The increasing number of patients suffering from atherosclerosis continues to drive research into cholesterol and triglyceride metabolism. Through a large number of investigations, the essentials of the control of cholesterol metabolism have been elucidated in the past two decades (see FIG. 1). The central system for the control of cholesterol metabolism requires two sets of separable pathways: 1) the endogenous pathway and 2) the exogenous cholesterol-entry pathways. Both of sets of pathways are modulated by the protein lysosomal acid lipase (LAL) [1]. In the former, the cell senses the need for endogenous cholesterol synthesis via the release of transcription factors, Sterol Regulatory Element Binding Proteins (SREBP1 and 2), whose precursors are bound to the nuclear membrane and endoplasmic reticulum. SREBPs up-regulate HMG-CoA reductase and other enzymes in the endogenous synthesis pathways [2-5]. This upregulation is derived from the cell's biochemical feedback mechanism sensing a low level of free cholesterol in the surrounding media and/or plasma that is derived from the receptor mediated endocytosis pathway; i.e., the exogenous pathway [6]. Low density lipoprotein receptors (LDLR) and other plasma membrane receptors participate in this uptake process. These LDLR-delivered and other lipoprotein associated lipids are presented to the lysosome for degradation by LAL. Once a deficient exogenous cholesterol supply is sensed, SREBP1 and 2 stimulate the transcription of a cascade of enzymes leading to the production of free intracellular cholesterol and fatty acids [7-10]. The cell then senses the adequacy of free cholesterol levels and, once exceeded, ACAT (acyl CoA: cholesterol acyltransferase) is directly activated by free cholesterol and ACAT synthesis is up regulated. The net effect is to remove free cholesterol by esterification to a cytoplasmic storage pool of cholesteryl esters that is not contained within membranes, i.e., non-lysosomal, and to remove free cholesterol and cholesteryl esters from the cells. Once the cell senses that sufficient free cholesterol is available, a steady state pool of free cholesterol is maintained [11].
Both SREBP1 and 2 are transcription factors that bind to Sterol Regulatory Elements (SREs) in the promoter regions of key genes in cholesterol and fatty acid synthesis. The SREBPs are activated by a two step proteolytic process that is mediated by proteases that are activated by free cholesterol sensing elements in the plasma membrane and, potentially, other components of the cell [12, 13]. These proteases cleave the endoplasmic recticulum (ER) resident SREBPs and release their active components which are then transported to the nucleus. SREBP2 has a single transcript whereas the SREBP-1 gene produces two transcripts and proteins, SREBP-1a and SREBP-1c. These alternative forms of SREBP1 arise from the use of transcription start sites resident in alternative first exons that are then spliced into a common second exon. In humans, the mRNAs for SREBP-1a/-1c also display alternative splicing at the 3′ end that leads to proteins that differ by 113 amino acids at the C-terminus [14, 15]. All three SREBP members share the same structural domains indicating their common function [16]. These domains include: 1) the NH2-terminal segment of 480 amino acids is a basic helix-loop-helix-leucine zipper-“like” transcription activator, 2) the middle segment of 80 amino acids comprises two membrane spanning sequences, and 3) the carboxy-terminal half of 590 amino acids that functions as a regulatory domain [17].
There are at least two pathways for the entrance of external cholesterol into monocyte/macrophage derived cells [18]: 1) the ldlr and ldlr-related protein systems [19]; and 2) the scavenger receptor system (e.g., SRA, SR-B and CD36) for lipoprotein bound cholesteryl esters (CE's) [20-24]. The SR-B1 pathway delivers cholesteryl esters into the cell via transfer of cholesteryl esters through SR-B1 without uptake of HDL [25, 26].
In the LDL-CE (cholesteryl ester) or -TG (triglyceride) pathway, the complexes are taken up into cells following receptor-mediated recognition. The endosomal pathway delivers these lipids to the lysosomes after uncoupling the LDL-lipid complexes from the receptor in the late endosomal acidified compartment. Once the LDL-lipid particle is delivered to the lysosome, the lipids are liberated, possible after degradation of the LDL particle, via proteolysis or by simultaneous attack through proteolysis and by LAL [27]. This derived free cholesterol is then transported out of the lysosome into the cytosol by one or more proteins resident in, or at, the lysosomal membrane. Once it exits the lysosome, free cholesterol moves to the inner surface of the plasma membrane and directly to the endoplasmic reticulum. Free cholesterol from the inner surface of the plasma membrane is then transported to the endoplasmic reticulum and participates in the feedback control of the endogenous synthetic pathway. Thus, from this simplified overview of cholesterol and triglyceride metabolism in cells, it is clear that LAL occupies a central position in the control of endogenous cholesterol synthesis since, without its activity, neither free cholesterol nor free fatty acids (FFA) derived from the LDL pathway can be liberated from the lysosome to control these critical pathways.
The importance of LAL in cholesterol and triglyceride metabolism is underscored by the human phenotypes resulting from inherited deficiencies of LAL. These two rare diseases, Wolman Disease and Cholesteryl Ester Storage Disease, are early and late onset diseases, respectively [28]. Wolman disease results in the massive accumulation of cholesteryl esters and triglycerides in lysosomes of a variety of tissues and cells including those of the liver (hepatocytes and Kupffer cells), spleen, adrenal gland and epithelium of the small intestine. This leads to a severe phenotype characterized by hepatosplenomegaly, adrenal calcification, and a thickened and dilated small intestine. In comparison, cholesteryl ester storage disease is a much more heterogeneous disease with onset from early childhood to late adolescence, and even adulthood with isolated hepatomegaly and/or progressive cirrhosis and primarily storage of cholesteryl esters.
The inventor has discovered that additional circumstantial evidence has implicated lower LAL activities in monocytes and/or plaques from patients with atherosclerosis or carotid artery atheromata. This evidence indicates that polymorphic variants could lead to differential activity of LAL in various tissues and may predispose to, or be an additional risk factor in, the development of atherosclerotic disease in humans [29]. In accordance with this invention, this suggests that supplementation of LAL activity in cells of pathologic involvement in athero-/arterio-sclerosis may provide a means to diminish the accumulated, pathologic cholesteryl esters and triglycerides that are causally related to these diseases.
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USPTO Backgrounds