β1 Integrins Mediate Tubule Formation Induced by Supernatants Derived from KSHV-Infected Cells

Abstract

Objective: Angiogenesis is defined as the formation of new blood vessels. In a recently concluded study, we identified Kaposi’s sarcoma-associated herpesvirus (KSHV)-infected cells derived from primary effusion lymphoma (PEL) to overexpress vascular endothelial growth factor (VEGF) that had the propensity to mediate tubule formation on a Matrigel, an indicator of angiogenesis. The objective of this study was to determine the receptor molecules that mediate the tubule formation induced by the supernatant derived from KSHV-infected PEL cells. Methods: The identity of receptor(s) that play a role in mediating tubule formation driven by PEL supernatant was determined by the classical in vitro angiogenesis assay conducted on a Matrigel. Results: RGD peptides, antibodies, and siRNA specific to β1 integrins significantly lowered the ability of the PEL supernatants to induce tubule formation by endothelial cells. β1 Integrins mediated tubule formation to comparable levels in endothelial cells that were incubated with supernatants derived from uninduced or TPA-induced PEL cells. Interestingly, the β1 integrins did not seem to have a major role in cellular attachment. Conclusion: We report for the first time a critical role for β1 integrins in angiogenesis supported by the supernatant from KSHV-infected PEL cells.

Latent KSHV Infection of Endothelial Cells Induces Integrin Beta3 to Activate Angiogenic Phenotypes

“…These activities are necessary for vascularization of solid tumors and contribute to the promotion of tumor formation and metastasis. As has been described by others, KSHV is likely to promote angiogenesis in a paracrine fashion to promote new blood vessel growth in the KS tumor [10], [12], [19]–[21], [28], [41], [59]. Here we found that KSHV also directly induces angiogenic phenotypes in latently infected cells.…”

Cell Membrane-bound Kaposi's Sarcoma-associated Herpesvirus-encoded Glycoprotein B Promotes Virus Latency by Regulating Expression of Cellular Egr-1

“…Quantitative RT-PCR (qRT-PCR)-The qRT-PCR was performed using the synthesized cDNA in a 25-l reaction volume to analyze the expression of ORF50, egr-1, and ␤-actin as per earlier protocols (24). (25).…”

“…The gB-and gL-positive cells were sorted using a flow cytometer based on the expression of GFP. The time at which the mixture of cells was seeded onto 12-well plates was considered as 0 h. At different time points (1,4,8,12,24,48,72,96, and 120 h) post-seeding, the cells were lysed, RNA was extracted, cDNA was prepared, and the expression of ORF50 was monitored by qRT-PCR using specific primers. The relative copy numbers of the ORF50 transcripts were calculated from the standard graph plotted using the Ct values for different dilutions of in vitro transcribed ORF50 (using ORF50/pGEM-T plasmid).…”

“…Western Blotting-Equal amounts of protein were used in Western blotting experiments as per earlier studies (24). We used 60 g (3 times more) of protein in this experiment for clarity of the bands.…”

KSHV-transformed primary effusion lymphoma cells induce a VEGF-dependent angiogenesis and establish functional gap junctions with endothelial cells

“…[27][28][29] It was shown that KSHV-infected cells secrete VEGF and induce in vitro angiogenesis. 30 Recent studies reported that b-integrins 31 or induction of angiopoietin-2 32 mediates PEL-induced tube formation. Cells influence their immediate microenvironment either through paracrine mechanisms or through direct cell-cell interaction, such as adhesion and gap junction-mediated communication.…”