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. 2008 Jun 15;68(12):4640-8.
doi: 10.1158/0008-5472.CAN-07-5988.

Kaposi's sarcoma-associated herpesvirus confers a survival advantage to endothelial cells

Ling Wang  1 , Blossom Damania
Affiliations

Kaposi's sarcoma-associated herpesvirus confers a survival advantage to endothelial cells

Ling Wang et al. Cancer Res. .

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with three different human malignancies, including Kaposi's sarcoma (KS), primary effusion lymphoma, and multicentric Castleman's disease. The KS lesion is of endothelial cell in origin and is highly dependent on autocrine and paracrine factors for survival and growth. In this study, we show that KSHV infection of endothelial cells induces the activation of the prosurvival phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin pathway. KSHV infection of endothelial cells augmented cell survival in the presence of apoptotic inducers, including etoposide and staurosporine, and under conditions of serum deprivation. We found that KSHV infection of endothelial cells also increased the ability of these cells to form an in vitro tubular network under conditions of stress and growth factor deprivation. Finally, we show that the nuclear factor-kappaB and PI3K pathways are also required for endothelial tubular network formation. Collectively, these results suggest that KSHV infection of endothelial cells modulates cell signaling pathways and induces cell survival and angiogenesis, thereby contributing to the pathogenesis induced by KSHV.

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Figures

Figure 1
Figure 1. KSHV infection induces activation of the PI3K/Akt/mTOR pathway
HUVEC and KSHV-HUVEC were harvested 48 hours post-seeding, and equal micrograms of cell lysates were subjected to SDS-PAGE. The gel was transferred to nitrocellulose and western blots were performed with the indicated antibodies. KSHV infection of endothelial cells increased phosphorylation of the p85 sub-unit of PI3K, phosphorylation of Akt, and mTOR. We also found that KSHV-HUVEC displayed a decrease in the phosphorylation of AMPK.
Figure 2
Figure 2. KSHV infection confers endothelial cell survival upon serum deprivation
HUVEC and KSHV-HUVEC were grown in serum-free media for five days. (A) Images of the HUVEC and KSHV-HUVEC cells were taken under bright field microscopy. Additionally images of the KSHV-HUVEC (which express GFP) were taken under fluorescence microscopy. Time-points included were 24, 48, 72, 96 and 120 hours post-serum starvation. Images shown were taken at 4X magnification. (B) Graph of the percent of live cells at different time points. The number of live cells in ten different fields were counted and averaged.
Figure 3
Figure 3. KSHV infection suppresses apoptosis induced by etoposide and staurosporine
(A) HUVEC and KSHV-HUVEC were treated with endothelial media, EBM-2, containing 500 ng/ml TNFα, 100 μM etoposide or 20nM staurosporine for 24 hours. Cells were then harvested and Caspase-3 activity was measured using the Caspase-3 substrate, DEVD-AFC. Etoposide and staurosporine, but not TNFα, induced Caspase-3 activation in the HUVEC and KSHV-HUVEC. However, Caspase-3 activity induced in KSHV-infected HUVEC was lower than that induced in uninfected HUVEC. (B) HUVEC and KSHV-HUVEC were incubated in EGM-2 media containing 10%FBS, with either 100 μM etoposide, 100 μM etoposide plus 50μM Z-VAD-FMK, 20nM staurosporine, or 20nM staurosporine plus 50μM Z-VAD-FMK for 24 hours. Cells were then harvested and Caspase-3 activity was measured as above. Z-VAD-FMK, a Caspase-3 inhibitor, dramatically suppressed Caspase-3 activity induced by etoposide and staurosporine in HUVEC and KSHV-HUVEC. (C) HUVEC and KSHV-HUVEC were incubated in EBM-2 media, containing either 100μM etoposide or 20nM staurosporine for 24 hours. Cells were then harvested and PARP-1 activity was measured using a Colorimetric PARP Assay kit (Trevigen). KSHV-HUVEC treated with etoposide maintained higher PARP-1 activity compared to uninfected HUVEC. D) HUVEC and KSHV-HUVEC were incubated in EGM-2 media containing 10%FBS, with either 100 μM etoposide, 100 μM etoposide plus 50μM Z-VAD-FMK, 20nM staurosporine, or 20nM staurosporine plus 50μM Z-VAD-FMK for 24 hours. Cells were then harvested and PARP-1 activity was measured as above. Z-VAD-FMK dramatically increased PARP-1 activity in HUVEC and KSHV-HUVEC stimulated with etoposide and staurosporine.
Figure 4
Figure 4. Tubule formation assay for HUVEC and KSHV-HUVEC
(A) HUVEC and KSHV-HUVEC were plated on growth factor reduced matrigel in the presence of media with serum for 24 hours and allowed to form capillary-like tubules. (B) KSHV-negative BJAB and KSHV-positive BCBL-1 cells were incubated in plain RPMI media without serum for 48 hours at 37°C, and then the conditioned media from both cell lines were harvested for tubule formation assays. HUVEC and KSHV-HUVEC were seeded on matrigel to form capillary-like tubules in the presence of media without serum, or conditioned media from BJAB or BCBL-1 cells. Images of the HUVEC and KSHV-HUVEC cells were taken under bright field microscopy. Additionally images of the KSHV-HUVEC (which express GFP) were taken under fluorescence microscopy. KSHV-HUVEC cells were capable of forming more tubules than the uninfected HUVEC under all three conditions. (C) Depicted is the angiogenic index, which reflects the number of branch points formed under each condition. Shown is the average number of branch points in five different fields per sample.
Figure 5
Figure 5. KSHV infection of endothelial cells allows tubule formation in the absence of VEGF and bFGF
(A) Tubule formation assays were performed with HUVEC and KSHV-HUVEC in endothelial cell complete media, media without VEGF, or media without bFGF for four days. Images of the HUVEC and KSHV-HUVEC cells were taken under bright field microscopy. Additionally images of the KSHV-HUVEC (which express GFP) were taken under fluorescence microscopy. The following time-points were taken: 24 hours (panel A), 48 hours (panel B), 72 hours (panel C), and 96 hours (panel D) hours. Images shown were taken at 10X magnification. The tubules formed by KSHV-HUVEC appeared to be maintained on matrigel for a longer period of time compared with those formed by HUVEC. (B) Tubule formation assays were performed with HUVEC and KSHV-HUVEC in serum-free EBM-2 media, with or without anti-VEGF antibody. Images were taken after 16 hours at 4X magnification. Uninfected HUVEC could not form tubules in the presence of the blocking VEGF antibody. Although overall numbers of tubules was greatly reduced, KSHV-infected HUVEC could still form a few tubules in the presence of the blocking antibody.
Figure 6
Figure 6. NF-κB and PI3K pathways are essential for tubule formation by KSHV-HUVEC
(A) The tubule formation assays were performed on matrigel with HUVEC and KSHV-HUVEC in EBM-2 media containing 50nM Rapamycin (mTOR inhibitor), 150μM Ciglitazone (AMPK activator), 2.5μM SU6656 (Src inhibitor), 20μM Piceatannol (Syk inhibitor), 5μM Bay11-7085 (NF-κB inhibitor) or 20μM LY294002 (PI3K inhibitor). Images of the HUVEC and KSHV-HUVEC cells under bright field were taken at 24, 48, 72 and 96 hours. KSHV-HUVEC were also imaged under fluorescence microscopy. Images shown were taken at 4X magnification. (B) Depicted is the angiogenic index, which reflects the number of branch points formed under each condition. Shown is the average number of branch points in five different fields per sample.
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