Human Endothelial Colony Forming Cells Express Intracellular CD133 that Modulates their Vasculogenic Properties.

Stem cells at the origin of endothelial progenitor cells and in particular endothelial colony forming cells (ECFCs) subtype have been largely supposed to be positive for the CD133 antigen, even though no clear correlation has been established between its expression and function in ECFCs. We postulated that CD133 in ECFCs might be expressed intracellularly, and could participate to vasculogenic properties. ECFCs extracted from cord blood were used either fresh (n = 4) or frozen (n = 4), at culture days <30, to investigate the intracellular presence of CD133 by flow cytometry and confocal analysis. Comparison with HUVEC and HAEC mature endothelial cells was carried out. Then, CD133 was silenced in ECFCs using specific siRNA (siCD133-ECFCs) or scramble siRNA (siCtrl-ECFCs). siCD133-ECFCs (n = 12), siCtrl-ECFCs (n = 12) or PBS (n = 12) were injected in a hind-limb ischemia nude mouse model and vascularization was quantified at day 14 with H&E staining and immunohistochemistry for CD31. Results of flow cytometry and confocal microscopy evidenced the positivity of CD133 in ECFCs after permeabilization compared with not permeabilized ECFCs (p < 0.001) and mature endothelial cells (p < 0.03). In the model of mouse hind-limb ischemia, silencing of CD133 in ECFCs significantly abolished post-ischemic revascularization induced by siCtrl-ECFCs; indeed, a significant reduction in cutaneous blood flows (p = 0.03), capillary density (CD31) (p = 0.01) and myofiber regeneration (p = 0.04) was observed. Also, a significant necrosis (p = 0.02) was observed in mice receiving siCD133-ECFCs compared to those treated with siCtrl-ECFCs. In conclusion, our work describes for the first time the intracellular expression of the stemness marker CD133 in ECFCs. This feature could resume the discrepancies found in the literature concerning CD133 positivity and ontogeny in endothelial progenitors.

Interleukin-8 release by endothelial colony-forming cells isolated from idiopathic pulmonary fibrosis patients might contribute to their pathogenicity.

INTRODUCTION: Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by obliteration of alveolar architecture, resulting in declining lung function and ultimately death. Pathogenic mechanisms involve a concomitant accumulation of scar tissue together with myofibroblasts activation and a strong abnormal vascular remodeling. Endothelial progenitor cells (ECFC subtype) have been investigated in several human lung diseases as a potential actor in IPF. We previously demonstrated that ECFCs are down-regulated in IPF in contrast to healthy controls. We postulated here that ECFCs might behave as a liquid biopsy in IPF patients and that they exert modified vasculogenic properties. METHODS AND RESULTS: ECFCs isolated from controls and IPF patients expressed markers of the endothelial lineage and did not differ concerning adhesion, migration, and differentiation in vitro and in vivo. However, senescent and apoptotic states were increased in ECFCs from IPF patients as shown by galactosidase staining, p16 expression, and annexin-V staining. Furthermore, conditioned medium of IPF-ECFCs had increased level of interleukin-8 that induced migration of neutrophils in vitro and in vivo. In addition, an infiltration by neutrophils was shown in IPF lung biopsies and we found in a prospective clinical study that a high level of neutrophils in peripheral blood of IPF patients was associated to a poor prognosis. CONCLUSION: To conclude, our study shows that IPF patients have a senescent ECFC phenotype associated with an increased IL-8 secretion potential that might contribute to lung neutrophils invasion during IPF.

Treprostinil treatment decreases circulating platelet microvesicles and their procoagulant activity in pediatric pulmonary hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) results from pulmonary vascular disease and may eventually lead to right heart failure and death. Vasodilator therapy has greatly improved PAH prognosis. Circulating microvesicles are considered as surrogate markers of endothelial and hematopoietic cell activation. AIM: Thus, our purpose was to determine if MVs are upregulated in pediatric PAH such as reported in adult patients, and to analyze the impact of vasodilator therapies on MV count and function. PATIENTS: Population study consisted of 26 patients of median age 6.09 years, with Congenital Heart Disease (CHD) and elevated pulmonary vascular resistance (CHD-PAH) or idiopathic PAH (iPAH). RESULTS: Compared to healthy controls, all circulating MV subpopulations were found higher in untreated PAH patients. No significant differences of annexin-V+ total MV, endothelial, or leukocyte derived-MV counts were found between untreated patients and those receiving oral vasodilator therapies. Conversely, platelet MVs were significantly lower in the group treated with SC-treprostinil compared with both untreated PAH and oral therapy groups (P = 0.01), and exhibited a significant decrease of phospholipid procoagulant activity. Control samples treated in vitro with treprostinil at therapeutic concentrations showed as expected a significant decrease of platelet aggregation but also a reduced spontaneous MV generation. CONCLUSION: Our results suggest that treprostinil, besides vasodilation, might exert its beneficial effect through an inhibition of platelet activation, resulting in a decreased number and procoagulant activity of circulating MVs.

Endothelial Microparticles are Associated to Pathogenesis of Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by obliteration of alveolar architecture, resulting in declining lung function and ultimately death. Pathogenic mechanisms remain unclear but involve a concomitant accumulation of scar tissue together with myofibroblasts activation. Microparticles (MPs) have been investigated in several human lung diseases as possible pathogenic elements, prognosis markers and therapeutic targets. We postulated that levels and cellular origins of circulating MPs might serve as biomarkers in IPF patients and/or as active players of fibrogenesis. Flow cytometry analysis showed a higher level of Annexin-V positive endothelial and platelet MPs in 41 IPF patients compared to 22 healthy volunteers. Moreover, in IPF patients with a low diffusing capacity of the lung for carbon monoxide (DLCO<40%), endothelial MPs (EMPs) were found significantly higher compared to those with DLCO>40% (p = 0.02). We then used EMPs isolated from endothelial progenitor cells (ECFCs) extracted from IPF patients or controls to modulate normal human lung fibroblast (NHLF) properties. We showed that EMPs did not modify proliferation, collagen deposition and myofibroblast transdifferentiation. However, EMPs from IPF patients stimulated migration capacity of NHLF. We hypothesized that this effect could result from EMPs fibrinolytic properties and found indeed higher plasminogen activation potential in total circulating MPs and ECFCs derived MPs issued from IPF patients compared to those isolated from healthy controls MPs. Our study showed that IPF is associated with an increased level of EMPs in the most severe patients, highlighting an active process of endothelial activation in the latter. Endothelial microparticles might contribute to the lung fibroblast invasion mediated, at least in part, by a fibrinolytic activity.

Co-injection of mesenchymal stem cells with endothelial progenitor cells accelerates muscle recovery in hind limb ischemia through an endoglin-dependent mechanism.

Endothelial colony-forming cells (ECFCs) are progenitor cells committed to endothelial lineages and have robust vasculogenic properties. Mesenchymal stem cells (MSCs) have been described to support ECFC-mediated angiogenic processes in various matrices. However, MSC-ECFC interactions in hind limb ischemia (HLI) are largely unknown. Here we examined whether co-administration of ECFCs and MSCs bolsters vasculogenic activity in nude mice with HLI. In addition, as we have previously shown that endoglin is a key adhesion molecule, we evaluated its involvement in ECFC/MSC interaction. Foot perfusion increased on day 7 after ECFC injection and was even better at 14 days. Co-administration of MSCs significantly increased vessel density and foot perfusion on day 7 but the differences were no longer significant at day 14. Analysis of mouse and human CD31, and in situ hybridization of the human ALU sequence, showed enhanced capillary density in ECFC+MSC mice. When ECFCs were silenced for endoglin, coinjection with MSCs led to lower vessel density and foot perfusion at both 7 and 14 days (p<0.001). Endoglin silencing in ECFCs did not affect MSC differentiation into perivascular cells or other mesenchymal lineages. Endoglin silencing markedly inhibited ECFC adhesion to MSCs. Thus, MSCs, when combined with ECFCs, accelerate muscle recovery in a mouse model of hind limb ischemia, through an endoglin-dependent mechanism.