Journal: Circulation Research

Article Title: Interleukin-1β Causes Acute Lung Injury via αvβ5 and αvβ6 Integrin–Dependent Mechanisms

doi: 10.1161/circresaha.107.161067

Figure Lengend Snippet: Figure 8. Schematics of the effect IL-1 on the alveolar capillary barrier. Our model diagrams the IL-1 signaling pathway that leads to an increase in lung epithelial and endothelial permeability. IL-1 causes an increase in RhoA activity (a), leading to TGF- activation via the v6 integrin (b).26 TGF- induces an increase in per- meability (c)13 and an inhibition of the Na-driven fluid transport (d) in alveolar epithelial cells.12 Subsequent binding of TGF- on its receptor on endothelial cells induces an increase in lung endothelial permeability via a RhoA-dependent phos- phorylation of the VE-cadherin (f)31 and a formation of actin stress fibers (i). We fur- ther show that IL-1 activates RhoA (g) and increases lung endothelial permeabil- ity via the phosphorylation and endocyto- sis of -catenin (h) and v5 integrin– dependent stress fiber formation in lung endothelial cells (i).

Article Snippet: Mink lung epithelial cells stably transfected with a luciferase reporter plasmid under the control of the plasminogen activator inhibitor-1 promoter (TMLC cells7) were obtained from Daniel Rifkin (New York University, New York, NY) and used in co-culture with ATII cells to measure TGF-β activation as we have previously published.4 Bovine pulmonary arterial endothelial cells (BPAEC; ATCC, CCL-209; passages all < 8) were used for lung endothelial studies.2 Measurement of transepithelial and transendothelial albumin fluxes.

Techniques: Permeability, Activity Assay, Activation Assay, Inhibition, Binding Assay, Phospho-proteomics

Journal: American Journal of Physiology-Cell Physiology

Article Title: Inhibition of VRAC by c-Src tyrosine kinase targeted to caveolae is mediated by the Src homology domains

doi: 10.1152/ajpcell.2001.281.1.c248

Figure Lengend Snippet: Fig. 1. Inhibition of swelling-activated Cl2 current (ICl,swell) in calf pulmonary artery endothelial (CPAE) cells by c-Src Ser3Cys and c-Src Ser3Cys/Tyr527Phe is accompanied by a prolonged time for half-maximal activation (t1/2). A: control CPAE cells and CPAE cells transfected with, respectively, c-Src, c-Src Ser3Cys, c-Src Tyr527Phe, or c-Src Ser3Cys/Tyr527Phe, as indicated, were subjected to a 25% hypotonic solution (HTS), and ICl,swell was measured. Mean difference currents (maximal HTS-triggered current 2 basal current in isotonic medium) at 1100 mV are plotted for the different conditions. The mean current densities for the latter 3 conditions are significantly different from those in control and c-Src-transfected CPAE cells. The mean current density for CPAE cells transfected with c-Src Tyr527Phe also differs significantly from those in CPAE cells transfected with c-Src Ser3Cys or c-Src Ser3Cys/Tyr527Phe. B: t1/2 of ICl,swell at 1100 mV is plotted for the same 5 conditions. ICl,swell in CPAE cells transfected with c-Src Ser3Cys or c-Src Ser3Cys/Tyr527Phe mutants has a significantly prolonged t1/2 compared with control or c-Src-transfected CPAE cells. In addition, activation of ICl,swell in c-Src Ser3Cys/Tyr527Phe- transfected cells is significantly slower than in c-Src Tyr527Phe cells. Error bars correspond to SE.

Article Snippet: We used single endothelial cells from an established calf pulmonary artery cell line (CPAE; American Type Culture Collection CCL 209).

Techniques: Inhibition, Activation Assay, Control, Transfection

Journal: Human Molecular Genetics

Article Title: Dystrophin conferral using human endothelium expressing HLA-E in the non-immunosuppressive murine model of Duchenne muscular dystrophy

doi: 10.1093/hmg/ddq458

Figure Lengend Snippet: In vitro characterization of hPAE cells. ( A ) Macroscopic views showing an explant culture method of hPAE cells. hPAE cells were dissected from isolated placenta arterial vessels (indicated by arrowheads) in human placenta. ( B ) Photos showing morphology of hPAE cells by phase contrast microscopy at primary stages at passage I (left panel: PD 0 and right panel: PD 3). ( C ) Proliferative capacity of hPAE cells. The number of cells was counted with ViCell (Beckman Coulter) at each passage. The total number of PDs (PD level or accumulative PDs) was calculated, using the formula log 10 (total number of cells/starting number of cells)/log 10 2. ( D ) Flow cytometric profiles indicating expression of several cell surface markers on hPAE cells. ( E ) Scores of peak intensity, compared with isotype controls. ‘++': strongly positive (10 times and above that of the isotype control), ‘+': weakly positive (<10 times and twice and above that of the isotype control), ‘−': negative (less than twice that of the isotype control). ( F ) RT–PCR analysis for endothelial marker expression in hPAE cells at passage VI, IX and XX. The cells were cultured without any inductive stimuli. RNAs from HUVECs and H 2 O serve as positive (P) and negative (N) controls, respectively. ( G ) Immunocytochemical analyses of CD31 and vWF in hPAE cells. ( H ) Phase contrast micrograph of in vitro endothelial network formation of hPAE cells. hPAE cells were cultured on a basement membrane matrix gel. An ‘angiogenesis network' was formed 6 h after cultivation began.

Article Snippet: hPAE cells were implanted into the thigh muscle of 4- to 6-week-old BALB/c (Sankyo Labo Service Corporation, Hamamatsu, Japan) or mdx (C57BL/10ScSn-Dmdmdx/J, Jax Labs, Bar Harbor, ME, USA) mice.

Techniques: In Vitro, Isolation, Microscopy, Expressing, Control, Reverse Transcription Polymerase Chain Reaction, Marker, Cell Culture, Membrane

Journal: Human Molecular Genetics

Article Title: Dystrophin conferral using human endothelium expressing HLA-E in the non-immunosuppressive murine model of Duchenne muscular dystrophy

doi: 10.1093/hmg/ddq458

Figure Lengend Snippet: HLA-E mRNA and protein in hPAE cells upon treatment with tumor necrosis factor α (TNFα) and interferon γ (IFNγ). ( A ) RT–PCR showing a time-course of HLA-E expression in response to TNFα and IFNγ. 18S RNA was used as a loading control. M = size markers and N = a negative control in PCR with H 2 O. ( B ) Immunocytochemistry of HLA-E localization. The cells were incubated for 24 h with a combination of TNFα and IFNγ at the indicated concentrations (right). Left panel = untreated control. ( C ) Western blot analysis of cell lysates showing levels of HLA-E at 24 h after treatment with TNFα and IFNγ. Combination of two reagents induced more HLA-E at the protein level. Actin was used as a loading control. ( D ) Immunoprecipitation analysis of culture supernatants showing a soluble form of HLA-E (sHLA-E) with exposure to TNFα and IFNγ. sHLA-E level was determined by each signal intensity (mean ± SE). n = 3, * P < 0.05.

Article Snippet: hPAE cells were implanted into the thigh muscle of 4- to 6-week-old BALB/c (Sankyo Labo Service Corporation, Hamamatsu, Japan) or mdx (C57BL/10ScSn-Dmdmdx/J, Jax Labs, Bar Harbor, ME, USA) mice.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Negative Control, Immunocytochemistry, Incubation, Western Blot, Immunoprecipitation

Journal: Human Molecular Genetics

Article Title: Dystrophin conferral using human endothelium expressing HLA-E in the non-immunosuppressive murine model of Duchenne muscular dystrophy

doi: 10.1093/hmg/ddq458

Figure Lengend Snippet: Myogenic differentiation of hPAE cells under cell culture conditions. ( A ) Photos showing myogenic differentiation of hPAE cells detected by phase contrast microscopy (left) and by fluorescent microscopy (right) in an identical area. EGFP-labelled hPAE cells co-cultured with neonatal murine thymocytes for 21 days. ( B and C ) Immunocytochemistry of hPAE cells expressing myogenic markers, desmin (B) and skeletal myosin heavy chain (C, MY32). ( D ) Quantitative analysis of MY32-positive hPAE cells. MY32- and EGFP-double positive cells (no. of MY32+ EGFP+ cells) were counted in 35 mm dishes 3 weeks after induction (mean ± SE). n = 3, * P < 0.05. ( E ) RT–PCR showing myocyte-specific genes were expressed along with myogenic differentiation. RT–PCR analysis with PCR primers that amplify only human mRNAs of Myf5, myogenin, desmin and MyHC-IIx/d, but not murine mRNAs. RNAs from human muscle and H 2 O served as positive (P) and negative (N) controls, respectively.

Article Snippet: hPAE cells were implanted into the thigh muscle of 4- to 6-week-old BALB/c (Sankyo Labo Service Corporation, Hamamatsu, Japan) or mdx (C57BL/10ScSn-Dmdmdx/J, Jax Labs, Bar Harbor, ME, USA) mice.

Techniques: Cell Culture, Microscopy, Immunocytochemistry, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Human Molecular Genetics

Article Title: Dystrophin conferral using human endothelium expressing HLA-E in the non-immunosuppressive murine model of Duchenne muscular dystrophy

doi: 10.1093/hmg/ddq458

Figure Lengend Snippet: Implantation of hPAE cells into the thigh muscle of BALB/c mice. ( A ) Human periosteal cells (2 × 10 7 cells) were injected directly into the thigh muscles of BALB/c mice. Immunohistochemical analysis was performed on the muscle section using an antibody against vimentin. Upper panels: 2 days after injection and lower panels: 2 weeks after injection. ( B ) hPAE cells (2 × 10 7 cells) were injected directly into the thigh muscles of BALB/c mice. Upper panels: immunohistochemistry against vimentin. Lower panels: immunofluorescent analysis. DAPI (blue), vimentin (green), laminin (red) and MERGE (from left to right). ( C ) Immunohistochemical analysis of the thigh muscle sections at 2 days or 2 weeks after injection of human periosteal cells (hPeriosteal) and at 2 weeks after injection of hPAE cells, using antibodies against vimentin (upper panels: red and lower panels: green), leukocyte marker CD45 (green) and T cell marker CD3 (red). ( D ) Immunofluorescent analysis using an antibody against HLA-E (red) and human laminin (green) on the thigh muscle sections at 2 weeks after injection of hPAE cells. ( E ) Western blot analysis of muscle lysates showing levels of HLA-E, dystrophin and laminin. BALB/c mice were implanted with PBS or hPAE cells at the indicated weeks. The level of actin protein was used as a loading control. ( F ) Immunofluorescent analysis using an antibody against human dystrophin (green) on thigh muscle sections 3 weeks after direct injection of hPAE cells (middle and lower panels). PBS was injected into contralateral muscles as a control (upper panels). Dystrophin is totally absent in PBS-injected muscles (upper panels), whereas clusters of muscle fibres display peripheral localization of the dystrophin protein in mice injected with hPAE cells (middle and lower panels). Dystrophin (green), DAPI (blue) and MERGE (from left to right). ( G ) Immunofluorescent analysis using antibodies against laminin (green), human nuclei (HuNucl, red, arrows) and DAPI staining (blue, arrowheads) on thigh muscle sections 3 weeks after injection of hPAE cells.

Article Snippet: hPAE cells were implanted into the thigh muscle of 4- to 6-week-old BALB/c (Sankyo Labo Service Corporation, Hamamatsu, Japan) or mdx (C57BL/10ScSn-Dmdmdx/J, Jax Labs, Bar Harbor, ME, USA) mice.

Techniques: Injection, Muscles, Immunohistochemical staining, Immunohistochemistry, Marker, Western Blot, Control, Staining

Journal: Human Molecular Genetics

Article Title: Dystrophin conferral using human endothelium expressing HLA-E in the non-immunosuppressive murine model of Duchenne muscular dystrophy

doi: 10.1093/hmg/ddq458

Figure Lengend Snippet: Functional effect of HLA-E siRNA on immunosuppression. ( A ) Inhibition of HLA-E mRNA by siRNA. hPAE cells (1 × 10 4 ) grown on 6-well plates were transfected with either control siRNA or HLA-E-specific siRNA (20 μ m ) for 48 h. HLA-E mRNA levels were quantified using RT–PCR, normalized to β-actin (mean ± SE). n = 3, ** P < 0.01. ( B ) Inhibition of HLA-E protein by siRNA. Whole-cell protein extracts were analysed by SDS–PAGE immunoblotting with antibodies to HLA-E and actin. ( C – F ) siHLA-E-treated hPAE cells and control siRNA-treated hPAE cells were injected into the right and left thigh muscle of BALB/c mice, respectively. Mice were sacrificed 7 days after injection. (C) Injected sites are indicated by arrows (left: control siRNA and right: HLA-E-specific siRNA). (D) Microscopic view (HE stain and immunohistochemistry) of thigh muscles implanted with siHLA-E-treated (upper panels) or control siRNA-treated (lower panels) hPAE cells. (E and F) Immunohistochemical analysis of thigh muscle sections, after injection of siHLA-E-treated or control siRNA-treated hPAE cells and staining with antibodies against vimentin (E: red and F: green), leukocyte marker CD45 (E: green) and T cell marker CD3 (F: red). ( G ) Induction of xenoreactive lysis with spleen-derived lymphocytes. siHLA-E-treated hPAE cells or control siRNA-treated hPAE cells were co-cultured with spleen-derived lymphocytes and immunocytochemically stained for human vimentin. (G) Upper left: hPAE cells, upper right: siHLA-E-treated hPAE cells without any co-cultivation, lower left: control siRNA-treated hPAE cells co-cultured with primed lymphocytes, lower right: siHLA-E-treated hPAE cells co-cultured with primed lymphocytes. ( H ) Survival of hPAE cells after xenoreactive analysis. Vimentin-positive cells (no. of vimentin+ cells/mm 2 ) significantly decreased in siHLA-E-treated cells when compared with control siRNA-treated cells 3 days after co-incubation with primed lymphocytes. * P < 0.01, NS = not significant.

Article Snippet: hPAE cells were implanted into the thigh muscle of 4- to 6-week-old BALB/c (Sankyo Labo Service Corporation, Hamamatsu, Japan) or mdx (C57BL/10ScSn-Dmdmdx/J, Jax Labs, Bar Harbor, ME, USA) mice.

Techniques: Functional Assay, Inhibition, Transfection, Control, Reverse Transcription Polymerase Chain Reaction, SDS Page, Western Blot, Injection, H&E Stain, Immunohistochemistry, Muscles, Immunohistochemical staining, Staining, Marker, Lysis, Derivative Assay, Cell Culture, Incubation

Journal: Human Molecular Genetics

Article Title: Dystrophin conferral using human endothelium expressing HLA-E in the non-immunosuppressive murine model of Duchenne muscular dystrophy

doi: 10.1093/hmg/ddq458

Figure Lengend Snippet: Conferral of dystrophin to mdx myocytes by hPAE cells. ( A ) EGFP-labelled hPAE cells were injected into the thigh muscle of mdx mice. Immunohistochemical analysis revealed the incorporation of implanted cells into newly formed EGFP-positive myofibres (green), which expressed human dystrophin (red) 3 weeks after implantation. ( B ) PBS was injected into contralateral muscles as a control. ( C ) Quantitative analysis of human dystrophin-positive myotubes. The percentage of human EGFP- and dystrophin-positive myofibre areas (% double positive area) was calculated 3 weeks after injection of cells or PBS (mean ± SE). n = 3, * P = 0.05.

Article Snippet: hPAE cells were implanted into the thigh muscle of 4- to 6-week-old BALB/c (Sankyo Labo Service Corporation, Hamamatsu, Japan) or mdx (C57BL/10ScSn-Dmdmdx/J, Jax Labs, Bar Harbor, ME, USA) mice.

Techniques: Injection, Immunohistochemical staining, Muscles, Control