HHV-8 acute infection induces MCP-1–dependent capillary-like structure formation. Mock-infected (control) and HHV-8–infected HUVECs were harvested 24 hours after infection and plated on BME (5 × 10⁴ cell/well). Cells were incubated at 37°C in complete medium (i-ii) or medium containing 1 μg/mL anti–MCP-1 mAb (iii-iv), or unrelated mAb (v-vi). After 4 hours cells were observed by optical microscopy (Leica DM IRB, Wetzlar, Germany) and photographed with CCD optics (Hitachi Denshi Color Camera KP-D50E/K; Rodgau, Germany) using a digital analysis system (QWIN LITE version 2.3; Leica). Tubelike structure formation is evident in HHV-8–infected cells (compare panels i and ii). The addition of anti–MCP-1 antibody completely blocks the formation of capillary-like structures in HHV-8–infected HUVECs (compare panels ii and iv), whereas the unrelated mAb has no effect (compare panels iv and vi). Photographs (4×/0.10 NA Plan objective lens) from a representative experiment of 3 with similar results are shown; mAb indicates monoclonal antibody. (B) HUVECs were treated with 5 μM MG132 (carbobenzoxy-l-leucyl-l-leucyl-l-leucynal; Sigma-Aldrich; +) or control diluent (−). After 1 hour, cells were infected with HHV-8 or mock infected (C) and incubated in the presence or absence of MG132. At 4 hours after infection, MCP-1 levels in culture supernatants were determined by ELISA. Data represent the mean ± SD of triplicate samples. (C) In a parallel experiment, HHV-8–infected HUVECs were cultured in the presence (+) or absence (−) of MG132 (1 μM) for 24 hours, and then plated on BME (5 × 10⁴ cell/well) in fresh medium in the absence of MG132. Cells were observed under microscope for capillary-like structure formation at 4 hours after seeding. Photographs (original magnification × 40) from a representative experiment are shown. MG132 treatment during infection completely prevented endothelial cell alignment and tubule formation.