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（MAPCs）是表征良好的骨髓来源干细胞之一。我们的初步研究表明，氧化低密度脂蛋白（ox-LDL）通过选择性和显著降低丝氨酸/苏氨酸激酶Akt （Akt）磷酸化，显著抑制小鼠MAPCs的增殖及其内皮分化。众所周知，在高脂血症患者中，内皮祖细胞（EPCs）的数量和功能明显降低。本研究将验证ox-LDL减少骨髓内干细胞数量的假设，并由于Akt信号传导中断而损害其向EPCs的分化。具体目标是：1)。探讨ox-LDL对骨髓干细胞群的影响及其机制；和2)。探讨ox-LDL在MAPCs向EPCs分化中的作用及其机制。小鼠MAPCs将从高脂血症雄性LDL受体敲除（LDLR-/-）小鼠（动脉粥样硬化饮食）和ox-LDL灌注野生型（WT）正常C57BL/6小鼠中分离、培养和定量分析。我们将评估骨髓中MAPCs的数量、Oct-4的表达以及它们向EPCs的增殖和分化。为了进一步验证这一假设，将在LDLR（-/-）小鼠和ox- ldl输注的WT小鼠中进行eGFP标记MAPCs的骨髓移植。从动物中分离egfp阳性EPCs，定量分析EPCs的数量和功能。如果该假设成立，预计在ox- ldl输注动物和高脂血症LDLR（-/-）小鼠中，骨髓中MAPCs的数量和功能以及骨髓和外周血中EPCs的数量和功能将显著降低。我们还预计，从高脂血症LDLR（-/-）小鼠和ox-LDL-输注的WT小鼠中分离的MAPCs的Oct-4表达、细胞增殖和内皮分化将显著降低，MAPCs中Akt磷酸化水平也会显著降低。相反，在接受逆转录病毒Akt1载体转染eGFP阳性MAPCs并稳定过表达活性Akt的骨髓移植的动物（包括高脂血症小鼠和ox- ldl输注的WT小鼠）中，骨髓中eGFP阳性MAPCs以及骨髓和外周血中EPCs的数量和功能都会显著增加。此外，活性Akt的过表达会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灌注小鼠新内膜的形成来促进血管修复。本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.