Strategies for the clearance of age-related lysosomal residuals: 7-ketocholesterol and foam cell formation - Jacques Mathieu
Lysosomal storage diseases (LSDs) have traditionally been defined as a group of inherited metabolic disorders that are typically the consequence of a deficiency in a single lysosomal enzyme. Often overlooked, however, is lysosomal storage that occurs with increasing age, and that is the result of catabolic insufficiency. These forms of lysosomal storage occur in all individuals to some degree, are chronically rather than acutely cytotoxic, and are increasingly being associated with age-related degenerative diseases. Examples of such substances include lipofuscin, 7-ketocholesterol, and N-retinylidene-N-retinyl-ethanolamine (A2E). As with inborn errors of metabolism, the consequences of age-related lysosomal storage can include volume expansion, enzyme inhibition, reduced protein homeostasis, and altered membrane composition. This may result in the activation of a number of pathological cascades, including apoptosis, oxidative stress, inflammation, and endoplasmic reticulum stress. In an effort to discern the pathological consequences of such lysosomal storage, and provide novel therapeutic avenues for treatment, our group is developing methods to facilitate the selective elimination of unmetabolized substances from the lysosome.
To establish proof-of-concept that the selective removal of intralysosomal substances can help attenuate age-related disease, we first sought to determine the principle component of oxidized LDL responsible for foam cell formation. 7-ketocholesterol (7KC) is the major form of oxysterol found associated with oxidized LDL, and is highly concentrated in atherosclerotic plaques. Intracellularly, its propensity to concentrate in the lysosome hinders enzymatic transformations that reduce its toxicity and increases sensitivity to lysosomal membrane permeabilization. Using THP-1 monocytes, we have found that 7KC-loaded LDL causes significantly accelerated macrophage differentiation as well as a 50% increase in both neutral and polar lipid retention when compared to cells treated with normal or moderately oxidized LDL. Importantly, 7KC-differentiated macrophages are primarily of the spindle-shaped, M1 variety, which are considered pro-inflammatory. We assessed the potential to mitigate the effects of 7KC on foam cell formation by exposing THP-1 macrophages to the cyclic oligosaccharide hydroxypropyl-β-cyclodextrin (HβCD). We found that HβCD is effective at reducing monocyte to macrophage differentiation. Furthermore, HβCD is able to reverse 7KC-mediated lysosomal membrane permeabilization, as determined by lysosomal retention assays. Interestingly, while HβCD also significantly increased the viability of 7KC-exposed monocytes, it reduced the viability of monocytes exposed to high concentrations of LDL or oxLDL. In order to address this apparent discrepancy, we are also currently exploring the overexpression of both 7KC-active enzymes as well as oxysterol-specific sterol transporters to facilitate 7KC clearance from foam cells.