Toll-like receptor 3 regulates cord blood-derived endothelial cell function in vitro and in vivo.

Circulating endothelial progenitor cells (cEPC) are capable of homing to neovascularisation sites, in which they proliferate and differentiate into endothelial cells. Transplantation of cEPC-derived cells, in particular those isolated from umbilical cord blood (UCB), has emerged as a promising approach in the treatment of cardio-vascular diseases. After in vivo transplantation, these cells may be exposed to local or systemic inflammation or pathogens, of which they are a common target. Because Toll-like receptors (TLR) are critical in detecting pathogens and in initiating inflammatory responses, we hypothesized that TLR may govern UCB cEPC-derived cells function. While these cells expressed almost all TLR, we found that only TLR3 dramatically impaired cell properties. TLR3 activation inhibited cell proliferation, modified cell cycle entry, impaired the in vitro angiogenic properties and induced pro-inflammatory cytokines production. The anti-angiogenic effect of TLR3 activation was confirmed in vivo in a hind-limb ischemic mice model. Moreover, TLR3 activation consistently leads to an upregulation of miR-29b, -146a and -155 and to a deregulation of cytoskeleton and cell cycle regulator. Hence, TLR3 activation is likely to be a key regulator of cEPC-derived cells properties.

[Cord blood circulating endothelial progenitors: perspectives for clinical use in cardiovascular diseases]. / Les progéniteurs endothéliaux circulants du sang de cordon: perspectives thérapeutiques pour les maladies cardiovasculaires.

The discovery of circulating endothelial progenitor cells (EPCs) in adult peripheral blood has opened up many exciting possibilities in vascular biology. Several studies have confirmed the existence of EPCs, as well as their bone marrow origin and their ability to integrate into vascular structures at sites of neoangiogenesis. EPCs appear to be naturally involved in the prevention of ischemia by participating directly in the vascularization process. Given their tropism for sites of neoangiogenesis, EPCs have clear therapeutic potential for treating ischemic diseases. If associated with other cell therapy products, they could improve tissue regeneration by promoting graft vascularization. However, the use of EPCs as a cell therapy product is limited by their rarity in peripheral blood. Cord blood contains many more EPCs, which are functional and can be expanded in culture. Their clinical use will require expansion in strictly controlled conditions and rigorous validation in preclinical models. EPCs could also serve as a quality markerforfrozen cord blood, showing the presence of non hematopoietic stem cells.

Porous polysaccharide-based scaffolds for human endothelial progenitor cells.

Human ECFCs contribute to vascular repair. For this reason, they are considered as valuable cell therapy products in ischemic diseases. Porous scaffolds are prepared that are composed of natural polysaccharides, pullulan and dextran, by chemical crosslinking without use of organic solvents. These porous scaffolds, which have pores with an average size of 42 µm and a porosity of 21%, preserve the viability and the proliferation of cord-blood ECFCs. After 7 d of culture in porous scaffolds, ECFCs express endothelial markers (CD31 and vWf) and maintain endothelial functions. The cultured cells can be easily retrieved by enzymatic degradation of the porous scaffolds. In vitro results suggest that the porous scaffold could allow cell delivery of ECFCs for treatment of vascular diseases.

Impact of age and gender interaction on circulating endothelial progenitor cells in healthy subjects.

OBJECTIVE: To assess the level of circulating endothelial progenitor cells (CEPC) in cycling women compared with men and menopausal women. DESIGN: Controlled clinical study. SETTING: Healthy, nonsmoking volunteers. PATIENT(S): Twelve women, aged 18-40 years, with regular menstrual cycles, 12 menopausal women, and two groups of 12 age-matched men were recruited. Women did not receive any hormone therapy. INTERVENTION(S): Collection of 20 mL of peripheral blood. MAIN OUTCOME MEASURE(S): The number of CEPC, defined as (Lin-/7AAD-/CD34+/CD133+/KDR+) cells per 10(6) mononuclear cells (MNC), was measured by flow cytometry. RESULT(S): The number of CEPC was significantly higher in cycling women than in age-matched men and menopausal women (26.5 per 10(6) MNC vs. 10.5 per 10(6) MNC vs. 10 per 10(6) MNC, respectively). The number of CEPC was similar in menopausal women, age-matched, and young men. CONCLUSION(S): The number of CEPC is influenced by an age-gender interaction. This phenomenon may explain in part the better vascular repair and relative cardiovascular protection in younger women as compared with age-matched men.

In vitro and in vivo analysis of endothelial progenitor cells from cryopreserved umbilical cord blood: are we ready for clinical application?

Umbilical cord blood (CB) represents a main source of circulating endothelial progenitor cells (cEPCs). In view of their clinical use, in either the autologous or allogeneic setting, cEPCs should likely be expanded from CB kept frozen in CB banks. In this study, we compared the expansion, functional features, senescence pattern over culture, and in vivo angiogenic potential of cEPCs isolated from fresh or cryopreserved CB (cryoCB). cEPCs could be isolated in only 59% of cryoCB compared to 94% for fresh CB, while CB units were matched in terms of initial volume, nucleated and CD34(+) cell number. Moreover, the number of endothelial colony-forming cells was significantly decreased when using cryoCB. Once cEPCs culture was established, the proliferation, migration, tube formation, and acetylated-LDL uptake potentials were similar in both groups. In addition, cEPCs derived from cryoCB displayed the same senescence status and telomeres length as that of cEPCs derived from fresh CB. Karyotypic aberrations were found in cells obtained from both fresh and cryoCB. In vivo, in a hind limb ischemia murine model, cEPCs from fresh and cryoCB were equally efficient to induce neovascularization. Thus, cEPCs isolated from cryoCB exhibited similar properties to those of fresh CB in vitro and in vivo. However, the low frequency of cEPCs colony formation after cryopreservation shed light on the need for specific freezing conditions adapted to cEPCs in view of their future clinical use.