Journal: Biotechnology letters

Article Title: Ultrasensitive immuno-detection using viral nanoparticles with modular assembly using genetically-directed biotinylation

doi: 10.1007/s10529-014-1555-9

Figure Lengend Snippet: VEGF (26 aM - 2.6 pM) was spiked into 50% BAL fluid (in PBS), and incubated with polyclonal anti-VEGF antibody functionalized magnetic particles. The biotinylated antibody/Neutravidin/phage affinity reagent was added, followed by washing. The sample was then analyzed using real-time PCR with phage-specific primers (n=6, error bars = ±1 SD, Non-template control Ct ≥34). The No-VEGF control (Ct =22.67±0.55) is shown as a solid line with +/− one standard deviation represented by dotted lines.

Article Snippet: The particles were washed, resuspended in coating buffer (1×10 7 particles in 1 ml 0.1 M sodium borate, pH 9.5) and mixed with 240 μl polyclonal anti-human Vascular Endothelial Growth Factor (VEGF) antibody (1 mg/ml, AB-293-NA, R&D Systems) and 240 μl 3 M (NH 4 ) 2 SO 4 .

Techniques: Incubation, Real-time Polymerase Chain Reaction, Control, Standard Deviation

Journal: Stem Cells (Dayton, Ohio)

Article Title: Follistatin‐Like 3 Enhances the Function of Endothelial Cells Derived from Pluripotent Stem Cells by Facilitating β‐Catenin Nuclear Translocation Through Inhibition of Glycogen Synthase Kinase‐3β Activity

doi: 10.1002/stem.2820

Figure Lengend Snippet: Induced pluripotent stem cell differentiation toward endothelial cells. Human iPS Cells were differentiated using StemPro serum free media supplemented with BMP4, Activin A, fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF) for 5 days. The differentiated cells were seeded on collagen IV, while CD144 positive cells were magnetically sorted on day 6 using MicroBeads Kit (Miltenyi BIotec) and culturing in EGM‐2 media (LONZA). (A) : Images show morphology of iPS Cells (left panel) and of their differentiated EC counterparts (right panel) Scale bar: 50 μm. (B) : Real time polymerase chain reaction (PCR) data revealed significantly increased early and (C) late EC marker mRNA expression (Data is means ±SEM [ n = 3], *, p < .05; **, p < .01; ***, p < .001). (D) : Western blots showing protein expression of the EC‐specific marker CD144 in iPS‐ECs and (E) corresponding densitometry. (F) : Immunofluorescent confocal images showing that iPS‐ECs express typical EC markers, CD144, KDR, CD31, and vWF, also costained with DAPI (4′,6‐diamidino‐2‐phenylindole, dihydrochloride). (G) : iPS‐ECs stained positive for CD144, were able to uptake LDL and form tubes in vitro. Scale bar: 50 μm. The data presented are representative or means (±SEM) of three independent experiments. Abbreviations: DAPI, 4′,6‐diamidino‐2‐phenylindole; EC, endothelial cell; GAPDH, Glyceraldehyde 3‐phosphate dehydrogenase; iPS, induced pluripotent stem; LDL, low density lipoprotein.

Article Snippet: Antibodies against VE‐Cadherin (CD144) (ab33168 and STJ96234), VEGFR (ab9530), GAPDH (ab8245), OCT‐4 (ab19857 and STJ72238), KLF4 (ab72543) eNOS (ab76198), and FSTL3 (STJ112317) were purchased from Abcam, UK or St John's Laboratory, UK.

Techniques: Cell Differentiation, Real-time Polymerase Chain Reaction, Marker, Expressing, Western Blot, Staining, In Vitro

Journal: Stem Cells (Dayton, Ohio)

Article Title: Follistatin‐Like 3 Enhances the Function of Endothelial Cells Derived from Pluripotent Stem Cells by Facilitating β‐Catenin Nuclear Translocation Through Inhibition of Glycogen Synthase Kinase‐3β Activity

doi: 10.1002/stem.2820

Figure Lengend Snippet: FSTL3 is highly expressed in iPS‐ECs. (A) : FSTL3 was progressively induced during EC differentiation in iPS‐ECs at the mRNA level, both preselection (pre‐iPS‐ECs) and postselection with CD144 (iPS‐ECs) (Data is means ± SEM [ n = 3], *, p < .05). (B) : Western Blot showing FSTL3 protein being expressed in parallel with the EC‐specific marker CD144 and (C) corresponding densitometry. (D) : FSTL3 secretion was progressively induced in cell culture media, as shown by ELISA (Data is means ± SEM [ n = 3], *, p < .05). (E) : Immunofluorescent images of FSTL3 in iPS‐ECs, Scale bar: 50 µm. (F) : Immunofluorescent staining showing that FSTL3 is expressed in parallel with the EC‐specific marker CD144 in iPS‐ECs. Scale bar: 50 µm. The data presented are representative or means (±SEM) of three independent experiments. Abbreviations: DAPI, 4′,6‐diamidino‐2‐phenylindole; ECs, endothelial cells; FSTL3, follistatin‐like 3; iPS, induced pluripotent stem.

Article Snippet: Antibodies against VE‐Cadherin (CD144) (ab33168 and STJ96234), VEGFR (ab9530), GAPDH (ab8245), OCT‐4 (ab19857 and STJ72238), KLF4 (ab72543) eNOS (ab76198), and FSTL3 (STJ112317) were purchased from Abcam, UK or St John's Laboratory, UK.

Techniques: Western Blot, Marker, Cell Culture, Enzyme-linked Immunosorbent Assay, Staining

Journal: Stem Cells (Dayton, Ohio)

Article Title: Follistatin‐Like 3 Enhances the Function of Endothelial Cells Derived from Pluripotent Stem Cells by Facilitating β‐Catenin Nuclear Translocation Through Inhibition of Glycogen Synthase Kinase‐3β Activity

doi: 10.1002/stem.2820

Figure Lengend Snippet: FSTL3 regulates endothelial cell (EC) marker expression in induced pluripotent stem (iPS)‐ECs. iPS‐ECs were transfected with the EX‐FSTL3 plasmid carrying the red mCherry fluorophore, or an empty control vector plasmid (EX‐mCherry). Forty‐eight hours later, FSTL3 overexpression induced (A) mRNA and (B, C) protein expression of EC markers CD144, eNOS, and KDR (Data is means ± SEM [ n = 3], **, p < .01; ***, p < .001). FSTL3 knockdown by short hairpin RNA (shRNA) in iPS‐ECs resulted in suppression of EC markers at the (D) mRNA and (E, F) protein levels when the cells were harvested 72 hours later (Data is means ± SEM [ n = 3], ***, p < .001). (G) : Fluorescent microscope images of iPS‐ECs transfected with EX‐FSTL3 plasmid carrying the red mCherry fluorophore, or an empty control vector plasmid (EX‐mCherry). Scale bar: 50 µm. (H) : Treatment of the cells with 25–50 ng/ml of recombinant FSTL3 (R FSTL3) increased EC marker expression such as CD144, eNOS, and KDR compared to phosphate buffered saline (PBS) control (R CTL) (Data is means ± SEM [ n = 3], ***, p < .001). ELISA showing FSTL3 concentration in cell culture media after FSTL3 overexpression compared to control (Data is means ± SEM [ n = 3], **, p < .01). The data presented are representative or means (±SEM) of three independent experiments. Abbreviations: FSTL3, follistatin‐like 3; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase. (I) Enzyme‐linked immunosorbent assay (ELISA) showed increased concentration of FSTL3 in the media after overexpression with EX‐FSTL3 plasmid (Data is means ± SEM [n < 3], **, p < .01

Article Snippet: Antibodies against VE‐Cadherin (CD144) (ab33168 and STJ96234), VEGFR (ab9530), GAPDH (ab8245), OCT‐4 (ab19857 and STJ72238), KLF4 (ab72543) eNOS (ab76198), and FSTL3 (STJ112317) were purchased from Abcam, UK or St John's Laboratory, UK.

Techniques: Marker, Expressing, Transfection, Plasmid Preparation, Control, Over Expression, Knockdown, shRNA, Microscopy, Recombinant, Saline, Enzyme-linked Immunosorbent Assay, Concentration Assay, Cell Culture

Journal: Stem Cells (Dayton, Ohio)

Article Title: Follistatin‐Like 3 Enhances the Function of Endothelial Cells Derived from Pluripotent Stem Cells by Facilitating β‐Catenin Nuclear Translocation Through Inhibition of Glycogen Synthase Kinase‐3β Activity

doi: 10.1002/stem.2820

Figure Lengend Snippet: FSTL3 induced vascular tube formation in vivo and significantly improved angiogenesis, neovascularization, and blood flow recovery in the hind limb ischemic model. Human induced pluripotent stem (iPS)‐ECs overexpressing FSTL3 enhanced the formation of vascular‐like tubes in vivo. (A) : iPS‐ECs overexpressing FSTL3 (EX‐FSTL3), significantly induced angiogenesis, after subcutaneous injection in severe combined immunodeficiency (SCID) mice, in comparison to the control iPS‐ECs (PBS CTL) or control iPS‐ECs expressing mCherry (EX‐mCherry), as shown by H&E staining and (B) capillary density quantification (Data is means ± SEM [ n = 3], **, p < .01). Scale bar: 50 µm. (C) : phosphate buffered saline (PBS)‐CTL and iPS‐ECs overexpressing mCherry (EX‐mCherry) or FSTL3 (EX‐FSTL3) were injected intramuscularly in NOD.CB17‐Prkdcscid/NcrCrl SCID mice immediately after induction of hind limb ischemia. Laser Doppler images show blood flow (BF) in the lower limbs of mice in prone position on day 14. (D) : BF recovery in the ischemic foot (calculated as a percentage ratio between ischemic foot BF and the contralateral foot) for each of the conditions. Statistical analysis shows significantly higher BF recovery in the EX‐FSTL3 treated mice at 14 days in comparison to controls; (Data is means ± SEM [ n = 3], *, p < .05). (E) : Sections of adductor muscles were stained with CD144 antibody for each condition. (F) : Quantified capillary density of CD144‐stained cells, expressed as capillary number per mm 2 (Data is means ± SEM [ n = 3], *, p < .05). (G) Quantified capillary density of CD144‐ and mCherry‐stained cells (Data is means ± SEM; **, p < .01; quantification from 10 random microscopic fields at ×40, scale: 50 μm). The data presented are representative or means (±SEM) of three independent experiments. Abbreviations: DAPI, 4′,6‐diamidino‐2‐phenylindole; ECs, endothelial cells; FSTL3, follistatin‐like 3.

Article Snippet: Antibodies against VE‐Cadherin (CD144) (ab33168 and STJ96234), VEGFR (ab9530), GAPDH (ab8245), OCT‐4 (ab19857 and STJ72238), KLF4 (ab72543) eNOS (ab76198), and FSTL3 (STJ112317) were purchased from Abcam, UK or St John's Laboratory, UK.

Techniques: In Vivo, Injection, Comparison, Control, Expressing, Staining, Saline, Muscles

Journal: PLoS ONE

Article Title: The Effect of Vascular Endothelial Growth Factor on Osteoclastogenesis in Rheumatoid Arthritis

doi: 10.1371/journal.pone.0124909

Figure Lengend Snippet: (A) The synovial fluid samples of 32 RA patients were collected and their VEGF and soluble RANKL concentrations were determined by sandwich ELISA. (B) The serum samples of 32 RA patients were collected and their VEGF and soluble RANKL concentrations were determined by sandwich ELISA. Each dot expresses the results from an individual patient. (C) The synovial tissues of patients with RA and osteoarthritis (OA) were simultaneously labeled with anti-VEGF (green), anti-RANKL (red), and CD55 (white) antibodies and then photographed under appropriate filters. The merged image shows co-localization of the three markers (yellow). Sections were counterstained with DAPI staining. The figures are representative of three independent experiments (original magnification 400×).

Article Snippet: Anti-human VEGF, anti-human VEGFR1, Anti-human VEGFR2 and anti-CD55 antibodies were purchased from R&D Systems.

Techniques: Sandwich ELISA, Labeling, Staining

Journal: PLoS ONE

Article Title: The Effect of Vascular Endothelial Growth Factor on Osteoclastogenesis in Rheumatoid Arthritis

doi: 10.1371/journal.pone.0124909

Figure Lengend Snippet: (A) After RA synovial fibroblasts were cultured with 0–50 ng/ml of VEGF for 72 h, the RANKL mRNA expression determined by RT-PCR. Data were normalized to beta-actin and reported in relative expression units. The figure is representative of three experiments. (B) RA synovial fibroblasts were cultured with VEGF for 72 h, and RANKL concentration in the cultured media was measured by sandwich ELISA. (C) RA synovial fibroblasts were cultured with VEGF for 72 h and then stained with anti-RANKL antibodies (red) (original magnification 400×). The figures are representative of three independent experiments. (D) Triplicate wells of RA synovial fibroblasts were transfected with 1 μg of pGL3-RANKL reporter plasmids and 1 μg of pRLTk control plasmid. Both firefly and renilla luminescence were measured after 24 h incubation with 20ng/ml of VEGF. (E) After RA synovial fibroblasts were cultured with VEGF for 72 h, the concentrations of IL-1β, TNF-α, and IL-6 in the cultured media was determined by sandwich ELISA. The data represent the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01.

Article Snippet: Anti-human VEGF, anti-human VEGFR1, Anti-human VEGFR2 and anti-CD55 antibodies were purchased from R&D Systems.

Techniques: Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction, Concentration Assay, Sandwich ELISA, Staining, Transfection, Control, Plasmid Preparation, Incubation

Journal: PLoS ONE

Article Title: The Effect of Vascular Endothelial Growth Factor on Osteoclastogenesis in Rheumatoid Arthritis

doi: 10.1371/journal.pone.0124909

Figure Lengend Snippet: (A) RA synovial fibroblasts were pretreated with anti-VEGFR1 (20 ng/ml), anti-VEGFR2 (20 ng/ml), SB203580, a p38 MAPK inhibitor (10 nM), Src inhibitor (10 nM), or PKC inhibitor (5 nM) for 1 h, and then cultured with 20 ng/ml VEGF for 72 h. The expression of RANKL mRNA was determined by real time-PCR. Data were normalized to beta-actin and reported in relative expression units. (B) RA synovial fibroblasts were stimulated with 20 ng/ml VEGF, the phosphorylated forms of Src, PKC, and ERK were detected by western blotting. The figures are representative of three independent experiments. (C) Stimulation of RA synovial fibroblasts with VEGF activated the phosphorylation of p-Src, Src, p-PKC, PKC, p-ERK and ERK as detected by Western blotting and shown by the ratio of phosphorylated to total proteins. Data were normalized to beta-actin and reported in relative expression units. The figure represents one of three independent experiments. The data represent the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01.

Article Snippet: Anti-human VEGF, anti-human VEGFR1, Anti-human VEGFR2 and anti-CD55 antibodies were purchased from R&D Systems.

Techniques: Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Phospho-proteomics

Journal: PLoS ONE

Article Title: The Effect of Vascular Endothelial Growth Factor on Osteoclastogenesis in Rheumatoid Arthritis

doi: 10.1371/journal.pone.0124909

Figure Lengend Snippet: (A) CD14+ monocytes isolated from peripheral blood of RA patients were cultured with 25 ng/ml M-CSF and 0–50 ng/ml VEGF or 30 ng/ml RANKL. After maximal 21 days of culturing, TRAP-positive multinucleated cells were counted. The figures represent one of three independent experiments. (B) The gene expression of osteoclast markers such as TRAP, RANK, CTR, cathepsin K, and MMP-9 from differentiated osteoclasts measured by real-time PCR. Data were normalized to beta-actin and reported in relative expression units. The data represent the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01.

Article Snippet: Anti-human VEGF, anti-human VEGFR1, Anti-human VEGFR2 and anti-CD55 antibodies were purchased from R&D Systems.

Techniques: Isolation, Cell Culture, Gene Expression, Real-time Polymerase Chain Reaction, Expressing

Journal: PLoS ONE

Article Title: The Effect of Vascular Endothelial Growth Factor on Osteoclastogenesis in Rheumatoid Arthritis

doi: 10.1371/journal.pone.0124909

Figure Lengend Snippet: (A) CD14+ monocytes were cultured with M-CSF and 20 ng/ml of VEGF in the presence of 20ng/ml of anti-VEGFR1, 20ng/ml of anti-VEGFR2, or 10nM of p38 MAPK inhibitor. After 21 days of culturing, TRAP+ multinucleated cells were counted. The figure represents one of three independent experiments. (B) CD14+ monocytes were cultured with M-CSF and 20 ng/ml of VEGF in the presence of Src inhibitor (10 nM), or PKC inhibitor (5 nM). After 21 days of culturing, TRAP+ multinucleated cells were counted. The figure represents one of three independent experiments. (C) The gene expression of TRAP, RANK, CTR, cathepsin K, and MMP-9 from differentiated osteoclasts was measured by real-time PCR. Data were normalized to beta-actin and reported in relative expression units. The data represent the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01.

Article Snippet: Anti-human VEGF, anti-human VEGFR1, Anti-human VEGFR2 and anti-CD55 antibodies were purchased from R&D Systems.

Techniques: Cell Culture, Gene Expression, Real-time Polymerase Chain Reaction, Expressing

Journal: PLoS ONE

Article Title: The Effect of Vascular Endothelial Growth Factor on Osteoclastogenesis in Rheumatoid Arthritis

doi: 10.1371/journal.pone.0124909

Figure Lengend Snippet: (A) RA synovial fibroblasts were cultured with 20 ng/ml of VEGF for 72 h and RANKL production was quantified using ELISA in the cultured media. (B) RA synovial fibroblasts were preincubated with 20 ng/ml of VEGF for 72 h and Src inhibitor (10 nM) or PKC inhibitor (5 nM) and then cocultured with CD14+ monocytes from the peripheral blood in the presence of M-CSF. After 21 days of culturing, TRAP-positive multinucleated cells were counted. The figure represents one of three independent experiments. (B) The gene expressions of TRAP, RANK, CTR, cathepsin K, and MMP-9 from differentiated osteoclasts measured by real-time PCR. Data were normalized to beta-actin and reported in relative expression units. The data represent the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01.

Article Snippet: Anti-human VEGF, anti-human VEGFR1, Anti-human VEGFR2 and anti-CD55 antibodies were purchased from R&D Systems.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Expressing

Journal: PLoS ONE

Article Title: Attenuation of Choroidal Neovascularization by Histone Deacetylase Inhibitor

doi: 10.1371/journal.pone.0120587

Figure Lengend Snippet: Primer sequences used in real-time PCR.

Article Snippet: Bovine CECs (BCECs) or HUVECs grown in 6-well plates were starved for 4 h in endothelium basal medium (EBM, Lonza, Basel, Switzerland) with 1% FBS, and then treated with 0, 0.05, 0.1, 0.3, 0.5 or 0.7 μM TSA only for 24h or 48 h, or followed by stimulation with 20 ng/mL human recombinant VEGF (R&D Systems Inc Minneapolis, MN) for 10 min.

Techniques:

Journal: PLoS ONE

Article Title: Attenuation of Choroidal Neovascularization by Histone Deacetylase Inhibitor

doi: 10.1371/journal.pone.0120587

Figure Lengend Snippet: Antibodies used for RPE cells.

Article Snippet: Bovine CECs (BCECs) or HUVECs grown in 6-well plates were starved for 4 h in endothelium basal medium (EBM, Lonza, Basel, Switzerland) with 1% FBS, and then treated with 0, 0.05, 0.1, 0.3, 0.5 or 0.7 μM TSA only for 24h or 48 h, or followed by stimulation with 20 ng/mL human recombinant VEGF (R&D Systems Inc Minneapolis, MN) for 10 min.

Techniques:

Journal: PLoS ONE

Article Title: Attenuation of Choroidal Neovascularization by Histone Deacetylase Inhibitor

doi: 10.1371/journal.pone.0120587

Figure Lengend Snippet: Primer sequences used in ChIP assay.

Article Snippet: Bovine CECs (BCECs) or HUVECs grown in 6-well plates were starved for 4 h in endothelium basal medium (EBM, Lonza, Basel, Switzerland) with 1% FBS, and then treated with 0, 0.05, 0.1, 0.3, 0.5 or 0.7 μM TSA only for 24h or 48 h, or followed by stimulation with 20 ng/mL human recombinant VEGF (R&D Systems Inc Minneapolis, MN) for 10 min.

Techniques:

Journal: PLoS ONE

Article Title: Attenuation of Choroidal Neovascularization by Histone Deacetylase Inhibitor

doi: 10.1371/journal.pone.0120587

Figure Lengend Snippet: Real-time PCR (A) and Western blot assays (B) were performed on RPE cells treated with 0–0.5 μM TSA and 150 μM CoCl 2 . (A) Cells were treated with 0–0.5 μM TSA for 14 h and then co-treated with 150 μM CoCl 2 for 6 h for the analysis of gene expression by real-time PCR. Changes in HIF-1α mRNA levels were not statistically significant. CoCl 2 causes a fourfold enhancement of VEGF mRNA expression; but at 0.5 μM TSA, the mRNA level of VEGF reduces to less than half of that in cells treated with CoCl 2 only. TSA induces a statistically significant increase in the mRNA level of PEDF. (B) Cells were treated with 0–0.5 μM TSA for 18 h and then co-treated with 150 μM CoCl 2 for 6 h for Western blot analysis. TSA reduces the CoCl 2 -induced HIF-1α and VEGF protein levels by 4.3-fold and 5.7-fold, respectively, and up-regulates PEDF protein level by threefold. (C) Densitometry data for Western blot of HIF-1α, VEGF and PEDF. (*: t test p<0.05; **: t test p<0.01).

Article Snippet: Bovine CECs (BCECs) or HUVECs grown in 6-well plates were starved for 4 h in endothelium basal medium (EBM, Lonza, Basel, Switzerland) with 1% FBS, and then treated with 0, 0.05, 0.1, 0.3, 0.5 or 0.7 μM TSA only for 24h or 48 h, or followed by stimulation with 20 ng/mL human recombinant VEGF (R&D Systems Inc Minneapolis, MN) for 10 min.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Gene Expression, Expressing

Journal: PLoS ONE

Article Title: Attenuation of Choroidal Neovascularization by Histone Deacetylase Inhibitor

doi: 10.1371/journal.pone.0120587

Figure Lengend Snippet: After RPE cells were harvested, chromatin fixed with 1% formaldehyde and fragmented by sonication was immunoprecipitated with mouse IgG, anti-RNA polymerase II or anti-acetyl-histone H3 antibodies. Released chromatin was then amplified by PCR using primers targeting VEGF and PEDF encompassing the region from 200 bp upstream of the transcription start site to 200 bp downstream of the transcription start site. Amplified chromatin was then run on a 1% agarose gel. (A) For VEGF, less promoter opening was found in TSA and CoCl 2 -treated cells than in CoCl 2 -only-treated cells and untreated cells. For PEDF, more promoter opening was found in TSA and CoCl 2 -treated cells than in CoCl 2 -only-treated cells and untreated cells. (B) Densitometry of ChIP assay result normalized by input levels. (*: t test p<0.05).

Article Snippet: Bovine CECs (BCECs) or HUVECs grown in 6-well plates were starved for 4 h in endothelium basal medium (EBM, Lonza, Basel, Switzerland) with 1% FBS, and then treated with 0, 0.05, 0.1, 0.3, 0.5 or 0.7 μM TSA only for 24h or 48 h, or followed by stimulation with 20 ng/mL human recombinant VEGF (R&D Systems Inc Minneapolis, MN) for 10 min.

Techniques: Sonication, Immunoprecipitation, Amplification, Agarose Gel Electrophoresis

Journal: PLoS ONE

Article Title: Attenuation of Choroidal Neovascularization by Histone Deacetylase Inhibitor

doi: 10.1371/journal.pone.0120587

Figure Lengend Snippet: 1 × 10 4 of HUVECs treated with 0–1 μM TSA for 24 h were transferred in 150 μL of endothelium basal medium + 1% FBS with or without 25 ng/mL of human recombinant VEGF and the corresponding concentrations of TSA then incubated on 50 μL of Geltrex Reduced Growth Factor Basement Membrane Matrix gel in 96-well plates for 2 h. (A) Phase-contrast microscopy documented that tube formation induced by VEGF was inhibited by TSA in a dose-dependent manner. (B) Quantification of the amount of tube formation under different treatment conditions. (*: t test p<0.05) (bar = 200 μm).

Article Snippet: Bovine CECs (BCECs) or HUVECs grown in 6-well plates were starved for 4 h in endothelium basal medium (EBM, Lonza, Basel, Switzerland) with 1% FBS, and then treated with 0, 0.05, 0.1, 0.3, 0.5 or 0.7 μM TSA only for 24h or 48 h, or followed by stimulation with 20 ng/mL human recombinant VEGF (R&D Systems Inc Minneapolis, MN) for 10 min.

Techniques: Recombinant, Incubation, Membrane, Microscopy

Journal: PLoS ONE

Article Title: Attenuation of Choroidal Neovascularization by Histone Deacetylase Inhibitor

doi: 10.1371/journal.pone.0120587

Figure Lengend Snippet: Real-time PCR (A) and Western blot (B) were performed on BCECs (left panel) and HUVECs (right panel) treated with 0–0.7 μM TSA for 24h or 48 h. (A-B) TSA exerts a dose-dependent reduction effect on the mRNA and protein levels of VEGFR2. (C) Densitometry data for Western blot result of VEGFR2 normalized by GAPDH levels for BCECs (left panel) and HUVECs (right panel). (*: t test p<0.05, **: t test p<0.01) (D) HUVECs were treated with 0–0.7 μM TSA for 48 h, and then stimulated with 25 ng/mL of human recombinant VEGF for 10 min, followed by Western blot analysis (left panel). VEGF significantly induces the phosphorylation of VEGFR2, but the phosphorylation was attenuated by TSA at 0.7 μM, concomitant with a down-regulation of VEGFR2 total protein level. Densitometry data (right panel) for Western blot result of VEGFR2 and phospho-VEGFR2 normalized by GAPDH levels. (*: t test p<0.05) (E) ChIP assay was performed as described in on HUVECs treated with 0.5 μM TSA for 48 h. Released chromatin was then amplified by PCR using primers targeting VEGFR2 encompassing the region from 200 bp upstream of the transcription start site to 200 bp downstream of the transcription start site. Amplified chromatin was then run on a 1% agarose gel. Less promoter opening was found in TSA-treated cells than in untreated cells. Densitometry of ChIP assay result normalized by input levels. (*: t test p<0.05).

Article Snippet: Bovine CECs (BCECs) or HUVECs grown in 6-well plates were starved for 4 h in endothelium basal medium (EBM, Lonza, Basel, Switzerland) with 1% FBS, and then treated with 0, 0.05, 0.1, 0.3, 0.5 or 0.7 μM TSA only for 24h or 48 h, or followed by stimulation with 20 ng/mL human recombinant VEGF (R&D Systems Inc Minneapolis, MN) for 10 min.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Recombinant, Phospho-proteomics, Amplification, Agarose Gel Electrophoresis

Journal: PLoS ONE

Article Title: Attenuation of Choroidal Neovascularization by Histone Deacetylase Inhibitor

doi: 10.1371/journal.pone.0120587

Figure Lengend Snippet: Immunohistochemical staining was performed on murine retinal cryostat sections in CNV lesions day 7 post-laser for (A-B) VEGF, (C-D) VEGFR2 and (E-F) α-SMA. Figures on the left panel (A, C and E) are from PBS control mice, and figures on the right panel (B, D and F) are from TSA-treated mice. TSA reduced the amount of cells stained positively for (A) VEGF, (C) VEGFR2 and (E) α-SMA, when compared to PBS controls (B, D and F). (G) Quantification of positively stained area for each protein normalized by the size of the CNV lesions. (*: t test p<0.05; **: t test p<0.01; n = 4/group; bar = 50 μm).

Article Snippet: Bovine CECs (BCECs) or HUVECs grown in 6-well plates were starved for 4 h in endothelium basal medium (EBM, Lonza, Basel, Switzerland) with 1% FBS, and then treated with 0, 0.05, 0.1, 0.3, 0.5 or 0.7 μM TSA only for 24h or 48 h, or followed by stimulation with 20 ng/mL human recombinant VEGF (R&D Systems Inc Minneapolis, MN) for 10 min.

Techniques: Immunohistochemical staining, Staining, Control

Journal: Translational vision science & technology

Article Title: Malondialdehyde-Modified Photoreceptor Outer Segments Promote Choroidal Neovascularization in Mice.

doi: 10.1167/tvst.11.1.12

Figure Lengend Snippet: Figure 4. MDA-modified POS induced VEGF expression. (A) Real- time polymerase chain reaction showed that VEGF messenger RNA in ARPE-19 cells under regular culture conditions was elevated by POS treatment, while the difference in VEGF messenger RNA levels between the unmodified and MDA-modified POS groups was not significant. VEGF in RPE cells grown under not only regular culture conditions (B) but also polarized culture conditions (C) was also detected by enzyme e-linked immunosorbent assay. VEGF secreted into the medium was increased in both groups compared with the control group. Data are shown as the mean ± standard error of the mean. *P < 0.05, **P < 0.01 by the Kruskal–Wallis test with the post hoc Steel–Dwass test.

Article Snippet: Vascular endothelial growth factor (VEGF) levels in the cell culture medium were measured using a human VEGF ELISA kit (R&D Systems, Minneapolis,MN) following themanufacturer’s instructions.

Techniques: Modification, Expressing, Real-time Polymerase Chain Reaction, Control

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Effect of Ionizing Radiation Induced Damage of Endothelial Progenitor Cells in Vascular Regeneration

doi: 10.1161/atvbaha.111.237651

Figure Lengend Snippet: Figure 3. Stress response of mouse endothelial progenitor cells (EPCs) under radiation exposure. Mouse EPCs isolated from bone marrow mononuclear cells (BM-MNCs) were irradiated with 5 Gy of X-ray. A, -H2AX level was determined by immunofluorescence staining analysis. DAPI was used for nuclear counterstaining. B, Lin positive cells (Lin) and Lin negative (Lin) cells were stained with -H2AX and Sca1 antibodies. White arrow indicates Lin/Sca1 cell. C, The -H2AX positive cells after 3Gy of Ray at each condition (Lin/Sca1, Lin/Sca1-, or Lin/Sca1) were counted and their percentage is presented as a graph (left panel). D, mRNA level of p21Cip1 (left panel) and VEGF (right panel) of mEPCs at indicated times after irradiation were determined by qRT-PCR.

Article Snippet: CD34 cells were cultured in methylcellulose-containing medium SF H4236 (StemCell Technologies, Vancouver, Canada) with 100 ng/mL stem cell-derived factor (Kirin, Tokyo, Japan), 50 ng/mL 344 Arterioscler Thromb Vasc Biol February 2012 vascular endothelial growth factor (R&D Systems, Minneapolis, MN), 20 ng/mL interleukin-3 (Kirin), 50 ng/mL basic fibroblast growth factor (Wako, Osaka, Japan), 50 ng/mL epidermal growth factor receptor (Wako), 50 ng/mL insulin-like growth factor-1 (Wako), 2 U/mL heparin (Ajinomoto.

Techniques: Isolation, Irradiation, Staining, Quantitative RT-PCR

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Effect of Ionizing Radiation Induced Damage of Endothelial Progenitor Cells in Vascular Regeneration

doi: 10.1161/atvbaha.111.237651

Figure Lengend Snippet: Figure 4. Defective human cord blood endothelial progenitor cells (EPCs) under ionizing radiation (IR) (A) CD34 cells, isolated from human cord blood, were irradiated by 5 Gy of X-ray. Colony forming unit (CFU) of EPCs from CD34 cord blood stem cells with and without IR exposure was determined (right panel); the percentage of colony number formed during culture is represented as a graph (left panel). B, Proliferation rate of cultured human EPCs with and without IR exposure was determined by cell counting on the indi- cated day (NT: control, IR: 5 Gy of X-rays). C, The senescent cells (indicated by arrows) were identified by -galactosidase activity assay 4 days after IR. D, Tubule formation of EPCs with (5 Gy, 24 hours after IR) and without IR on the Matrigel (left panels) was determined by assessing the number of tubule junctions (top panel) and average tubule length (bottom panel); the percentage is rep- resented as a graph. E, Migration level of EPCs with (5 Gy, 24 hours after IR) and without IR was determined by measuring the re-en- dothelialized area after scratching (left panel). Percentage of re-endothelialized area is represented as a graph (right panel). F, Blood flow rate of hind-limb ischemic mice model was measured after cell transplantation with dPBS, control EPCs (NT_EPCs) or irradiated EPCs (IR_EPCs) respectively. ***P0.001 (left panel). Representative laser Doppler images of blood flow rate are displayed (right panel). G, Quantification of capillary density by staining with CD31, an endothelial marker, in ischemic tissues after cell transplantation with dPBS, control EPCs (NT_EPCs) or irradiated EPCs (IR_EPCs), respectively. **P0.01, ***P0.001. H, p53, p21, and -H2AX pro- tein level by IR was determined by immunoblotting analysis. ERK2 levels were used as the equal loading control (bottom panel). I, mRNA level of p21Cip1 (left panel) and VEGF (right panel) were determined by qRT-PCR analysis, and the fold change is presented as a graph.

Article Snippet: CD34 cells were cultured in methylcellulose-containing medium SF H4236 (StemCell Technologies, Vancouver, Canada) with 100 ng/mL stem cell-derived factor (Kirin, Tokyo, Japan), 50 ng/mL 344 Arterioscler Thromb Vasc Biol February 2012 vascular endothelial growth factor (R&D Systems, Minneapolis, MN), 20 ng/mL interleukin-3 (Kirin), 50 ng/mL basic fibroblast growth factor (Wako, Osaka, Japan), 50 ng/mL epidermal growth factor receptor (Wako), 50 ng/mL insulin-like growth factor-1 (Wako), 2 U/mL heparin (Ajinomoto.

Techniques: Isolation, Irradiation, Cell Culture, Cell Counting, Control, Activity Assay, Migration, Transplantation Assay, Staining, Marker, Western Blot, Quantitative RT-PCR

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Effect of Ionizing Radiation Induced Damage of Endothelial Progenitor Cells in Vascular Regeneration

doi: 10.1161/atvbaha.111.237651

Figure Lengend Snippet: Figure 5. Functional defects in ionizing radiation (IR) exposed endothelial progenitor cells (EPCs) is mediated by p53. Human EPCs (hEPCs) were transfected with control siRNA (siNC) or p53 siRNA (siTP53), and p53 knock-down was achieved 24 hours after siRNA introduction. A, p53 mRNA level of hEPCs with control siRNA (siNC) or p53 siRNA (siTP53) was determined by semiquantitative real- time PCR analysis (left panel) and RT-PCR (right panel). B, Proliferation rate of hEPCs after IR with control or p53 siRNA was deter- mined by cell counting. Fold change is presented as a graph. ***P0.001. C, -galactosidase positive cells (indicated by arrows) 3 days after IR with control or p53 siRNA was displayed. D, Tubule formation of EPCs 24 hours after IR with control or p53 siRNA on the Matrigel (left panels) was determined by counting the number of tubule junctions (top panel), and average tubule length (bottom panel); its percentage is represented as a graph. E, Migration level of EPCs 24 hours after IR with control or p53 siRNA was deter- mined by measuring the distance after scratching (left panel). Percentage of the re-endothelialized area is represented as a graph (right panel). F, p21Cip1 protein level (top panel), p53 protein level (middle panel) of hEPCs 6 hs after IR with control or p53 siRNA was determined by immunoblotting assay. ERK2 was used as the equal loading control. G, H, p21mRNA level and H, VEGF mRNA level were determined by semiquantitative real-time PCR analysis; the fold change is presented as a graph.

Article Snippet: CD34 cells were cultured in methylcellulose-containing medium SF H4236 (StemCell Technologies, Vancouver, Canada) with 100 ng/mL stem cell-derived factor (Kirin, Tokyo, Japan), 50 ng/mL 344 Arterioscler Thromb Vasc Biol February 2012 vascular endothelial growth factor (R&D Systems, Minneapolis, MN), 20 ng/mL interleukin-3 (Kirin), 50 ng/mL basic fibroblast growth factor (Wako, Osaka, Japan), 50 ng/mL epidermal growth factor receptor (Wako), 50 ng/mL insulin-like growth factor-1 (Wako), 2 U/mL heparin (Ajinomoto.

Techniques: Functional Assay, Transfection, Control, Knockdown, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Cell Counting, Migration, Western Blot