Howard S. Kruth

Inhibition of Atherosclerosis Development in Cholesterol-Fed Human Apolipoprotein A-I–Transgenic Rabbits

BACKGROUND Prospective epidemiological studies support the hypothesis that high levels of high-density lipoprotein (HDL) cholesterol and apolipoprotein (apo) A-I limit atherosclerosis development. However, more data from studies with animal models of atherosclerosis that resemble the human disease are required to demonstrate the effect of apo A-I in the inhibition of atherogenesis. The rabbit is a good animal model for human atherosclerosis. METHODS AND RESULTS Human apo A-I-transgenic rabbits have been produced, and we have evaluated the effect of apo A-I on the development of atherosclerosis in transgenic rabbits fed a cholesterol-rich diet for 14 weeks. Plasma cholesterol levels of atherogenic apo B-containing lipoproteins were similar for transgenic and control rabbits (> 1000 mg/dL), while plasma levels of HDL cholesterol in the transgenic group were always about twice that of the control group (68 +/- 11 versus 37 +/- 3 mg/dL at 14 weeks; P < .001). At the end of the experiment, the amount of aortic surface area covered by lesions as well as the amount of lipid accumulation in the aorta were significantly less in transgenic rabbits compared with the control group (15 +/- 12% versus 30 +/- 8%, P < .0027 for the surface area of the thoracic aorta; 116 +/- 31 versus 247 +/- 39 mumol/g aorta, P < .0068 for cholesterol content in total aorta). CONCLUSIONS Overexpression of human apo A-I in rabbits inhibits the development of atherosclerosis in this animal model that resembles, in many respects, human atherosclerosis.

Prevention of bioprosthetic heart valve calcification by ethanol preincubation: Efficacy and mechanisms

BACKGROUND Calcification of the cusps of bioprosthetic heart valves fabricated from either glutaraldehyde cross-linked porcine aortic valves or bovine pericardium frequently causes the clinical failure of these devices. Our investigations studied ethanol pretreatment of glutaraldehyde cross-linked porcine aortic valves as a new approach to prevent cuspal calcification. The hypothesis governing this approach holds that ethanol pretreatment inhibits calcification resulting from protein structural alterations and lipid extraction. METHODS AND RESULTS Results demonstrated complete inhibition of calcification of glutaraldehyde-pretreated porcine bioprosthetic aortic valve cusps by 80.0% ethanol in rat subdermal implants (60-day ethanol-pretreated calcium level, 1.87 +/- 0.29 micrograms/mg tissue compared with control calcium level, 236.00 +/- 6.10 micrograms/mg tissue) and in sheep mitral valve replacements (ethanol-pretreated calcium level, 5.22 +/- 2.94 micrograms/mg tissue; control calcium level, 32.50 +/- 11.50 micrograms/mg tissue). The mechanism of ethanol inhibition may be explained by several observations: ethanol pretreatment resulted in an irreversible alteration in the amide I band noted in the infrared spectra for both purified type I collagen and glutaraldehyde cross-linked porcine aortic leaflets. Ethanol pretreatment also resulted in nearly complete extraction of leaflet cholesterol and phospholipid. CONCLUSIONS Ethanol pretreatment of glutaraldehyde cross-linked porcine aortic valve bioprostheses represents a highly efficacious and mechanistically based approach and may prevent calcific bioprosthetic heart valve failure.

Heterogeneity of human macrophages in culture and in atherosclerotic plaques.

Research suggests that monocytes differentiate into unique lineage-determined macrophage subpopulations in response to the local cytokine environment. The present study evaluated the atherogenic potential of two divergent lineage-determined human monocyte-derived macrophage subpopulations. Monocytes were differentiated for 7 days in the presence of alternative macrophage development cytokines: granulocyte-macrophage colony-stimulating factor to produce granulocyte-macrophage-CSF macrophages (GM-Mac), or macrophage colony-stimulating factor (M-CSF) to produce M-Mac. Gene chip analyses of three monocyte donors demonstrated differential expression of inflammatory and cholesterol homeostasis genes in the macrophage subpopulations. Quantitative PCR confirmed a fivefold elevation in the expression of genes that promote reverse cholesterol transport (PPAR-gamma, LXR-alpha, and ABCG1) and macrophage emigration from lesions (CCR7) in GM-Mac compared to that in M-Mac. Immunocytochemistry confirmed enhanced expression of the proinflammatory marker CD14 in M-Mac relative to GM-Mac. M-Mac spontaneously accumulated cholesterol when incubated with unmodified low-density lipoprotein whereas GM-Mac only accumulated similar levels of cholesterol after protein kinase C activation. Immunostained human coronary arteries showed that macrophages with similar antigen expression to that of M-Mac (CD68(+)/CD14(+)) were predominant within atherosclerotic lesions whereas macrophages with antigen expression similar to GM-Mac (CD68(+)/CD14(-)) were predominant in areas devoid of disease. The identification of macrophage subpopulations with different gene expression patterns and, thus, different potentials for promoting atherosclerosis has important experimental and clinical implications and could prove to be a valuable finding in developing therapeutic interventions in diseases dependent on macrophage function.

APOLIPOPROTEIN E PRODUCED BY HUMAN MONOCYTE-DERIVED MACROPHAGES MEDIATES CHOLESTEROL EFFLUX THAT OCCURS IN THE ABSENCE OF ADDED CHOLESTEROL ACCEPTORS

Human monocyte-derived macrophages can efflux accumulated cholesterol without exogenously added cholesterol acceptors (Kruth, H. S., Skarlatos, S. I., Gaynor, P. M., and Gamble, W. (1994) J. Biol. Chem. 269, 24511-24518). Most of the effluxed cholesterol accumulates in the medium as apolipoprotein E-discoidal lipid particles. In the current study, we determined whether and to what degree cholesterol efflux from human monocyte-macrophages depended on apolipoprotein E secretion. Unexpectedly, 2-week-old differentiated monocyte-macrophages secreted similar amounts of apolipoprotein E without or with cholesterol enrichment. Apolipoprotein E mRNA levels in these macrophages were not increased by cholesterol enrichment and were comparable with levels in HepG2 cells. Without cholesterol enrichment, monocyte-macrophages secreted lipid-poor apolipoprotein E with a density >1.21 g/ml. By contrast, cholesterol enrichment of monocyte-macrophages induced the association of apoE with phospholipid and cholesterol to form discoidal particles that floated at densities of 1.08-1.10 g/ml. An anti-apolipoprotein E monoclonal antibody added to the culture medium significantly inhibited cholesterol and phospholipid efflux from the monocyte-macrophages. This showed that apolipoprotein E was required for most of the cholesterol efflux, and that apolipoprotein E did not leave macrophages with lipid but rather associated with lipid after it was secreted. Thus, 1) apolipoprotein E was constitutively secreted by differentiated human monocyte-macrophages, 2) apolipoprotein E only formed discoidal particles following macrophage cholesterol enrichment, 3) apolipoprotein E was necessary for cholesterol efflux to occur in the absence of added cholesterol acceptors and, in addition 4) the level of macrophage unesterified cholesterol was not rate-limiting for this cholesterol efflux, and 5) net phospholipid synthesis occurred in macrophages secondary to apoE-mediated loss of macrophage phospholipid. In conclusion, apolipoprotein E functions in an autocrine pathway that mediates cholesterol efflux from human monocyte-derived macrophages.

Fluorescent pegylated nanoparticles demonstrate fluid-phase pinocytosis by macrophages in mouse atherosclerotic lesions

The uptake of lipoproteins by macrophages is a critical step in the development of atherosclerotic lesions. Cultured monocyte-derived macrophages take up large amounts of native LDL by receptor-independent fluid-phase pinocytosis, either constitutively or in response to specific activating stimuli, depending on the macrophage phenotype. We therefore sought to determine whether fluid-phase pinocytosis occurs in vivo in macrophages in atherosclerotic lesions. We demonstrated that fluorescent pegylated nanoparticles similar in size to LDL (specifically nontargeted Qtracker quantum dot and AngioSPARK nanoparticles) can serve as models of LDL uptake by fluid-phase pinocytosis in cultured human monocyte-derived macrophages and mouse bone marrow-derived macrophages. Using fluorescence microscopy, we showed that atherosclerosis-prone Apoe-knockout mice injected with these nanoparticles displayed massive accumulation of the nanoparticles within CD68+ macrophages, including lipid-containing foam cells, in atherosclerotic lesions in the aortic arch. Similar results were obtained when atherosclerotic mouse aortas were cultured with nanoparticles in vitro. These results show that macrophages within atherosclerotic lesions can take up LDL-sized nanoparticles by fluid-phase pinocytosis and indicate that fluid-phase pinocytosis of LDL is a mechanism for macrophage foam cell formation in vivo.

Colocalization of cholesterol and hydroxyapatite in human atherosclerotic lesions

SummaryCholesterol and calcium phosphate, the latter in the form of hydroxyapatite, accumulate in atherosclerotic lesions. In this report, we demonstrate that these organic and inorganic constitutents of lesions can accumulate together, closely associated in crystal agglomerates. Using the fluorescent cholesterol probe, filipin, we identified unesterified cholesterol that was associated with calcium granules in tissue sections of lesions. We also have shown that small crystallites of cholesterol can associate with preformed hydroxyapatite crystals in vitro. Scanning electron microscopy couple with energy-dispersive X-ray analysis demonstrated the physical association of many small crystallites of cholesterol with larger crystals of hydroxyapatite. These small crystallites of cholesterol associated with hydroxyapatite stained with filipin. This contrasted with the lack of filipin staining of unassociated larger cholesterol crystals or hydroxyapatite alone. How cholesterol and calcium come to be closely associated in crystal agglomerates within atherosclerotic lesions remains to be determined.

Glycosphingolipid Accumulation in the Aortic Wall Is Another Feature of Human Atherosclerosis

High accumulation of lipids is a typical feature of an atherosclerotic lesion. We have previously identified the chemical structure of the major glycosphingolipids (GSLs) of human aorta; however, quantification of the absolute concentration of GSLs was not carried out. In the present study, for the first time we have performed a quantitative comparative analysis of GSL composition in the media and two sublayers of the intima taken from normal regions, fatty streaks, and atherosclerotic plaques of the human aorta. The intimal tissue containing fatty streaks and atherosclerotic plaques accumulated GSLs, predominantly glucosylceramide (GlcCer), lactosylceramide (LacCer), and ganglioside GM3. GSL levels in plaques were highest: GlcCer was 18- and 8-fold, LacCer was 8- and 7-fold, and GM3 was 2.5- and 12-fold higher than in musculoelastic and elastic-hyperplastic intimal layers of normal regions, respectively. We did not observe a significant increase in other GSLs. An increase in the content of gangliosides GD3 and GD1a was detected in the media underlying atherosclerotic lesions. On the basis of an analysis of the ratio of GlcCer, LacCer, and GM3 accumulated in the tissue and cells of the elastic-hyperplastic layer of intima, we have concluded that the accumulation of the above-mentioned GSLs occurs mainly in the extracellular space of the intima. In this study, we have also demonstrated that extracellular lipid liposomes, which appear in the early stages of atherogenesis, are one locus of GSL accumulation in the extracellular space of the intima.(ABSTRACT TRUNCATED AT 250 WORDS)

Constitutive Receptor-independent Low Density Lipoprotein Uptake and Cholesterol Accumulation by Macrophages Differentiated from Human Monocytes with Macrophage-Colony-stimulating Factor (M-CSF)

Recently, we have shown that macrophage uptake of low density lipoprotein (LDL) and cholesterol accumulation can occur by nonreceptor mediated fluid-phase macropinocytosis when macrophages are differentiated from human monocytes in human serum and the macrophages are activated by stimulation of protein kinase C (Kruth, H. S., Jones, N. L., Huang, W., Zhao, B., Ishii, I., Chang, J., Combs, C. A., Malide, D., and Zhang, W. Y. (2005) J. Biol. Chem. 280, 2352–2360). Differentiation of human monocytes in human serum produces a distinct macrophage phenotype. In this study, we examined the effect on LDL uptake of an alternative macrophage differentiation phenotype. Differentiation of macrophages from human monocytes in fetal bovine serum with macrophage-colony-stimulating factor (M-CSF) produced a macrophage phenotype demonstrating constitutive fluid-phase uptake of native LDL leading to macrophage cholesterol accumulation. Fluid-phase endocytosis of LDL by M-CSF human macrophages showed non-saturable uptake of LDL that did not down-regulate over 48 h. LDL uptake was mediated by continuous actin-dependent macropinocytosis of LDL by these M-CSF-differentiated macrophages. M-CSF is a cytokine present within atherosclerotic lesions. Thus, macropinocytosis of LDL by macrophages differentiated from monocytes under the influence of M-CSF is a plausible mechanism to account for macrophage foam cell formation in atherosclerotic lesions. This mechanism of macrophage foam cell formation does not depend on LDL modification or macrophage receptors.

Dominant Late-onset Retinal Degeneration with Regional Variation of Sub-Retinal Pigment Epithelium Deposits, Retinal Function, and Photoreceptor Degeneration

PURPOSE To clarify the pathogenesis of late-onset retinal degeneration (L-ORD), an autosomal dominant disorder characterized by thick deposits of lipid-rich material between the retinal pigment epithelium (RPE) and Bruchs membrane. STUDY DESIGN Comparative clinicopathologic case report and case series. TISSUES: Eyes of an 82-year-old L-ORD eye donor and an age-matched control. SUBJECTS Five descendants of the eye donor and his affected sister. METHODS The eyes were processed for histopathologic examination, including electron microscopy and immunohistochemistry. Family members were examined clinically and with retinal function tests. RESULTS The L-ORD eye had sub-RPE deposits that were positive for lipid, including esterified and unesterified cholesterol. The deposits were thinnest in the macula, which retained the highest percentage of photoreceptors. In the periphery, RPE thinning and photoreceptor loss correlated with thickness of the sub-RPE deposits. The eye donor was asymptomatic until his late 50s, when he developed problems with adapting to darkness. At age 68, the eye donor had normal acuity but a midperipheral scotoma and subnormal electroretinograms (ERGs); visual loss was progressive. The five descendants (at the time of examination ages 44-58) of the eye donor and his affected sister, who were at 50/50 risk of inheriting L-ORD, had normal ERGs, but four showed defects in dark adaptation. The dark adaptation abnormalities had a distribution similar to the thickness of the sub-RPE deposits in the eye donor, with slow kinetics in the midperiphery and normal kinetics centrally. CONCLUSIONS The L-ORD donor eye differed from a previous case in the regional distribution of sub-RPE deposits and photoreceptors. In the next generation of this L-ORD family, the first expression of disease, abnormal dark adaptation, mirrored the regional distribution of the deposits in the donor eye. The fine structure and staining characteristics of the sub-RPE deposits in L-ORD resemble those in age-related macular degeneration and Sorsby fundus dystrophy.

Aggregated Low Density Lipoprotein Induces and Enters Surface-connected Compartments of Human Monocyte-Macrophages UPTAKE OCCURS INDEPENDENTLY OF THE LOW DENSITY LIPOPROTEIN RECEPTOR

Aggregation of low density lipoprotein (LDL) stimulates its uptake by macrophages. We have now shown by electron microscopic and chemical experiments that aggregated LDL (produced by vortexing (VxLDL) or treatment with phospholipase C) induced and became sequestered in large amounts within surface-connected compartments (SCC) of human monocyte-derived macrophages. This occurred through a process different from phagocytosis. Formation of SCC and accumulation of aggregated LDL in SCC are cell-mediated processes that were temperature-dependent (10 × greater cell association at 37 °C than at 4 °C) and blocked by cytochalasin D but not by nocodazole. Because of the surface connections of SCC, trypsin could release aggregated LDL from SCC. Degradation of125I-VxLDL through the SCC pathway showed delayed and a lower rate of degradation (10–55%) compared with nonaggregated125I-acetylated LDL that did not enter SCC. However, similar to 125I-acetylated LDL degradation,125I-VxLDL degradation occurred through a chloroquine-sensitive pathway. Uptake of VxLDL into SCC was not mediated by the LDL receptor. Methylation of LDL prevents its binding to the LDL receptor. However, methylated LDL still entered SCC after it was aggregated by vortexing. On the other hand, degradation of125I-VxLDL was substantially decreased by methylation of LDL and by cholesterol enrichment of macrophages, which decreases macrophage LDL receptor expression. The results suggest that whereas uptake of aggregated LDL into SCC occurs independently of the LDL receptor, movement of aggregated LDL from SCC to lysosomes may depend in part on LDL receptor function. Sequestration into SCC is a novel endocytosis pathway for uptake of aggregated LDL that allows the macrophage to store large amounts of this lipoprotein before it is further processed.