Journal: Investigative Ophthalmology & Visual Science

Article Title: VEGF-B Is an Autocrine Gliotrophic Factor for Müller Cells under Pathologic Conditions

doi: 10.1167/iovs.61.11.35

Figure Lengend Snippet: Concentration of Compounds Used for Functionality Assays and to Induce Different Stress Conditions

Article Snippet: VEGF-B murine recombinant , , , , 100 ng/mL , Cloud-Clone Corp. , rpa144muo1.

Techniques: Concentration Assay, Recombinant

Journal: Investigative Ophthalmology & Visual Science

Article Title: VEGF-B Is an Autocrine Gliotrophic Factor for Müller Cells under Pathologic Conditions

doi: 10.1167/iovs.61.11.35

Figure Lengend Snippet: The expression of VEGF family members and relevant receptors in Müller cells. The expression of VEGF family member and receptor was assessed at mRNA and protein levels by qPCR and ELISA in murine QMMuC-1 Müller cells, PMCs, and human MIO-M1 cells under normal culture conditions. ( A – C ), mRNA expression of VEGF-A, -B, -C, -D, and PlGF in QMMuC-1 ( A ), PMCs ( B ), and human MIO-M1 ( C ). ( D ) Protein quantification by ELISA of VEGF-A and VEGF-B in QMMuC-1 Müller cell lysates. ( E – H ), VEGF receptor expression in Müller cells. mRNA expression of VEGFR1, VEGFR2, and coreceptor NRP1 in murine Müller cell line QMMuC-1 cells ( E ), PMCs ( F ), and in human MIO-M1 Müller cells ( G ). ( H ) ELISA measurement of VEGFR1, VEGFR2 and NRP1 in QMMuC-1 cell lysates. n = 3 per group in PCR data and n = 4 in protein. • P < 0.05; •• P < 0.01 Student t test when two groups are compared. **** P < 0.001; *** P < 0.005; ** P < 0.01, * P < 0.05. One-way ANOVA followed by Newmann-Keuls post hoc test.

Article Snippet: VEGF-B murine recombinant , , , , 100 ng/mL , Cloud-Clone Corp. , rpa144muo1.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: VEGF-B Is an Autocrine Gliotrophic Factor for Müller Cells under Pathologic Conditions

doi: 10.1167/iovs.61.11.35

Figure Lengend Snippet: Expression of VEGF-B and its receptors VEGFR1 and NRP1 under different stress conditions. QMMuC-1 cells cultured under hypoxia (HYP, 1% O 2 ), high glucose (HG, 30 mM), or mannitol (MANN), IL-1β (20 ng/mL) and 4-HNE (10 µM) for 72 hours (for HYP and HG/MANN) or 24 hours (for IL-1β and 4-HNE) and the mRNA expression of VEGF-B ( A ), coreceptor NRP1 ( D ), and VEGFR1 ( F ) was determined by qPCR. Protein expression of VEGF-B in QMMuC-1 cell lysates ( B ) and supernatant ( C ), NRP1 and VEGFR1 ( E and G , respectively) and the soluble receptor sFLT-1 was assessed by ELISA. n = 3–5 per group in PCR, n = 5-8 in ELISA. * P < 0.05; ** P < 0.01; *** P < 0.005 Student t test, compared with respective control.

Article Snippet: VEGF-B murine recombinant , , , , 100 ng/mL , Cloud-Clone Corp. , rpa144muo1.

Techniques: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: VEGF-B Is an Autocrine Gliotrophic Factor for Müller Cells under Pathologic Conditions

doi: 10.1167/iovs.61.11.35

Figure Lengend Snippet: The effect of VEGF-B and its receptors on Müller cell viability under oxidative stress and hypoxic conditions. ( A ) Effect of the neutralization of VEGF-A and its receptors (VEGFR2 and NRP1) and VEGF-B and its receptors (VEGFR1 and NRP1) on QMMuC-1 Müller cell viability under hypoxic conditions (1% O 2 for 72 hours). * P < .05; ** P < .01 compared with control (hypoxia only), one-way ANOVA followed by Dunnett's multiple comparison test. ( B ) Effect of VEGF-A or VEGF-B neutralization on 4-HNE–induced QMMuC-1 Müller cell viability. C , Effect of the neutralization of VEGFR1 and NRP1 on QMMuC-1 and their receptor (VEGFR1 and NRP1) on 4-HNE–induced QMMuC-1 damage. ( D ) The effect of recombinant VEGF-B and VEGF-A on 4-HNE–induced cell death in QMMuC-1 and PMC Müller cells. The viability of cells was determined by Alamar Blue methods. ( A ) n = 5–9, ( B and C ) n = 4–6, ( D ) n = 5–10 per group. * P < 0.05; ** P < 0.01; *** P < 0.005 and **** P < 0.001, one-way ANOVA followed by Newmann-Keuls post hoc test. Antibody, AB. A and B are VEGF-A and VEGF-B respectively. R1 and R2 are VEGFR1 and VEGFR2. Anti-VEGF-A, VEGFR2 500 ng/mL and 10 µg/mL, respectively; NRP1 antibody, 30 µg/mL; VEGF-B and VEGFR1, 500 ng/mL and 10 µg/mL, respectively; recombinant VEGF-A and VEGF-B, 100 ng/mL; 4-HNE 20 µM.

Article Snippet: VEGF-B murine recombinant , , , , 100 ng/mL , Cloud-Clone Corp. , rpa144muo1.

Techniques: Neutralization, Recombinant

Journal: Investigative Ophthalmology & Visual Science

Article Title: VEGF-B Is an Autocrine Gliotrophic Factor for Müller Cells under Pathologic Conditions

doi: 10.1167/iovs.61.11.35

Figure Lengend Snippet: The effect of VEGF-B blockade on caspase-3 expression in Müller cells under hypoxia and oxidative stress conditions. ( A ) Expression of caspase-3 mRNA by qPCR in QMMuC-1 cells cultured under hypoxic conditions with or without VEGF-B neutralizing antibody (for 72 hours). ( B ) Caspase-3 mRNA expression in QMMuC-1 Müller cells treated with 4-HNE with or without VEGF-B neutralizing antibody. ( C ) Western Blot showing cleaved caspase-3 in different groups. ( D and E ) the effect of VEGF-B receptor blockage (anti-VEGFR1 and anti-NRP1) on the expression of caspase-3/7 in PMCs determined by immunocytochemistry. ( E ) Representative images showing caspase-3/7 expression in control and 4-HNE + VEGFR blockage group. Anti–VEGF-B and VEGFR1 antibodies, 500 ng/mL and 10 µg/mL, respectively; NRP1 antibody, 30 µg/mL; 4-HNE 10 µM. * P < 0.05; ** P < 0.01; *** P < 0.005, one-way ANOVA followed by a Newmann-Keuls post hoc test. Experiments performed at least twice, n = 3–4 per group in qPCR, and n = 4–6 in immunocytochemistry and Western Blot. Scale bar = 50 µM.

Article Snippet: VEGF-B murine recombinant , , , , 100 ng/mL , Cloud-Clone Corp. , rpa144muo1.

Techniques: Expressing, Cell Culture, Western Blot, Immunocytochemistry

Journal: Investigative Ophthalmology & Visual Science

Article Title: VEGF-B Is an Autocrine Gliotrophic Factor for Müller Cells under Pathologic Conditions

doi: 10.1167/iovs.61.11.35

Figure Lengend Snippet: The effect of VEGF-B on GS, Kir4.1 and AQP4 expression in Müller cells under hypoxia and oxidative stress. ( A ) QMMuC-1 cells were treated with recombinant VEGF-A or VEGF-B and the expression of GS was measured by Western blotting. ( B ) Western Blot analysis of GS expression in QMMuC-1 cells cultured in hypoxic conditions for 72 hours with or without recombinant VEGF-A or VEGF-B. ( C and D ) Kir4.1 mRNA ( C ) and protein ( D ) expression in hypoxia (1% O 2 for 72 hours) treated QMMuC-1 ( C , D , left ) and PMCs ( D , right ) with or without VEGF-B neutralizing antibody. ( E and F ) AQP4 mRNA ( E ) and protein ( F ) expression in QMMuC-1 cells in hypoxia condition with or without VEGF-B neutralization. ( G ) GS expression in QMMuC-1 cells under oxidative conditions (4-HNE) with/without VEGF-B neutralization. ( H and I ) AQP4 mRNA ( H ) and protein ( I ) expression in 4-HNE treated QMMuC-1 with or without VEGF-B neutralization. VEGF-B antibody, 500 ng/mL. 4-HNE, 10 µM. Data normalized to control values. n = 3–5/group in qPCR; n = 4–10 in Western Blot. * P < 0.05; ** P < 0.01; *** P < 0.005; **** P < 0.001. One-way ANOVA followed by Newmann-Keuls post hoc test. AB, antibody. Recomb, recombinant.

Article Snippet: VEGF-B murine recombinant , , , , 100 ng/mL , Cloud-Clone Corp. , rpa144muo1.

Techniques: Expressing, Recombinant, Western Blot, Cell Culture, Neutralization

Journal: Investigative Ophthalmology & Visual Science

Article Title: VEGF-B Is an Autocrine Gliotrophic Factor for Müller Cells under Pathologic Conditions

doi: 10.1167/iovs.61.11.35

Figure Lengend Snippet: Effect of VEGF-B on TRPV4 expression and functionality under oxidative stress. ( A – C ) QMMuC-1 cells were treated with 4-HNE (10 µM) with or without recombinant VEGF-B (100 ng/mL) for 24 hours. The expression of TRPV4 was examined by immunofluorescence staining ( A, B ) and Western Blot ( C ). ( A ) Representative image of TRPV4 ( green ) and phalloidin ( red ) in untreated control, 4-HNE treated and 4-HNE + recombinant VEGF-B (100 ng/mL) treated QMMuC-1 cells. Arrow in control indicates cluster aggregation of TRPV4. Arrows in 4-HNE–treated group indicates contracted cell body. ( B ) Quantitative analysis of different types of TRPV4-expressing QMMuC-1 cells (A, spread cell with evenly distributed TRPV4; B, spread cell with aggregated or uneven TRPV4 expression; C, contracted cell with aggregated TRPV4 expression). **** P < 0.001; n = 4 wells per experiment. Two-way ANOVA followed by Bonferroni post hoc test. ( C ) Western Blot of TRPV4 expression on 4-HNE–treated QMMuC-1 cells with/without recombinant VEGF-B, n = 6–7. ( D and E ) Effect of TRPV4 and VEGF-B on intracellular Ca 2+ concentration in 4-HNE–treated QMMuC-1 cells and its representative plots ( D ). Intracellular Ca 2+ levels were measured using the FlexStation. The y -axis was the 340 nm/380 nm fluorescence ratio (ΔR340 nm/380 nm). ( D and E ) TRPV4 agonist GSK101 (100 nM) alone did not elicit a measurable calcium response in QMMuC-1 cells. A 24 hours 4-HNE (10 µM) treatment significantly increased the GSK101-evoked response, which was fully blocked by the TRPV4 antagonist HC06 (10 µM) and by recombinant VEGF-B (100 ng/mL). Mean ± SD, n = 4–8 **** P < 0.001; one-way ANOVA followed by Newmann-Keuls post hoc test. All experiments were performed at least twice.

Article Snippet: VEGF-B murine recombinant , , , , 100 ng/mL , Cloud-Clone Corp. , rpa144muo1.

Techniques: Expressing, Recombinant, Immunofluorescence, Staining, Western Blot, Concentration Assay, Fluorescence