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Pulmonary arterial hypertension (PAH) is a chronic and progressive disorder characterized by vascular remodeling of the small pulmonary arteries, resulting in elevated pulmonary vascular resistance and ultimately, right ventricular failure. Expanded understanding of PAH pathophysiology as it pertains to the nitric oxide (NO), prostacyclin (prostaglandin I2) (PGI2) and endothelin-1 pathways has led to recent advancements in targeted drug development and substantial improvements in morbidity and mortality. There are currently several classes of drugs available to target these pathways including phosphodiesterase-5 inhibitors (PDE5i), soluble guanylate cyclase (sGC) stimulators, prostacyclin class agents and endothelin receptor antagonists (ERAs). Combination therapy in PAH, either upfront or sequentially, has become a widely adopted treatment strategy, allowing for simultaneous targeting of more than one of these signaling pathways implicated in disease progression. Much of the current treatment landscape has focused on initial combination therapy with ambrisentan and tadalafil, an ERA and PDE5I respectively, following results of the AMBITION study demonstrating combination to be superior to either agent alone as upfront therapy. Consequently, clinicians often consider combination therapy with other drugs and drug classes, as deemed clinically appropriate, for patients with PAH. An alternative regimen that targets the NO and PGI2 pathways has been adopted by some clinicians as an effective and sometimes preferred therapeutic combination for PAH. Although there is a paucity of prospective data, preclinical data and results from secondary data analysis of clinical studies targeting these pathways may provide novel insights into this alternative combination as a reasonable, and sometimes preferred, alternative approach to combination therapy in PAH. This review of preclinical and clinical data will discuss the current understanding of combination therapy that simultaneously targets the NO and PGI2 signaling pathways, highlighting the clinical advantages and theoretical biochemical interplay of these agents.

Natural or native molecular hydrogen (H2) can be a major component in natural gas, and yet its role in the global energy sector’s usage as a clean energy carrier is not normally considered. Here, we update the scarce reporting of hydrogen in Australian natural gas with new compositional and isotopic analyses of H2 undertaken at Geoscience Australia. The dataset involves ~1000 natural gas samples from 470 wells in both sedimentary and non-sedimentary basins with reservoir rocks ranging in age from the Neoarchean to Cenozoic. Pathways to H2 formation can involve either organic matter intermediates and its association with biogenic natural gas or chemical synthesis and its presence in abiogenic natural gas. The latter reaction pathway generally leads to H2-rich (>10mol% H2) gas in non-sedimentary rocks. Abiogenic H2 petroleum systems are described within concepts of source–migration–reservoir–seal but exploration approaches are different to biogenic natural gas. Rates of abiogenic H2 generation are governed by the availability of specific rock types and different mineral catalysts, and through chemical reactions and radiolysis of accessible water. Hydrogen can be differently trapped compared to hydrocarbon gases; for example, pore space can be created in fractured basement during abiogenic reactions, and clay minerals and evaporites can act as effective adsorbents, traps and seals. Underground storage of H2 within evaporites (specifically halite) and in depleted petroleum reservoirs will also have a role to play in the commercial exploitation of H2. Estimated H2 production rates mainly from water radiolysis in mafic–ultramafic and granitic rocks and serpentinisation of ultramafic–mafic rocks gives a H2 inferred resource potential between ~1.6 and ~58MMm3 year−1 for onshore Australia down to a depth of 1km. The prediction and subsequent identification of subsurface H2 that can be exploited remains enigmatic and awaits robust exploration guidelines and targeted drilling for proof of concept.

Synopsis We present measured absolute double differential cross section (DDCS) of electron emission from fluorene (C13H10) molecule upon 3.5 MeV/u Si8+ ion impact. We have compared the energy and angular distributions of the DDCS with CB1 (Ist Born with correct boundary conditions) calculations. The e-DDCS shows an indication of collective excitation peak in low energy part of the spectrum at a few backward angles. The shape of the distribution has also been compared with that for a smaller molecule i.e. CH4.

Monocytes are able to undergo homotypic fusion to produce different types of multinucleated giant cells, such as Langhans giant cells in response to M. tuberculosis infection or foreign body giant cells in response to implanted biomaterials. Monocyte fusion is highly coordinated and complex, with various soluble, intracellular, and cell-surface components mediating different stages of the process. Tetraspanins, such as CD9, CD63, and CD81, are known to be involved in cell:cell fusion and have been suggested to play a role in regulating homotypic monocyte fusion. However, peripheral human monocytes are not homogenous: they exist as a heterogeneous population consisting of three subsets, classical (CD14++CD16−), intermediate (CD14++CD16+), and non-classical (CD14+CD16+), at steady state. During infection with mycobacteria, the circulating populations of intermediate and non-classical monocytes increase, suggesting they may play a role in the disease outcome. Human monocytes were separated into subsets and then induced to fuse using concanavalin A. The intermediate monocytes were able to fuse faster and form significantly larger giant cells than the other subsets. When antibodies targeting tetraspanins were added, the intermediate monocytes responded to anti-CD63 by forming smaller giant cells, suggesting an involvement of tetraspanins in fusion for at least this subset. However, the expression of fusion-associated tetraspanins on monocyte subsets did not correlate with the extent of fusion or with the inhibition by tetraspanin antibody. We also identified a CD9High and a CD9Low monocyte population within the classical subset. The CD9High classical monocytes expressed higher levels of tetraspanin CD151 compared to CD9Low classical monocytes but the CD9High classical subset did not exhibit greater potential to fuse and the role of these cells in immunity remains unknown. With the exception of dendrocyte-expressed seven transmembrane protein, which was expressed at higher levels on the intermediate monocyte subset, the expression of fusion-related proteins between the subsets did not clearly correlate with their ability to fuse. We also did not observe any clear correlation between giant cell formation and the expression of pro-inflammatory or fusogenic cytokines. Although tetraspanin expression appears to be important for the fusion of intermediate monocytes, the control of multinucleate giant cell formation remains obscure.

Responses to angiotensin II (Ang II) were investigated in anaesthetised CD1 mice. Injections of Ang II caused dose-related increases in systemic arterial pressure that were antagonised by candesartan. Responses to Ang II were not altered by PD 123319. At the lowest dose studied (20 µg/kg i.v.), the inhibitory effects of candesartan were competitive, whereas at the highest dose (100 µg/kg i.v.), the dose-response curve for Ang II was shifted to the right in a non-parallel manner. The inhibitory effects of candesartan were selective and were similar in animals pretreated with enalaprilat to reduce endogenous Ang II production. Pressor responses to Ang II were not altered by propranolol, phentolamine or atropine, but were enhanced by hexamethonium. Increases in total peripheral resistance were inhibited by the AT1-receptor antagonist (ARB) but were not altered by AT2-receptor, alpha- or beta-receptor antagonists. These results suggest that pressor responses to Ang II are mediated by AT 1-receptors, are buffered by the baroreceptors, are not modulated by effects on AT2receptors, and that activation of the sympathetic nervous system plays little role in mediating rapid haemodynamic responses to the peptide in anaesthetised mice.

Pulmonary arterial hypertension is a progressive disorder in which endothelial dysfunction and vascular remodeling obstruct small pulmonary arteries, resulting in increased pulmonary vascular resistance and pulmonary pressures. This leads to reduced cardiac output, right heart failure, and ultimately death. In this review, we attempt to answer some important questions commonly asked by patients diagnosed with pulmonary arterial hypertension pertaining to the disease, and aim to provide an explanation in terms of classification, diagnosis, pathophysiology, genetic causes, demographics, and prognostic factors. Furthermore, important molecular pathways that are central to the pathogenesis of pulmonary arterial hypertension are reviewed, including nitric oxide, prostacyclin, endothelin-1, reactive oxygen species, and endothelial and smooth muscle proliferation.

Right ventricular (RV) function is the major determinant of mortality in pulmonary arterial hypertension and male sex is a strong predictor of mortality in this disease. The effects of testosterone on RV structure and function in load stress are presently unknown. We tested whether testosterone levels affect RV hypertrophic responses, fibrosis, and function. Male C57BL/6 mice underwent castration or sham followed by pulmonary artery banding (PAB) or sham. After recovery, testosterone pellets were placed in a subset of the castrated mice and mice were maintained for at least two weeks, when they underwent hemodynamic measurements and tissues were harvested. Plasma levels of testosterone were reduced by castration and repleted by testosterone administration. In PAB, castration resulted in lower right ventricle/left ventricle + septum (RV/LV+S), and myocyte diameter (P < 0.05). Replacement of testosterone normalized these parameters and increased RV fibrosis (P < 0.05). Two weeks of PAB resulted in increased RV systolic pressure in all groups with decreased markers of RV systolic and diastolic function, specifically reduced ejection fraction and increased time constant, and dPdt minimum (P < 0.05), though there was minimal effect of testosterone on hemodynamic parameters. Survival was improved in mice that underwent castration with PAB compared with PAB alone (P < 0.05). Testosterone affects RV hypertrophic response to load stress through increased myocyte size and increased fibrosis in mice. Castration and testosterone replacement are not accompanied by significant alterations in RV in vivo hemodynamics, but testosterone deprivation appears to improve survival in PAB. Further study of the role of testosterone in RV dysfunction is warranted to better understand these findings in the context of human disease.

Pulmonary vessel constriction results from an imbalance between vasodilator and vasoconstrictor factors released by the endothelium including nitric oxide, endothelin, prostanoids, and reactive oxygen species (ROS). ROS, generated by a variety of enzymatic sources (such as mitochondria and NADPH oxidases, a.k.a. Nox), appear to play a pivotal role in vascular homeostasis, whereas elevated levels effect vascular disease. The pulmonary circulation is very sensitive to changes in the partial pressure of oxygen and differs from the systemic circulation in its response to this change. In fact, the pulmonary vessels contract in response to low oxygen tension, whereas systemic vessels dilate. Growing evidence suggests that ROS production and ROS-related pathways may be key factors that underlie this differential response to oxygen tension. A major emphasis of our laboratory is the role of Nox isozymes in cardiovascular disease. In this review, we will focus our attention on the role of Nox-derived ROS in the control of pulmonary vascular tone.

Arginase expression and activity have been noted to be heightened in conditions associated with erectile dysfunction, including aging. Previously, arginase inhibition by chronic administration of the arginase inhibitor 2-(S)-amino-6-boronohexanoic acid (ABH) has been shown to improve endothelial dysfunction in aged rats. The objective of this study was to assess whether chronic oral ABH administration affects cavernosal erectile function. Rats were divided into 4 groups: young control, young treated with arginase inhibitor, aged control, and aged treated with arginase inhibitor. Arginase activity was measured and presented as a proportion of young untreated rats. In vivo erectile responses to cavernous nerve stimulation were measured in all cohorts. The cavernous nerve was stimulated with a graded electrical stimulus, and the intracavernosal/mean arterial pressure ratios and total intracavernosal pressure were recorded. Arginase activity was elevated in the aged rats compared with young controls; however, arginase activity was significantly decreased in aged rats treated with ABH. With the addition of ABH, erectile responses improved in the aged rats (P.05). Oral inhibition of arginase with ABH results in improved erectile function in aged rats, resulting in erectile hemodynamics similar to young rats. This represents the first documentation of systemic arginase inhibition positively affecting corporal cavernosal function.

Background Emerging evidence suggests that phosphoinositide 3-kinase (PI3K) may modulate cardiac inotropy; however, the underlying mechanism remains elusive. We hypothesized that β(2)-adrenergic receptor (AR)-coupled PI3K constrains increases in cardiac inotropy through cyclic adenosine monophosphate (cAMP)-dependent phosphodiesterase (PDE) activation. Methods We tested the effects of PI3K and PDE4 inhibition on myocardial contractility by using isolated murine cardiac myocytes to study physiologic functions (sarcomere shortening [SS] and intracellular Ca(+) transients), as well as cAMP and PDE activity. Results PI3K inhibition with the reversible inhibitor LY294002 (LY) resulted in a significant increase in SS and Ca(2+) handling, indicating enhanced contractility. This response depended on G(iα) protein activity, because incubation with pertussis toxin (an irreversible G(iα) inhibitor) abolished the LY-induced hypercontractility. In addition, PI3K inhibition had no greater effect on SS than both a PDE3,4 inhibitor (milrinone) and LY combined. Furthermore, LY decreased PDE4 activity in a concentration-dependent manner (58.0% of PDE4 activity at LY concentrations of 10 μM). Notably, PI3K(γ) coimmunoprecipitated with PDE4D. The β(2)-AR inverse agonist, ICI 118,551 (ICI), abolished induced increases in contractility. Conclusions PI3K modulates myocardial contractility by a cAMP-dependent mechanism through the regulation of the catalytic activity of PDE4. Furthermore, basal agonist-independent activity of the β(2)-AR and its resultant cAMP production and enhancement of the catalytic activity of PDE4 through PI3K represents an example of integrative cellular signaling, which controls cAMP dynamics and thereby contractility in the cardiac myocyte. These results help to explain the mechanism by which milrinone is able to increase myocardial contractility in the absence of direct β-adrenergic stimulation and why it can further augment contractility in the presence of maximal β-adrenergic stimulation.

Pulmonary hypertension (PH) is a chronic pulmonary complication of sickle cell disease (SCD) and a leading cause of death in adults. The exact molecular causes of SCD-associated PH are unknown although pathologic hemolysis related to chronic nitric oxide depletion and oxidative stress are recognized as contributing factors. We have reported that the protein thrombospondin-1 (TSP1) is elevated in the plasma of patients with SCD and by interacting with its receptor CD47 limits vasodilation of distal pulmonary arteries ex vivo . We hypothesized that the TSP1-CD47 interaction may promote PH in the BERK sickle mouse model. To test this hypothesis, we inquired whether TSP1 and CD47 are upregulated in the lungs of BERK mice and patients with SCD-associated PH. We found that lungs of age matched male 13-14 months old BERK sickle mice overexpressed TSP1 and CD47 as compared to both C57BL/6J and Hemi BERK mice and also confirmed our prior findings that sickle mice develop PH as they age. Immunohistochemistry of lung sections from 6 patients with SCD-associated PH revealed increased levels of CD47 as compared to sections from patients without PH or overt lung disease. We interrogated the TSP1-CD47 axis in vivo by generating chimeric animals with a sickle erythropoiesis on a CD47KO background by transplanting BERK sickle bone marrow into C57BL/6J (n=24) and CD47 knockout (CD47KO, n=27) mice. Fully engrafted chimeric mice underwent pulmonary hemodynamic assessment after 6 months. Right ventricular (RV) pressures were lower in Sickle-to-CD47KO chimeras as compared to Sickle-to-C57BL/6J chimeras as shown by the reduced maximum pressure of the RV (22.1 ± 3.6 vs. 28.1 ± 8.8 mmHg, p=0.013) and mean pulmonary artery pressure (15.3 ± 2.6 vs. 18.8 ± 5.7 mmHg, p=0.020). The afterload of the Sickle-to-CD47KO chimeras was lower compared to Sickle-to-C57BL/6J chimeras as shown by the diminished pulmonary vascular resistance (1.2 ± 0.7 vs. 2.4 ± 2.2 Wood units, p=0.024) and RV effective arterial elastance (1.5 ± 0.9 vs. 2.7 ± 2.6 mmHg/µL, p=0.052). The RV diastolic function as measured by the RV dP/dtmin also showed a trend towards improvement as compared to Sickle-to-C57BL/6J chimeras (-1297.0 ± 318.2 vs. -1604.0 ± 668.2 mmHg/s, p=0.059). Finally, the Sickle-to-CD47KO chimeras had a tendency for less RV hypertrophy as evidenced by lower RV free wall weight as compared to Sickle-to-C57BL/6J chimeras (21.45 ± 5.0 vs. 24.63 ± 6.0 mg, p=0.056). Interestingly, plasma levels of soluble TSP1 were reduced in Sickle-to-CD47KO chimeras (61.45 ± 10.11 vs. 76.89 ± 10.84 pg/mL, p=0.012). On myography, aortic segments from Sickle-to-CD47KO chimeras had improved relaxation to the endothelial activator acetylcholine. We hypothesized that in SCD TSP1-CD47 signaling promotes PH, in part, by increasing ROS generation. Treatment with exogenous TSP1 (2.2x10-9 M, a concentration found in the plasma of SCD patients) stimulated increased levels of superoxide and hydrogen peroxide in human pulmonary artery endothelial cells by cytochrome C and Amplex Red assays, respectively. Furthermore, lungs of CD47KO chimeras had decreased oxidative damage as measured by reduced 4-hydroxynonenal and 3-nitrotyrosine staining vs. Sickle-to-C57BL/6J chimeras. Finally, suppression of CD47 with a morpholino (10 mg/kg body weight i.p. weekly) for 8 weeks (n=8 animals) also reduced pulmonary pressures in BERK mice. Our results show that genetic deletion or suppression of CD47 ameliorates SCD-associated PH, which may be due, in part, to decreased ROS levels. Targeting TSP1-CD47 may provide a new molecular approach to the treatment of SCD-associated PH. Disclosures Isenberg: Tioma Therapeutics: Equity Ownership; Radiation Control Technologies, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

There is increasing evidence that upregulation of arginase contributes to impaired endothelial function in aging. In this study, we demonstrate that arginase upregulation leads to endothelial nitric oxide synthase (eNOS) uncoupling and that in vivo chronic inhibition of arginase restores nitroso-redox balance, improves endothelial function, and increases vascular compliance in old rats. Arginase activity in old rats was significantly increased compared with that shown in young rats. Old rats had significantly lower nitric oxide (NO) and higher superoxide (O2−) production than young. Acute inhibition of both NOS, with NG-nitro-l-arginine methyl ester, and arginase, with 2( S)-amino- 6-boronohexanoic acid (ABH), significantly reduced O2− production in old rats but not in young. In addition, the ratio of eNOS dimer to monomer in old rats was significantly decreased compared with that shown in young rats. These results suggest that eNOS was uncoupled in old rats. Although the expression of arginase 1 and eNOS was similar in young and old rats, inducible NOS (iNOS) was significantly upregulated. Furthermore, S-nitrosylation of arginase 1 was significantly elevated in old rats. These findings support our previously published finding that iNOS nitrosylates and activates arginase 1 (Santhanam et al., Circ Res 101: 692–702, 2007). Chronic arginase inhibition in old rats preserved eNOS dimer-to-monomer ratio and significantly reduced O2− production and enhanced endothelial-dependent vasorelaxation to ACh. In addition, ABH significantly reduced vascular stiffness in old rats. These data indicate that iNOS-dependent S-nitrosylation of arginase 1 and the increase in arginase activity lead to eNOS uncoupling, contributing to the nitroso-redox imbalance, endothelial dysfunction, and vascular stiffness observed in vascular aging. We suggest that arginase is a viable target for therapy in age-dependent vascular stiffness.

Erectile dysfunction (ED) is a common men's health problem characterized by the consistent inability to sustain an erection sufficient for sexual intercourse. Basic science research on erectile physiology has been devoted to investigating the pathogenesis of ED and has led to the conclusion that ED is predominately a disease of vascular origin and/or neurogenic dysfunction. The constitutive forms of nitric oxide synthase [NOS; endothelial NOS (eNOS) and neuronal NOS (nNOS)] are important enzymes involved in the production of nitric oxide (NO) and thus regulate penile vascular homeostasis. Given the impact of endothelial- and neuronal-derived NO in penile vascular biology, a great deal of research over the past decade has focused on the role of NO synthesis from the endothelium and nitrergic nerve terminal in normal erectile physiology as well as in disease states. Loss of the functional integrity of the endothelium and subsequent endothelial dysfunction plays an integral role in the occurrence of ED. Therefore, molecular mechanisms involved in dysregulation of these NOS isoforms in the development of ED are essential to discovering the pathogenesis of ED in various disease states. This communication reviews the role of eNOS and nNOS in erectile physiology and discusses the alterations in eNOS and nNOS via post-translation modification in various vascular diseases of the penis.

We determined the effect of passive secondhand cigarette smoke on 1) erectile function in vivo, 2) molecular mechanisms involved in penile vascular function, and 3) erectile function and penile molecular signaling in the presence of phosphodiesterase type 5 inhibitor therapy.Four groups of mice were used, including group 1--controls, group 2--mice exposed to 3 weeks of secondhand smoke (5 hours per day for 5 days per week), group 3--control plus sildenafil (100 mg/kg per day) and group 4--smoke exposed plus sildenafil (100 mg/kg per day). Cavernous nerve electrical stimulation and intracavernous injection of acetylcholine were done to assess erectile function. Constitutive and inducible nitric oxide synthase activity, reactive oxygen species generation, nitrotyrosine formation and superoxide anion levels were assessed.Decreased erectile responses to cavernous nerve electrical stimulation and impaired endothelium dependent erectile responses to ACh in mice exposed to secondhand smoke were observed. Superoxide anion was increased in endothelial and corporeal smooth muscle cells of smoking mouse penises. In mice exposed to secondhand smoke constitutive nitric oxide synthase activity was decreased, and inducible nitric oxide synthase activity, reactive oxygen species generation and nitrotyrosine formation increased. Sildenafil therapy restored constitutive nitric oxide synthase activity in the penis of smoking mice, decreased inducible nitric oxide synthase activity, reactive oxygen species generation and nitrotyrosine formation, and improved erectile responses to cavernous nerve electrical stimulation and acetylcholine.Short-term exposure to secondhand smoke impairs erectile function through excessive penile reactive oxygen species signaling and inducible nitric oxide synthase activity. Decreased penile constitutive nitric oxide synthase activity may be attributable to the decreased endothelial nitric oxide synthase activity resulting from increased oxidative stress. Sildenafil therapy restored nitric oxide synthase activity and decreased reactive oxygen species signaling, resulting in improved erectile function.

Background— Pulmonary hypertension (PH) secondary to left heart failure portends a poor prognosis and is a relative contraindication to heart transplantation at many centers. We tested the hypothesis that when PH persists after adequate left ventricle unloading via recent left ventricular assist device (LVAD) therapy, phosphodiesterase type 5A inhibition would decrease PH in this population. Methods and Results— We performed an open-label clinical trial using control patients not receiving therapy. Between 1999 and 2007, 138 consecutive patients undergoing cardiac transplantation evaluation with advanced left ventricular dysfunction, an elevated pulmonary capillary wedge pressure, and PH (defined by a pulmonary vascular resistance (PVR) >3 Woods Units), were treated with LVAD therapy. Fifty-eight of these patients reduced their pulmonary capillary wedge pressure to a value P P Conclusions— In patients with persistent PH after recent LVAD placement, phosphodiesterase type 5A inhibition in this open-label trial resulted in a significant decrease in PVR when compared with control patients.

Oxidized low-density lipoproteins increase arginase activity and reciprocally decrease endothelial NO in human aortic endothelial cells. Here, we demonstrate that vascular endothelial arginase activity is increased in atherogenic-prone apolipoprotein E–null (ApoE −/− ) and wild-type mice fed a high cholesterol diet. In ApoE −/− mice, selective arginase II inhibition or deletion of the arginase II gene (Arg II −/− mice) prevents high-cholesterol diet–dependent decreases in vascular NO production, decreases endothelial reactive oxygen species production, restores endothelial function, and prevents oxidized low-density lipoprotein–dependent increases in vascular stiffness. Furthermore, arginase inhibition significantly decreases plaque burden. These data indicate that arginase II plays a critical role in the pathophysiology of cholesterol-mediated endothelial dysfunction and represents a novel target for therapy in atherosclerosis.

The free radical peroxynitrite (ONOO-) is formed in biological systems from the reaction of nitric oxide (NO) with superoxide (O2-) and can react with protein and nonprotein thiol groups to produce tissue injury. However, these pathologic actions of (ONOO-) may have been overemphasized, in that (ONOO-) has vasorelaxant properties through activation of soluble guanylate cyclase; inhibits leukocyte-endothelial cell interactions; and reduces ischemia-reperfusion injury in the heart, lung, and liver. It has been reported that tolerance develops to the vasodilator actions of (ONOO-) and that (ONOO-) impairs vascular function. However, little, if anything, is known about responses to (ONOO-) in the hindlimb circulation of the cat. To better understand the effects of (ONOO-) on responses to vasoactive agonists and the mechanism by which (ONOO-) induces vasodilation, the effects of short-term exposure to (ONOO-) were investigated under constant-flow conditions in the hindlimb vascular bed of the cat. In these studies, direct intraarterial injections of (ONOO-) produced dose-dependent decreases in hindquarters perfusion pressure. The vasodilator responses to (ONOO-) were rapid in onset, were short in duration, and could be repeated without exhibiting tachyphylaxis. Vasodilator responses to (ONOO-) were not changed in the presence of inhibitors of nitric-oxide synthase, cyclooxygenase, or K+-ATP (adenosine triphosphate-sensitive potassium) channels. Furthermore, responses to (ONOO-) were enhanced in duration by the type 5-cGMP (cyclic guanosine monophosphate) phosphodiesterase inhibitor zaprinast, whereas rolipram, a type 4-cGMP phosphodiesterase inhibitor, was without effect. Repeated administration of (ONOO-) had no significant effect on responses to vasoconstrictor or to vasodilator agents including acetylcholine. These results show that (ONOO-) has significant vasodilator activity in the hindlimb vascular bed of the cat and suggest that the response is mediated by a cGMP- dependent mechanism. The results of experiments with repeated injections of (ONOO-) indicate that (ONOO-) does not impair vasoconstrictor and endothelium-dependent or endothelium-independent vasodilator responses. Furthermore, tolerance did not develop with repeated short-term exposure to (ONOO-). Moreover, the results of experiments with inhibitors suggest that responses to (ONOO-) are not dependent on K-ATP (adenosine triphosphate-sensitive potassium) channel activation, increased NOS activity, or the formation of products in the cyclooxygenase pathway. The results of these studies are consistent with the hypothesis that (ONOO-) is rapidly converted in the hindlimb circulation to a substance that has the properties of an NO donor. These studies suggest that under physiologic conditions, the cytotoxic effects of (ONOO-) on a short-term basis may have been overemphasized.

Nitric oxide regulation of the cardiovascular system involves both cGMP-dependent and independent mechanisms. The former directly interacts with the family of catabolic phosphodiesterases (PDEs) that control cGMP levels and thus distal effects such as protein kinase G stimulation. Growing evidence supports an important role of several PDEs, including PDE1, PDE2, and PDE5, in the regulation of cGMP in both vascular smooth muscle and cardiac myocytes. These PDEs have relatively little impact on resting function, but they can potently modulate acute contractile tone in cells stimulated by external agonists such as angiotensin or catecholamines. Regulation by PDEs is compartmentalized, with selective interactions occurring between a given source of cGMP and PDE hydrolysis. PDE1 and/or PDE5 are also reportedly up-regulated in chronic disease conditions such as atherosclerosis or cardiac pressure-load stress and heart failure as well as in response to long-term exposure to nitrates. Such up-regulation is thought to contribute to vascular and cardiac pathophysiology and to drug tolerance. Recent studies utilizing selective PDE5 inhibitors support significant cross-signaling with NO-cGMP synthetic pathways that may be particularly helpful in treating certain disease states.

Background Cardiac allograft vasculopathy (CAV) poses the greatest threat to the long-term survival of cardiac transplant recipients, and these individuals often exhibit elevated levels of uric acid (UA), a stimulator of T cells. We hypothesized that hyperuricemia is associated with CAV in cardiac transplant recipients. Methods UA levels were measured in cardiac transplant recipients between January 2003 and January 2005. Surveillance cardiac catheterizations performed 3 months to 1 year after UA measurement were reviewed. The relationship between UA and CAV was adjusted for possible confounders with propensity scores and confirmed with goodness-of-fit tests. Results The 105 patients included in this study were a median 63.3 months post-transplant and their left heart catheterizations were performed a median 5.6 months after UA measurement. Focal stenosis was evident in 25 angiograms and 31 showed distal pruning of the coronary arteries. Compared with the lowest quartile of UA, the highest quartile had an increased risk of CAV: odds ratio (OR) 6.11 (95% CI 1.47 to 25.5; p = 0.013) for focal stenosis and OR 4.60 (95% CI 1.34 to 15.8; p = 0.015) for distal pruning. After adjustment, this relationship persisted for both focal stenosis (OR 5.53, 95% confidence interval [CI] 1.29 to 23.7; p = 0.021) and distal pruning (OR 4.21, 95% CI 1.15 to 15.4; p = 0.029). Conclusions Elevated UA confers an increased risk of CAV. This association may be causal, with pathophysiologic implications for the role of hyperuricemia in allograft failure and, if substantiated, could have clinical implications for the use of xanthine oxidase inhibitors in cardiac transplant recipients.

Since both increased nitric oxide (NO) synthase (NOS) abundance and diminished NO signaling have been reported in the aging penis, the role of NO in the adaptations of aging remains controversial. Here we tested the hypothesis that arginase, an enzyme that competes with NOS for the substrate l-arginine, contributes to erectile dysfunction with advanced age in the B6/129 mouse strain. Arginase protein abundance, mRNA expression, and enzyme activity were elevated in aged compared with young penile endothelial cells. In addition, endothelial NOS (NOS3) protein abundance was greater in aged versus young penile endothelial cells, whereas NOS activity and cGMP levels were reduced. Calcium-dependent l-arginine-to-l-citrulline conversion and cGMP formation increased significantly in aged mouse penes in the presence of the arginase inhibitor 2( S)-amino-6-boronohexanoic acid (ABH). However, there was no effect on l-arginine-to-l-citrulline conversion or cGMP accumulation in the endothelium from young mouse penes. To assess the functional role of arginase in the inhibition of NOS pathway responsiveness in the penis, we evaluated the effects of ABH and an adeno-associated virus encoding an antisense sequence to arginase I (AAVanti-arginase) on erectile function in vivo. ABH and AAVanti-arginase enhanced endothelium-dependent erectile responses in the aged mice without altering endothelium-independent responses. Paralleling our in vitro observations, ABH or AAVanti-arginase did not affect vascular responses in the young mice. Inhibition of the arginase pathway improves endothelial function in the aging penile circulation, suggesting that the arginase pathway may be exploited to improve erectile dysfunction associated with aging.

Introduction Recent research suggests that priapism in sickle cell disease (SCD) is due to dysregulation of penile erection homeostasis including alteration of nitric oxide synthase (NOS) and phosphodiesterase type 5 (PDE5) activities by excessive levels of reactive oxygen species (ROS) released during hemolysis. It is unknown if subacute exposure to hemolysis is sufficient or if chronic reconditioning of erectile tissues is required for perturbation of homeostatic pathways and whether PDE5 inhibitor (PDE5I) treatment can restore erectile homeostasis in the subacute setting. Aims The aim of this study was to investigate the effects of subacute hemolysis (3-month exposure) on priapism and NO pathway regulation. Methods Mice underwent bone marrow transplantation with either SCD (BM-SS) or wild-type (WT) bone marrow. BM-SS mice were treated with sildenafil 100 mg/kg/day. We measured intracavernous pressure (ICP) measurements with or without cavernous nerve stimulation following bone marrow transplantation to assess for priapism. Main Outcome Measures ICP and frequency of erections were assessed. Penile tissues were analyzed for NOS, protein kinase G (PKG), PDE5, and ROS activities. Results BM-SS mice demonstrated a priapism phenotype. PDE5I treatment reduced the frequency of erections in BM-SS mice (1.7 ± 1.1 vs. 5.5 ± 2.8 erections per hour, P < 0.05). Penile tissues from BM-SS mice demonstrated decreased NOS, PKG, PDE5 and elevated ROS activities compared with that of control mice. PDE5I treatment increased NOS (11.6 ± 1.3% vs. 7.8 ± 2.3%, P < 0.05) and PDE5 (76.3 ± 9.8% vs. 52.3 ± 11.1%, P < 0.05) activities and decreased ROS activity (137.8 ± 12.1% vs. 199.1 ± 11.3%, P < 0.05) compared with non-PDE5I treated BM-SS mice. PKG activity was increased beyond control levels with PDE5I treatment (158.4 ± 10.3%, P < 0.05). Conclusion Short-term hemolysis is sufficient to establish a priapism phenotype and results in loss of erectile function. PDE5I treatment ameliorates priapism, in part, because of restored NO balance with decreased ROS generation and increased PDE5 activity.

To evaluate interactions between human endothelial cells (ECs) and smooth muscle cells (SMCs) for the development of tissue-engineered vessels, we examined the adhesion and key cell properties of human ECs grown on quiescent human aortic SMCs. ECs attached to SMCs spread more slowly than ECs attached to fibronectin surfaces, and ECs aligned along the direction of the SMCs. ECs attached firmly and less than 5% of the cells were removed by shear stresses as high as 300 dyn cm(-2). Unlike porcine SMCs and co-cultures, human SMCs or co-cultures do not contract under flow, and the human ECs and SMCs in co-culture align toward the direction of flow. A confluent endothelium could be maintained in co-culture for over 30 days, and some of the ECs reoriented perpendicular to the SMCs after 9 days in static culture. Surface tissue factor levels in ECs and SMCs were less in co-culture than in monoculture. Co-culture induced an increase in calponin expression in SMCs. These findings show that human co-cultures can be maintained for long culture periods, where the endothelium remains confluent and responds to long-term exposure to flow, and EC-SMC interactions lead to an increase in SMC differentiation and an EC surface that is less thrombotic.

Pulmonary hypertension is a highly prevalent complication of sickle cell disease and is a strong risk factor for early mortality. However, the pathophysiological mechanisms by which sickle hemoglobin leads to pulmonary vasculopathy remain unclear. Transgenic mice provide opportunities for mechanistic studies of vascular pathophysiology in an animal model of severe sickle cell disease. Using micro-cardiac catheterization we found that all mice expressing exclusively human sickle hemoglobin develop pulmonary hypertension. Recognizing that the NO pathways can have complex abnormalities in other conditions of pulmonary hypertension, the NO axis in sickle mice was assessed by multiple methods. From a mechanistic standpoint the mice exhibit profound pulmonary and systemic endothelial dysfunction and vascular instability characterized by diminished responses to authentic nitric oxide (NO), NO donors and endothelium-dependent pulmonary vasodilators, and enhanced responses to vaso-constrictors. However, endothelium-independent vasodilation in the sickle mice was normal. Mechanisms of vasculopathy in sickle mice involve global dysregulation of the NO-axis: impaired constitutive nitric oxide synthase activity with loss of eNOS coupling (dimerization), increased NO scavenging by plasma hemoglobin and superoxide, increased arginase activity, and depleted intravascular nitrite reserves. Consistent with a functional rather than structural defect, light microscopy and computed tomography of the lungs revealed no plexogenic arterial remodeling, thrombi/emboli, or inflammation. Transplanting sickle marrow into wild-type mice conferred the same phenotype. Similar pathobiology was observed in a non-sickle mouse model of acute alloimmune hemolysis, supporting a major role of hemolysis as a mechanism for this dysregulation of NO and vasculopathy. In this study, alloimmune hemolytic mice were chosen for comparison in order to generalize beyond hemoglobinopathies. Future analogous studies with thalassemic mice may be useful to model pulmonary hypertension in human thalassemia intermedia. In conclusion, this animal model extends the evidence for global impairment in NO responsiveness and NO production in sickle cell disease, and suggests that hemolytic anemia is associated with endothelial dysfunction and pulmonary hypertension.

From a mouse model of hypoxia-induced pulmonary hypertension, we previously found a highly upregulated protein in the lung that we named hypoxia-induced mitogenic factor (HIMF), also known as found in inflammatory zone 1 (FIZZ1), and resistin-like molecule α (RELMα). However, the mechanisms of HIMF in the pulmonary vascular remodeling remain unknown. We now demonstrate that HIMF promoted cell proliferation, migration, and the production of vascular endothelial growth factor (VEGF) and monocyte chemotactic protein-1 (MCP-1) in pulmonary endothelial cells as well as the production of reactive oxygen species in murine monocyte/macrophage cells. HIMF-induced CD31-positive cell infiltrate in in vivo Matrigel plugs was significantly suppressed by VEGF receptor-2 (VEGFR2) blockade. In ex vivo studies, HIMF stimulated the production of VEGF, MCP-1, and stromal cell-derived factor-1 (SDF-1) in the lung resident cells, and VEGFR2 neutralization significantly suppressed HIMF-induced MCP-1 and SDF-1 production. Furthermore, intravenous injection of HIMF showed marked increase of CD68-positive inflammatory cells in the lungs, and these events were attenuated by VEGFR2 neutralization. Intravenous injection of HIMF also downregulated the expression of VEGFR2 in the lung. These results suggest that HIMF plays critical roles in pulmonary inflammation as well as angiogenesis.

Previous studies indicate that endothelial nitric oxide synthase (eNOS) function is impaired in diabetes as a result of increased vascular generation of reactive oxygen species. We hypothesized that eNOS gene therapy would augment NO· bioavailability and protect against hepatic ischemia-reperfusion (I-R) injury in type 2 diabetes mellitus. We developed a transgenic (Tg) diabetic mouse in which eNOS is systemically overexpressed. We also examined the effects of hepatic eNOS adenovirus therapy in diabetic mice. Diabetic (db/db) and nondiabetic mice were subjected to hepatic I-R injury. In nondiabetic mice, genetic overexpression of eNOS (both eNOS-Tg and eNOS adenovirus) resulted in hepatoprotection. In contrast, hepatic I-R injury was significantly increased in the db/db eNOS-Tg mouse, as serum alanine aminotransaminase (ALT) levels were increased by 3.3-fold compared with diabetic controls. Similarly, eNOS adenovirus treatment resulted in a 3.2-fold increase in serum ALT levels as compared with diabetic controls. We determined that hepatic eNOS was dysfunctional in the db/db mouse and increased genetic expression of eNOS resulted in greater production of peroxynitrite. Treatment with the eNOS cofactor tetrahydrobiopterin (BH 4 ) or the BH 4 precursor sepiapterin resulted in a significant decrease in serum ALT levels following I-R injury. We present clear examples of the protective and injurious nature of NO· therapy in I-R. Our data indicate that eNOS exists in an “uncoupled” state in the setting of diabetes and that “recoupling” of the eNOS enzyme with cofactor therapy is beneficial.

Basic science research on erectile physiology has been devoted to investigating the pathogenesis of erectile dysfunction (ED) and has led to the conclusion that ED is predominately a disease of vascular origin. It is well recognized that the incidence of ED dramatically increases in men who suffer from diabetes mellitus, hypercholesterolemia, and cardiovascular disease. Endothelial nitric oxide synthase (eNOS) is an important factor in cardiovascular homeostasis, angiogenesis, and erectile function. Given the impact of endothelial-derived nitric oxide (NO) in vascular biology, a great deal of research over the past decade has focused on the role of NO synthesis from the endothelium in normal erectile physiology as well as in disease states. Loss of the functional integrity of the endothelium and subsequent endothelial dysfunction plays an integral role in the occurrence of ED. Therefore, a likely target of gene therapy for the treatment of ED is eNOS. This communication reviews the role of eNOS in erectile physiology and discusses the alterations in eNOS expression in various vascular diseases of the penis. Putative gene therapy interventions to restore eNOS expression and subsequent endothelial function may represent an exciting new therapeutic strategy for the future treatment of ED.

Conflicting information exists regarding the long-term efficacy of phosphodiesterase-5 (PDE5) inhibitor therapy for erectile dysfunction, particularly in regard to whether therapeutic resistance occurs. We investigated the erectile response, and cavernous PDE5 expression and activity after continuous long-term administration of sildenafil at verified therapeutic plasma concentrations, applying an in vivo rat model of age related erectile dysfunction.Male Fisher 344 young (4 months old) and aged (19 months old) rats (National Institute of Aging, Bethesda, Maryland) were injected with sildenafil mesylate (20 mg/kg) or saline subcutaneously every 8 hours for 3 weeks. After a 10 to 18-hour washout period electrical stimulation of the cavernous nerve was performed to assess penile erection. Penes were excised to measure PDE5 protein expression and activity, and blood was collected for sildenafil measurement. Responses were compared with those determined 30 minutes after a single sildenafil injection.Chronic sildenafil treatment increased the detumescence phase in young and aged rats (p0.05), although aged rats showed a greater increase than young rats. Baseline cavernous PDE5 expression and activity were greater in aged vs young rats (p0.05). After chronic sildenafil treatment cavernous PDE5 expression was increased in young (p0.05) but not in aged rats. Chronic and acute sildenafil treatment similarly inhibited PDE5 activity in the penis of young and aged rats (p0.05), coincident with its free plasma concentrations equivalent to clinically therapeutic ranges.Pharmacological PDE5 inhibitor therapy with sildenafil chronically does not result in treatment resistance. Rather, therapeutic efficacy is maintained and apparently more pronounced with erectile impairment than with normal erectile ability.

Decreased nitric oxide (NO) is considered an important pathogenetic mechanism in pulmonary arterial hypertension (PAH), but clear evidence is lacking.We used multiple techniques to assess endogenous NO in 10 patients with untreated PAH (8 idiopathic and 2 anorexigen-associated PAH) and 12 control subjects.After a nitrite/nitrate-restricted diet, NO metabolites (NOx) were assayed in 24-hour urine collections and exhaled NO (FE(NO)) determined at multiple expiratory flows. Analysis of the relation between FE(NO) and flow allowed derivation of three flow-independent parameters: airway wall concentration (C(W)), diffusing capacity (D(NO)), and alveolar concentration (C(A)). Seven patients underwent follow-up testing after 3 months of bosentan treatment.At baseline, FE(NO) was markedly decreased at the two lowest expiratory flows in PAH: 21 +/- 4 versus 36 +/- 4 ppb at 18 ml/second and 11 +/- 2 versus 17 +/- 2 ppb at 50 ml/second, for subjects with PAH and control subjects, respectively (p0.05). C(W) was 33 +/- 11 ppb in subjects with PAH versus 104 +/- 34 in control subjects (p = 0.04). Urinary NOx was also reduced in PAH (42 +/- 6 microM NOx/mM creatinine versus 62 +/- 7 in control subjects; p = 0.04). After bosentan, FE(NO), C(W), and urine NOx increased to control values (p0.05). Exclusion of the two anorexigen cases did not alter these results.FE(NO) at low expiratory flows was decreased in PAH due to reduced C(W). Bosentan reversed these abnormalities, suggesting that suppression of NO in PAH may have been caused by endothelin.

Introduction Superoxide anion may contribute to erectile dysfunction (ED) in diabetes mellitus by reducing cavernosal nitric oxide (NO) bioavailability. The purpose of this study was to determine if gene transfer of extracellular superoxide dismutase (EC‐SOD) can reduce superoxide anion formation and determine if this reactive oxygen species may contribute to diabetes‐related ED in an experimental model of diabetes. Methods Three groups of animals were utilized: (1) control; (2) streptozotocin (STZ)‐diabetic rats [60 mg/kg intraperitoneally (ip)] intracavernosally injected with AdCMVβgal (negative control); and (3) STZ‐rats intracavernosally injected with AdCMVEC‐SOD. Two months after ip injection of STZ, groups 2 and 3 were transfected with the adenoviruses and 2 days after transfection, all animals underwent cavernosal nerve stimulation (CNS) to assess erectile function. Confocal microscopy for superoxide anion and von Willebrand Factor (vWF) was performed in the STZ‐diabetic rat. Superoxide anion production, total SOD activity, and cyclic guanosine monophosphate (cGMP) levels were measured in each experimental group of rats. Results 248 Confocal microscopy demonstrated superoxide in smooth muscle and endothelial cells of the STZ‐rat cavernosum and colocalized with vWF in the endothelium. Higher superoxide anion levels and decreased cGMP levels were found in the penis of STZ‐rats at a time when erectile function was reduced. Two days after administration of AdCMVEC‐SOD, superoxide anion levels were significantly lower in the penis of STZ‐rats. Total SOD activity and cavernosal cGMP was increased in the penis of EC‐SOD‐transfected rats. STZ‐rats transfected with AdCMVEC‐SOD had a peak intracavernosal pressure (ICP) and total ICP to CNS that was similar to control rats. Conclusions These data demonstrate that in vivo adenoviral gene transfer of EC‐SOD can reduce corporal superoxide anion levels and raise cavernosal cGMP levels by increasing NO bioavailability thus restoring erectile function in the STZ‐diabetic rat.

The molecular mechanism for priapism is not well characterized. Although the nitric oxide (NO) pathway is known to mediate penile erection under normal conditions, we hypothesized that the mechanism of priapism rests in aberrant downstream signaling of this pathway based on our previous findings that mice lacking the gene for endothelial nitric oxide synthase (eNOS —/— ) and mice lacking both neuronal NOS (nNOS) and eNOS (nNOS —/— , eNOS —/— ) have a tendency for priapic activity. We investigated the role of downstream guanylate cyclase and phosphodiesterase type 5 (PDE5A) expression and function in mediating these responses in eNOS —/— and nNOS —/— , eNOS —/— mice. Erectile responses to both cavernous nerve stimulation and intracavernosal injection of the NO donor diethylamine-NONOate were augmented in eNOS —/— and nNOS —/— , eNOS —/— mice but not in WT or nNOS —/— mice. PDE5A protein expression and activity and cGMP levels were significantly lower in eNOS —/— and nNOS —/— , eNOS —/— mice, and this effect was reproduced in WT corpus cavernosum exposed to NOS inhibitors. Moreover, cavernous nerve stimulation was associated with a marked augmentation of cavernosal cGMP levels, suggesting that, although lower at baseline, the production of cGMP is unchecked in eNOS —/— and nNOS —/— , eNOS —/— mice upon neurostimulation. Transfection of eNOS —/— mice with an adenovirus encoding eNOS resulted in a normalization of PDE5A protein and activity as well as a correction of priapic activity. Coupled with the observation that sickle cell disease mice (which show a priapism phenotype) evince dysregulated PDE5A expression/activity, these data suggest that PDE5A dysregulation is a fundamental mechanism for priapism.

Significant impairment in endothelial-derived nitric oxide is present in the diabetic corpus cavernosum. RhoA/Rho-kinase may suppress endothelial nitric oxide synthase (eNOS). Here, we tested the hypothesis that RhoA/Rho-kinase contributes to diabetes-related erectile dysfunction and down-regulation of eNOS in the streptozotocin (STZ)-diabetic rat penis. Colocalization of Rho-kinase and eNOS protein was present in the endothelium of the corpus cavernosum. RhoA/Rho-kinase protein abundance and MYPT-1 phosphorylation at Thr-696 were elevated in the STZ-diabetic rat penis. In addition, eNOS protein expression, cavernosal constitutive NOS activity, and cGMP levels were reduced in the STZ-diabetic penis. To assess the functional role of RhoA/Rho-kinase in the penis, we evaluated the effects of an adeno-associated virus encoding the dominant-negative RhoA mutant (AAVTCMV19NRhoA) on RhoA/Rho-kinase and eNOS and erectile function in vivo in the STZ-diabetic rat. STZ-diabetic rats transfected with AAVCMVT19NRhoA had a reduction in RhoA/Rho-kinase and MYPT-1 phosphorylation at a time when cavernosal eNOS protein, constitutive NOS activity, and cGMP levels were restored to levels found in the control rats. There was a significant decrease in erectile response to cavernosal nerve stimulation in the STZ-diabetic rat. AAVT19NRhoA gene transfer improved erectile responses in the STZ-diabetic rat to values similar to control. These data demonstrate a previously undescribed mechanism for the down-regulation of penile eNOS in diabetes mediated by activation of the RhoA/Rho-kinase pathway. Importantly, these data imply that inhibition of RhoA/Rho-kinase improves eNOS protein content and activity thus restoring erectile function in diabetes.

Nitric oxide (NO) functions principally as a diffusible paracrine effector. The exception is in cardiomyocytes where both NO synthases (NOS) and target proteins coexist, allowing NO to work in an autocrine/intracrine fashion. However, the most abundant myocyte isoform (NOS3) is far more expressed in vascular endothelium; thus, the in vivo contribution of myocyte-NOS3 remains less clear. The present study tested this role by transfecting whole hearts of NOS3-null (NOS3 −/− ) mice with adenovirus-expressing NOS3 coupled to a α-MHC promoter (AdV NOS3 ), comparing results to hearts transfected with marker-gene β-galactosidase (AdVβ gal ). Total myocardial NOS3 protein and activity were restored to near wild-type (WT) levels in NOS3 −/− +AdV NOS3 hearts, and NOS3 relocalized normally with caveolin-3. Ejection function by pressure-volume analysis was enhanced in NOS3 −/− +AdVβ gal over WT or NOS3 −/− +AdV NOS3 . More prominently, isoproterenol (ISO)-stimulated systolic and diastolic function in WT was amplified in NOS3 −/− +AdVβ gal , whereas NOS3 −/− +AdV NOS3 returned the response to control. ISO-activated systolic function was inhibited 85% by concomitant muscarinic stimulation (carbachol) in NOS3 −/− +AdV NOS3 but not NOS3 −/− +AdVβ gal hearts. Lastly, NOS3 −/− +AdVβ gal mice displayed enhanced inotropy and lusitropy over WT at slower heart rates but a blunted rate augmentation versus controls. A more positive rate response was restored in NOS3 −/− +AdV NOS3 ( P

Erectile dysfunction associated with diabetes mellitus is caused in part by disordered endothelial smooth muscle relaxation, neuropathy, and a decrease in cavernosal nitric oxide synthase (NOS) activity. The purpose of this study was to determine whether a combination of sildenafil and adenoviral gene transfer of endothelial NOS (eNOS) could enhance the erectile response in diabetic rats. Five groups of animals were utilized: (1) age-matched control rats, (2) streptozotocin (STZ)-induced diabetic rats (60 mg/kg i.p.), (3) STZ-rats + sildenafil (2 mg/kg i.v.), (4) STZ-rats transfected with AdCMVbetagal or AdCMVeNOS, and (5) STZ-rats transfected with AdCMVeNOS +sildenafil (2 mg/kg i.v.). At 2 months after i.p. injection of STZ, groups 4 and 5 were transfected with the adenoviruses and 1-2 days after transfection, all animals underwent cavernosal nerve stimulation (CNS) to assess erectile function. Cyclic 3',5'-guanosine monophosphate (cGMP) levels were assessed in the cavernosal tissue. STZ-diabetic rats had a significant decrease in erectile function as determined by the peak intracavernosal pressure (ICP) and total ICP (area under the erectile curve; AUC) after CNS when compared to control rats. STZ-diabetic rats+AdCMVeNOS had a peak ICP and AUC, which were similar to control animals. STZ-diabetic rats administered sildenafil demonstrated a significant increase in peak ICP at the 5 and 7.5 V settings, while the AUC was significantly increased at all voltage (V) settings. The increase in both ICP and AUC of STZ-diabetic rats transfected with AdCMVeNOS at all V settings was greater than STZ-diabetic rats transfected with AdCMVbetagal. STZ-diabetic rats transfected with AdCMVeNOS and administered sildenafil had a significant increase in total ICP that was greater than eNOS gene therapy alone. Cavernosal cGMP levels were significantly decreased in STZ-diabetic rats, but were increased after transfection with AdCMVeNOS to values greater than control animals. In conclusion, overexpression of eNOS and cGMP in combination with sildenafil significantly increased both the peak ICP and total ICP to CNS in the STZ-diabetic rat, which was similar to the response observed in control rats. Moreover, the total erectile response was greater in STZ-diabetic rats receiving eNOS gene therapy plus sildenafil than STZ-rats receiving sildenafil or eNOS gene therapy alone.

This study compares uncertainties of equivalent doses after internal contamination by 125 I, 129 I or 131 I. Uncertainties were calculated using reported distributions of physiological parameters and Monte Carlo simulation. In adults, uncertainties increase from 131 I to 125 I and 129 I with 1% of the population receiving 3.9, 4.0 and 7.2 times the median dose for the respective isotopes. In newboms, these values were 7.5, 12.3 and 19.0 for 131 I, 125 I and 129 I respectively. The ratio of the beta dose delivered to the epithelium versus a homogeneous distributed dose was estimated for different iodine concentrations in colloid, epithelium and interstitium. In adults, for 131 I, about 40% of the beta dose was delivered to the epithelial cells whereas this fraction varied depending on the concentration for 125 I and 129 I, i.e. 20-30% at a relative epithelial concentration of 20% and 7-14% at a concentration of 3%. Small variations were observed depending on age.

We determined whether adenoviral gene transfer of endothelial nitric oxide synthase (eNOS) to the penis of streptozotocin induced diabetic rats could improve the impaired erectile response.Two experimental groups of animals were transfected with adenoviruses, including streptozotocin (Sigma Chemical Company, St. Louis, Missouri) diabetic rats with AdCMVbetagal and streptozotocin diabetic rats with AdCMVeNOS. At 1 to 2 days after transfection these study animals underwent cavernous nerve stimulation to assess erectile function and their responses were compared with those of age matched control rats. In control and transfected streptozotocin diabetic rats eNOS and neuronal NOS (nNOS) were examined by Western blot analysis. Constitutive and inducible NOS activities were evaluated in the presence and absence of calcium by L-arginine to L-citrulline conversion and nitrate plus nitrite levels were measured. In control and streptozotocin diabetic penes beta-galactosidase activity and localization were determined.After transfection with AdCMVbetagal beta-galactosidase was localized to the endothelium and smooth muscle cells of the streptozotocin diabetic rat penis. Streptozotocin diabetic rats had a significant decrease in erectile function, as determined by peak and total intracavernous pressure (area under the curve) after cavernous nerve stimulation compared with control rats. Streptozotocin diabetic rats transfected with AdCMVeNOS had peak intracavernous pressure and area under the curve similar to those in control animals. This change in erectile function was a result of eNOS over expression with an increase in eNOS protein expression and constitutive NOS activity as well as an increase in nitric oxide biosynthesis, as reflected by an increase in cavernous nitrate plus nitrite formation. There was no change in nNOS protein expression or calcium independent conversion of NOS (inducible NOS activity).Adenoviral gene transfer of eNOS significantly increased peak and total intracavernous pressure to cavernous nerve stimulation in streptozotocin diabetic rats to a value similar to the response observed in control rats. Our results suggest that eNOS contributes significantly to the physiology of penile erection. These data demonstrate that in vivo adenoviral gene transfer of eNOS can physiologically improve erectile function in the streptozotocin diabetic rat.

Increased superoxide anion (O[Formula: see text]·) may contribute to vascular dysfunction in aging. In aged cavernosal tissue, lucigenin-enhanced chemiluminescence demonstrated a threefold increase in superoxide formation, and the oxidative fluorescent probe hydroethidine indicated higher superoxide levels throughout the aged penis. This increase in superoxide was associated with impaired cavernosal nerve-mediated and agonist-induced erectile responses, increased nitrotyrosine staining, and lower cGMP levels, but no compensatory change in cavernosal extracellular (EC)-superoxide dismutase (EC-SOD) mRNA or protein. In vivo adenoviral (Ad) gene transfer of EC-SOD to the penis resulted in higher expression of EC-SOD mRNA, protein, SOD activity, cGMP levels, and lower nitrotyrosine staining. Transfection with AdCMVEC-SOD resulted in a significant increase in erectile response to cavernosal nerve stimulation, ACh, and zaprinast to a magnitude similar to young rats. These data provide evidence in support of the hypothesis that erectile dysfunction associated with aging is related in part to an increase in cavernosal O[Formula: see text]· formation. Gene-transfer of EC-SOD reduces superoxide formation and restores age-associated erectile function and may represent a novel therapeutic target for the treatment of erectile dysfunction.

Nitric oxide (NO) plays critical roles in the regulation of integrated cardiac and vascular function and homeostasis. An understanding of the physiologic role and relative contribution of the three NO synthase isoforms (neuronal--NOS1, inducible--NOS2, and endothelial--NOS3) is imperative to comprehend derangements of the NO signaling pathway in the failing cardiovascular system. Several theories of NO and its regulation have developed as explanations for the divergent observations from studies in health and disease states. Here we review the physiologic and pathophysiologic influence of NO on cardiac function, in a framework that considers several theories of altered NO signaling in heart failure. We discuss the notion of spatial compartmentalization of NO signaling within the myocyte in an effort to reconcile many controversies about derangements in the influences of NO in the heart and vasculature.

Peyronie's disease is an idiopathic, localized connective tissue disorder of the penis which involves the tunica albuginea of the corpus cavernosum and the adjacent areolar space. Peyronie's disease is characterized by local changes in the collagen and elastic fiber composition of the tunica albuginea. The formation of fibrotic plaques alters penile anatomy and can cause different degrees of bending and narrowing, as well as penile pain and erectile dysfunction. Though long recognized as an important clinical entity of the male genitalia, the etiology of this disease has remained poorly understood. Until recently there have been no studies to examine the role nitric oxide (NO) and nitric oxide synthase (NOS) isoforms may play in the onset and progression of Peyronie's disease. NO is a potent biological mediator with diverse physiological and pathophysiological roles. The purpose of this review is to describe each of the NOS isoforms and their potential roles in the pathophysiology of Peyronie's disease, with particular emphasis on the regulation of endothelial and inducible NOS isoforms.

Erectile dysfunction in the aging male is caused, in part, by inadequate relaxation of the corpora cavernosal smooth musculature. Calcitonin gene-related peptide (CGRP), a peptide neurotrasmitter localized in the corpora cavernosa, is down-regulated in the aging rat penis. We examined the hypothesis that this reduction in CGRP may contribute to decreased cavernosal smooth muscle relaxation. Therefore, we sought to determine whether adenoviral-mediated gene transfer of prepro-CGRP (AdRSVCGRP) could enhance erectile responses in aged rats. We found a significant decrease in CGRP concentrations and in cAMP and cGMP levels in aged rat cavernosal tissue compared to younger rats. Aged rats also had significantly lower erectile function as determined by cavernosal nerve stimulation compared to younger rats. Five days after transfection with AdRSVCGRP, these aged rats had an approximately threefold increase in cavernosal CGRP levels compared to animals transfected with adenoviruses encoding nuclear-targeted beta-galactosidase (AdRSV beta gal). The AdRSVCGRP-transfected animals also demonstrated an increase in CGRP mRNA and immunohistochemical localization of CGRP in the smooth muscle of the corpora cavernosa. In addition, cAMP levels in the corpora cavernosa were significantly increased, whereas cGMP levels remained unchanged. Adenoviral transduction efficiency of beta-galactosidase reporter gene was measured by chemiluminescence and was observed in cavernosal tissue 5 days after transfection with AdRSV beta gal. More importantly, 5 days after administration of AdRSVCGRP, a significant increase was observed in the erectile response to cavernosal nerve stimulation in the aged rat, similar to the response observed in younger rats. These data suggest that in vivo adenoviral gene transfer of CGRP can physiologically improve erectile function in the aged rat.

Responses to human calcitonin gene-related peptide (hCGRP) and human adrenomedullin (hADM) hAmylin were investigated in isolated mesenteric resistance arteries from the rat. The results of the present investigation show that hCGRP, hAmylin, and hADM induce dose-related vasodilator responses in isolated resistance arteries from the rat mesenteric vascular bed. Vasodilator responses to hCGRP and hAmylin were not altered after denuding the vascular endothelium, after administration of the nitric oxide synthase inhibitor L-NA, or after administration of the soluble guanylate cyclase inhibitor ODQ, suggesting that vasodilator responses to hCGRP and hAmylin are not mediated by the release of nitric oxide from the vascular endothelium and the subsequent increase in cGMP. Vasodilator responses to hCGRP, hAmylin, and hADM were not altered by the vascular selective K+ATP channel antagonist U-37883A. The role of the CGRP1 receptor was investigated and responses to hCGRP and hAmylin, but not hADM, were significantly reduced following administration of hCGRP-(8–37). Moreover, vasodilator responses to hCGRP and hAmylin, but not hADM, were significantly reduced by hAmylin-(8–37), suggesting that an hAmylin-(8–37)-sensitive receptor mediates responses to hCGRP and hAmylin in the rat mesenteric artery. These data suggest that hCGRP and hAmylin have direct vasodilator effects in the isolated mesenteric resistance artery that are mediated by hAmylin-(8–37)- and hCGRP-(8–37)-sensitive receptors.

Background: This study was designed to examine the responses to and the mechanism by which purinergic agonists decrease vascular resistance in the mesenteric vascular bed of the cat. Methods and Results: Injections of ATP, UTP, and 2-MethylThioATP (2-MetSATP) into the mesenteric perfusion circuit elicited dose-dependent decreases in perfusion pressure while injections of P,y-MethylATP (f3,y-MetATP) produced a biphasic response with an initial vasopressor response followed by a vasodilator response. The order of potency of the vasodilator response was 2-MetSATP > ATP > UTP > P,y-MetATP. The vasodilator responses to ATP, UTP, 2-MetSATP, and P,y-MetATP were increased in duration by the cAMP phosphodiesterase inhibitor, rolipram. However, vasodilator responses were not altered by the adminstration of a nitric oxide synthase inhibitor, a cGMP phosphodiesterase inhibitor, or a cyclooxygenase inhibitor. Treatment with PPADS, a P2X,, P2Y, and P2Y4 receptor antagonist, did not alter vasodilator responses to the purinergic agonists; however, the vasopressor component of the response to P,y-MetATP was decreased. Conclusions: These data suggest that ATP, UTP, 2-MetSATP, and P,Y-MetATP dilate the mesentary vascular bed in the cat by a cAMP dependent mechanism, and that nitric oxide or prostaglandin release, cGMP accumulation, or activation of P2X,, P2Y, or P2Y4 receptors play little or no role in mediating vasodilator responses to the purinergic agonists in this regional vascular bed. In addition, these results suggest that the pressor component of the response to P,y-MetATP is mediated by the activation of P2X, receptors.

Controlled release of nitric oxide (NO*) may be useful in the treatment of a variety of vascular disorders. NO* donors of the diazeniumdiolate family with different rates of spontaneous NO* release have been synthesized. In the current study responses to seven diazeniumdiolate NO* donors (DEA/NO*, DETA/NO*, OXI/NO*, PIPERAZI/NO*, PROLI/NO*, SPER/NO*, and SULFI/NO*) were investigated in the hindquarters vascular bed of the cat. Intravenous injections of all NO* donors caused dose-dependent decreases in systemic arterial pressure and the rank order of potency was SNP > DEA/NO* > PIPERAZI/NO* > SPER/NO* > PROLI/NO* > OXI/NO*. Injections of all NO* donors into the hindlimb perfusion circuit caused dose-related decreases in hindquarters perfusion pressure that were similar to the order of potency in decreasing systemic arterial pressure. The rank order of the time required for the response to return to 50% of the maximal decrease in pressure (T(1/2)) and total duration of action of the NO* donors was SPER/NO* > PIPERAZI/NO* > DEA/NO* > OXI/NO* > DETA/NO* > PROLI/NO* > SULFI/NO*. After treatment with the NO* synthase inhibitor, N(omega)-nitro-L-arginine methyl ester (100 mg/kg, i.v.), hindlimb vasodilator responses to the NO* donors were not significantly different, but vasodilator responses to acetylcholine were significantly reduced. After treatment with zaprinast (2 mg/kg, i.v.), a type V cyclic 3',5'-guanosine monophosphate-specific phosphodiesterase inhibitor, the duration of vasodilator responses to the NO* donors, as measured by T(1/2), was increased significantly, whereas the duration of the response to the beta2-adrenergic receptor agonist albuterol was unchanged. These data suggest that diazeniumdiolate NO* donors are endothelium-independent, directly stimulate soluble guanylate cyclase, and decrease vascular resistance by increasing cyclic 3',5'-guanosine monophosphate levels in the hindquarters vascular bed of the cat.

Purpose: Zaprinast, dipyridamole and sildenafil were injected into the corpora cavernosa of cats to determine whether changes in the steady state level of cyclic guanosine monophosphate (cGMP) induced by inhibiting type 5 phosphodiesterase would cause an erectile response.Materials and Methods: Increases in intracavernous pressure, penile length and erectile response duration were determined after intracavernous injection of the type 5 cGMP specific phosphodiesterase inhibitors zaprinast, dipyridamole and sildenafil as well as combined zaprinast and prostaglandin E1 (PGE1), and zaprinast and sodium nitroprusside. Systemic arterial pressure was concurrently assessed in these experiments. All responses to phosphodiesterase inhibitors were compared to a control triple drug combination of 1.65 mg. papaverine, 0.5 μg. PGE1 and 25 μg. phentolamine.Results: Each selective type 5 phosphodiesterase inhibitor caused dose related increases in intracorporeal pressure and penile length. However, none of the compounds ...

Responses to the nonpeptide angiotensin II agonist 5, 7-Dimethyl-2-ethyl-3-[[2'-([butyloxycarbonyl) aminosulfonyl]-5'-(3-methyoxybenzyl)-[1, 1'-biphenyl]-4-yl]methyl]-3H-imidazo[4,5-b]pyridine (L-163,491) were investigated and compared with responses to angiotensin II, angiotensin IV and norepinephrine in the hindquarters vascular bed of the cat under constant-flow conditions. Injections of L-163,491 into the hindquarter perfusion circuit caused dose-related increases in hindquarters perfusion pressure. In relative terms, angiotensin II was more potent than norepinephrine, which was more potent than angiotensin IV and L-163,491 in increasing hindlimb vascular resistance. The slope of the dose-response curve for L-163,491 was flat, while the apparent affinity of the compound for angiotensin AT(1) receptors was slightly greater than angiotensin IV. Responses to L-163,491 were inhibited by the angiotensin AT(1) receptor antagonist DuP 532 (2-propyl-4-pentafluoroethyl-1-[2'-(1H-tetrazol-5-yl)bipheny l-4-yl)me thyl]imidazole-5-carboxylic acid) and were not altered by the angiotensin AT(2) receptor antagonist PD123,319 (S(+)-1-[[4-(Dimethylamino)-3-methylphenyl]methyl]-5-(diphenylacetyl+ ++) -4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid ditribluoroacetate). However, the increase in hindlimb perfusion pressure in response to angiotensin II and angiotensin IV was significantly decreased following injection of L-163,491. These data suggest that the nonpeptide angiotensin analog L-163,491 has partial agonist activity, which is dependent on the stimulation of angiotensin AT(1) receptors in the hindquarters vascular bed of the cat.

The effects of the purinergic agonists, ATP, ATPγS, UTP, and 2-Met-Thio AP, were investigated in the hindlimb vascular bed of the cat. Under constant-flow conditions, injections of the purinergic agonists into the perfusion circuit elicited dose-related decreases in perfusion pressure. The order of potency was 2-Met-Thio ATP > ATPγS > ATP > UTP. In contrast, injections of GTPγS, cAMP, UDP, and UMP had no effect. Vasodilator responses to ATP, ATPγS, UTP, and 2-Met-Thio ATP were increased in duration by the cAMP phosphodiesterase inhibitor rolipram, whereas the cGMP phosphodiesterase inhibitor zaprinast had no effect. Responses to the purinergic agonists were not altered by nitric oxide synthase inhibitors, K+ATP channel antagonists, cyclooxygenase inhibitors, or agents that interfere with the actions of the adrenergic nervous system. These data suggest that ATP, ATPγS, UTP, and 2-Met-Thio ATP dilate the hindlimb vascular bed by a direct cAMP-dependent mechanism, and that the release of nitric oxide, vasodilator prostaglandins, K+ATP channel opening, or an inhibitory effect on the adrenergic nervous system play little, if any, role in mediating or modulating responses to the purinergic agonists in the hindlimb circulation of the cat.Key words: purinergic agonists, P2 purinergic receptors, cAMP-dependent vasodilator activity, adrenergic system, nitric oxide prostaglandins.

Purpose: Our objective was to assess erectile function in saline-injected, transforming growth factor-beta 1 (TGF-β1)-injected, and surgical injury rats after six weeks and to determine the role of nitric oxide in this rat model of Peyronie’s disease.Materials and Methods: Fifty-four adult male CD rats were divided into three groups: 1) saline-injected (0.1 ml.) into the tunica albuginea; 2) TGF-β1 (0.5 μg.) injected into the tunica albuginea; and 3) surgical injury to the tunica albuginea. All groups underwent electrical stimulation of the cavernosal nerve and pharmacological stimulation with acetylcholine, an endothelium-dependent vasodilator, after six weeks. In a separate group of animals, aminoguanidine (5 mg./kg. i.v.), a specific iNOS inhibitor, was administered and cavernosal nerve stimulation was performed. Cavernosal tissue was homogenized and constitutive and inducible NOS enzyme activity were measured by L-arginine to L-citrulline conversion in the presence and absence of calcium after 2 days,...

Nitric oxide (NO) is an important mediator in the relaxation of cavernosal smooth muscle. The aim of this study was to investigate the in vivo feline erectile response after transurethral administration of sodium nitroprusside (SNP), a NO donor drug. Erectile responses after administration of transurethral SNP were compared with those elicited by an intracavernosal control triple-drug combination (1.65 mg papaverine, 25 microg phentolamine, and 0.5 microg prostaglandin E1). SNP was administered via a 20-gauge Jelco intravenous catheter in a volume of 200 microl and changes in intracavernosal pressure, penile length, and systemic blood pressure were monitored. The control triple-drug combination was administered via a 30-gauge needle at the end of each experiment to serve as a control reference. Transurethral administration of SNP (1-4 mg) induced penile erection in a dose-dependent manner with minimal changes in systemic blood pressure. The maximum increase in intracavernosal pressure and penile length after transurethral administration of SNP (4 mg) was significantly less than after the intracavernosal injection of the control triple-drug combination (P0.01). These data suggest that transurethral administration of SNP can induce an erectile response in cats with minimal side effects.

Purpose:: Phosphodiesterases (PDEs) are an important component of the signal transduction pathway during the erectile response. To determine the PDE isoforms in the corpora cavernosa in the cat and to establish the functional presence of PDE 4 in human cavernosal tissue, the erectile response to intracavernosal phosphodiesterase (PDE) inhibitors alone and the combination of PDE inhibitors and prostaglandin E1 (PGE1) was evaluated in the anesthetized cat. The in vitro formation of cAMP and cGMP in human cavernosal smooth muscle cells (HCSMCs) treated with PGE1 and rolipram in primary culture was also measured.Materials and Methods:: In pentobarbital-anesthetized cats, increases in intracavernosal pressure, penile length, and duration of erectile response were determined after intracavernosal injections of (i) the type 3 cAMP-specific, cGMP-inhibitable PDE inhibitor, milrinone, (ii) the type 4 cAMP-specific PDE inhibitor, rolipram, (iii) the type 5 cGMP-specific PDE inhibitor, zaprinast, and (iv) the combin...

Purpose: The effects of novel nitric oxide (NO) donors administered intracavernosally and transurethrally on erectile function in the anesthetized cat were evaluated.Materials and Methods: In pentobarbital-anesthetized cats, increases in intracavernosal pressure, penile length, and duration of erectile response were determined after intracavernosal and transurethral injections of novel NO donors (MAHMA/NO, PAPA/NO, DEA/NO, PIPERAZI/NO and PROLI/NO). All parameters were measured after administration of NO donors intracavernosally via a 30-gauge needle and urethrally via a Jelco i.v. catheter in a volume of 200 micro l. Systemic arterial pressure was also assessed in these experiments. All NO donors were compared with a triple-drug control combination comprised of papaverine (1.65 mg.), prostaglandin E1 (0.5 micro g.), and phentolamine (25 micro g.).Results: MAHMA/NO, PAPA/NO, DEA/NO, PIPERAZI/NO and PROLI/NO induced dose dependent increases in intracavernosal pressure and penile length (p