ENHPATHY is a multidisciplinary science consortium bringing together a world-leading expertise on enhancer biology, epigenomics, bioinformatics and medical genetics to increase scientific knowledge on the molecular basis of human enhanceropathies and to open new diagniostic and therapeutic avenues for patients. ENHPATHY brings together academic institutions, beneficiaries and partners from the non-academic sector, innovative SMEs focused on technological development, bioinformatics companies, communication and management companies, European organisations along with prestigious cultural institutions. To achieve its main objective, ENHPATHY has set up an innovative, integrated and disease-focused research programme. Mutations within coding genes have traditionally been considered the major genetic cause of human diseases. However, it is becoming increasingly clear that the genetic, structural and/or epigenetic disruption of enhancers and their landscapes represent major etiological factors in numerous human diseases (i.e. enhanceropathies), ranging from rare congenital disorders to common diseases associated with ageing (e.g. cancer, diabetes). Although changes in enhancer activity are predicted to have broad pathological and therapeutic implications, we currently have a limited mechanistic understanding of human enhanceropathies. This reflects, at least partly, our still primitive and partial understanding of the mechanisms whereby enhancers can control gene expression. We hypothesize that enhancers are a diverse group of regulatory sequences that can utilize different mechanisms to control gene expression at the transcriptional and/or post-transcriptional level. Consequently, human enhanceropathies are likely to display an equally diverse molecular basis that, we believe, can only be uncovered using highly multidisciplinary systems biology approaches. Chiefly, elucidating the molecular basis of human enhanceropathies has far reaching clinical applications, especially considering the pandemic proportions that some of these disorders are acquiring in recent years. Therefore, the major goal of the ENHPATHY network is to provide early-stage researchers (ESRs) with a multidisciplinary training in which cutting-edge genomic and genetic engineering approaches are combined with various in vitro and in vivo disease models. Moreover, together with our private partners we aim at translating our molecular findings into new diagnostic and therapeutic strategies. Earlier this year, the ENHPATHY consortium submitted a proposal in the frame of the H2020-MSCA-ITN-2018's call which received a total score of 85% and a positive operational capacity notification. In order to increase the success rate of the resubmitted form of ENHPATHY planned for 2019, funding is requested to address the reviewer’s critics related to the importance and the great multidisciplinarity of its network.

The overall goal of the proposal is to define peptides being related in atherosclerotic patients to progression of atherosclerosis towards atherothrombosis or towards aneurysm formation, to obtain assay systems to determine levels of these peptides in circulation, to use these peptides for imaging and to analyze a possible role of these peptides for the disease using animal models. Specifically (i) to determine pepdides by means of proteomic analysis differently present in stable vs. unstable vs. thrombotic plaque and normal vs. aneurysm vessel walls; (ii) use these peptides to obtain specific monoclonal antibodies and to develop suitable assay systems to determine levels of these peptides in plasma and tissue samples; (iii) use peptides and specific antibodies to design reagents for functional imaging; (iv) use the assays and reagents for analyzing human samples and patients and (v) design and use animal experiments to analyze the relevance of these peptides, the substrates where the peptides originate from and the proteases releasing the peptides to in a later stage develop interfering drugs.

Pulmonary arterial hypertension (PAH) is a rare disease characterized by the obstructive elevation in pulmonary vascular resistance (PVR) and subsequent right-heart failure and death. There is currently no cure for PAH that has a very poor prognosis with a mean survival of 2.8 yr. Although the exact mechanisms leading to the onset and progression of PAH are still largely unclear, many predisposing and contributing factors have been identified, such as mutations in the bone morphogenetic protein-receptor (BMP-R)2 gene, inflammation and endothelial dysfunction, characterized by increased vasoconstriction, remodeling of pulmonary arterial wall of distal vessels and thrombosis. My primary research for the past several years seeks to provide a better understanding of the role played by pulmonary endothelial cells (P-ECs) in idiopathic PAH (iPAH) pathophysiology and identify target genes potentially involved in endothelial dysfunction. During my postdoctoral research, I have found that a selective disruption of the endothelial peroxisome proliferators activated receptor (PPAR)-gamma signaling in mice is sufficient to cause mild PAH and impair recovery from chronic hypoxia-induced PAH. More recently, we have observed paracrine overproduction of serotonin, endothelin-1 and fibroblast growth factor (FGF)-2 from iPAH P-ECs that contribute to increased smooth muscle cell proliferation. Moreover, our data have revealed that iPAH P-ECs display phenotypic abnormalities including a greater proliferative response to growth factors and apoptosis resistance. Although our observations support the hypothesis that pulmonary endothelial dysfunction is a major participant in the pathogenesis of PAH, its exact contribution to the disease remains unknown. CD74 (invariant chain, Ii) is an integral membrane protein which, beside its role as an MHC class II chaperone, has recently been found to play an additional role as an accessory-signaling molecule. CD74 demonstrates high-affinity binding to the proinflammatory cytokine macrophage migration-inhibitory factor (MIF), and the MIF homologue D-dopachrome tautomerase (DDT) that initiates a signaling cascade leading to cell proliferation and survival. Signal transduction from CD74/MIF or CD74/DDT occurs through two different modes: a) in a CD44/Src dependent pathway in which CD74 interacts with CD44. CD44 is a transmembrane protein whose phosphorylation leads to activation of Src-family kinase and MAPK/ERK, PI3K/Akt and NF-kappa B pathway and to apoptotic resistance by increasing the anti-apoptotic factors BCL2, BCL-xL and by inhibiting p53; b) in a CD44 independent pathway in which CD74 cytosolic region is cleaved by a two-step process: translocation of CD74 cytosolic fragment (CD74-ICD) to the cell nucleus resulting in NF-kappa B activation, and induction of a survival cascade via up-regulation of BCL2. Moreover, CD74 is known to interact with angiotensin AT1-receptor and nitric-oxide synthase 2. Taken together, these results strongly suggest that endothelial CD74 is a key molecule at the crossroad of inflammation and endothelial dysfunction, and thus may play an important role in the pathogenesis of PAH. Some of our recent data have shown that endothelial CD74 and MIF protein levels are markedly increased in the endothelium of muscularized pulmonary arteries from PAH patients compared to control subjects (unpublished results). Interestingly, in vitro exposure of P-ECs to MIF stimulation resulted in a 2.5-fold increase in IL-6 levels. This effect was significantly abolished by the MIF antagonist ISO-1. To expand our knowledge on this topic in iPAH, we propose to: •determine whether endothelial CD74 is involved in the acquisition/maintenance of endothelial dysfunction (Aim 1) •study the relationship between CD74 and inflammation (Aim 2) •investigate the mechanism(s) underlying the over-expression of endothelial CD74 (Aim 3)

Establish the global gene expression profile (approximately 40,000 genes) of cells in the presence of multiple stimuli._x000D_ Define dominance of each stimulus based on global analysis tools. Analyze the geometry and architecture of this complex data sets._x000D_ Analyze the gene-by-gene interactions induced by the multiple stimuli for each of the 40,000 genes. Develop appropriate algorithms and statistical methods. Identify additivity, synergies, inhibition, dominance and emergence._x000D_ Model cellular pathways in response to multiple stimuli as compared to individual stimuli. Analyze interaction between pathways by in silico experiments._x000D_ Built dynamic models based on kinetics (time-course) data sets. Use these models to predict interactions between cell signaling pathways._x000D_ In the end, built predictive tools of the behaviour of a human cell-type in a complex environment that would best reflect physiological or pathological conditions._x000D_ Analyze cell behaviour to combinations of drugs as a service to the pharmaceutical industry. Use the models to predict efficacy and toxicity of drugs on human cells._x000D_ Identify new therapeutic targets critically involved in cellular responses to inflammatory environments.