Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Contribution of glibenclamide-sensitive, ATP-dependent K+ channel activation to acetophenone analogues-mediated in vitro pulmonary artery relaxation of rat

Compared to the currently available therapeutic drugs for peripheral vascular diseases, agents that are selective for relaxing pulmonary circulation are scarce. The present study was undertaken, using isometric tension change measurement and whole-cell patch-clamp electrophysiology methods, to evaluate the vascular relaxation effect and the underlying mechanisms involved of two naturally found alkaloids: paeonol (2-hydroxy-4-methoxy-acetophenone), acetovanillone (4-hydroxy-3-methoxy-acetophenone) and the non-substituted analogue acetophenone on pulmonary artery of Sprague-Dawley rats. Cumulative administration (3 μM–1 mM) of acetophenone analogues resulted in a concentration-dependent relaxation of phenylephrine (1 μM) pre-contracted pulmonary artery. A relative order of inhibitory potency, estimated by comparing the concentration at which a 50% relaxation of phenylephrine-induced contraction observed was: acetovanillone > paeonol > acetophenone. Endothelial denudation and inhibition of nitric oxide synthase (with 20 μM NG-nitro-l-arginine methyl-ester) only moderately suppressed (17.6 ± 4.2%) acetovanillone- but not paeonol- or acetophenone-mediated maximum relaxation. Glibenclamide (3 μM, an ATP-sensitive K+ (IKATP) channel blocker) markedly attenuated all acetophenone analogues-mediated endothelium-independent relaxation. Neither cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine (MDL 12330A, 10 μM), iberiotoxin (300 nM), 4-aminopyridine (3 mM), (±)-propranolol (1 μM, a non-selective β-adrenoceptor blocker) nor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (3 μM, a guanylate cyclase inhibitor) altered endothelium-independent relaxation. In electrophysiological experiments using single pulmonary artery smooth muscle cells, acetovanillone, paeonol, acetophenone and cromakalim activated glibenclamide-sensitive, IKATP channels. In conclusion, our results demonstrate that acetophenone analogues caused pulmonary artery relaxation through opening of IKATP channels. In addition, acetovanillone-mediated pulmonary artery relaxation is partly depended on nitric oxide released from endothelium

Inhibitory effect of nonsteroidal anti-inflammatory drugs on adenosine transport in vascular smooth muscle cells

It is generally accepted that the clinical efficacy of nonsteroidal anti-inflammatory drugs (NSAIDs) arises mainly from the inhibition of cyclooxygenase (COX). However, more evidence has suggested that certain pharmacological actions of NSAIDs may be mediated by COX-independent mechanisms. The present study investigated the effects of NSAIDs on adenosine uptake in human aortic smooth muscle cells (HASMCs). Among the NSAIDs tested (all at 100 μM), aspirin, ibuprofen and naproxen had no effect on [3H]adenosine uptake. Piroxicam inhibited [3H]adenosine uptake by 30%, while etodolac, indomethacin, ketoprofen, mefenamic acid and sulindac inhibited [3H]adenosine by 13-18%. Sulindac sulfide, an active metabolite of sulindac, inhibited [3H]adenosine uptake and [3H]nitrobenzylmercaptopurine ribonucleoside (NBMPR) binding of HASMCs with IC50 values of 40.67 ± 4.82 and 24.19 ± 3.76 μM, respectively. Kinetic studies revealed that sulindac sulfide was a competitive inhibitor of adenosine uptake. Using the nucleoside-transporter-deficient PK15NTD cells that stably express equilibrative nucleoside transport (ENT) 1 and ENT2, it was found that the inhibitory effect of sulindac sulfide on ENT1 was greater than that on ENT2. Sulindac sulfide increased the extracellular adenosine level. In addition, it inhibited the proliferation of HASMCs and this anti-proliferative effect could be abolished by adenosine A2B receptor antagonist. Our results suggest that sulindac sulfide may exert pharmacological effects through the inhibition of adenosine uptake, which modulates the availability of adenosine in the vicinity of adenosine receptors

14,15-Epoxyeicosatrienoic acid induces vasorelaxation through the prostaglandin EP(2) receptors in rat mesenteric artery

Epoxyeicosatrienoic acids (EETs) induce vasorelaxation, probably through G protein-coupled receptors. The identity of these receptors is unclear, but it has been reported that EETs may bind to peroxisome proliferator activated receptors (PPARs) and E-prostanoid (EP) receptors. Therefore, we studied whether PPARs or EP receptors were involved in 14,15-EET-induced vasorelaxation. Isometric tensions of rat mesenteric arteries were measured. The vasorelaxant effect of 14,15-EET was inhibited by NF449 (Gs-protein inhibitor), Rp-cAMP (cAMP antagonist) and KT5720 (PKA inhibitor), suggesting that the effect of 14,15-EET was mediated through Gs protein-coupled receptors which were linked to the cAMP/PKA-dependent pathway. Pretreatments with MK886 (PPARα antagonist) and GW9662 (PPARγ antagonist) did not influence 14,15-EET-induced vasorelaxation. The vasorelaxant effect of 14,15-EET was inhibited by AH6809 (EP2 receptor antagonist), whereas SC19220 (EP1 receptor antagonist), L798106 (EP3 receptor antagonist) and GW627368X (EP4 receptor antagonist) had no effect. The effect of 14,15-EET and the mechanism involved was mimicked by prostaglandin E2 (an EP2 receptor agonist). The 14,15-EET-induced relaxation was slightly potentiated in the presence of indomethacin (cyclooxygenase inhibitor which block PGE2 synthesis). Binding study showed that the amount of 14,15-EET bound to the cell membrane of rat mesenteric arterial smooth muscle cells was much higher than that bound to the nuclear membrane. The binding of 14,15-EET to the cell membrane was attenuated by AH6809 and siRNA against EP2 receptors. In conclusion, our study has demonstrated that 14,15-EET exerts relaxant effects on rat mesenteric arteries, at least partly via the stimulation of EP2 receptors. This subsequently leads to activation of cAMP/PKA-dependent pathway in vascular smooth muscle cells

Inhibitory effect of nonsteroidal anti-inflammatory drugs on adenosine transport in vascular smooth muscle cells

It is generally accepted that the clinical efficacy of nonsteroidal anti-inflammatory drugs (NSAIDs) arises mainly from the inhibition of cyclooxygenase (COX). However, more evidence has suggested that certain pharmacological actions of NSAIDs may be mediated by COX-independent mechanisms. The present study investigated the effects of NSAIDs on adenosine uptake in human aortic smooth muscle cells (HASMCs). Among the NSAIDs tested (all at 100 µM), aspirin, ibuprofen and naproxen had no effect on [3H]adenosine uptake. Piroxicam inhibited [3H]adenosine uptake by 30%, while etodolac, indomethacin, ketoprofen, mefenamic acid and sulindac inhibited [3H]adenosine by 13–18%. Sulindac sulfide, an active metabolite of sulindac, inhibited [3H]adenosine uptake and [3H]nitrobenzylmercaptopurine ribonucleoside (NBMPR) binding of HASMCs with IC50 values of 40.67 ± 4.82 and 24.19 ± 3.76 μM, respectively. Kinetic studies revealed that sulindac sulfide was a competitive inhibitor of adenosine uptake. Using the nucleoside-transporter-deficient PK15NTD cells that stably express equilibrative nucleoside transport (ENT) 1 and ENT2, it was found that the inhibitory effect of sulindac sulfide on ENT1 was greater than that on ENT2. Sulindac sulfide increased the extracellular adenosine level. In addition, it inhibited the proliferation of HASMCs and this anti-proliferative effect could be abolished by adenosine A2B receptor antagonist. Our results suggest that sulindac sulfide may exert pharmacological effects through the inhibition of adenosine uptake, which modulates the availability of adenosine in the vicinity of adenosine receptors

Consumption of dried fruit of Crataegus pinnatifida (hawthorn) suppresses high cholesterol diet-induced hypercholesterolemia in rats

The hypocholesterolemic and atheroscleroprotective potentials of dietary consumption of hawthorn (dried fruit of Crataegus pinnatifida, Shan Zha) were investigated by monitoring plasma lipid profiles and aortic relaxation in Sprague–Dawley rats fed with either normal diet, high-cholesterol diet (HCD) or HCD supplemented with hawthorn powder (2%, w/w) (4 weeks). In HCD-fed rats, an increased plasma total cholesterol and LDL-cholesterol with a decreased HDL-cholesterol was observed, and consumption of hawthorn markedly suppressed the elevated total cholesterol and LDL-lipoprotein levels plus an increased HDL-cholesterol level. The blunted acetylcholine-induced, endothelium-dependent relaxation of isolated aortas of HCD-fed rats was improved by hawthorn. The development of fatty liver, an increased nitric oxide synthase (NOS) activity and an elevated oxidative stress (as estimated by the attenuated levels of anti-oxidant enzymes) associated with HCD were attenuated by hawthorn. Thus, the results demonstrated that hawthorn consumption provides overall beneficial effects on reversing HCD associated detrimental changes

Mitochondrial monoamine oxidase-A-mediated hydrogen peroxide generation enhances 5-hydroxytryptamine-induced contraction of rat basilar artery

BACKGROUND AND PURPOSE We evaluated the role(s) of monoamine oxidase (MAO)-mediated H2O2 generation on 5-hydroxytryptamine (5-HT)-induced tension development of isolated basilar artery of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. EXPERIMENTAL APPROACH Basilar artery (endothelium-denuded) was isolated for tension measurement and Western blots. Enzymically dissociated single myocytes from basilar arteries were used for patch-clamp electrophysiological and confocal microscopic studies. KEY RESULTS Under resting tension, 5-HT elicited a concentration-dependent tension development with a greater sensitivity (with unchanged maximum tension development) in SHR compared with WKY (EC50: 28.4 ± 4.1-nM vs. 98.2 ± 9.4-nM). The exaggerated component of 5-HT-induced tension development in SHR was eradicated by polyethylene glycol-catalase, clorgyline and citalopram whereas exogenously applied H2O2 enhanced the 5-HT-elicited tension development in WKY. A greater protein expression of MAO-A was detected in basilar arteries from SHR than in those from WKY. In single myocytes and the entire basilar artery, 5-HT generated (clorgyline-sensitive) a greater amount of H2O2 in SHR compared with WKY. Whole-cell iberiotoxin-sensitive Ca2+-activated K+ (BKCa) amplitude measured in myocytes of SHR was approximately threefold greater than that in WKY (at +60-mV: 7.61 ± 0.89-pA.pF-1 vs. 2.61 ± 0.66-pA.pF-1). In SHR myocytes, 5-HT caused a greater inhibition (clorgyline-, polyethylene glycol-catalase- and reduced glutathione-sensitive) of BKCa amplitude than in those from WKY. CONCLUSIONS AND IMPLICATIONS 5-HT caused an increased generation of mitochondrial H2O2 via MAO-A-mediated 5-HT metabolism, which caused a greater inhibition of BKCa gating in basilar artery myocytes, leading to exaggerated basilar artery tension development in SHR

Folic acid supplementation modifies b-adrenoceptor-mediated in vitro lipolysis of obese/diabetic (+db/+db) mice

The effects of folic acid (5.7 and 71 μg/kg, 4 weeks) consumption on the β-adrenoceptors (β-ARs)–elicited lipolysis in vitro of the abdominal adipocytes of lean/control (+m/+db) and obese/diabetic (+db/+db) mice (female) were investigated. β-AR agonists (salbutamol, a β2-AR agonist; BRL 37344 and CGP 12177, β3-AR agonists; adrenaline, a β-AR agonist)–mediated lipolysis, β2-, and β3-ARs protein expression of the adipose tissues after folic acid consumption were evaluated. Our results demonstrate that a smaller magnitude of the basal (spontaneous) and the β-AR agonists–triggered lipolysis was observed in +db/+db mice, and folic acid supplementation (71 μg/kg) resulted in an improvement of both the baseline and the β-ARs–mediated lipolysis. In controls, a lower β2-and β3-ARs protein expression of the adipose tissues was detected in +db/+db mice, compared to +m/+db mice. In both strains fed with folic acid (71 μg/kg), a reduction of β2-AR protein expression was observed compared to the respective controls. In +db/+db mice, folic acid (5.7 and 71 μg/kg) consumption caused a dose-dependent increase of β3-AR protein expression compared to controls. We demonstrate that lipolysis elicited by β-AR (β2- and β3-ARs) agonists was blunted in +db/+db mice. Folic acid consumption has significant modulatory effects on β-ARs protein expression and lipolysis

Folic acid consumption reduces resistin level and restores blunted acetylcholine-induced aortic relaxation in obese/diabetic mice

Folic acid supplementation provides beneficial effects on endothelial functions in patients with hyperhomocysteinemia. However, its effects on vascular functions under diabetic conditions are largely unknown. Therefore, the effect(s) of folic acid (5.7 and 71 μg/kg/day for 4 weeks) on aortic relaxation was investigated using obese/diabetic (+db/+db) mice and lean littermate (+db/+m) mice. Acetylcholine-induced relaxation in +db/+db mice was less than that observed in +db/+m mice. The reduced relaxation in +db/+db mice was restored by consumption of 71 μg/kg folic acid. Acetylcholine-induced relaxation (with and without folic acid treatment) was sensitive to NG-nitro-l-arginine methyl ester, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one, geldanamycin and triciribine. In addition, acetylcholine-induced relaxation was attenuated by resistin. The plasma level of resistin in +db/+db mice was sevenfold higher than that measured in +db/+m mice, and the elevated plasma level of resistin in +db/+db mice was reduced by 25% after treatment with 71 μg/kg folic acid. Folic acid slightly increased the ratio of reduced glutathione to oxidized glutathione in +db/+db mice. Moreover, folic acid caused a reduction in PTEN (phosphatase and tensin homolog deleted on chromosome 10) expression, an increase in the phosphorylation of endothelial nitric oxide synthase (eNOSSer1177) and AktSer473, and an enhanced interaction of heat shock protein 90 (HSP90) with eNOS in both strains, with greater magnitude observed in +db/+db mice. In conclusion, folic acid consumption improved blunted acetylcholine-induced relaxation in +db/+db mice. The mechanism may be, at least partly, attributed to enhancement of PI3K/HSP90/eNOS/Akt cascade, reduction in plasma resistin level, down-regulation of PTEN and slight modification of oxidative state