Journal: Cardiovascular Research

Article Title: Human epicardial cell-conditioned medium contains HGF/IgG complexes that phosphorylate RYK and protect against vascular injury

doi: 10.1093/cvr/cvv168

Figure Lengend Snippet: HGF is a key vaso-protective factor in EPI CdM. (A) EPI CdM protected cultured primary human coronary artery endothelial cells for 48 h under simulated ischemia, as measured by MTS assay (n = 5 donors). Data are mean ± S.D. (B) EPI CdM protected cultured human microvascular endothelial cells for 48 h under simulated ischemia, as measured by CyQuant assay (n = 3 donors). Data are mean ± S.D. In both experiments, survival of cells in growth medium (GM) was considered as 100%. (C) Receptor tyrosine kinase (RTK) array demonstrates 30 min exposure to 1× EPI CdM induces phosphorylation of multiple growth factor receptors of coronary artery endothelial cells compared with MEM treatment (n = 2). (D) HGF ELISA data are for 5 different EPI CdM donors (D1–D5). Measurements are in duplicate. (E) Pull down (PD) of HGF from EPI CdM with an HGF-specific antibody (2 µg/ml) decreased EPI CdM-mediated protection of microvascular endothelial cells during simulated ischemia (48 h) compared to PD with a non-specific IgG antibody (*P ≤ 0.05 Control IgG vs. Anti-HGF). In contrast, PD of SDF-1, ANG1 or VEGFA from EPI CdM did not alter its protection of microvascular endothelial cells (all antibodies used at 2 µg/ml) (n = 3). Cell numbers were determined by CyQuant assay (dye binding of nucleic acids). Data are mean ± S.D. One way ANOVA for A,B, and E: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Scale bar = 100 µM.

Article Snippet: EPI CdM protects human cardiac endothelial cells during simulated ischaemia To investigate EPI CdM-mediated protection of cardiac endothelial cells, human cardiac endothelial cells were purchased from Lonza (Catalog # CC-2585 and CC-7030, Passage 1).

Techniques: Cell Culture, MTS Assay, CyQUANT Assay, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Control, Binding Assay

Journal: Cardiovascular Research

Article Title: Human epicardial cell-conditioned medium contains HGF/IgG complexes that phosphorylate RYK and protect against vascular injury

doi: 10.1093/cvr/cvv168

Figure Lengend Snippet: HGF/IgG complexes enhance vascular protection by interacting with RYK. (A) Stable producer cell lines and purification of bioactive recombinant human HGF. (Left, top) Phase contrast images of HEK 293 cells with genomic integration of pIRES-puro vector expressing human HGF-His tag-F2A-GFP. (Left, bottom) Epifluorescence image showing GFP expression (FITC channel). (Right) Gel electrophoresis illustrating purification of HGF. Lane 1, electrophoresis of eluted material under non-reducing conditions demonstrates purity and molecular weight of single chain pro-HGF (90 kDa). Gel stained with Coomassie Brilliant Blue. Lane 2, purified protein run under reducing conditions (beta-mercaptoethanol) demonstrates the active heterodimer of human HGF (see bands at 65 kDa and 32 kDa). (B) CyQuant assay demonstrates enhanced protective effects of HGF/IgG complexes when compared with free HGF (alone) or free HGF with IgG (Both free, unconcentrated factors). For greater detail, please refer to Methods. Cell number was assayed after 48 h of simulated ischemia (MEM, 69 ± 24.7% of HGF; free HGF alone, 100 ± 7.3%; free HGF with IgG [Free], 114 ± 3.1% of HGF; HGF with IgG [Complex], 169 ± 4.0% of HGF [n = 4 for all]). Free HGF with IgG (Free) conferred protection (P ≤ 0.01, vs. MEM). However, HGF/IgG complexes (Complex) protected better than did free HGF with IgG (Free) (P ≤ 0.001). (C) Treatment of coronary endothelial cells with HGF/IgG complexes (Complex, top), free HGF and IgG (Free, middle) or MEM (bottom) affects the level of phosphorylation for RYK, but not c-Met. Positive control signal (pos-ctrl) has been included for each membrane to compare for normalization (n = 2). (D) Blocking RYK reduced protection conferred by HGF/IgG complexes (P ≤ 0.01, n = 4) but not protection by free HGF with IgG (P = 0.09, n = 4). Note: coronary artery endothelial cells were incubated under simulated ischemia for 24 h. (E) Intra-arterial treatment with HGF/IgG complexes significantly decreased FITC-albumin extravasation in the LV wall of rats at 24 hr after reperfusion (MEM, n = 4; free HGF with IgG [Free], n = 8; HGF/IgG complexes [Complex], n = 11; P ≤ 0.01). (F and F′) Epifluorescent image showing localization of His-HGF outside of blood vessels (by anti-His antibody) in animals treated with free HGF (F) and complex HGF (F′). HGF/IgG complexes were localized to large blood vessels (F and F′) and the capillary network at the borders of infarcted regions (F″). We confirmed a similar staining pattern in hearts of 3 different animals. Note: the presence of HGF not localized to capillaries is likely indicative of leakage from damaged or dead blood vessels in the capillary bed. Data are mean ± S.D. One way ANOVA for B,D, and E: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Scale bar = 100 µM.

Article Snippet: EPI CdM protects human cardiac endothelial cells during simulated ischaemia To investigate EPI CdM-mediated protection of cardiac endothelial cells, human cardiac endothelial cells were purchased from Lonza (Catalog # CC-2585 and CC-7030, Passage 1).

Techniques: Purification, Recombinant, Plasmid Preparation, Expressing, Nucleic Acid Electrophoresis, Electrophoresis, Molecular Weight, Staining, CyQUANT Assay, Phospho-proteomics, Positive Control, Membrane, Blocking Assay, Incubation

Journal: Investigative Ophthalmology & Visual Science

Article Title: MicroRNA 9 Is a Regulator of Endothelial to Mesenchymal Transition in Diabetic Retinopathy

doi: 10.1167/iovs.64.7.13

Figure Lengend Snippet: High glucose promoted EndMT and suppressed miR-9 in vitro. Exposure to HG (25 mM for 48 hours) reduced the expressions of endothelial markers ( A ) PECAM1 and ( B ) CDH5 and increased the expressions of mesenchymal markers ( C ) TAGLN and ( D ) S100A4 in HRECs. HG also reduced expression of ( E ) miR-9. Exposure to high L-glucose (OSM, 25 mM for 48 hours) produced no significance compared to cells grown in NG (5 mM for 48 hours) (n = 6/group; data presented as ratio to β-actin mRNA or U6 snRNA and normalized to NG; * P < 0.05, n.s., not significant).

Article Snippet: Human retinal endothelial cells (HREC; Olaf Pharmaceuticals, Worcester, MA, USA) were cultured with endothelial cell growth basal medium-2 (Lonza Group, Basel, Switzerland) supplemented with microvascular endothelial growth medium-2 (Lonza Group), 10% v/v fetal-bovine serum, and 100 μg/mL penicillin/streptomycin in a humidified hood held at 37°C with 4% CO 2 .

Techniques: In Vitro, Expressing, Produced

Journal: Investigative Ophthalmology & Visual Science

Article Title: MicroRNA 9 Is a Regulator of Endothelial to Mesenchymal Transition in Diabetic Retinopathy

doi: 10.1167/iovs.64.7.13

Figure Lengend Snippet: MiR-9 mimics prevented high glucose-induced EndMT changes in vitro. HG (25 mM for 48 hours) significantly reduced mRNA expressions of endothelial markers ( A ) PECAM1 and ( B ) CDH5 and increased mRNA expressions of mesenchymal markers ( C ) TAGLN and ( D ) S100A4 in cells transfected with scrambled RNA (SCR). Protein expressions reflect a similar trend, expression of ( E ) PECAM1 was decreased and expression of ( F ) S100A4 was increased in SCR-HG compared with SCR cells cultured under NG (5 mM for 48 hours) conditions. MiR-9 mimic transfection (miR-9) prevented high glucose-induced EndMT changes at both mRNA and protein levels (n = 6/group for mRNA and n = 3/group for protein; data presented as ratio to β-actin mRNA/protein and normalized to NG; * P < 0.05).

Article Snippet: Human retinal endothelial cells (HREC; Olaf Pharmaceuticals, Worcester, MA, USA) were cultured with endothelial cell growth basal medium-2 (Lonza Group, Basel, Switzerland) supplemented with microvascular endothelial growth medium-2 (Lonza Group), 10% v/v fetal-bovine serum, and 100 μg/mL penicillin/streptomycin in a humidified hood held at 37°C with 4% CO 2 .

Techniques: In Vitro, Transfection, Expressing, Cell Culture

Journal: Investigative Ophthalmology & Visual Science

Article Title: MicroRNA 9 Is a Regulator of Endothelial to Mesenchymal Transition in Diabetic Retinopathy

doi: 10.1167/iovs.64.7.13

Figure Lengend Snippet: MiR-9 transgenic mice showed endothelial specific miR-9 overexpression and inhibition of EndMT. MiR-9 expression in ( A ) retinal ECs are considerably higher in M9 mice compared with WT mice, whereas miR-9 expression in ( B ) retinal non-ECs showed no significant difference between M9 and WT. ( C ) The endothelial marker CD31 ( green arrows ) showed strong fluorescence in the capillaries of all nondiabetic retinas, as well as in the capillaries of diabetic M9 retinas, and weak fluorescence in the WT diabetic retinas. The mesenchymal marker α-SMA ( orange arrow ) showed strong fluorescence throughout the retinas of diabetic WT mice, and little to no fluorescence in the capillaries of the other groups. There was considerable overlap between red and green fluorescence in diabetic WT retinas. (n = 6/group for miR-9 analysis and n = 4/group for imaging; miR-9 expression presented as ratio to U6 snRNA and normalized to WT; * P < 0.05, n.s., not significant; fluorescence images were uniformly adjusted to reduce background noise; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; white bar: 10 µm].

Article Snippet: Human retinal endothelial cells (HREC; Olaf Pharmaceuticals, Worcester, MA, USA) were cultured with endothelial cell growth basal medium-2 (Lonza Group, Basel, Switzerland) supplemented with microvascular endothelial growth medium-2 (Lonza Group), 10% v/v fetal-bovine serum, and 100 μg/mL penicillin/streptomycin in a humidified hood held at 37°C with 4% CO 2 .

Techniques: Transgenic Assay, Over Expression, Inhibition, Expressing, Marker, Fluorescence, Imaging

Journal: Investigative Ophthalmology & Visual Science

Article Title: MicroRNA 9 Is a Regulator of Endothelial to Mesenchymal Transition in Diabetic Retinopathy

doi: 10.1167/iovs.64.7.13

Figure Lengend Snippet: MiR-9 prevented diabetes-induced EndMT and diabetes-induced vascular leakage in mouse retinas. Streptozotocin-induced diabetes significantly reduced retinal mRNA expressions of endothelial markers ( A ) Pecam1 and ( B ) Cdh5 , and increased expressions of mesenchymal markers ( C ) Acta2 and ( D ) S100a4 in male WT mice. Such changes were prevented in diabetic M9 mice. Protein expressions of ( E ) Pecam1 and ( F ) Acta2 also followed the same pattern. ( G ) IgG staining was seen within the retinal capillaries of all mice ( arrow ). Diabetes caused leakage of IgG into the retinal layers of WT diabetic mice, resulting in intense (+++) staining throughout the retina when compared with the nondiabetic mice (+). Diabetic M9 mice showed minimal leakage of IgG into the retina and had staining intensity comparable to non-diabetic M9 mice (+). ND, nondiabetic; D, diabetic; M9, miR-9 transgenic (n = 6/group for mRNA, n = 3/group for protein, and n = 3/group for immunohistochemistry; molecular data normalized to WT-ND, mRNA data presented as ratio to β-actin mRNA, and protein data presented as ratio to total protein; * P < 0.05).

Article Snippet: Human retinal endothelial cells (HREC; Olaf Pharmaceuticals, Worcester, MA, USA) were cultured with endothelial cell growth basal medium-2 (Lonza Group, Basel, Switzerland) supplemented with microvascular endothelial growth medium-2 (Lonza Group), 10% v/v fetal-bovine serum, and 100 μg/mL penicillin/streptomycin in a humidified hood held at 37°C with 4% CO 2 .

Techniques: Staining, Transgenic Assay, Immunohistochemistry