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Caveolae and caveolin-1 mediate endocytosis and transcytosis of oxidized low density lipoprotein in endothelial cells.

Sun SW, Zu XY, Tuo QH, Chen LX, Lei XY, Li K, Tang CK, Liao DF - Acta Pharmacol. Sin. (2010)

Bottom Line: Caveolae inhibitors - carrageenan (250 μg/mL), filipin (5 μg/mL), and nocodazole (33 μmol/L)-decreased the transport of ox-LDL across the monolayer by 48.9%, 72.4%, and 79.8% as compared to the control group.In addition, they effectively decreased ox-LDL uptake and inhibited the efflux of ox-LDL.Caveolin-1 and LOX-1 were up-regulated by ox-LDL in a time-dependent manner and decreased gradually after depletion of ox-LDL (P<0.05).

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacy and Pharmacology, University of South China, Hengyang, China.

ABSTRACT

Aim: To explore the mechanisms involved in ox-LDL transcytosis across endothelial cells and the role of caveolae in this process.

Methods: An in vitro model was established to investigate the passage of oxidized low density lipoprotein (ox-LDL) through a tight monolayer of human umbilical vein endothelial cells (HUVEC) cultured on a collagen-coated filter. Passage of DiI-labeled ox-LDL through the monolayer was measured using a fluorescence spectrophotometer. The uptake and efflux of ox-LDL by HUVEC were determined using fluorescence microscopy and HPLC.

Results: Caveolae inhibitors - carrageenan (250 μg/mL), filipin (5 μg/mL), and nocodazole (33 μmol/L)-decreased the transport of ox-LDL across the monolayer by 48.9%, 72.4%, and 79.8% as compared to the control group. In addition, they effectively decreased ox-LDL uptake and inhibited the efflux of ox-LDL. Caveolin-1 and LOX-1 were up-regulated by ox-LDL in a time-dependent manner and decreased gradually after depletion of ox-LDL (P<0.05). After treatment HUVEC with ox-LDL and silencing caveolin-1, NF-κB translocation to the nucleus was blocked and LOX-1 expression decreased (P<0.05).

Conclusion: Caveolae can be a carrier for ox-LDL and may be involved in the uptake and transcytosis of ox-LDL by HUVEC.

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Related in: MedlinePlus

Effects of caveolae-specific inhibitors on ox-LDL efflux by HUVECs. HUVECs were incubated with ox-LDL (40 μg/mL) for 24 h. After lipid loading, HUVECs were washed twice with PBS, and incubated for another 6, 12, and 24 h in an ox-LDL depleted medium with or without the caveolae inhibitors. After incubation, the cholesterol in the cytoplasm (A) and cholesterol in medium (B) were analyzed by HPLC. Similar results were obtained in six independent experiments. Data are mean±SD. bP<0.05 vs corresponding groups at 0 h.
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fig3: Effects of caveolae-specific inhibitors on ox-LDL efflux by HUVECs. HUVECs were incubated with ox-LDL (40 μg/mL) for 24 h. After lipid loading, HUVECs were washed twice with PBS, and incubated for another 6, 12, and 24 h in an ox-LDL depleted medium with or without the caveolae inhibitors. After incubation, the cholesterol in the cytoplasm (A) and cholesterol in medium (B) were analyzed by HPLC. Similar results were obtained in six independent experiments. Data are mean±SD. bP<0.05 vs corresponding groups at 0 h.

Mentions: As described in the Introduction, we hypothesized that caveolae are involved in transcytosis of ox-LDL. If that is the case, efflux of ox-LDL is also expected to be slow when caveolae are inhibited. To examine whether the inhibition of caveolae decreased ox-LDL efflux, the cholesterol content in the medium and cellular cholesterol was determined after treatment with inhibitors. We incubated HUVEC with ox-LDL for 24 h. After lipid loading, HUVEC were washed twice with PBS and incubated for another 6, 12, and 24 h in an ox-LDL depleted medium in the absence or presence of the caveolae inhibitors. After incubation, the cholesterol content in the medium and cellular cholesterol were analyzed by HPLC. Caveolae inhibitors significantly reversed the decrease in cellular cholesterol and the increase in the cholesterol content in the medium (Figure 3). These data suggest that caveolae are involved in the efflux of ox-LDL by HUVEC.


Caveolae and caveolin-1 mediate endocytosis and transcytosis of oxidized low density lipoprotein in endothelial cells.

Sun SW, Zu XY, Tuo QH, Chen LX, Lei XY, Li K, Tang CK, Liao DF - Acta Pharmacol. Sin. (2010)

Effects of caveolae-specific inhibitors on ox-LDL efflux by HUVECs. HUVECs were incubated with ox-LDL (40 μg/mL) for 24 h. After lipid loading, HUVECs were washed twice with PBS, and incubated for another 6, 12, and 24 h in an ox-LDL depleted medium with or without the caveolae inhibitors. After incubation, the cholesterol in the cytoplasm (A) and cholesterol in medium (B) were analyzed by HPLC. Similar results were obtained in six independent experiments. Data are mean±SD. bP<0.05 vs corresponding groups at 0 h.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4012904&req=5

fig3: Effects of caveolae-specific inhibitors on ox-LDL efflux by HUVECs. HUVECs were incubated with ox-LDL (40 μg/mL) for 24 h. After lipid loading, HUVECs were washed twice with PBS, and incubated for another 6, 12, and 24 h in an ox-LDL depleted medium with or without the caveolae inhibitors. After incubation, the cholesterol in the cytoplasm (A) and cholesterol in medium (B) were analyzed by HPLC. Similar results were obtained in six independent experiments. Data are mean±SD. bP<0.05 vs corresponding groups at 0 h.
Mentions: As described in the Introduction, we hypothesized that caveolae are involved in transcytosis of ox-LDL. If that is the case, efflux of ox-LDL is also expected to be slow when caveolae are inhibited. To examine whether the inhibition of caveolae decreased ox-LDL efflux, the cholesterol content in the medium and cellular cholesterol was determined after treatment with inhibitors. We incubated HUVEC with ox-LDL for 24 h. After lipid loading, HUVEC were washed twice with PBS and incubated for another 6, 12, and 24 h in an ox-LDL depleted medium in the absence or presence of the caveolae inhibitors. After incubation, the cholesterol content in the medium and cellular cholesterol were analyzed by HPLC. Caveolae inhibitors significantly reversed the decrease in cellular cholesterol and the increase in the cholesterol content in the medium (Figure 3). These data suggest that caveolae are involved in the efflux of ox-LDL by HUVEC.

Bottom Line: Caveolae inhibitors - carrageenan (250 μg/mL), filipin (5 μg/mL), and nocodazole (33 μmol/L)-decreased the transport of ox-LDL across the monolayer by 48.9%, 72.4%, and 79.8% as compared to the control group.In addition, they effectively decreased ox-LDL uptake and inhibited the efflux of ox-LDL.Caveolin-1 and LOX-1 were up-regulated by ox-LDL in a time-dependent manner and decreased gradually after depletion of ox-LDL (P<0.05).

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacy and Pharmacology, University of South China, Hengyang, China.

ABSTRACT

Aim: To explore the mechanisms involved in ox-LDL transcytosis across endothelial cells and the role of caveolae in this process.

Methods: An in vitro model was established to investigate the passage of oxidized low density lipoprotein (ox-LDL) through a tight monolayer of human umbilical vein endothelial cells (HUVEC) cultured on a collagen-coated filter. Passage of DiI-labeled ox-LDL through the monolayer was measured using a fluorescence spectrophotometer. The uptake and efflux of ox-LDL by HUVEC were determined using fluorescence microscopy and HPLC.

Results: Caveolae inhibitors - carrageenan (250 μg/mL), filipin (5 μg/mL), and nocodazole (33 μmol/L)-decreased the transport of ox-LDL across the monolayer by 48.9%, 72.4%, and 79.8% as compared to the control group. In addition, they effectively decreased ox-LDL uptake and inhibited the efflux of ox-LDL. Caveolin-1 and LOX-1 were up-regulated by ox-LDL in a time-dependent manner and decreased gradually after depletion of ox-LDL (P<0.05). After treatment HUVEC with ox-LDL and silencing caveolin-1, NF-κB translocation to the nucleus was blocked and LOX-1 expression decreased (P<0.05).

Conclusion: Caveolae can be a carrier for ox-LDL and may be involved in the uptake and transcytosis of ox-LDL by HUVEC.

Show MeSH
Related in: MedlinePlus