Florence Lecerf

Endothelial-to-Mesenchymal Transition in Pulmonary Hypertension

Background— The vascular remodeling responsible for pulmonary arterial hypertension (PAH) involves predominantly the accumulation of &agr;-smooth muscle actin–expressing mesenchymal-like cells in obstructive pulmonary vascular lesions. Endothelial-to-mesenchymal transition (EndoMT) may be a source of those &agr;-smooth muscle actin–expressing cells. Methods and Results— In situ evidence of EndoMT in human PAH was obtained by using confocal microscopy of multiple fluorescent stainings at the arterial level, and by using transmission electron microscopy and correlative light and electron microscopy at the ultrastructural level. Findings were confirmed by in vitro analyses of human PAH and control cultured pulmonary artery endothelial cells. In addition, the mRNA and protein signature of EndoMT was recognized at the arterial and lung level by quantitative real-time polymerase chain reaction and Western blot analyses. We confirmed our human observations in established animal models of pulmonary hypertension (monocrotaline and SuHx). After establishing the first genetically modified rat model linked to BMPR2 mutations (BMPR2&Dgr;140Ex1/+ rats), we demonstrated that EndoMT is linked to alterations in signaling of BMPR2, a gene that is mutated in 70% of cases of familial PAH and in 10% to 40% of cases of idiopathic PAH. We identified molecular actors of this pathological transition, including twist overexpression and vimentin phosphorylation. We demonstrated that rapamycin partially reversed the protein expression patterns of EndoMT, improved experimental PAH, and decreased the migration of human pulmonary artery endothelial cells, providing the proof of concept that EndoMT is druggable. Conclusions— EndoMT is linked to alterations in BPMR2 signaling and is involved in the occlusive vas cular remodeling of PAH, findings that may have therapeutic implications.

Establishment of a human thymic myoid cell line. Phenotypic and functional characteristics.

The subset of myoid cells is a normal component of the thymic stroma. To characterize these cells, we immortalized stromal cells from human thymus by using a plasmid vector encoding the SV40 T oncogene. Among the eight cell lines obtained, one had myoid characteristics including desmin and troponin antigens. This new line was designated MITC (myoid immortalized thymic cells). These cells expressed both the fetal and adult forms of muscle acetylcholine receptor (AChR) at the mRNA level, as well as the myogenic transcription factor MyoD1. alpha-Subunit AChR protein expression was detected by flow cytometry and the AChR was functional in patch-clamp studies. In addition, AChR expression was down-modulated by myasthenia gravis sera or by monoclonal antibody anti-AChR on MITC line similarly to TE671 rhabdomyosarcoma cells, making the MITC line an interesting tool for AChR antigenic modulation experiments. Finally, the MITC line expressed LFA-3, produced several cytokines able to act on T cells, and protected total thymocytes from spontaneous apoptosis in vitro. These results are compatible with a role of thymic myoid cells in some steps of thymocyte development. Therefore MITC line appears to be a useful tool to investigate the physiological role of thymic myoid cells.

A Critical Role for p130Cas in the Progression of Pulmonary Hypertension in Humans and Rodents

RATIONALE Pulmonary arterial hypertension (PAH) is a progressive and fatal disease characterized by pulmonary arterial muscularization due to excessive pulmonary vascular cell proliferation and migration, a phenotype dependent upon growth factors and activation of receptor tyrosine kinases (RTKs). p130(Cas) is an adaptor protein involved in several cellular signaling pathways that control cell migration, proliferation, and survival. OBJECTIVES We hypothesized that in experimental and human PAH p130(Cas) signaling is overactivated, thereby facilitating the intracellular transmission of signal induced by fibroblast growth factor (FGF)2, epidermal growth factor (EGF), and platelet-derived growth factor (PDGF). MEASUREMENTS AND MAIN RESULTS In patients with PAH, levels of p130(Cas) protein and/or activity are higher in the serum, in the walls of distal pulmonary arteries, in cultured smooth muscle cells (PA-SMCs), and in pulmonary endothelial cells (P-ECs) than in control subjects. These abnormalities in the p130(Cas) signaling were also found in the chronically hypoxic mice and monocrotaline-injected rats as models of human PAH. We obtained evidence for the convergence and amplification of the growth-stimulating effect of the EGF-, FGF2-, and PDGF-signaling pathways via the p130(Cas) signaling pathway. We found that daily treatment with the EGF-R inhibitor gefitinib, the FGF-R inhibitor dovitinib, and the PDGF-R inhibitor imatinib started 2 weeks after a subcutaneous monocrotaline injection substantially attenuated the abnormal increase in p130(Cas) and ERK1/2 activation and regressed established pulmonary hypertension. CONCLUSIONS Our findings demonstrate that p130(Cas) signaling plays a critical role in experimental and idiopathic PAH by modulating pulmonary vascular cell migration and proliferation and by acting as an amplifier of RTK downstream signals.

Emergence of inflammatory alveolar macrophages during rejection or infection after lung transplantation.

Local activation of macrophages may play an important role in immune complications following lung transplantation. To document such a phenomenon, we have investigated the possible changes of alveolar macrophage surface antigen expression after lung transplantation. Using immunocytofluorometry, we have analyzed the phenotype of alveolar macrophages from 41 bron-choalveolar lavage fluids obtained from 19 lung transplant recipients displaying various complications. The strong expression of HLA-DR observed on almost all alveolar macrophages was similar among groups I (no complication), II (minimal acute rejection), and III (mild to severe acute rejection), but was enhanced in group IV (bronchial infection) (P<0.03). We observed no significant variation in the monocyte lineage CD14 antigen expression among the 4 groups, and about 83% of alveolar macrophages expressed this marker strongly. Membrane expression of the 27E10 antigen that characterizes infiltrating macrophages in acute inflammatory lesions was significantly higher during mild to severe rejection episodes than in controls (P<0.02) and during bronchial infections (P<0.05) but not during minimal rejection. Double staining experiments confirmed that 27E10-positive cells in groups III and IV belonged to the macrophage lineage. In addition, the expression of the 27E10 antigen on cultured alveolar macrophages was found to be increased after stimulation by bacterial lipopolysaccharide or IFN-γ.

Impact of cyclosporine A on magnesium homeostasis: clinical observation in lung transplant recipients and experimental study in mice.

Background. Hypomagnesemia is a common finding in patients receiving cyclosporine A (CsA) therapy. The relationship between CsA-induced hypomagnesemia and nephrotoxicity and the effects of oral magnesium (Mg) supplementation remain unclear. After a retrospective analysis of the time-course of plasma Mg and creatinine levels in lung allograft recipients treated with both CsA and oral Mg supplementation, we investigated the effects of CsA treatment on Mg homeostasis in mice with normal or Mg-deficient diet and the effects of oral Mg supplementation on plasma Mg levels. Methods. Thirty lung-allograft recipients entered the retrospective study. One thousand two hundred twenty-eight blood samples were analyzed for blood and creatinine levels. Cyclosporine A (50 mg/kg/day by intraperitoneal injection) was administered to mice maintained on normal diet (1400 ppm) or Mg-deficient (50 ppm) diet. Magnesium levels were determined in plasma, urine, feces and femur, and creatinine levels were determined in plasma and urine. Results. Plasma Mg concentration declines from the day of transplantation in 36.7% of the patients despite Mg supplementation, without correlation with creatinine changes. In mice, CsA induced an early moderate hypomagnesemia, which could not be ameliorated by oral Mg supplementation and was aggravated by low-Mg dietary, late increase in plasma creatinine and decrease in urine creatinine without histological signs of renal injury, decrease in intestinal Mg absorption and Mg mobilization from bone. Conclusion. Cyclosporine A treatment may induce moderate hypomagnesemia that is aggravated by inadequate Mg intake and is not ameliorated by Mg supplementation. Because of the clinical complications of hypomagnesemia, Mg should be monitored regularly in allograft recipients receiving CsA.

Differential Effects of Cyclosporin A and Tacrolimus on Magnesium Influx in Caco2 Cells

PURPOSE Hypomagnesemia with urinary magnesium (Mg) wasting is a well acknowledged side effect of cyclosporin A (CsA) and tacrolimus (FK506) treatments. TRPM6, TRPM7 and MagT1 are involved in the active transcellular Mg transport processes in intestine and kidney. Since Mg homeostasis is tightly controlled by the dynamic action of intestinal absorption of dietary Mg and renal excretion of Mg, we investigated whether CsA and FK506 in commercially available solutions for clinical use decrease the expression and the function of TRPM6, TRPM7 or MagT1 in the intestinal epithelial cell line Caco2. METHODS Changes of intracellular free Mg concentrations were measured by Mag-fura-2 imaging in Mg-free medium after the addition of 1 mM MgCl2. TRPM6, TRPM7 and MagT1 were evidenced in cells by immunofluorescence. Proteins and mRNAs were quantified after 18 hours of treatment with CsA or FK506 by western-blot and real-time RT-PCR analyses, respectively. RESULTS TRPM6 and MagT1 were evidenced on all cell membranes, TRPM7 only on the inner membranes. CsA was responsible for a profound decrease in Mg2+ influx in intestinal epithelial cells, which may result in a decrease of intestinal Mg absorption, whereas FK506 was responsible for a marked increase in Mg2+ influx. Neither CsA nor FK506 altered TRPM6, TRPM7 or MagT1 mRNA levels or MagT1 protein level. CONCLUSIONS In Caco2 cells, Mg2+ influx was inhibited by CsA solutions whereas enhanced by FK506 solutions, without alteration of MagT1, TRPM6 and TRPM7 expression, leading to the conclusion that CsA and FK506 have opposite effects in the functional activity of the Mg transporters herein examined. In clinical use, FK506 should be preferred for patients at risk for hypomagnesemia.

The Beneficial Effect of Suramin on Monocrotaline-Induced Pulmonary Hypertension in Rats

Background Pulmonary hypertension (PH) is a progressive disorder characterized by an increase in pulmonary artery pressure and structural changes in the pulmonary vasculature. Several observations indicate that growth factors play a key role in PH by modulating pulmonary artery smooth muscle cell (PA-SMC) function. In rats, established monocrotaline-induced PH (MCT-PH) can be reversed by blocking platelet-derived growth factor receptors (PDGF-R), epidermal growth factor receptors (EGF-R), or fibroblast growth factor receptors (FGF-R). All these receptors belong to the receptor tyrosine kinase (RTK) family. Methods and Results We evaluated whether RTK blockade by the nonspecific growth factor inhibitor, suramin, reversed advanced MCT-PH in rats via its effects on growth-factor signaling pathways. We found that suramin inhibited RTK and ERK1/2 phosphorylation in cultured human PA-SMCs. Suramin inhibited PA-SMC proliferation induced by serum, PDGF, FGF2, or EGF in vitro and ex vivo. Treatment with suramin from day 1 to day 21 after monocrotaline injection attenuated PH development, as shown by lower values for pulmonary artery pressure, right ventricular hypertrophy, and distal vessel muscularization on day 21 compared to control rats. Treatment with suramin from day 21 to day 42 after monocrotaline injection reversed established PH, thereby normalizing the pulmonary artery pressure values and vessel structure. Suramin treatment suppressed PA-SMC proliferation and attenuated both the inflammatory response and the deposition of collagen. Conclusions RTK blockade by suramin can prevent MCT-PH and reverse established MCT-PH in rats. This study suggests that an anti-RTK strategy that targets multiple RTKs could be useful in the treatment of pulmonary hypertension.

Neuroprotective gene profile in the brain of magnesium-deficient mice.

BACKGROUND Magnesium (Mg) deficiency may lead to serious metabolic, biological and organic dysfunctions, and cause various clinical disorders. In the current study, we explore endothelial cell activation, inflammation and cell death induced in the brain of adult mice by Mg-deficient diet. METHODS AND RESULTS Neither TNFalpha, substance P, sTNFRI, sTNFRII proteins (ELISA), nor TNFalpha, adherence molecules and prolactin mRNAs, nor NK1R (immunohistochemistry on brain sections) were up-regulated. No inflammatory infiltrates and no apoptotic cells were observed. Using cDNA assay, we showed a neuroprotective, anti-apoptotic and neurotrophic gene expression profile in the brain at early stage of hypomagnesemia. As a model for neuronal injury, mild sound stimulation of Mg-deficient mice without convulsive seizures triggers neither the release of substance P, nor the development of an inflammatory process or cell death in the brain. CONCLUSION Our results suggest that Mg-deficiency in mice favours the development of a neuroprotective environment in the brain.

Telomere Maintenance Is a Critical Determinant in the Physiopathology of Pulmonary Hypertension.

Idiopathic pulmonary arterial hypertension (iPAH) is a rare disease that occurs sporadically and in which pulmonary arterial pressure elevation leads to right heart failure and death. Although the fundamental causes remain elusive, vascular remodeling due to increased proliferation of pulmonary

Effect of hypomagnesemia on allogeneic activation in mice

INTRODUCTION Magnesium (Mg) plays an essential role in a wide range of fundamental cellular reactions. It has been reported that in rodents Mg-deficient diet-induced hypomagnesemia results in an early inflammation. We have previously shown that chronic severe hypomagnesemia was associated neither with endothelial cell activation nor with an inflammatory process which are crucial in the allograft rejection process. T cell allogeneic stimulation activates the phosphatase calcineurin which triggers the signaling pathways leading to IL-2 synthesis and lymphocyte proliferation. Full activation of calcineurin requires Mg. Surveys suggest that a significant number of people consume less Mg than the international dietary reference intakes leading to hypomagnesemia in 2.5% to 15% of the general population. OBJECTIVE The aim of the study was to investigate the effects of hypomagnesemia on lymphocyte allogeneic activation and proliferation in a murine model of dietary-induced hypomagnesemia. METHODS C57BL/6J (H-2(b), Mls(b)) mice were given normal Mg-containing diet (1400 ppm Mg, control mice), or synthetic Mg-deficient diets containing either 50 ppm Mg or 150 ppm Mg for 28 days. Serum Mg levels were determined at days 5, 14 and 28. In parallel, complete urine and faeces were collected by using metabolic cages during a 24 h period for Mg determinations. Splenocytes from C57BL/6 mice fed either normal diet or 50 ppm Mg-diet were used as responder cells in mixed lymphocyte reaction (MLR) performed with splenocytes from C3H/He mice (H-2(k), Mls(IIa)) and C57BL/6 mice fed normal diet as stimulators for allogeneic and isogeneic conditions, respectively. TGF-beta and IL-2 productions were quantified in the supernates of mixed splenocytes cultures. 3x10(6) splenocytes from mice fed 50 ppm Mg-diet were used for calcineurin activity determination at day 28. RESULTS In mice fed 150 ppm Mg-diet, moderate hypomagnesemia was observed from day 5 to day 28. Oral supplementation with Mg pidolate (5 or 20 mg Mg/kg/day) could not restore normal serum Mg levels. Serum Mg concentration early decreased in mice fed 50 ppm Mg-diet to achieve stabilized severe hypomagnesemia at days 14 and 28. Urine Mg concentration early dramatically fell down then stabilized in mice fed Mg-deficient diets. In MLR performed at day 28 with splenocytes from mice fed 50 ppm Mg-diet, proliferation and IL-2 production in allogeneic conditions were similar to control mice. No TGF-beta production was detected in any group. Lastly, calcineurin activity measured at day 28 was significantly lower in splenocyes from mice fed 50 ppm Mg-diet than in mice fed control diet. CONCLUSION Mg-deficiency does not alter splenocyte allogeneic activation and proliferation and IL-2 production in vitro, although it partially inhibits calcineurin activity. We hypothesize that the remaining activity is sufficient for IL-2 gene normal activation. Alternatively, Mg-deficiency may trigger other signaling pathways leading to IL-2 production.