Circulating endothelial progenitor cells in periodontitis.

BACKGROUND Several biologically plausible mechanisms have been proposed to mediate the association between periodontitis and atherosclerotic vascular disease (AVD), including adverse effects on vascular endothelial function. Circulating endothelial progenitor cells (cEPCs) are known to contribute to vascular repair, but limited data are available regarding the relationship between cEPC levels and periodontitis. The aims of this cross-sectional study are to investigate the levels of hemangioblastic and monocytic cEPCs in patients with periodontitis and periodontally healthy controls and to associate cEPC levels with the extent and severity of periodontitis. METHODS A total of 112 individuals (56 patients with periodontitis and 56 periodontally healthy controls, aged 26 to 65 years; mean age: 43 years) were enrolled. All participants underwent a full-mouth periodontal examination and provided a blood sample. Hemangioblastic cEPCs were assessed using flow cytometry, and monocytic cEPCs were identified using immunohistochemistry in cultured peripheral blood mononuclear cells. cEPC levels were analyzed in the entire sample, as well as in a subset of 50 pairs of patients with periodontitis/periodontally healthy controls, matched with respect to age, sex, and menstrual cycle. RESULTS Levels of hemangioblastic cEPCs were approximately 2.3-fold higher in patients with periodontitis than periodontally healthy controls, after adjustments for age, sex, physical activity, systolic blood pressure, and body mass index (P = 0.001). A non-significant trend for higher levels of monocytic cEPCs in periodontitis was also observed. The levels of hemangioblastic cEPCs were positively associated with the extent of bleeding on probing, probing depth, and clinical attachment loss. Hemangioblastic and monocytic cEPC levels were not correlated (Spearman correlation coefficient 0.03, P = 0.77), suggesting that they represent independent populations of progenitor cells. CONCLUSION These findings further support the notion that oral infections have extraoral effects and document that periodontitis is associated with a mobilization of EPCs from the bone marrow, apparently in response to systemic inflammation and endothelial injury.

The secretome of endothelial progenitor cells promotes brain endothelial cell activity through PI3-kinase and MAP-kinase

BACKGROUND Angiogenesis and vascular remodelling are crucial events in tissue repair mechanisms promoted by cell transplantation. Current evidence underscores the importance of the soluble factors secreted by stem cells in tissue regeneration. In the present study we investigated the effects of paracrine factors derived from cultured endothelial progenitor cells (EPC) on rat brain endothelial cell properties and addressed the signaling pathways involved. METHODS Endothelial cells derived from rat brain (rBCEC4) were incubated with EPC-derived conditioned medium (EPC-CM). The angiogenic response of rBCEC4 to EPC-CM was assessed as effect on cell number, migration and tubular network formation. In addition, we have compared the outcome of the in vitro experiments with the effects on capillary sprouting from rat aortic rings. The specific PI3K/AKT inhibitor LY294002 and the MEK/ERK inhibitor PD98059 were used to study the involvement of these two signaling pathways in the transduction of the angiogenic effects of EPC-CM. RESULTS Viable cell number, migration and tubule network formation were significantly augmented upon incubation with EPC-CM. Similar findings were observed for aortic ring outgrowth with significantly longer sprouts. The EPC-CM-induced activities were significantly reduced by the blockage of the PI3K/AKT and MEK/ERK signaling pathways. Similarly to the outcome of the rBCEC4 experiments, inhibition of the PI3K/AKT and MEK/ERK pathways significantly interfered with capillary sprouting induced by EPC-CM. CONCLUSION The present study demonstrates that EPC-derived paracrine factors substantially promote the angiogenic response of brain microvascular endothelial cells. In addition, our findings identified the PI3K/AKT and MEK/ERK pathways to play a central role in mediating these effects.

Endothelial progenitor cells as a biological marker of peripheral artery disease.

The role of endothelial progenitor cells (EPCs) in peripheral artery disease (PAD) remains unclear. We hypothesized that EPC mobilization and function play a central role in the development of endothelial dysfunction and directly influence the degree of atherosclerotic burden in peripheral artery vessels. The number of circulating EPCs, defined as CD34(+)/KDR(+) cells, were assessed by flow cytometry in 91 subjects classified according to a predefined sample size of 31 non-diabetic PAD patients, 30 diabetic PAD patients, and 30 healthy volunteers. Both PAD groups had undergone endovascular treatment in the past. As a functional parameter, EPC colony-forming units were determined ex vivo. Apart from a broad laboratory analysis, a series of clinical measures using the ankle-brachial index (ABI), flow-mediated dilatation (FMD) and carotid intima-media thickness (cIMT) were investigated. A significant reduction of EPC counts and proliferation indices in both PAD groups compared to healthy subjects were observed. Low EPC number and pathological findings in the clinical assessment were strongly correlated to the group allocation. Multivariate statistical analysis revealed these findings to be independent predictors of disease appearance. Linear regression analysis showed the ABI to be a predictor of circulating EPC number (p=0.02). Moreover, the functionality of EPCs was correlated by linear regression (p=0.017) to cIMT. The influence of diabetes mellitus on EPCs in our study has to be considered marginal in already disease-affected patients. This study demonstrated that EPCs could predict the prevalence and severity of symptomatic PAD, with ABI as the determinant of the state of EPC populations in disease-affected groups.

Endothelial Progenitor Cells in Breast Cancer

Editorial

Directing migration of endothelial progenitor cells with applied DC electric fields

Naturally-occurring, endogenous electric fields (EFs) have been detected at skin wounds, damaged tissue sites and vasculature. Applied EFs guide migration of many types of cells, including endothelial cells to migrate directionally. Homing of endothelial progenitor cells (EPCs) to an injury site is important for repair of vasculature and also for angiogenesis. However, it has not been reported whether EPCs respond to applied EFs. Aiming to explore the possibility to use electric stimulation to regulate the progenitor cells and angiogenesis, we tested the effects of direct-current (DC) EFs on EPCs. We first used immunofluorescence to confirm the expression of endothelial progenitor markers in three lines of EPCs. We then cultured the progenitor cells in EFs. Using time-lapse video microscopy, we demonstrated that an applied DC EF directs migration of the EPCs toward the cathode. The progenitor cells also align and elongate in an EF. Inhibition of vascular endothelial growth factor (VEGF) receptor signaling completely abolished the EF-induced directional migration of the progenitor cells. We conclude that EFs are an effective signal that guides EPC migration through VEGF receptor signaling in vitro. Applied EFs may be used to control behaviors of EPCs in tissue engineering, in homing of EPCs to wounds and to an injury site in the vasculature.

Evaluation of circulating endothelial progenitor cells by multicolor flow cytometry in chronic kidney disease patients

Endothelial dysfunction and impaired endothelial regenerative capacity play a key role in the pathogenesis of cardiovascular disease, which is one of the major causes of mortality in chronic kidney disease (CKD) patients. Circulating endothelial cells (CEC) may be an indicator of vascular damage, while circulating endothelial progenitor cells (EPC) may be a biomarker for vascular repair. However, the simultaneously evaluation of CEC and EPC circulating levels and its relation were not previously examined in CKD population. A blood sample (18ml) of healthy subjects (n=10), early CKD (n=10) and advanced CKD patients (n=10) was used for the isolation of early and late EPCs, CECs, and hematopoietic cells, identified by flow cytometry (BD FACSCanto™ II system) using a combination of fluorochrome-conjugated primary antibodies: CD31-PE, CD45-APC Cy7, CD34-FITC, CD117-PerCp Cy5.5, CD133-APC, CD146-Pacific Blue, and CD309-PECy7. Exclusion of dead cells was done according to a fixable viability dye staining. This eightcolor staining flow cytometry optimized protocol allowed us to accurate simultaneously identify EPCs, CECs and hematopoietic cells. In addition, it was also possible to distinguish the two subpopulations of EPCs, early and late EPCs subpopulation, by CD45intCD31+CD34+CD117-CD133+CD309-CD146- and CD45intCD31+CD34+CD117-CD133-CD309+CD146- multiple labeling, respectively. Moreover, the identification of CECs and hematopoietic cells was performed by CD45-CD31+CD34-/lowCD117-CD133-CD309-CD146+ and CD34+CD117+, respectively. The levels of CECs were non-significantly increased in early CKD (312.06 ± 91.34) and advanced CKD patients (191.43±49.86) in comparison with control group (103.23±24.13). By contrast, the levels of circulating early EPCs were significantly reduced in advanced CKD population (17.03±3.23) in comparison with early CKD (32.31±4.97), p=0.04 and control group (36.25 ± 6.16), p=0.03. In addition the levels of late EPCs were significantly reduced in both advanced (6.60±1.89), p=0.01, and early CKD groups (8.42±2.58), p=0.01 compared with control group (91.54±29.06). These results were accompanied by a dramatically reduction in the recruitment, differentiation and regenerative capacity indexes in CKD population. Taken together, these results suggest an imbalance in the process of endothelial repairment in CKD population, and further propose that the indexes of recruitment, differentiation and regenerative capacity of EPCs, may help to select the patients to benefit from guiding intervention strategies to improve cardiovascular health by inducing vascular protection.

Mécanismes de défense immunitaire innée impliqués dans l’hépatite aiguë induite par le virus de l’hépatite murine de type 3

Le virus de l’hépatite murine de type 3 (MHV3) est un excellent modèle animal pour l’étude des différents désordres immunologiques lors d’infections virales. L’hépatite aiguë fulminante induite par ce virus chez la souris susceptible C57BL/6 se caractérise par la présence de plusieurs foyers nécrotiques et inflammatoires dans le foie associée à une immunodéficience en lymphocytes B et T, tuant les souris entre 3 et 5 jours post-infection. L’évolution rapide de cette maladie virale suggère un débalancement dans les mécanismes de l’immunité naturelle sous le contrôle des cellules NK et NK-T et un bris de l’équilibre entre la tolérance hépatique et la réponse inflammatoire. Afin d’élucider les rôles respectifs des différents mécanismes de la défense innée impliqués dans le développement de l’hépatite aiguë, des infections in vivo ont été réalisées chez des souris C57BL/6 avec la souche pathogène L2-MHV3 ou avec des variants du virus MHV3. Ces derniers possèdent des tropismes différents pour les cellules endothéliales sinusoïdales hépatiques et les cellules de Kupffer, tels que les virus faiblement atténué 51.6-MHV3, fortement atténué CL12-MHV3 et non pathogène YAC-MHV3. Ces études in vivo ont montré une diminution des cellules NK spléniques et myéloïdes suite à une infection avec le virus MHV3. Cette chute en cellules NK spléniques reflète un recrutement de ces cellules au niveau du foie. Par contre, les cellules NK se sont avérées permissives à la réplication virale entraînant un processus d’apoptose suite à la formation de syncétia induits par le virus. Les niveaux de recrutement et d’apoptose des cellules NK et NK-T dans le foie reflètent la pathogénicité des variants MHV3 durant les trois premiers jours de l’infection virale bien que les cellules NK recrutées au niveau du foie maintiennent leur activité cytotoxique. L’ajout des IL-12 et IL-18, qui sont normalement diminués lors de l’hépatite aiguë, provoque une production synergique d’IFN-g par les cellules NK, résultant d’une interaction entre l’activation de la voie p38 MAPK et la réplication virale. Par ailleurs, le récepteur viral CEACAM1a (carcinoembryonic antigen cell adhesion molecule 1a) serait essentiel à cette synergie, mais exercerait aussi une action inhibitrice dans la production de l’IFN-g. D’autre part, les niveaux de production des cytokines immunosuppressives IL-10, TGF-b et PGE2, impliquées dans la tolérance hépatique et particulièrement produites par les cellules de Kupffer et les cellules endothéliales sinusoïdales, sont en relation inverse avec le degré de pathogénicité des variants du virus MHV3. Finalement, le virus pathogène L2-MHV3 déclenche la production de cytokines inflammatoires par les macrophages, tels que l’IL-6 et le TNF-a. L’induction de ces cytokines par les macrophages serait indépendante de la présence de la molécule CEACAM1a. Cette stimulation est plutôt reliée à la fixation des particules virales sur des récepteurs TLR2, en association avec les régions riches en héparanes sulfates. Tous ces résultats mettent en évidence de nouveaux mécanismes par lesquels le virus MHV3 peut diminuer l’efficacité des mécanismes de l’immunité naturelle sous le contrôle des cellules NK et NK-T intrahépatiques, suite à une stimulation de l’inflammation résultant du bris de la tolérance hépatique.

Expression of inducible nitric oxide synthase in bovine corneal endothelial cells and keratocytes in vitro after lipopolysaccharide and cytokines stimulation.

PURPOSE: To determine whether bovine corneal endothelial (BCE) cells and keratocytes express the inducible form of nitric oxide synthase (NOS) after exposure to cytokines and lipopolysaccharide (LPS), and to study the regulation of NOS by growth factors. METHODS: Cultures of bovine corneal endothelial cells and keratocytes were exposed to increasing concentrations of LPS, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha). At selected intervals after exposure, nitrite levels in the supernatants were evaluated by the Griess reaction. Total RNA was extracted from the cell cultures, and messenger RNA levels for inducible NOS (NOS-2) were measured by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Exposure of BCE cells and keratocytes to LPS and IFN-gamma resulted in an increase of nitrite levels that was potentiate by the addition of TNF-alpha. Analysis by RT-PCR demonstrated that nitrite release was correlated to the expression of NOS-2 messenger RNA in BCE cells and keratocytes. Stereoselective inhibitors of NOS and cycloheximide inhibited LPS-IFN-gamma-induced nitrite release in both cells, whereas transforming growth factor-beta (TGF-beta) slightly potentiated it. Fibroblast growth factor-2 (FGF-2) inhibited LPS-IFN-gamma-induced nitrite release and NOS-2 messenger RNA accumulation in keratocytes but not in BCE cells. CONCLUSIONS: The results demonstrate that in vitro activation of keratocytes and BCE cells by LPS and cytokines induces NOS-2 expression and release of large amounts of NO. The high amounts of NO could be involved in inflammatory corneal diseases in vivo.

Endothelial cells as key determinants of the tumor microenvironment: interaction with tumor cells, extracellular matrix and immune killer cells.

Besides tumor cells, the tumor microenvironment harbors a variety of host-derived cells, such as endothelial cells, fibroblasts, innate and adaptive immune cells. It is a complex and highly dynamic environment, providing very important cues to tumor development and progression. Tumor-associated endothelial cells play a key role in this process. On the one hand, they form tumor-associated (angiogenic) vessels through sprouting from locally preexisting vessels or recruitment of bone marrow-derived endothelial progenitor cells, to provide nutritional support to the growing tumor. On the other hand, they are the interface between circulating blood cells, tumor cells and the extracellular matrix, thereby playing a central role in controlling leukocyte recruitment, tumor cell behavior and metastasis formation. Hypoxia is a critical parameter modulating the tumor microenvironment and endothelial/tumor cell interactions. Under hypoxic stress, tumor cells produce factors that promote tumor angiogenesis, tumor cell motility and metastasis. Among these factors, VEGF, a main angiogenesis modulator, can also play a critical role in the control of immune tolerance. This review discusses some aspects of the role of endothelial cells within tumor microenvironment and emphasizes their interaction with tumor cells, the extracellular matrix and with immune killer cells. We will also address the role played by circulating endothelial progenitor cells and illustrate their features and mechanism of recruitment to the tumor microenvironment and their role in tumor angiogenesis.

Bone marrow-derived cells are implicated as a source of lymphatic endothelial progenitors in human breast cancer.

Bone marrow-derived endothelial progenitor cells (EPCs) infiltrate into sites of neovascularization in adult tissues and mature into functional blood endothelial cells (BECs) during a process called vasculogenesis. Human marrow-derived EPCs have recently been reported to display a mixed myeloid and lymphatic endothelial cell (LEC) phenotype during inflammation-induced angiogenesis; however, their role in cancer remains poorly understood. We report the in vitro differentiation of human cord blood CD133(+)CD34(+) progenitors into podoplanin(+) cells expressing both myeloid markers (CD11b, CD14) and the canonical LEC markers vascular endothelium growth factor receptor 3 (VEGFR-3), lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), and prospero homeobox 1 (PROX-1). These podoplanin(+) cells displayed sprouting behavior comparable to that of LECs in vitro and a dual hemangiogenic and lymphangiogenic activity in vivo in an endothelial cell sprouting assay and corneal vascularization assay, respectively. Furthermore, these cells expressed vascular endothelium growth factor (VEGF) family members A, -C, and -D. Thus, bone-marrow derived EPCs stimulate hemangiogenesis and lymphangiogenesis through their ability to differentiate into LECs and to produce angiogenic factors. Importantly, plasma from patients with breast cancer induced differentiation of CD34(+) cord blood progenitors into hemangiogenic and lymphangiogenic CD11b(+) myeloid cells, whereas plasma from healthy women did not have this effect. Consistent with these findings, circulating CD11b(+) cells from breast cancer patients, but not from healthy women, displayed a similar dual angiogenic activity. Taken together, our results show that marrow-derived EPCs become hemangiogenic and lymphangiogenic upon exposure to cancer plasma. These newly identified functions of bone-marrow derived EPCs are expected to influence the diagnosis and treatment of breast cancer.