Fig 2: Early pathological features of hypervitaminosis A in young rats. a) Plasma levels of Vegfa, Tnfa and soluble Icam 1 (sIcam1) protein at day 1, 2 and 7 into hypervitaminosis A, determined by ELISA. n = 4–5/group (day 1 and day 2) and n = 9–10/group (day 7). Student's t-test; p < 0.05 * compared to control day 1. b) Representative picture of immunohistochemical staining (brown) for Vegfa protein around diaphyseal bone at day 1 and 2 of hypervitaminosis A. Brackets indicate thickness of periosteum and endosteal cell layer. Bar 25 µm. p = periost and e = endost. c) Representative pictures of hematoxylin and eosin (HTX) stained humerus sections from day 1 of hypervitaminosis A. Pictures show tissue infiltration of red blood cells (microbleeding), immediately below the growth plate at day 1 of hypervitaminosis A. Right panel show pictures at higher magnification from boxed area. Arrows indicate endothelial cells lining blood vessels in controls. Arrowheads show contracted endothelial cells lining leaky endothelium in rats with hypervitaminosis A. Bar 25 µm. d) Representative pictures of HTX stained diaphyseal bone from day 2 of hypervitaminosis A and controls. Thickened periosteum show numerous engorged blood vessels whereas the thickened cell layer at the endosteal site show microbleeding. Brackets indicate thickness of periosteum and endosteal cell layer. Arrowheads indicate small intact blood vessel in controls. Bar 25 µm. b = bone and mu = muscle. Data of periosteal thickness is an average of 3 measurements per animal from n = 4 rats/group. Student's t-test; p < 0.01 ** compared to control. e) Representative pictures of Pecam1 stained diaphyseal bone from day 7 of hypervitaminosis A and controls. Brackets indicate thickness of periosteal cell layer. Arrow indicate small blood vessel in controls. f) Representative photograph of intact tibia from day 14 of hypervitaminosis A and control. Results are presented as mean ± SD.

Fig 3: Normal human endothelial cell and osteoblast response to retinoic acid (RA). a) CYP26B1 mRNA expression in human dermal microvascular endothelial cells (HDMEC) and human primary osteoblasts (HOB) treated with or without 400 nM RA for 24 h (h). b) VEGFA mRNA expression in HDMEC and HOB cells treated with or without 400 nM RA for 24 h or 48 h. c) Representative pictures of HDMEC cells treated with or without 400 nM RA for 24 and 48 h. Yellow arrowheads indicate RA induced cell contraction/slimming noticed at both time points. RA did not induce apoptosis as determined by caspase-3 activity. d) Immunofluorescent VE-Cadherin staining of HDMEC cells treated with or without 400 nM RA for 48 h. White arrowheads indicate RA induced gaps in VE-cadherin staining at cell-cell junctions. RA reduced VE-Cadherin staining between endothelial cells. n = 4/treatment. Bar 25 µm. Results are presented as mean ± SD. Student's t-test; p < 0.05 *, p < 0.01 ** and p < 0.001 *** compared to control.