Heterozygous disruption of Becn1 enhances hypoxia-induced tumor growth and angiogenesis. B16F10 melanoma cells were injected subcutaneously in Becn1^+/+ and Becn1^+/− mice which were subsequently exposed to normoxia or hypoxia for 3 weeks. (A) Macroscopic appearance and (B) volume of B16F10 tumors from Becn1^+/+ and Becn1^+/− mice after 3 weeks normoxia or hypoxia. Scale bar, 5 mm. The graph shows mean tumor volume ± SD, (*p < 0.05, Student's t-test), n = 7 mice per treatment group. (C) H&E and (D) CD31 staining of B16F10 tumor sections from Becn1^+/+ or Becn1^+/− mice exposed to hypoxia for 3 weeks. Scale bars, 10 µm. (E) Blood vessel density in tumors from Becn1^+/+ or Becn1^+/− mice. Vessel density was quantified by counting the number of vessels per mm² in CD31 stained sections. Data represent the mean ± SD, n = 5–6 tumors per group (*p < 0.05, Student's t-test). (F) HMB45 staining of melanoma cells in lung sections from tumor-bearing mice after 3 weeks of hypoxia exposure. Red arrows indicate HMB45 positive cells. Scale bars, 50 µm (left part) and 10 µm (right part). (G) Quantification of HMB45 stained cells in lung tissues from B16F10-injected Becn1^+/+ and Becn1^+/− mice exposed to normoxia or hypoxia for 3 weeks. Data represent mean ± SD. (*p < 0.05, Student's t-test), n = 5−6 lungs per condition.

Beclin 1 deficiency results in enhanced levels of EPO production during hypoxia exposure in mice. Quantification of (A) VEGF and (B) EPO production in Becn1^+/+ and Becn1^+/− mouse plasma was determined by Quantikine™ ELISA after hypoxia exposure for 3 weeks. Data are means ± SEM. (*p < 0.05, Student's t-test), (n = 10 per group). (C and D) Circulating levels of VEGF and EPO from the plasma of B16F10-injected mice exposed to hypoxia for 3 weeks. (*p < 0.05, Student's t-test), n = 7 per group.

Decreased autophagy phenotype in Becn1^+/− mice. The Becn1^+/+ and Becn1^+/− mouse were exposed to hypoxia or normoxia for 3 weeks. (A) Lung tissue homogenates were analyzed for the expression of LC3B-I/-II and Beclin 1 by western immunoblot analysis. β-actin served as the standard. (B) Lung tissue sections were fixed in glutaraldehyde and analyzed by electron microscopy for immature (AVi) or degradative (AVd) autophagosome formation. Total autophagosome formation per 100 µm² area is quantitated from N = 15 sections.

Heterozygous disruption of Becn1 promotes hypoxia-induced endothelial cell proliferation, migration and tube formation. (A) The proliferation of Becn1^+/+ and Becn1^+/− mLECs exposed to normoxia was assessed by crystal violet staining. (B and C) Proliferation indices of mLECs exposed to hypoxia (1% O₂) were assessed by crystal violet and Trypan blue exclusion methods. Data represent means ± SEM from experiments in triplicate (*p < 0.05, Student's t-test). (D and E) PAECs or PASMC were transfected with control or Becn1 siRNA. Western (inserts) shows validation of Beclin 1 expression levels following control or Becn1 siRNA treatment. β-actin served as the standard. (D) Following addition of ET-1 (40 nM), PAEC cultures were exposed to hypoxia or normoxia for an additional 48 h. Data represent means ± SEM in triplicate experiments. (*p < 0.05, Student's t-test). (E) Following addition of PDGF-BB (20 ngml⁻¹) siRNA transfected PASMC cultures were exposed to hypoxia or normoxia for an additional 48 h. Data represent means ± SEM from three independent experiments. (*p < 0.05, Student's t-test).

(A) Chemotactic motility of mLECs induced by hypoxia after 12 h of incubation. (Scale bar, 2 mm). (B) Quantification of EC migration through the transwell. Data represent means ± SEM in triplicate experiments. (*p < 0.05, Student's t-test). (C) mLECs were plated on Matrigel-coated wells at a density of 1.8 × 10⁵ cells/well with hypoxia. After 20 h, photomicrographs were taken. (Scale bar, 50 µm). (D) Graphs show mean capillary-like network on the Matrigel. Images were quantified with ImageJ software. Data are means ± SEM in triplicate experiments. (*p < 0.05, Student's t-test).

Becn1^+/− mLEC display increased hypoxia-induced angiogenesis in vivo and vessel sprouting ex vivo. (A) Representative Matrigel plugs were photographed. (B) Plugs were stained for infiltrating ECs using an anti-CD31 antibody. (C) Quantitative assessment of CD31 positive ECs. Data are means ± SEM (*p < 0.05, Student's t-test), n = 7 per group. (D) Representative photographs of endothelial cell sprouts formed from the margin of vessel segments. Aortic rings harvested from Becn1^+/+ and Becn1^+/− were embedded in Matrigel and cultured under normoxia or hypoxia for 10 days. (E) Vessel sprouting was quantified by counting the number of vascular sprouts that directly originated from the mouse aorta. Data are means ± SEM (*p < 0.05, Student's t-test), n = 8 per group.

Western blot analysis of (A) Nrf-2 and Keap1 or (B) phospho (p)-p65 and IκBα levels in Becn1^+/+ and Becn1^+/− mLEC exposed to hypoxia (1% O₂) for the indicated times. The immunoblot shown is representative of three independent experiments. β-actin served as the standard.

Hypoxia-induced angiogenesis is mediated by a selective increase in HIF-2α expression in Becn1^+/− mLECs. (A) Tumor-bearing Becn1^+/+ and Becn1^+/− mice were subjected to hypoxia or normoxia (3 weeks). Tumor tissue was recovered for analysis of HIF-1α, HIF-1β and HIF-2α levels by western blot analysis. β-actin served as the standard. (B) Western blot analysis of total HIF-1α, HIF-1β and HIF-2α levels in Becn1^+/+ and Becn1^+/− mLEC exposed to hypoxia (1% O₂) for the indicated times. The immunoblot shown is representative of three independent experiments. β-actin served as the standard. (C) Western blot analysis of total HIF-1α and HIF-2α levels in Becn1^+/+ and Becn1^+/− mLEC exposed to mild hypoxia (5% O₂) for the indicated times. The positive control (lane 9) represents lysate from Becn1^+/+ mLEC exposed to 24 h hypoxia at 1% O₂. β-actin served as the standard. (D) The Becn1^+/+ and Becn1^+/− mLECs were transfected with Beclin 1 expression vector or vector control (pCMV) and then exposed to hypoxia (1% O₂) or normoxia for 24 h. Western blot analysis was performed to detect the levels of HIF-1α, HIF-2α and Beclin 1. The immunoblot shown is representative of three independent experiments. β-actin served as the standard. (E) The Becn1^+/+ and Becn1^+/− mLECs were subjected to hypoxia (4 h) and analyzed for Hif-2α mRNA levels by qRT-PCR. Data are expressed as fold difference over normoxic wild-type control. (F) The Becn1^+/+ and Becn1^+/− mLEC were exposed to hypoxia (1% O₂) for 24 hrs followed by treatment with cyclohexamide (CHX) for 0–90 minutes. HIF-1α and HIF-2α levels were determined by western immunoblot analysis. β-actin served as the standard.

(A) mLEC were infected with siRNA corresponding to Hif-1α and Hif-2α or control, and then assayed for HIF-1α and HIF-2α protein expression by western immunoblot analysis. β-actin served as the standard. (B–D) Becn1^+/+ and Becn1^+/− mLEC were infected with siRNA corresponding to Hif-1α and Hif-2α or control, and then exposed to hypoxia or normoxia. Graphs indicate the quantification of (B) relative cell proliferation (C) migration and (D) tube formation. Data represent means ± SEM from three independent experiments. (*p < 0.05, Student's t-test).