Decreased blood vessel density and endothelial cell subset dynamics during ageing of the endocrine system (2020)
Figure 6

Figure 6. Coupling of angiogenesis and β-cell expansion

A Representative 3D images of pancreas from adult (18-weeks-old) sunitinib-treated and control mice with Insulin, CD31 and Emcn immunostaining.

B Combined box and whiskers, and scatter plot (left) shows the quantification of CD31+ Emcnhi vessel density in sunitinib-treated and control pancreatic islets. Combo plot (right) shows ELISA based analysis of Insulin.

C Representative 3D images (upper panel) show CXCR4, Ki67 and VE-Cadherin in sunitinib-treated and control pancreatic islet. 3D images (bottom) show sunitinib-treated and control pancreatic islets immunolabeled with Insulin and VE-Cadherin.

D Combo plot (top) shows the percentage of CXCR4+ β-cells per islet in sunitinib-treated and control pancreatic islet. Combo plot (middle) shows the quantification of Ki67+ β-cells normalized to the total number of β-cell numbers. Combo plot (bottom) shows the percentage of Insulin+ β-cells per islet.

E Representative 3D images of pancreas from adult Vegfr2i∆EC mutant and control mice with Insulin, CD31 and Emcn immunostainings.

F Combo plot (left) with quantification of CD31+ Emcnhi vessel density in Vegfr2i∆EC and control pancreatic islet. Right combo plot with insulin estimation in pancreas by ELISA assay.

G Representative 3D images in the upper panel show CXCR4, Ki67 and VE-Cadherin in islets from Vegfr2i∆EC mutant and control mice. 3D images (lower panel) show Vegfr2i∆EC mutant and control islets immunolabeled with Insulin and VE-Cadherin.

H Combo plot (top) shows the percentage of CXCR4+ β-cells per islet in Vegfr2i∆EC mutant and control pancreatic islet. Combo plot (middle) shows the quantification of Ki67+ β-cells normalized to the number of β-cells. Combo plot (bottom) shows the percentage of Insulin+ β-cells per islet.

I Graph shows qPCR analysis of Pdgfa, Pdgfb, Igf1, Igf2, Cxcl12, Hgf and Kitl expression (normalized to Actb) in purified CD31+ Emcnhi ECs compared to CD31+ Emcnlo ECs.

J Bar graph with the quantification of Ki67+ β-cell numbers per islet (f.u) and Ki67+ EC numbers per islet (f.u). 45 pancreatic islets were analysed and quantified from 9 young mice.

K Bar graphs show qPCR analysis of Dll4, Emcn, Kdr, Flt4, Sele, Mmp9, Plxnd1, Robo4, Unc5b, Cdh5, Cldn5, Gja1, Gja4, Icam1, Icam2, Jam3, Ntn1, Ntn2, Ocln, Pecam1, Tjp1, Tjp2, Tjp3 and Vcam1 expression (normalized to Actb) in purified CD31+ Emcnhi ECs compared to CD31+ Emcnlo ECs from murine pancreas.

L Graph shows qPCR analysis of Gja1 expression (normalized to Actb) in purified CD31+ Emcnhi ECs from juvenile, adult and aged murine pancreas.

Data information: The white and dashed lines in panel A, C, E and G represent the outlines of pancreatic islets. Data represent mean ± s.d. (n=5), P-values, two-tailed unpaired t-tests in panel (B, D, F, H, I and K) and one-way ANOVA tests with Tukey's multiple comparisons tests in panel (J, L). *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001. Nuclei: DAPI or TO-PRO-3 as indicated. Is: pancreatic islet. f.u: fluorescence unit. For the Combo plots, the boxes represent mean ± s.d., line in the box is the median, and the lower and upper lines show the minimum and the maximum of the values. The line on the right side of these combo plots represent the sample distribution. Scale bars are 50 µm for all images.