Reactive oxygen species mediate angiotensin II-induced transcytosis of low-density lipoprotein across endothelial cells

Abstract

The retention of plasma low-density lipoprotein (LDL) particles to subendothelial spaces through transcytosis across the endothelium is the initial step of atherosclerosis (AS). Angiotensin II (Ang II), as the principal effector molecule of the renin-angiotensin system (RAS), is implicated in several important steps of AS development. However, whether or not Ang II can directly exert a pro‑atherogenic effect by promoting LDL transcytosis across endothelial barriers, has not been defined. In the present study, we found that Ang II upregulated intracellular reactive oxygen species (ROS) levels in endothelial cells (ECs) by measuring fluorescence of 2',7'-dichlorofluorescein (DCF‑DA). Based on our transcytosis model, we observed that Ang II significantly accelerated LDL transcytosis, whereas transcytosis inhibitor methyl-β-cyclodextrin (MβCD) and ROS inhibitor dithiothreitol (DTT), markedly blocked the Ang II-stimulated increase in LDL transcytosis. Confocal imaging analysis revealed that both LDL uptake by cells and LDL retention in human umbilical venous walls were highly elevated after Ang II exposure, while MβCD and DTT significantly inhibited the effects of Ang II. What is more, proteins involved in caveolae-mediated transcytosis, including LDL receptor (LDLR), caveolin-1 and cavin-1, were associated with Ang II-induced LDL transcytosis across the ECs. Nevertheless, this process was independent of clathrin in our study. Of note, ROS inhibitor, DTT, markedly decreased the expression levels of those proteins. Consequently, ROS are critical mediators in Ang II-induced LDL transcytosis. Hopefully, these findings will provide novel insight into the crosstalk between dyslipidemia and RAS in atherogenesis.

Figure 1

The established in vitro low-density lipoprotein (LDL) transcytosis model. Cells were seeded (~4×10⁴ cells/insert) on a polyester membrane of a Costar Transwell (6.5-mm diameter, 0.4-µm pore size) to form an integrated cell monolayer. Two inserts of cell monolayers with equal integrity were divided into the same group: the control insert and the naive insert, respectively. The control insert was stimulated with fluorescein isothiocyanate (FITC)-LDL to determine the total amount of LDL transport. Paracellular transport was analyzed by treatment with FITC-LDL and 6-fold excess of unlabeled LDL in the naive insert. After treatment, samples were collected from the outer chambers and further dialyzed against PBS to remove the free FITC. The relative fluorescence was measured via a fluorescence spectrophotometer.

Figure 2

(A) Reactive oxygen species (ROS) are involved in angiotensin II (Ang II)-induced low-density lipoprotein (LDL) transcytosis across human umbilical vein endothelial cells (HUVECs). HUVECs were pretreated with methyl-β-cyclodextrin (MβCD) or dithiothreitol (DTT) for 30 min, and then stimulated with fluorescein isothiocyanate (FITC)-LDL (and/or LDL) and Ang II for 24 h. After 24 h, samples were collected from the outer chambers and further dialyzed against PBS to remove the free FITC. (B) The relative fluorescence was measured via a fluorescence spectrophotometer. The amount of LDL transcytosis was normalized to that obtained in the control group. ^*P<0.05 vs. control; ^#P<0.05 vs. Ang II.

Figure 3

Angiotensin II (Ang II) stimulation increases the uptake of low-density lipoprotein (LDL) in human umbilical vein endothelial cells (HUVECs). HUVECs were first incubated with fluorescein isothiocyanate (FITC)-LDL for 24 h and then treated with or without Ang II, methyl-β-cyclodextrin (MβCD), and dithiothreitol (DTT) for 24 h at 37°C. (A) Confocal microscopy images of FITC-LDL uptake stimulated by Ang II alone or after pretreatment with MβCD/DTT in HUVECs. Scale bars, 50 µm. (B) Quantification summary of FITC-LDL uptake in HUVECs. ^*P<0.05 vs. control; ^#P<0.05 vs. Ang II.

Figure 4

Angiotensin II (Ang II) stimulation increases the retention of low-density lipoprotein (LDL) in human umbilical venous walls. The human umbilical venous rings were bubbled with a mixed gas (95% O₂, 5% CO₂) and incubated with fluorescein isothiocyanate (FITC)-LDL, and/or Ang II, methyl-β-cyclodextrin (MβCD), dithiothreitol (DTT) for 3 h at 37°C. (A) Confocal microscopy images of FITC-LDL retention stimulated by Ang II alone or after pretreatment with MβCD/DTT in human umbilical venous rings. Scale bars, 100 µm. (B) Quantification summary of FITC-LDL retention in vessels. ^*P<0.05 vs. control; ^#P<0.05 vs. Ang II.

Figure 5

Angiotensin II (Ang II) stimulates the expression of molecules involved in low-density lipoprotein (LDL) transcytosis. Human umbilical vein endothelial cells (HUVECs) were pretreated with methyl-β-cyclodextrin (MβCD) or dithiothreitol (DTT) for 30 min, and then stimulated with or without Ang II for 24 h. (A) Representative western blots showing the expression of LDL receptor (LDLR), caveolin-1, cavin-1 and clathrin. (B) Quantitative analysis of the expression of proteins. ^*P<0.05 vs. control; ^#P<0.05 vs. Ang II.