Identification of Novel Molecular Pathways Involved in Angiogenesis

In cancer and cardiovascular diseases (CVD), abnormal development of blood vessels plays a major role in disease progression. In case of cancer, abnormal angiogenesis promote tumour expansion and metastasis, which are characterized by formation of an abundant chaotic capillary network. In case of CVD, abnormal angiogenesis is characterised by obstruction of blood vessels, which results in hypoxia leading to cell apoptosis and necrosis in e.g. cardiac tissue. Many pathways of angiogenesis are still poorly understood and should be explored using gene function studies. In this thesis, we aim to identify novel molecular pathways involved in angiogenesis, which might shed light on failing gene therapy and offer novel perspectives on pro- and anti-angiogenic therapies. To identify new genetic factors involved in angiogenesis, a genome-wide microarray screen was performed on the transcriptome of murine embryo`s. A list of genes that were significantly upregulated in FLK-positive cells was validated using whole-mount in situ hybridization in zebrafish larvae. Genes with a corresponding expression pattern in the vasculature were selected and silenced in an in vivo model of angiogenesis, in developing zebrafish larvae and in a mural retina model. In vitro knockdown assays were also performed in 3D-collagen based co-culture of EC and pericytes to assess blood vessel formation. In this thesis CMTM3, CMTM4, CGNL1, THSD1, and CECR1 were selected as candidate genes and their molecular pathways were investigated. For the first time to our knowledge we concluded that their properties in ECs are involved in the angiogenesis process. We also investigated the angiogenic property of the nucleotide Up4A as it is synthesised in response to VEGFR2 stimulation during stress conditions, such as hypoxia or in cancer conditions. In conclusion, our fundamental findings of novel pathways involved in angiogenesis hold promising therapeutic perspectives to understand and treat vascular diseases

CECR1-mediated cross talk between macrophages and vascular mural cells promotes neovascularization in malignant glioma

Glioblastomas (glioblastoma multiforme, GBM) are most malignant brain tumors characterized by profound vascularization. The activation of macrophages strongly contributes to tumor angiogenesis during GBM development. Previously, we showed that extracellular adenosine deaminase protein Cat Eye Syndrome Critical Region Protein 1 (CECR1) is highly expressed by M2-like macrophages in GBM where it defines macrophage M2 polarization and contributes to tumor expansion. In this study, the effect of CECR1 in macrophages on tumor angiogenesis was investigated. Immunohistochemical evaluation of GBM tissue samples showed that the expression of CECR1 correlates with microvascular density in the tumors, confirming data from the TCGA set. In a three-dimensional co-culture system consisting of human pericytes, human umbilical vein endothelial cells and THP1-derived macrophages, CECR1 knockdown by siRNA and CECR1 stimulation of macrophages inhibited and promoted new vessel formation, respectively. Loss and gain of function studies demonstrated that PDGFB mRNA and protein levels in macrophages are modulated by CECR1. The proangiogenic properties of CECR1 in macrophages were partially mediated via paracrine activation of pericytes by PDGFB–PDGFRβ signaling. CECR1–PDGFB–PDGFRβ cross-activation between macrophages and pericytes promoted pericyte migration, shown by transwell migration assay, and enhanced expression and deposition of periostin, a matrix component with proangiogenic properties. CECR1 function in (M2-like) macrophages mediates cross talk between macrophages and pericytes in GBM via paracrine PDGFB–PDGFRβ signaling, promoting pericyte recruitment and migration, and tumor angiogenesis. Therefore, CECR1 offers a new portent target for anti-angiogenic therapy in GBM via immune modulation.Oncogene advance online publication, 22 May 2017; doi:10.1038/onc.2017.145

Endothelial loss of Fzd5 stimulates PKC/Ets1-mediated transcription of Angpt2 and Flt1

Aims: Formation of a functional vascular system is essential and its formation is a highly regulated process initiated during embryogenesis, which continues to play important roles throughout life in both health and disease. In previous studies, Fzd5 was shown to be critically involved in this process and here we investigated the molecular mechanism by which endothelial loss of this receptor attenuates angiogenesis. Methods and results: Using short interference RNA-mediated loss-of-function assays, the function and mechanism of signaling via Fzd5 was studied in human endothelial cells (ECs). Our findings indicate that Fzd5 signaling promotes neovessel formation in vitro in a collagen matrix-based 3D co-culture of primary vascular cells. Silencing of Fzd5 reduced EC proliferation, as a result of G0/G1 cell cycle arrest, and decreased cell migration. Furthermore, Fzd5 knockdown resulted in enhanced expression of the factors Angpt2 and Flt1, which are mainly known for their destabilizing effects on the vasculature. In Fzd5-silenced ECs, Angpt2 and Flt1 upregulation was induced by enhanced PKC signaling, without the involvement of canonical Wnt signaling, non-canonical Wnt/Ca2+-mediated activation of NFAT, and non-canonical Wnt/PCP-mediated activation of JNK. We demonstrated that PKC-induced transcription of Angpt2 and Flt1 involved the transcription factor Ets1. Conclusions: The current study demonstrates a pro-angiogenic role of Fzd5, which was shown to be involved in endothelial tubule formation, cell cycle progression and migration, and partly does so by repression of PKC/Ets1-mediated transcription of Flt1 and Angpt2

Transcriptome analysis reveals microvascular endothelial cell-dependent pericyte differentiation

Microvascular homeostasis is strictly regulated, requiring close interaction between endothelial cells and pericytes. Here, we aimed to improve our understanding of how microvascular crosstalk affects pericytes. Human-derived pericytes, cultured in absence, or presence of human endothelial cells, were studied by RNA sequencing. Compared with mono-cultured pericytes, a total of 6704 genes were differentially expressed in co-cultured pericytes. Direct endothelial contact induced transcriptome profiles associated with pericyte maturation, suppression of extracellular matrix production, proliferation, and morphological adaptation. In vitro studies confirmed enhanced pericyte proliferation mediated by endothelial-derived PDGFB and pericyte-derived HB-EGF and FGF2. Endothelial-induced PLXNA2 and ACTR3 upregulation also triggered pericyte morphological adaptation. Pathway analysis predicted a key role for TGFβ signaling in endothelial-induced pericyte differentiation, whereas the effect of signaling via gap- and adherens junctions was limited. We demonstrate that endothelial cells have a major impact on the transcriptional profile of pericytes, regulating endothelial-induced maturation, proliferation, and suppression of ECM production

Fuzzy Sliding Mode Control of DFIG applied to the WECS

8th International Conference on Systems and Control (ICSC), Univ Caddi Ayyad, Marrakesh, MOROCCO, OCT 23-25, 2019International audienceThis papers deal with a direct power control based on DFIG of 3 KW for WECS using FSMC. The strategy (FSMC) the most appropriate scheme for the reduction of high power ripple and the reduction of steadystate errors with the fact that the impact of the machine parameters variations does not modify the performances of the system. The proposed algorithm on a DFIG of 5 KW was made to evaluate the results of the capacity and the robustness of the proposed control scheme with the presence of various uncertainties

High-efficiency induction motor drives using type-2 fuzzy logic

International audienceIn this work we propose to develop an algorithm for improving the efficiency of an induction motor using type-2 fuzzy logic. Vector control is used to control this motor due to the high performances of this strategy. The type-2 fuzzy logic regulators are developed to obtain the optimal rotor flux for each torque load by minimizing the copper losses. We have compared the performances of our fuzzy type-2 algorithm with the type-1 fuzzy one proposed in the literature. The proposed algorithm is tested with success on the dSPACE DS1104 system even if there is parameters variance

Ets2 determines the inflammatory state of endothelial cells in advanced atherosclerotic lesions

RATIONALE: Neovascularization is required for embryonic development and plays a central role in diseases in adults. In atherosclerosis, the role of neovascularization remains to be elucidated. In a genome-wide microarray-screen of Flk1+ angioblasts during murine embryogenesis, the v-ets erythroblastosis virus E26 oncogene homolog 2 (Ets2) transcription factor was identified as a potential angiogenic factor. OBJECTIVES: We assessed the role of Ets2 in endothelial cells during atherosclerotic lesion progression toward plaque instability. METHODS AND RESULTS: In 91 patients treated for carotid artery disease, Ets2 levels showed modest correlations with capillary growth, thrombogenicity, and rising levels of tumor necrosis factor-alpha (TNFalpha), monocyte chemoattractant protein 1, and interleukin-6 in the atherosclerotic lesions. Experiments in ApoE(-/-) mice, using a vulnerable plaque model, showed that Ets2 expression was increased under atherogenic conditions and was augmented specifically in the vulnerable versus stable lesions. In endothelial cell cultures, Ets2 expression and activation was responsive to the atherogenic cytokine TNFalpha. In the murine vulnerable plaque model, overexpression of Ets2 promoted lesion growth with neovessel formation, hemorrhaging, and plaque destabilization. In contrast, Ets2 silencing, using a lentiviral shRNA construct, promoted lesion stabilization. In vitro studies showed that Ets2 was crucial for TNFalpha-induced expression of monocyte chemoattractant protein 1, interleukin-6, and vascular cell adhesion molecule 1 in endothelial cells. In addition, Ets2 promoted tube formation and amplified TNFalpha-induced loss of vascular endothelial integrity. Evaluation in a murine retina model further validated the role of Ets2 in regulating vessel inflammation and endothelial leakage. CONCLUSIONS: We provide the first evidence for the plaque-destabilizing role of Ets2 in atherosclerosis development by induction of an intraplaque proinflammatory phenotype in endothelial cells. [KEYWORDS: Analysis of Variance, Animals, Aortic Diseases/immunology/ metabolism/pathology/physiopathology, Apolipoproteins E/deficiency/genetics, Carotid Artery Diseases/immunology/ metabolism/pathology/physiopathology, Cells, Cultured, Chemokine CCL2/metabolism, Disease Models, Animal, Endothelial Cells/immunology/ metabolism, Hemorrhage/metabolism, Humans, Inflammation/immunology/ metabolism/pathology/physiopathology, Inflammation Mediators/metabolism, Interleukin-6/metabolism, Mice, Mice, I