Kevin Jon Williams

Subendothelial Lipoprotein Retention as the Initiating Process in Atherosclerosis Update and Therapeutic Implications

The key initiating process in atherogenesis is the subendothelial retention of apolipoprotein B–containing lipoproteins. Local biological responses to these retained lipoproteins, including a chronic and maladaptive macrophage- and T-cell–dominated inflammatory response, promote subsequent lesion development. The most effective therapy against atherothrombotic cardiovascular disease to date—low density lipoprotein–lowering drugs—is based on the principle that decreasing circulating apolipoprotein B lipoproteins decreases the probability that they will enter and be retained in the subendothelium. Ongoing improvements in this area include more aggressive lowering of low-density lipoprotein and other atherogenic lipoproteins in the plasma and initiation of low-density lipoprotein–lowering therapy at an earlier age in at-risk individuals. Potential future therapeutic approaches include attempts to block the interaction of apolipoprotein B lipoproteins with the specific subendothelial matrix molecules that mediate retention and to interfere with accessory molecules within the arterial wall that promote retention such as lipoprotein lipase, secretory sphingomyelinase, and secretory phospholipase A2. Although not the primary focus of this review, therapeutic strategies that target the proatherogenic responses to retained lipoproteins and that promote the removal of atherogenic components of retained lipoproteins also hold promise. The finding that certain human populations of individuals who maintain lifelong low plasma levels of apolipoprotein B lipoproteins have an ≈90% decreased risk of coronary artery disease gives hope that our further understanding of the pathogenesis of this leading killer could lead to its eradication.

The response-to-retention hypothesis of atherogenesis reinforced.

Many lines of evidence indicate that the key initiating event in early atherosclerosis is the subendothelial retention of cholesterol-rich, atherogenic lipoproteins. Once retained, these lipoproteins provoke a cascade of responses that lead to disease in a previously non-lesional artery. We review recent experimental work that has substantially reinforced this hypothesis. Lipoprotein retention has been shown to be a pivotal requirement in the murine model of atherosclerosis: low-density lipoprotein, engineered through site-directed mutagenesis of apolipoprotein-B100 to bind poorly to arterial proteoglycans, causes relatively few lesions in vivo, even during significant hyperlipidemia. In addition, many molecules in the arterial wall that are involved in the retention of atherogenic lipoproteins and in arterial responses to retained material have recently been characterized. Overall, the response-to-retention hypothesis can now be regarded as a central paradigm in our understanding of the pathogenesis of this deadly disease.

Lipid peroxidation and oxidant stress regulate hepatic apolipoprotein B degradation and VLDL production

How omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) lower plasma lipid levels is incompletely understood. We previously showed that marine omega-3 PUFAs (docosahexaenoic acid [DHA] and eicosapentaenoic acid) stimulate a novel pathway, post-ER presecretory proteolysis (PERPP), that degrades apolipoprotein B100 (ApoB100), thereby reducing lipoprotein secretion from liver cells. To identify signals stimulating PERPP, we examined known actions of omega-3 PUFA. In rat hepatoma or primary rodent hepatocytes incubated with omega-3 PUFA, cotreatment with the iron chelator desferrioxamine, an inhibitor of iron-dependent lipid peroxidation, or vitamin E, a lipid antioxidant, suppressed increases in thiobarbituric acid-reactive substances (TBARSs; a measure of lipid peroxidation products) and restored ApoB100 recovery and VLDL secretion. Moreover, omega-6 and nonmarine omega-3 PUFA, also prone to peroxidation, increased ApoB100 degradation via intracellular induction of TBARSs. Even without added fatty acids, degradation of ApoB100 in primary hepatocytes was blocked by desferrioxamine or antioxidant cotreatment. To extend these results in vivo, mice were infused with DHA, which increased hepatic TBARSs and reduced VLDL-ApoB100 secretion. These results establish a novel link between lipid peroxidation and oxidant stress with ApoB100 degradation via PERPP, and may be relevant to the hypolipidemic actions of dietary PUFAs, the basal regulation of ApoB100 secretion, and hyperlipidemias arising from ApoB100 overproduction.

The syndecan family of proteoglycans. Novel receptors mediating internalization of atherogenic lipoproteins in vitro.

Cell-surface heparan sulfate proteoglycans have been shown to participate in lipoprotein catabolism, but the roles of specific proteoglycan classes have not been examined previously. Here, we studied the involvement of the syndecan proteoglycan family. First, transfection of CHO cells with expression vectors for several syndecan core proteins produced parallel increases in the cell association and degradation of lipoproteins enriched in lipoprotein lipase, a heparan-binding protein. Second, a chimeric construct, FcR-Synd1, that consists of the ectodomain of the IgG Fc receptor Ia linked to the highly conserved transmembrane and cytoplasmic domains of syndecan-1 directly mediated efficient internalization, in a process triggered by ligand clustering. Third, internalization of lipase-enriched lipoproteins via syndecan-1 and of clustered IgGs via the chimera showed identical kinetics (t1/2 = 1 h) and identical dose-response sensitivities to cytochalasin B, which disrupts microfilaments, and to genistein, which inhibits tyrosine kinases. In contrast, internalization of the receptor-associated protein, which proceeds via coated pits, showed a t1/2 < 15 min, limited sensitivity to cytochalasin B, and complete insensitivity to genistein. Thus, syndecan proteoglycans can directly mediate ligand catabolism through a pathway with characteristics distinct from coated pits, and might act as receptors for atherogenic lipoproteins and other ligands in vivo.

Rabbit aorta and human atherosclerotic lesions hydrolyze the sphingomyelin of retained low-density lipoprotein. Proposed role for arterial-wall sphingomyelinase in subendothelial retention and aggregation of atherogenic lipoproteins.

Aggregation and retention of LDL in the arterial wall are key events in atherogenesis, but the mechanisms in vivo are not known. Previous work from our laboratories has shown that exposure of LDL to bacterial sphingomyelinase (SMase) in vitro leads to the formation of LDL aggregates that can be retained by extracellular matrix and that are able to stimulate macrophage foam cell formation. We now provide evidence that retained LDL is hydrolyzed by an arterial-wall SMase activity. First, we demonstrated that SMase-induced aggregation is caused by an increase in particle ceramide content, even in the presence of excess sphingomyelin (SM). This finding is compatible with previous data showing that lesional LDL is enriched in SM, though its ceramide content has not previously been reported. To address this critical compositional issue, the ceramide content of lesional LDL was assayed and, remarkably, found to be 10-50-fold enriched compared with plasma LDL ceramide. Furthermore, the ceramide was found exclusively in lesional LDL that was aggregated; unaggregated lesional LDL, which accounted for 20-25% of the lesional material, remained ceramide poor. When [3H]SM-LDL was incubated with strips of rabbit aorta ex vivo, a portion of the LDL was retained, and the [3H]SM of this portion, but not that of unretained LDL, was hydrolyzed to [3H]ceramide by a nonlysosomal arterial hydrolase. In summary, LDL retained in atherosclerotic lesions is acted upon by an arterial-wall SMase, which may participate in LDL aggregation and possibly other SMase-mediated processes during atherogenesis.

Accelerated transfer of cholesteryl esters in dyslipidemic plasma. Role of cholesteryl ester transfer protein.

Plasma cholesteryl esters, synthesized in the high density lipoproteins (HDL), may be transferred to other lipoproteins by a cholesteryl ester transfer protein (CETP). We found a twofold increase in mass transfer of cholesteryl ester from HDL to apoB-containing lipoproteins in incubated hypercholesterolemic rabbit plasma compared with control. There was a two- to fourfold increase in the activity of CETP, measured in an isotopic assay in hypercholesterolemic plasma. A CETP-like molecule was isolated in increased amounts from hypercholesterolemic plasma. Incubated plasma from four dysbetalipoproteinemic subjects also showed an increase (threefold) in cholesteryl ester mass transfer, compared with normolipidemic controls. There was a twofold increase in the activity of CETP, assayed in whole or lipoprotein-free plasma. Thus, there is increased transfer of cholesteryl esters from HDL to potentially atherogenic apoB-containing lipoproteins in dyslipidemic rabbit and human plasma. The enhanced transfer results in part from increased activity of CETP, possibly reflecting an increase in CETP mass.

Cell-surface heparan sulfate proteoglycans: dynamic molecules mediating ligand catabolism.

Though sometimes regarded as merely passive, space-filling components, proteoglycans are in fact metabolically active molecules with carbohydrate and protein domains that are highly conserved throughout evolution, indicating specific, crucial functions. Here we review recent evidence that heparan sulfate proteoglycans, particularly syndecans and perlecan, are able to mediate directly the internalization of lipoproteins and other ligands, without requiring the participation of LDL receptor family members. Thus, heparan sulfate proteoglycans can function as receptors. In the case of syndecan heparan sulfate proteoglycans, efficient internalization is triggered by clustering of the transmembrane and cytoplasmic domains and then proceeds through a noncoated pit pathway, possibly caveolae. The physiologic and pathophysiologic importance of these direct heparan sulfate proteoglycan-mediated catabolic pathways in the liver and in the arterial wall in vivo remains to be settled.

Sphingomyelinase, an Enzyme Implicated in Atherogenesis, Is Present in Atherosclerotic Lesions and Binds to Specific Components of the Subendothelial Extracellular Matrix

Atherosclerotic lesions contain an extracellular sphingomyelinase (SMase) activity that hydrolyzes the sphingomyelin of subendothelial low density lipoprotein (LDL). This SMase activity may promote atherosclerosis by enhancing subendothelial LDL retention and aggregation, foam cell formation, and possibly other atherogenic processes. The results of recent cell-culture studies have led to the hypothesis that a specific molecule called secretory SMase (S-SMase) is responsible for the SMase activity known to be in lesions, although its presence in atheromata had not been examined directly. Herein we provide immunohistochemical and biochemical support for this hypothesis. First, 2 different antibodies against S-SMase detected extracellular immunoreactive protein in the intima of mouse, rabbit, and human atherosclerotic lesions. Much of this material in lesions appeared in association with the subendothelial matrix. Second, binding studies in vitro demonstrated that (125)I-S-SMase adheres to the extracellular matrix of cultured aortic smooth muscle and endothelial cells, specifically to the laminin and collagen components. Third, in its bound state, S-SMase retains substantial enzymatic activity against lipoprotein substrates. Overall, these data support the hypothesis that S-SMase is an extracellular arterial wall SMase that contributes to the hydrolysis of the sphingomyelin of subendothelial LDL. S-SMase may therefore be an important participant in atherogenesis through local enzymatic effects that stimulate subendothelial retention and aggregation of atherogenic lipoproteins.

Presecretory oxidation, aggregation, and autophagic destruction of apoprotein-B: A pathway for late-stage quality control

Hepatic secretion of apolipoprotein-B (apoB), the major protein of atherogenic lipoproteins, is regulated through posttranslational degradation. We reported a degradation pathway, post-ER pre secretory proteolysis (PERPP), that is increased by reactive oxygen species (ROS) generated within hepatocytes from dietary polyunsaturated fatty acids (PUFA). We now report the molecular processes by which PUFA-derived ROS regulate PERPP of apoB. ApoB exits the ER; undergoes limited oxidant-dependent aggregation; and then, upon exit from the Golgi, becomes extensively oxidized and converted into large aggregates. The aggregates slowly degrade by an autophagic process. None of the oxidized, aggregated material leaves cells, thereby preventing export of apoB-lipoproteins containing potentially toxic lipid peroxides. In summary, apoB secretory control via PERPP/autophagosomes is likely a key component of normal and pathologic regulation of plasma apoB levels, as well as a means for remarkably late-stage quality control of a secreted protein.

Acid Sphingomyelinase Promotes Lipoprotein Retention Within Early Atheromata and Accelerates Lesion Progression

Objective—The key initial step in atherogenesis is the subendothelial retention of apolipoprotein B–containing lipoproteins. Acid sphingomyelinase (acid SMase), an enzyme present extracellularly within the arterial wall, strongly enhances lipoprotein retention in model systems in vitro, and retained lipoproteins in human plaques are enriched in ceramide, a product of SMase. We now sought to test a direct causative role for acid SMase in lipoprotein retention and atherogenesis in vivo. Methods and Results—We studied atherogenesis and lipoprotein retention in Asm−/− versus Asm+/+ mice on the Apoe−/− and Ldlr−/− backgrounds. Asm−/−;Apoe−/− mice had a ≈40% to 50% decrease in early foam cell aortic root lesion area compared with Asm+/+;Apoe−/− mice (P<0.05) despite no difference in plasma cholesterol or lipoproteins. To assay lipoprotein retention in vivo, the two groups of mice were injected with fluorescently labeled Apoe−/− lipoproteins. Early foam cell lesions of Asm−/−;Apoe−/− mice showed a striking 87% reduction in lipoprotein trapping (P<0.0001) compared with Asm+/+;Apoe−/− lesions. Similar results were obtained with Ldlr−/− mice, including an 81% reduction in lipoprotein retention within Asm−/−;Ldlr−/− lesions compared with Asm+/+;Ldlr−/− lesions (P<0.0005). Conclusions—These findings support a causal role for acid SMase in lipoprotein retention and lesion progression and provides further support for the response-to-retention model of atherogenesis.