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:
Aim1 a: 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 1 b: 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.
The most common cause of atherosclerosis in cardiovascular disease in the U.S. is development of plaque in the coronary arteries which leads to myocardial infarction and death. Inflammation induced by macrophages in this plaque area is considerable cause of development and progression of this disease. Recently, this has been identified there are two types of macrophages M1 and M2 which release detrimental (M1 macrophages) and beneficial (M2 macrophages) cytokines in this microenvironment. However, switch of this M1 vs. M2 macrophage polarization depends upon the cytokine present at the given time. Therefore, we made an effort to identify novel molecule, BMP-7 which favors in the increase of M2 macrophages from monocytes. Moreover, increased M2 macrophages release anti-inflammatory cytokines and inhibit the progression of atherosclerosis. If our project is successful this molecule can be used widely where macrophage switch is needed to decrease inflammation in the various inflammatory diseases.
|Singla, Dinender K; Singla, Reetu; Wang, Jing (2016) BMP-7 Treatment Increases M2 Macrophage Differentiation and Reduces Inflammation and Plaque Formation in Apo E-/- Mice. PLoS One 11:e0147897|
|Singla, Dinender K; Singla, Reetu D; Abdelli, Latifa S et al. (2015) Fibroblast growth factor-9 enhances M2 macrophage differentiation and attenuates adverse cardiac remodeling in the infarcted diabetic heart. PLoS One 10:e0120739|
|Urbina, Princess; Singla, Dinender K (2014) BMP-7 attenuates adverse cardiac remodeling mediated through M2 macrophages in prediabetic cardiomyopathy. Am J Physiol Heart Circ Physiol 307:H762-72|
|Singla, Reetu D; Wang, Jing; Singla, Dinender K (2014) Regulation of Notch 1 signaling in THP-1 cells enhances M2 macrophage differentiation. Am J Physiol Heart Circ Physiol 307:H1634-42|