Human neutrophil elastase induces endothelial cell apoptosis by activating the PERK-CHOP branch of the unfolded protein response.

Human neutrophil elastase impacts on atherosclerotic plaque stability by inducing apoptosis in endothelial cells. Our aim was to investigate the proapoptotic mechanism of elastase on endothelial cells and to evaluate the presence of elastase in human plaque material. Human endothelial cells were treated with purified human neutrophil elastase. Apoptosis was assayed by capsase-3/7 activation, TUNEL, and sub-G1 assay. Activation of unfolded protein response (UPR) effector molecules binding Ig protein, soluble X-binding protein-1, protein kinase RNA-like ER kinase (PERK), and C/EBP-homologous protein (CHOP) was analyzed by RT-PCR, immunocytochemistry, and Western blot. Genetic silencing of CHOP was achieved by small interfering RNA. Elastase induces autophagic-apoptotic forms of endothelial cell death in a time- and dose-dependent manner, in conjunction with a significant increase in phosphorylation/expression of the canonical UPR-activation markers PERK and CHOP. By using CHOP knockdown, we identified CHOP as a key mediator of elastase-induced endothelial cell death. Immunohistochemical analysis of human rupture-prone plaque specimens confirmed the presence of elastase and colocalization with apoptosis. We have demonstrated for the first time that the PERK-CHOP branch of the UPR is causally involved in elastase-induced apoptosis of endothelial cells. Ex vivo analysis of human rupture-prone plaques confirmed the presence of elastase and its colocalization with markers of apoptosis. This novel role of elastase underlines the potential of combined targeting of elastase and endoplasmic reticulum stress in the prevention of plaque progression and cardiovascular events.-Grechowa, I., Horke, S., Wallrath, A., Vahl, C.-F., Dorweiler, B. Human neutrophil elastase induces endothelial cell apoptosis by activating the PERK-CHOP branch of the unfolded protein response.

Synthetic (glyco-)peptides of the homophilic recognition domain of E-cadherin lead to increased E-cadherin mRNA synthesis and are inductors of cell differentiation in primary lung cancer cell lines.

E-cadherin is one of the critical molecules involved in the metastatic process in many types of cancer. Once combined, E-cadherin exceeds the amount of membranous E-cadherin on the cellular surface by activation of intracellular signaling cascades. Studies on transformed keratinocytes of the HaCat cell line showed induction of differentiation by synthetical partial structures of the homophilic binding region of E-cadherin. The knowledge of effects in lung cancer cells is sparse. Therefore, the effects in primary lung cancer cell lines were investigated. Four primary lung cancer cell lines were incubated for 3, 6, 12, 15, 18, and 24h with synthetic partial structures (peptide and glycopeptide). The control substance was sodium butyrate. mRNA was isolated, and relative quantification of E-cadherin was performed using the Real-Time PCR. During the stimulation period, morphologic pictures were taken, and immunohistochemical staining of membranous E-cadherin was performed. Life/dead assays were used to display cell vitality. The intracellular E-cadherin mRNA amount was increased after incubation with the synthetic partial structures. Life/dead assays showed improved survival and integrated cell/cell bindings after stimulation with the partial structures. Increased cell mortality was revealed after sodium butyrate incubation. An effect mediated via E-cadherin on the cellular surface is proposed. The two synthetic partial structures of the homophilic binding region of E-cadherin increased the intracellular E-cadherin mRNA amount, cell-cell bindings, and survival of the tumor cells. Extracellular binding by synthetic partial structures to the binding region may have a beneficial influence on tumor progression in the metastatic process.

Ultrathin polymer monolayers for promotion of cell growth on bioprosthetic materials -- evolution of a new concept to improve long term performance of biologic heart vales.

Reoperation of aldehyde tanned bioprotheses due to calcific degeneration remains their major drawback. Based on experiments studying mechanisms and factors that influence the time phase, extent and progression of calcification and evaluating the efficiency of anticalcification treatments and the effects of surface seeding with vital cells a new concept to avoid calcification emerged: masking aldehyde-residues with a covalently bound polymer that supports surface cell seeding. Different covalently bound polymers were tested for their suitability to grow cells. Dense cell growth was achieved on some polymers but without correlation to physico-chemical properties. Ultrathin coating of biological materials appears a promising approach to achieve lining with vital cells.