Cloning and expression of an anti-LDL(-) single-chain variable fragment, and its inhibitory effect on experimental atherosclerosis.
The in vivo modified forms of low-density lipoprotein (LDL) are important for the formation of foam cells and as mediators of the immuno-inflammatory process involved in the progression of atherosclerosis. Electronegative LDL, LDL(-), is a LDL subfraction with pro-inflammatory properties that is present in human blood. To investigate possible atheroprotective effects, an anti-LDL(-) single-chain variable fragment (scFv) was expressed in the methylotrophic yeast Pichia pastoris and its activity was evaluated in vitro against macrophages and in experimental atherosclerosis in Ldlr(-/-) mice. The recombinant 2C7 scFv was produced in a yield of 9.5 mg of protein/L. The specificity and affinity of purified 2C7 scFv against LDL(-) was confirmed by ELISA. To assess the activity of 2C7 scFv on foam cell formation, RAW 264.7 macrophages were exposed to LDL(-) in the presence or absence of 2C7 scFv. The 2C7 scFv inhibited the uptake of LDL(-) by macrophages in a dose-dependent manner, and internalization of LDL(-) by these cells was found to be mediated by the CD36 and CD14 receptor. In addition, compared with untreated cells, lipid accumulation in macrophages was decreased, and the expression of Cd36, Tlr-4 and Cox-2 was downregulated in macrophages treated with 2C7 scFv. Importantly, compared with untreated mice, the treatment of Ldlr(-/-) mice with 2C7 scFv decreased the atherosclerotic lesion area at the aortic sinus. In conclusion, our data show that 2C7 scFv inhibits foam cell formation and atherosclerotic plaque development by modulating the expression of genes relevant to atherogenesis. These results encourage further use of this antibody fragment in the development of new therapeutic strategies that neutralize the pro-atherogenic effects of LDL(-).
Department of Clinical Analysis; Faculty of Pharmaceutical Sciences; University of Sao Paulo; Sao Paulo, Brazil.
Soraya M
SM
Kazuma
Marcela F
MF
Cavalcante
Andréia E R
AE
Telles
Andrea Queiroz
AQ
Maranhão
Dulcineia S P
DS
Abdalla
D000818:Animals; D000915:Antibody Affinity / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D050197:Atherosclerosis / Q000235:genetics / Q000276:immunology / Q000517:prevention & control*; D018955:CD36 Antigens / Q000235:genetics / Q000276:immunology; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000276:immunology; D003001:Cloning, Molecular; D051546:Cyclooxygenase 2 / Q000235:genetics / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D005487:Foam Cells / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects / Q000276:immunology; D008055:Lipids / Q000032:analysis; D008077:Lipoproteins, LDL / Q000097:blood / Q000276:immunology*; D008264:Macrophages / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010843:Pichia / Q000235:genetics; D011973:Receptors, LDL / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011994:Recombinant Proteins / Q000276:immunology / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000276:immunology
D018955:CD36 Antigens; D008055:Lipids; D008077:Lipoproteins, LDL; D011973:Receptors, LDL; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; C099252:oxidized low density lipoprotein; C077623:Ptgs2 protein, mouse; D051546:Cyclooxygenase 2
Pichia pastoris; atherosclerosis; electronegative LDL; foam cell; macrophage; single-chain fragment variable