Neural deletion of Tgfbr2 impairs angiogenesis through an altered secretome.
Bottom Line: Blood vessels exhibited an atypical, clustered appearance were less in number and displayed reduced branching.HUVEC showed reduced migration towards CM of mutants compared with controls.These findings will be useful to further elucidate neurovascular interaction in general and to understand pathologies of the blood vessel system such as intracerebral haemorrhages, hereditary haemorrhagic telangiectasia, Alzheimeŕs disease, cerebral amyloid angiopathy or tumour biology.
Affiliation: Department of Molecular Embryology, Institute of Anatomy and Cell Biology, University of Freiburg, 79104 Freiburg, Germany, Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany.Show MeSH
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Mentions: To gain further insight into molecular mechanisms implicated in abnormal angiogenesis in Tgfbr2-cKO, we specifically analysed expression of (1) soluble factors produced by neural cells, which influence angiogenesis (VEGFA, FGF2, IGF1 and -2, IGFBPs, TGFβ); (2) a TGFβ target gene, which influences angiogenesis (ID1) and (3) proteins present on an angiogenesis protein array (AP-array) (e.g. THSB2 and ADAMTS1). Total expression of VEGFA isoforms did not change either on protein or mRNA levels in whole forebrain tissue samples and in neural cells that we cultured until DIV4 and 12, respectively (Fig. 2B, Supplementary Material, Fig. S6Aa–f). However, VEGFA appeared in protein aggregates in stained sections of Tgfbr2-cKO (Fig. 2Aa, b). These aggregates were enriched in all forebrain regions and mainly localized outside of vessels within the brain parenchyma (Fig. 2C, Supplementary Material, Fig. S6Ai). We detected aggregates already at E11.5 and E12.5 in Tgfbr2-cKO forebrains (Supplementary Material, Fig. S6Ag, h). We hypothesized that although expression levels of VEGFA did not change, altered VEGFA protein localization could be implicated in the observed phenotype of Tgfbr2-cKO. IGF1 and -2 protein levels were significantly increased in whole brain extracts (Fig. 2Ac–f, and B). On the mRNA level, we corroborated significantly higher IGF1 expression in neurons cultured from the VT until DIV12 and higher IGF2 expression from the VT and DT of Tgfbr2-cKO (Fig. 2D). qRTPCR further revealed higher expression of Igfbp2 and Igf2bp1 in VT-derived cultured cells of Tgfbr2-cKO compared with controls (Fig. 2D). Igfbp3-mRNA expression was reduced in cultured cells from the DT of Tgfbr2-cKO, although its expression level was unchanged on protein level (Fig. 2D, Supplementary Material, Fig. S6Bg, h). Immunostainings for IGF1 and -BP2 revealed that both proteins only partially co-localized in the VT of control and Tgfbr2-cKO hemispheres, albeit IGF2 overlapped substantially with IGF2BP1 in the VT (Fig. 2Ac–f). These data suggested that although amounts of IGF1 and -2 were increased in VT of Tgfbr2-cKO, their bioavailability might be limited owing to increased IGFBP2 and IGF2BP1 production.Figure 2.
Affiliation: Department of Molecular Embryology, Institute of Anatomy and Cell Biology, University of Freiburg, 79104 Freiburg, Germany, Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany.