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Introduction Black radiologists remain significantly underrepresented in the radiology workforce, despite a 1973 plea by Black radiologists of the National Medical Association to increase training programs for minority radiologists. Objective The authors provide a qualitative narrative that highlights the radiology residency programs of three historically Black schools of medicine (HBSOM) in the U.S., their contributions, and lessons learned from their closure. Methods Data from public repositories, interviews, and conversations were conflated to chronicle significant events and establish a timeline during these residency programs' existence. Results Radiology residencies at Howard University School of Medicine (1945), Meharry Medical College (1949), and Charles R. Drew University of Medicine and Science (1972) were established to train Black doctors to treat communities of color. These programs provided care to underserved and under-resourced areas of the country, where inequitable health care fueled a legacy of poor health outcomes. These radiology residency programs collapsed under the weight of suboptimal funding, strapped capital budgets, attrition of faculty, a declining hospital patient census, and failure to maintain other residency specialty programs.` Conclusion Understanding the history and impact of these programs, and of their closure, can be leveraged to develop strategies to increase the representation of racial and ethnic minorities in radiology. Possible reinstatement, with appropriate allocation of resources and creation of intentional policies to ensure sustained success, merits further investigation and may be a pathway to achieve optimal representation.

Aims: Peroxiredoxin 6 (Prdx6), a 1-cys Prdx has both peroxidase and phospholipase A2 activities, protecting against oxidative stress and regulating pulmonary surfactant phospholipid metabolism. This study determined the mechanism by which keratinocyte growth factor (KGF) and the glucocorticoid analogue, dexamethasone (Dex), induce increased Prdx6 expression. Results: Transcriptional activation by KGF in both A549 lung adenocarcinoma cells and rat lung alveolar epithelial type II (ATII) cells utilizes an antioxidant response element (ARE), located between 357 and 349 nucleotides before the PRDX6 translational start, that is also necessary for upregulation of the human PRDX6 promoter in response to oxidative stress. Activation is mediated by binding of the transcription factor, Nrf2, to the ARE as shown by experiments using siRNA against Nrf2 and by transfecting ATII cells isolated from lungs of Nrf2 null mice. KGF triggers the migration of Nrf2 from cytoplasm to nucleus where it binds to the PRDX6 promoter as shown by chromatin immunoprecipitation assays. Activation of transcription by Dex occurs through a glucocorticoid response element located about 750 nucleotides upstream of the PRDX6 translational start. Innovation: This study demonstrates that KGF can activate an ARE in a promoter without reactive oxygen species involvement and that KGF and Dex can synergistically activate the PRDX6 promoter and protect cells from oxidative stress. Conclusion: These two different activators work through different DNA elements. Their combined effect on transcription of the reporter gene is synergistic; however, at the protein level, the combined effect is additive and protects cells from oxidative damage. Antioxid. Redox Signal. 20, 391–402.

To compare stereoscopic digital mammography (DM) with standard DM for the rate of patient recall and the detection of cancer in a screening population at elevated risk for breast cancer.Starting in September 2004 and ending in December 2007, this prospective HIPAA-compliant, institutional review board-approved screening trial, with written informed consent, recruited female patients at elevated risk for breast cancer (eg, personal history of breast cancer or breast cancer in a close relative). A total of 1298 examinations from 779 patients (mean age, 58.6 years; range, 32-91 years) comprised the analyzable data set. A paired study design was used, with each enrolled patient serving as her own control. Patients underwent both DM and stereoscopic DM examinations in a single visit, findings of which were interpreted independently by two experienced radiologists, each using a Breast Imaging Reporting and Data System (BI-RADS) assessment (BI-RADS category 0, 1, or 2). All patients determined to have one or more findings with either or both modalities were recalled for standard diagnostic evaluation. The results of 1-year follow-up or biopsy were used to determine case truth.Compared with DM, stereoscopic DM showed significantly higher specificity (91.2% [1167 of 1279] vs 87.8% [1123 of 1279]; P = .0024) and accuracy (90.9% [1180 of 1298] vs 87.4% [1135 of 1298]; P = .0023) for detection of cancer. Sensitivity for detection of cancer was not significantly different for stereoscopic DM (68.4% [13 of 19]) compared with DM (63.2% [12 of 19], P .99). The recall rate for stereoscopic DM was 9.6% (125 of 1298) and that for DM was 12.9% (168 of 1298) (P = .0018).Compared with DM, stereoscopic DM significantly improved specificity for detection of cancer, while maintaining comparable sensitivity. The recall rate was significantly reduced with stereoscopic DM compared with DM.http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.12120382/-/DC1.

The Niemann-Pick C (NPC) pathway plays an essential role in the intracellular trafficking of cholesterol by facilitating the release of lipoprotein-derived sterol from the lumen of lysosomes. Regulation of cellular cholesterol homeostasis is of particular importance to lung alveolar type II cells because of the need for production of surfactant with an appropriate lipid composition. We performed microscopic and biochemical analysis of NPC proteins in isolated rat type II pneumocytes. NPC1 and NPC2 proteins were present in the lung, isolated type II cells in culture, and alveolar macrophages. The glycosylated and nonglycosylated forms of NPC1 were prominent in the lung and the lamellar body organelles. Immunocytochemical analysis of isolated type II pneumocytes showed localization of NPC1 to the limiting membrane of lamellar bodies. NPC2 and lysosomal acid lipase were found within these organelles, as confirmed by z-stack analysis of confocal images. All three proteins also were identified in small, lysosome-like vesicles. In the presence of serum, pharmacological inhibition of the NPC pathway with compound U18666A resulted in doubling of the cholesterol content of the type II cells. Filipin staining revealed a striking accumulation of cholesterol within lamellar bodies. Thus the NPC pathway functions to control cholesterol accumulation in lamellar bodies of type II pneumocytes and, thereby, may play a role in the regulation of surfactant cholesterol content.

Alveolar surfactant protein A (SP-A) is endocytosed by type II epithelial cells through clathrin-dependent uptake and targeted to lamellar bodies for resecretion. However, the mechanism for secretion of newly synthesized SP-A, whether regulated exocytosis of lamellar bodies or constitutive secretion, is unresolved. If it is the latter, lamellar body SP-A would represent endocytosed protein. Amantadine, an inhibitor of clathrin-coated vesicle budding, was used to evaluate the role of endocytosis in accumulation of SP-A in lamellar bodies. In isolated rat lungs, amantadine (10 mM) inhibited uptake of endotracheally instilled35S-labeled biosynthesized surfactant proteins by >80%. To study trafficking of newly synthesized SP-A, lungs were perfused for up to 6 h with [35S]methionine, and surfactant was isolated from lung lavage fluid and lamellar bodies were isolated from lung homogenate. With control lungs, the mean specific activity of [35S]SP-A (disintegrations per minute per microgram of SP-A) increased linearly with time of perfusion: it was significantly higher in isolated lamellar bodies than in surfactant and was increased in both compartments by 50–60% in the presence of 0.1 mM 8-bromo-cAMP. These results suggest a precursor-product relationship between lamellar body and extracellular [35S]SP-A. Specific activities in both compartments were unaffected by addition of amantadine (10 mM) to the lung perfusate, indicating that uptake from the alveolar space was not responsible for the increase in lamellar body [35S]SP-A. Thus the pathway for secretion of newly synthesized SP-A is by transfer from the site of synthesis to the storage/secretory organelle prior to lamellar body exocytosis.

This quality improvement initiative compared children who are sedated versus children who are not sedated for the quality of images and the amount of time it took to obtain a brain magnetic resonance imaging (MRI) scan on 4 to 7 year olds, with and without contrast, at Children's Hospital Boston. Results demonstrate that both nonsedated and sedated children who underwent brain MRI examinations received diagnostic quality studies. Although the number of studies with motion artifact and repeat studies were higher for the nonsedated group, the actual scan time did not significantly change.

We have recently described a putative receptor for lung surfactant protein-A (SP-A) on rat type II pneumocytes. The receptor, P63, is a 63-kDa type II transmembrane protein. Coincubation of type II cells with P63 antibody (Ab) reversed the inhibitory effect of SP-A on secretagogue-stimulated surfactant secretion from type II cells. To further characterize SP-A interactions with P63, we expressed recombinant P63 protein in Escherichia coli and generated antibodies to P63. Immunogold electron microscopy confirmed endoplasmic reticulum and plasma membrane localization of P63 in type II cells with prominent labeling of microvilli. Binding characteristics of iodinated SP-A to type II cells in the presence of P63 Ab were determined. Binding (4°C, 1 h) of125I-SP-A to type II cells demonstrated both specific (calcium-dependent) and nonspecific (calcium-independent) components. Ab to P63 protein blocked the specific binding of125I-SP-A to type II cells and did not change the nonspecific SP-A association. A549 cells, a pneumocyte model cell line, expressed substantial levels of P63 and demonstrated specific binding of125I-SP-A that was inhibited by the P63 Ab. The secretagogue (cAMP)-stimulated increase in calcium-dependent binding of SP-A to type II cells was blocked by the presence of P63 Ab. Transfection of type II cells with small interfering RNA to P63 reduced P63 protein expression, attenuated P63-specific SP-A binding, and reversed the ability of SP-A to prevent surfactant secretion from the cells. Our results further substantiate the role of P63 as an SP-A receptor protein localized on the surface of lung type II cells.

Mice gene targeted for ATP-binding cassette transporter A1 (ABCA1; Abca1−/−) have been shown to have low-serum high-density lipoprotein and abnormal lung morphology. We examined alterations in the structure and function of lungs from −/− mice (DBA1/J). Electron microscopy of the diseased mouse lung revealed areas of focal disease confirming previous results ( 47 ). Lipid analysis of the lung tissue of −/− mice showed a 1.2- and 1.4-fold elevation in total phospholipid (PL) and saturated phosphatidylcholine, respectively, and a marked 50% enrichment in total cholesterol content predominately due to a 17.5-fold increase in cholesteryl ester compared with wild type (WT). Lung surfactant in the −/− mice was characterized by alveolar proteinosis (161%), a slight increase in total PL (124%), and a marked increase in free cholesterol (155%) compared with WT. Alveolar macrophages were enriched in cholesterol (4.8-fold) due to elevations in free cholesterol (2.4-fold) and in cholesteryl ester (14.8-fold) compared with WT macrophages. More PL mass was cleared from the alveolar space of −/− mice lungs, measured using intratracheal installation of3H-PL liposomes. Compared with WT mice, the Abca1−/−mice demonstrated respiratory distress with rapid, shallow breathing. Thus the lungs of mice lacking ABCA1 protein demonstrated abnormal morphology and physiology, with alveolar proteinosis and cholesterol enrichment of tissue, surfactant, and macrophages. The results indicate that the activity of ABCA1 is important for the maintenance of normal lung lipid composition, structure, and function.

We developed a heterologous system to study the effect of mechanical deformation on alveolar epithelial cells. First, isolated primary rat alveolar type II (ATII) cells were plated onto silastic substrata coated with fibronectin and maintained in culture under conditions where they become alveolar type I-like (ATI) cells. This was followed by a second set of ATII cells labeled with the nontransferable, vital fluorescent stain 5-chloromethylfluorescein diacetate to distinguish them from ATI cells. By morphometric analysis, equibiaxial deformation (stretch) of the silastic substratum induced comparable changes in cell surface area for both ATII and ATI cells. Surfactant lipid secretion was measured using cells metabolically labeled with [3H]choline. In response to 21% tonic stretch for 15 min, ATII cells seeded with ATI cells secreted nearly threefold more surfactant lipid compared with ATII cells seeded alone. ATI cells did not secrete lipid in response to stretch. The enhanced lipid secretion by ATII plus ATI cocultures was inhibited by treatment with apyrase and adenosine deaminase, suggesting that ATP release by ATI cells enhanced surfactant lipid secretion at 21% stretch. This was confirmed using a luciferase assay where, in response to 21% stretch, ATI cells released fourfold more ATP than ATII cells. Because ATI cells release significantly more ATP at a lower level of stretch than ATII cells, this supports the hypothesis that ATI cells are mechanosensors in the lung and that paracrine stimulation of ATII cells by extracellular ATP released from ATI cells plays a role in regulating surfactant secretion.

Mind-body interventions can help facilitate pediatric procedures. This study evaluated the perceptions and attitudes of 49 staff members regarding the use of mind-body interventions to facilitate magnetic resonance imaging procedures for children. Reported obstacles to the use of such interventions included identification of children in need of intervention, identification of qualified personnel in the department to provide intervention, and limited time to provide intervention. Such obstacles, once noted, can be addressed to improve opportunities for use of interventions.

We evaluated the contribution of endocytotic pathways to pulmonary uptake of surfactant lipids from the alveolar space. Resting and stimulated 8-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP) uptake of unilamellar liposomes labeled with either [3H]dipalmitoylphosphatidylcholine ([3H]DPPC) or 1-palmitoyl-2-[12-(7-nitro-2–1,3-benzoxadiazol-4-yl) amino] dodecanoyl-phosphatidylcholine (NBD-PC) was studied in isolated perfused rat lungs and isolated type II cells. Amantadine and phenylarsine oxide, inhibitors of clathrin-mediated endocytosis, each decreased [3H]DPPC uptake under resting conditions by ∼40%; their combination had no additional effect. Cytochalasin D, an inhibitor of actin-dependent processes, reduced liposome uptake by 55% and potentiated the effect of either clathrin inhibitor alone. Relative inhibition for all agents was higher in the presence of 8-Br-cAMP. The effect of inhibitors was similar for liposomes labeled with [3H]DPPC or NBD-PC. By fluorescence microscopy, NBD-PC taken up by lungs was localized primarily to alveolar type II cells and was localized to lamellar bodies in both lungs and isolated cells. These studies indicate that both clathrin-mediated and actin-mediated pathways are responsible for endocytosis of DPPC-labeled liposomes by alveolar type II cells in the intact lung.

The role of surfactant protein-A (SP-A) in pulmonary uptake and metabolism of [3H]dipalmitoylphosphatidylcholine ([3H]DPPC) was studied in SP-A gene-targeted mice (SP-A −/−). Unilamellar liposomes were instilled into the trachea of anesthetized mice. Uptake was measured as dpm in lungs plus liver and kidney for in vivo experiments and in lungs and perfusate for isolated lung experiments. [3H]DPPC uptake increased with CO2-induced hyperventilation in wild-type mice (SP-A +/+) but was unchanged in SP-A −/−. Secretagogue treatment approximately doubled the uptake of [3H]DPPC in isolated lungs from SP-A +/+ but had no effect in SP-A −/−. Lungs degraded 23 ± 1.2% of internalized [3H]DPPC in SP-A +/+ and 36 ± 0.6% in SP-A −/−; degradation increased with 8-bromoadenosine 3′,5′-cyclic monophosphate in SP-A +/+ but was unchanged in SP-A −/−. Activity of lysosomal-type phospholipase A2(PLA2) was significantly greater in lungs from SP-A −/− compared with SP-A +/+. Thus SP-A is necessary for lungs to respond to hyperventilation or secretagogues with increased DPPC uptake and also modulates the PLA2-mediated degradation of internalized DPPC.

Lamellar bodies are specialized cellular organelles used for storage of surfactant by alveolar type II cells of the lung. We utilized monoclonal antibody (MAb) 3C9, which recognizes an integral lamellar body-limiting membrane protein of 180 kDa, to follow lamellar body trafficking.125I-labeled MAb 3C9 bound to the surface of type II cells and was internalized by the cells in a time- and concentration-dependent manner that was inhibitable by excess unlabeled antibody. The internalized antibody remained undegraded over a 4-h time period. The L2 rat lung cell line that does not have lamellar bodies did not bind iodinated 3C9. Exposure of type II cells to the secretagogues ATP, phorbol 12-myristate 13-acetate, and cAMP resulted in a 1.5- to 2-fold enhancement of binding and uptake of MAb 3C9. Calphostin C inhibited phorbol 12-myristate 13-acetate-stimulated phospholipid secretion and also reduced binding and uptake of MAb 3C9 by type II cells. Treatment of type II cells with phenylarsine oxide to obstruct clathrin-mediated endocytosis had no effect on the internalization of MAb 3C9 while markedly blocking the uptake of surfactant protein A and transferrin. An actin-mediated process was important for lamellar body membrane uptake because incubation with cytochalasin D partially inhibited MAb 3C9 incorporation by type II cells. These studies are compatible with enhanced lamellar body membrane turnover associated with surfactant secretion and indicate that this process can be monitored by the trafficking of the antigen reporter MAb 3C9.

Secretagogues stimulate both secretion and reuptake of surfactant components by pulmonary type II cells as well as enhance surfactant protein A (SP-A) binding. We have evaluated the possibility that the observed increase in SP-A binding is due to the movement of SP-A receptors from an intracellular pool to the plasma membrane. We utilized an anti-idiotypic monoclonal antibody, A2R, which recognizes an SP-A binding protein on type II cell membranes. Immunocytochemistry studies showed that A2R reacted with cellular antigens on type II cell membranes and paranuclear granules. A2R inhibited cell association of125I-SP-A to type II cells plated on Transwell membranes as well as those plated on plastic dishes and also inhibited the SP-A-stimulated incorporation of phosphatidylcholine liposomes into type II cells. On exposure to secretagogues, the binding of125I-A2R and125I-SP-A to type II cells increased in parallel. With permeabilized type II cells on Transwell membranes, one-sixth of the binding sites were located on the plasma membrane, with the remainder being intracellular; phorbol 12-myristate 13-acetate treatment increased the binding of A2R to the cell surface but did not affect the total binding of A2R. Ligand blots of type II cell plasma membranes showed that SP-A and A2R both bound proteins with molecular masses of ∼32 and 60 kDa, respectively, reduced. Under nonreducing conditions, the mass of the SP-A and A2R binding protein was ∼210 kDa, indicating that the SP-A receptor is composed of disulfide-linked subunits. The results support our hypothesis that secretagogues increase SP-A binding sites by accelerating recruitment of receptors to the cell surface.

The metabolism of iodinated lung surfactant protein A (SP-A) by alveolar macrophages in primary culture was examined to determine the role these cells play in the degradation of this surfactant protein. SP-A was isolated from lung lavage obtained from normal bovines, patients with alveolar proteinosis, and silica-treated rats. SP-A (0.5 microgram/ml) was incubated for 3 h with rat alveolar macrophages obtained by lung lavage. Cell association and degradation of human and rat SP-A was three times greater than that of bovine SP-A. During the 3-h period, 50% of total macrophage-associated SP-A was degraded. Degradation was time-, temperature-, and concentration-dependent after a 1-h lag period. SP-A degradation was intracellular, since NH4Cl inhibited degradation > 50%, and macrophage-conditioned medium was ineffective. Tenfold more SP-A was degraded by macrophages than by type II cells isolated after elastase digestion of rat lungs. There was little degradation of SP-A by HeLa cells. We conclude that alveolar macrophages take up and degrade SP-A and thus could contribute to the catabolism of SP-A in the lung.

A marked sequence homology has been noted between lung surfactant protein A (SP-A) and an inhibitor of phospholipase A 2 (PLA 2 ) isolated from the serum of Trimeresurus flavoviridis (Habu snake). This study evaluated the effect of SP-A on PLA 2 activity from several sources. SP-A was isolated from bovine or rat lung surfactant by extraction with 1-butanol and octyl β- d -glucopyranoside. The addition of SP-A produced a concentration-dependent inhibition of T. flavoviridis PLA 2 that indicated non-competitive kinetics with K i 5 μg/ml. Inhibition was reversed by heat inactivation, disulfide bond reduction or alkylation of SP-A, or by the presence of anti-SP-A antibody. Treatment of SP-A with endoglycosidase F or the presence of various monosaccharides or lectins did not alter SP-A inhibition. Binding of PLA 2 to SP-A was shown by ultrafiltration and was abolished by SP-A alkylation or the presence of SDS. The SP-A/PLA 2 complex recovered from the ultrafilter had essentially no enzymatic activity, but activity was restored by treatment with mercaptoethanol. SP-A had no effect on activity of PLA 2 from Naja naja , Crotalus atrox , or bovine pancreas. These results indicate that surfactant protein A selectively inhibits Trimeresurus phospholipase A 2 activity and suggest that binding to the enzyme is the mechanism for inhibition.

Macrophages store excess unesterified cholesterol (free, FC) in the form of cholesteryl ester (CE) in cytoplasmic lipid droplets. The hydrolysis of droplet-CE in peripheral foam cells is critical to HDL-promoted reverse cholesterol transport because it represents the first step in cellular cholesterol clearance, as only FC is effluxed from cells to HDL. Cytoplasmic lipid droplets move within the cell utilizing the cytoskeletal network, but, little is known about the influence of the cytoskeleton on lipid droplet formation. To understand this role we employed cytochalasin D (cyt.D) to promote actin depolymerization in J774 macrophages. Incubating J774 with acetylated LDL creates foam cells having a 4-fold increase in cellular cholesterol content (30-40% cholesterol present as cholesteryl ester (CE)) in cytoplasmic droplets. Lipid droplets formed in the presence of cyt.D are smaller in diameter. CE-deposition and -hydrolysis are decreased when cells are cholesterol-enriched in the presence of cyt.D or latrunculin A, another cytoskeleton disrupting agent. However, when lipid droplets formed in the presence of cyt.D are isolated and incubated with an exogenous CE hydrolase, the CE is more rapidly metabolized compared to droplets from control cells. This is apparently due to the smaller size and altered lipid composition of the droplets formed in the presence of cyt.D. Cytoskeletal proteins found on CE droplets influence droplet lipid composition and maturation in model foam cells. In J774 macrophages, cytoskeletal proteins are apparently involved in facilitating the interaction of lipid droplets and a cytosolic neutral CE hydrolase and may play a role in foam cell formation. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).

Surfactant protein B (SP-B) metabolism was studied in primary cultures of alveolar type II cells. Iodinated SP-B reconstituted with surfactant was incorporated rapidly into lung pneumocytes and degraded to trichloroacetic acid (TCA)-soluble products after a lag period of 1 h. Cellular degradation of SP-B occurred whether or not phospholipid liposomes or surfactant was added to the phospholipid-poor SP-B. Uptake and degradation of SP-B at 37 degrees C showed a linear increase up to 3 micrograms SP-B/ml after which the slope of the curve became less steep with increasing concentrations of SP-B in the media. After 4 h of incubation with SP-B, 35% of the SP-B processed was recovered as degradation products. Ninety-six percent of the degradation products were in the media and only 4% were recovered in the cell. The bulk of the breakdown of SP-B occurred inside the type II cells since degradation did not occur at 4 degrees C, showed a 1-h lag period, was proportional to the SP-B protein internalized by the cells, was inhibited 47% by ammonium chloride, was unaffected by the addition of protease inhibitors to the medium, and cell-conditioned medium produced only limited SP-B degradation. Alveolar macrophages also degraded SP-B, whereas other cell types degraded SP-B to a lesser extent. Thus the specificity of the metabolism of SP-B may be through the capability of lung cells to degrade SP-B.(ABSTRACT TRUNCATED AT 250 WORDS)

Surfactant protein A (SP-A) plays an important role in the maintenance of lung lipid homeostasis. Previously, an SP-A receptor, P63 (CKAP4), on type II pneumocyte plasma membranes (PM) was identified by chemical cross-linking techniques. An antibody to P63 blocked the specific binding of SP-A to pneumocytes and the ability of SP-A to regulate surfactant secretion. The current report shows that another biological activity of SP-A, the stimulation of surfactant uptake by pneumocytes, is inhibited by P63 antibody. cAMP exposure resulted in enrichment of P63 on the cell surface as shown by stimulation of SP-A binding, enhanced association of labeled P63 antibody with type II cells, and promotion of SP-A-mediated liposome uptake, all of which were inhibited by competing P63 antibody. Incubation of A549 and type II cells with SP-A also increased P63 localization on the PM. The phosphatidylinositol 3-kinase (PI3-kinase) signaling pathway was explored as a mechanism for the transport of this endoplasmic reticulum (ER)-resident protein to the PM. Treatment with LY-294002, an inhibitor of the PI3-kinase pathway, prevented the SP-A-induced PM enrichment of P63. Exposure of pneumocytes to SP-A or cAMP activated Akt (PKB). Blocking either PI3-kinase or Akt altered SP-A-mediated lipid turnover. The data demonstrate an important role for the PI3-kinase-Akt pathway in intracellular transport of P63. The results add to the growing body of evidence that P63 is critical for SP-A receptor-mediated interactions with type II pneumocytes and the resultant regulation of surfactant turnover.

Surfactant protein B (SP-B, mol wt 9,000, reduced) is a low-molecular-weight hydrophobic protein found in organic extracts of lung surfactant. The interaction of iodinated bovine SP-B (125I-SP-B) and isolated rat alveolar type II cells was examined. The association of SP-B with the lung cells was time and temperature dependent; type II cells exhibited time-dependent binding (at 4 degrees C) and uptake (at 37 degrees C) of SP-B. Binding of phospholipid-poor 125I-SP-B was linearly related to the external SP-B concentration from 0.25 to 60 microgram/ml and was not inhibited by a 60-fold excess of unlabeled SP-B. However, the binding of 125I-SP-B reconstituted with bovine surfactant or with phospholipid-containing liposomes occurred through a high-affinity, saturable process and could be inhibited with unlabeled SP-B. By Scatchard analysis, half-maximum binding in the presence of surfactant occurred at 3.1 +/- 0.7 micrograms SP-B/ml. Saturable binding of SP-B reconstituted with surfactant also occurred with other cell types. The results indicate that SP-B was bound and internalized by type II cells. The apparent lack of specificity in the absence of phospholipid may have been due to the self-association of SP-B. The reconstitution of SP-B with phospholipid altered the binding of phospholipid-poor SP-B from a nonspecific process to a high-affinity process consistent with a cell surface binding site.

Surfactant protein B (SP-B), a peptide found in organic solvent extracts of mammalian surfactant, has been isolated from surfactant previously by column chromatography and/or preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE). We have developed a method for isolation of SP-B from bovine surfactant utilizing differential organic extraction. Dried surfactant, isolated from lavage of excised cow lungs, was delipidated by extraction with diisopropyl ether-butanol (3:2). The aqueous layer, containing surfactant proteins, was dried and then was sequentially extracted with diethyl ether-ethanol (3:1) and CHCl3:MeOH:HCl (3:2:0.005 N). SP-B partitioned into chloroform-methanol, which was evaporated under N2. Purified SP-B, quantitated by Coomassie dye binding, represented 1% (wt/wt) of the original surfactant with a final phospholipid-to-protein ratio less than 1. Silver-stained SDS/PAGE of the SP-B extract revealed a single band at 9 kDa (reduced) and 18 kDa (nonreduced), which by immunoblotting reacted strongly with monospecific anti-SP-B antibody. Amino acid sequence analysis confirmed the presence of NH2 and N-1 terminal sequences of bovine SP-B. This procedure offers a rapid, reliable method for isolation of purified SP-B from whole surfactant.

15(S)-lipoxygenase-1 (15-LO-1) was present in the whole-cell homogenate of an acute human monocytic leukemia cell line (THP-1). Additionally, 15-LO-1 was detected on neutral lipid droplets isolated from THP-1 foam cells. To investigate if 15-LO-1 is active on lipid droplets, we used the mouse leukemic monocytic macrophage cell line (RAW 264.7), which are stably transfected with human 15-LO-1. The RAW 15-LO-1 cells were incubated with acetylated low density lipoprotein to generate foam cells. 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE], the major 15-LO-1 metabolite of arachidonic acid, was produced in the 15-LO-1 RAW but not in the mock transfected cells when incubated with arachidonic acid. Lipid droplets were isolated from the cells and incubated with arachidonic acid, and production of 15(S)-HETE was measured over 2 h. 15(S)-HETE was produced in the incubations with the lipid droplets, and this production was attenuated when the lipid droplet fraction was subjected to enzyme inactivation through heating. Efflux of 15(S)-HETE from cholesteryl ester-enriched 15-LO RAW cells, when lipid droplets are present, was significantly reduced compared with that from cells enriched with free cholesterol (lipid droplets are absent). We propose that 15-LO-1 is present and functional on cytoplasmic neutral lipid droplets in macrophage foam cells, and these droplets may act to accumulate the anti-inflammatory lipid mediator 15(S)-HETE.

Surfactant protein-A (SP-A) plays an important role in the clearance of surfactant from the lung alveolar space and in the regulation of surfactant secretion and uptake by type II pneumocytes in culture. Two pathways are important for the endocytosis of surfactant by type II cells and the intact lung, a receptor-mediated clathrin-dependent pathway and a non-clathrin, actin-mediated pathway. The critical role of the clathrin/receptor-mediated pathway in normal mice is supported by the finding that SP-A gene-targeted mice use the actin-dependent pathway to maintain normal clearance of surfactant. Addition of SP-A to the surfactant of the SP-A null mice “rescued” the phenotype, further emphasizing the essential role of the SP-A/receptor-mediated process in surfactant turnover. This review presents an overview of the structure of SP-A and its function in surfactant turnover. The evidence that the interaction of SP-A with type II cells is a receptor-mediated process is presented. A newly identified receptor for SP-A, P63/CKAP4, is described in detail, with elucidation of the specific structural features of this 63 kDa, nonglycosylated, highly coiled, transmembrane protein. The compelling evidence that P63 functions as a receptor for SP-A on type II cells is summarized. Regulation of P63 receptor density on the surface of pneumocytes may be a novel approach for the regulation of surfactant homeostasis by the lung.

Previous studies with the isolated perfused rat lung showed that both clathrin- and actin-mediated pathways are responsible for endocytosis of dipalmitoylphosphatidylcholine (DPPC)-labeled liposomes by granular pneumocytes in the intact lung. Using surfactant protein-A (SP-A) gene-targeted mice, we examined the uptake of [3H]DPPC liposomes by isolated mouse lungs under basal and secretagogue-stimulated conditions. Unilamellar liposomes composed of [3H]DPPC: phosphatidylcholine:cholesterol:egg phosphatidylglycerol (10:5:3:2 mol fraction) were instilled into the trachea of anesthetized mice, and the lungs were perfused (2 h). Uptake was calculated as percentage of instilled disintegrations per minute in the postlavaged lung. Amantadine, an inhibitor of clathrin and, thus, receptor-mediated endocytosis via clathrin-coated pits, decreased basal [3H]DPPC uptake by 70% in SP-A +/+ but only by 20% in SP-A −/− lung, data compatible with an SP-A/receptor-regulated lipid clearance pathway in the SP-A +/+ mice. The nonclathrin, actin-dependent process was low in the SP-A +/+ lung but accounted for 55% of liposome endocytosis in the SP-A −/− mouse. With secretagogue (8-bromoadenosine 3′,5′-cyclic monophosphate) treatment, both clathrin- and actin-dependent lipid clearance were elevated in the SP-A +/+ lungs while neither pathway responded in the SP-A −/− lungs. Binding of iodinated SP-A to type II cells isolated from both genotypes of mice was similar indicating a normal SP-A receptor status in the SP-A −/− lung. Inclusion of SP-A with instilled liposomes served to “rescue” the SP-A −/− lungs by reestablishing secretagogue-dependent enhancement of liposome uptake. These data are compatible with a major role for receptor-mediated endocytosis of DPPC by granular pneumocytes, a process critically dependent on SP-A.

The mechanisms used by alveolar type I pneumocytes for maintenance of the lipid homeostasis necessary to sustain these large squamous cells are unknown. The processes may involve the ATP-binding cassette transporter A1 (ABCA1), a transport protein shown to be crucial in apolipoprotein A-I (apoA-I)-mediated mobilization of cellular cholesterol and phospholipid. Immunohistochemical data demonstrated the presence of ABCA1 in lung type I and type II cells and in cultured pneumocytes. Type II cells isolated from rat lungs and cultured for 5 days in 10% serum trans-differentiated toward cells with a type I-like phenotype which reacted with the type I cell-specific monoclonal antibody VIIIB2. Upon incubation of the type I-like pneumocytes with agents that up-regulate the ABCA1 gene (9-cis-retinoic acid [9cRA] and 22-hydroxycholesterol [22-OH, 9cRA/22-OH]), ABCA1 protein levels were enhanced to maximum levels after 8 to 16 hours and remained elevated for 24 hours. In the presence of apoA-I and 9cRA/22-OH, efflux of radioactive phospholipid and cholesterol from pneumocytes was stimulated 3- to 20-fold, respectively, over controls. Lipid efflux was inhibited by Probucol. Sucrose density gradient analysis of the media from stimulated cells incubated with apoA-I identified heterogeneous lipid particles that isolated at a density between 1.063 and 1.210 g/ml, with low or high apoA-I content. Thus, pneumocytes with markers for the type I phenotype contained functional ABCA1 protein, released lipid to apoA-I protein, and were capable of producing particles resembling nascent high-density lipoprotein, indicating an important role for ABCA1 in the maintenance of lung lipid homeostasis.

Surfactant protein A (SP-A) binds to alveolar type II cells through a specific high-affinity cell membrane receptor, although the molecular nature of this receptor is unclear. In the present study, we have identified and characterized an SP-A cell surface binding protein by utilizing two chemical cross-linkers: profound sulfo-SBED protein-protein interaction reagent and dithiobis(succinimidylpropionate) (DSP). Sulfo-SBED-biotinylated SP-A was cross-linked to the plasma membranes isolated from rat type II cells, and the biotin label was transferred from SP-A to its receptor by reduction. The biotinylated SP-A-binding protein was identified on blots by using streptavidin-labeled horseradish peroxidase. By using DSP, we cross-linked SP-A to intact mouse type II cells and immunoprecipitated the SP-A-receptor complex using anti-SP-A antibody. Both of the cross-linking approaches showed a major band of 63 kDa under reduced conditions that was identified as the rat homolog of the human type II transmembrane protein p63 (CKAP4/ERGIC-63/CLIMP-63) by matrix-assisted laser desorption ionization and nanoelectrospray tandem mass spectrometry of tryptic fragments. Thereafter, we confirmed the presence of p63 protein in the cross-linked SP-A-receptor complex by immunoprobing with p63 antibody. Coimmunoprecipitation experiments and functional assays confirmed specific interaction between SP-A and p63. Antibody to p63 could block SP-A-mediated inhibition of ATP-stimulated phospholipid secretion. Both intracellular and membrane localized pools of p63 were detected on type II cells by immunofluorescence and immunobloting. p63 colocalized with SP-A in early endosomes. Thus p63 closely interacts with SP-A and may play a role in the trafficking or the biological function of the surfactant protein.

The human surfactant protein A (SP-A) locus consists of two functional genes, SP-A1 and SP-A2, with several alleles characterized for each gene. Functional variations between SP-A1 and SP-A2 variants either before or after ozone exposure have been observed. To understand the basis of these differences, we studied SP-A1 and SP-A2 variants by comparing coding sequences, oligomerization patterns under various conditions, composition of oligomers with regard to amino terminal sequence isoforms, biological activity (regulation of phosphatidylcholine (PC) secretion by alveolar type II cells), and the impact of ozone-induced oxidation. We found that (i) the SP-A1 (6A(4)) allele is the most divergent from all SP-A2 alleles, particularly from the SP-A2 (1A(1)). (ii) Differences exist in oligomerization among SP-A1, SP-A2, and coexpressed SP-A1/SP-A2, with higher order multimers (i.e., consisting of more subunits) observed for SP-A1 than for SP-A2 variants. Differences among SP-A1 or SP-A2 gene products are minimal. (iii) Amino acid variants in the amino terminal sequences are observed after signal peptide removal, including variants with an extra cysteine. (iv) Oxidation is observed after ozone exposure, involving several SP-A residues that include cysteine, methionine, and tryptophan. (v) The SP-A2 variant (1A(0)) and the coexpressed protein 1A(0)/6A(2) inhibit ATP-stimulated PC secretion from alveolar type II cells to a greater extent than SP-A1 (6A(2)), a biologic activity that was susceptible to ozone treatment.

Background Surfactant protein (SP)-D is an epithelial cell product of the distal air spaces that aids uptake and clearance of inhaled pathogens and allergens. Allergic airway inflammation significantly increases SP-D levels in the bronchoalveolar lavage fluid in asthmatic patients and mouse models, but the mechanisms involved remain unknown. Objective To investigate the effects of the T H 2-type cytokine IL-4 on SP-D production by isolated pulmonary epithelial cells. Methods Rat type II alveolar epithelial cells were purified and cultured with dexamethasone, cAMP, and isobutyl-1-methyl-xanthine (DCI). The effects of IL-4 on SP-D expression were investigated at the protein and mRNA levels by means of Western and Northern blot analyses. Results In contrast to a lamellar body protein ABCA3 and surfactant protein-A, expression of SP-D significantly declined when cells were cultured in medium alone for 24 hours. The presence of DCI in the culture medium restored SP-D levels, which were enhanced by 2-fold after addition of recombinant IL-4. The enhancing effects of IL-4 were concentration-dependent, with maximum effects observed at 20 ng/mL (1.43 nmol/L). IL-4 did not rescue cycloheximide-induced decrease of intracellular SP-D levels and did not inhibit extracellular release of SP-D. However, IL-4 significantly augmented DCI-induced SP-D mRNA expression by approximately 2.5-fold over control levels. Conclusions IL-4 selectively upregulates SP-D expression, and it may act at the level of mRNA in isolated pulmonary epithelial cells. Since SP-D has a potent anti-inflammatory function, this mechanism may be part of a negative feedback loop providing a regulatory link between adaptive and innate immunity during allergic inflammation.

Surfactant secretion by lung type II cells occurs when lamellar bodies (LBs) fuse with the plasma membrane and surfactant is released into the alveolar lumen. Surfactant protein A (SP-A) blocks secretagogue-stimulated phospholipid (PL) release, even in the presence of surfactant-like lipid. The mechanism of action is not clear. We have shown previously that an antibody to LB membranes (MAb 3C9) can be used to measure LB membrane trafficking. Although the ATP-stimulated secretion of PL was blocked by SP-A, the cell association of iodinated MAb 3C9 was not altered, indicating no effect on LB movement. FM1-43 is a hydrophobic dye used to monitor the formation of fusion pores. After secretagogue exposure, the threefold enhancement of the number of FM1-43 fluorescent LBs (per 100 cells) was not altered by the presence of SP-A. Finally, there was no evidence of a large PL pool retained on the cell surface through interaction with SP-A. Thus SP-A exposure does not affect these stages in the surfactant secretory pathway of type II cells.

A culture system designed to maintain the differentiated characteristics of rat type II cells based on protocols used for human fetal lung pneumocytes was investigated. Type II cells were isolated either from adult rats with elastase (adult type II cells) or from young rats (4–11 days postnatal) with collagenase and trypsin (young type II cells) and were incubated with dexamethasone (Dex, 10 nM) and cAMP (0.1 mM). By day 4 of culture with hormone treatment, the mRNA levels in adult type II cells were less than 3% of day 0 values, whereas surfactant protein (SP)-A protein content was 26%. However, young type II cells maintained lamellar bodies and microvilli and secreted phospholipid in response to ATP. SP-A, -B, and -C mRNA levels were elevated to 159, 350, and 39%, respectively, of day 0 values with a synergistic response to Dex and cAMP, whereas SP-A protein content rose to 119%. Surfactant mRNA and protein did not recover in cells cultured without hormones. This cell culture system restored surfactant components in rat type II cells.

The current study examined whether long-term culture of macrophages affects their metabolism of surfactant components. Compared with freshly isolated resting macrophages in culture for 1 h, macrophages attached to plastic dishes for 24 h showed evidence of conversion to a “primed” state with 1) an altered morphology characterized by a larger size, ruffled membranes, lamellipodia, and a “foamy” appearance after attachment to glass and 2) a fivefold greater respiratory burst in response to phorbol 12-myristate 13-acetate stimulation. On incubation with iodinated surfactant protein A (SP-A), the 24-h alveolar or tissue macrophages showed a 5- or a 23-fold greater increase in SP-A degradation, respectively, than macrophages cultured for 1 h. Conditioned media experiments demonstrated that the elevated rate of SP-A degradation after prolonged culture was not a result of proteases secreted by the macrophages. Incubation of cells with NH4Cl reduced the degradation of SP-A to a similar extent (to 33% of control values) in resting and primed tissue macrophages. On the other hand, length of time of cell culture did not affect macrophage uptake and degradation of [3H]dipalmitoylphosphatidylcholine in mixed unilamellar liposomes. Thus freshly isolated resting tissue and alveolar macrophages can be primed to specifically increase their rate of SP-A degradation. Activation of macrophages associated with lung disease may be important for SP-A metabolism and surfactant function.

Since secretagogues have been shown to increase the internalization of surfactant phospholipid and protein by lung cells, we postulated that their action occurred through a mechanism involving increased surfactant protein A (SP-A) receptor density. Therefore, we evaluated the influence of secretagogues on the binding of iodinated SP-A to alveolar type II cells. Type II cells were isolated from rat lung and maintained in primary culture for 18 h on Transwell membranes. Upon exposure to 8-bromo-cyclic AMP (cAMP, 0.1 mM), phorbol 12-myristate 13-acetate (PMA, 10 nM), terbutaline (0.1 mM), or ATP (1 mM), the binding of SP-A increased 1.5-2-fold. This stimulation was cell substrate-dependent since type II cells plated on plastic dishes did not show this effect. A time course of the stimulation of SP-A binding due to secretagogues showed that both cAMP and PMA increased SP-A binding by 2-fold after 20 min. With cAMP, binding remained elevated for 2 h, while binding in the presence of PMA had returned to control values. The effects of submaximal concentrations of cAMP and PMA on binding were additive. Inhibition of cellular protein synthesis with cycloheximide did not alter the increase of SP-A binding stimulated by the secretagogues. Type II cells pretreated with PMA responded to subsequent treatment with cAMP by increasing SP-A binding, while these cells were refractory to subsequent treatment with PMA. Both constitutive and regulated binding of SP-A to type II cells were sensitive to trypsin. The binding of SP-A to type II cells showed saturation at a concentration of 1 μg/ml SP-A under control and secretagogue-stimulated conditions, with both total and calcium-dependent binding showing a 2-fold increase upon secretagogue exposure. The data are consistent with the hypothesis that secretagogues stimulate surfactant uptake, at least in part, through recruitment of SP-A receptors to the type II cell surface, resulting in an increase in the number of SP-A binding sites.

We have investigated the internalization of surfactant by type II cells in primary culture. Previously, we demonstrated that type II cells cultured on microporous membranes [Transwell membranes (TM)] maintained the morphological characteristics of lung pneumocytes and took up surfactant protein and phospholipid in a fashion similar to that described for the uptake of surfactant by whole lung. In the present study, cells cultured on TM and exposed to equivalent amounts of phospholipid (PL) as either natural surfactant or liposomes incorporated fivefold greater amounts of natural surfactant PL. The evidence supported an important role for surfactant protein A (SP-A), as the incorporation of surfactant into type II cells on TM was reduced by anti-SP-A antisera and by pretreatment of the surfactant with beta-mercaptoethanol. In addition, the uptake of liposomes into type II cells on TM was augmented by SP-A. With the use of iodinated bovine SP-A reconstituted in rat surfactant, cells cultured on TM showed a 2.6-fold increase in binding and a 3.2-fold stimulation in uptake of SP-A over that seen with cells cultured on plastic. The change in the slope of the total binding curve of 125I-labeled bovine SP-A reconstituted with bovine surfactant to type II cells on the two substrates occurred at the same concentration of SP-A (17 micrograms/mg), but maximal binding was approximately eight times higher for cells on TM than for cells on plastic dishes. Thus, in a more physiological environment, i.e., SP-A in surfactant and cells on microporous membranes, SP-A plays an important role in the uptake of phospholipids by alveolar pneumocytes.

Pediatric neuro-oncology patients require serial MRI scans for years after diagnosis and medical treatment to monitor disease status. Managing anxiety and distress associated with MRI procedures is essential to improving the quality of the patient's repeated experience in radiology and ensuring the quality of the scan. Mind/body interventions such as relaxation, guided imagery, and hypnosis can be useful tools to facilitate MRI procedures. In a large pediatric radiology department, the investigators used an educational presentation regarding mind/body interventions and pre- and postpresentation surveys to evaluate staffs' attitudes and perceptions and to identify potential obstacles regarding the use of such interventions. A total of 49 radiology staff attended the educational presentation and completed the pre- and postpresentation surveys. Primary system obstacles to the use of mind/body interventions in radiology were identified as well as discipline differences regarding basic knowledge of mind/body interventions. A wide range of staff's attitudes and perceptions about mind/body interventions was recorded. Most participants reported finding the presentation "informative" and believed it would have a positive impact on their clinical work. In addition, based upon the study results, the investigators have developed and are piloting a screening measure to be used by nursing staff to facilitate proactive identification of patients in need of such interventions for subsequent scans. The investigators intend this study to serve as a template for nursing staff at other sites to evaluate staff perceptions and the need for mind/body interventions within their own radiology department.

The effects of high, low and very low density lipoprotein fractions from normal or hyperlipemic rhesus monkey serum on the accumulation or removal of cholesterol esters from rhesus monkey smooth muscle cells in tissue culture were determined. Serum or serum lipoproteins were labeled with [ l4 C] free cholesterol and adjusted to the same free cholesterol level in the incubation medium. Of the two normal lipoproteins examined, the LDL fraction caused more esterification than the HDL. Cells incubated in hyperlipemic serum showed a 2-fold stimulation in esterification as compared to cells in normal serum. This was contributed by hyperlipemic VLDL and LDL and led to a concomitant increase in cellular cholesterol ester content. Both hyperlipemic LDL and HDL stimulated esterification when compared to their normal counterparts. Cholesterol ester removal was examined by incubating the serum or lipoprotein fractions with cells enriched in cholesterol ester through a prior exposure to hyperlipemic serum. The cells incubated in normal or hyperlipemic HDL or lipoprotein-deficient serum had the lowest cholesterol ester content. Thus, the lipoprotein fractions which caused the lowest levels of cholesterol esterification were also the most efficient in the removal of cellular cholesterol esters.

Arterial smooth muscle cells synthesize matrix macromolecules in response to mechanical stimulation. Exposure to serum lipids also stimulates connective tissue fiber accumulation. To assess the effect of serum lipids on the biosynthetic response to tensile stress, we subjected rabbit aortic smooth muscle cells that were cultured on purified elastin membranes to cyclic stretching and relaxation 50 times per minute in the presence of serum-free medium (SFM), normolipemic serum (NLS), or hyperlipemic serum (HLS). Incorporation of 14C-proline into proline and into hydroxyproline was taken as a measure of protein and collagen synthesis. When cells were grown in plastic Petri dishes, exposure to NLS or HLS increased both protein and collagen production to the same extent compared to synthesis in SFM (1.7 times for NLS and 1.6 times for HLS; p less than 0.001 compared to SFM). For cells grown on stationary elastin membranes, NLS and HLS also increased protein and collagen synthesis compared to SFM. The effect of NLS was 1.35 times that of HLS for protein and 1.43 times greater for collagen (p less than 0.03). Cyclic stretching in SFM doubled synthesis for both protein (p less than 0.002) and collagen (p less than 0.002) compared to stationary controls, but had no effect on synthesis in NLS. In HLS, however, cyclic stretching elevated synthesis to the same level as was found in NLS (p less than 0.003). We conclude that the relative inhibition of synthesis on stationary membranes by HLS was not due to a toxic effect, since HLS increased synthesis both in Petri dishes and on elastin membranes, and the amplifying effect of cyclic stretching in HLS was similar to that seen in SFM.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of human serum and serum lipoproteins on cellular sterol flux and synthesis were measured in an L-cell mouse fibroblast tissue culture system. Since L cells synthesize desmosterol as their major sterol, endogenous cellular sterol (desmosterol) can easily be differentiated from exogenous serum lipoprotein sterol (cholesterol). Taking advantage of this unique property, influx, efflux and synthesis of sterol were measured. Therefore, following incubation with serum or serum lipoproteins, cellular incorporation of cholesterol was a measure of influx, and desmosterol released from the cells into the media was a measure of cellular efflux. The effect of sterol flux on cellular desmosterol synthesis was determined using the incorporation of [14C] acetate into sterol and measurement of net desmosterol synthesis. Time-course experiments demonstrated that after the addition of human serum, sterol flux was rapid, with influx and efflux approaching maximum values within 8 h, and cellular sterol synthesis was reduced by 50% within 24 h. Comparative determinations made at 4 and 24 h after the addition to the media of human high-density lipoproteins (HDL), low-density lipoproteins (LDL), or serum yielded the following results: (a) HDL at both sampling times promoted the greatest efflux of desmosterol from the cell; (b) influx of cholesterol was similar in the presence of both HDL and LDL; (c) in all instances an increase in cellular sterol occurred; (d) sterol synthesis was not related solely to cellular sterol content, and was most reduced in the presence of LDL.

The livers of both baboons and rhesus monkeys fed a high fat, high cholesterol diet secreted very low density lipoproteins (VLDL) that were enriched in cholesteryl ester and apoe as compared to VLDL secreted by the livers of chow-fed animals. Stimulation of macrophage cholesterol esterification by the experimental VLDL was compared to that produced by the standard beta-VLDL obtained from the plasma of a rhesus monkey fed 25% coconut oil plus 2% cholesterol. This standard beta-VLDL stimulated 7- to 10-fold more esterification than did the bovine albumin control. Hepatic VLDL from fat-fed animals stimulated esterification in J774 macrophages 50 to 150% as well as did the standard beta-VLDL, even though hepatic VLDL did not display beta electrophoretic mobility on agarose gel electrophoresis. Plasma VLDL from lard-fed baboons did not exhibit beta electrophoretic mobility but did stimulate esterification in macrophages. Baboons were divided into high and low responders based on the change in plasma cholesterol levels in response to a high fat, high cholesterol diet. Both plasma and hepatic VLDL from high responders stimulated cholesterol esterification, whereas hepatic VLDL obtained from low responders or chow-fed baboons did not stimulate cholesterol esterification in macrophages. There was a strong positive correlation (r = 0.866) between the number of apoE molecules per VLDL particle in VLDL obtained from chow-fed, lard-fed, or coconut oil-fed primates and the rate of cholesterol esterification in macrophages. Our results show that hepatic perfusate VLDL obtained from fat- and cholesterol-fed primates have compositional and functional properties usually ascribed to circulating beta-VLDL, without displaying beta mobility, and indicate that the liver may be an important source of atherogenic lipoproteins.

Monkey arterial smooth muscle cell binding, incorporation and degradation of low density lipoproteins (LDL) from normal or hyperlipemic rhesus monkey plasma were examined by labeling lipoproteins with 125 I. Specific and nonspecific binding of both lipoproteins occurred. Although the dissociation constant for the normal LDL was twice as high as for hyperlipemic LDL, indicating a higher binding affinity for hyperlipemic LDL, the maximal binding capacity of the smooth muscle cells for normal [ 125 I]LDL was 2-fold higher than for hyperlipemic [ 125 I]LDL. In competition experiments where the two types of LDL competed with normal [ 125 I]LDL for binding to smooth muscle cells, LDL isolated from hyperlipemic plasma was a more effective competitor than normal LDL. Receptors for normal LDL were reduced to a greater extent by preincubation with hyperlipemic LDL than by the same protein concentration of normal LDL. Under conditions where nonspecific uptake of lipoproteins would occur predominantly, the cells showed higher incorporation and degradation of normal LDL than hyperlipemic LDL. The corresponding cellular cholesterol ester levels showed an inverse pattern, with higher accumulation of cholesterol ester in the cells grown in hyperlipemic LDL. The results suggest that binding and incorporation of LDL by specific receptors or through the nonspecific pathway play a role in the changes of cellular cholesterol content but cannot totally account for the enhanced accumulation of cellular cholesterol esters produced by hyperlipemic monkey LDL.

Phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) were co-isolated with the low molecular weight rat surfactant-associated protein C (SP-C) of Mr approximately equal to 6,000. The contribution of these phospholipids to the incorporation of 3H-labeled phosphatidylcholine (PC) liposomes into rat alveolar type II cells stimulated by SP-C was examined. PG showed a concentration-dependent enhancement in the uptake of PC liposomes by the pneumocytes. PE alone had no effect but could inhibit the incorporation of liposomal PC stimulated by PG depending on the concentration of PG and the PG to PE ratio. SP-C augmented the cellular uptake of the PC liposomes only when the SP-C preparation had a protein to phospholipid ratio greater than 1 and a PG to PE ratio greater than 2. The results with the isolated SP-C could be reproduced using mixtures of PG and PE which reflected the phospholipid composition of the SP-C in the absence of SP-C protein. Thus, the ability of SP-C to stimulate liposomal PC uptake by rat type II cells could be accounted for by its phospholipid composition.