culture-macrophage-colony-stimulating-factor-differentiated-human

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

National Institutes of Health

In the present study, we tested the effect of cold water stress (CWS) on dopaminergic neurons in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease (PD) mouse model, and found that CWS pretreatment elicited less MPTP neurotoxicity. To understand the molecular mechanism underlying this phenomenon, we detected the expression of heat shock protein 70 (Hsp70) in the striatum of the experimental mice, and found that CWS pretreatment could significantly increase striatal Hsp70 in MPTP-treated mice. Furthermore, in parallel with the induction of Hsp70, the MPTP-induced increase of striatal α-synuclein was inhibited in the CWS + MPTP-treated mice. CWS pretreatment also significantly inhibited the reduction of anti-apoptotic molecule Bcl-2 expression in the striatum and enhanced Bcl-2 transcription in the substantia nigra of MPTP-treated mice. Taken together, these data indicated that Hsp70 might be an important intermediate for the neuroprotective effect of CWS against MPTP-induced dopaminergic toxicity.

Cold water stress attenuates dopaminergic neurotoxicity induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mice.

MicroRNA-34a (miR-34a) promotes apoptosis via down-regulating many anti-apoptotic proteins. Aldehyde dehydrogenase 2 (ALDH2) is an anti-apoptotic enzyme whose activity decline associates with myocardial injury. We tested hypothesis that miR-34a might play a pro-apoptotic role in myocardial infarction (MI) by down-regulating ALDH2. MiR-34a was highly increased while ALDH2 expression was decreased after experimental MI. Overexpression of miR-34a in neonatal rat cardiomyocyte could significantly enhance apoptosis and down-regulate ALDH2 expression. In 293 cells, luciferase reporter assay results demonstrated that ALDH2 was a direct target of miR-34a. Serum miR-34a levels in acute myocardial infarction (AMI) patients and rats were significantly higher than healthy subjects and sham rats. Our results proved that miR-34a could promote cardiomyocyte apoptosis via negatively regulating ALDH2 and circulating miR-34a was increased in the condition of MI. Thus, miR-34a may constitute a new therapeutic target and diagnostic marker for patients with MI.

MicroRNA-34a promotes cardiomyocyte apoptosis post myocardial infarction through down-regulating aldehyde dehydrogenase 2.

Naoxintong (NXT), a Chinese Materia Medica standardized product, extracted from 16 various kinds of Chinese traditional herbal medicines including Salvia miltiorrhiza, Angelica sinennsis, Astragali Radix, is clinically effective in treating atherosclerosisrelated diseases. Here, we tested the hypothesis that the anti-atherosclerosis effects of NXT might be mediated by suppressing maturation of dendritic cells (DCs) in a mice model of atherosclerosis. LDLR(-/-) mice fed a high-fat diet were treated with placebo, NXT (0.7 g/kg/d, oral diet) or simvastatin (100mg/kg/d, oral diet) for 8 weeks, respectively. NXT treatment significantly reduced plasma triglyceride (112 ± 18 mg/dl vs. 192 ± 68 mg/dl, P<0.05) and total cholesterol (944 ± 158 mg/dl vs. 1387 ± 208 mg/dl, P<0.05) compared to placebo treatment. Vascular lesions were significantly smaller and macrophage content and amount of DCs in plaques were significantly less in NXT and simvastatin groups than in placebo group (all P<0.05). In addition, expressions of splenic DC membrane molecules (CD40, CD86 and CD80) and the plasma level of IL-12p70 were significantly lower in NXT and simvastatin groups than in placebo group. In conclusion, NXT protects against atherosclerosis through lipid-lowering and inhibiting DCs maturation in this mice model of atherosclerosis.

Naoxintong protects against atherosclerosis through lipid-lowering and inhibiting maturation of dendritic cells in LDL receptor knockout mice fed a high-fat diet.

Previous studies have demonstrated that the ATP-binding cassette transporters (ABC)A1 and ABCG1 function in many aspects of cholesterol efflux from macrophages. In this current study, we continued our investigation of extracellular cholesterol microdomains that form during enrichment of macrophages with cholesterol. Human monocyte-derived macrophages and mouse bone marrow-derived macrophages, differentiated with macrophage colony-stimulating factor (M-CSF) or granulocyte macrophage colony-stimulation factor (GM-CSF), were incubated with acetylated LDL (AcLDL) to allow for cholesterol enrichment and processing. We utilized an anti-cholesterol microdomain monoclonal antibody to reveal pools of unesterified cholesterol, which were found both in the extracellular matrix and associated with the cell surface, that we show function in reverse cholesterol transport. Coincubation of AcLDL with 50 μg/ml apoA-I eliminated all extracellular and cell surface-associated cholesterol microdomains, while coincubation with the same concentration of HDL only removed extracellular matrix-associated cholesterol microdomains. Only at an HDL concentration of 200 µg/ml did HDL eliminate the cholesterol microdomains that were cell-surface associated. The deposition of cholesterol microdomains was inhibited by probucol, but it was increased by the liver X receptor (LXR) agonist TO901317, which upregulates ABCA1 and ABCG1. Extracellular cholesterol microdomains did not develop when ABCG1-deficient mouse bone marrow-derived macrophages were enriched with cholesterol. Our findings show that generation of extracellular cholesterol microdomains is mediated by ABCG1 and that reverse cholesterol transport occurs not only at the cell surface but also within the extracellular space.

ABCG1-mediated generation of extracellular cholesterol microdomains.

Noncoding gene variants at the SORT1 locus are strongly associated with low-density lipoprotein cholesterol (LDL-C) levels, as well as with coronary artery disease. SORT1 encodes a protein called sortilin, and hepatic sortilin modulates LDL metabolism by targeting apolipoprotein B-containing lipoproteins to the lysosome. Sortilin is also expressed in macrophages, but its role in macrophage uptake of LDL and in atherosclerosis independent of plasma LDL-C levels is unknown.

Macrophage sortilin promotes LDL uptake, foam cell formation, and atherosclerosis.

Probucol, an agent characterized by lipid-lowering and antioxidant property, retards atherosclerosis effectively. To test the hypothesis that probucol might act its antiatherosclerotic role by suppressing immune maturation of dendritic cells (DCs), 7-week-old LDLR mice were rendered diabetic with streptozotocin (STZ) and then fed either a high-fat diet only or added with 0.5% (wt/wt) probucol for 4 months, and human monocyte-derived dendritic cells were preincubated with or without probucol and stimulated by oxidized low-density lipoprotein. In STZ-induced diabetic LDLR mice, probucol treatment significantly lowered plasma total cholesterol and high-density lipoprotein-cholesterol levels; regressed aortic atherosclerotic lesions; reduced splenic CD40, CD80, CD86, MHC-II expression, and plasma IL-12p70 production; and decreased the expression of CD11c DCs within atherosclerotic lesions. In vitro, oxidized low-density lipoprotein promoted human monocyte-derived dendritic cells maturation; stimulated CD40, CD86, CD1a, HLA-DR expression; increased tumor necrosis factor-α production; and decreased IL-4 production. However, these effects were obviously inhibited by probucol pretreatment. In conclusion, our study indicated that probucol effectively retarded atherosclerosis at least partly through lipid-lowering and inhibiting immune maturation of CD11c DCs in STZ-induced diabetic LDLR mice.

Probucol Protects Against Atherosclerosis Through Lipid-lowering and Suppressing Immune Maturation of CD11c+ Dendritic Cells in STZ-induced Diabetic LDLR-/- Mice.

This technical report addresses the problem of endotoxin contamination of apolipoprotein reagents. Using a bromodeoxyuridine incorporation cell proliferation assay, we observed that human plasma ApoA-I as low as 1 μg/ml resulted in a >90% inhibition in macrophage proliferation. However, not all ApoA-I from different sources showed this effect. We considered the possibility that endotoxin contamination of the apolipoproteins contributed to the differential inhibition of macrophage cell proliferation. Endotoxin alone very potently inhibited macrophage proliferation (0.1 ng/ml inhibited macrophage proliferation>90%). Measurement of endotoxin levels in the apolipoprotein products, including an analysis of free versus total endotoxin, the latter which included endotoxin that was masked due to binding to protein, suggested that free endotoxin mediated inhibition of macrophage proliferation. Despite the use of an advanced endotoxin removal procedure and agents commonly used to inhibit endotoxin action, the potency of endotoxin precluded successful elimination of endotoxin effect. Our findings show that endotoxin contamination can significantly influence apparent apolipoprotein-mediated cell effects (or effects of any other biological products), especially when these products are tested on highly endotoxin-sensitive cells, such as macrophages.

Endotoxin contamination of apolipoprotein A-I: effect on macrophage proliferation--a cautionary tale.

LCAT, a plasma enzyme that esterifies cholesterol, has been proposed to play an antiatherogenic role, but animal and epidemiologic studies have yielded conflicting results. To gain insight into LCAT and the role of free cholesterol (FC) in atherosclerosis, we examined the effect of LCAT over- and underexpression in diet-induced atherosclerosis in scavenger receptor class B member I-deficient [Scarab(-/-)] mice, which have a secondary defect in cholesterol esterification. Scarab(-/-)×LCAT-null [Lcat(-/-)] mice had a decrease in HDL-cholesterol and a high plasma ratio of FC/total cholesterol (TC) (0.88 ± 0.033) and a marked increase in VLDL-cholesterol (VLDL-C) on a high-fat diet. Scarab(-/-)×LCAT-transgenic (Tg) mice had lower levels of VLDL-C and a normal plasma FC/TC ratio (0.28 ± 0.005). Plasma from Scarab(-/-)×LCAT-Tg mice also showed an increase in cholesterol esterification during in vitro cholesterol efflux, but increased esterification did not appear to affect the overall rate of cholesterol efflux or hepatic uptake of cholesterol. Scarab(-/-)×LCAT-Tg mice also displayed a 51% decrease in aortic sinus atherosclerosis compared with Scarab(-/-) mice (P < 0.05). In summary, we demonstrate that increased cholesterol esterification by LCAT is atheroprotective, most likely through its ability to increase HDL levels and decrease pro-atherogenic apoB-containing lipoprotein particles.

Increased plasma cholesterol esterification by LCAT reduces diet-induced atherosclerosis in SR-BI knockout mice.

We previously reported that cholesterol-enriched macrophages excrete cholesterol into the extracellular matrix. A monoclonal antibody that detects cholesterol microdomains labels the deposited extracellular particles. Macro-phage deposition of extracellular cholesterol depends, in part, on ABCG1, and this cholesterol can be mobilized by HDL components of the reverse cholesterol transport process. The objective of the current study was to determine whether ABCA1 also contributes to macrophage deposition of extracellular cholesterol. ABCA1 functioned in extracellular cholesterol deposition. The liver X receptor agonist, TO901317 (TO9), an ABCA1-inducing factor, restored cholesterol deposition that was absent in cholesterol-enriched ABCG1(-/-) mouse macrophages. In addition, the ABCA1 inhibitor, probucol, blocked the increment in cholesterol deposited by TO9-treated wild-type macrophages, and completely inhibited deposition from TO9-treated ABCG1(-/-) macrophages. Lastly, ABCA1(-/-) macrophages deposited much less extracellular cholesterol than wild-type macrophages. These findings demonstrate a novel function of ABCA1 in contributing to macrophage export of cholesterol into the extracellular matrix.

Xueting Jin

Experimental Atherosclerosis Section,
National Heart,
Lung,
and Blood Institute,
Experimental Atherosclerosis Section, National Heart, Lung, and Blood Institute

National Institutes of Health

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

Xueting Jin

Experimental Atherosclerosis Section,National Heart,Lung,and Blood Institute,Experimental Atherosclerosis Section, National Heart, Lung, and Blood Institute

National Institutes of Health

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

Experimental Atherosclerosis Section,
National Heart,
Lung,
and Blood Institute,
Experimental Atherosclerosis Section, National Heart, Lung, and Blood Institute