Journal: PLoS Pathogens

Article Title: Kaposi's Sarcoma Associated Herpes Virus (KSHV) Induced COX-2: A Key Factor in Latency, Inflammation, Angiogenesis, Cell Survival and Invasion

doi: 10.1371/journal.ppat.1000777

Figure Lengend Snippet: ( A ) Scheme of MMPs on MMP array-1. ( B ) Representative MMP arrays showing the signals for various MMPs in the conditioned medium obtained from serum starved uninfected HMVEC-d cells or cells infected with 30 DNA copies/ cell of KSHV for 4 h, 8 h, and 24 h. ( C ) Densitometric analysis of MMP array blots measuring the release of human MMPs. The values were normalized to identical background levels using the Ray Biotech Human MMP antibody array 1 analysis tool. The fold induction in MMP secretion was calculated by dividing the respective values obtained from infected-cell supernatants with the values obtained from uninfected-cell supernatants. Each point represents the average ± SD from three independent experiments. ( D, E, F and G ) MMP gene expression. MMP-1 ( D ), MMP-2 ( E ), MMP-9 ( F ), and MMP-10 ( G ) gene expression was measured by q-RT-PCR with cDNA prepared from serum starved (8h) HMVEC-d cells infected or NS-398 treated (1h), and then infected with KSHV for 4h, 8h, and 24h. Result shows the mean ± S.D of three independent experiments. % inhibition in gene expression upon NS-398 treatment was calculated using gene expression in the presence of KSHV infected HMVEC-d cells at different time points as 100%. ( H ) MMP-2 and MMP-9 gene expression in TIVE-LTC cells upon COX inhibitor treatment. Gene expression was measured by q-RT-PCR with cDNA prepared from serum starved (24h) TIVE-LTC cells untreated or treated with either 500 µM Indo or 75 µM NS-398 for 24h. Result shows the mean ± S.D of three independent experiments. % inhibition in gene expression upon inhibitor treatment was calculated using gene expression in untreated TIVE-LTC cells as 100%. *, **, ***-statistically significant at p<0.01, p<0.005 and p<0.001 respectively.

Article Snippet: Total soluble protein was quantified by bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL) prior to use ensuring equal protein concentration for studying the cytokine profile by human protein cytokine arrays 3.1 and 5.1 from Ray Biotech (Norcross, GA) and Ray Biotech human MMP antibody array-1 which detects 10 human MMPs in one experiment.

Techniques: Infection, Ab Array, Expressing, Reverse Transcription Polymerase Chain Reaction, Inhibition

Journal: PLoS Pathogens

Article Title: Kaposi's Sarcoma Associated Herpes Virus (KSHV) Induced COX-2: A Key Factor in Latency, Inflammation, Angiogenesis, Cell Survival and Invasion

doi: 10.1371/journal.ppat.1000777

Figure Lengend Snippet: In vitro KSHV infection of HMVEC-d cells involves binding of the virus to the cell surface heparan sulfate (HS) molecules via its envelope glycoproteins gpK8.1A and gB , , , , followed by interaction with integrins and xCT molecules. Virus interaction with target cell triggers pre-existing signal cascades facilitating virus entry, delivery of viral genome into the target cell nucleus and reprograms host gene expression required for various growth, angiogenic and invasive factors and one which being COX-2 . Data presented here show appreciable COX-2 gene expression at 5d PI of endothelial cells, in latently infected TIVE-LTC cells and in KS lesions. COX-2 catalyzes the synthesis of PGH2 from arachidonic acid (AA) through an unstable intermediate PGG2 . PGH2 is converted by mPGES to PGE2, which is released to the infected cell supernatant where it can mediate its downstream effects through either an autocrine or paracrine mechanism on a neighboring infected or uninfected cell via their interactions through the family of seven transmembrane G-protein-coupled rhodopsin-type EP (1–4) receptors. KSHV induced COX-2 regulates multiple events involved in KS pathogenesis such as secretion of pro-inflammatory cytokines, growth and angiogenic factors, anti-inflammatory cytokines, and MMPs and TIMPs. In addition, COX-2 induction also regulated infection related cell adhesion to the ECM, invasion through the matrix and angiogenesis related capillary tube formation. We demonstrated that KSHV infection induced COX-2/PGE2 also stimulates the induction of Rac1-GTPases in adhering endothelial cells. Interestingly, our study also demonstrates that KSHV infection induced COX-2 potentially modulates the survival and proliferation of latently infected endothelial cells. In summary, together with the down-regulation of viral latent gene expression upon COX-2 inhibition, our study suggests that KSHV hijacks host cellular machinery and manipulates cellular inducible angiogenic stress response gene COX-2 to its advantage to aggravate pathogenesis, cell survival and its persistence in the target cell.

Article Snippet: Total soluble protein was quantified by bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL) prior to use ensuring equal protein concentration for studying the cytokine profile by human protein cytokine arrays 3.1 and 5.1 from Ray Biotech (Norcross, GA) and Ray Biotech human MMP antibody array-1 which detects 10 human MMPs in one experiment.

Techniques: In Vitro, Infection, Binding Assay, Expressing, Inhibition