Constructing Lipoparticles Capable of Endothelial Cell-Derived Exosome-Mediated Delivery of Anti-miR-33a-5p to Cultured Macrophages.

Atherosclerosis is driven by intimal arterial macrophages accumulating cholesterol. Atherosclerosis also predominantly occurs in areas consisting of proinflammatory arterial endothelial cells. At time of writing, there are no available clinical treatments that precisely remove excess cholesterol from lipid-laden intimal arterial macrophages. Delivery of anti-miR-33a-5p to macrophages has been shown to increase apoAI-mediated cholesterol efflux via ABCA1 upregulation but delivering transgenes to intimal arterial macrophages is challenging due to endothelial cell barrier integrity. In this study, we aimed to test whether lipoparticles targeting proinflammatory endothelial cells can participate in endothelial cell-derived exosome exploitation to facilitate exosome-mediated transgene delivery to macrophages. We constructed lipoparticles that precisely target the proinflammatory endothelium and contain a plasmid that expresses XMOTIF-tagged anti-miR-33a-5p (LP-pXMoAntimiR33a5p), as XMOTIF-tagged small RNA demonstrates the capacity to be selectively shuttled into exosomes. The cultured cells used in our study were immortalized mouse aortic endothelial cells (iMAECs) and RAW 264.7 macrophages. From our results, we observed a significant decrease in miR-33a-5p expression in macrophages treated with exosomes released basolaterally by LPS-challenged iMAECs incubated with LP-pXMoAntimiR33a5p when compared to control macrophages. This decrease in miR-33a-5p expression in the treated macrophages caused ABCA1 upregulation as determined by a significant increase in ABCA1 protein expression in the treated macrophages when compared to the macrophage control group. The increase in ABCA1 protein also simulated ABCA1-dependent cholesterol efflux in treated macrophages-as we observed a significant increase in apoAI-mediated cholesterol efflux-when compared to the control group of macrophages. Based on these findings, strategies that involve combining proinflammatory-targeting lipoparticles and exploitation of endothelial cell-derived exosomes appear to be promising approaches for delivering atheroprotective transgenes to lipid-laden arterial intimal macrophages.

miR-33a Expression Attenuates ABCA1-Dependent Cholesterol Efflux and Promotes Macrophage-Like Cell Transdifferentiation in Cultured Vascular Smooth Muscle Cells.

Recent evidence suggests that the majority of cholesterol-laden cells found in atherosclerotic lesions are vascular smooth muscle cells (VSMC) that have transdifferentiated into macrophage-like cells (MLC). Furthermore, cholesterol-laden MLC of VSMC origin have demonstrated impaired ABCA1-dependent cholesterol efflux, but it is poorly understood why this occurs. A possible mechanism which may at least partially be attributed to cholesterol-laden MLC demonstrating attenuated ABCA1-dependent cholesterol efflux is a miR-33a expression, as a primary function of this microRNA is to silence ABCA1 expression, but this has yet to be rigorously investigated. Therefore, the VSMC line MOVAS cells were used to generate miR-33a knockout (KO) MOVAS cells, and we used KO and wild-type (WT) MOVAS cells to delineate any possible proatherogenic role of miR-33a expression in VSMC. When WT and KO MOVAS cells were cholesterol-loaded to convert into MLC, this resulted in the WT MOVAS cells to exhibit impaired ABCA1-dependent cholesterol efflux. In the cholesterol-loaded WT MOVAS MLC, we also observed a delayed restoration of the VSMC phenotype when these cells were exposed to the ABCA1 cholesterol acceptor, apoAI. These results imply that miR-33a expression in VSMC drives atherosclerosis by triggering MLC transdifferentiation via attenuated ABCA1-dependent cholesterol efflux.

The Impact of MiR-33a-5p Inhibition in Pro-Inflammatory Endothelial Cells.

Evidence suggests cholesterol accumulation in pro-inflammatory endothelial cells (EC) contributes to triggering atherogenesis and driving atherosclerosis progression. Therefore, inhibiting miR-33a-5p within inflamed endothelium may prevent and treat atherosclerosis by enhancing apoAI-mediated cholesterol efflux by upregulating ABCA1. However, it is not entirely elucidated whether inhibition of miR-33a-5p in pro-inflammatory EC is capable of increasing ABCA1-dependent cholesterol efflux. In our study, we initially transfected LPS-challenged, immortalized mouse aortic EC (iMAEC) with either pAntimiR33a5p plasmid DNA or the control plasmid, pScr. We detected significant increases in both ABCA1 protein expression and apoAI-mediated cholesterol efflux in iMAEC transfected with pAntimiR33a5p when compared to iMAEC transfected with pScr. We subsequently used polymersomes targeting inflamed endothelium to deliver either pAntimiR33a5p or pScr to cultured iMAEC and showed that the polymersomes were selective in targeting pro-inflammatory iMAEC. Moreover, when we exposed LPS-challenged iMAEC to these polymersomes, we observed a significant decrease in miR-33a-5p expression in iMAEC incubated with polymersomes containing pAntimR33a5p versus control iMAEC. We also detected non-significant increases in both ABCA1 protein and apoAI-mediated cholesterol in iMAEC exposed to polymersomes containing pAntimR33a5p when compared to control iMAEC. Based on our results, inhibiting miR-33a-5p in pro-inflammatory EC exhibits atheroprotective effects, and so precisely delivering anti-miR-33a-5p to these cells is a promising anti-atherogenic strategy.

Utilizing the LoxP-Stop-LoxP System to Control Transgenic ABC-Transporter Expression In Vitro.

ABCA1 and ABCG1 are two ABC-transporters well-recognized to promote the efflux of cholesterol to apoAI and HDL, respectively. As these two ABC-transporters are critical to cholesterol metabolism, several studies have assessed the impact of ABCA1 and ABCG1 expression on cellular cholesterol homeostasis through ABC-transporter ablation or overexpressing ABCA1/ABCG1. However, for the latter, there are currently no well-established in vitro models to effectively induce long-term ABC-transporter expression in a variety of cultured cells. Therefore, we performed proof-of-principle in vitro studies to determine whether a LoxP-Stop-LoxP (LSL) system would provide Cre-inducible ABC-transporter expression. In our studies, we transfected HEK293 cells and the HEK293-derived cell line 293-Cre cells with ABCA1-LSL and ABCG1-LSL-based plasmids. Our results showed that while the ABCA1/ABCG1 protein expression was absent in the transfected HEK293 cells, the ABCA1 and ABCG1 protein expression was detected in the 293-Cre cells transfected with ABCA1-LSL and ABCG1-LSL, respectively. When we measured cholesterol efflux in transfected 293-Cre cells, we observed an enhanced apoAI-mediated cholesterol efflux in 293-Cre cells overexpressing ABCA1, and an HDL2-mediated cholesterol efflux in 293-Cre cells constitutively expressing ABCG1. We also observed an appreciable increase in HDL3-mediated cholesterol efflux in ABCA1-overexpressing 293-Cre cells, which suggests that ABCA1 is capable of effluxing cholesterol to small HDL particles. Our proof-of-concept experiments demonstrate that the LSL-system can be used to effectively regulate ABC-transporter expression in vitro, which, in turn, allows ABCA1/ABCG1-overexpression to be extensively studied at the cellular level.

Polyploid giant cancer cells are dependent on cholesterol for progeny formation through amitotic division.

Polyploid Giant Cancer Cells (PGCC) are increasingly being recognized as drivers of cancer recurrence. Therapy stress promotes the formation of these cells, which upon stress cessation often successfully generate more aggressive progeny that repopulate the tumor. Therefore, identification of potential PGCC vulnerabilities is key to preventing therapy failure. We have previously demonstrated that PGCC progeny formation depends on the lysosomal enzyme acid ceramidase (ASAH1). In this study, we compared transcriptomes of parental cancer cells and PGCC in the absence or presence of the ASAH1 inhibitor LCL521. Results show that PGCC express less INSIG1, which downregulates cholesterol metabolism and that inhibition of ASAH1 increased HMGCR which is the rate limiting enzyme in cholesterol synthesis. Confocal microscopy revealed that ceramide and cholesterol do not colocalize. Treatment with LCL521 or simvastatin to inhibit ASAH1 or HMGCR, respectively, resulted in accumulation of ceramide at the cell surface of PGCC and prevented PGCC progeny formation. Our results suggest that similarly to inhibition of ASAH1, disruption of cholesterol signaling is a potential strategy to interfere with PGCC progeny formation.

Combined LXR and RXR Agonist Therapy Increases ABCA1 Protein Expression and Enhances ApoAI-Mediated Cholesterol Efflux in Cultured Endothelial Cells.

Endothelial ABCA1 expression protects against atherosclerosis and this atheroprotective effect is partially attributed to enhancing apoAI-mediated cholesterol efflux. ABCA1 is a target gene for LXR and RXR; therefore, treating endothelial cells with LXR and/or RXR agonists may increase ABCA1 expression. We tested whether treating cultured immortalized mouse aortic endothelial cells (iMAEC) with the endogenous LXR agonist 22(R)-hydroxycholesterol, synthetic LXR agonist GW3965, endogenous RXR agonist 9-cis-retinoic acid, or synthetic RXR agonist SR11237 increases ABCA1 protein expression. We observed a significant increase in ABCA1 protein expression in iMAEC treated with either GW3965 or SR11237 alone, but no significant increase in ABCA1 protein was observed in iMAEC treated with either 22(R)-hydroxycholesterol or 9-cis-retionic acid alone. However, we observed significant increases in both ABCA1 protein expression and apoAI-mediated cholesterol efflux when iMAEC were treated with a combination of either 22(R)-hydroxycholesterol and 9-cis-retinoic acid or GW3965 and SR11237. Furthermore, treating iMAEC with either 22(R)-hydroxycholesterol and 9-cis-retinoic acid or GW3965 and SR11237 did not trigger an inflammatory response, based on VCAM-1, ICAM-1, CCL2, and IL-6 mRNA expression. Based on our findings, delivering LXR and RXR agonists precisely to endothelial cells may be a promising atheroprotective approach.