Patrick Sips

Gender-specific hypertension and responsiveness to nitric oxide in sGCα1 knockout mice

AIM The effects of nitric oxide (NO) in the cardiovascular system are attributed in part to cGMP synthesis by the alpha1beta1 isoform of soluble guanylate cyclase (sGC). Because available sGC inhibitors are neither enzyme- nor isoform-specific, we generated knockout mice for the alpha1 subunit (sGCalpha1(-/-) mice) in order to investigate the function of sGCalpha1beta1 in the regulation of blood pressure and cardiac function. METHODS AND RESULTS Blood pressure was evaluated, using both non-invasive and invasive haemodynamic techniques, in intact and gonadectomized male and female sGCalpha1(-/-) and wild-type (WT) mice. Cardiac function was assessed with a conductance catheter inserted in the left ventricle of male and female sGCalpha1(-/-) and WT mice. Male sGCalpha1(-/-) mice developed hypertension (147 +/- 2 mmHg), whereas female sGCalpha1(-/-) mice did not (115 +/- 2 mmHg). Orchidectomy and treatment with an androgen receptor antagonist prevented hypertension, while ovariectomy did not influence the phenotype. Chronic testosterone treatment increased blood pressure in ovariectomized sGCalpha1(-/-) mice but not in WT mice. The NO synthase inhibitor Nomega-nitro-L-arginine methyl ester hydrochloride raised blood pressure similarly in male and female WT and sGCalpha1(-/-) mice. The ability of NO donor compounds to reduce blood pressure was slightly attenuated in sGCalpha1(-/-) male and female mice as compared to WT mice. The direct sGC stimulator BAY 41-2272 reduced blood pressure only in WT mice. Increased cardiac contractility and arterial elastance as well as impaired ventricular relaxation were observed in both male and female sGCalpha1(-/-) mice. CONCLUSION These findings demonstrate that sGCalpha1beta1-derived cGMP signalling has gender-specific and testosterone-dependent cardiovascular effects and reveal that the effects of NO on systemic blood pressure do not require sGCalpha1beta1.

Systemic NO production during (septic) shock depends on parenchymal and not on hematopoietic cells: in vivo iNOS expression pattern in (septic) shock

Septic shock is the leading cause of death in noncoronary intensive care units and the 10th leading cause of death overall. Several lines of evidence support an important role for the vasodilator NO in hypotension, a hallmark of septic shock. However, NO may also positively or negatively regulate inflammation, apoptosis, and oxidative stress. These dual effects of NO may relate to its isoform specific production but also to differences in cellular and/or temporal expression. Via bone marrow transplantations, we examined the contribution of hematopoietic cells to the dramatically elevated NO levels seen in (septic) shock. Surprisingly, hematopoietic cells are not responsible at all for the production of circulating NO after systemic tumor necrosis factor or lipopolysaccharide challenge and contribute only marginally in a bacteremic (Salmonella) model of septic shock. Immunohistochemistry identified the nonhematopoietic sources of NO as hepatocytes, paneth cells, and intestinal and renal epithelial cells. In contrast, during granulomatous Bacillus Calmette‐Guérin inflammation, the hematopoietic cell population represents the sole source of systemic NO. These mouse data demonstrate that, in contrast to the general conjecture, the dramatically elevated levels of NO during (septic) shock are not produced by hematopoietic cells such as monocytes/macrophages but rather by parenchymal cells in liver, kidney and gut.—Bultinck, J., Sips, P., Vakaet, L., Brouckaert, P., Cauwels, A. Systemic NO production during (septic) shock depends on parenchymal and not on hematopoietic cells: in vivo iNOS expression pattern in (septic) shock FASEB J. 20, E1619 –E1627 (2006)

Soluble Guanylate Cyclase-α1 Deficiency Selectively Inhibits the Pulmonary Vasodilator Response to Nitric Oxide and Increases the Pulmonary Vascular Remodeling Response to Chronic Hypoxia

Background— Nitric oxide (NO) activates soluble guanylate cyclase (sGC), a heterodimer composed of &agr;- and &bgr;-subunits, to produce cGMP. NO reduces pulmonary vascular remodeling, but the role of sGC in vascular responses to acute and chronic hypoxia remains incompletely elucidated. We therefore studied pulmonary vascular responses to acute and chronic hypoxia in wild-type (WT) mice and mice with a nonfunctional &agr;1-subunit (sGC&agr;1−/−). Methods and Results— sGC&agr;1−/− mice had significantly reduced lung sGC activity and vasodilator-stimulated phosphoprotein phosphorylation. Right ventricular systolic pressure did not differ between genotypes at baseline and increased similarly in WT (22±2 to 34±2 mm Hg) and sGC&agr;1−/− (23±2 to 34±1 mm Hg) mice in response to acute hypoxia. Inhaled NO (40 ppm) blunted the increase in right ventricular systolic pressure in WT mice (22±2 to 24±2 mm Hg, P<0.01 versus hypoxia without NO) but not in sGC&agr;1−/− mice (22±1 to 33±1 mm Hg) and was accompanied by a significant rise in lung cGMP content only in WT mice. In contrast, the NO-donor sodium nitroprusside (1.5 mg/kg) decreased systemic blood pressure similarly in awake WT and sGC&agr;1−/− mice as measured by telemetry (−37±2 versus −42±4 mm Hg). After 3 weeks of hypoxia, the increases in right ventricular systolic pressure, right ventricular hypertrophy, and muscularization of intra-acinar pulmonary vessels were 43%, 135%, and 46% greater, respectively, in sGC&agr;1−/− than in WT mice (P<0.01). Increased remodeling in sGC&agr;1−/− mice was associated with an increased frequency of 5′-bromo-deoxyuridine–positive vessels after 1 and 3 weeks (P<0.01 versus WT). Conclusions— Deficiency of sGC&agr;1 does not alter hypoxic pulmonary vasoconstriction. sGC&agr;1 is essential for NO-mediated pulmonary vasodilation and limits chronic hypoxia-induced pulmonary vascular remodeling.

Gastric motility in soluble guanylate cyclase α1 knock‐out mice

The principal target of the relaxant neurotransmitter nitric oxide (NO) is soluble guanylate cyclase (sGC). As the α1β1‐isoform of sGC is the predominant one in the gastrointestinal tract, the aim of this study was to investigate the role of sGC in nitrergic regulation of gastric motility in male and female sGCα1 knock‐out (KO) mice. In circular gastric fundus muscle strips, functional responses and cGMP levels were determined in response to nitrergic and non‐nitrergic stimuli. sGC subunit mRNA expression in fundus was measured by real‐time RT‐PCR; in vivo gastric emptying of a phenol red meal was determined. No changes were observed in sGC subunit mRNA levels between wild‐type (WT) and KO tissues. Nitrergic relaxations induced by short trains of electrical field stimulation (EFS) were abolished, while those by long trains of EFS were reduced in KO strips; the latter responses were abolished by 1H[1,2,4,]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ). The relaxations evoked by exogenous NO and the NO‐independent sGC activator BAY 41‐2272 were reduced in KO strips but still sensitive to ODQ. Relaxations induced by vasoactive intestinal peptide (VIP) and 8‐bromo‐cGMP were not influenced. Basal cGMP levels were decreased in KO strips but NO, long train EFS and BAY 41‐2272 still induced a moderate ODQ‐sensitive increase in cGMP levels. Gastric emptying, measured at 15 and 60 min, was increased at 15 min in male KO mice. sGCα1β1 plays an important role in gastric nitrergic relaxation in vitro, but some degree of nitrergic relaxation can occur via sGCα2β1 activation in sGCα1 KO mice, which contributes to the moderate in vivo consequence on gastric emptying.

Involvement of soluble guanylate cyclase α1 and α2, and SKCa channels in NANC relaxation of mouse distal colon

In distal colon, both nitric oxide (NO) and ATP are involved in non-adrenergic non-cholinergic (NANC) inhibitory neurotransmission. The role of the soluble guanylate cyclase (sGC) isoforms alpha(1)beta(1) and alpha(2)beta(1), and of the small conductance Ca(2+)-dependent K(+) channels (SK(Ca) channels) in the relaxation of distal colon by exogenous NO and by NANC nerve stimulation was investigated, comparing wild type (WT) and sGCalpha(1) knockout (KO) mice. In WT strips, the relaxation induced by electrical field stimulation (EFS) at 1 Hz but not at 2-8 Hz was significantly reduced by the NO-synthase inhibitor L-NAME or the sGC inhibitor ODQ. In sGCalpha(1) KO strips, the EFS-induced relaxation at 1 Hz was significantly reduced and no longer influenced by L-NAME or ODQ. The SK(Ca) channel blocker apamin alone had no inhibitory effect on EFS-induced relaxation, but combined with ODQ or L-NAME, apamin inhibited the relaxation induced by EFS at 2-8 Hz in WT strips and at 8 Hz in sGCalpha(1) KO strips. Relaxation by exogenous NO was significantly attenuated in sGCalpha(1) KO strips, but could still be reduced further by ODQ. Basal cGMP levels were lower in sGCalpha(1) KO strips but NO still significantly increased cGMP levels versus basal. In conclusion, in the absence of sGCalpha(1)beta(1), exogenous NO is able to partially act through sGCalpha(2)beta(1). NO, acting via sGCalpha(1)beta(1), is the principal neurotransmitter in EFS-evoked responses at 1 Hz. At higher stimulation frequencies, NO, acting at sGCalpha(1)beta(1) and/or sGCalpha(2)beta(1), functions together with another transmitter, probably ATP acting via SK(Ca) channels, with some degree of redundancy.

Role of the soluble guanylyl cyclase α1/α2 subunits in the relaxant effect of CO and CORM-2 in murine gastric fundus

Carbon monoxide (CO) has been shown to cause enteric smooth muscle relaxation by activating soluble guanylyl cyclase (sGC). In gastric fundus, the sGCα1β1 heterodimer is believed to be the most important isoform. The aim of our study was to investigate the role of the sGCα1/α2 subunits in the relaxant effect of CO and CORM-2 in murine gastric fundus using wild-type (WT) and sGCα1 knock-out (KO) mice. In WT mice, CO (bolus)-induced relaxations were abolished by the sGC inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), while CORM-2- and CO (infusion)-induced relaxations were only partially inhibited by ODQ. In sGCα1 KO mice, relaxant responses to CO and CORM-2 were significantly reduced when compared with WT mice, but ODQ still had an inhibitory effect. The sGC sensitizer 1-benzyl-3-(5′-hydroxymethyl-2′-furyl-)-indazol (YC-1) was able to potentiate CO- and CORM-2-induced relaxations in WT mice but lost this potentiating effect in sGCα1 KO mice. Both in WT and sGCα1 KO mice, CO-evoked relaxations were associated with a significant cGMP increase; however, basal and CO-elicited cGMP levels were markedly lower in sGCα1 KO mice. These data indicate that besides the predominant sGCα1β1 isoform, also the less abundantly expressed sGCα2β1 isoform plays an important role in the relaxant effect of CO in murine gastric fundus; however, the sGC stimulator YC-1 loses its potentiating effect towards CO in sGCα1 KO mice. Prolonged administration of CO—either by the addition of CORM-2 or by continuous infusion of CO—mediates gastric fundus relaxation in both a sGC-dependent and sGC-independent manner.

Small intestinal motility in soluble guanylate cyclase α1 knockout mice

Nitric oxide (NO) activates soluble guanylate cyclase (sGC) to produce guanosine-3′,5′-cyclic-monophosphate (cGMP). The aim of this study was to investigate the nitrergic regulation of jejunal motility in sGCα1 knockout (KO) mice. Functional responses to nitrergic stimuli and cGMP levels in response to nitrergic stimuli were determined in circular muscle strips. Intestinal transit was determined. Nitrergic relaxations induced by electrical field stimulation and exogenous NO were almost abolished in male KO strips, but only minimally reduced and sensitive to ODQ in female KO strips. Basal cGMP levels were decreased in KO strips, but NO still induced an increase in cGMP levels. Transit was not attenuated in male nor female KO mice. In vitro, sGCα1β1 is the most important isoform in nitrergic relaxation of jejunum, but nitrergic relaxation can also occur via sGCα2β1 activation. The latter mechanism is more pronounced in female than in male KO mice. In vivo, no important implications on intestinal motility were observed in male and female KO mice.

Targeting the NO – cGMP pathway: phenotyping of NO-insensitive sGCbeta1 H105F knockin mice

Address: 1Department of Molecular Biology, Faculty of Sciences, Ghent University, Gent, Belgium, 2Department of Molecular Biomedical Research, Molecular Pathology and Experimental Therapy unit, VIB, Gent-Zwijnaarde, Belgium, 3Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, USA and 4Department of General physiology and human physiology and pathopyphysiology, Ghent University, Gent, Belgium

Zebrafish type I collagen mutants faithfully recapitulate human type I collagenopathies

Significance Type I collagenopathies are a heterogenous group of connective tissue disorders, caused by genetic defects in type I collagen. Inherent to these disorders is a large clinical variability, of which the underlying molecular basis remains undefined. By systematically analyzing skeletal phenotypes in a large set of type I collagen zebrafish mutants, we show that zebrafish models are able to both genocopy and phenocopy different forms of human type I collagenopathies, arguing for a similar pathogenetic basis. This study illustrates the future potential of zebrafish as a tool to further dissect the molecular basis of phenotypic variability in human type I collagenopathies, to improve diagnostic strategies as well as promote the discovery of new targetable pathways for pharmacological intervention of these disorders. The type I collagenopathies are a group of heterogeneous connective tissue disorders, that are caused by mutations in the genes encoding type I collagen and include specific forms of osteogenesis imperfecta (OI) and the Ehlers–Danlos syndrome (EDS). These disorders present with a broad disease spectrum and large clinical variability of which the underlying genetic basis is still poorly understood. In this study, we systematically analyzed skeletal phenotypes in a large set of zebrafish, with diverse mutations in the genes encoding type I collagen, representing different genetic forms of human OI, and a zebrafish model resembling human EDS, which harbors a number of soft connective tissues defects, typical of EDS. Furthermore, we provide insight into how zebrafish and human type I collagen are compositionally and functionally related, which is relevant in the interpretation of human type I collagen-related disease models. Our studies reveal a high degree of intergenotype variability in phenotypic expressivity that closely correlates with associated OI severity. Furthermore, we demonstrate the potential for select mutations to give rise to phenotypic variability, mirroring the clinical variability associated with human disease pathology. Therefore, our work suggests the future potential for zebrafish to aid in identifying unknown genetic modifiers and mechanisms underlying the phenotypic variability in OI and related disorders. This will improve diagnostic strategies and enable the discovery of new targetable pathways for pharmacological intervention.

Transgenic mice with a NO-insensitive soluble guanylate cyclase

In order to distinguish between the physiological and pathogenic role of heme-dependent activation of sGC at one hand, and basal activity or heme-independent activation at the other hand, we generated knock-in mice with a H105F mutation of the beta1 subunit of sGC. These mice might furthermore be a good model for pathological situations in which the heme is functionally inactive due to oxidation, as has been suggested to be the case in a number of cardiovascular pathologies.