensitive myograph system. Laser-scanning confocal microscopy was used to determine the intracellular [Ca.sup.2+] concentration of fluo 3-loaded VSMCs. Adenosine (0.1-100 [micro]M) relaxed artery rings that were precontracted by phenylephrine in a concentration-dependent manner. In cultured mesenteric VSMCs, passive store depletion by thapsigargin and active store depletion by phenylephrine both induced [Ca.sup.2+] influx due to SOCE. Adenosine inhibited SOCE-mediated increases in cytosolic [Ca.sup.2+] levels evoked by the emptying of the stores. In isolated artery rings, adenosine inhibited SOCE-induced contractions due to store depletion. [A.sub.2A] receptor antagonism with SCH-58261 and adenylate cyclase inhibition with SQ-22536 largely attenuated adenosine responses. The cAMP analog 8-bromo-cAMP mimicked the effects of adenosine on SOCE. Our results indicate a novel mechanism of vasodilatation by adenosine that involves regulation of SOCE through the cAMP signaling pathway due to activation of adenosine [A.sub.2A] receptors. adenosine receptor; capacitative calcium ion entry; vasorelaxation

The cerebral arteries of hypertensive rats are depolarized and highly myogenic, suggesting a loss of [K.sup.+] channels in the vascular smooth muscle cells (VSMCs). The present study evaluated whether the dilator function of the prominent Shaker-type voltage-gated [K.sup.+] ([K.sub.v]1) channels is attenuated in middle cerebral arteries from two rat models of hypertension. Block of [K.sub.v]1 channels by correolide (1 [micro]mol/l) or psora-4 (100 nmol/l) reduced the resting diameter of pressurized (80 mmHg) cerebral arteries from normotensive rats by an average of 28 [+ or -] 3% or 26 [+ or -] 3%, respectively. In contrast, arteries from spontaneously hypertensive rats (SHR) and aortic-banded (Ao-B) rats with chronic hypertension showed enhanced [Ca.sup.2+]-dependent tone and failed to significantly constrict to correolide or psora-4, implying a loss of [K.sub.v]1 channel-mediated vasodilation. Patch-clamp studies in the VSMCs of SHR confirmed that the peak [K.sup.+] current density attributed to [K.sub.v]1 channels averaged only 5.47 [+ or -] 1.03 pA/pF, compared with 9.58 [+ or -] 0.82 pA/pF in VSMCs of control Wistar-Kyoto rats. Subsequently, Western blots revealed a 49 [+ or -] 7% to 66 [+ or -] 7% loss of the pore-forming [[alpha].sub.1.2]- and [[alpha].sub.1.5]-subunits that compose [K.sub.v]1 channels in cerebral arteries of SHR and Ao-B rats compared with control animals. In each case, the deficiency of [K.sub.v]1 channels was associated with reduced mRNA levels encoding either or both [alpha]-subunits. Collectively, these findings demonstrate that a deficit of [[alpha].sub.1.2]- and [[alpha].sub.1.5]-subunits results in a reduced contribution of [K.sub.v]1 channels to the resting diameters of cerebral arteries from two rat models of hypertension that originate from different etiologies. potassium channels; vascular smooth muscle; cerebral arteries; hypertension

Two-pore domain [K.sup.+] ([K.sub.2P]) channels are a new channel family. The goal of this study was to determine if [K.sub.2P] channels are activated by the nitric oxide (NO)/cGMP/PKG pathway in vascular smooth muscle. Relative levels of message for [K.sub.2P] channels were assessed in rat middle cerebral arteries (MCAs) using quantitative RT-PCR, and [K.sup.+] currents were measured in freshly dispersed vascular smooth muscle cells of the MCA. The rat MCA expresses a number of [K.sub.2P] channels. Message for TREK-1 was the most abundant [K.sub.2P] channel, followed by TASK-1 and TWIK-2, which were expressed at ~10% of the level of TREK-1. Message for other [K.sub.2P] channels was i% or less than that of TREK-1. A number of [K.sub.2P] channels, including TREK-1, TWIK-2, and TASK-1, have putative PKG phosphorylation sites in the intracellular domains. The NO donor sodium nitroprusside (100 [micro]M) or the membrane permeable analog of cGMP 8-bromo-cGMP (10 [micro]M) elicited transient increases in whole cell current of vascular smooth muscle from the rat MCA. However, after large-conductance [Ca.sup.2+]-activated [K.sup.+] channels had been blocked with 10 mM tetraethylammonium (TEA), no increase in whole cell current was observed. Since [K.sub.2P] channels are resistant to the blocking effects of TEA, we conclude that [K.sub.2P] channels in vascular smooth muscle were not activated by the NO/cGMP/PKG pathway. Although Kzp channels are highly expressed, [K.sub.2P] currents are not activated via the NO/cGMP pathway in rat MCA smooth muscle, despite the presence of numerous putative PKG phosphorylation sites. cerebrovascular circulation; nitric oxide/cGMP/protein kinase G; vasodilation

Vascular endothelial protein tyrosine phosphatase (VE-PTP) is an endothelial-specific receptor-type tyrosine phosphatase that associates with Tie-2 and VE-cadherin. VE-PTP gene disruption leads to embryonic lethality, vascular remodeling defects, and enlargement of vascular structures in extraembryonic tissues. We show here that antibodies against the extracellular part of VE-PTP mimic the effects of VE-PTP gene disruption exemplified by vessel enlargement in allantois explants. These effects require the presence of the angiopoietin receptor Tie-2. Analyzing the mechanism we found that anti--VE-PTP antibodies trigger endocytosis and selectively affect Tie-2-associated, but not VE-cadherin--associated VE-PTP. Dissociation of VE-PTP triggers the activation of Tie-2, leading to enhanced endothelial cell proliferation and enlargement of vascular structures through activation of Erk1/2. Importantly, the antibody effect on vessel enlargement is also observed in newborn mice. We conclude that VE-PTP is required to balance Tie-2 activity and endothelial cell proliferation, thereby controlling blood vessel development and vessel size.

Previously, we demonstrated that very low-frequency (VLF) blood pressure variability (BPV) depends on voltage-gated L-type [Ca.sup.2+]-channels, suggesting that autoregulation of blood flow and/or myogenic vascular function significantly contributes to VLF BPV. To further substantiate this possibility, we tested the hypothesis that the frequency response characteristic of whole body autoregulation of blood flow is consistent with the frequency range of VLF BPV (0.02-0.2 Hz) in rats. In anesthetized rats (n = 11), BPV (0.016-0.5 Hz) was induced by computer-regulated cardiac pacing while blood pressure, heart rate, and cardiac output (CO) were recorded during control conditions (NaCl, 1 ml/h iv) and during [[alpha].sub.1]-adrenergic receptor stimulation (phenylephrine, 1 mg x [ml.sup.-1] x [h.sup.-1] iv) that has been reported to facilitate myogenic vascular function. Baroreceptor-heart rate reflex responses were elicited to confirm a functional baroreflex despite anesthesia. During control conditions, transfer function analyses between mean arterial pressure (MAP) and CO, and between MAP and total vascular conductance (CO/MAP) indicated autoregulation of blood flow at 0.016 Hz, passive vascular responses between 0.033 and 0.2 Hz, and vascular responses compatible with baroreflex-mediated mechanisms at 0.333 and 0.5 Hz. Stimulation of [[alpha].sub.1]-adrenergic receptors extended the frequency range of autoregulation of blood flow to frequencies up to 0.033 Hz. In conclusion, depending on sympathetic vascular tone, whole body autoregulation of blood flow operates most effectively at frequencies below 0.05 Hz. This frequency range overlaps with the lower end of the frequency band of VLF BPV in rats. Baroreceptor reflex-like mechanisms contribute to LF (0.2-0.6 Hz) but not VLF BPV-induced vascular responses. cardiac pacing; blood pressure variability; myogenic vascular function

The free radical form of nitric oxide (N[O.sup.x]) is a well-known mediator of vascular tone. What is not so well recognized is that N[O.sup.x] exists in several different redox forms. There is considerable evidence that N[O.sup.x] and its one-electron reduction product, nitroxyl (HNO), have pharmacologically distinct actions that extend into the regulation of the vasculature. The aim of this study was to compare the vasorelaxation mechanisms of HNO and N[O.sup.x], including an examination of the ability of these redox variants to hyperpolarize and repolarize vascular smooth muscle cells from rat mesenteric arteries. The HNO donor Angeli's salt (0.1 nM-10 [micro]M) caused a concentration-dependent hyperpolarization of vessels at resting tone and a simultaneous, concentration-dependent vasorelaxation and repolarization of vessels precontracted and depolarized with methoxamine. Both vasorelaxation and repolarization responses to Angeli's salt were significantly attenuated by both the HNO scavenger L-cysteine (3 mM) and the voltage-dependent [K.sup.+] ([K.sub.v]) channel inhibitor 4-aminopyridine (4-AP; 1 mM) and virtually abolished by the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 [micro]M) or 30 mM [K.sup.+]. In contrast, N[O.sup..] (0.01-1 [micro]M) repolarized arteries to a lesser extent than HNO, and these responses were resistant to inhibition by ODQ (10 [micro]M) and 4-AP (1 mM). Blockade of [K.sub.v] channels (1 mM 4-AP) also significantly inhibited the repolarization response to YC-1 (0.l-10 [micro]M), confirming a role for sGC/cGMP in the activation of [K.sub.v] channels in this preparation. We conclude that HNO causes vasorelaxation via a cGMP-dependent activation of [K.sub.v] channels and that there are different profiles of vasorelaxant activity for the redox siblings HNO and N[O.sup.x]. nitroxyl; hyperpolarization; soluble guanylate cyclase; [K.sup.+] channels; nitric oxide

In response to cold and in fever, heat dissipation from the skin is reduced by sympathetic vasoconstriction. The preoptic region has been implicated in regulating basal, thermal, and febrile vasoconstriction of cutaneous vessels such as the rat's tail, but the neurons responsible for these functions have not been well localized. We recorded activity from single sympathetic nerve fibers supplying tail vessels in urethane-anesthetized rats, while microinjections of GABA (300 mM, 15-30 nl) were used to inhibit neurons in different parts of the preoptic region. Tail fiber activity increased promptly after GABA injections in two distinct regions: a rostromedial preoptic region (RMPO) centered around the organum vasculosum of the lamina terminalis, and a second region centered ~1 mm caudolaterally (CLPO). Responses to GABA within each region were similar. The febrile mediator, [PGE.sub.2] (0.2 or 1 ng in 15 nl) was then microinjected into GABA-sensitive preoptic sites. Injections of [PGE.sub.2] into the RMPO induced a rapid increase in tail fiber activity followed by a rise in core temperature; injections into the rostromedial part of CLPO gave delayed tail fiber responses; injections into the central and caudal parts of CLPO were without effect. These results indicate that neurons in two distinct preoptic regions provide tonic inhibitory drive to the tail vasoconstrictor supply, but febrile vasoconstriction is mediated by [PGE.sub.2] selectively inhibiting neurons in the rostromedial region. prostaglandin [E.sub.2]; gamma-aminobutyric acid; heat conservation

Mitochondria affect cerebrovascular tone by activation of mitochondrial ATP-sensitive [K.sup.+] (KATe) channels and generation of reactive oxygen species (ROS). Insulin resistance accompanying obesity causes mitochondrial dysfunction, but the consequences on the cerebral circulation have not been fully identified. We evaluated the mitochondrial effects of diazoxide, a putative mitochondrial [K.sub.ATP] channel activator, on cerebral arteries of Zucker obese (ZO) rats with insulin resistance and lean (ZL) controls. Diameter measurements showed diminished diazoxide-induced vasodilation in ZO compared with ZL rats. Maximal relaxation was 38 [+ or -] 3% in ZL vs. 21 [+ or -] 4% in ZO rats (P < 0.05). Iberiotoxin, a [Ca.sup.2+]-activated [K.sup.+] channel inhibitor, or manganese(Ill) tetrakis(4-benzoic acid)porphyrin chloride, an SOD mimetic, or endothelial denudation diminished vasodilation to diazoxide, implicating [Ca.sup.2+]-activated [K.sup.+] channels, ROS, and endothelial factors in vasodilation. Inhibition of nitric oxide synthase (NOS) in ZL rats diminished diazoxide-induced vasodilation in intact arteries, but vasodilation was unaffected in endothelium-denuded arteries. In contrast, NOS inhibition in ZO rats enhanced vasodilation in endotheliumdenuded arteries, but intact arteries were unaffected, suggesting that activity of endothelial NOS was abolished, whereas factors derived from nonendothelial NOS promoted vasoconstriction. Fluorescence microscopy showed decreased mitochondrial depolarization, ROS production, and nitric oxide generation in response to diazoxide in ZO arteries. Protein and mRNA measurements revealed increased expression of endothelial NOS and SODs in ZO arteries. Thus, cerebrovascular dilation to mitochondria-derived factors involves integration of endothelial and smooth muscle mechanisms. Furthermore, mitochondria-mediated vasodilation was diminished in ZO rats due to impaired mitochondrial [K.sub.ATP] channel activation, diminished mitochondrial ROS generation, increased ROS scavenging, and abnormal NOS activity. diazoxide; depolarization; mitochondrial ATP-sensitive potassium channels; Zucker obese rats