Fig 1: Vascular endothelial growth factor B (VEGFB) expression is negatively regulated by pigment epithelial‐derived factor (PEDF) in peripheral tissues. A, Comparison of serum VEGFB levels in participants with and without atherosclerotic cardiovascular disease (ASCVD). B, Correlation between serum PEDF levels and serum VEGFB levels. VEGFB protein expression in mouse aortic smooth muscle cells (MOVAS), mouse myoblasts (C2C12), and neonatal mouse cardiac cells after treatments with increasing concentration of human recombinant PEDF (0, 10, 25, 50, 100 and 200 nmol/L) for 24 hours (C) or with 100 nmol/L PEDF for increasing time (0, 0.5, 3, 6, 12 and 24 hours) (D) (n=3). E, Relative RNA expression of vegfb in white adipose tissue, heart, and skeletal muscle in wild‐type (WT), apolipoprotein E–deficient (ApoE−/−), and ApoE−/−/PEDF‐deficient mice fed high‐fat diet (HFD) for 24 weeks and age‐matched WT fed chow diet (CD). F, Immunohistochemical localization of VEGFB in the heart sections of WT (CD), WT, ApoE−/−, and ApoE−/−/PEDF −/− mice fed HFD for 36 weeks (n=3–4 per genotype) (bar=50 μm). Data are presented as mean±SE. *P<0.05, **P<0.01, ***P<0.001 vs WT CD; ## P<0.01, ### P<0.001 vs WT HFD; && P<0.01 vs ApoE−/− HFD.
Fig 2: Immunohistochemical staining of the retinal vascular proliferative membrane (A and B) VEGF-A expression in (A) the surgery group and (B) the drug pretreatment group. (C and D) VEGF-B expression in (C) the surgery group and (D) the drug pretreatment group. (E) Negative control group (scale bars, 50 µm). The drug group also received surgery.
Fig 3: Transplantation of 6F-ADRCs into acute myocardial infarcted tissues improves chronic cardiac function in vivo(A) Experimental scheme using 8-week-old C57Bl/6J mice subjected to intracardiac injection of 6F-ADRCs. ADRCs were harvested from mT/mG mice so that the implanted ADRCs could be identified. As the treatment group, 6F-ADRCs 1 week after viral induction were injected (6F-ADRC group). As the control group, ADRCs without viral induction were injected (Control ADRC group).(B) Survival plots up to 28 days after coronary artery ligation followed by the implantation of ADRCs (n = 22–32). Survival curves were analyzed using Kaplan–Meier estimators and log-rank (Mantel-Cox) tests.(C) Echocardiogram analysis of left ventricular fractional shortening.(D) Representative images of Masson’s trichrome staining were captured 4 weeks after transplantation. Scale bars, 1,000 µm. (E) Transplantation of 6F-ADRCs decreases the scar area (left) and cross-sectional area (middle) of the heart after MI. Relatively, the percent scar area was not significantly different (right).(F) Transplanted ADRCs labeled with tdTomato (enhanced with immunostaining of anti-RFP/Alexa 647) from mT/mG mice were identified in the border area 4 weeks after acute MI. Immunostaining showed that double-positive cells of tdTomato and cTnT (with Alexa 488) were frequently observed in the 6F-ADRCs group compared with the uninduced ADRCs group. Scale bars, 100 µm (low magnified images) and 50 µm (high magnified images) (G) The number of dtTomato-positive cells representing implanted ADRCs significantly increased in the 6F-ADRC group compared with the Control ADRC group (up right). Almost no cTnT-positive implanted cell was observed in Control ADRC group (up left); however, 13.36 ± 1.754% of implanted cells were positive for cTnT in the 6F-ADRC group (bottom) (n = 5).(H) Heatmap image of RNA-seq analysis illustrating 13 genes related to angiogenic paracrine factors among uninduced control ADRCs and GFP+ 6F-ADRCs. Log10(RPKM+1) values obtained from RNA-seq are shown (n = 2).(I) RNA-seq-based angiogenic paracrine signals including VEGF-A and VEGF-B expression levels of GFP+ 6F-ADRCs relative to uninduced ADRCs. RPKM values obtained from RNA-seq are shown (n = 2).(J) Transplanted 6F-ADRCs labeled with tdTomato and stained with cTnT were stained with VEGF-A and VEGF-B in the border area 4 weeks after acute MI surgery. The left row shows VEGF-A and the right row shows VEGF-B staining. Scale bars, 100 µm (low magnified images) and 50 µm (high magnified images). (K) CD31 immunofluorescence staining of section border zone of acute MI scar area, and CD31+ fraction area, compared with Control ADRCs treatment group and 6F-ADRCs treatment group (n = 6). Scale bars, 100 µm.Data are represented as mean ± SEM (C, E, G, I, K). Statistical significance was determined with the Student’s t-test between two groups (C, E, G, K).
Fig 4: Pigment epithelial‐derived factor (PEDF) inhibits expression of vascular endothelial growth factor B (VEGFB)–targeted receptors in endothelial cells. Representative images of heart sections visualized with anti–vascular endothelial growth factor receptor 1 (VEGFR1) (A) or anti–neuropilin 1 (NRP1) (B) antibody and colocalized with CD31 (endothelial marker) antibody from wild‐type (WT), apolipoprotein E–deficient (ApoE−/−), and ApoE−/−/PEDF‐deficient (PEDF −/−) mice fed high‐fat diet (HFD) for 36 weeks and age‐matched WT fed chow diet (CD) (n=3–4 per genotype). Merged images appear as yellow. C, Representative immunofluorescent images of human umbilical vein endothelial cells (HUVECs) treated with mock (unstimulated), 100 nmol/L PEDF alone, 100 ng/mL VEGFB alone, 100 ng/mL VEGFB+100 nmol/L PEDF, and 100 ng/mL VEGFB+100 nmol/L PEDF+PEDF neutralizing antibody (PEDF‐Ab) for 24 hours. D, Cultures were triple stained for VEGFR1 (red) or NRP1 (red) with CD31 (green) (magnification ×20; bar=50 μm). E, Protein expression of VEGFR1, NRP1, fatty acid transport protein (FATP) 3, and FATP4 in HUVECs after treatments with mock, increasing concentrations of VEGFB (25, 50, and 100 ng/mL), 100 ng/mL VEGFB+100 nmol/L PEDF, and 100 ng/mL VEGFB+100 nmol/L PEDF+PEDF‐Ab for 24 hours (n=4). F, 4,4‐Difluoro‐3a,4adiaza‐s‐indacene—fatty acid uptake into HUVECs after the same treatments as above (n=3). Data are presented as mean±SE. DAPI indicates 4′,6‐diamidino‐2‐phenylindole; PECAM, platelet endothelial cell adhesion molecule 1. *P<0.05, ***P<0.001.
Fig 5: Comparison of (A) VEGF-A and (B) VEGF-B levels in aqueous humor prior to and after conbercept treatment in the drug group (P=0.001 and P=0.022, respectively). **P<0.01 vs. after injection; *P<0.05 vs. after injection. The drug group also received surgery.
Supplier Page from Abcam for Anti-VEGFB antibody