Loss of Gucy1a3 Causes Poor Post-Stroke Recovery by Reducing Angiogenesis Via the HIF-1α/VEGFA Signaling Pathway in Mice

Ischemic stroke is a common and debilitating disease that can cause permanent neurological damage. Gucy1a3, which encodes the α1 subunit of soluble guanylyl cyclase, has been reported to be associated with functional recovery after ischemic stroke. However, the mechanism is still not well understood. In the present study, we investigated the effects of Gucy1a3 on (i) post-stroke recovery; (ii) vascular endothelial growth factor A (VEGFA) and hypoxia inducible factor 1 alpha (HIF-1α) expression; and (iii) angiogenesis after ischemic stroke. A permanent middle cerebral artery occlusion (pMCAO) model was established using wild-type and Gucy1a3 knockout C57BL/6J male mice. Neurological deficits, infarct volume, microvascular density, and VEGFA and HIF-1α expression levels of mice were evaluated. Our results suggest that loss of Gucy1a3 increased the infarct volume and aggravated neurological deficits after pMCAO. In addition, the Gucy1a3 knockout brains exhibited significantly lower microvessel densities and VEGFA and HIF-1α expression levels than the wild-type brains at 96 hours post-pMCAO. The study shows that the expression of GUCY1A3 after ischemic stroke may play a substantial role in neurological function recovery and is related to angiogenesis in the peri-infarct region. The beneficial effects of GUCY1A3 might be mediated through the HIF-1α/VEGFA signaling pathway.

The Involvement of l-Arginine-Nitric Oxide-cGMP-ATP-Sensitive K+ Channel Pathway in Antinociception of BBHC, a Novel Diarylpentanoid Analogue, in Mice Model

The present study focuses on the possible involvement of l-arginine-nitric oxide-cGMP-ATP-sensitive K+ channel pathway in the antinociceptive activity of a novel diarylpentanoid analogue, 2-benzoyl-6-(3-bromo-4-hydroxybenzylidene)cyclohexen-1-ol (BBHC) via a chemical nociceptive model in mice. The antinociceptive action of BBHC (1 mg/kg, i.p.) was attenuated by the intraperitoneal pre-treatment of l-arginine (a nitric oxide synthase precursor) and glibenclamide (an ATP-sensitive K+ channel blocker) in acetic acid-induced abdominal constriction tests. Interestingly, BBHC’s antinociception was significantly enhanced by the i.p. pre-treatment of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a selective inhibitor of soluble guanylyl cyclase (p < 0.05). Altogether, these findings suggest that the systemic administration of BBHC is able to establish a significant antinociceptive effect in a mice model of chemically induced pain. BBHC’s antinociception is shown to be mediated by the involvement of l-arginine-nitric oxide-cGMP-ATP-sensitive K+ channel pathway, without any potential sedative or muscle relaxant concerns.

Combination of strong anion exchange liquid chromatography with microchip capillary electrophoresis sodium dodecyl sulfate for rapid two-dimensional separations of complex protein mixtures

Two-dimensional separations provide a simple way to increase the resolution and peak capacity of complex protein separations. The feasibility of a recently developed instrumental approach for two-dimensional separations of proteins was evaluated. The approach is based on the general principle of two-dimensional gel electrophoresis. In the first dimension, semi-preparative strong anion exchange high-performance liquid chromatography is utilized and fractions are collected by means of a fraction collector. They are subsequently analyzed in the second dimension with microchip capillary electrophoresis sodium dodecyl sulfate. Microchip capillary electrophoresis provides the necessary speed (approximately 1 min/fraction) for short analysis. In this study, three different samples were investigated. Different constructs of soluble guanylyl cyclase were expressed in Sf9-cells using the baculovirus expression system. Cell lysates were analyzed and the resulting separations were compared. In our experimental setup, the soluble guanylyl cyclase was identified among hundreds of other proteins in these cell lysates, indicating its potential for screening, process control, or analysis. The results were validated by immunoblotting. Samples from Chinese hamster ovary cell culture before and after a purification step were investigated and approximately 9% less impurities could be observed. The separation patterns obtained for human plasma are closely similar to patterns obtained with two-dimensional gel electrophoresis and a total of 218 peaks could be observed. Overall, the approach was well applicable to all samples and, based on these results, further directions for improvements were identified. Graphical abstract .

Citral inhibits the nociception in the rat formalin test: Effect of metformin and blockers of opioid receptor and the NO-cGMP-K+ channel pathway

The objective of the present study was to scrutinize the effect of nitric oxide (NO), cGMP, potassium channel blockers and metformin on the citral-produced peripheral antinociception. The rat paw 1% formalin test was used to assess nociception and antinociception. Rats were treated with local peripheral administration of citral (10-100 µg/paw). The antinociception of citral (100 µg/paw) was evaluated with and without the local pretreatment of naloxone, NG-L-nitro-arginine methyl ester (L-NAME, a NO synthesis inhibitor), 1H-(1,2,4)-oxadiazolo(4,2-a)quinoxalin-1-one (ODQ, a soluble guanylyl cyclase inhibitor), metformin, opioid receptors antagonists, and K+ channel blockers. Injection of citral in the rat paw significantly decreased the nociceptive effect of formalin administration during the two phases of the test. Local pretreatment of the paws with L-NAME and ODQ did not reduced the citral-induced antinociception. Glipizide or glibenclamide (Kir6.1-2; ATP-sensitive K+ channel blockers), tetraethylammonium or 4-aminopyridine (KV; voltage-gated K+ channel blockers) or charybdotoxin (KCa1.1; big conductance calcium-activated K+ channel blocker) or apamin (KCa2.1-3; small conductance Ca2+-activated K+ channel antagonist), or metformin, but not the opioid antagonists, reduced the antinociception of citral. Citral produced peripheral antinociception during both phases of the formalin test. These effects were due to the activation of K+ channels and a biguanide-dependent mechanism.

Loss of soluble guanylyl cyclase in platelets contributes to atherosclerotic plaque formation and vascular inflammation

Aim: The role of platelets in atherosclerosis remains incompletely understood. Variants in genes encoding the soluble guanylyl cyclase (sGC) in platelets are associated with coronary artery disease (CAD) risk. Here we sought to investigate the contribution of platelet sGC to atherosclerosis and the therapeutic potential of targeting sGC in atherosclerosis. Methods and Results: We genetically deleted sGC in platelets of atherosclerosis-prone Ldlr-/- mice. By intravital fluorescence microscopy such Pf4-Cre+Gucy1b1flox/floxLdlr-/- mice displayed enhanced leukocyte adhesion to atherosclerotic plaques in comparison with their litter mates. Moreover, histological and flow cytometry analyses revealed more numerous inflammatory leukocytes and larger plaque sizes in aortic tissue of Ldlr-/- mice lacking sGC in platelets. In vitro, supernatant from activated platelets lacking sGC promoted leukocyte adhesion to endothelial cells (EC) via enhanced EC activation. Using cytokine profiling, we identified reduced angiopoietin-1 release by Pf4-Cre+Gucy1b1flox/flox and human GUCY1A1 risk allele carrier platelets to be responsible for enhanced activation of EC and subsequent leukocyte adhesion. Pharmacological sGC stimulation increased platelet angiopoietin-1 release in vitro and reduced recruitment of adoptively transferred leukocytes in Ldlr-/- mice fed a Western diet. Pharmacological sGC stimulation further reduced atherosclerotic plaque formation and vascular inflammation. Conclusion: Loss of sGC in platelets contributes to atherosclerotic plaque formation via reduced release of the soluble factor angiopoietin-1 and, subsequently, enhanced leukocyte recruitment. Pharmacological sGC stimulation might represent a novel therapeutic strategy to prevent and treat CAD.

Age Impairs Soluble Guanylyl Cyclase Function in Mouse Mesenteric Arteries

Endothelial dysfunction (ED) comes with age, even without overt vessel damage such as that which occurs in atherosclerosis and diabetic vasculopathy. We hypothesized that aging would affect the downstream signalling of the endothelial nitric oxide (NO) system in the vascular smooth muscle (VSM). With this in mind, resistance mesenteric arteries were isolated from 13-week (juvenile) and 40-week-old (aged) mice and tested under isometric conditions using wire myography. Acetylcholine (ACh)-induced relaxation was reduced in aged as compared to juvenile vessels. Pretreatment with L-NAME, which inhibits nitrix oxide synthases (NOS), decreased ACh-mediated vasorelaxation, whereby differences in vasorelaxation between groups disappeared. Endothelium-independent vasorelaxation by the NO donor sodium nitroprusside (SNP) was similar in both groups; however, SNP bolus application (10−6 mol L−1) as well as soluble guanylyl cyclase (sGC) activation by runcaciguat (10−6 mol L−1) caused faster responses in juvenile vessels. This was accompanied by higher cGMP concentrations and a stronger response to the PDE5 inhibitor sildenafil in juvenile vessels. Mesenteric arteries and aortas did not reveal apparent histological differences between groups (van Gieson staining). The mRNA expression of the α1 and α2 subunits of sGC was lower in aged animals, as was PDE5 mRNA expression. In conclusion, vasorelaxation is compromised at an early age in mice even in the absence of histopathological alterations. Vascular smooth muscle sGC is a key element in aged vessel dysfunction.

Empagliflozin Relaxes Resistance Mesenteric Arteries by Stimulating Multiple Smooth Muscle Cell Voltage-Gated K+ (KV) Channels

The antidiabetic drug empagliflozin is reported to produce a range of cardiovascular effects, including a reduction in systemic blood pressure. However, whether empagliflozin directly modulates the contractility of resistance-size mesenteric arteries remains unclear. Here, we sought to investigate if empagliflozin could relax resistance-size rat mesenteric arteries and the associated underlying molecular mechanisms. We found that acute empagliflozin application produces a concentration-dependent vasodilation in myogenic, depolarized and phenylephrine (PE)-preconstricted mesenteric arteries. Selective inhibition of smooth muscle cell voltage-gated K+ channels KV1.5 and KV7 abolished empagliflozin-induced vasodilation. In contrast, pharmacological inhibition of large-conductance Ca2+-activated K+ (BKCa) channels and ATP-sensitive (KATP) channels did not abolish vasodilation. Inhibition of the vasodilatory signaling axis involving endothelial nitric oxide (NO), smooth muscle cell soluble guanylyl cyclase (sGC) and protein kinase G (PKG) did not abolish empagliflozin-evoked vasodilation. Inhibition of the endothelium-derived vasodilatory molecule prostacyclin (PGI2) had no effect on the vasodilation. Consistently, empagliflozin-evoked vasodilation remained unaltered by endothelium denudation. Overall, our data suggest that empagliflozin stimulates smooth muscle cell KV channels KV1.5 and KV7, resulting in vasodilation in resistance-size mesenteric arteries. This study demonstrates for the first time a novel mechanism whereby empagliflozin regulates arterial contractility, resulting in vasodilation. Due to known antihypertensive properties, treatment with empagliflozin may complement conventional antihypertensive therapy.

Runcaciguat, a novel soluble guanylate cyclase activator, shows renoprotection in hypertensive, diabetic, and metabolic preclinical models of chronic kidney disease

Chronic kidney diseaQueryse (CKD) is associated with oxidative stress which can interrupt the nitric oxide (NO)/soluble guanylyl cyclase (sGC) signaling and decrease cyclic guanosine monophosphate (cGMP) production. Low cGMP concentrations can cause kidney damage and progression of CKD. The novel sGC activator runcaciguat targets the oxidized and heme-free form of sGC, restoring cGMP production under oxidative stress. The purpose of this study is to investigate if runcaciguat could provide an effective treatment for CKD. Runcaciguat was used for the treatment not only in rat CKD models with different etiologies and comorbidities, namely of hypertensive rats, the renin transgenic (RenTG) rat, and angiotensin-supplemented (ANG-SD) rat, but also in rats with diabetic and metabolic CKD, the Zucker diabetic fatty (ZDF) rat. The treatment duration was 2 to 42 weeks and runcaciguat was applied orally in doses from 1 to 10 mg/kg/bid. In these different rat CKD models, runcaciguat significantly reduced proteinuria (urinary protein to creatinine ratio; uPCR). These effects were also significant at doses which did not or only moderately decrease systemic blood pressure. Moreover, runcaciguat significantly decreased kidney injury biomarkers and attenuated morphological kidney damages. In RenTG rats, runcaciguat improved survival rates and markers of heart injury. These data demonstrate that the sGC activator runcaciguat exhibits cardio-renal protection at doses which did not reduce blood pressure and was effective in hypertensive as well as diabetic and metabolic CKD models. These data, therefore, suggest that runcaciguat, with its specific mode of action, represents an efficient treatment approach for CKD and associated CV diseases. Graphical abstract

sGC stimulation lowers elevated blood pressure in a new canine model of resistant hypertension

Therapy-resistant hypertension is a serious medical problem, causing end-organ damage, stroke, and heart failure if untreated. Since the standard of care fails in resistant hypertension patients, there is still a substantial unmet medical need for effective therapies. Active stimulation of soluble guanylyl cyclase via novel soluble guanylyl cyclase stimulators might provide an effective treatment option. To test this hypothesis, we established a new experimental dog model and investigated the effects of the soluble guanylyl cyclase-stimulator BAY 41-2272. In beagle dogs, a resistant hypertension phenotype was established by combining unilateral renal wrapping with the occlusion of the renal artery in the contralateral kidney. The most frequently used antihypertensive drugs were administered orally, either alone or in combination, and their acute effect on telemetric measured blood pressure was assessed and compared with that of BAY 41-2272. The chosen disease stimulus led to a moderate and stable increase in blood pressure. Even high doses of standard-of-care antihypertensives only slightly decreased blood pressure. In contrast, the administration of the soluble guanylyl cyclase stimulator BAY 41-2272 as standalone therapy led to a dose-dependent reduction in blood pressure (−14.1 ± 1.8 mmHg). Moreover, BAY 41-2272 could also further decrease blood pressure in addition to a triple combination of standard-of-care antihypertensives (−28.6 ± 13.2 mmHg). BAY 41-2272 was highly efficient as a standalone treatment in resistant hypertension but was also effective in addition to standard-of-care treatment. These data strongly suggest that soluble guanylyl cyclase stimulators might provide an effective pharmacologic therapy for patients with resistant hypertension.