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

Journal: Scientific Reports

Article Title: Endothelial glycocalyx shields the interaction of SARS-CoV-2 spike protein with ACE2 receptors

doi: 10.1038/s41598-021-91231-1

Figure Lengend Snippet: Differences in adhesive interactions between the S-proteins of SARS-CoV-2 and the surfaces of human bronchial epithelial cells (HBECs) and human pulmonary artery endothelial cells (HPAECs). ( a ) Histograms of the maximal detachment force F max for HBECs. Left: comparison of data for native and anti-ACE2 treated cells. Right: comparison of data for native and heparin treated system. ( b ) Comparison of mean values determined from histograms. ( c ) AFM height map measured for a single HBEC cell. ( d ) Corresponding adhesive maps measured for this cell. Left: native cell. Middle: anti-ACE2 treatment. Right: heparin treatment. ( e ) Fluorescent staining of ACE2 (green, left column), glycocalyx (Glx, red, middle column) and Merged (right column). ( f ) Quantitative data of ACE2 and Glx mean fluorescence intensity. ( g ) Histograms of the maximal detachment force F max for HPAECs. Left: comparison of data for native and anti-ACE2 treated cells. Right: comparison of data for native and heparin treated system. ( h ) Comparison of mean values determined from histograms. ( i ) AFM height map measured for a single HPAEC cell. ( j ) Corresponding adhesive maps measured for this cell. ( k ) Fluorescent staining of ACE2 (green, left column), Glx (red, middle column) and Merged (right column) ( l ) Quantitative data of ACE2 and Glx mean fluorescence intensity. Left: native cell. Middle: anti-ACE2 treatment. Right: heparin treatment. Statistics: p values were determined by one-way ANOVA followed by Tukey’s post-hoc test. Experimental details are listed in Supplementary Table . Source data are provided as a Source Data file. Figure created with OriginPro2021 ( https://www.originlab.com/2021 ), ImageJ 1.53e ( https://imagej.nih.gov/ij/ ) and JPK Data Processing 6.1.79 ( https://www.jpk.com/ ).

Article Snippet: Primary Human Pulmonary Artery Endothelial Cells (HPAECs, ATCC) were grown in Vascular Cell Basal Medium (Cat. No. PCS-100-030, ATCC), supplemented with Endothelial Cell Growth Kit-VEGF (Cat. No. PCS-100-041, ATCC) The cells were maintained in standard conditions at 37 °C, 5% CO 2 , and 95% humidity.

Techniques: Adhesive, Comparison, Staining, Fluorescence

Journal: Scientific Reports

Article Title: Endothelial glycocalyx shields the interaction of SARS-CoV-2 spike protein with ACE2 receptors

doi: 10.1038/s41598-021-91231-1

Figure Lengend Snippet: Endothelial cells stiffening after incubation with S-protein is more pronounced for cells with removed glycocalyx. ( a ) Elastic modulus of HPAECs for native cells (grey histogram) and for cells incubated with S-protein (magenta). ( b ) Mean fluorescence intensity of phalloidin (AlexaFluor488). ( c ) Examples of fluorescence images depict the actin structure in native HPAECs and after incubation with S-protein. Green—actin. Blue—nucleus. ( d ) Examples of AFM-QI images of native HPAECs and after incubation with S-protein. ( e ) Elastic modulus obtained for HPAECs pre-incubated with heparinase (green) and next incubated with S-protein (magenta). ( f ) Mean fluorescence intensity of phalloidin (AlexaFluor488) after removal of HS. ( g ) Fluorescence images show the actin polymerization that occurred after incubation with S-protein for HPAECs pre-incubated with heparinase. ( h ) Examples of AFM-QI images depict the changes of cell morphology and cortical actin network after incubation with S-protein for HPAECs pre-incubated with heparinase. Statistics: p values were determined by one-way ANOVA followed by Tukey’s post-hoc test. Figure created with OriginPro2021 ( https://www.originlab.com/2021 ), ImageJ 1.53e ( https://imagej.nih.gov/ij/ ) and JPK Data Processing 6.1.79 ( https://www.jpk.com/ ).

Article Snippet: Primary Human Pulmonary Artery Endothelial Cells (HPAECs, ATCC) were grown in Vascular Cell Basal Medium (Cat. No. PCS-100-030, ATCC), supplemented with Endothelial Cell Growth Kit-VEGF (Cat. No. PCS-100-041, ATCC) The cells were maintained in standard conditions at 37 °C, 5% CO 2 , and 95% humidity.

Techniques: Incubation, Fluorescence

Journal: American Journal of Physiology - Cell Physiology

Article Title: Pentastatin, a matrikine of the collagen IVα5, is a novel endogenous mediator of pulmonary endothelial dysfunction

doi: 10.1152/ajpcell.00391.2023

Figure Lengend Snippet: Pentastatin (PS) induces cell death in human pulmonary arterial endothelial cells (hPAECs). A : representative micrographs and quantification of hPAECs adhesion following treatment with veh or increasing concentration of PS (0.5–50 µg/mL) for 30 min. Scale bars: 50 µm. * P < 0.05; determined by one-way ANOVA for repeated measures followed by Tukey’s post hoc test; representative of n = 4 independent experiments from n = 4 different hPAECs. B : visualization of active caspase 3/7 in hPAECs after stimulation with either veh or PS (50 µg/mL) for 30 min. Scale bars: 20 µm. C – F : hPAECs were stimulated with veh or increasing concentration of PS (0.5–50 µg/mL) for 30 and 240 min. Active caspase 3/7 ( C and D ) and Annexin V/PI double staining ( E and F ) analyzed by flow cytometry. In D–G , * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001; determined by mixed-effect analysis of variance (split-plot ANOVA) with Dunnett’s post hoc test; in C and D , PS (50 µg/mL) vs. RP (50 µg/mL): ** P < 0.01 and **** P < 0.0001; determined by paired t test; n = 6 independent experiments from n = 4 different hPAECs. Error bars represent standard deviation in E and F . G : proliferation of hPAECs were assessed by 3 H-thymidine incorporation after 24 h stimulation of increasing concentration of PS (0.5–50 µg/mL). Thymidine counts were normalized to veh and presented as percentage (%). * P < 0.05; determined one-way ANOVA for repeated measures with Tukey’s post hoc test; n = 10 independent experiments from n = 7 different hPAECs. DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; AnxV/PI, Annexin V/propidium iodide.

Article Snippet: In addition, hPAECs were stimulated with VEGF (#293-VE, R&D Systems, Minnesota) and TGF-β (240-B, R&D Systems, Minnesota) at 70–80% confluency for 24 h.

Techniques: Concentration Assay, Double Staining, Flow Cytometry, Standard Deviation