Scavenger receptors of endothelial cells mediate the uptake and cellular proatherogenic effects of carbamylated LDL

Abstract

Objective: Carbamylated LDL (cLDL) has been recently shown to have robust proatherogenic effects on human endothelial cells in vitro, suggesting cLDL may have a significant role in atherosclerosis in uremia. The current study was designed to determine which receptors are used by cLDL and thus cause the proatherogenic effects.

Methods and results: In ex vivo or in vitro models as well as in intact animals, administration of cLDL was associated with endothelial internalization of cLDL and subendothelial translocation (transcytosis). In vitro recombinant LOX-1 and SREC-1 receptors showed the greatest cLDL binding. However, pretreatment of the endothelial cells with specific inhibiting antibodies demonstrated that cLDL binds mainly to LOX-1 and CD36 receptors. The transcytosis was dependent on SR-A1, SREC-1, and CD36 receptors whereas LOX-1 receptor was not involved. The cytotoxicity was mediated by several studied scavenger receptors, but cLDL-induced monocyte adhesion depended only on LOX-1. The cLDL-induced synthesis of LOX-1 protein significantly contributed to both cytotoxicity and accelerated monocyte adhesion to endothelial cells.

Conclusions: Our data suggest that cLDL uses a unique pattern of scavenger receptors. They show that LOX-1 receptor, and partially CD36, SREC-1, and SR-A1 receptors, are essential for the proatherogenic effects of cLDL on human endothelial cells.

Figure 1

Accumulation of ¹²⁵I-cLDL or ¹²⁵I-nLDL in tissues. The amounts of both LDLs remaining in plasma (A) or present in tissues (B) were measured 24 hours later. n=10; *P<0.05 as compared to 0 hours, #P<0.05 as compared to nLDL. Accumulation of AF488-labeled nLDL and AF488-labeled cLDL is shown in the aorta in vivo (C) and in the heart ex vivo (D). Scale, 150 μm. n=10; *P<0.05 as compared to control, #P<0.05 as compared to nLDL.

Figure 2

Endothelial internalization and subendothelial transfer of cLDL in vitro. Internalization of AF594-labeled LDLs to HCAECs was detected by confocal microscopy (A) and fluorimetry in a dose-dependent (B) and a time-dependent manner (C). Small attached plots are presented to illustrate the data legibility at small doses and early time-points. n=4 per time/dose point; *P<0.05 as compared to nLDL; #P<0.05 as compared to oxLDL. Transfer of LDLs (25 μg/ml, 24 hours) through endothelial monolayer (D). n=6 per point, *P<0.05 as compared to nLDL, #P<0.05 as compared to oxLDL.

Figure 3

Antibody inhibition (10 μg/ml) of endothelial internalization (A) and subendothelial translocation (B) of fluorescently-labeled LDLs (25 μg/ml, 4 hours). n=4 per point; *P<0.05 as compared to an nLDL; #P<0.05 as compared to pretreated with non-specific IgGs. Inhibition of cytotoxicity (C) and monocyte adhesion (D) induced by cLDL, nLDL or oxLDL (200 μg/ml, 16 or 24 hours for cytotoxicity and monocyte adhesion, respectively) using the antibodies to LOX-1, SREC-1, CD36 or SR-A1 (10 μg/ml). n=4 per point; *P<0.05 as compared to cells pretreated with IgGs; #P<0.05 as compared to vehicle (PBS, 200 μM EDTA)-treated cells; ¶P<0.05 as compared to nLDL-treated cells.

Figure 4

Cell ELISA measurements of the LDLR, VLDLR, LOX-1, SREC-1, CD36 and SR-A1 protein expressions in HCAECs after treatment with nLDL or cLDL at concentration of 12.5 μg/ml (A) and 200 μg/ml (B) for 2, 10 or 24 hours as compared to baseline level (dashed line at 100%). n = 3-4 per point; *P<0.05 as compared to 0 hours time point; #P<0.05 as compared to vehicle-treated cells; ¶P<0.05 as compared to nLDL-treated cells. (C) Representative images of LOX-1 protein expression after cLDL or nLDL treatment (200 μg/mL, 24 hours) as determined by immunocytochemistry. Scale bar, 10 μm. (D) Expression of LOX-1 mRNA in endothelial cells after cLDL or nLDL treatment (200 μg/mL, 24 hours) as measured by real-time RT-PCR. n = 3 per point; *P<0.05 as compared to vehicle-treated cells; #P<0.05 as compared to nLDL-treated cells.

Figure 5

Inhibition of cytotoxicity (A) and monocyte adhesion (B) to HCAECs induced by cLDL, nLDL or oxLDL (200 μg/ml, 16 or 24 hours for cytotoxicity and monocyte adhesion, respectively) using the siRNA to LOX-1 (50 nM, 48 hours). In the same experiment, ICAM-1 (C) and VCAM-1 (D) expression was measured by cell ELISA. n=4 per point; *P<0.05 as compared to cells treated with control siRNA; #P<0.05 as compared to vehicle-treated cells; ¶P<0.05 as compared to nLDL-treated cells.