IGF2 and IGF1R identified as novel tip cell genes in primary microvascular endothelial cell monolayers

Abstract

Tip cells, the leading cells of angiogenic sprouts, were identified in cultures of human umbilical vein endothelial cells (HUVECs) by using CD34 as a marker. Here, we show that tip cells are also present in primary human microvascular endothelial cells (hMVECs), a more relevant endothelial cell type for angiogenesis. By means of flow cytometry, immunocytochemistry, and qPCR, it is shown that endothelial cell cultures contain a dynamic population of CD34+ cells with many hallmarks of tip cells, including filopodia-like extensions, elevated mRNA levels of known tip cell genes, and responsiveness to stimulation with VEGF and inhibition by DLL4. Furthermore, we demonstrate that our in vitro tip cell model can be exploited to investigate cellular and molecular mechanisms in tip cells and to discover novel targets for anti-angiogenesis therapy in patients. Small interfering RNA (siRNA) was used to knockdown gene expression of the known tip cell genes angiopoietin 2 (ANGPT2) and tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE1), which resulted in similar effects on tip cells and sprouting as compared to inhibition of tip cells in vivo. Finally, we identified two novel tip cell-specific genes in CD34+ tip cells in vitro: insulin-like growth factor 2 (IGF2) and IGF-1-receptor (IGF1R). Knockdown of these genes resulted in a significant decrease in the fraction of tip cells and in the extent of sprouting in vitro and in vivo. In conclusion, this study shows that by using our in vitro tip cell model, two novel essential tip cells genes are identified.Electronic supplementary materialThe online version of this article (10.1007/s10456-018-9627-4) contains supplementary material, which is available to authorized users.

IGF-binding proteins 3 and 4 are regulators of sprouting angiogenesis

“…In our previous study, we found that IGF2 mRNA is enriched in CD34 + tip cells as compared to non-tip cells, and that knockdown of IGF2 gene expression by siRNA leads to a reduction in the fraction of tip cells in endothelial cell cultures, and in reduced sprouting from spheroids [2]. Now, we verified this finding in a novel 3D angiogenesis model [34] after knockdown of IGF2 by siRNA.…”

“…Previously, we have shown that knockdown of IGF1R reduces sprouting in vitro [2]. When studying the other receptors of the IGF family, we found that knockdown of INSR significantly decreased the number of sprout per spheroid but not their length, whereas knockdown of IGF2R did not change the number or length of sprouts ( Fig.…”

“…However, specific information about the exact role of IGF family members in sprouting angiogenesis was lacking until recently, when we reported that IGF2 and IGF1R are involved in tip cell maintenance and sprouting angiogenesis by means of a local autocrine growth-regulating signaling axis [2]. We now present evidence that suggests that other IGF family members besides IGF2 and IGF1R are involved in this process as well.…”

“…It is initiated by an array of growth factors such as vascular endothelial growth factor (VEGF) [1]. We have recently reported that insulin-like growth factor 2 (IGF2) and insulin-like growth factor 1 receptor (IGF1R) are essential for sprouting angiogenesis, because they enable maintenance of the tip cells, the leading cells in vessel sprouts [2,3]. Here, we have analyzed the role of other IGF family members in sprouting angiogenesis.…”

Nitroxoline: repurposing its antimicrobial to antitumor application

“…It enables spread of tumor cells to distant sites and formation of metastasis and tumor progression. Angiogenesis is a complex multistep process that includes proliferation of endothelial cells, degradation of extracellular matrix (ECM), migration of endothelial cells through remodeled ECM and tube formation by endothelial cells (Dallinga et al, 2018;Sato, 2004). MetAP2 has been shown to play an important role in endothelial cell proliferation (Ehlers et al, 2016;Sato, 2004).…”