LOXL2 expression, ECM localization and activity are induced by hypoxia in endothelial ECM in vitro. (A) Two-dimensional silver stained gels of ECM from HDMEC or HUVECs cultured in normoxia or hypoxia for 5 days. Areas corresponding to similar molecular weights and pI are shown. (B) LOXL2 and von Willebrand factor (VWF) were immunodetected in ECM extracts or secretion medium from HUVECs cultured in normoxia (N) or hypoxia (H). von Willebrand factor was used as a control ECM-binding protein secreted by EC, whose amount and distribution between the secretion medium and the ECM were not modified by hypoxia. (C) Epifluorescence of HUVECs cultured in normoxia or hypoxia and double-stained for LOXL2 and CD31. (D) Confocal images of HUVECs cultured in hypoxia. Double-immunofluorescence of LOXL2 and either collagen IV (Col IV), or fibronectin (Fn) or Cyr61 was performed. Nuclei were stained with TO-PRO-3. Bars: 20 μm (C and D). (E) Semiquantitative RT-PCR of loxl2 mRNA from HUVECs cultured in normoxia (N) or hypoxia (H) for 2 (D2) or 5 (D5) days. Actin was used as a control. The mean expression of 3 independent experiments is represented ± SD. *P < .05. (F-G) Lysyl oxidase activity was measured as the amine oxidase activity inhibited by β-APN. (F) HUVECs were cultured in normoxia (N) or hypoxia (H). (G) HUVECs silenced for either lox or loxl2 using siRNA were cultured in hypoxia. Lysyl oxidase activity was normalized to the mean of controls. Graphs represents the mean of 3 independent experiments values ± SEM. **P < .005 and ***P < .001. See also supplemental Figures 2-3.