LOXL2 increases tubulogenesis in 3D fibrin gel. (A-C) Culture of HUVECs in absence (HUVEC) or presence (Coculture) of fibroblasts. HUVECs were seeded in 6 well plates and fibroblasts in culture inserts above HUVECs (A-B). Cells were maintained in complete medium for 2 days (A) or 4 days (B). (A) Expression of loxl2 mRNA was measured by semiquantitative RT-PCR. Actin was used as control. A representative gel of 3 independent experiments is shown. (B) Distribution of LOXL2 and collagen IV was analyzed by immunofluorescence. Nuclei were stained with DAPI. Bar: 20 μm. (C) Single HUVEC suspension was embedded in fibrin gels and maintained in the presence (coculture) or absence (HUVEC) of fibroblasts on top of the gel. Immunofluorescence staining of collagen IV was performed. Bar: 50 μm. (D, F) HUVEC-coated Cytodex beads were embedded in fibrin gels and maintained in the presence of fibroblasts. Images were acquired at day 6 for sprout length measurement. For each experiment (n = 3), mean total length of sprouts per bead was normalized to the mean of control cells. Graph represents the mean of normalized values ± SEM. *P < .05, **P < .005, and ***P < .001. (D) HUVECs were infected with a control (ctl) or a loxl2 coding lentivirus (LOXL2). (E-F) HUVEC were infected with lentiviruses coding for either control (shctl), or lox (shlox) or loxl2 (shloxl2) shRNA. (E) Semiquantitative RT-PCR of lox, loxl2, and actin. (F) HUVEC were treated (+B) or not with β-APN. (G) Cocultures were treated with β-APN from day 6 to day 10 to follow sprout evolution. Bar: 200 μm. (H) Single cell suspensions of HUVEC infected with lentiviruses coding for either control (shctl), or lox (shlox), or loxl2 (shloxl2) shRNA were embedded in fibrin gel at 100 × 10³ cells per well under low cross-linking conditions. Cells were maintained in the presence of fibroblasts on top of the gel and stained with CMTMR CellTracker. Epifluorescence z-stacks (150 μm thick and 10 μm step) were acquired and treated with ImageJ Version 1.43. Projections are represented with a 0-150 μm color-code for depth in the z dimension. Bars: 100 μm (H). Graphs represent measurements from one experiment (duplicate wells and 4 fields per well). SEM *P < .05 and ***P < .001. See also supplemental Figure 6 and supplemental Videos 4-5.