Godfrey S. Getz

Site Specificity of Atherosclerosis: Site-Selective Responses to Atherosclerotic Modulators

Abstract—Atherosclerosis is a complex disease process that affects very specific sites of the vasculature. It is recognized that hemodynamic forces are largely responsible for dictating which vascular sites are either susceptible or resistant to developing atherosclerosis. In addition, a number of systemic and local factors also modulate the pathogenesis of the disease. By studying the development of atherosclerosis in mice, investigators have gained insights into the molecular mechanisms of this disease, although studies have largely focused on a single vascular site. Here, we review those recent studies in which vascular site-specific effects on atherosclerosis were reported when more than 1 site was examined. We assess the hypothesis that regional differences in the hemodynamic profile prime the endothelial phenotype to respond distinctly to such systemic risk factors as hypercholesterolemia, genetics, immune status, gender, and oxidative stress. Because a given treatment may differentially affect the development of atherosclerotic lesions throughout the vasculature, the sites chosen for study are critically important. By accounting for the complex interplay of factors that may operate at these different sites, a more complete understanding of the overriding mechanisms that control the initiation and progression of the atherosclerotic lesion may be realized.

Animal Models of Atherosclerosis

Atherosclerosis is a chronic inflammatory disorder that is the underlying cause of most cardiovascular disease. Both cells of the vessel wall and cells of the immune system participate in atherogenesis. This process is heavily influenced by plasma lipoproteins, genetics, and the hemodynamics of the blood flow in the artery. A variety of small and large animal models have been used to study the atherogenic process. No model is ideal as each has its own advantages and limitations with respect to manipulation of the atherogenic process and modeling human atherosclerosis or lipoprotein profile. Useful large animal models include pigs, rabbits, and nonhuman primates. Due in large part to the relative ease of genetic manipulation and the relatively short time frame for the development of atherosclerosis, murine models are currently the most extensively used. Although not all aspects of murine atherosclerosis are identical to humans, studies using murine models have suggested potential biological processes and interactions that underlie this process. As it becomes clear that different factors may influence different stages of lesion development, the use of mouse models with the ability to turn on or delete proteins or cells in tissue specific and temporal manner will be very valuable.

Lymphotoxin β receptor signaling promotes tertiary lymphoid organogenesis in the aorta adventitia of aged ApoE−/− mice

Atherosclerosis involves a macrophage-rich inflammation in the aortic intima. It is increasingly recognized that this intimal inflammation is paralleled over time by a distinct inflammatory reaction in adjacent adventitia. Though cross talk between the coordinated inflammatory foci in the intima and the adventitia seems implicit, the mechanism(s) underlying their communication is unclear. Here, using detailed imaging analysis, microarray analyses, laser-capture microdissection, adoptive lymphocyte transfers, and functional blocking studies, we undertook to identify this mechanism. We show that in aged apoE−/− mice, medial smooth muscle cells (SMCs) beneath intimal plaques in abdominal aortae become activated through lymphotoxin β receptor (LTβR) to express the lymphorganogenic chemokines CXCL13 and CCL21. These signals in turn trigger the development of elaborate bona fide adventitial aortic tertiary lymphoid organs (ATLOs) containing functional conduit meshworks, germinal centers within B cell follicles, clusters of plasma cells, high endothelial venules (HEVs) in T cell areas, and a high proportion of T regulatory cells. Treatment of apoE−/− mice with LTβR-Ig to interrupt LTβR signaling in SMCs strongly reduced HEV abundance, CXCL13, and CCL21 expression, and disrupted the structure and maintenance of ATLOs. Thus, the LTβR pathway has a major role in shaping the immunological characteristics and overall integrity of the arterial wall.

Diet and Murine Atherosclerosis

Lipid-enriched diets are often used to induce or accelerate the rate of atherosclerotic lesion development in murine models of atherosclerosis. It appears that the induction of persistent hypercholesterolemia to levels >≈300 mg/dL is required for the development of experimental atherosclerosis in the mouse. A variety of different diets have been used that vary in the level of cholesterol, the level and type of fatty acid, and the absence or presence of cholate. Each of these components as well as the protein source has been shown to influence lipoprotein level and/or atherosclerosis, with dietary cholesterol being the major proatherogenic component. In some instances the effects of these components on the expression of hepatic genes relevant to lipid homeostasis has been observed. An appreciation of the effect of the differences in diet composition on these processes is important to compare results from different atherosclerosis studies, so the composition of the diets used should always be reported or referenced. Cholate should not be used unless its effects are being specifically investigated.

Effect of Immune Deficiency on Lipoproteins and Atherosclerosis in Male Apolipoprotein E–Deficient Mice

Abstract—To determine whether T cells and B cells influence lipid metabolism and atherosclerosis, we crossed apolipoprotein E-deficient (apoE°) mice with recombination activating gene 2-deficient (RAG2°) mice. Total plasma cholesterol levels were ≈20% higher in male apoE° mice compared with the apoE°RAG2° mice at 8 weeks of age, and plasma triglyceride levels were 2.5-fold higher in the apoE° mice even when plasma cholesterol levels were similar. Male mice with plasma cholesterol levels between 400 and 600 mg/dL at 8 weeks of age were euthanized at 27 and 40 weeks of age. The aortic root lesion area in the apoE°RAG2° mice, compared with that in the immune-competent apoE° mice, was 81% and 57% smaller at 27 and 40 weeks of age, respectively. In contrast, there was no difference in the size of the brachiocephalic trunk lesions. Similar results were obtained with mice euthanized at 40 weeks of age that had 8-week cholesterol levels between 300 and 399 mg/dL. In apoE°RAG2° mice, aortic root atherosclerosis was more profoundly suppressed at lower cholesterol levels. Thus, T and B cells and their products differentially influence the development of atherosclerosis at different sites. We also demonstrate a profound effect of the immune system on plasma lipid homeostasis.

Lipoproteins in the central nervous system

Abstract: Although the synthesis and metabolism of plasma lipoproteins are well characterized, little is known about lipid delivery and clearance within the central nervous system (CNS). Our work has focused on characterizing the lipoprotein particles present in the cerebrospinal fluid (CSF) and the nascent particles secreted by astrocytes. In addition to carrying lipids, we have found that β‐amyloid (Ab) associates with lipoproteins, including the discoidal particles secreted by cultured astrocytes and the spherical lipoproteins found in CSF. We believe that association with lipoproteins provides a means of transport and clearance for Aβ. This process may be further influenced by an interaction between Ab and apoprotein E (apoE), the primary protein component of CNS lipoproteins. Specifically, we have investigated the formation and physiologic relevance of a SDS‐stable complex between apoE and Aβ. In biochemical assays, native apoE2 and E3 (associated with lipid particles) form an SDS‐stable complex with Ab that is 20‐fold more abundant than the apoE4:Aβ complex. In cell culture, native apoE3 but not E4 prevents Aβ‐induced neurotoxicity by a mechanism dependent on cell surface apoE receptors. In addition, apoE and the inhibition of apoE receptors prevent Aβ‐induced astrocyte activation. Therefore, we hypothesize that the protection from Aβ‐induced neurotoxicity afforded by apoE3 may result from clearance of the peptide by SDS‐stable apoE3:Aβ complex formation and uptake by apoE receptors.

Unique lipoproteins secreted by primary astrocytes from wild type, apoE (-/-), and human apoE transgenic mice.

Composition of central nervous system lipoproteins affects the metabolism of lipoprotein constituents within the brain. The ε4 allele of apolipoprotein E (apoE) is a risk factor for Alzheimer’s disease via an unknown mechanism(s). As glia are the primary central nervous system cell type that synthesize apoE, we characterized lipoproteins secreted by astrocytes from wild type (WT), apoE (−/−), and apoE transgenic mice expressing human apoE3 or apoE4 in a mouseapoE (−/−) background. Nondenaturing size exclusion chromatography demonstrates that WT, apoE3, and apoE4 astrocytes secrete particles the size of plasma high density lipoprotein (HDL) composed of phospholipid, free cholesterol, and protein, primarily apoE and apoJ. However, the lipid:apoE ratio of particles containing human apoE is significantly lower than WT. ApoE localizes across HDL-like particle sizes. ApoJ localizes to the smallest HDL-like particles.ApoE (−/−) astrocytes secrete little phospholipid or free cholesterol despite comparable apoJ expression, suggesting that apoE is required for normal secretion of astrocyte lipoproteins. Further, particles were not detected in apoE (−/−) samples by electron microscopy. Nondenaturing immunoprecipitation experiments indicate that apoE and apoJ reside predominantly on distinct particles. These studies suggest that apoE expression influences the unique structure of astrocyte lipoproteins, a process further modified by apoE species.

Paraoxonase, a cardioprotective enzyme: continuing issues.

Purpose of review The paraoxonase family consists of three members (PON1, PON2 and PON3) that share structural properties and enzymatic activities, among which is the ability to hydrolyze oxidized lipids in LDL. The exact function of the different family members is not clear although the conservation among the individual family members across species suggests a strong evolutionary pressure to preserve these functional differences. The purpose of this review is to highlight several problems with respect to the mechanism of action of paraoxonase and differences between the family members that merit further study. Recent findings PON1 transgenic mice are at lower risk for atherosclerosis, which is consistent with PON1 gene knockout studies in mice and human genetic polymorphism studies. The exact mechanism by which paraoxonase is cardioprotective is not clear, although it is likely to be related to its antioxidant properties especially on LDL. PON1 levels are influenced by a variety of environmental factors, including statins and cytokines. The preferential association of PON1 with HDL is mediated in part by its signal peptide and by desorption from the plasma membrane of expressing cells by HDL or phospholipid. Apolipoprotein A-I is not necessary for PON1 association with HDL, but its activity is stabilized in the presence of the apolipoprotein. Only in the absence of both lecithin cholesterol acyltransferase and apolipoprotein E is paraoxonase associated with non-HDL lipoproteins. The displacement of paraoxonase by serum amyloid A may explain in part the proinflammatory nature of HDL in the acute phase. The mechanism by which PON3 associates with HDL has not been studied. In addition to the ability to hydrolyze oxidized lipids in LDL, paraoxonase also alters the oxidative state of macrophages. Exogenous PON1 is able to reverse the oxidative stress in macrophages in aged apolipoprotein E deficient and PON1 deficient mice. The increase in oxidative stress in macrophages from PON1 deficient mice occurs despite the expression of PON2 and PON3 in macrophages. PON1 has recently been shown to contain phospholipase A2 activity, with the subsequent release of lysophosphatidylcholine that influences macrophage cholesterol biosynthesis. Summary PON1 mass and activity in the plasma significantly influence the risk of developing cardiovascular disease. This is likely mediated by its antioxidation properties on LDL and/or macrophages. The precise mechanism by which this HDL associated protein prevents or attenuates oxidation of LDL and the oxidative stress of macrophages remains to be clarified. The role of PON2 and PON3 in atherosclerosis and their antioxidant properties with respect to LDL and macrophages also merit further investigation.

The Metabolism of Proinsulin and Insulin by the Liver

The removal of bovine proinsulin by the isolated perfused rat liver has been studied and the results compared with the removal of insulin. At high concentrations of insulin (> 180 ng/ml) the removal process was saturated and the t(1/2) varied between 35 and 56 min. With low initial insulin levels the disappearance followed first-order kinetics, the mean regression coefficient being - 0.022, t(1/2) 13.8 min, and the hepatic extraction 4.0 ml/min. The results with proinsulin were in striking contrast to these findings. At both high and low concentrations the hepatic removal of proinsulin was considerably slower, averaging 10-15 times less than that of insulin. Specific immunoassay techniques and gel filtration of samples taken from perfusions to which both labeled and unlabeled proinsulin had been added did not show conversion to either insulin or the C-peptide. Bovine and rat (131)I-labeled proinsulins were degraded more slowly than bovine insulin-(131)I by bovine and rat liver homogenates. Both proinsulin and insulin inhibited the degradation of insulin-(131)I, equimolar quantities of proinsulin being 2-5 times less effective than insulin. These results indicate significant differences in the capacity of the liver to remove and degrade insulin and proinsulin. The low hepatic extraction of proinsulin may account for its prolonged half-life in vivo and contribute to its relatively high plasma concentration in the fasting state. Furthermore this finding will have to be taken into account in the interpretation of changes in the proinsulin:insulin ratios in peripheral blood in a variety of metabolich situations.

Atherosclerosis in the rhesus monkey fed three food fats.

Abstract Three groups of 6 male Rhesus monkeys were fed one of three diets, each containing 2% cholesterol and 25% lipid, either corn oil, butterfat, or peanut oil, over a period of 50 weeks. These diets produced prompt elevation of serum lipids. The highest serum cholesterol concentrations were observed in animals fed butterfat and the levels in animals fed peanut oil and corn oil were similar and much lower. Gross examination of the arteries revealed a definite difference in the degree and the characteristics of aortic atherosclerosis among the three dietary groups. The butterfat diet produced severe aortic lesions, characterized by abundant lipid deposition and relatively little cell proliferation or collagen deposition. The most widespread and advanced atherosclerosis was observed in monkeys fed peanut oil. The aortic lesions in these animals were characterized by thick, fibrous plaques which were elevated to the point of apparent narrowing of the ostia of various vessels. In addition, the peanut oil ration produced not only the highest incidence of coronary artery involvement but also the most severe coronary narrowing. This was apparently due to prominent intimal cell proliferation associated with a particularly high content of collagen. In spite of similar blood lipid elevations, the corn oil fed monkeys showed fewer gross atheromatous lesions in all parts of the arterial system and there was relatively little intimal thickening and much less lipid in the lesions. This study adds to the growing body of experimental evidence in several animal species that the tissue components and the severity of atherosclerotic lesions can be greatly influenced by the food fat fed and that peanut oil is an unusually atherogenic fat, mainly because of the severe intimal proliferation and fibrosis that occur.