Fig.2. Ccm3ECKO mice exhibit disrupted EC-PC and EC-EC junctions with increased ANGPT2.
a. P10 cerebellum sections for CD31 with NG2 (top: 10×; bottom: 40×) and connexin-43 (CX43) co-staining, respectively. b. P10 cerebellum sections for staining of CD31 with VE-cadherin or claudin-5. The boxed areas are shown in high power for VE-cadherin and CD31 staining. Representative images of normal vessels (arrowheads), CCM lesions (asterisks) and vessels between lobules (arrows) are shown. c. Quantifications of coverages of NG2, CX43, VE-cadherin and claudin-5 on CD31 vessels. d. ANGPT2 and CD31 staining in P5 and P10 cerebellum sections. Representative high power images of ANGPT2-positive vessels are from boxed areas of P5 cerebellum. e. Cerebellum, retina and lung tissues as well as blood from WT and Ccm3ECKO pups were collected. Protein levels of ANGPT2 were determined by ELISA. f–g. mRNA levels (f) and protein levels (g) of ANGPT2-TIE2 pathway in cerebellum were determined by qRT-PCR and Western blotting. Data represent fold changes with WT levels normalized to 1.0. h–i. ANGPT2-pTIE2 was upregulated in human CCM lesions. Human CCM specimens were immunostained for CD31 with ANGPT2 or p-TIE2 (h) or Claudin-5 (i). Representative images from one (#2) of 8 human CCM3 samples are shown. Arrowheads indicate normal vessels whereas asterisks indicate for lesions. Arrows indicate gain of ANGPT2/p-TIE2 staining but loss of claudin-5 in lesions. n=10, *P <0.05, **P <0.01 (unpaired two-tailed Student’s t-test). Error bars indicate s.e.m. Scale bar: a (top): 100 μm; others: 25 μm (yellow); 5 μm (white for boxed images).