anti vascular endothelial growth factor vegf a Search Results


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  • 99
    Thermo Fisher vascular endothelial growth factor vegf
    Vascular Endothelial Growth Factor Vegf, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 548 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti vegf
    Anti Vegf, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 144 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology vegf a
    Elevated TGF ‐β levels causally relate with reduced <t>PDGF</t> ‐Rα expression in nCLD patients and mice undergoing MV ‐O 2 , reducing downstream signaling and migration in pulmonary myofibroblasts Representative immunofluorescence images showing reduced expression of PDGF‐Rα (red) in the lungs of patients ( n = 7) developing nCLD (lower left panel, red stain; white arrows) together with increased pSMAD‐2 expression (lower middle panel, green stain; white arrows) as compared to lung sections from a non‐nCLD patient ( n = 1) (upper panel) (200×). Negative correlation between PDGF‐Rα and TGF‐β1 in transcriptome analysis 72 h after birth in preterms with nCLD ( n = 11) in contrast to non‐nCLD ( n = 9); z test of the difference of the Fisher's z transformed correlations divided by the standard error of the difference ( P = 0.048); scatter plots log2‐gene expression; linear regression (blue), with 95% CI (gray). MV‐O 2 reduced lung PDGF‐Rα (main: red stain; white arrows) and increased pSMAD‐2 levels (insert: red stain; white arrows) in ventilated neonatal WT (lower panel) when compared to control WT mice (upper panel); ( n = 4 mice/group; 10 images/mouse; 200×). Luciferase assay of CCL‐206 cells transfected with pGL4.14 containing PDGF‐Rα promoter revealing reduced promoter activity upon TGF‐β application (normalized to control) ( n = 3 experiments). Immunoblot analysis showing reduced PDGF‐Rα (E), <t>VEGF‐A</t> (F), and pERK/EKR (G) protein levels upon TGF‐β application alone in primary pulmonary myofibroblasts from 5–7‐day‐old WT mice ( n = 6–9 mice/group). Reduced migration of myofibroblasts (MFBs) from neonatal WT mice upon TGF‐β application alone ( n = 5 mice/group, 3 technical replicates). Translation of the results in fibroblasts isolated from tracheal aspirates of ventilated preterm infants (hMFBs) displayed reduced PDGF‐Rα levels (I) and migration assessed by Boyden chamber assay (J) upon TGF‐β application ( n = 3–5 patients/group). Representative phase contrast images (100×) of scratch migration assays in human lung fibroblasts (hMFBs) after 48 h of TGF‐β incubation indicating decreased wound closure (K) quantified by reduced velocity (L) and distance travelled (M) ( n = 3 patients/group). Data information: In (D–J) and (L, M), data are presented as mean ± SD and normalized to control. Statistical test used is two‐tailed unpaired Student's t ‐test or Mann–Whitney test ( P = 0.0002–0.039). *** P
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    vegf  (Abcam)
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    Abcam vegf
    (A) Immunohistochemical staining of <t>VEGF-A</t> (magnification, ×400) in fractures. (a) In PTHKO and (b) WT mice 1 week after fracture, VEGF-A expression was significantly reduced in PTHKO mice compared with in WT mice. (c) In PTHKO and (d) WT mice 2 weeks after fracture, VEGF-A expression was increased in PTHKO mice, but remained significantly lower than that in WT mice. (B) Immunohistochemical staining of <t>pVEGFR2</t> (magnification, ×400) in fractures. (a) In PTHKO and (b) WT mice 1 week after fracture, a significantly smaller number of pVEGFR2-positive cells was detected in the cartilaginous callus in PTHKO mice compared with in WT mice. (c) In PTHKO and (d) WT mice 2 weeks after fracture, a large number of pVEGFR2-positive cells was observed in the cartilaginous callus in WT mice, whereas a much lower level of angiogenesis was detected in PTHKO mice. (C) Immunohistochemical staining for HIF1α (magnification, ×400) in fractures. (a) In PTHKO and (b) WT mice 1 week after fracture, the expression levels of cytoplasmic HIF1α were significantly lower in PTHKO mice. (c) In PTHKO and (d) WT mice 2 weeks after fracture, HIF1α expression was increased in both groups; however, the expression remained lower in PTHKO mice compared with in WT mice. Black arrows indicate positive areas. (D) Protein expression levels of VEGF, pVEGFR2 and HIF1α were detected by western blot analysis. HIF1α, hypoxia inducible factor-1α; PTH, parathroid hormone; PTHKO, PTH knockout; pVEGFR, phosphorylated-VEGF receptor 2; VEGF, vascular endothelial growth factor; WT, wild-type.
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    R&D Systems mouse vegf r3 flt 4 antibody
    (A) Immunohistochemical staining of <t>VEGF-A</t> (magnification, ×400) in fractures. (a) In PTHKO and (b) WT mice 1 week after fracture, VEGF-A expression was significantly reduced in PTHKO mice compared with in WT mice. (c) In PTHKO and (d) WT mice 2 weeks after fracture, VEGF-A expression was increased in PTHKO mice, but remained significantly lower than that in WT mice. (B) Immunohistochemical staining of <t>pVEGFR2</t> (magnification, ×400) in fractures. (a) In PTHKO and (b) WT mice 1 week after fracture, a significantly smaller number of pVEGFR2-positive cells was detected in the cartilaginous callus in PTHKO mice compared with in WT mice. (c) In PTHKO and (d) WT mice 2 weeks after fracture, a large number of pVEGFR2-positive cells was observed in the cartilaginous callus in WT mice, whereas a much lower level of angiogenesis was detected in PTHKO mice. (C) Immunohistochemical staining for HIF1α (magnification, ×400) in fractures. (a) In PTHKO and (b) WT mice 1 week after fracture, the expression levels of cytoplasmic HIF1α were significantly lower in PTHKO mice. (c) In PTHKO and (d) WT mice 2 weeks after fracture, HIF1α expression was increased in both groups; however, the expression remained lower in PTHKO mice compared with in WT mice. Black arrows indicate positive areas. (D) Protein expression levels of VEGF, pVEGFR2 and HIF1α were detected by western blot analysis. HIF1α, hypoxia inducible factor-1α; PTH, parathroid hormone; PTHKO, PTH knockout; pVEGFR, phosphorylated-VEGF receptor 2; VEGF, vascular endothelial growth factor; WT, wild-type.
    Mouse Vegf R3 Flt 4 Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 95/100, based on 452 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology rabbit anti vegf a
    Fluorescence images of <t>VEGF</t> IR in detrusor smooth muscle in the bladder neck region in whole-mount preparations of urinary bladder in control rats ( A and B ) and rats treated with CYP for 4 h ( C and D ) and 48 h ( E and F ). VEGF IR in the detrusor smooth
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    Abcam anti vegf antibody
    Fluorescence images of <t>VEGF</t> IR in detrusor smooth muscle in the bladder neck region in whole-mount preparations of urinary bladder in control rats ( A and B ) and rats treated with CYP for 4 h ( C and D ) and 48 h ( E and F ). VEGF IR in the detrusor smooth
    Anti Vegf Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 450 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Genentech anti vegf antibody
    Long-term effects of <t>intravitreally</t> injected <t>anti-VEGF</t> antibody on BAT. (A) Quantitative analyses of the number of large lipid droplets ( > 50 μm 2 ) per field at x400 magnification ( n = 3–6). The effects of anti-VEGF antibody were quantitatively analyzed by comparison to the group treated with intravitreal PBS injection as 100%. (B) Quantitative analyses of vascularity of interscapular BAT demonstrated by isolectin B4 staining ( n = 3–6). The effects of anti-VEGF antibody were quantitatively analyzed by comparison to the group treated with intravitreal PBS injection as 100%. (C) The changes in body weight from P14 to P56. Anti-VEGF, anti-VEGF antibody. NS , not significant (two-tailed, unpaired T-test).
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    Long-term effects of <t>intravitreally</t> injected <t>anti-VEGF</t> antibody on BAT. (A) Quantitative analyses of the number of large lipid droplets ( > 50 μm 2 ) per field at x400 magnification ( n = 3–6). The effects of anti-VEGF antibody were quantitatively analyzed by comparison to the group treated with intravitreal PBS injection as 100%. (B) Quantitative analyses of vascularity of interscapular BAT demonstrated by isolectin B4 staining ( n = 3–6). The effects of anti-VEGF antibody were quantitatively analyzed by comparison to the group treated with intravitreal PBS injection as 100%. (C) The changes in body weight from P14 to P56. Anti-VEGF, anti-VEGF antibody. NS , not significant (two-tailed, unpaired T-test).
    Anti Vegf Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 95/100, based on 185 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems vegf
    Long-term effects of <t>intravitreally</t> injected <t>anti-VEGF</t> antibody on BAT. (A) Quantitative analyses of the number of large lipid droplets ( > 50 μm 2 ) per field at x400 magnification ( n = 3–6). The effects of anti-VEGF antibody were quantitatively analyzed by comparison to the group treated with intravitreal PBS injection as 100%. (B) Quantitative analyses of vascularity of interscapular BAT demonstrated by isolectin B4 staining ( n = 3–6). The effects of anti-VEGF antibody were quantitatively analyzed by comparison to the group treated with intravitreal PBS injection as 100%. (C) The changes in body weight from P14 to P56. Anti-VEGF, anti-VEGF antibody. NS , not significant (two-tailed, unpaired T-test).
    Vegf, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 4694 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam rabbit anti vegf
    Long-term effects of <t>intravitreally</t> injected <t>anti-VEGF</t> antibody on BAT. (A) Quantitative analyses of the number of large lipid droplets ( > 50 μm 2 ) per field at x400 magnification ( n = 3–6). The effects of anti-VEGF antibody were quantitatively analyzed by comparison to the group treated with intravitreal PBS injection as 100%. (B) Quantitative analyses of vascularity of interscapular BAT demonstrated by isolectin B4 staining ( n = 3–6). The effects of anti-VEGF antibody were quantitatively analyzed by comparison to the group treated with intravitreal PBS injection as 100%. (C) The changes in body weight from P14 to P56. Anti-VEGF, anti-VEGF antibody. NS , not significant (two-tailed, unpaired T-test).
    Rabbit Anti Vegf, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 315 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam antibodies against vegf a
    <t>VEGF-A</t> expression is activated in the retinas of OIR mice. (A) Immunoblot analysis of the protein expression levels of VEGF-A and HIF-1α in the retinas. (B) Quantification revealed an increase in the expression levels of VEGF-A and HIF-1α in the retinas of the OIR mice compared with WT mice. The relative protein expression level was normalized to GAPDH (n=3 mice per group). Data are presented as the mean ± standard deviation of the mean. *P
    Antibodies Against Vegf A, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems mouse vegf r2 kdr flk 1 antibody
    <t>VEGF-A</t> expression is activated in the retinas of OIR mice. (A) Immunoblot analysis of the protein expression levels of VEGF-A and HIF-1α in the retinas. (B) Quantification revealed an increase in the expression levels of VEGF-A and HIF-1α in the retinas of the OIR mice compared with WT mice. The relative protein expression level was normalized to GAPDH (n=3 mice per group). Data are presented as the mean ± standard deviation of the mean. *P
    Mouse Vegf R2 Kdr Flk 1 Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 294 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology rabbit polyclonal anti vegf a
    <t>VEGF-A</t> expression is activated in the retinas of OIR mice. (A) Immunoblot analysis of the protein expression levels of VEGF-A and HIF-1α in the retinas. (B) Quantification revealed an increase in the expression levels of VEGF-A and HIF-1α in the retinas of the OIR mice compared with WT mice. The relative protein expression level was normalized to GAPDH (n=3 mice per group). Data are presented as the mean ± standard deviation of the mean. *P
    Rabbit Polyclonal Anti Vegf A, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 89/100, based on 71 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems anti vegf
    Schematic illustration of the experimental procedure that was followed. (1) sutures were placed intrastromally into the temporal cornea, and immediately followed by topical application of eye drops (IgG, anti- <t>Vegf</t> or dexamethasone). Eye drops were applied until the 48 h time point. At t=48 h, IVCM and slit lamp data was collected and used for phenotypic characterisation. (2) cornea tissue was harvested and used for RNA extraction, and RNA quality verified. (3) high quality RNA was used for target preparation for microarray hybridisation on to GeneChip Rat 230 2.0 microarray chips. The microarray chips were scanned and image files acquired. (4) CEL files were normalised using expression console software. The generated CHP together with the CEL files were submitted to Gene Expression Omnibus repository.
    Anti Vegf, supplied by R&D Systems, used in various techniques. Bioz Stars score: 95/100, based on 235 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore vegf
    Schematic illustration of the experimental procedure that was followed. (1) sutures were placed intrastromally into the temporal cornea, and immediately followed by topical application of eye drops (IgG, anti- <t>Vegf</t> or dexamethasone). Eye drops were applied until the 48 h time point. At t=48 h, IVCM and slit lamp data was collected and used for phenotypic characterisation. (2) cornea tissue was harvested and used for RNA extraction, and RNA quality verified. (3) high quality RNA was used for target preparation for microarray hybridisation on to GeneChip Rat 230 2.0 microarray chips. The microarray chips were scanned and image files acquired. (4) CEL files were normalised using expression console software. The generated CHP together with the CEL files were submitted to Gene Expression Omnibus repository.
    Vegf, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1674 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Elevated TGF ‐β levels causally relate with reduced PDGF ‐Rα expression in nCLD patients and mice undergoing MV ‐O 2 , reducing downstream signaling and migration in pulmonary myofibroblasts Representative immunofluorescence images showing reduced expression of PDGF‐Rα (red) in the lungs of patients ( n = 7) developing nCLD (lower left panel, red stain; white arrows) together with increased pSMAD‐2 expression (lower middle panel, green stain; white arrows) as compared to lung sections from a non‐nCLD patient ( n = 1) (upper panel) (200×). Negative correlation between PDGF‐Rα and TGF‐β1 in transcriptome analysis 72 h after birth in preterms with nCLD ( n = 11) in contrast to non‐nCLD ( n = 9); z test of the difference of the Fisher's z transformed correlations divided by the standard error of the difference ( P = 0.048); scatter plots log2‐gene expression; linear regression (blue), with 95% CI (gray). MV‐O 2 reduced lung PDGF‐Rα (main: red stain; white arrows) and increased pSMAD‐2 levels (insert: red stain; white arrows) in ventilated neonatal WT (lower panel) when compared to control WT mice (upper panel); ( n = 4 mice/group; 10 images/mouse; 200×). Luciferase assay of CCL‐206 cells transfected with pGL4.14 containing PDGF‐Rα promoter revealing reduced promoter activity upon TGF‐β application (normalized to control) ( n = 3 experiments). Immunoblot analysis showing reduced PDGF‐Rα (E), VEGF‐A (F), and pERK/EKR (G) protein levels upon TGF‐β application alone in primary pulmonary myofibroblasts from 5–7‐day‐old WT mice ( n = 6–9 mice/group). Reduced migration of myofibroblasts (MFBs) from neonatal WT mice upon TGF‐β application alone ( n = 5 mice/group, 3 technical replicates). Translation of the results in fibroblasts isolated from tracheal aspirates of ventilated preterm infants (hMFBs) displayed reduced PDGF‐Rα levels (I) and migration assessed by Boyden chamber assay (J) upon TGF‐β application ( n = 3–5 patients/group). Representative phase contrast images (100×) of scratch migration assays in human lung fibroblasts (hMFBs) after 48 h of TGF‐β incubation indicating decreased wound closure (K) quantified by reduced velocity (L) and distance travelled (M) ( n = 3 patients/group). Data information: In (D–J) and (L, M), data are presented as mean ± SD and normalized to control. Statistical test used is two‐tailed unpaired Student's t ‐test or Mann–Whitney test ( P = 0.0002–0.039). *** P

    Journal: EMBO Molecular Medicine

    Article Title: Attenuated PDGF signaling drives alveolar and microvascular defects in neonatal chronic lung disease

    doi: 10.15252/emmm.201607308

    Figure Lengend Snippet: Elevated TGF ‐β levels causally relate with reduced PDGF ‐Rα expression in nCLD patients and mice undergoing MV ‐O 2 , reducing downstream signaling and migration in pulmonary myofibroblasts Representative immunofluorescence images showing reduced expression of PDGF‐Rα (red) in the lungs of patients ( n = 7) developing nCLD (lower left panel, red stain; white arrows) together with increased pSMAD‐2 expression (lower middle panel, green stain; white arrows) as compared to lung sections from a non‐nCLD patient ( n = 1) (upper panel) (200×). Negative correlation between PDGF‐Rα and TGF‐β1 in transcriptome analysis 72 h after birth in preterms with nCLD ( n = 11) in contrast to non‐nCLD ( n = 9); z test of the difference of the Fisher's z transformed correlations divided by the standard error of the difference ( P = 0.048); scatter plots log2‐gene expression; linear regression (blue), with 95% CI (gray). MV‐O 2 reduced lung PDGF‐Rα (main: red stain; white arrows) and increased pSMAD‐2 levels (insert: red stain; white arrows) in ventilated neonatal WT (lower panel) when compared to control WT mice (upper panel); ( n = 4 mice/group; 10 images/mouse; 200×). Luciferase assay of CCL‐206 cells transfected with pGL4.14 containing PDGF‐Rα promoter revealing reduced promoter activity upon TGF‐β application (normalized to control) ( n = 3 experiments). Immunoblot analysis showing reduced PDGF‐Rα (E), VEGF‐A (F), and pERK/EKR (G) protein levels upon TGF‐β application alone in primary pulmonary myofibroblasts from 5–7‐day‐old WT mice ( n = 6–9 mice/group). Reduced migration of myofibroblasts (MFBs) from neonatal WT mice upon TGF‐β application alone ( n = 5 mice/group, 3 technical replicates). Translation of the results in fibroblasts isolated from tracheal aspirates of ventilated preterm infants (hMFBs) displayed reduced PDGF‐Rα levels (I) and migration assessed by Boyden chamber assay (J) upon TGF‐β application ( n = 3–5 patients/group). Representative phase contrast images (100×) of scratch migration assays in human lung fibroblasts (hMFBs) after 48 h of TGF‐β incubation indicating decreased wound closure (K) quantified by reduced velocity (L) and distance travelled (M) ( n = 3 patients/group). Data information: In (D–J) and (L, M), data are presented as mean ± SD and normalized to control. Statistical test used is two‐tailed unpaired Student's t ‐test or Mann–Whitney test ( P = 0.0002–0.039). *** P

    Article Snippet: After measurement of protein concentrations (BCA, #23227, Pierce Scientific), immunoblots were performed using a Bis‐Tris or a Tris‐Acetate gel (#NP0321BOX, #EA0375BOX, Life Technologies) using the following antibodies: PDGF‐Rα (C‐20, Santa Cruz Biotechnology #338), VEGF‐A (147, Santa Cruz Biotechnology #507), VEGF‐R2 (Abcam, Cambridge, USA #Ab2349), VE‐cadherin (H‐72, Santa Cruz Biotechnology #28644), cleaved caspase‐3 (Cell Signaling Technology #9661), cleaved caspase‐9 (Cell Signaling Technologies #7237), eNOS (Cell Signaling Technologies #5880), phospho‐ERK (Cell Signaling Technologies #4370), total ERK (Cell Signaling Technologies #4695), RAS (Cell Signaling Technologies #8955), PI3K (Cell Signaling Technologies #13666), JAK‐2 (Cell Signaling Technologies #3230), STAT‐3 (Cell Signaling Technologies #9139).

    Techniques: Expressing, Mouse Assay, Migration, Immunofluorescence, Staining, Transformation Assay, Luciferase, Transfection, Activity Assay, Isolation, Boyden Chamber Assay, Incubation, Two Tailed Test, MANN-WHITNEY

    Supplemental PDGF ‐A rescues both the air sac and microvascular nCLD phenotypes induced by MV ‐O 2 in neonatal PDGF ‐Rα haploinsufficient mice Improved alveolar structure in 5–8‐day‐old PDGF‐Rα +/− mice undergoing 8 h of MV‐O 2 after intra‐tracheal treatment with PDGF‐A (10 μl/g bw, 25 ng/ml PDGF‐A) when compared to mice receiving sterile saline (200×), confirmed by quantitative image analysis with increased alveolar counts (B) as well as vessel number normalized to 100 alveoli (C) (20–100 μm; n = 2–4 mice/group). Immunoblot analysis of total lung homogenates showed increased PDGF‐Rα (D) together with increased JAK‐2 (E), STAT‐3 (F), VEGF‐A (G), VE‐cadherin (H), and AKT (I) protein levels in PDGF‐A‐treated PDGF‐Rα +/− mice after 8 h of MV‐O 2 when compared to WT littermates ( n = 3–4 mice/group). Panels (D, H) and (E, F) are from same blot hence having same β‐actin bands. Quantitative image analysis indicated increased VEGF‐A to PDGF‐Rα protein levels (J, M upper panel) together with an increase in CD31 expression in relation to total tissue (K, M lower panel) and a decrease in apoptotic (cleaved caspase‐3) CD31‐expressing cells (L) in the lungs of PDGF‐A‐treated PDGF‐Rα +/− mice when compared to saline‐treated controls after 8 h of MV‐O 2 ( n = 2–4 mice/group). Upper panel in (M) shows sections from PDGF‐Rα +/− mice treated with NaCl (left) or PDGF‐A (right) stained with VEGF‐A (green), PDGF‐Rα (red) dual positive (orange, white arrows), and inserts show VEGF‐A stain (green). Lower panel shows sections from PDGF‐Rα +/− mice treated with NaCl (left) or PDGF‐A (right) stained with cleaved caspase‐3 (green), CD31 (red) dual positive (orange, white arrows). Nucleus is stained with DAPI (blue). Treatment with PDGF‐A in ventilated neonatal PDGF‐Rα +/− mice led to improved lung compliance displayed as a function of airway pressure (Ptramax) and tidal volume when compared to untreated mice ( n = 4 mice/group). Data information: In (B–L, N), the data are presented as mean ± SD. *** P

    Journal: EMBO Molecular Medicine

    Article Title: Attenuated PDGF signaling drives alveolar and microvascular defects in neonatal chronic lung disease

    doi: 10.15252/emmm.201607308

    Figure Lengend Snippet: Supplemental PDGF ‐A rescues both the air sac and microvascular nCLD phenotypes induced by MV ‐O 2 in neonatal PDGF ‐Rα haploinsufficient mice Improved alveolar structure in 5–8‐day‐old PDGF‐Rα +/− mice undergoing 8 h of MV‐O 2 after intra‐tracheal treatment with PDGF‐A (10 μl/g bw, 25 ng/ml PDGF‐A) when compared to mice receiving sterile saline (200×), confirmed by quantitative image analysis with increased alveolar counts (B) as well as vessel number normalized to 100 alveoli (C) (20–100 μm; n = 2–4 mice/group). Immunoblot analysis of total lung homogenates showed increased PDGF‐Rα (D) together with increased JAK‐2 (E), STAT‐3 (F), VEGF‐A (G), VE‐cadherin (H), and AKT (I) protein levels in PDGF‐A‐treated PDGF‐Rα +/− mice after 8 h of MV‐O 2 when compared to WT littermates ( n = 3–4 mice/group). Panels (D, H) and (E, F) are from same blot hence having same β‐actin bands. Quantitative image analysis indicated increased VEGF‐A to PDGF‐Rα protein levels (J, M upper panel) together with an increase in CD31 expression in relation to total tissue (K, M lower panel) and a decrease in apoptotic (cleaved caspase‐3) CD31‐expressing cells (L) in the lungs of PDGF‐A‐treated PDGF‐Rα +/− mice when compared to saline‐treated controls after 8 h of MV‐O 2 ( n = 2–4 mice/group). Upper panel in (M) shows sections from PDGF‐Rα +/− mice treated with NaCl (left) or PDGF‐A (right) stained with VEGF‐A (green), PDGF‐Rα (red) dual positive (orange, white arrows), and inserts show VEGF‐A stain (green). Lower panel shows sections from PDGF‐Rα +/− mice treated with NaCl (left) or PDGF‐A (right) stained with cleaved caspase‐3 (green), CD31 (red) dual positive (orange, white arrows). Nucleus is stained with DAPI (blue). Treatment with PDGF‐A in ventilated neonatal PDGF‐Rα +/− mice led to improved lung compliance displayed as a function of airway pressure (Ptramax) and tidal volume when compared to untreated mice ( n = 4 mice/group). Data information: In (B–L, N), the data are presented as mean ± SD. *** P

    Article Snippet: After measurement of protein concentrations (BCA, #23227, Pierce Scientific), immunoblots were performed using a Bis‐Tris or a Tris‐Acetate gel (#NP0321BOX, #EA0375BOX, Life Technologies) using the following antibodies: PDGF‐Rα (C‐20, Santa Cruz Biotechnology #338), VEGF‐A (147, Santa Cruz Biotechnology #507), VEGF‐R2 (Abcam, Cambridge, USA #Ab2349), VE‐cadherin (H‐72, Santa Cruz Biotechnology #28644), cleaved caspase‐3 (Cell Signaling Technology #9661), cleaved caspase‐9 (Cell Signaling Technologies #7237), eNOS (Cell Signaling Technologies #5880), phospho‐ERK (Cell Signaling Technologies #4370), total ERK (Cell Signaling Technologies #4695), RAS (Cell Signaling Technologies #8955), PI3K (Cell Signaling Technologies #13666), JAK‐2 (Cell Signaling Technologies #3230), STAT‐3 (Cell Signaling Technologies #9139).

    Techniques: Mouse Assay, Expressing, Staining

    Enrichment of PDGF ‐Rα SNP s associated with reduced protein levels and migration of lung fibroblasts from ventilated preterm infants developing nCLD Decreased PDGF‐Rα expression in human lung fibroblasts (hMFBs) from preterms undergoing MV‐O 2 (21.7 ± 8 vs. 4.7 ± 1 day of life; serial samples 2 5, 3 6; n = 3 patients/group). Regional association plot showing −log10 P ‐values ( y ‐axis) of SNPs according to chromosomal positions ( x ‐axis). Light blue: estimated recombination rate (cM/Mb, HapMap CEU population); blue: most significant SNP (rs12506783); red: r 2 ≥ 0.8; orange: 0.8 > r 2 ≥ 0.5, yellow: 0.5 > r 2 ≥ 0.2, gray: 0.2 > r 2 ≥ 0. P ‐values were determined using R by case‐control analysis with a logistic regression model including case/control status, sex, gestational age at birth, status “small for gestational age”, and country of origin of the mother. Analysis was adjusted for relatedness to account for multiple births (R‐package GenABEL). Levels of PDGF‐Rα gene expression in patients ( n = 9), which are carrying at least one SNP (minor allele) compared to patients with no SNPs (major allele). Major alleles are given in the figure labels. Minor alleles in rs10022540 are A, in rs11133311 are T, and in rs12506783 are C . PDGF‐R (D) and VEGF‐A (E) protein levels in separate patient cohort ( n = 13) carrying at least one SNP (minor allele) at position rs12506783 compared to patients with no SNPs (major allele). Protein levels were quantified using SOMAlogic technique. Data are presented as mean ± SD. Two‐tailed unpaired Student's t ‐test (* P = 0.0336; # P = 0.0863). Representative PDGF‐Rα levels (F) and migratory potential assessed by Boyden chamber assay (G) in fibroblasts isolated from tracheal aspirates of patients with nCLD. The fibroblast carrying SNP at both alleles (homozygote minor allele) displayed reduced PDGF‐Rα levels and migration when compared to fibroblasts from patients carrying SNP at one allele (heterozygote minor allele). Data are presented as mean ± SD ( n = 3/4 replicates). Source data are available online for this figure.

    Journal: EMBO Molecular Medicine

    Article Title: Attenuated PDGF signaling drives alveolar and microvascular defects in neonatal chronic lung disease

    doi: 10.15252/emmm.201607308

    Figure Lengend Snippet: Enrichment of PDGF ‐Rα SNP s associated with reduced protein levels and migration of lung fibroblasts from ventilated preterm infants developing nCLD Decreased PDGF‐Rα expression in human lung fibroblasts (hMFBs) from preterms undergoing MV‐O 2 (21.7 ± 8 vs. 4.7 ± 1 day of life; serial samples 2 5, 3 6; n = 3 patients/group). Regional association plot showing −log10 P ‐values ( y ‐axis) of SNPs according to chromosomal positions ( x ‐axis). Light blue: estimated recombination rate (cM/Mb, HapMap CEU population); blue: most significant SNP (rs12506783); red: r 2 ≥ 0.8; orange: 0.8 > r 2 ≥ 0.5, yellow: 0.5 > r 2 ≥ 0.2, gray: 0.2 > r 2 ≥ 0. P ‐values were determined using R by case‐control analysis with a logistic regression model including case/control status, sex, gestational age at birth, status “small for gestational age”, and country of origin of the mother. Analysis was adjusted for relatedness to account for multiple births (R‐package GenABEL). Levels of PDGF‐Rα gene expression in patients ( n = 9), which are carrying at least one SNP (minor allele) compared to patients with no SNPs (major allele). Major alleles are given in the figure labels. Minor alleles in rs10022540 are A, in rs11133311 are T, and in rs12506783 are C . PDGF‐R (D) and VEGF‐A (E) protein levels in separate patient cohort ( n = 13) carrying at least one SNP (minor allele) at position rs12506783 compared to patients with no SNPs (major allele). Protein levels were quantified using SOMAlogic technique. Data are presented as mean ± SD. Two‐tailed unpaired Student's t ‐test (* P = 0.0336; # P = 0.0863). Representative PDGF‐Rα levels (F) and migratory potential assessed by Boyden chamber assay (G) in fibroblasts isolated from tracheal aspirates of patients with nCLD. The fibroblast carrying SNP at both alleles (homozygote minor allele) displayed reduced PDGF‐Rα levels and migration when compared to fibroblasts from patients carrying SNP at one allele (heterozygote minor allele). Data are presented as mean ± SD ( n = 3/4 replicates). Source data are available online for this figure.

    Article Snippet: After measurement of protein concentrations (BCA, #23227, Pierce Scientific), immunoblots were performed using a Bis‐Tris or a Tris‐Acetate gel (#NP0321BOX, #EA0375BOX, Life Technologies) using the following antibodies: PDGF‐Rα (C‐20, Santa Cruz Biotechnology #338), VEGF‐A (147, Santa Cruz Biotechnology #507), VEGF‐R2 (Abcam, Cambridge, USA #Ab2349), VE‐cadherin (H‐72, Santa Cruz Biotechnology #28644), cleaved caspase‐3 (Cell Signaling Technology #9661), cleaved caspase‐9 (Cell Signaling Technologies #7237), eNOS (Cell Signaling Technologies #5880), phospho‐ERK (Cell Signaling Technologies #4370), total ERK (Cell Signaling Technologies #4695), RAS (Cell Signaling Technologies #8955), PI3K (Cell Signaling Technologies #13666), JAK‐2 (Cell Signaling Technologies #3230), STAT‐3 (Cell Signaling Technologies #9139).

    Techniques: Migration, Expressing, Two Tailed Test, Boyden Chamber Assay, Isolation

    PDGF ‐Rα haploinsufficiency drives reduced pulmonary micro‐vessel density with increased endothelial cell apoptosis in neonatal mice undergoing MV ‐O 2 Histologic and immunoblot analysis displayed reduced small vessel number (20–100 μm diameter) normalized to 100 alveoli as well as reduced pulmonary VEGF‐R2, VE‐cadherin, and VEGF‐A protein levels, respectively ( n = 6–8 mice/group). Panels (B) and (C) are from same blot hence having same β‐actin bands. Immunofluorescence images of lung tissue (400×; merged) from neonatal PDGF‐Rα +/− mice indicating increased cleaved caspase‐3 (red, white arrows; lower panel) after 8 h of MV‐O 2 in contrast to WT mice (upper panel; green: CD31; blue: DAPI). Double stain revealed increased cleaved caspase‐3 + /CD31 + cells normalized to CD31 area in PDGF‐Rα +/− mice after 8 h of MV‐O 2 ( n = 4 mice/group, 4 sections/mice, and 10 images/section). Representative image confirming increased endothelial apoptosis in neonatal PDGF‐Rα +/− mice after 8 h of MV‐O 2 (lower panel; white arrows) with VE‐cadherin (red) and cleaved caspase‐3 (green) and nucleus stained with DAPI (blue) when compared to WT mice (upper panel) ( n = 2 mice/group). Increased caspase‐3 activation in HUVECs upon incubation with supernatants for 6 h obtained from lung mouse myofibroblasts after PDGF‐Rα siRNA treatment when compared to control siRNA ( n = 3 experiments). In vitro application of PDGF‐Rα siRNA to primary lung mouse myofibroblasts from WT mice diminished PDGF‐Rα (H) protein (normalized to control), associated with reduced VEGF‐A protein (I) ( n = 3 mice/group). Increased cleaved caspase‐9 and reduced eNOS protein levels in HUVECs upon incubation with supernatants for 6 h obtained from lung mouse myofibroblasts after PDGF‐Rα siRNA treatment when compared to control siRNA ( n = 3 mice/group). Data information: Data are presented as mean ± SD. *** P

    Journal: EMBO Molecular Medicine

    Article Title: Attenuated PDGF signaling drives alveolar and microvascular defects in neonatal chronic lung disease

    doi: 10.15252/emmm.201607308

    Figure Lengend Snippet: PDGF ‐Rα haploinsufficiency drives reduced pulmonary micro‐vessel density with increased endothelial cell apoptosis in neonatal mice undergoing MV ‐O 2 Histologic and immunoblot analysis displayed reduced small vessel number (20–100 μm diameter) normalized to 100 alveoli as well as reduced pulmonary VEGF‐R2, VE‐cadherin, and VEGF‐A protein levels, respectively ( n = 6–8 mice/group). Panels (B) and (C) are from same blot hence having same β‐actin bands. Immunofluorescence images of lung tissue (400×; merged) from neonatal PDGF‐Rα +/− mice indicating increased cleaved caspase‐3 (red, white arrows; lower panel) after 8 h of MV‐O 2 in contrast to WT mice (upper panel; green: CD31; blue: DAPI). Double stain revealed increased cleaved caspase‐3 + /CD31 + cells normalized to CD31 area in PDGF‐Rα +/− mice after 8 h of MV‐O 2 ( n = 4 mice/group, 4 sections/mice, and 10 images/section). Representative image confirming increased endothelial apoptosis in neonatal PDGF‐Rα +/− mice after 8 h of MV‐O 2 (lower panel; white arrows) with VE‐cadherin (red) and cleaved caspase‐3 (green) and nucleus stained with DAPI (blue) when compared to WT mice (upper panel) ( n = 2 mice/group). Increased caspase‐3 activation in HUVECs upon incubation with supernatants for 6 h obtained from lung mouse myofibroblasts after PDGF‐Rα siRNA treatment when compared to control siRNA ( n = 3 experiments). In vitro application of PDGF‐Rα siRNA to primary lung mouse myofibroblasts from WT mice diminished PDGF‐Rα (H) protein (normalized to control), associated with reduced VEGF‐A protein (I) ( n = 3 mice/group). Increased cleaved caspase‐9 and reduced eNOS protein levels in HUVECs upon incubation with supernatants for 6 h obtained from lung mouse myofibroblasts after PDGF‐Rα siRNA treatment when compared to control siRNA ( n = 3 mice/group). Data information: Data are presented as mean ± SD. *** P

    Article Snippet: After measurement of protein concentrations (BCA, #23227, Pierce Scientific), immunoblots were performed using a Bis‐Tris or a Tris‐Acetate gel (#NP0321BOX, #EA0375BOX, Life Technologies) using the following antibodies: PDGF‐Rα (C‐20, Santa Cruz Biotechnology #338), VEGF‐A (147, Santa Cruz Biotechnology #507), VEGF‐R2 (Abcam, Cambridge, USA #Ab2349), VE‐cadherin (H‐72, Santa Cruz Biotechnology #28644), cleaved caspase‐3 (Cell Signaling Technology #9661), cleaved caspase‐9 (Cell Signaling Technologies #7237), eNOS (Cell Signaling Technologies #5880), phospho‐ERK (Cell Signaling Technologies #4370), total ERK (Cell Signaling Technologies #4695), RAS (Cell Signaling Technologies #8955), PI3K (Cell Signaling Technologies #13666), JAK‐2 (Cell Signaling Technologies #3230), STAT‐3 (Cell Signaling Technologies #9139).

    Techniques: Mouse Assay, Immunofluorescence, Staining, Activation Assay, Incubation, In Vitro

    Model for how attenuated PDGF signaling and positive pressure ventilation interact to produce the distinct phenotypic manifestations of nCLD MV‐O 2 in vivo , a combination of O 2 , that is, oxygen and stretch (purple arrow) and/or TGF‐β alone or in combination with mechanical stretch in vitro (yellow arrow), reduces platelet‐derived growth factor receptor α (PDGF‐Rα) levels and its downstream signaling through JAK‐2 and STAT‐3 in the pulmonary myofibroblast (MFB). This reduction in turn abrogates vascular endothelial growth factor expression (VEGF‐A and VEGF‐R2), leading to increased apoptosis in pulmonary endothelial cells (EC). Whereas myofibroblast migration is diminished through reduced RAS and pERK/ERK signaling, stretch alone increases their proliferation, hence depicting the differential effect of the most important denominators of nCLD development in the premature lung undergoing MV‐O 2 . Application of PDGF‐A to premature lung increases PDGF‐Rα levels in an AKT‐dependent manner in turn activating the downstream cascade through JAK‐2, STAT‐3 signaling. This then activates VEGF‐A secretion and VEGF‐R2 activity reducing apoptosis in endothelial cells (ECs).

    Journal: EMBO Molecular Medicine

    Article Title: Attenuated PDGF signaling drives alveolar and microvascular defects in neonatal chronic lung disease

    doi: 10.15252/emmm.201607308

    Figure Lengend Snippet: Model for how attenuated PDGF signaling and positive pressure ventilation interact to produce the distinct phenotypic manifestations of nCLD MV‐O 2 in vivo , a combination of O 2 , that is, oxygen and stretch (purple arrow) and/or TGF‐β alone or in combination with mechanical stretch in vitro (yellow arrow), reduces platelet‐derived growth factor receptor α (PDGF‐Rα) levels and its downstream signaling through JAK‐2 and STAT‐3 in the pulmonary myofibroblast (MFB). This reduction in turn abrogates vascular endothelial growth factor expression (VEGF‐A and VEGF‐R2), leading to increased apoptosis in pulmonary endothelial cells (EC). Whereas myofibroblast migration is diminished through reduced RAS and pERK/ERK signaling, stretch alone increases their proliferation, hence depicting the differential effect of the most important denominators of nCLD development in the premature lung undergoing MV‐O 2 . Application of PDGF‐A to premature lung increases PDGF‐Rα levels in an AKT‐dependent manner in turn activating the downstream cascade through JAK‐2, STAT‐3 signaling. This then activates VEGF‐A secretion and VEGF‐R2 activity reducing apoptosis in endothelial cells (ECs).

    Article Snippet: After measurement of protein concentrations (BCA, #23227, Pierce Scientific), immunoblots were performed using a Bis‐Tris or a Tris‐Acetate gel (#NP0321BOX, #EA0375BOX, Life Technologies) using the following antibodies: PDGF‐Rα (C‐20, Santa Cruz Biotechnology #338), VEGF‐A (147, Santa Cruz Biotechnology #507), VEGF‐R2 (Abcam, Cambridge, USA #Ab2349), VE‐cadherin (H‐72, Santa Cruz Biotechnology #28644), cleaved caspase‐3 (Cell Signaling Technology #9661), cleaved caspase‐9 (Cell Signaling Technologies #7237), eNOS (Cell Signaling Technologies #5880), phospho‐ERK (Cell Signaling Technologies #4370), total ERK (Cell Signaling Technologies #4695), RAS (Cell Signaling Technologies #8955), PI3K (Cell Signaling Technologies #13666), JAK‐2 (Cell Signaling Technologies #3230), STAT‐3 (Cell Signaling Technologies #9139).

    Techniques: In Vivo, In Vitro, Derivative Assay, Expressing, Migration, Activity Assay

    Pronounced effect of TGF ‐β on pulmonary myofibroblasts from PDGF ‐Rα +/− mice and in concert with mechanical stretch on both mice and human myofibroblasts TGF‐β application (Th1) to myofibroblasts (MFBs) isolated from neonatal PDGF‐Rα +/− mice reduced PDGF‐Rα (A), JAK‐2 (B), STAT‐3 (C), VEGF‐A (D), and pERK/ERK (E) protein levels when compared to control ( n = 3–6 mice/group). Panels (A, E) and (B, C) are from same blot hence having same β‐actin bands. Reduced migration assessed by Boyden chamber in myofibroblasts (MFBs) isolated from PDGF‐Rα +/− mice compared to WT mice ( n = 5 mice/group). TGF‐β application in combination with mechanical stretch (S+Th1) in myofibroblasts (MFBs) isolated from neonatal WT mice showed reduced PDGF‐Rα (G) and VEGF‐A (H) protein levels as well as migratory RAS (I) and pERK/ERK (J) protein levels when compared to control myofibroblasts as assessed by immunoblot assay ( n = 6–9 mice/group). TGF‐β application (Th1) as an additional dose (Th2) on stretched myofibroblasts (MFBs) from WT mice reduced migration as assessed by Boyden chamber assay ( n = 5 mice/group). TGF‐β application in combination with stretch (S+Th1) reduced PDGF‐Rα protein levels in fibroblasts (hMFBs) isolated from tracheal aspirates of nCLD patients when compared to control (C) or stretched (S) myofibroblasts (L) and as an additional dose (Th2) to stretched fibroblasts reduced migration (M) ( n = 3–6 patients/group). Data information: Values are normalized to the respective controls except for (B–D). Data are presented as mean ± SD. Statistical test used in (A, D–F) is two‐tailed and in (B, C) is one‐tailed Student's t ‐test or Mann–Whitney test ( P = 0.004–0.05) and in (G–M) is ordinary one‐way ANOVA with Bonferroni's correction ( P = 0.0001–0.04). *** P

    Journal: EMBO Molecular Medicine

    Article Title: Attenuated PDGF signaling drives alveolar and microvascular defects in neonatal chronic lung disease

    doi: 10.15252/emmm.201607308

    Figure Lengend Snippet: Pronounced effect of TGF ‐β on pulmonary myofibroblasts from PDGF ‐Rα +/− mice and in concert with mechanical stretch on both mice and human myofibroblasts TGF‐β application (Th1) to myofibroblasts (MFBs) isolated from neonatal PDGF‐Rα +/− mice reduced PDGF‐Rα (A), JAK‐2 (B), STAT‐3 (C), VEGF‐A (D), and pERK/ERK (E) protein levels when compared to control ( n = 3–6 mice/group). Panels (A, E) and (B, C) are from same blot hence having same β‐actin bands. Reduced migration assessed by Boyden chamber in myofibroblasts (MFBs) isolated from PDGF‐Rα +/− mice compared to WT mice ( n = 5 mice/group). TGF‐β application in combination with mechanical stretch (S+Th1) in myofibroblasts (MFBs) isolated from neonatal WT mice showed reduced PDGF‐Rα (G) and VEGF‐A (H) protein levels as well as migratory RAS (I) and pERK/ERK (J) protein levels when compared to control myofibroblasts as assessed by immunoblot assay ( n = 6–9 mice/group). TGF‐β application (Th1) as an additional dose (Th2) on stretched myofibroblasts (MFBs) from WT mice reduced migration as assessed by Boyden chamber assay ( n = 5 mice/group). TGF‐β application in combination with stretch (S+Th1) reduced PDGF‐Rα protein levels in fibroblasts (hMFBs) isolated from tracheal aspirates of nCLD patients when compared to control (C) or stretched (S) myofibroblasts (L) and as an additional dose (Th2) to stretched fibroblasts reduced migration (M) ( n = 3–6 patients/group). Data information: Values are normalized to the respective controls except for (B–D). Data are presented as mean ± SD. Statistical test used in (A, D–F) is two‐tailed and in (B, C) is one‐tailed Student's t ‐test or Mann–Whitney test ( P = 0.004–0.05) and in (G–M) is ordinary one‐way ANOVA with Bonferroni's correction ( P = 0.0001–0.04). *** P

    Article Snippet: After measurement of protein concentrations (BCA, #23227, Pierce Scientific), immunoblots were performed using a Bis‐Tris or a Tris‐Acetate gel (#NP0321BOX, #EA0375BOX, Life Technologies) using the following antibodies: PDGF‐Rα (C‐20, Santa Cruz Biotechnology #338), VEGF‐A (147, Santa Cruz Biotechnology #507), VEGF‐R2 (Abcam, Cambridge, USA #Ab2349), VE‐cadherin (H‐72, Santa Cruz Biotechnology #28644), cleaved caspase‐3 (Cell Signaling Technology #9661), cleaved caspase‐9 (Cell Signaling Technologies #7237), eNOS (Cell Signaling Technologies #5880), phospho‐ERK (Cell Signaling Technologies #4370), total ERK (Cell Signaling Technologies #4695), RAS (Cell Signaling Technologies #8955), PI3K (Cell Signaling Technologies #13666), JAK‐2 (Cell Signaling Technologies #3230), STAT‐3 (Cell Signaling Technologies #9139).

    Techniques: Mouse Assay, Isolation, Migration, Boyden Chamber Assay, Two Tailed Test, One-tailed Test, MANN-WHITNEY

    ( A ) Heart’s specimen from normal mice obtained 7 day after exposure to hypoxia. The expression of VEGF returns to initial levels. ( B ) Heart’s specimen from mdx mice obtained 7 day after exposure to hypoxia. The expression of VEGF in myocardium remains elevated but returns to normal in vessel walls. Scale bar =100 μm.

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: Cardiomyopathy in the mouse model of Duchenne muscular dystrophy caused by disordered secretion of vascular endothelial growth factor

    doi: 10.12659/MSM.882043

    Figure Lengend Snippet: ( A ) Heart’s specimen from normal mice obtained 7 day after exposure to hypoxia. The expression of VEGF returns to initial levels. ( B ) Heart’s specimen from mdx mice obtained 7 day after exposure to hypoxia. The expression of VEGF in myocardium remains elevated but returns to normal in vessel walls. Scale bar =100 μm.

    Article Snippet: The membranes were blocked in 0.05%Tween 20 for 1 h, then incubated with primary anti-VEGF-A (Santa Cruz Biotechnology) at 3 μl/ml in 0.05% Tween 20 in a humid chamber with orbital shaking for 1.5 h, followed by incubation with the secondary biotinylated antibody under similar conditions for 1 h. Proteins were visualized using the horseradish peroxidase-diaminobenzidine system (DAB Substrate Kit for Peroxidase; SK-4100, Vector Laboratories).

    Techniques: Mouse Assay, Expressing

    In situ hybridization analysis of VEGF mRNA expression in cardiac vessel endothelial cells ( A ) and in cardiac myocytes ( B ) from normal (C57BI/10ScSn) and mdx (C57BI/10ScSn) mice (values are optical densities expressed as percentage of the signal for control mice). N – 5 samples from each group, 5 fields of myocardium in 6 preparations from each mouse were examined together 150 analysis. * Statistically significant compared to normal – healthy mice.

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: Cardiomyopathy in the mouse model of Duchenne muscular dystrophy caused by disordered secretion of vascular endothelial growth factor

    doi: 10.12659/MSM.882043

    Figure Lengend Snippet: In situ hybridization analysis of VEGF mRNA expression in cardiac vessel endothelial cells ( A ) and in cardiac myocytes ( B ) from normal (C57BI/10ScSn) and mdx (C57BI/10ScSn) mice (values are optical densities expressed as percentage of the signal for control mice). N – 5 samples from each group, 5 fields of myocardium in 6 preparations from each mouse were examined together 150 analysis. * Statistically significant compared to normal – healthy mice.

    Article Snippet: The membranes were blocked in 0.05%Tween 20 for 1 h, then incubated with primary anti-VEGF-A (Santa Cruz Biotechnology) at 3 μl/ml in 0.05% Tween 20 in a humid chamber with orbital shaking for 1.5 h, followed by incubation with the secondary biotinylated antibody under similar conditions for 1 h. Proteins were visualized using the horseradish peroxidase-diaminobenzidine system (DAB Substrate Kit for Peroxidase; SK-4100, Vector Laboratories).

    Techniques: In Situ Hybridization, Expressing, Mouse Assay

    Immunohistochemical analysis of VEGF expression in cardiac vessel endothelial cells ( A ) and in cardiac myocytes ( B ) from normal (C57BI/10ScSn) and mdx (C57BI/10ScSn) mice (values are optical densities expressed as percentage of the signal for control mice).). N – 5 samples from each group, 5 fields of myocardium in 6 preparations from each mouse were examined together 150 analysis. * Statistically significant compared to normal – healthy mice.

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: Cardiomyopathy in the mouse model of Duchenne muscular dystrophy caused by disordered secretion of vascular endothelial growth factor

    doi: 10.12659/MSM.882043

    Figure Lengend Snippet: Immunohistochemical analysis of VEGF expression in cardiac vessel endothelial cells ( A ) and in cardiac myocytes ( B ) from normal (C57BI/10ScSn) and mdx (C57BI/10ScSn) mice (values are optical densities expressed as percentage of the signal for control mice).). N – 5 samples from each group, 5 fields of myocardium in 6 preparations from each mouse were examined together 150 analysis. * Statistically significant compared to normal – healthy mice.

    Article Snippet: The membranes were blocked in 0.05%Tween 20 for 1 h, then incubated with primary anti-VEGF-A (Santa Cruz Biotechnology) at 3 μl/ml in 0.05% Tween 20 in a humid chamber with orbital shaking for 1.5 h, followed by incubation with the secondary biotinylated antibody under similar conditions for 1 h. Proteins were visualized using the horseradish peroxidase-diaminobenzidine system (DAB Substrate Kit for Peroxidase; SK-4100, Vector Laboratories).

    Techniques: Immunohistochemistry, Expressing, Mouse Assay

    ( A ) Heart’s specimen from normal mice obtained 1 day after exposure to hypoxia. Increased expression of VEGF in both myocardium and vessel walls. ( B ) Heart’s specimen from mdx mice obtained 1 day after exposure to hypoxia. Low expression of VEGF in myocardium and an enhanced signal in vessel walls. Scale bar =100 μm.

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: Cardiomyopathy in the mouse model of Duchenne muscular dystrophy caused by disordered secretion of vascular endothelial growth factor

    doi: 10.12659/MSM.882043

    Figure Lengend Snippet: ( A ) Heart’s specimen from normal mice obtained 1 day after exposure to hypoxia. Increased expression of VEGF in both myocardium and vessel walls. ( B ) Heart’s specimen from mdx mice obtained 1 day after exposure to hypoxia. Low expression of VEGF in myocardium and an enhanced signal in vessel walls. Scale bar =100 μm.

    Article Snippet: The membranes were blocked in 0.05%Tween 20 for 1 h, then incubated with primary anti-VEGF-A (Santa Cruz Biotechnology) at 3 μl/ml in 0.05% Tween 20 in a humid chamber with orbital shaking for 1.5 h, followed by incubation with the secondary biotinylated antibody under similar conditions for 1 h. Proteins were visualized using the horseradish peroxidase-diaminobenzidine system (DAB Substrate Kit for Peroxidase; SK-4100, Vector Laboratories).

    Techniques: Mouse Assay, Expressing

    ( A ) Western analysis of VEGF expression in the heart following hypoxia normal mice. Control group (1); immediately after (2); 1 d ay after (3); 3 days after (4); 7 days after (5), and 21 days after (6) hypoxia. ( B ) Western analysis of VEGF expression in the heart following hypobaric hypoxia in dystrophic mice. Control group (1); immediately after (2); 1 day after (3); 3 days after (4); 7 days after (5), and 21 days after (6) hypoxia. ( C ) Quantitative analysis of western blot signals in normal and mdx mice. Note the difference in the timing of maximum VEGF expression between normal and mdx mice. The control group signal is set to 100%. * Statistically significant compared to healthy mice. In each group was 10 mice.

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: Cardiomyopathy in the mouse model of Duchenne muscular dystrophy caused by disordered secretion of vascular endothelial growth factor

    doi: 10.12659/MSM.882043

    Figure Lengend Snippet: ( A ) Western analysis of VEGF expression in the heart following hypoxia normal mice. Control group (1); immediately after (2); 1 d ay after (3); 3 days after (4); 7 days after (5), and 21 days after (6) hypoxia. ( B ) Western analysis of VEGF expression in the heart following hypobaric hypoxia in dystrophic mice. Control group (1); immediately after (2); 1 day after (3); 3 days after (4); 7 days after (5), and 21 days after (6) hypoxia. ( C ) Quantitative analysis of western blot signals in normal and mdx mice. Note the difference in the timing of maximum VEGF expression between normal and mdx mice. The control group signal is set to 100%. * Statistically significant compared to healthy mice. In each group was 10 mice.

    Article Snippet: The membranes were blocked in 0.05%Tween 20 for 1 h, then incubated with primary anti-VEGF-A (Santa Cruz Biotechnology) at 3 μl/ml in 0.05% Tween 20 in a humid chamber with orbital shaking for 1.5 h, followed by incubation with the secondary biotinylated antibody under similar conditions for 1 h. Proteins were visualized using the horseradish peroxidase-diaminobenzidine system (DAB Substrate Kit for Peroxidase; SK-4100, Vector Laboratories).

    Techniques: Western Blot, Expressing, Mouse Assay

    The 3′UTR of VEGF-A mRNA mediates translation inhibition. ( A ) Schematic of VEGF-A mRNA and chimeric luciferase constructs used for in vitro translation (top panel). The m 7 G cap is indicated by an open circle, the IRES by a light gray rectangle, the putative GAIT element by a black rectangle, and the AREs by dark gray rectangles. Capped, FLuc-VEGF-A 3′UTR (11–900) -A 30 RNA was translated in RRL containing [ 35 S]Met, and in absence or presence of cytosolic extracts from U937 cells treated with IFN-γ for up to 24 h (middle panel). Capped, RLuc RNA lacking the GAIT element was co-translated in each reaction as control. Translation reactions were resolved on SDS–10% polyacrylamide gel. The same RNAs were translated in the presence of cytosolic extract from 24-h, IFN-γ-treated U937 cells, and in the presence of 10- and 50-fold molar excess of in vitro transcribed VEGF-A 3′UTR RNA as competitor (bottom panel). ( B ) Schematic of chimeric luciferase constructs used for in vitro translation (top panel). In vitro translation, in presence of IFN-γ-treated U937 cytosolic extracts, of capped FLuc-VEGF-A 3′UTR (324–455) -A 30 encompassing the putative GAIT element (middle panel), and FLuc-VEGF-A 3′UTR (441–560) -A 30 (bottom panel). RLuc RNA was co-translated in each reaction.

    Journal: The EMBO Journal

    Article Title: A post-transcriptional pathway represses monocyte VEGF-A expression and angiogenic activity

    doi: 10.1038/sj.emboj.7601774

    Figure Lengend Snippet: The 3′UTR of VEGF-A mRNA mediates translation inhibition. ( A ) Schematic of VEGF-A mRNA and chimeric luciferase constructs used for in vitro translation (top panel). The m 7 G cap is indicated by an open circle, the IRES by a light gray rectangle, the putative GAIT element by a black rectangle, and the AREs by dark gray rectangles. Capped, FLuc-VEGF-A 3′UTR (11–900) -A 30 RNA was translated in RRL containing [ 35 S]Met, and in absence or presence of cytosolic extracts from U937 cells treated with IFN-γ for up to 24 h (middle panel). Capped, RLuc RNA lacking the GAIT element was co-translated in each reaction as control. Translation reactions were resolved on SDS–10% polyacrylamide gel. The same RNAs were translated in the presence of cytosolic extract from 24-h, IFN-γ-treated U937 cells, and in the presence of 10- and 50-fold molar excess of in vitro transcribed VEGF-A 3′UTR RNA as competitor (bottom panel). ( B ) Schematic of chimeric luciferase constructs used for in vitro translation (top panel). In vitro translation, in presence of IFN-γ-treated U937 cytosolic extracts, of capped FLuc-VEGF-A 3′UTR (324–455) -A 30 encompassing the putative GAIT element (middle panel), and FLuc-VEGF-A 3′UTR (441–560) -A 30 (bottom panel). RLuc RNA was co-translated in each reaction.

    Article Snippet: The cells were metabolically labeled for 1 h with [35 S]Met/Cys, and conditioned media and cell lysates were subjected to immunoprecipitation with anti-VEGF-A antibody (Santa Cruz) coupled to protein A-Sepharose CL (Sigma) in RIPA buffer.

    Techniques: Inhibition, Luciferase, Construct, In Vitro

    Translational silencing of VEGF-A expression in vivo. ( A ) RT–PCR analysis of total RNA from U937 cells treated with IFN-γ for 0, 8, or 24 h. RT–PCR was done using primers specific for VEGF-A (top panel) and GAPDH (bottom panel). Real-time PCR results indicating the increase in VEGF-A mRNA expression in IFN-γ-treated cells compared to untreated cells are included below the top panel (expressed as fold-increase normalized to β-actin). ( B ) Cell lysates from U937 cells treated with IFN-γ for 0, 8, or 24 h were processed in Laemlli gel-loading buffer in absence of reducing agent. Lysates were subjected to immunoblotting with anti-VEGF-A (top) and anti-GAPDH (bottom panel) antibodies. ( C ) RT–PCR analysis of total RNA from human PBMCs treated with IFN-γ for 0, 8, or 24 h. RT–PCR was performed using primers specific for VEGF-A (top panel) and GAPDH (bottom panel). ( D ) Cell lysates from PBMCs treated with IFN-γ for 0, 8, or 24 h were processed in Laemlli gel-loading buffer in absence of reducing agent. Lysates were subjected to immunoblotting with anti-VEGF-A (top) and anti-GAPDH (bottom panel) antibodies. ( E ) U937 cells were treated with IFN-γ for up to 24 h. At the end of each interval, cells were metabolically labeled with [ 35 S]Met/Cys for 1 h. Conditioned media and cell lysates were immunoprecipitated with anti-VEGF-A antibody and resolved by electrophoresis on SDS–10% polyacrylamide gel (top panel). Monomeric and dimeric VEGF-A forms are indicated by arrows. The same samples were subjected to electrophoresis without immunoprecipitation (bottom). ( F ) U937 cells were treated with IFN-γ for 8 or 24 h and cytosolic extracts were fractionated into polysomal and non-polysomal, RNP fractions by ultracentrifugation on a 20% sucrose cushion in the presence or absence of 10 mM EDTA. RNA associated with each fraction was isolated and subjected to RT–PCR using primers specific for VEGF-A (top panel) and GAPDH (bottom panel).

    Journal: The EMBO Journal

    Article Title: A post-transcriptional pathway represses monocyte VEGF-A expression and angiogenic activity

    doi: 10.1038/sj.emboj.7601774

    Figure Lengend Snippet: Translational silencing of VEGF-A expression in vivo. ( A ) RT–PCR analysis of total RNA from U937 cells treated with IFN-γ for 0, 8, or 24 h. RT–PCR was done using primers specific for VEGF-A (top panel) and GAPDH (bottom panel). Real-time PCR results indicating the increase in VEGF-A mRNA expression in IFN-γ-treated cells compared to untreated cells are included below the top panel (expressed as fold-increase normalized to β-actin). ( B ) Cell lysates from U937 cells treated with IFN-γ for 0, 8, or 24 h were processed in Laemlli gel-loading buffer in absence of reducing agent. Lysates were subjected to immunoblotting with anti-VEGF-A (top) and anti-GAPDH (bottom panel) antibodies. ( C ) RT–PCR analysis of total RNA from human PBMCs treated with IFN-γ for 0, 8, or 24 h. RT–PCR was performed using primers specific for VEGF-A (top panel) and GAPDH (bottom panel). ( D ) Cell lysates from PBMCs treated with IFN-γ for 0, 8, or 24 h were processed in Laemlli gel-loading buffer in absence of reducing agent. Lysates were subjected to immunoblotting with anti-VEGF-A (top) and anti-GAPDH (bottom panel) antibodies. ( E ) U937 cells were treated with IFN-γ for up to 24 h. At the end of each interval, cells were metabolically labeled with [ 35 S]Met/Cys for 1 h. Conditioned media and cell lysates were immunoprecipitated with anti-VEGF-A antibody and resolved by electrophoresis on SDS–10% polyacrylamide gel (top panel). Monomeric and dimeric VEGF-A forms are indicated by arrows. The same samples were subjected to electrophoresis without immunoprecipitation (bottom). ( F ) U937 cells were treated with IFN-γ for 8 or 24 h and cytosolic extracts were fractionated into polysomal and non-polysomal, RNP fractions by ultracentrifugation on a 20% sucrose cushion in the presence or absence of 10 mM EDTA. RNA associated with each fraction was isolated and subjected to RT–PCR using primers specific for VEGF-A (top panel) and GAPDH (bottom panel).

    Article Snippet: The cells were metabolically labeled for 1 h with [35 S]Met/Cys, and conditioned media and cell lysates were subjected to immunoprecipitation with anti-VEGF-A antibody (Santa Cruz) coupled to protein A-Sepharose CL (Sigma) in RIPA buffer.

    Techniques: Expressing, In Vivo, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Metabolic Labelling, Labeling, Immunoprecipitation, Electrophoresis, Isolation

    The GAIT complex binds the VEGF-A GAIT element and causes translational silencing. ( A ) RNA EMSA using 32 P-labeled Cp and VEGF-A GAIT element probes. The riboprobes were incubated with cytosolic extracts from U937 cells treated with IFN-γ for up to 24 h. RNA–protein complexes were resolved by electrophoresis on a nondenaturing 5% polyacrylamide gel. ( B ) RNA–protein complexes formed between 32 P-labeled VEGF-A GAIT element RNA and lysates from 24-h, IFN-γ-treated U937 cells were supershifted with antibodies against GAIT complex components. The cell lysate was incubated with the respective antibodies or non-immune IgG before incubation with the riboprobe. ( C ) Lysate from U937 cells treated with IFN-γ for 24 h was incubated with protein-A Sepharose beads coupled to anti-EPRS antibody (or to pre-immune serum, Pre-im.) to immunodeplete the GAIT complex. The beads were pelleted, and the supernatant subjected to immunoblotting with anti-EPRS antibody to verify effective immunodepletion. ( D ) At 24-h, IFN-γ-treated U937 cell lysates, immunodepleted with anti-EPRS antibody or pre-immune serum, were added to in vitro translation reactions containing FLuc-VEGF-A 3′UTR (11–900) -A 30 and RLuc RNAs.

    Journal: The EMBO Journal

    Article Title: A post-transcriptional pathway represses monocyte VEGF-A expression and angiogenic activity

    doi: 10.1038/sj.emboj.7601774

    Figure Lengend Snippet: The GAIT complex binds the VEGF-A GAIT element and causes translational silencing. ( A ) RNA EMSA using 32 P-labeled Cp and VEGF-A GAIT element probes. The riboprobes were incubated with cytosolic extracts from U937 cells treated with IFN-γ for up to 24 h. RNA–protein complexes were resolved by electrophoresis on a nondenaturing 5% polyacrylamide gel. ( B ) RNA–protein complexes formed between 32 P-labeled VEGF-A GAIT element RNA and lysates from 24-h, IFN-γ-treated U937 cells were supershifted with antibodies against GAIT complex components. The cell lysate was incubated with the respective antibodies or non-immune IgG before incubation with the riboprobe. ( C ) Lysate from U937 cells treated with IFN-γ for 24 h was incubated with protein-A Sepharose beads coupled to anti-EPRS antibody (or to pre-immune serum, Pre-im.) to immunodeplete the GAIT complex. The beads were pelleted, and the supernatant subjected to immunoblotting with anti-EPRS antibody to verify effective immunodepletion. ( D ) At 24-h, IFN-γ-treated U937 cell lysates, immunodepleted with anti-EPRS antibody or pre-immune serum, were added to in vitro translation reactions containing FLuc-VEGF-A 3′UTR (11–900) -A 30 and RLuc RNAs.

    Article Snippet: The cells were metabolically labeled for 1 h with [35 S]Met/Cys, and conditioned media and cell lysates were subjected to immunoprecipitation with anti-VEGF-A antibody (Santa Cruz) coupled to protein A-Sepharose CL (Sigma) in RIPA buffer.

    Techniques: Labeling, Incubation, Electrophoresis, In Vitro

    Functional identification of the VEGF-A 3′UTR GAIT element. ( A ) Folding structures of the Cp (nt 78–106) and the putative VEGF-A GAIT (nt 358–386) elements as predicted by the Mfold algorithm. Base pairing between A7:U23 and U8:A22 was disallowed while folding the VEGF-A GAIT element. ( B ) Chimeric luciferase constructs containing wild-type or mutant VEGF-A 3′UTR GAIT elements. Capped and poly-A tailed RNAs, containing the putative VEGF-A GAIT element (FLuc-VEGF-A GAIT-A 30 ) or a mutant (U10C) GAIT element (FLuc-VEGF-A GAIT mut -A 30 ), downstream of FLuc (top panel), were subjected to in vitro translation in presence of cytosolic extracts from IFN-γ-treated U937 cells (bottom panel). RLuc RNA was co-translated in each reaction. ( C ) Capped and poly-A tailed RNAs, containing the putative VEGF-A GAIT element or a mutant GAIT element as in (B), were subjected to in vitro translation in presence of cytosolic extracts from IFN-γ-treated human PBMC (top panel). RLuc RNA was co-translated in each reaction. Fluc was quantified by densitometry, normalized to Rluc, and expressed as per cent of control condition without cell lysate (bottom). ( D ) U937 cells were transfected with eukaryotic, CMV-driven expression vectors containing the FLuc gene upstream of either wild-type (CMV-FLuc-VEGF-A GAIT-A 30 ) or mutant VEGF-A GAIT element (CMV-FLuc-VEGF-A GAIT mut -A 30 ) or lacking any GAIT element (CMV-FLuc). Cells were co-transfected with a vector containing RLuc gene under the SV40 promoter. Following transfection, cells were treated with IFN-γ for 8 (gray bars) or 24 h (black bars), or with medium alone (hatched bars). Luciferase activity in cell lysates was measured by dual luciferase assay. Results show mean and standard deviation of values from three independent experiments.

    Journal: The EMBO Journal

    Article Title: A post-transcriptional pathway represses monocyte VEGF-A expression and angiogenic activity

    doi: 10.1038/sj.emboj.7601774

    Figure Lengend Snippet: Functional identification of the VEGF-A 3′UTR GAIT element. ( A ) Folding structures of the Cp (nt 78–106) and the putative VEGF-A GAIT (nt 358–386) elements as predicted by the Mfold algorithm. Base pairing between A7:U23 and U8:A22 was disallowed while folding the VEGF-A GAIT element. ( B ) Chimeric luciferase constructs containing wild-type or mutant VEGF-A 3′UTR GAIT elements. Capped and poly-A tailed RNAs, containing the putative VEGF-A GAIT element (FLuc-VEGF-A GAIT-A 30 ) or a mutant (U10C) GAIT element (FLuc-VEGF-A GAIT mut -A 30 ), downstream of FLuc (top panel), were subjected to in vitro translation in presence of cytosolic extracts from IFN-γ-treated U937 cells (bottom panel). RLuc RNA was co-translated in each reaction. ( C ) Capped and poly-A tailed RNAs, containing the putative VEGF-A GAIT element or a mutant GAIT element as in (B), were subjected to in vitro translation in presence of cytosolic extracts from IFN-γ-treated human PBMC (top panel). RLuc RNA was co-translated in each reaction. Fluc was quantified by densitometry, normalized to Rluc, and expressed as per cent of control condition without cell lysate (bottom). ( D ) U937 cells were transfected with eukaryotic, CMV-driven expression vectors containing the FLuc gene upstream of either wild-type (CMV-FLuc-VEGF-A GAIT-A 30 ) or mutant VEGF-A GAIT element (CMV-FLuc-VEGF-A GAIT mut -A 30 ) or lacking any GAIT element (CMV-FLuc). Cells were co-transfected with a vector containing RLuc gene under the SV40 promoter. Following transfection, cells were treated with IFN-γ for 8 (gray bars) or 24 h (black bars), or with medium alone (hatched bars). Luciferase activity in cell lysates was measured by dual luciferase assay. Results show mean and standard deviation of values from three independent experiments.

    Article Snippet: The cells were metabolically labeled for 1 h with [35 S]Met/Cys, and conditioned media and cell lysates were subjected to immunoprecipitation with anti-VEGF-A antibody (Santa Cruz) coupled to protein A-Sepharose CL (Sigma) in RIPA buffer.

    Techniques: Functional Assay, Luciferase, Construct, Mutagenesis, In Vitro, Transfection, Expressing, Plasmid Preparation, Activity Assay, Standard Deviation

    Ablation of the GAIT complex in vivo prevents translational silencing of VEGF-A. ( A ) Lysates from U937 cells stably transfected with pSUPER vector (U937-pSUPER) or pSUPER encoding a short hairpin RNA targeting L13a (U937-L13a-SHR) were immunoblotted with anti-L13a antibody. ( B ) Lysates from the stably transfected cell lines in (A) were treated with IFN-γ for 0, 8, or 24 h and processed in Laemlli gel-loading buffer in absence of reducing agent. Lysates were subjected to immunoblotting with anti-VEGF-A (top panel) and anti-GAPDH (bottom panel) antibodies. ( C ) Total RNA was isolated from the stably transfected cell lines treated with IFN-γ for 0, 8, or 24 h, and analyzed by RT–PCR using primers specific for VEGF-A (top panel) and β-actin (bottom panel). Real-time PCR results indicating increased VEGF-A mRNA expression in IFN-γ-treated cells compared to untreated cells (expressed as fold-increase normalized to β-actin) are inserted below the top panel. ( D ) The cell lines described in (A) were treated with IFN-γ for 24 h and lysates immunoprecipitated with anti-EPRS antibody, followed by RT–PCR with VEGF-A-specific primers.

    Journal: The EMBO Journal

    Article Title: A post-transcriptional pathway represses monocyte VEGF-A expression and angiogenic activity

    doi: 10.1038/sj.emboj.7601774

    Figure Lengend Snippet: Ablation of the GAIT complex in vivo prevents translational silencing of VEGF-A. ( A ) Lysates from U937 cells stably transfected with pSUPER vector (U937-pSUPER) or pSUPER encoding a short hairpin RNA targeting L13a (U937-L13a-SHR) were immunoblotted with anti-L13a antibody. ( B ) Lysates from the stably transfected cell lines in (A) were treated with IFN-γ for 0, 8, or 24 h and processed in Laemlli gel-loading buffer in absence of reducing agent. Lysates were subjected to immunoblotting with anti-VEGF-A (top panel) and anti-GAPDH (bottom panel) antibodies. ( C ) Total RNA was isolated from the stably transfected cell lines treated with IFN-γ for 0, 8, or 24 h, and analyzed by RT–PCR using primers specific for VEGF-A (top panel) and β-actin (bottom panel). Real-time PCR results indicating increased VEGF-A mRNA expression in IFN-γ-treated cells compared to untreated cells (expressed as fold-increase normalized to β-actin) are inserted below the top panel. ( D ) The cell lines described in (A) were treated with IFN-γ for 24 h and lysates immunoprecipitated with anti-EPRS antibody, followed by RT–PCR with VEGF-A-specific primers.

    Article Snippet: The cells were metabolically labeled for 1 h with [35 S]Met/Cys, and conditioned media and cell lysates were subjected to immunoprecipitation with anti-VEGF-A antibody (Santa Cruz) coupled to protein A-Sepharose CL (Sigma) in RIPA buffer.

    Techniques: In Vivo, Stable Transfection, Transfection, Plasmid Preparation, shRNA, Isolation, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Expressing, Immunoprecipitation

    VEGF-A mRNA interacts with the GAIT complex. ( A ) Secondary structure and sequence features of the human Cp GAIT element (top panel). The query pattern, based on the secondary structure and sequence features of the Cp GAIT element, was used to search a nonredundant 3′UTR database using the PatSearch program (bottom panel). Following the syntax of the PatSearch algorithm, allowed base-pairs are represented by r number and patterns defined by p number. The GAIT element-specific stems and loops are shown below. ( B ) PatSearch result predicted the presence of GAIT elements in Cp and VEGF-A 3′UTR. UTRdb ID refers to the sequence entry in the UTR database, and sequence position refers to the 3′UTR position of the sequence encoding the predicted GAIT element. ( C ) To show VEGF-A mRNA interaction with the GAIT complex in vivo , U937 cells were treated with IFN-γ for 8 or 24 h, and lysates were immunoprecipitated (IP) with anti-EPRS antibody to isolate GAIT complex, or with control pre-immune (Pre-im.) serum. RNA associated with the GAIT complex, or present in the non-immunoprecipitated supernatant (Sup.), was subjected to RT–PCR using primers specific for VEGF-A or β-actin mRNA, and products were resolved in 1.6% agarose gels. ( D ) To verify antibody specificity, lysate from U937 cells treated with IFN-γ for 24 h was immunoprecipitated with polyclonal anti-human EPRS antibody and immunoblotted with the same antibody, or with pre-immune serum as control.

    Journal: The EMBO Journal

    Article Title: A post-transcriptional pathway represses monocyte VEGF-A expression and angiogenic activity

    doi: 10.1038/sj.emboj.7601774

    Figure Lengend Snippet: VEGF-A mRNA interacts with the GAIT complex. ( A ) Secondary structure and sequence features of the human Cp GAIT element (top panel). The query pattern, based on the secondary structure and sequence features of the Cp GAIT element, was used to search a nonredundant 3′UTR database using the PatSearch program (bottom panel). Following the syntax of the PatSearch algorithm, allowed base-pairs are represented by r number and patterns defined by p number. The GAIT element-specific stems and loops are shown below. ( B ) PatSearch result predicted the presence of GAIT elements in Cp and VEGF-A 3′UTR. UTRdb ID refers to the sequence entry in the UTR database, and sequence position refers to the 3′UTR position of the sequence encoding the predicted GAIT element. ( C ) To show VEGF-A mRNA interaction with the GAIT complex in vivo , U937 cells were treated with IFN-γ for 8 or 24 h, and lysates were immunoprecipitated (IP) with anti-EPRS antibody to isolate GAIT complex, or with control pre-immune (Pre-im.) serum. RNA associated with the GAIT complex, or present in the non-immunoprecipitated supernatant (Sup.), was subjected to RT–PCR using primers specific for VEGF-A or β-actin mRNA, and products were resolved in 1.6% agarose gels. ( D ) To verify antibody specificity, lysate from U937 cells treated with IFN-γ for 24 h was immunoprecipitated with polyclonal anti-human EPRS antibody and immunoblotted with the same antibody, or with pre-immune serum as control.

    Article Snippet: The cells were metabolically labeled for 1 h with [35 S]Met/Cys, and conditioned media and cell lysates were subjected to immunoprecipitation with anti-VEGF-A antibody (Santa Cruz) coupled to protein A-Sepharose CL (Sigma) in RIPA buffer.

    Techniques: Sequencing, In Vivo, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

    Silencing of VEGF-A translation in monocytic cells inhibits angiogenic activity. ( A ) EC proliferation was measured in presence of medium conditioned by IFN-γ-treated U937 cells. U937 cells were pre-treated with IFN-γ for up to 24 h, and then fresh medium was added for an additional 2 h. The conditioned medium was added to 50% confluent ECs, and proliferation measured by MTT assay. Cells were treated with recombinant VEGF-A (rVEGF-A, 10 ng/ml) as a positive control. Stimulation of proliferation was expressed as fold-increase compared to cells treated with medium alone (gray bars). Parallel wells contained conditioned medium pre-incubated with anti-VEGF-A antibody (black bars). Shown are the mean and standard deviation from three independent experiments. ( B ) Tube-formation by ECs on growth factor-depleted matrigel was determined after 12 h in presence of conditioned medium from U937 cells treated with IFN-γ for 8, 16, or 24 h, or with recombinant human VEGF-A (10 ng/ml). ( C ) EC tube formation was quantitated by computer-assisted tracing. Shown are the mean and standard deviation from three representative fields, for three independent experiments. ( D ) IFN-γ activates the transcription of VEGF-A, Cp, and other pro-inflammatory genes in macrophages at the site of chronic inflammation. Subsequently, IFN-γ activates the GAIT complex that binds to the GAIT element in the 3′UTR of VEGF-A, Cp, and possibly other transcripts, and silences their translation. This mechanism prevents persistent expression of these inflammatory proteins and reduces or resolves chronic inflammation and tissue injury.

    Journal: The EMBO Journal

    Article Title: A post-transcriptional pathway represses monocyte VEGF-A expression and angiogenic activity

    doi: 10.1038/sj.emboj.7601774

    Figure Lengend Snippet: Silencing of VEGF-A translation in monocytic cells inhibits angiogenic activity. ( A ) EC proliferation was measured in presence of medium conditioned by IFN-γ-treated U937 cells. U937 cells were pre-treated with IFN-γ for up to 24 h, and then fresh medium was added for an additional 2 h. The conditioned medium was added to 50% confluent ECs, and proliferation measured by MTT assay. Cells were treated with recombinant VEGF-A (rVEGF-A, 10 ng/ml) as a positive control. Stimulation of proliferation was expressed as fold-increase compared to cells treated with medium alone (gray bars). Parallel wells contained conditioned medium pre-incubated with anti-VEGF-A antibody (black bars). Shown are the mean and standard deviation from three independent experiments. ( B ) Tube-formation by ECs on growth factor-depleted matrigel was determined after 12 h in presence of conditioned medium from U937 cells treated with IFN-γ for 8, 16, or 24 h, or with recombinant human VEGF-A (10 ng/ml). ( C ) EC tube formation was quantitated by computer-assisted tracing. Shown are the mean and standard deviation from three representative fields, for three independent experiments. ( D ) IFN-γ activates the transcription of VEGF-A, Cp, and other pro-inflammatory genes in macrophages at the site of chronic inflammation. Subsequently, IFN-γ activates the GAIT complex that binds to the GAIT element in the 3′UTR of VEGF-A, Cp, and possibly other transcripts, and silences their translation. This mechanism prevents persistent expression of these inflammatory proteins and reduces or resolves chronic inflammation and tissue injury.

    Article Snippet: The cells were metabolically labeled for 1 h with [35 S]Met/Cys, and conditioned media and cell lysates were subjected to immunoprecipitation with anti-VEGF-A antibody (Santa Cruz) coupled to protein A-Sepharose CL (Sigma) in RIPA buffer.

    Techniques: Activity Assay, MTT Assay, Recombinant, Positive Control, Incubation, Standard Deviation, Expressing

    Schematic diagram of KLF4 mediated VEGF signaling pathway. KLF4 binds to the VEGF promoter and induces VEGF expression, subsequently phosphorylates VEGFR2 and activates downstream ERK1/2 and AKT to promote cell proliferation, migration and angiogenesis.

    Journal: PLoS ONE

    Article Title: KLF4 Promotes Angiogenesis by Activating VEGF Signaling in Human Retinal Microvascular Endothelial Cells

    doi: 10.1371/journal.pone.0130341

    Figure Lengend Snippet: Schematic diagram of KLF4 mediated VEGF signaling pathway. KLF4 binds to the VEGF promoter and induces VEGF expression, subsequently phosphorylates VEGFR2 and activates downstream ERK1/2 and AKT to promote cell proliferation, migration and angiogenesis.

    Article Snippet: Immunofluorescent staining To detect the expression of VEGF and KLF4 in HRMECs, KLF4-expressing and control cells were fixed for 10 min using 4% PFA, washed three times with 0.1% Tween-20 in PBS (PBST), and incubated with blocking buffer (5% normal goat serum, 3% bovine serum albumin, and 0.1% Triton-X 100 in PBS) for 1 h. The fixed cells were incubated with primary antibodies against VEGF and KLF4 (1:200 dilution, Santa Cruz, Dallas, Texas), overnight.

    Techniques: Expressing, Migration

    KLF4 promotes VEGF-induced tube formation and enhances angiogenesis in vivo. A.B. Tube formation assays were performed in KLF4 expressing and knockdown HRMECs, respectively. The angiogenic effect of KLF4 on VEGF induced tube formation was determined by counting nodes and sprouts of tube-like structures from at least three different fields of three independent experiments and normalized to vehicle treated control cells. Significance was compared between KLF4 expressing and control cells with or without VEGF treatment (*p

    Journal: PLoS ONE

    Article Title: KLF4 Promotes Angiogenesis by Activating VEGF Signaling in Human Retinal Microvascular Endothelial Cells

    doi: 10.1371/journal.pone.0130341

    Figure Lengend Snippet: KLF4 promotes VEGF-induced tube formation and enhances angiogenesis in vivo. A.B. Tube formation assays were performed in KLF4 expressing and knockdown HRMECs, respectively. The angiogenic effect of KLF4 on VEGF induced tube formation was determined by counting nodes and sprouts of tube-like structures from at least three different fields of three independent experiments and normalized to vehicle treated control cells. Significance was compared between KLF4 expressing and control cells with or without VEGF treatment (*p

    Article Snippet: Immunofluorescent staining To detect the expression of VEGF and KLF4 in HRMECs, KLF4-expressing and control cells were fixed for 10 min using 4% PFA, washed three times with 0.1% Tween-20 in PBS (PBST), and incubated with blocking buffer (5% normal goat serum, 3% bovine serum albumin, and 0.1% Triton-X 100 in PBS) for 1 h. The fixed cells were incubated with primary antibodies against VEGF and KLF4 (1:200 dilution, Santa Cruz, Dallas, Texas), overnight.

    Techniques: In Vivo, Expressing

    KLF4 promotes VEGF induced cell proliferation. A, B. Cell proliferation was examined at different time points in KLF4 expressing and knocking down HRMECs. Cells were treated with VEGF or vehicle (Veh) following 24h serum-free media culture before measuring cell proliferation using a MTT assay. Significance was observed between KLF4 expressing and control cells with or without VEGF induction. C . Cell apoptosis was examined using ELISA from HRMECs transduced with lentiviral KLF4shRNA1, 2 and Scramble controls. D. One representative Western blot was shown on the active caspase3 expression at indicated time points in KLF4 knockdown and control cells following 12h serum starvation. Significance was compared between KLF4 knockdown and control cells at the indicated time points. Data are presented as mean ±S.E. from 3 independent experiments, (*p

    Journal: PLoS ONE

    Article Title: KLF4 Promotes Angiogenesis by Activating VEGF Signaling in Human Retinal Microvascular Endothelial Cells

    doi: 10.1371/journal.pone.0130341

    Figure Lengend Snippet: KLF4 promotes VEGF induced cell proliferation. A, B. Cell proliferation was examined at different time points in KLF4 expressing and knocking down HRMECs. Cells were treated with VEGF or vehicle (Veh) following 24h serum-free media culture before measuring cell proliferation using a MTT assay. Significance was observed between KLF4 expressing and control cells with or without VEGF induction. C . Cell apoptosis was examined using ELISA from HRMECs transduced with lentiviral KLF4shRNA1, 2 and Scramble controls. D. One representative Western blot was shown on the active caspase3 expression at indicated time points in KLF4 knockdown and control cells following 12h serum starvation. Significance was compared between KLF4 knockdown and control cells at the indicated time points. Data are presented as mean ±S.E. from 3 independent experiments, (*p

    Article Snippet: Immunofluorescent staining To detect the expression of VEGF and KLF4 in HRMECs, KLF4-expressing and control cells were fixed for 10 min using 4% PFA, washed three times with 0.1% Tween-20 in PBS (PBST), and incubated with blocking buffer (5% normal goat serum, 3% bovine serum albumin, and 0.1% Triton-X 100 in PBS) for 1 h. The fixed cells were incubated with primary antibodies against VEGF and KLF4 (1:200 dilution, Santa Cruz, Dallas, Texas), overnight.

    Techniques: Expressing, MTT Assay, Enzyme-linked Immunosorbent Assay, Transduction, Western Blot

    KLF4 transcriptionally activates VEGF expression. A. Luciferase reporter assays were performed to assess KLF4 activation of the VEGF promoter. Luciferase activities in KLF4 expressing and control HRMECs were measured at 24h after transfection with 1.5kb and 0.2kbVEGF promoter luciferase constructs in serum-free conditions. Data were presented as the mean ± SE from three independent experiments. Significance of luciferase activity was found between KLF4 expressing and control cells when 1.5Kb VEGF promoter was transfected in both cells (**p

    Journal: PLoS ONE

    Article Title: KLF4 Promotes Angiogenesis by Activating VEGF Signaling in Human Retinal Microvascular Endothelial Cells

    doi: 10.1371/journal.pone.0130341

    Figure Lengend Snippet: KLF4 transcriptionally activates VEGF expression. A. Luciferase reporter assays were performed to assess KLF4 activation of the VEGF promoter. Luciferase activities in KLF4 expressing and control HRMECs were measured at 24h after transfection with 1.5kb and 0.2kbVEGF promoter luciferase constructs in serum-free conditions. Data were presented as the mean ± SE from three independent experiments. Significance of luciferase activity was found between KLF4 expressing and control cells when 1.5Kb VEGF promoter was transfected in both cells (**p

    Article Snippet: Immunofluorescent staining To detect the expression of VEGF and KLF4 in HRMECs, KLF4-expressing and control cells were fixed for 10 min using 4% PFA, washed three times with 0.1% Tween-20 in PBS (PBST), and incubated with blocking buffer (5% normal goat serum, 3% bovine serum albumin, and 0.1% Triton-X 100 in PBS) for 1 h. The fixed cells were incubated with primary antibodies against VEGF and KLF4 (1:200 dilution, Santa Cruz, Dallas, Texas), overnight.

    Techniques: Expressing, Luciferase, Activation Assay, Transfection, Construct, Activity Assay

    KLF4 enhances VEGF mediated angiogenesis signaling pathway. A.B. VEGF signaling pathway was examined in KLF4 expressing and knockdown HRMECs following serum starvation and VEGF treatment using Western blot. Data were presented from three separate experiments by measuring band density using the Image J program. Western blot was shown from one representative experiment. Significance was compared between KLF4 expressing and control cells at different time points of VEGF treatment (*p

    Journal: PLoS ONE

    Article Title: KLF4 Promotes Angiogenesis by Activating VEGF Signaling in Human Retinal Microvascular Endothelial Cells

    doi: 10.1371/journal.pone.0130341

    Figure Lengend Snippet: KLF4 enhances VEGF mediated angiogenesis signaling pathway. A.B. VEGF signaling pathway was examined in KLF4 expressing and knockdown HRMECs following serum starvation and VEGF treatment using Western blot. Data were presented from three separate experiments by measuring band density using the Image J program. Western blot was shown from one representative experiment. Significance was compared between KLF4 expressing and control cells at different time points of VEGF treatment (*p

    Article Snippet: Immunofluorescent staining To detect the expression of VEGF and KLF4 in HRMECs, KLF4-expressing and control cells were fixed for 10 min using 4% PFA, washed three times with 0.1% Tween-20 in PBS (PBST), and incubated with blocking buffer (5% normal goat serum, 3% bovine serum albumin, and 0.1% Triton-X 100 in PBS) for 1 h. The fixed cells were incubated with primary antibodies against VEGF and KLF4 (1:200 dilution, Santa Cruz, Dallas, Texas), overnight.

    Techniques: Expressing, Western Blot

    KLF4 promotes VEGF induced cell migration. A, B. Transwell migration assays were performed in HRMECs following KLF4 expression or knockdown. Migrated cells were stained with Crystal Violet and then counted. KLF4 expression in HRMECs significantly increased cell migration while knockdown of KLF4 reduced it as compared to control cells with and without VEGF treatment. The data were presented from three independent experiments in triplicate as mean+S.E and normalized by comparing it to vehicle treated control cells (* p

    Journal: PLoS ONE

    Article Title: KLF4 Promotes Angiogenesis by Activating VEGF Signaling in Human Retinal Microvascular Endothelial Cells

    doi: 10.1371/journal.pone.0130341

    Figure Lengend Snippet: KLF4 promotes VEGF induced cell migration. A, B. Transwell migration assays were performed in HRMECs following KLF4 expression or knockdown. Migrated cells were stained with Crystal Violet and then counted. KLF4 expression in HRMECs significantly increased cell migration while knockdown of KLF4 reduced it as compared to control cells with and without VEGF treatment. The data were presented from three independent experiments in triplicate as mean+S.E and normalized by comparing it to vehicle treated control cells (* p

    Article Snippet: Immunofluorescent staining To detect the expression of VEGF and KLF4 in HRMECs, KLF4-expressing and control cells were fixed for 10 min using 4% PFA, washed three times with 0.1% Tween-20 in PBS (PBST), and incubated with blocking buffer (5% normal goat serum, 3% bovine serum albumin, and 0.1% Triton-X 100 in PBS) for 1 h. The fixed cells were incubated with primary antibodies against VEGF and KLF4 (1:200 dilution, Santa Cruz, Dallas, Texas), overnight.

    Techniques: Migration, Expressing, Staining

    Concentrations of VEGF 165a and VEGF 165b plotted against the secretion ratio of VEGF 165b to total VEGF 165 isoforms in normal (left column), blood (middle column) and disease compartments (right column). Each row represents the variation of secretion ratio of VEGF 165b to total VEGF 165 in ( A ) normal, ( B ) blood, and ( C ) disease compartment.

    Journal: Scientific Reports

    Article Title: A multiscale computational model predicts distribution of anti-angiogenic isoform VEGF165b in peripheral arterial disease in human and mouse

    doi: 10.1038/srep37030

    Figure Lengend Snippet: Concentrations of VEGF 165a and VEGF 165b plotted against the secretion ratio of VEGF 165b to total VEGF 165 isoforms in normal (left column), blood (middle column) and disease compartments (right column). Each row represents the variation of secretion ratio of VEGF 165b to total VEGF 165 in ( A ) normal, ( B ) blood, and ( C ) disease compartment.

    Article Snippet: Antibodies VEGF165b antibody was purchased from Millipore (Clone 56/1, Cat No: MABC595), VEGF-A antibody was purchased from Santa Cruz Biotech (Cat No: SC-7269) and β-Actin was purchased from Sigma (Cat No: A2103).

    Techniques:

    Molecular Interactions of VEGF 165a , VEGF 165b and VEGF 121 .

    Journal: Scientific Reports

    Article Title: A multiscale computational model predicts distribution of anti-angiogenic isoform VEGF165b in peripheral arterial disease in human and mouse

    doi: 10.1038/srep37030

    Figure Lengend Snippet: Molecular Interactions of VEGF 165a , VEGF 165b and VEGF 121 .

    Article Snippet: Antibodies VEGF165b antibody was purchased from Millipore (Clone 56/1, Cat No: MABC595), VEGF-A antibody was purchased from Santa Cruz Biotech (Cat No: SC-7269) and β-Actin was purchased from Sigma (Cat No: A2103).

    Techniques:

    Western blot analysis of VEGF 165b , VEGF-A and Actin in non-ischemic (NGA) and ischemic gastrocnemius muscle (IGA) at day 3 and day 7 post hindlimb ischemia (HLI). (n = 4. One-way ANOVA with Dunnetts post-test. p

    Journal: Scientific Reports

    Article Title: A multiscale computational model predicts distribution of anti-angiogenic isoform VEGF165b in peripheral arterial disease in human and mouse

    doi: 10.1038/srep37030

    Figure Lengend Snippet: Western blot analysis of VEGF 165b , VEGF-A and Actin in non-ischemic (NGA) and ischemic gastrocnemius muscle (IGA) at day 3 and day 7 post hindlimb ischemia (HLI). (n = 4. One-way ANOVA with Dunnetts post-test. p

    Article Snippet: Antibodies VEGF165b antibody was purchased from Millipore (Clone 56/1, Cat No: MABC595), VEGF-A antibody was purchased from Santa Cruz Biotech (Cat No: SC-7269) and β-Actin was purchased from Sigma (Cat No: A2103).

    Techniques: Western Blot

    ( A ) VEGF distribution and ( B ) VEGFR occupancy in human. The bars in y-axis represent the percentage of each species for each VEGF ligand VEGF 165a , VEGF 165b and VEGF 121 in ( A ) and each receptor VEGFR1, VEGFR2, NRP1, and sVEGFR1 in ( B ).

    Journal: Scientific Reports

    Article Title: A multiscale computational model predicts distribution of anti-angiogenic isoform VEGF165b in peripheral arterial disease in human and mouse

    doi: 10.1038/srep37030

    Figure Lengend Snippet: ( A ) VEGF distribution and ( B ) VEGFR occupancy in human. The bars in y-axis represent the percentage of each species for each VEGF ligand VEGF 165a , VEGF 165b and VEGF 121 in ( A ) and each receptor VEGFR1, VEGFR2, NRP1, and sVEGFR1 in ( B ).

    Article Snippet: Antibodies VEGF165b antibody was purchased from Millipore (Clone 56/1, Cat No: MABC595), VEGF-A antibody was purchased from Santa Cruz Biotech (Cat No: SC-7269) and β-Actin was purchased from Sigma (Cat No: A2103).

    Techniques:

    Local sensitivity analysis for VEGF 165a , VEGF 165b and VEGF 121 . The results show that VEGF 165a in the disease compartment and VEGF 165b in the blood compartment are more sensitive to VEGFR2 than VEGFR1, whereas VEGF 121 in the disease and blood compartment is sensitive to both VEGFR1 and VEGFR2. VEGF 165a , VEGF 165b , VEGF 121 are not sensitive to NRP1. The sensitivity analysis demonstrates the importance of VEGF 165b -VEGFR2 binding.

    Journal: Scientific Reports

    Article Title: A multiscale computational model predicts distribution of anti-angiogenic isoform VEGF165b in peripheral arterial disease in human and mouse

    doi: 10.1038/srep37030

    Figure Lengend Snippet: Local sensitivity analysis for VEGF 165a , VEGF 165b and VEGF 121 . The results show that VEGF 165a in the disease compartment and VEGF 165b in the blood compartment are more sensitive to VEGFR2 than VEGFR1, whereas VEGF 121 in the disease and blood compartment is sensitive to both VEGFR1 and VEGFR2. VEGF 165a , VEGF 165b , VEGF 121 are not sensitive to NRP1. The sensitivity analysis demonstrates the importance of VEGF 165b -VEGFR2 binding.

    Article Snippet: Antibodies VEGF165b antibody was purchased from Millipore (Clone 56/1, Cat No: MABC595), VEGF-A antibody was purchased from Santa Cruz Biotech (Cat No: SC-7269) and β-Actin was purchased from Sigma (Cat No: A2103).

    Techniques: Binding Assay

    ( A ) VEGF distribution and ( B ) VEGFR occupancy in mouse. The bars represent the percentage of each species for each VEGF ligand in ( A ) and each receptor in ( B ), respectively, same as Fig. 5 .

    Journal: Scientific Reports

    Article Title: A multiscale computational model predicts distribution of anti-angiogenic isoform VEGF165b in peripheral arterial disease in human and mouse

    doi: 10.1038/srep37030

    Figure Lengend Snippet: ( A ) VEGF distribution and ( B ) VEGFR occupancy in mouse. The bars represent the percentage of each species for each VEGF ligand in ( A ) and each receptor in ( B ), respectively, same as Fig. 5 .

    Article Snippet: Antibodies VEGF165b antibody was purchased from Millipore (Clone 56/1, Cat No: MABC595), VEGF-A antibody was purchased from Santa Cruz Biotech (Cat No: SC-7269) and β-Actin was purchased from Sigma (Cat No: A2103).

    Techniques:

    ELISA showing the specificity and sensitivity of VEGF 165a (VEGFA) and VEGF 165b antibodies.

    Journal: Scientific Reports

    Article Title: A multiscale computational model predicts distribution of anti-angiogenic isoform VEGF165b in peripheral arterial disease in human and mouse

    doi: 10.1038/srep37030

    Figure Lengend Snippet: ELISA showing the specificity and sensitivity of VEGF 165a (VEGFA) and VEGF 165b antibodies.

    Article Snippet: Antibodies VEGF165b antibody was purchased from Millipore (Clone 56/1, Cat No: MABC595), VEGF-A antibody was purchased from Santa Cruz Biotech (Cat No: SC-7269) and β-Actin was purchased from Sigma (Cat No: A2103).

    Techniques: Enzyme-linked Immunosorbent Assay

    Three-compartment model of VEGF in peripheral arterial disease. 121: VEGF 121 . 165a: VEGF 165a . 165b: VEGF 165b . N: neuropilin-1. 1: soluble and membrane-bound VEGFR1. 2: VEGFR2. GAG: glycosaminoglycan. α2M: alpha-2 macroglobulin.

    Journal: Scientific Reports

    Article Title: A multiscale computational model predicts distribution of anti-angiogenic isoform VEGF165b in peripheral arterial disease in human and mouse

    doi: 10.1038/srep37030

    Figure Lengend Snippet: Three-compartment model of VEGF in peripheral arterial disease. 121: VEGF 121 . 165a: VEGF 165a . 165b: VEGF 165b . N: neuropilin-1. 1: soluble and membrane-bound VEGFR1. 2: VEGFR2. GAG: glycosaminoglycan. α2M: alpha-2 macroglobulin.

    Article Snippet: Antibodies VEGF165b antibody was purchased from Millipore (Clone 56/1, Cat No: MABC595), VEGF-A antibody was purchased from Santa Cruz Biotech (Cat No: SC-7269) and β-Actin was purchased from Sigma (Cat No: A2103).

    Techniques:

    (A) Immunohistochemical staining of VEGF-A (magnification, ×400) in fractures. (a) In PTHKO and (b) WT mice 1 week after fracture, VEGF-A expression was significantly reduced in PTHKO mice compared with in WT mice. (c) In PTHKO and (d) WT mice 2 weeks after fracture, VEGF-A expression was increased in PTHKO mice, but remained significantly lower than that in WT mice. (B) Immunohistochemical staining of pVEGFR2 (magnification, ×400) in fractures. (a) In PTHKO and (b) WT mice 1 week after fracture, a significantly smaller number of pVEGFR2-positive cells was detected in the cartilaginous callus in PTHKO mice compared with in WT mice. (c) In PTHKO and (d) WT mice 2 weeks after fracture, a large number of pVEGFR2-positive cells was observed in the cartilaginous callus in WT mice, whereas a much lower level of angiogenesis was detected in PTHKO mice. (C) Immunohistochemical staining for HIF1α (magnification, ×400) in fractures. (a) In PTHKO and (b) WT mice 1 week after fracture, the expression levels of cytoplasmic HIF1α were significantly lower in PTHKO mice. (c) In PTHKO and (d) WT mice 2 weeks after fracture, HIF1α expression was increased in both groups; however, the expression remained lower in PTHKO mice compared with in WT mice. Black arrows indicate positive areas. (D) Protein expression levels of VEGF, pVEGFR2 and HIF1α were detected by western blot analysis. HIF1α, hypoxia inducible factor-1α; PTH, parathroid hormone; PTHKO, PTH knockout; pVEGFR, phosphorylated-VEGF receptor 2; VEGF, vascular endothelial growth factor; WT, wild-type.

    Journal: International Journal of Molecular Medicine

    Article Title: Lack of endogenous parathyroid hormone delays fracture healing by inhibiting vascular endothelial growth factor-mediated angiogenesis

    doi: 10.3892/ijmm.2018.3614

    Figure Lengend Snippet: (A) Immunohistochemical staining of VEGF-A (magnification, ×400) in fractures. (a) In PTHKO and (b) WT mice 1 week after fracture, VEGF-A expression was significantly reduced in PTHKO mice compared with in WT mice. (c) In PTHKO and (d) WT mice 2 weeks after fracture, VEGF-A expression was increased in PTHKO mice, but remained significantly lower than that in WT mice. (B) Immunohistochemical staining of pVEGFR2 (magnification, ×400) in fractures. (a) In PTHKO and (b) WT mice 1 week after fracture, a significantly smaller number of pVEGFR2-positive cells was detected in the cartilaginous callus in PTHKO mice compared with in WT mice. (c) In PTHKO and (d) WT mice 2 weeks after fracture, a large number of pVEGFR2-positive cells was observed in the cartilaginous callus in WT mice, whereas a much lower level of angiogenesis was detected in PTHKO mice. (C) Immunohistochemical staining for HIF1α (magnification, ×400) in fractures. (a) In PTHKO and (b) WT mice 1 week after fracture, the expression levels of cytoplasmic HIF1α were significantly lower in PTHKO mice. (c) In PTHKO and (d) WT mice 2 weeks after fracture, HIF1α expression was increased in both groups; however, the expression remained lower in PTHKO mice compared with in WT mice. Black arrows indicate positive areas. (D) Protein expression levels of VEGF, pVEGFR2 and HIF1α were detected by western blot analysis. HIF1α, hypoxia inducible factor-1α; PTH, parathroid hormone; PTHKO, PTH knockout; pVEGFR, phosphorylated-VEGF receptor 2; VEGF, vascular endothelial growth factor; WT, wild-type.

    Article Snippet: RUNX2 (ab76956; 1:600), HIF1α (ab8366; 1:600), pVEGF receptor 2 (pVEGFR2; ab131241; 1:200), proliferating cell nuclear antigen (PCNA; ab92552; 1:400), VEGF (ab46154; 1:200), PKA (ab75991) and pAKT monoclonal antibodies (ab81283) were purchased from Abcam (Cambridge, MA, USA).

    Techniques: Immunohistochemistry, Staining, Mouse Assay, Expressing, Western Blot, Knock-Out

    Fluorescence images of VEGF IR in detrusor smooth muscle in the bladder neck region in whole-mount preparations of urinary bladder in control rats ( A and B ) and rats treated with CYP for 4 h ( C and D ) and 48 h ( E and F ). VEGF IR in the detrusor smooth

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Upregulation of vascular endothelial growth factor isoform VEGF-164 and receptors (VEGFR-2, Npn-1, and Npn-2) in rats with cyclophosphamide-induced cystitis

    doi: 10.1152/ajprenal.90305.2008

    Figure Lengend Snippet: Fluorescence images of VEGF IR in detrusor smooth muscle in the bladder neck region in whole-mount preparations of urinary bladder in control rats ( A and B ) and rats treated with CYP for 4 h ( C and D ) and 48 h ( E and F ). VEGF IR in the detrusor smooth

    Article Snippet: Briefly, sections were incubated with 400 μl of rabbit anti-VEGF-A (1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) in 1% goat serum and 0.1 M phosphate buffer overnight at room temperature.

    Techniques: Fluorescence

    VEGF immunoreactivity (IR) in cryostat sections of urothelium after CYP treatment. Urothelium (U) was outlined (green) and measured in total-pixels area ( A , D , G , and J ). A threshold encompassing an intensity range of 100–250 gray-scale values

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Upregulation of vascular endothelial growth factor isoform VEGF-164 and receptors (VEGFR-2, Npn-1, and Npn-2) in rats with cyclophosphamide-induced cystitis

    doi: 10.1152/ajprenal.90305.2008

    Figure Lengend Snippet: VEGF immunoreactivity (IR) in cryostat sections of urothelium after CYP treatment. Urothelium (U) was outlined (green) and measured in total-pixels area ( A , D , G , and J ). A threshold encompassing an intensity range of 100–250 gray-scale values

    Article Snippet: Briefly, sections were incubated with 400 μl of rabbit anti-VEGF-A (1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) in 1% goat serum and 0.1 M phosphate buffer overnight at room temperature.

    Techniques:

    Regulation of urinary bladder VEGF co-receptor neuropilin-1 (Npn-1) transcript level in control rats, after 2–6 h of CYP treatment, and after chronic (10 day) CYP-induced bladder inflammation. Top : semiquantitative RT-PCR showing urinary bladder

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Upregulation of vascular endothelial growth factor isoform VEGF-164 and receptors (VEGFR-2, Npn-1, and Npn-2) in rats with cyclophosphamide-induced cystitis

    doi: 10.1152/ajprenal.90305.2008

    Figure Lengend Snippet: Regulation of urinary bladder VEGF co-receptor neuropilin-1 (Npn-1) transcript level in control rats, after 2–6 h of CYP treatment, and after chronic (10 day) CYP-induced bladder inflammation. Top : semiquantitative RT-PCR showing urinary bladder

    Article Snippet: Briefly, sections were incubated with 400 μl of rabbit anti-VEGF-A (1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) in 1% goat serum and 0.1 M phosphate buffer overnight at room temperature.

    Techniques: Reverse Transcription Polymerase Chain Reaction

    Regulation of urinary bladder VEGF co-receptor neuropilin-2 (Npn-2) transcript level in control rats, after 2–6 h of CYP treatment, and after chronic (10 day) CYP-induced bladder inflammation. Top : semiquantitative RT-PCR showing urinary bladder

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Upregulation of vascular endothelial growth factor isoform VEGF-164 and receptors (VEGFR-2, Npn-1, and Npn-2) in rats with cyclophosphamide-induced cystitis

    doi: 10.1152/ajprenal.90305.2008

    Figure Lengend Snippet: Regulation of urinary bladder VEGF co-receptor neuropilin-2 (Npn-2) transcript level in control rats, after 2–6 h of CYP treatment, and after chronic (10 day) CYP-induced bladder inflammation. Top : semiquantitative RT-PCR showing urinary bladder

    Article Snippet: Briefly, sections were incubated with 400 μl of rabbit anti-VEGF-A (1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) in 1% goat serum and 0.1 M phosphate buffer overnight at room temperature.

    Techniques: Reverse Transcription Polymerase Chain Reaction

    VEGF protein expression in the whole bladder with cyclophosphamide (CYP)-induced cystitis measured by ELISAs. Increases in VEGF protein expression were observed at all time points [2–48 h and 10 days (chronic)], but acute (2–4

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Upregulation of vascular endothelial growth factor isoform VEGF-164 and receptors (VEGFR-2, Npn-1, and Npn-2) in rats with cyclophosphamide-induced cystitis

    doi: 10.1152/ajprenal.90305.2008

    Figure Lengend Snippet: VEGF protein expression in the whole bladder with cyclophosphamide (CYP)-induced cystitis measured by ELISAs. Increases in VEGF protein expression were observed at all time points [2–48 h and 10 days (chronic)], but acute (2–4

    Article Snippet: Briefly, sections were incubated with 400 μl of rabbit anti-VEGF-A (1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) in 1% goat serum and 0.1 M phosphate buffer overnight at room temperature.

    Techniques: Expressing

    Semiquantitative analysis of VEGF IR in urothelium with CYP-induced cystitis. Values are means ± SE ( n = 6 for each group). * P ≤ 0.05.

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Upregulation of vascular endothelial growth factor isoform VEGF-164 and receptors (VEGFR-2, Npn-1, and Npn-2) in rats with cyclophosphamide-induced cystitis

    doi: 10.1152/ajprenal.90305.2008

    Figure Lengend Snippet: Semiquantitative analysis of VEGF IR in urothelium with CYP-induced cystitis. Values are means ± SE ( n = 6 for each group). * P ≤ 0.05.

    Article Snippet: Briefly, sections were incubated with 400 μl of rabbit anti-VEGF-A (1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) in 1% goat serum and 0.1 M phosphate buffer overnight at room temperature.

    Techniques:

    Fluorescence images of VEGF IR in suburothelial vasculature in the bladder neck region in whole-mount preparations of urinary bladder in control rats ( A and B ) and rats treated with CYP for 4 h ( C and D ) and 48 h ( E and F ). VEGF IR in the vasculature

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Upregulation of vascular endothelial growth factor isoform VEGF-164 and receptors (VEGFR-2, Npn-1, and Npn-2) in rats with cyclophosphamide-induced cystitis

    doi: 10.1152/ajprenal.90305.2008

    Figure Lengend Snippet: Fluorescence images of VEGF IR in suburothelial vasculature in the bladder neck region in whole-mount preparations of urinary bladder in control rats ( A and B ) and rats treated with CYP for 4 h ( C and D ) and 48 h ( E and F ). VEGF IR in the vasculature

    Article Snippet: Briefly, sections were incubated with 400 μl of rabbit anti-VEGF-A (1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) in 1% goat serum and 0.1 M phosphate buffer overnight at room temperature.

    Techniques: Fluorescence

    Long-term effects of intravitreally injected anti-VEGF antibody on BAT. (A) Quantitative analyses of the number of large lipid droplets ( > 50 μm 2 ) per field at x400 magnification ( n = 3–6). The effects of anti-VEGF antibody were quantitatively analyzed by comparison to the group treated with intravitreal PBS injection as 100%. (B) Quantitative analyses of vascularity of interscapular BAT demonstrated by isolectin B4 staining ( n = 3–6). The effects of anti-VEGF antibody were quantitatively analyzed by comparison to the group treated with intravitreal PBS injection as 100%. (C) The changes in body weight from P14 to P56. Anti-VEGF, anti-VEGF antibody. NS , not significant (two-tailed, unpaired T-test).

    Journal: PLoS ONE

    Article Title: Intravitreally Injected Anti-VEGF Antibody Reduces Brown Fat in Neonatal Mice

    doi: 10.1371/journal.pone.0134308

    Figure Lengend Snippet: Long-term effects of intravitreally injected anti-VEGF antibody on BAT. (A) Quantitative analyses of the number of large lipid droplets ( > 50 μm 2 ) per field at x400 magnification ( n = 3–6). The effects of anti-VEGF antibody were quantitatively analyzed by comparison to the group treated with intravitreal PBS injection as 100%. (B) Quantitative analyses of vascularity of interscapular BAT demonstrated by isolectin B4 staining ( n = 3–6). The effects of anti-VEGF antibody were quantitatively analyzed by comparison to the group treated with intravitreal PBS injection as 100%. (C) The changes in body weight from P14 to P56. Anti-VEGF, anti-VEGF antibody. NS , not significant (two-tailed, unpaired T-test).

    Article Snippet: Long-term effects of intravitreally injected anti-VEGF antibody on BAT. (A) Representative images of H & E staining of interscapular BAT after intravitreal injection of PBS or anti-VEGF antibody.

    Techniques: Injection, Staining, Two Tailed Test

    Ocular and systemic consequences of intravitreally injected anti-VEGF antibody. (A) Effects of intravitreally injected anti-VEGF antibody (1 μg/eye) on retinal neovascularization in OIR mice ( n = 6). Neovascular tufts were highlighted with yellow pseudocolor on representative images of isolectin B4-stained retina. The area of neovascular tufts was normalized to total retinal area; then, the effects of anti-VEGF antibody were quantified and normalized to the control (intravitreal PBS injection). Scale bar, 200 μm. (B) Retinal VEGF concentrations at P17 with intravitreal injection of PBS or anti-VEGF antibody ( n = 3). The level of VEGF was normalized to total amounts of proteins in the retina. (C) Serum concentrations of anti-VEGF antibody after intravitreal injection at P14, P17, and P21 ( n = 3–6). (D) Serum VEGF concentrations after intravitreal injection of anti-VEGF antibody at P17, P21, and P28 ( n = 3–6). Data are presented as mean ± SEM in graphs. Anti-VEGF, anti-VEGF antibody. NS , not significant; **, P

    Journal: PLoS ONE

    Article Title: Intravitreally Injected Anti-VEGF Antibody Reduces Brown Fat in Neonatal Mice

    doi: 10.1371/journal.pone.0134308

    Figure Lengend Snippet: Ocular and systemic consequences of intravitreally injected anti-VEGF antibody. (A) Effects of intravitreally injected anti-VEGF antibody (1 μg/eye) on retinal neovascularization in OIR mice ( n = 6). Neovascular tufts were highlighted with yellow pseudocolor on representative images of isolectin B4-stained retina. The area of neovascular tufts was normalized to total retinal area; then, the effects of anti-VEGF antibody were quantified and normalized to the control (intravitreal PBS injection). Scale bar, 200 μm. (B) Retinal VEGF concentrations at P17 with intravitreal injection of PBS or anti-VEGF antibody ( n = 3). The level of VEGF was normalized to total amounts of proteins in the retina. (C) Serum concentrations of anti-VEGF antibody after intravitreal injection at P14, P17, and P21 ( n = 3–6). (D) Serum VEGF concentrations after intravitreal injection of anti-VEGF antibody at P17, P21, and P28 ( n = 3–6). Data are presented as mean ± SEM in graphs. Anti-VEGF, anti-VEGF antibody. NS , not significant; **, P

    Article Snippet: Long-term effects of intravitreally injected anti-VEGF antibody on BAT. (A) Representative images of H & E staining of interscapular BAT after intravitreal injection of PBS or anti-VEGF antibody.

    Techniques: Injection, Mouse Assay, Staining

    Effects of intravitreally injected anti-VEGF antibody on BAT of neonatal mice. (A) Concentrations of VEGF in interscapular BAT at P21 and P28. The level of VEGF was normalized to total amounts of proteins in BAT ( n = 3–6). (B) Representative images of H E staining of interscapular BAT after intravitreal injection of PBS or anti-VEGF antibody show enlarged lipid droplets. Scale bar, 20 μm. (C) Quantitative analyses of vascularity of interscapular BAT based on isolectin B4 staining ( n = 3–6). The effects of anti-VEGF antibody were quantified and normalized to the control (intravitreal PBS injection). (D) Relative expression of Ucp1 and Ppargc1a in interscapular BAT ( n = 3–6). Data are presented as mean ± SEM in graphs. Anti-VEGF, anti-VEGF antibody. *, P

    Journal: PLoS ONE

    Article Title: Intravitreally Injected Anti-VEGF Antibody Reduces Brown Fat in Neonatal Mice

    doi: 10.1371/journal.pone.0134308

    Figure Lengend Snippet: Effects of intravitreally injected anti-VEGF antibody on BAT of neonatal mice. (A) Concentrations of VEGF in interscapular BAT at P21 and P28. The level of VEGF was normalized to total amounts of proteins in BAT ( n = 3–6). (B) Representative images of H E staining of interscapular BAT after intravitreal injection of PBS or anti-VEGF antibody show enlarged lipid droplets. Scale bar, 20 μm. (C) Quantitative analyses of vascularity of interscapular BAT based on isolectin B4 staining ( n = 3–6). The effects of anti-VEGF antibody were quantified and normalized to the control (intravitreal PBS injection). (D) Relative expression of Ucp1 and Ppargc1a in interscapular BAT ( n = 3–6). Data are presented as mean ± SEM in graphs. Anti-VEGF, anti-VEGF antibody. *, P

    Article Snippet: Long-term effects of intravitreally injected anti-VEGF antibody on BAT. (A) Representative images of H & E staining of interscapular BAT after intravitreal injection of PBS or anti-VEGF antibody.

    Techniques: Injection, Mouse Assay, Staining, Expressing

    Schematic depiction of RPE cell structure / functions with associated retinal disorders Cellular retinol binding protein (CRBP) binds all-trans-retinol (atROL) and directs it the smooth endoplasmic reticulum where lecithin:retinol acyltransferase (LRAT) converts all- trans -retinol to all- trans -retinyl ester which is then converted to 11- cis -retinol (11cROL) by RPE65 and to 11-cis retinal (11cRAL) by 11-cis-retinol dehydrogenase (11cRDH). Visual cycle proteins associated with forms of retinal degeneration include RPE65 (Leber congenital amaurosis, LCA; severe retinitis pigmentosa, RP); CRALBP and 11cRDH (encoded by gene retinol dehydrogenase 5, RDH5). The sodium/potassium adenosine triphosphatase (Na + /K + -ATPase) mediates outflux of sodium (Na + ) and influx of potassium (K + ). The c-mer tyrosine kinase (MERTK) receptor is responsible for internalization of shed out segment membrane. Mutations in MERTK are associated with severe retinitis pigmentosa (RP). Melanin pigment is contained within melanosomes; disruptions in melanin or melanosome biogenesis leads to albinism. Lipofuscin accumulates with excess in ABCA4-related disease, Stargardt-like macular dystrophy (STGD3), Best vitelliform macular dystrophy (BVMD) and adult-onset vitelliform macular dystrophy (AVMD); lipofuscin is also implicated in the pathogenesis of AMD. RPE cell secretion of vascular endothelial growth factor (VEGF) is implicated in neovascular age-related macular degeneration (NV-AMD). Junctional complexes between adjacent RPE cells constitute the outer blood-retinal barrier. Mutations in the protein bestrophin (hBEST1) are associated with Best vitelliform macular dystrophy (BVMD) and adult-onset vitelliform macular dystrophy (AVMD). The basal surface of the RPE rests on a basement membrane - Bruch’s membrane - that is implicated in age-related macular degeneration (AMD) and Sorby’s fundus dystrophy (SFD). See text for details.

    Journal: Current molecular medicine

    Article Title: The Retinal Pigment Epithelium in Health and Disease

    doi:

    Figure Lengend Snippet: Schematic depiction of RPE cell structure / functions with associated retinal disorders Cellular retinol binding protein (CRBP) binds all-trans-retinol (atROL) and directs it the smooth endoplasmic reticulum where lecithin:retinol acyltransferase (LRAT) converts all- trans -retinol to all- trans -retinyl ester which is then converted to 11- cis -retinol (11cROL) by RPE65 and to 11-cis retinal (11cRAL) by 11-cis-retinol dehydrogenase (11cRDH). Visual cycle proteins associated with forms of retinal degeneration include RPE65 (Leber congenital amaurosis, LCA; severe retinitis pigmentosa, RP); CRALBP and 11cRDH (encoded by gene retinol dehydrogenase 5, RDH5). The sodium/potassium adenosine triphosphatase (Na + /K + -ATPase) mediates outflux of sodium (Na + ) and influx of potassium (K + ). The c-mer tyrosine kinase (MERTK) receptor is responsible for internalization of shed out segment membrane. Mutations in MERTK are associated with severe retinitis pigmentosa (RP). Melanin pigment is contained within melanosomes; disruptions in melanin or melanosome biogenesis leads to albinism. Lipofuscin accumulates with excess in ABCA4-related disease, Stargardt-like macular dystrophy (STGD3), Best vitelliform macular dystrophy (BVMD) and adult-onset vitelliform macular dystrophy (AVMD); lipofuscin is also implicated in the pathogenesis of AMD. RPE cell secretion of vascular endothelial growth factor (VEGF) is implicated in neovascular age-related macular degeneration (NV-AMD). Junctional complexes between adjacent RPE cells constitute the outer blood-retinal barrier. Mutations in the protein bestrophin (hBEST1) are associated with Best vitelliform macular dystrophy (BVMD) and adult-onset vitelliform macular dystrophy (AVMD). The basal surface of the RPE rests on a basement membrane - Bruch’s membrane - that is implicated in age-related macular degeneration (AMD) and Sorby’s fundus dystrophy (SFD). See text for details.

    Article Snippet: Therapeutic approaches to the treatment of neovascular AMD include three anti-VEGF agents, ranibizumab (Lucentis, Genentech Inc, San Francisco CA), bevacizumab (Avastin; Genentech Inc) and pegaptanib (Macugen, Eyetech Pharmaceuticals, New York) [ ].

    Techniques: Binding Assay

    VEGF-A expression is activated in the retinas of OIR mice. (A) Immunoblot analysis of the protein expression levels of VEGF-A and HIF-1α in the retinas. (B) Quantification revealed an increase in the expression levels of VEGF-A and HIF-1α in the retinas of the OIR mice compared with WT mice. The relative protein expression level was normalized to GAPDH (n=3 mice per group). Data are presented as the mean ± standard deviation of the mean. *P

    Journal: Molecular Medicine Reports

    Article Title: Oxidative stress, autophagy and pyroptosis in the neovascularization of oxygen-induced retinopathy in mice

    doi: 10.3892/mmr.2018.9759

    Figure Lengend Snippet: VEGF-A expression is activated in the retinas of OIR mice. (A) Immunoblot analysis of the protein expression levels of VEGF-A and HIF-1α in the retinas. (B) Quantification revealed an increase in the expression levels of VEGF-A and HIF-1α in the retinas of the OIR mice compared with WT mice. The relative protein expression level was normalized to GAPDH (n=3 mice per group). Data are presented as the mean ± standard deviation of the mean. *P

    Article Snippet: Following electrophoresis and wet transfer to a polyvinylidene difluoride (PVDF) membrane, the membrane was blocked in 5% skim milk in TBS with Tween-20 (TBST) at room temperature for 1 h. Immunostaining was conducted using antibodies against VEGF-A (cat. no. ab52917; 1:500), caspase-1 (cat. no. ab1872; 1:1,000), pro-caspase-1 (cat. no. ab179515; 1:1,000), interleukin (IL)-1β (cat. no. ab2105; 1:500), pro-IL-1β (cat. no. ab2105; 1:500), microtubule associated protein 1 light chain 3α (LC3; cat. no. ab48394; 1:500) (Abcam, Cambridge, MA, USA), HIF-1α (cat. no. 36169; 1:1,000), autophagy protein (Atg)5 (cat. no. 12994; 1:1,000), Atg7 (cat. no. 8558; 1:1,000), Atg12 (cat. no. 4180; 1:1,000), Beclin1 (cat. no. 3495; 1:1,000), p62 (cat. no. 23214; 1:1,000), NOD-like receptor family pyrin domain-containing 3 (NLRP3; cat. no. 15101; 1:1,000) and GAPDH (cat. no. 5174; 1:1,000; Cell Signaling Technology, Inc, Danvers, MA, USA) at 4°C overnight.

    Techniques: Expressing, Mouse Assay, Standard Deviation

    Schematic illustration of the experimental procedure that was followed. (1) sutures were placed intrastromally into the temporal cornea, and immediately followed by topical application of eye drops (IgG, anti- Vegf or dexamethasone). Eye drops were applied until the 48 h time point. At t=48 h, IVCM and slit lamp data was collected and used for phenotypic characterisation. (2) cornea tissue was harvested and used for RNA extraction, and RNA quality verified. (3) high quality RNA was used for target preparation for microarray hybridisation on to GeneChip Rat 230 2.0 microarray chips. The microarray chips were scanned and image files acquired. (4) CEL files were normalised using expression console software. The generated CHP together with the CEL files were submitted to Gene Expression Omnibus repository.

    Journal: Scientific Data

    Article Title: Genome-wide expression datasets of anti-VEGF and dexamethasone treatment of angiogenesis in the rat cornea

    doi: 10.1038/sdata.2017.111

    Figure Lengend Snippet: Schematic illustration of the experimental procedure that was followed. (1) sutures were placed intrastromally into the temporal cornea, and immediately followed by topical application of eye drops (IgG, anti- Vegf or dexamethasone). Eye drops were applied until the 48 h time point. At t=48 h, IVCM and slit lamp data was collected and used for phenotypic characterisation. (2) cornea tissue was harvested and used for RNA extraction, and RNA quality verified. (3) high quality RNA was used for target preparation for microarray hybridisation on to GeneChip Rat 230 2.0 microarray chips. The microarray chips were scanned and image files acquired. (4) CEL files were normalised using expression console software. The generated CHP together with the CEL files were submitted to Gene Expression Omnibus repository.

    Article Snippet: Treatment regime Three groups of six rats each were treated with one of three topical treatments: IgG (Cat. No. 108-C, R & D Systems) at 20 μgml−1 , anti-Vegf (Cat. No AF 564, a neutralizing rat-specific goat polyclonal pan-VEGFA antibody, R & D Systems, Minneaplois MN, USA) at 20 μgml−1 or dexamethasone (Opnol, Clean Chemical, Sweden AB, Borlänge, Sweden) at 1 mgml−1 .

    Techniques: RNA Extraction, Microarray, Hybridization, Expressing, Software, Generated

    A correlation analysis within treatment group. ( a – d ) are signal intensity values correlated between control, IgG, anti- Vegf and dexamethasone treated samples respectively. ( e ) is an example of pathway enrichment analysis and ( f ) is a display of the genes involved in a selected pathway (PI3K-Akt signalling pathway).

    Journal: Scientific Data

    Article Title: Genome-wide expression datasets of anti-VEGF and dexamethasone treatment of angiogenesis in the rat cornea

    doi: 10.1038/sdata.2017.111

    Figure Lengend Snippet: A correlation analysis within treatment group. ( a – d ) are signal intensity values correlated between control, IgG, anti- Vegf and dexamethasone treated samples respectively. ( e ) is an example of pathway enrichment analysis and ( f ) is a display of the genes involved in a selected pathway (PI3K-Akt signalling pathway).

    Article Snippet: Treatment regime Three groups of six rats each were treated with one of three topical treatments: IgG (Cat. No. 108-C, R & D Systems) at 20 μgml−1 , anti-Vegf (Cat. No AF 564, a neutralizing rat-specific goat polyclonal pan-VEGFA antibody, R & D Systems, Minneaplois MN, USA) at 20 μgml−1 or dexamethasone (Opnol, Clean Chemical, Sweden AB, Borlänge, Sweden) at 1 mgml−1 .

    Techniques: