Allogeneic Hematopoietic Stem Cell Transplantation (alloHSCT) Following Blinatumomab-Induced Remission in Pediatric Patients with Relapsed/Refractory (r/r) B-Precursor Acute Lymphoblastic Leukemia (ALL): Preliminary Results from a Phase I/II Stud

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Role of the TGF-b/Alk1 pathway in human and experimental pulmonary arterial hypertension (PAH)

Mon projet porte d'une part sur le rôle de la voie TGF-b/Alk1 dans l’hypertension artérielle (HTAP) humaine et expérimentale. Le but est d’évaluer in vitro (i) l’expression du TGF-b et de ses récepteurs ALK1/Endogline dans les cellules endothéliales d’artères pulmonaires (CE-AP) de patients atteints d’HTAP idiopathique (HTAPi), (ii) les conséquence de l’activation de la voie TGF-b/ALK1 des CE-AP dans la synthèse de facteurs capables d’induire la prolifération des cellules musculaires lisses d’artères pulmonaires (CML-AP), (iii) identifier par une analyse protéomique différentielle la nature de ses facteurs paracrines, (iv) évaluer chez la souris la conséquence de la déficience en Endogline, co-récepteur de ALK1 sur le développement de l’hypertension artérielle pulmonaire.My project relates to the role of the TGF-b/Alk1 pathway in human and experimental pulmobnary arterial hypertension (PAH). The goal is to evaluate in vitro (I) the expression of TGF-b and its receptors ALK1/Endoglin in pulmonary arterial endothelial (P-EC) of patients reached of idiopathic PAH (iPAH), (II) the consequence of the activation of the TGF-b/ALK1 pathway on the P-EC in the synthesis of factors able to induce the proliferation of the pulmonary arterial smooth muscle cell (PA-SMC), (III) to identify by a differential proteomic analysis the nature of its factors paracrines, (iv) to evaluate in the mouse the consequence of deficiency in Endoglin (co-receptor of ALK1) on the development of PAH

Physiopathologie de l'hypertension artérielle pulmonaire (rôles de la voie de signalisation TGF-b/ALK1/Endogline et de p53)

Mon projet porte d'une part sur le rôle de la voie TGF-b/Alk1 dans l hypertension artérielle (HTAP) humaine et expérimentale. Le but est d évaluer in vitro (i) l expression du TGF-b et de ses récepteurs ALK1/Endogline dans les cellules endothéliales d artères pulmonaires (CE-AP) de patients atteints d HTAP idiopathique (HTAPi), (ii) les conséquence de l activation de la voie TGF-b/ALK1 des CE-AP dans la synthèse de facteurs capables d induire la prolifération des cellules musculaires lisses d artères pulmonaires (CML-AP), (iii) identifier par une analyse protéomique différentielle la nature de ses facteurs paracrines, (iv) évaluer chez la souris la conséquence de la déficience en Endogline, co-récepteur de ALK1 sur le développement de l hypertension artérielle pulmonaire.My project relates to the role of the TGF-b/Alk1 pathway in human and experimental pulmobnary arterial hypertension (PAH). The goal is to evaluate in vitro (I) the expression of TGF-b and its receptors ALK1/Endoglin in pulmonary arterial endothelial (P-EC) of patients reached of idiopathic PAH (iPAH), (II) the consequence of the activation of the TGF-b/ALK1 pathway on the P-EC in the synthesis of factors able to induce the proliferation of the pulmonary arterial smooth muscle cell (PA-SMC), (III) to identify by a differential proteomic analysis the nature of its factors paracrines, (iv) to evaluate in the mouse the consequence of deficiency in Endoglin (co-receptor of ALK1) on the development of PAH.PARIS-EST-Université (770839901) / SudocSudocFranceF

Figure 5

<p>Mouse lung PECAM-1 immunostaining (<b>A</b>). (<b>B</b>) Number of vessels per 100 alveoli. (<b>C</b>) Mouse lung F4/80 immunostaining. (<b>D</b>) Number of macrophages per 100 alveoli. Values are mean±SEM. *<i>P</i><0.02 compared with wild-type mice exposed to normoxia and ^σ<i>P</i><0.02 compared with wild-type mice exposed to hypoxia.</p

Key Role of the Endothelial TGF-β/ALK1/Endoglin Signaling Pathway in Humans and Rodents Pulmonary Hypertension

<div><p>Mutations affecting transforming growth factor-beta (TGF-β) superfamily receptors, activin receptor-like kinase (ALK)-1, and endoglin (ENG) occur in patients with pulmonary arterial hypertension (PAH). To determine whether the TGF-β/ALK1/ENG pathway was involved in PAH, we investigated pulmonary TGF-β, ALK1, ALK5, and ENG expressions in human lung tissue and cultured pulmonary-artery smooth-muscle-cells (PA-SMCs) and pulmonary endothelial cells (PECs) from 14 patients with idiopathic PAH (iPAH) and 15 controls. Seeing that ENG was highly expressed in PEC, we assessed the effects of TGF-β on Smad1/5/8 and Smad2/3 activation and on growth factor production by the cells. Finally, we studied the consequence of ENG deficiency on the chronic hypoxic-PH development by measuring right ventricular (RV) systolic pressure (RVSP), RV hypertrophy, and pulmonary arteriolar remodeling in ENG-deficient (<i>Eng^+/−</i>) and wild-type (<i>Eng^+/+</i>) mice. We also evaluated the pulmonary blood vessel density, macrophage infiltration, and cytokine expression in the lungs of the animals. Compared to controls, iPAH patients had higher serum and pulmonary TGF-β levels and increased ALK1 and ENG expressions in lung tissue, predominantly in PECs. Incubation of the cells with TGF-β led to Smad1/5/8 phosphorylation and to a production of FGF2, PDGFb and endothelin-inducing PA-SMC growth. Endoglin deficiency protected mice from hypoxic PH. As compared to wild-type, <i>Eng^+/−</i> mice had a lower pulmonary vessel density, and no change in macrophage infiltration after exposure to chronic hypoxia despite the higher pulmonary expressions of interleukin-6 and monocyte chemoattractant protein-1. The TGF-β/ALK1/ENG signaling pathway plays a key role in iPAH and experimental hypoxic PH via a direct effect on PECs leading to production of growth factors and inflammatory cytokines involved in the pathogenesis of PAH.</p></div

Figure 2

<p>Effect of increasing TGF-β doses on Smad 1,5,8 and Smad 2,3 phosphorylation, respectively, in pulmonary endothelial cells (PECs) from controls and from patients with idiopathic pulmonary hypertension (iPAH) (<b>A</b> and <b>B</b>). Protein levels were normalized for β-actin. Values are means±SEM normalized for results without TGF-β. *<i>P</i><0.05 compared to relevant controls without TGF-β, ^§<i>P</i><0.05 compared to control PECs under the same conditions. (<b>C</b>) Growth of pulmonary-artery smooth-muscle cells (PA-SMCs) from controls in response to serum-free media derived from cultured PECs from controls and stimulated by TGF-β with or without anti-ENG antibody (ENG Ab).Values are mean±SEM. *<i>P</i><0.05 compared to basal condition, ^§<i>P</i><0.05 compared to PA-SMCs stimulated with PEC medium.</p

Effect of pulmonary endothelial cells (PECs) from controls incubated with TGF-β with or without anti-ENG antibody (ENG Ab) on mRNA expression of A) <i>preproET-1</i>, (B) <i>PDGFa</i>, (C) <i>PDGFb</i>, (D) <i>FGF2</i>, (E) <i>EGF</i>, (F) <i>MCP-1</i>, and (G) <i>IL-6</i>.

<p>Values are mean±SEM.*<i>P</i><0.05 compared to PECs without TGF-β treatment.</p

Figure 5

<p>Mouse lung PECAM-1 immunostaining (<b>A</b>). (<b>B</b>) Number of vessels per 100 alveoli. (<b>C</b>) Mouse lung F4/80 immunostaining. (<b>D</b>) Number of macrophages per 100 alveoli. Values are mean±SEM. *<i>P</i><0.02 compared with wild-type mice exposed to normoxia and ^σ<i>P</i><0.02 compared with wild-type mice exposed to hypoxia.</p

The open bars indicate the results in controls and the closed bars in patients with idiopathic pulmonary hypertension (iPAH).

<p>TGF-β was assayed using an ELISA in serum and lung homogenates (controls, n = 15; iPAH patients, n = 14) (<b>A</b> and <b>B</b>). (<b>C</b>) TGF-β mRNA measured in pulmonary endothelial cells (PECs) and pulmonary-artery smooth-muscle cells (PA-SMCs; controls, n = 7; iPAH patients, n = 7). (<b>D</b>) TGF-β receptor expression: <i>ALK1, ALK5</i>, and <i>ENG</i> mRNA measured in PECs and PA-SMCs (controls, n = 7; iPAH patients, n = 7). (<b>E</b>): ENG, ALK1, and ALK5 protein expression in LUNG, PECs and PA-SMCs. Protein levels were normalized for β-actin (controls, n = 7; iPAH patients, n = 7). Values are mean±SEM. *<i>P</i><0.05 and **<i>P</i><0.01 compared with controls.</p