IGF-binding proteins 3 and 4 are regulators of sprouting angiogenesis |
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Authors: | Dallinga Marchien G Habani Yasmin I Kayser Richelle P Van Noorden Cornelis J F Klaassen Ingeborg Schlingemann Reinier O |
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Institution: | 1.Ocular Angiogenesis Group, Departments of Ophthalmology and Medical Biology, Amsterdam Cardiovascular Sciences, Cancer Center Amsterdam, Amsterdam UMC, University of Amsterdam, Meibergdreef 9, Amsterdam, The Netherlands ;2.Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Ljubljana, Slovenia ;3.Department of Ophthalmology, University of Lausanne, Jules Gonin Eye Hospital, Fondation Asile des Aveugles, Lausanne, Switzerland ;4.Ocular Angiogenesis Group, Department of Medical Biology, Amsterdam UMC, Meibergdreef 15, Room L3-154, 1105 AZ, Amsterdam, The Netherlands ; |
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Abstract: | Purpose We have previously identified insulin-like growth factor 2 (IGF2) and insulin-like growth factor 1 receptor (IGF1R) as essential proteins for tip cell maintenance and sprouting angiogenesis. In this study, we aim to identify other IGF family members involved in endothelial sprouting angiogenesis. MethodsEffects on sprouting were analyzed in human umbilical vein endothelial cells (HUVECs) using the spheroid-based sprouting model, and were quantified as mean number of sprouts per spheroid and average sprout length. RNA silencing technology was used to knockdown gene expression. Recombinant forms of the ligands (IGF1 and IGF2, insulin) and the IGF-binding proteins (IGFBP) 3 and 4 were used to induce excess effects. Effects on the tip cell phenotype were analyzed by measuring the fraction of CD34+ tip cells using flow cytometry and immunohistochemistry in a 3D angiogenesis model. Experiments were performed in the presence and absence of serum. ResultsKnockdown of IGF2 inhibited sprouting in HUVECs, in particular when cultured in the absence of serum, suggesting that components in serum influence the signaling of IGF2 in angiogenesis in vitro. We then determined the effects of IGFBP3 and IGFBP4, which are both present in serum, on IGF2-IGF1R signaling in sprouting angiogenesis in the absence of serum: knockdown of IGFBP3 significantly reduced sprouting angiogenesis, whereas knockdown of IGFBP4 resulted in increased sprouting angiogenesis in both flow cytometry analysis and immunohistochemical analysis of the 3D angiogenesis model. Other IGF family members except INSR did not affect IGF2-IGF1R signaling. ConclusionsSerum components and IGF binding proteins regulate IGF2 effects on sprouting angiogenesis. Whereas IGFBP3 acts as co-factor for IGF2-IGF1R binding, IGFBP4 inhibits IGF2 signaling. |
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