BMP-7 induced Macrophage Polarization in Atherosclerosis

描述（由申请人提供）：最近出现了一个新的概念，表明单核细胞在浸润到动脉后，可以在动脉粥样硬化（ATH）的发展过程中分化为两种主要的经典(M)或替代（M2）巨噬细胞。体外初步数据显示，BMP-7刺激的单核细胞（THP-1细胞）分化为M2极化巨噬细胞，M2特异性标志物CD206升高，IL-10释放上调，证实了这一点。体内初步数据显示，在冠状动脉部分结痂（PLCA）的ApoE KO小鼠中静脉注射BMP-7，与假对照组相比，两周随访时，M2极化巨噬细胞增加，单核细胞和M2极化巨噬细胞上BMP-7R增加，M2巨噬细胞特异性释放的IL-10和IL-1受体拮抗剂增加，血流速度增加。因此，我们提出单核细胞在ATH发展的不同阶段在BMP-7的影响下分化为M2巨噬细胞，这是一种抗炎表型。此外，我们打算了解M-CSF是否也需要与BMP-7一起作用于单核细胞向M2极化巨噬细胞的分化，并利用回交op小鼠（M-CSF缺陷）和ApoE缺陷小鼠获得的杂合小鼠来关联BMP-7对ATH中M1和M2巨噬细胞的存在和影响。我们还提出建立BMP-7影响巨噬细胞极化的机制。具体目的如下：Aim1a：检测ApoE KO动物ATH发展过程中病变中BMP-7的存在，并将其与M1和M2型巨噬细胞的存在联系起来。我们将使用免疫组织化学和实时PCR技术来确定这一点，并测量促炎和抗炎细胞因子水平，炎症基因的特异性表达，以及M1和M2表型巨噬细胞的特异性基因，如精氨酸酶和清除率受体。此外，将确定特定巨噬细胞表型的存在和优势。目的1b：我们将使用siRNA方法和BMP-7抑制剂来测定巨噬细胞的极化。目的2：确定BMP-7诱导的单核细胞M2极化是单独通过BMP-7介导的，还是由单核细胞释放的M-CSF提供的。我们将确定是a) BMP-7诱导单核细胞分化为M2巨噬细胞，还是b) BMP-7和M-CSF都诱导单核细胞分化为M2极化巨噬细胞。M-CSF基因缺失特异性基因改变的op小鼠将与ApoE KO小鼠回交，杂合小鼠将进行PLCA手术，所有参数将按照Aim 1的建议建立。目的3：我们将确定smad依赖性和p38， JNK和ERK途径及其与特定表型相关基因表达的释放/表达的相关性。这些研究的结果将明确BMP-7对M2极化巨噬细胞、抗炎细胞因子的影响及其对基因表达的影响，以及ATH进展中的分子机制。

DESCRIPTION (provided by applicant): A novel concept has recently emerged demonstrating that monocytes, following infiltration into arteries, can differentiate into two major classical (M) or alternative (M2) macrophages during the development of atherosclerosis (ATH). In vitro preliminary data indicates that BMP-7 stimulated monocytes (THP-1 cells) differentiate into M2 polarized macrophages, which was confirmed with increased M2-specific marker CD206 and up regulation of released IL-10. In vivo preliminary data suggests that intravenous injection of BMP-7 in ApoE KO mice experiencing a partial ligated coronary artery (PLCA) have increased M2 polarized macrophages, increased BMP-7R on monocytes and M2 polarized macrophages, increased M2 macrophage- specific released IL-10 and IL-1 receptor antagonist, and increased blood velocity compared with sham controls at two weeks follow up. Accordingly, we propose that monocytes differentiate into M2 macrophages, an anti-inflammatory phenotype, under the influence of BMP-7 at different stages of ATH development. Additionally, we propose to understand whether M-CSF action is also required along with BMP-7 on monocyte differentiation to M2 polarized macrophages and to correlate the presence and influence of BMP-7 on M1 and M2 macrophages in ATH using heterozygous mice obtained from backcrossing op mice (M-CSF deficient) and ApoE deficient mice. We also propose to establish the mechanisms by which BMP-7 would affect macrophage polarization. Specific aims are as follows: Aim1a: To examine the presence of BMP-7 in the lesions during the development of ATH in ApoE KO animals and to correlate its presence with M1 and M2 types of macrophages. We will determine this using immunohistochemical and real-time PCR techniques and measuring pro-and anti- inflammatory cytokine levels, specific expression of inflammatory genes, and specific genes for M1 and M2 phenotype macrophages such as arginase and scavenger receptors. Furthermore, the presence and preponderance of specific macrophage phenotypes will be identified. Aim 1b: We will use loss of function of BMP-7R using siRNA methodology and inhibitors for BMP-7 to determine macrophage polarization. Aim 2: To determine if BMP-7 induced monocytes to M2 polarization is mediated via BMP-7 alone or if influence is provided by released M-CSF from the monocytes. We will establish whether a) BMP-7 induces differentiation of monocytes into M2 macrophages, or whether b) BMP-7 and M-CSF both induce monocytes into M2 polarized macrophages. Genetically altered op mice specific for M-CSF gene deletion will be backcrossed with the ApoE KO mice, heterozygous mice will be subjected to PLCA surgery, and all parameters will be established as proposed in Aim 1. Aim 3: We will determine SMAD-dependent and p38, JNK, and ERK pathways and their correlation with the release/expression of specific phenotype-associated gene expressions. Results of these studies will define the impact of BMP-7 on M2 polarized macrophages, anti-inflammatory cytokines, their effects on gene expression, and molecular mechanisms in the progression of ATH.