BMP-7 induced Macrophage Polarization in Atherosclerosis

BMP-7 诱导动脉粥样硬化中的巨噬细胞极化

• 批准号: 8896859
• 负责人: Sampath Parthasarathy
• 金额: $ 35.78万
• 依托单位: UNIVERSITY OF CENTRAL FLORIDA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-19 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8896859
• 关键词: Address; Affect; Animals; Anti-Inflammatory Agents; Anti-inflammatory; Apolipoprotein E; Area; Arterial Fatty Streak; Arteries; Atherosclerosis; BMP7 gene; Backcrossings; Biological Models; Blood; Blood Vessels; Bone Morphogenetic Proteins; Cardiovascular Diseases; Carotid Arteries; Cell Culture Techniques; Cells; Cessation of life; Clinical; Colony-Stimulating Factor Gene; Coronary artery; Data; Development; Disease; Disease Progression; Gene Deletion; Gene Expression; Generations; Genes; Growth Factor; Health; Human; In Vitro; Infiltration; Inflammation; Inflammatory; Interleukin-1; Interleukin-10; Knock-out; Knockout Mice; Left; Lesion; Ligation; MAP2K1 gene; MAPK14 gene; MAPK8 gene; Macrophage Colony-Stimulating Factor; Measures; Mediating; Methodology; Mitogen-Activated Protein Kinases; Modeling; Molecular; Mononuclear; Mus; Myocardial Infarction; Operative Surgical Procedures; Osteoblasts; Osteoporosis; Pathway interactions; Phenotype; Play; Proto-Oncogene Proteins c-akt; Research; Role; Small Interfering RNA; Smooth Muscle Myocytes; Staging; Techniques; Testing; Thromboplastin; Time; Up-Regulation; arginase; base; bone morphogenetic protein 7; calcification; cytokine; experience; follow-up; improved; in vivo; inhibitor/antagonist; intravenous injection; loss of function; macrophage; monocyte; novel; prevent; receptor; receptor function; scavenger receptor; shear stress; success; transdifferentiation

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.

描述(申请人提供)：最近出现了一个新的概念，表明在动脉粥样硬化(ATH)的发展过程中，单核细胞在渗入动脉后可以分化为两种主要的经典(M)或替代(M2)巨噬细胞。体外初步数据表明，BMP-7刺激的单核细胞(THP-1细胞)分化为M2极化的巨噬细胞，M2特异性标记CD206的增加和IL-10释放的上调证实了这一点。体内初步数据表明，静脉注射BMP-7可使部分结扎冠状动脉(PLCA)的ApoE KO小鼠M2极化的巨噬细胞增多，单核细胞和M2极化的巨噬细胞表面BMP-7R增多，M2巨噬细胞特异性释放IL-10和IL-1受体拮抗剂增多，血流速度加快。因此，我们认为在ATH发育的不同阶段，单核细胞在BMP-7的影响下分化为M2巨噬细胞，这是一种抗炎表型。此外，我们建议了解在单核细胞分化为M2极化巨噬细胞的过程中是否也需要M-CSF和BMP-7的作用，并使用从回交操作小鼠(M-CSF缺陷)和载脂蛋白E缺陷小鼠获得的杂合子小鼠来关联BMP-7对ATH中M1和M2巨噬细胞的存在和影响。我们还建议建立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发生发展过程中皮损中BMP-7的表达，并探讨其与巨噬细胞M1、M2型的关系。我们将使用免疫组织化学和实时聚合酶链式反应技术，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，如精氨酸酶和清道夫受体，来确定这一点。此外，还将确定特定巨噬细胞表型的存在和优势。目的1b：我们将利用siRNA技术中BMP-7R的功能丧失和BMP-7的抑制剂来确定巨噬细胞的极化。目的：探讨骨形态发生蛋白-7(BMP-7)是否单独通过BMP-7介导单核细胞向M2极化，或单核细胞释放M-CSF对其影响。我们将确定a)BMP-7是否诱导单核细胞分化为M2巨噬细胞，或者b)BMP-7和M-CSF是否都诱导单核细胞分化为M2极化巨噬细胞。针对M-CSF基因缺失的基因改变的OP小鼠将与ApoE KO小鼠回交，杂合子小鼠将接受PLCA手术，所有参数将按照目标1的建议建立。目标3：我们将确定SMAD依赖和p38、JNK和ERK通路及其与特定表型相关基因表达的释放/表达的相关性。这些研究的结果将确定BMP-7对M2极化巨噬细胞的影响，抗炎细胞因子，它们对基因表达的影响，以及ATH进展的分子机制。