Vascular balancing act: EGFL7 and Notch

In this issue of Blood, Nichol and colleagues make the interesting and important observation that EGFL7 negatively regulates Notch activity in endothelial cells (ECs) to control blood vessel development.¹ 

Previous work demonstrates that EGFL7 is expressed in a highly restricted manner in endothelial cells during developmental vascularization and plays a role in controlling the patterning and remodeling of vascular tubes during these events.^2-4  This work supports the work of other groups who have similarly identified a role for EGFL7 protein and mRNA in vascular morphogenesis.^5,6  Interestingly, the EGFL7 mRNA carries an embedded micro- RNA in intron 7 called miR-126 which affects vascularization by decreasing expression of Spred-1 and PI3KR2, 2 proteins that negatively regulate vascular endothelial growth factor (VEGF)–dependent signaling.⁷ 

EGFL7 is an ∼ 30 kDa secreted protein that contain an Emilin-like (EMI) domain (a multimerization motif), and 2 epidermal growth factor (EGF) domains, one of which binds calcium.⁶  Based on these domains, it has been hypothesized that EGFL7 may self-assemble like extracellular matrix (ECM) proteins and, thus, could incorporate into ECM.⁶  EGFL7 has been reported to stimulate cell adhesion as well as motility in a manner similar to ECM proteins.^3,5  EGFL7 has been shown to be primarily expressed by developing ECs but also by primordial germ cells and some central nervous system neurons. Interestingly, EGFL7 expression markedly decreases in ECs in postnatal life, but can be strongly up-regulated after various tissue injuries that lead to increased angiogenic responses.³ 

Support for a possible role of EGFL7 in ECM assembly and function is recent data showing that it binds lysyl oxidases and decreases enzyme activity through interactions with the enzyme catalytic site.⁸  Lysyl oxidases are known to cross-link key interstitial matrix proteins including collagen type I and elastin which play a major role during large vessel maturation (ie, particularly arteries) which is necessary for stability of these vessels.⁹  Interestingly, EGFL7 was reported to inhibit elastogenesis in large arteries when overexpressed in transgenic mice.⁸  Furthermore, small interfering RNA (siRNA) suppression of EGFL7 in human ECs markedly stimulates elastin deposition in a manner dependent on lysyl oxidase enzymes.⁸  Thus, EGFL7 appears to regulate ECM deposition and cross-linking of interstitial matrix proteins that are known to be critical for maturation and stabilization of the developing and postnatal vasculature. These functions are particularly important in the arterial system, the major source of vascular Notch expression.

The new study by Nichol and colleagues demonstrates that increased expression of EGFL7 during vascular development or in the postnatal retina leads to vessel changes reminiscent of Notch inhibition.¹  siRNA suppression of EGFL7 in human ECs leads to decreased proliferation, decreased sprouting, and reduced EC motility, all cell behaviors observed with increased Notch signaling. The EGFL7-expressing transgenic mice demonstrate hemorrhages indicative of vascular abnormalities including altered vascular tube patterning and remodeling. In addition, the postnatal retina in these mice shows increased EC sprouting, a response that is consistent with Notch inhibition.¹  In support of this conclusion, Nichol and colleagues demonstrate a direct binding interaction of EGFL7 with Notch proteins using yeast 2-hybrid analysis. Furthermore, they demonstrate—using Notch reporter assays—that EGFL7 inhibits Notch-dependent transcriptional activation.¹  Similar findings were recently reported with respect to a potential role for EGFL7 in suppression of Notch activity during neural stem cell differentiation.¹⁰ 

Notch is well known to negatively regulate EC proliferation and vascular morphogenesis, where it strongly inhibits EC sprouting responses.¹  EC tip cells have been shown to express delta-like-4, which negatively regulates adjacent ECs through expression of Notch. This in turn keeps these trailing ECs from sprouting, further allowing them to serve other functions such as forming lumens behind the invading sprout. When Notch is inhibited, marked increases in EC sprouting responses are observed, a result seen by Nichol and colleagues in EGFL7-transgenic mice.¹  Interestingly, EGFL7 acts as a soluble Notch inhibitor^1,10  in contrast to the typical Notch inhibitory molecules which are expressed by adjacent cells as transmembrane proteins (eg, DLL-4, Jagged).

Overall, these new data strongly suggest that one function of EGFL7 is to suppress Notch activity within the vasculature. Interestingly, because ECs are the primary source of EGFL7 and the protein is secreted (and most likely deposited in the ECM), Notch activity could be inhibited in both ECs and nearby perivascular cells such as pericytes, vascular smooth muscle cells, or mononuclear leukocytes which accumulate around vessels. One intriguing possibility is that EGFL7 expression controls the timing of vessel maturation events during developmental processes. It is important that the vasculature forms and remodels vascular networks before maturation stimuli such as that contributed by recruited mural cells.¹¹  For example, pericyte recruitment to developing tubes leads to vascular basement membrane matrix assembly, a key event in vessel maturation and stabilization events.¹¹  Perhaps EGFL7 is capable of modulating such events in part through inhibition of Notch activity.

Suppression of Notch activity by secreted or ECM-deposited EGFL7 may be critical to negatively balance this activity so that differentiation and maturation events are appropriately controlled in both ECs and perivascular cells. The ability of EGFL7 to inhibit lysyl oxidase activity may represent a similar phenomenon, in that lysyl oxidase activity is particularly involved in maturation of large arteries in late fetal development and early postnatal life.⁹  Interestingly, EGFL7 is primarily expressed during vascular development,²  a time when collagen and elastin cross-linking is less prominent. Thus, it is possible that these findings are functionally coupled in some manner. It is also interesting that through their cross-linking function, lysyl oxidases can affect the mechanical properties of ECM. Recently, variations in ECM elasticity have been shown to mediate stem cell differentiation along distinct lineages.¹²  Could EGFL7 play a role in such properties of the developing vascular ECM and together with its ability to inhibit Notch activity contribute to the precise timing and sequence of events necessary to assemble vascular networks and control vascular cell differentiation within the vascular wall? In conclusion, this important study by Nichol and colleagues reveals a new manner in which signaling in the developing vasculature is balanced through negative and positive signals, where EC-expressed EGFL7 negatively regulates vascular Notch activity to control the formation and patterning of developing blood vessels.