David K Crossman

University of Alabama at Birmingham, Birmingham, Alabama, United States

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Publications (23)127.73 Total impact

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    ABSTRACT: Although most hypocalcemia with hypomagenesemia in the neonatal period is due to transient neonatal hypoparathyroidism, magnesium channel defects should also be considered. We report a case of persistent hypomagnesemia in an 8-day-old Hispanic male who presented with generalized seizures. He was initially found to have hypomagnesemia, hypocalcemia, hyperphosphatemia and normal parathyroid hormone. Serum calcium normalized with administration of calcitriol and calcium carbonate. Serum magnesium improved with oral magnesium sulfate. However, 1 week after magnesium was discontinued, serum magnesium declined to 0.5 mg/dL. Magnesium supplementation was immediately restarted, and periodic seizure activity resolved after serum magnesium concentration was maintained above 0.9 mg/dL. The child was eventually weaned off oral calcium and calcitriol with persistent normocalemia. However, supraphysiologic oral magnesium doses were necessary to prevent seizures and maintain serum magnesium at the low limit of normal. As his clinical presentation suggested primary renal magnesium wastage, TRPM6 gene mutations were suspected; subsequent genetic testing revealed the child to be compound heterozygous for TRPM6 mutations. Two novel TRPM6 mutations are described with a new geographic and ethnic origin. This case highlights the importance of recognizing disorders of magnesium imbalance and describing new genetic mutations.
    07/2015; DOI:10.1515/jpem-2014-0394
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    ABSTRACT: Systemic lupus erythematosus (SLE) is a severe autoimmune disease that is associated with increased circulating apoptotic cell autoantigens (AC-Ags) as well as increased type I IFN signaling. Here, we describe a pathogenic mechanism in which follicular translocation of marginal zone (MZ) B cells in the spleens of BXD2 lupus mice disrupts marginal zone macrophages (MZMs), which normally clear AC debris and prevent follicular entry of AC-Ags. Phagocytosis of ACs by splenic MZMs required the megakaryoblastic leukemia 1 (MKL1) transcriptional coactivator-mediated mechanosensing pathway, which was maintained by MZ B cells through expression of membrane lymphotoxin-alpha1beta2 (mLT). Specifically, type I IFN-induced follicular shuttling of mLT-expressing MZ B cells disengaged interactions between these MZ B cells and LTbeta receptor-expressing MZMs, thereby downregulating MKL1 in MZMs. Loss of MKL1 expression in MZMs led to defective F-actin polymerization, inability to clear ACs, and, eventually, MZM dissipation. Aggregation of plasmacytoid DCs in the splenic perifollicular region, follicular translocation of MZ B cells, and loss of MKL1 and MZMs were also observed in an additional murine lupus model and in the spleens of patients with SLE. Collectively, the results suggest that lupus might be interrupted by strategies that maintain or enhance mechanosensing signaling in the MZM barrier to prevent follicular entry of AC-Ags.
    The Journal of clinical investigation 06/2015; DOI:10.1172/JCI81059. · 13.77 Impact Factor
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    ABSTRACT: The apolipoprotein (apo)A-I mimetic peptide 4F favors the differentiation of human monocytes to an alternatively-activated M2 phenotype. The goal of the current study was to test whether the 4F-mediated differentiation of monocyte-derived macrophages (MDMs) requires the induction of an oxidative metabolic program. 4F treatment induced several genes in MDMs that play an important role in lipid metabolism, including peroxisome proliferator-activated receptor γ (PPARγ) and CD36. Addition of 4F was associated with a significant increase in fatty acid (FA) uptake and oxidation compared to vehicle treatment. Mitochondrial respiration was assessed by measurement of oxygen consumption rate (OCR). 4F increased basal and ATP-linked OCR as well as maximal uncoupled mitochondrial respiration. These changes were associated with a significant increase in mitochondrial membrane potential (ΔΨm). The increase in metabolic activity in 4F-treated MDMs was attenuated by etomoxir, an inhibitor of mitochondrial FA uptake. Finally, addition of the PPARγ antagonist T0070907 to 4F-treated MDMs reduced the expression of CD163 and CD36, cell surface markers for M2 macrophages, and reduced basal and ATP-linked OCR. These results support our hypothesis that the 4F-mediated differentiation of MDMs to an anti-inflammatory phenotype is due, in part, to an increase in FA acid uptake and mitochondrial oxidative metabolism.
    Biochemical Journal 03/2015; DOI:10.1042/BJ20131635 · 4.78 Impact Factor
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    ABSTRACT: Polyunsaturated fatty acids (PUFA) intake has increased over the last 100 years, contributing to the current obesogenic environment. Obesity and aging are prominent risk factors for myocardial infarction (MI). How obesity interacts with aging to alter post-MI response, however, is unclear. We tested the hypothesis that obesity in aging mice would impair the resolution of post-MI inflammation. PUFA diet (PUFA aging) feeding to 12 months-old C57BL/6J mice for 5 months showed higher fat mass compared to standard lab chow (LC) fed young (LC young; 3-5 months-old) or aging alone control mice (LC aging). LC young, LC aging and PUFA aging mice were subjected to coronary artery ligation to induce MI. Despite similar infarct areas post-MI, plasma proteomic profiling revealed higher vascular cell adhesion molecule-1 in PUFA aging compared to LC young and LC aging, leading to increased neutrophil infiltration in the PUFA aging group (p<0.05). Macrophage inflammatory protein-1γ and CD40 were also increased at d1, while myeloperoxidase remained elevated at d5, an observation consistent with delayed wound healing in PUFA aging. Lipidomic analysis showed higher levels of the arachidonic acid and 12(S)hydroxyeicosatetraenoic acid at d1 post-MI in the PUFA aging compared to LC aging (all p<0.05), thereby mediating neutrophil extravasation in PUFA aging. The inflammation resolving enzymes 5-lipoxygenase, cyclooxygenase-2 and heme oxyegnase-1 were altered to delay wound healing post-MI in PUFA aging compared to LC young and LC aging. PUFA aging magnifies post-MI inflammatory response and impairs healing response by stimulating prolonged neutrophil trafficking and pro-inflammatory lipid mediators. Copyright © 2014, American Journal of Physiology - Heart and Circulatory Physiology.
    AJP Heart and Circulatory Physiology 12/2014; 308(4):ajpheart.00604.2014. DOI:10.1152/ajpheart.00604.2014 · 4.01 Impact Factor
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    ABSTRACT: Genetic changes occurring in different stages of pre-cancer lesions reflect causal events initiating and promoting the progression to cancer. Co-existing pre-cancerous lesions including low- and high-grade squamous intraepithelial lesion (LGSIL and HGSIL), and adjacent "normal" cervical epithelium from six formalin-fixed paraffin-embedded samples were selected. Tissues from these 18 samples were isolated using laser-capture microdissection, RNA was extracted and sequenced. RNA-sequencing generated 2.4 billion raw reads in 18 samples, of which ~50.1% mapped to known and annotated genes in the human genome. There were 40 genes up-regulated and 3 down-regulated (normal to LGSIL) in at least one-third of the sample pairs (same direction and FDR p < 0.05) including S100A7 and KLK6. Previous studies have shown that S110A7 and KLK7 are up-regulated in several other cancers, whereas CCL18, CFTR, and SLC6A14, also differentially expressed in two samples, are up-regulated specifically in cervical cancer. These differentially expressed genes in normal to LGSIL progression were enriched in pathways related to epithelial cell differentiation, keratinocyte differentiation, peptidase, and extracellular activities. In progression from LGSIL to HGSIL, two genes were up-regulated and five down-regulated in at least two samples. Further investigations using co-existing samples, which account for all internal confounders, will provide insights to better understand progression of cervical pre-cancer.
    Frontiers in Oncology 11/2014; 4:339. DOI:10.3389/fonc.2014.00339
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    ABSTRACT: Rationale: DNA methylation, a major epigenetic mechanism, may regulate coordinated expression of multiple genes at specific time points during alveolar septation in lung development. Objectives: To identify genes regulated by methylation during normal septation in mice, and during disordered septation in bronchopulmonary dysplasia. Methods: Mouse: Newborn lungs (pre-septation) and adult lungs (post-septation) were evaluated by microarray analysis of gene expression and immunoprecipitation of methylated DNA followed by sequencing (MeDIP-Seq). Human: Microarray gene expression data were integrated with genome-wide DNA methylation data from bronchopulmonary dysplasia versus preterm and term lung. Genes with reciprocal changes in expression and methylation, suggesting regulation by DNA methylation, were identified. Measurements and Main Results: Mouse: 95 genes with inverse correlation between expression and methylation during normal septation were identified. In addition to genes known to be important in lung development (Wnt signaling, Angpt2, Sox9, etc.) and its extracellular matrix (Tnc, Eln, etc), genes involved with immune and antioxidant defense (Stat4, Sod3, Prdx6, etc.) were also observed. Human: 23 genes were differentially methylated with reciprocal changes in expression in bronchopulmonary dysplasia compared to preterm or term lung. Genes of interest included those involved with detoxifying enzymes (Gstm3) and transforming growth factor beta signaling (Bmp7). Overlap: 20 genes and three pathways methylated during mouse lung development also demonstrated changes in methylation between preterm and term human lung. Conclusions: Changes in methylation correspond to altered expression of a number of genes associated with lung development, suggesting that DNA methylation of these genes may regulate normal and abnormal alveolar septation.
    American Journal of Respiratory Cell and Molecular Biology 11/2014; Epub ahead of print(PMID: 25387348). DOI:10.1165/rcmb.2014-0160OC · 4.11 Impact Factor
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    ABSTRACT: Historically, in order to study microbes, it was necessary to grow them in the laboratory. It was clear though that many microbe communities were refractory to study because none of the members could be grown outside of their native habitat. The development of culture-independent methods to study microbiota using high-throughput sequencing of the 16S ribosomal RNA gene variable regions present in all prokaryotic organisms has provided new opportunities to investigate complex microbial communities. In this unit, the process for a microbiome analysis is described. Many of the components required for this process may already exist. A pipeline is described for acquisition of samples from different sites on the human body, isolation of microbial DNA, and DNA sequencing using the Illumina MiSeq sequencing platform. Finally, a new analytical workflow for basic bioinformatics data analysis, QWRAP, is described, which can be used by clinical and basic science investigators. Curr. Protoc. Hum. Genet. 82:18.8.1-18.8.29. © 2014 by John Wiley & Sons, Inc.
    Current protocols in human genetics / editorial board, Jonathan L. Haines ... [et al.] 01/2014; 82:18.8.1-18.8.29. DOI:10.1002/0471142905.hg1808s82
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    ABSTRACT: Constitutional SMARCB1 mutations at 22q11.23 have been found in ∼50% of familial and <10% of sporadic schwannomatosis cases. We sequenced highly conserved regions along 22q from eight individuals with schwannomatosis whose schwannomas involved somatic loss of one copy of 22q, encompassing SMARCB1 and NF2, with a different somatic mutation of the other NF2 allele in every schwannoma but no mutation of the remaining SMARCB1 allele in blood and tumor samples. LZTR1 germline mutations were identified in seven of the eight cases. LZTR1 sequencing in 12 further cases with the same molecular signature identified 9 additional germline mutations. Loss of heterozygosity with retention of an LZTR1 mutation was present in all 25 schwannomas studied. Mutations segregated with disease in all available affected first-degree relatives, although four asymptomatic parents also carried an LZTR1 mutation. Our findings identify LZTR1 as a gene predisposing to an autosomal dominant inherited disorder of multiple schwannomas in ∼80% of 22q-related schwannomatosis cases lacking mutation in SMARCB1.
    Nature Genetics 12/2013; 46(2). DOI:10.1038/ng.2855 · 29.65 Impact Factor
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    ABSTRACT: Aim: Neoplastic transformation provides one of the few existing opportunities to analyze molecular changes in real time during the initiation and progression of breast cancer. Materials & methods: Human mammary epithelial cells underwent neoplastic reprogramming, generating one line of semitransformed, premalignant cells and two separate, temporal lines of fully transformed human mammary epithelial cells (THMECs). An Illumina Infinium HumanMethylation27 BeadChip was used to analyze DNA methylation alterations in 27,578 CpG loci at three consecutive time points over an 80-day (d) transformation period. Results: The mean β value for semitransformed human mammary epithelial cells CpG loci (0.245) was much greater than for either THMEC-40d (0.055) or THMEC-80d (0.066), indicating a large loss of methylation after neoplastic induction. In addition, 54% of CpG loci were hypermethylated during the THMEC-40d to THMEC-80d transition. We observed that the CpG loci exhibiting DNA methylation changes during early oncogenesis were enriched for biological functions like cellular movement; this was distinctly different than in the later, more progressive stages of the transformation process enriched for processes involving differentiation. Conclusion: The timing of major methylomic changes may be important in directing the cell toward a more cancerous phenotype. In addition, gene-specific hypermethylation appears to silence developmentally related genes, leading to dedifferentiation.
    Epigenomics 04/2013; 5(2):155-65. DOI:10.2217/epi.13.6 · 5.22 Impact Factor
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    ABSTRACT: Wnt5a is a non-canonical signaling Wnt. Low expression of WNT5A is correlated with poor prognosis in breast cancer patients. The highly invasive breast cancer cell lines, MDA-MB-231 and 4T1, express very low levels of WNT5A. To determine if enhanced expression of WNT5A would affect metastatic behavior, we generated WNT5A expressing cells from the 4T1 and MDA-MB-231 parental cell lines. WNT5A expressing cells demonstrated cobblestone morphology and reduced in vitro migration relative to controls. Cell growth was not altered. Metastasis to the lung via tail vein injection was reduced in the 4T1-WNT5A expressing cells relative to 4T1-vector controls. To determine the mechanism of WNT5A action on metastasis, we performed microarray and whole-transcriptome sequence analysis (RNA-seq) to compare gene expression in 4T1-WNT5A and 4T1-vector cells. Analysis indicated highly significant alterations in expression of genes associated with cellular movement. Down-regulation of a subset of these genes, Mmp13, Nos2, Il1a, Cxcl2, and Lamb3, in WNT5A expressing cells was verified by semi-quantitative RT-PCR. Significant differences in transcript splicing were also detected in cell movement associated genes including Cd44. Cd44 is an adhesion molecule with a complex genome structure. Variable exon usage is associated with metastatic phenotype. Alternative spicing of Cd44 in WNT5A expressing cells was confirmed using RT-PCR. We conclude that WNT5A inhibits metastasis through down-regulation of multiple cell movement pathways by regulating transcript levels and splicing of key genes like Cd44.
    PLoS ONE 03/2013; 8(3):e58329. DOI:10.1371/journal.pone.0058329 · 3.23 Impact Factor
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    ABSTRACT: The apolipoprotein A-I (apoA-I) mimetic peptide 4F favors the differentiation of human monocytes to an anti-inflammatory phenotype and attenuates lipopolysaccharide (LPS)-induced inflammatory responses. We investigated the effects of LPS on the Toll-like receptor (TLR) signaling pathway in 4F-differentiated monocyte-derived macrophages. Monocyte-derived macrophages were pretreated with 4F or vehicle for 7 days. 4F downregulated cell-surface TLRs (4, 5, and 6) as determined by flow cytometry. 4F attenuated the LPS-dependent upregulation of genes encoding TLR1, 2, and 6 and genes of the MyD88-dependent (CD14, MyD88, TRAF6, interleukin-1 receptor-associated kinase 4, and inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta) and MyD88-independent (interferon regulatory factor 3, TANK-binding kinase 1, and Toll-interleukin 1 receptor domain-containing adaptor-inducing interferon-β) pathways as determined by microarray analysis and quantitative reverse transcriptase polymerase chain reaction. Functional analyses of monocyte-derived macrophages showed that 4F reduced LPS-dependent TLR4 recycling, phosphorylation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha, activation and translocation of nuclear factor-κB and inhibited the secretion of tumor necrosis factor-α and interleukin-6 induced by LPS or lipoteichoic acid. These changes were associated with depletion of cellular cholesterol and caveolin, components of membrane lipid rafts. These data suggest that disruption of rafts by 4F alters the assembly of TLR-ligand complexes in cell membranes and inhibits proinflammatory gene expression in monocyte-derived macrophages, thus attenuating the responsiveness of macrophages to LPS.
    Arteriosclerosis Thrombosis and Vascular Biology 09/2012; 32(11):2631-9. DOI:10.1161/ATVBAHA.112.300167 · 5.53 Impact Factor
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    ABSTRACT: Rationale: DNA methylation is an important epigenetic mechanism, which often occurs in response to environmental stimuli and is crucial in regulating gene expression. It is likely that epigenetic alterations contribute to pathogenesis in idiopathic pulmonary fibrosis (IPF). Objectives: To determine the DNA methylation changes in IPF and their effects on gene expression. Methods: Total DNA methylation and DNA methyltransferase expression were compared in IPF and normal control lung tissues. IPF and normal tissues were subjected to comparative analysis of genome-wide DNA methylation and RNA expression using DNA hybridization to the Illumina HumanMethylation27 BeadChip and RNA hybridization to Illumina HumanHT-12 BeadChip. Functional analyses of differentially expressed and differentially methylated genes were done. Selected genes were validated at DNA, RNA, and protein levels. Measurements and Main Results: DNA methylation status was altered in IPF. IPF samples demonstrated higher DNA methyltransferase expression without observed alterations in global DNA methylation. Genome-wide differences in DNA methylation status and RNA expression were demonstrated by array hybridization. Among the genes whose DNA methylation status and RNA expression were both significantly altered, 16 genes were hypermethylated in DNA associated with decreased mRNA expression or vice versa. We validated CLDN5, ZNF467, TP53INP1, and DDAH1 genes at the level of DNA methylation status, RNA, and protein-level expression. Conclusions: Changes in DNA methylation correspond to altered mRNA expression of a number of genes, some with known and others with previously uncharacterized roles in IPF, suggesting that DNA methylation is important in the pathogenesis of IPF.
    American Journal of Respiratory and Critical Care Medicine 06/2012; 186(6):525-35. DOI:10.1164/rccm.201201-0077OC · 11.99 Impact Factor
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    ABSTRACT: Renal injury induced by brain death is characterized by ischemia and inflammation, and limiting it is a therapeutic goal that could improve outcomes in kidney transplantation. Brain death resulted in decreased circulating nitrite levels and increased infiltrating inflammatory cell infiltration into the kidney. Since nitrite stimulates nitric oxide signaling in ischemic tissues, we tested whether nitrite therapy was beneficial in a rat model of brain death followed by kidney transplantation. Nitrite, administered over 2 h of brain death, blunted the increased inflammation without affecting brain death-induced alterations in hemodynamics. Kidneys were transplanted after 2 h of brain death and renal function followed over 7 days. Allografts collected from nitrite-treated brain-dead rats showed significant improvement in function over the first 2 to 4 days after transplantation compared with untreated brain-dead animals. Gene microarray analysis after 2 h of brain death without or with nitrite therapy showed that the latter significantly altered the expression of about 400 genes. Ingenuity Pathway Analysis indicated that multiple signaling pathways were affected by nitrite, including those related to hypoxia, transcription, and genes related to humoral immune responses. Thus, nitrite therapy attenuates brain death-induced renal injury by regulating responses to ischemia and inflammation, ultimately leading to better post-transplant kidney function.
    Kidney International 04/2012; 82(3):304-13. DOI:10.1038/ki.2012.116 · 8.52 Impact Factor
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    ABSTRACT: To identify endoplasmic reticulum (ER) stress-induced microRNAs (miRNA) that govern ER protein influx during the adaptive phase of unfolded protein response, we performed miRNA microarray profiling and analysis in human airway epithelial cells following ER stress induction using proteasome inhibition or tunicamycin treatment. We identified miR-346 as the most significantly induced miRNA by both classic stressors. miR-346 is encoded within an intron of the glutamate receptor ionotropic delta-1 gene (GRID1), but its ER stress-associated expression is independent of GRID1. We demonstrated that the spliced X-box-binding protein-1 (sXBP1) is necessary and sufficient for ER stress-associated miR-346 induction, revealing a novel role for this unfolded protein response-activated transcription factor. In mRNA profiling arrays, we identified 21 mRNAs that were reduced by both ER stress and miR-346. The target genes of miR-346 regulate immune responses and include the major histocompatibility complex (MHC) class I gene products, interferon-induced genes, and the ER antigen peptide transporter 1 (TAP1). Although most of the repressed mRNAs appear to be indirect targets because they lack specific seeding sites for miR-346, we demonstrate that the human TAP1 mRNA is a direct target of miR-346. The human TAP1 mRNA 3'-UTR contains a 6-mer canonical seeding site for miR-346. Importantly, the ER stress-associated reduction in human TAP1 mRNA and protein levels could be reversed with an miR-346 antagomir. Because TAP function is necessary for proper MHC class I-associated antigen presentation, our results provide a novel mechanistic explanation for reduced MHC class I-associated antigen presentation that was observed during ER stress.
    Journal of Biological Chemistry 12/2011; 286(48):41862-70. DOI:10.1074/jbc.M111.304956 · 4.57 Impact Factor
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    ABSTRACT: Aberrant activation of the hedgehog (Hh) signaling pathway is implicated widely in both pediatric and adult malignancies. Inactivation of the Hh regulator PTCH is responsible for the Gorlin cancer predisposition syndrome. The spectrum of tumors found in Gorlin Syndrome includes basal cell carcinoma, medulloblastoma, and rarely, rhabdomyosarcoma (RMS). A previous report utilizing in situ hybridization has provided initial evidence for the expression of Hh targets GLI1 and PTCH in RMS tumors. To investigate the role of Hh pathway signaling in pediatric RMS and undifferentiated sarcoma (US) tumors, the expression of Hh pathway targets GLI1 and PTCH was measured. RNA was extracted from archival human tumor specimens collected from pediatric patients enrolled on Intergroup Rhabdomyosarcoma Study III and IV, and subjected to quantitative reverse transcriptase-polymerase chain reaction. Expression of GLI1 with or without PTCH was detected in substantial subsets of embryonal RMS (ERMS) and US tumors but only rarely in alveolar RMS tumors. Neither PTCH mutations nor activating SMO mutations were detected in ERMS tumors with high GLI1 expression. Microarray analysis demonstrated relative overexpression of downstream Hh targets in ERMS tumors with high or intermediate GLI1 expression. Unlike a recent report, Hh pathway activity in ERMS tumors did not correlate with a unique clinical phenotype. Our findings support a role for Hh pathway activation in the genesis of a subset of ERMS and US tumors. Hh signaling may represent a novel therapeutic target in affected tumors.
    Pediatric Blood & Cancer 12/2011; 57(6):930-8. DOI:10.1002/pbc.23174 · 2.56 Impact Factor
  • Free Radical Biology and Medicine 12/2010; 49. DOI:10.1016/j.freeradbiomed.2010.10.112 · 5.71 Impact Factor
  • Scientific Sessions on Arteriosclerosis, Thrombosis and Vascular Biology; 11/2010
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    ABSTRACT: Drusen are extracellular lesions characteristic of aging and age-related maculopathy, a major retinal disease of the elderly. We determined the relative proportions of lipids and proteins in drusen capped with retinal pigment epithelium (RPE) and in RPE isolated from non-macular regions of 36 human retinas with grossly normal maculas obtained <6 hr after death. Druse pellets were examined by light and electron microscopy. Component proteins were extracted using novel methods for preserved tissues, separated, subjected to tryptic digestion and LC-MS(MS)(2) analysis using an ion trap mass spectrometer, and identified with reference to databases. Lipid classes were separated using thin layer chromatography and quantified by densitometry. Major druse components were esterified cholesterol (EC), phosphatidylcholine (PC), and protein (37.5+/-13.7, 36.9+/-12.9, and 43.0+/-11.5 ng/druse, respectively). Lipid-containing particles (median diameter, 77 nm) occupied 37-44% of druse volume. Major proteins include vitronectin, complement component 9, apoE, and clusterin, previously seen in drusen, and ATP synthase subunit beta, scavenger receptor B2, and retinol dehydrogenase 5, previously seen in RPE. Drusen and RPE had similar protein profiles, with higher intensities and greater variability in drusen. C8, part of the complement membrane attack complex, was localized in drusen by immunofluorescence. At least 40% of druse content is comprised by lipids dominated by EC and PC, 2 components that are potentially accounted for by just one pathway, the secretion of lipoproteins by RPE. Manipulating genes encoding apolipoprotein pathways would be a fruitful approach to producing drusen with high EC content in laboratory animals. Therapies that directly mitigate drusen should prepare for the substantial volume of neutral lipids. The catalog of major druse proteins is nearing completion.
    PLoS ONE 04/2010; 5(4):e10329. DOI:10.1371/journal.pone.0010329 · 3.23 Impact Factor