Journal: Developmental Cell

Article Title: Macrophages trigger cardiomyocyte proliferation by increasing epicardial vegfaa expression during larval zebrafish heart regeneration

doi: 10.1016/j.devcel.2022.05.014

Figure Lengend Snippet: Vegfaa drives cardiomyocyte proliferation by endocardial notch signaling (A) LSFM images of Tg(myl7:h2b-GFP) larvae at 24 hpi treated with PBS 0.1% BSA or zfVegfaa 0.1% BSA injection. (B) Ventricular cardiomyocyte number in Tg(myl7:h2b-GFP) larvae at 24 and 48 hpi treated with PBS 0.1% BSA or zfVegfaa 0.1% BSA injection, n = 20. Unpaired t test. (C) Images of injured ventricles from Tg(myl7:h2b-GFP) larvae, EdU stained and bathed in vehicle or AV951, imaged at 48 hpi. Non-myocardial EdU signal is excluded post-acquisitionally. (D) Percentage of EdU+ cardiomyocyte nuclei from uninjured and injured ventricles from Tg(myl7:h2b-GFP) larvae, EdU stained and bathed in vehicle or AV951, n = 13–36. Unpaired t test. (E) Images of injured Tg(myl7:nlsDsRed) larvae treated with vehicle or DAPT, acquired at 48 hpi by LSFM. (F) Ventricular cardiomyocyte number in uninjured and injured Tg(myl7:h2b-GFP) larvae at 48 hpi treated with vehicle or DAPT, n = 24–40. Unpaired t test. (G) Images of injured Tg(myl7:nlsDsRed) larvae treated with vehicle or AG1478, acquired at 48 hpi by LSFM. (H) Ventricular cardiomyocyte number in uninjured and injured Tg(myl7:h2b-GFP) larvae at 48 hpi treated with vehicle or AG1478, n = 24. Unpaired t test. (I) Treatment strategy for the injection of uninjured larvae with zfVegfaa and continuous bathing in AG1478 solution. (J) Hypothesized signaling pathway active in uninjured and injured larval hearts driving cardiomyocyte proliferation. (K) LSFM-acquired z plane showing notch expression colocalizing with endocardium in Tg(Tp1:venus-PEST;kdrl:hsa.HRAS-mCherry) , abbreviated in the figure to Tg(Tp1:venus-PEST;kdrl:mCherry) . AG1478 abbreviated to AG; white box, zoom panel. (L) Proportion of larvae with notch+ endocardium at 6, 24, and 48 hpt following zfVegfaa injection and bathing in AG1478, n = 28. Fisher’s exact test. (M) Treatment strategy for the lasering of larvae and continuous bathing in AG1478 solution. (N) Representative z plane images of uninjured, injured, and injured AG-treated ventricles from Tg(tp1:venus-PEST) larvae at 48 hpi. BA, bulbous arteriosus; AVV, atrioventricular valve; white arrowheads, laterally inhibited cardiomyocytes; cyan arrowheads, notch+ endocardium; cyan box, zoom panel. Fisher’s exact test. (O) Proportion of larvae with notch+ endocardium at 6, 24, and 48 hpt following laser injury and bathing in AG1478, n = 18. (P) Cardiomyocyte number at 48 hpi following injection with recombinant Vegfaa and continuous bathing in DAPT or AG1478, n = 22–25. One-way ANOVA followed by Holms-Sidak’s multiple comparison post-hoc tests. All images are maximum intensity projections of 3D LSFM stacks unless otherwise stated. Scale bars, 50 μm. Data are represented as mean ± SEM, ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, and ∗∗∗∗ p ≤ 0.0001.

Article Snippet: zfVegfaa , Kingfisher Biotech , Cat # RP1040Z-025.

Techniques: Injection, Staining, Expressing, Recombinant, Comparison

Journal: Developmental Cell

Article Title: Macrophages trigger cardiomyocyte proliferation by increasing epicardial vegfaa expression during larval zebrafish heart regeneration

doi: 10.1016/j.devcel.2022.05.014

Figure Lengend Snippet:

Article Snippet: zfVegfaa , Kingfisher Biotech , Cat # RP1040Z-025.

Techniques: Recombinant, In Situ, Imaging, Software

Journal: REPRODUCTION

Article Title: Possible action of vasohibin-1 as an inhibitor in the regulation of vascularization of the bovine corpus luteum

doi: 10.1530/rep-11-0465

Figure Lengend Snippet: Figure 6 VEGFA and VASH1-induced capillary-like tube formation by LyECs. The effects of VEGFA and VASH1 on capillary-like tube formation of cultured iliac LyECs in vitro in a matrigel assay are shown in figure. VEGFA at 10 and 100 ng/ml significantly stimulated capillary- like tube structure in cultured iliac LyECs relative to that by control cells at 8 h posttreatment (A). VASH1 had no individual effect on capillary- like tube formation, but it significantly inhibited VEGFA-induced capillary-like tube structure. Typical images of tube formed by cultured iliac LyECs in each experimental group are shown in B, C, D, E, Fand G. All values are shown as meanGS.E.M. (nZ4/group). Different super- script letters (a and b) indicate significant differences (P!0.05) as determined by ANOVA followed by Bonferroni’s multiple comparison test.

Article Snippet: DMEM/nutrient mixture and Ham’s F-12 medium (1:1), PBS, amphotericin B, gentamicin, Tween-20, anti-b-actin mouse monoclonal (clone AC-15 antibody), ECL Western Blotting Detection System, and 0.3% H2O2 were purchased from Sigma; BSA was purchased from Wako (Osaka, Japan); TRIzol reagent was purchased from Invitrogen Corporation; cell culture plates (24, 48, or 96 wells/plate) were purchased from Nunc (Roskilde, Denmark); optimal cutting temperature (OCT) compound was purchased from Sakura Finetechnical (Tokyo, Japan); GNRH was purchased from Intervet (Unterschleibheim, Germany); DNase using a commercial kit was purchased from SV total RNA Isolation System: Promega Co.; THE RNA storage Solution was purchased from Ambion, Inc. (Austin, TX, USA); DNA purification kit SUPRECTM-01 was purchased from TaKaRa Bio., Inc. (Otsu, Japan); 4% Block Ace Powder was purchased from DS Pharma Biomedical (Osaka, Japan); antimouse-LYVE1 rabbit polyclonal antibody was purchased from Abcam (Cambridge, UK); BD matrigel basement membrane was purchased from BD Biosciences (Bedford, MA, USA); recombinant bovine VEGFA was purchased from Kingfisher Biotech, Inc. (St Paul, MN, USA); Protease Inhibitor Cocktail and WST-1 were purchased from Roche; 70 mm filter (Cell Straner, REF 352350) was purchased from BD Falcon (Franklin Lakes, NJ, USA); PVDF membranes were purchased from Bio-Rad Laboratories (Hercules, CA, USA); lymphoprep was purchased from Axis-Shield (Oslo, Norway); HRP-conjugated anti-rabbit IgG antibodies were purchased from GE Healthcare (Ltd, Buckinghamshire, UK); HRP-conjugated anti-mouse IgG antibodies were purchased from Rockland Immunochemicals, Inc. (Gilbertsville, PA, USA); anti-human VWF rabbit polyclonal antibody (A0082) was purchased from Dako Denmark A/S (Glostrup, Denmark); biotinylated goat anti-rabbit BA-1000 www.reproduction-online.org IgG and avidin–biotin reagent (PK-6100, Vectastain ABC kit) were purchased from Vector Laboratories, Inc. (Burlingame, CA, USA); anti-human-VASH1 rabbit polyclonal antibody was purchased from Phoenix Pharmaceuticals, Inc. (Burlingame, CA, UK); VASH1 recombinant protein was purchased from Abnova Corporation (Taipei, Taiwan).

Techniques: Cell Culture, In Vitro, Matrigel Assay, Control, Comparison

Journal: REPRODUCTION

Article Title: Possible action of vasohibin-1 as an inhibitor in the regulation of vascularization of the bovine corpus luteum

doi: 10.1530/rep-11-0465

Figure Lengend Snippet: Figure 5 VEGFA and VASH1-induced capillary-like tube formation by LECs. The effects of VEGFA and VASH1 on capillary-like tube formation by cultured LECs in vitro in a matrigel assay are shown. VEGFA at 10 and 100 ng/ml significantly stimulated the capillary-like tube structure formation by cultured LECs relative to that by control cells at 8 h posttreatment (A). VASH1 had no individual effect on capillary-like tube formation, but it significantly inhibited VEGFA-induced capillary- like tube structure formation. Typical images of tube formed by cultured LECs in each experimental group are shown in B, C, D, E, F and G. All values are shown as meanGS.E.M. (nZ4/group). Different superscript letters (a and b) indicate significant differences (P!0.05) as determined by ANOVA followed by Bonferroni’s multiple comparison test.

Article Snippet: DMEM/nutrient mixture and Ham’s F-12 medium (1:1), PBS, amphotericin B, gentamicin, Tween-20, anti-b-actin mouse monoclonal (clone AC-15 antibody), ECL Western Blotting Detection System, and 0.3% H2O2 were purchased from Sigma; BSA was purchased from Wako (Osaka, Japan); TRIzol reagent was purchased from Invitrogen Corporation; cell culture plates (24, 48, or 96 wells/plate) were purchased from Nunc (Roskilde, Denmark); optimal cutting temperature (OCT) compound was purchased from Sakura Finetechnical (Tokyo, Japan); GNRH was purchased from Intervet (Unterschleibheim, Germany); DNase using a commercial kit was purchased from SV total RNA Isolation System: Promega Co.; THE RNA storage Solution was purchased from Ambion, Inc. (Austin, TX, USA); DNA purification kit SUPRECTM-01 was purchased from TaKaRa Bio., Inc. (Otsu, Japan); 4% Block Ace Powder was purchased from DS Pharma Biomedical (Osaka, Japan); antimouse-LYVE1 rabbit polyclonal antibody was purchased from Abcam (Cambridge, UK); BD matrigel basement membrane was purchased from BD Biosciences (Bedford, MA, USA); recombinant bovine VEGFA was purchased from Kingfisher Biotech, Inc. (St Paul, MN, USA); Protease Inhibitor Cocktail and WST-1 were purchased from Roche; 70 mm filter (Cell Straner, REF 352350) was purchased from BD Falcon (Franklin Lakes, NJ, USA); PVDF membranes were purchased from Bio-Rad Laboratories (Hercules, CA, USA); lymphoprep was purchased from Axis-Shield (Oslo, Norway); HRP-conjugated anti-rabbit IgG antibodies were purchased from GE Healthcare (Ltd, Buckinghamshire, UK); HRP-conjugated anti-mouse IgG antibodies were purchased from Rockland Immunochemicals, Inc. (Gilbertsville, PA, USA); anti-human VWF rabbit polyclonal antibody (A0082) was purchased from Dako Denmark A/S (Glostrup, Denmark); biotinylated goat anti-rabbit BA-1000 www.reproduction-online.org IgG and avidin–biotin reagent (PK-6100, Vectastain ABC kit) were purchased from Vector Laboratories, Inc. (Burlingame, CA, USA); anti-human-VASH1 rabbit polyclonal antibody was purchased from Phoenix Pharmaceuticals, Inc. (Burlingame, CA, UK); VASH1 recombinant protein was purchased from Abnova Corporation (Taipei, Taiwan).

Techniques: Cell Culture, In Vitro, Matrigel Assay, Control, Comparison

Journal: Developmental cell

Article Title: Macrophages trigger cardiomyocyte proliferation by increasing epicardial vegfaa expression during larval zebrafish heart regeneration.

doi: 10.1016/j.devcel.2022.05.014

Figure Lengend Snippet: Figure 7. Vegfaa drives cardiomyocyte proliferation by endocardial notch signaling (A) LSFM images of Tg(myl7:h2b-GFP) larvae at 24 hpi treated with PBS 0.1% BSA or zfVegfaa 0.1% BSA injection. (B) Ventricular cardiomyocyte number inTg(myl7:h2b-GFP) larvae at 24and 48hpi treated with PBS 0.1% BSA or zfVegfaa 0.1% BSA injection, n = 20. Unpaired t test. (C) Images of injured ventricles from Tg(myl7:h2b-GFP) larvae, EdU stained and bathed in vehicle or AV951, imaged at 48 hpi. Non-myocardial EdU signal is excluded post-acquisitionally. (D) Percentage of EdU+ cardiomyocyte nuclei from uninjured and injured ventricles from Tg(myl7:h2b-GFP) larvae, EdU stained and bathed in vehicle or AV951, n = 13–36. Unpaired t test. (E) Images of injured Tg(myl7:nlsDsRed) larvae treated with vehicle or DAPT, acquired at 48 hpi by LSFM. (F) Ventricular cardiomyocyte number in uninjured and injured Tg(myl7:h2b-GFP) larvae at 48 hpi treated with vehicle or DAPT, n = 24–40. Unpaired t test.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins Phenylthiourea Thermo Fisher Scientific Cat #L06690.09 Tricaine methanesulfonate Sigma Aldrich Cat #E10521 Metronidazole Thermo Fisher Scientific Cat #210340050 DMSO Sigma Aldrich Cat #20-139 Propidium iodide Thermo Fisher Scientific Cat #P1304MP zfIFN-g-rel (IFN-1.1) Kingfisher Biotech Cat #RP1058Z-025 zfVegfaa Kingfisher Biotech Cat # RP1040Z-025 Pacific blue 500kDa dextran Fina Biosolutions https://www.finabio.net/ (custom made upon request) AV-951 Stratech Scientific Cat #A2251-APE DAPT Cambridge Bioscience Cat #CAY13197-10 AG-1478 Cambridge Bioscience Cat #CAY10010244-10 Critical commercial assays RedTaq ReadyMix Sigma Aldrich Cat #R2523 ApopTag Red In situ kit MilliporeSigma Cat #S7165 EdU Imaging Kit with Alexa Fluor 594 Invitrogen Cat #C10339 RNeasy Plus Micro Kit Qiagen Cat #74034 Deposited data RNA-seq data This paper (Supplementary file 1) Raw data are publicly available at Array express: E-MTAB-10860.

Techniques: Injection, Staining