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. 2020 Mar 29;9(4):824. doi: 10.3390/cells9040824

Figure 4.

Figure 4

Loss of hyaluronan in ECs increases angiogenesis and endothelial destabilization. To test the function of HA in vitro, a short hairpin RNA (HAS2-shRNA) was used to silence HAS2 expression in ECs. (A) Representative images of EC-pericyte coculture angiogenesis plexus assay stained with CD31 (green) and α-SMA (red) (scale bar = 100 µm). Quantification of both (B) vascular area and (C) vascular branches show increased angiogenesis with silencing of HAS2 (n = 3). (D) Representative images of EC-pericyte coculture (n = 4, scale bar = 20 µm) and (E) its side view stained for VE-Cadherin/CD144 (green) and F-actin (red), which ECs (VE-cadherin positive) stay on top of pericytes (scale bar = 10 µm). (F) Quantification of linear adherens junctions shows endothelial destabilization with silencing of HAS2 (n = 4). (G) Images and (H) quantification of western blot for p-Tie2, Tie2 and GAPDH show a reduced Tie2 activation with silencing of Has2 (n = 4) compared to control (n = 4). (I) An increased ICAM mRNA expression is shown with silencing of Has2 (n = 4), indicates ECs activation after loss of HA. Values are given as mean ± SD. * p < 0.05.