Christophe Jayle

Amniotic Fluid-Derived Mesenchymal Stem Cells Prevent Fibrosis and Preserve Renal Function in a Preclinical Porcine Model of Kidney Transplantation

It is well known that ischemia/reperfusion injuries strongly affect the success of human organ transplantation. Development of interstitial fibrosis and tubular atrophy is the main deleterious phenomenon involved. Stem cells are a promising therapeutic tool already validated in various ischemic diseases. Amniotic fluid‐derived mesenchymal stem cells (af‐MSCs), a subpopulation of multipotent cells identified in amniotic fluid, are known to secrete growth factors and anti‐inflammatory cytokines. In addition, these cells are easy to collect, present higher proliferation and self‐renewal rates compared with other adult stem cells (ASCs), and are suitable for banking. Consequently, af‐MSCs represent a promising source of stem cells for regenerative therapies in humans. To determine the efficiency and the safety of af‐MSC infusion in a preclinical porcine model of renal autotransplantation, we injected autologous af‐MSCs in the renal artery 6 days after transplantation. The af‐MSC injection improved glomerular and tubular functions, leading to full renal function recovery and abrogated fibrosis development at 3 months. The strong proof of concept generated by this translational porcine model is a first step toward evaluation of af‐MSC‐based therapies in human kidney transplantation.

Transient Receptor Potential Canonical Channel 6 Links Ca2+ Mishandling to Cystic Fibrosis Transmembrane Conductance Regulator Channel Dysfunction in Cystic Fibrosis

In cystic fibrosis (CF), abnormal control of cellular Ca(2+) homeostasis is observed. We hypothesized that transient receptor potential canonical (TRPC) channels could be a link between the abnormal Ca(2+) concentrations in CF cells and cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction. We measured the TRPC and CFTR activities (using patch clamp and fluorescent probes) and interactions (using Western blotting and co-immunoprecipitation) in CF and non-CF human epithelial cells treated with specific and scrambled small interfering RNA (siRNA). The TRPC6-mediated Ca(2+) influx was abnormally increased in CF compared with non-CF cells. After correction of abnormal F508 deletion (del)-CFTR trafficking in CF cells, the level of TRPC6-dependent Ca(2+) influx was also normalized. In CF cells, siRNA-TRPC6 reduced this abnormal Ca(2+) influx. In non-CF cells, siRNA-TRPC6 reduced the Ca(2+) influx and activity wild-type (wt)-CFTR. Co-immunoprecipitation experiments revealed TRPC6/CFTR and TRPC6/F508 del-CFTR interactions in CF or non-CF epithelial cells. Although siRNA-CFTR reduced the activity of wt-CFTR in non-CF cells and of F508 del-CFTR in corrected CF cells, it also enhanced TRPC6-dependent Ca(2+) influx in non-CF cells, mimicking the results obtained in CF cells. Finally, this functional and reciprocal coupling between CFTR and TRPC6 was also detected in non-CF ciliated human epithelial cells freshly isolated from lung samples. These data indicate that TRPC6 and CFTR are functionally and reciprocally coupled within a molecular complex in airway epithelial human cells. Because this functional coupling is lost in CF cells, the TRPC6-dependent Ca(2+) influx is abnormal.

The heart rate-lowering agent ivabradine inhibits the pacemaker current I(f) in human atrial myocytes.

Introduction: It has been speculated that pacemaker current (If) in human atria could play a role in causing ectopic atrial automaticity. Ivabradine is a novel selective and specific If inhibitor in the sinus node that reduces heart rate without any negative inotropic effect. The aim of the study was to explore possible effects of ivabradine on If in atrial myocytes.

Flail Chest in Polytraumatized Patients: Surgical Fixation Using Stracos Reduces Ventilator Time and Hospital Stay

Objectives. Conservative management of patients with flail chest is the treatment of choice. Rib fracture repair is technically challenging; however, with the advent of specially designed molding titanium clips, surgical management has been simplified. Surgical stabilization has been used with good outcomes. We are reporting on our institutional matched-case-control study. Methods. Between April 2010 and April 2011, ten polytraumatized patients undergoing rib stabilization for flail chest were matched 1 : 1 to 10 control patients by age ±10 years, sex, neurological or vertebral trauma, abdominal injury, and arm and leg fractures. Surgery was realized in the first 48 hours. Results. There were no significant differences between groups for matched data and prognostic scores: injury severity score, revised trauma score, and trauma injury severity score. Ventilator time (142 ± 224 versus 74 ± 125 hours, P = 0.026) and overall hospital stay (142 ± 224 versus 74 ± 125 hours, P = 0.026) were significantly lower for the surgical group after adjustment on prognostic scores. There was a trend towards shorter ICU stay for operative patients (12.3 ± 8.5 versus 9.0 ± 4.3 days, P = 0.076). Conclusions. Rib fixation with Stracos is feasible and decreases the length of ventilation and hospital stay. A multicenter randomized study is warranted so as to confirm these results and to evaluate impact on pulmonary function status, pain, and quality of life.

Bronchorelaxation of the human bronchi by CFTR activators.

The airway functions are profoundly affected in many diseases including asthma, COPD and cystic fibrosis (CF). CF the most common lethal autosomal recessive genetic disease is caused by mutations of the CFTR (Cystic Fibrosis transmembrane Conductance Regulator) gene, which normally encodes a multifunctional and integral membrane cAMP regulated and ATP gated Cl(-) channel expressed in airway epithelial cells. Using human lung tissues obtained from patients undergoing surgery for lung cancer, we demonstrated that CFTR participates in bronchorelaxation. Using human bronchial smooth muscle cells (HBSMC), we applied iodide influx assay to analyze the CFTR-dependent ionic transport and immunofluorescence technique to localize CFTR proteins. Moreover, the relaxation was studied in isolated human bronchial segments after pre-contraction with carbachol to determine the implication of CFTR in bronchodilation. We found in HBSMC that the pharmacology and regulation of CFTR is similar to that of its epithelial counterpart both for activation (using forskolin/genistein or a benzo[c]quinolizinium derivative) and for inhibition (CFTR(inh)-172 and GPinh5a). With human bronchial rings, we observed that whatever the compound used including salbutamol, the activation of muscular CFTR leads to a bronchodilation after constriction with carbachol. Altogether, these observations revealed that CFTR in the human airways is expressed in bronchial smooth muscle cells and can be pharmacologically manipulated leading to the hypothesis that this ionic channel could contribute to bronchodilation in human.

Influence of nephron mass and a phosphorylated 38 mitogen-activated protein kinase inhibitor on the development of early and long-term injury after renal warm ischaemia

Renal ischaemia is accompanied by acute and chronic complications. Tumour necrosis factor (TNF) α production via p38 mitogen‐activated protein kinase (MAPK) is one of the pivotal mechanisms linking ischaemia to inflammation and could be a therapeutic target. FR167653 (FR), an inhibitor of p38 MAPK and TNF‐α production, may ameliorate renal damage through its effects on TNF‐α.

Pleural lipoma: a non-surgical lesion?

Pleural lipomas are benign tumours that develop at the expense of adipose tissues, and they never evolve towards liposarcoma. Located usually at the mediastinal, bronchial and pulmonary levels, a pleural situation is extremely rare. Chest X-rays usually detect them and computed tomography scans confirm the diagnosis. As complications occur, a wait-and-see policy is common. We report our pleural lipoma surgical exeresis experience since 1999. We have operated on five cases of pleural lipomas among nearly 1800 cases of thoracic exeresis: three male and two female patients, without obesity (in all cases, body mass index (BMI) < 28). The mean age was 54.6 years (range 35-72 years). Four patients were electively operated and one in emergency, three with video-assisted thoracic surgery (VATS) procedure and two with open chest surgery, without recurrent cases. Advancements in VATS have greatly reduced the morbidity rate of these benign tumours especially if exeresis is performed early on a small, uncomplicated adhesion-free tumour. On the other hand, the operation may be deleterious, complicated by the presence of a large lipoma or in a complicating situation. In our opinion, we should revise the wait-and-see policy when facing these lesions considering their evolutionary potential. We should advise VATS in pleural lipomas.

ANO1 contributes to Angiotensin-II-activated Ca2+-dependent Cl− current in human atrial fibroblasts

Cardiac fibroblasts are an integral part of the myocardial tissue and contribute to its remodelling. This study characterises for the first time the calcium-dependent chloride channels (CaCC) in the plasma membrane of primary human atrial cardiac fibroblasts by means of the iodide efflux and the patch clamp methods. The calcium ionophore A23187 and Angiotensin II (Ang II) activate a chloride conductance in cardiac fibroblasts that shares pharmacological similarities with calcium-dependent chloride channels. This chloride conductance is depressed by RNAi-mediated selective Anoctamine 1 (ANO1) but not by Anoctamine 2 (ANO2) which has been revealed as CaCC and is inhibited by the selective ANO1 inhibitor, T16inh-A01. The effect of Ang II on anion efflux is mediated through AT1 receptors (with an EC50 = 13.8 ± 1.3 nM). The decrease of anion efflux by calphostin C and bisindolylmaleimide I (BIM I) suggests that chloride conductance activation is dependent on PKC. We conclude that ANO1 contributes to CaCC current in human cardiac fibroblasts and that this is regulated by Ang II acting via the AT1 receptor pathway.

A functional tandem between transient receptor potential canonical channels 6 and calcium-dependent chloride channels in human epithelial cells.

TRPC6 plays important human physiological functions, notably in artery and arterioles constriction, in regulation of vascular volume and in bronchial muscle constriction. It is implicated in pulmonary hypertension, cardiovascular disease, and focal segmental glomerulosclerosis and seems to play a role in cancer development. Previously, we identified Guanabenz, an α2-adrenergic agonist used for hypertension treatment (Wytensin®), as an activator of calcium-dependent chloride channels (CaCC) in human Cystic Fibrosis (CF) nasal epithelial cells by transiently increasing [Ca2+]i via an influx of extracellular Ca2+. In this study, using assays to measure chloride channel activity, we show that guanabenz is an activator of CaCC in freshly dissociated human bronchial epithelial cells from three CF patients with various genotypes (F508del/F508del, F508del/R1066C, F508del/H1085R). We further characterised the effect of guanabenz and show that it is independent of α-adrenergic receptors, is inhibited by the TRPC family inhibitor SKF-96365 but not by the TRPV family inhibitor ruthenium red. Using western-blotting, Ca2+ measurements and iodide efflux assay, we found that TRPC1 siRNA has no effect on guanabenz induced responses whereas TRPC6 siRNA prevented the guanabenz-dependent Ca2+ influx and the CaCC-dependent activity stimulated by guanabenz. In conclusion, we show that TRPC6 channel is pivotal for the activation of CaCC by guanabenz through a α2-adrenergic-independent pathway in human airway epithelial cells. We suggest propose a functional coupling between TRPC6 and CaCC and guanabenz as a potential TRPC6 activator for exploring TRPC6 and CaCC channel functions and corresponding channelopathies.

Cystic fibrosis bronchial epithelial cells are lipointoxicated by membrane palmitate accumulation.

The F508del-CFTR mutation, responsible for Cystic Fibrosis (CF), leads to the retention of the protein in the endoplasmic reticulum (ER). The mistrafficking of this mutant form can be corrected by pharmacological chaperones, but these molecules showed limitations in clinical trials. We therefore hypothesized that important factors in CF patients may have not been considered in the in vitro assays. CF has also been associated with an altered lipid homeostasis, i. e. a decrease in polyunsaturated fatty acid levels in plasma and tissues. However, the precise fatty acyl content of membrane phospholipids from human CF bronchial epithelial cells had not been studied to date. Since the saturation level of phospholipids can modulate crucial membrane properties, with potential impacts on membrane protein folding/trafficking, we analyzed this parameter for freshly isolated bronchial epithelial cells from CF patients. Interestingly, we could show that Palmitate, a saturated fatty acid, accumulates within Phosphatidylcholine (PC) in CF freshly isolated cells, in a process that could result from hypoxia. The observed PC pattern can be recapitulated in the CFBE41o− cell line by incubation with 100 µM Palmitate. At this concentration, Palmitate induces an ER stress, impacts calcium homeostasis and leads to a decrease in the activity of the corrected F508del-CFTR. Overall, these data suggest that bronchial epithelial cells are lipointoxicated by hypoxia-related Palmitate accumulation in CF patients. We propose that this phenomenon could be an important bottleneck for F508del-CFTR trafficking correction by pharmacological agents in clinical trials.