Mel-CAM (CD146) is a cell-cell adhesion protein found in normal and tumoral tissues. The aim of this study was to analyse Mel-CAM expression in mucoepidermoid carcinoma (MEC), and assess its importance in prognosis and its utility in differentiating high-grade MEC from squamous cell carcinoma (SCC). Immunohistochemical expression of Mel-CAM in 41 parotid MEC was correlated with clinical parameters. Ten cases of oral cavity SCC were included for comparison. Mel-CAM expression was found in 92.7% of the MEC but was not expressed by the SCC. Mel-CAM expression was greater in intermediate/high grade tumors, was weaker in patients that presented local recurrence, regional and distant metastasis, but no correlation between Mel-CAM and clinical stage and survival of the patients was found. Decreased Mel-CAM expression can impair cellular contact properties, facilitating growth, cell spreading and metastasis in MEC. Mel-CAM can also be useful in differentiating high grade MEC from SCC.

OBJECTIVE

To study the expression and regulatory effects of CD146 protein in colorectal cancer and the correlation between CD146 protein expression and the prognosis of colorectal cancer.

METHODS

The CD146 protein level was detected by immunohistochemistry staining. The relationship between CD146 expression and clinicopathological parameters of colorectal cancer was determined.

RESULTS

It was observed that 216 (20.00%) of the 1080 cases positively expressed CD146 protein. Univariate analyses indicated that CD146 expression was related to histological grade, Duke's stage, and liver metastasis (p=0.001, 0.001, and 0.001, respectively). Spearman correlation analysis showed that CD146 expression has line correlation to histological grade, Duke's stage, and liver metastasis (p=0.02, 0.01 and 0.001, respectively). After multivariate analysis, Duke's stage and CD146 were related to liver metastasis (p=0.01 and 0.001, respectively). In the Cox regression test, histological grade, Duke's stage, and CD146 were detected as the independent prognostic factors (p=0.045, 0.01, and 0.001, respectively).

CONCLUSIONS

CD146 protein may be a potential biomarker for the postoperative liver metastasis of colorectal cancer.

During culture expansion, multipotent mesenchymal stromal cells (MSCs) differentially express aldehyde dehydrogenase (ALDH), an intracellular detoxification enzyme that protects long-lived cells against oxidative stress. Thus, MSC selection based on ALDH-activity may be used to reduce heterogeneity and distinguish MSC subsets with improved regenerative potency. After expansion of human bone marrow-derived MSCs, cell progeny was purified based on low versus high ALDH-activity (ALDHhi ) by fluorescence-activated cell sorting, and each subset was compared for multipotent stromal and provascular regenerative functions. Both ALDHl ° and ALDHhi MSC subsets demonstrated similar expression of stromal cell (>95% CD73+ , CD90+ , CD105+ ) and pericyte (>95% CD146+ ) surface markers and showed multipotent differentiation into bone, cartilage, and adipose cells in vitro. Conditioned media (CDM) generated by ALDHhi MSCs demonstrated a potent proliferative and prosurvival effect on human microvascular endothelial cells (HMVECs) under serum-free conditions and augmented HMVEC tube-forming capacity in growth factor-reduced matrices. After subcutaneous transplantation within directed in vivo angiogenesis assay implants into immunodeficient mice, ALDHhi MSC or CDM produced by ALDHhi MSC significantly augmented murine vascular cell recruitment and perfused vessel infiltration compared with ALDHl ° MSC. Although both subsets demonstrated strikingly similar mRNA expression patterns, quantitative proteomic analyses performed on subset-specific CDM revealed the ALDHhi MSC subset uniquely secreted multiple proangiogenic cytokines (vascular endothelial growth factor beta, platelet derived growth factor alpha, and angiogenin) and actively produced multiple factors with chemoattractant (transforming growth factor-β, C-X-C motif chemokine ligand 1, 2, and 3 (GRO), C-C motif chemokine ligand 5 (RANTES), monocyte chemotactic protein 1 (MCP-1), interleukin [IL]-6, IL-8) and matrix-modifying functions (tissue inhibitor of metalloprotinase 1 & 2 (TIMP1/2)). Collectively, MSCs selected for ALDHhi demonstrated enhanced proangiogenic secretory functions and represent a purified MSC subset amenable for vascular regenerative applications. Stem Cells 2017;35:1542-1553.

Self-renewing, multipotent progenitors of skeletal tissues are found within skeletal segments (skeletal stem cells) and coincide with adventitial reticular cells of bone marrow vessels in situ and with explanted clonogenic stromal cells ex vivo. These cells, which can be identified and prospectively isolated based on a minimal surface phenotype noted for expression of CD146, CD105 and alkaline phosphatase, are established during bone development through interactions with developing sinusoids. They represent a crucial crossroad of skeletal and hematopoietic physiology, as well as of endocrine regulation of bone growth and remodeling. In addition, they are central to major endocrine functions of bone itself, such as regulation of renal phosphate handling. Skeletal stem cells represent a central model system for investigating skeletal diseases, as tools for in vitro and in vivo models, for cell therapy-based strategies, or as targets for drugs.

BACKGROUND

Circulating endothelial cells (CECs) have been studied as a biomarker for tumour progression and monitoring therapeutic effects. The CellSearch system is a semi-automated system that allows standardised analysis of CECs. This study assessed the clinical implications of CECs determined by the CellSearch system in breast cancer patients.

METHODS

Seventy-six consecutive breast cancer patients (53 operable and 23 metastatic or recurrent) were enrolled for the study. Thirty-five patients with operable breast cancer received preoperative chemotherapy with a regimen based on anthracycline and/or taxane. CECs are defined as CD146(+)CD105(+)CD45(-)DAPI(+) cells in the system. CD34 expression was examined using the additional channel in the system.

RESULTS

A majority (4539 of 5183 cells, 88%) of CECs from patients with operable breast cancer were CD34-positive. Triple-negative cancers showed higher baseline CEC and CD34(+)CEC counts than the other types (P=0.0387 and 0.0377, respectively). Low baseline CEC and CD34(+)CEC counts, and a low CD34 positive rate were associated with pathological complete response (pCR) of preoperative chemotherapy in patients with primary breast cancer (P=0.046, 0.027 and 0.01, respectively). In multivariate analyses, the CD34 positive rate was significant for pCR (P=0.021). During preoperative chemotherapy, CEC and CD34(+)CEC counts before each cycle of chemotherapy increased with taxane-based regimens (P=0.0018 and 0.0008, respectively) but not with anthracycline-based regimens.

CONCLUSIONS

Baseline CEC, in particular CD34(+)CEC, counts and the CD34 positive rate might be useful for the prediction of treatment response of preoperative chemotherapy in patients with operable breast cancer.

In the search for an ideal minimally-invasive multipotent postnatal stem cells' source, the aim of the present study was to isolate and characterize multipotent postnatal stem/progenitor cells from the human alveolar bone proper tissue of the oral cavity. Cells were isolated from human alveolar bone parts, immunomagnetically sorted using STRO-1 antibodies and characterized flow cytometrically for the expression of CD14, CD34, CD45, CD73, CD90, CD105, CD146/MUC18 and STRO-1 surface markers. Colony-formation and multilineage differentiation potential were tested. Mineralized tissue marker expression was examined using real time polymerase chain reaction (PCR). The cells were plastic-adherent and showed colony-formation. Cells expressed the surface markers CD73, CD90, CD105, STRO-1 and CD146/MUC18, while lacking the expression of the hematopoietic markers CD14, CD34 and CD45. Cells could be differentiated into osteoblastic, adipocytic and chondroblastic lineages. Unstimulated cells expressed alkaline phosphatase (ALP), type I, III and V collagens, osteonectin and osteocalcin in a very distinctive pattern. This study presents a practical and minimally-invasive scheme for the isolation of multipotent postnatal stem/progenitor cells from the human alveolar bone tissue of the oral cavity.

Increased numbers of circulating endothelial cells (CEC) in peripheral blood have been observed in diseases with vascular involvement, and are considered a promising surrogate marker for vascular damage. It was the objective of this study to evaluate the correlation between putative soluble markers of endothelial injury, activation, and endothelial proliferation, and absolute numbers of CEC. CEC were evaluated in 125 healthy donors and 40 patients with metastatic carcinoma by automated CD146 driven immunomagnetic isolation. Plasma concentrations of E-selectin, endoglin, and thrombomodulin were assessed by ELISA in plasma obtained from 40 healthy donors and 40 patients. CEC numbers in blood were positively correlated with plasma thrombomodulin levels, but not with levels of E-selectin and endoglin. Multivariate analysis demonstrated a significant increase in CEC numbers with age. The levels of plasma biomarkers were not influenced by age. Higher levels of thrombomodulin and E-selectin were observed in males when compared to females. In conclusion, CEC numbers correlate positively with plasma levels of thrombomodulin.

OBJECTIVE

Idiopathic inflammatory myopathies (IIM) are a heterogeneous group of diseases characterized by chronic inflammation of muscles. We investigated the role of cellular adhesion molecules implicated in the cohesion of endothelial cells in IIM.

METHODS

In 22 patients with IIM we investigated plasma concentrations of soluble junctional adhesion molecules [platelet-endothelial cell adhesion molecule (sPECAM-1) and sCD146] and cellular adhesion molecules [sP-selectin, sE-selectin, intercellular adhesion molecule (sICAM-1), and vascular cell adhesion molecule (sVCAM-1)] implicated in leukocyte/endothelial cell interactions. Results were compared to a control group. Muscle biopsy samples from 8 out of 22 IIM patients were studied by immunohistochemistry for tissue expression of these molecules and compared to normal muscle samples. PECAM-1 and CD146 expression was also studied using immunoblots from muscle biopsies from 5 patients and 2 controls.

RESULTS

We observed distinct patterns of soluble levels and in situ expression between dermatomyositis (DM), polymyositis (PM), and sporadic inclusion body myositis (s-IBM). PM samples showed significantly increased levels of sCD146, sPECAM-1, and s-ICAM1 and increased expression of CD146, CD31, and ICAM-1 in endothelial cells, whereas CD146 and ICAM-1 were also recorded in some muscle fibers. In DM, sE-selectin, sP-selectin, and sPECAM-1 were significantly increased, with abnormal expression of ICAM-1 in endothelial cells and perifascicular muscle fibers. In the small group of s-IBM samples, results were similar to PM, but the only significant increase was the level of sPECAM-1. Immunoblots confirmed increased expression of PECAM-1 and CD146 in all IIM muscles in comparison to controls, with the highest expression in PM and IBM samples.

CONCLUSIONS

We observed abnormal increases of soluble levels of adhesion molecules implicated in endothelial cell junctions in PM (sCD146, sPECAM-1) and to a lesser extent in DM and s-IBM (sPECAM-1). We conclude that the distinctly different profiles between PM/s-IBM and DM reflect differences in the pathophysiological background of these diseases.

BACKGROUND

Brain microvascular endothelial cells (BMVECs) constitute the primary limitation for passage of ions and molecules from the blood into the brain through the blood brain barrier. Numerous multi-step procedures for isolating and culturing BMVECs have been described. However, each one demonstrates major limitations in purity of culture and/or low proliferation rate. Our goal was to study the efficiency of our pending patent medium, Endothelial Proliferation Medium (EndoPM), on the isolation and purification of human and murine BMVECs.

METHODS

BMVECs, cultured in EndoPM were compared to those cultured in a commercial medium EBM. Cultures were characterized by flow cytometric analysis, lineage differentiation, the ability to form tube-like structure, immunofluorescence, molecular analyses and also in an in vivo model assay. Moreover permeability was assayed by monitoring the passage of Dextran-FITC through a tight monolayer of BMVECs grown to confluence in Boyden chambers. One way Anova two-tailed test was utilized for all statistical analyses.

RESULTS

The properties of ECs in human and murine BMVECs is confirmed by the expression of endothelial markers (CD31, CD105, CD146, Tie-2 and vWF), of representative proangiogenic genes (ICAM1, VCAM1 and integrin ITGAV), of considerable tube-forming ability, with low-density lipoprotein uptake, eNOS and GLUT-1 expression. Furthermore cells are able to express markers of the junctional architecture as VE-cadherin, β-catenin and Claudin-5 and greatly reduce dextran permeability as barrier functional test. Moreover BMVECs spontaneously organize in vascular-like structures and maintain the expression of endothelial markers in an in vivo xenograft model assay. The significant effect of EndoPM is confirmed by the study of proliferation index, survival index and the behaviour of BMVECs and fibroblasts in co-culture conditions.

CONCLUSIONS

Herein we describe a simple and reproducible method for the isolation and expansion of human and mouse BMVECs, based on a newly formulated medium (EndoPM) with optimized concentration of growth factors (EGF, FGF-2 and Bovine Brain Extract-BBE). This procedure should facilitate the isolation and expansion of human and mouse BMVECs with extended lifetime, good viability and purity. This approach may provide an effective strategy to aid phenotypical and functional studies of brain vessels under physiological and pathological conditions.

OBJECTIVE

Intraplaque neovascularization and foam cell infiltration contribute to the development of unstable plaque, leading to thromboembolism and stroke. Cell adhesion molecules (CAMs) have been reported to be involved in the progression of atherosclerosis and plaque vulnerability. The aim of this study was to assess the association of adhesion molecule CD146 with carotid plaque instability.

METHODS

We collected forty atherosclerotic plaques from 40 patients undergoing carotid endarterectomy. The clinical information of each patient was obtained, and the plaque morphology and characteristics were examined by the ultrasound. The CD146 expressions of the plaques were graded by using semiquantitative scales. The serum level of soluble form of CD146 was detected by enzyme-linked immunosorbent assay (ELISA).

RESULTS

CD146 expression was mainly on the intraplaque blood vessels and infiltrated macrophages. The CD146 expression was strongly correlated with the matrix metalloproteinase-9(MMP-9)expressions (P < 0.001) in the plaques. Soluble CD146 (sCD146) was also elevated in patients with atherosclerotic plaques. There was significant correlation between the increased CD146 expression and sCD146 level (P = 0.0057). sCD146 correlated well with serum MMP-9 (P < 0.0044), IL-6 (P = 0.0044) and high sensitivity C-reactive protein (hsCRP) (P = 0.005).

CONCLUSIONS

Adhesion molecules CD146 and its soluble form strongly correlated with the development of inflammation of atherosclerosis and plaque instability. CD146 may be a promising biomarker for monitoring the development and instability of atherosclerotic plaque in patients with carotid diseases.

The objective of is this article is to review murine xenotransplantation models for myelodysplastic syndromes (MDS). The difficulties in achieving sustained engraftment of MDS cells in immunodeficient mice may lie in innate characteristics of the MDS clones and microenvironmental factors. Engraftment of very low numbers of CD45(+) clonal MDS cells has been achieved with intravenous injection; higher rates of engraftment are obtained via the intramedullary route. Coinjection of certain stroma components with hematopoietic cells overcomes limitations of intravenous (IV) administration, allowing for engraftment of high proportions of human CD45(+) cells in mouse spleen and marrow. Expression of CD146 on stroma cells conveys an engraftment-facilitating effect. Clonal MDS cells have been propagated for periods beyond 6 months and have been transplanted successfully into secondary recipients. Engraftment of human clonal MDS cells with stem cell characteristics in immunodeficient mice is greatly facilitated by coinjection of stroma/mesenchymal cells, particularly with IV administration. CD146 expression on stroma is an essential factor; however, no model develops the laboratory and clinical features of human MDS. Additional work is needed to determine cellular and noncellular factors required for the full evolution of MDS.

Angiogenesis plays an important role in vertebrate development and tumor growth. In this process, gicerin, which is known as a kind of cell adhesion molecule, has recently been reported to play an important role but its in vivo function is still unclear in developing vasculature. To address this issue, we used gain-of-function and loss-of-function analyses of gicerin in zebrafish. In the gain of function experiments using enforced expression of various domains of gicerin constructs, extracellular domain induced angiogenic sprouting defects, most notably in the intersegmental vessels, whereas the cytoplasmic domain of gicerin did not affect angiogenic sprouting. Moreover, morpholino-mediated knockdown of gicerin in embryos resulted in angiogenic sprouting defects in intersegmental vessels. Mechanistically, the angiogenic function of gicerin was found to be genetically linked to VEGF signaling in the knock-down experiments using vegf-a mRNA, VEGFR inhibitor and gicerin morpholino. In addition to the physiological angiogenesis during development, gicerin morphants efficiently blocked the tumor angiogenesis in zebrafish. Thus, knock-down of gicerin might have an important implication in controlling tumor angiogenesis.

OBJECTIVE

Overexpression of CD146 in solid tumors has been linked to disease progression, invasion, and metastasis. We describe the generation of a 64Cu-labeled CD146-specific antibody and its use for quantitative immunoPET imaging of CD146 expression in six lung cancer models.

METHODS

The anti-CD146 antibody (YY146) was conjugated to 1,4,7-triazacyclononane-triacetic acid (NOTA) and radiolabeled with 64Cu. CD146 expression was evaluated in six human lung cancer cell lines (A549, NCI-H358, NCI-H522, HCC4006, H23, and NCI-H460) by flow cytometry and quantitative western blot studies. The biodistribution and tumor uptake of 64Cu-NOTA-YY146 was assessed by sequential PET imaging in athymic nude mice bearing subcutaneous lung cancer xenografts. The correlation between CD146 expression and tumor uptake of 64Cu-NOTA-YY146 was evaluated by graphical software while ex vivo biodistribution and immunohistochemistry studies were performed to validate the accuracy of PET data and spatial expression of CD146.

RESULTS

Flow cytometry and western blot studies showed similar findings with H460 and H23 cells showing high levels of expression of CD146. Small differences in CD146 expression levels were found among A549, H4006, H522, and H358 cells. Tumor uptake of 64Cu-NOTA-YY146 was highest in CD146-expressing H460 and H23 tumors, peaking at 20.1 ± 2.86 and 11.6 ± 2.34 %ID/g at 48 h after injection (n = 4). Tumor uptake was lowest in the H522 model (4.1 ± 0.98 %ID/g at 48 h after injection; n = 4), while H4006, A549 and H358 exhibited similar uptake of 64Cu-NOTA-YY146. A positive correlation was found between tumor uptake of 64Cu-NOTA-YY146 (%ID/g) and relative CD146 expression (r 2 = 0.98, p < 0.01). Ex vivo biodistribution confirmed the accuracy of the PET data.

CONCLUSIONS

The strong correlation between tumor uptake of 64Cu-NOTA-YY146 and CD146 expression demonstrates the potential use of this radiotracer for imaging tumors that elicit varying levels of CD146. In the future, this tool may promote enhanced monitoring of therapeutic response and improved patient stratification.

BACKGROUND

endothelial damage and dysfunction play a crucial role in the pathophysiology of coronary artery disease (CAD). The quantification of circulating endothelial cells (CEC) in the peripheral blood is a novel method for assessing endothelial damage.

OBJECTIVE

to evaluate the possible diagnostic use of single quantification of CEC in peripheral blood by flow cytometry in patients with CAD.

METHODS

we examined 48 patients with CAD, including 23 patients with acute myocardial infarction (AMI) and 25 patients with stable angina (SA). The control group consisted of 20 healthy subjects without symptoms of CAD. The CEC count was evaluated by flow cytometry using antibodies against CD31, CD146, and CD45. Plasma biochemical markers of endothelial damage (von Willebrand Factor [vWF], thrombomodulin [TM]) were measured by ELISA. Serum concentrations of troponin I (TnI) and lipid parameters were also included in the statistical analysis.

RESULTS

A significant increase in the CEC count was found in patients with AMI compared to the control group (p < 0.05) and SA patients (p < 0.05). However, no difference was found in the CEC count between patients with SA and the control group. Increased vWF activity was found in both groups of CAD patients compared to the control group (AMI: p < 0.001, SA: p , 0.01), and vWF activity was significantly higher in AMI patients compared to SA patients (p < 0.001). Thrombomodulin concentration did not differ significantly between any patient groups and the control group. The CEC count correlated positively with vWF activity (r = 0.3852, p < 0.05) and the atherogenic index TC/HDL-C (r = 0.3844, p < 0.05) in all patients with CAD (AMI + SA). The sensitivity of CEC count for the diagnosis of an acute coronary syndrome was lower than that of TnI level on admission (39% vs 69%).

CONCLUSIONS

we confirmed that CEC count in peripheral blood can be determined by flow cytometry in CAD patients with both AMI and SA. The CEC count in AMI was increased in comparison to healthy subjects and SA patients in one third of all cases. To determine whether CEC count could be used to improve the diagnosis of an acute coronary syndrome in patients with CAD, additional studies in larger patient groups would be required.

BACKGROUND

An anti-CD146 monoclonal antibody, AA98, has been identified as an inhibitor of tumor angiogenesis. To overcome the inherent immunogenicity of murine antibody as well as to facilitate immunotoxin construction, a single chain AA98 V(H)/L with three-domain fragments was constructed and expressed in mammalian cells.

METHODS

The genes of the AA98 heavy chain variable region and the light chain were linked with a modified 12 amino acid sequence that was derived from the heavy chain C(H)1 region, thus constituting the three-domain antibody V(H)/L. Soluble AA98 V(H)/L was produced by mammalian cells and purified by affinity chromatography. The specificity of AA98 V(H)/L for the CD146 molecule was detected by ELISA, immunofluorescence staining and flow cytometry.

RESULTS

AA98 V(H)/L alone showed anti-angiogenic properties in a chicken chorioallantoic membrane (CAM) assay as the parent mAb AA98 did.

CONCLUSIONS

This newly generated AA98 V(H)/L antibody displays a therapeutic potential for tumor and other angiogenesis disorders, as well as providing a new strategy for antibody engineering for clinical applications.

Adipogenic potential was evaluated in uncultured stromal vascular fraction (SVF) loaded onto porous 3D collagen matrix and gelatin sponge scaffolds with predefined shapes. The SVF was isolated from 16 freshly lipectomized fat. Mean cell number was 6.0±4.68×10(7) cells/mL, and mean cell viability was 72%. Flow cytometric analysis revealed adipose-derived stromal cells (CD31(-), CD34(-/+), CD45(-), CD90(+), CD105(-), CD146(-), and CD166(+)) in the SVF. Three hours after harvest of fat, 200 μL of isolated SVF was loaded onto an experimental scaffold (4 mg in weight) and cultured in Dulbecco's modified Eagle's medium. Examination of the construct under an inverted light microscope and a scanning electron microscope demonstrated adequate seeding and active proliferation of the SVF cells on pore surfaces. Cells grew to varying sizes in clusters or in strands. On day 28, histologic study of the constructs by H&E staining revealed viable adipocytes in the microstructure. Positive Oil-Red O stain confirmed lipid accumulation in mature adipocytes. The presence of human adipocytes was further assessed by the presence of genomic DNA detected by GAPDH gene and by the mRNA expression of peroxisome proliferator activated receptor-γ, and lipoprotein lipase. The results demonstrated that new adipose tissue can be regenerated by seeding freshly isolated, uncultured SVF on 3D porous collagen matrix and gelatin sponge scaffolds.

Clopidogrel enhances the levels of endothelial nitric oxide and prostacyclin in tissue culture. We have previously described a marked increase in circulating endothelial cells (CECs), an ex vivo indicator of vascular injury, in patients with type 2 diabetes mellitus. We hypothesized that clopidogrel treatment would result in a decrease in CEC number and increased activity of endothelial progenitor cell recruitment signaling pathways in diabetic patients. CECs were isolated from the peripheral blood of 9 patients with type 2 diabetes using anti-<em>CD146</em>-coated Dynabeads. The cells were stained with acridine orange and counted by fluorescence microscopy. Endothelial progenitor cells were isolated in a similar fashion using anti-CD34 and anti-CD133 and assayed for expression of phosphorylated Akt and phosphorylated adenosine monophosphate kinase. The patients were then treated with clopidogrel 75 mg/day for 30 days, after which repeat blood specimens were analyzed. As previously observed, diabetic patients had an elevated number of CECs (mean 79 +/- 15 cells/ml peripheral blood), which was reduced by clopidogrel treatment (mean 10 +/- 4 cells/ml; p <0.001). This was associated with a significant increase in the expression of both phosphorylated Akt and phosphorylated adenosine monophosphate kinase (p </=0.05). In conclusion, clopidogrel reduces endothelial cell sloughing and increases expression of endothelial progenitor cell phosphorylated Akt and phosphorylated adenosine monophosphate kinase in the peripheral blood of patients with type 2 diabetes mellitus. This represents a novel mechanism by which this agent can promote improved vascular function, protect against oxidative stress, inhibit apoptosis, and attenuate vascular damage in patients with diabetes mellitus.

Endothelial progenitor cells (EPCs) have been implicated in the pathogenesis and treatment of cardiovascular disease. By use of quantitative uptake of DiLDL and lectin staining, EPCs have been characterized reliably. However, the exact nature and function of this cell population still remains poorly defined. In an attempt to further clarify the cell surface characteristics of EPCs, mononuclear cells (MNCs) were isolated from human blood and cell surface expression patterns were defined by FACS analysis before and after differentiation for 1-10 days in cell culture. "Classical" double staining for DiLDL and Ulex europaeus increases to 89.2 /- 0.05 after 10 days in culture. Looking at EPC-specific markers by FACS analysis, 0.18 +/- 0.11% of freshly isolated MNCs express CD34, 0.13 +/- 0.08% CD133, 0.59 +/-0.51% VEGFr2, 0.01 +/- 0.02% CD34/VEGFr2, 0.09 +/- 0.05% CD34/CD133, 0.58 +/- 0.13% CD34/CD31, and 0.02 +/- 0.01% CD34/CD146, respectively. Induction of the endothelial phenotype is evidenced by positive staining for VEGFr2, CD146, and CD31, and occurs in co-expression with stem cell markers in less than 2 +/- 0.52% of cultured cells. Expression of CD34 increases to 0.38 +/- 0.10% after 10 days, whereas the CD133(+) cell population shows an initial peak at 24h (0.29 +/- 0.18%) before decreasing to 0.15 +/- 0.02% at day 10. EPCs co-expressing CD34/CD133 increase to 0.19 +/- 0.09% after 10 days, and EPCs double-positive for CD34/VEGFr2 increase to 1.45 +/- 1.03%. Looking at leukocyte, lymphocyte, and monocyte lineage markers, 56.27 +/- 0.15% of freshly isolated MNCs express CD45, 7.13 +/- 0.02% CD14, and 38.65 +/- 0.01% CD3. Over the 10-day culture period, expression of CD45 decreases to 28.48 +/- 0.18%, CD3 to 23.11 +/- 0.02%, and CD14 to 0.09 +/- 0.02%. Cells co-expressing CD3/CD45 decrease from 38.88 +/- 0.33% to 24.86 +/- 2.49% after 10 days in culture. These findings extend present knowledge by showing that human MNCs differentiate at a very low rate to EPCs, while a majority of the cultured cell population remain committed to the leukocyte or lymphocyte lineage. Careful surface marker analysis might be necessary when using in vitro EPC differentiation systems.

Microparticles (MPs) could be considered biomarkers of cell damage and activation as well as novel signalling structures. Since rheumatoid arthritis (RA) is characterized by immune and endothelial activation, the main aim of the present study was to analyse MP counts in RA patients. Citrated-blood samples were obtained from 114 RA patients, 33 healthy controls (HC) and 72 individuals with marked cardiovascular (CV) risk without autoimmune manifestations (CVR). MPs were analysed in platelet-poor plasma (PPP) and different subsets were identified by their surface markers: platelet- (CD41+), endothelial- (CD146+), granulocyte- (CD66+), monocyte- (CD14+) and Tang- (CD3+CD31+) derived. Disease activity score (DAS28), clinical and immunological parameters as well as traditional CV risk factors (diabetes, hypertension, dyslipidaemia and obesity) were registered from clinical records and all data were integrated using Principal Component Analysis (PCA). Absolute MP number was increased in RA patients compared with HC and positively correlated with traditional CV risk factors, similar to that of CVR subjects. In addition, frequency of the different MP subsets was different in RA patients and significantly associated with disease features. Moreover, in vitro assays revealed that MPs isolated from RA patients were able to promote endothelial activation and exhibited detrimental effects on human microvascular endothelial cells (HMEC-I) endothelial cell functionality. Circulating MPs from RA patients displayed quantitative and qualitative alterations that are the result of both disease-specific and traditional CV risk factors. Accordingly, this MP pool exhibited in vitro detrimental effects on endothelial cells, thus supporting their role as biomarkers of vascular damage.

The effect of cryopreservation on mesenchymal stromal cell (MSC) therapeutic properties has become highly controversial. However, data thus far have indiscriminately involved the assessment of different types of MSCs with distinct production processes. This study assumed that MSC-based products are affected differently depending on the tissue source and manufacturing process and analyzed the effect of cryopreservation on a specific population of umbilical cord tissue-derived MSCs (UC-MSCs), UCX®.

Cell phenotype was assessed by flow cytometry through the evaluation of the expression of relevant surface markers such as CD14, CD19, CD31, CD34, CD44, CD45, CD90, CD105, CD146, CD200, CD273, CD274 and HLA-DR. Immunomodulatory activity was analyzed in vitro through the ability to inhibit activated T cells and in vivo by the ability to reverse the signs of inflammation in an adjuvant-induced arthritis (AIA) model. Angiogenic potential was evaluated in vitro using a human umbilical vein endothelial cell-based angiogenesis assay, and in vivo using a mouse model for hindlimb ischemia.

Phenotype and immunomodulatory and angiogenic potencies of this specific UC-MSC population were not impaired by cryopreservation and subsequent thawing, both in vitro and in vivo.

This study suggests that potency impairment related to cryopreservation in a given tissue source can be avoided by the production process. The results have positive implications for the development of advanced-therapy medicinal products.

Placenta cretas are defined as abnormal adherences or ingrowths of placental tissue, but their pathogenetic mechanism has not been fully explained. During histologic examination of postpartum uteri, we noticed that the number of implantation site intermediate trophoblasts was increased in the placental bed of placenta cretas. To validate our observation and to address the pathogenetic role of implantation site intermediate trophoblasts in placenta cretas, we examined postpartum uteri with placenta cretas (n=34) and noncretas (n=22), obtained from Cesarean or immediate postpartum hysterectomy specimens. Using antibody to CD146, a marker for implantation site intermediate trophoblasts, we found that placenta cretas had significantly thicker layer of implantation site intermediate trophoblasts (2300+/-1200 mum) than noncretas (1500+/-1200 microm, P<0.025). We also observed that the confluent distribution of cells was more frequent in placenta cretas (97%) than noncretas samples (45%, P<0.001), and that the total number of implantation site intermediate trophoblasts within the superficial myometrium of the placental bed was significantly higher in placenta cretas than noncretas. Using antibodies to Ki-67, Bcl-2 and cleaved caspase-3 to determine the proliferative index and apoptotic rates of implantation site intermediate trophoblasts, we found that they were close to zero in both groups and did not differ significantly. These findings suggest that the increased number of implantation site intermediate trophoblasts observed in placenta cretas may be related to the pathogenesis of placental ingrowth, but the mechanism by which the increase in implantation site intermediate trophoblasts causes placenta cretas remains to be clarified.

The purpose of this study was to determine whether the expression of melanoma cell adhesion molecule (MCAM/CD146), like most other endothelial adhesion molecules, is reduced in the pregnancy disorder pre-eclampsia, and whether it is associated with the invasiveness of trophoblast. We used immunohistochemical approaches and analyzed 42 placentas from control and pre-eclamptic patients using an anti-CD146 monoclonal antibody. Our data show that in normal placentas, CD146 staining was specifically detected in intermediate trophoblasts that are most invasive and migratory, but not detected in noninvasive cytotrophoblasts or syncytiotrophoblasts. However in pre-eclampsia, CD146 was no longer present in the intermediate trophoblasts, although it was detectable in the blood vessels. At the same time, we tested CD31/PECAM-1, another endothelial adhesion molecule, and found its negative staining for all kinds of trophoblasts in both normal and pre-eclamptic placentas. The different staining patterns of CD146 in the normal and pre-eclamptic placentas provide the first evidence that in pre-eclampsia, intermediate trophoblasts fail to express CD146, implicating that CD146 plays an important role in trophoblast invasion.

A body of growing evidence now implicates white adipose tissue as a relevant source of stromal progenitor cells recruited to the tumor microenvironment to form supportive tumor stroma. While the role of periprostatic (PP) adipose tissue in prostate cancer progression has been barely appreciated, we sought to determine the progenitor cell population in PP adipose tissue and the association with prostate cancer. We isolated and characterized CD31(-)CD34(+)CD45(-)CD146(-) progenitor cells (adipose-derived stem cells [ASC]) in paired samples of PP and preperitoneal visceral adipose tissue from prostate tissue and peripheral blood mononuclear cells of prostate cancer and nodular prostatic hyperplasia patients. ASC were quantified by flow cytometry and confirmed through target gene expression. Here we show a significantly higher amount of ASC in PP than in visceral adipose tissue, independent of body mass index and prostatic disease. In the prostate, ASC are increased in cancer compared with prostatic nodular hyperplasia patients. Concordantly, adipsin gene (CFD) expression, which is known to be up-regulated in adipose stem cells, was overexpressed in PP adipose tissue, in the prostate of cancer patients and in prostate CD31(-)CD34(+)CD45(-)CD146(-) sorted cells. ASC were found at higher levels in the blood of prostate cancer patients simultaneously overweight/obese. Present findings indicate that PP adipose tissue is a reservoir of progenitor cells with the potential to migrate towards prostate tumors, although its clinical significance merits further evaluation.

Tumour cells can find in bone marrow (BM) a niche rich in growth factors and cytokines that promote their self-renewal, proliferation and survival. In turn, tumour cells affect the homeostasis of the BM and bone, as well as the balance among haematopoiesis, osteogenesis, osteoclastogenesis and bone-resorption. As a result, growth and survival factors normally sequestered in the bone matrix are released, favouring tumour development. Mesenchymal stem cells (MSCs) from BM can become tumour-associated fibroblasts, have immunosuppressive function, and facilitate metastasis by epithelial-to-mesenchymal transition. Moreover, MSCs generate osteoblasts and osteocytes and regulate osteoclastogenesis. Therefore, MSCs can play an important pro-tumorigenic role in the formation of a microenvironment that promotes BM and bone metastasis. In this study we showed that BM MSCs from untreated advanced breast and lung cancer patients, without bone metastasis, had low osteogenic and adipogenic differentiation capacity compared to that of healthy volunteers. In contrast, chondrogenic differentiation was increased. Moreover, MSCs from patients had lower expression of CD146. Finally, our data showed higher levels of Dkk-1 in peripheral blood plasma from patients compared with healthy volunteers. Because no patient had any bone disorder by the time of the study we propose that the primary tumour altered the plasticity of MSCs. As over 70 % of advanced breast cancer patients and 30-40 % of lung cancer patients will develop osteolytic bone metastasis for which there is no total cure, our findings could possibly be used as predictive tools indicating the first signs of future bone disease. In addition, as the MSCs present in the BM of these patients may not be able to regenerate bone after the tumour cells invasion into BM/bone, it is possible that they promote the cycle between tumour cell growth and bone destruction.