1. Kringle 5 of human plasminogen, an angiogenesis inhibitor, induces both autophagy and apoptotic death in endothelial cells
by Tri Minh Bui Nguyen, Indira V. Subramanian, Ameeta Kelekar, and Sundaram Ramakrishnan
Blood
Volume 109(11):
June 1, 2007
©2007 by American Society of Hematology

2. Effects of rK5 on endothelial cells.
Effects of rK5 on endothelial cells. (A) The effect of rK5 on VEGF-induced cell proliferation. BrdU incorporation was used as an index of proliferation. Each data point represents a mean of 3 independent experiments using triplicate cultures. HUVECs (□), human foreskin fibroblast (■), OVCAR-5 (●), and OVCAR-3 (▵). Purified rAAV-GFP CM (3 μg/mL)–treated HUVECs (▴) was used as negative control. Inset: anti-His Tag immunoblot analysis of purified recombinant AAV-K5 (rK5) in fractions eluted by 100 mM imidazole (lanes 2-5) and CM before purification (lane 6). A major band of approximately 10 kDa was detected. Lane 1 shows CM of nontransduced HEK-293 cells; lane 7 shows endostatin with a C-terminal poly-His–tagged (positive control); and lane 8 shows purified rAAV-GFP CM. (B) The effect of rK5 on endothelial cell migration in Boyden chamber assay. The migration of endothelial cells was determined by a method previously described.18 Endothelial cells were prelabeled with 5.0 μM 56-CFDA and induced to migrate toward the bottom chamber containing 40 ng/mL VEGF. The number of cells migrated to the bottom side of the membrane was counted using a PixCell II LCM system at 40× magnification. Data represent values from 3 independent experiments using 3 wells per concentration. M199 medium (5% FBS; ▵) was used to determine basal level of migration. P125A endostatin (■) was used as a positive control. rK5 induced concentration-dependent inhibition of cell migration (□). (C) The effect of rK5 on angiogenesis was evaluated by using VEGF-stimulated tube formation in a Matrigel assay.18 Representative images of endothelial cell tube formation are shown. (Ci) Basal level of tube formation. (Cii-iv) Tube formation induced by 40 ng/mL VEGF. (Ciii) rK5. (Civ) P125A endostatin (300 ng/mL). Bright-field images were recorded at 40× magnification (top row) and processed for analysis (Cv-viii) as described in “Materials and methods” (bottom row). (D) Morphometric analysis of tube formation. Ends (□), branch points (■), and tube length (▵) are shown. Values represent data from 3 independent experiments using 3 wells per sample. VEGF-induced tube formation was considered as 100% tube formation. Values are shown as means ± SE. (E) rK5 induced endothelial cell apoptosis under nutrient-rich culture conditions. HUVECs were cultured in the presence of 40 ng/mL VEGF. Cells were treated with 1.5 μg/mL rK5 for 24 hours and then labeled with annexin V FLUOS and DAPI. Images were recorded at 40× magnification and processed by the Image Processing Toolkit and Adobe Photoshop. Percentage of apoptotic cells was determined by the ratio of annexin V+ cells over the total number of nuclei per field. Purified rAAV-GFP CM was used as a negative control at a similar concentration. Values represent data from 2 independent experiments using 3 wells per sample. Error bars denote SE (*P < .05). (F) HUVECs were stimulated with VEGF and treated with different concentrations of rK5. In one set of cultures, rK5 was withdrawn after 24 hours of exposure (■) and replaced with fresh media. The second set of cultures was continuously exposed to rK5 (□). Cells from both treatment groups were labeled with TUNEL concurrently with DAPI after 72 hours of treatment. Percentage of apoptotic HUVECs was determined by the ratio of TUNEL+ cells over the total number of nuclei observed per field. Purified rAAV-GFP CM (3 μg/mL; ▴) and 0.02% H2O2 (♦) were used as a negative and positive control, respectively. Values represent data from 3 independent experiments using triplicate wells per concentration. Values are shown as means ± SE.
Tri Minh Bui Nguyen et al. Blood 2007;109:
©2007 by American Society of Hematology

3. rK5 transduces apoptotic signal via an intrinsic pathway.
rK5 transduces apoptotic signal via an intrinsic pathway. (A) Mitochondrial membrane depolarization following 24-hour treatment of endothelial cells with rK5 in the presence or absence of VEGF. HUVECs were incubated with the ΔψM-sensitive mitochondrial dye, TMRE (40 nM), for 15 minutes prior to harvesting and immediately analyzed by FACS. The profiles of unstained and etoposide (10 μg/mL)–treated HUVECs are included with each histogram. (B) Western blot analysis of full-length caspase-7 and cleaved caspase-7 in rK5-treated HUVECs. Total cell lystates from rK5-treated HUVECs were collected at different time points and subjected to Western blot analysis. β-actin was used to normalize the amount of loaded proteins. (C) Representative Western blot analysis of cleaved caspase-3 and β-actin in rK5-treated HUVECs. Total cell lystates from rK5-treated HUVECs were collected at different time points and subjected to Western blot analysis. (D) Densitometric analysis was used to quantify the levels of caspase-3 activation. Values were normalized against β-actin and presented as fold increase compared with the basal level (0 hours of treatment). Data are shown as means ± SE.
Tri Minh Bui Nguyen et al. Blood 2007;109:
©2007 by American Society of Hematology

4. rK5 induces autophagy in HUVECs
rK5 induces autophagy in HUVECs. (A) HUVECs cultured in different conditions (5% FBS in M199, 5% FBS in M199 supplemented with 40 ng/mL VEGF, and 10% FBS in M199) were treated with 1.5 μg/mL rK5 for 24 hours and stained with neutral red as described previou...
rK5 induces autophagy in HUVECs. (A) HUVECs cultured in different conditions (5% FBS in M199, 5% FBS in M199 supplemented with 40 ng/mL VEGF, and 10% FBS in M199) were treated with 1.5 μg/mL rK5 for 24 hours and stained with neutral red as described previously.27 Phase-contrast images were recorded at 400× magnification using an Olympus BX60 upright microscope (Olympus, Center Valley, PA). There was an increase in the intensity and size of neutral red–positive vesicles in rK5-treated cells compared with control cultures. (B) Kinetic study of rK5-induced autophagy in endothelial cells. HUVECs were treated with 1.5 μg/mL of rK5 for the indicated time periods (□). Autophagic vesicles were labeled by 0.05 mM MDC and recorded by confocal microscopy at 600× magnification at different time points. The number of autophagic vesicles per cell was determined by an image analysis program as described in “Materials and methods.” Untreated cultures (■) and cultures treated with 1.5 μg/mL purified rAAV-GFP CM (▴). Values are shown as means ± SE. Inset: representative grayscale image of MDC-labeled HUVECs; digital centroid images are shown. Cells were treated by rK5 in the presence of VEGF. Images were recorded after 24 hours of treatment with rK5. (C) TEM analysis of HUVECs treated with 1.5 μg/mL rK5. Treated and control HUVECs were processed for TEM as described previously.20 Images were recorded at 5000× magnification (column 1) and 20 000× magnification (column 2) using a JEOL 1200 EX transmission electron microscope (JEOL, Peabody, MA). Note the presence of double-membrane autophagosomes and single-membrane autolysosomes that contained disintegrated materials clustering at the perinuclear sites (arrows). The nucleus and the cellular membrane structures of both control and treated cell remained intact. (D) rK5-induced autophagy was detected by LC3-GFP. HUVECs were transfected with LC3-GFP using Lipofectamine 2000 (Invitrogen). Transiently transfected cells were treated by 1.5 μg/mL rK5 in the presence of VEGF (40 ng/mL; column 3). Transfected HUVECs cultured with 40 ng/mL VEGF (column 2) or without (column 1) was used as control. After 24 hours of treatment, LC3-GFP–labeled cells (top row) were colocalized with mitochondria labeled with Mitotracker Red (bottom row). Confocal images were obtained at 600× magnification.
Tri Minh Bui Nguyen et al. Blood 2007;109:
©2007 by American Society of Hematology

5. rK5-induced autophagy in HUVECs is independent of nutrient deprivation-induced stress response.
rK5-induced autophagy in HUVECs is independent of nutrient deprivation-induced stress response. (A) LAMP1-GFP–transfected HUVECs were treated with different concentrations of rK5 cultured in M199 containing 5% FBS and supplemented with 40 ng/mL VEGF (□). After 24 hours of treatment, random fields of confocal microscopy images were recorded at 600× magnification and processed as described in “Materials and methods” to determine the number of autolysosomes per cell. Transfected HUVECs in serum-reduced medium (■) was used as a positive control. Values are shown as means ± SE. (B) LAMP1-GFP–transfected HUVECs cultured either in 5% FBS supplemented with VEGF or 10% FBS medium were treated with rK5 in the presence or absence of 3-MA. The number of LAMP1-GFP+ vesicles per cell was quantified. Values are presented as fold increase when compared with the control group (**P < .001; n = 3). The number of vesicles per cell in the control group was considered the basal level (1.0). Values are shown as means ± SE. (C) Representative confocal images of LAMP1-GFP–transfected HUVECs cultured in 5% FBS with (Cii-v) or without (Ci) VEGF. Transfected cells were treated with rK5 (1.5 μg/mL) in the presence (Ciii) or absence (Civ) of 5 mM 3-MA. LAMP1-GFP+ vesicles (left column) were colocalized with mitochondria (Mitotracker Red staining). Confocal images were recorded at 600× magnification. (D) Representative confocal images of HUVECs cultured in 10% FBS in the absence (Di) or presence (Diii) of rK5. As a control, heat-inactivated rK5 (Dii) was used. Control (Dv) and rK5-treated cultures (Div) were exposed to 5 mM 3-MA. LAMP1-GFP–labeled cells from different treatment groups (left column) were merged with the DAPI and Mitotracker Red images.
Tri Minh Bui Nguyen et al. Blood 2007;109:
©2007 by American Society of Hematology

6. rK5-induced autophagy is Beclin 1 dependent.
rK5-induced autophagy is Beclin 1 dependent. (A) Representative immunoprecipitation of Beclin 1 in rK5-treated HUVECs. Cell lysates (200 μg/mL) collected at different time points were immunoprecipitated by anti–Beclin 1 antibody and blotted with anti–Beclin 1 antibody. (B) Confocal microscopy images of HUVECs showing endogenous Beclin 1 levels at different time points after rK5 treatment. Nuclei were labeled by DAPI. Beclin 1 expression (red) was detected using an anti–Beclin 1 monoclonal antibody. Alexa Fluor 647 goat anti–mouse IgG was used as secondary antibody. rK5-treated HUVEC (16 hours) sample without the primary antibody treatment was used as a negative control. (C) HUVECs were grown to subconfluent conditions. Scrambled shRNA or shRNA specific to Beclin 1 were transfected using Lipofectamine Top row shows total-cell lysates of untransfected HUVECs (lane 1), scrambled shRNA-transfected HUVECs (lane 2), and shRNA specific to Beclin 1–transfected HUVECs (lane 3) that were analyzed to determine Beclin 1 levels by Western blot. Bottom row shows confocal images of shRNA-transfected HUVECs showing Beclin 1 levels (red) 36 hours after transfection. Nuclei were stained with DAPI (blue). (D) HUVECs were cotransfected with LC3-GFP (green) and shRNA. Transfected HUVECs were cultured in 5% FBS in M199 with or without 40 ng/mL VEGF. Scrambled shRNA and shRNA specific to Beclin 1–transfected cells were exposed to 1.5 μg/mL rK5 for 24 hours. Treated HUVECs were labeled with Mitotracker Red. Confocal microscopy images were obtained at 600× magnification.
Tri Minh Bui Nguyen et al. Blood 2007;109:
©2007 by American Society of Hematology

7. Effects of rK5 on endothelial cells are mediated via Beclin 1/Bcl-2 interactions.
Effects of rK5 on endothelial cells are mediated via Beclin 1/Bcl-2 interactions. (A) Representative Western blot analysis for Beclin 1, Bcl-2, and β-actin in rK5-treated HUVECs. Total-cell lysates from rK5-treated HUVECs were collected at different time points and subjected to Western blot analysis. (B) Densitometric analysis of Beclin 1 and Bcl-2 (■) levels. Values were normalized against β-actin and presented as fold increase compared with the basal level (0 hours of treatment). Data represent means of 3 independent blots. Error bars denote SE (*P < .05). (C) Coimmunoprecipitation of Beclin 1 and Bcl-2 in HUVECs. Cells were grown in 5% FBS in M199 medium and supplemented with 40 ng/mL VEGF in the presence of 1.5 μg/mL rK5. Cell lysates from rK5-treated HUVECs were collected at different time points. The lysates (200 μg per sample) were incubated with anti–Beclin 1 antibodies covalently coupled to protein A/G Plus–Sepharose. The immunoprecipitates were analyzed for Beclin 1 and Bcl-2 by immunoblotting. (D) Densitometric analysis of Bcl-2 (□) and Beclin 1 (■) levels. Coimmunoprecipitation experiments were repeated 3 times. Values are presented as fold increase compared with the basal level (0 hours of treatment; *P < .05). Data are shown as means ± SE. (E) HUVECs were treated with 1.5 μg/mL rK5 in the presence of 25 μM zVAD-fmk. Cells were labeled with 0.05 mM MDC (red) and 1 μM Mitotracker Green. Images were recorded using confocal microscopy at 600× magnification and processed as described previously to quantify the number of autophagy vesicles per cell. (F) Values of MDC+ vesicles in rK5-treated HUVECs in the presence or absence of 25 μM zVAD-fmk. Data represent values from 2 independent experiments using triplicate cultures; bars denote SE.
Tri Minh Bui Nguyen et al. Blood 2007;109:
©2007 by American Society of Hematology

8. Flow cytometric analysis of HUVECs showing increased levels of activated caspases.
Flow cytometric analysis of HUVECs showing increased levels of activated caspases. HUVECs were cotransfected with a transposon expressing DsRed 2 plasmid and shRNA specific to Beclin 1 or a scrambled shRNA, using Lipofectamine After 36 hours, transfected HUVECs were treated with 1.5 μg/mL rK5. Treated cells were labeled with green fluorescent-labeled inhibitor of caspases (FLICA) and analyzed by FACS. Transfected HUVECs were gated for DsRed+ cell populations and scored for FAM-VAD-FMK+ cells. (A) Histogram shows FAM-VAD-FMK+ cells in the control, untransfected, scrambled shRNA-transfected, and shRNA specific to Beclin 1–transfected HUVECs. (B) Histogram of rK5-treated HUVECs that were transfected with either shRNA specific to Beclin 1 or scrambled shRNA. (C) Summary of the data from the flow cytometric analyses is depicted as a percentage (mean ± SE) of transfected HUVECs (DsRed 2) positive for the activation of caspases. Each value is a mean of triplicate determinations.
Tri Minh Bui Nguyen et al. Blood 2007;109:
©2007 by American Society of Hematology