Low density lipoprotein like (LDE) nanoemulsion cell uptake: chemical composition and lipoprotein receptor expression

Author(s)

Cristina Pio de Almeida

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina (FM/SBD)
• Defense date: 2010-08-11

Examining board members

Raul Cavalcante Maranhao; Sergio Paulo Bydlowski; Fabio Bessa Lima; Heraldo Possolo de Souza; Claudete Justina Valduga

• Advisor: Raul Cavalcante Maranhao

Abstract

With fat composition similar to natural LDL, the LDE nanoemulsion can be used to study the metabolism of LDL. Other studies have shown that LDE is uptaken by cells by LDL-r receptors. Other receptors such as LRP-1, CD36 and CD38 may also be involved in the uptake. The objectives of this study were to investigate the uptake of LDE by endothelial and tumor cells, fibroblasts, monocytes and macrophages, to identify those receptors involved in this process and to evaluate the effects on LDE uptake by changing its chemical composition. A labeled LDE with [3H]-cholesterol and [14C]- cholesteryl ester was incubated for 4 hours with cells, after which LDE uptake and competition tests were evaluated. LDL-r, LRP, CD36 and CD38 were evaluated by using immunocytochemistry methods, cytometric flow and real time PCR. To investigate the effects of LDE chemical composition modifications, cholesteryl oleate (LDE-CO) was replaced with cholesteryl linoleate (LDE-CL) and cholesterol stearate (LDE-CE). These were then tested for stability, cellular uptake, lipoperoxidation and citotoxitity. Results showed that all cells internalized [3H]-cholesterol and [14C]-cholesteryl ester proportionally to incubated LDE-CO concentrations albeit with some saturation differences. LDE-CO lipid uptake had a higher cholesterol uptake than the cholesteryl ester uptake. Furthermore, monocytes (THP-1) had a higher LDE-CO uptake than other cells. LDE-CO uptake decreased (r2 -0.73) in the presence of natural LDL, suggesting that these two particles may be competing for the same receptors. LDE-CO appeared to inhibit LDL protein receptor expression in HUVEC (3.98 times), in monocytes (6.25 times) and in fibroblasts (3.70 times), as well as the gene expression in monocytes and HUVEC. A decrease in LDL-r expression in both H292 and fibroblasts was also observed. LDE-CO increased the protein expression in HUVEC 3.75 times while in monocytes, it was able to decrease gene and protein expression of LRP-1, 3.15 times. In macrophages and H292, there was an increase in genetic expression of LRP-1. LDE-CO increased the CD36 in HUVEC gene and protein expressions 3.1 times, decreased the macrophage protein expression 4.34 times and decreased the H292 gene and protein expression 2.94 times. LDE increased protein expression 2.09 times in CD68 in H292 and increase gene expression in both monocytes and macrophages. Fibroblasts presented the highest survival rate in the presence of LDE-CO of all cells studied. The LDE chemical modification effect studies, presented smaller sized LDE-CO and less lipoperoxidation than LDE-CL and LDE-CE presented no stability modifications in less than 30 days. Cells presented higher lipoperoxidation in the presence of LDE-CL when compared to the presence of LDE-CO and LDE-CE. [3H]-cholesterol was greater than cholesteryl ester for all three LDE types in all the studied cells. LDE-CO presented favorable characteristics in terms of particle size and susceptibility to peroxidation. Cholesterol cell uptake was higher than that of cholesteryl ester for all LDEs of all the studied cells which suggests that that cholesterol may be capable of disassociating itself from LDE and being uptaken by cells through non-specific pathways. The results of this study can help to better understand the mechanisms of uptake by cells, the effects of stability and LDE system adequation for therapeutic and diagnostic applications. (AU)