A lipidomic screen of hyperglycemia-treated HRECs links 12/15-Lipoxygenase to microvascular dysfunction during diabetic retinopathy via NADPH oxidase

Retinal hyperpermeability and subsequent macular edema is a cardinal feature of early diabetic retinopathy (DR). Here, we investigated the role of bioactive lipid metabolites, in particular 12/15-lipoxygenase (LOX)-derived metabolites, in this process. LC/MS lipidomic screen of human retinal endothelial cells (HRECs) demonstrated that 15-HETE was the only significantly increased metabolite (2.4 ± 0.4-fold, P = 0.0004) by high glucose (30 mM) treatment. In the presence of arachidonic acid, additional eicosanoids generated by 12/15-LOX, including 12- and 11-HETEs, were significantly increased. Fluorescein angiography and retinal albumin leakage showed a significant decrease in retinal hyperpermeability in streptozotocin-induced diabetic mice lacking 12/15-LOX compared with diabetic WT mice. Our previous studies demonstrated the potential role of NADPH oxidase in mediating the permeability effect of 12- and 15-HETEs, therefore we tested the impact of intraocular injection of 12-HETE in mice lacking the catalytic subunit of NADPH oxidase (NOX2). The permeability effect of 12-HETE was significantly reduced in NOX2−/− mice compared with the WT mice. In vitro experiments also showed that 15-HETE induced HREC migration and tube formation in a NOX-dependent manner. Taken together our data suggest that 12/15-LOX is implicated in DR via a NOX-dependent mechanism.National Institutes of Health Grant 5R01EY023315 and National Priorities Research Program Grant 4-1046-3-284 from the Qatar National Research Fund (a member of Qatar Foundation). This study was also supported in part by the National Center for Research Resources, National Institutes of Health Grant S10RR027926

The Role of Antioxidation and Immunomodulation in Postnatal Multipotent Stem Cell-Mediated Cardiac Repair

Oxidative stress and inflammation play major roles in the pathogenesis of coronary heart disease including myocardial infarction (MI). The pathological progression following MI is very complex and involves a number of cell populations including cells localized within the heart, as well as cells recruited from the circulation and other tissues that participate in inflammatory and reparative processes. These cells, with their secretory factors, have pleiotropic effects that depend on the stage of inflammation and regeneration. Excessive inflammation leads to enlargement of the infarction site, pathological remodeling and eventually, heart dysfunction. Stem cell therapy represents a unique and innovative approach to ameliorate oxidative stress and inflammation caused by ischemic heart disease. Consequently, it is crucial to understand the crosstalk between stem cells and other cells involved in post-MI cardiac tissue repair, especially immune cells, in order to harness the beneficial effects of the immune response following MI and further improve stem cell-mediated cardiac regeneration. This paper reviews the recent findings on the role of antioxidation and immunomodulation in postnatal multipotent stem cell-mediated cardiac repair following ischemic heart disease, particularly acute MI and focuses specifically on mesenchymal, muscle and blood-vessel-derived stem cells due to their antioxidant and immunomodulatory properties

Nitric Oxide-cGMP Signaling Stimulates Erythropoiesis through Multiple Lineage-Specific Transcription Factors: Clinical Implications and a Novel Target for Erythropoiesis.

Much attention has been directed to the physiological effects of nitric oxide (NO)-cGMP signaling, but virtually nothing is known about its hematologic effects. We reported for the first time that cGMP signaling induces human γ-globin gene expression. Aiming at developing novel therapeutics for anemia, we examined here the hematologic effects of NO-cGMP signaling in vivo and in vitro. We treated wild-type mice with NO to activate soluble guanylate cyclase (sGC), a key enzyme of cGMP signaling. Compared to untreated mice, NO-treated mice had higher red blood cell counts and total hemoglobin but reduced leukocyte counts, demonstrating that when activated, NO-cGMP signaling exerts hematopoietic effects on multiple types of blood cells in vivo. We next generated mice which overexpressed rat sGC in erythroid and myeloid cells. The forced expression of sGCs activated cGMP signaling in both lineage cells. Compared with non-transgenic littermates, sGC mice exhibited hematologic changes similar to those of NO-treated mice. Consistently, a membrane-permeable cGMP enhanced the differentiation of hematopoietic progenitors toward erythroid-lineage cells but inhibited them toward myeloid-lineage cells by controlling multiple lineage-specific transcription factors. Human γ-globin gene expression was induced at low but appreciable levels in sGC mice carrying the human β-globin locus. Together, these results demonstrate that NO-cGMP signaling is capable of stimulating erythropoiesis in both in vitro and vivo settings by controlling the expression of multiple lineage-specific transcription factors, suggesting that cGMP signaling upregulates erythropoiesis at the level of gene transcription. The NO-cGMP signaling axis may constitute a novel target to stimulate erythropoiesis in vivo

Sp1 Transcription Factor as a Molecular Target for Nitric Oxide– and Cyclic Nucleotide–Mediated Suppression of cGMP-Dependent Protein Kinase-Iα Expression in Vascular Smooth Muscle Cells

cGMP-dependent protein kinase (PKG) expression is highly variable and decreases in cultured vascular smooth muscle cells (VSMCs), exposure of cells to nitric oxide (NO), or in response to balloon catheter injury in vivo. In this study, the mechanisms of human type I PKG-α (PKG-Iα) gene expression were examined. Three structurally unrelated NO donors decreased PKG-Iα promoter activity after transfection of a promoter/luciferase construct in VSMCs. Promoter deletion analysis demonstrated that (1) a 120-bp promoter containing tandem Sp1 sites was sufficient to drive basal PKG-Iα promoter activity, and (2) NO was inhibitory at this site. Cyclic nucleotide analogues also suppressed PKG-Iα promoter activity with cAMP being more potent than cGMP. The effects of cyclic nucleotides to suppress PKG-Iα promoter activity were attenuated by a specific cAMP-dependent protein kinase (PKA) inhibitor. Single or double mutation of Sp1 binding sites abolished PKG-Iα expression. Moreover, Sp1 binding activity on the PKG-Iα promoter was detected in A7r5 cells, and this binding was inhibited by NO and cyclic nucleotides. These results indicate that PKG-Iα gene expression is driven by an Sp1 transcription mechanism, and that NO and cAMP inhibit Sp1-mediated PKG-Iα gene expression through separate mechanisms

Effect of the sGC-cGMP pathway on expression of lineage cell-specific transcription factors in murine and human BM progenitors.

<p><b>(A to E)</b> Altered expression of erythroid- and myeloid cell-specific transcription factors in sGC transgenic mice. Transcription factor expression was determined by RT-PCR as described in Materials & Methods using spleen-derived erythroid cells and peripheral blood leukocytes from sGC mice (sGC-5 and sGC-7, n = 4) and Tg(-) littermates (n = 5). Expression level of transcription factors in Tg(-) littermates (n = 4) was set to 1. P values: *, <i>P</i><0.05; **, <i>P</i><0.01 compared with Tg(-) littermates. <b>(F to J)</b> Effect of 8-bromo-cGMP on expression of erythroid- and myeloid cell-specific transcription factors in human BMMNC. Human BM cells (4 × 10⁵ cells) were mixed with Methocult in the presence or absence of various concentrations of 8-bromo-cGMP (0.01 to 20 μM). Cells were isolated from semi-solid colony plates and total RNAs were purified. Expression levels of transcription factors were determined by real time-PCR. P values: *, <i>P</i><0.05; **, <i>P</i><0.01 compared with control cultures.</p

cGMP-dependent protein kinase and the regulation of vascular smooth muscle cell gene expression: possible involvement of Elk-1 sumoylation

Although the regulation of smooth muscle cell (SMC) gene expression by cGMP-dependent protein kinase (PKG) is now recognized, the mechanisms underlying these effects are not fully understood. In this study, we report that PKG-I stimulates myocardin/serum response factor (SRF)-dependent gene expression in vascular SMCs. The expression of PKG in PKG-deficient cells enhanced myocardin-induced SM22 promoter activity in a concentration-dependent fashion. However, neither SRF nor myocardin expression was affected. To investigate alternative mechanisms, we examined whether PKG affects the phosphorylation of E26-like protein-1 (Elk-1), a SRF/myocardin transcription antagonist. The activation of PKG caused an increase in a higher molecular mass form of phospho-Elk-1 that was determined to be small ubiquitin-related modifier (sumo)ylated Elk-1. PKG increased Elk-1 sumoylation twofold compared with the PKG-deficient cells, and Elk-1 sumoylation was reduced using dominant-negative sumo-conjugating enzyme, DN-Ubc9, confirming PKG-dependent sumoylation of phospho-Elk-1 in vascular SMCs. In addition, PKG stimulated Elk-1 sumoylation in COS-7 cells overexpressing Elk-1, sumo-1, and PKG-I. The increased expression of PKG in vascular SMCs inhibited Elk-1 binding to SMC-specific promoters, SM22 and smooth muscle myosin heavy chain, as measured by EMSA and chromatin immunoprecipitation assay, and PKG suppressed the Elk-1 inhibition of SM22 reporter gene expression. Taken together, these data suggest that PKG-I decreases Elk-1 activity by sumo modification of Elk-1, thereby increasing myocardin-SRF activity on SMC-specific gene expression

Generation and characterization of mice that overexpress rat sGC subunits in blood cells.

<p><b>(A)</b> Structure of the transgenes used to generate sGC transgenic mice. Human β-globin gene promoter (β-pro) is ligated with a mini β-LCR (open box), cDNA encoding either rat sGCα or sGCβ (black box), and an SV40 intron poly(A) signal sequence (SV40pA) (gray box). Black bar is equivalent to 1 kilo-base (kb). <b>(B)</b> Transgene expression in sGC transgenic mouse lines. Expression was examined by reverse transcriptase-PCR using 1 μg total RNA prepared from BM cells. PCR products of rat sGCα (R-sGCα), rat sGCβ (R-sGCβ), mouse sGCα (M-sGCα), and mouse sGCβ (M-sGCβ) are shown in (B)-(D). Lanes: 1, sGC-5; 2, sGC-7; 3, sGC-8; 4, sGC-9; 5, non-transgenic (Tg(-)) mice; M, molecular weight marker. In (B)-(D), mouse hypoxanthine phosphoribosyltransferase (HPRT) was used as an internal control for equal loading. Note: Mouse endogenous sGC mRNAs were undetectable in sGC-5 mice (Lane 1) by RT-PCR. <b>(C)</b> Transgene expression in various tissues of sGC-5 mice. Reverse transcriptase-PCR was performed using 1 μg total RNA prepared from various tissues; heart (lane 1); lung (2); liver (3); spleen (4); BM cells (5); peripheral RBCs (6); and peripheral leukocytes (7); M, molecular weight marker. <b>(D)</b> Developmental stage-specific expression of transgenes in sGC-7 mice and non-transgenic littermates. Reverse transcriptase-PCR was performed using 1 μg total RNA of yolk sac at 10.5 dpc (lanes 1&4); fetal liver at 14.5 dpc (2&5); and adult BM cells (3&6).</p

NO-cGMP signaling modulates hematopoiesis <i>in vivo</i>.

<p><b>(A-E)</b> NO upregulates erythropoiesis but suppresses myelopoiesis. Wild-type mice (n = 4) were treated with 8 ppm NO for 8 weeks. Intracellular cGMP levels of RBCs (A), peripheral blood leukocytes (B), total hemoglobin levels (C), hematocrit (D), and leukocyte count (E) were determined. P values are shown above the figures. <b>(F-I)</b> NO affects cell-lineage populations in BM. Mouse BMMNC, with or without 8 weeks of NO inhalation treatment, were examined for CD antigens. Results are shown for erythroid-lineage cells (F&G) and leukocytes (H&I). Experiments were repeated 3 times using BMMNCs from 3 defferent mice and representative results are shown. <b>(J&K)</b> NO affects cell differentiation of hematopoietic progenitors. Semi-solid colony assays were performed with BMMNC prepared from mice treated with 2 or 6 ppm NO for 8 weeks (J). The cells were plated on semi-solid plates with various SNP concentrations (K). The numbers of erythroid- (pink) and myeloid-lineage (blue) cells are shown. <b>(L)</b> NO donor upregulates βb-globin expression. Murine globin expression in erythroblasts prepared from semi-solid cultures was analyzed by immunoblotting. Cellular extracts (10 μg) were loaded to gels and murine βb-globin was detected by antibody.</p

Model of the molecular actions of NO-cGMP signaling and HU.

<p>While the current study has shown hematologic effects of NO-cGMP signaling on RBCs and leukocytes, the molecular actions of HU are yet to be elucidated. NO may be generated when HU is metabolized in the liver (pathway 1). HU may reduce leukocyte counts by inhibiting DNA synthesis through ribonucleotide reductase (pathway 2) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144561#pone.0144561.ref058" target="_blank">58</a>] or GM-CSF (pathway 3) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144561#pone.0144561.ref056" target="_blank">56</a>]. cGMP induces HbF expression [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144561#pone.0144561.ref021" target="_blank">21</a>] and inhibition of ribonucleotide reductase may account in part for leukocyte reduction and HbF induction [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144561#pone.0144561.ref061" target="_blank">61</a>]. This study suggests that cGMP may also be associated with a rise in total hemoglobin (Hb) and a decrease in leukocyte count. Solid lines represent molecular actions noted in the literature and dotted lines indicate those examined in this study.</p