Robert L. Reddick

Atherosclerosis in mice lacking apo E. Evaluation of lesional development and progression.

Apolipoprotein E-deficient mice have spontaneous elevations of total plasma cholesterol and triglycerides and reduced high-density lipoprotein. The mice develop arterial lesions in a time-dependent manner. Lesional distribution was centered in the aortic sinus in young mice, and the lesions were widely distributed throughout the arterial tree in mice at 8 to 9 months of age. In young mice, subendothelial foam cell deposits were present in the aortic sinus adjacent to valve-attachment sites. By 5 months of age, foam cell deposits, free cholesterol, and admixed smooth muscle cells composed the developing atherosclerotic lesions. After 8 to 9 months of age, the arterial lesions showed increased complexity, and fibrous cap lesions were present. Transmission electron microscopy showed foam cells, smooth muscle cells (both contractile and synthetic varieties), cellular debris, and acicular cholesterol deposits within the plaques. By scanning electron microscopy, subendothelial collections of foam cells were present within the aortic sinus and ascending aorta. The results show that the complexity of the atherosclerotic lesions that develop in these apo E deficient-mice are similar to those described in other species and therefore represent an important model for studies of genetic and environmental influences on the atherosclerotic process.

Targeted Replacement of the Mouse Apolipoprotein E Gene with the Common Human APOE3 Allele Enhances Diet-induced Hypercholesterolemia and Atherosclerosis

Apolipoprotein (apo) E, a constituent of several lipoproteins, is a ligand for the low density lipoprotein receptor, and this interaction is important for maintaining cholesterol and triglyceride homeostasis. We have used a gene replacement strategy to generate mice that express the human apoE3 isoform in place of the mouse protein. The levels of apoE mRNA in various tissues are virtually the same in the human apoE3 homozygous (3/3) mice and their littermates having the wild type mouse allele (+/+). Total cholesterol and triglyceride levels in fasted plasma from the 3/3 mice were not different from those in the +/+ mice, when maintained on a normal (low fat) chow diet. We found, however, notable differences in the distribution of plasma lipoproteins and apolipoprotein E between the two groups: β-migrating lipoproteins and plasma apoB100 levels are decreased in the 3/3 mice, and the apoE distribution is shifted from high density lipoproteins to larger lipoprotein particles. In addition, the fractional catabolic rate of exogenously administered remnant particles without apoE was 6-fold slower in the 3/3 mice compared with the +/+ mice. When the 3/3 and +/+ animals were fed a high fat/high cholesterol diet, the 3/3 animals responded with a dramatic increase (5-fold) in total cholesterol compared with the +/+ mice (1.5-fold), and after 12 weeks on this same diet the 3/3 animals developed significantly (at least 13-fold) larger atherosclerotic plaques in the aortic sinus area than the +/+ animals. Thus the structural differences between human APOE3 and mouse ApoE proteins are sufficient to cause an increased susceptibility to dietary-induced hypercholesterolemia and atherosclerosis in the 3/3 mice.

Enhanced atherosclerosis and kidney dysfunction in eNOS–/–Apoe–/– mice are ameliorated by enalapril treatment

Hypertension and atherosclerosis are each important causes of morbidity and mortality in the developed world. We have investigated the interaction between these conditions by breeding mice that are atherosclerotic due to lack of apolipoprotein (apo) E with mice that are hypertensive due to lack of endothelial nitric oxide synthase (eNOS). The doubly deficient mice (nnee) have higher blood pressure (BP) and increased atherosclerotic lesion size but no change in plasma lipoprotein profiles compared with normotensive but atherosclerotic (NNee) mice. The nnee mice also develop kidney damage, evidenced by increased plasma creatinine, decreased kidney weight/body weight ratio, and glomerular lipid deposition and calcification. Enalapril treatment abolishes the deleterious effects of eNOS deficiency on BP, atherosclerosis, and kidney dysfunction in nnee mice. In striking contrast, a genetic lack of inducible NOS, which does not affect BP, has no effect on the development of atherosclerotic lesions in Apoe(-/-) mice. We also observed a positive relationship between BP and size of atherosclerotic lesions These results suggest that the atherogenic effects of eNOS deficiency can be partially explained by an increase in BP and reemphasize the importance of controlling hypertension in preventing atherosclerosis.

Classification of Proliferative Pulmonary Lesions of the Mouse Recommendations of the Mouse Models of Human Cancers Consortium

Rapid advances in generating new mouse genetic models for lung neoplasia provide a continuous challenge for pathologists and investigators. Frequently, phenotypes of new models either have no precedents or are arbitrarily attributed according to incongruent human and mouse classifications. Thus, comparative characterization and validation of novel models can be difficult. To address these issues, a series of discussions was initiated by a panel of human, veterinary, and experimental pathologists during the Mouse Models of Human Cancers Consortium (NIH/National Cancer Institute) workshop on mouse models of lung cancer held in Boston on June 20–22, 2001. The panel performed a comparative evaluation of 78 cases of mouse and human lung proliferative lesions, and recommended development of a new practical classification scheme that would (a) allow easier comparison between human and mouse lung neoplasms, (b) accommodate newly emerging mouse neoplasms, and (c) address the interpretation of benign and preinvasive lesions of the mouse lung. Subsequent discussions with additional experts in pulmonary pathology resulted in the current proposal of a new classification. It is anticipated that this classification, as well as the complementary digital atlas of virtual histological slides, will help investigators and pathologists in their characterization of new mouse models, as well as stimulate further research aimed at a better understanding of proliferative lesions of the lung.

Diet-induced atherosclerosis in mice heterozygous and homozygous for apolipoprotein E gene disruption.

With the aim of establishing whether a genetically reduced capability of producing apolipoprotein E (apo E) can affect atherogenesis, we have compared the consequences of dietary stress on normal mice and on mice heterozygous or homozygous for a disrupted apo E gene. A dramatically accelerated development of lesions occurred in the vasculature of the homozygous mutants as a result of feeding an atherogenic diet for 12 wk, and extensive deposition of lipid-filled macrophages was found outside the cardiovascular system. In nine heterozygotes fed the atherogenic diet for 12 wk, the amount of apo E in their total plasma lipoproteins increased to a level comparable to normal, but all nine developed much larger foam cell lesions in their proximal aorta than those found in 3 of 9 normal mice fed the same diet. The other six normals had no lesions. Our study demonstrates that heterozygous mice with only one functional apo E gene are more susceptible to diet-induced atherosclerosis than are normal, two-copy mice. Genetically determined quantitative limitations of apo E could, therefore, have similar effects in humans when they are stressed by an atherogenic diet.

A strategy to increase the donor pool: use of cadaver lungs for transplantation.

A shortage of suitable donors is a serious obstacle to the widespread application of isolated lung transplantation for end-stage lung disease. We hypothesized that lung tissue likely remains viable for a sufficient period of time to allow for safe postmortem retrieval of lungs for transplantation. Studies were conducted in a nonsurvival model of canine lung allotransplantation. Donor animals were sacrificed, and subsequent lung harvest was delayed for 1 hour, 2 hours, or 4 hours. Pulmonary retrieval was then performed in a standard fashion, flushing the lung block with modified Euro-Collins solution. Lungs were then stored for 4 hours before single allotransplantation. Recipient animals were maintained anesthetized, and followed up for 8 hours. By occlusion of the pulmonary artery and bronchus to the native lung, recipient animals were forced to survive solely on the transplanted lung, with a constant inspired oxygen fraction of 0.40. All 5 recipient animals of 1-hour cadaver lungs survived the 8-hour observation period with excellent hemodynamics and gas exchange. Two of 5 recipients of 2-hour cadaver lungs survived the observation period, whereas a third animal survived for 5 hours with excellent gas exchange. One of 4 animals transplanted with a 4-hour cadaver lung survived the observation period. Retrieval of lungs from cadavers whose hearts are not beating may prove to be a safe and effective method to increase the pulmonary donor pool.

Estrogen receptor α is a major mediator of 17β-estradiol’s atheroprotective effects on lesion size in Apoe–/– mice

The inhibitory effects of estrogen (17β-estradiol) on atherosclerosis have been well documented in numerous animal models, and epidemiological evidence supports this protective effect in humans. The detailed mechanisms for this protection are not understood, but most are thought to be mediated through estrogen receptors (ERs), of which two are known (ERα and ERβ). To investigate the role of ERα in the atheroprotective effect of 17β-estradiol (E2), we ovariectomized female mice that lack apoE (AAee) or lack both apoE and ERα (ααee), and treated half of them with E2 for three months. E2 treatment of ovariectomized AAee females dramatically reduced the size of the lesions as well as their histological complexity. Plasma cholesterol was significantly reduced in this group, although the observed extent of protection by E2 was greater than could be explained solely by the change in lipid levels. In contrast, E2 treatment of ovariectomized ααee females caused minimal reduction in lesion size and no reduction in total plasma cholesterol compared with ααee mice without E2, demonstrating that ERα is a major mediator of the atheroprotective effect of E2. Nevertheless, E2 treatment significantly reduced the complexity of plaques in the ααee females, although not to the same degree as in AAee females, suggesting the existence of ERα-independent atheroprotective effects of E2.

Lack of apoA-I is not associated with increased susceptibility to atherosclerosis in mice.

The consequences of the lack of apolipoprotein A-I (apoA-I) were evaluated in mice made to lack apoA-I by gene targeting. Inbred strain 129 mice homozygous for the inactive Apoa1 gene and maintained on regular mouse chow had markedly reduced total cholesterol (26% normal) and high-density lipoprotein (HDL) cholesterol (25% normal) levels in their plasma. Their plasma lipoproteins lacked apoA-I and were reduced in all other apolipoproteins but apoE. ApoE comprises about one third of the protein of HDL particles in homozygotes, whereas it is present in only trace amounts in normal HDL. Despite the reduction of HDL cholesterol, no atherosclerotic lesions were observed in any of the homozygous mice evaluated (up to 15 months of age). After being maintained on an atherogenic diet for 4 weeks, total plasma cholesterol of the homozygous mutants increased by 20 mg/dL, while that of normals increased by 60 mg/dL. Mice with mixed 129 and C57BL/6J genetic backgrounds were fed the atherogenic diet for 20 weeks. A small number of foam cells were found attached to the aortic surface in some of the animals, but the extent and occurrence of these depositions were not related to the apoA-I genotype. Our results demonstrate that a lack of apoA-I does not by itself cause atherosclerosis in mice.

CCR5 deficiency is not protective in the early stages of atherogenesis in apoE knockout mice

The accumulation of macrophages and T lymphocytes in vessel walls is a hallmark of atherogenesis. It has recently been demonstrated in mouse models of atherosclerosis that full disease potential is dependent on several regulators of leukocyte trafficking, including the chemokine monocyte chemotactic protein 1 (MCP-1) and the chemokine receptors CCR2 and CXCR2. A possible role for the chemokine receptor CCR5 in atherogenesis has been suggested by CCR5 expression on macrophages, T cells, coronary endothelial cells and aortic smooth muscle cells and by the presence of CCR5 ligands in atherosclerotic plaques. Moreover, individuals who are naturally deficient in CCR5 were reported to be at reduced risk for severe coronary artery disease (CAD) and early myocardial infarction (MI). To investigate whether CCR5 is pro-atherogenic in mice, we generated CCR5-deficient mice and crossed them with atherosclerosis-prone apoE-deficient mice. Although CCR5-deficient mice exhibit defects in induced macrophage trafficking, mean atherosclerotic lesion area did not differ significantly between apoE-deficient mice and apoE/CCR5-deficient mice after 16 weeks on a diet of normal chow. Ribonuclease protection assays (RPA) on RNA isolated from plaques from both apoE-deficient and apoE/CCR5-deficient animals showed strong signals for the macrophage marker F4/80 but no evidence for expression of prominent markers of T and B lymphocytes. These results indicate that the early stages of plaque formation in this model of lipid-mediated atherogenesis do not depend on CCR5.

Paradoxical enhancement of atherosclerosis by probucol treatment in apolipoprotein E-deficient mice.

Dietary administration of probucol (0.5%, wt/wt) efficiently reduced total plasma cholesterol levels in apolipoprotein E-deficient mice (apoE-/-) by 40%, with decreases in high density lipoprotein (HDL) and apoAI by 70 and 50%, respectively. Paradoxically, however, aortic atherosclerotic plaques in the probucol-treated apoE-/- mice formed more rapidly than in the untreated apoE-/- mice, and the lesions were two to four times larger and more mature regardless of sex, age, and genetic background (P < 10(-)6). Histologically, lesions in probucol-treated mice contained increased fibrous materials and cells other than foam cells, and were commonly associated with focal inflammation and aneurysmal dilatation. Probucol treatment also accelerated lesion development in apoE+/- mice fed an atherogenic diet, indicating that the adverse effect is not dependent on the complete absence of apoE. Furthermore, mice lacking apoE and apoAI have plasma lipoprotein profiles very similar to the probucol-treated apoE-/- mice, but do not have accelerated plaque development. Thus, the enhanced atherosclerosis in the probucol-treated animals is unlikely to be caused by the reduction of HDL and apoAI levels. Our data indicate that a reduction in plasma cholesterol caused by probucol does not necessarily lead to an antiatherogenic effect.