Hyaluronan Accumulation Is Elevated in Cultures of Low Density Lipoprotein Receptor-deficient Cells and Is Altered by Manipulation of Cell Cholesterol Content

Michael G. Kinsella,Yann Goueffic,Pamela Y. Johnson,Susan Potter-Perigo,Michael E. Rosenfeld,Kathleen R. Braun,Sana W. Sakr,Thomas N. Wight

doi:10.1074/jbc.m807772200

Abstract

The extracellular matrix molecule hyaluronan (HA) accumulates in human atherosclerotic lesions. Yet the reasons for this accumulation have not been adequately addressed. Because abnormalities in lipid metabolism promote atherosclerosis, we have asked whether disrupted cholesterol homeostasis alters HA accumulation in low density lipoprotein receptor-deficient cell cultures. Cultured aortic smooth muscle cells (ASMC) from Watanabe heritable hyperlipidemic (WHHL) rabbits and skin fibroblasts from homozygous patients with familial hypercholesterolemia accumulated 2-4-fold more HA than corresponding cells from age- and sex-matched normolipidemic rabbits and individuals. This occurred in both cell-associated and secreted HA fractions and was independent of cell density or medium serum concentration. WHHL ASMC cultures synthesized twice the proportion of high molecular mass HA (>2x10(6) Da) as normal rabbit ASMC but showed a lower capacity to degrade exogenous [3H]HA. Most importantly, cholesterol depletion or blocking cholesterol synthesis markedly reduced HA accumulation in WHHL ASMC cultures, whereas cholesterol replenishment or stimulation of cholesterol synthesis restored elevated HA levels. We conclude the following: 1) maintaining normal HA levels in cell cultures requires normal cell cholesterol homeostasis; 2) HA degradation may contribute to but is not the predominant mechanism to increase high molecular mass HA accumulation in low density lipoprotein receptor-deficient WHHL ASMC cultures; and 3) elevated accumulation of HA depends on cellular or membrane cholesterol content and, potentially, intact cholesterol-rich microdomains.

Highlights

Considerable work indicates that HA, a widely distributed component of the extracellular matrix (ECM) of vertebrate tissues [1, 2], accumulates in human atherosclerotic lesions [13,14,15,16,17,18] and in animal models of vascular injury and atherosclerosis (19 –21)
Watanabe heritable hyperlipidemic (WHHL) aortic smooth muscle cells (ASMC) Cultures Accumulate Elevated Levels of Hyaluronan and Grow More Slowly than Controls—In vivo accumulation of HA is a feature of atherosclerosis [16], a disease that commonly occurs in low density lipoprotein (LDL) receptor-deficient WHHL rabbits [24, 26, 39]
WHHL ASMC cultures had higher HA levels than those of New Zealand White (NZW) ASMC even in the absence of high serum (Fig. 1A, day 0), indicating that this difference is not dependent upon serum stimulation of the cells. These results showed for the first time that lacking a functional LDL receptor can be associated with enhanced HA accumulation in cell cultures over a wide range of incubation periods

Summary

Introduction

Considerable work indicates that HA, a widely distributed component of the extracellular matrix (ECM) of vertebrate tissues [1, 2], accumulates in human atherosclerotic lesions [13,14,15,16,17,18] and in animal models of vascular injury and atherosclerosis (19 –21). Characterization of Hyaluronan by Hydrodynamic Size— WHHL and NZW rabbit ASMC, seeded and growth-arrested as described above, were labeled for 6 h with 50 ␮Ci/ml [3H]glucosamine ([3H]GlcN) spiked at 0-, 6-, 12-, and 18-h time points in 0.5% or 10% FBS-containing medium.

Results

Conclusion