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  • Enhanced wound healing, kinase and stem cell marker expression in diabetic organ-cultured human corneas upon MMP-10 and cathepsin F gene silencing. 24255036

    Diabetic corneas overexpress proteinases including matrix metalloproteinase-10 (M10) and cathepsin F (CF). Our purpose was to assess if silencing M10 and CF in organ-cultured diabetic corneas using recombinant adenovirus (rAV)-driven small hairpin RNA (rAV-sh) would normalize slow wound healing, and diabetic and stem cell marker expression.Sixteen pairs of organ-cultured autopsy human diabetic corneas (four per group) were treated with rAV-sh. Proteinase genes were silenced either separately, together, or both, in combination (Combo) with rAV-driven c-met gene overexpression. Fellow control corneas received rAV-EGFP. Quantitative RT-PCR confirmed small hairpin RNA (shRNA) silencing effect. Ten days after transfection, 5-mm epithelial wounds were made with n-heptanol and healing time recorded. Diabetic, signaling, and putative stem cell markers were studied by immunofluorescence of corneal cryostat sections.Proteinase silencing reduced epithelial wound healing time versus rAV-enhanced green fluorescent protein (EGFP) control (23% for rAV-shM10, 31% for rAV-shCF, and 36% for rAV-shM10 + rAV-shCF). Combo treatment was even more efficient (55% reduction). Staining patterns of diabetic markers (α₃β₁ integrin and nidogen-1), and of activated epidermal growth factor receptor and its signaling target activated Akt were normalized upon rAV-sh treatment. Combo treatment also restored normal staining for activated p38. All treatments, especially the combined ones, increased diabetes-altered staining for putative limbal stem cell markers, ΔNp63α, ABCG2, keratins 15 and 17, and laminin γ3 chain.Small hairpin RNA silencing of proteinases overexpressed in diabetic corneas enhanced corneal epithelial and stem cell marker staining and accelerated wound healing. Combined therapy with c-met overexpression was even more efficient. Specific corneal gene therapy has a potential for treating diabetic keratopathy.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • 5-Aza-2'-deoxycytidine sensitizes busulfan-resistant myeloid leukemia cells by regulating expression of genes involved in cell cycle checkpoint and apoptosis. 19732952

    Busulfan (Bu) is a DNA-alkylating drug used in myeloablative pretransplant conditioning therapy for patients with myeloid leukemia (ML). A major obstacle to successful treatment is cellular Bu-resistance. To investigate the possible contribution of DNA hypermethylation to Bu-resistance, we examined the cytotoxic activity of combined 5-aza-2'-deoxycytidine (DAC) and Bu. Exposure of Bu-resistant B5/Bu250(6) ML cells to 0.5muM DAC resulted in G2-arrest and apoptosis. The observed G2-arrest was associated with hypomethylation and subsequent expression of epigenetically controlled genes including p16(INK4A), activation of the p53 pathway, and phosphorylation of CDC2. The DAC-mediated apoptosis was partly due to hypomethylation and up-regulation of XAF1, which resulted in down-regulation of the anti-apoptotic proteins XIAP, cIAP1 and cIAP2. The pro-apoptotic PUMA and BNIP3 proteins were up-regulated while pro-survival STAT3 and c-MYC were suppressed. Combination of 0.05muM DAC and 5mug/ml Bu resulted in synergistic cytotoxicity, which was associated with PARP1 cleavage and activation of caspases 3 and 8, suggesting induction of an apoptotic response. P53 inhibition in B5/Bu250(6) cells using pifithrin-alpha alleviated these effects, suggesting a role for p53 therein; this observation was supported by the relative resistance of p53-null K562 cells to [DAC+Bu] combinations and by the effects of an anti-p53 shRNA on the OCI-AML3 cell line. We conclude that the synergistic effects of [DAC+Bu] are p53-dependent and involve cell cycle arrest, apoptosis induction and down-regulation of pro-survival genes. Our results suggest that, depending on tumor p53 status, incorporation of DAC might synergistically improve the cytoreductive efficacy of Bu-based pretransplant regimen in patients with ML.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Implication of lipoprotein associated phospholipase A2 activity on oxLDL uptake by macrophages. 20332422

    Recognition and uptake of oxLDL by scavenger receptors of macrophages and foam cell formation are mediated by the oxidatively modified ApoB and lipid moiety of oxLDL. A great amount of oxidized phosphatidylcholine (oxPC) of oxLDL is hydrolyzed at the sn-2 position by lipoprotein associated phospholipase A2 (Lp-PLA2) to lysophosphatidylcholine (lysoPC) and small oxidation products. This study examines the involvement of Lp-PLA2 in the uptake of oxLDL by mouse peritoneal macrophages. LDL with intact Lp-PLA2 activity [LDL (+)] and LDL with completely inhibited Lp-PLA2 activity [LDL (-)] were subjected to oxidation with 5muM CuSO4 for 6h (moderately oxidized LDL, MoxLDL), or 24h (heavily oxidized LDL, HoxLDL) and peritoneal macrophages were incubated with these preparations. The uptake of MoxLDL(-) was about 30% increased, compared to that of MoxLDL(+) while HoxLDL(-) uptake was about 20% increased, compared to that of HoxLDL(+). Inhibition of Lp-PLA2 activity had no effect on the uptake of ApoB-liposomes conjugates with ApoB isolated from MoxLDL(-), MoxLDL(+), HoxLDL(-) and HoxLDL(+). Liposomes prepared from the lipid extract of MoxLDL(-), MoxLDL(+), HoxLDL(-) and HoxLDL(+) exhibited a similar pattern to that observed in the uptake of the corresponding intact lipoproteins. This study suggests that the progressive inactivation of Lp-PLA2 during LDL oxidation leads to an increased uptake of oxLDL by macrophages, which could be primarily attributed to the increased uptake of the oxidized phospholipids enriched lipid moiety of oxLDL.
    Document Type:
    Reference
    Product Catalog Number:
    AB153