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: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Expression of CD70 Modulates Nitric Oxide and Redox Status in Endothelial Cells

doi: 10.1161/ATVBAHA.122.317866

Figure Lengend Snippet: CD70 knockdown reduced cellular NO levels and eNOS (endothelial nitric oxide synthase) expression. Real-time intracellular levels of NO in human aortic endothelial cells (HAECs) and human pulmonary artery endothelial cells (HPAECs) were measured following stimulation with 30 μM ATP. A and B , Show representative averaged time curves for baseline fluorescence, ATP treatment, and washout, with greater fluorescence change (%ΔF intensity ) correlating with greater NO levels. NO levels were reduced following siRNA-mediated CD70 (siCD70) knockdown compared with control-siRNA-treated cells (siCtrl) for HAECs and HPAECs ( C and G ). For HAECs, data represent the median of 89 cells for siCtrl and 129 cells for siCD70, collected over 3 independent experiments. For HPAECs, data represent the median of 105 cells for siCtrl and 106 cells for siCD70 collected over 4 independent experiments. Protein levels ( D and H ) mRNA ( E and I ) for eNOS were decreased after treatment with siCD70 compared with siCtrl. Protein expression of the eNOS chaperone Hsp90 (heat shock protein 90) was reduced with siCD70 treatment in HAECs and HPAECs ( F and J ). For eNOS and Hsp90, representative Western blots are shown along with corresponding densitometry, with values normalized to β-actin levels and expressed as fold change compared with siCtrl. For eNOS mRNA, data are normalized to β-actin mRNA and expressed as fold change compared with siCtrl. Data presented as mean±SE. **** P <0.0001.

Article Snippet: Primary human aortic endothelial cells (HAECs) and pulmonary artery endothelial cells (HPAECs) were purchased from Lonza (Walkersville, MD) and grown in EBM-2 media with EGM-2 supplements (Lonza) but without an antimicrobial additive.

Techniques: Expressing, Fluorescence, Western Blot

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Expression of CD70 Modulates Nitric Oxide and Redox Status in Endothelial Cells

doi: 10.1161/ATVBAHA.122.317866

Figure Lengend Snippet: 3-Nitrotyrosine and cellular superoxide are increased and cGMP levels are decreased after CD70 knockdown. Human aortic endothelial cells (HAECs) and human pulmonary artery endothelial cells (HPAECs) were treated with control (siCtrl) vs CD70-directed siRNA (siCD70). Immunoblotting for 3-nitrotyrosine adducts revealed two major species in endothelial cells: a higher molecular weight band corresponding to ≈ 45 kD and a lower molecular weight band corresponding to ≈ 20 kD ( A ). Both bands were increased in siCD70 cells as compared with siCtrl in HAECs ( B and C ) and HPAECs ( D and E ). Total intracellular cGMP levels were likewise decreased in siCD70 cells compared with siCtrl ( F and G ). CD70-knockdown HPAECs loaded with dihydroethidium (DHE) demonstrated increased basal fluorescence in the expected spectrum (excitation 488 nm, emission 588 nm) for 2-hydroethidium, the product of superoxide’s reaction with DHE ( H ). Cells loaded with DHE and then subsequently stimulated with menadione showed increased fluorescence in the setting of CD70 knockdown; furthermore, the reduction in fluorescence with PEG-SOD (pegylated superoxide dismutase) was greater in these cells compared with control cells, supporting the specificity of this increased fluorescence to enhanced superoxide levels. Nitrite/nitrate levels are unchanged in control vs CD70-knockdown cells ( J ). For 3-nitrotyrosine, representative Western blots are shown along with corresponding densitometry, with values normalized to β-actin levels and expressed as fold change compared with siCtrl. Data presented as mean±SE. **** P <0.0001.

Article Snippet: Primary human aortic endothelial cells (HAECs) and pulmonary artery endothelial cells (HPAECs) were purchased from Lonza (Walkersville, MD) and grown in EBM-2 media with EGM-2 supplements (Lonza) but without an antimicrobial additive.

Techniques: Western Blot, Molecular Weight, Fluorescence

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Expression of CD70 Modulates Nitric Oxide and Redox Status in Endothelial Cells

doi: 10.1161/ATVBAHA.122.317866

Figure Lengend Snippet: Reduced CD70 expression leads to increased cytosolic and caveolar hydrogen peroxide levels in endothelial cells. Human pulmonary artery endothelial cells (HPAECs) were treated with control (siCtrl) vs CD70-directed siRNA (siCD70) and subsequently transfected with the genetically encoded biosensor Hyper7.2 for real-time intracellular monitoring of hydrogen peroxide (H 2 O 2 ) in the cytosol ( A , B , and E ) and plasmalemmal caveolae ( C , D , and F ). Hydrogen peroxide was assessed by the ratio of fluorescence emission following excitation at 490 nm and 420 nm normalized to baseline fluorescence (normalized R/R 0 ). Following treatment with 1 μM auranofin, cells were treated with 25 μM H 2 O 2 as a positive control. The rate of rise of intracellular H 2 O 2 , calculated as the slope of the linear portion of the auranofin-treatment curve over the final 300 seconds of drug treatment (ΔF intensity /s), was greater in siCD70-treated cells compared with control cells for both cytosolic ( A and B ) and caveolar H 2 O 2 ( C and D ). Treatment with histamine similarly led to greater cytosolic ( E ) and caveolar H 2 O 2 ( F ) levels in CD70-knockdown cells compared with control. All panels were analyzed by unpaired Mann-Whitney U test. *** P <0.001, **** P <0.0001.

Article Snippet: Primary human aortic endothelial cells (HAECs) and pulmonary artery endothelial cells (HPAECs) were purchased from Lonza (Walkersville, MD) and grown in EBM-2 media with EGM-2 supplements (Lonza) but without an antimicrobial additive.

Techniques: Expressing, Transfection, Fluorescence, Positive Control, MANN-WHITNEY

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Expression of CD70 Modulates Nitric Oxide and Redox Status in Endothelial Cells

doi: 10.1161/ATVBAHA.122.317866

Figure Lengend Snippet: CD70 knockdown is associated with enhanced NOX (NADPH oxidase) expression. Following CD70 knockdown (siCD70), human pulmonary artery endothelial cells (HPAECs) demonstrated increased NOX (NADPH oxidase) 1 transcript ( A ) and protein ( B ) expression, and NOXA1 mRNA ( C ). Transcript levels of gp91phox, the catalytic subunit of NOX2, were enhanced ( D ), along with the corresponding protein levels of gp91phox ( E ). For NOX1 and gp91phox protein levels, representative Western blots are shown along with corresponding densitometry, with values normalized to β-actin levels and expressed as fold change compared with siCtrl. For NOX1 and NOXA1, mRNA levels are normalized to β-actin mRNA, and for gp91phox, mRNA levels are normalized to RNA polymerase II subunit A mRNA; data are expressed as fold change compared with siCtrl. Data presented as mean±SE.

Article Snippet: Primary human aortic endothelial cells (HAECs) and pulmonary artery endothelial cells (HPAECs) were purchased from Lonza (Walkersville, MD) and grown in EBM-2 media with EGM-2 supplements (Lonza) but without an antimicrobial additive.

Techniques: Expressing, Western Blot

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Expression of CD70 Modulates Nitric Oxide and Redox Status in Endothelial Cells

doi: 10.1161/ATVBAHA.122.317866

Figure Lengend Snippet: Reduction in CD70 alters endothelial anti-oxidant enzyme expression. In comparison to control-treated cells (siCtrl), CD70-knockdown cells (siCD70) showed enhanced SOD1 (superoxide dismutase 1) mRNA ( A and H ) and protein ( B and I ) levels in both human aortic endothelial cells (HAECs) and human pulmonary artery endothelial cells (HPAECs). Catalase protein expression was reduced in siCD70 samples ( C and J ); conversely, GPx-1 (glutathione peroxidase 1) mRNA ( D and K ) and protein levels ( E and L ) were elevated. Total intracellular GPx activity was augmented after treatment with siCD70 ( F and M ). Nrf2 (nuclear factor-erythroid factor 2-related factor 2) transcript levels were elevated in siCD70-treated cells ( G and N ). For SOD1, catalase and GPx-1 protein levels, representative Western blots are shown along with corresponding densitometry, with values normalized to β-actin levels and expressed as fold change compared with siCtrl. For SOD1, GPx-1, and Nrf2 mRNA levels, data are normalized to β-actin mRNA and expressed as fold change compared with siCtrl. Data presented as mean±SE.

Article Snippet: Primary human aortic endothelial cells (HAECs) and pulmonary artery endothelial cells (HPAECs) were purchased from Lonza (Walkersville, MD) and grown in EBM-2 media with EGM-2 supplements (Lonza) but without an antimicrobial additive.

Techniques: Expressing, Activity Assay, Western Blot

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Expression of CD70 Modulates Nitric Oxide and Redox Status in Endothelial Cells

doi: 10.1161/ATVBAHA.122.317866

Figure Lengend Snippet: CD70 knockdown leads to increased mitochondrial hydrogen peroxide levels. Following treatment with CD70 siRNA, human pulmonary artery endothelial cells (HPAECs) showed faster accumulation of hydrogen peroxide in the mitochondrial matrix in response to treatment with 1 mmol/L auranofin ( A and B ). SOD2 transcript ( C and E ) and protein ( D and F ) expression were correspondingly increased after CD70 knockdown compared with control cells in human aortic endothelial cells (HAECs) and HPAECs. Representative Western blots are shown for SOD2 along with corresponding densitometry, with values normalized to β-actin levels and expressed as fold change compared with siCtrl. For SOD2 mRNA, data are normalized to β-actin mRNA and expressed as fold change compared with siCtrl. Data presented as mean±SE. **** P <0.0001.

Article Snippet: Primary human aortic endothelial cells (HAECs) and pulmonary artery endothelial cells (HPAECs) were purchased from Lonza (Walkersville, MD) and grown in EBM-2 media with EGM-2 supplements (Lonza) but without an antimicrobial additive.

Techniques: Expressing, Western Blot