Lymphatic dysfunction in transgenic mice expressing KSHV k-cyclin under the control of the VEGFR-3 promoter

Abstract

Kaposi sarcoma-associated herpesvirus (KSHV) infects endothelial cells within KS tumors, and these cells express the KSHV latent-cycle gene k-cyclin (kCYC) as well as vascular endothelial growth factor receptor 3 (VEGFR-3), a marker for lymphatic endothelium. To further understand KSHV-mediated pathogenesis, we generated transgenic mice expressing kCYC under the control of the VEGFR-3 promoter. kCYC mRNA and functional protein expression within tissue correlated with VEGFR-3 expression and were most abundantly detected within lung tissue. Clinically, most transgenic mice died within 6 months of age secondary to progressive accumulation of chylous pleural fluid. In skin, edema was detected by magnetic resonance imaging and mice demonstrated persistent erythema of the ears following trauma. Histologically, erythematous skin showed extravasation of erythrocytes and accumulation of erythrocytes within lymphatic lumens. In addition, lymphatic drainage of injected contrast dyes was markedly impaired in transgenic mice. Karyomegaly, a feature observed in kCYC-expressing cells in IntroductionKaposi sarcoma (KS) is the most common cancer in individuals with AIDS as well as the most common cancer in the general population of many sub-Saharan countries where Kaposi sarcomaassociated herpesvirus (KSHV) infection is endemic. 1 Clinically, KS most commonly involves the skin, where lesions appear as dark red-to-purple patches, plaques, nodules, and tumors. Skin lesions are often preceded by or accompanied by localized tissue edema, especially when they are present on the lower extremities. Histologically, KS tumor cells within skin are localized to the dermis where they demonstrate a spindle cell morphology and form irregular, slitlike vascular structures. Numerous inflammatory cells and extravasated erythrocytes are also commonly present within KS tumors. To date, KS tumor cells have been shown to express specific markers for lymphatic endothelium, including vascular endothelial growth factor receptor 3 (VEGFR-3), 2-6 podoplanin, 5,9 and Prox-1. 9,10 In all clinical forms of KS, tumor cells are infected with the ␥-herpesvirus KSHV. 3,[11][12][13][14] Although the KSHV genome encompasses over 80 open reading frames (ORFs), 15 the expression of KSHV genes in the vast majority of KS tumor spindle cells is restricted to relatively few latent-cycle genes, including latencyassociated nuclear antigen (LANA), k-cyclin (kCYC), and viral FLICE inhibitory protein (vFLIP). 3,16-20 kCYC, which is encoded by ORF72, is a cellular cyclin D2 homologue that possesses a number of functional properties believed to be important in KS pathogenesis. It interacts with all types of cyclin-dependent kinases (Cdks), although it preferentially binds to Cdk6. 21-24 kCYC/Cdk6 complexes phosphorylate and inactivate both retinoblastoma (Rb) protein 24,25 and the Cdk inhibitor p27, 26,27 which leads to unregulated progression through the cell cycle. In the presence of normal p53 function, kCYC also sensitizes cells to undergo apoptosis, 24,28,29 whereas in ...

Regulation of Microfilament Organization by Kaposi Sarcoma-associated Herpes Virus-cyclin·CDK6 Phosphorylation of Caldesmon

“…Human vascular endothelial cells, which normally form cobblestone-like cell arrays, are known to respond to KSHV infection with a striking shape change leading to narrow, light-refractive cell bodies, loss of cell junctions, and decreased substratum adhesion (72,73); this is quite reminiscent of the response that arises in the U2OS cells upon K-cyclin expression, as shown above (72,73). This morphological transformation is also reproduced in transgenic mice with endothelium selective K-cyclin expression (74). The work presented here provides a possible molecular explanation of how these morphological alterations are achieved.…”

Oncostatin M Enhances CCL21 Expression by Microvascular Endothelial Cells and Increases the Efficiency of Dendritic Cell Trafficking to Lymph Nodes

“…Lymphatic transport of Evans blue dye to draining LN normally occurs within minutes, and this technique has been used to document defects in lymphatic drainage in skin (28). Compared with PBS mock treatment, injection with OSM significantly increased the accumulation of dye in the LN by ϳ3-fold ( p ϭ 0.0291) (Fig.…”

“…C, Evans blue dye was injected into mouse footpads that had been treated with either rmOSM (1 g) or PBS 24 h earlier. One minute after injection of dye, mice were euthanized, and the Evans blue dye was extracted from draining LN for quantification of OD measurement at 630 nm (28). The y-axis indicates the concentration of dye in the LN as determined by comparison with a standard curve of known amounts of dye.…”

CCL11–CCR3 Interactions Promote Survival of Anaplastic Large Cell Lymphoma Cells via ERK1/2 Activation

“…cDNA was synthesized using TaqMan Reverse Transcription Reagents (Applied Biosystems). Quantitative reverse-transcriptase (RT)-PCR was performed as described previously (27). Primers for human genes were as follows: CCR3 forward, 5 0 -TCG TTC TCC CTC TGC TCG TT-3 0 and reverse, 5 0 -GCC GGA TGG CCT TGT ACT TT-3 0 ; CCL11 forward, 5 0 -GGG CCA GCT TCT GTC CCA AC-3 0 and reverse, 5 0 -TTA TGG CTT TGG AGT TGG AGA TTT-3 0 ; and GAPDH forward, 5 0 -ACC CAC TCC TCC ACC TTT GA-3 0 and reverse, 5 0 -CAT ACC AGG AAA TGA GCT TGA CAA-3 0 .…”