**5. Ligand and cell signalization**

Historically, the first molecule interacting with the extracellular portion of CD146 is NOF (neurite outgrowth factor). A stable transfection of complementary DNA encoding for CD146 induces an adhesion of neuronal cells on a NOF matrix [32]. More recently, laminin-8 has been identified as a new vascular ligand of CD146 expressed by TH17 lymphocytes. In this study, it has been demonstrated that the laminin-411/CD146 interaction favor adhesion and tissue transmigration of these lymphocytes, leading to an increased inflammation [52]. Furthermore, one study showed that CD146 DNA transfection in the CD146 deficient melanoma cell line Mel-888 induced an increased aggregation between these cells and cells which do not express CD146 suggesting that there are other still unidentified partners [53].

The existence of a homophilic interaction for CD146 is controversial. One study showed that CD146 transfection in neuronal cells induced their aggregation, suggesting that CD146 could create homophilic bonds [32]. Another *in vitro* study demonstrated that the neurite growth of PC12 cells is increased when cells are in a chimeric CD146 protein substrate. In addition, under these conditions, the use of an anti-CD146 antibody blocks neurite growth. This inhibition would be associated with an inhibition of CD146-CD146 homophilic interaction [54]. CD146 dimerization at the cell membrane following stimulation with an activating CD146 antibody (clone AA98) was also demonstrated using fluorescence resonance energy transfer (FRET) and pull-down. The use of an NFkB signaling pathway inhibitor reduced this dimerization [55]. Finally, a recent study highlighted dimerization after stimulation with VEGF [56].

Conversely, other studies could not replicate the homophilic interaction, in particular, between soluble CD146 and CD146-Fc [33].

Recently, new ligands for CD146 were identified. A direct and strong interaction between CD146 and VEGFR-2 was demonstrated in endothelial cells and this association was important for VEGFR-2 phosphorylation by VEGF. These results were confirmed in a CD146 KO mouse model where the absence of CD146 inhibited vessel formation induced by VEGF. Experiments in mouse models of pancreas and melanoma cancer cell xenograft have shown that the combined use of anti-VEGF antibody (bevacizumab) and anti-CD146 antibody (AA98) displayed a synergistic effect on tumor development [57].

Another work identified galectin 1 as a new CD146 ligand on the endothelium [58]. This protein induced apoptosis of endothelial cells and specifically bound to CD146 via extracellular glycosylations. This interaction is specific for galectin 1 since it is not found with galectin 2. Using siRNA or antibodies able to block CD146 resulted in an increased cell apoptosis, suggesting a protective role of CD146 against apoptosis.

Different factors have been shown to modulate CD146 expression:



**5. Ligand and cell signalization**

438 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

partners [53].

with VEGF [56].

cell surface [33].

between soluble CD146 and CD146-Fc [33].

(AA98) displayed a synergistic effect on tumor development [57].

Different factors have been shown to modulate CD146 expression:

gesting a protective role of CD146 against apoptosis.

Historically, the first molecule interacting with the extracellular portion of CD146 is NOF (neurite outgrowth factor). A stable transfection of complementary DNA encoding for CD146 induces an adhesion of neuronal cells on a NOF matrix [32]. More recently, laminin-8 has been identified as a new vascular ligand of CD146 expressed by TH17 lymphocytes. In this study, it has been demonstrated that the laminin-411/CD146 interaction favor adhesion and tissue transmigration of these lymphocytes, leading to an increased inflammation [52]. Furthermore, one study showed that CD146 DNA transfection in the CD146 deficient melanoma cell line Mel-888 induced an increased aggregation between these cells and cells which do not express CD146 suggesting that there are other still unidentified

The existence of a homophilic interaction for CD146 is controversial. One study showed that CD146 transfection in neuronal cells induced their aggregation, suggesting that CD146 could create homophilic bonds [32]. Another *in vitro* study demonstrated that the neurite growth of PC12 cells is increased when cells are in a chimeric CD146 protein substrate. In addition, under these conditions, the use of an anti-CD146 antibody blocks neurite growth. This inhibition would be associated with an inhibition of CD146-CD146 homophilic interaction [54]. CD146 dimerization at the cell membrane following stimulation with an activating CD146 antibody (clone AA98) was also demonstrated using fluorescence resonance energy transfer (FRET) and pull-down. The use of an NFkB signaling pathway inhibitor reduced this dimerization [55]. Finally, a recent study highlighted dimerization after stimulation

Conversely, other studies could not replicate the homophilic interaction, in particular,

Recently, new ligands for CD146 were identified. A direct and strong interaction between CD146 and VEGFR-2 was demonstrated in endothelial cells and this association was important for VEGFR-2 phosphorylation by VEGF. These results were confirmed in a CD146 KO mouse model where the absence of CD146 inhibited vessel formation induced by VEGF. Experiments in mouse models of pancreas and melanoma cancer cell xenograft have shown that the combined use of anti-VEGF antibody (bevacizumab) and anti-CD146 antibody

Another work identified galectin 1 as a new CD146 ligand on the endothelium [58]. This protein induced apoptosis of endothelial cells and specifically bound to CD146 via extracellular glycosylations. This interaction is specific for galectin 1 since it is not found with galectin 2. Using siRNA or antibodies able to block CD146 resulted in an increased cell apoptosis, sug-



Membrane CD146 activates multiple signaling pathways, leading to the activation of the NFkB pathway. CD146 dimerization has been described in the membrane of endothelial cells following the addition of culture medium of tumor cells (A375 cell line). Inhibition of the NFkB pathway (by BAY11-7082 compound) causes a reduction of the nuclear translocation of NFKB but also inhibits the dimerization of CD146 [63, 64]. The junctional molecules involved in adhesion such as VE-cadherin or claudins are also involved in a phenomenon of actin cytoskeleton reorganization. CD146 is also connected to the actin cytoskeleton.

Indeed, CD146 targeting with the S-ENDO1 antibody led to FAK phosphorylation and an increase in the release of intracellular calcium and extracellular calcium entry. This mechanism of action of calcium flux was mediated by the recruitment and activation of Fyn leading to the phosphorylation of PLC gamma. Calcium entry also caused the recruitment of PYK2 and p130. On the other hand, FAK activation led to signaling pathways involved in the reorganization of the actin cytoskeleton and also modulated transcription factors involved in cells survival and migration. In these studies, there was no evidence of direct interaction between CD146, paxillin, and FAK. It seems therefore important to identify the intermediate partners [65, 66].

Recently, a study confirmed the role of CD146 in the migration and induction of signals related to the actin cytoskeleton. Indeed, CD146 displays direct physical interaction with the ezrin-radixin-moesin (ERM) proteins, allowing the recruitment of ERM at the protrusions of melanoma cells. This phenomenon induces the elongation and expansion of microvilli at these protrusions [67].

Recruitment by CD146 allows the sequestration of a RhoA inhibitor (Rho guanine nucleotide dissociation inhibitory factor 1) leading to RhoA activation and an increased cell motility. Another study showed that CD146 is redistributed in a polarized structure named W-RAMP (Wnt5a-mediated actin-myosin receptor-polarity) in subconfluent melanoma cells stimulated with Wnt5a. W-RAMP is involved in membrane retraction and the direction of cell migration with an intervention of Rho-A [68].

In another study that focused on the priming of ECFC with soluble CD146 in order to improve the therapeutic potential of these cells *in vivo*, the authors showed that a priming of these cells

**Figure 3.** Mechanism of actions of endothelial progenitor cells (EPC) activation leads to their recruitment after inflammatory or angiogenic stimulation.

with soluble CD146 did not modify the number of engrafted ECFC in the ischemic muscle but improved their survival capacity leading to an enhanced revascularization [39]. They showed that in ECFC, it exists a signalosome that is located in a particular region of cell membrane called lipid rafts. This signalosome contains soluble CD146, the short isoform of CD146 (shCD146), presenilin-1 but also the two VEGF receptor called flt1 and flk1. The mechanism of action is characterized by a sequential proteolytic cleavage, induced by soluble CD146, with an extracellular shedding of the short CD146 followed by an intramembrane cleavage which is mediated by both the ADAM/matrix metalloproteases (MMP) and the gamma-secretase protein. The consequences of this shedding involved a nuclear translocation of the new intracellular peptide of shCD146 which binds to the transcription factor CSL and is associated with a modulation of gene transcription leading to angiogenesis (eNOS) and cell survival (FADD, Bcl-xl). The association between CD146 and VEGFR2 was described in a previous paper and based on these results the authors showed that the effect of soluble CD146 on EFCF is dependent on VEGFR2 but also VEGFR1 which are phosphorylated by soluble CD146. All these findings show that the stimulation of this cell by soluble CD146 and the proteolytic cleavage of shCD146 is a promising pathway to increase the regenerative properties of endothelial progenitor cells for the treatment of cardiovascular diseases (**Figure 3**).
