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High-density lipoprotein (HDL) and its main protein, apolipoprotein AI (apoAI), have established benefits in various cells, but whether these cytoprotective effects of HDL pertain to renal cells is unclear. We investigated the in vitro consequences of exposing damaged podocytes to normal apoAI, HDL, and apoAI mimetic (L-4F), and the in vivo effects of L-4F on kidney and atherosclerotic injury in a podocyte-specific injury model of proteinuria. In vitro, primary mouse podocytes were injured by puromycin aminonucleoside (PAN). Cellular viability, migration, production of reactive oxygen species (ROS), apoptosis, and the underlying signaling pathway were assessed. In vivo, we used a proteinuric model, Nphs1-hCD25 transgenic (NEP25+) mice, which express human CD25 on podocytes. Podocyte injury was induced by using immunotoxin (LMB2) and generated a proteinuric atherosclerosis model, NEP25+:apoE−/−mice, was generated by mating apoE-deficient (apoE−/−) mice with NEP25+mice. Animals received L-4F or control vehicle. Renal function, podocyte injury, and atherosclerosis were assessed. PAN reduced podocyte viability, migration, and increased ROS production, all significantly lessened by apoAI, HDL, and L-4F. L-4F attenuated podocyte apoptosis and diminished PAN-induced inactivation of Janus family protein kinase-2/signal transducers and activators of transcription 3. In NEP25+mice, L-4F significantly lessened overall proteinuria, and preserved podocyte expression of synaptopodin and cell density. Proteinuric NEP25+:apoE−/−mice had more atherosclerosis than non-proteinuric apoE−/−mice, and these lesions were significantly decreased by L-4F. Normal human apoAI, HDL, and apoAI mimetic protect against podocyte damage. ApoAI mimetic provides in vivo beneficial effects on podocytes that culminate in reduced albuminuria and atherosclerosis. The results suggest supplemental apoAI/apoAI mimetic may be a novel candidate to lessen podocyte damage and its complications.

Macrophage apoptosis and efferocytosis are key determinants of atherosclerotic plaque inflammation and necrosis. Bone marrow transplantation studies in ApoE- and LDLR-deficient mice revealed that hematopoietic scavenger receptor class B type I (SR-BI) deficiency results in severely defective efferocytosis in mouse atherosclerotic lesions, resulting in a 17-fold higher ratio of free to macrophage-associated dead cells in lesions containing SR-BI−/−cells, 5-fold more necrosis, 65.2% less lesional collagen content, nearly 7-fold higher dead cell accumulation, and 2-fold larger lesion area. Hematopoietic SR-BI deletion elicited a maladaptive inflammatory response [higher interleukin (IL)-1β, IL-6, and TNF-α lower IL-10 and transforming growth factor β]. Efferocytosis of apoptotic thymocytes was reduced by 64% in SR-BI−/−versus WT macrophages, both in vitro and in vivo. In response to apoptotic cells, macrophage SR-BI bound with phosphatidylserine and induced Src phosphorylation and cell membrane recruitment, which led to downstream activation of phosphoinositide 3-kinase (PI3K) and Ras-related C3 botulinum toxin substrate 1 (Rac1) for engulfment and clearance of apoptotic cells, as inhibition of Src decreased PI3K, Rac1-GTP, and efferocytosis in WT cells. Pharmacological inhibition of Rac1 reduced macrophage efferocytosis in a SR-BI-dependent fashion, and activation of Rac1 corrected the defective efferocytosis in SR-BI−/−macrophages. Thus, deficiency of macrophage SR-BI promotes defective efferocytosis signaling via the Src/PI3K/Rac1 pathway, resulting in increased plaque size, necrosis, and inflammation.

Tissue cholesterol accumulation, macrophage infiltration, and inflammation are features of atherosclerosis and some forms of dermatitis. HDL and its main protein, apoAI, are acceptors of excess cholesterol from macrophages; this process inhibits tissue inflammation. Recent epidemiologic and clinical trial evidence questions the role of HDL and its manipulation in cardiovascular disease. We investigated the effect of ectopic macrophage apoAI expression on atherosclerosis and dermatitis induced by the combination of hypercholesterolemia and absence of HDL in mice. Hematopoietic progenitor cells were transduced to express human apoAI and transplanted into lethally irradiated LDL receptor−/−/apoAI−/−mice, which were then placed on a high-fat diet for 16 weeks. Macrophage apoAI expression reduced aortic CD4+T-cell levels (−39.8%), lesion size (−25%), and necrotic core area (−31.6%), without affecting serum HDL or aortic macrophage levels. Macrophage apoAI reduced skin cholesterol by 39.8%, restored skin morphology, and reduced skin CD4+T-cell levels. Macrophage apoAI also reduced CD4+T-cell levels (−32.9%) in skin-draining lymph nodes but had no effect on other T cells, B cells, dendritic cells, or macrophages compared with control transplanted mice. Thus, macrophage apoAI expression protects against atherosclerosis and dermatitis by reducing cholesterol accumulation and regulating CD4+T-cell levels, without affecting serum HDL or tissue macrophage levels.

Background:Postoperative delirium is associated with increased perioperative morbidity and mortality. Poor cerebrovascular health may increase delirium risk. We hypothesized that higher HDL concentration is associated with less postoperative delirium.Methods:After IRB approval, we prospectively recruited 65 adults undergoing major, elective cardiac and vascular surgery. Research staff performed preoperative dementia screening using the Mini Mental Status Exam (MMSE) and the Trail Making Test Part B. Postoperative Richmond Agitation Scale Scores and Confusion Assessment Method tool scores were collected from the medical record on postoperative days 1 and 2. Because HDL function often correlates with disease better than cholesterol level, HDL cholesterol efflux capacity (CEC) was measured in J774 macrophages loaded with 3H-cholesterol in acetylated-LDL and then incubated with ApoB-depleted patient serum for 24 hours. Multivariate logistic regression was used to assess the association between HDL-C and HDL CEC and postoperative delirium, adjusted for age, preoperative MMSE score, preoperative Trail making score, and cardiopulmonary bypass exposure.Results:HDL-C was correlated with HDL CEC (Spearman’s R=0.37, p=0.002). One patient in the first (lowest) tertile, one patient in the second, and six patients in the third HDL-C tertile developed delirium. In contrast, four patients in the first tertile, three patients in the second, and one patient in the third tertile of CEC developed delirium. A higher preoperative HDL-C was independently associated with a higher odds of postoperative delirium (odds ratio 1.14 (95% CI 1.03, 1.26), p=0.01). In contrast, a higher preoperative HDL CEC was associated with a lower odds of delirium (odds ratio 0.82 (95% CI 0.68-0.99), p=0.04).Conclusions:Higher HDL CEC, but not HDL-C, was associated with less postoperative delirium. In this patient population, HDL function may play an important role in the development of postoperative delirium.

Mice deficient in scavenger receptor class B type I (SR-BI) and apolipoprotein E (apoE) [double knockout (DKO) mice] develop dyslipidemia, accelerated atherosclerosis, and myocardial infarction, and die prematurely. We examined effects of apoE and SR-BI deficiency on macrophage cholesterol homeostasis. DKO macrophages had increased total cholesterol (TC) stores (220–380 μg/mg protein) compared with apoE−/−cells (40 μg/mg), showed significant lysosomal lipid engorgement, and increased their TC by 34% after exposure to HDL. DKO macrophages from apoE−/−mice reconstituted with DKO bone marrow showed less cholesterol accumulation (89 μg/mg), suggesting that the dyslipidemia of DKO mice explains part of the cellular cholesterol defect. However, analyses of DKO and apoE−/−macrophages from transplanted apoE−/−mice revealed a role for macrophage SR-BI, inasmuch as the TC in DKO macrophages increased by 10% in the presence of HDL, whereas apoE−/−macrophage TC decreased by 33%. After incubation with HDL, the free cholesterol (FC) increased by 29% in DKO macrophages, and decreased by 8% in apoE−/−cells, and only DKO cells had FC in large peri-nuclear pools. Similar trends were observed with apoA-I as an acceptor. Thus, the abnormal cholesterol homeostasis of DKO macrophages is due to the plasma lipid environment of DKO mice and to altered trafficking of macrophage cholesterol. Both factors are likely to contribute to the accelerated atherosclerosis in DKO mice.

Mice deficient in scavenger receptor class B type I (SR-BI) and apolipoprotein E (apoE) [double knockout (DKO) mice] develop dyslipidemia, accelerated atherosclerosis, and myocardial infarction, and die prematurely. We examined effects of apoE and SR-BI deficiency on macrophage cholesterol homeostasis. DKO macrophages had increased total cholesterol (TC) stores (220–380 µg/mg protein) compared with apoE–/–cells (40 µg/mg), showed significant lysosomal lipid engorgement, and increased their TC by 34% after exposure to HDL. DKO macrophages from apoE–/–mice reconstituted with DKO bone marrow showed less cholesterol accumulation (89 µg/mg), suggesting that the dyslipidemia of DKO mice explains part of the cellular cholesterol defect. However, analyses of DKO and apoE–/–macrophages from transplanted apoE–/–mice revealed a role for macrophage SR-BI, inasmuch as the TC in DKO macrophages increased by 10% in the presence of HDL, whereas apoE–/–macrophage TC decreased by 33%. After incubation with HDL, the free cholesterol (FC) increased by 29% in DKO macrophages, and decreased by 8% in apoE–/–cells, and only DKO cells had FC in large peri-nuclear pools. Similar trends were observed with apoA-I as an acceptor. Thus, the abnormal cholesterol homeostasis of DKO macrophages is due to the plasma lipid environment of DKO mice and to altered trafficking of macrophage cholesterol. Both factors are likely to contribute to the accelerated atherosclerosis in DKO mice.

We have investigated apolipoprotein E (apoE) recycling in Chinese hamster ovary (CHO) cells, a peripheral cell that does not produce lipoproteins or express apoE. Using a pulse-chase protocol in which cells were pulsed with 125I-apoE-VLDL and chased for different periods, ∼30% of the apoE internalized during the pulse was resecreted within a 4 h chase in a relatively lipid-free state. The addition of lysosomotropic agents or brefeldin A had no effect on apoE recycling. Unlike previous results with hepatocytes and macrophages, neither apoA-I nor upregulation of ABCA1 stimulated apoE recycling. However, cyclodextrin, which extracts cholesterol from plasma membrane lipid rafts, increased recycling. Confocal studies revealed that apoE, internalized during a 1 h pulse, colocalizes with early endosomal antigen-1, Rab5, Rab11a, and lysobisphosphatidic acid but not with lysosomal-associated membrane protein-1. Colocalization of apoE and Rab11a persisted even after cells had been chased for 1 h, suggesting a pool of apoE within the endosomal recycling compartment (ERC). Our data suggest that apoE recycling in CHO cells is linked to cellular cholesterol removal via the ERC and phospholipid-containing acceptors in a pathway alternative to the ABCA1-apoA-I axis.

Alagille syndrome is associated with bile duct paucity resulting in liver disease. Patients can be divided into mildly and severely icteric groups, with both groups having altered lipoproteins. The incidence of ischemic heart disease is rare in severely cholestatic children despite increased total cholesterol and decreased high density lipoprotein cholesterol (HDL-C). The present studies examine the impact of altered lipid and lipoproteins on scavenger receptor class B type I (SR-BI)- and ABCA1-mediated efflux to serum from both groups. Efflux was compared with serum from 29 patients (15 with normal plasma cholesteryl ester, 14 with low cholesteryl ester). Efflux via SR-BI and ABCA1 was studied using cell systems having either low or high expression levels of these receptors. SR-BI efflux was lower (P = 0.04) with serum from severely icteric patients (3.9 +/- 1.4%) compared with serum from mildly icteric patients (5.1 +/- 1.4%) and was positively correlated with HDL-C and its apolipoproteins. SR-BI-mediated efflux was not correlated with any particular mature HDL but was negatively correlated with small lipid-poor prebeta-1 HDL. Consistent with severely icteric patients having high prebeta-1 HDL levels, the ABCA1 efflux was significantly higher with their serum (4.8 +/- 2.2%) compared with serum from mildly icteric patients (2.0 +/- 0.6%) and was positively correlated with prebeta-1 HDL. These studies demonstrated that prebeta-1 HDL is the preferred acceptor for ABCA1 efflux, whereas many particles mediate SR-BI efflux.

The effects of in vivo modulation of HDL phospholipid (PL) on scavenger receptor class BI (SR-BI)- and ATP binding cassette transporter 1 (ABCA1)-mediated efflux were examined by overexpressing either endothelial lipase (EL) or phosphatidylserine phospholipase (PS-PLA1) in human apolipoprotein A-I (apoA-I) transgenic mice. Overexpression of EL led to large reductions in the serum PL/apoA-I ratio (−60%), total cholesterol (TC; −89%), and HDL cholesterol (−91%). Relative to the serum before overexpression of EL, the efflux potential of the serum via SR-BI decreased by 90% and ABCA1-mediated efflux increased by 63%. In contrast to overexpression of EL, overexpression of PS-PLA1led to increases in the PL/apoA-I ratio (88%), TC (78%), HDL cholesterol (57%), and HDL size. The efflux potential of the serum increased by 60% via SR-BI and decreased by 57% via ABCA1. There were significant positive correlations between SR-BI-mediated efflux and a number of serum parameters, including PL/apoA-I ratio, PL, TC, free cholesterol (FC), and HDL cholesterol. In striking contrast, the same correlations were seen with ABCA1-mediated efflux, but the relationships were inverse.

Low levels of transgenic mouse apolipoprotein E (apoE) suppress atherosclerosis in apoE knockout (apoE−/−) mice without normalizing plasma cholesterol. To test whether this is due to facilitation of cholesterol efflux from the vessel wall, we produced apoA-I−/−/apoE−/−mice with or without the transgene. Even without apoA-I and HDL, apoA-I−/−/apoE−/−mice had the same amount of aorta cholesteryl ester as apoE−/−mice. Low apoE in the apoA-I−/−/apoE−/−transgenic mice reduced aortic lesions by 70% versus their apoA-I−/−/apoE−/−siblings. To define the free cholesterol (FC) efflux capacity of lipoproteins from the various genotypes, sera were assayed on macrophages expressing ATP-binding cassette transporter A1 (ABCA1). Surprisingly, ABCA1 FC efflux was twice as high to sera from the apoA-I−/−/apoE−/−or apoE−/−mice compared with wild-type mice, and this activity correlated with serum apoA-IV. Immunodepletion of apoA-IV from apoA-I−/−/apoE−/−serum abolished ABCA1 FC efflux, indicating that apoAI-V serves as a potent acceptor for FC efflux via ABCA1. With increasing apoE expression, apoA-IV and FC acceptor capacity decreased, indicating a reciprocal relationship between plasma apoE and apoA-IV.

Microscopy has played a critical role in first identifying and then defining the role of lysosomes in formation of atherosclerotic foam cells. We review the evidence implicating lysosomal lipid accumulation as a factor in the pathogenesis of atherosclerosis with reference to the role of microscopy. In addition, we explore mechanisms by which lysosomal lipid engorgement occurs. Low density lipoproteins which have become modified are the major source of lipid for foam cell formation. These altered lipoproteins are taken into the cell via receptor-mediated endocytosis and delivered to lysosomes. Under normal conditions, lipids from these lipoproteins are metabolized and do not accumulate in lysosomes. In the atherosclerotic foam cell, this normal metabolism is inhibited so that cholesterol and cholesteryl esters accumulate in lysosomes. Studies of cultured cells incubated with modified lipoproteins suggests this abnormal metabolism occurs in two steps. Initially, hydrolysis of lipoprotein cholesteryl esters occurs normally, but the resultant free cholesterol cannot exit the lysosome. Further lysosomal cholesterol accumulation inhibits hydrolysis, producing a mixture of cholesterol and cholesteryl esters within swollen lysosomes. Various lipoprotein modifications can produce this lysosomal engorgement in vitro and it remains to be seen which modifications are most important in vivo.

Foam cells in the atherosclerotic lesion have substantial cholesterol stores within large, swollen lysosomes. This feature is mimicked by incubating THP-1 macrophages with mildly oxidized low density lipoprotein (LDL). Incubation of THP-1 cells with acetylated LDL produces cytoplasmic cholesteryl ester accumulation rather than lysosomal storage. The differences could be due to differences in uptake and delivery of lipoprotein to lysosomes or to lysosomal and post-lysosomal processing events. We compared uptake and lysosomal trafficking of acetylated and oxidized LDL using colloidal gold-labeled lipoproteins. Labeling did not alter cellular cholesterol accumulation. We found that uptake and delivery to lysosomes are not different for acetylated and oxidized LDL. In fact, both oxidized and acetylated LDL can be delivered to the same lysosomes. Sequential incubation with oxidized LDL followed by acetylated LDL showed that the lipid-engorged lysosomes are long-lived structures, continuously accepting newly ingested lipoprotein. Comparison of acetylated and oxidized LDL in mouse peritoneal macrophages, a cell which does not accumulate substantial lysosomal lipid, also revealed no differences in uptake. This indicates that in THP-1 cells, the differences in metabolism of oxidized and acetylated LDL are due to cell-specific lysosomal or post-lysosomal events not present in B6C3F1 mouse macrophages.

The role of high density lipoprotein (HDL) phospholipid in scavenger receptor BI (SR-BI)-mediated free cholesterol flux was examined by manipulating HDL3phosphatidylcholine and sphingomyelin content. Both phosphatidylcholine and sphingomyelin enrichment of HDL enhanced the net efflux of cholesterol from SR-BI-expressing COS-7 cells but by two different mechanisms. Phosphatidylcholine enrichment of HDL increased efflux, whereas sphingomyelin enrichment decreased influx of HDL cholesterol. Although similar trends were observed in control (vector-transfected) COS-7 cells, SR-BI overexpression amplified the effects of phosphatidylcholine and sphingomyelin enrichment of HDL 25- and 2.8-fold, respectively. By using both phosphatidylcholine-enriched and phospholipase A2-treated HDL to obtain HDL with a graded phosphatidylcholine content, we showed that SR-BI-mediated cholesterol efflux was highly correlated (r2= 0.985) with HDL phosphatidylcholine content. The effects of varying HDL phospholipid composition on SR-BI-mediated free cholesterol flux were not correlated with changes in either the KdorBmaxvalues for high affinity binding to SR-BI. We conclude that SR-BI-mediated free cholesterol flux is highly sensitive to HDL phospholipid composition. Thus, factors that regulate cellular SR-BI expression and the local modification of HDL phospholipid composition will have a large impact on reverse cholesterol transport.

In this study, we compared cholesterol efflux mediated by either high density lipoproteins (HDL3) or β-cyclodextrins, cyclic oligosaccharides that are able to dissolve lipids in their hydrophobic core. β-Cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, and methyl-β-cyclodextrin at 10 mM induced the release of 50-90% of L-cell [3H]cholesterol after 8 h of incubation, with a major portion of this cholesterol being released in the first 1-2 h of incubation. The cholesterol efflux kinetics are different if cells are incubated with HDL3, which induces a relatively constant rate of release of cholesterol throughout an 8-h incubation. Cholesterol efflux to cyclodextrins was much greater than phospholipid release. To test the hypothesis that maximal efflux rate constants for a particular cell are independent of the type of acceptor, we estimated the maximal rate constants for efflux (Vmax) of cellular cholesterol from L-cells, Fu5AH cells, and GM3468A fibroblasts. The rate constant for HDL3-mediated efflux varied among cell lines in the order Fu5AH > L-cells > fibroblasts. However, these differences were not evident when cyclodextrins were used as cholesterol acceptors. The estimated Vmaxvalues for cyclodextrin-mediated efflux were 3.5-70-fold greater than for HDL3for the three cell lines. The very high efficiency of cyclodextrins in stimulating cell cholesterol efflux suggests that these compounds can be used in two general ways for studies of atherosclerosis: 1) as research tools to probe mechanisms of cholesterol transport and aspects of membrane structure or 2) as potential pharmacological agents that could modify in vivocholesterol metabolism and influence the development of the atherosclerotic plaque.

The efflux of cholesterol from cells in culture to cyclodextrin acceptors has been reported to be substantially more rapid than efflux induced by other known acceptors of cholesterol (Kilsdonk, E. P. C., Yancey, P., Stoudt, G., Bangerter, F. W., Johnson, W. J., Phillips, M. C., and Rothblat, G. H. (1995) J. Biol. Chem.270, 17250-17256). In this study, we compared the kinetics of cholesterol efflux from cells with 2-hydroxypropyl-β-cyclodextrins and with discoidal high density lipoprotein (HDL) particles to probe the mechanisms governing the remarkably rapid rates of cyclodextrin-mediated efflux. The rate of cholesterol efflux was enhanced by shaking cells growing in a monolayer and further enhanced by placing cells in suspension to achieve maximal efflux rates. The extent of efflux was dependent on cyclodextrin concentration, and maximal efflux was observed at concentrations >50 mM. For several cell types, biexponential kinetics of cellular cholesterol efflux were observed, indicating the existence of two kinetic pools of cholesterol: a fast pool (half-time (t1/2) ∼19-23 s) and a slow pool with t1/2 of 15–30 min. Two distinct kinetic pools of cholesterol were also observed with model membranes (large unilamellar cholesterol-containing vesicles), implying that the cellular pools are in the plasma membrane. Cellular cholesterol content was altered by incubating cells with solutions of cyclodextrins complexed with increasing levels of cholesterol. The number of kinetic pools was unaffected by raising the cellular cholesterol content, but the size of the fast pool increased. After depleting cells of the fast pool of cholesterol, this pool was completely restored after a 40-min recovery period. The temperature dependence of cyclodextrin-mediated cholesterol efflux from cells and model membranes was compared; the activation energies were 7 kcal/mol and 2 kcal/mol, respectively. The equivalent activation energy observed with apo-HDL-phospholipid acceptor particles was 20 kcal/mol. It seems that cyclodextrin molecules are substantially more efficient than phospholipid acceptors, because cholesterol molecules desorbing from a membrane surface can diffuse directly into the hydrophobic core of a cyclodextrin molecule without having to desorb completely into the aqueous phase before being sequestered by the acceptor.

Kidney disease affects intestinal structure and function. Although intestinal lymphatics are central in absorption and remodeling of dietary and synthesized lipids/lipoproteins, little is known about how kidney injury impacts the intestinal lymphatic network, or lipoproteins transported therein. To study this, we used puromycin aminoglycoside-treated rats and NEP25 transgenic mice to show that proteinuric injury expanded the intestinal lymphatic network, activated lymphatic endothelial cells and increased mesenteric lymph flow. The lymph was found to contain increased levels of cytokines, immune cells, and isolevuglandin (a highly reactive dicarbonyl) and to have a greater output of apolipoprotein AI. Plasma levels of cytokines and isolevuglandin were not changed. However, isolevuglandin was also increased in the ileum of proteinuric animals, and intestinal epithelial cells exposed to myeloperoxidase produced more isolevuglandin. Apolipoprotein AI modified by isolevuglandin directly increased lymphatic vessel contractions, activated lymphatic endothelial cells, and enhanced the secretion of the lymphaniogenic promoter vascular endothelial growth factor-C by macrophages. Inhibition of isolevuglandin synthesis by a carbonyl scavenger reduced intestinal isolevuglandin adduct level and lymphangiogenesis. Thus, our data reveal a novel mediator, isolevuglandin modified apolipoprotein AI, and uncover intestinal lymphatic network structure and activity as a new pathway in the crosstalk between kidney and intestine that may contribute to the adverse impact of kidney disease on other organs.

Oxidative stress promotes acute kidney injury (AKI). Higher HDL cholesterol concentrations are associated with less AKI. To test the hypothesis that HDL antioxidant activity is associated with AKI after cardiac surgery, we quantified HDL particle (HDL-P) size and number, paraoxonase-1 (PON-1) activity, and isofuran concentrations in 75 patients who developed AKI and 75 matched control patients. Higher preoperative HDL-P was associated with less AKI (OR: 0.80; 95% CI, 0.71–0.91; P = 0.001), higher PON-1 activity ( P< 0.001), and lower plasma concentrations of isofurans immediately after surgery (P = 0.02). Similarly, higher preoperative small HDL-P was associated with less AKI, higher PON-1 activity, and lower isofuran concentrations. Higher intraoperative particle losses were associated with less AKI (OR: 0.79; 95% CI 0.67–0.93; P = 0.005), and with decreased postoperative isofuran concentrations (P= 0.04) . Additionally, higher preoperative small HDL-P and increased intraoperative small particle loss were associated with improved long-term renal function (P = 0.003, 0.01, respectively). In conclusion, a higher preoperative concentration of HDL-P, particularly small particles, is associated with lower oxidative damage and less AKI. Perioperative changes in HDL-P concentrations are also associated with AKI. Small HDL-P may represent a novel modifiable risk factor for AKI.

The development of high and intermediate voltage electron microscopes provides the ability to investigate the ultrastructure of thick biological samples. This produces a unique view of the 3-D interrelationships of cells and organelles. However, the analysis and display of this 3-D information has presented challenges, particularly to quantitative biologists. Major advances in adapting computed tomography (CT) to electron microscopy have provided the major means of 3-D analysis of cellular ultrastructure (1,2). In particular, tremendous gains in determining the structure of macromolecules have occurred with advances in electron microscopy CT (3). However, CT remains an arduous undertaking and one not well suited to analysis of the structural alterations within large populations of cells requiring many reconstructions. We have adapted an alternative approach, Tuned Aperture Computer Tomography (TACT®), for use with high and intermediate voltage electron microscopes. Although with EM specimens TACT®does not usually produce as high Z axis resolution as CT, the simplicity of the TACT®algorithms lend themselves to the rapid analysis of multiple samples. TACT®is already gaining acceptance in radiology (4, 5). Like CT, TACT®uses a series of images of the sample taken from different angular perspectives. However, unlike CT, for TACT®only a few projections are required and thus good reconstructions of beam sensitive material are possible. The quality of the reconstruction is determined by the quality of the input images, the total angular spread, and the individual tilt increments between images. Better images, a greater angular spread, and smaller angles between images will produce more accurate results.