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1.
FIG. 4.

FIG. 4. From: Kaposi's Sarcoma-Associated Herpesvirus Upregulates Angiogenin during Infection of Human Dermal Microvascular Endothelial Cells, Which Induces 45S rRNA Synthesis, Antiapoptosis, Cell Proliferation, Migration, and Angiogenesis .

ANG expression and secretion is elevated upon LANA-1 expression. 293T cells were transfected with increasing concentrations of LANA-1 expression plasmids, and cDNA from these cells was used in quantitative real-time RT-PCRs to measure LANA-1 (A) and ANG (B) gene expression (results were normalized to tubulin levels). (C) ANG-ELISA for supernatants from serum-starved 293T cells transfected with increasing concentrations of LANA-1 expression plasmids. Each reaction was done in duplicate, and each point represents the average ± standard deviation of data from three independent experiments. ** and ***, statistically significant at P < 0.01 and P < 0.001, respectively.

Sathish Sadagopan, et al. J Virol. 2009 April;83(7):3342-3364.
2.
FIG. 2.

FIG. 2. From: Kaposi's Sarcoma-Associated Herpesvirus Upregulates Angiogenin during Infection of Human Dermal Microvascular Endothelial Cells, Which Induces 45S rRNA Synthesis, Antiapoptosis, Cell Proliferation, Migration, and Angiogenesis .

Viral gene expression augments ANG secretion. (A) Supernatants from serum-starved HFF and HMVEC-d cells (\∋7ε30% confluence) infected with live KSHV or UV-KSHV (MOI of 10) for 48 h and supernatants from uninfected cells were analyzed by ANG-ELISA. (B) ANG gene expression in uninfected TIVE cells and KSHV-infected (TIVE-LTC) cells were compared by quantitative real-time RT-PCR using the SYBR green detection protocol. The ANG level normalized to tubulin in TIVE cells was assigned a value of 1 for comparisons. (C) Supernatants from serum-starved TIVE cells and TIVE-LTC (KSHV) were analyzed by ANG-ELISA. (D) Equal numbers of BJAB and BCBL cells were seeded in T25 flasks, and the supernatant harvested from these cells after centrifugation was used for ANG-ELISA. For TPA treatment, identical numbers of BCBL cells in T25 flasks were induced with 20 ng of TPA/ml. At 48 h postinduction, supernatants were harvested and used for ANG-ELISA after centrifugation.

Sathish Sadagopan, et al. J Virol. 2009 April;83(7):3342-3364.
3.
FIG. 3.

FIG. 3. From: Kaposi's Sarcoma-Associated Herpesvirus Upregulates Angiogenin during Infection of Human Dermal Microvascular Endothelial Cells, Which Induces 45S rRNA Synthesis, Antiapoptosis, Cell Proliferation, Migration, and Angiogenesis .

KSHV latency-associated gene ORF73 and lytic genes ORF74 and ORF50 expression augments ANG secretion. (A) cDNA prepared from HMVEC-d cells transduced with lentivirus harboring viral genes was used for quantitative real-time RT-PCR to measure ANG gene expression and normalized to tubulin levels using the SYBR green protocol. The ANG level in pSIN-transduced HMVEC-d cells was assigned a value of 1 for comparisons. ** and ***, statistically significant at P < 0.01 and P < 0.001, respectively. (B) Supernatants from HMVEC-d cells transduced with lentivirus expressing the indicated viral genes and control pSIN were used for ANG-ELISA. Each reaction was done in duplicate, and each point represents the average ± standard deviation of data from three independent experiments. (C) cDNA prepared from HMVEC-d cells infected for 48 h with Ad (MOI of 1) harboring GFP or ORF50 was used for quantitative real-time RT-PCR to measure ANG gene expression and normalized to tubulin levels using the SYBR green protocol. (D) Serum-starved HMVEC-d cells were infected with AdGFP (MOI of 1) or AdORF50 (MOI of 1); at 48 h p.i., supernatants were harvested and assayed for ANG expression by ANG-ELISA.

Sathish Sadagopan, et al. J Virol. 2009 April;83(7):3342-3364.
4.
FIG. 5.

FIG. 5. From: Kaposi's Sarcoma-Associated Herpesvirus Upregulates Angiogenin during Infection of Human Dermal Microvascular Endothelial Cells, Which Induces 45S rRNA Synthesis, Antiapoptosis, Cell Proliferation, Migration, and Angiogenesis .

Immunofluorescence analysis of ANG in KSHV-infected HMVEC-d and HFF cells. (a and b) Serum-starved HMVEC-d cells (>50 to 60% confluence) were either left uninfected (a) or infected with KSHV (MOI of 10) (b) for 48 h and stained for ANG (green) and actin (phalloidin) (red) without permeabilization. (c and d) Serum-starved HMVEC-d cells (>80% confluence) were either left uninfected (c) or infected with KSHV for 48 h (d) and stained for tubulin (red) and ANG (green) after permeabilization. (e and f) Serum-starved semiconfluent HMVEC-d cells (>50 to 60% confluence) were either left uninfected (e) or infected with KSHV for 48 h (f) and stained for ANG (green) and actin (phalloidin) (red) after permeabilization. (g and h) Serum-starved semiconfluent HFF cells were either left uninfected (g) or infected with KSHV for 48 h (h) and stained for ANG (green) and actin (red). Magnification, ×40. Insets show a single cell in each of panels b, d, f, and h. Magnification, ×80. (I to l) Serum-starved semiconfluent HMVEC-d cells were infected with KSHV for 48 h and stained for ANG (green) (i) and LANA-1 (red) (j) after permeabilization. DAPI (blue) (k) was used as a nuclear stain and merged with LANA-1 and ANG (l) staining. Magnification, ×40. In the panels to the right of panels b to h, the yellow arrow indicates actin-ANG interaction, the green arrows indicate cytoplasmic localization of ANG, and the cyan arrow shows nuclear translocation of ANG.

Sathish Sadagopan, et al. J Virol. 2009 April;83(7):3342-3364.
5.
FIG. 6.

FIG. 6. From: Kaposi's Sarcoma-Associated Herpesvirus Upregulates Angiogenin during Infection of Human Dermal Microvascular Endothelial Cells, Which Induces 45S rRNA Synthesis, Antiapoptosis, Cell Proliferation, Migration, and Angiogenesis .

Localization of ANG in the nuclei and nucleoli of infected cells. (A) Serum-starved semiconfluent (50 to 60% confluence) (a to e) or subconfluent (30 to 40% confluence) (f to j) HMVEC-d cells were infected with KSHV for 48 h and stained for ANG (green) (b and g), fibrillarin (red) (c and h), and DAPI (d and i). Magnification, ×20. Insets show a single cell in panels e and j. Magnification, ×80. Panels a and f show bright-field images of semiconfluent and subconfluent HMVEC-d cells, respectively. (B) Confocal microscopic analysis demonstrating nuclear (a and b) and nucleolar (c) localization of ANG. (a) Serum-starved semiconfluent HMVEC-d cells were infected with KSHV at an MOI of 10 for 48 h and stained for ANG (green) after permeabilization. Topro (blue) was used as a nuclear stain and merged with ANG. (b) Semiconfluent HMVEC-d cells were infected, fixed, permeabilized, and stained for ANG (green) and fibrillarin (red), and the images were merged. (c) Subconfluent HMVEC-d cells were infected and stained for ANG (green) and fibrillarin (red). Magnification, ×100. (C) Serum-starved HMVEC-d cells (30 to 40% confluence) left untreated or treated with exogenous ANG (250 ng/ml for 48 h) or pretreated with neomycin (Neo; 100 μM for 1 h) were either left uninfected or infected with KSHV (MOI of 10) for 48 h, and nuclear extracts were prepared and Western blotted for ANG (top panel). The blots were stripped and reprobed for lamin B (middle panel) and tubulin (bottom panel). (D) Serum-starved HMVEC-d cells (30 to 40% confluence) were left untreated or pretreated with neomycin and infected with KSHV (MOI of 10) for 48 h, and supernatants were harvested and assayed for ANG secretion by ANG -ELISA.

Sathish Sadagopan, et al. J Virol. 2009 April;83(7):3342-3364.
6.
FIG. 11.

FIG. 11. From: Kaposi's Sarcoma-Associated Herpesvirus Upregulates Angiogenin during Infection of Human Dermal Microvascular Endothelial Cells, Which Induces 45S rRNA Synthesis, Antiapoptosis, Cell Proliferation, Migration, and Angiogenesis .

Schematic representation of the potential consequences of the presence of KSHV-induced ANG for the infected endothelial cell population. KSHV infection induces signal pathway activation leading to viral entry, nuclear delivery of the genome, and viral gene expression necessary for the activation of several growth factors, cytokines, and angiogenic factors (32). ANG is one of the angiogenic factors, and the results presented here demonstrate that it could act either in an autocrine fashion on the same infected cell or in a paracrine fashion on neighboring infected or uninfected cells to elicit multiple responses, including 45S rRNA synthesis, antiapoptosis, cell proliferation, VEGF-C expression, and tube formation mediated both by ANG and VEGF. In addition, ANG also interacts with infected cell surface actin to activate uPA, which is responsible for the plasmin generation required for the dissolution of basement membrane, cell migration, and tube formation. Collectively, the data strongly indicate a critical role for ANG in KSHV infection of endothelial cells and that this effect could be influencing the angioproliferative nature of KS lesions.

Sathish Sadagopan, et al. J Virol. 2009 April;83(7):3342-3364.
7.
FIG. 8.

FIG. 8. From: Kaposi's Sarcoma-Associated Herpesvirus Upregulates Angiogenin during Infection of Human Dermal Microvascular Endothelial Cells, Which Induces 45S rRNA Synthesis, Antiapoptosis, Cell Proliferation, Migration, and Angiogenesis .

KSHV infection and the presence of ANG increase HMVEC-d cell survival and block apoptosis. (A) HMVEC-d cells were serum starved using EBM-2, left untreated or pretreated with anti-ANG antibody for 1 h followed by incubation for 48 h with growth factors or with EBM-2 plus the indicated growth factors or with the indicated HMVEC-d cell culture supernatants, and assayed for the percentages of cell survival by MTT assays. Each reaction was performed in duplicate, and each bar represents the average ± standard deviation of data from three independent experiments. ** and ***, statistically significant at P < 0.01 and P < 0.001, respectively. (B) Serum-starved HMVEC-d cells (30 to 40% confluence) were either left uninfected or infected with live KSHV or UV-KSHV (MOI of 10) or treated with ANG (250 ng/ml for 48 h). At 48 h p.i., cells were washed, stained using Mitotracker, and visualized under a fluorescent microscope. Red fluorescence represents live cells where MitoSensor has aggregated, and green fluorescence represents apoptotic cells with cytoplasmic accumulation of monomeric MitoSensor. Magnification, ×20. (C) Serum-starved HMVEC-d cells (30 to 40% confluence) were pretreated with neomycin (100 μM for 1 h) or paromomycin (100 μM for 1 h) followed by infection with KSHV (MOI of 10) for 48 h or ANG treatment (250 ng/ml for 48 h). Cells were washed and stained using Mitotracker and visualized. Magnification, ×20. Each reaction was done in duplicate, and three different fields were counted for each treatment. The percentages of cells that fluoresced green or red were calculated by counting the total number of cells in a field by use of phase-contrast microscopy and dividing the number of green or red cells by the total number of cells.

Sathish Sadagopan, et al. J Virol. 2009 April;83(7):3342-3364.
8.

FIG. 10. From: Kaposi's Sarcoma-Associated Herpesvirus Upregulates Angiogenin during Infection of Human Dermal Microvascular Endothelial Cells, Which Induces 45S rRNA Synthesis, Antiapoptosis, Cell Proliferation, Migration, and Angiogenesis .

ANG secretion induces increased tube formation. (A) Serum-starved HMVEC-d cells (30 to 40% confluence) incubated with or without anti-ANG antibody (200 μg/ml) were mixed with EBM-2 containing ANG (250 ng/ml) (a and b) or VEGF (100 ng/ml) (c and d) or uninfected HMVEC-d cell supernatant (e and f) or supernatants from KSHV-infected (48 h) HMVEC-d cells (g and h), layered on Matrigel, and incubated for 16 h. Tube formation was observed using a phase-contrast microscope. (B) Control rabbit IgG (a) or anti-total ERK2 IgG (b) or EBM-2 (c) mixed with serum-starved HMVEC-d cells was used for Matrigel assays. (C) Serum-starved HMVEC-d cells left untreated or pretreated with 100 μM neomycin were mixed with EBM-2 and ANG (a and b) or VEGF (c and d) or supernatant from KSHV-infected cells (e and f) and layered over Matrigel. (D) Representation of the angiogenic index for Matrigel assays. The angiogenic index was calculated based on the number of branch points from each node formed due to various treatments and is represented in comparison to the results determined with supernatant from uninfected cells. (E) Quantitative real-time PCR analysis showing the expression of VEGF-C RNA in HMVEC-d cells that were either left uninfected or infected with KSHV for 48 h or ANG treated (250 ng/ml for 48 h) after pretreatment with neomycin (Neo) or paromomycin (Paro) (100 μM for 1 h). VEGF-C expression levels in uninfected HMVEC-d cells normalized to tubulin were assigned a value of 1 for comparisons. (F) Serum-starved HMVEC-d cells with or without neomycin pretreatment were either left uninfected or infected with KSHV (MOI of 10) or treated with 250 ng/ml ANG for 48 h. After 48 h p.i., cells were trypsinized, washed, and subjected to FACS analysis using VEGF-C antibody. (a) FACS analysis showing VEGF-C expression after ANG treatment compared to uninfected cell results. (b) VEGF-C expression upon KSHV infection without neomycin pretreatment and upon KSHV infection after neomycin pretreatment. Data represented as VEGF-C AF488 in x axis and cell counts in y axis.

Sathish Sadagopan, et al. J Virol. 2009 April;83(7):3342-3364.
9.
FIG. 9.

FIG. 9. From: Kaposi's Sarcoma-Associated Herpesvirus Upregulates Angiogenin during Infection of Human Dermal Microvascular Endothelial Cells, Which Induces 45S rRNA Synthesis, Antiapoptosis, Cell Proliferation, Migration, and Angiogenesis .

Increases in uPA activation and cell migration upon KSHV infection. (A) Cell lysates from serum-starved HMVEC-d cells (30 to 40% confluence) left uninfected or infected with KSHV (MOI of 10) for the indicated time periods were prepared. Lysates (100 μg) were used to assay for increases in uPA activity levels by use of active uPA ELISA, and the results were normalized to total uPA levels. The amount of active uPA normalized to total uPA in uninfected cells was assigned a value of 1 for comparisons. Each bar represents the average ± standard deviation of data from three independent experiments. (B) Serum-starved HMVEC-d cells left untreated or pretreated with neomycin (Neo) or paromomycin (Paro) were either left uninfected or infected with KSHV (MOI of 10) for 48 h. Cells treated with ANG (250 ng/ml for 48 h) were used as a positive control. Lysate (100 μg) was used for active uPA ELISA followed by normalization of the results to total uPA data. (C) A total of 20 μg of the indicated lysates was resolved using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotted with antiplasmin antibody (top panel). The blots were stripped and probed for β-actin (bottom panels). Asterisks and stars indicate the cleaved forms of plasmin heavy chain and plasmin light chain, respectively. (D) HMVEC-d cells with or without neomycin or paromomycin treatment were seeded in the upper chambers for cell migration assays, and cell culture supernatants from uninfected, KSHV-infected, and ANG-treated HMVEC-d cells were used as a chemoattractant in the bottom chambers. Cells that migrated to the bottom were suspended in cell detachment buffer and read at 528 nm emission and 485 nm excitation and expressed as relative fluorescence units (RFU). Each bar represents the average ± standard deviation of data from three independent experiments.

Sathish Sadagopan, et al. J Virol. 2009 April;83(7):3342-3364.
10.
FIG. 7.

FIG. 7. From: Kaposi's Sarcoma-Associated Herpesvirus Upregulates Angiogenin during Infection of Human Dermal Microvascular Endothelial Cells, Which Induces 45S rRNA Synthesis, Antiapoptosis, Cell Proliferation, Migration, and Angiogenesis .

KSHV-induced ANG binds to the 45S rRNA promoter and upregulates transcription of 45S rRNA. (A) Nuclear extracts prepared from subconfluent serum-starved HMVEC-d cells were either left uninfected (lane 1) or infected (lane 3) with KSHV (MOI of 10) for 72 h, incubated with ANG-specific probe, and analyzed by EMSA. The specificity of DNA-protein interactions was assessed by induction with ANG (250 ng/ml) (lane 4) and competitive EMSAs using a 100× molar excess of unlabeled double-stranded oligonucleotide ABE probe (Cold probe; lane 2). Each EMSA data point is representative of the results of at least three independent experiments. The solid arrow shows the shift in mobility. (B) HEK 293 cells were transfected with control pGL3E or pGL3E-ABE promoter-luciferase constructs. After 24 h, cells were either left uninfected or infected with KSHV (MOI of 10) for 48 h or treated with exogenous ANG (250 ng/ml for 48 h). Cells were harvested, lysed, and assayed for firefly luciferase activity. The data represent the averages + standard deviations (SD) of the results of three experiments and the mean relative luciferase units of results determined after normalizing to cotransfected Renilla luciferase activity levels. (C) HMVEC-d cells (30 to 40% confluence) were left uninfected or infected with KSHV for the indicated time periods, and total RNA was extracted, cDNA was prepared, and 45S rRNA gene expression was quantitated by real-time PCR. (D) HMVEC-d cells either left untreated or treated with neomycin (Neo; 100 μM for 1 h) were left uninfected or infected with KSHV for 48 h or treated with exogenous ANG (250 ng/ml for 48 h). Real-time PCR was performed for 45S rRNA gene expression determinations. The results shown in panels C and D represent the averages ± standard deviations of data from three independent experiments that were normalized to the HPRT expression levels. The 45S rRNA level normalized to uninfected cells was assigned a value of 1 for comparisons. ** and ***, statistically significant at P < 0.01 and P < 0.001, respectively.

Sathish Sadagopan, et al. J Virol. 2009 April;83(7):3342-3364.
11.
FIG. 1.

FIG. 1. From: Kaposi's Sarcoma-Associated Herpesvirus Upregulates Angiogenin during Infection of Human Dermal Microvascular Endothelial Cells, Which Induces 45S rRNA Synthesis, Antiapoptosis, Cell Proliferation, Migration, and Angiogenesis .

Increases in ANG protein and mRNA levels in KSHV-infected HMVEC-d cells. (A) HMVEC-d cells grown to ∼30 to 40% confluence (subconfluent cells) were serum starved for 8 h and infected with KSHV at an MOI of 10. At various times p.i., the levels of ANG and IL-8 proteins released in the cell-free culture supernatants were measured by ANG-ELISA and IL-8 ELISA, respectively. The results were normalized to a 1 mg/ml total protein concentration in the supernatant. TNF, tumor necrosis factor. (B) Serum-starved HMVEC-d cells (∼30% confluence; subconfluent cells) were infected with KSHV at different MOIs or with KSHV (MOI of 10) preincubated at 37°C for 1 h with DMEM containing 100 μg of soluble heparin/ml. After 48 h of infection at 37°C, ANG levels released in the supernatants were measured by ANG-ELISA. (C) RNA from KSHV (MOI of 10)-infected serum-starved HMVEC-d cells (∼30% confluence) was extracted at different days p.i., cDNA was prepared and used in real-time RT-PCR for ANG gene expression determinations performed using the SYBR green detection protocol, and the results were normalized to HPRT expression levels. Each vertical bar represents the average ± standard deviation of the results from three independent experiments. The ANG level normalized to HPRT in the uninfected cells was assigned a value of 1 for comparisons. ** and ***, statistically significant at P < 0.01 and P < 0.001, respectively. (D) Various concentrations (in micrograms/milliliter) of supernatants from serum-starved uninfected HMVEC-d cells (∼30% confluence) or from cells infected with KSHV for 8 and 24 h (top panel) or 10 μg of supernatant from serum-starved HMVEC-d cells infected with KSHV (MOI of 10) for different time periods (bottom panel) were adsorbed onto a nylon membrane and immunoblotted with polyclonal antibody against ANG. (E) Lymph node tissue sections from a KS patient and from a healthy individual were stained for ANG (brown) and counterstained using hematoxylin. Arrows indicate tissue staining positive for ANG.

Sathish Sadagopan, et al. J Virol. 2009 April;83(7):3342-3364.

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