Date of Award

2002

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College

College of Graduate Studies

First Advisor

Maria Lopes-Virella

Second Advisor

Gabriel Virella

Third Advisor

Philippe Arnaud

Fourth Advisor

Robert Boackle

Fifth Advisor

Deyi Zheng

Abstract

Mounting evidence suggests that two major vascular complications of diabetes mellitus, atherosclerosis and nephropathy, are pathogenically analogous processes, with atherosclerosis occurring primarily in the microvasculature and nephropathy involving the microvasculature. Additionally, several lines of evidence point to oxidized low-density lipoprotein (oxLDL) as a major participant in both processes. OxLDL is found deposited in both types of vascular lesions, and appears to influence several events. It causes influx of macrophages, which ingest the oxLDL and are transformed into foam cells - a key component of atherosclerosis and glomerulosclerosis. OxLDL also appears to promote the proliferation of smooth muscle cells in atherosclerosis and mesangial cells in glomerulosclerosis; and expansion of the extracellular matrix in both lesions. Previous work in our laboratory has focused on the immunogenicity of oxLDL, and we have shown that heterogeneous LDL-containing immune complexes (LDL-IC), made with human LDL and rabbit LDL-hyperimmune antisera, are unmatched in their ability to drive the transformation of human monocyte-derived macrophages into foam cells. Recently, we undertook a three-part endeavor to better understand the pathogenic role of LDL-IC in microvascular and macrovascular complications of diabetes. Our goals were to 1) further characterize oxLDL antibodies that we isolated from serum and immune complexes; 2) considering the similarities between nephropathy and atherosclerosis, we wished to determine if LDL-IC are associated with diabetic nephropathy, as is observed in macrovascular disease; 3) determine if artificially prepared homogeneous oxLDL-IC, made with oxLDL and human oxLDL antibodies, produce similar results in macrophages to those observed with the use of heterogeneous LDL-IC. Our characterization of oxLDL antibodies revealed that free and complexed antibodies, purified from serum and IC were predominately of the lgG isotype, with lgG1 and lgG2 subclasses predominating. Interestingly, we found that the avidity of oxLDL antibodies isolated from ICs was significantly higher than that of free oxLDL antibodies. These findings suggest that oxLDL-ICs form stable circulating complexes and are composed of lgG subclasses that may render them pro-inflammatory. To investigate the potential association of LDL-IC with diabetic nephropathy, we compared the concentrations of free oxLDL antibodies, avidities of free and IC-bound oxLD antibodies, and concentrations of surrogate markers of LDL-ICs (IC-cholesterol and IC-apolipoprotein 8) in polyethylene glycol-precipitated ICs in three nephropathy classes of type 1 diabetic subjects. Subjects were classified according to their levels of urinary albuminuria, with the normoalbuminurics demonstrating a urinary albumin excretion (UAE) of <30mg/day; microalbuminurics had a UAE of 30-300mg/day; and macroalbuminurics had a UAE of greater than 300mg/day. We observed no significant difference in serum oxLDL antibody levels between groups, but identified a significant increase in IC-cholesterol and IC-apoliprotein B in the microalbuminuric and macroalbuminuric groups when compared to the normoalbuminuric group. Also, avidity levels of oxLDL isolated from immune complexes appeared to increase as nephropathy worsened. We interpreted these data to indicate a positive association between concentrations of LDL-ICs, and diabetic nephropathy, and that stable immune complexes formed with antibodies of higher affinity may play a role in the pathogenesis of renal disease in type 1 diabetes. Since these studies, in conjunction with our previous studies of heterogeneous LDL-IC-induced foam cell formation in human macrophages implicated LDL-IC as a potential participant in atherosclerosis and glomerulosclerosis, but required validation with homogeneous LDL-IC. To meet this objective, we purified human oxLDL antibodies and synthesized oxLDL-IC by mixing these antibodies with oxLDL. We then precipitated the oxLDL-IC using 4% polyethylene glycol, and incubated the oxLDL-IC with THP-1 macrophages (a human monocyte-derived macrophage cell line). After 48 hours incubation, we extracted the cholesterol from these cells, and measured intracellular cholesterol ester accumulation. We observed intracellular cholesterol ester accumulation in cells incubated with oxLDL-IC that exceeded that of cells incubated with oxLDL alone, and these levels approached that observed in cells incubated with heterogeneous LDL-IC. These findings validate the continued use of heterogeneous LDL-IC in studies of foam cell formation. In conclusion, these findings demonstrate a positive association of diabetic microvascular disease and LDL-ICs that is reminiscent of that observed between LDL-IC and macrovascular disease. We have shown that homogeneous oxLDL-IC causes foam cell formation in macrophages, therefore are likely participants in microvascular (nephropathy) and macrovacular (atherosclerosis) diseases processes. Lastly, we have validated the continued use of heterogeneous LDL­ ICs in studies of macrophage: LDL-IC interactions.

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