Consequences of GCN2 deficiency in murine and human : implications in the pathophysiology of pulmonary veno-occlusive disease

La maladie veino-occlusive pulmonaire (MVOP) est une forme rare d’hypertension pulmonaire. La forme héréditaire de la maladie (MVOPh) est liée à des mutations bialléliques du gène EIF2AK4. Ces mutations conduisent à la perte de fonction de la protéine GCN2. A ce jour, les mécanismes physiopathologiques de la MVOPh liés à la perte de fonction de GCN2 sont à élucider. L’objectif de cette thèse est d’explorer les mécanismes moléculaires et cellulaires mis en place chez le rat Gcn2-/- déclenchés par un stress métabolique et chez le patient atteint de MVOPh. Ce travail établit le rôle essentiel de la protéine GCN2 dans le maintien de l’homéostasie et la protection contre l’inflammation pulmonaire lors d’une carence en glutamine et asparagine chez le rat. Une étude cellulaire a permis de mettre en évidence la présence d’une population de lymphocyte T caractérisée par une expression accrue de LAG3 unique aux rats Gcn2-/- en condition basale. En parallèle, nous avons exploré les similitudes et les différences des transcriptomes de patients atteints d’hypertension artérielle pulmonaire idiopathique (HTAPi) et ceux souffrant de MVOPh. Nous montrons que la baisse de l’inflammation est un processus dicriminant la MVOPh de l’HTAPi. En résumé, ce travail renforce le statut protecteur de GCN2 contre l’inflammation lors d’une carence en acide aminé et révèle un nouveau rôle dans la régulation du système immunitaire adaptatif en condition basale. Les différences entre la MVOP et l’HTAP ouvrent de nouvelles perspectives permettant de différencier les deux maladies tandis que les similitudes aident à mieux comprendre les mécanismes du développement de la MVOPh.Pulmonary veno-occlusive disease (PVOD) is a rare form of pulmonary hypertension. The hereditary form of the disease (hPVOD) is linked to biallelic mutations in the EIF2AK4 gene. These mutations lead to loss of function of the GCN2 protein. To date, the pathophysiological mechanisms of hPVOD related to the loss of function of GCN2 remain to be elucidated. The objective of this thesis is to explore the molecular and cellular mechanisms in the Gcn2-/- rat triggered by metabolic stress and in the hPVOD patient. This work establishes the essential role of GCN2 protein in the maintenance of homeostasis and protection against pulmonary inflammation during glutamine and asparagine deficiency in rats. A cellular study revealed the presence of a T cell population characterized by an increased expression of LAG3 unique to Gcn2-/- rats in basal condition. In parallel, we explored the similarities and differences in the transcriptomes of patients with idiopathic pulmonary arterial hypertension (iPAH) and those with hPVOD. We show that decreased inflammation is a process that dicriminates hPVOD from iPAH. In summary, this work reinforces the protective status of GCN2 against inflammation during amino acid deficiency and reveals a novel role in the regulation of the adaptive immune system under basal conditions. The differences between PVOD and PAH provide new insights to differentiate the two diseases, while the similarities help to better understand the mechanisms of hPVOD development

Conséquences du déficit en GCN2 chez le murin et l’Homme : implications en physiopathologie de la maladie veino-occlusive pulmonaire

Pulmonary veno-occlusive disease (PVOD) is a rare form of pulmonary hypertension. The hereditary form of the disease (hPVOD) is linked to biallelic mutations in the EIF2AK4 gene. These mutations lead to loss of function of the GCN2 protein. To date, the pathophysiological mechanisms of hPVOD related to the loss of function of GCN2 remain to be elucidated. The objective of this thesis is to explore the molecular and cellular mechanisms in the Gcn2-/- rat triggered by metabolic stress and in the hPVOD patient. This work establishes the essential role of GCN2 protein in the maintenance of homeostasis and protection against pulmonary inflammation during glutamine and asparagine deficiency in rats. A cellular study revealed the presence of a T cell population characterized by an increased expression of LAG3 unique to Gcn2-/- rats in basal condition. In parallel, we explored the similarities and differences in the transcriptomes of patients with idiopathic pulmonary arterial hypertension (iPAH) and those with hPVOD. We show that decreased inflammation is a process that dicriminates hPVOD from iPAH. In summary, this work reinforces the protective status of GCN2 against inflammation during amino acid deficiency and reveals a novel role in the regulation of the adaptive immune system under basal conditions. The differences between PVOD and PAH provide new insights to differentiate the two diseases, while the similarities help to better understand the mechanisms of hPVOD development.La maladie veino-occlusive pulmonaire (MVOP) est une forme rare d’hypertension pulmonaire. La forme héréditaire de la maladie (MVOPh) est liée à des mutations bialléliques du gène EIF2AK4. Ces mutations conduisent à la perte de fonction de la protéine GCN2. A ce jour, les mécanismes physiopathologiques de la MVOPh liés à la perte de fonction de GCN2 sont à élucider. L’objectif de cette thèse est d’explorer les mécanismes moléculaires et cellulaires mis en place chez le rat Gcn2-/- déclenchés par un stress métabolique et chez le patient atteint de MVOPh. Ce travail établit le rôle essentiel de la protéine GCN2 dans le maintien de l’homéostasie et la protection contre l’inflammation pulmonaire lors d’une carence en glutamine et asparagine chez le rat. Une étude cellulaire a permis de mettre en évidence la présence d’une population de lymphocyte T caractérisée par une expression accrue de LAG3 unique aux rats Gcn2-/- en condition basale. En parallèle, nous avons exploré les similitudes et les différences des transcriptomes de patients atteints d’hypertension artérielle pulmonaire idiopathique (HTAPi) et ceux souffrant de MVOPh. Nous montrons que la baisse de l’inflammation est un processus dicriminant la MVOPh de l’HTAPi. En résumé, ce travail renforce le statut protecteur de GCN2 contre l’inflammation lors d’une carence en acide aminé et révèle un nouveau rôle dans la régulation du système immunitaire adaptatif en condition basale. Les différences entre la MVOP et l’HTAP ouvrent de nouvelles perspectives permettant de différencier les deux maladies tandis que les similitudes aident à mieux comprendre les mécanismes du développement de la MVOPh

Conséquences du déficit en GCN2 chez le murin et l’Homme : implications en physiopathologie de la maladie veino-occlusive pulmonaire

Pulmonary veno-occlusive disease (PVOD) is a rare form of pulmonary hypertension. The hereditary form of the disease (hPVOD) is linked to biallelic mutations in the EIF2AK4 gene. These mutations lead to loss of function of the GCN2 protein. To date, the pathophysiological mechanisms of hPVOD related to the loss of function of GCN2 remain to be elucidated. The objective of this thesis is to explore the molecular and cellular mechanisms in the Gcn2-/- rat triggered by metabolic stress and in the hPVOD patient. This work establishes the essential role of GCN2 protein in the maintenance of homeostasis and protection against pulmonary inflammation during glutamine and asparagine deficiency in rats. A cellular study revealed the presence of a T cell population characterized by an increased expression of LAG3 unique to Gcn2-/- rats in basal condition. In parallel, we explored the similarities and differences in the transcriptomes of patients with idiopathic pulmonary arterial hypertension (iPAH) and those with hPVOD. We show that decreased inflammation is a process that dicriminates hPVOD from iPAH. In summary, this work reinforces the protective status of GCN2 against inflammation during amino acid deficiency and reveals a novel role in the regulation of the adaptive immune system under basal conditions. The differences between PVOD and PAH provide new insights to differentiate the two diseases, while the similarities help to better understand the mechanisms of hPVOD development.La maladie veino-occlusive pulmonaire (MVOP) est une forme rare d’hypertension pulmonaire. La forme héréditaire de la maladie (MVOPh) est liée à des mutations bialléliques du gène EIF2AK4. Ces mutations conduisent à la perte de fonction de la protéine GCN2. A ce jour, les mécanismes physiopathologiques de la MVOPh liés à la perte de fonction de GCN2 sont à élucider. L’objectif de cette thèse est d’explorer les mécanismes moléculaires et cellulaires mis en place chez le rat Gcn2-/- déclenchés par un stress métabolique et chez le patient atteint de MVOPh. Ce travail établit le rôle essentiel de la protéine GCN2 dans le maintien de l’homéostasie et la protection contre l’inflammation pulmonaire lors d’une carence en glutamine et asparagine chez le rat. Une étude cellulaire a permis de mettre en évidence la présence d’une population de lymphocyte T caractérisée par une expression accrue de LAG3 unique aux rats Gcn2-/- en condition basale. En parallèle, nous avons exploré les similitudes et les différences des transcriptomes de patients atteints d’hypertension artérielle pulmonaire idiopathique (HTAPi) et ceux souffrant de MVOPh. Nous montrons que la baisse de l’inflammation est un processus dicriminant la MVOPh de l’HTAPi. En résumé, ce travail renforce le statut protecteur de GCN2 contre l’inflammation lors d’une carence en acide aminé et révèle un nouveau rôle dans la régulation du système immunitaire adaptatif en condition basale. Les différences entre la MVOP et l’HTAP ouvrent de nouvelles perspectives permettant de différencier les deux maladies tandis que les similitudes aident à mieux comprendre les mécanismes du développement de la MVOPh

Progenitor/Stem Cells in Vascular Remodeling during Pulmonary Arterial Hypertension

International audiencePulmonary arterial hypertension (PAH) is characterized by an important occlusive vascular remodeling with the production of new endothelial cells, smooth muscle cells, myofibroblasts, and fibroblasts. Identifying the cellular processes leading to vascular proliferation and dysfunction is a major goal in order to decipher the mechanisms leading to PAH development. In addition to in situ proliferation of vascular cells, studies from the past 20 years have unveiled the role of circulating and resident vascular in pulmonary vascular remodeling. This review aims at summarizing the current knowledge on the different progenitor and stem cells that have been shown to participate in pulmonary vascular lesions and on the pathways regulating their recruitment during PAH. Finally, this review also addresses the therapeutic potential of circulating endothelial progenitor cells and mesenchymal stem cells

From islet of Langerhans transplantation to the bioartificial pancreas

Type 1 diabetes is a disease resulting from autoimmune destruction of the insulin-producing beta cells in the pancreas. When type 1 diabetes develops into severe secondary complications, in particular end-stage nephropathy, or life-threatening severe hypoglycemia, the best therapeutic approach is pancreas transplantation, or more recently transplantation of the pancreatic islets of Langerhans. Islet transplantation is a cell therapy procedure, that is minimally invasive and has a low morbidity, but does not display the same rate of functional success as the more invasive pancreas transplantation because of suboptimal engraftment and survival. Another issue is that pancreas or islet transplantation (collectively known as beta cell replacement therapy) is limited by the shortage of organ donors and by the need for lifelong immunosuppression to prevent immune rejection and recurrence of autoimmunity. A bioartificial pancreas is a construct made of functional, insulin-producing tissue, embedded in an anti-inflammatory, immunomodulatory microenvironment and encapsulated in a perm-selective membrane allowing glucose sensing and insulin release, but isolating from attacks by cells of the immune system. A successful bioartificial pancreas would address the issues of engraftment, survival and rejection. Inclusion of unlimited sources of insulin-producing cells, such as xenogeneic porcine islets or stem cell-derived beta cells would further solve the problem of organ shortage. This article reviews the current status of clinical islet transplantation, the strategies aiming at developing a bioartificial pancreas, the clinical trials conducted in the field and the perspectives for further progress.</p

T-cell dysregulation and inflammatory process in Gcn2 ( Eif2ak4 −/− ) deficient rats in basal and stress conditions

International audienceHereditary pulmonary veno-occlusive disease (hPVOD) is a severe form of autosomal recessive pulmonary hypertension and is due to biallelic loss of function of the EIF2AK4 gene (alias GCN2) coding for GCN2. GCN2 is a stress kinase that belongs to the integrated stress response pathway (ISR). Three rat lines carrying biallelic Gcn2 mutation were generated and found phenotypically normal and did not spontaneously develop a PVOD-related disease. We submitted these rats to amino acid deprivation to document the molecular and cellular response of the lungs and to identify phenotypic changes that could be involved in PVOD pathophysiology. Gcn2 −/− rat lungs were analyzed under basal conditions and 3 days after a single administration of PEG-asparaginase (ASNase). Lung mRNAs were analyzed by RNAseq and single-cell RNAseq (scRNA-seq), flow cytometry, tissue imaging, and Western blots. The ISR was not activated after ASNase treatment in Gcn2 −/− rat lungs, and apoptosis was increased. Several proinflammatory and innate immunity genes were overexpressed, and inflammatory cells infiltration was also observed in the perivascular area. Under basal conditions, scRNA-seq analysis of Gcn2 −/− rat lungs revealed increases in two T-cell populations, a LAG3 + T-cell population and a proliferative T-cell population. Following ASNase administration, we observed an increase in calprotectin expression involved in TLR pathway activation and neutrophil infiltration. In conclusion, under basal and asparagine and glutamine deprivation induced by asparaginase administration, Gcn2 −/− rats display molecular and cellular signatures in the lungs that may indicate a role for Gcn2 in immune homeostasis and provide further clues to the mechanisms of hPVOD development

Intercellular contacts affect secretion and biosynthesis of pancreatic islet cells

Cell protein biosynthesis is regulated by different factors, but implication of intercellular contacts on alpha and beta cell protein biosynthesis activity has not been yet investigated. Islet cell biosynthetic activity is essential in regulating not only the hormonal reserve within cells but also in renewing all the proteins involved in the control of secretion. Here we aimed to assess whether intercellular interactions affected similarly secretion and protein biosynthesis of rat alpha and beta cells. Insulin and glucagon secretion were analyzed by ELISA or reverse hemolytic plaque assay, and protein biosynthesis evaluated at single cell level using bioorthogonal noncanonical amino acid tagging. Regarding beta cells, we showed a positive correlation between insulin secretion and protein biosynthesis. We also observed that homologous contacts increased both activities at low or moderate glucose concentrations. By contrast, at high glucose concentration, homologous contacts increased insulin secretion and not protein biosynthesis. In addition, heterogeneous contacts between beta and alpha cells had no impact on insulin secretion and protein biosynthesis. Regarding alpha cells, we showed that when they were in contact with beta cells, they increased their glucagon secretion in response to a drop of glucose concentration but, on the other hand, they decreased their protein biosynthesis under any glucose concentrations. All together, these results emphasize the role of intercellular contacts on the function of islet cells, showing that intercellular contacts increased protein biosynthesis in beta cells, except at high glucose, and decreased protein biosynthesis in alpha cells even when glucagon secretion is stimulated

Disruption of GCN2 Pathway Aggravates Vascular and Parenchymal Remodeling During Pulmonary Fibrosis.

Pulmonary fibrosis (PF) and pulmonary hypertension (PH) are chronic diseases of the pulmonary parenchyma and circulation, respectively, which may coexist, but underlying mechanisms remain elusive. Mutations in the GCN2 gene (EIF2AK4) were recently associated with pulmonary veno-occlusive disease. This study aims to explore the involvement of the GCN2/eIF2α pathway in the development of PH during PF, in both human disease and in an experimental animal model. Lung tissue from PF patients with or without PH were collected at the time of lung transplantation, and controls were obtained from tumor resection surgery. Experimental lung disease was induced in either male wild-type or EIF2AK4-mutated Sprague-Dawley rats, randomly receiving a single intratracheal instillation of bleomycin or saline. Hemodynamic studies, as well as organ collection were performed 3 weeks post-instillation. Only significant results (p<0.05) are given. In PF lung tissue, GCN2 protein expression was decreased, when compared with controls. GCN2 expression was reduced in CD31+ endothelial cells. In line with human data, GCN2 protein expression was decreased in the lung of bleomycin rats when compared with saline. EIF2AK4-mutated rats treated with bleomycin showed increased parenchymal fibrosis (hydroxyproline levels) and vascular remodeling (media wall thickness) as well as increased right ventricular systolic pressure when compared to wild-type animals. Our data shows that GCN2 is dysregulated in both human and in an animal model of combined PF+PH. The possibility of a causative implication of GCN2 dysregulation in PF and/or PH development should be further studied

Comparison of Human and Experimental Pulmonary Veno-Occlusive Disease

International audiencePulmonary veno-occlusive disease (PVOD) occurs in humans either as heritable form (hPVOD) due to biallelic inactivating mutations of EIF2AK4 (encoding GCN2), or as a sporadic form at older age (sPVOD). The chemotherapeutic agent Mitomycin C is a potent inducer of PVOD in humans and in rats (MMC-PVOD). Here we compared human hPVOD and sPVOD, and MMC-PVOD pathophysiology at the histological, cellular and molecular levels to unravel common altered pathomechanisms. MMC-exposure in rats was primarily associated with arterial and microvessels remodeling and secondarily followed by venous remodeling, when PVOD became symptomatic. In all forms of PVOD tested, there were convergent GCN2-dependent but eIF2α-independent pulmonary protein overexpression of heme oxygenase 1 (HO-1) and CCAAT-enhancer-binding protein (C/EBP) homologous protein (CHOP), two downstream effectors of GCN2 signaling and endoplasmic reticulum (ER) stress. In human PVOD samples, CHOP immunohistochemical staining mainly labeled endothelial cells in remodeled veins and arteries. Strong HO-1 staining was observed only within capillary hemangiomatosis foci, where intense microvascular proliferation occurs. HO-1 and CHOP stainings were not observed in control and pulmonary arterial hypertension lung tissues, supporting the specificity for CHOP and HO-1 involvement in PVOD pathobiology. In vivo loss of GCN2 (EIF2AK4 mutations carriers and Eif2ak4-/- rats) or in vitro GCN2 inhibition in cultured pulmonary artery endothelial cells (PAECs) using pharmacological and siRNA approaches demonstrated that GCN2 loss-of-function negatively regulates BMP-dependent SMAD1/5/9 signaling. Exogenous BMP9 was still able to reverse GCN2 inhibition-induced PAECs proliferation. In conclusion, we identified CHOP and HO-1 inhibition, and BMP9 as potential therapeutic options for PVOD

Comparison of Human and Experimental Pulmonary Veno-Occlusive Disease

Pulmonary veno-occlusive disease (PVOD) occurs in humans either as a heritable form (hPVOD) due to biallelic inactivating mutations of EIF2AK4 (encoding GCN2) or as a sporadic form in older age (sPVOD). The chemotherapeutic agent mitomycin C (MMC) is a potent inducer of PVOD in humans and in rats (MMC-PVOD). Here, we compared human hPVOD and sPVOD, and MMC-PVOD pathophysiology at the histological, cellular, and molecular levels to unravel common altered pathomechanisms. MMC exposure in rats was associated primarily with arterial and microvessel remodeling, and secondarily by venous remodeling, when PVOD became symptomatic. In all forms of PVOD tested, there was convergent GCN2-dependent but eIF2α-independent pulmonary protein overexpression of HO-1 (heme oxygenase 1) and CHOP (CCAAT-enhancer-binding protein [C/EBP] homologous protein), two downstream effectors of GCN2 signaling and endoplasmic reticulum stress. In human PVOD samples, CHOP immunohistochemical staining mainly labeled endothelial cells in remodeled veins and arteries. Strong HO-1 staining was observed only within capillary hemangiomatosis foci, where intense microvascular proliferation occurs. HO-1 and CHOP stainings were not observed in control and pulmonary arterial hypertension lung tissues, supporting the specificity for CHOP and HO-1 involvement in PVOD pathobiology. In vivo loss of GCN2 (EIF2AK4 mutations carriers and Eif2ak4 -/- rats) or in vitro GCN2 inhibition in cultured pulmonary artery endothelial cells using pharmacological and siRNA approaches demonstrated that GCN2 loss of function negatively regulates BMP (bone morphogenetic protein)-dependent SMAD1/5/9 signaling. Exogenous BMP9 was still able to reverse GCN2 inhibition-induced proliferation of pulmonary artery endothelial cells. In conclusion, we identified CHOP and HO-1 inhibition, and BMP9, as potential therapeutic options for PVOD.status: publishe