In systemic sclerosis (SSc), the development of fibrosis seems to be a consequence of the initial ischemic process related to an endothelial injury. The initial trigger event in SSc is still unknown, but circulating progenitor cells (CPCs) might play a key role. Such cells have the ability to traffic into injury sites, exhibiting inflammatory features of macrophages, tissue remodeling properties of fibroblasts, and vasculogenesis functions of endothelial cells. The different subsets of CPCs described thus far in SSc arise from a pool of circulating monocyte precursors (CD14 (+) cells) and probably correspond to a different degree of differentiation of a single cell of origin. Several subsets of CPCs have been described in patients with SSc, all have a monocytic origin but may or may not express CD14, and all of these cells have the ability to give origin to endothelial cells, or collagen (Col)-producing cells, or both. We were able to identify six subsets of CPCs: pluripotent stem cells (CD14 (+), CD45 (+), and CD34 (+)), monocyte-derived multipotential cells (MOMCs) or monocyte-derived mesenchymal progenitors (CD14 (+), CD45 (+), CD34 (+), Col I (+), CD11b (+), CD68 (+), CD105 (+), and VEGFR1 (+)), early endothelial progenitor cells (EPCs) or monocytic pro-angiogenic hematopoietic cells or circulating hematopoietic cells (CD14 (+), CD45 (+), CD34 (low/-), VEGFR2 (+/-), CXCR4 (+), c-kit (+), and DC117 (+)), late EPCs (CD14 (-), CD133 (+), VEGFR2 (+), CD144 (+) [VE-cadherin (+)], and CD146 (+)), fibroblast-like cells (FLCs)/circulating Col-producing monocytes (CD14 (+), CD45 (+), CD34 (+/-), and Col I (+)), and fibrocytes (CD14 (-), CD45 (+), CD34 (+), Col I (+), and CXCR4 (+)). It has been demonstrated that circulating CD14 (+) monocytes with an activated phenotype are increased in patients with SSc when compared with normal subjects. CD14 (+), CD34 (+), and Col I (+) spindle-shaped cells have been found in increased numbers in lungs of SSc patients with interstitial lung disease. Elevated blood amounts of early EPCs have been found in patients with SSc by different groups of researchers and such levels correlate directly with the interstitial lung involvement. The prevalence of hematopoietic markers expressed by CPCs that migrate from blood into injury sites in SSc differs and changes according to the degree of differentiation. CXCR4 is the most commonly expressed marker, followed by CD34 and CD45 at an end stage of differentiation. Such difference also indicates a continuous process of cell differentiation that might relate to the SSc clinical phenotype (degree of fibrosis and vascular involvement). A deeper understanding of the role of each subtype of CPCs in the development of the disease will help us to better classify patients in order to offer them targeted approaches in the future.

Previously, the perivascular characteristics of dental pulp stem cells (DPSCs) were reported, which suggested the potential application of DPSCs as perivascular cell source. In this study, we investigated whether DPSCs had angiogenic capacity by coinjection with human umbilical vein endothelial cells (HUVECs) in vivo; in addition, we determined the role of stromal cell-derived factor 1-α (SDF-1α) and C-X-C chemokine receptor type 4 (CXCR4) axis in the mutual interaction between DPSCs and HUVECs. Primarily isolated DPSCs showed mesenchymal stem cell- (MSC-) like characteristics. Moreover, DPSCs expressed perivascular markers such as NG2, α-smooth muscle actin (α-SMA), platelet-derived growth factor receptor β (PDGFRβ), and CD146. In vivo angiogenic capacity of DPSCs was demonstrated by in vivo Matrigel plug assay. We could observe microvessel-like structures in the coinjection of DPSCs and HUVECs at 7 days postinjection. To block SDF-1α and CXCR4 axis between DPSCs and HUVECs, AMD3100, a CXCR4 antagonist, was added into Matrigel plug. No significant microvessel-like structures were observed at 7 days postinjection. In conclusion, DPSCs have perivascular characteristics that contribute to in vivo angiogenesis. The findings of this study have potential applications in neovascularization of engineered tissues and vascular diseases.

OBJECTIVE

To analyze the potential of human dental pulp stem cells (hDPSCs) for maintaining their undifferentiated status and osteogenic differentiation capacity when arranged in cell sheets (CSs) for future application in bone replacement.

METHODS

CSs were formed after being induced for 10-15 days by clonogenic medium containing additional vitamin C (20 μg/ml). The cell viability of hDPSC4s in the CSs was followed until 96 h using the Live/Dead® assay. The cells of the CSs were enzymatically dissociated and then compared with the original hDPSC4s. The two cell types were characterized immunophenotypically by flow cytometry using specific mesenchymal stem cell-associated markers (CD105, CD146, CD44, STRO-1, and OCT3/4) and non-associated markers (CD34, CD45, and CD14). Osteogenic differentiation was analyzed with the Alizarin red assay.

RESULTS

Living cells were observed until 96 h in the CSs. Both cell types exhibited osteogenic differentiation and expressed the specific undifferentiated MSC-associated markers. Cells spontaneously detached from the CSs attached and proliferated at the bottom of the culture dishes.

CONCLUSIONS

Cells in the hDPSC4s cell sheets survived for at least 96 h. Moreover, the cells in the cell sheets retained their stemness and their osteogenic differentiation potential.

CONCLUSIONS

Cell sheets of hDPSCs could be employed as natural tri-dimensional structures for treating bone loss. This technique would be useful particularly for critical bone defects or any type of bone defects in patients carrying diseases that impair bone regeneration, such as diabetes mellitus, medication-related osteonecrosis of the jaw (MRONJ), and osteoporosis.

BACKGROUND

Recent advancement in cancer research has shown that tumors are highly heterogeneous, and multiple phenotypically different cell populations are found in a single tumor. Cancer development and tumor growth are driven by specific types of cells-stem cell-like cancer cells (SCLCCs)-which are also responsible for metastatic spread and drug resistance. This research was designed to verify the presence of SCLCCs in renal cell cancer cell lines. Subsequently, we aimed to characterize phenotype and cell biology of CD105+ cells, defined previously as renal cell carcinoma tumor-initiating cells. The main goal of the project was to describe the gene-expression profile of stem cell-like cancer cells of primary tumor and metastatic origin.

METHODS

Real-time PCR analysis of stemness genes (Oct-4, Nanog and Ncam) and soft agar colony formation assay were conducted to check the stemness properties of renal cell carcinoma (RCC) cell lines. FACS analysis of CD105+ and CD133+ cells was performed on RCC cells. Isolated CD105+ cells were verified for expression of mesenchymal markers-CD24, CD146, CD90, CD73, CD44, CD11b, CD19, CD34, CD45, HLA-DR and alkaline phosphatase. Hanging drop assay was used to investigate CD105+ cell-cell cohesion. Analysis of free-floating 3D spheres formed by isolated CD105+ was verified, as spheres have been hypothesized to contain undifferentiated multipotent progenitor cells. Finally, CD105+ cells were sorted from primary (Caki-2) and metastatic (ACHN) renal cell cancer cell lines. Gene-expression profiling of sorted CD105+ cells was performed with Agilent's human GE 4x44K v2 microarrays. Differentially expressed genes were further categorized into canonical pathways. Network analysis and downstream analysis were performed with Ingenuity Pathway Analysis.

RESULTS

Metastatic RCC cell lines (ACHN and Caki-1) demonstrated higher colony-forming ability in comparison to primary RCC cell lines. Metastatic RCC cell lines harbor numerous CD105+ cell subpopulations and have higher expression of stemness genes (Oct-4 and Nanog). CD105+ cells adopt 3D grape-like floating structures under handing drop conditions. Sorted CD105+ cells are positive for human mesenchymal stem cell (MSC) markers CD90, CD73, CD44, CD146, and alkaline phosphatase activity, but not for CD24 and hematopoietic lineage markers CD34, CD11b, CD19, CD45, and HLA-DR. 1411 genes are commonly differentially expressed in CD105+ cells (both from primary [Caki-2] and metastatic RCC [ACHN] cells) in comparison to a healthy kidney epithelial cell line (ASE-5063). TGF-β, Wnt/β-catenine, epithelial-mesenchymal transition (EMT), Rap1 signaling, PI3K-Akt signaling, and Hippo signaling pathway are deregulated in CD105+ cells. TGFB1, ERBB2, and TNF are the most significant transcriptional regulators activated in these cells.

CONCLUSIONS

All together, RCC-CD105+ cells present stemlike properties. These stem cell-like cancer cells may represent a novel target for therapy. A unique gene-expression profile of CD105+ cells could be used as initial data for subsequent functional studies and drug design.

Disorganized vessels in the tumor vasculature lead to impaired perfusion, resulting in reduced accessibility to immune cells and chemotherapeutic drugs. In the breast tumor-stroma interplay, paracrine factors such as interleukin-6 (IL-6) often facilitate disordered angiogenesis. We show here that epigenetic mechanisms regulate the cross-talk between IL-6 and vascular endothelial growth factor receptor 2 (VEGFR2) signaling pathways in myoepithelial (CD10+) and endothelial (CD31+, CD105+, CD146+, and CD133-) cells isolated from malignant and non-malignant tissues of clinically characterized human breast tumors. Tumor endothelial (Endo-T) cells in 3D cultures exhibited higher VEGFR2 expression levels, accelerated migration, invasion, and disorganized sprout formation in response to elevated IL-6 levels secreted by tumor myoepithelial (Epi-T) cells. Constitutively, compared with normal endothelial (Endo-N) cells, Endo-T cells differentially expressed DNA methyltransferase isoforms and had increased levels of IL-6 signaling intermediates such as IL-6R and signal transducer and activator of transcription 3 (STAT3). Upon IL-6 treatment, Endo-N and Endo-T cells displayed altered expression of the DNA methyltransferase 1 (DNMT1) isoform. Mechanistic studies revealed that IL-6 induced proteasomal degradation of DNMT1, but not of DNMT3A and DNMT3B and subsequently led to promoter hypomethylation and expression/activation of VEGFR2. IL-6 induced VEGFR2 up-regulation was inhibited by overexpression of DNMT1. Transfection of a dominant-negative STAT3 mutant, but not of STAT1, abrogated VEGFR2 expression. Our results indicate that in the breast tumor microenvironment, IL-6 secreted from myoepithelial cells influences DNMT1 stability, induces the expression of VEGFR2 in endothelial cells via a promoter methylation-dependent mechanism, and leads to disordered angiogenesis.

Keywords: DNA methyltransferase; Tumor microenvironment; VEGFR2; angiogenesis; breast cancer; interleukin 6 (IL-6); signal transduction.

Bone healing depends of a transient inflammatory response, involving selective migration of leukocytes under the control of chemokine system. CCR2 has been regarded as an essential receptor for macrophage recruitment to inflammation and healing sites, but its role in the intramembranous bone healing on craniofacial region remains unknown. Therefore, we investigated the role of CCR2 on F4/80+ cells migration and its consequences to the intramembranous healing outcome. C57BL/6 wild-type (WT) and CCR2KO mice were subjected to upper right incisor extraction, followed by micro-computed tomography, histological, immunological, and molecular analysis along experimental periods. CCR2 was associated with F4/80+ cells influx to the intramembranous bone healing in WT mice, and CCR2+ cells presented a kinetics similar to F4/80+ and CCR5+ cells. By contrast, F4/80+ and CCR5+ cells were significantly reduced in CCR2KO mice. The absence of CCR2 did not cause major microscopic changes in healing parameters, while molecular analysis demonstrated differential genes expression of several molecules between CCR2KO and WT mice. The mRNA expression of TGFB1, RUNX2, and mesenchymal stem cells markers (CXCL12, CD106, OCT4, NANOG, and CD146) was decreased in CCR2KO mice, while IL6, CXCR1, RANKL, and ECM markers (MMP1, 2, 9, and Col1a2) were significantly increased in different periods. Finally, immunofluorescence and FACS revealed that F4/80+ cells are positive for both CCR2 and CCR5, suggesting that CCR5 may account for the remaining migration of the F4/80+ cells in CCR2KO mice. In summary, these results indicate that CCR2+ cells play a primary role in F4/80+ cells migration along healing in intramembranous bones, but its deficiency does not critically impact healing outcome.

OBJECTIVE

Adipose-derived stem cells (ASCs) have angiogenic potential owing to their differentiation into endothelial cells and their release of angiogenic growth factors to elicit paracrine effects. In addition, control-released basic fibroblast growth factor (bFGF) sustained with a gelatin hydrogel also supports effective angiogenesis. We sought to determine if coadministration of ASCs and control-released bFGF into murine ischemic limbs facilitates angiogenesis.

METHODS

Levels of growth factors in the conditioned media of ASCs cultured with or without control-released bFGF were measured by enzyme-linked immunosorbent assays. A murine ischemic hind limb model was generated and intramuscularly injected with the following: gelatin hydrogel (group 1), a high number of ASCs (group 2), control-released bFGF (group 3), a small number of ASCs and control-released bFGF (group 4), and a high number of ASCs and control-released bFGF (group 5). Macroscopic and microscopic vascular changes were evaluated until day 7 by laser Doppler perfusion imaging and histologic analyses, respectively.

RESULTS

Secretion of hepatocyte growth factor, vascular endothelial growth factor, and transforming growth factor-β1 was enhanced by control-released bFGF. Vascular improvement was achieved in groups 4 and 5 according to laser Doppler perfusion imaging. Hematoxylin and eosin staining and CD31 immunohistochemical staining demonstrated an increase in the vascular density, vessel diameter, and thickness of vessel walls in groups 4 and 5. Cells positively stained for CD146, α-smooth muscle actin, and transforming growth factor-β1 were observed around vessel walls in groups 4 and 5.

CONCLUSIONS

These findings suggest that coadministration of ASCs and control-released bFGF facilitates angiogenesis in terms of vessel maturation in a murine ischemic hind limb model.

Telocytes (TCs) are morphologically defined as small-sized cells with long, thin, moniliform processes called telopodes (Tps). Numerous papers imply that TCs are a distinctive cell type, and that transmission electron microscopy (TEM) is the gold standard tool for their identification. We aimed to reproduce previous studies on myocardial TCs to check their validity. For this purpose we performed an immunohistochemical study on human cardiac samples from six autopsied donor cadavers, using antibodies against CD10, CD31, CD34, CD146, Ki67, alpha-smooth muscle actin (α-SMA), Platelet-Derived Growth Factor Receptor-alpha (PDGFRα) and laminin. Additionally we performed a TEM study on cardiac samples from three human autopsied donor cadavers and five adult Sprague-Dawley rats. We found endothelial cells (ECs), cords, and filopodia-projecting endothelial tip cells (ETCs) that expressed CD10, CD31, CD34, CD146, and PDGFR-α. Often, endothelial cells closely neighbored the sarcolemmal basal laminae. Endothelial progenitor cells, as well as nascent capillaries, were CD31+/CD34+. Proliferative endothelial cells expressed Ki67. In larger vessels we found pericytes that expressed CD146 and α-SMA; scarce α-SMA-expressing spindle-shaped cells lining cardiomyocytes were suggestive of a pericytic role in angiogenic sprout guidance. The TEM study showed that endothelial tubes are almost exclusively found in the narrow myocardial interstitia. ECs that built them up appeared identical to the cells that previous TEM studies have suggested to be myocardial telocytes. A subset of stromal cells with TC-like phenotype and telopodes-like processes actually seem to configure blood vessels, and therefore belong to the endothelial lineage. This study shows that data presented in previous studies on myocardial telocytes is not enough to allow the reproducibility of the results. At least a subset of cells considered to be TCs might belong to the endothelial lineage.

Telocytes (TCs) are stromal cells defined by the presence of long and slender prolongations (telopodes). They are a biologically and functionally heterogeneous population that has not been previously investigated in the sclera. The purpose of this study is to investigate the presence and characteristics of scleral telocytes through a combined immunohistochemical and transmission electron microscopy (TEM) study using samples from ten adult patients. Stromal cells with a TC-like morphology expressed CD34, CD45, CD105, vimentin and occasionally CD68 but were negative for collagen III, CD31, CD133, and CD146. Conjunctival epithelial cells expressed CD45, CD105, CD146, and vimentin. These phenotypes support a scleral niche with immune TCs and haematopoietic stem cells (HSCs). In TEM, we often found spindle-shaped stromal cells projecting telopodes or filopodes, with extremely long nuclei extended even within those prolongations. We separated these cells into a light subtype, which contained a complete set of organelles, and a dark subtype, consisting of undifferentiated stem/progenitor cells. The light cells contained dense vesicles, Weibel-Palade bodies, and rounded α-granule-like structures. These storage areas for the von Willebrand factor (vWF) are known to express selectins that are critically involved in HSC homing and could also indicate endothelial progenitors. The dark cells were scarcely myoid, populated the episcleral perivascular niches and the scleral stroma, and were equipped with lipid storage areas such as lamellar bodies and lipid droplets (LDs). Previously, unreported intranuclear LDs were found in these cells, which is characteristic of an HSC population. It appears that the human scleral stroma is a niche harbouring TC-like cells with immune and HSC phenotypes, and the mere presence or characteristics of telopodes are not enough to differentiate them.

The most widely used technique for isolation of human bone marrow stromal cells (hBMSCs) from bone marrow includes density gradient centrifugation, recovery of the mononuclear cell population, and subsequent isolation of hBMSCs by virtue of their plastic adherence. During subsequent in vitro cultivation, they may lose their original characteristics since in vitro the stem cell niche cannot yet be properly mimicked. To further characterize these culture-induced changes in regard to mRNA and extra- and intracellular protein expression, as well as potential differences between hBMSCs from different donors, we investigated a panel of CD antigens for their presence on in vitro cultured hBMSCs. Interestingly, after culture-induced downregulation of their extracellular expression, both CD146 and CD271 persist intracellularly, which hints at the possibility that culture-induced changes may be reversed by appropriate stimuli. Further, CD34-a protein whose expression on hBMSCs is still controversial-is expressed at the intracellular level in hBMSCs of all donors independently of passage number. CD34 mRNA levels are significantly higher in female than in male donors. In summary, we further elucidate phenotypical changes induced by in vitro culture of hBMSCs, highlight interindividual differences in the phenotype of these cells and for the first time show the intracellular expression of CD34.

Melanoma cell adhesion molecule (MCAM) was originally reported to be involved in the invasion and progression of melanoma. It was also shown to be responsible for the attachment of cells to endothelial cells. In this study, we demonstrated by immunohistochemistry that immunoreactive MCAM was not expressed on granulosa cells in the pre-ovulatory follicle, but it was clearly detected in large luteal cells in corpora lutea from the mid-luteal phase of the menstrual cycle. Northern blotting analysis confirmed the expression of MCAM mRNA in corpus luteum. MCAM was weakly detected by immunocytochemical staining in human luteinizing granulosa cells isolated from patients undergoing IVF treatment. Its expression was found to be increased during time in culture of these cells. Flow cytometry and Northern blot analysis revealed that MCAM expression on luteinizing granulosa cells was enhanced when the cells were cultured for 5 days in the presence of hCG (1 IU/ml) or cytokines such as interleukin-1alpha (10 ng/ml) and tumour necrosis factor-alpha (10 ng/ml). No significant difference of MCAM expression was observed between the cultures under normoxic (20% oxygen) and hypoxic (1% oxygen) conditions. These results indicate that luteinizing granulosa cells express MCAM and that MCAM expression is regulated by LH/hCG and cytokines during luteinization. Since MCAM has been reported to mediate cellular interaction with endothelial cells, this molecule may play a role in neovascularization during corpus luteum formation in the human ovary.

OBJECTIVE

The objective of the present study was to evaluate whether fibrotic markers and endometrial stem cell markers were abnormal expressed in endometrium of intrauterine adhesions and a female mouse model for intrauterine adhesions.

METHODS

We revaluated endometrial fibrosis using Masson's stain. We detected the expression of endometrium stem cell markers (CD146 and CD140b) and fibrosis markers (TGF-Beta, CTGF, collagen protein I and collagen protein III) in endometrial tissue with intrauterine adhesions using real-time PCR and S-P (Streptavidin-Peroxidase) immunohistochemistry. We create a female mouse model for intrauterine adhesions using mechanical injury, and then revalue the expression of endometrial stem cell markers and fibrosis markers in endometrial tissue of mouse model for intrauterine adhesions.

RESULTS

The ratio of the area with endometrial fibrosis to total endometrial area in intrauterine adhesious significantly increased compared with the normal endometrial tissue (P < 0.05); The expression levels of fibrotic markers and endometrial stem cell markers were higher in the endometrial tissue with intrauterine adhesious compared to normal endometrial tissue (P < 0.05). The animal experiments showed that the ratio of the area with endometrial fibrosis to total endometrial area significantly increased compared with the control group (P < 0.05); The expression levels of fibrotic markers and endometrial stem cell markers were higher in the endometrial tissue compared to the control group (P < 0.05).

CONCLUSIONS

Aberrant activation of fibrosis may be involved in the pathology of intrauterine adhesious.

Regeneration of periodontal tissues, particularly cementum, is key to regaining periodontal attachment and health. Human periodontal ligament stem cells (hPDLSCs) have been shown to be a good cell source to regenerate periodontal tissues. However, their subpopulations and the differentiation induction in relation to cementogenic lineages is unclear. Thus, we aim to examine the expression of cementum-associated genes in PDLSC subpopulations and determine the effect of broadly used osteogenic stimulus or vitamin C (VC) on the expression of cementogenic and osteogenic genes in PDLSCs. Our real-time quantitative polymerase chain reaction (qPCR) analysis showed that cementogenic marker cementum attachment protein (CAP) expressed only slightly higher in STRO-1+/CD146+, STRO-1-/CD146+ and STRO-1-/CD146- subpopulations than in the original cell pool, while cementum protein 1 (CEMP1) expression in these subpopulations was not different from the original pool. Notably, under the stimulation with osteogenic differentiation medium, CAP and CEMP1 were downregulated while osteogenic markers bone sialoprotein (BSP) and osteocalcin (OCN) were upregulated. Both CAP and CEMP1 were upregulated by VC treatment. Transplantation of VC-treated PDLSCs into immunocompromised mice resulted in forming significantly more ectopic cementum- and bone-like mineral tissues in vivo. Immunohistochemical analysis of the ectopic growth showed that CAP and CEMP1 were mainly expressed in the mineral tissue and in some cells of the fibrous tissues. We conclude that osteogenic stimulation is not inductive but appears to be inhibitory of cementogenic pathways, whereas VC induces cementogenic lineage commitment by PDLSCs and may be a useful stimulus for cementogenesis in periodontal regeneration.

BACKGROUND

No study has examined the possible factors associated with different characteristics of stem-like cells derived from anterior cruciate ligament (ACL) remnants. And the purpose of the study is to elucidate whether demographic factors are associated with healing potential of stem-like cells derived from the ACL remnants tissue.

METHODS

Thirty-six ACL remnants were harvested from patients who received primary arthroscopic ACL reconstruction. Interval from injury to surgery, age, sex, and combined meniscal or chondral injuries were analyzed. Cells were isolated from remnant tissues and their healing potential was evaluated by: 1) characterization of surface markers (CD34, CD44, CD45, CD146, CD29, and Stro-1), 2) cell expansion, 3) osteogenic differentiation, and 4) endothelial differentiation. Finally, using multivariable logistic regression to evaluate the relation between demographic factors and healing potential parameters. Adjusted odds ratios (OR) were calculated, and the significant difference was set at p < 0.05.

RESULTS

ACL remnant tissue harvested less than 90 days after injury predicted higher fractions of stem-like cells [CD34+ (OR = 6.043, p = 0.025), CD44 + (OR = 8.440, p = 0.011), CD45+ (OR = 16.144, p = 0.015), and CD146+ (OR = 9.246, p = 0.015)] and higher expansion potential (passage 3: OR = 9.755, p = 0.034; passage 10: OR = 33.245, p = 0.003). Regarding osteogenic differentiation, higher gene expression of Osteocalcin (OR = 22.579, p = 0.009), Alkaline phosphatase (OR = 6.527, p = 0.022), and Runt-related transcription factor 2 (OR = 5.247, p = 0.047) can also be predicted. Younger age predicted higher CD34+ levels (20 ≤ age < 30 years, OR = 2.020, p = 0.027) and higher expansion potential at passage 10 (10 ≤ age < 20 years, OR = 25.141, p = 0.026). There was no significant relation found between meniscal or chondral injuries and ACL healing potential.

CONCLUSIONS

Our results indicated that the ACL remnant tissue harvested within 3-months after injury yields higher healing potential, suggesting early surgical intervention may achieve better clinical results.

Mast cells (MC) and blood basophils (Ba) are multifunctional effector cells of the immune system and accumulate in areas of ongoing disease. However, despite of similar morphology, MC and Ba differ from each other in terms of cell surface receptor expression, mediator content, and tissue distribution. In order to gain new insights into mechanisms and molecules responsible for the distribution and accumulation of MC and Ba, we have investigated expression of homing receptors on primary human MC (lung, n=28; uterus, n=17), Ba (healthy donors, n=64), the mast cell line HMC-1, and the basophil line KU-812. Expression of cell surface antigens on MC and Ba was analyzed by mAb and indirect immunofluorescence staining techniques. In addition to previous findings, Ba were found to react with mAb against the selectin-ligands sLe(x) (CD15s) and PSGL-1 (CD162), L-selectin (CD62L), beta7-integrin, the 'matrix-receptor' neurothelin (CD147), platelet-endothelial cell tetraspan antigen-3 (PETA-3=CD151), and BST-1 (CD157). Novel antigens detectable on MC (lung and uterus) were CD147, CD151, CD157 and CD49c (VLA-3alpha). By contrast, MC were not recognized by mAb to sLe(x), PSGL-1, L-selectin, or beta7 integrin. No reactivity of Ba or MC with mAb to syndecan-1 (CD138), VE-cadherin (CD144), MUC18/MCAM (CD146), MGC-24 (CD164), or ALCAM (CD166) was found. The cell lines HMC-1 and KU-812 expressed a similar profile of antigens when compared to primary cells. In summary, Ba and MC express a unique profile of homing molecules. Apparently, Ba differ from MC in expression of recognition receptors relevant for binding to endothelium and consecutive transmigration.

The restoration of damaged endothelium is promising to reduce side effects, including restenosis and thrombosis, in the stent treatment for vascular diseases. Current technologies based on drug delivery for these complications still do not satisfy patients due to invariant recurrence rate. Recently, even if one approach was applied to clinical trial to develop the firstly commercialized stent employing circulating endothelial progenitor cells (EPCs) in blood vessels, it resulted in failure in clinical trial. Based on instruction of the failed case, we designed an advanced EPC-capture stent covered with anti-CD146 antibody (Ab) immobilized silicone nanofilament (SiNf) for the highly efficient and specific capture of not early but late stage of EPCs. In vitro cell capture test demonstrates enhanced capture efficiency and adhesion morphology of late EPCs on the modified substrate. The modified substrates could capture 8 times more late EPCs and even 3 times more mesenchymal stem cells (MSCs) as compared to unmodified one. A porcine model with high similarity to human reproduced in vivo results ideally translated from in vitro cell capture results. As restenosis indicators, lumen area, neointimal rate and stenosis area for modified stents were reduced at the range of 30-60% as compared to those for bare metal stent (BMS). Fibrin score indicating thrombosis was lowered less than half as comparing to that on BMS. These inspiring results are attributed to ~2-fold increased endothelial coverage, determined by immuno-histological staining. Taken together, the CD146 Ab-armed nanofilamentous stent could show great performance in the reduction of thrombosis and restenosis through re-endothelialization due to highly efficient specific cell capture. STATEMENT OF SIGNIFICANCE: Stents have been developed from simple metal stents to functionalized stents for past decades. However, they have still risks to relapse the occlusion in stented arteries. In this paper, we describe the fabrication and optimization of cell capturing stents to maximize the effective re-endothelialization through the serial coating of silicone nanofilaments and anti-CD146 antibody. The nanofilaments increase the amount of coated antibodies and provide the anchoring points of circulating angiogenic cells for strong focal adhesion. We demonstrate high immobilizing ability of circulating angiogenic cells (endotheliali progenitor cells and mesenchymal stem cells) in vitro under similar shear stress to coronary arteries (15 dyne/cm²). Also, we show accelerating re-endothelialization and the efficient prevention of restenosis in porcine coronary arteries in vivo.

BACKGROUND

Very late antigen 4 (VLA-4; integrin α4β1) is critical for transmigration of T helper (TH) 1 cells into the central nervous system (CNS) under inflammatory conditions such as multiple sclerosis (MS). We have previously shown that VLA-4 and melanoma cell adhesion molecule (MCAM) are important for trans-endothelial migration of human TH17 cells in vitro and here investigate their contribution to pathogenic CNS inflammation.

METHODS

Antibody blockade of VLA-4 and MCAM is assessed in murine models of CNS inflammation in conjunction with conditional ablation of α4-integrin expression in T cells. Effects of VLA-4 and MCAM blockade on lymphocyte migration are further investigated in the human system via in vitro T cell transmigration assays.

RESULTS

Compared to the broad effects of VLA-4 blockade on encephalitogenic T cell migration over endothelial barriers, MCAM blockade impeded encephalitogenic T cell migration in murine models of MS that especially depend on CNS migration across the choroid plexus (CP). In transgenic mice lacking T cell α4-integrin expression (CD4::Itga4-/-), MCAM blockade delayed disease onset. Migration of MCAM-expressing T cells through the CP into the CNS was restricted, where laminin 411 (composed of α4, β1, γ1 chains), the proposed major ligand of MCAM, is detected in the endothelial basement membranes of murine CP tissue. This finding was translated to the human system; blockade of MCAM with a therapeutic antibody reduced in vitro transmigration of MCAM-expressing T cells across a human fibroblast-derived extracellular matrix layer and a brain-derived endothelial monolayer, both expressing laminin α4. Laminin α4 was further detected in situ in CP endothelial-basement membranes in MS patients' brain tissue.

CONCLUSIONS

Our findings suggest that MCAM-laminin 411 interactions facilitate trans-endothelial migration of MCAM-expressing T cells into the CNS, which seems to be highly relevant to migration via the CP and to potential future clinical applications in neuroinflammatory disorders.

OBJECTIVE

To detect and quantify circulating tumour cells (CTCs) in peripheral blood of patients with uveal melanoma primary non-metastatic tumours, and to analyze the possible relationship between CTCs and clinical risk factors.

METHODS

Prospective study with two clinical groups: 4 patients diagnosed with choroidal nevus and 8 patients with choroidal melanoma prior to treatment. A single sample of 7.5 mL of peripheral blood was taken and the CTCs were isolated using a CellSearch system that captures positive cells for the CD146 antigen (MUC18).

RESULTS

None of the patients with choroidal nevus showed CTCs in peripheral blood. More than one CTC/7.5 mL was detected in 50 % of patients with choroidal melanoma prior to treatment. The higher level of CTC cells in peripheral blood (3/7.5 mL) was detected in the patient with the larger choroidal melanoma which also presented extrascleral extension and epithelioid pathology.

CONCLUSIONS

Performing an analysis with the CellSearch system allows to quantify the choroidal melanoma CTCs in peripheral blood. This finding highlights the potential usefulness of this technique to achieve the correct stratification and monitoring of the treatment.

Recent observation identifies subendothelial (mural) cells expressing MCAM, a specific system of clonogenic, self-renewing, osteoprogenitors (a.k.a, "mesenchymal stem cells") in the microvascular compartment of post-natal human bone marrow (BM). In this study, we used MCAM/CD146, as a marker to localize, isolate and assay subendothelial clonogenic cells from the microvasculature of postnatal human skeletal muscle. We show here that these cells share with their BM counterpart, anatomic position (subendothelial/adventitial) and ex vivo clonogenicity (CFU-Fs). When assayed under the stringent conditions, these cells display a high spontaneous myogenic potential (independent of co-culture with myoblasts or of in vivo fusion with local myoblasts), which is otherwise only attained in cultures of satellite cells. These muscle-derived mural cells activated a myogenic program in culture. Cultured CD146+ cells expressed the myogenic factors (Pax7, Pax3 and Myf5), NCAM/CD56, desmin as well as proteins characteristic of more advanced myogenic differentiation, such as myosin heavy chain. In vivo, these cells spontaneously generate myotubes and myofibrils. These data identify the anatomy and phenotype of a novel class of committed myogenic progenitor in human post-natal skeletal muscle of subendothelial cells associated with the abluminal surface of microvascular compartment distinct from satellite cells.

Recent works demonstrated the presence of a multipotent epithelial cell population in the bulge region of adult human hair follicles. These cells can be cultured in vitro, thus leading to the preparation of dermal-epidermal substitutes which are applicable in the treatment of burns and ulcers. We evaluated the main marker expression in cells obtained from stripped human hair follicles. A pool of hair follicles were incubated at 37 degrees C and 5% CO(2) in a growth medium. The cells were then labelled with antibodies (anti-CD34, anti-CD38, anti-CD45, anti-CD90, anti-CD133, anti-CD146) and analysed by cytometry. We also used hair follicles for immunohistochemical studies, employing antibodies such as CD34, Actin Smooth Muscle, Filaggrin, Desmin, Vimentin, Glial Fibrillary Acidic Protein, Ki-67, PanCytokeratin, CK15, CK19. The cytometry results revealed that a part of bulge cells were CD34+ (1-2%). CD34+ population comprises both large, CD45-, CD133-, CD146- cells and small, CD45+, CD133+, CD146+ cells. Thus, a part of CD34+ cells present a mature endothelial marker (CD146). An expression of the proliferation marker Ki-67 and the stem cell marker CD34 is present in the follicle bulge region. In conclusion, we observed that the stripped hair follicle has the same multipotent cell population as adult and fetal scalp hair follicles.

Characterization of the cell surface marker phenotype of ex vivo cultured cells growing out of human fibrovascular epiretinal membranes (fvERMs) from proliferative diabetic retinopathy (PDR) can give insight into their function in immunity, angiogenesis, and retinal detachment. FvERMs from uneventful vitrectomies due to PDR were cultured adherently ex vivo. Surface marker analysis, release of immunity- and angiogenesis-pathway-related factors upon TNF α activation and measurement of the intracellular calcium dynamics upon mechano-stimulation using fluorescent dye Fura-2 were all performed. FvERMs formed proliferating cell monolayers when cultured ex vivo, which were negative for endothelial cell markers (CD31, VEGFR2), partially positive for hematopoietic- (CD34, CD47) and mesenchymal stem cell markers (CD73, CD90/Thy-1, and PDGFR β ), and negative for CD105. CD146/MCAM and CD166/ALCAM, previously unreported in cells from fvERMs, were also expressed. Secretion of 11 angiogenesis-related factors (DPPIV/CD26, EG-VEGF/PK1, ET-1, IGFBP-2 and 3, IL-8/CXCL8, MCP-1/CCL2, MMP-9, PTX3/TSG-14, Serpin E1/PAI-1, Serpin F1/PEDF, TIMP-1, and TSP-1) were detected upon TNF α activation of fvERM cells. Mechano-stimulation of these cells induced intracellular calcium propagation representing functional viability and role of these cells in tractional retinal detachment, thus serving as a model for studying tractional forces present in fvERMs in PDR ex vivo.

OBJECTIVE

Behçet's disease (BD) is a systemic disorder characterised by vasculitis. Endothelial progenitor cells are derived from the bone marrow and contribute to new vessel formation. The aim of this study was to investigate the level of endothelial progenitor cells in BD and BD-associated conditions.

METHODS

A total of 74 subjects were included in this study, of whom 44 and 30 subjects were patients with BD or healthy subjects, respectively. Endothelial progenitor cells were defined and measured by flow cytometry according to the expression of CD146, CD31 and CD34. We separated BD patients according to the active disease, pathergy test results, thrombosis and gender. MedCalc 12.5 software programme was used for statistical analyses.

RESULTS

The level of endothelial progenitor cells was comparable in patients with BD and healthy subjects (p=0.849). It was also comparable in patients with active or inactive BD (p=0.320). The level of endothelial progenitor cells was higher in patients with thrombosis (p=0.04). There was no statistical significant difference between pathergy positive and negative patients (p=0.969). The level of endothelial progenitor cells was not correlated with age, C-reactive protein, erythrocyte sedimentation rate, white blood cells and disease duration (p>0.05).

CONCLUSIONS

The level of endothelial progenitor cells was significantly higher in BD patients with thrombosis. On the other hand, they were not associated with disease activity, pathergy test and other conditions. EPCs may be a useful marker for thrombosis in patients with BD. In our opinion, this is the most expected result in this study.

Although the primary cell cultures from dental pulp and other oral tissue are frequently used to study osteogenic potential and stem cell responses, few systematic and comparative studies on stemness for the dentinogenic differentiation of these cells have been conducted. In the present study, to investigate the stemness of oral primary cells during extended culture, human adult dental pulp cells (hDPCs), periodontal ligament stem cells (hPDLSCs) and gingival fibroblasts (hGFs) were obtained and cultured from pulp tissue, periodontal ligaments, and marginal and attached gingival tissue of extracted third molars, respectively. As shown by fluorescence-activated cell sorting analysis and immunophenotyping, the mesenchymal stem cell markers, CD44, CD73, CD90, CD146 and CD166, were highly expressed in early passage hDPCs, hPDLSCs and hGFs. However, when the cells were treated with osteogenic additives, mineralization markedly increased in the hDPCs and hPDLSCs, but not in the hGFs. Moreover, the expression of dentinogenic markers, such as dentin sialophosphoprotein and dentin matrix protein-1, appeared to decrease during extended culture past passage number 8 of the hDPCs and hPDLSCs. These data suggest that hDPCs and hPDLSCs may have differentiation potential during the early passages, and that their progenitor potential is diminished during extended culture. The hGFs did not show differentiation capability during culture, even though they contained general mesenchymal stem cell surface proteins. The transcriptional expression of dentinogenic markers in hDPCs was not affected by co-culture with hPDLSCs and/or hGFs.