Numbers of circulating endothelial cells (CECs) are increased in cancer patients with progressive disease. Also, cancer patients have an increased risk for thrombotic events, being negatively associated with prognosis. Tissue factor (TF), the physiological initiator of coagulation, is present on the surface of many extravascular cells. In 34 samples from cancer patients and in seven from volunteers, CECs were quantified (with endothelium-specific anti-CD146 beads), and TF-activity assessed with a chromogenic assay. All samples displayed very limited TF-activity (patients: 1.6+/-3.1 microU; volunteers: 0.94+/-1.7 microU FXa/100 CECs, P = 0.30 by Mann-Whitney test). After in vitro TNFalpha-stimulation, CECs from both cancer patients and volunteers showed substantially increased TF-activity (endogenous activity: 17.3+/-6.4 microU; after TNFalpha-stimulation: 73.8+/-34.3 microU FXa; P = 0.028, Wilcoxon signed ranks test), reflecting the potential of CECs to generate biologically active TF. As the chromogenic assay determines a mean cellular TF-activity, we also analyzed immunohistochemical TF-antigen expression on CEC subsets. TF-antigen expression was undetectable. CECs isolated from cancer patients do not express TF. CECs can generate functional TF after stimulation and may therefore play a role in (intratumoral) coagulation induction and tumor angiogenesis.

The thymic microenvironment is thought to play a critical role in T-lymphocyte development, providing signals both via cell surface molecules such as adhesion molecules and soluble molecules. The present investigation is focused on immunoelectron microscopical analysis of distribution patterns of CD146 adhesion molecules (MUC18 or MCAM) in the microenvironment of normal human thymus, using the pre-embedding indirect immunoperoxidase technique. The anti-CD146 monoclonal antibody (mAb) revealed strong membraneous labelling of immature thymocytes at both the light and electron microscopical level. Proliferating thymocytes, most of the epithelial cells, macrophages, endothelial cells and smooth musle cells of small vessels and capillaries showed both membraneous and cytoplasmic labelling with anti-CD146 mAb as was demonstrated by electron microscopy. In contrast, these cells displayed a strong cytoplasmic immunoreactivity at the light microscopical level. The extracellular matrix was also stained with the anti-CD146 mAb. No labelling was observed in interdigitating cells. Interestingly, the CD146 molecule was strongly expressed on apical and lateral membranes of endothelial cells as was demonstrated electron microscopically. This selective CD146 labelling of capillary endothelium mainly localized at the cortico-medullary junction may be manifestations of lymphocyte transmembrane migration and lymphocyte homing. In conclusion, the present study suggests that CD146 is expressed by most elements of the microenvironment of normal human thymus. Therefore, it may be a pan-antigen which is essential for the maintenance of thymic architecture and function.

BACKGROUND

Circulating endothelial cells (CECs) have been proposed to predict patient response to antiangiogenic cancer therapy. However, contradictory reports and inconsistency in the phenotypic identification of CECs have led us to compare three cell populations with partially overlapping phenotype in cancer patients receiving chemotherapy and the antiangiogenic agent bevacizumab.

METHODS

Patients (n = 20) with locally advanced pancreatic cancer were monitored during 16 weeks of neoadjuvant treatment with gemcitabine and bevacizumab. Detection of circulating cell populations was based on the marker combination CD45, CD31, and CD146; levels of viable and dead (7-aminoactinomycin D-positive) cells were evaluated by flow cytometry in 2-week intervals.

RESULTS

We were able to discriminate and concomitantly monitor three cell populations elevated in cancer patients. Whereas CECs were defined as CD45(-) CD31(+) CD146(+), the distinct populations of CD45(-) CD31(-) CD146(+) and CD45(-) CD31(high) CD146(-) cells were partly positive for CD3 and CD41, respectively. CECs and CD45(-) CD31(-) CD146(+) cells increased during therapy; the rise in dead cells was positively correlated with patient response or survival. Conversely, CD45(-) CD31(high) CD146(-) cells decreased in neoadjuvant treatment. A highly significant correlation was established for improved patient response and a minor decrease in viable cell counts.

CONCLUSIONS

Flow cytometric CEC analysis based on CD45, CD31, and CD146 requires careful discrimination between blood cell populations with overlapping phenotype showing hallmarks of activated T cells and large platelets. However, these three cell populations show distinct regulation during cancer therapy, and their concomitant analysis may offer extended prognostic and predictive information.

Progenitors derived from the stromal vascular fraction (SVF) of white adipose tissue (WAT) possess the ability to form clonal populations and differentiate along multiple lineage pathways. However, the literature continues to vacillate between defining adipocyte progenitors as "stromal" or "stem" cells. Recent studies have demonstrated that a nonpericytic subpopulation of adipose stromal cells, which possess the phenotype, CD45(-) /CD31(-) /CD146(-) /CD34(+) , are mesenchymal, and suggest this may be an endogenous progenitor subpopulation within adipose tissue. We hypothesized that an adipose progenitor could be sorted based on the expression of CD146, CD34, and/or CD29 and when implanted in vivo these cells can persist, proliferate, and regenerate a functional fat pad over serial transplants. SVF cells and culture expanded adipose stromal/stem cells (ASC) ubiquitously expressing the green fluorescent protein transgene (GFP-Tg) were fractionated by flow cytometry. Both freshly isolated SVF and culture expanded ASC were seeded in three-dimensional silk scaffolds, implanted subcutaneously in wild-type hosts, and serially transplanted. Six-week WAT constructs were removed and evaluated for the presence of GFP-Tg adipocytes and stem cells. Flow cytometry, quantitative polymerase chain reaction, and confocal microscopy demonstrated GFP-Tg cell persistence, proliferation, and expansion, respectively. Glycerol secretion and glucose uptake assays revealed GFP-Tg adipose was metabolically functional. Constructs seeded with GFP-Tg SVF cells or GFP-Tg ASC exhibited higher SVF yields from digested tissue, and higher construct weights, compared to nonseeded controls. Constructs derived from CD146(-) CD34(+) -enriched GFP-Tg ASC populations exhibited higher hemoglobin saturation, and higher frequency of GFP-Tg cells than unsorted or CD29(+) GFP-Tg ASC counterparts. These data demonstrated successful serial transplantation of nonpericytic adipose-derived progenitors that can reconstitute adipose tissue as a solid organ. These findings have the potential to provide new insights regarding the stem cell identity of adipose progenitor cells.

BACKGROUND

Circulating endothelial cells (CECs) have been detected at increased numbers in patients with solid cancers. CECs have not been systematically evaluated in patients with osteosarcoma.

METHODS

Patients 12 months to 30 years of age with newly diagnosed high-grade osteosarcoma were eligible for this prospective cohort study. Patients provided a single blood sample at study entry for CEC quantification by flow cytometry at a single reference laboratory. CECs were defined as CD146+, CD31+, CD45-, and CD133-. CEC progenitor cells (CEPs) were defined as CD146+, CD31+, CD45-, and CD133+.

RESULTS

Eighteen patients enrolled (11 males; median age 16 years; range 5-21 years). CEC counts did not differ between patients with osteosarcoma compared to seven pediatric healthy controls (median 645 cells/ml, range 60-5,320 cells/ml vs. 1,670 cells/ml, range 330-4,700 cells/ml, respectively; P = 0.12). CEP counts did not differ between patients compared to controls (median 126 cells/ml, range 0-5,320 cells/ml vs. median 260 cells/ml, range 0-10,670 cells/ml, respectively; P = 0.69). CEC and CEP counts did not correlate with metastatic status, tumor size, or histologic response to neoadjuvant chemotherapy.

CONCLUSIONS

CEC and CEP levels are not increased in patients with osteosarcoma compared to healthy controls. CECs and CEPs do not correlate with clinical features of osteosarcoma. Alternative novel markers of disease burden and response are needed in this disease.

BACKGROUND

It has been suggested that the immunopathology of rheumatoid nodules parallels that of inflamed synovium in rheumatoid arthritis (RA).

OBJECTIVE

To analyse the effect of infliximab on the immunopathology of rheumatoid nodules in order to provide new insights into the relationship between synovial inflammation and rheumatoid nodules.

METHODS

Nodules were present at baseline in six patients with RA and after infliximab treatment in five patients, including paired nodules before and after treatment in three patients. In one patient, the nodule appeared during treatment. Paraffin sections were used for histological analysis. Frozen sections were stained by immunohistochemistry for cellular markers (CD3, CD4, CD8, CD16, CD20, CD68), blood vessels (CD146, vWF, alphavbeta3), and adhesion molecules (E-selectin, VCAM-1, ICAM-1).

RESULTS

No manifest immunopathological differences were found between the nodules before and after infliximab treatment. All nodules depicted the classical structure with a central necrotic zone, surrounding the palisade layer, and an outer connective tissue zone. Immunohistochemistry showed the presence of CD68+ and CD16+ macrophages in the palisade and the connective tissue zone, as well as a small number of CD3+, CD4+ T lymphocytes in the perivascular areas. Small vessels were seen in the connective tissue and were sometimes positive for the neovascularisation marker alphavbeta3. They expressed no VCAM-1, E-selectin weakly, but ICAM-1 strongly. ICAM-1 was also strongly expressed on palisade cells.

CONCLUSIONS

Despite an improvement of articular symptoms, infliximab treatment had no distinct effect on the histopathology of rheumatoid nodules, suggesting that different pathogenetic mechanisms mediate the two disease manifestations in RA.

Identification of cell types in tumor-associated stroma that are involved in the development of melanoma is hampered by their heterogeneity. The authors used flow cytometry and immunohistochemistry to demonstrate that anti-MART-1 antibodies can discriminate between melanoma and stroma cells. They investigated the cellular composition of the MART-1-, non-hematopoietic melanoma-associated stroma, finding it consisted mainly of Sca-1+ and CD146+ cells. These cell types were also observed in the skin and muscle adjacent to developing melanomas. The Sca-1+ cell population was observed distributed in the epidermis, hair follicle bulges, and tumor capsule. The CD146+ population was found distributed within the tumor, mainly associated with blood vessels in a perivascular location. In addition to a perivascular distribution, CD146+ cells expressed α-smooth muscle actin, lacked expression of endothelial markers CD31 and CD34, and were therefore identified as pericytes. Pericytes were found to be associated with CD31+ endothelial cells; however, some pericytes were also observed associated with CD31-, MART-1+ B16 melanoma cells that appeared to form blood vessel structures. Furthermore, the authors observed extensive nuclear expression of HIF-1α in melanoma and stroma cells, suggesting hypoxia is an important factor associated with the melanoma microenvironment and vascularization. The results suggest that pericytes and Sca-1+ stroma cells are important contributors to melanoma development.

Malignant mesothelioma (MM), which is associated with asbestos exposure, is one of the most deadly tumors in humans. Early MM is concealed in the serosal cavities and lacks specific clinical symptoms. For better treatment, early detection and prognostic markers are necessary. Recently, CD146 and insulin-like growth factor 2 mRNA-binding protein 3 (IMP3) were reported as possible positive markers of MM to distinguish from reactive mesothelia in humans. However, their application on MM of different species and its impact on survival remain to be elucidated. To disclose the utility of these molecules as early detection and prognostic markers of MM, we injected chrysotile or crocidolite intraperitoneally to rats, thus obtaining 26 peritoneal MM and establishing 11 cell lines. We immunostained CD146 and IMP3 using paraffin-embedded tissues and cell blocks and found CD146 and IMP3 expression in 58% (15/26) and 65% (17/26) of MM, respectively, but not in reactive mesothelia. There was no significant difference in both immunostainings for overexpression among the three histological subtypes of MM and the expression of CD146 and IMP3 was proportionally associated. Furthermore, the overexpression of CD146 and/or IMP3 was proportionally correlated with shortened survival. These results suggest that CD146 and IMP3 are useful diagnostic and prognostic markers of MM.

Mesenchymal stromal cells (MSCs) are cultured cells that can give rise to mature mesenchymal cells under appropriate conditions and secrete a number of biologically relevant molecules that may play an important role in regenerative medicine. Evidence indicates that pericytes (PCs) correspond to mesenchymal stem cells in vivo and can give rise to MSCs when cultured, but a comparison between the gene expression profiles of cultured PCs (cPCs) and MSCs is lacking. We have devised a novel methodology to isolate PCs from human adipose tissue and compared cPCs to MSCs obtained through traditional methods. Freshly isolated PCs expressed CD34, CD140b, and CD271 on their surface, but not CD146. Both MSCs and cPCs were able to differentiate along mesenchymal pathways in vitro, displayed an essentially identical surface immunophenotype, and exhibited the ability to suppress CD3(+) lymphocyte proliferation in vitro. Microarray expression data of cPCs and MSCs formed a single cluster among other cell types. Further analyses showed that the gene expression profiles of cPCs and MSCs are extremely similar, although MSCs differentially expressed endothelial cell (EC)-specific transcripts. These results confirm, using the power of transcriptomic analysis, that PCs give rise to MSCs and suggest that low levels of ECs may persist in MSC cultures established using traditional protocols.

OBJECTIVE

Mesenchymal stromal cells (MSCs) have great potential for use in cell-based therapies for restoration of structure and function of many tissue types including smooth muscle.

METHODS

We compared proliferation, immunophenotype, differentiation capability and gene expression of bone marrow-derived MSCs expanded in different media containing human serum, plasma and platelet lysate in combination with commonly used protocols for myogenic, osteogenic, chondrogenic and adipogenic differentiation. Moreover, we developed a xenogenic-free protocol for myogenic differentiation of MSCs.

RESULTS

Expansion of MSCs in media complemented with serum, serum + platelet lysate or plasma + platelet lysate were multipotent because they differentiated toward four mesenchymal (myogenic, osteogenic, chondrogenic, adipogenic) lineages. Addition of platelet lysate to expansion media increased the proliferation of MSCs and their expression of CD146. Incubation of MSCs in medium containing human serum or plasma plus 5% human platelet lysate in combination with smooth muscle cell (SMC)-inducing growth factors TGFβ1, PDGF and ascorbic acid induced high expression of ACTA2, TAGLN, CNN1 and/or MYH11 contractile SMC markers. Osteogenic, adipogenic and chondrogenic differentiations served as controls.

CONCLUSIONS

Our study provides novel data on the myogenic differentiation potential of human MSCs toward the SMC lineage using different xenogenic-free cell culture expansion media in combination with distinct differentiation medium compositions. We show that the choice of expansion medium significantly influences the differentiation potential of human MSCs toward the smooth muscle cell, as well as osteogenic, adipogenic and chondrogenic lineages. These results can aid in designing studies using MSCs for tissue-specific therapeutic applications.

Medullary nephrocalcinosis is a hallmark of medullary sponge kidney (MSK). We had the opportunity to study a spontaneous calcification process in vitro by utilizing the renal cells of a patient with MSK who was heterozygous for the c.-27 + 18G>A variant in the GDNF gene encoding glial cell-derived neurotrophic factor. The cells were obtained by collagenase digestion of papillary tissues from the MSK patient and from two patients who had no MSK or nephrocalcinosis. These cells were typed by immunocytochemistry, and the presence of mineral deposits was studied using von Kossa staining, scanning electron microscopy analysis and an ALP assay. Osteoblastic lineage markers were studied using immunocytochemistry and RT-PCR. Staminality markers were also analysed using flow cytometry, magnetic cell separation technology, immunocytochemistry and RT-PCR. Starting from p2, MSK and control cells formed nodules with a behaviour similar to that of calcifying pericytes; however, Ca₂PO₄ was only found in the MSK cultures. The MSK cells had morphologies and immunophenotypes resembling those of pericytes or stromal stem cells and were positive for vimentin, ZO1, αSMA and CD146. In addition, the MSK cells expressed osteocalcin and osteonectin, indicating an osteoblast-like phenotype. In contrast to the control cells, GDNF was down-regulated in the MSK cells. Stable GDNF knockdown was established in the HK2 cell line and was found to promote Ca₂PO₄ deposition when the cells were incubated with calcifying medium by regulating the osteonectin/osteopontin ratio in favour of osteonectin. Our data indicate that the human papilla may be a perivascular niche in which pericyte/stromal-like cells can undergo osteogenic differentiation under particular conditions and suggest that GDNF down-regulation may have influenced the observed phenomenon.

Endothelial colony-forming cells (ECFC) constitute an endothelial progenitor fraction with a promising interest for the treatment of ischaemic cardiovascular diseases. As soluble CD146 (sCD146) is a new factor promoting angiogenesis, we examined whether sCD146 priming could improve the therapeutic potential of ECFC and defined the involved mechanism.

We investigated the effects of sCD146 priming on regenerative properties of ECFC in vivo. In a mouse model of hindlimb ischaemia, the homing of radiolabelled cells to ischaemic tissue was assessed by SPECT-CT imaging. Soluble CD146 priming did not modify the number of engrafted ECFC but improved their survival capacity, leading to an enhanced revascularization. The mechanism of action of sCD146 on ECFC was studied in vitro. We showed that sCD146 acts in ECFC through a signalosome, located in lipid rafts, containing angiomotin, the short isoform of CD146 (shCD146), VEGFR1, VEGFR2, and presenilin-1. Soluble CD146 induced a sequential proteolytic cleavage of shCD146, with an extracellular shedding followed by an intramembrane cleavage mediated by matrix metalloprotease (MMP)/ADAM and presenilin-1, respectively. The generated intracellular part of shCD146 was directed towards the nucleus where it associated with the transcription factor CSL and modulated the transcription of genes involved in cell survival (FADD, Bcl-xl) and angiogenesis (eNOS). This effect was dependent on both VEGFR1 and VEGFR2, which were rapidly phosphorylated by sCD146.

These findings establish that activation of the proteolytic processing of shCD146, in particular by sCD146, constitutes a promising pathway to improve endothelial progenitors' regenerative properties for the treatment of cardiovascular diseases.

Adipose tissue is a rich source of multipotent mesenchymal stem-like cells, located in the perivascular niche. Based on their surface markers, these have been assigned to two main categories: CD31- /CD45- /CD34+ /CD146- cells (adventitial stromal/stem cells [ASCs]) and CD31- /CD45- /CD34- /CD146+ cells (pericytes [PCs]). These populations display heterogeneity of unknown significance. We hypothesized that aldehyde dehydrogenase (ALDH) activity, a functional marker of primitivity, could help to better define ASC and PC subclasses. To this end, the stromal vascular fraction from a human lipoaspirate was simultaneously stained with fluorescent antibodies to CD31, CD45, CD34, and CD146 antigens and the ALDH substrate Aldefluor, then sorted by fluorescence-activated cell sorting. Individual ASCs (n = 67) and PCs (n = 73) selected from the extremities of the ALDH-staining spectrum were transcriptionally profiled by Fluidigm single-cell quantitative polymerase chain reaction for a predefined set (n = 429) of marker genes. To these single-cell data, we applied differential expression and principal component and clustering analysis, as well as an original gene coexpression network reconstruction algorithm. Despite the stochasticity at the single-cell level, covariation of gene expression analysis yielded multiple network connectivity parameters suggesting that these perivascular progenitor cell subclasses possess the following order of maturity: (a) ALDHbr ASC (most primitive); (b) ALDHdim ASC; (c) ALDHbr PC; (d) ALDHdim PC (least primitive). This order was independently supported by specific combinations of class-specific expressed genes and further confirmed by the analysis of associated signaling pathways. In conclusion, single-cell transcriptional analysis of four populations isolated from fat by surface markers and enzyme activity suggests a developmental hierarchy among perivascular mesenchymal stem cells supported by markers and coexpression networks. Stem Cells 2017;35:1273-1289.

Lymphatic malformations (LMs) are vascular anomalies thought to arise from dysregulated lymphangiogenesis. These lesions impose a significant burden of disease on affected individuals. LM pathobiology is poorly understood, hindering the development of effective treatments. In the present studies, immunostaining of LM tissues revealed that endothelial cells lining aberrant lymphatic vessels and cells in the surrounding stroma expressed the stem cell marker, CD133, and the lymphatic endothelial protein, podoplanin. Isolated patient-derived CD133+ LM cells expressed stem cell genes (NANOG, Oct4), circulating endothelial cell precursor proteins (CD90, CD146, c-Kit, VEGFR-2), and lymphatic endothelial proteins (podoplanin, VEGFR-3). Consistent with a progenitor cell identity, CD133+ LM cells were multipotent and could be differentiated into fat, bone, smooth muscle, and lymphatic endothelial cells in vitro. CD133+ cells were compared to CD133- cells isolated from LM fluids. CD133- LM cells had lower expression of stem cell genes, but expressed circulating endothelial precursor proteins and high levels of lymphatic endothelial proteins, VE-cadherin, CD31, podoplanin, VEGFR-3 and Prox1. CD133- LM cells were not multipotent, consistent with a differentiated lymphatic endothelial cell phenotype. In a mouse xenograft model, CD133+ LM cells differentiated into lymphatic endothelial cells that formed irregularly dilated lymphatic channels, phenocopying human LMs. In vivo, CD133+ LM cells acquired expression of differentiated lymphatic endothelial cell proteins, podoplanin, LYVE1, Prox1, and VEGFR-3, comparable to expression found in LM patient tissues. Taken together, these data identify a novel LM progenitor cell population that differentiates to form the abnormal lymphatic structures characteristic of these lesions, recapitulating the human LM phenotype. This LM progenitor cell population may contribute to the clinically refractory behavior of LMs.

OBJECTIVE

To evaluate numbers of circulating endothelial cells (CECs) in ANCA associated vasculitis and compare vasculitic relapse with limited granulomatous disease.

METHODS

Sixteen patients with vasculitic relapse of ANCA associated vasculitis and 12 patients with limited granulomatous disease due to Wegener's granulomatosis (WG) were studied. Six patients with newly diagnosed vasculitic disease and six patients with vasculitis with infectious complications were also studied. Twenty two patients in remission were studied, as were 20 healthy controls. Counting of CECs was performed with anti-CD146 driven immunomagnetic isolation and staining with Ulex Europaeus lectin 1(UEA-1).

RESULTS

Patients with vasculitic relapse had markedly increased numbers of circulating endothelial cells (12-800 cells/ml, median 88 cells/ml) as did patients with newly diagnosed systemic vasculitis (20-216 cells/ml, median 56 cells/ml). Patients with limited granulomatous disease due to WG had only slightly increased cell numbers (4-44 cells/ml, median 20 cells/ml), which were similar to those of patients in remission (4-36 cells/ml, median 16 cells/ml). Numbers of CECs in patients with granulomatous disease were significantly lower than in those patients with relapse or new onset vasculitis (p<0.001). Cell numbers in patients with relapse and new onset vasculitis declined with immunosuppressive treatment. Patients with infection had 4-36 cells/ml (median 10 cells/ml). A cut off value of 20 cells/ml for a positive result yielded 64% specificity and 95% sensitivity for active systemic vasculitis; the positive predictive value was 63% and the negative predictive value 95%.

CONCLUSIONS

Markedly increased numbers of CECs discriminate active vasculitis from limited granulomatous disease and remission. These findings add further proof to the concept of CECs as a marker of ANCA associated small vessel vasculitis.

OBJECTIVE

Adipose tissue in vocal fold lipoinjection is currently used to treat patients affected by laryngeal hemiplegia or anatomical defects. The aim of this study has been to evaluate the efficacy of this clinical strategy, by long-term follow-up of the patients and to investigate whether the fat samples used to treat them contain a stem cell population with a wide differentiation potential.

METHODS

Fat samples harvested from 12 patients affected by severe breathy dysphonia who had undergone vocal fold lipoinjection were analysed by immunocytochemistry, by flow cytometry and reverse transcription-polymerase chain reaction, and the isolated adipose derived mesenchymal stem cells (ADMSCs) were evaluated in order to define their ability to produce soluble factors possibly involved in tissue regeneration, and to differentiate towards different lineages.

RESULTS

ADMSCs were efficiently and successfully isolated from all of the samples. They were positive for SSEA-4, an embryonic marker recently identified on bone marrow MSCs and which could explain their high differentiation plasticity. Molecular analysis showed that these cells also expressed Oct-4, Runx-1 and ABCG-2, which characterize the stem cell state, and a number of other specific lineage markers. Flow cytometry revealed mesenchymal markers expressed on ADMSCs and identified a subpopulation characterized by CD146(+)/34(-)/45(-) cells consistent with perivascular/pericyte-like cells. Osteogenic, adipogenic and endothelial tissue differentiation were obtained.

CONCLUSIONS

Our results confirmed the therapeutic efficacy of this clinical approach and showed that adipose tissue, administered to patients in order to restore glottic competence, contains mesenchymal stem cells.

Several factors may influence the analysis of endothelial cells (ECs) by flow cytometry: separation of mononuclear cell, washing and centrifugation steps, panel of monoclonal antibodies, and the lack of standardization of gating technique. Therefore, the reliable quantification of ECs remains a technical challenge. The purpose of this study is to define a new flow cytometric protocol to characterize and quantitate ECs. In previous investigations, increased numbers of circulating ECs have been found in sickle cell disease. The patients with sickle cell disease might provide useful material for the study. We performed flow cytometry on whole blood from 20 normal controls and 31 patients with sickle cell disease (20 patients with steady-state disease and 11 patients with vaso-occlusive crises) using a lyse/no-wash procedure, specific and nonspecific antibody combinations (CD146, CD144, CD34, and CD117), and broad gating. This protocol produced much higher values for the number of circulating ECs (a mean of 2,396.55 +/- 658.37 ECs/mL in controls vs 6,709.60 +/- 1,772.32 ECs/mL in the steady-state group, or 18,213.50 +/- 8,451 in the vaso-occlusive crises group, P < 0.001 for both), and also showed variable EC size and granularity, which may reflect activated, or early release ECs. This novel protocol performed comparably in terms of reproducibility, reliability, and dilution linearity with a previously described protocol. This approach has significant advantages for the characterization and quantitation ECs compatible with the pathophysiology. Using the specific antibodies, CD146 and CD144, together may give more informative EC data than the general approach used.

Dental pulp stem cells (DPSCs) have the capacity to give rise to cells with neuronal-like phenotypes, suggesting their use in brain cell therapies. In the present work, we wanted to address the phenotypic fate of adult genetically unmodified human DPSCs cultured in Neurocult^TM (Stem Cell Technologies), a cell culture medium without serum which can be alternatively supplemented for the expansion and/or differentiation of adult neural stem cells (NSCs). Our results show that non-genetically modified human adult DPSCs cultured with Neurocult NS-A proliferation supplement generated neurosphere-like dentospheres expressing the NSC markers Nestin and glial fibrillary acidic protein (GFAP), but also the vascular endothelial cell marker CD31. Remarkably, 1 month after intracranial graft into athymic nude mice, human CD31+/CD146+ and Nestin+ DPSC-derived cells were found tightly associated with both the endothelial and pericyte layers of brain vasculature, forming full blood vessels of human origin which showed an increased laminin staining. These results are the first demonstration that DPSC-derived cells contributed to the generation of neovasculature within brain tissue, and that Neurocult and other related serum-free cell culture media may constitute a fast and efficient way to obtain endothelial cells from human DPSCs.

OBJECTIVE

Evaluation of the effect of Propolis as a bioactive material on quality of dentin and presence of dental pulp stem cells.

METHODS

For conducting this experimental split-mouth study,a total of 48 maxillary and mandibular incisors of male guinea pigs were randomly divided into an experimental Propolis group and a control calcium hydroxide group. Cutting the crowns and using Propolis or calcium hydroxide to cap the pulp, all of the cavities were sealed. Sections of the teeth were obtained after sacrificing 4 guinea pigs from each group on the 10(th), 15(th) and 30(th) day. After they had been stained by hematoxylin and eosin (H&E), specimens underwent a histological evaluation under a light microscope for identification of the presence of odontoblast-like cells, pulp vitality, congestion, inflammation of the pulp and the presence of remnants of the material used. The immunohistochemistry (IHC) method using CD29 and CD146 was performed to evaluate the presence of stem cells and the results were statistically evaluated by Kruskal-Wallis, Chi Square and Fisher tests.

RESULTS

In H&E stained specimens, there was no difference between the two groups in the presence of odontoblast-like cells, pulp vitality, congestion, inflammation of the pulp and the presence of remnants of used material(p>0.05). There was a significant difference between the quality of regenerative dentin on the 15(th) and 30(th) days (p<0.05): all of the Propolis cases presented tubular dentin while 14% of the calcium hydroxide cases produced porous dentin. There was no significant difference between Propolis and calcium hydroxide in stimulation of dental pulp stem cells (DPSCs).

CONCLUSIONS

This study which is the first one that documented the stimulation of stem cells by Propolis, provides evidence that this material has advantages over calcium hydroxide as a capping agent in vital pulp therapy. In addition to producing no pulpal inflammation, infection or necrosis this material induces the production of high quality tubular dentin.

Tissue injury is inevitably accompanied by disruption of the endothelium and exposure of the subendothelial matrix. To generate a guidance molecule directing progenitor cells to sites of vascular lesions, we designed a bifunctional protein. The protein consists of the soluble platelet collagen receptor glycoprotein VI and an antibody to CD133 (hereafter called GPVI-CD133). In vitro and in vivo, this construct substantially mediates endothelial progenitor cell (EPC) homing to vascular lesions. Exposure of EPCs to GPVI-CD133 did not impair their capability to differentiate toward mature endothelial cells as verified by the formation of colony-forming units, the upregulation of endothelial markers CD31 and CD146 analyzed by flow cytometry or von Willebrand factor and endoglin assessed by immunofluorescence microscopy, as well as the presence of Weibel-Palade bodies using transmission electron microscopy. In vivo, GPVI-CD133 augments reendothelialization of vascular lesions. Thus, this bifunctional protein could be a potential new therapeutic option for cardiovascular diseases.

Adventitial microvessels, vasa vasorum in the vessel walls, have an active role in the vascular remodeling, although its mechanisms are still unclear. It has been reported that microvascular pericytes (PCs) possess mesenchymal plasticity. Therefore, microvessels would serve as a systemic reservoir of stem cells and contribute to the tissues remodeling. However, most aspects of the biology of multipotent PCs (mPCs), in particular of pathological microvessels are still obscure because of the lack of appropriate methods to detect and isolate these cells. In order to examine the characteristics of mPCs, we established immortalized cells residing in adventitial capillary growing at the injured vascular walls. We recently developed in vivo angiogenesis to observe adventitial microvessels using collagen-coated tube (CCT), which also can be used as an adventitial microvessel-rich tissue. By using the CCT, CD146- or NG2-positive cells were isolated from the adventitial microvessels in the injured arteries of mice harboring a temperature-sensitive SV40 T-antigen gene. Several capillary-derived endothelial cells (cECs) and PCs (cPCs) cell lines were established. cECs and cPCs maintain a number of key endothelial and PC features. Co-incubation of cPCs with cECs formed capillary-like structure in Matrigel. Three out of six cPC lines, termed capillary mPCs demonstrated both mesenchymal stem cell- and neuronal stem cell-like phenotypes, differentiating effectively into adipocytes, osteoblasts, as well as schwann cells. mPCs differentiated to ECs and PCs, and formed capillary-like structure on their own. Transplanted DsRed-expressing mPCs were resident in the capillary and muscle fibers and promoted angiogenesis and myogenesis in damaged skeletal muscle. Adventitial mPCs possess transdifferentiation potential with unique phenotypes, including the reconstitution of capillary-like structures. Their phenotype would contribute to the pathological angiogenesis associated with vascular remodeling. These cell lines also provide a reproducible cellular tool for high-throughput studies on angiogenesis, vascular remodeling, and regeneration as well.

OBJECTIVE

As angiogenic and lymphangiogenic key players, endothelial cells (ECs) are promising candidates for vascular regenerative therapies. To culture ECs in vitro, fetal calf serum (FCS) is most often used. However, some critical aspects of FCS usage, such as possible internalization of xenogeneic proteins and prions, must be considered. Therefore, the aim of this project was to determine if human platelet lysate (hPL) is a suitable alternative to FCS as medium supplement for the culture of blood vascular and lymphatic endothelial cells.

METHODS

The usability of hPL was tested by analysis of endothelial surface marker expression, metabolic activity and vasculogenic potential of outgrowth ECs (OECs), human umbilical vein ECs (HUVECs), and lymphatic ECs (LECs).

RESULTS

Expression of EC markers CD31, VEGFR2, VE-cadherin and CD146 did not differ significantly between the EC types cultured in FCS or hPL. In addition, OECs, HUVECs and LECs formed tube-like structures on Matrigel when cultured in hPL and FCS. With the use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid assays, we found that the metabolic activity of OECs and LECs was slightly decreased when hPL was used. However, HUVECs and LECs did not show a significant decrease in metabolic activity, and HUVECs showed a slightly higher activity at low seeding densities.

CONCLUSIONS

The use of hPL on different EC types did not reveal any substantial negative effects on EC behavior. Thus, hPL appears to be a favorable candidate to replace FCS as a medium supplement in the culture of ECs.

Programmed cell death protein-1 (PD-1) is a prominent immune checkpoint receptor interacting with its ligand, programmed cell death protein ligand-1 (PD-L1, B7-H1). The PD-1/PD-L1 interaction induces functional exhaustion of tumor-reactive cytotoxic T cells and, thus, interferes with antitumor T-cell immunity. In addition, PD-1/PD-L1 interaction promotes tumorigenesis via the mTOR signaling pathway in a group of cancers including melanoma. Based on the dual functions of PD-1/PD-L1 interactions in tumor progression, we hypothesize that siRNA targeting PD-L1 (siPD-L1) will suppress melanoma growth, acting on both immune checkpoint and intrinsic tumorigenesis pathways. We tested this hypothesis by delivering siPD-L1 with a polymeric carrier ("pd") consisting of disulfide-cross-linked polyethylenimine (CLPEI) and dermatan sulfate (DS), which we previously found to have a specific interaction with CD146-positive B16F10 melanoma cells. The siPD-L1/pd suppressed the expression of PD-L1 in the interferon-γ (IFN-γ)-challenged B16F10 melanoma cells in a cell-type dependent manner and attenuated the expression of tumor-specific genes in B16F10 cells. siPD-L1/pd suppressed the B16F10 melanoma growth in C57BL/6 immune-competent mice with increased tumor-specific immunity. siPD-L1/pd also suppressed melanoma growth in immune-compromised nude mice. Both animals showed a positive correlation between PD-L1 and p-S6k (a marker of mTOR pathway activation) expression in tumors. These results indicate that the siPD-L1/pd complex attenuates melanoma growth in both T-cell-dependent and independent mechanisms.

BACKGROUND

Pulmonary vasodilators in general and prostacyclin analogues in particular have improved the outcome of patients with pulmonary arterial hypertension (PAH). Endothelial dysfunction is a key feature of PAH and we previously described that circulating endothelial cell (CEC) level could be used as a biomarker of endothelial dysfunction in PAH. We now hypothesized that an efficient PAH-specific vasodilator therapy might decrease CEC level.

RESULTS

CECs were prospectively quantified by immunomagnetic separation with mAb CD146-coated beads in peripheral blood from children with idiopathic PAH (iPAH, n = 30) or PAH secondary to congenital heart disease (PAH-CHD, n = 30): before, after treatment and during follow up. Controls were 23 children with reversible PAH. Oral treatment with endothelin receptor antagonists (ERA) and/or phosphodiesterase 5 inhibitors (PDE5) significantly reduced CEC counts in children. In 10 children with refractory PAH despite oral combination therapy, subcutaneous (SC) treprostinil was added and we observed a significant decrease in CEC counts during the first month of such treatment. CECs were quantified during a 6 to 36 month-follow-up after initiation of SC treprostinil and we found that CEC counts changed over time, with rising counts always preceding clinical deterioration.

CONCLUSIONS

CECs might be useful as a biomarker during follow-up of pediatric iPAH and PAH-CHD to assess response to treatment and to anticipate clinical worsening.