Mechanism Of Macrophage Foam Cell Formation And Migration Pathway

Atherosclerosis is a chronic disease in which the inside of an artery narrows due to the build up of plaque.The atherosclerosis contributes to major mortality and modality of cardiovascular diseases in Western countries.When severe,it can result in coronary artery disease,stroke,peripheral artery disease,or kidney problems depending on the arteries which are affected.Atherosclerosis is characterized by the accumulation of lipids,macrophages,vascular smooth muscle cells,T lymphocytes and fibrous tissue.Lipid-laden macrophage accumulation in the subendothelial area of the arterial wall is a hallmark of atherosclerosis.These lipid-laden cells promote inflammatory responses in the arterial wall and lead to multiple fatal pathological consequences,such as hemorrhage,rupture,and calcification,which are called foam cells.Foam cell formation is the key process in the development of atherosclerosis.In early fatty streak lesions,macrophages that are differentiated from monocytes become matured and gather low-density lipoprotein(LDL)and modified LDL,such as oxidized low-density lipoprotein(ox LDL).Macrophages play important roles in all stages of atherosclerosis.The primary origin of macrophages is myeloid progenitor cells in bone marrow.Recent lineage-tracing studies reveal that a large portion of macrophage marker-positive cells in mouse and human atherosclerotic lesions are vascular SMC derived Cells.These cells will be further activated become the center of atherosclerosis.However,the molecular mechanism of foam cell formation is still largely unknown.Lysophosphatidic acid(LPA)is one of the simplest phospholipids,it exerts diverse biological functions on a wide variety of living cells.LPA highly accumulates in atherosclerotic lesions.Elevated LPA levels in plasma could affect a divergent function of endothelial cells(ECs),smooth muscle cells(SMCs),monocytes,and macrophages,influencing physiology and pathology of vascular cells and promoting vascular diseases.Lipopolysaccharide(LPS),is also referred as an endotoxin,which has numerous biological activities and is responsible for many pathological conditions caused by Gram-negative bacteria.As the highly inflammatory constituent of the outer membrane of Gram-negative bacteria,LPS induces macrophage foam cell formation in vitro and leads to the formation of aortic lesions in vivo.However,how LPA or LPS induces foam cell formation and macrophage migration remain elusive.We first report here that LPA enhances LPS-induced ox LDL uptake in macrophages.Our data revealed that both LPA and LPS highly induce the CD14 expression at messenger RNA and protein levels in macrophages.The role of CD14,one component of the LPS receptor cluster,in LPA-induced biological functions has been unknown.We took several steps to examine the role of CD14 in LPA signaling pathways.Knockdown of CD14 expression nearly completely blocked LPA/LPSinduced ox LDL uptake in macrophages,demonstrating for the first time that CD14 is a key mediator responsible for both LPA-and LPS-induced ox LDL uptake/foam cell formation.To determine the molecular mechanism mediating CD14 function,we demonstrated that both LPA and LPS significantly induce the expression of scavenger receptor class A type I(SR-AI),which has been implicated in lipid uptake process,and depletion of CD14 levels blocked LPA/LPS-induced SR-AI expression.We further showed that the SR-AI–specific antibody,which quenches SR-AI function,blocked LPA-and LPS-induced foam cell formation.Thus,SR-AI is the downstream mediator of CD14 in regulating LPA-,LPS-,and LPA/LPS-induced foam cell formation.Mitogen-activated protein(MAP)kinases have been implicated in the development of atherosclerosis.However,whether these pathways are involved in LPS-induced ox LDL uptake/foam cell formation has been currently unrevealed.To determine the upstream mediator of LPS-induced foam cell formation,we employed pharmaceutical and gene knockdown approaches.Our results demonstrated that the specific c-Jun N-terminal kinase(JNK)pathway inhibitor,SP600125,but neither the specific inhibitor of extracellular signaling-regulated kinase(ERK)kinase MEK1/2,U0126,nor the specific inhibitor of p38 MAPK,SB203580,significantly blocks LPSinduced ox LDL uptake,suggesting that the JNK pathway is the upstream mediator of LPS-induced ox LDL uptake/foam cell formation.To address whether JNK pathway mediates LPS-induced ox LDL uptake is due to JNK pathway-regulated CD14 and SR-AI expression,we assessed whether the pharmaceutical inhibitor of JNK influences LPS-induced expression of CD14 and SR-AI.Our results indicate that JNK pathway mediates LPS-induced CD14 and SR-AI expression.To conclusively address the isoform role of JNK family,we depleted JNK isoforms using the JNK isoformspecific si RNA.Our data showed that the depletion of JNK1,but not JNK2 blocked LPS-induced CD14/SR-AI expression and foam cell formation.Macrophage emigration has been shown to occur in early atherosclerotic plaques,after the formation of foam cells,they gradually lose the capacity for emigration during progression of the atherosclerotic lesion,and this will contribute the development of atherosclerosis.There is growing evidence supporting the notion that the balance between retention and emigration signals contributes to the accumulation of macrophages in atherosclerotic lesions.In our study,we found LPA can block LPS-induced migration in BMDMs.Our data revealed that LPA can decrease LPS-induced AKT phosphorylation in BMDMs.It has been demonstrated AKT family has three members,AKT1,AKT2 and AKT3 and they play key roles in cell migration for a variety of cell types.To identify the specific roles of AKTs in BMDM migration,Knockdown approach was applied.We found the phosphorylation of AKT2 and AKT3 induced by LPS was blocked by LPA,and the depletion of them blocked LPS-induced BMDM migration,suggesting a key role of AKT2 and AKT3 in BMDM migration.To further explore the molecular mechanism involved in the blocking effect of LPA on BMDM migration,we isolated BMDMs from WT,LPA receptors knockout mice and used the knockdown technique.Our results indicate that LPA through LPA5 and LPA6,blocked the phosphorylation of AKT2 and AKT3,which in turn contribute LPS-induced migration in BMDMs.The novel LPALPA5/LPA6-AKT2/AKT3 pathway,contribute to the retention of macrophage in atherosclerotic lesion.Taken together,our results demonstrate that LPA and LPS play a regulatory role in foam cell formation and macrophage retention during atherosclerosis development.We identify that CD14 is a novel connecting molecule linking both LPA and LPS pathways and is a key mediator responsible for LPA/LPS-induced foam cell formation.JNK1 is the upstream mediator of LPS-induced CD14 and SR-AI expression in macrophages,leading to LPS-induced ox LDL uptake/foam cell formation.Besides the augment effect of LPA on LPS induced foam cell formation,we are the first time indicate that LPA has the blocking effect on macrophage migration.The novel LPA-LPA5/LPA6-AKT2/AKT3 pathway contribute to the retention of macrophage,which increase the macrophage number in lesion area.The molecular mechanism involved in LPA and LPS pathway might be the new convergent pathway,contributing to the worsening of atherosclerosis.