Retention Of Atherogenic Lipoproteins Research Articles

Lipid accumulation and novel insight into vascular smooth muscle cells in atherosclerosis.

Atherosclerosis is a chronic and progressive process. It is the most important pathological basis of cardiovascular disease and stroke. Vascular smooth muscle cells (VSMCs) are an essential cell type in atherosclerosis. Previous studies have revealed that VSMCs undergo phenotypic transformation in atherosclerosis to participate in the retention of atherogenic lipoproteins as well as the formation of the fibrous cap and the underlying necrotic core in plaques. The emergence of lineage-tracing studies indicates that the function and number of VSMCs in plaques have been greatly underestimated. In addition, recent studies have revealed that VSMCs make up at least 50% of the foam cell population in human and mouse atherosclerotic lesions. Therefore, understanding the formation of lipid-loaded VSMCs and their regulatory mechanisms is critical to elucidate the pathogenesis of atherosclerosis and to explore potential therapeutic targets. Moreover, combination of many complementary technologies such as lineage tracing, single-cell RNA sequencing (scRNA-seq), flow cytometry, and mass cytometry (CyTOF) with immunostaining has been performed to further understand the complex VSMC function. Correct identification of detrimental and beneficial processes may reveal successful therapeutic treatments targeting VSMCs and their derivatives during atherosclerosis. The purpose of this review is to summarize the process of lipid-loaded VSMC formation in atherosclerosis and to describe novel insight into VSMCs gained by using multiple advanced methods.

Smooth Muscle Cell-Proteoglycan-Lipoprotein Interactions as Drivers of Atherosclerosis.

In humans, smooth muscle cells (SMCs) are the main cell type in the artery medial layer, in pre-atherosclerotic diffuse thickening of the intima, and in all stages of atherosclerotic lesion development. SMCs secrete the proteoglycans responsible for the initial binding and retention of atherogenic lipoproteins in the artery intima, with this retention driving foam cell formation and subsequent stages of atherosclerosis. In this chapter we review current knowledge of the extracellular matrix generated by SMCs in medial and intimal arterial layers, their relationship to atherosclerotic lesion development and stabilization, how these findings correlate with mouse models of atherosclerosis, and potential therapies aimed at targeting the SMC matrix-lipoprotein interaction for atherosclerosis prevention.

Abstract 612: Low Expression of Lysosomal Acid Lipase in Smooth Muscle Cells Relative to Macrophages Provides New Insights into Foam Cell Lipid Accumulation

Background and Hypothesis: Smooth muscle cells (SMCs) are the predominant cell type within the intima of human atherosclerosis-prone arteries and promote initial retention of atherogenic lipoproteins in the deep intima. We previously found that ≥50% of foam cells in intermediate coronary atheromas are of SMC origin and that intimal SMCs have reduced expression of the ATP-binding cassette transporter A1 (ABCA1). Previously we also found that ABCA1 expression is acutely dependent on the flux of cholesterol out of lysosomes and subsequent generation of oxysterols for promotion of gene transcription by the nuclear liver X receptor (LXR). Hypothesis: In the present studies we tested the hypothesis that SMCs have lysosomal dysfunction that contributes to foam cell formation. Methods and Results: Human monocyte-derived macrophages (HMMs) and arterial SMCs were treated with aggregated LDL (agLDL) to increase intracellular cholesterol. Unlike HMMs, agLDL treatment failed to upregulate ABCA1 expression in SMCs and did not significantly increase 27-hydroxycholesterol levels. Also in contrast to HMMs, SMCs did not downregulate new cholesterol synthesis and displayed minimal increases in cholesteryl ester formation with agLDL loading. This data suggested retention of lipids within lysosomal compartments. Indeed, confocal microscopy revealed retention of lipids identified by the neutral lipid dye BODIPY within lysosomal compartments stained with LAMP1 in SMCs, while HMMs showed mostly cytosolic lipid droplets [Pearson correlation of colocalization of +0.3197±0.0123 in SMCs and -0.0897±0.0143 in HMMs (p<0.0001, avg±SEM, n>100 cells)]. LIPA mRNA levels and LAL protein were markedly reduced in SMCs relative to HMMs, with LAL activity being 23.4-times higher in HMMs compared to SMCs (p<0.0001, n=4). Similarly, we found reduced LAL levels in mouse SMCs relative to macrophages. Incubation of SMCs with medium containing LAL reduced lysosomal lipid accumulation, resulting in decreased new cholesterol synthesis and increased cholesterol efflux. Conclusions: We find that arterial SMCs have a relative deficiency in LAL expression resulting in defects in downstream sterol regulatory events compared to macrophages. Our results provide a novel reason for SMC foam cell formation and a potential therapeutic target.

The role of oxidative stress and autophagy in atherosclerosis.

Atherosclerosis is a multifactorial, multistep disorder of large- and medium-sized arteries involving, in addition to age, gender and menopausal status, a complex interplay between lifestyle and genetic risk factors. Atherosclerosis usually begins with the diffusion and retention of atherogenic lipoproteins into the subendothelial space of the artery wall where they become oxidized by local enzymes and accumulate, leading to the formation of a cushion called atheroma or atheromatous or fibrofatty plaque, composed of a mixture of macrophages, lymphocytes, smooth muscle cells (SMCs), cholesterol cleft, necrotic debris, and lipid-laden foam cells. The pathogenesis of atherosclerosis still remains incompletely understood but emerging evidence suggests that it may involve multiple cellular events, including endothelial cell (EC) dysfunction, inflammation, proliferation of vascular SMCs, matrix (ECM) alteration, and neovascularization. Actually, a growing body of evidence indicates that autophagy along with the chronic and acute overproduction of reactive oxygen species (ROS) is integral to the development and progression of the disease and may represent fruitful avenues for biological investigation and for the identification of new therapeutic targets. In this review, we give an overview of ROS and autophagy in atherosclerosis as background to understand their potential role in this vascular disease.

Increased atherosclerosis in mice with increased vascular biglycan content

ObjectiveThe response to retention hypothesis of atherogenesis proposes that atherosclerosis is initiated via the retention of atherogenic lipoproteins by vascular proteoglycans. Co-localization studies suggest that of all the vascular proteoglycans, biglycan is the one most closely co-localized with LDL. The goal of this study was to determine if over-expression of biglycan in hyperlipidemic mice would increase atherosclerosis development. MethodsTransgenic mice were developed by expressing biglycan under control of the smooth muscle actin promoter, and were crossed to the LDL receptor deficient (C57BL/6 background) atherosclerotic mouse model. Biglycan transgenic and non-transgenic control mice were fed an atherogenic Western diet for 4–12 weeks. ResultsLDL receptor deficient mice overexpressing biglycan under control of the smooth muscle alpha actin promoter had increased atherosclerosis development that correlated with vascular biglycan content. ConclusionIncreased vascular biglycan content predisposes to increased lipid retention and increased atherosclerosis development.

Macrophages, Atherosclerosis and the Potential of netrin-1 As a Novel Target for Future Therapeutic Intervention

Atherosclerosis is a chronic inflammatory disease that is distinguished by the persistence of cholesterol-laden macrophages in plaques of the arterial wall[1]. Based on observational studies that extend into the era of modern atherosclerosis research (which dates to the introduction in the early 90's of mouse models of the disease), the prevailing view has been that the recruitment of monocytes into atherosclerotic plaques is an unproductive exercise, in which these cells are fated to become macrophage foam cells that persist in this site, establishing chronic inflammation, until eventually dying by either necrosis or apoptosis. Unlike in other tissues, macrophages that accumulate in plaques appear to have a diminished capacity to migrate[2–5], and go from being chemotactic to chemostatic, thereby contributing to a failure to resolve the inflammatory process in arteries set in motion by the retention of atherogenic lipoproteins. Moreover, the impaired efferocytotic ability of these macrophages prevents the clearance of their dying compatriots[6], further contributing to the pro-inflammatory milieu of the plaque and ultimately resulting in the formation of a necrotic core. In such unstable plaques, inflammasome-activating cholesterol crystals[7] and pro-thrombotic tissue factor accumulate in this graveyard of dead macrophages, further adding to the mayhem.

Thiazolidinediones reduce the LDL binding affinity of non-human primate vascular cell proteoglycans.

Retention of atherogenic lipoproteins in the artery wall by proteoglycans is a key step in the development of atherosclerosis. Thiazolidinediones have been shown to reduce atherosclerosis in mouse models. The aim of this study was to determine whether thiazolidinediones modify vascular proteoglycan synthesis in a way that decreases LDL binding. Primate aortic smooth muscle cells were exposed to troglitazone or rosiglitazone, or no stimulus at all for a 24-hour steady-state labelling period. Sulphate incorporation, size and LDL binding affinity of proteoglycans were determined. Proteoglycans secreted by cells in the presence or absence of troglitazone were separated into large and small classes by size exclusion chromatography, and LDL binding affinity was determined. Proteoglycans synthesised by cells exposed to troglitazone or rosiglitazone were smaller, with decreased sulphate incorporation and decreased LDL binding affinity. However, troglitazone had a greater effect than rosiglitazone. Troglitazone reduced the LDL binding affinities of both the large and small proteoglycans compared with control. The binding differences persisted when glycosaminoglycan chains released from proteoglycans were incubated with LDL, indicating that troglitazone affects the glycosaminoglycan synthetic machinery of these cells. Thiazolidinediones decrease the LDL binding affinity of the proteoglycans synthesised by primate aortic smooth muscle cells. This could, in part, account for the reduced atherosclerosis observed in animal models.

Lipoprotein-matrix interactions in macrovascular disease in diabetes.

The retention of atherogenic lipoproteins in the artery wall through their interactions with the arterial extracellular matrix is a critical step in the development of atherosclerosis, as outlined in the 'response to retention' hypothesis. Lipoprotein retention by vascular proteoglycans is thought to be the principle means of lipoprotein retention, although lipoprotein binding to other components of the extracellular matrix has been reported. The interactions of lipoproteins and proteoglycans can be direct through ionic interactions between the negatively charged glycosaminoglycan chains of proteoglycans and positively charged residues of apolipoproteins B and E, or can be mediated through bridging molecules such as lipoprotein lipase. Retention of atherogenic lipoproteins within the artery wall environment leads to pathophysiologically important modifications of the lipoproteins, including oxidation. Oxidation of lipoproteins leads to increased uptake by macrophages, leading to the formation of foam cells. This article reviews the scientific evidence in support of the response to retention hypothesis, with a specific focus on the effects of diabetes to modify lipoprotein retention.

Retention of atherogenic lipoproteins in atherogenesis.

Atherosclerosis is a multifactorial disease whose pathogenesis is still unclear. Mounting evidence, however, supports the concept that subendothelial retention of apoB100-containing lipoproteins is the initiating event in atherogenesis. Subsequently, a series of biological responses to this retained material leads to specific molecular and cellular processes that promote lesion formation.

Fatal Attraction: Scientists expose molecular liaison that incites cholesterol buildup in arteries (Atherosclerosis)

The artery-plugging gunk that provokes a fatal stroke or heart attack can start amassing during childhood, yet no one has known what sets off this insidious accumulation. New research suggests that the attraction between gobbets of cholesterol in the blood and particular molecules in artery walls might foment the buildup. The study reveals the earliest stages of America's number one killer and might allow doctors to pinpoint more people who are at risk for stroke or heart attack. Epidemiological studies have linked atherosclerosis to many factors, including high blood concentrations of low-density lipoproteins (LDLs), the so-called bad form of cholesterol. Cardiovascular researchers disagree over what sparks the formation of fatty atherosclerotic lesions. Some champion inflammation of the arterial lining caused by injury or infection, an idea that's garnered a lot of popular interest. Others argue that oxidants such as hydrogen peroxide attack LDLs, making them targets for immune cells that then glom onto the arterial lining. Evidence has been accruing, however, that atherosclerosis begins when LDL particles themselves lodge in the artery lining, says Kevin Jon Williams of Thomas Jefferson University in Philadelphia, Pennsylvania. A molecule on the surface of LDL particles called apolipoprotein B, or apoB, can adhere to support molecules in the arterial wall that help the blood vessel hold its shape. According to the "response to retention" hypothesis, this tête-à-tête might detain LDL in the arterial wall and set off the chain of events that leads to atherosclerosis. To test whether the attraction between these two molecules instigates fat buildup, Skålén and colleagues created three types of mice, each of which carries a different form of apoB that doesn't bind to the support molecules. After allowing the rodents to gorge on a high-fat diet for 20 weeks, the researchers killed the animals and measured the extent of fatty buildup plastering the lining of the aorta, the large vessel leading from the heart. In the three groups with altered apoB, the area covered by the lesions was less than half that in the control group, which sported normal apoB. Although the findings do not rule out a role for inflammation and oxidation, they argue that LDL attachment comes first, says cell biologist Jan Borén of Göteborg University in Sweden, who led the research team: "The vicious cycle [of atherosclerosis] could start here." The study "is the first direct proof of this hypothesis," says atherosclerosis expert Ira Tabas of Columbia University in New York City. Genetic differences in apoB among people might explain why some patients have what seem like perilously high LDL concentrations but don't develop atherosclerosis, whereas others with normal concentrations die from heart attacks. Testing for apoB variants might allow early identification of people likely to develop atherosclerosis. Researchers might also develop drugs that keep apoB from gluing LDL to arteries. Williams also lauds the work, saying that the findings should remind everyone that, contrary to the popular thinking that links inflammation with atherosclerosis, controlling LDL levels is the key to thwarting cardiovascular disease. So put down that doughnut and get off the couch. --Mitch Leslie K. Skålén, M. Gustafsson, E. K. Rydberg, L. M. Hultén, O. Wiklund, T. L. Innerarity, J. Borén, Subendothelial retention of atherogenic lipoproteins in early atherosclerosis. Nature 417 , 750-754 (2002). [Abstract] [Full Text]

Arterial smooth muscle cell proteoglycans synthesized in the presence of glucosamine demonstrate reduced binding to LDL

Atherosclerosis is the main cause of morbidity and mortality in diabetes, yet the underlying mechanisms remain unclear. Retention of atherogenic lipoproteins by vascular proteoglycans is thought to play a key role in the development of atherosclerotic lesions. High glucose levels cause a variety of diabetic complications by several mechanisms, including upregulation of the hexosamine pathway. Glucosamine, a component of the hexosamine pathway, is a precursor for the synthesis of glycosaminoglycan components of proteoglycans. This study evaluated whether high glucose or glucosamine supplementation of vascular smooth muscle cells would increase proteoglycan synthesis, leading to increased lipoprotein retention. Aortic smooth muscle cells were exposed to physiologic (5.6 mM) or high (25 mM) glucose levels, such as seen in diabetes, or to glucosamine (12 mM). Extracellular proteoglycans were characterized by sulfate incorporation, molecular sieve chromatography, and SDS-PAGE. LDL interactions were assessed by affinity chromatography and gel mobility shift assay. Proteoglycans synthesized in the presence of high glucose demonstrated no differences in size, sulfate incorporation, or LDL binding affinity compared with proteoglycans synthesized under physiological glucose conditions. However, proteoglycans synthesized in the presence of glucosamine had smaller glycosaminoglycan chains than control proteoglycans with a corresponding decrease in lipoprotein retention.Thus, glucose and glucosamine have different effects on proteoglycan biosynthesis and different effects on lipoprotein retention.

Oxidative modifications of LDL increase its binding to extracellular matrix from human aortic intima: influence of lesion development, lipoprotein lipase and calcium.

Retention of atherogenic lipoproteins in the arterial intima by extracellular matrix (ECM) is assumed to occur during early atherogenesis and its further development. Low density lipoprotein (LDL) trapped in the intima may undergo oxidative modifications, which initiate a chain reaction in atherogenesis. Lipoprotein lipase (LPL) has been found to mediate the binding of native and oxidized LDL to ECM produced by cultured cells and to contribute to foam cell formation by mildly oxidized LDL. In this study ECM, isolated from human aortic intima with different atherosclerotic lesions, was used for the first time to measure the binding to it in vitro of native and differently oxidized 125I-LDL. Oxidation of 125I-LDL increased its binding to the ECM, which was most prominent with the material isolated from intima at the early stage of atherogenesis. With the progression of atherosclerosis, the ability of the isolated intimal ECM to bind native and oxidized 125I-LDL decreased, and strongly oxidized 125I-LDL decreased more than native and moderately oxidized 125I-LDL. LPL increased the binding of moderately oxidized 125I-LDL to the ECM more than native 125I-LDL, while it had only a small effect on strongly oxidized 125I-LDL. LPL-mediated binding of native and oxidized 125I-LDL decreased with the development of atherosclerotic lesions. Calcium ions also increased the binding of LDL to the ECM. This enhanced binding increased with the extent of LDL oxidation, especially at the early stage of atherogenesis, and decreased with lesion progression. These data suggest that the ability of ECM to retain LDL in arterial intima depends on LDL oxidation status and changes with the progression of atherogenesis. In addition, LPL and calcium ions may participate in the retention of LDL in vivo.

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.

Subendothelial proteoglycan synthesis and transforming growth factor beta distribution correlate with susceptibility to atherosclerosis.

Coronary bypass vessels, saphenous vein (SV) and internal thoracic artery (ITA), differ in susceptibility to atherosclerosis and medium- to long-term patency. Whereas most ITA remain patent (90% at 10 years), 20% of SV grafts fail in the first year and approximately 45% fail within 10 years. Reasons for these differences are not fully understood. Loss of SV patency may reflect early metabolic events, particularly increased proteoglycan (PG) synthesis which contributes to intimal volume and promotes atherogenesis through retention of atherogenic lipoproteins. We determined, in vitro, the PG metabolic activity of SV, ITA, and human coronary arteries through autoradiographic detection of incorporated [3H]glucosamine. SV had significantly higher levels of PG synthesis than ITA, especially in the subendothelial zone and after time (7 days) in culture. Patterns of synthesis in coronary vessels were similar to SV with high levels of incorporation in the subendothelial zone of thickened intima (> 100 microm). Increased subendothelial labelling in SV was due to increased PG synthesis, not decreased degradation. ITA showed no propensity for upregulation of subendothelial PG synthesis. Immunohistochemistry showed TGF-beta1 and TGF-beta2 localised primarily to the subendothelial zone of SV and coronary arteries. With time in culture immunostaining increased in parallel with increased PG synthesis. Subendothelial TGF-beta1 and TGF-beta2 were absent in ITA. A panspecific TGF-beta neutralising antibody reduced subendothelial PG synthesis in SV and coronary arteries by 50 and 60%, respectively. These results support the idea that vessels susceptible to atherosclerosis show increased accumulation of subendothelial PG mediated by TGF-beta.