Adrian J Hobbs

Publications of Adrian J Hobbs

• Dietary Nitrate Ameliorates Pulmonary Hypertension: Cytoprotective Role for Endothelial Nitric Oxide Synthase and Xanthine Oxidoreductase.

  Authors: Reshma S Baliga, Alexandra B Milsom, Suborno M Ghosh, Sarah L Trinder, Raymond J Macallister, Amrita Ahluwalia, Adrian J Hobbs

  Circulation. 05/2012;

  BACKGROUND: Pulmonary hypertension (PH) is a multi-factorial disease characterized by increased pulmonary vascular resistance and right ventricular failure; morbidity and mortality remainBACKGROUND: Pulmonary hypertension (PH) is a multi-factorial disease characterized by increased pulmonary vascular resistance and right ventricular failure; morbidity and mortality remain unacceptably high. Loss of nitric oxide (NO) bioactivity is thought to contribute to the pathogenesis of PH and agents that augment pulmonary NO signaling are clinically effective in the disease. Inorganic nitrate (NO(3)-) and nitrite (NO(2)-) elicit a reduction in systemic blood pressure in healthy individuals; this effect is underpinned by endogenous and sequential reduction to NO. Herein, we determined whether dietary nitrate and nitrite might be preferentially reduced to NO by the hypoxia associated with PH, and thereby offer a convenient, inexpensive method of supplementing NO functionality to reduce disease severity. METHODS AND RESULTS: Dietary nitrate reduced the right ventricular pressure and hypertrophy, and pulmonary vascular re-modeling, in wild-type mice exposed to 3 weeks hypoxia; this beneficial activity was mirrored largely by dietary nitrite. The cytoprotective effects of dietary nitrate were associated with increased plasma & lung concentrations of nitrite and cGMP. The beneficial effects of dietary nitrate and nitrite were reduced in mice lacking endothelial NO synthase (eNOS) or treated with the xanthine oxidoreductase (XOR) inhibitor allopurinol. CONCLUSIONS: These data demonstrate that dietary nitrate, and to a lesser extent dietary nitrite, elicit pulmonary dilatation, prevent pulmonary vascular remodeling, and reduce the RVH characteristic of PH. This favorable pharmacodynamic profile is dependent on eNOS and XOR -catalyzed reduction of nitrite to NO. Exploitation of this mechanism (i.e. dietary nitrate/nitrite supplementation) represents a viable, orally-active therapy for PH.

• Distinct endothelial pathways underlie sexual dimorphism in vascular autoregulation.

  Authors: Melissa V Chan, Kristen J Bubb, Alastair Noyce, Inmaculada C Villar, Johan Duchene, Adrian J Hobbs, Ramona S Scotland, Amrita Ahluwalia

  British journal of pharmacology. 04/2012;

  Background and purpose:  Pre-menopausal females have a lower incidence of cardiovascular disease compared to age-matched males. Differences in the mechanisms/pathways regulating vasoactivity haveBackground and purpose:  Pre-menopausal females have a lower incidence of cardiovascular disease compared to age-matched males. Differences in the mechanisms/pathways regulating vasoactivity have been implicated. Small arteries constrict in response to rises in intraluminal pressure; a response termed myogenic tone that is a major determinant of vascular resistance. Endothelium-derived dilators, particularly nitric oxide (NO), tonically moderate myogenic tone and, since the endothelium is an important target for female sex hormones, we investigated whether NO-mediated moderation of myogenic tone differs between the sexes. Experimental approach:  Pressure-diameter or relaxation concentration-response curves to NO donor Spermine-NO or soluble guanylate cyclase (sGC) stimulation (BAY41-2272) were constructed before and following drug intervention in murine mesenteric resistance arteries. Hypotensive responses to activators of the NO-sGC pathway were determined. Quantitative PCR and western blotting were used for expression analysis. Key results:  NO synthase inhibition enhanced myogenic tone of arteries of both sexes whilst block of endothelium-derived hyperpolarising factor (EDHF) enhanced responses in arteries of females only. Spermine-NO caused concentration-dependent relaxation of mesenteric arteries isolated from either sex. However, whilst inhibition of sGC activity attenuated responses of arteries from male mice only, endothelium-denudation attenuated responses of arteries from female mice only. BAY41-2272 and Spermine-NO-induced vasodilatation and hypotension were enhanced in males vs females. Conclusions and implications:  These data demonstrate that NO moderates myogenic tone in arteries of male mice via an sGC-dependent pathway whilst EDHF is the predominant endothelial regulator in arteries of females. We have identified a potentially important sexual dimorphism in NO-mediated reactivity and further implicate EDHF as the predominant endothelial vasodilator in female resistance arteries. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

• Manganese carbonyls bearing tripodal polypyridine ligands as photoactive carbon monoxide-releasing molecules.

  Authors: Margarita A Gonzalez, Melanie A Yim, Stephanie Cheng, Amie Moyes, Adrian J Hobbs, Pradip K Mascharak

  Inorganic chemistry. 12/2011; 51(1):601-8.

  The recently discovered cytoprotective action of CO has raised interest in exogenous CO-releasing materials (CORMs) such as metal carbonyls (CO complexes of transition metals). To achieve control onThe recently discovered cytoprotective action of CO has raised interest in exogenous CO-releasing materials (CORMs) such as metal carbonyls (CO complexes of transition metals). To achieve control on CO delivery with metal carbonyls, we synthesized and characterized three Mn(I) carbonyls, namely, [Mn(tpa)(CO)(3)]ClO(4) [1, where tpa = tris(2-pyridyl)amine], [Mn(dpa)(CO)(3)]Br [2, where dpa = N,N-bis(2-pyridylmethyl)amine], and [Mn(pqa)(CO)(3)]ClO(4) [3, where pqa = (2-pyridylmethyl)(2-quinolylmethyl)amine], by crystallography and various spectroscopic techniques. All three carbonyls are sensitive to light and release CO when illuminated with low-power UV (5-10 mW) and visible (λ > 350 nm, ~100 mW) light. The sensitivity of 1-3 to light has been assessed with respect to the number of pyridine groups in their ligand frames. When a pyridine ring is replaced with quinoline, extended conjugation in the ligand frame increases the absorptivity and makes the resulting carbonyl 3 more sensitive to visible light. These photosensitive CORMs (photoCORMs) have been employed to deliver CO to myoglobin under the control of light. The superior stability of 3 in aqueous media makes it a photoCORM suitable for inducing vasorelaxation in mouse aortic muscle rings.

• LXRα regulates macrophage arginase 1 through PU.1 and interferon regulatory factor 8.

  Authors: Benoit Pourcet, Jonathan E Feig, Yuliya Vengrenyuk, Adrian J Hobbs, Diane Kepka-Lenhart, Michael J Garabedian, Sidney M Morris, Edward A Fisher, Inés Pineda-Torra

  Circulation research. 08/2011; 109(5):492-501.

  Activation of liver X receptors (LXRs) inhibits the progression of atherosclerosis and promotes regression of existing lesions. In addition, LXRα levels are high in regressive plaques. MacrophageActivation of liver X receptors (LXRs) inhibits the progression of atherosclerosis and promotes regression of existing lesions. In addition, LXRα levels are high in regressive plaques. Macrophage arginase 1 (Arg1) expression is inversely correlated with atherosclerosis progression and is markedly decreased in foam cells within the lesion. To investigate LXRα regulation of Arg1 expression in cultured macrophages and atherosclerotic regressive lesions. We found that Arg1 expression is enhanced in CD68+ cells from regressive versus progressive lesions in a murine aortic arch transplant model. In cultured macrophages, ligand-activated LXRα markedly enhances basal and interleukin-4-induced Arg1 mRNA and protein expression as well as promoter activity. This LXRα-enhanced Arg1 expression correlates with a reduction in nitric oxide levels. Moreover, Arg1 expression within regressive atherosclerotic plaques is LXRα-dependent, as enhanced expression of Arg1 in regressive lesions is impaired in LXRα-deficient CD68+ cells. LXRα does not bind to the Arg1 promoter but instead promotes the interaction between PU.1 and interferon regulatory factor (IRF)8 transcription factors and induces their binding of a novel composite element. Accordingly, knockdown of either IRF8 or PU.1 strongly impairs LXRα regulation of Arg1 expression in macrophage cells. Finally, we demonstrate that LXRα binds the IRF8 locus and its activation increases IRF8 mRNA and protein levels in these cells. This work implicates Arg1 in atherosclerosis regression and identifies LXRα as a novel regulator of Arg1 and IRF8 in macrophages. Furthermore, it provides a unique molecular mechanism by which LXRα regulates macrophage target gene expression through PU.1 and IRF8.

• Natriuretic peptide receptor-3 underpins the disparate regulation of endothelial and vascular smooth muscle cell proliferation by C-type natriuretic peptide.

  Authors: Rayomand S Khambata, Catherine M Panayiotou, Adrian J Hobbs

  British journal of pharmacology. 04/2011; 164(2b):584-97.

  C-type natriuretic peptide (CNP) is an endothelium-derived vasorelaxant, exerting anti-atherogenic actions in the vasculature and salvaging the myocardium from ischaemic injury. The cytoprotectiveC-type natriuretic peptide (CNP) is an endothelium-derived vasorelaxant, exerting anti-atherogenic actions in the vasculature and salvaging the myocardium from ischaemic injury. The cytoprotective effects of CNP are mediated in part via the G(i) -coupled natriuretic peptide receptor (NPR)3. As GPCRs are well-known to control cell proliferation, we investigated if NPR3 activation underlies effects of CNP on endothelial and vascular smooth muscle cell mitogenesis. Proliferation of human umbilical vein endothelial cells (HUVEC), rat aortic smooth muscle cells (RAoSMC) and endothelial and vascular smooth muscle cells from NPR3 knockout (KO) mice was investigated in vitro. CNP (1 pM-1 µM) facilitated HUVEC proliferation and inhibited RAoSMC growth concentration-dependently. The pro- and anti-mitogenic effects of CNP were blocked by the NPR3 antagonist M372049 (10 µM) and the extracellular signal-regulated kinase (ERK) 1/2 inhibitor PD98059 (30 µM) and were absent in cells from NPR3 KO mice. Activation of ERK 1/2 by CNP was inhibited by Pertussis toxin (100 ng·mL⁻¹) and M372049 (10 µM). In HUVEC, ERK 1/2 activation enhanced expression of the cell cycle promoter, cyclin D1, whereas in RAoSMC, ERK 1/2 activation increased expression of the cell cycle inhibitors p21(waf1/cip1) and p27(kip1) . A facet of the vasoprotective profile of CNP is mediated via NPR3-dependent ERK 1/2 phosphorylation, resulting in augmented endothelial cell proliferation and inhibition of vascular smooth muscle growth. This pathway may offer an innovative approach to reversing the endothelial damage and vascular smooth muscle hyperplasia that characterize many vascular disorders.

• Suppression of endothelial P-selectin expression contributes to reduced cell trafficking in females: an effect independent of NO and prostacyclin.

  Authors: Inmaculada C Villar, Ramona S Scotland, Rayomand S Khambata, Melissa Chan, Johan Duchene, Andre L Sampaio, Mauro Perretti, Adrian J Hobbs, Amrita Ahluwalia

  Arteriosclerosis, thrombosis, and vascular biology. 02/2011; 31(5):1075-83.

  Sex hormones underlie the lower incidence of cardiovascular disease in premenopausal women. Vascular inflammation is involved in the pathogenesis of several cardiovascular diseases and it has beenSex hormones underlie the lower incidence of cardiovascular disease in premenopausal women. Vascular inflammation is involved in the pathogenesis of several cardiovascular diseases and it has been reported that sex hormones modulate inflammatory responses but mechanisms responsible for these effects are not yet fully established. Herein, we assessed whether sex differences in leukocyte recruitment might exist and investigated the underlying mechanisms involved in this response. Treatment with interleukin-1β (IL-1β) or tumor necrosis factor-α caused leukocyte rolling, adhesion, and emigration in mesenteric postcapillary venules in vivo that was substantially reduced in female mice compared with male mice; this difference was abolished by ovariectomy and partially restored by estrogen replacement. Deletion of endothelial nitric oxide (NO) synthase or cyclooxygenase-1 alone or in combination did not alter the leukocyte recruitment in IL-1β-treated females but significantly enhanced this response in male mice. Treatment of murine pulmonary endothelial cells with IL-1β increased expression of P-selectin in male but not female cells. We have demonstrated a profound estrogen-dependent and NO and prostacyclin-independent suppression of leukocyte recruitment in females.

• New perspectives for the treatment of pulmonary hypertension.

  Authors: Reshma S Baliga, Raymond J MacAllister, Adrian J Hobbs

  British journal of pharmacology. 12/2010; 163(1):125-40.

  Pulmonary hypertension (PH) is a debilitating disease with a poor prognosis. Therapeutic options remain limited despite the introduction of prostacyclin analogues, endothelin receptor antagonists andPulmonary hypertension (PH) is a debilitating disease with a poor prognosis. Therapeutic options remain limited despite the introduction of prostacyclin analogues, endothelin receptor antagonists and phosphodiesterase 5 inhibitors within the last 15 years; these interventions address predominantly the endothelial and vascular dysfunctionS associated with the condition, but simply delay progression of the disease rather than offer a cure. In an attempt to improve efficacy, emerging approaches have focused on targeting the pro-proliferative phenotype that underpins the pulmonary vascular remodelling in the lung and contributes to the impaired circulation and right heart failure. Many novel targets have been investigated and validated in animal models of PH, including modulation of guanylate cyclases, phosphodiesterases, tyrosine kinases, Rho kinase, bone morphogenetic proteins signalling, 5-HT, peroxisome proliferator activator receptors and ion channels. In addition, there is hope that combinations of such treatments, harnessing and optimizing vasodilator and anti-proliferative properties, will provide a further, possibly synergistic, increase in efficacy; therapies directed at the right heart may also offer an additional benefit. This overview highlights current therapeutic options, promising new therapies, and provides the rationale for a combination approach to treat the disease.

• Phospholemman Ser69 phosphorylation contributes to sildenafil-induced cardioprotection against reperfusion injury.

  Authors: Melanie Madhani, Andrew R Hall, Friederike Cuello, Rebecca L Charles, Joseph R Burgoyne, William Fuller, Adrian J Hobbs, Michael J Shattock, Philip Eaton

  American journal of physiology. Heart and circulatory physiology. 09/2010; 299(3):H827-36.

  The phosphodiesterase type-5 inhibitor sildenafil has powerful cardioprotective effects against ischemia-reperfusion injury. PKG-mediated signaling has been implicated in this protection, althoughThe phosphodiesterase type-5 inhibitor sildenafil has powerful cardioprotective effects against ischemia-reperfusion injury. PKG-mediated signaling has been implicated in this protection, although the mechanism and the downstream targets of this kinase remain to be fully elucidated. In this study we assessed the role of phospholemman (PLM) phosphorylation, which activates the Na(+)/K(+)-ATPase, in cardioprotection afforded by sildenafil administered during reperfusion. Isolated perfused mouse hearts were optimally protected against infarction (indexed by tetrazolium staining) by 0.1 muM sildenafil treatment during the first 10 min of reperfusion. Extended sildenafil treatment (30, 60, or 120 min at reperfusion) did not alter the degree of protection provided. This protection was PKG dependent, since it was blocked by KT-5823. Western blot analysis using phosphospecific antibodies to PLM showed that sildenafil at reperfusion did not modulate PLM Ser63 or Ser68 phosphorylation but significantly increased Ser69 phosphorylation. The treatment of isolated rat ventricular myocytes with sildenafil or 8-bromo-cGMP (PKG agonist) enhanced PLM Ser69 phosphorylation, which was bisindolylmaleimide (PKC inhibitor) sensitive. Patch-clamp studies showed that sildenafil treatment also activated the Na(+)/K(+)-ATPase, which is anticipated in light of PLM Ser69 phosphorylation. Na(+)/K(+)-ATPase activation during reperfusion would attenuate Na(+) overload at this time, providing a molecular explanation of how sildenafil guards against injury at this time. Indeed, using flame photometry and rubidium uptake into isolated mouse hearts, we found that sildenafil enhanced Na(+)/K(+)-ATPase activity during reperfusion. In this study we provide a molecular explanation of how sildenafil guards against myocardial injury during postischemic reperfusion.

• Resistance to endotoxic shock in mice lacking natriuretic peptide receptor-A.

  Authors: Catherine M Panayiotou, Reshma Baliga, Raymond Stidwill, Valerie Taylor, Mervyn Singer, Adrian J Hobbs

  British journal of pharmacology. 08/2010; 160(8):2045-54.

  Excessive production of nitric oxide (NO) by inducible NO synthase (iNOS) is thought to underlie the vascular dysfunction, systemic hypotension and organ failure that characterize endotoxic shock.Excessive production of nitric oxide (NO) by inducible NO synthase (iNOS) is thought to underlie the vascular dysfunction, systemic hypotension and organ failure that characterize endotoxic shock. Plasma levels of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) are raised in animal models and humans with endotoxic shock and correlate with the associated cardiovascular dysfunction. Since both NO and natriuretic peptides play important roles in cardiovascular homeostasis via activation of guanylate cyclase-linked receptors, we used mice lacking natriuretic peptide receptor (NPR)-A (NPR1) to establish if natriuretic peptides contribute to the cardiovascular dysfunction present in endotoxic shock. Wild-type (WT) and NPR-A knockout (KO) mice were exposed to lipopolysaccharide (LPS) and vascular dysfunction (in vitro and in vivo), production of pro-inflammatory cytokines, and iNOS expression and activity were evaluated. LPS-treated WT animals exhibited a marked fall in mean arterial blood pressure (MABP) whereas NPR-A KO mice maintained MABP throughout. LPS administration caused a greater suppression of vascular responses to the thromboxane-mimetic U46619, ANP, acetylcholine and the NO-donor spermine-NONOate in WT versus NPR-A KO mice. This differential effect on vascular function was paralleled by reduced pro-inflammatory cytokine production, iNOS expression and activity (plasma [NO(x)] and cyclic GMP). These observations suggest that NPR-A activation by natriuretic peptides facilitates iNOS expression and contributes to the vascular dysfunction characteristic of endotoxic shock. Pharmacological interventions that target the natriuretic peptide system may represent a novel approach to treat this life-threatening condition.

• Inorganic nitrate supplementation lowers blood pressure in humans: role for nitrite-derived NO.

  Authors: Vikas Kapil, Alexandra B Milsom, Michael Okorie, Sheiva Maleki-Toyserkani, Farihah Akram, Farkhanda Rehman, Shah Arghandawi, Vanessa Pearl, Nigel Benjamin, Stavros Loukogeorgakis, Raymond Macallister, Adrian J Hobbs, Andrew J Webb, Amrita Ahluwalia

  Hypertension. 08/2010; 56(2):274-81.

  Ingestion of dietary (inorganic) nitrate elevates circulating and tissue levels of nitrite via bioconversion in the entero-salivary circulation. In addition, nitrite is a potent vasodilator inIngestion of dietary (inorganic) nitrate elevates circulating and tissue levels of nitrite via bioconversion in the entero-salivary circulation. In addition, nitrite is a potent vasodilator in humans, an effect thought to underlie the blood pressure-lowering effects of dietary nitrate (in the form of beetroot juice) ingestion. Whether inorganic nitrate underlies these effects and whether the effects of either naturally occurring dietary nitrate or inorganic nitrate supplementation are dose dependent remain uncertain. Using a randomized crossover study design, we show that nitrate supplementation (KNO(3) capsules: 4 versus 12 mmol [n=6] or 24 mmol of KNO(3) (1488 mg of nitrate) versus 24 mmol of KCl [n=20]) or vegetable intake (250 mL of beetroot juice [5.5 mmol nitrate] versus 250 mL of water [n=9]) causes dose-dependent elevation in plasma nitrite concentration and elevation of cGMP concentration with a consequent decrease in blood pressure in healthy volunteers. In addition, post hoc analysis demonstrates a sex difference in sensitivity to nitrate supplementation dependent on resting baseline blood pressure and plasma nitrite concentration, whereby blood pressure is decreased in male volunteers, with higher baseline blood pressure and lower plasma nitrite concentration but not in female volunteers. Our findings demonstrate dose-dependent decreases in blood pressure and vasoprotection after inorganic nitrate ingestion in the form of either supplementation or by dietary elevation. In addition, our post hoc analyses intimate sex differences in nitrate processing involving the entero-salivary circulation that are likely to be major contributing factors to the lower blood pressures and the vasoprotective phenotype of premenopausal women.

• C-type natriuretic peptide (CNP): cardiovascular roles and potential as a therapeutic target.

  Authors: Natalie G Lumsden, Rayomand S Khambata, Adrian J Hobbs

  Current pharmaceutical design. 01/2010; 16(37):4080-8.

  Natriuretic peptides play a fundamental role in cardiovascular homeostasis by modulation of fluid and electrolyte balance and vascular tone. C-type natriuretic peptide (CNP) represents the paracrineNatriuretic peptides play a fundamental role in cardiovascular homeostasis by modulation of fluid and electrolyte balance and vascular tone. C-type natriuretic peptide (CNP) represents the paracrine element of the natriuretic peptide axis which complements the endocrine actions of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). CNP is produced by the endothelium and the heart and appears to play a prominent role in vascular and cardiac function, both physiologically and pathologically. This provides a rationale for the therapeutic potential of pharmacological interventions targeted to CNP signalling. This article provides an overview of the biology and pharmacology of CNP, with emphasis on the cardiovascular system, and discusses pathologies in which drugs designed to manipulate CNP signalling maybe of clinical benefit.

• Laminar Shear Stress Regulates Endothelial Kinin B1 Receptor Expression and Function. Potential Implication in Atherogenesis.

  Authors: Johan Duchene, Cécile Cayla, Sandrine Vessillier, Ramona Scotland, Kazuo Yamashiro, Florence Lecomte, Irfan Syed, Phuong Vo, Alessandra Marrelli, Costantino Pitzalis, Francesco Cipollone, Joost Schanstra, Jean-Loup Bascands, Adrian J Hobbs, Mauro Perretti, Amrita Ahluwalia

  Arteriosclerosis, thrombosis, and vascular biology. 09/2009;

  OBJECTIVE: The proinflammatory phenotype induced by low laminar shear stress (LSS) is implicated in atherogenesis. The kinin B1 receptor (B1R), known to be induced by inflammatory stimuli, exertsOBJECTIVE: The proinflammatory phenotype induced by low laminar shear stress (LSS) is implicated in atherogenesis. The kinin B1 receptor (B1R), known to be induced by inflammatory stimuli, exerts many proinflammatory effects including vasodilatation and leukocyte recruitment. We investigated whether low LSS is a stimulus for endothelial B1R expression and function. METHODS AND RESULTS: Human and mouse atherosclerotic plaques expressed high level of B1R mRNA and protein. In addition, B1R expression was upregulated in the aortic arch (low LSS region) of ApoE(-/-) mice fed a high-fat diet compared to vascular regions of high LSS and animals fed normal chow. Of interest, a greater expression of B1R was noticed in endothelial cells from regions of low LSS in aortic arch of ApoE(-/-) mice. B1R was also upregulated in human umbilical vein endothelial cells (HUVECs) exposed to low LSS (0 to 2 dyn/cm(2)) compared to physiological LSS (6 to 10 dyn/cm(2)): an effect similarly evident in murine vascular tissue perfused ex vivo. Functionally, B1R activation increased prostaglandin and CXCL5 expression in cells exposed to low, but not physiological, LSS. IL-1beta and ox-LDL induced B1R expression and function in HUVECs, a response substantially enhanced under low LSS conditions and inhibited by blockade of NFkappaB activation. CONCLUSIONS: Herein, we show that LSS is a major determinant of functional B1R expression in endothelium. Furthermore, whereas physiological high LSS is a powerful repressor of this inflammatory receptor, low LSS at sites of atheroma are associated with substantial upregulation, identifying this receptor as a potential therapeutic target.

• The effects of nitroxyl (HNO) on soluble guanylate cyclase activity: Interactions at ferrous heme and cysteine thiols.

  Authors: Tomas W Miller, Melisa M Cherney, Andrea J Lee, Nestor E Francoleone, Patrick J Farmer, S Bruce King, Adrian J Hobbs, Katrina M Miranda, Judith N Burstyn, Jon M Fukuto

  The Journal of biological chemistry. 07/2009;

  It has been previously proposed that nitric oxide (NO) is the only biologically relevant nitrogen oxide capable of activating the enzyme soluble guanylate cyclase (sGC). However, recent reportsIt has been previously proposed that nitric oxide (NO) is the only biologically relevant nitrogen oxide capable of activating the enzyme soluble guanylate cyclase (sGC). However, recent reports implicate HNO as another possible activator of sGC. Herein, we examine the affect of HNO donors on the activity of purified bovine lung sGC and find that, indeed, HNO is capable of activating this enzyme. Like NO, HNO activation appears to occur via interaction with the regulatory ferrous heme on sGC. Somewhat unexpectedly, HNO does not activate the ferric form of the enzyme. Finally, HNO-mediated cysteine thiol modification appears to also affect enzyme activity leading to inhibition. Thus, sGC activity can be regulated by HNO via interactions at both the regulatory heme and cysteine thiols.

• Mechanisms Underlying Erythrocyte and Endothelial Nitrite Reduction to Nitric Oxide in Hypoxia. Role for Xanthine Oxidoreductase and Endothelial Nitric Oxide Synthase.

  Authors: Andrew J Webb, Alexandra B Milsom, Krishnaraj S Rathod, Wai Lum Chu, Shehla Qureshi, Matthew J Lovell, Florence M J Lecomte, David Perrett, Carmello Raimondo, Espeed Khoshbin, Zubair Ahmed, Rakesh Uppal, Nigel Benjamin, Adrian J Hobbs, Amrita Ahluwalia

  Circulation research. 10/2008;

  Reduction of nitrite (NO2(-)) provides a major source of nitric oxide (NO) in the circulation, especially in hypoxemic conditions. Our previous studies suggest that xanthine oxidoreductase (XOR) isReduction of nitrite (NO2(-)) provides a major source of nitric oxide (NO) in the circulation, especially in hypoxemic conditions. Our previous studies suggest that xanthine oxidoreductase (XOR) is an important nitrite reductase in the heart and kidney. Herein, we have demonstrated that conversion of nitrite to NO by blood vessels and RBCs was enhanced in the presence of the XOR substrate xanthine (10 micromol/L) and attenuated by the XOR inhibitor allopurinol (100 micromol/L) in acidic and hypoxic conditions only. Whereas endothelial nitric oxide synthase (eNOS) inhibition had no effect on vascular nitrite reductase activity, in RBCs L-NAME, L-NMMA, and L-arginine inhibited nitrite-derived NO production by >50% (P<0.01) at pH 7.4 and 6.8 under hypoxic conditions. Western blot and immunohistochemical analysis of RBC membranes confirmed the presence of eNOS and abundant XOR on whole RBCs. Thus, XOR and eNOS are ideally situated on the membranes of RBCs and blood vessels to generate intravascular vasodilator NO from nitrite during ischemic episodes. In addition to the proposed role of deoxyhemoglobin, our findings suggest that the nitrite reductase activity within the circulation, under hypoxic conditions (at physiological pH), is mediated by eNOS; however, as acidosis develops, a substantial role for XOR becomes evident.

• Synergy Between Natriuretic Peptides and PDE5 Inhibitors Ameliorates Pulmonary Arterial Hypertension.

  Authors: Reshma S Baliga, Lan Zhao, Melanie Madhani, Belen Lopez-Torondel, Cristina Visintin, David Selwood, Martin R Wilkins, Raymond J MacAllister, Adrian J Hobbs

  American journal of respiratory and critical care medicine. 09/2008;

  Rationale/ OBJECTIVES: Phosphodiesterase (PDE) 5 inhibitors (e.g. Sildenafil) are selective pulmonary vasodilators in patients with pulmonary arterial hypertension (PAH). The mechanism(s) underlyingRationale/ OBJECTIVES: Phosphodiesterase (PDE) 5 inhibitors (e.g. Sildenafil) are selective pulmonary vasodilators in patients with pulmonary arterial hypertension (PAH). The mechanism(s) underlying this specificity remains unclear, but studies in genetically-modified animals intimate it might be dependent on natriuretic peptide bioactivity. Herein, we explored the interaction between PDE5 inhibitors and the natriuretic peptide system in vitro and in vivo, including an animal model of PH, to elucidate the (patho)physiological relationship between these two cGMP-regulating systems and potential of a combination therapy exploiting these cooperative pathways. Methods/ MAIN RESULTS: In vitro pharmacological studies using rat vascular tissue revealed that Sildenafil augments vasodilator responses to nitric oxide (NO) in both pulmonary and systemic conduit and resistance arteries whereas identical relaxant responses to ANP are enhanced only in pulmonary vessels. This differential activity was mirrored in vivo where Sildenafil increases the hypotensive actions of ANP in the pulmonary, but not systemic, vasculature. In an animal model of (hypoxia-induced) PH, combination treatment with Sildenafil plus the neutral endopeptidase (NEP) inhibitor Ecadotril (increases endogenous natriuretic peptide levels) acted synergistically, in a cGMP-dependent manner, to reduce many indices of disease severity (e.g. pulmonary artery pressure, right ventricular hypertrophy and pulmonary vascular re-modeling) without significantly affecting systemic blood pressure. CONCLUSIONS: These data demonstrate that PDE5 is a key regulator of cyclic GMP (cGMP)-mediated vasodilatation by ANP in the pulmonary, but not systemic, vasculature, thereby explaining the pulmonary-selectivity of PDE5 inhibitors. Furthermore, exploitation of this mechanism (i.e. PDE5 and NEP inhibition) represents a novel, orally-active combination therapy for PAH.

• Characterization of the Human {alpha}1{beta}1 Soluble Guanylyl Cyclase Promoter: KEY ROLE FOR NF-{kappa}B(p50) AND CCAAT-BINDING FACTORS IN REGULATING EXPRESSION OF THE NITRIC OXIDE RECEPTOR.

  Authors: Martín L. Marro, Concepción Peiró, Catherine M Panayiotou, Reshma S Baliga, Sabine Meurer, Harald H H W Schmidt, Adrian J Hobbs

  The Journal of biological chemistry. 08/2008; 283(29):20027-36.

  Soluble guanylyl cyclase (sGC) is the principal receptor for NO and plays a ubiquitous role in regulating cellular function. This is exemplified in the cardiovascular system where sGC governs smoothSoluble guanylyl cyclase (sGC) is the principal receptor for NO and plays a ubiquitous role in regulating cellular function. This is exemplified in the cardiovascular system where sGC governs smooth muscle tone and growth, vascular permeability, leukocyte flux, and platelet aggregation. As a consequence, aberrant NO-sGC signaling has been linked to diseases including hypertension, atherosclerosis, and stroke. Despite these key (patho)physiological roles, little is known about the expressional regulation of sGC. To address this deficit, we have characterized the promoter activity of human alpha(1) and beta(1) sGC genes in a cell type relevant to cardiovascular (patho)physiology, primary human aortic smooth muscle cells. Luciferase reporter constructs revealed that the 0.3- and 0.5-kb regions upstream of the transcription start sites were optimal for alpha(1) and beta(1) sGC promoter activity, respectively. Deletion of consensus sites for c-Myb, GAGA, NFAT, NF-kappaB(p50), and CCAAT-binding factor(s) (CCAAT-BF) revealed that these are the principal transcription factors regulating basal sGC expression. In addition, under pro-inflammatory conditions, the effects of the strongest alpha(1) and beta(1) sGC repressors were enhanced, and enzyme expression and activity were reduced; in particular, NF-kappaB(p50) is pivotal in regulating enzyme expression under such conditions. NO itself also elicited a cGMP-independent negative feedback effect on sGC promoter activity that is mediated, in part, via CCAAT-BF activity. In sum, these data provide a systematic characterization of the promoter activity of human sGC alpha(1) and beta(1) subunits and identify key transcription factors that govern subunit expression under basal and pro-inflammatory (i.e. atherogenic) conditions and in the presence of ligand NO.

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• Sex differences in vascular function: implication of endothelium-derived hyperpolarizing factor.

  Authors: Inmaculada C Villar, Adrian J Hobbs, Amrita Ahluwalia

  The Journal of endocrinology. 07/2008; 197(3):447-62.

  The vascular endothelium plays a crucial role in the regulation of vascular homeostasis by controlling vascular tone, coagulation, and inflammatory responses. These actions are exerted by endothelialThe vascular endothelium plays a crucial role in the regulation of vascular homeostasis by controlling vascular tone, coagulation, and inflammatory responses. These actions are exerted by endothelial factors including nitric oxide, prostacyclin, and endothelium-derived hyperpolarizing factor (EDHF). The greater incidence of cardiovascular disease (CVD) in men and postmenopausal women compared with premenopausal women implies a vasoprotective phenotype of females, which may be influenced by sex hormones. These hormones, particularly estrogen, have modulatory effects on the endothelium and circulating cells that have been implicated in vascular inflammation and in the development of CVD. EDHF seems to be the predominant endothelial factor in the resistance vasculature of females and this mediator could afford the beneficial cardiovascular risk profile observed in premenopausal woman. In this review, we discuss sex differences in EDHF biology and how sex hormones can modulate EDHF responses. We also review the implication of sex hormone-dependent regulation of EDHF in inflammatory processes, platelet function, and repair after vascular damage, each of which have a critical role in several aspects of the pathogenesis of CVD.

• Circulating endothelial progenitor cells in patients with Eisenmenger syndrome and idiopathic pulmonary arterial hypertension.

  Authors: Gerhard-Paul Diller, Sven van Eijl, Darlington O Okonko, Luke S Howard, Omar Ali, Thomas Thum, Stephen J Wort, Elisabeth Bédard, J Simon R Gibbs, Johann Bauersachs, Adrian J Hobbs, Martin R Wilkins, Michael A Gatzoulis, John Wharton

  Circulation. 06/2008; 117(23):3020-30.

  Impaired endothelial homeostasis underlies the pathophysiology of pulmonary arterial hypertension (PAH). We speculated that PAH patients are deficient in circulating endothelial progenitor cellsImpaired endothelial homeostasis underlies the pathophysiology of pulmonary arterial hypertension (PAH). We speculated that PAH patients are deficient in circulating endothelial progenitor cells (EPCs), potentially contributing to endothelial dysfunction and disease progression. We recruited 41 patients with Eisenmenger syndrome (13 with Down syndrome), 55 with idiopathic PAH, and 47 healthy control subjects. Flow cytometry and in vitro assays were used to quantify EPCs and to assess cell function. The number of circulating CD34+, CD34+/AC133+, CD34+/KDR+, and CD34+/AC133+/KDR+ progenitor cells was low in Eisenmenger patients compared with healthy control subjects, and those with Down syndrome displayed even fewer EPCs. Reductions in EPC numbers correlated with New York Heart Association functional class, 6-minute walk distance, and plasma brain-type natriuretic peptide levels. The capacity of cultured peripheral blood mononuclear cells to form colonies and incorporate into tube-like structures was impaired in Eisenmenger patients. Idiopathic PAH patients had reduced numbers of EPCs, and the number of circulating EPCs correlated with invasive hemodynamic parameters in this cohort. Levels of immune inflammatory markers, cGMP, stable nitric oxide oxidation products, and asymmetric dimethylarginine were abnormal in patients with PAH and related to numbers of EPCs. Within the idiopathic PAH population, treatment with the phosphodiesterase inhibitor sildenafil was associated with a dose-dependent rise in EPC numbers, resulting in levels consistently above those found with other therapies. Circulating EPC numbers are reduced in 2 well-characterized forms of PAH, which also exhibit raised levels of inflammatory mediators. Sildenafil treatment may represent a pharmacological means of increasing circulating EPC numbers long-term.

• Acute blood pressure lowering, vasoprotective, and antiplatelet properties of dietary nitrate via bioconversion to nitrite.

  Authors: Andrew J Webb, Nakul Patel, Stavros Loukogeorgakis, Mike Okorie, Zainab Aboud, Shivani Misra, Rahim Rashid, Philip Miall, John Deanfield, Nigel Benjamin, Raymond MacAllister, Adrian J Hobbs, Amrita Ahluwalia

  Hypertension. 04/2008; 51(3):784-90.

  Diets rich in fruits and vegetables reduce blood pressure (BP) and the risk of adverse cardiovascular events. However, the mechanisms of this effect have not been elucidated. Certain vegetablesDiets rich in fruits and vegetables reduce blood pressure (BP) and the risk of adverse cardiovascular events. However, the mechanisms of this effect have not been elucidated. Certain vegetables possess a high nitrate content, and we hypothesized that this might represent a source of vasoprotective nitric oxide via bioactivation. In healthy volunteers, approximately 3 hours after ingestion of a dietary nitrate load (beetroot juice 500 mL), BP was substantially reduced (Delta(max) -10.4/8 mm Hg); an effect that correlated with peak increases in plasma nitrite concentration. The dietary nitrate load also prevented endothelial dysfunction induced by an acute ischemic insult in the human forearm and significantly attenuated ex vivo platelet aggregation in response to collagen and ADP. Interruption of the enterosalivary conversion of nitrate to nitrite (facilitated by bacterial anaerobes situated on the surface of the tongue) prevented the rise in plasma nitrite, blocked the decrease in BP, and abolished the inhibitory effects on platelet aggregation, confirming that these vasoprotective effects were attributable to the activity of nitrite converted from the ingested nitrate. These findings suggest that dietary nitrate underlies the beneficial effects of a vegetable-rich diet and highlights the potential of a "natural" low cost approach for the treatment of cardiovascular disease.

• Definitive role for natriuretic peptide receptor-C in mediating the vasorelaxant activity of C-type natriuretic peptide and endothelium-derived hyperpolarising factor.

  Authors: Inmaculada C Villar, Catherine M Panayiotou, Adil Sheraz, Melanie Madhani, Ramona S Scotland, Muriel Nobles, Barbara Kemp-Harper, Amrita Ahluwalia, Adrian J Hobbs

  Cardiovascular research. 06/2007; 74(3):515-25.

  C-type natriuretic peptide (CNP) has recently been suggested to represent an endothelium-derived hyperpolarising factor (EDHF) in the mammalian resistance vasculature and, as such, important in theC-type natriuretic peptide (CNP) has recently been suggested to represent an endothelium-derived hyperpolarising factor (EDHF) in the mammalian resistance vasculature and, as such, important in the regulation of local blood flow and systemic blood pressure. Additionally, this peptide has been shown to protect against ischaemia-reperfusion injury and inhibits leukocyte and platelet activation. Herein, we use a novel, selective natriuretic peptide receptor-C (NPR-C) antagonist (M372049) to highlight the pivotal contribution of CNP/NPR-C signalling in the EDHF-dependent regulation of vascular tone and investigate the mechanism(s) underlying the release and biological activity of CNP. In vitro pharmacological investigation was conducted in rat (Sprague-Dawley) aorta and mesenteric resistance arteries. Relaxant responses to CNP, atrial natriuretic peptide (ANP), the nitric oxide donor spermine-NONOate (SPER-NO) and the endothelium-dependent vasodilator, acetylcholine (ACh) were examined in the absence and presence of M372049 or inhibitor cocktails shown previously to block endothelium-dependent dilatation in the resistance vasculature. RT-PCR was employed to characterize the expression of NPR subtypes in the vessels studied. M372049 produced concentration-dependent inhibition of the vasorelaxant activity of CNP in rat isolated mesenteric resistance arteries but not aorta; in contrast, M372049 did not affect relaxations to ANP or SPER-NO in either vessel. M372049 or ouabain alone produced small, significant inhibition of EDHF-dependent relaxations in mesenteric arteries and in combination acted synergistically to abolish such responses. A combination of M372049 with established inhibitors of EDHF-dependent relaxation revealed that multiple, distinct pathways coordinate the bioactivity of EDHF in the resistance vasculature, and that CNP/NPR-C signalling represents a major component. These data substantiate CNP/NPR-C signalling as a fundamental pathway underlying EDHF-dependent regulation of vascular tone in the rat mesenteric resistance vasculature. An increased understanding of the physiological roles of CNP/NPR-C signalling in the vasculature (now facilitated by the identification of a selective NPR-C antagonist) should aid determination of the (patho)physiological importance of EDHF and might provide the rationale for the design of novel therapeutics.