BACKGROUND

The endothelium and angiogenesis are therapeutic targets in cancer. Response to treatment may be assessed by laboratory plasma markers such as circulating endothelial cells (CECs), endothelial progenitor cells (EPCs), von Willebrand factor (vWf), soluble E selectin, vascular endothelial growth factor (VEGF) and angiogenin. We hypothesised that these markers, obtained before surgery, would predict 2-year outcome after surgery with or without anti-angiogenic therapy for colorectal cancer (CRC).

METHODS

We recruited 154 patients with CRC, of whom 51 were treated with surgery alone, 74 were treated with standard chemotherapy (5-fluorouracil) and 29 were treated with standard chemotherapy plus anti-VEGF therapy (Avastin). Peripheral blood was taken before surgery. CD34(+)/CD45(-)/CD146(+) CECs and CD34(+)/CD45(-)/CD309 [KDR](+) EPCs were measured by flow cytometry and plasma markers by ELISA.

RESULTS

After a mean of 2.1 years follow-up (range 1.9-2.3 years), 52 of the patients (33.7 %) experienced a poor outcome (radiological and/or histological evidence of tumour spread or recurrence, or death [n = 26]). In univariate analysis, poor outcome was linked to Dukes' stage (p < 0.001), American Joint Committee on Cancer (AJCC) stage (p < 0.001), type of treatment (surgery alone, standard chemotherapy with or without anti-antigenic therapy) (p = 0.047), CECs (p < 0.02) and EPCs (p < 0.01). In subsequent binary logistic regression analysis, only Dukes' stage (hazard ratio 2.3, 95 % confidence interval 1.0-5.3, p = 0.047) and modified AJCC stage (4.62, 1.88-11.33, p < 0.001) predicted a poor outcome.

CONCLUSIONS

Endothelial cell markers (CECs, EPCs, vWf, soluble E selectin) and growth factors (VEGF and angiogenin), measured before surgery, have nothing extra to offer in predicting 2-year outcome in colorectal cancer when compared to Dukes' or AJCC stage.

Human mesenchymal stem cells (MSCs) possess well-known reparative abilities, but any defect of the immunomodulatory activity and/or the differentiation process may determine the development of human diseases, including those affecting the vascular wall. MSCs residing within the human aortic wall represent a potential cell mediator of atherosclerotic aneurysm development.

MSCs isolated from healthy and aneurysm aortas were characterized by flow cytometer and tested for differentiation properties. Healthy aorta (ha)-MSCs were then subjected to inflammatory stimuli to evaluate the microenvironmental impact on their function and involvement in vascular remodelling.

Abdominal aortic aneurysm (AAA)-MSCs were isolated from calcified and inflamed aortas of 12 patients with high serum levels of MMP-9 protein. AAA-MSCs expressed typical mesenchymal markers and, in line with the histological analysis, elevated levels of OPN, an osteogenic marker also involved in vascular remodelling. AAA-MSCs were highly osteogenic and underwent intense calcium deposition under proper stimulation; moreover, AAA-MSCs were able to differentiate into tubule-like structures in Matrigel, even if the lack of CD146 and the reduced structural stability suggested an inefficient maturation process. We further demonstrated an association between osteogenesis and inflammation; indeed, ha-MSCs cultured with either cytokines (TNF-α, IL-1β) or AAA-PBMCs showed increased expression of MMP-9 and osteogenic markers, to the detriment of the adipogenic regulator PPAR-γ. Interestingly, the culture with inflammatory cells highly stimulated ha-MSCs towards the osteogenic commitment.

AAA-MSCs displayed high osteogenic potential and pathological angiogenesis that represent crucial steps for AAA progression; we showed that the inflammatory process critically addresses human vascular MSCs towards a pathological behaviour, inducing vascular bone matrix deposition and remodelling. Inhibition of this pathway may represent a pharmacological approach against arterial calcification.

OBJECTIVE

to detect the levels of CEC and expression of NFκB in the three groups of subjects with certain conditions.

METHODS

this study is an exploratory study using human peripheral blood samples. The study subjects comprised three groups, the group of 23 healthy people, a group of 35 people that has one or more risk factors for vascular disease and a group of 15 vascular disease patients (coronary heart disease, diabetes mellitus, stroke). CEC were isolated from peripheral blood mononuclear cells (PBMC). CEC level is identified through the measurement of CD45 and CD146 by flowcytometry method. NFκB expression is recognized by ELISA method (imgenex, USA).

RESULTS

the highest average levels of CEC were found in the sick group (28.6%). The highest average expression of NFκB (924.9495) is found in the group with risk factors. The lowest average expression of NFκB and CEC is found in the healthy group. Statistical analysis of ANOVA at the interval confidence of 95% shows a significant difference (p=0.00) levels of CEC and NFκB expression between the healthy group with the group with the risk of cardiovascular disease (CVD) and patients with known CVD.

CONCLUSIONS

increase of level CEC and NFκB expression has a strong relationship with vascular disease and its risk factors.

The present study investigated the role of runt-related transcription factor 2 (Runx2) in regulating the differentiation of human dental pulp stem cells (hDPSCs) into odontoblasts under the mediation of the Rho/Rho-associated protein kinase (ROCK) signaling pathway. hDPSCs and human bone marrow mesenchymal stem cells (hBMSCs) were mineralized to induce differentiation. The expression levels of odontoblast- and osteoblast-specific proteins, dentin sialophosphoprotein (DSPP), osteocalcin (OCN) and Runx2, were measured using western blot analysis. The hDPSCs were treated with Rho/ROCK signaling pathway inhibitor, C3 exoenzyme, and mineralized prior to determining the protein expression levels of RhoA, ROCK, Runx2, OCN, DSPP, and mRNA expression levels of early mineralization genes, including alkaline phosphatase, collagen type I, Msh homeobox 2 and distal-less homeobox 2, and late mineralization genes, including DSPP, dentin matrix protein-1 (DMP-1), bone sialoprotein (BSP) and OCN. Flow cytometry data indicated that 95% of the isolated hDPSCs were positive for mesenchymal stem cell markers, including cluster of differentiation (CD)29, CD90 or CD105, and vascular endothelial cell marker, CD146, whereas <5% of the hDPSCs were positive for hematopoietic stem cell markers, CD34 and CD45. The expression levels of DSPP in hDPSCs and OCN in hBMSCs were significantly upregulated with increased time in mineralization medium (P<0.01), which suggested that hDPSCs and hBMSCs were differentiated into odontoblasts and osteoblasts, respectively. During the osteogenic process, Runx2 protein was highly expressed in mesenchymal stem cells following stimulation with mineralization medium compared with cells that received no stimulation. During odontoblast differentiation in hDPSCs, Runx2 protein was highly expressed in the early stage; however, the expression declined in the late stage. Furthermore, treatment with C3 exoenzyme significantly downregulated the expression of RhoA, ROCK and Runx2 compared with the control in hDPSCs (P<0.01). Additionally, in mineralization solution, C3 exoenzyme also significantly downregulated the expression of Runx2 (P<0.01); however, the Rho/ROCK signaling pathway inhibitor did not significantly impact the expression of early mineralization genes. By contrast, C3 exoenzyme significantly upregulated the expression of DSPP and DMP-1, and downregulated the expression of BSP and OCN (P<0.01). The present findings suggested that odontoblast differentiation in hDPSCs may be regulated by Rho/ROCK signaling pathway-mediated downregulation of Runx2.

Previous research has indicated that purified perivascular stem cells (PSCs) have increased chondrogenic potential compared to conventional mesenchymal stem cells (MSCs) derived in culture. This study aimed to develop an autologous large animal model for PSC transplantation and to specifically determine if implanted cells are retained in articular cartilage defects. Immunohistochemistry and fluorescence-activated cell sorting were used to ascertain the reactivity of anti-human and anti-ovine antibodies, which were combined and used to identify and isolate pericytes (CD34-CD45-CD146+) and adventitial cells (CD34+CD45-CD146-). The purified cells demonstrated osteogenic, adipogenic, and chondrogenic potential in culture. Autologous ovine PSCs (oPSCs) were isolated, cultured, and efficiently transfected using a green fluorescence protein (GFP) encoding lentivirus. The cells were implanted into articular cartilage defects on the medial femoral condyle using hydrogel and collagen membranes. Four weeks following implantation, the condyle was explanted and confocal laser scanning microscopy demonstrated the presence of oPSCs in the defect repaired with the hydrogel. These data suggest the testability in a large animal of native MSC autologous grafting, thus avoiding possible biases associated with xenotransplantation. Such a setting will be used in priority for indications in orthopedics, at first to model articular cartilage repair.

OBJECTIVE

It has been established that endothelial dysfunction (ED) occurs after coronary artery bypass grafting (CABG). The aim of the study was to assess whether adiponectin may act as a novel marker of ED and its potential relations with new markers of ED: novel cell adhesion molecule CD146, a natural anti-thrombin glycoprotein - thrombomodulin (TM) and the well-established ED marker - Von Willebrand factor (VWF) in coronary artery disease (CAD) patients undergoing CABG.

METHODS

45 CAD patients undergoing elective CABG were included in the study.

RESULTS

In the study group the concentration of adiponectin and CD146 before the surgery were significantly lower than in the control group - 6.06 μg/ml ± 3.06 vs. 19.0 μg/ml ± 6.4 and 303.2 ng/ml ± 28.7 vs. 328.1 ng/ml ± 22.6 (p<0.05). Significant increase of adiponectin and CD146 concentration 3 months after CABG vs. before the surgery was found. Adiponectin concentration 3 months after CABG correlated with VWF, TM, CD146, and a number of grafts. CD146 before and 3 months after CABG correlated significantly with adiponectin, VWF activity as well as the statins therapy after the surgery.

CONCLUSIONS

In CAD patients undergoing CABG new markers of endothelial cell dysfunction as adiponectin and CD146 are significantly lower compared to healthy volunteers. Significant increase in adiponectin and CD146 concentration 3 months after CABG vs. before the surgery was found. However adiponectin concentrations 3 months after CABG were still significantly lower compared to healthy individuals, whereas CD146 concentration returned to the values comparable to the control.

Peripheral artery disease is an atherosclerotic occlusive disease that causes limb ischemia and has few effective noninterventional treatments. Stem cell therapy is promising, but concomitant diabetes may limit its effectiveness. We evaluated the therapeutic potential of skeletal muscle pericytes to augment postischemic neovascularization in wild-type and type 2 diabetic (T2DM) mice. Wild-type C57BL/6J and leptin receptor spontaneous mutation db/db T2DM mice underwent unilateral femoral artery excision to induce limb ischemia. Twenty-four hours after ischemia induction, CD45-CD34-CD146+ skeletal muscle pericytes or vehicle controls were transplanted into ischemic hindlimb muscles. At postoperative day 28, pericyte transplantation augmented blood flow recovery in wild-type mice (79.3 ± 5% vs. 61.9 ± 5%; P = 0.04), but not in T2DM mice (48.6% vs. 46.3 ± 5%; P = 0.51). Pericyte transplantation augmented collateral artery enlargement in wild-type (26.7 ± 2 μm vs. 22.3 ± 1 μm, P = 0.03), but not T2DM mice (20.4 ± 1.4 μm vs. 18.5 ± 1.2 μm, P = 0.14). Pericyte incorporation into collateral arteries was higher in wild-type than in T2DM mice ( P = 0.002). Unexpectedly, pericytes differentiated into Schwann cells in vivo. In vitro, Insulin increased Nox2 expression and decreased tubular formation capacity in human pericytes. These insulin-induced effects were reversed by N-acetylcysteine antioxidant treatment. In conclusion, T2DM impairs the ability of pericytes to augment neovascularization via decreased collateral artery enlargement and impaired engraftment into collateral arteries, potentially via hyperinsulinemia-induced oxidant stress. While pericytes show promise as a unique form of stem cell therapy to increase postischemic neovascularization, characterizing the molecular mechanisms by which T2DM impairs their function is essential to achieve their therapeutic potential.

Angiogenesis inhibition is a promising approach for treating metastatic colorectal cancer (mCRC). Recent evidences support the seemingly counterintuitive ability of certain antiangiogenic drugs to promote normalization of residual tumor vessels with important clinical implications. Lenalidomide is an oral drug with immune-modulatory and anti-angiogenic activity against selected hematologic malignancies but as yet little is known regarding its effectiveness for solid tumors. The aim of this study was to determine whether lenalidomide can normalize colorectal cancer neo-vessels in vivo, thus reducing tumor hypoxia and improving the benefit of chemotherapy.

We set up a tumorgraft model with NOD/SCID mice implanted with a patient-derived colorectal cancer liver metastasis. The mice were treated with oral lenalidomide (50 mg/Kg/day for 28 days), intraperitoneal 5-fluorouracil (5FU) (20 mg/Kg twice weekly for 3 weeks), combination (combo) of lenalidomide and 5FU or irrelevant vehicle. We assessed tumor vessel density (CD146), pericyte coverage (NG2; alphaSMA), in vivo perfusion capability of residual vessels (lectin distribution essay), hypoxic areas (HP2-100 Hypoxyprobe) and antitumor activity in vivo and in vitro.

Treatment with lenalidomide reduced tumor vessel density (p = 0.0001) and enhanced mature pericyte coverage of residual vessels (p = 0.002). Perfusion capability of tumor vessels was enhanced in mice treated with lenalidomide compared to controls (p = 0.004). Accordingly, lenalidomide reduced hypoxic tumor areas (p = 0.002) and enhanced the antitumor activity of 5FU in vivo. The combo treatment delayed tumor growth (p = 0.01) and significantly reduced the Ki67 index (p = 0.0002). Lenalidomide alone did not demonstrate antitumor activity compared to untreated controls in vivo or against 4 different mCRC cell lines in vitro.

We provide the first evidence of tumor vessel normalization and hypoxia reduction induced by lenalidomide in mCRC in vivo. This effect, seemingly counterintuitive for an antiangiogenic compound, translates into indirect antitumor activity thus enhancing the therapeutic index of chemotherapy. Our findings suggest that further research should be carried out on synergism between lenalidomide and conventional therapies for treating solid tumors that might benefit from tumor vasculature normalization.

The blood-brain barrier (BBB) dysfunction is an initial event of various neuroinflammatory diseases. However, the absence of reliable markers and mechanisms for BBB damage greatly limits the diagnosis and treatment of neuroinflammatory diseases. Soluble CD146 (sCD146) is mainly derived from vascular endothelial cells (ECs) and highly elevated in inflammatory settings. Based on a small cohort, our previous study showed that sCD146 is elevated in the cerebrospinal fluid (CSF) of multiple sclerosis (MS), which is accompanied with BBB damage. Nevertheless, whether sCD146 monitors and regulates the BBB dysfunction remains unknown. Methods: Coupled serum and CSF samples from patients with or without neuroinflammatory diseases were collected via multicenter collaborations. sCD146 was measured by sandwich ELISA using anti-CD146 antibodies AA1 and AA98, both of which were generated in our laboratory. The correlations between sCD146 and other clinical parameters or inflammatory factors were analyzed by Spearman's correlation coefficient analysis. The role of sCD146 on BBB function was examined in an in vitro BBB model. Results: Between July 20, 2011, and February 31, 2017, we collected coupled serum and CSF samples from 823 patients, of which 562 (68.3%) had neuroinflammatory diseases, 44 (5.3%) had remitting MS, and 217 (26.4%) had non-inflammatory neurological diseases (NIND). We found that sCD146 in CSF, but not in serum, is abnormally elevated in neuroinflammatory diseases (37.3 ± 13.3 ng/mL) compared with NIND (4.7 ± 2.9 ng/mL) and remitting MS (4.6 ± 3.5 ng/mL). Abnormally elevated CSF sCD146 is significantly correlated with the hyperpermeability-related clinical parameters of BBB and neuroinflammation-related factors. Moreover, CSF sCD146 shows higher sensitivity and specificity for evaluating BBB damage. Using an in vitro BBB model, we found that sCD146 impairs BBB function by promoting BBB permeability via an association with integrin αvβ1. Blocking integrin αvβ1 significantly attenuates sCD146-induced hyperpermeability of the BBB. Conclusion: Our study provides convincing evidence that CSF sCD146 is a sensitive marker of BBB damage and neuroinflammation. Furthermore, sCD146 is actively involved in BBB dysfunction.

Dysregulated expression of the cell surface protein, CD146, has been implicated in various types of cancer in humans, including in lung cancer. The present study aimed to clarify the mechanism underlying abnormal CD146 expression in human pulmonary large cell neuroendocrine carcinoma (LCNEC) cell lines (NCI-H460 and NCI-H810). The functions of CD146 were investigated by measuring cell migration and viability following CD146 knockdown or overexpression via small interference RNA and plasmid transfection. The findings demonstrated that decreased protein expression of CD146 could inhibit migration and viability in LCNEC cells. Furthermore, CD146 was determined to influence the expression of epithelial-mesenchymal transition markers (epithelial cadherin, vimentin and Snail) and promoted AKT phosphorylation. The present results imply CD146 may function in the migration and proliferation of pulmonary LCNEC cells.

BACKGROUND

Endothelial cells (ECs) are an important component of the blood coagulation system because it maintains blood fluid. Because in patients with venous thromboembolic disease (VTD) a thrombophilic condition is not found sometimes, we investigated if endothelial colony-forming cells (ECFCs) from these patients have biological and functional abnormalities.

METHODS

Human mononuclear cells (MNCs) were obtained from peripheral blood from patients with VTD and controls to obtain ECFCs. These cells were assayed for their immunophenotype and electron microscopy characteristics and their ability to form capillary-like structures and to produce pro-inflammatory and pro-angiogenic cytokines and reactive oxygen species (ROS).

RESULTS

ECFCs appeared at 7 and 21 days of culture in VTD patients and controls, respectively. ECFCs increased 8-fold in patients and emerged 1 week earlier. No differences in the size of the colonies of ECFCs were found. Numbers and time of appearance of ECFCs was different between groups. ECFC-derived ECs (ECFC-ECs) of both groups expressed CD31, CD34, CD146, and CD-309 but none expressed CD45, CD14, or CD90. Interest CD34 was highly expressed in ECFC-ECs from patients. In both groups, ECFC-ECs showed similar capacity to form capillary-like structures but ECFC-ECs from patients had significant abnormalities in the mitochondrial membrane. We found a significant increase in ROS production in ECFC-ECs from patients. There were significant differences in cytokine profiles between VTD patients and controls.

CONCLUSIONS

We found a dysfunctional state in ECFC from VTD patients resembling some characteristics of dysfunctional ECs. These findings may help to understand some pathophysiological aspects of VTD.

CD146 (MCAM, MUC18, S-Endo1) is a transmembrane glycoprotein belonging to both CAM and mucin families. It exists as different splice variants and is cleaved from the membrane by metalloproteases to generate a soluble form. CD146 is expressed by numerous cancer cells as well as being one of the numerous proteins expressed by the vascular endothelium. It has also been identified on smooth muscle cells, pericytes, and some immune cells. This protein was initially described as an actor involved in tumor growth and metastatic dissemination processes. Some recent works highlighted the role of CD146 in angiogenesis. Interestingly, this knowledge allowed the development of therapeutic and diagnostic tools specifically targeting the different CD146 variants. The first anti-CD146 antibody designed to study the function of this molecule, MUC18, was described by the Pr. J.P. Jonhson in 1987. In this review, we will discuss the 30 following years of research focused on the detection, study, and blocking of this protein in physiological and pathological processes.

BACKGROUND

CD146 is a cell adhesion molecule belonging to the immunoglobulin superfamily. It plays a critical role in cell proliferation and activation, and contributes to inflammatory reactions. We studied the association between CD146 and renal tubular epithelial cells (RTECs) under normal (NG) and high glucose (HG) to understand its role in the pathogenesis of diabetic nephropathy.

METHODS

Human proximal tubular epithelial (HK-2) cells were cultured in different glucose media (NG5mM, HG15mM and HG40mM). CD146 mRNA expression was determined by RT-PCR. CD146 protein expression was assessed by fluorescence immunoassay, enzymatic immunohistochemistry and flow cytometry. A soluble form of CD146 was evaluated by ELISA.

RESULTS

We found weak expression of CD146 mRNA and protein in the NG group. Faint labeling of CD146 protein was detected mainly at the cell-cell junction, with sparse staining in cytoplasm and nucleus. In the HG group, both membrane and intracellular expression of CD146 were significantly increased by high glucose, while osmotic pressure had no effect on CD146 expression in cultured HK-2 cells. A soluble form of CD146, sCD146, was detected in the supernatant of HK-2 cells in the NG group which was elevated in the HG40mM group (29.33 -/+ 1.22 ng/mL vs. 61.87 -/+ 9.10 ng/mL, p<0.05).

CONCLUSIONS

CD146 might be a novel marker for RTECs, correlating with cell proliferation and cell-cell conjunctions. High glucose up-regulates both CD146 and sCD146 expression. CD146 may be a biomarker of activated RTECs, and sCD146 may have clinical value for determining severity and prognosis of diabetic nephropathy.

In this study, mesenchymal stem cells were isolated from rat adipose tissue (AD-MSCs) to characterize and differentiate them into endothelial-like cells. AD-MSCs were isolated by mechanical and enzymatic treatments, and their identity was verified by colony-forming units (CFU) test and by differentiation into cells of mesodermal lineages. The endothelial differentiation was induced by plating another aliquot of cells in EGM-2 medium, enriched with specific endothelial growth factors. Five subcultures were performed. The expression of stemness genes (OCT4, SOX2 and NANOG) was investigated. The presence of CD90 and the absence of the CD45 were evaluated by flow cytometry. The endothelial-like cells were characterized by the evaluation of morphological changes and gene expression analysis for endothelial markers (CD31, CD144, CD146). Characterization of AD-MSCs showed their ability to form clones, to differentiate in vitro and the OCT-4, SOX-2, NANOG genes expression. Immunophenotypic characterization showed the CD90 presence and the CD45 absence. The endothelial-like cells showed morphological changes, the expression of CD31, CD144, CD146 genes and the presence of CD31 membrane receptor. Matrigel assay showed their ability to form network and vessels-like structures. This study lays the foundations for future evaluation of the potential AD-MSCs pro-angiogenic and therapeutic role.

Acute graft-versus-host disease (aGVHD) remains a major complication of allogeneic hematopoietic cell transplantation (HCT). CD146 and CCR5 are proteins that mark activated T helper 17 (Th17) cells. The Th17 cell phenotype is promoted by the interaction of the receptor ICOS on T cells with ICOS ligand (ICOSL) on dendritic cells (DCs). We performed multiparametric flow cytometry in a cohort of 156 HCT recipients and conducted experiments with aGVHD murine models to understand the role of ICOSL⁺ DCs. We observed an increased frequency of ICOSL⁺ plasmacytoid DCs, correlating with CD146⁺CCR5⁺ T cell frequencies, in the 64 HCT recipients with gastrointestinal aGVHD. In murine models, donor bone marrow cells from ICOSL-deficient mice compared to those from wild-type mice reduced aGVHD-related mortality. Reduced aGVHD resulted from lower intestinal infiltration of pDCs and pathogenic Th17 cells. We transplanted activated human ICOSL⁺ pDCs along with human peripheral blood mononuclear cells into immunocompromised mice and observed infiltration of intestinal CD146⁺CCR5⁺ T cells. We found that prophylactic administration of a dual human ICOS/CD28 antagonist (ALPN-101) prevented aGVHD in this model better than did the clinically approved belatacept (CTLA-4-Fc), which binds CD80 (B7-1) and CD86 (B7-2) and interferes with the CD28 T cell costimulatory pathway. When started at onset of aGVHD signs, ALPN-101 treatment alleviated symptoms of ongoing aGVHD and improved survival while preserving antitumoral cytotoxicity. Our data identified ICOSL⁺-pDCs as an aGVHD biomarker and suggest that coinhibition of the ICOSL/ICOS and B7/CD28 axes with one biologic drug may represent a therapeutic opportunity to prevent or treat aGVHD.

BACKGROUND

Circulating endothelial cells (CECs) are widely reported as a promising biomarker of endothelial damage/dysfunction in coronary artery disease (CAD). The two popular methods of CEC quantification include the use of immunomagnetic beads separation (IB) and flow cytometry analysis (FC); however, they suffer from two main shortcomings that affect their diagnostic and prognostic responses: non-specific bindings of magnetic beads to non-target cells and a high degree of variability in rare cell identification, respectively. We designed a microfluidic chip with spatially staggered micropillars for the efficient harvesting of CECs with intact cellular morphology in an attempt to revisit the diagnostic goal of CEC counts in CAD patients with angina pectoris.

METHODS

A label-free microfluidic assay that involved an in-situ enumeration and immunofluorescent identification (DAPI+/CD146+/VEGFR1+/CD45-) of CECs was carried out to assess the CEC count in human peripheral blood samples. A total of 55 CAD patients with angina pectoris [16 with chronic stable angina (CSA) and 39 with unstable angina (UA)], together with 15 heathy controls (HCs) were enrolled in the study.

RESULTS

CEC counts are significantly higher in both CSA and UA groups compared to the HC group [respective medians of 6.9, 10.0 and 1.5 cells/ml (p < 0.01)]. Further, a significant elevation of CEC count was observed in the three UA subgroups [low risk (5.3) vs. intermediate risk (10.8) vs. high risk (18.0) cells/ml, p < 0.001) classified in accordance to the TIMI NSTEMI/UA risk score system. From the receiver-operating characteristic curve analysis, the AUCs for distinguishing CSA and UA from HC were 0.867 and 0.938, respectively. The corresponding sensitivities were 87.5% and 84.6% and the specificities were 66.7% and 86.7%, respectively.

CONCLUSIONS

Our microfluidic assay system is efficient and stable for CEC capture and enumeration. The results showed that the CEC count has the potential to be a promising clinical biomarker for the assessment of endothelial damage/dysfunction in CAD patients with angina pectoris.

BACKGROUND

Human periodontal ligament stem cells (hPDLSCs) have been shown to be a reliable source of mesenchymal stem cells (MSCs). On the other hand, rabbits have been commonly used in preclinical trials for musculoskeletal research. However, there is a lack of sufficient data on using rabbit periodontal ligament stem cells (rPDLSCs) for regenerative dentistry. This study, for the first time, comprehensively compared rPDLSCs against hPDLSCs in terms of clonogenicity, growth potential, multi-differential capacity and surface antigens.

METHODS

Periodontal ligament (PDL) was obtained from the rabbit and human teeth. rPDL and hPDL cells were isolated from PDL using enzymatic digestion method. After culturing for 2 weeks, the cells were first analyzed microscopically. STRO-1+CD146+ PDLSCs were then sorted from PDL cells by fluorescence-activated cell sorting (FACS) followed by examination of CD34, CD45, CD90, vimentin and desmin markers. The cells were also evaluated by immunohistocytochemical and multi-differentiation potential tests. The clonogenicity and growth of PDL cells were analyzed by Independent T test and 2-way repeated measures ANOVA respectively.

RESULTS

rPDL cells were broader and less elongated as compared to hPDL cells. STRO-1+CD146+ hPDLSCs were isolated from hPDL cells but not from the rPDL cells. Therefore, heterogeneous population of rabbit and human PDL cells were subsequently used for latter comparative studies. FACS analysis and immunohistocytochemistry revealed that rPDL cells were partially positive for STRO-1 as compared to hPDL cells. Furthermore, both rPDL cells and hPDL cells were positive for CD146, CD90, vimentin, and desmin, while negative for CD34 and CD45. No difference in clonogenicity between rPDL and hPDL cells was found (p>> 0.05). The proliferative potential of rPDL cells displayed significantly slower growth as compared to hPDL cells (p < 0.05). Osteogenic, adipogenic, and chondrogenic differentiation potential was comparatively less in rPDL cells than that of hPDL cells, but the neurogenic differential potential was similar.

CONCLUSIONS

Although rPDL cells manifested variable differences in expression of stem cell markers and multi-differential potential as compared to hPDL cells, they demonstrated the attributes of stemness. Further studies are also required to validate if the regenerative potential of rPDL cells is similar to rPDLSCs.

BACKGROUND

Mesenchymal stem cells (MSCs) are multipotent cells capable of self-renewal and multilineage differentiation. Their multipotential capacity and immunomodulatory properties have led to an increasing interest in their biological properties and therapeutic applications. Currently, the definition of MSCs relies on a combination of phenotypic, morphological and functional characteristics which are typically evaluated upon in vitro expansion, a process that may ultimately lead to modulation of the immunophenotypic, functional and/or genetic features of these cells. Therefore, at present there is great interest in providing markers and phenotypes for direct in vivo and ex vivo identification and isolation of MSCs.

METHODS

Multiparameter flow cytometry immunophenotypic studies were performed on 65 bone marrow (BM) samples for characterization of CD13(high) CD105(+) CD45(-) cells. Isolation and expansion of these cells was performed in a subset of samples in parallel to the expansion of MSCs from mononuclear cells following currently established procedures. The protein expression profile of these cells was further assessed on (paired) primary and in vitro expanded BM MSCs, and their adipogenic, chondrogenic and osteogenic differentiation potential was also determined.

RESULTS

Our results show that the CD13(high) CD105(+) CD45(-) immunophenotype defines a minor subset of cells that are systematically present ex vivo in normal/reactive BM (n = 65) and that display immunophenotypic features, plastic adherence ability, and osteogenic, adipogenic and chondrogenic differentiation capacities fully compatible with those of MSCs. In addition, we also show that in vitro expansion of these cells modulates their immunophenotypic characteristics, including changes in the expression of markers currently used for the definition of MSCs, such as CD105, CD146 and HLA-DR.

CONCLUSIONS

BM MSCs can be identified ex vivo in normal/reactive BM, based on a robust CD13(high) CD105(+) and CD45(-) immunophenotypic profile. Furthermore, in vitro expansion of these cells is associated with significant changes in the immunophenotypic profile of MSCs.

Scleraxis is a basic helix-loop-helix transcription factor that plays a central role in promoting tenocyte proliferation and matrix synthesis during embryonic tendon development. However, the role of scleraxis in the growth and adaptation of adult tendons is not known. We hypothesized that scleraxis is required for tendon growth in response to mechanical loading and that scleraxis promotes the specification of progenitor cells into tenocytes. We conditionally deleted scleraxis in adult mice using a tamoxifen-inducible Cre-recombinase expressed from the Rosa26 locus (ScxΔ) and then induced tendon growth in Scx+ and ScxΔ adult mice via plantaris tendon mechanical overload. Compared with the WT Scx+ group, ScxΔ mice demonstrated blunted tendon growth. Transcriptional and proteomic analyses revealed significant reductions in cell proliferation, protein synthesis, and extracellular matrix genes and proteins. Our results indicate that scleraxis is required for mechanically stimulated adult tendon growth by causing the commitment of CD146+ pericytes into the tenogenic lineage and by promoting the initial expansion of newly committed tenocytes and the production of extracellular matrix proteins.

Keywords: Cell Biology; Extracellular matrix; Pericytes; Stem cells.

BACKGROUND

Hyperoxia is a frequently employed therapy for prematurely born infants, induces lung injury and contributes to development of bronchopulmonary dysplasia (BPD). BPD is characterized by decreased cellular proliferation, cellular migration, and failure of injury repair systems. Actin binding proteins (ABPs) such as VASP, cofilin1, and profilin1 regulate cell proliferation and migration via modulation of actin dynamics. Lung mesenchymal stem cells (L-MSCs) initiate repair processes by proliferating, migrating, and localizing to sites of injury. These processes have not been extensively explored in hyperoxia induced lung injury and repair.

METHODS

ABPs and CD146+ L-MSCs were analyzed by immunofluorescence in human lung autopsy tissues from infants with and without BPD and by western blot in lung tissue homogenates obtained from our murine model of newborn hyperoxic lung injury.

RESULTS

Decreased F-actin content, ratio of VASPpS157/VASPpS239, and profilin 1 expression were observed in human lung tissues but this same pattern was not observed in lungs from hyperoxia-exposed newborn mice. Increases in cofilin1 expression were observed in both human and mouse tissues at 7d indicating a dysregulation in actin dynamics which may be related to altered growth. CD146 levels were elevated in human and newborn mice tissues (7d).

CONCLUSIONS

Altered phosphorylation of VASP and expression of profilin 1 and cofilin 1 in human tissues indicate that the pathophysiology of BPD involves dysregulation of actin binding proteins. Lack of similar changes in a mouse model of hyperoxia exposure imply that disruption in actin binding protein expression may be linked to interventions or morbidities other than hyperoxia alone.