Alia Khalil1, Philippe Poelvoorde2, Mohammad Fayyad-Kazan2, Alexandre Rousseau3, Vincent Nuyens3, Sophie Uzureau4, Patrick Biston3, Yolla El-Makhour5, Bassam Badran5, Pierre Van Antwerpen6, Karim Zouaoui Boudjeltia3, Luc Vanhamme7. 1. Laboratory of Experimental Medicine (ULB 222 Unit), CHU de Charleroi, A. Vésale Hospital, Université Libre de Bruxelles, Montigny-le-Tilleul, Belgium; Laboratory of Cancer Biology and Molecular Immunology, Lebanese University, Faculty of Sciences, Hadath-Beirut, Lebanon. 2. Laboratory of Molecular Biology of Inflammation, IBMM, Faculty of Sciences, Université Libre de Bruxelles, Gosselies, Belgium. 3. Laboratory of Experimental Medicine (ULB 222 Unit), CHU de Charleroi, A. Vésale Hospital, Université Libre de Bruxelles, Montigny-le-Tilleul, Belgium. 4. Laboratory of Molecular Parasitology, IBMM, Faculty of Sciences, Université Libre de Bruxelles, Gosselies, Belgium. 5. Laboratory of Cancer Biology and Molecular Immunology, Lebanese University, Faculty of Sciences, Hadath-Beirut, Lebanon. 6. Laboratory of Pharmaceutical Chemistry and Analytical Platform of the Faculty of Pharmacy, Faculty of Pharmacy, Université Libre de Bruxelles, Brussels, Belgium. 7. Laboratory of Molecular Biology of Inflammation, IBMM, Faculty of Sciences, Université Libre de Bruxelles, Gosselies, Belgium. Electronic address: luc.vanhamme@ulb.ac.be.
Abstract
BACKGROUND AND AIMS: Endothelial cells are main actors in vascular homeostasis as they regulate vascular pressure and permeability as well as hemostasis and inflammation. Disturbed stimuli delivered to and by endothelial cells correlate with the so-called endothelial dysfunction and disrupt this homeostasis. As constituents of the inner layer of blood vessels, endothelial cells are also involved in angiogenesis. Apolipoprotein Ls (APOL) comprise a family of newly discovered apolipoproteins with yet poorly understood function, and are suggested to be involved in inflammatory processes and cell death mechanisms. Here we investigate the role of APOLs in endothelial cells stimulated with factors known to be involved in atherogenesis and their possible contribution to endothelial dysfunction with an emphasis on inflammation driven-angiogenesis in vitro. METHODS: Using the CRISPR/Cas9 technique, we analyzed the effect of APOL3 gene knock out in HMEC-1 endothelial cells on cell migration, tubulogenesis, endothelial permeability, intracellular signal transduction as assessed by kinase phosphorylation, and angiogenesis gene expression (measured by qRT-PCR). RESULTS: Our results indicate that among the family, APOL3 was the only member induced by myeloperoxidase, oxidized LDL, VEGF and FGF treatments. APOL3 invalidation increased endothelial permeability, reduced wound repair and tubule formation in vitro, the latter only in MPO and VEGF-induced conditions. Accordingly, some pro-angiogenic signaling pathways (ERK1/2 and FAK but not Akt) and some pro-angiogenic genes were partially inhibited in APOL3 knock out cells. CONCLUSIONS: These findings suggest the involvement of APOL3 in angiogenesis in vitro and as a modulator of MAPK and FAK signaling in endothelial cells.
BACKGROUND AND AIMS: Endothelial cells are main actors in vascular homeostasis as they regulate vascular pressure and permeability as well as hemostasis and inflammation. Disturbed stimuli delivered to and by endothelial cells correlate with the so-called endothelial dysfunction and disrupt this homeostasis. As constituents of the inner layer of blood vessels, endothelial cells are also involved in angiogenesis. Apolipoprotein Ls (APOL) comprise a family of newly discovered apolipoproteins with yet poorly understood function, and are suggested to be involved in inflammatory processes and cell death mechanisms. Here we investigate the role of APOLs in endothelial cells stimulated with factors known to be involved in atherogenesis and their possible contribution to endothelial dysfunction with an emphasis on inflammation driven-angiogenesis in vitro. METHODS: Using the CRISPR/Cas9 technique, we analyzed the effect of APOL3 gene knock out in HMEC-1 endothelial cells on cell migration, tubulogenesis, endothelial permeability, intracellular signal transduction as assessed by kinase phosphorylation, and angiogenesis gene expression (measured by qRT-PCR). RESULTS: Our results indicate that among the family, APOL3 was the only member induced by myeloperoxidase, oxidized LDL, VEGF and FGF treatments. APOL3 invalidation increased endothelial permeability, reduced wound repair and tubule formation in vitro, the latter only in MPO and VEGF-induced conditions. Accordingly, some pro-angiogenic signaling pathways (ERK1/2 and FAK but not Akt) and some pro-angiogenic genes were partially inhibited in APOL3 knock out cells. CONCLUSIONS: These findings suggest the involvement of APOL3 in angiogenesis in vitro and as a modulator of MAPK and FAK signaling in endothelial cells.