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Neutral lipid alterations in human herpesvirus 8-infected HUVEC cells and their possible involvement in neo-angiogenesis.

Angius F, Uda S, Piras E, Spolitu S, Ingianni A, Batetta B, Pompei R - BMC Microbiol. (2015)

Bottom Line: In particular, triglyceride synthesis increases in the lytic phase, whereas cholesteryl ester synthesis rises in the latent one.Moreover, the inhibition of cholesterol esterification reduces neo-tubule formation mainly in latently infected cells.We suggest that a reprogramming of cholesteryl ester metabolism is involved in regulating neo-angiogenesis in HHV8-infected cells and plays a likely role in the high metastatic potential of derived-tumours.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, University of Cagliari, via Porcell 4, Cagliari, 09124, Italy. fangius@unica.it.

ABSTRACT

Background: Human Herpesvirus 8 (HHV8), the causative agent of Kaposi's sarcoma, induces an intense modification of lipid metabolism and enhances the angiogenic process in endothelial cells. In the present study, neutral lipid (NL) metabolism and angiogenesis were investigated in HHV8-infected HUVEC cells. The viral replication phases were verified by rtPCR and also by K8.1 and LANA immunostaining.

Results: Lipid droplets (Nile Red) were higher in all phases and NL staining (LipidTOX) combined with viral-antigen detection (immunofluorescence) demonstrated a NL content increase in infected cells. In particular, triglyceride synthesis increases in the lytic phase, whereas cholesteryl ester synthesis rises in the latent one. Moreover, the inhibition of cholesterol esterification reduces neo-tubule formation mainly in latently infected cells.

Conclusions: We suggest that a reprogramming of cholesteryl ester metabolism is involved in regulating neo-angiogenesis in HHV8-infected cells and plays a likely role in the high metastatic potential of derived-tumours.

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Related in: MedlinePlus

Neutral lipid content in lipid droplets in HHV8-infected HUVEC cells. HUVEC cells were infected with HHV8 as described in Figure 1. 24 h before the indicated times, cells were seeded at a density of 2.0 × 105 in 35 mm glass-bottomed dishes and cultured at 37°C in a 5% CO2 incubator in a growth medium. On days 3, 14 and 24 post infection, cells were fixed and stained with Nile Red (for details see Methods), reported as green/yellow dots in the figure (panel A). The bar in the figure is 30 μm. Panel B represents the quantitative analysis of Nile Red green fluorescence intensity. At least 200 cells were individually selected and analyzed for each experimental group. Normalized data represent the percentage of the mean density value (intensity per pixel) ± standard error (SE). Significance was set up when p < 0.05 (*) or p < 0.01(**) vs. respective control (ANOVA and Fischer’s LSD as post hoc test).
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Fig2: Neutral lipid content in lipid droplets in HHV8-infected HUVEC cells. HUVEC cells were infected with HHV8 as described in Figure 1. 24 h before the indicated times, cells were seeded at a density of 2.0 × 105 in 35 mm glass-bottomed dishes and cultured at 37°C in a 5% CO2 incubator in a growth medium. On days 3, 14 and 24 post infection, cells were fixed and stained with Nile Red (for details see Methods), reported as green/yellow dots in the figure (panel A). The bar in the figure is 30 μm. Panel B represents the quantitative analysis of Nile Red green fluorescence intensity. At least 200 cells were individually selected and analyzed for each experimental group. Normalized data represent the percentage of the mean density value (intensity per pixel) ± standard error (SE). Significance was set up when p < 0.05 (*) or p < 0.01(**) vs. respective control (ANOVA and Fischer’s LSD as post hoc test).

Mentions: Figure 2A shows a remarkable increase of neutral lipids in LDs in all the HHV8-infected cells when compared to the respective control. As demonstrated by imaging analysis, the highest increase was observed on day 3 (Figure 2B). As is evident from the images, there is a heterogeneous distribution of LDs throughout the cells, probably due to the mixed population of infected/uninfected cells. In order to ascertain whether the neutral lipid increase was a peculiarity of the infected cells, we next used a double stain for neutral lipids (LipidTOX, red) combined with FITC-conjugated antibodies (green) for the detection of viral-antigens, namely K8.1 on day 3 and LANA on days 14 and 24 (Figure 3A). Imaging analysis of the merged images demonstrated that LDs were definitely higher in infected-cells (Figure 3B). In particular, when the LDs were only evaluated in infected cells, the strong increase of LDs was more evident on day 14 (p < 0.001). The differences between the infected and control cells were statistically significant (p < 0.05).Figure 2


Neutral lipid alterations in human herpesvirus 8-infected HUVEC cells and their possible involvement in neo-angiogenesis.

Angius F, Uda S, Piras E, Spolitu S, Ingianni A, Batetta B, Pompei R - BMC Microbiol. (2015)

Neutral lipid content in lipid droplets in HHV8-infected HUVEC cells. HUVEC cells were infected with HHV8 as described in Figure 1. 24 h before the indicated times, cells were seeded at a density of 2.0 × 105 in 35 mm glass-bottomed dishes and cultured at 37°C in a 5% CO2 incubator in a growth medium. On days 3, 14 and 24 post infection, cells were fixed and stained with Nile Red (for details see Methods), reported as green/yellow dots in the figure (panel A). The bar in the figure is 30 μm. Panel B represents the quantitative analysis of Nile Red green fluorescence intensity. At least 200 cells were individually selected and analyzed for each experimental group. Normalized data represent the percentage of the mean density value (intensity per pixel) ± standard error (SE). Significance was set up when p < 0.05 (*) or p < 0.01(**) vs. respective control (ANOVA and Fischer’s LSD as post hoc test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4384337&req=5

Fig2: Neutral lipid content in lipid droplets in HHV8-infected HUVEC cells. HUVEC cells were infected with HHV8 as described in Figure 1. 24 h before the indicated times, cells were seeded at a density of 2.0 × 105 in 35 mm glass-bottomed dishes and cultured at 37°C in a 5% CO2 incubator in a growth medium. On days 3, 14 and 24 post infection, cells were fixed and stained with Nile Red (for details see Methods), reported as green/yellow dots in the figure (panel A). The bar in the figure is 30 μm. Panel B represents the quantitative analysis of Nile Red green fluorescence intensity. At least 200 cells were individually selected and analyzed for each experimental group. Normalized data represent the percentage of the mean density value (intensity per pixel) ± standard error (SE). Significance was set up when p < 0.05 (*) or p < 0.01(**) vs. respective control (ANOVA and Fischer’s LSD as post hoc test).
Mentions: Figure 2A shows a remarkable increase of neutral lipids in LDs in all the HHV8-infected cells when compared to the respective control. As demonstrated by imaging analysis, the highest increase was observed on day 3 (Figure 2B). As is evident from the images, there is a heterogeneous distribution of LDs throughout the cells, probably due to the mixed population of infected/uninfected cells. In order to ascertain whether the neutral lipid increase was a peculiarity of the infected cells, we next used a double stain for neutral lipids (LipidTOX, red) combined with FITC-conjugated antibodies (green) for the detection of viral-antigens, namely K8.1 on day 3 and LANA on days 14 and 24 (Figure 3A). Imaging analysis of the merged images demonstrated that LDs were definitely higher in infected-cells (Figure 3B). In particular, when the LDs were only evaluated in infected cells, the strong increase of LDs was more evident on day 14 (p < 0.001). The differences between the infected and control cells were statistically significant (p < 0.05).Figure 2

Bottom Line: In particular, triglyceride synthesis increases in the lytic phase, whereas cholesteryl ester synthesis rises in the latent one.Moreover, the inhibition of cholesterol esterification reduces neo-tubule formation mainly in latently infected cells.We suggest that a reprogramming of cholesteryl ester metabolism is involved in regulating neo-angiogenesis in HHV8-infected cells and plays a likely role in the high metastatic potential of derived-tumours.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, University of Cagliari, via Porcell 4, Cagliari, 09124, Italy. fangius@unica.it.

ABSTRACT

Background: Human Herpesvirus 8 (HHV8), the causative agent of Kaposi's sarcoma, induces an intense modification of lipid metabolism and enhances the angiogenic process in endothelial cells. In the present study, neutral lipid (NL) metabolism and angiogenesis were investigated in HHV8-infected HUVEC cells. The viral replication phases were verified by rtPCR and also by K8.1 and LANA immunostaining.

Results: Lipid droplets (Nile Red) were higher in all phases and NL staining (LipidTOX) combined with viral-antigen detection (immunofluorescence) demonstrated a NL content increase in infected cells. In particular, triglyceride synthesis increases in the lytic phase, whereas cholesteryl ester synthesis rises in the latent one. Moreover, the inhibition of cholesterol esterification reduces neo-tubule formation mainly in latently infected cells.

Conclusions: We suggest that a reprogramming of cholesteryl ester metabolism is involved in regulating neo-angiogenesis in HHV8-infected cells and plays a likely role in the high metastatic potential of derived-tumours.

Show MeSH
Related in: MedlinePlus