at day 7, the presence of populations of bone marrow-derived cells displaying double labeling of Sca-1+/CD31+, Sca-1+/CD45+, Sca-1+/CD14+ and F4/80+/ CD31+ whose levels were enhanced compared to those at day 0. It should be noted that the percentages of bone marrowderived cells expressing Sca-1+/Flk-1+ and CD133+/Flk-1+ were not significantly different at day 0 and 7. This characterization indicates that, at day 7, bone marrow-derived cells are mainly cells expressing monocytic and endothelial markers; and moreover that the EPC subpopulation belongs to the so-called ��early EPCs”. To investigate the effects of PPARa carried by MPs on in vitro bone marrow-derived cell differentiation, circulating MPs isolated from PPARa-deficient or WT mice were added to adherent bone marrowderived cells from WT mice. Thereafter, EPCs were identified by DilAcetyl-LDL-uptake with concomitant lectin binding, as previously described, and double expression of Sca-1 and Flk-1 determined. After 7 days, isolated bone marrow-derived cells cultured in the presence of MPsPPARa+/+ displayed enhanced adhesion but with no changes in cell proliferation. At day 7, confocal microscopy characterization of adherent cells incubated with MPsPPARa+/+, but not with MPsPPARa2/2, also demonstrated increased Dil-ac-LDL/lectin uptake typical for endothelial lineage. As illustrated in Pharmacological PPARa activation induces EPC differentiation To evaluate the presence of a functional PPARa pathway in bone marrow-derived cells and also whether direct activation of PPARa leads to EPC differentiation, 12600694 we first analyzed PPARa expression in bone marrow-derived cells and secondly, we used a selective PPARa agonist, 27027724 WY-14643 to stimulate EPC differentiation of cells isolated from WT mice. As shown by Western blot, PPARa was expressed on bone marrow-derived cells at day 7 of culture and that PPARa levels were not modified in bone marrow-derived cells after incubation with 3 August 2010 | Volume 5 | Issue 8 | e12392 MPs-PPAR & Endothelial Cells MPsPPARa+/+, suggesting that PPARa transfer between MPs and bone marrow-derived cells did not take place. Interestingly, the PPARa agonist significantly increased EPC differentiation from WT but not from PPARa-null mice as assessed by Sca-1+/Flk-1+ staining. In addition, when bone marrow-derived cells from PPARa-null mice were treated with MPs from WT mice, no changes in EPC differentiation were Tedizolid (phosphate) site observed. Altogether, these results demonstrated the presence of functional PPARa receptors on bone marrow-derived cells from WT mice that induce differentiation and that it was unlikely 
that PPARa protein was transferred from MPs to bone marrow-derived cells. differentiation marker genes profiles by q-PCR. Mice endothelial markers and angiogenic gene expression were analyzed using a panel of 52 transcripts. The data in MPsPPARa+/+-induced EPC differentiation is associated with up-regulation of pro-angiogenic factor mRNA levels To confirm and extend the results obtained by immunofluorescence and flow cytometry, we further analyzed the expression of MPsPPARa+/+ enhance EC-specific marker expression and Akt activity We investigated the molecular mechanisms underlying the effects of MPs on EPC differentiation. To further confirm the 4 August 2010 | Volume 5 | Issue 8 | e12392 MPs-PPAR & Endothelial Cells effects of MPs harboring PPARa on bone marrow-derived cells differentiation at day 7 of growth, we examined the protein expression levels in cellular extracts
