Enzymatically Modified Low-Density Lipoprotein Promotes Foam Cell Formation in Smooth Muscle Cells via Macropinocytosis and Enhances Receptor-Mediated Uptake of Oxidized Low-Density Lipoprotein

Abstract

Objective: Enzyme-modified nonoxidized low-density lipoprotein (ELDL) is present in human atherosclerotic lesions. Our objective is to understand the mechanisms of ELDL uptake and its effects on vascular smooth muscle cells (SMC).

Approach and results: Transformation of murine aortic SMCs into foam cells in response to ELDL was analyzed. ELDL, but not acetylated or oxidized LDL, was potent in inducing SMC foam cell formation. Inhibitors of macropinocytosis (LY294002, wortmannin, amiloride) attenuated ELDL uptake. In contrast, inhibitors of receptor-mediated endocytosis (dynasore, sucrose) and inhibitor of caveolae-/lipid raft-mediated endocytosis (filipin) had no effect on ELDL uptake in SMC, suggesting that macropinocytosis is the main mechanism of ELDL uptake by SMC. Receptor for advanced glycation end products (RAGE) is not obligatory for ELDL-induced SMC foam cell formation, but primes SMC for the uptake of oxidized LDL in a RAGE-dependent manner. ELDL increased intracellular reactive oxygen species, cytosolic calcium, and expression of lectin-like oxidized LDL receptor-1 in wild-type SMC but not in RAGE(-/-) SMC. The macropinocytotic uptake of ELDL is regulated predominantly by intracellular calcium because ELDL uptake was completely inhibited by pretreatment with the calcium channel inhibitor lacidipine in wild-type and RAGE(-/-) SMC. This is in contrast to pretreatment with PI3 kinase inhibitors which completely prevented ELDL uptake in RAGE(-/-) SMC, but only partially in wild-type SMC.

Conclusions: ELDL is highly potent in inducing foam cells in murine SMC. ELDL endocytosis is mediated by calcium-dependent macropinocytosis. Priming SMC with ELDL enhances the uptake of oxidized LDL.

Keywords: amiloride; atherosclerosis; endocytosis; foam cells; wortmannin.

Figure 1. ELDL, but not acetylated or oxidized LDL, induces foam cells in cultured mouse aortic SMC

A. Peritoneal macrophages (top row) or aortic SMC (bottom row) were incubated with 10 μg/ml (based on lipoprotein protein) of native LDL, acetylated LDL (AcLDL), oxidized LDL (OxLDL) or Enzyme-modified LDL (trypsin and cholesterol esterase modified LDL, ELDL) for 24h. The cells were stained for lipids with Oil Red O. B. Total cellular cholesterol/protein from experiments shown in A. Data are represented as mean ± SD. (** p<0.01, AcLDL, OxLDL & ELDL vs. native LDL) C. Aortic SMC were incubated with 10, 25 and 50 μg/ml (based on protein) of ELDL, 2000 μg/ml native LDL, 500 μg/ml AcLDL, and 200 μg/ml OxLDL and stained for lipids with Oil Red O. D. Total cellular cholesterol/protein from experiments shown in C. Data are represented as mean ± SD. p<0.01 ELDL (all tested concentrations of 10, 25 and 50 μg/ml) vs. native LDL (2000 μg/ml) or OxLDL (200 μg/ml). p< 0.01 ELDL (50 μg/ml) vs AcLDL(500 μg/ml), p<0.05 AcLDL (500 μg/ml) vs LDL (2000 μg/ml) or OxLDL (200 μg/ml). P<0.05 for native LDL 2000 μg/ml vs 0 or 10 μg/ml native LDL.

Figure 2. Aggregated/fused ELDL is preferentially taken up by aortic SMC

A. Agarose gel electrophoresis of native LDL, ELDL (trypsin and cholesterol esterase modified LDL), CLDL (cholesterol esterase modified LDL), TLDL (trypsin modified LDL), AcLDL and OxLDL. Arrows indicate a LDL with reduced mobility that is indicative of fused LDLs or lower negative charge. B. Protein, cholesterol and phospholipid content in ELDL fractions after separation by FPLC. C. Foam cell formation in aortic SMC incubated with 5 μg/ml of the indicated FPLC fractions; fraction numbers are shown in the panels. Cells were stained for lipids with Oil Red O. D. Total cellular cholesterol/mg protein from experiments shown in C. Data are represented as mean ± SD (** p<0.01, Fractions 8 &10 vs 16, 18, 22 and 24).

Figure 3. ELDL up regulates expression of oxidized LDL receptor (LOX-1) in aortic SMC and promotes uptake of OxLDL

A. qRT PCR analysis of scavenger receptor gene expression in mouse aortic SMCs; wild type SMC were incubated with 10 μg/ml of native LDL, ELDL, OxLDL, or AcLDL for 24h (expression is relative to levels in cells incubated with native LDL). Data are represented as mean ± SD, for LOX-1 * p<0.05 for OxLDL vs native LDL, ** p<0.01 for ELDL vs native LDL. B. Semi-quantitative expression of LOX-1 protein expression in SMC treated as indicated in Panel A, ** p<0.01 for ELDL vs native LDL. C. LOX-1 protein expression in WT and RAGE−/− SMC upon pretreatment with ELDL as indicated and densitometric analysis. ** p<0.01 either 50, 25 or 10 μg/ml vs 0 μg/ml. D. qRT PCR analysis of mRNA in WT and RAGE^−/− SMC incubated with 10 μg/ml ELDL versus control (10 μg/ml native LDL) ** p<0.01 for ELDL vs native LDL. E. Fluorescent images of WT or RAGE^−/− SMC pretreated with bovine serum albumin (BSA) or ELDL for 24h, followed by incubation for 24h with DiI-labeled LDL (red color) or DiI-labeled OxLDL (red color). ELDL was removed prior to adding DiI-labelled LDL or OxLDL. Nuclei were stained in blue with Hoechst. F. Fluorescence intensity from experiments in E was quantified using Image J as a function of total pixel density per total area of cells in frame (refer methods for details). G. Foam cell formation assessed by Oil red O staining in RAGE^−/− SMC incubated for 24h with 10 μg/ml ELDL or native LDL (negative control). H. Total cellular cholesterol/protein from experiments shown in G.

Figure 4. ELDL endocytosis by aortic SMC is mediated by macropinocytosis and is not receptor/clathrin mediated or lipid raft/caveolae dependent

A. Foam cell formation assessed by Oil red O staining in aortic SMC incubated for 24h with 10 μg/ml ELDL (positive control) or native LDL as negative control. Prior to incubation with lipoproteins, cells were pretreated for 1h with inhibitors of receptor mediated endocytosis (100 μM dynasore or 0.1M sucrose). Pharmacologic inhibitors were not removed from media until end of experiment. B. Aortic SMC pre-incubated for 1h in serum free medium with 5 μM filipin to inhibit lipid raft/caveolae mediated endocytosis followed by incubation with 10 μg/ml ELDL or native LDL for 24h. C. Aortic SMC were pre-incubated for 1h with 3 mM amiloride to inhibit macropinocytosis, followed by incubation with 10 μg/ml ELDL or native LDL for 24h (amiloride was not removed from the media prior to adding ELDL). D. Total cellular cholesterol/protein from experiments shown in A, B and C. Data are represented as mean ± SD (** p<0.01 ELDL untreated vs. ELDL amiloride treated)

Figure 5. PIP-independent macropinocytotic uptake of ELDL in WT SMC

Foam cell formation assessed by Oil red O staining in A. WT SMC and B. RAGE^−/− SMC incubated for 24h with 10 μg/ml ELDL or native LDL (negative control). The cells were pretreated for 1h with PI3K inhibitors (20 to 300 μM LY294002 or 1 to 2 μM wortmannin) prior to adding ELDL; inhibitors were not removed from media until end of experiment. C. Total cellular cholesterol/protein in cells from panels A and B. Data are represented as mean ± SD.

Figure 6. Macropinocytotic uptake of ELDL by SMC: role for ROS and intracellular calcium

A. WT and RAGE^−/− SMC were incubated with 10 μg/ml ELDL, and ROS expression was analyzed in live cells by staining with H₂DCFDA at time points as indicated. B. WT and RAGE^−/− SMC were incubated with 10 μg/ml ELDL and cytosolic calcium was analyzed in live cells by staining with Fluo-8AM at time points as indicated. WT SMC were also pretreated with amiloride for 1h prior to adding ELDL (amiloride was not removed prior to the addition of ELDL). C/D. Fluorescence intensity from A and B quantified using Image J. Data are represented as mean ± SD. E. WT and RAGE^−/− SMC were incubated with 10 μg/ml ELDL after pretreated for 1h with the calcium channel inhibitor lacidipine (30 μM). Foam cell formation was analyzed by Oil Red O staining at 4h and 24h after ELDL loading. F. Total cellular cholesterol/protein from experiments shown in E. Data are represented as mean ± SD. (** p<0.01,* p<0.05). G. Schematic model of the role of RAGE in ELDL endocytosis in mouse aortic smooth muscle cells. PIP2 and PIP3 are plasma membrane inositides. PIP2 is phosphorylated to PIP3 by PI3K, and blocking PIP3 formation with PI3K inhibitors (LY294002, wortmannin) are known strategies to impair macropinocytosis. PI3K inhibitors partially prevented macropinocytosis of ELDL in SMC in contrast to the complete inhibition of macropinocytosis of ELDL in SMC deficient for RAGE treated with the PI3K inhibitors. ELDL-RAGE signaling down regulates PIP3; nevertheless, ROS and cellular Ca²⁺ levels are upregulated by ELDL in the SMC. Ca²⁺ is also an important determinant of macropinocytosis whose mechanism of action is unknown. Despite PIP3 inhibition by ELDL, we see unhindered macropinocytosis of ELDL, which suggests elevated cellular Ca²⁺ is potent enough to override the inhibition of PIP3 by ELDL via some unknown mechanisms.