EGF repeats of epidermal growth factor­like domain 7 promote endothelial cell activation and tumor escape from the immune system.

The tumor blood vessel endothelium forms a barrier that must be crossed by circulating immune cells in order for them to reach and kill cancer cells. Epidermal growth factor­like domain 7 (Egfl7) represses this immune infiltration by lowering the expression levels of leukocyte adhesion receptors on the surface of endothelial cells. However, the protein domains involved in these properties are not completely understood. Egfl7 is structurally composed of the predicted EMI­, EGF­ and C­terminal domains. The present study aimed to investigate the roles of these different domains in tumor development by designing retroviruses coding for deletion mutants and then infecting 4T1 breast cancer cell populations, which consequently overexpressed the variants. By performing in vitro soft­agar assays, it was found that Egfl7 and its deletion variants did not affect cell proliferation or anchorage­independent growth. When 4T1 cells expressing either the wild­type Egfl7 protein or Egfl7 domain variants were implanted in mice, Egfl7 expression markedly promoted tumor development and deletion of the EGF repeats decreased the tumor growth rate. By contrast, deleting any other domain displayed no significant effect on tumor development. The overexpression of Egfl7 also decreased T cell and natural killer cell infiltration in tumors, as determined by immunofluorescence staining of tumor sections, whereas deletion of the EGF repeats inhibited this effect. Reverse transcription­quantitative PCR analysis of the mechanisms involved revealed that deleting the EGF repeats partially restored the expression levels of vascular cell adhesion molecule 1 and E­selectin, which were suppressed by overexpression of Egfl7 in endothelial cells in vitro. This resulted in a higher number of lymphocytes bound to HUVEC expressing Egfl7­ΔEGF compared with HUVEC expressing wild­type Egfl7, as assessed by fluorescent­THP­1 adhesion assays onto endothelial cells. Overall, the present study demonstrated that the EGF repeats may participate in the protumoral and anti­inflammatory effects of Egfl7.

MAGP-1 and fibronectin control EGFL7 functions by driving its deposition into distinct endothelial extracellular matrix locations.

The extracellular matrix (ECM) is essential to provide mechanical support to tissues but is also a bioactive edifice which controls cell behavior. Cell signaling generated by ECM components through integrin-mediated contacts, modulates cell biological activity. In addition, by sequestrating or releasing growth factors, the ECM is an active player of physiological and pathological processes such as vascular development. EGFL7 is mainly expressed during blood vessel development and is deposited in the ECM after secretion by endothelial cells. While EGFL7 is known to control various endothelial cell molecular mechanisms [i.e., the repression of endothelial-derived lysyl oxidase (LOX) enzyme, the regulation of the Notch pathway, and the expression of leukocyte adhesion molecules and of RHOA by endothelial cells], it is not established whether EGFL7 functions when bound to the ECM. Here, we show that microfibrillar-associated glycoprotein-1 (MAGP-1) and fibronectin drive the deposition of EGFL7 into both fibers and individual aggregates in endothelial ECM. Although EGFL7 does not need to be docked into the ECM to control endothelial adhesion molecule expression, the ECM accumulation of EGFL7 is required for its regulation of LOX activity and of HEY2 expression along the Notch pathway. The interaction of EGFL7 with MAGP-1 is necessary for LOX activity repression by EGFL7 while it does not participate in the control of the Notch pathway by this protein. Altogether, this study highlights the roles played by EGFL7 in controlling various endothelial molecular mechanisms upon its localization and shows how the ECM can modulate its functions.

Endothelial Cell Activation Is Regulated by Epidermal Growth Factor-like Domain 7 (Egfl7) during Inflammation.

Activation of the blood vessel endothelium is a critical step during inflammation. Endothelial cells stimulated by pro-inflammatory cytokines play an essential part in the adhesion and extravasation of circulating leukocytes into inflamed tissues. The endothelial egfl7 gene (VE-statin) represses endothelial cell activation in tumors, and prior observations suggested that it could also participate in the regulation of endothelial cell activation during inflammation. We show here that Egfl7 expression is strongly repressed in mouse lung endothelial cells during LPS- and TNFα-induced inflammation in vivo LPS have a limited effect on Egfl7 expression by endothelial cells in vitro, whereas the pro-inflammatory cytokine TNFα strongly represses Egfl7 expression in endothelial cells. TNFα regulates the egfl7 gene promoter through regions located between -7585 and -5550 bp ahead of the main transcription start site and via an NF-κB-dependent mechanism. Conversely, Egfl7 regulates the response of endothelial cells to TNFα by restraining the induced expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin, resulting in a decreased adhesion of leukocytes onto endothelial cells stimulated by TNFα. Egfl7 regulates the expression of these adhesion molecules through the NF-κB and MEK/Erk pathways, in particular by preventing the proteasome-mediated degradation of IkBα both in non-activated endothelial cells and during activation. Egfl7 is thus an endogenous and constitutive repressor of blood vessel endothelial cell activation in normal and inflammatory conditions and participates in a loop of regulation of activation of these cells by pro-inflammatory cytokines.