Microparticles Carrying Peroxisome Proliferator-Activated Receptor Alpha Restore the Reduced Differentiation and Functionality of Bone Marrow-Derived Cells Induced by High-Fat Diet

Abstract

Metabolic pathologies such as diabetes and obesity are associated with decreased level of circulating and bone marrow (BM)-derived endothelial progenitor cells (EPCs). It is known that activation of peroxisome proliferator-activated receptor alpha (PPARα) may stimulate cell differentiation. In addition, microparticles (MPs), small membrane vesicles produced by activated and apoptotic cells, are able to reprogram EPCs. Here, we evaluated the role of MPs carrying PPARα on both phenotype and function of progenitor cells from mice fed with a high-fat diet (HFD). HFD reduced circulating EPCs and, after 7 days of culture, BM-derived EPCs and monocytic progenitor cells from HFD-fed mice displayed impaired differentiation. At the same time, we show that MPs bearing PPARα, MPs^PPARα+/+ , increased the differentiation of EPCs and monocytic progenitors from HFD-fed mice, whereas MPs taken from PPARα knockout mice (MPs^PPARα-/- ) had no effect on the differentiation of all types of progenitor cells. Furthermore, MPs^PPARα+/+ increased the ability of progenitor cells to promote in vivo angiogenesis in mice fed with HFD. The in vitro and in vivo effects of MPs^PPARα+/+ were abolished in presence of MK886, a specific inhibitor of PPARα. Collectively, these data highlight the ability of MPs carrying PPARα to restore the failed differentiation and functionality of BM-derived cells induced by HFD. Stem Cells Translational Medicine 2018;7:135-145.

Keywords: Angiogenesis; Bone marrow-derived cells; Differentiation; High-fat diet; Microparticles.

Figure 1

HFD impairs mobilization of bone marrow (BM)‐derived cells. At sacrifice day, circulating EPCs in peripheral blood (A) and total numbers of progenitors from BM (B) of mice was performed by flow cytometry. Count of cells was determined using double‐positive staining for Sca1⁺/Flk1⁺ (EPCs). (C, D): At day 0 and after 7 days of culture, flow cytometer characterization of BM‐derived cells was performed. Cells with double‐positive staining for Sca1⁺/Flk1⁺ were identified as differentiating EPCs, Sca1⁺/CD14⁺ were identified as monocytes, Sca1⁺/CD45⁺ were identified as leukocytes. Data are shown as mean values ± SEM (n = 8–16) *, p < .05. Abbreviations: EPCs, endothelial progenitor cells; HFD, high‐fat diet; Sca1, stem cell antigen 1; SD, standard diet.

Figure 2

Peroxisome proliferator‐activated receptor alpha (PPARα) carried by MPs increases the number of bone marrow (BM)‐derived progenitor cells undergoing in vitro differentiation in mice normal‐ and HFD‐fed diet. (A): Confocal microscopy characterization of mice BM‐derived endothelial progenitor cell (EPC) differentiation: cells were incubated in the absence or in the presence of either MPs^PPARα+/+ or MPs^{PPARαα–/–}. After 7 days in culture, cells with endothelial phenotype were characterized as adherent cells with double‐positive staining for Dil‐acetylated‐LDL (red) and lectin (green). Nuclei were counterstained with 4′,6‐diamidino‐2‐phenylindole (blue). (B): Histograms showing the percentage of BM‐derived cells labeled with DiI‐Ac‐LDL and lectin in the absence or in the presence of either MPs^PPARα+/+ or MPs^{PPARα–/–}. (C): Representative flow cytometric plots for EPCs from normal‐ and HFD‐fed mice after 7 days of treatment. (D): Percentage of BM‐derived EPCs pre‐treated with GW7647 (PPARα agonist), MPs^PPARα+/+, MPs^{PPARα–/–}, in the absence or in the presence of MK886. Data are shown as mean values ± SEM of at least 5 independent experiments for each condition. *, p< .05; **, p < .01; ***, p < .001. Abbreviations: Dil‐Ac‐LDL, 19‐dioctadecyl‐3,3,39,39‐tetramethyl‐indocarbocyanine‐labeled acetylated‐LDL; HFD, high‐fat diet; MPs, microparticles; SD, standard diet.

Figure 3

MP treatment increases the number of bone marrow (BM)‐derived myeloid progenitors in normal‐ and HFD‐fed diet mice. (A, B): Flow cytometer characterization of BM‐derived monocyte differentiation: cells were incubated either in the absence or in the presence of GW7647 (PPARα agonist), MPs^PPARα+/+ or MPs^{PPARα–/–}, in the absence or in the presence of MK886. After 7 days in culture, cells with double‐positive staining for Sca1⁺/CD14⁺ were identified as monocytes. Data are shown as mean values ± SEM of at least 6 independent experiments for each condition. *, p < .05; **, p < .01. Abbreviations: HFD, high‐fat diet; MPs, microparticles; Sca1, stem cell antigen 1; SD, standard diet.

Figure 4

MPs harboring PPARα promote in vivo bone marrow (BM)‐derived EPC‐mediated angiogenesis. (A): BM‐derived cells from normal‐ and HFD‐fed mice were treated with GW7647 (1 µM), MPs^PPARα+/+ or MPs^{PPARα–/–} in the absence or in the presence of MK866. Then, cells were detached, mixed with Matrigel and basic fibroblast growth factor (bFGF) and injected subcutaneously into mice fed with normal or HFD. (B): Quantitative measurement of Hb was reported as absorbance/weight of plugs. Data are shown as mean values ± SEM of at least 5 independent experiments for each condition. *, p < .05. Abbreviations: Hb, hemoglobin; HFD, high‐fat diet; MPs, microparticles; SD, standard diet.

Figure 5

MPs^PPARα+/+ induce activation of PPARα DNA‐binding domains. (A): Concentration‐response of mouse PPARα Assay using reference agonist GW590735. (B): Reporter cells expressing mouse PPARα were incubated either in the absence or in the presence of MPs^PPARα+/+, MPs^{PPARα–/–} in the absence or tin the presence of MK886 or the specific activator GW7647 for 24 hours. Luciferase activity was determined as described in Methods. Data are presented as the as mean values ± SEM of eight independent measurements. *, p < .05. Abbreviations: PPARα, peroxisome proliferator‐activated receptor α; RLU, relative light units.