Glucagon and a glucagon-GLP-1 dual-agonist increases cardiac performance with different metabolic effects in insulin-resistant hearts

BACKGROUND AND PURPOSE

The prevalence of heart disease continues to rise, particularly in subjects with insulin resistance (IR), and improved therapies for these patients is an important challenge. In this study we evaluated cardiac function and energy metabolism in IR JCR:LA-cp rat hearts before and after treatment with an inotropic compound (glucagon), a glucagon-like peptide-1 (GLP-1) receptor agonist (ZP131) or a glucagon-GLP-1 dual-agonist (ZP2495).

EXPERIMENTAL APPROACH

Hearts from IR and lean JCR:LA rats were isolated and perfused in the working heart mode for measurement of cardiac function and metabolism before and after addition of vehicle, glucagon, ZP131 or ZP2495. Subsequently, cardiac levels of nucleotides and short-chain CoA esters were measured by HPLC.

KEY RESULTS

Hearts from IR rats showed decreased rates of glycolysis and glucose oxidation, plus increased palmitate oxidation rates, although cardiac function and energy state (measured by ATP/AMP ratios) was normal compared with control rats. Glucagon increased glucose oxidation and glycolytic rates in control and IR hearts, but the increase was not enough to avoid AMP and ADP accumulation in IR hearts. ZP131 had no significant metabolic or functional effects in either IR or control hearts. In contrast, ZP2495 increased glucose oxidation and glycolytic rates in IR hearts to a similar extent to that of glucagon but with no concomitant accumulation of AMP or ADP.

CONCLUSION AND IMPLICATIONS

Whereas glucagon compromised the energetic state of IR hearts, glucagon-GLP-1 dual-agonist ZP2495 appeared to preserve it. Therefore, a glucagon-GLP-1 dual-agonist may be beneficial compared with glucagon alone in the treatment of severe heart failure or cardiogenic shock in subjects with IR.     

Irbesartan treatment up-regulates hepatic expression of PPAR�� and its target genes in obese Koletsky (fak/fak) rats: a link to amelioration of hypertriglyceridaemia

BACKGROUND AND PURPOSE

Hypertriglyceridaemia is associated with an increased risk of cardiovascular disease. Irbesartan, a well-established angiotensin II type 1 receptor (AT₁) blocker, improves hypertriglyceridaemia in rodents and humans but the underlying mechanism of action is unclear.

EXPERIMENTAL APPROACH

Male obese Koletsky (fa^k/fa^k) rats, which exhibit spontaneous hypertension and metabolic abnormalities, received irbesartan (40 mg·kg⁻¹·day⁻¹) or vehicle by oral gavage over 7 weeks. Adipocyte-derived hormones in plasma were measured by ELISA. Gene expression in liver and other tissues was assessed by real-time PCR and Western immunoblotting.

KEY RESULTS

In Koletsky (fa^k/fa^k) rats irbesartan lowered plasma concentrations of triglycerides and non-esterified fatty acids, and decreased plasma insulin concentrations and the homeostasis model assessment of insulin resistance index. However, this treatment did not affect food intake, body weight, epididymal white adipose tissue weight, adipocyte size and plasma leptin concentrations, although plasma adiponectin was decreased. Irbesartan up-regulated hepatic expression of mRNAs corresponding to peroxisome proliferator-activated receptor (PPAR)α and its target genes (carnitine palmitoyltransferase-1a, acyl-CoA oxidase and fatty acid translocase/CD36) that mediate hepatic fatty acid uptake and oxidation; the increase in hepatic PPARα expression was confirmed at the protein level. In contrast, irbesartan did not affect expression of adipose PPARγ and its downstream genes or hepatic genes that mediate fatty acid synthesis.

CONCLUSIONS AND IMPLICATIONS

These findings demonstrate that irbesartan treatment up-regulates PPARα and several target genes in liver of obese spontaneously hypertensive Koletsky (fa^k/fa^k) rats and offers a novel insight into the lipid-lowering mechanism of irbesartan.     

Sevoflurane post-conditioning protects isolated rat hearts against ischemia-reperfusion injury via activation of the ERK1/2 pathway

Aim:

To investigate the role of extracellular signal-regulated kinases (ERKs) in sevoflurane post-conditioning induced cardioprotection in vitro.

Methods:

Isolated rat hearts were subjected to 30 min ischemia followed by 120 min reperfusion (I/R). Sevoflurane post-conditioning was carried out by administration of O₂-enriched gas mixture with 3% sevoflurane (SEVO) for 15 min from the onset of reperfusion. Cardiac functions, myocardial infarct size, myocardial ATP and NAD⁺ contents, mitochondrial ultrastructure, and anti-apototic and anti-oncosis protein levels were measured.

Results:

Sevoflurane post-conditioning significantly improved the heart function, decreased infarct size and mitochondria damage, and increased myocardial ATP and NAD⁺ content in the I/R hearts. Furthermore, sevoflurane post-conditioning significantly increased the levels of p-ERK and p-p70S6K, decreased the levels of porimin, caspase-8, cleaved caspase-3, and cytosolic cytochrome c in the I/R hearts. Co-administration of the ERK1/2 inhibitor PD98059 (20 μmol/L) abolished the sevoflurane-induced protective effects against myocardial I/R.

Conclusion:

Sevoflurane post-conditioning protects isolated rat hearts against myocardial I/R injury and inhibits cell oncosis and apoptosis via activation of the ERK1/2 pathway.     

Inhibition of nitric oxide generation unmasks vascular dysfunction in insulin-resistant,obese JCR:LA-cp rats

1. The effects of nitric oxide (NO) on vascular reactivity and platelet function in the obese (cp/cp) and lean (+/?) JCR:LA-cp rats were investigated.
2. Phenylephrine (PE; 0.1 nM–10 μM) induced contraction of isolated aortic rings in both genotypes (cp/cp and +/?) of JCR:LA-cp rats. The sensitivity to contraction with PE was enhanced in cp/cp compared with +/? rings. Rings from both genotypes showed an increased contraction upon removal of the endothelium.
3. Acetylcholine (ACh; 0.1 nM–10 μM)-induced endothelium-dependent relaxation of rings was not significantly different in the two genotypes. Both were inhibited to a similar extent by N^G-nitro-L-arginine methyl ester (L-NAME; 0.01–1 mM) when administered in vitro.
4. The nitric oxide synthase (NOS) inhibitor (L-NAME; 0.3, 1 or 3 mg ml⁻¹, p.o.) when administered in vivo increased blood pressure in cp/cp rats but not in +/? rats.
5. L-NAME resulted in greater inhibition of ACh-induced relaxation in cp/cp rings compared with +/? rings.
6. L-NAME treatment in vivo caused a decrease in cyclic GMP and NOS activity in rings from cp/cp but not +/? rats.
7. The NO donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP; 0.1 nM–10 μM)-induced relaxation of rings from +/? rats, an effect enhanced by the treatment with L-NAME in vivo.
8. Oral administration of L-NAME did not enhance the vasorelaxant effect of SNAP on rings of aorta from cp/cp animals.
9. Platelet aggregation and NOS activity were similar in both genotypes and were not modified by oral administration of L-NAME.
10. These results show that unimpaired generation of NO is crucial for maintenance of vascular tone particularly under conditions of vascular insult exemplified by insulin resistance, obesity and dyslipidemia detected in cp/cp rats.

     

Fungal transformation of androsta-1,4-diene-3,17-dione by Aspergillus brasiliensis

Background

The biotransformation of steroids by fungal biocatalysts has been recognized for many years. There are numerous fungi of the genus Aspergillus which have been shown to transform different steroid substances. The possibility of using filamentous fungi Aspergillus brasiliensis cells in the biotransformation of androsta-1,4-diene-3,17-dione, was evaluated.

Methods

The fungal strain was inoculated into the transformation medium which supplemented with androstadienedione as a substrate and fermentation continued for 5 days. The metabolites were extracted and isolated by thin layer chromatography. The structures of these metabolites were elucidated using ¹H-NMR, broadband decoupled ¹³C-NMR, EI Mass and IR spectroscopies.

Results

The fermentation yielded one reduced product: 17β-hydroxyandrost-1,4-dien-3-one and two hydroxylated metabolites: 11α-hydroxyandrost-1,4-diene-3,17-dione and 12β-hydroxyandrost-1,4-diene-3,17-dione.

Conclusions

The results obtained in this study show that A. brasiliendsis could be considered as a biocatalyst for producing important derivatives from androstadienedione.     

The negative inotropic action of canrenone is mediated by L-type calcium current blockade and reduced intracellular calcium transients

Background and purpose:

Adding spironolactone to standard therapy in heart failure reduces morbidity and mortality, but the underlying mechanisms are not fully understood. We analysed the effect of canrenone, the major active metabolite of spironolactone, on myocardial contractility and intracellular calcium homeostasis.

Experimental approach:

Left ventricular papillary muscles and cardiomyocytes were isolated from male Wistar rats. Contractility of papillary muscles was assessed with force transducers, Ca²⁺ transients by fluorescence and Ca²⁺ fluxes by electrophysiological techniques.

Key results:

Canrenone (300–600 µmol·L⁻¹) reduced developed tension, maximum rate of tension increase and maximum rate of tension decay of papillary muscles. In cardiomyocytes, canrenone (50 µmol·L⁻¹) reduced cell shortening and L-type Ca²⁺ channel current, whereas steady-state activation and inactivation, and reactivation curves were unchanged. Canrenone also decreased the Ca²⁺ content of the sarcoplasmic reticulum, intracellular Ca²⁺ transient amplitude and intracellular diastolic Ca²⁺ concentration. However, the time course of [Ca²⁺]_i decline during transients evoked by caffeine was not affected by canrenone.

Conclusion and implications:

Canrenone reduced L-type Ca²⁺ channel current, amplitude of intracellular Ca²⁺ transients and Ca²⁺ content of sarcoplasmic reticulum in cardiomyocytes. These changes are likely to underlie the negative inotropic effect of canrenone.     

Simvastatin inhibited cardiac hypertrophy and fibrosis in apolipoprotein E-deficient mice fed a “Western-style diet” by increasing PPAR α and γ expression and reducing TC,MMP-9, and Cat S levels

Aim:

The examine the cardiac hypertrophy and fibrosis in apolipoprotein E-deficient mice (ApoE−/− mice) fed a “Western-style diet” and the effect of simvastatin intervention.

Methods:

Male ApoE−/− mice (n=36) were fed a “Western-style diet” from the age of 8 weeks. After 16 weeks, they were randomly given either simvastatin (25 mg·kg⁻¹·d⁻¹) or normal saline (control group) by gavage for 8, 16, or 24 weeks. The left ventricular (LV) wall thickness and diameter of the myocardial cells were determined with Hematoxylin-Eosin stain, and the level of fibrosis of the myocardial matrix was assessed with Masson stain. Real-time quantitative polymerase chain reaction and Western blotting analysis were used to determine the mRNA and protein expression of matrix metalloproteinase-9 (MMP-9), Cathepsin S (Cat S), and the peroxisome proliferator-activated receptors (PPARs) in the myocardium of ApoE−/− mice.

Results:

ApoE−/− mice fed a “Western-style diet” showed an significant age-dependent increase in total cholesterol (TC), LV wall thickness, myocardial cell diameter and LV collagen content (P<0.05). The simvastatin treatment group showed significantly reduced LV wall thickness, myocardial cell diameters and LV collagen content at 40 weeks when compared with the control group (P<0.05). Furthermore, treatment with simvastatin also significantly inhibited the mRNA and protein expressions of MMP-9 and Cat S as well as increased the mRNA and protein expressions of PPAR alpha and PPAR gamma at 32 and 40 weeks compared with the control group (P<0.05).

Conclusion:

ApoE−/− mice fed a “Western-style diet” had cardiac hypertrophy and fibrosis, which worsened with age. Simvastatin treatment inhibits the development of cardiac hypertrophy and fibrosis, and this effect may be mediated through increased levels of PPAR alpha and PPAR gamma and reduced levels of TC, MMP-9, and Cat S.     

Pretreatment with a combination of ligustrazine and berberine improves cardiac function in rats with coronary microembolization

Aim:

We have shown that a combination of ligustrazine and berberine produces more effective inhibition on platelet activation and inflammatory reactions in rat acute myocardial infarction compared with either agent alone. In this study we evaluated the beneficial effects of a combination of ligustrazine and berberine in a rat model of coronary microembolization (CME).

Methods:

SD rats were treated with ligustrazine, berberine, ligustrazine+berberine, or clopidogrel for 2 weeks. When the treatment completed, CME was induced by injection of sodium laurate into the left ventricular, while obstructing the ascending aorta. All rats were intubated for hemodynamic measurements. Blood samples were collected for biochemical analyses, flow cytometry, and ELISAs. Heart tissues were isolated for histopathology and subsequent protein analyses.

Results:

Pretreatment with the combination of ligustrazine (27 mg·kg⁻¹·d⁻¹) and berberine (90 mg·kg⁻¹·d⁻¹) significantly improved cardiac function, and decreased myocardial necrosis, inflammatory cell infiltration, microthrombosis and serum CK-MB levels in CME rats. In addition, this combination significantly decreased plasma ET-1 levels and von Willebrand factor, inhibited ADP-induced platelet activation, and reduced TNFα, IL-1β, ICAM-1 and RANTES levels in serum and heart tissues. The protective effects of this combination were more prominent than those of ligustrazine or berberine alone, but comparable to those of a positive control clopidogrel (6.75 mg·kg⁻¹·d⁻¹).

Conclusion:

The combination of ligustrazine and berberine significantly improved cardiac function in rat CME model via a mechanism involving antiplatelet and anti-inflammatory effects.     

Chronic nicotine treatment enhances vascular smooth muscle relaxation in rats

Aim:

To investigate the effect of chronic nicotine treatment on vascular function and to identify the underlying mechanisms.

Methods:

Adult rats were treated with nicotine (3 mg·kg⁻¹·d⁻¹, sc) for 6 weeks. After the rats were sacrificed, aortic rings were prepared for detecting vascular reactivity, and thoracic aorta and periaortic fat samples were collected for histological and molecular biology studies.

Results:

Chronic nicotine treatment significantly reduced periaortic fat, and specifically enhanced smooth muscle relaxation without altering the aortic adventitial fat and endothelium function. Pretreatment with the soluble guanylyl cyclase inhibitor ODQ (3 μmol/L) or PKG inhibitor Rp-8-Br-PET-cGMP (30 μmol/L) abolished the nicotine-induced enhancement of smooth muscle relaxation, whereas the cGMP analogue 8-Br-cGMP could mimic the nicotine-induced enhancement of smooth muscle relaxation. However, the chronic nicotine treatment did not alter PKG protein expression and activity in aortic media.

Conclusion:

Chronic nicotine treatment enhances vascular smooth muscle relaxation of rats via activation of PKG pathway.     

Modelling of α1-adrenoceptor-mediated temporal dynamics of inotropic response in rat heart to assess ligand binding and signal transduction parameters

Background and purpose:

In order to use the transient response to an antagonist (prazosin) to evaluate properties of agonist interactions with the α₁-adrenoceptor system, an integrative mechanistic model of cardiac uptake of prazosin and its competitive interaction with phenylephrine at the receptor site was developed. Based on the operational model of agonism, the aim was to evaluate both the receptor binding and signal transduction process as determinants of the inotropic effect of phenylephrine.

Experimental approach:

In Langendorff-perfused rat hearts, prazosin outflow concentration and left ventricular developed pressure were measured, first in the presence of 12.3 µmol·L⁻¹ phenylephrine following a 1 min infusion of 1.27 nmol [³H]-prazosin, and second, when after 30 min the phenylephrine concentration in perfusate was reduced to 6.1 µmol·L⁻¹, the 1 min infusion of 1.27 nmol [³H]-prazosin was repeated.

Key results:

The kinetic model accounted for cardiac uptake and receptor binding kinetics of prazosin (dissociation constant, mean ± SD: 0.057 ± 0.012 nmol·L⁻¹), assuming that the competitive displacement of phenylephrine (dissociation constant: 101 ± 13 nmol·L⁻¹) reduced the receptor occupation by the agonist and, consequently, contractility. This competitive binding process appeared to be the rate-determining step in response generation. The relationship between receptor occupancy and inotropic response was described by an efficacy parameter (τ, ratio of receptor density and coupling efficiency) of 4.9.

Conclusions and implications:

Mechanistic pharmacodynamic modelling of the kinetics of antagonism by prazosin allows quantitative assessment of the α₁-adrenoceptor system both at the receptor and post-receptor levels.     

Characterization of AQX-1125, a small-molecule SHIP1 activator: Part 2. Efficacy studies in allergic and pulmonary inflammation models in vivo

Background

The efficacy of AQX-1125, a small-molecule SH2-containing inositol-5′-phosphatase 1 (SHIP1) activator and clinical development candidate, is investigated in rodent models of inflammation.

Experimental Approach

AQX-1125 was administered orally in a mouse model of passive cutaneous anaphylaxis (PCA) and a number of rodent models of respiratory inflammation including: cigarette smoke, LPS and ovalbumin (OVA)-mediated airway inflammation. SHIP1 dependency of the AQX-1125 mechanism of action was investigated by comparing the efficacy in wild-type and SHIP1-deficient mice subjected to an intrapulmonary LPS challenge.

Results

AQX-1125 exerted anti-inflammatory effects in all of the models studied. AQX-1125 decreased the PCA response at all doses tested. Using bronchoalveolar lavage (BAL) cell counts as an end point, oral or aerosolized AQX-1125 dose dependently decreased the LPS-mediated pulmonary neutrophilic infiltration at 3–30 mg kg⁻¹ and 0.15–15 μg kg⁻¹ respectively. AQX-1125 suppressed the OVA-mediated airway inflammation at 0.1–10 mg kg⁻¹. In the smoke-induced airway inflammation model, AQX-1125 was tested at 30 mg kg⁻¹ and significantly reduced the neutrophil infiltration of the BAL fluid. AQX-1125 (10 mg kg⁻¹) decreased LPS-induced pulmonary neutrophilia in wild-type mice but not in SHIP1-deficient mice.

Conclusions

The SHIP1 activator, AQX-1125, suppresses leukocyte accumulation and inflammatory mediator release in rodent models of pulmonary inflammation and allergy. As shown in the mouse model of LPS-induced lung inflammation, the efficacy of the compound is dependent on the presence of SHIP1. Pharmacological SHIP1 activation may have clinical potential for the treatment of pulmonary inflammatory diseases.

Linked Article

This article is accompanied by Stenton et al., pp. 1506–1518 of this issue. To view this article visit http://dx.doi.org/10.1111/bph.12039     

Mast cell degranulation – a mechanism for the anti-arrhythmic effect of endothelin-1?

Background and purpose:

The aim of this study was to investigate whether the previously reported anti-arrhythmic effect of endothelin-1 (ET-1) is mediated by degranulation of cardiac mast cells prior to myocardial ischaemia.

Experimental approach:

Male Sprague-Dawley rats received either ET-1 (1.6 nmol·kg⁻¹) in the presence or absence of disodium cromoglycate (DSCG; 20 mg·kg⁻¹·h⁻¹) prior to coronary artery occlusion (CAO). In separate experiments rats were given compound 48/80 (50 µg·kg⁻¹) to compare the effects of ET-1 with those of a known mast cell degranulator. Ischaemia-induced ventricular arrhythmias were detected through continuous monitoring of a lead I electrocardiogram. After 30 min of CAO, the hearts were removed and mast cell degranulation determined by histological analysis. A parallel series of sham groups were performed to determine the direct effects of ET-1 and compound 48/80 on mast cell degranulation in the absence of ischaemia.

Key results:

ET-1 and compound 48/80 both exerted profound anti-arrhythmic effects, significantly reducing the total number of ventricular ectopic beats (P < 0.001) and the incidence of ventricular fibrillation (P < 0.05). These anti-arrhythmic effects were abolished by concomitant DSCG infusion prior to CAO. In sham animals ET-1 and compound 48/80 both induced mast cell degranulation (P < 0.001), an effect which was abolished by DSCG, confirming their ability to induce degranulation of mast cells.

Conclusions and implications:

These results demonstrate for the first time that when given prior to ischaemia ET-1 mediates its anti-arrhythmic effects, at least in part, via cardiac mast cell degranulation.     

A comparison of 4β-hydroxycholesterol : cholesterol and 6β-hydroxycortisol : cortisol as markers of CYP3A4 induction

Aim

To compare plasma 4β-hydroxycholesterol : cholesterol with urinary 6β-hydroxycortisol : cortisol as markers of cytochrome P4503A4 activity before and after treatment with rifampicin for 2 weeks.

Method

6β-hydroxycortisol and cortisol were determined by liquid chromatography tandem mass spectrometry and 4β-hydroxycholesterol was determined by gas chromatography–mass spectrometry in three groups of healthy volunteers.

Results

Induction ratios for 6β-hydroxycortisol : cortisol were 1.8, 3.9 and 4.5 for 20 mg day⁻¹, 100 mg day⁻¹ or 500 mg day⁻¹ of rifampicin, respectively. The corresponding ratios for 4β-hydroxycholesterol : cholesterol were 1.5, 2.4 and 3.8.

Conclusions

Plasma 4β-hydroxycholesterol : cholesterol gave similar induction ratios to urinary 6β-hydroxycortisol : cortisol.     

Response of mesenchymal stem cells to shear stress in tissue-engineered vascular grafts

Aim:

Recent studies have demonstrated that mesenchymal stem cells (MSCs) can differentiate into endothelial cells. The effect of shear stress on MSC differentiation is incompletely understood, and most studies have been based on two-dimensional systems. We used a model of tissue-engineered vascular grafts (TEVGs) to investigate the effects of shear stress on MSC differentiation.

Methods:

MSCs were isolated from canine bone marrow. The TEVG was constructed by seeding MSCs onto poly-ɛ-caprolactone and lactic acid (PCLA) scaffolds and subjecting them to shear stress provided by a pulsatile bioreactor for four days (two days at 1 dyne/cm² to 15 dyne/cm² and two days at 15 dyne/cm²).

Results:

Shear stress significantly increased the expression of endothelial cell markers, such as platelet-endothelial cell adhesion molecule-1 (PECAM-1), VE-cadherin, and CD34, at both the mRNA and protein levels as compared with static control cells. Protein levels of alpha-smooth muscle actin (α-SMA) and calponin were substantially reduced in shear stress-cultured cells. There was no significant change in the expression of α-SMA, smooth muscle myosin heavy chain (SMMHC) or calponin at the mRNA level.

Conclusion:

Shear stress upregulated the expression of endothelial cell-related markers and downregulated smooth muscle-related markers in canine MSCs. This study may serve as a basis for further investigation of the effects of shear stress on MSC differentiation in TEVGs.     

Relationship between plasma,atrial and ventricular perhexiline concentrations in humans: insights into factors affecting myocardial uptake

AIM

Little is known regarding the steady-state uptake of drugs into the human myocardium. Perhexiline is a prophylactic anti-anginal drug which is increasingly also used in the treatment of heart failure and hypertrophic cardiomyopathy. We explored the relationship between plasma perhexiline concentrations and its uptake into the myocardium.

METHODS

Blood, right atrium ± left ventricle biopsies were obtained from patients treated with perhexiline for a median of 8.5 days before undergoing coronary surgery in the perhexiline arm of a randomized controlled trial. Perhexiline concentrations in plasma and heart tissue were determined by HPLC.

RESULTS

Atrial biopsies were obtained from 94 patients and ventricular biopsies from 28 patients. The median plasma perhexiline concentration was within the therapeutic range at 0.24 mg l⁻¹ (IQR 0.12–0.44), the median atrial concentration was 6.02 mg kg^–1 (IQR 2.70–9.06) and median ventricular concentration was 10.0 mg kg^–1 (IQR 5.76–13.1). Atrial (r² = 0.76) and ventricular (r² = 0.73) perhexiline concentrations were closely and directly correlated with plasma concentrations (both P < 0.001). The median atrial : plasma ratio was 21.5 (IQR 18.1–27.1), ventricular : plasma ratio was 34.9 (IQR 24.5–55.2) and ventricular : atrial ratio was 1.67 (IQR 1.39–2.22). Using multiple regression, the best model for predicting steady-state atrial concentration included plasma perhexiline, heart rate and age (r² = 0.83). Ventricular concentrations were directly correlated with plasma perhexiline concentration and length of therapy (r² = 0.84).

CONCLUSIONS

This study demonstrates that plasma perhexiline concentrations are predictive of myocardial drug concentrations, a major determinant of drug effect. However, net myocardial perhexiline uptake is significantly modulated by patient age, potentially via alteration of myocardial:extracardiac drug uptake.     

Reduction of cerebral infarct volume by apocynin requires pretreatment and is absent in Nox2-deficient mice

Background and purpose:

Reactive oxygen species (ROS) derived from Nox2-containing reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity is reportedly detrimental in cerebrovascular disease. However, ROS generation by other Nox isoforms may have a physiological role. No Nox2-selective inhibitors have yet been identified, and thus it is unclear whether isoform non-selective Nox inhibitors would necessarily improve outcome after stroke. We assessed the effect of apocynin on cerebrovascular ROS production and also on outcome following cerebral ischaemia when administered either before ischaemia or after cerebral reperfusion. The involvement of Nox2-containing NADPH oxidase in the effects of apocynin was assessed using Nox2^−/− mice.

Experimental approach:

Transient cerebral ischaemia was induced by 0.5 h middle cerebral artery occlusion followed by 23.5 h reperfusion. Mice received apocynin (2.5 mg·kg⁻¹, i.p.) either 0.5 h before ischaemia or 1 h after reperfusion. In situ superoxide production after cerebral ischaemia-reperfusion was measured in brain sections of wild-type mice at 24 h using dihydroethidium fluorescence.

Key results:

Treatment with apocynin 0.5 h before ischaemia reduced total infarct volume, neurological impairment and mortality in wild-type but not Nox2^−/− mice. Conversely, treatment with apocynin 1 h after initiation of reperfusion had no protective effect. Cerebral ischaemia and reperfusion increased superoxide production in the brain at 24 h, and pretreatment but not posttreatment with apocynin reduced superoxide levels.

Conclusions and implications:

Apocynin improves outcome following stroke when administered before ischaemia in wild-type but not Nox2^−/− mice.     

Brain protection against ischemic stroke using choline as a new molecular bypass treatment

Aim:

To determine whether administration of choline could attenuate brain injury in a rat model of ischemic stroke and the underlying mechanisms.

Methods:

A rat model of ischemic stroke was established through permanent middle cerebral artery occlusion (pMCAO). After the surgery, the rats were treated with choline or choline plus the specific α7 nAChR antagonist methyllycaconitine (MLA), or with the control drug nimodipine for 10 days. The neurological deficits, brain-infarct volume, pial vessel density and the number of microvessels in the cortex were assessed. Rat brain microvascular endothelial cells (rBMECs) cultured under hypoxic conditions were used in in vitro experiments.

Results:

Oral administration of choline (100 or 200 mg·kg⁻¹·d⁻¹) or nimodipine (20 mg·kg⁻¹·d⁻¹) significantly improved neurological deficits, and reduced infarct volume and nerve cell loss in the ischemic cerebral cortices in pMCAO rats. Furthermore, oral administration of choline, but not nimodipine, promoted the pial arteriogenesis and cerebral-cortical capillary angiogenesis in the ischemic regions. Moreover, oral administration of choline significantly augmented pMCAO-induced increases in the expression levels of α7 nAChR, HIF-1α and VEGF in the ischemic cerebral cortices as well as in the serum levels of VEGF. Choline-induced protective effects were prevented by co-treatment with MLA (1 mg·kg⁻¹·d⁻¹, ip). Treatment of rBMECs cultured under hypoxic conditions in vitro with choline (1, 10 and 100 μmol/L) dose-dependently promoted the endothelial-cell proliferation, migration and tube formation, as well as VEGF secretion, which were prevented by co-treatment with MLA (1 μmol/L) or by transfection with HIF-1α siRNA.

Conclusion:

Choline effectively attenuates brain ischemic injury in pMCAO rats, possibly by facilitating pial arteriogenesis and cerebral-cortical capillary angiogenesis via upregulating α7 nAChR levels and inducing the expression of HIF-1α and VEGF.     

Sirt1 is involved in decreased bone formation in aged apolipoprotein E-deficient mice

Aim:

Apolipoprotein E (ApoE) plays an important role in the transport and metabolism of lipids. Recent studies show that bone mass is increased in young apoE^−/− mice. In this study we investigated the bone phenotype and metabolism in aged apoE^−/− mice.

Methods:

Femurs and tibias were collected from 18- and 72-week-old apoE^−/− mice and their age-matched wild-type (WT) littermates, and examined using micro-CT and histological analysis. Serum levels of total cholesterol, oxidized low-density lipoprotein (ox-LDL) and bone turnover markers were measured. Cultured bone mesenchymal stem cells (BMSCs) from tibias and femurs of 18-week-old apoE^−/− mice were used in experiments in vitro. The expression levels of Sirt1 and Runx2 in bone tissue and BMSCs were measured using RT-PCR and Western blot analysis.

Results:

Compared with age-matched WT littermates, young apoE^−/− mice exhibited high bone mass with increased bone formation, accompanied by higher serum levels of bone turnover markers OCN and TRAP5b, and higher expression levels of Sirt1, Runx2, ALP and OCN in bone tissue. In contrast, aged apoE^−/− mice showed reduced bone formation and lower bone mass relative to age-matched WT mice, accompanied by lower serum OCN levels, and markedly reduced expression levels of Sirt1, Runx2, ALP and OCN in bone tissue. After BMSCs were exposed to ox-LDL (20 μg/mL), the expression of Sirt1 and Runx2 proteins was significantly increased at 12 h, and then decreased at 72 h. Treatment with the Sirt1 inhibitor EX527 (10 μmol/L) suppressed the expression of Runx2, ALP and OCN in BMSCs.

Conclusion:

In contrast to young apoE^−/− mice, aged apoE^−/− mice showe lower bone mass than age-matched WT mice. Long-lasting exposure to ox-LDL decreases the expression of Sirt1 and Runx2 in BMSCs, which may explain the decreased bone formation in aged apoE^−/− mice.     

Relationship between the receptor occupancy profile and pleiotropic effects of angiotensin II receptor blockers

Aim

To investigate whether (i) angiotensin receptor occupying profiles of angiotensin II receptor blockers (ARBs) vary among the drugs and (ii) such differences contribute to the degree of their pleiotropic effects.

Methods

In a randomized, three phase crossover study, nine hypertensive patients received repeated doses (each recommended starting dose for 7 days and then each maximum recommended dose for 20 days) of irbesartan, valsartan and candesartan. The time course profiles and trough level of receptor occupancy were determined on days 7 and 28, respectively. The pleiotropic effect related parameters were measured on days 0 and 28 in each trial.

Results

Of the pleiotropic effect related parameters investigated, urinary 8-isoprostane, fasting serum insulin and homeostasis model assessment of insulin resistance index were more suppressed after 4 weeks treatment with irbesartan than after candesartan and valsartan therapy, respectively. The maximum, area under the curve and trough values of receptor occupancy significantly differed between the ARBs [geometric mean (and 95% CI) of trough value 18.1 (12.9, 25.3) for irbesartan, 9.6 (6.0, 15.3) for valsartan and 5.5 (2.8, 10.8) for candesartan, respectively] and were negatively correlated with the change in urinary 8-isoprostane (r = −0.46 − −0.55, P < 0.05), but not the markers of insulin resistance (r = 0.02–0.15, P = 0.46–0.94).

Conclusions

Our results demonstrate that the receptor occupying profiles are different among the ARBs. This class of drugs might have both receptor occupancy dependent and independent pleiotropic effects.     

Antagonism of endogenous nociceptin/orphanin FQ inhibits infarction-associated ventricular arrhythmias via PKC-dependent mechanism in rats

BACKGROUND AND PURPOSE

Evidence indicates nociceptin/orphanin FQ (N/OFQ) may participate in the pathology of cardiac arrhythmias associated with myocardial infarction. But the role of N/OFQ in the arrhythmogenesis in acute myocardial infarction is unclear. The aim of this study was to investigate the effects of endogenous N/OFQ on infarction-associated arrhythmias.

EXPERIMENTAL APPROACH

The expression of N/OFQ, PKC activity and ventricular arrhythmias in presence and absence of UFP-101, a specific antagonist of N/OFQ receptor, were examined following permanent coronary artery occlusion in anaesthetized rats. The effect of N/OFQ on action potential duration was examined in isolated rat cardiomyocytes.

KEY RESULTS

It was observed that N/OFQ was increased by 41% in the myocardium after coronary artery occlusion (P < 0.01 vs. control). Pretreatment with UFP-101 (10⁻⁷ mol·kg⁻¹, i.v.) reduced the incidence of ventricular ectopic beats by 70% and ventricular tachycardia by 51% respectively (all P < 0.05 vs. control). Meanwhile, PKC activity was elevated in the rats treated with UFP-101 (by 35%, P < 0.05 vs. control). A selective PKC inhibitor, calphostin C, completely abolished the anti-arrhythmic effects of UFP-101 (P < 0.01). N/OFQ (at 10⁻¹¹, 10⁻⁹ and 1 × 10⁻⁷ mol·L⁻¹) shortened the action potential duration by 3% (P > 0.05), 10% (P < 0.05) and 22% (P < 0.01), respectively, via N/OFQ receptor.

CONCLUSIONS AND IMPLICATIONS

Antagonism of endogenous N/OFQ produces anti-arrhythmic effects on ventricular arrhythmias in acute myocardial infarction, possibly via modulating PKC activity and action potential of myocytes.