Cell-based therapies constitute a real hope for the treatment of ischaemic diseases. One of the sources of endothelial progenitors for autologous cell therapy is Endothelial Colony Forming Cells (ECFC) that can be isolated from peripheral blood. However, their use is limited by their low number in the bloodstream and the loss of their stem cell phenotype associated with the acquisition of a senescent phenotype in culture. We hypothesized that adding soluble CD146, a novel endothelial growth factor with angiogenic properties, during the isolation and growth procedures could improve their number and therapeutic potential. Soluble CD146 increased the number of isolated peripheral blood ECFC colonies and lowered their onset time. It prevented cellular senescence, induced a partial mesenchymal phenotype and maintained a stem cell phenotype by stimulating the expression of embryonic transcription factors. These different effects were mediated through the induction of mature miR-21. When injected in an animal model of hindlimb ischaemia, sCD146-primed ECFC isolated from 40 ml of blood from patients with peripheral arterial disease were able to generate new blood vessels and restore blood flow. Treatment with sCD146 could thus constitute a promising strategy to improve the use of autologous cells for the treatment of ischaemic diseases.

Endothelial colony-forming cells (ECFCs) are promising candidates for cell therapy of ischemic diseases, as less than 10% of patients with an ischemic stroke are eligible for thrombolysis. We previously reported that erythropoietin priming of ECFCs increased their in vitro and in vivo angiogenic properties in mice with hindlimb ischemia. The present study used SPECT/CT to evaluate whether priming of ECFCs with erythropoietin could enhance their homing to the ischemic site after transient middle cerebral artery occlusion (MCAO) followed by reperfusion in rats and potentiate their protective or regenerative effect on blood-brain barrier (BBB) disruption, cerebral apoptosis, and cerebral blood flow (CBF).

METHODS

Rats underwent a 1-h MCAO followed by reperfusion and then 1 d after MCAO received an intravenous injection of either PBS (control, n = 10), PBS-primed ECFCs (ECFCPBS, n = 13), or erythropoietin-primed ECFCs (ECFCEPO, n = 10). ECFC homing and the effect on BBB disruption, cerebral apoptosis, and CBF were evaluated by SPECT/CT up to 14 d after MCAO. The results were expressed as median ± interquartile range for ipsilateral-to-contralateral ratio of the activity in middle cerebral artery-vascularized territories in each hemisphere. Histologic evaluation of neuronal survival and astrocytic proliferation was performed on day 14.

RESULTS

Erythropoietin priming increased homing of ECFCs to the ischemic hemisphere (ECFCPBS, 111.0% ± 16.0%; ECFCEPO, 146.5% ± 13.3%). BBB disruption was significantly reduced (control, 387% ± 153%; ECFCPBS, 151% ± 46% [P < 0.05]; ECFCEPO, 112% ± 9% [P < 0.001]) and correlated negatively with ECFC homing (Pearson r = -0.6930, P = 0.0002). Cerebral apoptosis was significantly reduced (control, 161% ± 10%; ECFCPBS, 141% ± 9% [P < 0.05]; ECFCEPO,118% ± 5% [P < 0.001]) and correlated negatively with ECFC homing (r = -0.7251, P < 0.0001). CBF was significantly restored with ECFCs and almost totally so with erythropoietin priming (control, 72% ± 2%; ECFCPBS, 90% ± 4% [P < 0.01]; ECFCEPO, 99% ± 4% [P < 0.001]) and correlated positively with ECFC homing (r = 0.7348, P < 0.0001). Immunoblocking against the CD146 receptor on ECFCs highlighted its notable role in ECFC homing with erythropoietin priming (ECFCEPO, 147% ± 14%, n = 4; ECFCEPO with antibody against CD146, 101% ± 12%, n = 4 [P < 0.05]).

CONCLUSIONS

Priming with erythropoietin before cell transplantation is an efficient strategy to amplify the migratory and engraftment capacities of ECFCs and their beneficial impact on BBB disruption, apoptosis, and CBF.

BACKGROUND

Impaired glucose tolerance (IGT) following acute myocardial infarction (AMI) increases the incidence of major adverse cardiac events. We hypothesized that endothelial damage following AMI, as assessed by levels of von Willebrand factor (vWF) and circulating endothelial cells (CECs), would be more pronounced in patients with IGT compared to those with normal glucose tolerance (NGT).

METHODS

We studied non-diabetic patients with AMI (n=125; 107 (86%) male; mean age 59 years (SD 12.5)) who underwent oral glucose tolerance testing 3-5 days after admission. We measured vWF (enzyme-linked immunosorbent assay) and CECs (CD146 immunobead capture) in the fasting state and at 2 h post glucose load.

RESULTS

Base-line vWF and CEC levels were higher in IGT patients versus those with NGT and healthy controls (HC) (P<0.001). The acute increase in vWF and CECs in response to the glucose load was significantly higher in the IGT group compared to those with NGT and HC (P<0.01)-an increase on a par with that seen in newly diagnosed diabetics.

CONCLUSIONS

The degree of endothelial damage post AMI in patients with IGT is greater than NGT, and comparable to that seen in frank diabetes mellitus. Subjects with IGT therefore need to be as actively sought and managed.

Various mesenchymal cell types have been identified as critical components of the hematopoietic stem/progenitor cell (HSPC) niche. Although several groups have described the generation of mesenchyme from human pluripotent stem cells (hPSCs), the capacity of such cells to support hematopoiesis has not been reported. Here, we demonstrate that distinct mesenchymal subpopulations co-emerge from mesoderm during hPSC differentiation. Despite co-expression of common mesenchymal markers (CD73, CD105, CD90, and PDGFRβ), a subset of cells defined as CD146hiCD73hi expressed genes associated with the HSPC niche and supported the maintenance of functional HSPCs ex vivo, while CD146loCD73lo cells supported differentiation. Stromal support of HSPCs was contact dependent and mediated in part through high JAG1 expression and low WNT signaling. Molecular profiling revealed significant transcriptional similarity between hPSC-derived CD146++ and primary human CD146++ perivascular cells. The derivation of functionally diverse types of mesenchyme from hPSCs opens potential avenues to model the HSPC niche and develop PSC-based therapies.

Recently, we developed a small molecule responsive hyperactive Mpl-based Cell Growth Switch (CGS) that drives erythropoiesis associated with macrophages in the absence of exogenous cytokines. Here, we compare the physical, cellular and molecular interaction between the macrophages and erythroid cells in CGS expanded CD34+ cells harvested from cord blood, marrow or G-CSF-mobilized peripheral blood. Results indicated that macrophage based erythroid islands could be generated from cord blood and marrow CD34+ cells but not from G-CSF-mobilized CD34+ cells. Additional studies suggest that the deficiency resides with the G-CSF-mobilized CD34+ derived monocytes. Gene expression and proteomics studies of the in vitro generated erythroid islands detected the expression of erythroblast macrophage protein (EMP), intercellular adhesion molecule 4 (ICAM-4), CD163 and DNASE2. 78% of the erythroblasts in contact with macrophages reached the pre reticulocyte orthochromatic stage of differentiation within 14 days of culture. The addition of conditioned medium from cultures of CD146+ marrow fibroblasts resulted in a 700-fold increase in total cell number and a 90-fold increase in erythroid cell number. This novel CD34+ cell derived erythroid island may serve as a platform to explore the molecular basis of red cell maturation and production under normal, stress and pathological conditions.

OBJECTIVE

The aim of this study was to compare the cytotoxic effect of a methacrylate-based and a silorane-based composite on the human dental pulp stem cells (DPSCs) versus human dental pulp fibroblasts (DPFs).

METHODS

Samples of the Filtek Z250 and P90 were polymerized and immersed in the culture medium to obtain extracts after incubation for one, seven and 14 days. Magnetic cell sorting based on the CD146 expression was performed to purify DPSCs and DPFs. After incubation of both cells with the extracts, cytotoxicity was determined using the MTT test.

RESULTS

For the extracts of first and seventh day, both composites showed significantly lower cytotoxicity on DPSCs than DPFs (p=0.003). In addition, there was a significant difference in the time-group interaction of both materials indicating different cytotoxic behaviours (p=0.014). In contrast to Z250, exposure to the 14th day extract of P90 resulted in higher cell viability compared to that of day seven.

CONCLUSIONS

DPSCs are less susceptible to the cytotoxic effect of the composites than DPFs. Compared to Z250, the cytotoxic effect of silorane-based composite decreases as the time passes on. This difference should be considered, particularly in deep cavities, in order to preserve the regenerative capacity of the pulp.

The CD146 (or MUC18/MEL-CAM) antigen is a cell adhesion molecule of the immunoglobulin superfamily. Besides in melanoma, expression of CD146 antigen has been demonstrated in breast epithelia and hair follicles. We studied its expression by human keratinocytes in culture as well as in neoplastic and inflammatory skin diseases. Staining of primary cultured keratinocytes revealed expression of CD146 on the cell membrane, preferentially on cell-cell contact sites. Western blot analysis of keratinocytes detected a band of approximately 113 kDa, corresponding to the CD146 protein. In contrast to primary keratinocytes, neither CD146 protein nor mRNA expression was found in the keratinocyte-derived cell lines A431 and HaCaT. Treatment of keratinocytes with the proinflammatory cytokines interleukin-1 and interleukin-6, tumor necrosis factor-alpha, and interferon-gamma, resulted in no change of CD146 expression and incubation with phorbol 12-myristate 13-acetate led to a reduction of CD146 on keratinocytes. By contrast, when culturing keratinocytes in medium devoid of growth supplements, a distinct upregulation was observed as compared with culture in fully supplemented medium. In normal human epidermis expression of the CD146 antigen was not detectable. It was strongly upregulated, however, on suprabasal keratinocytes in psoriasis, in lichen planus, in the epidermis overlying skin neoplasms, and in viral warts. In squamous cell carcinomas and basal cell carcinomas only a minority of tumor cells expressed CD146. Our findings suggest that the CD146 antigen represents an activation marker of keratinocytes and may be involved in cutaneous inflammatory tissue reaction.

Current vascularization strategies for Tissue Engineering constructs, in particular cell sheet-based, are limited by time-consuming and expensive endothelial cell isolation and/or by the complexity of using extrinsic growth factors. Herein, we propose an alternative strategy using angiogenic cell sheets (CS) obtained from the stromal vascular fraction (SVF) of adipose tissue that can be incorporated into more complex constructs. Cells from the SVF were cultured in normoxic and hypoxic conditions for up to 8days in the absence of extrinsic growth factors. Immunocytochemistry against CD31 and CD146 revealed spontaneous organization in capillary-like structures, more complex after hypoxic conditioning. Inhibition of HIF-1α pathway hindered capillary-like structure formation in SVF cells cultured in hypoxia, suggesting a role of HIF-1α. Moreover, hypoxic SVF cells showed a trend for increased secretion of angiogenic factors, which was reflected in increased network formation by endothelial cells cultured on matrigel using that conditioned medium. In vivo implantation of SVF CS in a mouse hind limb ischemia model revealed that hypoxia-conditioned CS led to improved restoration of blood flow. Both in vitro and in vivo data suggest that SVF CS can be used as simple and cost-efficient tools to promote functional vascularization of TE constructs.

Neovascularization after implantation is a major obstacle for producing clinically viable cell sheet-based tissue engineered constructs. Strategies using endothelial cells and extrinsic angiogenic growth factors are expensive and time consuming and may raise concerns of tumorigenicity. In this manuscript, we describe a simplified approach using angiogenic cell sheets fabricated from the stromal vascular fraction of adipose tissue. The strong angiogenic behavior of these cell sheets, achieved without the use of external growth factors, was further stimulated by low oxygen culture. When implanted in an in vivo model of hind limb ischemia, the angiogenic cell sheets contributed to blood flux recovery. These cell sheets can therefore be used as a straightforward tool to increase the neovascularization of cell sheet-based thick constructs.

BACKGROUND

Sufficient uterine blood supply is essential for the fetus to develop normally in the uterus. Several mechanisms are involved in the process of vessel development in deciduas and villus. We focus on whether first-trimester decidua side population (SP) cells contain cells capable of differentiating into endothelial cells.

METHODS

Eight decidua samples were collected from healthy women, 22- to 30-years old, undergoing elective terminations of early pregnancy (six to eight gestational weeks). The cell suspensions from human deciduas were stained by Hoechst 33342 and sorted by flow cytometry, further cultured under differentiation conditions and analyzed for specific markers. These cells were implanted into ischemic limbs of nude mice to test the capacity of angiogenesis in vivo by DiI tracers and immunohistochemistry.

RESULTS

Decidua CD31(-)CD146(-) SP cells of first-trimester human pregnancy can differentiate into endothelial cells, express the corresponding specific markers of endothelial cells, such as CD31 and CD146, and form tube-like structures on Matrigel and part of newly formed vessels in the ischemic limbs of nude mice. Vascular endothelial growth factor was more effective in promoting proliferation of CD31(-)CD146(-)SP cells compared with other growth factors, and estrogen and progesterone at a final concentration of 10 μmol/L and 30 μmol/L, respectively, promoted the migration of CD31()-CD146(-)SP cells in a dose-dependent manner.

CONCLUSIONS

CD31(-)CD146(-) SP cells may be involved in the formation of new vessels in the maternal aspect of the placenta in the first trimester.

Appropriate cell sources, bioactive factors and biomaterials for generation of functional and integrated annulus fibrosus (AF) tissue analogues are still an unmet need. In the present study, the AF cell markers, collagen type I, cluster of differentiation 146 (CD146), mohawk (MKX) and smooth muscle protein 22α (SM22α) were found to be suitable indicators of functional AF cell induction. In vitro 2D culture of human AF cells showed that transforming growth factor β1 (TGF-β1) upregulated the expression of the functional AF markers and increased cell contractility, indicating that TGF-β1-pre-treated AF cells were an appropriate cell source for AF tissue regeneration. Furthermore, a tissue engineered construct, composed of polyurethane (PU) scaffold with a TGF-β1-supplemented collagen type I hydrogel and human AF cells, was evaluated with in vitro 3D culture and ex vivo preclinical bioreactor-loaded organ culture models. The collagen type I hydrogel helped maintaining the AF functional phenotype. TGF-β1 supplement within the collagen I hydrogel further promoted cell proliferation and matrix production of AF cells within in vitro 3D culture. In the ex vivo IVD organ culture model with physiologically relevant mechanical loading, TGF-β1 supplement in the transplanted constructs induced the functional AF cell phenotype and enhanced collagen matrix synthesis. In conclusion, TGF-β1-containing collagen-PU constructs can induce the functional cell phenotype of human AF cells in vitro and in situ. This combined cellular, biomaterial and bioactive agent therapy has a great potential for AF tissue regeneration and rupture repair.

OBJECTIVE

Angiotensin II (AII) is a powerful splanchnic vasoconstrictor with pro-inflammatory and pro-fibrotic properties. Angiotensin converting enzyme (ACE) inhibitors and AII Receptor Antagonists (ARBs) are therapeutic in animal models of colitis. The aim of this case-control study is to determine the expression of angiotensinogen and related genes in human ileal Crohn's disease.

METHODS

Using quantitative real-time polymerase chain reaction (RT-PCR), we measured mRNA expression levels of angiotensinogen (AGT), hypoxia inducible factor (HIF)1α and melanoma cell adhesion molecule (MCAM; CD146) in 101 human samples (69 biopsy, 12 resection) from affected ileum (inflamed CD cases, n=36) and unaffected ileum (non-inflamed CD cases, n=45 and controls, n=20). Immunohistochemistry for angiotensinogen was also performed. The study was of case-control design in a tertiary care setting.

RESULTS

Ileal expression of AGT was lower in CD cases compared to controls (p<0.0001), in inflamed CD samples (p=0.017) and current smokers (p=0.02). HIF1α expression was lower in non-inflamed CD biopsy samples than controls (p=0.006). The presence of disease-associated NOD2 variants was associated with increased expression of markers of angiogenesis (HIF1α p=0.009; MCAM p=0.007) in inflamed CD samples. Angiotensinogen immunohistochemical staining supported the quantitative RT-PCR expression findings.

CONCLUSIONS

Angiotensinogen expression is down regulated in human ileal CD, particularly in the presence of inflammation and current cigarette smoking, implicating the mesenteric vasculature and mucosal hypoxia as co-factors in ileal CD pathogenesis. A novel reduction in HIF1α expression in non-inflamed ileal mucosa in CD patients was also demonstrated.

OBJECTIVE

To explore the differences of CD146 expression in adult and children's acute B cell lymphoblastic leukemia(B-ALL), and its relation with clinical features, molecular biological and cytogenctic claracteristics.

METHODS

The expression of CD146 in bone marrow samples from adult and children's B-ALL patients were detected by flow cytometry (FCM) and the relation of CD146 abnormal high expression with the patients' clinical features, molecular biological and cytogenetical characteristics, as well as other antigens were analyzed.

RESULTS

The abnormal high expression rates of CD146 in adult and children's B-ALL patients were 29.17% and 9.09% respectively, showing that the expression rate of CD146 in adult patients was higher than that in children's patients(P<0.05). In adult B-ALL, CD146 was positively related with CD64 and CD117, while in children's B-ALL CD146 was positively related with CD71 and CD58 (P<0.05). After 1 course of standardized chemotherapy, the complete remission rates in adult and children's B-ALL patients with abnormal high expression of CD146 both were low as compared with adult and children's B-ALL without abnormal high expression of CD146 (P<0.05).

CONCLUSIONS

The expression rate of CD146 in adult B-ALL is higher than that in children's B-ALL. The CD146 positively relates with poor prognostic antigens, the CD146 may be one poor prognosis marker.

BACKGROUND

Increased concentrations of circulating endothelial cells (CECs) are thought to be a biomarker of vascular injury in human patients with cardiovascular disease, neoplasia, vasculitis, sickle cell anemia, shock, and sepsis. Immunomagnetic isolation is a technique currently used to enumerate human CECs and can detect low numbers of cells.

OBJECTIVE

The purpose of this study was to determine whether a standard protocol for immunomagnetic isolation could be used to obtain and enumerate CECs and a subpopulation of endothelial progenitor cells (EPCs) from canine whole blood.

METHODS

Cultured canine aortic endothelial cells were stained immunohistochemically with von Willebrand factor to verify morphology and number. Using magnetic beads conjugated with anti-CD146, CECs/EPCs were isolated in culture and in canine whole blood. CD146-positive cells were stained with fluorescein-conjugated Ulex europaeus agglutinin 1 (UEA-1) to confirm endothelial origin and cells were counted manually using a fluorescent microscope. The method was then applied to EDTA-anticoagulated whole blood samples from 10 healthy client-owned dogs.

RESULTS

The anti-CD146-coated magnetic beads (>5/cell) bound the cultured canine aortic endothelial cells. Only rare UEA-1-positive cells were obtained from whole blood, while >85-90% of cultured canine aortic endothelial cells were UEA-1 positive. The percentage recovery of cultured canine aortic endothelial cells was >86%. CECs in canine whole blood had >8 beads attached to the surface and were 10-40 microm in size. Using immunomagnetic isolation, 43.4 +/- 15.6 CECs/mL (range 24-70/mL) were isolated from canine whole blood samples.

CONCLUSIONS

Immunomagnetic isolation is an acceptable method for enumerating canine CECs/EPCs in whole blood. Further studies are warranted to evaluate the clinical significance of CEC/EPC concentration in different canine diseases.

Endothelial colony-forming cells (ECFCs) have been implicated in the process of vascularization, which includes vasculogenesis and angiogenesis. Vasculogenesis is a de novo formation of blood vessels, and is an essential physiological process that occurs during embryonic development and tissue regeneration. Angiogenesis is the growth of new capillaries from pre-existing blood vessels, which is observed both prenatally and postnatally. The placenta is an organ composed of a variety of fetal-derived cells, including ECFCs, and therefore has significant potential as a source of fetal ECFCs for tissue engineering.

To investigate the possibility of isolating clonal ECFCs from human early gestation chorionic villi (CV-ECFCs) of the placenta, and assess their potential for tissue engineering.The early gestation chorionic villus tissue was dissociated by enzyme digestion. Cells expressing CD31 were selected using magnetic-activated cell sorting, and plated in endothelial-specific growth medium. After 2-3 wks in culture, colonies displaying cobblestone-like morphology were manually picked using cloning cylinders. We characterized CV-ECFCs by flow cytometry, immunophenotyping, tube formation assay, and Dil-Ac-LDL uptake assay. Viral transduction of CV-ECFCs was performed using a Luciferase/tdTomato-containing lentiviral vector, and transduction efficiency was tested by fluorescent microscopy and flow cytometry. Compatibility of CV-ECFCs with a delivery vehicle was determined using an FDA approved, small intestinal submucosa extracellular matrix scaffold.

RESULTS

After four passages in 6-8 wks of culture, we obtained a total number of 1.8 × 10⁷ CV-ECFCs using 100 mg of early gestational chorionic villus tissue. Immunophenotypic analyses by flow cytometry demonstrated that CV-ECFCs highly expressed the endothelial markers CD31, CD144, CD146, CD105, CD309, only partially expressed CD34, and did not express CD45 and CD90. CV-ECFCs were capable of acetylated low-density lipoprotein uptake and tube formation, similar to cord blood-derived ECFCs (CB-ECFCs). CV-ECFCs can be transduced with a Luciferase/tdTomato-containing lentiviral vector at a transduction efficiency of 85.1%. Seeding CV-ECFCs on a small intestinal submucosa extracellular matrix scaffold confirmed that CV-ECFCs were compatible with the biomaterial scaffold.

In summary, we established a magnetic sorting-assisted clonal isolation approach to derive CV-ECFCs. A substantial number of CV-ECFCs can be obtained within a short time frame, representing a promising novel source of ECFCs for fetal treatments.

In this study, we used a large non-human primate model, the baboon, to establish a step-wise protocol to generate CD34+ endothelial progenitor cells (EPCs) from embryonic stem cells (ESCs) and to demonstrate their reparative effects. Baboon ESCs were sequentially differentiated from embryoid body cultures for 9 days and then were specified into EPCs by culturing them in monolayer for 12 days. The resulting EPCs expressed CD34, CXCR4 and UEA-1, but neither CD31 nor CD117. The EPCs were able to form intact lumen structures when seeded on Matrigel, took up Dil-LDL, and responded to TNF-α. Angioblasts specified in EGM-2 medium and ECGS medium had 6.41 ± 1.16% (n = 3) and 9.32 ± 3.73% CD34+ cells (n = 3). The efficiency of generating CD34+ EPCs did not differ significantly from ECGS to EGM-2 culture media, however, angioblasts specified in ECGS medium expressed a higher percentage of CD34+/CXCR4+ cells (3.49 ± 1.32%, n = 3) than those specified in EGM-2 medium (0.49 ± 0.52%, n = 3). To observe their reparative capacity, we purified CD34+ progenitors after specification by EGM-2 medium; inoculated fluorescently labelled CD34+ EPCs into an arterial segment denuded of endothelium in an ex vivo system. After 14 days of ex vivo culture, the grafted cells had attached and integrated to the denuded surface; in addition, they had matured further and expressed terminally differentiated endothelial markers including CD31 and CD146. In conclusion, we have proved that specified CD34+ EPCs are promising therapeutic agents for repairing damaged vasculature.

OBJECTIVE

Circulating endothelial cells (CECs) in patients with hematological malignancies are assessed as a noninvasive marker of angiogenesis. The aim of this study was to evaluate the numbers of CECs and their subsets during mobilization of hematopoietic stem cells.

METHODS

Thirty-eight patients were enrolled to the study (19 females and 19 males) at median age of 56.5 years. The group consisted of patients with multiple myeloma (26), lymphoma (10), and acute myeloid leukemia (2). Blood samples were collected before chemotherapy (0), 1 day after chemotherapy (Cht+1), on the day G-CSF commenced (G0), after 1 day of G-CSF (G+1), and on the day of the first apheresis. CECs were evaluated by four-color flow cytometry. Circulating progenitor cells were defined as CD45-/CD34+/CD31+/CD133+. Apoptotic CECs (ApoCECs) were defined as CD146+/AnnexinV+.

RESULTS

Median (Me) CECs number was 10.5/µl and it decreased after chemotherapy (Me = 8.3/µl, P < 0.001 when compared with baseline). Based on the number of aphereses needed to obtain 2 × 10(6)/kg CD34+ cells, patients were divided into "highly efficient" (one apheresis) and "poorly efficient" mobilizers (two or more aphereses). Median ApoCEC at Day G+1 was lower in highly efficient than in poorly efficient mobilizers (Me = 3.1/µl vs. Me = 5.1/µl, P = 0.02). ApoCEC at Day G+1 correlated with the number of aphereses (r = 0.48, P = 0.03). In multivariate analysis, ApoCEC at Day G+1 was an independent factor for successful mobilization during one apheresis.

CONCLUSIONS

CECs and their subsets change significantly during mobilization of HSCs. ApoCECs measured at the time of G-CSF commencement can predict the efficacy of HSC collection.

Survivors of Hodgkin's disease as well as of breast and lung cancer are at risk of radiation-associated cardiovascular disease. Recent studies demonstrated a correlation between cardiovascular risk factors and circulating endothelial microparticles (EMP) and thereby suggest increased EMP levels in circulation to be an early biomarker of endothelial dysfunction and cardiovascular risk. This prompted us to analyze the amount of EMP released by human aortic endothelial cells (HAEC) after exposure to different doses of X-ray (0.4, 2, 4, 6, and 20 Gy) using antibodies against the endothelial cell markers CD31, CD144, and CD146 by flow cytometry. In this pilot experiment only CD146 proved appropriate for quantification of HAEC-derived EMP. Exposure of HAEC to different doses of X-ray did not significantly influence formation of CD146-positive EMP. However, low doses (0.4 Gy) tended to decrease EMP formation, whereas higher doses (2 or 4 Gy) slightly increased release of CD146-positive EMP. By contrast, inflammatory activation of HAEC by TPA significantly increased EMP release about 15-fold (P < 0.01). In conclusion, under the present experimental conditions EMP did not prove a suitable biomarker for radiation-induced endothelial dysfunction in vitro.

Recent data suggest that CD146 may be involved in tumor development and may influence tumor prognosis in a number of systemic tumors besides gall bladder carcinomas. For instance, CD146 augments the development and progression of gastric carcinomas. It performs this function by accentuating epithelial mesenchymal transition (Liu et al., Int J Mol Sci 13:6399-6406, 2012). CD146 thus points towards a poor clinical outcome in gastric malignancies. An increase in gastric tissue vimentin is seen with an increase in CD146 levels. Similarly, around 8.7 % of lung adenocarcinomas express CD146. A decreased 5-year overall survival rate is seen following lung resection of pulmonary adenocarcinomas in male patients who express CD146 (Zeng et al., Proc Natl Acad Sci USA 109:1127-1132; 2012).

Eph and ephrin proteins are essential cell guidance cues that orchestrate cell navigation and control cell-cell interactions during developmental tissue patterning, organogenesis and vasculogenesis. They have been extensively studied in animal models of embryogenesis and adult tissue regeneration, but less is known about their expression and function during human tissue and organ regeneration. We discovered the hypoxia inducible factor (HIF)-1α-controlled expression of EphA3, an Eph family member with critical functions during human tumour progression, in the vascularised tissue of regenerating human endometrium and on isolated human endometrial multipotent mesenchymal stromal cells (eMSCs), but not in other highly vascularised human organs. EphA3 affinity-isolation from human biopsy tissue yielded multipotent CD29+/CD73+/CD90+/CD146+ eMSCs that can be clonally propagated and respond to EphA3 agonists with EphA3 phosphorylation, cell contraction, cell-cell segregation and directed cell migration. EphA3 silencing significantly inhibited the ability of transplanted eMSCs to support neovascularisation in immunocompromised mice. In accord with established roles of Eph receptors in mediating interactions between endothelial and perivascular stromal cells during mouse development, our findings suggest that HIF-1α-controlled expression of EphA3 on human MSCs functions during the hypoxia-initiated early stages of adult blood vessel formation.

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.

The present study was designed in order to explore the association between the early metastasis of renal cell carcinoma (RCC) and biological markers of tumor cells. A total of 200 patients with RCC, who received a nephrectomy between January 2015 and October 2015, were enrolled in the present study, while 100 healthy patients served as controls. The expression of cytokeratin 19 (CK19), endoglin (CD105) and cluster of differentiation 146 (CD146) were detected using immunohistochemical staining and western blotting. All three markers were highly expressed in tumor tissues compared with adjacent normal tissues. Subsequently, an enzyme-linked immunosorbent assay was used to detect the differential expression of CK19, CD105 and CD146. The results revealed that there was a statistically significant difference in the expression of CK19 and CD105 between the two groups (P<0.05), whereas CD146 did not exhibit a statistically significant difference. The results of further experiments revealed no significant difference between four time points (Q1, 1 day pre-operation; Q2, 1 day post-operation; Q3, 1 week post-operation; and Q4, 1 month post-operation). Then, subgroup analysis was performed based on whether patients were circulating tumor cell (CTC)-positive or not, and the difference between the Q1 time point and other three time points (Q2-4). The results revealed no difference between the CTC-positive and -negative groups, and no difference between the time points Q1 and Q2. However, the expression of CK19 and CD105 exhibited a significant difference between CTC-positive and CTC-negative groups according to the difference between the time points Q1 and Q3. Furthermore, on the basis of the difference between Q1 and Q4, the expression of CK19, CD105 and CD146 were significantly different (P<0.05). Taken together, the results suggested that CK19, CD105 and CD146 markers of peripheral blood may be considered to be effective tools to evaluate the early metastasis in a CTC-positive condition. CK19, CD105 and CD146 may be useful for CTC in evaluating the prognosis of patients with RCC, although a larger sample size is necessary for further investigation.

The proteins MUC18 and Mader have been identified as markers of tumor progression in melanoma cells. MUC18, also known as MCAM (melanoma cell adhesion molecule) and as CD146 (endothelial antigen), is a cell adhesion molecule belonging to the immunoglobulin superfamily. Mader is a transcriptional regulator shown to negatively regulate EGR-1. As it is known that neoplastic cells of neuroectodermal origin frequently express neuron-specific molecules, we studied whether these melanoma-associated antigens are found in normal CNS tissue. We investigated the expression of MUC18/MCAM and Mader in adult human post mortem CNS tissue by immunohistochemistry, immunoblot and two-dimensional gel electrophoresis. Our results show that Mader is preferentially expressed on neurons and glial cells and that the adhesion protein MUC18/MCAM is mainly expressed on vasculature within the CNS. These observations may have important implications for further studies investigating their possible roles in cell adhesion and proliferation control within the CNS.

The authors report a unique case of a primary ovarian placental site trophoblastic tumor (PSTT) in a 30-month-old girl who presented with isosexual precocious puberty of 1 month duration. Laboratory studies revealed mildly elevated beta human chorionic gonadotropin (37.5 mIU/ml; reference range <3.0 mIU/ml) and estradiol (74 pg/ml; reference range 0 to 56 pg/ml) serum levels. A 3.5-cm right ovarian mass was detected radiographically. The tumor was confined to the ovary as proven by the preoperative staging workup and the exploratory laparotomy. Microscopically, it was composed of intermediate trophoblastic cells with angioinvasive growth and deposition of fibrinoid material. The tumor cells were diffusely positive for human leukocyte antigen G, melanoma cell adhesion molecule (CD146), and cytokeratins (AE1/AE3, CK18, and CAM 5.2). Stains for human chorionic gonadotropin, alpha-inhibin, and human placental lactogen showed focal immunoreactivity. Serum markers returned to normal postoperatively. The patient remains disease-free at 24-months follow-up. We propose that tumors morphologically identical to uterine PSTT may rarely occur as gonadal germ cell tumors in children.