BACKGROUND

The purpose of this study was to investigate the diagnostic and prognostic value of circulating endothelial cell (CEC), a potential surrogate of tumor angiogenesis, in malignant pleural mesothelioma (MPM).

METHODS

We prospectively evaluated CEC count in 4.0 mL of peripheral blood sampled from patients with a suspicion of MPM. An automated system was used to capture CECs with an anti-CD146 antibody.

RESULTS

Of 109 eligible patients, 30 were finally diagnosed with non-malignant diseases, and 79 were with MPM. CEC count was significantly higher in MPM patients than in NM patients (mean CEC count, 120.3 and 39.9, respectively; P = 0.001), and a receiver operating characteristic (ROC) curve analysis showed that CEC provided a significant diagnostic performance in discrimination between MPM and nonmalignant diseases with an area under curve (AUC-ROC) of 0.700 (95 % confidence interval [95 % CI], 0.595-0.806; P = 0.001). Among MPM patients, CEC count was positively correlated with intratumoral microvessel density (MVD), a measurement of tumor angiogenesis (Spearman correlation coefficiency [r] = 0.444; P = 0.001). Higher CEC count (>50) was significantly associated with a poor prognosis (median overall survival, 11.4 months [95 % CI, 7.6-15.2] for higher CEC count patients versus 20.1 months [95 % CI, 16.0-24.2] for lower CEC count patients; P = 0.028). A multivariate analysis showed that higher CEC count was a significant and independent factor to predict a poor prognosis (hazard ratio [HR], 2.24, [95 % CI, 1.24-4.43]; P = 0.009).

CONCLUSIONS

CEC, as a surrogate of tumor angiogenesis, was a promising marker in diagnosis and prediction of prognosis in MPM.

The aim of this study was to determine whether the inflammatory milieu and/or hypoxia induces the dedifferentiation of synovial cells into mesenchymal stem-like cells, which may contribute to the tumor-like growth of synovial cells. Expression of mesenchymal stem cell markers (CD24, CD44, CD90, CD106, CD146 and Stro-1) was compared among cultured fibroblast-like synoviocytes (FLSs) from patients with rheumatoid arthritis (RA) or osteoarthritis (OA), bone marrow mesenchymal stem cells (BM MSCs) and normal dermal fibroblasts. After the cells were stimulated with pro-inflammatory cytokines for 3 days under hypoxia or normoxia, the stem cell markers were analyzed by FACS. CD44 and CD90 were expressed constitutively in all four cell types. Only the BM MSCs strongly expressed CD146. The expression of stem cell markers was similar between FLSs from RA and those from OA patients. In addition, the expression levels in FLSs were similar to those in normal dermal fibroblasts. The stimulation of FLSs and dermal fibroblasts with IL-1β or a mixture of cytokines under hypoxia did not induce a marked change in the expression of stem cell markers. These results indirectly suggest that the pro-inflammatory milieu may be not sufficient to induce the dedifferentiation of FLSs in arthritic joints.

We describe an epithelioid trophoblastic tumor (ETT) metastatic to the vagina in a 30-year-old Japanese woman. A polypoid tumor in the vaginal orifice was composed of nests of intermediate trophoblastic cells that showed a striking epithelioid appearance. In the hysterectomy specimen, a tumor infiltrated through the myometrium and showed histologic findings similar to those of the vaginal tumor. The tumor cells were positive for cytokeratin, inhibin-alpha, and melanoma cell adhesion molecule (Mel-CAM, CD146) but were only focally positive for human placental lactogen. Electron microscopic examination revealed bundles of well-developed, intermediate-type filaments surrounding the nuclei.

Endothelial colony-forming progenitor cells (ECFCs) are promising candidates for cell therapies. However, ECFC translation to the clinic requires optimized isolation and manufacture technologies according to good manufacturing practice (GMP).

ECFCs were manufactured from steady-state peripheral blood (PB) leukapheresis (11 donors), using GMP-compliant technologies including pooled human platelet (PLT) lysate, and compared to human umbilical cord endothelial cells, human aortic endothelial cells, and human cerebral microvascular endothelial cells. Specific variables assessed were growth kinetics, phenotype, trophic factors production, stimulation of tube formation, and Dil-AcLDL uptake.

ECFCs could be isolated from PB leukapheresis units with mean processed volume of 5411 mL and mean white blood cell (WBC) concentration factor of 8.74. The mean frequency was 1.44 × 10-8 ECFCs per WBC, corresponding to a mean of 177.8 ECFCs per apheresis unit. Expandable for up to 12 cumulative population doublings, calculated projection showed that approximately 730 × 103 ECFCs could be manufactured from 1 apheresis unit. ECFCs produced epidermal growth factor, hepatocyte growth factor, vascular endothelial growth factor (VEGF)-A, PLT-derived growth factor-B, interleukin-8, and monocyte chemoattractant protein-1, featured high potential for capillary-like tubes formation, and showed no telomerase activity. They were characterized by CD29, CD31, CD44, CD105, CD117, CD133, CD144, CD146, and VEGF-R2 expression, with the most common subpopulation CD34+CD117-CD133-. Compared to controls, ECFCs featured greater Dil-AcLDL uptake and higher expression of CD29, CD31, CD34, CD44, CD144, and VEGF-R2.

Here we show that isolation of ECFCs with proangiogenic profile from steady-state PB leukapheresis is feasible, marking a first step toward ECFC product manufacture according to GMP.

For non-invasive stem cells tracking through MRI, it is important to understand the efficiency of in vitro labeling of stem cells with iron oxide with regard to its relaxation behavior. In this study, we have carried out a pilot study of labeling mice mesenchymal stem cells (mMSCs) with ultrasmall superparamagnetic iron oxide (USPIO) entrapped with poly-L-lysine (PLL) in different ratios and incubated with different times. Our results demonstrated that 50:1.5 µg/ml of iron oxide and PLL at an incubation time of 6h with 10% serum concentration are sufficient enough for effective labeling. Optimized labeling showed that >98% of viability and <3% toxicity were observed at a total iron content of 11.8 pg/cell. In vitro relaxometry study showed that almost a 6.6 fold reduction in transverse relaxation time (T2) was observed after labeling as compared to unlabeled. IO-PLL complex was more effective than iron oxide alone in labeling and a detectable lower limit found to be hundred with optimized concentration. Significant increase in Oct-4 expression on day-3 after labeling was observed, whereas CD146 expression remains unchanged in real time RT-PCR. This optimized labeling method of MSCs may be very useful for cellular MRI and stem cells tracking studies.

OBJECTIVE

VAP-1 (vascular adhesion protein-1) is a copper-containing SSAO (semicarbazide sensitive amine oxidase) secreted by vascular smooth muscle cells, adipocytes, endothelial cells with functional monoamine oxidase activity. The oxidation process generates harmful products that may be involved in atherosclerosis and vascular damage. Elevation of SSAO activity is observed in atherosclerosis, diabetes mellitus and obesity. On the other hand, renalase, with possible monoamine oxidase activity, which breaks down catecholamines like SSAO, is also expressed in the endothelium as well as in the kidney. The aim of the study was to assess VAP-1 levels and its correlations with endothelial injury markers and renalase in 50 kidney allograft recipients.

METHODS

Hemoglobin, urea, creatinine, rate were studied by standard laboratory method in the hospital central laboratory. We assessed markers of endothelial function/injury: vWF, thrombomodulin, ICAM, VCAM, CD40L, CD44, CD146, inflammation: hsCRP, and IL-6 and adipocytokines: leptin, adiponectin, visfatin, apelin with commercially available assays.

RESULTS

The mean serum VAP-1 in Tx was significantly higher comparing to the control group. In kidney transplant recipients VAP-1 correlated with BMI (r=0.39, p<0.01), CD44 (r=0.27, p<0.05), hsCRP (r=0.28, p<0.05), serum creatinine (r=0.29, p<0.05), eGFR (CKD-EPI formula r=-0.27, p<0.05, MDRD r=-0.27,p<0.05, Cockcroft-Gault r=-0.35,p<0.01), serum urea (r=0.27, p<0.05), CD146 (r=0.49, p<0.001), CD40L (r=0.26, p<0.06), and renalase (r=0.34, p<0.05). In multiple regression analysis VAP-1 was predicted 80% by serum creatinine (beta value 0.33, p=0.01), and CD146 (beta value 43, p=0.0005).

CONCLUSIONS

VAP-1, elevated in kidney transplant recipients, is predominantly dependent on endothelial damage and kidney function, which deteriorated with time after kidney transplantation.

Perivascular epithelioid cell tumors (PEComas) are an uncommon family of soft tissue tumors with dual myoid-melanocytic differentiation. Although PEComa family tumors commonly demonstrate a perivascular growth pattern, pericyte antigen expression has not yet been examined among this unique tumor group. Previously, we demonstrated that a subset of perivascular soft tissue tumors exhibit a striking pericytic immunophenotype, with diffuse expression of αSMA, CD146, and PDGFRβ. Here, we describe the presence of pericyte antigens across a diverse group of PEComa family tumors (n = 19 specimens). Results showed that pericyte antigens differed extensively by histological appearance. Typical angiomyolipoma (AML) specimens showed variable expression of pericyte antigens among both perivascular and myoid-appearing cells. In contrast, AML specimens with a predominant spindled morphology showed diffuse expression of pericyte markers, including αSMA, CD146, and PDGFRβ. AML samples with predominant epithelioid morphology showed a marked reduction in or the absence of immunoreactivity for pericyte markers. Lymphangiomyoma samples showed more variable and partial pericyte marker expression. In summary, pericyte antigen expression is variable among PEComa family tumors and largely varies by tumor morphology. Pericytic marker expression in PEComa may represent a true pericytic cell of origin, or alternatively aberrant pericyte marker adoption. Markers of pericytic differentiation may be of future diagnostic utility for the evaluation of mesenchymal tumors, or identify actionable signaling pathways for future therapeutic intervention.

Expression of the adhesion molecule, CD146/MCAM/MelCAM, on T cells has been associated with recent activation, memory subsets and T helper type 17 (Th17) effector function, and is elevated in inflammatory arthritis. Th17 cells have been implicated in the pathogenesis of rheumatoid arthritis (RA) and spondyloarthritides (SpA). Here, we compared the expression of CD146 on CD4(+) T cells between healthy donors (HD) and patients with RA and SpA [ankylosing spondylitis (AS) or psoriatic arthritis (PsA)] and examined correlations with surface markers and cytokine secretion. Peripheral blood mononuclear cells (PBMC) were obtained from patients and controls, and synovial fluid mononuclear cells (SFMC) from patients. Cytokine production [elicited by phorbol myristate acetate (PMA)/ionomycin] and surface phenotypes were evaluated by flow cytometry. CD146(+) CD4(+) and interleukin (IL)-17(+) CD4(+) T cell frequencies were increased in PBMC of PsA patients, compared with HD, and in SFMC compared with PBMC. CD146(+) CD4(+) T cells were enriched for secretion of IL-17 [alone or with IL-22 or interferon (IFN)-γ] and for some putative Th17-associated surface markers (CD161 and CCR6), but not others (CD26 and IL-23 receptor). CD4(+) T cells producing IL-22 or IFN-γ without IL-17 were also present in the CD146(+) subset, although their enrichment was less marked. Moreover, a majority of cells secreting these cytokines lacked CD146. Thus, CD146 is not a sensitive or specific marker of Th17 cells, but rather correlates with heterogeneous cytokine secretion by subsets of CD4(+) helper T cells.

To study the effects of the donor age on the application potential of human urine-derived stem cells (hUSCs) in bone tissue engineering, by comparing proliferation, senescence and osteogenic differentiation of hUSCs originated from volunteers with different ages. The urine samples were collected from 19 healthy volunteers (6 cases from children group aged from 5 to 14, 5 cases from middle-aged group aged from 30 to 40, and 8 cases from the elder group aged from 65 to 75), and hUSCs were isolated and cultured. The cell morphology was observed by microscope and the cell surface markers were identified by flow cytometry. Their abilities to undergo osteogenic, adipogenic and chondrogenic differentiation were determined in vitro, and cell proliferation analyses were performed using Cell Counting Kit-8 (CCK8) Assay. The senescence of hUSCs among three groups was assessed by senescence-associated β galactosidase staining. After osteogenic differentiation, the alkaline phosphatase (ALP) activity of hUSCs was measured and expression of osteogenic-related runt-related transcription factor 2 (RUNX2) and osteocalcin (OCN) was determined by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. The hUSCs isolated from urine samples were adherent cells displayed "rice gain"-like and "spindle-shaped" morphology, expressing surface markers of mesenchymal stem cells (MSCs) (CD73, CD90, CD105) and the peripheral cell marker (CD146), but not hematopoietic stem cell markers (CD34, CD45) or the embryonic stem cell marker (OCT3/4). The obtained hUSCs could be induced into osteogenic, adipogenic or chondrogenic differentiation. The hUSCs from the children group showed higher proliferation and lower tendency to senescence than those from the middle-aged and elder groups. After osteogenic induction, the ALP activity and RUNX2 and OCN expression of hUSCs from the children group were higher than those from the elder group. While no significant differences were observed when comparing the middle-aged group with the children group or the elder group. Donor age could influence the potency of hUSCs on proliferation, senescence and capacity of osteogenic differentiation. hUSCs from children group have shown higher proliferation, lower tendency to senescence, and stronger osteogenic capacity, which means to be more suitable for basic research and have better clinical application. Furthermore, hUSCs from all groups suggest the application potential in bone tissue engineering as seed cells.

Clear cell renal cell carcinoma is the most prevalent of all reported kidney cancer cases, and currently there are no markers for early diagnosis. This has stimulated great research interest recently because early detection of the disease can significantly improve the low survival rate. Combining the proteome, glycoproteome, and N-glycome data from clear cell renal cell carcinoma plasma has the potential of identifying candidate markers for early diagnosis and prognosis and/or to monitor disease recurrence. Here, we report on the utilization of a multi-dimensional fractionation approach (12P-M-LAC) and LC-MS/MS to comprehensively investigate clear cell renal cell carcinoma plasma collected before (disease) and after (non-disease) curative nephrectomy (n = 40). Proteins detected in the subproteomes were investigated via label-free quantification. Protein abundance analysis revealed a number of low-level proteins with significant differential expression levels in disease samples, including HSPG2, CD146, ECM1, SELL, SYNE1, and VCAM1. Importantly, we observed a strong correlation between differentially expressed proteins and clinical status of the patient. Investigation of the glycoproteome returned 13 candidate glycoproteins with significant differential M-LAC column binding. Qualitative analysis indicated that 62% of selected candidate glycoproteins showed higher levels (upregulation) in M-LAC bound fraction of disease samples. This observation was further confirmed by released N-glycans data in which 53% of identified N-glycans were present at different levels in plasma in the disease vs non-disease samples. This striking result demonstrates the potential for significant protein glycosylation alterations in clear cell renal cell carcinoma cancer plasma. With future validation in a larger cohort, information derived from this study may lead to the development of clear cell renal cell carcinoma candidate biomarkers.

Cancer extracellular vesicles (EVs) shuttle at distance and fertilize pre-metastatic niches facilitating subsequent seeding by tumor cells. However, the link between EV secretion mechanisms and their capacity to form pre-metastatic niches remains obscure. Using mouse models, we show that GTPases of the Ral family control, through the phospholipase D1, multi-vesicular bodies homeostasis and tune the biogenesis and secretion of pro-metastatic EVs. Importantly, EVs from RalA or RalB depleted cells have limited organotropic capacities in vivo and are less efficient in promoting metastasis. RalA and RalB reduce the EV levels of the adhesion molecule MCAM/CD146, which favors EV-mediated metastasis by allowing EVs targeting to the lungs. Finally, RalA, RalB and MCAM/CD146, are factors of poor prognosis in breast cancer patients. Altogether, our study identifies RalGTPases as central molecules linking the mechanisms of EVs secretion and cargo loading to their capacity to disseminate and induce pre-metastatic niches in a CD146 dependent manner.

Keywords: cancer biology; cell biology; human; mouse; zebrafish.

OBJECTIVE

Visfatin is an adipocytokine that has recently generated much interest. The aim of the study was to assess visfatin in correlation with markers of endothelial damage and inflammation in haemodialyzed and peritoneally dialyzed patients.

METHODS

Visfatin, leptin, apelin and adiponectin, markers of coagulation (thrombin-antithrombin complexes (TAT), prothrombin fragments 1+2 (F1+2)), fibrinolysis (tissue plasminogen activator (tPA), plasminogen activator inhibitor type 1 (PAI-1)), endothelial function/injury (Von Willebrand factor (vWF), thrombomodulin, intracellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), CD146) and inflammation (high-sensitivity C-reactive protein (hsCRP), tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-6) were assessed.

RESULTS

Triglycerides, hsCRP, creatinine, IL-6, TNF-alpha, vWF, F1+2, TAT, thrombomodulin, ICAM, VCAM, CD146, PAI-1, leptin, adiponectin and visfatin were elevated in dialyzed patients over controls. Visfatin correlated significantly, in univariate analysis, in haemodialyzed patients with markers of endothelial damage/inflammation (CD146, ICAM, IL-6), other adipocytokines, Kt/V and dialysis vintage, and tended to correlate with hsCRP. In peritoneally dialyzed patients, visfatin correlated significantly with haemoglobin, and markers of endothelial damage. In the healthy volunteers visfatin correlated significantly with ICAM, creatinine and IL-6. In multiple regression analysis in HD patients visfatin was only independently related to Kt/V, dialysis vintage and IL-6.

CONCLUSIONS

Elevated visfatin related to markers of inflammation might represent a novel link between inflammation and adipocytokines in dialyzed patients. Time on dialyses and dialysis adequacy may influence visfatin in dialyzed patients due to the decreased clearance of visfatin.

Mesenchymal stem/stromal cells (MSCs) have been used in human and equine regenerative medicine, and interest in exploiting their potential has increased dramatically over the years. Despite significant effort to characterize equine MSCs, the actual origin of these cells and how much of their native phenotype is maintained in culture have not been determined. In this study, we investigated the relationship between MSCs, derived from adipose tissue (AT) and bone marrow (BM), and pericytes in the horse. Both pericyte (CD146, NG2, and αSMA) and MSC (CD29, CD90, and CD73) markers were detected in equine AT and colocalized around blood vessels. Importantly, as assessed by flow cytometry, both pericyte (CD146, NG2, and αSMA) and MSC (CD29, CD44, CD90, and CD105) markers were present in a majority (≥90%) of cells in cultures of AT-MSCs and BM-MSCs; however, levels of pericyte markers were variable within each of those populations. Moreover, the expression of pericyte markers was maintained for at least eight passages in both AT-MSCs and BM-MSCs. Hematopoietic (CD45) and endothelial (CD144) markers were also detected at low levels in MSCs by quantitative polymerase chain reaction (qPCR). Finally, in coculture experiments, AT-MSCs closely associated with networks produced by endothelial cells, resembling the natural perivascular location of pericytes in vivo. Our results indicate that equine MSCs originate from perivascular cells and moreover maintain a pericyte-like phenotype in culture. Therefore, we suggest that, in addition to classical MSC markers, pericyte markers such as CD146 could be used when assessing and characterizing equine MSCs.

The objective of this study was to determine the potential of human bone marrow derived mesenchymal stem cells (hBMSCs) as gene carriers for improving the outcome of human islet transplantation. hBMSCs were characterized for the expression of phenotypic markers and transduced with Adv-hVEGF-hIL-1Ra to overexpress human vascular endothelial growth factor (hVEGF) and human interleukin-1 receptor antagonist (hIL-1Ra). Human islets were co-cultured with hBMSCs overexpressing hVEGF and hIL-1Ra. Islet viability was determined by membrane fluorescent method and glucose stimulation test. Transduced hBMSCs and human islets were co-transplanted under the kidney capsule of NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl) /SzJ (NSG) diabetic mice and blood glucose levels were measured over time to demonstrate the efficacy of genetically modified hBMSCs. At the end of study, immunofluorescent staining of kidney section bearing islets was performed for insulin and von Willebrand Factor (vWF). hBMSCs were positive for the expression of CD73, CD90, CD105, CD146 and Stro-1 surface markers as determined by flow cytometry. Transduction of hBMSCs with adenovirus did not affect their stemness and differentiation potential as confirmed by mRNA levels of stem cell markers and adipogenic differentiation of transduced hBMSCs. hBMSCs were efficiently transduced with Adv-hVEGF-hIL-1Ra to overexpress hVEGF and hIL-1Ra. Live dead cell staining and glucose stimulation test have shown that transduced hBMSCs improved the viability of islets against cytokine cocktail. Co-transplantation of human islets with genetically modified hBMSCs improved the glycemic control of diabetic NSG mice as determined by mean blood glucose levels and intraperitoneal glucose tolerance test. Immunofluorescent staining of kidney sections was positive for human insulin and vWF. In conclusion, our results have demonstrated that hBMSCs may be used as gene carriers and nursing cells to improve the outcome of islet transplantation.

BACKGROUND

Angiogenesis is one of the hallmarks of cancer driving tumour growth and ultimately metastasis. Circulating endothelial cells (CECs) and circulating endothelial progenitor (CEPs) cells have been reported as candidate surrogate markers for tumour vascularisation. Our aim was to investigate the potential use of these circulating cells levels as predictors of prostate cancer treatment failure and metastasis.

METHODS

We examined the levels of CD31(+)CD45(-) cells (CECs) and CD31(+)CD45(-)CD117(+) (CEPs) in s.c. and orthotopic models of human prostate cancers and correlated measurements with tumour size, volume and microvessel density (MVD). We then performed a prospective cohort study in 164 men with localised prostate cancer undergoing prostatectomy. The CD31(+)CD45(-), CD31(+)CD45(-)CD146(+) (CECs) and CD31(+)CD45(intermediate)CD133(+) (CEPs) populations were quantified and subsequently enriched for further characterisation.

RESULTS

In preclinical models, levels of CD31(+)CD45(-) cells, but not CEPs, were significantly elevated in tumour-bearing mice and correlated with tumour size, volume and MVD. In our human prospective cohort study, the levels of CD31(+)CD45(-) cells were significantly higher in men who experienced treatment failure within the first year, and on logistic regression analysis were an independent predictor of treatment failure, whereas neither levels of CECs or CEPs had any prognostic utility. Characterisation of the isolated CD31(+)CD45(-) cell population revealed an essentially homogenous population of large, immature platelets representing <0.1% of circulating platelets.

CONCLUSIONS

Elevated levels of a distinct subpopulation of circulating platelets were an independent predictor for early biochemical recurrence in prostate cancer patients within the first year from prostatectomy.

OBJECTIVE

Although histone deacetylase inhibitors (HDACi) are emerging as a new class of anticancer agents, one of the most significant concerns is that interactions with a wide array of substrates using these agents might initiate both therapeutic and undesired protective responses. Here, we sought to identify the potential protective reactions initiated by HDACi and determine whether targeting these reactions would enhance the antitumoral activity of HDACi.

METHODS

Gene expression profiles were analyzed by cDNA microarray in Molt-4 cells before and after treatment of vorinostat. Induction of CD146 by vorinostat was examined in a wide range of tumors and nonmalignant cells. AA98, an anti-CD146 monoclonal antibody, was used to target CD146 function. Synergistic antitumoral and antiangiogenic effects between AA98 and vorinostat were examined both in vitro and in vivo. The potential effect of combined AA98 and vorinostat treatment on the AKT pathway was determined by Western blotting.

RESULTS

The induction of CD146 is a common phenomenon in vorinostat-treated cancer but not in nonmalignant cells. Targeting of CD146 with AA98 substantially enhanced vorinostat-induced killing via the suppression of activation of AKT pathways in cancer cells. Moreover, AA98 in combination with vorinostat significantly inhibited angiogenesis. In vivo, AA98 synergized with vorinostat to inhibit tumor growth and metastasis.

CONCLUSIONS

The present study provided the first evidence that an undesired induction of CD146 could serve as a protective response to offset the antitumor efficacy of vorinostat. On the other hand, targeting CD146 in combination with vorinostat could be exploited as a novel strategy to more effectively kill cancer cells.

In our previous studies, we constructed the Bifidobacterium infantis thymidine kinase/nucleoside analogue ganciclovir (BI-TK/GCV) system, which was proven to have a sustainable antitumor activity in an in vivo bladder cancer rodent model. In this article, a proteomic approach of isobaric tags for relative and absolute quantification (iTRAQ) and followed by liquid chromatography-tandem mass spectrometry was used to understand the molecular mechanisms of this system. iTRAQ identified 192 downregulated and 210 upregulated proteins after treatment with BI-TK/GCV in Sprague-Dawley rats. Downregulations of proliferating cell nuclear antigen (PCNA), pyruvate kinase isozymes M2 (PKM2), hexokinase 1 (HXK-1), 6-phosphofructokinase (PFK-B), and cell surface glycoprotein (CD146) in bladder cancer after treatment were confirmed by Western blot analysis and validated by immunohistochemistry. Furthermore, the networks of cancer proliferation associated with PCNA, glycolysis associated with PKM2, HXK-1, and PFK-B, and invasion associated with CD146 were illustrated using Ingenuity Pathway Analysis. This study represents the successful application of iTRAQ technology to reveal the molecular mechanisms of BI-TK/GCV treatment system and provides the theoretical support for the effectiveness of our successful treatment system.

To precisely characterize CD146 in adipose stromal/progenitor cells (ASCs) we sorted the stromal vascular faction (SVF) of human abdominal subcutaneous white adipose tissue (sWAT) according to cell surface (cs) expression of CD146, DLK1 and CD34. This test identified three main SVF cell populations: ~50% cs-DLK1-/cs-CD34+/cs-CD146- ASCs, ~7.5% cs-DLK1+/cs-CD34dim/+/cs-CD146+ and ~7.5% cs-DLK1+/cs-CD34dim/+/cs-CD146- cells. All cells contained intracellular CD146. Whole mount fluorescent IHC staining of small vessels detected CD146+ endothelial cells (CD31+/CD34+/CD146+) and pericytes (CD31-/CD34-/CD146+ ASCs). The cells in the outer adventitial layer showed the typical ASC morphology, were strongly CD34+ and contained low amounts of intracellular CD146 protein (CD31-/CD34+/CD146+). Additionally, we detected wavy CD34-/CD146+ and CD34dim/CD146+ cells. CD34dim/CD146+ cells were slightly more bulky than CD34-/CD146+ cells. Both CD34-/CD146+ and CD34dim/CD146+ cells were detached from the inner pericyte layer and protruded into the outer adventitial layer. Cultured early passage ASCs contained low levels of CD146 mRNA, which was expressed in two different splicing variants, at a relatively high amount of the CD146-long form and at a relatively low amount of the CD146-short form. ASCs contained low levels of CD146 protein, which consisted predominantly long form and a small amount of short form. The CD146 protein was highly stable, and the majority of the protein was localized in the Golgi apparatus. In conclusion, the present study contributes to a better understanding of the spatial localization of CD34+/CD146+ and CD34-/CD146+ cells in the adipose niche of sWAT and identifies CD146 as intracellular protein in cs-DLK1-/cs-CD34+/cs-CD146- ASCs.

NOTCH plays a role in regulating stem cell function and fate decision. It is involved in tooth development and injury repair. Information regarding NOTCH expression in human dental root apical papilla (AP) and its residing stem cells (SCAP) is limited. Here we investigated the expression of NOTCH3, its ligand JAG1, and mesenchymal stem cell markers CD146 and STRO-1 in the AP or in the primary cultures of SCAP isolated from AP. Our in situ immunostaining showed that in the AP NOTCH3 and CD146 were co-expressed and associated with blood vessels having NOTCH3 located more peripherally. In cultured SCAP, NOTCH3 and JAG1 were co-expressed. Flow cytometry analysis showed that 7%, 16% and 98% of the isolated SCAP were positive for NOTCH3, STRO-1 and CD146, respectively with a rare 1.5% subpopulation of SCAP co-expressing all three markers. The expression level of NOTCH3 reduced when SCAP underwent osteogenic differentiation. Our findings are the first step towards defining the regulatory role of NOTCH3 in SCAP fate decision.

Dental pulp is a highly vascularized tissue requiring adequate blood supply for successful regeneration. In this study, we investigated the functional role of stem cells from human exfoliated deciduous teeth (SHEDs) as a perivascular source for in vivo formation of vessel-like structures. Primarily isolated SHEDs showed mesenchymal stem cell (MSC)-like characteristics including the expression of surface antigens and in vitro osteogenic and adipogenic differentiation potentials. Moreover, SHEDs were positive for NG2, α-smooth muscle actin (SMA), platelet-derived growth factor receptor beta (PDGFRβ), and CD146 as pericyte markers. To prove feasibility of SHEDs as perivascular source, SHEDs were transplanted into immunodeficient mouse using Matrigel with or without human umbilical vein endothelial cells (HUVECs). Transplantation of SHEDs alone or HUVECs alone resulted in no formation of vessel-like structures with enough red blood cells. However, when SHEDs and HUVECs were transplanted together, extensive vessel-like structures were formed. The presence of murine erythrocytes within lumens suggested the formation of anastomoses between newly formed vessel-like structures in Matrigel plug and the host circulatory system. To understand underlying mechanisms of in vivo angiogenesis, the expression of angiogenic cytokine and chemokine, their receptors, and MMPs was compared between SHEDs and HUVECs. SHEDs showed higher expression of VEGF, SDF-1α, and PDGFRβ than HUVECs. On the contrary, HUVECs showed higher expression of VEGF receptors, CXCR4, and PDGF-BB than SHEDs. This differential expression pattern suggested reciprocal interactions between SHEDs and HUVECs and their involvement during in vivo angiogenesis. In conclusion, SHEDs could be a feasible source of perivascular cells for in vivo angiogenesis.

For melanoma resistant to molecularly targeted therapy and immunotherapy, new treatment strategies are urgently needed. A molecularly targeted theranostic pair may thus be of importance, where the diagnostic probe facilitates patient stratification and the therapeutic companion treats the selected cases. For this purpose, flow cytometry is used to assess the CD146 level in melanoma cells. Based on YY146, a CD146-specific monoclonal antibody, an imaging probe ⁸⁹Zr-Df-YY146 is synthesized and its diagnostic performance is evaluated by positron emission tomography (PET) imaging. Furthermore, a photoimmunotherapy (PIT) agent IR700-YY146 is developed and the therapeutic effect of IR700-YY146 PIT is assessed comprehensively. CD146 is highly expressed in A375 and SK-MEL-5 cells. ⁸⁹Zr-Df-YY146 PET readily detects CD146-positive A375 melanomas. Tumor accumulation of ⁸⁹Zr-Df-YY146 peaks at 72 h with an uptake value of 26.48 ± 3.28%ID g^-1, whereas the highest uptake of the nonspecific ⁸⁹Zr-Df-IgG is 4.80 ± 1.75%ID g^-1. More importantly, IR700-YY146 PIT effectively inhibits the growth of A375 tumors, owing to production of reactive oxygen species, decreased glucose metabolism, and reduced expression of CD146. To conclude, ⁸⁹Zr-Df-YY146 and IR700-YY146 are a promising theranostic pair with the former revealing CD146 expression in melanoma as a PET probe and the latter specifically treating CD146-positive melanoma as an effective PIT agent.

It has been reported that granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophage-colony stimulating factor (GM-CSF) can mobilize endothelial progenitor cells (EPCs) in bone marrow cells (BMCs) into peripheral blood (PB) in vivo. Previously, we also reported that macrophage-colony stimulating factor (M-CSF) can mobilize EPCs into PB, which results in the rapid recovery of blood flow in induced-ischemia limbs by augmenting the number of intramuscular capillaries in vivo. In the present study, we demonstrate that M-CSF and/or G-CSF can increase EPCs from lineage (CD3, B220, Gr-1, Mac-1, CD11c, Ter119, NK1.1 or CD31)-negative BMCs in vitro. Lineage-negative BMCs were cultured with or without M-CSF and/or G-CSF. Three days after culture with M-CSF and/or G-CSF, the number of Flk-1+/CD45-, Sca-1+/CD45-, CD31+/CD45- or CD146+/CD45- cells increased in comparison with no cytokines. When the cultured BMCs with or without G-CSF and/or M-CSF were intravenously injected into ischemia-induced hindlimbs of mice, the number of intramuscular capillaries in the ischemia-induced legs increased; BMCs cultured with G-CSF and/or M-CSF were more effective than those of cytokine non-treated BMCs. These results suggest that M-CSF and/or G-CSF can induce the differentiation of BMCs into EPCs, even in vitro.

OBJECTIVE

Human dental mesenchymal stem cells (hDMSCs) have been isolated from extracted human teeth and proven to have different proliferation and differentiation abilities among the subtypes. Despite increasing interest in the clinical use of hDMSCs, a well-defined specific marker has been absent for these stem cells. In this study, a comparative analysis with known mesenchymal stem cell surface markers such as STRO-1, CD90, CD146, CD34 and TfR (CD71) was performed.

METHODS

Four subtypes of the hDMSCs were obtained and cultured. The hDMSCs were processed by flow cytometric analysis, fluorescence immunocytostaining for in vitro study and in situ immunohistochemical staining for in vivo study.

CONCLUSIONS

The previously known positive and negative MSC markers, such as STRO-1, CD90, CD146 and CD34 showed comparative expression profiles of hDMSC subtypes. TfR was highly positive in hDMSCs compared with the control cells; therefore, TfR was suggested as a new marker for hDMSCs in this study.

Adult stem cells are characterised by longer telomeres compared to mature cells from the same tissue. In this study, candidate CD146 (+) umbilical cord (UC) mesenchymal stem cells (MSCs) were purified by cell sorting from UC tissue digests and their telomere lengths were measured in comparison to donor-matched CD146-negative fraction. UC tissue fragments were enzymatically treated with collagenase and the cells were used for cell sorting, colony-forming fibroblast (CFU-F) assay or for long-term MSC cultivation. Telomere lengths were measured by qPCR in both culture-expanded MSCs and candidate native UC MSCs. Immunohistochemistry was undertaken to study the topography of CD146 (+) cells. Culture-expanded UC MSCs had a stable expression of CD73, CD90 and CD105, whereas CD146 declined in later passages which correlated with the shortening of telomeres in the same cultures. In three out of four donors, telomeres in candidate native UC MSCs (CD45 (-)CD235α (-)CD31 (-)CD146 (+)) were longer compared to donor-matched CD146 (-) population (CD45 (-)CD235α (-)CD31 (-)CD146 (-)). The frequency of CD45 (-)CD235α (-)CD31 (-)CD146 (+) cells measured by flow cytometry was ~1000-fold above that of donor-matched CFU-Fs (means 10.4% and 0.01%, respectively). CD146 (+) cells were also abundant in situ having a broad topography including high levels of positivity in muscle areas in addition to vessels.