Effects of Oxidized Low-density Lipoprotein on Bone Marrow Stem Cell

描述(申请人提供)：成人骨髓多能祖细胞(MAPC)是一种特性良好的骨髓源性干细胞。我们的初步研究表明，氧化型低密度脂蛋白(OX-LDL)显著抑制小鼠MAPC的增殖及其内皮分化，并选择性地显著降低丝氨酸/苏氨酸激酶Akt(Akt)的磷酸化。众所周知，高脂血症患者内皮祖细胞的数量和功能明显降低。目前的研究将验证这一假说，即OX-LDL减少了骨髓中的干细胞数量，并由于Akt信号中断而损害了它们向内皮祖细胞的分化。具体目标是：1)。2)探讨氧化低密度脂蛋白对骨髓干细胞数量的影响及其机制；探讨氧化低密度脂蛋白(ox-LDL)在MAPC向EPC分化过程中的作用及其机制。从高脂血症性雄性低密度脂蛋白受体基因敲除(LDLR-/-)小鼠(用致动脉粥样硬化的饮食)和注入OX-低密度脂蛋白(WT)的野生型(WT)正常C57BL/6小鼠中分离、培养和定量分析小鼠MAPC。将评估骨髓中MAPC的数量、OCT-4的表达以及它们的增殖和分化为内皮祖细胞。为了进一步验证这一假设，将在LDLR(-/-)小鼠和注射ox-LDL的WT小鼠身上进行带有EGFP标记的MAPC的骨髓移植。将从动物中分离出EGFP阳性的EPC，并对EPC的数量和功能进行定量分析。如果这一假说是真的，那么在注射ox-LDL的动物和高脂血症LDLR(-/-)小鼠中，骨髓中的MAPC以及骨髓和外周血中的EPC的数量和功能将显著减少。还预计，从高脂血症LDLR(-/-)小鼠和ox-LDL注射的WT小鼠分离的MAPC的Oct-4表达、细胞增殖和内皮分化将显著降低，同时MAPC中Akt的磷酸化显著减少。相反，骨髓中EGFP阳性MAPC的数量和功能将显著增加，骨髓和外周血中的EPC将显著增加(无论是高脂血症小鼠还是OX-LDL注入的WT小鼠)，接受骨髓移植的EGFP阳性MAPC的数量和功能将显著增加，并稳定过表达活性Akt。此外，活性Akt的过表达将减少高脂血症小鼠动脉粥样硬化斑块的形成，并通过减少ox-LDL注射的小鼠的新生内膜形成来促进血管修复。这项研究的数据将为高脂血症患者动脉粥样硬化的发生机制提供新的信息，并有助于探索预防和治疗心血管疾病的新方法。

DESCRIPTION (provided by applicant): Adult bone marrow multipotent progenitor cells (MAPCs) are one of well characterized bone marrow- derived stem cells. Our initial studies show that oxidized low-density lipoprotein (ox-LDL) significantly inhibits the proliferation of murine MAPCs and their endothelial differentiation in association with a selective and dramatic reduction of serine/threonine kinase Akt (Akt) phosphorylation. It is well known that the number and function of endothelial progenitor cells (EPCs) are markedly reduced in the patients with hyperlipidemia. The present study will test the hypothesis that ox-LDL reduces stem cell population within the bone marrow, and impairs their differentiation into EPCs due to interrupted Akt signaling. The specific aims are: 1). To evaluate the effect of ox-LDL on the population of stem cells within bone marrow and the underlying mechanisms; and 2). To investigate the role of ox-LDL in the differentiation of MAPCs into EPCs and the underlying mechanisms. Murine MAPCs will be isolated, cultured and quantitatively analyzed from both hyperlipidemic male LDL receptor knock out (LDLR-/-) mice (with atherogenic diet) and ox-LDL-infused wild-type (WT) normal C57BL/6 mice. The number of MAPCs within the bone marrow, Oct-4 expression, and their proliferation and differentiation into EPCs will be evaluated. To further test the hypothesis, bone marrow transplantation with eGFP- labeled MAPCs will be performed in LDLR(-/-) mice and ox-LDL-infused WT mice. The eGFP-positive EPCs will be isolated and quantitatively analyzed from the animals for EPC number and function. If the hypothesis is true, it is expected that the population and function of MAPCs within the bone marrow and EPCs in the bone marrow and peripheral blood will be significantly decreased in ox-LDL-infused animals and in hyperlipidemic LDLR(-/-) mice. It is also anticipated that Oct-4 expression, cell proliferation, and endothelial differentiation of the MAPCs isolated from the hyperlipidemic LDLR(-/-) mice and ox-LDL- infused WT mice will be significantly decreased along with a significant reduction in Akt phosphorylation in MAPCs. Conversely, the number and function of eGFP-positive MAPCs within the bone marrow and EPCs in the bone marrow and peripheral blood will increase significantly in the animals (both hyperlipidemic mice and ox-LDL-infused WT mice) that receive bone marrow transplantation with eGFP- positive MAPCs transfected with retroviral Akt1 vectors, and stably over-expressing active Akt. In addition, overexpression of active Akt will decrease atherosclerotic plaque formation in hyperlipidemic mice, and enhance vascular repair with reduced neointima formation in ox-LDL-infused mice. The data from this study will provide novel information on the mechanisms for the development of atherosclerosis in patients with hyperlipidemia, and help explore new approaches to preventing and treating cardiovascular diseases.