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1.  Complementary precursor ion and neutral loss scan mode tandem mass spectrometry for the analysis of glycerophosphatidylethanolamine lipids from whole rat retina 
A “shotgun” tandem mass spectrometry (MS) approach involving the use of multiple lipid-class-specific precursor ion and neutral loss scan mode experiments has been employed to identify and characterize the glycerophosphatidylethanolamine (GPEtn) lipids that were present within a crude lipid extract of a normal rat retina, obtained with minimal sample handling prior to analysis. Characterization of these lipids was performed by complementary analysis of their protonated and deprotonated precursor ions, as well as their various ionic adducts (e.g., Na+, Cl−), using a triple-quadrupole mass spectrometer. Notably, the application of novel precursor ion and neutral loss scans of m/z 164 and m/z 43, respectively, for the specific identification of sodiated GPEtn precursor ions following the addition of 500 μM NaCl to the crude lipid extracts was demonstrated. The use of these novel MS/MS scans in parallel provided simplified MS/MS spectra and enhanced the detection of 1-alkenyl, 2-acyl (plasmenyl) GPEtn lipids relative to the positive ion mode neutral loss m/z 141 commonly used for GPEtn analysis. Furthermore, the novel use of a “low energy” neutral loss scan mode experiment to monitor for the exclusive loss of 36m/z (HCl) from [M+Cl]-GPEtn adducts was demonstrated to provide a more than 25-fold enhancement for the detection of GPEtn lipids in negative ion mode analysis. Subsequent “high-energy” pseudo MS3 product ion scans on the precursor ions identified from this experiment were then employed to rapidly characterize the fatty acyl chain substituents of the GPEtn lipids.
PMCID: PMC4112091  PMID: 19277613
Retina; Lipidomics; Glycerophosphatidylethanolamine; Electrospray ionization; Tandem mass spectrometry
2.  Examining the role of lipid mediators in diabetic retinopathy 
Clinical lipidology  2012;7(6):661-675.
Diabetic retinopathy is the most disabling complication of diabetes, affecting 65% of patients after 10 years of the disease. Current treatment options for diabetic retinopathy are highly invasive and fall short of complete amelioration of the disease. Understanding the pathogenesis of diabetic retinopathy is critical to the development of more effective treatment options. Diabetic hyperglycemia and dyslipidemia are the main metabolic insults that affect retinal degeneration in diabetes. Although the role of hyperglycemia in inducing diabetic retinopathy has been studied in detail, much less attention has been paid to dyslipidemia. Recent clinical studies have demonstrated a strong association between dyslipidemia and development of diabetic retinopathy, highlighting the importance of understanding the exact changes in retinal lipid metabolism in diabetes. This review describes what is known on the role of dyslipidemia in the development of diabetic retinopathy, with a focus on retinal-specific lipid metabolism and its dysregulation in diabetes.
PMCID: PMC3640872  PMID: 23646066
acid sphingomyelinase; cholesterol; diabetes; fatty acid; fatty acid elongase; phospholipid; retinopathy; sphingolipid
3.  The Unconventional Role of Acid Sphingomyelinase in Regulation of Retinal Microangiopathy in Diabetic Human and Animal Models 
Diabetes  2011;60(9):2370-2378.
Acid sphingomyelinase (ASM) is an important early responder in inflammatory cytokine signaling. The role of ASM in retinal vascular inflammation and vessel loss associated with diabetic retinopathy is not known and represents the goal of this study.
Protein and gene expression profiles were determined by quantitative RT-PCR and Western blot. ASM activity was determined using Amplex Red sphingomyelinase assay. Caveolar lipid composition was analyzed by nano-electrospray ionization tandem mass spectrometry. Streptozotocin-induced diabetes and retinal ischemia-reperfusion models were used in in vivo studies.
We identify endothelial caveolae-associated ASM as an essential component in mediating inflammation and vascular pathology in in vivo and in vitro models of diabetic retinopathy. Human retinal endothelial cells (HREC), in contrast with glial and epithelial cells, express the plasma membrane form of ASM that overlaps with caveolin-1. Treatment of HREC with docosahexaenoic acid (DHA) specifically reduces expression of the caveolae-associated ASM, prevents a tumor necrosis factor-α–induced increase in the ceramide-to-sphingomyelin ratio in the caveolae, and inhibits cytokine-induced inflammatory signaling. ASM is expressed in both vascular and neuroretina; however, only vascular ASM is specifically increased in the retinas of animal models at the vasodegenerative phase of diabetic retinopathy. The absence of ASM in ASM−/− mice or inhibition of ASM activity by DHA prevents acellular capillary formation.
This is the first study demonstrating activation of ASM in the retinal vasculature of diabetic retinopathy animal models. Inhibition of ASM could be further explored as a potential therapeutic strategy in treating diabetic retinopathy.
PMCID: PMC3161322  PMID: 21771974
5.  Inhibition of Cytokine Signaling in Human Retinal Endothelial Cells through Downregulation of Sphingomyelinases by Docosahexaenoic Acid 
DHA downregulates basal and cytokine-induced ASMase and NSMase activity in human retinal endothelial cells, and inhibition of sphingomyelinases in endothelial cells prevents cytokine-induced inflammatory response.
The authors have previously demonstrated that DHA inhibits cytokine-induced inflammation in human retinal endothelial cells (HRECs), the resident vasculature affected by diabetic retinopathy. However, the anti-inflammatory mechanism of docosahexaenoic acid (DHA) is still not well understood. Sphingolipids represent a major component of membrane microdomains, and ceramide-enriched microdomains appear to be a prerequisite for inflammatory cytokine signaling. Acid sphingomyelinase (ASMase) and neutral sphingomyelinase (NSMase) are key regulatory enzymes of sphingolipid metabolism, promoting sphingomyelin hydrolysis to proinflammatory ceramide. The authors address the hypothesis that DHA inhibits cytokine-induced inflammatory signaling in HRECs by downregulating sphingomyelinases.
ASMase and NSMase activity was determined by sphingomyelinase assay in primary cultures of HRECs. The expression of ASMase, NSMase, ICAM-1, and VCAM-1 was assessed by quantitative PCR and Western blot analysis. Gene silencing of ASMase and NSMase was obtained by siRNA treatment.
Inflammatory cytokines TNFα and IL-1β induced cellular adhesion molecule (CAM) expression and rapid increase in ASMase and NSMase activity in HRECs. DHA decreased basal and cytokine-induced ASMase and NSMase expression and activity and the upregulation of CAM expression. Anti-inflammatory effects of DHA on cytokine-induced CAM expression were mimicked by inhibition/gene silencing of ASMase and NSMase. The sphingomyelinase pathway rather than ceramide de novo synthesis pathway was important for inflammatory signaling in HRECs.
This study provides a novel potential mechanism for the anti-inflammatory effect of DHA in HRECs. DHA downregulates the basal and cytokine-induced ASMase and NSMase expression and activity level in HRECs, and inhibition of sphingomyelinases in endothelial cells prevents cytokine-induced inflammatory response.
PMCID: PMC2891477  PMID: 20071681
6.  Diabetic retinopathy is associated with bone marrow neuropathy and a depressed peripheral clock 
The Journal of Experimental Medicine  2009;206(13):2897-2906.
The present epidemic of diabetes is resulting in a worldwide increase in cardiovascular and microvascular complications including retinopathy. Current thinking has focused on local influences in the retina as being responsible for development of this diabetic complication. However, the contribution of circulating cells in maintenance, repair, and dysfunction of the vasculature is now becoming appreciated. Diabetic individuals have fewer endothelial progenitor cells (EPCs) in their circulation and these cells have diminished migratory potential, which contributes to their decreased reparative capacity. Using a rat model of type 2 diabetes, we show that the decrease in EPC release from diabetic bone marrow is caused by bone marrow neuropathy and that these changes precede the development of diabetic retinopathy. In rats that had diabetes for 4 mo, we observed a dramatic reduction in the number of nerve terminal endings in the bone marrow. Denervation was accompanied by increased numbers of EPCs within the bone marrow but decreased numbers in circulation. Furthermore, denervation was accompanied by a loss of circadian release of EPCs and a marked reduction in clock gene expression in the retina and in EPCs themselves. This reduction in the circadian peak of EPC release led to diminished reparative capacity, resulting in the development of the hallmark feature of diabetic retinopathy, acellular retinal capillaries. Thus, for the first time, diabetic retinopathy is related to neuropathy of the bone marrow. This novel finding shows that bone marrow denervation represents a new therapeutic target for treatment of diabetic vascular complications.
PMCID: PMC2806461  PMID: 19934019
7.  Anti-inflammatory Effect of Docosahexaenoic Acid on Cytokine-Induced Adhesion Molecule Expression in Human Retinal Vascular Endothelial Cells 
Docosahexaenoic acid (DHA22:6n3), the principal n3-polyunsaturated fatty acid (PUFA) in the retina, has been shown to have a pronounced anti-inflammatory effect in numerous in vivo and in vitro studies. Despite the importance of vascular inflammation in diabetic retinopathy, the anti-inflammatory role of DHA22:6n3 in cytokine-stimulated human retinal vascular endothelial cells (hRVECs) has not been addressed.
Cytokine-induced expression of cell adhesion molecules (CAMs) was assessed by Western blot. The effect of DHA22:6n3 on cytokine-induced nuclear factor (NF)-κB signaling was analyzed by Western blot analysis and electrophoretic mobility shift assay (EMSA).
Stimulation of hRVECs with VEGF165, TNFα, or IL-1β for 6 to 24 hours caused significant induction of intracellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 expression. Pretreatment of the cells with 100 μM of BSA-bound DHA22:6n3 for 24 hours remarkably inhibited cytokine-induced CAM expression. IL-1β, TNFα, and VEGF165 induced nuclear translocation and binding of p65 and p50 NF-κB isoforms to the VCAM-1 promoter. DHA22:6n3 pretreatment inhibited cytokine-induced NF-κB binding by 25% to 40%. Moreover, DHA22:6n3 diminished IL-1β induced phosphorylation of the inhibitor of nuclear factor (NF)-κB (I-κBα), thus preventing its degradation.
IL-1β, TNFα, and VEGF165 induced CAM expression in hRVECs through activation of the NF-κB pathway. DHA22:6n3 inhibited cytokine induced CAM expression through suppression of NF-κB nuclear translocation and upstream I-κBα phosphorylation and degradation. DHA22:6n3 could be an important anti-inflammatory agent in the face of increased cytokine production and CAM expression in the diabetic retina.
PMCID: PMC1378111  PMID: 16249517
8.  Inhibition of Cytokine Signaling in Human Retinal Endothelial Cells through Modification of Caveolae/Lipid Rafts by Docosahexaenoic Acid 
Docosahexaenoic acid (DHA22:6,n3) is the principal n3 polyunsaturated fatty acid (PUFA) in the retina. The authors previously demonstrated that DHA22:6,n3 inhibited cytokine-induced adhesion molecule expression in primary human retinal vascular endothelial (hRVE) cells, the target tissue affected by diabetic retinopathy. Despite the importance of vascular inflammation in diabetic retinopathy, the mechanisms underlying anti-inflammatory effects of DHA22:6,n3 in vascular endothelial cells are not understood. In this study the authors address the hypothesis that DHA22:6,n3 acts through modifying lipid composition of caveolae/lipid rafts, thereby changing the outcome of important signaling events in these specialized plasma membrane microdomains.
hRVE cells were cultured in the presence or absence of DHA22:6,n3. Isolated caveolae/lipid raft–enriched detergent-resistant membrane domains were prepared using sucrose gradient ultracentrifugation. Fatty acid composition and cholesterol content of caveolae/lipid rafts before and after treatment were measured by HPLC. The expression of Src family kinases was assayed by Western blotting and immunohistochemistry.
Disruption of the caveolae/lipid raft structure with a cholesterol-depleting agent, methyl-cyclodextrin (MCD), diminished cytokine-induced signaling in hRVE cells. Growth of hRVE cells in media enriched in DHA22:6,n3 resulted in significant incorporation of DHA22:6,n3 into the major phospholipids of caveolae/lipid rafts, causing an increase in the unsaturation index in the membrane microdomain. DHA22:6,n3 enrichment in the caveolae/raft was accompanied by a 70% depletion of cholesterol from caveolae/lipid rafts and displacement of the SFK, Fyn, and c-Yes from caveolae/lipid rafts. Adding water-soluble cholesterol to DHA22:6,n3-treated cells replenished cholesterol in caveolae/lipid rafts and reversed the effect of DHA22:6,n3 on cytokine-induced signaling.
Incorporation of DHA22:6,n3 into fatty acyl chains of phospholipids in caveolae/lipid rafts, followed by cholesterol depletion and displacement of important signaling molecules, provides a potential mechanism for anti-inflammatory effect of DHA22:6,n3 in hRVE cells.
PMCID: PMC1975816  PMID: 17197511
9.  Identification of APOBEC3DE as Another Antiretroviral Factor from the Human APOBEC Family▿  
Journal of Virology  2006;80(21):10522-10533.
A tandem arrayed gene cluster encoding seven cytidine deaminase genes is present on human chromosome 22. These are APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3DE, APOBEC3F, APOBEC3G, and APOBEC3H. Three of them, APOBEC3G, APOBEC3F, and APOBEC3B, block replication of human immunodeficiency virus type 1 (HIV-1) and many other retroviruses. In addition, APOBEC3A and APOBEC3C block intracellular retrotransposons and simian immunodeficiency virus (SIV), respectively. In opposition to APOBEC genes, HIV-1 and SIV contain a virion infectivity factor (Vif) that targets APOBEC3F and APOBEC3G for polyubiquitylation and proteasomal degradation. Herein, we studied the antiretroviral activities of the human APOBEC3DE and APOBEC3H. We found that only APOBEC3DE had antiretroviral activity for HIV-1 or SIV and that Vif suppressed this antiviral activity. APOBEC3DE was encapsidated and capable of deaminating cytosines to uracils on viral minus-strand DNA, resulting in disruption of the viral life cycle. Other than GG-to-AG and AG-to-AA mutations, it had a novel target site specificity, resulting in introduction of GC-to-AC mutations on viral plus-strand DNA. Such mutations have been detected previously in HIV-1 clinical isolates. In addition, APOBEC3DE was expressed much more extensively than APOBEC3F in various human tissues and it formed heteromultimers with APOBEC3F or APOBEC3G in the cell. From these studies, we concluded that APOBEC3DE is a new contributor to the intracellular defense network, resulting in suppression of retroviral invasion.
PMCID: PMC1641744  PMID: 16920826
10.  Lineage Switch Macrophages Can Present Antigen 
Developmental Immunology  1992;2(4):249-261.
Recent reports of “lineage switching” from a lymphoid to macrophage phenotype have left unresolved the question of whether such cells are functional macrophages or nonfunctional products of differentiation gone awry. This study demonstrates that several “macrophage-like” cell lines derived from v-Ha-ras-transformed pre-B cells have gained the capacity to effectively present antigen in MHC-restricted fashion. Using an assay involving the cocultivation of putative antigen-presenting cells with chicken ovalbumin (cOVA) and a cOVA-specific T-cell hybridoma, “lineage switch” cell lines were found to present antigen as effectively as macrophage-containing peritoneal exudates. Neither the original pre-B-cell precursors nor B-cell lymphomas derived from them present antigen. Thus, we have demonstrated that these “lineage switch” macrophages are capable of antigen presentation, a mature differentiated function. While gaining macrophage characteristics, these cells have also rearranged their kappa light-chain immunoglobulin locus, suggesting that macrophage differentiation and immunoglobulin rearrangement are not mutually exclusive processes. The existence of both lymphoid and myeloid characteristics in a cell fully capable of antigen presentation suggests greater plasticity in hematopoietic lineage commitment than conventionally thought to be the case.
PMCID: PMC2275870  PMID: 1343095
Lineage switch; macrophage; antigen presentation

Results 1-10 (10)