Mel-CAM (also termed CD146 or MUC18) is a cell-adhesion molecule belonging to the immunoglobulin gene superfamily. It mediates cell-cell interaction through heterophilic Mel-CAM/ligand adhesion. Previous studies showed Mel-CAM immunoreactivity in normal and neoplastic human tissues, but an extensive assessment of Mel-CAM distribution was not performed because of the lack of a Mel-CAM-specific monoclonal antibody that could be used in routinely processed, formalin-fixed, paraffin-embedded tissues. Therefore, we developed a mouse monoclonal antibody, MN-4, that specifically recognized a fixation-resistant epitope in the second extracellular (C2) domain of Mel-CAM. We performed immunohistochemical staining on 467 paraffin-embedded tissue samples with this MN-4 antibody and avidin-biotin peroxidase. MN-4 immunoreactivity was seen in normal tissues, including endothelium, smooth muscle, epithelial and myoepithelial cells of the breast, Schwann cells, ganglion cells, cerebellar cortex, implantation-site intermediate trophoblast, external root sheath of hair follicles, basal cells of bronchial epithelium, parathyroid glands, subcapsular epithelium of thymus, follicular dendritic reticulum cells, skeletal muscles, epithelium of lens, and glial cell fibers in the central nervous system in early embryos. In malignant tumors, MN-4 immunostaining was consistently present in all melanomas, angiosarcomas, Kaposi's sarcomas, leiomyosarcomas, mucoepidermoid carcinomas of salivary gland, placental-site trophoblastic tumors, and choriocarcinomas. Three of 8 squamous cell carcinomas, 2 of 2 small cell carcinomas of the lung, 2 of 11 of infiltrating breast carcinomas, and 4 of 11 of germinomas were focally and weakly positive for MN-4 antibody. In contrast, a wide variety of other carcinomas, sarcomas, lymphomas, leukemias, and neuroendocrine tumors failed to show MN-4 immunoreactivity. In conclusion, MN-4 is a specific monoclonal antibody that recognizes Mel-CAM on paraffin sections. This antibody is useful in retrospective studies of Mel-CAM expression in archival tissue sections and might provide a diagnostic and prognostic marker for human neoplasms.

BACKGROUND

Mature circulating endothelial cells (CECs) are surrogate markers of endothelial damage/dysfunction. A lack of standardized assays and consensus on CEC phenotype has resulted in a wide variation of reported CEC numbers (4-1300 per mL).

OBJECTIVE

Given the need for a quick, reliable, robust and validated CEC assay at an affordable price, we present a novel approach to enumerate CECs using a multi-parameter flow cytometric (FCM) method without immunological pre-enrichment.

METHODS

CECs were defined as CD34+, CD45neg, CD146+ and DNA+ events based on the immunophenotype of endothelial cells from vein-wall dissections. As CECs express high levels of CD34, we based our assay on absolute CD34 counts after analyzing all CD34 positive events in a total blood volume of 4 mL needed for a precise enumeration of CECs at a frequency of < 1 cell μL(-1).

RESULTS

The endothelial origin of CECs was confirmed by morphology, immunohistochemistry and gene expression. The new FCM assay was tested in parallel with a validated assay (i.e. CellSearch). CEC levels ranged from 4 to 79 CEC mL(-1) in healthy individuals and were significantly higher in patients with advanced solid malignancies (P = 0.0008) and in patients with hematological malignancies (P < 0.0001).

CONCLUSIONS

This flow cytometric method should be useful as a fast and economical assay to enumerate and characterize CECs.

The identification of skeletal progenitor cells in the human bone marrow (so-called mesenchymal stem cells) by anatomy and phenotype (CD146-expressing, adventitial reticular cells) has coincided with the recognition that the ability to transfer the hematopoietic microenvironment is an inherent property of skeletal progenitor cells. Inasmuch as these cells generate osteoblasts, associate with sinusoids (the assembly of which they dynamically direct), and coincide with, and self-renew into, stromal reticular cells, these cells are pivotal organizers of the hematopoietic microenvironment. Their nature as osteogenic cells and sinusoidal location reconcile the dual view of endosteal surfaces and sinusoidal walls as the hematopoietic stem cell "niches", and highlight the dynamic nature of a niche/microenvironment essentially maintained by cells with properties of progenitors/stem cells for skeletal tissues. This view brings the long recognized, and somewhat mysterious, interaction between bone and bone marrow into a new perspective, where two stem cells interact with each other at the same niche.

BACKGROUND

Crohn's disease (CD) and ulcerative colitis (UC), the 2 major forms of inflammatory bowel diseases (IBD), have been associated with disturbances in vascular physiology, including permeability and angiogenesis, that are in part regulated by the endothelial intercellular junctions. These junctions are composed of several adhesion molecules including the platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) and the more recently described CD146 (S-Endo1 Ag, MUC18).

OBJECTIVE

To study the expression of tissue and soluble form of CD146 in patients with CD or UC in relation to disease activity and location. This study was made in comparison with the soluble form of CD31 (sCD31).

RESULTS

In active disease, a high expression of CD146 was observed on endothelial cells in intestinal biopsies from both CD and UC. In addition, we observed a decrease of sCD146 in relation to active disease and extensive location of CD and UC. Lower levels of sCD31 were also detected in active and extensive location of UC, but no difference could be observed in CD.

CONCLUSIONS

sCD146 is a novel marker of the endothelial intercellular junction that reflects endothelial remodeling more effectively than soluble CD31. Further studies are warranted to determine whether sCD146 will provide a serological assay reflecting alterations in vascular permeability and vessel proliferation in the inflamed IBD intestine.

The acquisition of the metastatic melanoma phenotype is associated with increased expression of the melanoma cell adhesion molecule MCAM/MUC18 (CD146). However, the mechanism by which MUC18 contributes to melanoma metastasis remains unclear. Herein, we stably silenced MUC18 expression in two metastatic melanoma cell lines, A375SM and C8161, and conducted cDNA microarray analysis. We identified and validated that the transcriptional regulator, inhibitor of DNA binding-1 (Id-1), previously shown to function as an oncogene in several malignancies, including melanoma, was downregulated by 5.6-fold following MUC18 silencing. Additionally, we found that MUC18 regulated Id-1 expression at the transcriptional level via ATF-3, which itself was upregulated by 6.9-fold in our cDNA microarray analysis. ChIP analysis showed increased binding of ATF-3 to the Id-1 promoter after MUC18 silencing. To complement these studies, we rescued the expression of MUC18, which reversed the expression patterns of Id-1 and ATF-3. Moreover, we showed that MUC18 promotes melanoma invasion through Id-1, as overexpression of Id-1 in MUC18-silenced cells resulted in increased MMP-2 expression and activity. To our knowledge, this is the first demonstration that MUC18 is involved in cell signaling regulating the expression of Id-1 and ATF-3, thus contributing to melanoma metastasis.

To investigate the role of miRNA in controlling human embryonic stem (hES) cell differentiation toward the endothelial lineage and chick embryonic blood vessel formation, undifferentiated hES cells were first cultured on Matrigel-coated flasks and in endothelial cell growth medium-2 (EGM-2) to initiate endothelial cell (EC) differentiation. CD146(+) cells were isolated from differentiating hES cells and expanded in vitro. The in vitro expanded CD146(+) cells were positive for EC markers, capable of Ac-LDL uptake, lectin binding, and the formation of vascular structures in vitro and in vivo. miRNA gain/loss-of-function analyses revealed that miR-150 and miR-200c were crucial in EC differentiation. Transcriptional repressor zinc finger E-box-binding homeobox 1 (ZEB1) was identified as the communal target gene of miRNA-200C and -150, and inhibition of ZEB1 was required for miRNA-200C or -150 mediated EC gene expressions. Moreover, we demonstrated that ZEB1 could transcriptionally repress EC gene expression through direct binding to promoters of EC genes. Finally, we also demonstrated that miRNA-200c and -150 played an important role in chick embryonic blood vessel formation by in vivo inhibition of miRNA-200C or -150 in developing chick embryos, and blocking ZEB1 signaling in CD146-positive cells could rescue the inhibitory effects of miR-200c inhibiton in in vivo vasculogenesis. Our findings revealed that miR-150 and miR-200c play an important role in human endothelial lineage specification and chick embryonic vasculogenesis by targeting ZEB1.

OBJECTIVE

Enzastaurin targets the protein kinase C and phosphatidylinositol 3-kinase/AKT pathways to reduce tumor angiogenesis and cell proliferation and to induce cell death. A phase I trial was conducted to evaluate the feasibility of combining enzastaurin with gemcitabine and cisplatin.

METHODS

Patients with advanced cancer received a 14-day lead-in treatment with oral enzastaurin followed by subsequent 21-day cycles of daily enzastaurin, gemcitabine on days 1 and 8, and cisplatin on day 1. Enzastaurin doses were escalated between 350 mg once daily to 500 mg twice daily, whereas gemcitabine doses were either 1,000 or 1,250 mg/m(2) and cisplatin doses were either 60 or 75 mg/m(2). Circulating endothelial cell numbers and CD146 and CD133 mRNA expression were evaluated as pharmacodynamic markers.

RESULTS

Thirty-three patients (median age, 58 years) were enrolled in seven dose levels. The maximum tolerated dose was not identified. Two dose-limiting toxicities (grade 2 QT interval corrected for heart rate prolongation and grade 3 fatigue) were reported. Other toxicities included grade 3/4 neutropenia (3 of 6 patients), thrombocytopenia (1 of 6 patients), grade 3 leukopenia (2 patients), and fatigue (5 patients). Enzastaurin twice daily >> or =250 mg) resulted in more discontinuations and low-grade toxicities. In the combination, enzastaurin exposures decreased slightly but remained above the target of 1,400 nmol/L, whereas gemcitabine/cisplatin exposures were unaltered. Three patients (9.1%) had partial responses and 13 (39.4%) had stable disease. Measurement of circulating endothelial cell numbers and CD146 and CD133 mRNA expression did not contribute to decision-making on dose escalation.

CONCLUSIONS

Recommended phase II dose is 500 mg enzastaurin once daily, 1,250 mg/m(2) gemcitabine, and 75 mg/m(2) cisplatin. This regimen is well tolerated with no significant alterations in the pharmacokinetic variables of any drug.

BACKGROUND

Damage of microvascular endothelial cells is a salient feature of acute vascular rejection and chronic allograft nephropathy, yet specific blood markers of ongoing endothelial injury are currently unavailable. Circulating endothelial cells have recently been established as a novel marker of endothelial damage in a variety of vascular disorders.

METHODS

We studied 129 renal transplant recipients who underwent percutaneous graft biopsy. Circulating endothelial cells were isolated with immunomagnetic anti-CD146-coated Dynabeads. Cells were stained with acridine and counted. To verify their endothelial origin, staining for Ulex europaeus lectin 1 (UEA-1) was performed in parallel. Twenty-one healthy controls were also studied.

RESULTS

On biopsy, seven patients had acute vascular rejection, 15 patients had acute tubulointerstitial rejection, 14 patients had borderline rejection, and 93 patients had no rejection. Patients with acute vascular rejection had the highest cell numbers (72+/-39 cells/mL) when compared with all other patients (P<0.02). Regardless of their biopsy findings, however, all other renal transplant recipients had significantly higher numbers of circulating endothelial cells (25+/-20 cells/mL) than healthy controls (7+/-5 cells/mL, P<0.001). Finally, there was a significant correlation of cell numbers and serum cyclosporine A trough levels. By contrast, there was no correlation with serum creatinine, age, or the number of immunosuppressive drugs.

CONCLUSIONS

The number of circulating endothelial cells is a novel marker of endothelial damage in renal transplant recipients. Further studies must now evaluate the origin of these cells, corroborate the clinical significance of our findings, and delineate the influence of calcineurin inhibitors.

Mesenchymal stem cells (MSCs) are one of the most promising stem cell types due to their availability and relatively simple requirements for in vitro expansion and genetic manipulation. Besides the well-characterized MSCs derived from bone marrow, there is growing evidence suggesting that dental pulp and the umbilical cord matrix both contain a substantial amount of cells having properties similar to those of MSCs. In order to assess the potential of dental pulp-derived MSCs (DPSC) and umbilical cord-derived MSCs (UCSC) in future clinical applications, it is essential to gain more insight into their differentiation capacity and to evaluate the tissues formed by these cells. In the present study, the morphological and ultrastructural characteristics of DPSC and UCSC induced towards osteogenic, adipogenic, and chondrogenic lineages were investigated. Cultured DPSC and UCSC showed a similar expression pattern of antigens characteristic of MSCs including CD105, CD29, CD44, CD146, and STRO-1. Under appropriate culture conditions, both DPSC and UCSC showed chondrogenic and osteogenic potential. Adipogenesis could be only partially induced in DPSC resulting in the de novo expression of fatty acid binding protein (FABP), whereas UCSC expressed FABP combined with a very high accumulation of lipid droplets in the cytoplasm. Our results demonstrate, at the biochemical and ultrastructural level, that DPSC display at least bilineage potential, whereas UCSC, which are developmentally more primitive cells, show trilineage potential. We emphasize that transmission electron microscopical analysis is useful to elucidate detailed structural information and provides indisputable evidence of differentiation. These findings highlight their potential therapeutic value for cell-based tissue engineering.

Regenerative therapy using stem cells is a promising approach for the treatment of stroke. Recently, we reported that CD31⁻/CD146⁻ side population (SP) cells from porcine dental pulp exhibit highly vasculogenic potential in hindlimb ischemia. In this study, we investigated the influence of CD31⁻/CD146⁻ SP cells after transient middle cerebral artery occlusion (TMCAO). Adult male Sprague-Dawley rats were subjected to 2 h of TMCAO. Twenty-four hours after TMCAO, CD31⁻/CD146⁻ SP cells were transplanted into the brain. Motor function and infarct volume were evaluated. Neurogenesis and vasculogenesis were determined with immunochemical markers, and the levels of neurotrophic factors were assayed with real-time reverse transcription-polymerase chain reaction. In the cell transplantation group, the number of doublecortin-positive cells increased twofold, and the number of NeuN-positive cells increased eightfold, as compared with the control phosphate-buffered saline group. The vascular endothelial growth factor level in the ischemic brain with transplanted cells was 28 times higher than that in the normal brain. In conclusion, CD31⁻/CD146⁻ SP cells promoted migration and differentiation of the endogenous neuronal progenitor cells and induced vasculogenesis, and ameliorated ischemic brain injury after TMCAO.

Although mesenchymal stem cells (MSC) isolated from bone marrow and adipose tissues are known to be subjected to in vitro culture-related alterations in their stem cell properties, such data have not been reported in human tonsil-derived MSC (T-MSC). Here, we investigated the culture-related changes of phenotypes, the senescence, and the differentiation potential of T-MSC. T-MSC were serially passaged by a standard protocol, and their characteristics were assessed, including MSC-specific surface antigen profiles, the senescence, and the differentiation potentials into adipocytes, chondrocytes and osteocytes. Up to at least passage 15, we found no alterations in either MSC-specific surface marker, CD14, CD34, CD45, CD73 and CD90, or the mRNA expression of embryonic stem cell gene markers, Nanog, Oct4-A and Sox-2. However, the expression of CD146, recently identified another MSC marker, dramatically decreased with increasing passages from ~ 23% at passage 3 to ~ 1% at passage 15. The average doubling time increased significantly from ~ 38 h at passage 10 to ~ 46 h at passage 15. From passage 10, the cell size increased slightly and SA-β-gal staining was evident. Both Alizarin Red S staining and osteocalcin expression showed that the osteogenic differentiation potential increased up to passage 10 and decreased thereafter. However, the adipogenic and chondrogenic differentiation potential decreased passage-dependently from the start, as evidenced by staining of Oil Red O and Alcian Blue, respectively. Consistent with a passage-dependent osteogenic differentiation, the expression of CCN1, an angiogenic protein known to be related to both senescence and osteogenesis, also increased up to passage 10. Furthermore, ectopic expression of small interfering RNA against CCN1 at passage 10 significantly reversed Alizarin Red S staining and osteocalcin expression. Altogether, our study demonstrates the characterization of long-term in vitro cultured T-MSC and that CCN1 may be involved in mediating a passage-dependent increase in osteogenic potential of T-MSC.

BACKGROUND

Identification of surface markers for prospective isolation of functionally homogenous populations of human skeletal (stromal, mesenchymal) stem cells (hMSCs) is highly relevant for cell therapy protocols. Thus, we examined the possible use of CD146 to subtype a heterogeneous hMSC population.

METHODS

Using flow cytometry and cell sorting, we isolated two distinct hMSC-CD146(+) and hMSC-CD146(-) cell populations from the telomerized human bone marrow-derived stromal cell line (hMSC-TERT). Cells were examined for differences in their size, shape and texture by using high-content analysis and additionally for their ability to differentiate toward osteogenesis in vitro and form bone in vivo, and their migrational ability in vivo and in vitro was investigated.

RESULTS

In vitro, the two cell populations exhibited similar growth rate and differentiation capacity to osteoblasts and adipocytes on the basis of gene expression and protein production of lineage-specific markers. In vivo, hMSC-CD146(+) and hMSC-CD146(-) cells formed bone and bone marrow organ when implanted subcutaneously in immune-deficient mice. Bone was enriched in hMSC-CD146(-) cells (12.6 % versus 8.1 %) and bone marrow elements enriched in implants containing hMSC-CD146(+) cells (0.5 % versus 0.05 %). hMSC-CD146(+) cells exhibited greater chemotactic attraction in a transwell migration assay and, when injected intravenously into immune-deficient mice following closed femoral fracture, exhibited wider tissue distribution and significantly increased migration ability as demonstrated by bioluminescence imaging.

CONCLUSIONS

Our studies demonstrate that CD146 defines a subpopulation of hMSCs capable of bone formation and in vivo trans-endothelial migration and thus represents a population of hMSCs suitable for use in clinical protocols of bone tissue regeneration.

The respective abundance of circulating endothelial cells and endothelial progenitor cells may reflect the balance between vascular injury and repair. As pulmonary arterial hypertension (PAH) and chronic thromboembolic pulmonary hypertension (CTEPH) can share features of pulmonary remodelling, we postulated that the two disorders might be associated with different types of pulmonary endothelial dysfunction. We studied 25 consecutive patients undergoing cardiac catheterisation for suspected pulmonary hypertension. Nine patients had PAH, nine had CTEPH, and seven had normal pulmonary arterial pressure and served as controls. Circulating endothelial cells were isolated with CD146-coated beads. CD34(+)CD133(+) cell and endothelial progenitor cell numbers were respectively determined by flow cytometry and cell culture, in peripheral vein and pulmonary artery blood. Plasma levels of soluble vascular endothelial growth factor (VEGF), soluble E-selectin and soluble vascular cell adhesion molecule (sVCAM) were measured by ELISA. No difference in progenitor counts or VEGF levels was found across the three groups. Compared to controls, circulating endothelial cell numbers were significantly increased in PAH but not in CTEPH, in keeping with the elevated soluble E-selectin and sVCAM levels found in PAH alone. In conclusion, PAH, in contrast to CTEPH, is associated with markers of vascular injury (circulating endothelial cells, soluble E-selectin and sVCAM) but not with markers of remodelling (endothelial progenitor cells, CD34(+)CD133(+) cells and VEGF).

The presence of circulating tumor cells (CTCs) in the blood of ovarian cancer patients was shown to correlate with decreased overall survival, whereby CTCs with epithelial-mesenchymal-transition (EMT) or stem-like traits are supposed to be involved in metastatic progression and recurrence. Thus, investigating the transcriptional profiles of CTCs might help to identify therapy resistant tumor cells and to overcome treatment failure. For this purpose, we established a multi-marker panel for the molecular characterization of single CTCs, detecting epithelial (EpCAM, Muc-1, CK5/7), EMT (N-cadherin, Vimentin, Snai1/2, CD117, CD146, CD49f) and stem cell (CD44, ALDH1A1, Nanog, SOX2, Notch1/4, Oct4, Lin28) associated transcripts. First primer specificity and PCR-performance of the multiplex-RT-PCRs were successfully validated on genomic DNA and cDNA isolated from OvCar3 cells. The assay sensitivity of the epithelial panel was evaluated by adding defined numbers of tumor cells into the blood of healthy donors and performing a subsequent immunomagnetic tumor cell enrichment (AdnaTest OvarianCancerSelect), resulting in a 100% concordance for the epithelial markers EpCAM and Muc-1 to the AdnaTest OvarianCancerDetect. Additionally, by processing blood from ovarian cancer patients, high assay sensitivity could be verified. In blood of healthy donors no signals for epithelial markers were detected, for EMT and stem cell markers, however, signals were obtained mainly originating from leukocytes which calls for single cell analysis. To that aim by using the ovarian cancer cell line OvCar3, we successfully established a workflow enabling the characterization of single CTCs. It consists of a density gradient-dependent enrichment for nucleated cells, a depletion of CD45-positive cells of hematopoietic origin followed by immunofluorescent labeling of CTCs by EpCAM and Muc-1. Single CTCs are then isolated by micromanipulation and processed for panel gene expression profiling. Finally, fifteen single CTCs from three ovarian cancer patients were analyzed and found to be positive for stem cell (CD44, ALDH1A1, Nanog, Oct4) and EMT markers (N-cadherin, Vimentin, Snai2, CD117, CD146). Albeit, inter-cellular and intra/inter-patient heterogeneity and co-expression of epithelial, mesenchymal and stem cell transcripts on the same CTC was observed. We have established a robust workflow to perform sensitive single cell panel gene expression analysis without the need of pre-amplification steps. Our data point towards a heterogeneous expression of stem cell and EMT associated transcripts in ovarian cancer CTCs.

Our previous study showed that an anti-CD146 monoclonal antibody (mAb), AA98, which was raised against the vascular endothelial cells stimulated by a conditioned medium from hepatocarcinoma SMMC 7721 cells (SMMC 7721-CM), inhibited cell migration, angiogenesis, and tumor growth. However, the underlying mechanism was not elucidated. The objective of this study was to understand the mechanism by which mAb AA98 inhibits the endothelial cell migration and angiogenesis that is induced by SMMC 7721-CM. Using confocal imaging and biochemical studies, we found that SMMC 7721-CM induced nuclear factor kappaB (NF-kappaB) activation through the upstream p38 mitogen-activated protein kinase pathway, leading to the up-regulation of matrix metalloproteinase 9 and intercellular adhesion molecule 1 expression. Interestingly, all these activities stimulated by SMMC 7721-CM could be effectively inhibited by mAb AA98 in a dose- and time-dependent manner. Our data showed that the engagement of mAb AA98 with membrane protein CD146 inhibited p38 mitogen-activated protein kinase phosphorylation, suppressed NF-kappaB activation, and down-regulated matrix metalloproteinase 9 and intercellular adhesion molecule 1 expression, suggesting that the suppression of NF-kappaB is a critical point for the inhibitory function of mAb AA98 on endothelial cell migration, angiogenesis, and tumor metastasis. These results will provide clues for a better understanding of the mechanisms underlying tumor angiogenesis as well as antiangiogenesis therapy.

OBJECTIVE

CD146 (MUC18/MCAM/S-Endo) is a marker of tumor progression and metastasis formation in human melanoma. This molecule has also been identified in smooth muscle, endothelial cells, and activated T lymphocytes. We measured the synovial fluid levels of soluble CD146 in various human joint diseases, including rheumatoid arthritis (RA). In addition, we studied the distribution of CD146 in normal and RA synovial tissues.

METHODS

CD146 was isolated from MEL-OH melanoma cells and characterized by Coomassie blue staining and Western blotting. Soluble CD146 was measured by competitive enzyme-linked immunosorbent assay in synovial fluids of 3 healthy individuals and 7 cadavers (controls), as wells as in patients with traumatic joint injury (n = 10), osteoarthritis (OA; n = 10), psoriatic arthritis (PsA; n = 10), other non-RA polyarthritis (NRAP; n = 10), and RA (n = 31). Immunohistochemistry was performed on 3 normal and 3 RA synovial tissues. Flow cytometric, reverse transcription-polymerase chain reaction, and Western blot analyses were performed on enzymatically separated RA synovial tissue cells.

RESULTS

Compared with controls (mean +/- SD 10 +/- 2 ng/ml), significantly elevated synovial fluid levels of soluble CD146 were detected in patients with OA, PsA, and RA (17 +/- 7, 21 +/- 11, and 39 +/- 16 ng/ml, respectively; P < 0.02-0.001), but not in patients with traumatic joint injury or NRAP. Patients with early RA (<1 year after diagnosis) revealed the highest levels (51 +/- 15 ng/ml, n = 10; P < 0.001 versus controls). In RA, soluble CD146 correlated significantly with morning stiffness (P < 0.001), the number of tender joints (P < 0.02), and the number of swollen joints (P < 0.005), but not with the erythrocyte sedimentation rate (P = 0.07) or the C-reactive protein level (P = 0.57).

CONCLUSIONS

Since CD146 is expressed almost exclusively by vascular endothelium, high levels of soluble CD146 found in RA synovial fluid, particularly in patients with early disease, could reflect increased activity of endothelial cells and angiogenesis.

The ability to selectively block the entry of leukocytes into the central nervous system (CNS) without compromising the immune system is an attractive therapeutic approach for treating multiple sclerosis (MS). Using endothelial CD146-deficienct mice as a MS model, we found that endothelial CD146 plays an active role in the CNS-directed extravasation of encephalitogenic T cells, including CD146(+) TH1 and TH17 lymphocytes. Moreover, treating both active and passive MS models with the anti-CD146 antibody AA98 significantly decreased the infiltrated lymphocytes in the CNS and decreased neuroinflammation. Interestingly, the ability of AA98 to inhibit the migration of CD146(+) lymphocytes was dependent on targeting endothelial CD146, but not lymphocytic CD146. These results suggest a key molecular target located on the blood-brain barrier endothelium that mediates the extravasation of inflammatory cells into the CNS. In addition, our data suggest that the AA98 is a promising candidate for treating MS and other CNS autoimmune diseases.

BACKGROUND

Bone marrow aspirate concentrate (BMAC) including high densities of stem cells and progenitor cells may possess a stronger bone regenerative capability compared with Platelet-rich plasma (PRP), which contains enriched growth factors. The objective of this study was to evaluate the effects of human BMAC and PRP in combination with β-tricalcium phosphate (β-TCP) on promoting initial bone augmentation in an immunodeficient mouse model.

RESULTS

BMAC and PRP were concentrated with an automated blood separator from the bone marrow and peripheral blood aspirates. β-TCP particles were employed as a scaffold to carry cells. After cell counting and FACS characterization, three groups of nude mice (BMAC+TCP, PRP+TCP, and a TCP control) were implanted with graft materials for onlay placement on the cranium. Samples were harvested after 4 weeks, and serial sections were prepared. We observed the new bone on light microscopy and performed histomorphometric analysis. After centrifugation, the concentrations of nucleated cells and platelets in BMAC were increased by factors of 2.8 ± 0.8 and 5.3 ± 2.4, respectively, whereas leucocytes and platelets in PRP were increased by factors of 4.1 ± 1.8 and 4.4 ± 1.9, respectively. The concentrations of CD34-, CD271-, CD90-, CD105-, and CD146-positive cells were markedly increased in both BMAC and PRP. The percentage of new bone in the BMAC group (7.6 ± 3.9%) and the PRP group (7.2 ± 3.8%) were significantly higher than that of TCP group (2.7 ± 1.4%). Significantly more bone cells in the new bone occurred in sites transplanted with BMAC (552 ± 257) and PRP (491 ± 211) compared to TCP alone (187 ± 94). But the difference between the treatment groups was not significant.

CONCLUSIONS

Both human BMACs and PRP may provide therapeutic benefits in bone tissue engineering applications. These fractions possess a similar ability to enhance early-phase bone regeneration.

Since the discovery and isolation of a mesenchymal stem cell population from within the stromal vascular fraction (SVF) of adipose tissue, there has been a concerted effort to discover the characteristics of these cells. Particular attention has been paid to their morphology, selfrenewal capacity, multi-lineage differentiation capabilities and, as is of greatest interest in this instance, their cell surface profile.

The purpose of this study is to analyze and summarize the available literature that pertains to the cell surface characterization of adipose-derived stem cells (ASCs). The identification of a common set of positive and negative cell surface markers would allow for a much more consistent and reliable method of identifying this stem cell population both in vitro and in vivo.

The keywords "adipose-derived stem cells; stromal cells; surface markers" were searched in the following electronic databases: Medline, PubMed, ZETOC, Web of Knowledge, AMED, EMBASE, Ovid in process & other non-indexed citations and PsychINFO.

The most commonly reported positive markers were found to be CD90, CD44, CD29, CD105, CD13, CD34, CD73, CD166, CD10, CD49e and CD59, while the most commonly found negative markers were CD31, CD45, CD14, CD11b, CD34, CD19, CD56 and CD146. In addition, a number of other markers appeared in the literature including HLA-ABC, HLA-DR, SH2, SH3, STRO-1, VEGF2, vWF, ABCG2, SSEA-1 (CD15), PDGFR, alpha- SMA, c-Kit (CD117), OCT4+ and CCR5X (CD195).

A minimum panel of positive and negative markers for identifying ASCs can be recommended. The following markers should be positive: CD90, CD44, CD29, CD105, CD13, CD34, CD73, CD166, CD10, CD49e and CD59. The following markers should be negative: CD31, CD45, CD14, CD11b, CD19, CD56 and CD146. In addition to this, the positive expression of HLA- ABC and STRO- 1 should be seen along with the negative expression on HLA-DR. This review, however, has also found that there are a number of disagreements over the expression and existence of various markers, namely CD31, CD34, c- Kit (CD117) and STRO-1.

Using the Bmp2 floxed/3.6Col1a1-Cre (Bmp2-cKO(od)) mouse model, we have observed severe defects in odontogenesis and dentin formation with the removal of the Bmp2 gene in early-polarizing odontoblasts. The odontoblasts in the Bmp2-cKO(od) do not mature properly and fail to form proper dentin with normal dentinal tubules and activate terminal differentiation, as reflected by decreased Osterix, Col1a1, and Dspp expression. There is less dentin, and the dentin is hypomineralized and patchy. We also describe an indirect effect of the Bmp2 gene in odontoblasts on formation of the vascular bed and associated pericytes in the pulp. This vascular niche and numbers of CD146+ pericytes are likely controlled by odontogenic and Bmp2-dependent VegfA production in odontoblasts. The complex roles of Bmp2, postulated to be both direct and indirect, lead to permanent defects in the teeth throughout life, and result in teeth with low quantities of dentin and dentin of poor quality.

CD146/melanoma cell adhesion molecule is an adhesion molecule expressed by endothelial cells and by a small fraction of activated T and B lymphocytes in humans. In order to analyze the pattern of CD146 expression in mouse leukocytes at steady-state conditions, we generated a set of novel rat anti-mouse CD146 monoclonal antibodies. CD146 expression was undetectable on monocytes, dendritic cells, T cells or B cells, but was expressed on about 30% of neutrophils and 60% of NK cells. Within murine lymphocytes, CD146 was defined as a novel NK-specific surface molecule. An increased percentage of CD146+ cells was found in the most mature CD27(-)CD11b+ NK cell subpopulation, which also displays higher expression of Ly49C/I, Ly49D and KLRG1 and lower expression of NKG2A/C/E molecules. CD146+ NK cells were found to be less cytotoxic and produce less IFN-gamma than CD146(-) NK cells upon stimulation with target cells or activating antibodies. These findings define CD146 as a marker of mouse NK cell maturation that may be used as an alternative to the combined use of CD27 and CD11b staining to detect final stages of NK cell maturation.

Bone never forms without vascular interactions. Although this is a very simple and obvious statement, the biological, clinical, and pharmacologic implications are incompletely appreciated. The vasculature is not only the conduit for nutrient-metabolite exchange and the rate-limiting "point-of-reference" for Haversian bone formation, but also provides the sustentacular niche for the self-renewing osteoprogenitor. This past year, significant advances have been made in our understanding of the osteogenic-angiogenic interface that are immediately germane to osteoporosis disease biology and fracture management. The critical contributions of the osteoblast oxygen-sensing machinery, paracrine vascular endothelial growth factor and placental growth factor signaling, fracture-mobilized circulating osteoprogenitors, and the osteogenic CD146(+) marrow sinusoid stem cell have been recently discovered. This brief review recounts these revelations, highlighting the potential impact to human bone health and fracture repair.

Mel-cell adhesion molecule (CAM), also known as MUC18 and CD146, is a novel member of the immunoglobulin supergene family. Mel-CAM was first identified as an integral membrane glycoprotein in human melanoma and is also abundantly expressed by endothelial cells of various origins. In a previous study (I. M. Shih et al., Cancer Res., 54: 2514-2520, 1994), we showed that Mel-CAM is a cell-cell adhesion molecule with a possible role in melanoma invasion and metastasis. Here, we define the molecular mechanism responsible for cell-cell adhesion of Mel-CAM and demonstrate its role in melanoma-endothelial cell interactions. Most of human melanoma cells, including Mel-CAM-negative SBcl-2 cells, adhered to nitrocellulose-immobilized Mel-CAM produced by baculovirus recombinants. This adhesion can be blocked by full-length Mel-CAM or polyclonal antiserum against Mel-CAM. Adhesion is not affected by the presence of EDTA, truncated Mel-CAM extracellular domain, or heparan sulfate proteoglycan. In cell aggregation assays, Mel-CAM-negative SBcl-2 cells cluster with U937TM cells (U937 transfected with Mel-CAM cDNA) but not with control nontransfectants, suggesting that SBcl-2 cells express the ligand for Mel-CAM. SBcl-2 cells also form heterotypic aggregates with Mel-CAM-positive human endothelial cells but not with Mel-CAM-negative but ligand-positive smooth muscle cells. Taken together, our results show that Mel-CAM mediates cell-cell adhesion through heterophilic adhesion to an as yet unidentified ligand present on melanoma but not on endothelial cells. Thus, melanoma-endothelial interactions during metastasis may occur through this novel mechanism.

BACKGROUND

Metastasis is an important step in tumor progression leading to a disseminated and often incurable disease. First steps of metastasis include down-regulation of cell adhesion molecules, alteration of cell polarity and reorganization of cytoskeleton, modifications associated with enhanced migratory properties and resistance of tumor cells to anoikis. Such modifications resemble Epithelial to Mesenchymal Transition (EMT). In breast cancer CD146 expression is associated with poor prognosis and enhanced motility.

RESULTS

On 4 different human breast cancer cell lines, we modified CD146 expression either with shRNA technology in CD146 positive cells or with stable transfection of CD146 in negative cells. Modifications in morphology, growth and migration were evaluated. Using Q-RT-PCR, we analyzed the expression of different EMT markers. We demonstrate that high levels of CD146 are associated with loss of cell-cell contacts, expression of EMT markers, increased cell motility and increased resistance to doxorubicin or docetaxel. Experimental modulation of CD146 expression induces changes consistent with the above described characteristics: morphology, motility, growth in anchorage independent conditions and Slug mRNA variations are strictly correlated with CD146 expression. These changes are associated with modifications of ER (estrogen receptor) and Erb receptors and are enhanced by simultaneous and opposite modulation of JAM-A, or exposure to heregulin, an erb-B4 ligand.

CONCLUSIONS

CD146 expression is associated with an EMT phenotype. Several molecules are affected by CD146 expression: direct or indirect signaling contributes to EMT by increasing Slug expression. CD146 may also interact with Erb signaling by modifying cell surface expression of ErbB3 and ErbB4 and increased resistance to chemotherapy. Antagonistic effects of JAM-A, a tight junction-associated protein, on CD146 promigratory effects underline the complexity of the adhesion molecules network in tumor cell migration and metastasis.