integrin β5 sirna  (Santa Cruz Biotechnology)

Journal: Critical Care

Article Title: Mechanical ventilation enhances extrapulmonary sepsis-induced lung injury: role of WISP1–αvβ5 integrin pathway in TLR4-mediated inflammation and injury

doi: 10.1186/s13054-018-2237-0

Figure Lengend Snippet: MTV enhances CLP-induced lung damage via TLR4-dependent WISP1–integrin β5 pathway. As shown in Additional file : Figure S1, mice lung tissue samples in eight mice groups were fixed and stained with hematoxylin–eosin for histological analysis ( a ) and lung injury score ( b ). Gross lung image in each group ( c ) and vascular permeability evaluated by Evans blue dye ( d ). Mice receiving combination of CLP + MTV (two-hit model) compared to mice subjected to CLP alone for 18 h or sham operation followed by 6 h of MTV. Two-hit model in wildtype mice compared to subgroup of TLR4 −/− mice or wildtype mice that received intratracheally neutralizing antibodies to either integrin β5 (β5 Ab) or WISP1 (WISP1 Ab) or a control antibody (IgG Ab) during mechanical ventilation. * P < 0.05; ** P < 0.01; *** P < 0.001. CLP cecal ligation and puncture, MTV moderate tidal ventilation, TLR4 toll-like receptor 4, WISP1 WNT1 inducible secreted protein

Article Snippet: Cells were treated with LPS and/or costimulated with recombinant WISP1 (rWISP1) and cytokine production was assessed in conditioned medium of wildtype cells or after transfection with 50 nM small interfering RNA (siRNA) for integrin β5 (sc-35681; Santa Cruz) or scrambled siRNA.

Techniques: Staining, Permeability, Control, Ligation

Journal: Critical Care

Article Title: Mechanical ventilation enhances extrapulmonary sepsis-induced lung injury: role of WISP1–αvβ5 integrin pathway in TLR4-mediated inflammation and injury

doi: 10.1186/s13054-018-2237-0

Figure Lengend Snippet: MTV enhances CLP-induced WISP1 and integrin β5 expression via TLR4-dependent pathway. WISP1 protein level ( a ) and integrin β5 expression ( b ) in lungs from each group of mice shown by western blot. MTV did not affect both levels and CLP led to small increases but two-hit model increased very significantly. Integrin β5 expression in lungs induced by CLP demonstrated in time-dependent fashion from wildtype mice (C57BL/6) but not TLR4 −/− mice ( c ). Corresponding actin identified for normalizing densitometry. * P < 0.05; ** P < 0.01; *** P < 0.001. CLP cecal ligation and puncture, MTV moderate tidal ventilation, TLR4 toll-like receptor 4, WISP1 WNT1 inducible secreted protein

Article Snippet: Cells were treated with LPS and/or costimulated with recombinant WISP1 (rWISP1) and cytokine production was assessed in conditioned medium of wildtype cells or after transfection with 50 nM small interfering RNA (siRNA) for integrin β5 (sc-35681; Santa Cruz) or scrambled siRNA.

Techniques: Expressing, Western Blot, Ligation

Journal: Critical Care

Article Title: Mechanical ventilation enhances extrapulmonary sepsis-induced lung injury: role of WISP1–αvβ5 integrin pathway in TLR4-mediated inflammation and injury

doi: 10.1186/s13054-018-2237-0

Figure Lengend Snippet: MTV increases circulating levels of cytokines and chemokines. Cytokines (TNF-α and IL-6) and chemokines (MIP-2 and MCP-1) in plasma detected by ELISA. Mice receiving combination of CLP + MTV (two-hit model) compared to mice subjected to CLP alone or sham operation followed by MTV. Two-hit model in wildtype mice compared to subgroup of TLR4 −/− mice (TLK4 KO) or wildtype (WT) mice that received intratracheally neutralizing antibodies to either integrin β5 (β5 Ab) or WISP1 (WISP1 Ab) or a control antibody (IgG) followed with CLP 12, 14, 16 and 18 h and MTV 0, 2, 4 and 6 h, respectively. CLP alone induced expected increase in circulating cytokines and chemokines but not induced by MTV alone. Two-hit model (CLP + MTV) increased cytokines and chemokines by MTV in time-dependent manner whereas deletion of TLR4 prevented these increases and inhibition of WISP1 or integrin β5 with neutralizing antibodies also blocked increases induced by MTV. * P < 0.05 compared with CLP alone at 16 h; ** P < 0.05 compared with two-hit WT at 16 h; *** P < 0.05 compared with two-hit WT; # P < 0.05 compared with CLP alone at 18 h; ## P < 0.05 compared with two-hit WT at 18 h; ### P < 0.05 compared with two-hit WT at 18 h. CLP cecal ligation and puncture, IL interleukin, MCP-1 monocyte chemoattractant protein-1, MIP-2 macrophage inflammatory protein-1, MTV moderate tidal ventilation, TLR4 toll-like receptor 4, TNF-α tumor necrosis factor alpha, WISP1 WNT1 inducible secreted protein

Article Snippet: Cells were treated with LPS and/or costimulated with recombinant WISP1 (rWISP1) and cytokine production was assessed in conditioned medium of wildtype cells or after transfection with 50 nM small interfering RNA (siRNA) for integrin β5 (sc-35681; Santa Cruz) or scrambled siRNA.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Control, Inhibition, Ligation

Journal: Critical Care

Article Title: Mechanical ventilation enhances extrapulmonary sepsis-induced lung injury: role of WISP1–αvβ5 integrin pathway in TLR4-mediated inflammation and injury

doi: 10.1186/s13054-018-2237-0

Figure Lengend Snippet: MTV increases PMN infiltration in lungs of septic mice. Neutrophil immigration in lung detected by flow cytometry. Mice receiving combination of CLP + MTV (two-hit model) compared to mice subjected to CLP alone for 18 h or sham operation followed by 6 h of MTV. Two-hit model in wildtype mice compared to subgroup of TLR4 null mice or wildtype mice that received intratracheally neutralizing antibodies to either integrin β5 (β5 Ab) or WISP1 (WISP1 Ab) (or control antibodies (IgG Ab)). Neutrophil influx by quantifying percent of CD11b + and Ly6G + cells in CD45 + cell population isolated from lung homogenates. CLP significantly increased percent of PMN while MTV had no impact on percent PMN in lung; CLP and MTV (two-hit model) significantly further increased neutrophil immigration in lung whereas TLR4 deletion completely prevented change in percentage of PMN induced by CLP + MTV compared to this two-hit model in wildtype mice. Blocking either WISP1 or integrin β5 partially prevented increase in percentage of PMN induced in two-hit model compared to combined CLP and MTV in mice receiving control IgG antibody. * P < 0.05; ** P < 0.01; *** P < 0.001. CLP cecal ligation and puncture, MTV moderate tidal ventilation, TLR4 toll-like receptor 4, WISP1 WNT1 inducible secreted protein

Article Snippet: Cells were treated with LPS and/or costimulated with recombinant WISP1 (rWISP1) and cytokine production was assessed in conditioned medium of wildtype cells or after transfection with 50 nM small interfering RNA (siRNA) for integrin β5 (sc-35681; Santa Cruz) or scrambled siRNA.

Techniques: Flow Cytometry, Control, Isolation, Blocking Assay, Ligation

Journal: Critical Care

Article Title: Mechanical ventilation enhances extrapulmonary sepsis-induced lung injury: role of WISP1–αvβ5 integrin pathway in TLR4-mediated inflammation and injury

doi: 10.1186/s13054-018-2237-0

Figure Lengend Snippet: WISP1-induced cytokine and chemokine production in LPS-primed peritoneal macrophages requires integrin β5. Peritoneal macrophages (PM) treated with LPS (0.1 μg/ml) followed by WISP1 (10 μg/ml) exposure at 4 h. Supernatants collected at 2 h intervals from 4 to 10 h following WISP1 stimulation. Cytokines (TNF-α and IL-6) and chemokines (MIP-2 and MCP-1) in supernatant detected by ELISA. PM receiving combination of LPS + WISP1 (L + W) from wildtype mice (WT) compared to PM from subgroup of either TLR4 −/− mice or wildtype mice receiving integrin β5 siRNA transfection (or control siRNA) subjected to LPS or WISP1. LPS-induced increases in IL-6, TNF-α, MIP-2 and MCP-1 evident within 4–10 h and addition of WISP1 induced further increases in all four mediators. Suppression of integrin β5 expression prevented WISP1-induced enhancement of LPS induction in four mediators. * P < 0.05 compared with LPS alone at 8 h; ** P < 0.05 compared with L + W WT at 8 h; *** P < 0.05 compared with L + W WT; # P < 0.05 compared with LPS alone at 10 h; ## P < 0.05 compared with L + W WT at 10 h; ### P < 0.05 compared with L + W WT at 10 h. Ctl, control, IL interleukin, KO knockout, LPS lipopolysaccharide, MCP-1 monocyte chemoattractant protein-1, MIP-2 macrophage inflammatory protein-1, PBS phosphate buffered saline, siRNA small interfering RNA, TNF-α tumor necrosis factor alpha, WISP1 WNT1 inducible secreted protein

Article Snippet: Cells were treated with LPS and/or costimulated with recombinant WISP1 (rWISP1) and cytokine production was assessed in conditioned medium of wildtype cells or after transfection with 50 nM small interfering RNA (siRNA) for integrin β5 (sc-35681; Santa Cruz) or scrambled siRNA.

Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Control, Expressing, Knock-Out, Saline, Small Interfering RNA