**3. CD146 localization**

All the data concerning the expression of the different isoforms of CD146 and their functions are summarized in **Figures 1** and **2**.

## **3.1. Localization in cancer cells**

CD146 has been identified for the first time in melanoma where it plays an important role in disease progression. Thereafter, CD146 has been shown to be expressed in various cancers, such as pancreatic/breast/prostate/ovarian/lung/kidney cancers, osteosarcoma, Kaposi sarcoma, angiosarcoma, Schwann cell tumors, or leiomyosarcoma (**Figure 1**). The mechanism of this neo-expression is still largely unknown but, in prostate cancer, it was reported that high expression of CD146 resulted from hypermethylation at the promoter of the CD146 gene [19].

However, almost nothing is known on the differential expressions and localizations of the different isoforms of CD146 in these cells. A recent study has shown that many cancer cells expressing CD146 were able to secrete soluble CD146 through a metalloprotease-dependant shedding [20].


**Figure 1.** Summary table for the different isoforms of CD146 expressed in several organs and cells related to their functions, pathologies, and references associated.

MCAM and its Isoforms as Novel Targets in Angiogenesis Research and Therapy http://dx.doi.org/10.5772/66765 433

**Figure 2.** Expression, cell localization, and functions of the different isoforms of CD146 by endothelial cells (EC) and blood circulating cells.

## **3.2. Vascular localization**

**3. CD146 localization**

are summarized in **Figures 1** and **2**.

432 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

functions, pathologies, and references associated.

**3.1. Localization in cancer cells**

gene [19].

shedding [20].

All the data concerning the expression of the different isoforms of CD146 and their functions

CD146 has been identified for the first time in melanoma where it plays an important role in disease progression. Thereafter, CD146 has been shown to be expressed in various cancers, such as pancreatic/breast/prostate/ovarian/lung/kidney cancers, osteosarcoma, Kaposi sarcoma, angiosarcoma, Schwann cell tumors, or leiomyosarcoma (**Figure 1**). The mechanism of this neo-expression is still largely unknown but, in prostate cancer, it was reported that high expression of CD146 resulted from hypermethylation at the promoter of the CD146

However, almost nothing is known on the differential expressions and localizations of the different isoforms of CD146 in these cells. A recent study has shown that many cancer cells expressing CD146 were able to secrete soluble CD146 through a metalloprotease-dependant

**Figure 1.** Summary table for the different isoforms of CD146 expressed in several organs and cells related to their

CD146 is expressed on the whole vascular tree whatever the vessel anatomical location and caliber. The localization of the long and short isoforms of CD146 is different. The induction of long CD146 expression in the CHO cell line (which does not constitutively express CD146) results in the expression of the protein at the intercellular junctions. Costaining of CD146 with VE-cadherin, focal adhesion kinase (FAK), PECAM, and the complex catenin/cadherin shows no colocalization, suggesting that CD146 is not located in the adherent junctions, tight junctions, or focal adhesions sites [21, 22].

Overexpression of the long form of CD146 in the MDCK cell line (Madin-Darby canine kidney) leads to a basolateral localization of the protein. A dileucine motif on its intracytoplasmic peptide sequence is necessary for this localization [21]. An immunohistochemical staining of long CD146 in endothelial colony-forming cells (ECFC) confirmed this junctional localization of the protein. In addition, the presence of a cytoplasmic pool of long CD146 that can be redistributed to the cell membrane was also described in Ref. [23].

The short isoform of CD146 does not share the same cellular localization. Transfection shows an apical localization of the protein in MDCK cells [21] that was confirmed in ECFC in a culture with a specific antibody generated against this isoform [23].

The confluence state of endothelial cells appears to regulate the spatial distribution of the two isoforms. Indeed, the long CD146 isoform was not detected at the junction in nonconfluent endothelial cells. Under this condition, the long CD146 isoform was intracytoplasmic and the short CD146 isoform was essentially nuclear and at the migration front [23]. In other experiments performed in chickens, it was shown a preferential localization of the long isoform of CD146 in the microvilli where the protein plays a role in their formation. Overexpression of CD146 increased the size of these microvilli [24].

## **3.3. Localization on immune cells**

On peripheral blood of healthy patients approximately 1% of blood mononuclear cells express CD146. An analysis by flow cytometry of different lymphocyte populations showed an expression of CD146 on B and T lymphocytes in humans [25].

Research has shown that about 1% of B lymphocytes cells express CD146 and its expression is upregulated by a factor 5 following stimulation with IL-4 and CD40. Moreover, CD146 can be neo-expressed on some cell populations after stimulation [25]. The generation of two antibodies by rat immunization using cells from the T lymphocytic cell line HUT102 deepened these studies and shown that 2% of CD3+, CD3+/CD4+, and CD3+/CD8+ lymphocytes express CD146.

Moreover, stimulations with IL-2 [25] and PHA (phytohemagglutinin) [26] increases the amount of CD146+ T lymphocytes. The cells are also found *in vivo* in the synovial fluid of patients with rheumatoid arthritis [26].

In mice, a leucocytes screening was carried out which demonstrated that CD146 is not detectable on T/B lymphocyte populations, monocytes, and dendritic cells while 30% of neutrophils and 60% of NK cells express CD146. CD146 expression was correlated with an increased expression of CD11b and CD27 reflecting the maturity of NK. These CD146+ NK cells have a decreased cytotoxicity and produce gamma interferon in smaller quantities [27].

## **3.4. Bone marrow environment**

In adults, hematopoiesis takes place in the bone marrow located in long bones of the human body. It is composed of a dense network of discontinuous capillaries allowing easy passage of cells produced in the bone marrow into the blood. A vascular sinus network which is mainly composed of stromal cells (reticular, endothelial, adipocyte, and osteoblast) serves to support the hematopoiesis process.

In one particular study, a subpopulation of bone marrow stroma cells was shown to express CD146 and to display characteristics of mural cells. They were characterized as a subpopulation of advential reticular cells which are abundant in the bone marrow and are able to generate bone tissue and a hematopoietic environment after isolation and implantation into an immunodeficient mouse [28].

Furthermore, angiopoietin -1 is regulated by CD146+ stromal cells. A decrease in the expression of CD146 by siRNA or FGF-2 (CD146 and Ang-1 regulator) reduces the capacity of these cells to participate in the remodeling and the assembly of pseudovascular structures *in vitro*

and to form hematopoietic microenvironment *in vivo*. From data on the spatial location of adventitial reticular cells and the expression of Tie-2 (the angiopoietin-1 receptor), it was suggested that CD146 and angiopoietin-1 are involved in the interaction between endothelial and stromal cells [29].

#### **3.5. Localization in the central nervous system (CNS)**

The confluence state of endothelial cells appears to regulate the spatial distribution of the two isoforms. Indeed, the long CD146 isoform was not detected at the junction in nonconfluent endothelial cells. Under this condition, the long CD146 isoform was intracytoplasmic and the short CD146 isoform was essentially nuclear and at the migration front [23]. In other experiments performed in chickens, it was shown a preferential localization of the long isoform of CD146 in the microvilli where the protein plays a role in their formation. Overexpression of

On peripheral blood of healthy patients approximately 1% of blood mononuclear cells express CD146. An analysis by flow cytometry of different lymphocyte populations showed

Research has shown that about 1% of B lymphocytes cells express CD146 and its expression is upregulated by a factor 5 following stimulation with IL-4 and CD40. Moreover, CD146 can be neo-expressed on some cell populations after stimulation [25]. The generation of two antibodies by rat immunization using cells from the T lymphocytic cell line HUT102 deepened these studies and shown that 2% of CD3+, CD3+/CD4+, and CD3+/CD8+ lymphocytes express CD146.

Moreover, stimulations with IL-2 [25] and PHA (phytohemagglutinin) [26] increases the amount of CD146+ T lymphocytes. The cells are also found *in vivo* in the synovial fluid of

In mice, a leucocytes screening was carried out which demonstrated that CD146 is not detectable on T/B lymphocyte populations, monocytes, and dendritic cells while 30% of neutrophils and 60% of NK cells express CD146. CD146 expression was correlated with an increased expression of CD11b and CD27 reflecting the maturity of NK. These CD146+ NK cells have a

In adults, hematopoiesis takes place in the bone marrow located in long bones of the human body. It is composed of a dense network of discontinuous capillaries allowing easy passage of cells produced in the bone marrow into the blood. A vascular sinus network which is mainly composed of stromal cells (reticular, endothelial, adipocyte, and osteoblast) serves to support

In one particular study, a subpopulation of bone marrow stroma cells was shown to express CD146 and to display characteristics of mural cells. They were characterized as a subpopulation of advential reticular cells which are abundant in the bone marrow and are able to generate bone tissue and a hematopoietic environment after isolation and implantation into an

Furthermore, angiopoietin -1 is regulated by CD146+ stromal cells. A decrease in the expression of CD146 by siRNA or FGF-2 (CD146 and Ang-1 regulator) reduces the capacity of these cells to participate in the remodeling and the assembly of pseudovascular structures *in vitro*

decreased cytotoxicity and produce gamma interferon in smaller quantities [27].

CD146 increased the size of these microvilli [24].

an expression of CD146 on B and T lymphocytes in humans [25].

434 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

**3.3. Localization on immune cells**

patients with rheumatoid arthritis [26].

**3.4. Bone marrow environment**

the hematopoiesis process.

immunodeficient mouse [28].

CD146 is found in the central nervous system (CNS). It is expressed during fetal development of the embryo but decreases after birth. Studies performed in chickens and rats have shown an expression of CD146 in the cerebellum, hippocampus, Purkinje cells, and sensorimotor cells of the spinal cord [30]. In chickens, CD146 binds NOF (neurite outgrowth factor), causing neurite extension [31], and increases the extension of the optic tectum process [32].
