Erectile Dysfunction in Heme-Deficient Nitric Oxide-Unresponsive Soluble Guanylate Cyclase Knock-In Mice.

INTRODUCTION: The nitric oxide (NO), soluble guanylate cyclase (sGC), and cyclic guanosine monophosphate (cGMP) pathway is the leading pathway in penile erection. AIM: To assess erectile function in a mouse model in which sGC is deficient in heme (apo-sGC) and unresponsive to NO. METHODS: Mutant mice (sGCß1ki/ki) that express an sGC enzyme that retains basal activity but fails to respond to NO because of heme deficiency (apo-sGC) were used. Isolated corpora cavernosa from sGCß1ki/ki and wild-type mice were mounted in vitro for isometric tension recordings in response to sGC-dependent and -independent vasorelaxant agents. In addition, the erectile effects of some of these agents were tested in vivo at intracavernosal injection. MAIN OUTCOME MEASURES: In vitro and in vivo recordings of erectile responses in sGCß1ki/ki and wild-type mice after stimulation with sGC-dependent and -independent vasorelaxant agents. RESULTS: NO-induced responses were abolished in sGCß1ki/ki mice in vitro and in vivo. The ability of the heme-dependent, NO-independent sGC stimulator BAY 41-2272 to relax the corpora cavernosa was markedly attenuated in sGCß1ki/ki mice. In contrast, the relaxation response to the heme- and NO-independent sGC activator BAY 58-2667 was significantly enhanced in sGCß1ki/ki mice. The relaxing effect of sGC-independent vasorelaxant agents was similar in wild-type and sGCß1ki/ki mice, illustrating that the observed alterations in vasorelaxation are limited to NO-sGC-cGMP-mediated processes. CONCLUSION: Our results suggest that sGC is the sole target of NO in erectile physiology. Furthermore, this study provides indirect evidence that, in addition to sGCα1ß1, sGCα2ß1 is important for erectile function. In addition, the significant relaxation observed in sGCß1ki/ki mice with the cumulative addition of the sGC activator BAY 58-2667 indicates that sGC activators might offer value in treating erectile dysfunction.

At the cross-point of connexins, calcium, and ATP: blocking hemichannels inhibits vasoconstriction of rat small mesenteric arteries.

AIMS: Connexins form gap-junctions (GJs) that directly connect cells, thereby coordinating vascular cell function and controlling vessel diameter and blood flow. GJs are composed of two hemichannels contributed by each of the connecting cells. Hemichannels also exist as non-junctional channels that, when open, lead to the entry/loss of ions and the escape of ATP. Here we investigated cross-talk between hemichannels and Ca2+/purinergic signalling in controlling blood vessel contraction. We hypothesized that hemichannel Ca2+ entry and ATP release contributes to smooth muscle cell (SMC) Ca2+ dynamics, thereby influencing vessel contractility. We applied several peptide modulators of hemichannel function and inhibitors of Ca2+ and ATP signalling to investigate their influence on SMC Ca2+ dynamics and vessel contractility. METHODS AND RESULTS: Confocal Ca2+ imaging studies on small mesenteric arteries (SMAs) from rat demonstrated that norepinephrine-induced SMC Ca2+ oscillations were inhibited by blocking IP3 receptors with xestospongin-C and by interfering with hemichannel function, most notably by the specific Cx43 hemichannel blocking peptide TAT-L2 and by TAT-CT9 that promotes Cx43 hemichannel opening. Evidence for hemichannel involvement in SMC function was supported by the fact that TAT-CT9 significantly increased SMC resting cytoplasmic Ca2+ concentration, indicating it facilitated Ca2+ entry, and by the observation that norepinephrine-triggered vessel ATP release was blocked by TAT-L2. Myograph tension measurements on isolated SMAs showed significant inhibition of norepinephrine-triggered contractility by the ATP receptor antagonist suramin, but the strongest effect was observed with TAT-L2 that gave ∼80% inhibition at 37 °C. TAT-L2 inhibition of vessel contraction was significantly reduced in conditional Cx43 knockout animals, indicating the effect was Cx43 hemichannel-dependent. Computational modelling suggested these results could be explained by the opening of a single hemichannel per SMC. CONCLUSIONS: These results indicate that Cx43 hemichannels contribute to SMC Ca2+ dynamics and contractility, by facilitating Ca2+ entry, ATP release, and purinergic signalling.

Cardiovascular and pharmacological implications of haem-deficient NO-unresponsive soluble guanylate cyclase knock-in mice.

Oxidative stress, a central mediator of cardiovascular disease, results in loss of the prosthetic haem group of soluble guanylate cyclase (sGC), preventing its activation by nitric oxide (NO). Here we introduce Apo-sGC mice expressing haem-free sGC. Apo-sGC mice are viable and develop hypertension. The haemodynamic effects of NO are abolished, but those of the sGC activator cinaciguat are enhanced in apo-sGC mice, suggesting that the effects of NO on smooth muscle relaxation, blood pressure regulation and inhibition of platelet aggregation require sGC activation by NO. Tumour necrosis factor (TNF)-induced hypotension and mortality are preserved in apo-sGC mice, indicating that pathways other than sGC signalling mediate the cardiovascular collapse in shock. Apo-sGC mice allow for differentiation between sGC-dependent and -independent NO effects and between haem-dependent and -independent sGC effects. Apo-sGC mice represent a unique experimental platform to study the in vivo consequences of sGC oxidation and the therapeutic potential of sGC activators.

Relaxant and antioxidant capacity of the red wine polyphenols, resveratrol and quercetin, on isolated mice corpora cavernosa.

INTRODUCTION: The red wine polyphenols resveratrol and quercetin are known for their vasorelaxant and antioxidant capacity, which is assumed to rely on the activation of the nitric oxide (NO)/soluble guanylyl cyclase (sGC) pathway. Vasodilators as well as antioxidants can regulate penile erection and be beneficial for the treatment of erectile dysfunction (ED). AIMS: The goal of this study was to evaluate the NO/sGC dependency of the relaxant effect of resveratrol and quercetin on mice aorta and corpora cavernosa (CC), as well as to explore their influence on oxidative stress-induced ED. METHODS: Isolated mice aorta and CC were mounted for isometric tension recordings into organ baths. Cumulative concentration-response curves were constructed for resveratrol and quercetin in the absence/presence of inhibitors of the NO/sGC pathway. In addition, in CC the effect of resveratrol and quercetin was studied on NO-mediated relaxations using acetylcholine (Ach), sodium nitroprusside (SNP), and electrical field stimulation (EFS). In certain experiments, corporal tissues were exposed to oxidative stress using palmitic acid (PA, 0.5 mM). MAIN OUTCOME MEASURES: Corporal responses to resveratrol and quercetin were measured in the presence/absence of inhibitors of different molecular pathways. The effect of resveratrol and quercetin incubation on Ach-, SNP-, or EFS-mediated responses was explored in the presence/absence of PA. RESULTS: While both polyphenols are potent vasodilators of mice aorta, only resveratrol relaxes mice CC. The relaxation response to resveratrol on aorta was diminished in sGCα1 (-/-) mice, but not on CC. The polyphenols did not influence Ach-, SNP-, or EFS-mediated relaxations as such. Resveratrol, but not quercetin, was able to significantly reverse PA-induced decrease of EFS relaxations. CONCLUSION: The red wine compound resveratrol, but not quercetin, relaxes isolated mice CC concentration-dependently through mechanisms independent of the NO/sGC pathway. Resveratrol is a more potent antioxidant than quercetin, being able to restore decreased neuronal NO responses in mice CC.

NO-donating oximes relax corpora cavernosa through mechanisms other than those involved in arterial relaxation.

INTRODUCTION: Erectile dysfunction (ED), as well as many cardiovascular diseases, is associated with impaired nitric oxide (NO) bioavailability. Recently, oxime derivatives have emerged as vasodilators due to their NO-donating capacities. However, whether these oximes offer therapeutic perspectives as an alternative NO delivery strategy for the treatment of ED is unexplored. AIMS: This study aims to analyze the influence of formaldoxime (FAL), formamidoxime (FAM), and cinnamaldoxime (CAOx) on corporal tension and to elucidate the underlying molecular mechanisms. METHODS: Organ bath studies were carried out measuring isometric tension on isolated mice corpora cavernosa (CC), thoracic aorta, and femoral artery. After contraction with norepinephrine (NOR), cumulative concentration-response curves of FAL, FAM, and CAOx (100 nmol/L-1 mmol/L) were performed. MAIN OUTCOME MEASURES: FAL-/FAM-induced relaxations were evaluated in the absence/presence of various inhibitors of different molecular pathways. RESULTS: FAL, FAM, and CAOx relax isolated CC as well as aorta and femoral artery from mice. ODQ (soluble guanylyl cyclase-inhibitor), diphenyliodonium chloride (nonselective flavoprotein inhibitor), and 7-ethoxyresorufin (inhibitor of CYP450 1A1 and NADPH-dependent reductases) substantially blocked the FAL-/FAM-induced relaxation in the arteries but not in CC. Only a small inhibition of the FAM response in CC was observed with ODQ. CONCLUSIONS: This study shows for the first time that NO-donating oximes relax mice CC. Therefore, oximes are a new group of molecules with potential for the treatment of ED. However, the underlying mechanism(s) of the FAL-/FAM-induced corporal relaxation clearly differ(s) from the one(s) involved in arterial vasorelaxation.

Perivascular adipose tissue, inflammation and vascular dysfunction in obesity.

Adipocytes are no longer considered just as cells related to storage of energy and thermoregulation. Now we know that they release a huge number of paracrine and endocrine biologically active molecules. This is also the case for perivascular adipose tissue (PVAT) that surrounds almost all blood vessels in the organism. PVAT secretes the so-called adipo(cyto)kines that, because of its proximity, can easily influence vascular smooth muscle cells. The role of PVAT on vascular function can be both protective and deleterious. Normal healthy PVAT, as present in lean subjects, helps to keep the blood vessels dilated as its presence diminishes the effect of vasocontractile agents. Obesity is associated with an increased mass in PVAT. Excessive adipocyte hypertrophy may result in "adiposopathy" in which PVAT attracts macrophages and becomes a more inflammatory phenotype. This leads to a change in profile of the released adipo(cyto)kines, resulting in a decreased vasorelaxing effect of PVAT, which may be linked to obesity-induced hypertension. It also results in smooth muscle cell migration and proliferation and the development of atherosclerotic lesions. The increased knowledge of PVAT function brings up new targets that can be useful to develop novel therapeutic and preventive strategies for obesity-related cardiovascular diseases. This mini-review presents a general overview of the actual knowledge on the role of PVAT on vascular function and dysfunction in obesity.

Adipocytokines in relation to cardiovascular disease.

Adipose tissue can be considered as a huge gland producing paracrine and endocrine hormones, the adipo(cyto)kines. There is growing evidence that these adipo(cyto)kines may link obesity to cardiovascular diseases. The excessive adipocyte hypertrophy in obesity induces hypoxia in adipose tissue. This leads to adiposopathy, the process that converts "healthy" adipose tissue to "sick" adipose tissue. This is accompanied by a change in profile of adipo(cyto)kines released, with less production of the "healthy" adipo(cyto)kines such as adiponectin and omentin and more release of the "unhealthy" adipo(cyto)kines, ultimately leading to the development of cardiovascular diseases. The present review provides a concise and general overview of the actual concepts of the role of adipo(cyto)kines in endothelial dysfunction, hypertension, atherosclerosis and heart diseases. The knowledge of these concepts may lead to new tools to improve health in the next generations.

Divergent molecular mechanisms underlay CO- and CORM-2-induced relaxation of corpora cavernosa.

INTRODUCTION: Similar to nitric oxide (NO), the principal mediator of penile erection, carbon monoxide (CO) possesses vasodilator capacities. However, whether CO could be a therapeutic target for treating erectile dysfunction (ED) is unexplored. The danger associated with systemic administration of CO has led to the development of CO-releasing molecules (CORMs), releasing CO in a local, safe and controlled way. These CORMs have shown positive outcomes in cardiovascular studies. More knowledge on the (patho)physiological functions of CO in erectile function and the potential therapeutic role of CORMs is required. AIM: The present study aims the assessment of the effect of CO and CO donor CORM-2 on the corporal tension and the underlying molecular mechanisms. METHODS: Organ bath studies were performed measuring isometric tension on isolated mice corpora cavernosa (CC) strips. Responses to CO (10-300 µmol/L) and CORM-2 (10-100 µmol/L) were measured in the presence/absence of activators/inhibitors of different molecular pathways. MAIN OUTCOME MEASURES: CO and CORM-2 relax corporal strips concentration dependently, although the molecular mechanisms behind the corporal relaxation seem to differ completely. RESULTS: CO induces corporal relaxation by activating soluble guanylyl cyclase (sGC), increasing cyclic guanosine monophosphate (cGMP) concentrations. The molecular mechanism involved in CORM-2-induced corporal relaxation is not related to sGC activation and remains obscure. CONCLUSIONS: Both CO and CORM-2 induce corporal relaxation, although the underlying molecular mechanisms show no resemblance. That CO induces corporal relaxation through a mechanism similar to that of NO could be of importance as it indirectly offers the possibility that endogenous CO might serve as a backup system for insufficient NO availability in cases of ED. Whether CORM-2 possesses the same capacity remains questionable and requires further research.

Genetic modifiers of hypertension in soluble guanylate cyclase α1-deficient mice.

Nitric oxide (NO) plays an essential role in regulating hypertension and blood flow by inducing relaxation of vascular smooth muscle. Male mice deficient in a NO receptor component, the α1 subunit of soluble guanylate cyclase (sGCα1), are prone to hypertension in some, but not all, mouse strains, suggesting that additional genetic factors contribute to the onset of hypertension. Using linkage analyses, we discovered a quantitative trait locus (QTL) on chromosome 1 that was linked to mean arterial pressure (MAP) in the context of sGCα1 deficiency. This region is syntenic with previously identified blood pressure-related QTLs in the human and rat genome and contains the genes coding for renin. Hypertension was associated with increased activity of the renin-angiotensin-aldosterone system (RAAS). Further, we found that RAAS inhibition normalized MAP and improved endothelium-dependent vasorelaxation in sGCα1-deficient mice. These data identify the RAAS as a blood pressure-modifying mechanism in a setting of impaired NO/cGMP signaling.

Adipose tissue as regulator of vascular tone.

Adipokines secreted by visceral, subcutaneous, and perivascular adipocytes are involved in the regulation of vascular tone by acting as circulatory hormones (leptin, adiponectin, omentin, visfatin, angiotensin II, resistin, tumor necrosis factor-α, interleukin-6, apelin) and/or via local paracrine factors (perivascular adipocyte-derived relaxing and contractile factors). Vascular tone regulation by adipokines is compromised in obesitas and obesity-related disorders. Hypoxia created in growing adipose tissue dysregulates synthesis of vasoactive adipokines in favor of harmful proinflammatory adipokines, while the levels of the cardioprotective adipokines adiponectin and omentin decrease. Considering the potential of the role of adipokines in obesity-related vascular diseases, strategies to counter these diseases by targeting the adipokines are discussed.

Divergent mechanisms involved in CO and CORM-2 induced vasorelaxation.

Carbon monoxide (CO) may play an important physiological role in regulation of the vascular tone. CO-releasing molecule (CORM-2) is frequently used as a CO-donor to evaluate (patho)physiological properties of CO and its potential therapeutic applications. The aim of this study was to examine the molecular mechanisms underlying the vasodilatory properties of CORM-2 as this has not yet been extensively explored. Isometric tension recordings were performed using mice and rat isolated aortic ring segments as well as mice femoral artery ring segments. Responses to CO (10 µmol/l-300 µmol/l) and CORM-2 (30 µmol/l-600 µmol/l) were evaluated in the presence/absence of activators/inhibitors of different molecular pathways. CO was unable to relax mice blood vessels, whereas it induced concentration-dependent relaxations in rat aorta. The response to CO was inhibited by both the soluble guanylyl cyclase (sGC) inhibitor ODQ (10 µmol/l) and potassium (K(+)) channel blocker tetraethyl-ammonium chloride (3 mmol/l). CORM-2 relaxed both mice and rat isolated blood vessels in a concentration-dependent manner, however this response was only partially blocked by ODQ and tetraethyl-ammonium chloride. Interestingly, 4-aminopyridine (3 mmol/l) inhibited the CORM-2 induced vasodilatation whereas iberiotoxin (100 nmol/l) had no influence. The molecular mechanisms underlying CORM-2 induced relaxation differ from those of CO-induced relaxation. While CO relaxes vessels through activation of sGC and/or calcium-activated K(+)-channels, CORM-2 exerts its vasodilatory properties only partially through sGC or K(+)-channels activation. CORM-2 induced vasodilatation seems to involve voltage-dependent rather than calcium-activated K(+)-channels.

New therapeutic targets for the treatment of erectile dysfunction.

INTRODUCTION: Despite the high efficacy and safety rates of the currently available treatments for erectile dysfunction, basic research reveals numerous new targets that are explored for therapeutic use. AIM: To overview potential new targets and to review available animal and human studies focusing on the potential of these targets for effective therapy for treating erectile dysfunction. METHODS: A comprehensive literature search was conducted using the PubMed and Medline database, and citations were selected based on relevance. MAIN OUTCOME MEASURES: Data are presented based on the analysis of the selected scientific information and published clinical trials. RESULTS: Fundamental research has, in the past decade, increased the understanding in both the physiological and the pathophysiological pathways that play a role in erectile function. As this information increases each day, new targets to treat erectile dysfunction are frequently presented. Currently a number of new therapeutic targets have been published. Some of them target the nitric oxide/cyclic guanosine monophosphate relaxation pathway as the phosphodiesterase type 5 inhibitors do, others primarily target pathways involved in contraction. Also, targets within the central nervous system currently receive much attention. Some of these targets have already been used in clinical trials to test their efficacy and safety, with either disappointing or promising results. CONCLUSIONS: This review overviews potential therapeutic targets and summarizes animal as well as human studies evaluating their perspectives for the treatment of erectile dysfunction.

In vitro and in vivo studies on the importance of the soluble guanylyl cyclase α1 subunit in penile erection.

PURPOSE: Soluble guanylyl cyclase (sGC), which plays a pivotal role in penile erection, is a heterodimer build up by an α and a ß subunit. For both subunits two isoforms have been characterized, but only the sGCα(1)ß(1) and sGCα(2)ß(1) isoforms seem to be functionally active. To elucidate the functional role of the sGCα(1)ß(1) heterodimer in the mechanism of erection, experiments were performed in vivo and on isolated corpora cavernosa (CC) using sGCα(1)(-/-) mice. MATERIALS AND METHODS: For the in vivo study sGC-dependent and -independent vasorelaxing agents were injected intracavernosally in sGCα(1)(-/-) and sGCα(1)(+/+) mice and the rise in intracavernosal pressure was recorded. For the in vitro study, isolated CC tissues from sGCα(1)(-/-) and sGCα(1)(+/+) mice were mounted in organ baths for isometric tension recording and concentration-dependent curves were obtained for sGC-dependent and -independent vasorelaxing agents. These experiments were performed on 2 different mice strains (129SvEvS7 and C57BL6/J) to determine potential strain differences. RESULTS: The responses in sGCα(1)(-/-) after administration of the NO-donors, sodium nitroprusside (SNP) and spermine-NO, and to electrical stimulation are significantly reduced although not completely abolished. Responses to sGC-independent vasorelaxing agents are similar in sGCα(1)(-/-) and sGCα(1)(+/+) mice from both strains suggesting that the decreased potential of smooth muscle relaxation is not related to structural changes or changes in the pathway downstream sGC. CONCLUSION: This study illustrates the strain-independent importance of the sGCα(1)ß(1) heterodimer, although remaining vasorelaxing responses in the sGCα(1)(-/-) mice suggest a complementary role for the sGCα(2)ß(1) isoform or (an) sGC-independent mechanism(s).