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.

Pulmonary arterial hypertension (PAH) is a lethal disease characterized by excessive proliferation of pulmonary vascular cells, such as endothelial cells (ECs). Hereditary (H) PAH is mainly caused by ―loss-of-function‖ mutations in the gene coding for the bone morphogenetic protein type II receptor (BMPR2). However, the mechanisms by which these mutations cause PAH remain unclear. The hypothesis of this thesis was that BMPR2 mutations produce an imbalance in EC protein expression and/or activity that is integrally related to the development of abnormalities in lung vascular function and structure in HPAH. Patient-specific blood-outgrowth endothelial cells (BOECs) expanded ex vivo from peripheral blood mononuclear cells from patients with HPAH and healthy subjects were used to examine the consequences of BMPR2 mutations on the BOEC protein expression profile as well as on their functionality. Functional analyses of the BOECs revealed that HPAH-derived BOECs are more susceptible to apoptosis and more proliferative compared with healthy controls. Protein isolates of BOECs from patients with HPAH and from healthy subjects were subjected to 2-D gel electrophoresis and stained for total proteins and phosphoproteins, and to a quantitative computerassisted analysis. Differentially regulated proteins were identified by mass spectrometry (LC-MS/MS). Of the 416 total proteins detected under basal conditions, 11 were significantly downregulated in HPAH-derived BOECs and 11, including the translationally controlled tumor protein (TCTP), were upregulated. TCTP has previously been shown to be involved in systemic arteriolar remodeling, inflammation and growth. Therefore, the potential role of TCTP in PAH was studied in vivo in the SU5416 rat model of severe angioproliferative PAH. Immunofluorescence staining revealed high expression of TCTP in arteriolar ECs of PAH lungs tightly localized to proliferating cells within occlusive intimal lesions; whereas, only minimal TCTP expression was seen in vascular ECs of normal lungs. Similarly, abundant TCTP immunostaining was also seen in human PAH lung sections, again associated with complex vascular lesions. In BOECs, TCTP was found to participate in cell growth and survival. These data suggest that TCTP could play an important role in PAH by mediating pro-survival and growth signaling in vascular cells, contributing to occlusive pulmonary vascular remodeling triggered by EC apoptosis.

Pulmonary arterial hypertension (PAH) is a lethal disease characterized by excessive proliferation of pulmonary vascular cells, such as endothelial cells (ECs). Hereditary (H) PAH is mainly caused by ―loss-of-function‖ mutations in the gene coding for the bone morphogenetic protein type II receptor (BMPR2). However, the mechanisms by which these mutations cause PAH remain unclear. The hypothesis of this thesis was that BMPR2 mutations produce an imbalance in EC protein expression and/or activity that is integrally related to the development of abnormalities in lung vascular function and structure in HPAH. Patient-specific blood-outgrowth endothelial cells (BOECs) expanded ex vivo from peripheral blood mononuclear cells from patients with HPAH and healthy subjects were used to examine the consequences of BMPR2 mutations on the BOEC protein expression profile as well as on their functionality. Functional analyses of the BOECs revealed that HPAH-derived BOECs are more susceptible to apoptosis and more proliferative compared with healthy controls. Protein isolates of BOECs from patients with HPAH and from healthy subjects were subjected to 2-D gel electrophoresis and stained for total proteins and phosphoproteins, and to a quantitative computerassisted analysis. Differentially regulated proteins were identified by mass spectrometry (LC-MS/MS). Of the 416 total proteins detected under basal conditions, 11 were significantly downregulated in HPAH-derived BOECs and 11, including the translationally controlled tumor protein (TCTP), were upregulated. TCTP has previously been shown to be involved in systemic arteriolar remodeling, inflammation and growth. Therefore, the potential role of TCTP in PAH was studied in vivo in the SU5416 rat model of severe angioproliferative PAH. Immunofluorescence staining revealed high expression of TCTP in arteriolar ECs of PAH lungs tightly localized to proliferating cells within occlusive intimal lesions; whereas, only minimal TCTP expression was seen in vascular ECs of normal lungs. Similarly, abundant TCTP immunostaining was also seen in human PAH lung sections, again associated with complex vascular lesions. In BOECs, TCTP was found to participate in cell growth and survival. These data suggest that TCTP could play an important role in PAH by mediating pro-survival and growth signaling in vascular cells, contributing to occlusive pulmonary vascular remodeling triggered by EC apoptosis.

Background: Pulmonary arterial hypertension (PAH) is a multifaceted disease characterized by elevated pulmonary arterial pressure, right ventricular hypertrophy, and a poor prognosis. Pathological hallmarks of PAH include pulmonary vascular remodelling, pre-capillary arterial obliteration, and plexiform lesions. Over the past 15 years, pulmonary endothelial cell (EC) apoptosis has been repeatedly implicated as a key trigger of occlusive arterial remodelling in PAH. While it has been hypothesized that pulmonary EC apoptosis gives rise to the emergence of growth-dysregulated, apoptosis- resistant ECs involved in arterial remodelling, the molecular mechanisms linking these two events has not yet been fully elucidated. Recently, our lab identified translationally controlled tumour protein (TCTP) as one of several significantly dysregulated proteins in culture-derived blood-outgrowth endothelial cells (BOECs) isolated from hereditable PAH (HPAH) patients harbouring mutations in the gene encoding for bone morphogenetic protein receptor type 2. Immunohistological analyses indicated that TCTP expression was associated with intra-luminal pulmonary ECs and inflammatory cells in the remodelled vessels of both human PAH patients and SU5416 rats. Furthermore, TCTP silencing abrogated excessive HPAH BOEC proliferation and promoted apoptosis in vitro. Hypothesis: We hypothesized that TCTP represents a central molecular mechanism linking pulmonary arterial EC damage and apoptosis to the emergence of growth- dysregulated lung vascular cells and complex arterial remodelling in PAH.Purpose: The purpose of the present thesis was to examine the effects TCTP inhibition on EC survival and TCTP abundance in vitro as well as on pulmonary hemodynamic changes and arterial remodelling in vivo using a well-validated rat model of severe PAH. Methods: Inhibition of TCTP was accomplished using two TCTP small molecule inhibitors, sertraline and thioridazine. In vitro, rat lung microvascular ECs (RLMVECs) were exposed to thioridazine and assayed for TCTP abundance, survival, and markers of apoptosis. In vivo, PAH was induced in male Sprague Dawley rats using SU5416 combined with 3 weeks of chronic hypoxia (SU/CH). After 4 weeks, right ventricle systolic pressure (RVSP) was measured by direct catheterization and osmotic pumps containing either thioridazine or sertraline were implanted subcutaneously. Following 3 weeks of small molecule delivery, RVSP was re-evaluated, cardiac function/structure was determined using transthoracic echocardiography, and histological analyses of vascular remodelling and inflammation were performed. Results: Our in vitro experiments demonstrated that thioridazine was able to significantly down-regulate TCTP levels and induce an apoptotic phenotype in RLMVECs. In the SU/CH rat model of severe PAH, both thioridazine and sertraline failed to have any effect on pulmonary hemodynamics, right ventricle structure/function, or vascular remodelling. Moreover, neither small molecule was able to detectably down-regulate TCTP levels in the lungs of SU/CH rats. Immunofluorescence staining revealed that TCTP expression occasionally corresponded with the expression of macrophage/monocyte marker CD68 in the lungs of SU/CH rats, consistent with its expression by inflammatory cells; however, no significant differences were found in adventitial cell clearance in the presence or absence of the inhibitors. Conclusions: Our findings support previous reports that thioridazine is able to significantly down-regulate TCTP and induce apoptosis in vitro. In contrast, both small molecule inhibitors failed to down-regulate lung TCTP levels or have any beneficial effects on the progression of PAH in SU/CH rats.

No description available.

Yes / Pulmonary arterial hypertension (PAH) is a rare cardiovascular disorder caused by narrowing of blood vessels in the lung and in the absence of therapy leads to right heart failure and death. No cure for this devastating disorder is known. The major objective of the current treatments is to improve symptoms and these therapies were developed prior to the discovery that this disease has substantial genetic components. In this review, we discuss molecular genetic basis of PAH together with pathobiology, current and future therapeutic interventions.

Pulmonary arterial hypertension (PAH) alters pulmonary hemodynamics by changing the vascular wall mechanics. Currently, pulmonary vascular resistance is used clinically to assess the disease. However, the invasive nature of pressure measurements, needed for calculating the resistance, prevents longitudinal monitoring of patients during therapy. This work employs wave reflection as an alternative measure of the downstream stiffness and proposes a new method for wave reflection assessment using only non-invasive phase contrast magnetic resonance (PCMR) flow data. The feasibility of the proposed method was investigated in a numerical model of blood flow in the right pulmonary artery. Furthermore, it was validated experimentally using a flow phantom and compared with an existing invasive technique. Finally, the feasibility of the method was tested in a study of the right pulmonary artery of a volunteer. This approach may provide a non-invasive method to evaluate PAH and its response to therapy.

Pulmonary arterial hypertension (PAH) alters pulmonary hemodynamics by changing the vascular wall mechanics. Currently, pulmonary vascular resistance is used clinically to assess the disease. However, the invasive nature of pressure measurements, needed for calculating the resistance, prevents longitudinal monitoring of patients during therapy. This work employs wave reflection as an alternative measure of the downstream stiffness and proposes a new method for wave reflection assessment using only non-invasive phase contrast magnetic resonance (PCMR) flow data. The feasibility of the proposed method was investigated in a numerical model of blood flow in the right pulmonary artery. Furthermore, it was validated experimentally using a flow phantom and compared with an existing invasive technique. Finally, the feasibility of the method was tested in a study of the right pulmonary artery of a volunteer. This approach may provide a non-invasive method to evaluate PAH and its response to therapy.

Systemic sclerosis (SSc; scleroderma) is a chronic multisystem autoimmune disease that includes prominent skin involvement in all patients and is characterized by fibrosis, inflammation, and microvascular injury of the skin and internal organs. Clinical trial design for patients with systemic sclerosis (SSc) has been confounded by the heterogeneity of disease progression and lack of objective outcome measures. This has hampered identification of therapies for patients who have frequently fatal fibrotic complications. Direct pulmonary complications are the leading cause of death in SSc. For clinical trials in patients with diffuse cutaneous SSc, identification of a pharmacodynamic biomarker associated with clinical improvement would allow for alternative approaches to trial design. Furthermore, identification of a diagnostic biomarker for SSc complicated by pulmonary arterial hypertension (SSc-PAH) would provide a reliable non-invasive method for diagnosis of pulmonary arterial hypertension. Through the combination of high throughput technologies and clinical information we have identified three preliminary biomarkers for SSc: i) Two pharmacodynamic biomarkers for diffuse skin disease (dcSSc), one in using mRNA from skin biopsies and one using proteomic profiles from sera; ii) a serum based proteomic classifier for the screening and diagnostic evaluation of pulmonary arterial hypertension in systemic sclerosis. We show these biomarkers can be applied to assess changes in skin disease in dSSc patients over time and with further development could be used to supplement or replace the physical examination assessment (Modified Rodnan Skin Score, MRSS) as an outcome measure in clinical trials for dcSSc patients. Routine use of these biomarkers in SSc clinical trial design could expand treatment options for a patient population that currently has few if any treatment options that slow progression of disease. Furthermore we identified a serum biomarker for the major SSc pulmonary complication, SSc-PAH. This diagnostic SSc-PAH biomarker has the potential to be used as a screening tool in order to reduce the need for unnecessary invasive diagnostic procedures. This non-invasive screening method could lead to early diagnosis thus improving patient survival and aid in clinical management of a complication for which there are several treatments but which is still frequently fatal. / 2018-06-15T00:00:00Z

Background Pulmonary arterial hypertension (PAH) is a rare progressive disease with no known cure. Of various aetiologies, PAH in association with connective tissue disease (PAH-CTD) is the most rapidly progressive and difficult to treat. Management of PAH has evolved significantly in the past ten years since the introduction of oral therapies. Evidence for the efficacy of these agents outside randomised controlled trials is limited, but guidelines exist. Aim To measure the impact of first-line monotherapy with bosentan or sildenafil and the introduction of prescribing guidelines on outcomes in PAH-CTD. Methods Following a retrospective analysis of consecutive, incident, treatment-naive PAH-CTD cases identified by the ASPIRE registry, influences on outcome measures have been compared. First-line monotherapy episodes for 247 patients was analysed against four distinct endpoints: change in exercise capacity, WHO functional class, time on monotherapy and all-cause mortality. Results Treatment with bosentan or sildenafil resulted in clinical stability at 2 years for nearly 1/4 patients. No difference was identified between the groups in terms of either exercise capacity or WHO functional class. Sildenafil patients were found to remain on monotherapy longer than those prescribed bosentan. Patients prescribed sildenafil have improved survival over those treated with bosentan. Unexpected baseline differences in between groups may confound the results as the haemodynamics of the bosentan patients were more severe. Conclusions A significant number of patients with PAH-CTD remain clinically stable on monotherapy at 2 years. Both agents seem equally effective in this aggressive form of PAH. A novel endpoint “TOM” may be of value in future research assessing response to treatment.