Inhibition of vascular endothelial growth factor receptor 2–mediated endothelial cell activation by Axl tyrosine kinase receptor

Abstract

GAS6, the product of a growth arrest specific (GAS) gene, is the ligand of the tyrosine kinase receptor Axl. GAS6 and Axl are both expressed in endothelial cells, where they are involved in many processes such as leukocyte transmigration through capillaries and neointima formation in injured vessels. Here IntroductionAxl is a 140-kDa protein which belongs to a subfamily of tyrosine kinase receptor (RTK) that includes also Mer and Rse. These receptors are characterized by the presence of 2 immunoglobulinlike domains and 2 fibronectin-type III domains in the extracellular region and a distinctive intracellular kinase domain. 1-3 Axl was first isolated from the DNA of patients with chronic myelogenous leukemia, but subsequently its expression was demonstrated in mammalian cells of vascular, immune, and reproductive systems. 1,[4][5][6] Axl is involved in cell-cell aggregation, 7 in the maintenance of a wide variety of cell types in adult tissues, 6 and in homeostatic regulation of immune system. 8 GAS6, a protein codified by a growth arrest specific gene, is a member of the vitamin K-dependent protein family. GAS6 has 42% amino acid identity to protein S, a serum protein that negatively regulates blood coagulation. GAS6 is composed of characteristic structural motifs: a ␥-carboxylated aminoterminus domain followed by 4 epidermal growth factor-like domains and by a set of tandem G domains similar to those of sex hormone binding globulin. 9 GAS6 binds with different affinities and activates the kinase activity of each Axl family member. 10-12 GAS6 shows the higher affinity for Axl, and its interaction generally triggers antiapoptotic signals, resulting in an augmented cell survival. In some cell types it regulates homotypic and heterotypic adhesion, 4,13 promotes proliferation, 5,14 survival, [15][16][17][18] and motility 19,20 and amplifies the activity of extracellular stimuli. 21,22 Vasculature may be a target of GAS6/Axl axis, being both molecules expressed by endothelial cells (ECs), 4,9 pericytes, 23 and smooth muscle cells. 24 GAS6/Axl interaction is involved in leukocyte transmigration through capillaries, 4,8 in neointima formation in injured vessels, 24 and in EC survival after tumor necrosis factor ␣ (TNF-␣) treatment or acidification. 16,18 Mice lacking the 3 tyrosine kinase receptors, which bind GAS6, show alteration in vasculature survival. 6 By activating specific and partially overlapping genetic programs, ECs play key roles in many processes, as regulation of vascular tone, lipid metabolism, hematopoiesis, inflammation, antigen recognition, thrombosis, hemostasis, and angiogenesis. 25 Angiogenesis refers to the remodeling and maturation of the primitive vascular plexus formed by vasculogenesis during the development and to the sprouting and subsequent stabilization by pericytes of new capillaries from preexisting ones in adult life. 26 Physiologic angiogenesis results from a fine-tuning balance between inducers and inhibitors and achieves a network hierarchically and spatially organized to provide a...

Emodin inhibits vascular endothelial growth factor‐A‐induced angiogenesis by blocking receptor‐2 (KDR/Flk‐1) phosphorylation

“…54 In addition, some SH2-containing phosphatases also have been shown to be involved in down-regulating KDR/Flk-1 activation. 55,56 Therefore, it is possible that blocking of VEGF-A-mediated KDR/Flk-1 activation by emodin is through the induction of phosphatases that affect KDR/Flk-1 phosphorylation by VEGF-A.…”

The vitamin K–dependent anticoagulant factor, protein S, inhibits multiple VEGF-A–induced angiogenesis events in a Mer- and SHP2-dependent manner

“…It was reported that Axl stimulation by the structural homolog of ProS, Gas6, activates SHP2, thereby inhibiting VEGF-A-dependent activation of both VEGFR2 and the angiogenic program in vascular endothelial cells. 29 More recently, Ruan and Kazlauskas demonstrated that VEGF-A-dependent activation of VEGFR2 and recruitment of Src family kinases engages the receptor tyrosine kinase Axl to trigger ligand-independent autophosphorylation that promotes activation of Akt. 41 The evidence we provide for the existence of a ProS/Mer/SHP2 axis, which inhibits VEGF-A-mediated VEGFR2, MAPK Erk1/2, and Akt activation, suggests that, besides Axl, Mer tyrosine kinase receptors is also a .…”

“…29 ECs cultured on extracellular matrix preparations, such as Matrigel, spontaneously differentiate into capillary-like structures. 29 To ascertain further the inhibitory effect of ProS on angiogenesis, we next tested its effects in an in vitro morphogenesis assay in which human ECs were cultured on Matrigel. Typical capillary-like structures, formed by ECs cultured on Matrigel, for 24 hours, are represented in Figure 2Ai (by arrows, control).…”

“…As shown in Figure 7A, silencing of either Axl or Tyro3 did not alter the inhibitory action of human ProS on VEGF-A-induced EC proliferation, whereas silencing of Mer completely suppressed it, suggesting that ProS by activating Mer tyrosine kinase receptor inhibits VEGF-A-mitogenic signaling. Furthermore, we used the in vitro morphogenesis assay described in Figure 2, which is considered as the closest to in vivo angiogenesis 29 to assess the consequences of Mer silencing on the ability of human ProS to inhibit capillary-like structure formation. Figure 7B and C show that Mer silencing suppressed ProS inhibitory action on capillarylike structure formation.…”

“…29 Matrigel was supplemented with human ProS, human endostatin, or human albumin at the indicated concentrations, coated to each well of a 24-well plate, and kept at room temperature for 4 hours to allow gel formation. HUVECs (10 5 cells/ well) were then plated onto the Matrigel in growth factor-depleted medium (EBM-2 containing 0.5% FCS).…”