Dissecting the Impact of Vascular Smooth Muscle Cell ABCA1 versus ABCG1 Expression on Cholesterol Efflux and Macrophage-like Cell Transdifferentiation: The Role of SR-BI

Abstract

Cholesterol-laden macrophages are recognized as a major contributor to atherosclerosis. However, recent evidence indicates that vascular smooth muscle cells (VSMC) that accumulate cholesterol and transdifferentiate into a macrophage-like cell (MLC) phenotype also play a role in atherosclerosis. Therefore, removing cholesterol from MLC may be a potential atheroprotective strategy. The two transporters which remove cholesterol from cells are ABCA1 and ABCG1, as they efflux cholesterol to apoAI and HDL, respectively. In this study, the well-characterized immortalized VSMC line MOVAS cells were edited to generate ABCA1- and ABCG1-knockout (KO) MOVAS cell lines. We cholesterol-loaded ABCA1-KO MOVAS cells, ABCG1-KO MOVAS cells, and wild-type MOVAS cells to convert cells into a MLC phenotype. When we measured apoAI- and HDL-mediated cholesterol efflux in these cells, we observed a drastic decrease in apoAI-mediated cholesterol efflux within ABCA1-KO MOVAS MLC, but HDL-mediated cholesterol efflux was only partially reduced in ABCG1-KO MOVAS cells. Since SR-BI also participates in HDL-mediated cholesterol efflux, we assessed SR-BI protein expression in ABCG1-KO MOVAS MLC and observed SR-BI upregulation, which offered a possible mechanism explaining why HDL-mediated cholesterol efflux remains maintained in ABCG1-KO MOVAS MLC. When we used lentivirus for shRNA-mediated knockdown of SR-BI in ABCG1-KO MOVAS MLC, this decreased HDL-mediated cholesterol efflux when compared to ABCG1-KO MOVAS MLC with unmanipulated SR-BI expression. Taken together, these major findings suggest that SR-BI expression in MLC of a VSMC origin plays a compensatory role in HDL-mediated cholesterol efflux when ABCG1 expression becomes impaired and provides insight on SR-BI demonstrating anti-atherogenic properties within VSMC/MLC.

Inhibition of miR-33a-5p in Macrophage-like Cells In Vitro Promotes apoAI-Mediated Cholesterol Efflux

“…We previously reported that lentivirus is capable of efficiently transducing MOVAS cells [ 28 ]. Therefore, we transduced control MOVAS cells with LV-Scr, and to inhibit miR-33a-5p, we also transduced cultured cells with LV-AntimiR33a5p.…”

“…We chose lentivirus to introduce anti-miR-33a-5p to MOVAS cells, since we and others [ 28 , 44 ] have already shown lentiviral vectors efficiently transduce this cell line and cultured VSMC. As with our prior report [ 28 ], we observed effective gene manipulation when transducing MOVAS cells.…”

“…We chose lentivirus to introduce anti-miR-33a-5p to MOVAS cells, since we and others [ 28 , 44 ] have already shown lentiviral vectors efficiently transduce this cell line and cultured VSMC. As with our prior report [ 28 ], we observed effective gene manipulation when transducing MOVAS cells. However, quantification of lentiviral transduction efficiency using flow cytometry [ 45 ] would likely be needed when initial transductions in VSMC/MLC do not provide intended efficacy, to better gauge if higher lentiviral transduction efficiency may be achieved or if other viral vectors which demonstrate robust VSMC transduction efficiency [ 46 ] should be selected instead for enhancing transduction potential.…”

“…Both vectors also express GFP [ 27 ]. MOVAS cells were transduced with lentivirus (MOI = 5) for 24 h [ 27 , 28 ], washed with PBS, and utilized for subsequent experiments. To evaluate lentiviral transduction efficiency, transduced MOVAS cells were plated in four-chamber tissue-culture slides (Corning) and used for imaging analyses, which was conducted at the Clemson Light Imaging Facility (CLIF).…”

“…We used the following procedures to measure cellular cholesterol efflux [ 28 ]. Briefly, transduced MOVAS cells were trans-differentiated into MLC using our published protocol [ 12 ] also described above.…”