**6. CD146 in pathology**

## **6.1. Obstetrical pathologies**

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

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

440 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

progenitor cells for the treatment of cardiovascular diseases (**Figure 3**).

inflammatory or angiogenic stimulation.

About 2% of fertile women are affected by spontaneous fetal loss. The mechanism of this fetal loss is not yet understood.

One study showed that CD146 is highly expressed during the implantation window. During the following steps, the level of CD146 decreased rapidly and CD146 blocking with an antibody caused abortion [69]. CD146 is expressed by the intermediate trophoblasts (or extravillous) in humans but is not detected on the syncytiotrophoblasts and cytotrophoblasts [70].

After this work, soluble CD146 was described as a novel physiological factor with angiogenic properties involved in the regulation of placenta vascular development by acting on extravillous trophoblast (EVT). Using placenta explants, soluble CD146 was demonstrated to inhibit the growth of extravillous trophoblasts and the ability of EVT to migrate and form pseudocapillary tubes on Matrigel. A clinical study on the role of soluble CD146 in 50 pregnant women was also conducted. A physiological decrease of plasmatic soluble CD146 was observed in pregnant women as compared to nongestational women. These results inspired the authors to study the effects of prolonged administration of soluble CD146 in a pregnant rat model. Repeated systemic injection of soluble CD146 after mating caused a significant decrease in the pregnancy rate and the number of embryos. Histological studies of placenta showed a decreased migration of glycogen cells (cells that are similar to the EVT in rat) in female rat treated with soluble CD146.

In mice the use of a specific antibody blocking CD146 (AA98) caused a decrease in the blastocysts adhesion on a uterine epithelium cell monolayer and a decrease in the growth of trophoblastic cells. In addition, injection of this antibody in the uterine horn of the mouse at 3.5 dpc (days post coitum) resulted in a decrease of embryo implantation at 7.5 dpc. Histological analysis showed that the embryos were present but smaller and in poor condition [69]. Two clinical studies were in line with these observations. A first clinical study showed that the rate of membrane CD146 expression was lower on intermediate trophoblasts in the placenta of preeclamptic patients when compared to patients with nonpathological pregnancies [70]. In a second study, two populations of women have been used to compare the blood level of soluble CD146. In this study, the authors used 100 blood samples which were taken 2 months after the last obstetrical events between women with no pregnancy lost which have at least one living child and 100 blood samples from women with at least two consecutive losses at/ or before 21 weeks of gestation. They found an increase in the level of soluble CD146 in the second population compared to the first control population [71]. In this study, the two populations used are age matched.

Thus, in view of these results soluble CD146 may represent an attractive biomarker of vascular placental development as well as a therapeutic target in obstetric complications.

## **6.2. Inflammatory diseases**

Endothelial functions are altered in inflammatory diseases.

In inflammatory kidney disease, biopsies of patients with nephropathy show an increased expression of membrane CD146 on endothelial cells, but also on the mesangial cells and a neo-expression of CD146 on tubular cells [72]. In addition, there is a correlation between CD146 expression and proteinuria, endocapillary proliferation and inflammatory syndrome. The serum level of soluble CD146 is also modulated. Thus, an increase in CD146 secretion was observed in chronic renal failure which was correlated with the severity of this disease in type 2 diabetic nephropathy patients [73].

In a second type of inflammatory disease, CD146 has also been identified in primary cultures of keratinocytes while its expression was not observed on healthy skin. An increase in the expression of CD146 has been observed in various skin diseases. For example, CD146 is expressed in suprabasals keratinocytes of psoriasis patients [74]. CD146 is also detected in Kaposi's sarcoma, lichen planus, on the skin overlying skin neoplasms or in chronic and acute chronic dermatitis [74]. On the other hand, the expression of CD146 is not increased in other skin diseases such as lupus erythematosus.

#### **6.3. Tumor pathologies**

CD146 is expressed in many cancers, such as melanoma, prostate cancer, breast cancer, pancreatic cancer, lung cancer, Kaposi sarcoma, angiosarcoma, Schwann cells tumors, or leiomyosarcoma.

The role of tumor CD146 was first studied in melanoma. A direct correlation has been demonstrated between the increase in metastasizing capacities and the increased expression of CD146 [75]. The level of expression of CD146 by human melanoma cell lines has been shown to correlate with their ability to form tumors and metastasis in a mouse xenograft model in immunodeficient nude or SCID mice [76]. In addition, CD146 increases the number of lung metastases following intravenous injection of melanoma cells in nude mice *in vivo* [77]. These observations were confirmed through the use of interfering RNA directed against CD146 leading to a decrease in migration, proliferation, and invasion *in vitro* [78].

CD146 immunohistochemistry staining was performed on human primary melanoma tissue, showing a CD146 expression on tumor-associated endothelium and on smooth muscle cells [79]. The role of CD146 in tumor angiogenesis has been described in particular thanks to the use of the AA98 antibody [37, 64] that is able to block tumor angiogenesis and decrease tumor growth of human melanoma xenograft model in immunodeficient mice.

Currently, mechanisms involved in melanoma progression are unclear. A study showed that a particular population of B lymphocyte cells, the B1 lymphocytes, has a prometastatic potential. Indeed, depletion of this population caused a decrease of tumor growth and metastasis dissemination in mice in an experimental metastasis model, following a B16 cell line injection. The decrease in metastases dissemination involved homophilic interactions between B1 and B16 cells thanks to CD146 [80]. In addition, coculture of B1 cells with melanoma cells increased the expression of CD146 at the cell membrane of cancer cells, increasing the number of metastases *in vivo*.

A clinical study was conducted on a cohort of patients with skin cancer. Patients were divided into two groups: early and late stage melanoma, in order to analyze the presence of different commonly used cancer markers including CD146. Analysis in the blood of patients showed that CD146 was the only protein correlated with the advanced stages of the disease [81]. Another study confirmed this finding by demonstrating that CD146 is a marker of poor prognosis and survival in melanoma patients. CD146 constitutes a better marker than biopsies analysis of sentinel lymph node [82].

## **6.4. Angiogenesis-related diseases and therapeutic approaches**

**6.2. Inflammatory diseases**

2 diabetic nephropathy patients [73].

skin diseases such as lupus erythematosus.

**6.3. Tumor pathologies**

leiomyosarcoma.

*vitro* [78].

Endothelial functions are altered in inflammatory diseases.

442 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

In inflammatory kidney disease, biopsies of patients with nephropathy show an increased expression of membrane CD146 on endothelial cells, but also on the mesangial cells and a neo-expression of CD146 on tubular cells [72]. In addition, there is a correlation between CD146 expression and proteinuria, endocapillary proliferation and inflammatory syndrome. The serum level of soluble CD146 is also modulated. Thus, an increase in CD146 secretion was observed in chronic renal failure which was correlated with the severity of this disease in type

In a second type of inflammatory disease, CD146 has also been identified in primary cultures of keratinocytes while its expression was not observed on healthy skin. An increase in the expression of CD146 has been observed in various skin diseases. For example, CD146 is expressed in suprabasals keratinocytes of psoriasis patients [74]. CD146 is also detected in Kaposi's sarcoma, lichen planus, on the skin overlying skin neoplasms or in chronic and acute chronic dermatitis [74]. On the other hand, the expression of CD146 is not increased in other

CD146 is expressed in many cancers, such as melanoma, prostate cancer, breast cancer, pancreatic cancer, lung cancer, Kaposi sarcoma, angiosarcoma, Schwann cells tumors, or

The role of tumor CD146 was first studied in melanoma. A direct correlation has been demonstrated between the increase in metastasizing capacities and the increased expression of CD146 [75]. The level of expression of CD146 by human melanoma cell lines has been shown to correlate with their ability to form tumors and metastasis in a mouse xenograft model in immunodeficient nude or SCID mice [76]. In addition, CD146 increases the number of lung metastases following intravenous injection of melanoma cells in nude mice *in vivo* [77]. These observations were confirmed through the use of interfering RNA directed against CD146 leading to a decrease in migration, proliferation, and invasion *in* 

CD146 immunohistochemistry staining was performed on human primary melanoma tissue, showing a CD146 expression on tumor-associated endothelium and on smooth muscle cells [79]. The role of CD146 in tumor angiogenesis has been described in particular thanks to the use of the AA98 antibody [37, 64] that is able to block tumor angiogenesis and decrease tumor

Currently, mechanisms involved in melanoma progression are unclear. A study showed that a particular population of B lymphocyte cells, the B1 lymphocytes, has a prometastatic potential. Indeed, depletion of this population caused a decrease of tumor growth and metastasis dissemination in mice in an experimental metastasis model, following a B16 cell line injection. The decrease in metastases dissemination involved homophilic interactions between B1 and B16 cells thanks to CD146 [80]. In addition, coculture of B1 cells with melanoma cells

growth of human melanoma xenograft model in immunodeficient mice.

Recent studies revealed that both isoforms of CD146 are involved in angiogenesis with a promigratory and a proproliferative role of the short CD146 and a vessel stabilization role of long CD146, which is also described in this chapter. Soluble CD146 secreted by both endothelial and cancer cells is also able to stimulate angiogenesis. These different forms are involved in physiological angiogenesis but also in pathological angiogenesis, in particular in tumor angiogenesis.

Therefore, different antibodies have been generated to block its functions. The first one was ABX-MA1, an antibody recognizing the human form of this molecule. This antibody was able to inhibit the formation of spheroids containing melanoma cells, reducing metastasis, tumorigenicity, and vascularization of the tumor *in vivo*. This reduction was related to the inhibition of MMP-2 expression which is heavily involved in metastasis formation [83].

Another team-generated monoclonal antibody specifically directed against the vascular endothelium of tumors. During the screening of these antibodies, the authors focused on the AA98 antibody. This antibody recognizes CD146 localized in the intratumoral vasculature but not recognizes CD146 expressed on blood vessels in healthy tissues [37]. This antibody inhibits both *in vitro* and *in vivo* angiogenic properties of CD146 in human tumors xenografted in immunodeficient mice. In addition, it was demonstrated that the AA98 antibody is a potential diagnostic and therapeutic agent in vascular and cancer diseases. Following this work, it was shown that AA98 antibody inhibits phosphorylation of p38/MAPK, suppresses NFkB activation, and inhibits MMP-9 and ICAM-1 expression. This suggests that deleting NFkB is a pivotal point of the inhibitory effects of the antibody on endothelial cell migration, angiogenesis, and development of tumor metastases [64].

Of interest, this antibody displays additive inhibitory effects when used in combination with the anti-VEGF antibody bevacizumab in xenografted models of human pancreatic tumors and melanoma [57]. In addition, it reduces significantly the chronic inflammation in the colon in a mouse model and prevents the development of cancer associated with this chronic inflammation [38].

Recently, a novel antibody was generated against the soluble form of CD146 [20]. The authors demonstrated that this antibody was able to decrease tumor angiogenesis and growth but to also induce apoptosis of human melanoma and pancreatic tumors xenografted in immunodeficient mice. Of interest, this antibody cannot bind membrane CD146, a property that should limit the side effects that could be observed with antibodies targeting the membrane form.

**Figure 4.** Functions of the different isoforms of CD146 and the inhibitory antibodies associated during tumor growth and angiogenesis.

Functions of the different CD146 isoforms and the inhibitory antibodies associated are summarized in **Figure 4**.

## **Author details**

Jimmy Stalin1 \*, Lucie Vivancos<sup>2</sup> , Nathalie Bardin3 , Françoise Dignat-George<sup>3</sup> and Marcel Blot-Chabaud<sup>3</sup>

\*Address all correspondence to: jimmy.stalin@unige.ch

1 Department of Pathology and Immunology, University Medical Center, Geneva University, Switzerland

2 Pediatric Hematology-Oncology Research Laboratory, Pediatric Division, University Hospital CHUV, Lausanne, Switzerland

3 INSERM UMR-S 1076, Aix-Marseille University, UFR Pharmacy, Marseille, France
