Difference in LDL Receptor Feedback Regulation in Macrophages and Vascular Smooth Muscle Cells: Foam Cell Transformation Under Inflammatory Stress

Abstract

Macrophages and vascular smooth muscle cells (VSMCs) are the major cell types involved in foam cell formation associated with atherosclerosis. The aim of this experiment was to clarify cell-specific regulation of LDL receptor in THP-1 macrophages and human VSMCs under physiological and inflammatory conditions and its potential mechanisms. Inflammatory stress was induced by adding lipopolysaccharide (LPS) to human THP-1 macrophages and human VSMCs. Intracellular total cholesterol, free cholesterol, and cholesterol ester were measured by an enzymic assay. Oil Red O staining was used to visualize lipid droplet accumulation in cells. Total cellular RNA was isolated from cells for detecting LDL receptor, sterol regulatory element binding protein (SREBP)-2 and SREBP cleavage-activating protein (SCAP) mRNA levels using real-time quantitative polymerase chain reaction. LDL receptor, SREBP-2 and SCAP protein expression were examined by Western blotting. The translocation of SCAP from ER to Golgi was detected by confocal microscopy. LDL loading increased intracellular cholesterol level, reducing LDL receptor mRNA level in both THP-1 macrophages and VSMCs under physiological conditions. The IC50 in VSMCs was 11.25 μg/ml, which is much lower than 18.125 μg/ml in THP-1 macrophages. With the increase in concentration of LPS (0-400 ng/ml), the LDL receptor mRNA levels were upregulated in both cells, but the curve of LDL receptor mRNA in VSMCs exhibited a flatter profile than that of THP-1 macrophages. Under the treatment of 200 ng/ml of LPS, the upregulation fold of the LDL receptor mRNA in THP-1 macrophages was much higher than that of VSMCs (0.33 vs 0.04). LDL receptor blocking agent heparin decreased lipid droplets induced by LPS significantly in THP-1 macrophages and VSMCs. LDL loading reduced the SREBP2 and SCAP protein expression under physiological conditions. Exposure to LPS caused overexpression of SREBP2 and SCAP despite a high concentration of LDL in the culture medium, and increased translocation of SCAP from the ER to the Golgi in the presence of 25 μg/ml of LDL. Inflammatory stress disrupts LDL receptor negative feedback regulation induced by intracellular cholesterol in both cell types, to a greater degree in THP-1 macrophages, which could be one reason why THP-1 macrophages are more prone to become foam cells under inflammatory stress.

High‐fat diet‐induced GAIT element‐mediated translational silencing of mRNAs encoding inflammatory proteins in macrophage protects against atherosclerosis

“…Therefore, macrophage LDLR expression may have a relevant impact in modulation of foam cell formation. The significance of this observation is highlighted by prior studies demonstrating the role of LDLR in inflammation‐induced unmodified LDL accumulation, 40,41 foam cell formation 39 and insensitivity of HFD‐induced atherosclerosis in response to loss of SR‐A and CD36 pathways 38 . In addition, our finding of increased phagocytic activity of BMM from L13a‐deficient mice vs controls (Figure 4D) is notable given the fact that phagocytosis is clearly involved in the pathogenesis of atherosclerosis, albeit with roles that are complex and currently not fully defined 42 …”

“…However, an alternative, scavenger receptor‐independent, 38 mechanism of lipid uptake, and foam cell formation mediated by native low‐density lipoprotein (LDL) has also been reported 39 . Supporting the potential importance of this pathway, several recent studies demonstrated a significant role of inflammatory stress in the native LDL uptake and LDL receptor (LDLR) mediated pathways of foam cell formation 40 and in disruption of a LDLR negative feedback loop 41 …”

Role of the Scavenger Receptor CD36 in Accelerated Diabetic Atherosclerosis

“…However, CD36 is not the only mediator of oxLDL uptake. Other scavenger receptors for lipids, such as lectin-like oxidized LDL receptor-1 (LOX-1), low-density lipoprotein receptor-related protein (LRP1), and class A scavenger receptor (SR-A), internalize oxLDL and may be upregulated in VSMC by oxLDL [ 14 , 36 , 37 , 38 ]. On the contrary, the silencing of CD36 did not affect the upregulated expression of pro-inflammatory markers by HG and oxLDL, suggesting that the effects observed by the exposure to HG with or without the presence of oxLDL cannot be explained by the unique action of CD36-derived signaling.…”

Ruxolitinib and Polycation Combination Treatment Overcomes Multiple Mechanisms of Resistance of Pancreatic Cancer Cells to Oncolytic Vesicular Stomatitis Virus

“…Other tested conditions were cell starvation (0% fetal bovine serum [FBS] medium), which could increase LDLR levels (47), and the addition of unlabeled LDL, which could decrease LDLR levels (48)(49)(50). After 24 h of treatment, cell monolayers were incubated with VSV for 1 h at 4°C to examine VSV attachment by using Western blotting.…”