Journal: International journal of molecular sciences

Article Title: Finerenone, a Non-Steroidal Mineralocorticoid Receptor Antagonist, Reduces Vascular Injury and Increases Regulatory T-Cells: Studies in Rodents with Diabetic and Neovascular Retinopathy.

doi: 10.3390/ijms24032334

Figure Lengend Snippet: Figure 3. Finerenone reduced VEGF and vascular leakage in (mRen-2)27 after 12 weeks of diabetes. D, diabetic. Veh, vehicle. Per, perindopril. Fin, finerenone. (A) Three-micrometer paraffin sections of retina immunolabeled with VEGF and counterstained with hematoxylin. GCL, ganglion cell layer. IPL, inner plexiform layer. INL, inner nuclear layer. Scale bar = 40 µm. Ganglion cells denoted by single asterisk (*). Glial cells denoted by double asterisks (**). (B) Quantitation of VEGF immunolabeling in the retina. * p < 0.05 to diabetic+vehicle. n = 4 to 5 rats per group. (C) Vitreal levels of albumin (ELISA). * p < 0.05 to non-diabetic and diabetic+vehicle. # p < 0.05 to non-diabetic. n = 7 to 8 rats per group. Values are mean ± SEM.

Article Snippet: Immunohistochemistry for VEGF was performed by incubating three-micrometer paraffin sections with normal donkey serum for 1 h (D9663, Sigma-Aldrich), and then goat anti-rat VEGF (1:500, 564-RV, R&D Systems, Minneapolis, Minnesota, USA) overnight at 4 ◦C.

Techniques: Immunolabeling, Quantitation Assay, Enzyme-linked Immunosorbent Assay

Journal: International journal of molecular sciences

Article Title: Finerenone, a Non-Steroidal Mineralocorticoid Receptor Antagonist, Reduces Vascular Injury and Increases Regulatory T-Cells: Studies in Rodents with Diabetic and Neovascular Retinopathy.

doi: 10.3390/ijms24032334

Figure Lengend Snippet: Figure 5. Finerenone reduced retinal neovascularization, VEGF, and vascular leakage in mice with OIR at postnatal day 18. RA, room air control. Veh, vehicle. Fin, finerenone. (A) Retinal wholemounts labeled with FITC-isolectin to delineate retinal blood vessels in green. Top panels show whole retina. A quadrant of retina (yellow box) is magnified in the lower panel. Scale bar = 500 µm. Asterisks denote vaso-obliteration. Arrows denote neovascularization. (B) Quantitation of retinal neovascularization. * p < 0.05 to OIR+vehicle. n = 5 to 7 mice per group from 2 to 3 liters per group. (C) Retinal VEGF protein levels (ELISA). (D) Retinal VEGF mRNA levels. (E) Retinal vascular leakage (albumin ELISA). ** p < 0.01, **** p < 0.0001 to RA. # p < 0.05, ### p < 0.001 to OIR + vehicle (Kruskal–Wallis test). n = 5 to 8 mice per group from 2 to 3 litters of mice per group. Values are mean ± SEM.

Article Snippet: Immunohistochemistry for VEGF was performed by incubating three-micrometer paraffin sections with normal donkey serum for 1 h (D9663, Sigma-Aldrich), and then goat anti-rat VEGF (1:500, 564-RV, R&D Systems, Minneapolis, Minnesota, USA) overnight at 4 ◦C.

Techniques: Control, Labeling, Quantitation Assay, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Platelet-Derived Growth Factor-BB Controls Epithelial Tumor Phenotype by Differential Growth Factor Regulation in Stromal Cells

doi: 10.2353/ajpath.2006.060120

Figure Lengend Snippet: Paracrine induction of angiogenesis. A: In vitro, PDGF-BB treatment (50 ng/ml) has no effect on VEGFR-1 and -2 mRNA expression in human microvascular endothelial cells, as demonstrated by RT-PCR. GAPDH was used as internal standard (top). Endothelial cells (ECs) do not express the PDGFR subunits, in contrast to human dermal fibroblasts (HDFs) (bottom). B and C: In vivo, indirect immunofluorescence of 2-week-old PDGF-B transplants confirmed the lack of the PDGFR-β (green) on CD31-positive blood vessels (red) by lack of a yellow overlay color, nuclei stained with Hoechst/bisbenzimide (blue). D and E: Macrophage staining with the ERMP-23 antibody (red) at week 3 reveals partial overlap with signals for PDGFR-β (green), macrophages marked by arrows. F: VEGF-A mRNA is strongly up-regulated in human dermal fibroblasts in response to short-term stimulation with 50 ng/ml of PDGF-BB for 72 hours, as analyzed by RT-PCR. GAPDH was used as an internal standard. G: PDGF-BB (50 ng/ml)-stimulated fibroblast cultures for 72 hours demonstrate strongly increased amounts of VEGF-A protein in supernatants, as determined by ELISA. Each bar represents the mean ± SD from three independent experiments, *P < 0.05. Scale bars = 200 μm (B, C); 50 μm (D, E).

Article Snippet: Primary antibodies used were: rat monoclonal antibody against mouse CD31 (BD PharMingen, Heidelberg, Germany), guinea pig pan-keratin antiserum (Progen, Heidelberg, Germany), sheep polyclonal antibody against BrdU (NatuTec, Frankfurt, Germany), rabbit collagen type IV antibody (Novotec, Lyon, France), biotinylated mouse monoclonal antibody against α-smooth muscle actin (α-SMA) (Progen), rat monoclonal antibody against macrophages (ERMP-23; Acris, Bad Nauheim, Germany), rat monoclonal antibody against mouse neutrophil granulocytes (Serotec, Düsseldorf, Germany), rat affinity-purified polyclonal antibody sc-432 against PDGFR-β (Santa-Cruz Biotechnology, Inc., Heidelberg, Germany), goat affinity-purified antibody against murine VEGF (R&D Systems, Wiesbaden, Germany), goat affinity-purified antibody against murine HGF (R&D Systems), and goat affinity-purified antibody against murine endostatin (R&D Systems).

Techniques: In Vitro, Expressing, Reverse Transcription Polymerase Chain Reaction, In Vivo, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Platelet-Derived Growth Factor-BB Controls Epithelial Tumor Phenotype by Differential Growth Factor Regulation in Stromal Cells

doi: 10.2353/ajpath.2006.060120

Figure Lengend Snippet: Effects of PDGF-BB on growth factor expression by fibroblasts. A–C: Human dermal fibroblasts were stimulated with different concentrations of PDGF-BB for 16 days, stimulation was performed every second day. Conditioned media were analyzed by ELISA, and the cell number was determined. The mRNA expression of HGF was analyzed by RT-PCR. The graphs show mean values of representative experiments ± SD. At least two independent experiments with duplicate values were performed, *P < 0.05. A: VEGF-A protein was down-regulated by long-term treatment with PDGF-BB in a dose-dependent manner from day 4 to day 16. B: Determination of the cell number demonstrated dose-dependent mitogenic effects of PDGF-BB on fibroblasts throughout 16 days of stimulation. C: HGF protein expression (graph) and mRNA (figure) were dramatically increased on long-term PDGF-BB treatment for 10 or 16 days. D: The receptor for HGF, c-met, is constitutively expressed in normal HaCaT cells (lane 1), HaCaT control transfectants (lane 2), and HaCaT PDGF-B transfectants (lane 3) as analyzed by RT-PCR. GAPDH was used as internal control.

Article Snippet: Primary antibodies used were: rat monoclonal antibody against mouse CD31 (BD PharMingen, Heidelberg, Germany), guinea pig pan-keratin antiserum (Progen, Heidelberg, Germany), sheep polyclonal antibody against BrdU (NatuTec, Frankfurt, Germany), rabbit collagen type IV antibody (Novotec, Lyon, France), biotinylated mouse monoclonal antibody against α-smooth muscle actin (α-SMA) (Progen), rat monoclonal antibody against macrophages (ERMP-23; Acris, Bad Nauheim, Germany), rat monoclonal antibody against mouse neutrophil granulocytes (Serotec, Düsseldorf, Germany), rat affinity-purified polyclonal antibody sc-432 against PDGFR-β (Santa-Cruz Biotechnology, Inc., Heidelberg, Germany), goat affinity-purified antibody against murine VEGF (R&D Systems, Wiesbaden, Germany), goat affinity-purified antibody against murine HGF (R&D Systems), and goat affinity-purified antibody against murine endostatin (R&D Systems).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction

Journal:

Article Title: Platelet-Derived Growth Factor-BB Controls Epithelial Tumor Phenotype by Differential Growth Factor Regulation in Stromal Cells

doi: 10.2353/ajpath.2006.060120

Figure Lengend Snippet: PDGF-BB has a transient stimulatory effect on stromal VEGF production and a sustained enhancing effect on stromal HGF production. A–D, I, J, M: Immunofluorescence staining for murine VEGF (red) in frozen sections of 2- and 6-week-old transplants. Counterstaining of macrophages (A–D, I, J) and PDGFR-β (M) in green, of nuclei by Hoechst/bisbenzimide. Note the strong staining for mVEGF in the stroma of 2-week-old PDGF transplants (A, I) in contrast to the controls (B) and the reduced VEGF expression at week 6 (C, J), nearly to control level (D). Stromal macrophages are mostly negative for VEGF staining (A, C, I, J), whereas signals overlap with the PDGFR-β (M), resulting in a yellow overlay color. E–H, K, L, N: Immunofluorescence staining for murine HGF (red) in frozen sections of transplants at weeks 3 and 6. Macrophages (E, F, K), neutrophil granulocytes (G, H, L) and the PDGFR-β (N) are stained in green, nuclei with Hoechst/bisbenzimide in blue. The expression of stromal HGF is clearly enhanced in 3-week-old (E, K) and 6-week-old (G, L) PDGF transplants compared with age-matched controls (F, H). No overlap was detected with the signals for macrophages (E, F, K) and neutrophil granulocytes (G, H, L) in contrast to the overlap with the PDGFR-β (yellow) (N). Scale bars = 100 μm (A–H, I–L); 50 μm (M, N).

Article Snippet: Primary antibodies used were: rat monoclonal antibody against mouse CD31 (BD PharMingen, Heidelberg, Germany), guinea pig pan-keratin antiserum (Progen, Heidelberg, Germany), sheep polyclonal antibody against BrdU (NatuTec, Frankfurt, Germany), rabbit collagen type IV antibody (Novotec, Lyon, France), biotinylated mouse monoclonal antibody against α-smooth muscle actin (α-SMA) (Progen), rat monoclonal antibody against macrophages (ERMP-23; Acris, Bad Nauheim, Germany), rat monoclonal antibody against mouse neutrophil granulocytes (Serotec, Düsseldorf, Germany), rat affinity-purified polyclonal antibody sc-432 against PDGFR-β (Santa-Cruz Biotechnology, Inc., Heidelberg, Germany), goat affinity-purified antibody against murine VEGF (R&D Systems, Wiesbaden, Germany), goat affinity-purified antibody against murine HGF (R&D Systems), and goat affinity-purified antibody against murine endostatin (R&D Systems).

Techniques: Immunofluorescence, Staining, Expressing

Journal:

Article Title: Platelet-Derived Growth Factor-BB Controls Epithelial Tumor Phenotype by Differential Growth Factor Regulation in Stromal Cells

doi: 10.2353/ajpath.2006.060120

Figure Lengend Snippet: Model for dual time-dependent effects of PDGF-BB. PDGF-BB exerts dual time-dependent effects on stromal fibroblasts that provide a possible explanation for the benign tumor phenotype. In the early phase (left), PDGF-BB secreted by the epithelium activates PDGFR-positive fibroblasts, leading to the up-regulation of VEGF-A and HGF. VEGF-A in turn activates the dermal endothelial cells devoid of PDGFR, up-regulates their VEGFR expression, and induces angiogenesis, possibly also supported by the action of HGF. HGF as potent stimulatory factor for epithelial cells is considered to promote epithelial proliferation. During late phase (right), prolonged exposure to PDGF-BB down-regulates VEGF secretion by dermal fibroblasts, an effect that could be mediated by endostatin up-regulation, induces their differentiation into myofibroblasts and promotes the association of SMA-positive cells with blood vessel, thus promoting vessel maturation. Blood vessel maturation is accompanied by VEGFR down-regulation. At the same time, the strong and ongoing increase in HGF secretion continues to stimulate the proliferation of the epithelial cells.

Article Snippet: Primary antibodies used were: rat monoclonal antibody against mouse CD31 (BD PharMingen, Heidelberg, Germany), guinea pig pan-keratin antiserum (Progen, Heidelberg, Germany), sheep polyclonal antibody against BrdU (NatuTec, Frankfurt, Germany), rabbit collagen type IV antibody (Novotec, Lyon, France), biotinylated mouse monoclonal antibody against α-smooth muscle actin (α-SMA) (Progen), rat monoclonal antibody against macrophages (ERMP-23; Acris, Bad Nauheim, Germany), rat monoclonal antibody against mouse neutrophil granulocytes (Serotec, Düsseldorf, Germany), rat affinity-purified polyclonal antibody sc-432 against PDGFR-β (Santa-Cruz Biotechnology, Inc., Heidelberg, Germany), goat affinity-purified antibody against murine VEGF (R&D Systems, Wiesbaden, Germany), goat affinity-purified antibody against murine HGF (R&D Systems), and goat affinity-purified antibody against murine endostatin (R&D Systems).

Techniques: Expressing

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: Transient phenotypic changes in endothelial cells and pericytes in neonatal mouse retina following short-term blockade of vascular endothelial growth factor receptors.

doi: 10.1002/dvdy.24614

Figure Lengend Snippet: Fig. 10. Evaluation of VEGF expression in the retinas. A: Reverse transcription (RT)-PCR analysis to detect VEGF120 or VEGF164 mRNA. Bar graphs show the expression levels of VEGF120 or VEGF164 mRNA. Each column with a vertical bar represents the mean 6 SE for 4–5 animals. B: In situ hybridization for VEGF was combined with immunostaining for type IV collagen on retinal flat-mounts (a–c). C: Retinal flat-mounts stained for VEGF/type IV collagen (a, c, e) and PDGFRa/VEGF (b, d, f) from P6 mice of control group, P10 mice of P0/1 group, and P10 mice of P4/5 group. Scale bar, 100 mm in a (applies to b–f). Scale bar, B ¼ 100 mm. Scale bar, C ¼ 100 mm.

Article Snippet: The primary antibodies used were as follows: rat monoclonal anti-PECAM-1 antibody (1:500, clone MEC 13.3; BD Biosciences, San Diego, CA), hamster monoclonal anti-PECAM-1 antibody (1:500, clone 2H8; Chemicon, Temecula, CA, USA), rabbit polyclonal anti–type IV collagen (1:8000; Cosmo Bio, Tokyo, Japan), rabbit polyclonal anti-NG2 chondroitin sulfate proteoglycan (NG2, 1:500; EMD Millipore, Temecula, CA), Cy3-conjugated mouse monoclonal anti-aSMA antibody (1:500; Sigma-Aldrich, St. Louis, MO), rabbit monoclonal anti-PDGFRb antibody (1:500; Cell Signaling, Danvers, MA), and goat polyclonal anti-VEGF164 antibody (1:500; R&D Systems, Minneapolis, MN).

Techniques: Expressing, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, In Situ Hybridization, Immunostaining, Staining, Control