Reverse-D-4F Improves Endothelial Progenitor Cell Dysfunctions In High Fat Diet Mice

Atherosclerosis(Atherosclerosis, AS) is one of the most serious diseases of cardiovascular and cerebrovascular diseases. Therefore, it has become a hot spot in the research of basic medicine and clinical medicine to prevent the progression of AS. Diet has been shown to play an important role in the development of cardiovascular disease, which remains the major cause of death in western countries. It has been shown that a high fat diet contributes to the impairment of vascular function both in healthy subjects and in patients with cardiovascular disease(CVD), as well as the reduction of the circulating levels of EPCs after critical limb ischemia and the increase of the level of leukocytes in the peripheral blood. Recently Zhang et al. displayed that D-4F, one of apolipoprotein A-I mimetic peptide, promotes human EPC proliferation,migration, and adhesion through e NOS/NO pathway. Reverse-D-4F(Rev-D4F),a modified D4 F with reverse order, can also decrease aortic sinus atherosclerotic lesion area and lesion macrophage content by inhibiting endothelial inflammatory/oxidative events and improving the high density lipoprotein(HDL) function. However, the effect of Rev-D4 F on peripheral blood cell populations in C57BL/6J mice treated with high fat diets remains unclear. Therefore, in this study, we assessed the effect and the mechanism of Rev-D4 F on the number of different cell populations in the peripheral blood such as EPCs, lymphocytes,neutrophils, and monocytes, the inflammation of the arterial wall induced by a high fat diet,and the improvement of EPC functions impaired by the inflammatory factor TNF-α.1. The condition of mice bone marrow-derived MNCs induced to differentiate into EPCs and identification of EPCsThe different biological functions were studied in mouse bone marrow-derived endothelial progenitor cells isolated by differential time attachment to obtain the optimal adherent time in this study. Density gradient centrifugation-isolated bone marrow mononuclear cells were seeded on the fibronectin-coated dish. The 1-day cultured unattached cells were seeded on the second dish for 2 more days. Then unattached cells in the second dish were seeded on the third dish. The cells on 3 dishes were defined as 1-day adherent cells, 3-day adherent cells and3-day unattached cells, respectively. After 20-day culture, the biological functions, such as the percentage of biomarkers, the ability of adhesion, and the ability of forming tubes in vitro were analyzed. The results showed that the percentages of positive CD34, FLK-1, and CD34/FLK-1 expressions in 1-day attached cells were significantly increased compared to those in the 3-day adherent or unattached cells(P < 0.01), which showed the strongest adhesion ability. The expression of e NOS in 1- or 3-day adherent cells was significantly higher than that in 3-day u nattached cells(P < 0.01). The expression of VEGF in 3-day adherent cells was significantly higher than that in 1-day adherent cells or 3-day unattached cells(P < 0.01). These results sug gest the biological functions of 1-day adherent cells are significantly stronger than that of 3-da y adherent or unattached cells. VEGF expression in 3-day adherent cells is higher than that in1-day adherent cells or 3-day unattached cells. The expression of e NOS in 1-day adherent cell s or 3-day adherent cells is higher than that in 3-day unattached cells. The optimal adherent ti me to obtain mouse bone marrow-derived endothelial progenitor cells is 1–3 d.2. Apolipoprotein A-I mimetic peptide reverse D-4F improves the biological functions of mouse bone marrow-derived late EPCs.Apolipoprotein A-I(Apo A-I) mimetic peptide inhibits the development of atherosclerosis(AS) in apolipoprotein E-deficient mice; however, the underlying mechanism remains unclear. Endothelial progenitor cells(EPCs) can prevent AS progression through repairing proatherogenic factors impaired endothelium. In the present study, we examined the effect of reverse D-4F, one of apo A-I mimetic peptide on the proliferation, migration, and tube formation of mouse bone marrow-derived late EPCs. The present study showed that reverse D-4F(10–100 lg/ml) significantly improved the proliferation, migration, and tube formation of EPCs in a dose-dependent manner, and activated phospho-AKT at serine residue 473 and phospho-e NOS at serine residue 1177. LY294002(PI3-kinase inhibitor) and L-NAME(NOS inhibitor) significantly inhibited reverse D-4F mediated improvement of EPCs biological functions, and LY294002 significantly decreased reverse D-4F stimulated activation of phospho-AKT(473) and phospho-e NOS(1177). The results indicate that reverse D-4F mediated improvement of EPCs functions is dependent on the PI3K/AKT/e NOS pathway.3. Reverse-D-4F Increases the Number of Endothelial Progenitor Cells and Improves Endothelial Progenitor Cell Dysfunctions in High Fat Diet MiceAlthough high density lipoprotein(HDL) improves the functions of endothelial progenitor cells(EPCs), the effect of HDL Apo AI mimetic peptide reverse-D-4F(Rev-D4F) on EPC mobilization and repair of EPC dysfunctions remains to be studied. In this study, we investigated the effects of Rev-D4 F on peripheral blood cell subpopulations in C57 mice treated with a high fat diet and the mechanism of Rev-D4 F in improving the function of EPCs impaired by tumor necrosis factor-α(TNF-α). The high fat diet significantly decreased the number of EPCs, EPC migratory functions, and the percentage of lymphocytes in the white bloo-d cells. However, it significantly increased the number of white blood cells, the percentage of monocytes in the white blood cells, and the level of vascular endothelial growth factor(VEGF) and TNF-α in the plasma. Rev-D4 F clearly inhibited the effect of the high fat diet on the qu-antification of peripheral blood cell subpopulations and cytokine levels, and increased stroma-l cell derived factor 1α(SDF-1α) in the plasma.We provided in vitro evidence that TNF-α im-paired EPC proliferation, migration, and tube formation through inactive AKT and e NOS, which was restored by Rev-D4 F treatment. In contrast, both the PI3-kinase(PI3K) inhibitor(LY294002) and AKT inhibitor(perifosine) obviously inhibited the restoration of Rev-4F on EP-Cs impaired by TNF-α. Our results suggested that Rev-D4 F increases the quantity of endothe-lial progenitor cells through increasing the SDF-1α levels and decreasing the TNF-α level of peripheral blood in high fat diet-induced C57BL/6J mice, and restores TNF-α induced dysfunctions of EPCs partly through stimulating the PI3K/AKT signal pathway.