PMC

Expression of PHB1 in the vascular system. (A) Mouse aortas were paraffin embedded and sectioned and PHB1 protein expression was analyzed by immunofluorescence (red) microscopy compared with PECAM-1 (as an EC marker, green) with a 20× objective. Arrows and arrowheads depict PHB1 in SMCs and ECs, respectively. Bars, 10 μm. (B) Localization of PHB1 (green) was analyzed in ECs by indirect immunofluorescence compared with MitoTracker (red) and colocalization is shown (orange; blue, DAPI). Bars, 10 μm.(C) Western blot analysis of PHB1 in lysates from BAECs and mouse aorta and carotid arteries.

Loss of PHB reduces angiogenesis in vivo. Matrigel angiogenesis assays in vivo were performed using gels mixed with control or PHB1 knockdown constructs. Gels were dissected, sectioned, and analyzed immunohistochemically (n = 5). (A) Both control and PHB1 knockdown gel slides were stained for PHB1 to show successful knockdown and images were taken using a 20× objective. Bars, 10 μm. (B) After hematoxylin and eosin staining, images were taken using a 4× objective to quantify total cells invading the gels. Representative images of control and a PHB1 knockdown gel are shown on the bottom. n = 5; *, P < 0.05. Bars, 100 μm. (C) Angiogenic ECs in both control and PHB1 knockdown gels were quantified by staining the slides for PECAM-1 after taking images with a 10× objective. An example of PECAM-1–positive structures (arrows) in a control gel is shown on the right. n = 5; *, P < 0.05. Bar, 50 μm. (D) Red blood cell–positive vascular structures (indicated by arrows in the representative example from a control gel shown on the right) were quantified in both control and PHB1 knockdown gels after staining with hematoxylin and eosin and taking images with a 10× objective. Error bars show the calculated standard deviation. n = 5; *, P < 0.05. Bar, 50 μm.

Loss of PHB provokes an antiangiogenic phenotype by influencing the cytoskeletal balance in vitro. (A) Actin stress fiber formation (stained with phalloidin) was examined via indirect immunofluorescence analysis using control (upper left) and PHB1 knockdown (top right and bottom) cells stimulated with VEGF (all) and PEG-catalase (bottom) and stained with rhodamin-phalloidin (yellow) and DAPI (blue). Bars, 10 μm. (B) EC morphology after knockdown of PHB1 (left) and incubation with PEG-catalase (right) using life imaging phase contrast microscopy. Insets show a higher magnification. Bars, 100 μm. (C) Migration of control and PHB1 knockdown cells pretreated with and without PEG-catalase was examined via migration assay in the presence and absence of VEGF. n = 4; *, P < 0.05. (D) Tube formation assays on matrigel plugs were performed and quantified using control and PHB1 knockdown cells. Representative examples of tubes are shown on the right. Error bars show the calculated standard deviation. n = 3; *, P < 0.05. Bars, 100 μm.

RNAi-mediated knockdown of PHB1 causes a senescence-like phenotype in ECs. (A) Western blot analysis of PHB1 and β-actin in EC lysates from control and PHB1 knockdown cells. (B) Cell morphology was analyzed by live imaging phase contrast microscopy. Bars, 50 μm. (C) Proliferation assays were performed after knockdown of PHB1 for the indicated time points and compared with control cells. n = 6; *, P < 0.05. (D) Expression of p21, p16, and β-actin in PHB1 knockdown and control HUVECs was determined by Western blotting. (E) BAECs transfected with control or PHB1-specific siRNA were stained for β-galactosidase with x-gal at pH 6 and images were taken using a 10× phase contrast objective. Bars, 100 μm. (F) β-galactosidase–positive cells were quantified by counting positive and negative cells in three random vision fields and averaging the results. Error bars show the calculated standard deviation. n = 3; *, P < 0.05.

PHB1 regulates Akt and Rac1 activity. (A) The level of phosphorylated (P473) and total Akt (after 100-ng/ml VEGF stimulation for different time points) was examined via Western blotting after PHB1 knockdown in ECs (middle) in comparison to control cells (left) and PHB1 knockdown cells preincubated with PEG-catalase (right). (B) H₂O₂ activates Akt phosphorylation, as shown by Western blotting. (C) Rac1 activity assays were performed using control and PHB1 knockdown cells, and active Rac1 pulldowns as well as total cell lysates were Western blotted against Rac1 and PHB1. Cells were treated with 100 ng/ml VEGF and/or PI3-K inhibitors wortmannin (50 nM) and LY294002 (30 μM) as indicated. (D) Phospho-Akt (P473) and active Rac1 were monitored in control and PHB1 knockdown cells upon 100-ng/ml VEGF stimulation for the indicated time points. (E) Quantification of Akt phosphorylation (top) and active Rac1 (bottom) normalized to total Akt and Rac, respectively, from three independent experiments. *, P < 0.05. (F) Rac1 activity in ECs pretreated with VEGF and/or a Rac1 inhibitor as indicated by Western blot analysis of total cell lysates for p-Akt, total Rac1, PHB1, and active Rac1. Error bars show the calculated standard deviation.

PHB1 regulates mitochondrial function. (A) ROS production was analyzed by FACS using the dye CM-H₂-DCFDH. Control RNAi cells are depicted in gray, PHB1 RNAi cells are depicted in red, and PHB1 RNAi cells preincubated with PEG-catalase are depicted in green. Positive control cells were preincubated with H₂O₂ (blue). (B) Respirating mitochondria were stained with reduced MitoTracker CM-H₂ XRos (red) in BAECs (top) and rho⁰ ECs (bottom) and immunofluorescence analyses were performed (blue, DAPI). Bars, 10 μm. (C) ROS production of rho⁰ ECs transfected with control (red) or PHB1 siRNA (blue) was analyzed by FACS using CM-H₂-DCFDH. (D) Control (top) and PHB1-depleted BAECs (bottom) were stained with the radiometric dye JC-1 to monitor depolarization of the mitochondrial membrane and intensity of red (left) and green (right) fluorescence was analyzed by indirect immunofluorescence. Bar, 10 μm. (E) Control cells (top left) and PHB1 knockdown cells pretreated with (bottom right) or without (bottom left) PEG-catalase were stained with JC-1 and both FL-1 and FL-2 fluorescence was analyzed by FACS. Valinomycin-treated cells served as a positive control for depolarization of the mitochondrial membrane (top right). As a mathematical indicator for mitochondrial membrane depolarization, the ratio of means between red and green fluorescence was calculated. (F and G) Complex I and III activity of PHB1-depleted cells was calculated and normalized to control cells by measuring oxygen consumption in the presence and absence of rotenone, antimycin-A, and azide. Error bars show the calculated standard deviation. n = 3; *, P < 0.05.