Premium
Anti‐atherogenic Effect of T0901317 Nanovesicles in THP‐1 Derived Macrophages
Author(s) -
LIU JIE,
Li Chuan,
Wang Shu
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.966.35
Subject(s) - scavenger receptor , cd36 , liver x receptor , macrophage , chemistry , cholesterol , foam cell , mannose receptor , microbiology and biotechnology , receptor , biochemistry , biology , lipoprotein , in vitro , nuclear receptor , transcription factor , gene
Atherosclerotic cardiovascular disease is the leading cause of death in the United States. Intimal macrophages are determinant cells in the atherosclerotic lesion formation by facilitating cholesterol accumulation and increasing the inflammatory response in blood vessel walls. T0901317 can prevent and dramatically regress atherosclerosis by increasing cholesterol efflux and inhibiting inflammatory response via activating macrophage liver X receptors (LXR) . However its side effects including hypertriglyceridemia and hepatic steatosis hinder its application in clinics. We have successfully made T0901317 encapsulated nanovesicles (Tnano) and ligand‐incorporated Tnano (LTnano). Nanovesicles without T0901317 is Vnano, ligand Vnano is LVnano. This target ligand has a high binding affinity to macrophage scavenger receptor CD36. The object of the project is to investigate the anti‐atherogenic effect of LTnano in macrophages. Methods We used THP‐1 derived macrophages in this study. CD36 siRNA and Lipofectamine® RNAiMAX Transfection Reagent were used to knock down the CD36 in these macrophages. Scrambled SiRNA were used as a control. Fluorescence 1‐Oleoyl‐2‐[12‐[(7‐nitro‐2‐1,3‐benzoxadiazol‐4‐yl)amino]dodecanoyl]‐sn‐Glycero‐3‐Phosphocholine (NBD) were used to label all nanovesicles. THP‐1 derived macrophages and CD36 knockdown macrophages were treated with NBD‐labeled nanovesicles at 37 °C for 2 hours. After washing, cell nuclei were stained with 4′,6‐diamidino‐2‐phenylindole dihydrochloride (DAPI). Macrophage binding and uptake of nanovesicles were observed under an EVOS® fluorescence microscope. To investigate the cholesterol lowering effect, THP‐1 derived macrophages were treated with Tnano, LTnano, free T0901317 at 1μM and controls (Vnano, LVnano, PBS). Total cholesterol and free cholesterol were measured using a HPLC method. Cholesterol ester was calculated as the difference between total and free cholesterol. Gene expression of scavenger receptors and some cytokines were measured using real time PCR. Results LTnano had a higher binding affinity to macrophages than Tnano. CD36 knockdown diminished LTnano binding to and uptake by macrophages. LTnano also decreased macrophage total and free cholesterol content compare to free T0901317 or Tnano. LTnano increased the mRNA levels of ATP Binding Cassette Subfamily A Member 1 and ATP‐binding cassette sub‐family G member 1 . Expression of LXR, tumor necrosis factor alpha, monocyte chemoattractant protein‐1, and interleukin 6, macrophage scavenger receptor 1, scavenger receptor class B member 1, CD36 will be determined. Conclusions LTnano targeted macrophages via their CD36 binding sites. LTnano decreased macrophage total and free cholesterol content, which correlated with increased expression of ABCA1 and ABCG1. Additional anti‐atherogenic mechanisms are under investigation. Support or Funding Information Grant Funding Source: NIH 1R15AT007013‐01