Cloning and expression of an anti-LDL(-) single-chain variable fragment, and its inhibitory effect on experimental atherosclerosis

Abstract

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(-).

Keywords: :

• single-chain fragment variable
• Pichia pastoris
• atherosclerosis
• electronegative LDL
• macrophage
• foam cell

Disclosure of Potential Conflicts of Interest

No potential conflict of interest was disclosed.

Acknowledgments

The authors wish to thank M.Sc. Alejandro Esteban Cuevas Villegas for adjustment of the images, M.Sc. Elaine Moura Augusto for the isolation of LDL(-) and M.Sc. Renata Albuquerque for collaboration with flow cytometry. This research was supported by FAPESP (Fundação de Amparo à Pesquisa de São Paulo) and INCT_if/CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico). SMK and MFC received scholarships from FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo).