CD90+CD146+ identifies a pulmonary mesenchymal cell subtype with both immune modulatory and perivascular-like function in postnatal human lung.

Our understanding of mesenchymal cell subsets and their function in human lung affected by aging and in certain disease settings remains poorly described. We use a combination of flow cytometry, prospective cell-sorting strategies, confocal imaging, and modeling of microvessel formation using advanced microfluidic chip technology to characterize mesenchymal cell subtypes in human postnatal and adult lung. Tissue was obtained from patients undergoing elective surgery for congenital pulmonary airway malformations (CPAM) and other airway abnormalities including chronic obstructive pulmonary disease (COPD). In microscopically normal postnatal human lung, there was a fivefold higher mesenchymal compared with epithelial (EpCAM+) fraction, which diminished with age. The mesenchymal fraction composed of CD90+ and CD90+CD73+ cells was enriched in CXCL12 and platelet-derived growth factor receptor-α (PDGFRα) and located in close proximity to EpCAM+ cells in the alveolar region. Surprisingly, alveolar organoids generated from EpCAM+ cells supported by CD90+ subset were immature and displayed dysplastic features. In congenital lung lesions, cystic air spaces and dysplastic alveolar regions were marked with an underlying thick interstitium composed of CD90+ and CD90+PDGFRα+ cells. In postnatal lung, a subset of CD90+ cells coexpresses the pericyte marker CD146 and supports self-assembly of perfusable microvessels. CD90+CD146+ cells from COPD patients fail to support microvessel formation due to fibrinolysis. Targeting the plasmin-plasminogen system during microvessel self-assembly prevented fibrin gel degradation, but microvessels were narrower and excessive contraction blocked perfusion. These data provide important new information regarding the immunophenotypic identity of key mesenchymal lineages and their change in a diverse setting of congenital lung lesions and COPD.

Transcriptome and proteome profiles of human umbilical cord vein CD146+ stem cells.

In this study we used two different techniques in order to isolate pericytes from the wall of human umbilical cord vein and get two different groups of cells were named as "pellet and primer cells". These groups were compared with each other according to their morphologies and stem cell marker expressions. Also, these two different populations were compared with each other and with human bone marrow mesenchymal stem cells (BM-MSCs) according to their transcriptomic profiles. Then, pellet cells proteomic profiles were determined. Our results showed that morphologies and cell surface marker expressions of pellet cells and primer cells are similar. On the other hand, according to immunofluorescence staining results, in contrast to primer cells, pellet cells showed positive NG2 and PDGFR-ß staining. As a result of gene expression profiling, pellet cells have upregulated genes related with muscle, neural and immune cell differentiation, development and pluripotency. On the other hand, primer cells have upregulated adhesion pathway-related genes. In addition to differences between pellet and primer cells, the gene expression profiles of these cell groups are also different from BM-MSCs. The results of transcriptome and proteome analysis of pellet cells were in consistent with each other.

Blockade of MCAM/CD146 impedes CNS infiltration of T cells over the choroid plexus.

BACKGROUND: Very late antigen 4 (VLA-4; integrin α4ß1) is critical for transmigration of T helper (TH) 1 cells into the central nervous system (CNS) under inflammatory conditions such as multiple sclerosis (MS). We have previously shown that VLA-4 and melanoma cell adhesion molecule (MCAM) are important for trans-endothelial migration of human TH17 cells in vitro and here investigate their contribution to pathogenic CNS inflammation. METHODS: Antibody blockade of VLA-4 and MCAM is assessed in murine models of CNS inflammation in conjunction with conditional ablation of α4-integrin expression in T cells. Effects of VLA-4 and MCAM blockade on lymphocyte migration are further investigated in the human system via in vitro T cell transmigration assays. RESULTS: Compared to the broad effects of VLA-4 blockade on encephalitogenic T cell migration over endothelial barriers, MCAM blockade impeded encephalitogenic T cell migration in murine models of MS that especially depend on CNS migration across the choroid plexus (CP). In transgenic mice lacking T cell α4-integrin expression (CD4::Itga4-/-), MCAM blockade delayed disease onset. Migration of MCAM-expressing T cells through the CP into the CNS was restricted, where laminin 411 (composed of α4, ß1, γ1 chains), the proposed major ligand of MCAM, is detected in the endothelial basement membranes of murine CP tissue. This finding was translated to the human system; blockade of MCAM with a therapeutic antibody reduced in vitro transmigration of MCAM-expressing T cells across a human fibroblast-derived extracellular matrix layer and a brain-derived endothelial monolayer, both expressing laminin α4. Laminin α4 was further detected in situ in CP endothelial-basement membranes in MS patients' brain tissue. CONCLUSIONS: Our findings suggest that MCAM-laminin 411 interactions facilitate trans-endothelial migration of MCAM-expressing T cells into the CNS, which seems to be highly relevant to migration via the CP and to potential future clinical applications in neuroinflammatory disorders.

ImmunoPET Imaging of CD146 in Murine Models of Intrapulmonary Metastasis of Non-Small Cell Lung Cancer.

CD146 has been identified as an excellent biomarker for lung cancer as its overexpression in solid tumors has been linked to disease progression, invasion, and metastasis. Previously, our group described a positive correlation between 64Cu-labeled YY146 uptake and increased expression of CD146 in six human lung cancer cell lines using subcutaneous tumor models. In this study, we investigate a monoclonal antibody called YY146 for immunoPET imaging of CD146 in two intrapulmonary metastasis models of non-small cell lung cancer (NSCLC). The binding and immunoreactivity of the tracer were assessed by in vitro assays. Radiolabeling of YY146 with positron emitting Cu-64 (64Cu-NOTA-YY146) enabled PET imaging of intrapulmonary metastasis. Mice were intravenously injected with two million tumor cells, and CT imaging was used to verify the presence of lung metastases. 64Cu-NOTA-YY146 was injected into tumor-bearing mice, and animals were subjected to PET/CT imaging at 4, 24, and 48 h postinjection. Both the average and maximum lung PET signal intensities were quantified and compared between high and low CD146-expressing metastases. Further validation was accomplished through immunofluorescence imaging of resected tissues with CD31 and CD146. In flow cytometry, YY146 revealed strong binding to CD146 in H460 cells due to its high expression with minimal binding to CD146-low expressing H358 cells. Both YY146 and NOTA-YY146 showed similar binding, suggesting that NOTA conjugation did not elicit any negative effects on its binding affinity. Imaging of 64Cu-NOTA-YY146 in H460 tumor-bearing mice revealed rapid, persistent, and highly specific tracer accumulation. Uptake of 64Cu-NOTA-YY146 in the whole lung was calculated for H460 and H358 as 7.43 ± 0.38 and 3.95 ± 0.47% ID/g at 48 h postinjection (n = 4, p < 0.05), and the maximum lung signals were determined to be 13.85 ± 1.07 (H460) and 6.08 ± 0.73% ID/g (H358) at equivalent time points (n = 4, p < 0.05). To ensure the specificity of the tracer, a nonspecific antibody was injected into H460 tumor-bearing mice. Ex vivo biodistribution and immunofluorescence imaging validated the PET findings. In summary, 64Cu-NOTA-YY146 allowed for successful imaging of CD146-expressing intrapulmonary metastases of NSCLC in mice. This preliminary study provides evidence supporting the future clinical utilization of 64Cu-NOTA-YY146 for possible treatment monitoring of CD146-targeted therapy or improving patient stratification.

MUC18 regulates IL-13-mediated airway inflammatory response.

OBJECTIVE: To evaluate the effects of MUC18 on IL-13-mediated airway inflammatory responses in human airway epithelial cells and in mice. MATERIALS: Primary normal human tracheobronchial epithelial (HTBE) cells, wild-type (WT) and Muc18 knockout (KO) mice, and mouse tracheal epithelial cells (mTECs) were utilized. TREATMENT: Cultured HTBE cells treated with MUC18 siRNA or MUC18 expressing lentivirus were incubated with IL-13 (10 ng/mL) for 24 h. Mice were intranasally instilled with 500 ng of IL-13 for 3 days. mTECs were treated with IL-13 (10 ng/mL) for 3 days. METHODS: PCR was used to measure mRNA expression. Western Blot and ELISAs were used to quantify protein expression. Cytospins of bronchoalveolar lavage (BAL) cells were used to obtain leukocyte differentials. RESULTS: MUC18 siRNA reduced IL-13-mediated eotaxin-3 (183 ± 44 vs. 380 ± 59 pg/mL, p < 0.05), while MUC18 overexpression increased IL-13-mediated eotaxin-3 (95 ± 3 vs. 58 ± 3 pg/mL, p < 0.05) in HTBE cells. IL-13-treated Muc18 KO mice had a lower percentage of neutrophils in BAL than WT mice (25 ± 3 vs. 35 ± 3%, p = 0.0565). CONCLUSIONS: These results implicate MUC18 as a potential enhancer of airway inflammation in a type 2 cytokine (e.g., IL-13) milieu.

Endothelial progenitor cells, defined by the simultaneous surface expression of VEGFR2 and CD133, are not detectable in healthy peripheral and cord blood.

Circulating endothelial cells (CEC) and their progenitors (EPC) are restricted subpopulations of peripheral blood (PB), cord blood (CB), and bone marrow (BM) cells, involved in the endothelial homeostasis maintenance. Both CEC and EPC are thought to represent potential biomarkers in several clinical conditions involving endothelial turnover/remodeling. Although different flow cytometry methods for CEC and EPC characterization have been published so far, none of them have reached consistent conclusions, therefore consensus guidelines with respect to CEC and EPC identification and quantification need to be established. Here, we have carried out an in depth investigation of CEC and EPC phenotypes in healthy PB, CB and BM samples, by optimizing a reliable polychromatic flow cytometry (PFC) panel. Results showed that the brightness of CD34 expression on healthy PB and CB circulating cells represents a key benchmark for the identification of CEC (CD45neg/CD34bright/CD146pos) respect to the hematopoietic stem cell (HSC) compartment (CD45dim/CD34pos/CD146neg). This approach, combined with a dual-platform counting technique, allowed a sharp CEC enumeration in healthy PB (n = 38), and resulting in consistent CEC counts with previously reported data (median = 11.7 cells/ml). In parallel, by using rigorous PFC conditions, CD34pos/CD45dim/CD133pos/VEGFR2pos EPC were not found in any healthy PB or CB sample, since VEGFR2 expression was never detectable on the surface of CD34pos/CD45dim/CD133pos cells. Notably, the putative EPC phenotype was observed in all analyzed BM samples (n = 12), and the expression of CD146 and VEGFR2, on BM cells, was not restricted to the CD34bright compartment, but also appeared on the HSC surface. Altogether, our findings suggest that the previously reported EPC antigen profile, defined by the simultaneous expression of VEGFR2 and CD133 on the surface of CD45dim/CD34pos cells, should be carefully re-evaluated and further studies should be conducted to redefine EPC features in order to translate CEC and EPC characterization into clinical practice.

ImmunoPET for assessing the differential uptake of a CD146-specific monoclonal antibody in lung cancer.

PURPOSE: 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. CONCLUSION: 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.

Inhibiting CD146 by its Monoclonal Antibody AA98 Improves Radiosensitivity of Cervical Cancer Cells.

BACKGROUND Cervical cancer is one of the major causes of cancer death of females worldwide. Radiotherapy is considered effective for cervical cancer treatment, but the low radiosensitivity found in some cases severely affects therapeutic outcomes. This study aimed to reveal the role of CD146, an important adhesion molecule facilitating tumor angiogenesis, in regulating radiosensitivity of cervical cancer cells. MATERIAL AND METHODS CD146 protein expression was compared in normal cells, cervical cancer cells with lower radiosensitivity, and cervical cancer cells with higher sensitivity from cervical squamous cell carcinoma patients. Anti-CD146 monoclonal antibody AA98 was used to inhibit CD146 in human cervical cancer SiHa cells with relatively low radiosensitivity, and then the cell survival and apoptosis changes after radiation were detected by colony formation assay and flow cytometry. RESULTS CD146 protein was significantly up-regulated in cervical cancer cells (P<0.001), especially in cancer cells with lower radiosensitivity. The SiHa cells treated with AA98 showed more obvious inhibition in cell survival (P<0.05) and promotion in cell apoptosis (P<0.01) after radiation, compared to the untreated cells. More dramatic changes in apoptotic factors Caspase 3 and Bcl-XL were also detected in AA98-treated cells. CONCLUSIONS These results indicate that inhibiting CD146 improves the effect of radiation in suppressing SiHa cells. This study shows the potential of CD146 as a target for increasing radiosensitivity of cervical cancer cells, which might allow improvement in treatment outcome in cervical cancer. Further studies are necessary for understanding the detailed mechanism of CD146 in regulating radiosensitivity.

CRISPR-Cas9-mediated gene knockout in primary human airway epithelial cells reveals a proinflammatory role for MUC18.

Targeted knockout of genes in primary human cells using CRISPR-Cas9-mediated genome-editing represents a powerful approach to study gene function and to discern molecular mechanisms underlying complex human diseases. We used lentiviral delivery of CRISPR-Cas9 machinery and conditional reprogramming culture methods to knockout the MUC18 gene in human primary nasal airway epithelial cells (AECs). Massively parallel sequencing technology was used to confirm that the genome of essentially all cells in the edited AEC populations contained coding region insertions and deletions (indels). Correspondingly, we found mRNA expression of MUC18 was greatly reduced and protein expression was absent. Characterization of MUC18 knockout cell populations stimulated with TLR2, 3 and 4 agonists revealed that IL-8 (a proinflammatory chemokine) responses of AECs were greatly reduced in the absence of functional MUC18 protein. Our results show the feasibility of CRISPR-Cas9-mediated gene knockouts in AEC culture (both submerged and polarized), and suggest a proinflammatory role for MUC18 in airway epithelial response to bacterial and viral stimuli.

An ultrasensitive electrochemical immunosensor for the detection of CD146 based on TiO2 colloidal sphere laden Au/Pd nanoparticles.

An ultrasensitive electrochemical immunosensor for the detection of cluster of differentiation 146 antigen (CD146) based on TiO2 colloidal sphere laden Au/Pd nanoparticles (Au/Pd@TiO2) was developed. In this work, reduced graphene oxide-tetraethylene pentamine (rGO-TEPA) was applied as an electrode modifying material to modify the surface of a glassy carbon electrode (GCE). Au/Pd@TiO2 was used as the secondary-antibody (Ab2) label for the fabrication of the immunosensor. Amperometric response of the immunosensor for electrocatalytic reduction of hydrogen peroxide (H2O2) was recorded. Electrochemical impedance spectroscopy (EIS) proved that fabrication of the immunosensor was successful. The anti-CD146 primary antibody (Ab1) was immobilized on the rGO-TEPA modified GCE by a cross-linking reagent of glutaraldehyde (GA). With Ab1 immobilized onto the rGO-TEPA modified GCE and Ab2 linked with Au/Pd@TiO2, the immunosensor displayed a wide linear range (0.0050-20 ng mL(-1)), a low detection limit (1.6 pg mL(-1)), good reproducibility, good selectivity and acceptable stability. The designed sensing strategy may provide a potential application in the detection of other tumor markers.

Secretion of interleukin-17 by CD8+ T cells expressing CD146 (MCAM).

Interleukin-17 (IL-17) has been associated with the pathogenesis of numerous autoimmune diseases. CD4+ T cells secreting IL-17 are termed Th17 cells. CD8+ T cells, designated Tc17 cells, are also capable of secreting IL-17. Here we describe a population of Tc17 cells characterized by the expression of surface CD146, an endothelial adhesion molecule. These cells display signatures of a human Tc17 genotype and phenotype. Circulating CD8+CD146+ T cells are present in low levels in healthy adults. Elevations in CD8+CD146+ T cells are found in Behcet's disease and birdshot retinochoroidopathy, which have been reported to have HLA class I associations. Sarcoidosis does not have a class I association and displays an increase in CD4+ CD146+ T cells but not in CD8+CD146+ T cells. CD146 on these cells may facilitate their ability to bind to, and migrate through, endothelium, as has been reported for CD4+CD146+ T cells.

Characterization and quantification of porcine circulating endothelial cells.

BACKGROUND: Endothelial damage is a critical step in the development of (xeno) transplantation-related and cardiovascular pathology. In humans, the amount of circulating endothelial cells (CEC) correlates to disease intensity and functions as a valuable damage marker. While (xeno) transplantation and cardiovascular research is regularly performed in porcine models, the paucity of antibodies against porcine endothelium epitopes hinders the use of CEC as damage marker. OBJECTIVE: This study aimed to develop a method for porcine CEC detection using anti-human antibodies against porcine endothelium epitopes. METHODS: Human umbilical vein endothelial cells (HUVEC, control) and their swine equivalent (SUVEC) were used to assess the cross-species immunoreactivity of fluorescently labeled anti-human CD31/CD51/CD54/CD62E/CD105/CD106/CD144/CD146/PAL-E/lectin-1/vWF antibodies by isotype-controlled fluorescence-activated cell sorting (FACS) and confocal microscopy. Next, reactivity was ascertained with mature porcine kidney-derived endothelial cells (PKEC), and a FACS-based whole blood CEC quantification method was employed using osmotic erythrolysis and CD105 and CD146 double staining after CD45 exclusion. RESULTS: Of the 21 assayed antibodies, the MEM-229 clone of CD105 and P1H12 clone of CD146 showed immunoreactivity with SUVEC and PKEC. Double staining showed baseline porcine CEC count of 673.1 ± 551.4 CEC/ml, while the first 7.5 ml of drawn blood (representative of vascular damage) contained 1118 ± 661.4 CEC/ml (n = 14, P = 0.04). A second experiment (n = 5) including CD45 exclusion identified only 14.5 ± 10.8% double-positive CD105-146 events per ml blood. CONCLUSION: Porcine endothelium can be specifically labeled using anti-human CD146 and CD105 antibodies. These antibodies can therefore be used for the identification and quantification of CEC in porcine whole blood by FACS after osmotic erythrolysis.

Identity and ranking of colonic mesenchymal stromal cells.

Although ongoing clinical trials utilize systemic administration of bone-marrow mesenchymal stromal cells (BM-MSCs) in Crohn's disease (CD), nothing is known about the presence and the function of mesenchymal stromal cells (MSCs) in the normal human bowel. MSCs are bone marrow (BM) multipotent cells supporting hematopoiesis with the potential to differentiate into multiple skeletal phenotypes. A recently identified new marker, CD146, allowing to prospectively isolate MSCs from BM, renders also possible their identification in different tissues. In order to elucidate the presence and functional role of MSCs in human bowel we analyzed normal adult colon sections and isolated MSCs from them. In colon (C) sections, resident MSCs form a net enveloping crypts in lamina propria, coinciding with structural myofibroblasts or interstitial stromal cells. Nine sub-clonal CD146(+) MSC lines were derived and characterized from colon biopsies, in addition to MSC lines from five other human tissues. In spite of a phenotype qualitative identity between the BM- and C-MSC populations, they were discriminated and categorized. Similarities between C-MSC and BM-MSCs are represented by: Osteogenic differentiation, hematopoietic supporting activity, immune-modulation, and surface-antigen qualitative expression. The differences between these populations are: C-MSCs mean intensity expression is lower for CD13, CD29, and CD49c surface-antigens, proliferative rate faster, life-span shorter, chondrogenic differentiation rare, and adipogenic differentiation completely blocked. Briefly, BM-MSCs, deserve the rank of progenitors, whereas C-MSCs belong to the restricted precursor hierarchy. The presence and functional role of MSCs in human colon provide a rationale for BM-MSC replacement therapy in CD, where resident bowel MSCs might be exhausted or diverted from their physiological functions.

Knockdown of CD146 promotes endothelial-to-mesenchymal transition via Wnt/ß-catenin pathway.

PURPOSE: Cardiac fibrosis is characterized by the excessive deposition of extracellular matrix (ECM) proteins and leads to the maladaptive changes in myocardium. Endothelial cells (ECs) undergoing mesenchymal transition contributes to the occurrence and development of cardiac fibrosis. CD146 is an adhesion molecule highly expressed in ECs. The present study was performed to explore the role of CD146 in modulating endothelial to mesenchymal transition (EndMT). METHODS: C57BL/6 mice were subjected to subcutaneous implantation of osmotic minipump infused with angiotensin II (Ang Ⅱ). Adenovirus carrying CD146 short hairpin RNA (shRNA) or CD146 encoding sequence were infected into cultured human umbilical vein endothelial cells (HUVECs) followed by stimulation with Ang II or transforming growth factor-ß1 (TGF-ß1). Differentially expressed genes were revealed by RNA-sequencing (RNA-Seq) analysis. Gene expression was measured by quantitative real-time PCR, and protein expression and distribution were determined by Western blot and immunofluorescence staining, respectively. RESULTS: CD146 was predominantly expressed by ECs in normal mouse hearts. CD146 was upregulated in ECs but not fibroblasts and myocytes in hearts of Ang II-infused mice and in HUVECs stimulated with Ang Ⅱ. RNA-Seq analysis revealed the differentially expressed genes related to EndMT and Wnt/ß-catenin signaling pathway. CD146 knockdown and overexpression facilitated and attenuated, respectively, EndMT induced by Ang II or TGF-ß1. CD146 knockdown upregulated Wnt pathway-related genes including Wnt4, LEF1, HNF4A, FOXA1, SOX6, and CCND3, and increased the protein level and nuclear translocation of ß-catenin. CONCLUSIONS: Knockdown of CD146 exerts promotional effects on EndMT via activating Wnt/ß-catenin pathway and the upregulation of CD146 might play a protective role against EndMT and cardiac fibrosis.

CD146+ Endometrial-Derived Mesenchymal Stem/Stromal Cell Subpopulation Possesses Exosomal Secretomes with Strong Immunomodulatory miRNA Attributes.

The perivascular localization of endometrial mesenchymal stem/stromal cells (eMSC) allows them to sense local and distant tissue damage, promoting tissue repair and healing. Our hypothesis is that eMSC therapeutic effects are largely exerted via their exosomal secretome (eMSC EXOs) by targeting the immune system and angiogenic modulation. For this purpose, EXOs isolated from Crude and CD146+ eMSC populations were compared for their miRNA therapeutic signatures and immunomodulatory functionality under inflammatory conditions. eMSC EXOs profiling revealed 121 in Crude and 88 in CD146+ miRNAs, with 82 commonly present in both populations. Reactome and KEGG analysis of miRNAs highly present in eMSC EXOs indicated their involvement among others in immune system regulation. From the commonly present miRNAs, four miRNAs (hsa-miR-320e, hsa-miR-182-3p, hsa-miR-378g, hsa-let-7e-5p) were more enriched in CD146+ eMSC EXOs. These miRNAs are involved in macrophage polarization, T cell activation, and regulation of inflammatory cytokine transcription (i.e., TNF-α, IL-1ß, and IL-6). Functionally, stimulated macrophages exposed to eMSC EXOs demonstrated a switch towards an alternate M2 status and reduced phagocytic capacity compared to stimulated alone. However, eMSC EXOs did not suppress stimulated human peripheral blood mononuclear cell proliferation, but significantly reduced secretion of 13 pro-inflammatory molecules compared to stimulated alone. In parallel, two anti-inflammatory proteins, IL-10 and IL-13, showed higher secretion, especially upon CD146+ eMSC EXO exposure. Our study suggests that eMSC, and even more, the CD146+ subpopulation, possess exosomal secretomes with strong immunomodulatory miRNA attributes. The resulting evidence could serve as a foundation for eMSC EXO-based therapeutics for the resolution of detrimental aspects of tissue inflammation.

The effects of high dose interferon-ß1a on plasma microparticles: correlation with MRI parameters.

OBJECTIVES: We previously reported a correlation between levels of micro particles carrying CD31 (PMP(CD31+)) and disease activity in MS. However, the effects of long term (12 month) treatment with high dose, high frequency interferon-ß1a (Rebif™) on plasma levels of PMP(CD31+), PMP(CD146+), and PMP(CD54+) and MRI measures of disease activity have not yet been assessed. METHODS: During this prospective 1-year study, we used flow cytometry to measure changes in plasma micro particles (PMP) bearing CD31 (PMP(CD31+)), CD146 (PMP(CD146+)), and CD54/ICAM-1 (PMP(CD54+)) in 16 consecutive patients with relapsing-remitting MS (RRMS) before and after 3, 6, and 12 months of subcutaneous therapy with interferon-beta1a (44 micrograms, 3X weekly). At each visit, clinical exams and expanded disability status scale (EDSS) scores were recorded. RESULTS: Plasma levels of PMP(CD31+), and PMP(CD54+) were significantly reduced by treatment with IFN-ß1a. PMP(CD146+) appeared to decrease only at 3 months and did not persist at 6 and 12 months (p = 0.0511). In addition, the decrease in plasma levels of PMP(CD31+) and PMP(CD54+) levels at 12 months were associated with a significant decrease in the number and volume of contrast enhancing T1-weigthed lesions. CONCLUSION: Our data suggest that serial measurement of plasma micro particles (PMP), particularly in the initial stages of MS (when neuro-inflammatory cascades are more intense), may serve as reliable and reproducible surrogate markers of response to IFN-ß1a therapy for MS. In addition, the progressive decline in plasma levels of PMP(CD31+) and PMP(CD54+) further supports the concept that IFN-ß1a exerts stabilizing effect on the cerebral endothelial cells during pathogenesis of MS.

MCAM-expressing CD4(+) T cells in peripheral blood secrete IL-17A and are significantly elevated in inflammatory autoimmune diseases.

Th17 cells are a subset of CD4(+) T cells characterized by production of IL-17 and are known to be key participants in inflammatory reactions and various autoimmune diseases. In this study we found that a subset of human CD4(+) T cells expressing MCAM (CD146) have higher mRNA levels of RORC2, IL-23R, IL-26, IL-22, IL-17A, but not IFN-γ, compared to CD4(+) T cell not expressing CD146. Upon TCR stimulation with CD3/CD28, CD4(+)CD146(+) T cells secrete significantly more IL-17A, IL-6, and IL-8 than do CD4(+)CD146(-) T cells. Low frequencies of CD4(+)CD146(+) T cells are found in the circulation of healthy adults, but the frequency of these cells is significantly increased in the circulation of patients with inflammatory autoimmune diseases including Behcet's, sarcoidosis and Crohn's disease. Patterns of gene expression and cytokine secretion in these cells are similar in healthy and disease groups. In Crohn's disease, the increase in CD4(+)CD146(+) cells in the circulation correlates with disease severity scores. These data indicate that expression of CD146 on CD4(+) T cells identifies a population of committed human Th17 cells. It is likely the expression of CD146, an endothelial adhesion molecule, facilitates adherence and migration of Th17 cells through the endothelium to sites of inflammation.

Up-regulation of METCAM/MUC18 promotes motility, invasion, and tumorigenesis of human breast cancer cells.

BACKGROUND: Conflicting research has identified METCAM/MUC18, an integral membrane cell adhesion molecule (CAM) in the Ig-like gene super-family, as both a tumor promoter and a tumor suppressor in the development of breast cancer. To resolve this, we have re-investigated the role of this CAM in the progression of human breast cancer cells. METHODS: Three breast cancer cell lines were used for the tests: one luminal-like breast cancer cell line, MCF7, which did not express any METCAM/MUC18, and two basal-like breast cancer cell lines, MDA-MB-231 and MDA-MB-468, which expressed moderate levels of the protein.MCF7 cells were transfected with the human METCAM/MUC18 cDNA to obtain G418-resistant clones which expressed the protein and were used for testing effects of human METCAM/MUC18 expression on in vitro motility and invasiveness, and in vitro and in vivo tumorigenesis. Both MDA-MB-231 and MDA-MB-468 cells already expressed METCAM/MUC18. They were directly used for in vitro tests in the presence and absence of an anti-METCAM/MUC18 antibody. RESULTS: In MCF7 cells, enforced METCAM/MUC18 expression increased in vitro motility, invasiveness, anchorage-independent colony formation (in vitro tumorigenesis), and in vivo tumorigenesis. In both MDA-MB-231 and MDA-MB-468 cells, the anti-METCAM/MUC18 antibody inhibited both motility and invasiveness. Though both MDA-MB-231 and MDA-MB-468 cells established a disorganized growth in 3D basement membrane culture assay, the introduction of the anti-METCAM/MUC18 antibody completely destroyed their growth in the 3D culture. CONCLUSION: These findings support the notion that human METCAM/MUC18 expression promotes the progression of human breast cancer cells by increasing their motility, invasiveness and tumorigenesis.

CD146 bound to LCK promotes T cell receptor signaling and antitumor immune responses in mice.

Initiation of T cell receptor (TCR) signaling involves the activation of the tyrosine kinase LCK; however, it is currently unclear how LCK is recruited and activated. Here, we have identified the membrane protein CD146 as an essential member of the TCR network for LCK activation. CD146 deficiency in T cells substantially impaired thymocyte development and peripheral activation, both of which depend on TCR signaling. CD146 was found to directly interact with the SH3 domain of coreceptor-free LCK via its cytoplasmic domain. Interestingly, we found CD146 to be present in both monomeric and dimeric forms in T cells, with the dimerized form increasing after TCR ligation. Increased dimerized CD146 recruited LCK and promoted LCK autophosphorylation. In tumor models, CD146 deficiency dramatically impaired the antitumor response of T cells. Together, our data reveal an LCK activation mechanism for TCR initiation. We also underscore a rational intervention based on CD146 for tumor immunotherapy.

CD146 and its soluble form regulate monocyte transendothelial migration.

OBJECTIVES: During inflammation, cell adhesion molecules are modulated or redistributed for leukocyte transmigration. Among molecules at the interendothelial junction, CD146 is involved in cell-cell cohesion and permeability, but its role in monocyte transmigration is unknown. METHODS AND RESULTS: TNF enhanced CD146 expression at the junction and apical membrane of human umbilical veins endothelial cells (HUVECs) through CD146 synthesis and intracellular store redistribution. In addition, TNF increased the release of a soluble form (sCD146) through a metalloproteinase-dependent mechanism. The redistribution of CD146 to the junction led us to investigate its role in monocyte transmigration using THP1 and freshly isolated monocytes. Evidence that CD146 contributes to monocyte transmigration was provided by inhibition experiments using anti-CD146 antibodies and CD146 siRNA in HUVECs. In addition, sCD146 specifically bound both monocytes and HUVECs and dose-dependently increased monocyte transmigration. Assessment of sCD146 binding on immobilized CD146 failed to evidence any homophilic interaction. Together, our data suggest endothelial CD146 binds heterophilically with a yet unknown ligand on monocytes. CONCLUSIONS: Our results demonstrate that CD146 is regulated by the inflammatory cytokine TNF and that CD146 and sCD146 are both involved in monocyte transendothelial migration during inflammation.