Hypoxic preconditioning in renal ischaemia-reperfusion injury: a review in pre-clinical models.

Ischaemia-reperfusion injury (IRI) is a major cause of acute kidney injury (AKI) and chronic kidney disease, which consists of cellular damage and renal dysfunction. AKI is a major complication that is of particular concern after cardiac surgery and to a lesser degree following organ transplantation in the immediate post-transplantation period, leading to delayed graft function. Because effective therapies are still unavailable, several recent studies have explored the potential benefit of hypoxic preconditioning (HPC) on IRI. HPC refers to the acquisition of increased organ tolerance to subsequent ischaemic or severe hypoxic injury, and experimental evidences suggest a potential benefit of HPC. There are three experimental forms of HPC, and, for better clarity, we named them as follows: physical HPC, HPC via treated-cell administration and stabilised hypoxia-inducible factor (HIF)-1α HPC, or mimicked HPC. The purpose of this review is to present the latest developments in the literature on HPC in the context of renal IRI in pre-clinical models. The data we compiled suggest that preconditional activation of hypoxia pathways protects against renal IRI, suggesting that HPC could be used in the treatment of renal IRI in transplantation.

Homocysteine concentration and adenosine A2A receptor production by peripheral blood mononuclear cells in coronary artery disease patients.

Hyperhomocysteinemia is associated with coronary artery disease (CAD). The mechanistic aspects of this relationship are unclear. In CAD patients, homocysteine (HCy) concentration correlates with plasma level of adenosine that controls the coronary circulation via the activation of adenosine A2A receptors (A2A R). We addressed in CAD patients the relationship between HCy and A2A R production, and in cellulo the effect of HCy on A2A R function. 46 patients with CAD and 20 control healthy subjects were included. We evaluated A2A R production by peripheral blood mononuclear cells using Western blotting. We studied in cellulo (CEM human T cells) the effect of HCy on A2A R production as well as on basal and stimulated cAMP production following A2A R activation by an agonist-like monoclonal antibody. HCy concentration was higher in CAD patients vs controls (median, range: 16.6 [7-45] vs 8 [5-12] µM, P < 0.001). A2A R production was lower in patients vs controls (1.1[0.62-1.6] vs 1.53[0.7-1.9] arbitrary units, P < 0.001). We observed a negative correlation between HCy concentration and A2A R production (r = -0.43; P < 0.0001), with decreased A2A R production above 25 µM HCy. In cellulo, HCy inhibited A2A R production, as well as basal and stimulated cAMP production. In conclusion, HCy is negatively associated with A2A R production in CAD patients, as well as with A2A R and cAMP production in cellulo. The decrease in A2A R production and function, which is known to hamper coronary blood flow and promote inflammation, may support CAD pathogenesis.

Adenosine and Its Receptors: An Expected Tool for the Diagnosis and Treatment of Coronary Artery and Ischemic Heart Diseases.

Adenosine is an endogenous nucleoside which strongly impacts the cardiovascular system. Adenosine is released mostly by endothelial cells and myocytes during ischemia or hypoxia and greatly regulates the cardiovascular system via four specific G-protein-coupled receptors named A1R, A2AR, A2BR, and A3R. Among them, A2 subtypes are strongly expressed in coronary tissues, and their activation increases coronary blood flow via the production of cAMP in smooth muscle cells. A2A receptor modulators are an opportunity for intense research by the pharmaceutical industry to develop new cardiovascular therapies. Most innovative therapies are mediated by the modulation of adenosine release and/or the activation of the A2A receptor subtypes. This review aims to focus on the specific exploration of the adenosine plasma level and its relationship with the A2A receptor, which seems a promising biomarker for a diagnostic and/or a therapeutic tool for the screening and management of coronary artery disease. Finally, a recent class of selective adenosine receptor ligands has emerged, and A2A receptor agonists/antagonists are useful tools to improve the management of patients suffering from coronary artery disease.

Extracellular vesicles with ubiquitinated adenosine A2A receptor in plasma of patients with coronary artery disease.

Extracellular vesicles (EV) can transfer cellular molecules for specific intercellular communication with potential relevance in pathological conditions. We searched for the presence in plasma from coronary artery disease (CAD) patients of EV containing the adenosine A2A receptor (A2A R), a signalling receptor associated with myocardial ischaemia and whose expression is related to homocysteine (HCy) metabolism. Using protein organic solvent precipitation for plasma EV preparation and Western blotting for protein identification, we found that plasma from CAD patients contained various amounts of EV with ubiquitin bound to A2A R. Interestingly, the presence of ubiquitinated A2A R in EV from patients was dependent on hyperhomocysteinemia, the amount being inversely proportional to A2A R expression in peripheral mononuclear cells in patients with the highest levels of HCy. CEM, a human T cell line, was also found to released EV containing various amounts of ubiquitinated A2A R in stimulated conditions depending on the hypoxic status and HCy level of culture medium. Together, these data show that ubiquitinated A2A R-containing EV circulate in the plasma of CAD patients and that this presence is related to hyperhomocysteinemia. A2A R in plasma EV could be a useful tool for diagnosis and a promising drug for the treatment of CAD.

Adenosine plasma level in patients with paroxysmal or persistent atrial fibrillation and normal heart during ablation procedure and/or cardioversion.

The mechanism of atrial fibrillation (AF) in patients with normal heart remains unclear. While exogenous adenosine can trigger AF, nothing is known about the behavior of endogenous adenosine plasma level (APL) at the onset of AF and during ablation procedure. Ninety-one patients (68 with paroxysmal AF: 40 males, 66 ± 16 years; 23 with persistent AF: 14 males, 69 ± 11 years) and 18 controls were included. Among paroxysmal patients: i) medical therapy alone was performed in 45 cases and ablation procedure in 23. AF was spontaneously resolutive in 6 cases; ii) 23 underwent ablation procedure and blood was collected simultaneously in a brachial vein and in the left atrium; 17 were spontaneously in sinus rhythm while 6 were in sinus rhythm after direct current cardioversion. Among persistent patients: i) in 17 patients, blood samples were collected in a brachial vein before and after direct current cardioversion; ii) in 6 patients, blood samples were collected simultaneously in a brachial vein and in left atrium before and after cardioversion during ablation procedure. CV-APL was higher in patients with persistent AF vs patients with paroxysmal AF (median [range]: 0.9[0.6-1.1] vs 0.7[0.4-1.1] µM; p < 0.001). In patients with paroxysmal AF, LA-APL increased during the AF episode (0.95[0.85-1.4] vs 2.7[1.5-7] µM; p = 0.03) and normalized in sinus rhythm after DCCV. In patients with persistent AF, LA-APL was higher than CV-APL (1.2[0.7-1.8] vs 0.9[0.6-1.1] µM; p < 0.001), and both normalized in sinus rhythm (CV-APL: 0.8[0.6-1.1] vs 0.75[0.4-1] µM; p = 0.03), (LA-APL: 1.95[1.3-3] vs 1[0.5-1.15] µM; p = 0.03). The occurrence of AF is associated with a strong increase of APL in the atrium. The cause of this increase needs further investigations.

Fatty acid/monoglyceride type and amount modulate fat-soluble vitamin absorption from mixed assemblies in mice.

We investigated the effects of fatty acid/ monoglyceride type and amount on the absorption of fat-soluble vitamins. Micelles or vesicles made with either caprylic acid (CA) + monocaprylin (MC) or oleic acid (OA) + monoolein (MO) at low or high concentrations were infused in bile duct-ligated mice. Retinol + retinyl ester and γ-tocopherol intestinal mucosa contents were higher in mice infused with CA + MC than with OA + MO (up to + 350 % for vitamin A and up to + 62 %, for vitamin E; p < 0.05). Cholecalciferol intestinal mucosa content was the highest in mice infused with micelles with CA + MC at 5 mg/mL (up to + 105 %, p < 0.05). Retinyl ester plasma response was higher with mixed assemblies formed at low concentration of FA + MG compared to high concentration (up to + 1212 %, p < 0.05), while no difference in cholecalciferol and γ-tocopherol plasma responses were measured. No correlation between size or zeta potential and vitamin absorption was found. The impact of FA and MG on fat-soluble vitamin absorption thus differs from one vitamin to another and should be considered to formulate adequate vitamin oral or enteral supplements.

Carotenoids in familial hypobetalipoproteinemia disorders: Malabsorption in Caco2 cell models and severe deficiency in patients.

BACKGROUND: Familial hypobetalipoproteinemias (FHBL) are rare genetic diseases characterized by lipid malabsorption. We focused on abetalipoproteinemia (FHBL-SD1) and chylomicron retention disease (FHBL-SD3), caused by mutations in microsomal triglyceride transfer protein (MTTP) and SAR1B genes, respectively. Treatments include a low-fat diet and high-dose fat-soluble vitamin supplementations. However, patients are not supplemented in carotenoids, a group of lipid-soluble pigments essential for eye health. OBJECTIVE: Our aim was to evaluate carotenoid absorption and status in the context of hypobetalipoproteinemia. METHODS: We first used knock-out Caco-2/TC7 cell models of FHBL-SD1 and FHBL-SD3 to evaluate carotenoid absorption. We then characterized FHBL-SD1 and FHBL-SD3 patient status in the main dietary carotenoids and compared it to that of control subjects. RESULTS: In vitro results showed a significant decrease in basolateral secretion of α- and ß-carotene, lutein, and zeaxanthin (-88.8 ± 2.2 % to -95.3 ± 5.8 %, -79.2 ± 4.4 % to -96.1 ± 2.6 %, -91.0 ± 4.5 % to -96.7 ± 0.3 % and -65.4 ± 3.6 % to -96.6 ± 1.9 %, respectively). Carotenoids plasma levels in patients confirmed significant deficiencies, with decreases ranging from -89 % for zeaxanthin to -98 % for α-carotene, compared to control subjects. CONCLUSION: Given the continuous loss in visual function despite fat-soluble vitamin treatment in some patients, carotenoid supplementation may be of clinical utility. Future studies should assess the correlation between carotenoid status and visual function in aging patients and investigate whether carotenoid supplementation could prevent their visual impairment.

Impact of pulses, starches and meat on vitamin D and K postprandial responses in mice.

In vitro experiments showed that i) phytates, tannins and saponins from pulses can alter vitamin D and K bioavailability and ii) meat decreased vitamin D bioaccessibility by impairing its stability during digestion. We aimed to confirm these results in vivo by force-feeding mice with emulsions containing either potatoes or semolina or chickpeas or meat. Vitamin D and K plasma responses decreased after a gavage with chickpeas or meat compared with potatoes (-62 % and -67 %, respectively for vitamin D, -40 % and -64 %, respectively for vitamin K; p < 0.05). Vitamin D and K intestinal contents were also reduced in mice force-fed with chickpeas or meat compared with potatoes (from -64 to -83 % and from -76 to -84 %, respectively for vitamin D and from -7 to -59 % and from -7 to -90 %, respectively for vitamin K; p < 0.05). The results confirm that chickpea and meat compounds can decrease vitamin D and K bioavailability.

Validation of Knock-Out Caco-2 TC7 Cells as Models of Enterocytes of Patients with Familial Genetic Hypobetalipoproteinemias.

Abetalipoproteinemia (FHBL-SD1) and chylomicron retention disease (FHBL-SD3) are rare recessive disorders of lipoprotein metabolism due to mutations in MTTP and SAR1B genes, respectively, which lead to defective chylomicron formation and secretion. This results in lipid and fat-soluble vitamin malabsorption, which induces severe neuro-ophthalmic complications. Currently, treatment combines a low-fat diet with high-dose vitamin A and E supplementation but still fails in normalizing serum vitamin E levels and providing complete ophthalmic protection. To explore these persistent complications, we developed two knock-out cell models of FHBL-SD1 and FHBL-SD3 using the CRISPR/Cas9 technique in Caco-2/TC7 cells. DNA sequencing, RNA quantification and Western blotting confirmed the introduction of mutations with protein knock-out in four clones associated with i) impaired lipid droplet formation and ii) defective triglyceride (-57.0 ± 2.6% to -83.9 ± 1.6%) and cholesterol (-35.3 ± 4.4% to -60.6 ± 3.5%) secretion. A significant decrease in α-tocopherol secretion was also observed in these clones (-41.5 ± 3.7% to -97.2 ± 2.8%), even with the pharmaceutical forms of vitamin E: tocopherol-acetate and tocofersolan (α-tocopheryl polyethylene glycol succinate 1000). MTTP silencing led to a more severe phenotype than SAR1B silencing, which is consistent with clinical observations. Our cellular models thus provide an efficient tool to experiment with therapeutic strategies and will allow progress in understanding the mechanisms involved in lipid metabolism.

Pharmacological profile of adenosine A2A receptors in patients with lower extremity peripheral artery disease and associated coronary artery disease: A pilot study.

BACKGROUND: Altered blood flow occurs in patients with low extremity peripheral artery disease (LE-PAD). LE-PAD is mostly associated with coronary artery disease (CAD). Adenosine is an endogenous nucleoside that affects both coronary and limb artery blood flow, mostly via the adenosine A2A receptor (A2AR). We evaluated A2AR expression and function in peripheral blood mononuclear cells (PBMCs) and the femoral artery tissues of patients with LE-PAD. METHODS: Artery tissues and PBMCs were sampled in 24 patients with intermittent claudication, and compared with PBMCs in 24 healthy subjects. Expression and function of A2AR was studied, using a A2AR monoclonal antibody with agonist properties, allowing determination of A2AR affinity (KD) and cAMP production (ie.EC50). RESULTS: A2AR expression on PBMCs was lower in patients than controls (median1.3 [range 0.6-1.8] vs 1.75 [1.45-2.1] arbitrary units; P < 0.01), and correlated with A2AR expression in artery tissues (Pearson's r = 0.71; P < 0.01). Basal and maximally stimulated cAMP production of PBMCs was lower in patients vs controls: 172 [90-310] vs 244 [110-380] pg/106 cells (P < 0.05) and 375 [160-659] vs 670 [410-980] pg/106 cells (P < 0.05), respectively. A high KD/EC50 ratio, characteristic of spare receptors, was observed in CAD with inducible-myocardial-ischemia. CONCLUSION: A2AR expression in the arteries of patients, correlated with their expression in PBMCs. A2AR expression was lower in patients than in controls. A single blood sample (for measurement of A2AR expression on PBMCs) may help to screen patients with LE-PAD, whereas the presence of spare receptors may help with risk stratification before vascular surgery in CAD patients with high risk of myocardial ischemia.

Troponins in scuba divers with immersion pulmonary edema.

Immersion pulmonary edema (IPE) is a serious complication of water immersion during scuba diving. Myocardial ischemia can occur during IPE that worsens outcome. Because myocardial injury impacts the therapeutic management, we aim to evaluate the profile of cardiac markers (creatine phosphokinase (CPK), brain natriuretic peptide (BNP), highly sensitive troponin T (TnT-hs) and ultrasensitive troponin I (TnI-us) of divers with IPE. Twelve male scuba divers admitted for suspected IPE were included. The collection of blood samples was performed at hospital entrance (T0) and 6 h later (T0 + 6 h). Diagnosis was confirmed by echocardiography or computed-tomography scan. Mean ± S.D. BNP (pg/ml) was 348 ± 324 at T0 and 223 ± 177 at T0 + 6 h (P<0.01), while mean CPK (international units (IUs)), and mean TnT-hs (pg/ml) increased in the same times 238 ± 200 compared with 545 ± 39, (P=0.008) and 128 ± 42 compared with 269 ± 210, (P=0.01), respectively; no significant change was observed concerning TnI-us (pg/ml): 110 ± 34 compared with 330 ± 77, P=0.12. At T0 + 6 h, three patients had high TnI-us, while six patients had high TnT-hs. Mean CPK was correlated with TnT-hs but not with TnI-us. Coronary angiographies were normal. The increase in TnT during IPE may be secondary to the release of troponin from non-cardiac origin. The measurement of TnI in place of TnT permits in some cases to avoid additional examinations, especially unnecessary invasive investigations.

Towards Addressing the Body Electrolyte Environment via Sweat Analysis:Pilocarpine Iontophoresis Supports Assessment of Plasma Potassium Concentration.

Electrolyte concentration in sweat depends on environmental context and physical condition but also on the pathophysiological status. Sweat analyzers may be therefore the future way for biological survey although how sweat electrolyte composition can reflect plasma composition remains unclear. We recruited 10 healthy subjects and 6 patients to have a broad range of plasma electrolyte concentrations (chloride, potassium and sodium) and pH. These variables were compared to those found in sweat produced following cycling exercise or pilocarpine iontophoresis, a condition compatible with operating a wearable device. We found no correlation between plasma and sweat parameters when exercise-induced sweat was analyzed, and we could identify a correlation only between plasma and sweat potassium concentration (R = 0.78, p < 0.01) when sweat was induced using pilocarpine iontophoresis. We tested measurement repeatability in sweat at 24hr-interval for 3 days in 4 subjects and found a great intra-individual variability regarding all parameters in exercise-induced sweat whereas similar electrolyte levels were measured in pilocarpine-induced sweat. Thus, electrolyte concentration in sweat sampled following physical activity does not reflect concentration in plasma while pilocarpine iontophoresis appears to be promising to reproducibly address sweat electrolytes, and to make an indirect evaluation of plasma potassium concentration in chronic kidney disease and arrhythmia.

Expressions of adenosine A2A receptors in coronary arteries and peripheral blood mononuclear cells are correlated in coronary artery disease patients.

BACKGROUND: Altered coronary blood flow occurs in patients with coronary artery disease (CAD). Adenosine strongly impacts blood flow mostly via adenosine A2A receptor (A2AR) expressed in coronary tissues. As part of a systemic regulation of the adenosinergic system, we compared A2AR expression in situ, and on peripheral blood mononuclear cells (PBMC) in CAD patients. METHODS AND RESULTS: Aortic and coronary tissues, and PBMC were sampled in 20 CAD patients undergoing coronary artery bypass surgery and consecutively included. Controls were PBMC obtained from 15 healthy subjects. Expression and activity of A2AR were studied by Western blotting and cAMP measurement, respectively. A2AR expression on PBMC was lower in patients than in controls (0.83±0.31 vs 1.2±0.35 arbitrary units; p<0.01), and correlated with A2AR expression in coronary and aortic tissues (Pearson's r: 0.77 and 0.59, p<0.01, respectively). Basal and maximal cAMP productions following agonist stimulation of PBMC were significantly lower in patients than in controls (120±42 vs 191±65 and 360±113 vs 560±215pg/106 cells, p<0.05, respectively). In CAD patients, the increase from basal to maximal cAMP production in PBMC and aortic tissues was similar (+300% and +246%, respectively). CONCLUSION: Expression of A2AR on PBMC correlated with those measured in coronary artery and aortic tissues in CAD patients, A2AR activity of PBMC matched that observed in aorta, and A2AR expression and activity in PBMC were found reduced as compared to controls. Measuring the expression level of A2AR on PBMC represents a good tool to address in situ expression in coronary tissues of CAD patients.

Pathophysiological and diagnostic implications of cardiac biomarkers and antidiuretic hormone release in distinguishing immersion pulmonary edema from decompression sickness.

Immersion pulmonary edema (IPE) is a misdiagnosed environmental illness caused by water immersion, cold, and exertion. IPE occurs typically during SCUBA diving, snorkeling, and swimming. IPE is sometimes associated with myocardial injury and/or loss of consciousness in water, which may be fatal. IPE is thought to involve hemodynamic and cardiovascular disturbances, but its pathophysiology remains largely unclear, which makes IPE prevention difficult. This observational study aimed to document IPE pathogenesis and improve diagnostic reliability, including distinguishing in some conditions IPE from decompression sickness (DCS), another diving-related disorder.Thirty-one patients (19 IPE, 12 DCS) treated at the Hyperbaric Medicine Department (Ste-Anne hospital, Toulon, France; July 2013-June 2014) were recruited into the study. Ten healthy divers were recruited as controls. We tested: (i) copeptin, a surrogate marker for antidiuretic hormone and a stress marker; (ii) ischemia-modified albumin, an ischemia/hypoxia marker; (iii) brain-natriuretic peptide (BNP), a marker of heart failure, and (iv) ultrasensitive-cardiac troponin-I (cTnI), a marker of myocardial ischemia.We found that copeptin and cardiac biomarkers were higher in IPE versus DCS and controls: (i) copeptin: 68% of IPE patients had a high level versus 25% of DCS patients (P < 0.05) (mean ±â€Šstandard-deviation: IPE: 53 ±â€Š61 pmol/L; DCS: 15 ±â€Š17; controls: 6 ±â€Š3; IPE versus DCS or controls: P < 0.05); (ii) ischemia-modified albumin: 68% of IPE patients had a high level versus 16% of DCS patients (P < 0.05) (IPE: 123 ±â€Š25 arbitrary-units; DCS: 84 ±â€Š25; controls: 94 ±â€Š7; IPE versus DCS or controls: P < 0.05); (iii) BNP: 53% of IPE patients had a high level, DCS patients having normal values (P < 0.05) (IPE: 383 ±â€Š394 ng/L; DCS: 37 ±â€Š28; controls: 19 ±â€Š15; IPE versus DCS or controls: P < 0.01); (iv) cTnI: 63% of IPE patients had a high level, DCS patients having normal values (P < 0.05) (IPE: 0.66 ±â€Š1.50 µg/L; DCS: 0.0061 ±â€Š0.0040; controls: 0.0090 ±â€Š0.01; IPE versus DCS or controls: P < 0.01). The combined "BNP-cTnI" levels provided most discrimination: all IPE patients, but none of the DCS patients, had elevated levels of either/both of these markers.We propose that antidiuretic hormone acts together with a myocardial ischemic process to promote IPE. Thus, monitoring of antidiuretic hormone and cardiac biomarkers can help to make a quick and reliable diagnosis of IPE.