An exquisite cross-control mechanism among endothelial cell fate regulators directs the plasticity and heterogeneity of lymphatic endothelial cells.

Arteriovenous-lymphatic endothelial cell fates are specified by the master regulators, namely, Notch, COUP-TFII, and Prox1. Whereas Notch is expressed in the arteries and COUP-TFII in the veins, the lymphatics express all 3 cell fate regulators. Previous studies show that lymphatic endothelial cell (LEC) fate is highly plastic and reversible, raising a new concept that all 3 endothelial cell fates may co-reside in LECs and a subtle alteration can result in a reprogramming of LEC fate. We provide a molecular basis verifying this concept by identifying a cross-control mechanism among these cell fate regulators. We found that Notch signal down-regulates Prox1 and COUP-TFII through Hey1 and Hey2 and that activated Notch receptor suppresses the lymphatic phenotypes and induces the arterial cell fate. On the contrary, Prox1 and COUP-TFII attenuate vascular endothelial growth factor signaling, known to induce Notch, by repressing vascular endothelial growth factor receptor-2 and neuropilin-1. We show that previously reported podoplanin-based LEC heterogeneity is associated with differential expression of Notch1 in human cutaneous lymphatics. We propose that the expression of the 3 cell fate regulators is controlled by an exquisite feedback mechanism working in LECs and that LEC fate is a consequence of the Prox1-directed lymphatic equilibrium among the cell fate regulators.

Embryonic vascular endothelial cells are malleable to reprogramming via Prox1 to a lymphatic gene signature.

BACKGROUND: In vivo studies demonstrate that the Prox1 transcription factor plays a critical role in the development of the early lymphatic system. Upon Prox1 expression, early lymphatic endothelial cells differentiate from the cardinal vein and begin to express lymphatic markers such as VEGFR-3, LYVE-1 and Podoplanin. Subsequent in vitro studies have found that differentiated vascular endothelial cells can be reprogrammed by Prox1 to express a lymphatic gene profile, suggesting that Prox1 can initiate the expression of a unique gene signature during lymphangiogenesis. While the in vitro data suggest that gene reprogramming occurs upon Prox1 expression, it is not clear if this is a direct result of Prox1 in vascular endothelial cells in vivo. RESULTS: Overexpression of Prox1 in vascular endothelial cells during embryonic development results in the reprogramming of genes to that of a more lymphatic signature. Consequent to this overexpression, embryos suffer from gross edema that results in embryonic lethality at E13.5. Furthermore, hemorrhaging and anemia is apparent along with clear defects in lymph sac development. Alterations in junctional proteins resulting in an increase in vascular permeability upon Prox1 overexpression may contribute to the complications found during embryonic development. CONCLUSION: We present a novel mouse model that addresses the importance of Prox1 in early embryonic lymphangiogenesis. It is clear that there needs to be a measured pattern of expression of Prox1 during embryonic development. Furthermore, Prox1 reprograms vascular endothelial cells in vivo by creating a molecular signature to that of a lymphatic endothelial cell.

ROCK Inhibitor Enhances Adhesion and Wound Healing of Human Corneal Endothelial Cells.

Maintenance of corneal transparency is crucial for vision and depends mainly on the endothelium, a non-proliferative monolayer of cells covering the inner part of the cornea. When endothelial cell density falls below a critical threshold, the barrier and "pump" functions of the endothelium are compromised which results in corneal oedema and loss of visual acuity. The conventional treatment for such severe disorder is corneal graft. Unfortunately, there is a worldwide shortage of donor corneas, necessitating amelioration of tissue survival and storage after harvesting. Recently it was reported that the ROCK inhibitor Y-27632 promotes adhesion, inhibits apoptosis, increases the number of proliferating monkey corneal endothelial cells in vitro and enhance corneal endothelial wound healing both in vitro and in vivo in animal models. Using organ culture human cornea (N = 34), the effect of ROCK inhibitor was evaluated in vitro and ex vivo. Toxicity, corneal endothelial cell density, cell proliferation, apoptosis, cell morphometry, adhesion and wound healing process were evaluated by live/dead assay standard cell counting method, EdU labelling, Ki67, Caspase3, Zo-1 and Actin immunostaining. We demonstrated for the first time in human corneal endothelial cells ex vivo and in vitro, that ROCK inhibitor did not induce any toxicity effect and did not alter cell viability. ROCK inhibitor treatment did not induce human corneal endothelial cells proliferation. However, ROCK inhibitor significantly enhanced adhesion and wound healing. The present study shows that the selective ROCK inhibitor Y-27632 has no effect on human corneal endothelial cells proliferative capacities, but alters cellular behaviours. It induces changes in cell shape, increases cell adhesion and enhances wound healing ex vivo and in vitro. Its absence of toxicity, as demonstrated herein, is relevant for its use in human therapy.

Phosphorylation regulates FOXC2-mediated transcription in lymphatic endothelial cells.

One of the key mechanisms linking cell signaling and control of gene expression is reversible phosphorylation of transcription factors. FOXC2 is a forkhead transcription factor that is mutated in the human vascular disease lymphedema-distichiasis and plays an essential role in lymphatic vascular development. However, the mechanisms regulating FOXC2 transcriptional activity are not well understood. We report here that FOXC2 is phosphorylated on eight evolutionarily conserved proline-directed serine/threonine residues. Loss of phosphorylation at these sites triggers substantial changes in the FOXC2 transcriptional program. Through genome-wide location analysis in lymphatic endothelial cells, we demonstrate that the changes are due to selective inhibition of FOXC2 recruitment to chromatin. The extent of the inhibition varied between individual binding sites, suggesting a novel rheostat-like mechanism by which expression of specific genes can be differentially regulated by FOXC2 phosphorylation. Furthermore, unlike the wild-type protein, the phosphorylation-deficient mutant of FOXC2 failed to induce vascular remodeling in vivo. Collectively, our results point to the pivotal role of phosphorylation in the regulation of FOXC2-mediated transcription in lymphatic endothelial cells and underscore the importance of FOXC2 phosphorylation in vascular development.

High-dose sequential chemotherapy (ICE) under circulating progenitor cells (CPC) protection in patients with small cell lung carcinoma (SCLC). Preliminary report on a multicentric study

Starting in February 1994, 20 patients (pt) with a median age of 50 years(range 41-63) from 7 European centers have been included. Completedata were obtained in 16 patients so far. CPC were mobilized with chemo(Epirubicine 75 mg/m2 /d, 01 + 02) followed by G-CSF 5 p.gfkg/d for14 days. HD chemo consisted in 3 sequential courses of ICE regimen(UOs. 10 g/m2 , Carbo. 1200 mg/m2 and Etop. 1200 mg/m2 ) underCPC protection and G-CSF 5 p.g/kg/d. Out of the 16 pt, 12 completedfull program (3 cycles). One pt died of septic shock before receivingany ICE course. One pt died during the first ICE of renal insufficiency.Two pt had only 2 courses because of toxicity. Among the 16 pt, responserate (RR) was: 7 CR, 6 PR, 1 PO; 3 pt are not evaluable dueto early withdrawal (overall RR: 13/16 = 81 %). Thirty-nine cycles ofHD chemo were given with a median hematological recovery of 9 days(range 7-12) until neutro. counts> 1.0 x 109 /1 and 9 days (range 717)until thrombo. > 20 x 109 /1. No cumulative, hematological toxicitywas seen. Accrual of patients is still ongoing and updated results will bepresented.

Modulation of cdk2, cyclin D1, p16INK4a, p21WAF and p27Kip1 expression in endothelial cells by TNF/IFN gamma.

BACKGROUND: Regional administration of high doses of tumor necrosis factor (TNF) and interferon gamma (IFN gamma) to metastatic melanoma patients causes selective disruption of the tumor vasculature. This effect is paralleled by decreased endothelial cell proliferation and suppressed integrin alpha V beta 3-mediated adhesion in vitro. Overexpression of the cyclin-dependent kinase (cdk) inhibitory protein p16INK4a was reported to interfere with integrin alpha V beta 3-dependent melanoma cell adhesion. MATERIALS AND METHODS: TNF- and IFN gamma-treated HUVEC were analyzed for cell cycle progression and for protein expression by flow cytometry and Western blotting, respectively. p16INK4a was overexpressed by transient transfection, and HUVEC adhesion was tested in short-term adhesion assays. RESULTS: TNF and IFN gamma synergistically induced a G1 arrest associated with reduced levels of cyclin D1 and cdk2, and increased expression of the cdk inhibitors p16INK4a, p21WAF and p27Kip1. p16INK4a overexpression, however, had no effect on alpha V beta 3-mediated adhesion. CONCLUSION: These results implicate the down-regulation of cyclin D1 and cdk-2, and up-regulation of p16INK4a, p21WAF and p27Kip1 in the suppression of endothelial cell proliferation induced by TNF/IFN gamma and demonstrate that increased p16INK4a levels are not sufficient to suppress alpha V beta 3-mediated endothelial cell adhesion.

Manganese-induced integrin affinity maturation promotes recruitment of alpha V beta 3 integrin to focal adhesions in endothelial cells: evidence for a role of phosphatidylinositol 3-kinase and Src.

Integrin activity is controlled by changes in affinity (i.e. ligand binding) and avidity (i.e. receptor clustering). Little is known, however, about the effect of affinity maturation on integrin avidity and on the associated signaling pathways. To study the effect of affinity maturation on integrin avidity, we stimulated human umbilical vein endothelial cells (HUVEC) with MnCl(2) to increase integrin affinity and monitored clustering of beta 1 and beta 3 integrins. In unstimulated HUVEC, beta 1 integrins were present in fibrillar adhesions, while alpha V beta 3 was detected in peripheral focal adhesions. Clustered beta 1 and beta 3 integrins expressed high affinity/ligand-induced binding site (LIBS) epitopes. MnCl(2)-stimulation promoted focal adhesion and actin stress fiber formation at the basal surface of the cells, and strongly enhanced mAb LM609 staining and expression of beta 3 high affinity/LIBS epitopes at focal adhesions. MnCl(2)-induced alpha V beta 3 clustering was blocked by a soluble RGD peptide, by wortmannin and LY294002, two pharmacological inhibitors of phosphatidylinositol 3-kinase (PI 3-K), and by over-expressing a dominant negative PI 3-K mutant protein. Conversely, over-expression of active PI 3-K and pharmacological inhibiton of Src with PP2 and CGP77675, enhanced basal and manganese-induced alpha V beta 3 clustering. Transient increased phosphorylation of protein kinase B/Akt, a direct target of PI 3K, occurred upon manganese stimulation. MnCl(2) did not alter beta 1 integrin distribution or beta1 high-affinity/LIBS epitope expression. Based on these results, we conclude that MnCl(2)-induced alpha V beta 3 integrin affinity maturation stimulates focal adhesion and actin stress fiber formation, and promotes recruitment of high affinity alpha V beta 3 to focal adhesions. Affinity-modulated alpha V beta 3 clustering requires PI3-K signaling and is negatively regulate by Src.

Sensitization of endothelial cells to TNFα-induced death

RESUME L'angiogénèse tumorale est un processus essentiel au développement des tumeurs. Les intégrines, molécules d'adhésions transmembranaires, sont d'importants effecteurs de l'angiogenèse. En permettant l'adhésion à la matrice extra-cellulaire, les intégrines transmettant des signaux de survie, de migration, et de prolifération. Le facteur de nécrose tumorale α (TNFα) est utilisé pour le traitement régional de cancers chez l'homme. II agit en détruisant sélectivement les vaisseaux angiogéniques. Cependant, son administration systémique chez l'homme est limitée par les réactions de vaso-dilatation sévères qu'il provoque. Le but de mon travail fut de rechercher des conditions permettant la sensibilisation des cellules endothéliales au TNFα et qui pourraient être applicables en clinique, ceci afin d'accroître l'efficacité de cette molécule. Nous avons testé la possibilité d'interférer avec les signaux de survie provenant des intégrines. Pour cela, des cellules endothéliales furent cultivées dans des conditions d'adhésion ou en suspension, ou alors exposées dans des conditions d'adhésion au zoledronate (biphosphonate contenant du nitrogène). Dans ces conditions, les effets du TNFα sur les cellules endothéliales furent étudiés, en particulier l'induction de la mort cellulaire. Dans ce travail, nous montrons que le zoledronate sensibilise les cellules endothéliales à la nécrose induite par TNFα. Cet effet s'accompagne de l'inhibition de la phosphorylation de FAK, PKB, et JNK, ainsi que de l'inhibition de la prénylation des protéines. En revanche, l'activation de NF-kB et p38 n'est pas perturbée. La restoration de la prénylation des protéines empêche la mort des HUVEC traitées par zoledronate et TNFα, et rétablit la phosphorylation de FAK, PKB, et JNK. Des essais d'angiogénèse in vivo montrent que le zoledronate inhibe l'angiogénèse induite par FGF-2. Le zoledronate encapsulé dans des liposomes permet de ralentir la croissance tumorale et synergise avec le TNFα en l'inhibant. L'inihibtion de la prénylation des protéines est un des mécanismes de sensibilisation du zoledronate au TNFα. In vivo, la synergie de leur association sur la croissance tumorale est efficace. Ces résultats encouragent la poursuite de l'étude des effets de ces deux drogues sur la croissance tumorale. SUMMARY The formation of tumor-associated vessels is essential for tumor progression. Cell adhesion molecules of the integrin family are important mediators of angiogenesis, by providing adhesive signals necessary for endothelial cell migration, proliferation and survival. Anti-angiogenic therapies are currently considered as highly promising in the treatment of human cancer. Tumor Necrosis Factor α (TNFα) is used for the regional treatment of human cancer, whose mechanisms of action involved selective disruption of angiogenic tumor vessels. Systemic administration of TNFα in humans, however, induces a severe inflammatory condition that prevents its use far the treatments of tumors localized outside of limbs. The aim of my work was to find strategies to sensitize angiogenic endothelial cells to TNFα-induced death, which could be potentially translated into clinical setting to improve the therapeutic efficacy of TNFα. We specifically tested the hypothesis whether interference with integrin-mediated adhesion and signaling may sensitize endothelial cells to TNFα-induced death. To test this hypothesis we cultured endothelial cells (EC) under conditions of cell-matrix or cell-cell adhesion or exposed matrix-adherent EC to the nitrogen-containing bisphosphonate zoledronate, and characterized the effect on TNFα-mediated signaling events and cell death. We show that zoledronate sensitizes HUVEC to TNFα-induced necrosis-like programmed cell death. This effect was associated with suppression of sustained phosphorylation of PKB and JNK and decreased protein prenylation, whereas TNFα-induced activation of NF-kB and p38 were not inhibited. Restoration of protein prenylation rescued HUVEC from zoledronate and TNFα-induced death, and restored FAK, PKB and JNK phosphorylation. By using in vivo angiogenesis assay we showed that zoledronate suppressed FGF-2-induced angiogenesis. Liposome-encapulated zoledronate partially inhibited tumor growth and synergized with TNFα to fully suppress tumor growth. Taken together, this work has identified protein prenylation as a mechanisms by which zoledronate sensitizes endothelial cells to TNFα-induced death in vitro and provides initial evidence that zoledronate synergizes with TNFα in vivo resulting in improved anti-tumor activity. These results warrant further study of the anti-tumor effects of zoledronate and TNFα and should be further studies in view of their clinical relevance.

Zoledronate sensitizes endothelial cells to tumor necrosis factor-induced programmed cell death: evidence for the suppression of sustained activation of focal adhesion kinase and protein kinase B/Akt.

Bisphosphonates are potent inhibitors of osteoclast function widely used to treat conditions of excessive bone resorption, including tumor bone metastases. Recent evidence indicates that bisphosphonates have direct cytotoxic activity on tumor cells and suppress angiogenesis, but the associated molecular events have not been fully characterized. In this study we investigated the effects of zoledronate, a nitrogen-containing bisphosphonate, and clodronate, a non-nitrogen-containing bisphosphonate, on human umbilical vein endothelial cell (HUVEC) adhesion, migration, and survival, three events essential for angiogenesis. Zoledronate inhibited HUVEC adhesion mediated by integrin alphaVbeta3, but not alpha5beta1, blocked migration and disrupted established focal adhesions and actin stress fibers without modifying cell surface integrin expression level or affinity. Zoledronate treatment slightly decreased HUVEC viability and strongly enhanced tumor necrosis factor (TNF)-induced cell death. HUVEC treated with zoledronate and TNF died without evidence of enhanced annexin-V binding, chromatin condensation, or nuclear fragmentation and caspase dependence. Zoledronate inhibited sustained phosphorylation of focal adhesion kinase (FAK) and in combination with TNF, with and without interferon (IFN) gamma, of protein kinase B (PKB/Akt). Constitutive active PKB/Akt protected HUVEC from death induced by zoledronate and TNF/IFNgamma. Phosphorylation of c-Src and activation of NF-kappaB were not affected by zoledronate. Clodronate had no effect on HUVEC adhesion, migration, and survival nor did it enhanced TNF cytotoxicity. Taken together these data demonstrate that zoledronate sensitizes endothelial cells to TNF-induced, caspase-independent programmed cell death and point to the FAK-PKB/Akt pathway as a novel zoledronate target. These results have potential implications to the clinical use of zoledronate as an anti-angiogenic or anti-cancer agent.

Integrin-mediated adhesion and soluble ligand binding stabilize COX-2 protein levels in endothelial cells by inducing expression and preventing degradation.

Cyclooxygenase-2 (COX-2), a key enzyme in prostaglandin synthesis, is highly expressed during inflammation and cellular transformation and promotes tumor progression and angiogenesis. We have previously demonstrated that endothelial cell COX-2 is required for integrin alphaVbeta3-dependent activation of Rac-1 and Cdc-42 and for endothelial cell spreading, migration, and angiogenesis (Dormond, O., Foletti, A., Paroz, C., and Ruegg, C. (2001) Nat. Med. 7, 1041-1047; Dormond, O., Bezzi, M., Mariotti, A., and Ruegg, C. (2002) J. Biol. Chem. 277, 45838-45846). In this study, we addressed the question of whether integrin-mediated cell adhesion may regulate COX-2 expression in endothelial cells. We report that cell detachment from the substrate caused rapid degradation of COX-2 protein in human umbilical vein endothelial cells (HUVEC) independent of serum stimulation. This effect was prevented by broad inhibition of cellular proteinases and by neutralizing lysosomal activity but not by inhibiting the proteasome. HUVEC adhesion to laminin, collagen I, fibronectin, or vitronectin induced rapid COX-2 protein expression with peak levels reached within 2 h and increased COX-2-dependent prostaglandin E2 production. In contrast, nonspecific adhesion to poly-L-lysine was ineffective in inducing COX-2 expression. Furthermore, the addition of matrix proteins in solution promoted COX-2 protein expression in suspended or poly-L-lysine-attached HUVEC. Adhesion-induced COX-2 expression was strongly suppressed by pharmacological inhibition of c-Src, phosphatidylinositol 3-kinase, p38, extracellular-regulated kinase 1/2, and, to a lesser extent, protein kinase C and by the inhibition of mRNA or protein synthesis. In conclusion, this work demonstrates that integrin-mediated cell adhesion and soluble integrin ligands contribute to maintaining COX-2 steady-state levels in endothelial cells by the combined prevention of lysosomal-dependent degradation and the stimulation of mRNA synthesis involving multiple signaling pathways.

Improved IL-2 immunotherapy by selective stimulation of IL-2 receptors on lymphocytes and endothelial cells.

IL-2 immunotherapy is an attractive treatment option for certain metastatic cancers. However, administration of IL-2 to patients can lead, by ill-defined mechanisms, to toxic adverse effects including severe pulmonary edema. Here, we show that IL-2-induced pulmonary edema is caused by direct interaction of IL-2 with functional IL-2 receptors (IL-2R) on lung endothelial cells in vivo. Treatment of mice with high-dose IL-2 led to efficient expansion of effector immune cells expressing high levels of IL-2Rbetagamma, including CD8(+) T cells and natural killer cells, which resulted in a considerable antitumor response against s.c. and pulmonary B16 melanoma nodules. However, high-dose IL-2 treatment also affected immune cell lineage marker-negative CD31(+) pulmonary endothelial cells via binding to functional alphabetagamma IL-2Rs, expressed at low to intermediate levels on these cells, thus causing pulmonary edema. Notably, IL-2-mediated pulmonary edema was abrogated by a blocking antibody to IL-2Ralpha (CD25), genetic disruption of CD25, or the use of IL-2Rbetagamma-directed IL-2/anti-IL-2 antibody complexes, thereby interfering with IL-2 binding to IL-2Ralphabetagamma(+) pulmonary endothelial cells. Moreover, IL-2/anti-IL-2 antibody complexes led to vigorous activation of IL-2Rbetagamma(+) effector immune cells, which generated a dramatic antitumor response. Thus, IL-2/anti-IL-2 antibody complexes might improve current strategies of IL-2-based tumor immunotherapy.

Cornea graft endothelial cells undergo apoptosis by way of an alternate (caspase-independent) pathway.

PURPOSE: To look for apoptosis pathways involved in corneal endothelial cell death during acute graft rejection and to evaluate the potential role of nitric oxide in this process. MATERIALS AND METHODS: Corneal buttons from Brown-Norway rats were transplanted into Lewis rat corneas. At different time intervals after transplantation, apoptosis was assessed by diamino-2-phenylindol staining and annexin-V binding on flat-mount corneas, and by terminal transferase dUTP nick end labeling (TUNEL), caspase-3 dependent and leukocyte elastase inhibitor (LEI)/LDNase II caspase-independent pathways on sections. Inducible nitric oxide synthase (NOS-II) expression and the presence of nitrotyrosine were assayed by immunohistochemistry. RESULTS: Graft endothelial cells demonstrated nuclear fragmentation and LEI nuclear translocation, annexin-V binding, and membranes bleb formation. Apoptosis associated with caspase-3 activity or TUNEL-positive reaction was not observed at any time either in the graft or in the recipient corneal endothelial cells. During 14 days posttransplantation, the recipient corneal endothelial cells remained unaltered and their number unchanged in all studied corneas. NOS-II was expressed in infiltrating cells present within the graft. This expression was closely associated with the presence of nitrotyrosine in endothelial and infiltrating cells. CONCLUSION: During the time course of corneal graft rejection, graft endothelial cells undergo apoptosis. Apoptosis is caspase 3 independent and TUNEL negative and is, probably, carried out by an alternative pathway driven by an LEI/L-Dnase II. Peroxynitrite formation may be an additional mechanism for cell toxicity and programmed cell death of the graft endothelial cells during the rejection process in this model.

Fibronectin-binding proteins and clumping factor A in Staphylococcus aureus experimental endocarditis: FnBPA is sufficient to activate human endothelial cells.

Surface molecules of Staphylococcus aureus are involved in the colonization of vascular endothelium which is a crucial primary event in the pathogenesis of infective endocarditis (IE). The ability of these molecules to also launch endothelial procoagulant and proinflammatory responses, which characterize IE, is not known. In the present study we investigated the individual capacities of three prominent S. aureus surface molecules; fibronectin-binding protein A (FnBPA) and B (FnBPB) and clumping factor A (ClfA), to promote bacterial adherence to cultured human endothelial cells (ECs) and to activate phenotypic and functional changes in these ECs. Non-invasive surrogate bacterium Lactococcus lactis, which, by gene transfer, expressed staphylococcal FnBPA, FnBPB or ClfA molecules were used. Infection of ECs increased 50- to 100-fold with FnBPA- or FnBPB-positive recombinant lactococci. This coincided with EC activation, interleukin-8 secretion and surface expression of ICAM-1 and VCAM-1 and concomitant monocyte adhesion. Infection with ClfA-positive lactococci did not activate EC. FnBPA-positive L. lactis also induced a prominent tissue factor-dependent endothelial coagulation response that was intensified by cell-bound monocytes. Thus S. aureus FnBPs, but not ClfA, confer invasiveness and pathogenicity to non-pathogenic L. lactis organisms indicating that bacterium-EC interactions mediated by these adhesins are sufficient to evoke inflammation as well as procoagulant activity at infected endovascular sites.

Viral inhibitor of apoptosis vFLIP/K13 protects endothelial cells against superoxide-induced cell death.

Human herpesvirus 8 (HHV-8) is the etiological agent of Kaposi's sarcoma (KS). HHV-8 encodes an antiapoptotic viral Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein (vFLIP/K13). The antiapoptotic activity of vFLIP/K13 has been attributed to an inhibition of caspase 8 activation and more recently to its capability to induce the expression of antiapoptotic proteins via activation of NF-kappaB. Our study provides the first proteome-wide analysis of the effect of vFLIP/K13 on cellular-protein expression. Using comparative proteome analysis, we identified manganese superoxide dismutase (MnSOD), a mitochondrial antioxidant and an important antiapoptotic enzyme, as the protein most strongly upregulated by vFLIP/K13 in endothelial cells. MnSOD expression was also upregulated in endothelial cells upon infection with HHV-8. Microarray analysis confirmed that MnSOD is also upregulated at the RNA level, though the differential expression at the RNA level was much lower (5.6-fold) than at the protein level (25.1-fold). The induction of MnSOD expression was dependent on vFLIP/K13-mediated activation of NF-kappaB, occurred in a cell-intrinsic manner, and was correlated with decreased intracellular superoxide accumulation and increased resistance of endothelial cells to superoxide-induced death. The upregulation of MnSOD expression by vFLIP/K13 may support the survival of HHV-8-infected cells in the inflammatory microenvironment in KS.