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* Interdepartmental Program in Vascular Biology and Transplantation, Boyer Center for Molecular Medicine, and Departments of
Pathology and
Dermotology, Yale University School of Medicine, New Haven, CT 06510
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Abstract |
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30% of cultured EC to die, as assessed by propidium iodide uptake. Death is apoptotic, as assessed by Annexin V staining, 4',6'-diamidino-2-phenylindole staining, and DNA fragment ELISA. EC death is increased by cotreatment with cycloheximide but significantly reduced by caspase inhibitors or transduced dominant-negative Fas-associated death domain protein. In surviving cells, TRAIL activates NF-
B, induces expression of E-selectin, ICAM-1, and IL-8, and promotes adhesion of leukocytes. Injection of TRAIL into human skin xenografts promotes focal EC injury accompanied by limited neutrophil infiltration. These data suggest that TRAIL is an inducer of tissue injury in humans, an outcome that may influence antitumor therapy with TRAIL. |
Introduction |
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B (11, 12), leading to transcription of genes known to antagonize the FADD/caspase-8 death pathway and/or to promote inflammation. NF-B activation may be initiated by recruitment of an alternative death domain-containing adapter molecule, known as receptor-interacting protein (13). TRAIL ligation on human glioma tumor cells also leads to activation of transcription factor AP-1 (14). Cumulatively, these observations suggest that TRAIL, like TNF, may in parallel activate transcription/translation-independent pathways of apoptosis as well as antiapoptotic/proinflammatory pathways that depend upon NF-B- and/or AP-1-mediated new gene expression. The outcome of TRAIL binding to any given cell type may depend upon the relative strengths or kinetics of activation of these competing responses (15, 16).
In a variety of rodent tumor models, including those involving human xenografted tumor cells, human TRAIL can selectively kill the tumor cells without appreciable host toxicity (17, 18, 19). This wide therapeutic window has raised hopes that TRAIL can be useful in the treatment of human cancers. Human TNF initially showed a favorable therapeutic window in mouse tumor models but has generally proven to be too toxic for human therapy (20). The increased toxicity of TNF observed in the clinic may be related to the fact that human TNF can act on human cells through two receptors, TNFR1 (CD120a) and TNFR2 (CD120b) (21), but can act on mouse cells only through TNFR1 (22, 23). Mouse TNF, which can engage both TNFR types in rodents, is much more toxic for mice than is human TNF (23). Mouse TRAIL-Rs are incompletely characterized, and the capacity of human TRAIL to interact with different mouse receptors is unknown. Much of the antitumor efficacy of TNF is mediated through effects on vascular endothelial cells (EC) (24, 25). In cultured human EC, human TNF causes apoptosis, especially in the presence of RNA or protein synthesis inhibitors such as actinomycin D or cycloheximide (CHX), respectively. TNF also activates both NF-B and AP-1 in EC, leading to the expression of proinflammatory proteins, such as E-selectin (CD62E), ICAM-1 (CD54), and IL-8. The balance of TNF responses generally favors activation rather than apoptosis. We wondered whether TRAIL would also act on human EC, and if so, what the responses to TRAIL signaling might be.
In this study, we report the effects of recombinant human TRAIL on cultured human EC, including both human umbilical vein EC (HUVEC) and human dermal microvascular EC (HDMEC), as well as upon resting human EC in human skin xenografts transplanted on SCID/beige mice. We find that cultured human EC express death domain-containing TRAIL-Rs, and that recombinant human TRAIL protein triggers FADD-caspase-8-dependent apoptosis in EC. TRAIL also activates NF-B, resulting in E-selectin, ICAM-1, and IL-8 expression and increased leukocyte binding. TRAIL is more potent than TNF as an inducer of apoptosis, but less potent than TNF as an activator of EC inflammatory gene expression. Importantly, TRAIL injected into human skin xenografts results in focal endothelial damage with little accompanying neutrophil recruitment. These data suggest that TRAIL may be an inducer of normal tissue injury in humans.
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Materials and Methods |
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Recombinant human TRAIL, TNF-
, recombinant human Fas-Fc, recombinant human TRAIL-R2-Fc, caspase-8 selective inhibitor Z-Ile-Glu-Thr-Asp-fluoromethyl ketone, and caspase-3 selective inhibitor Z-Asp-Glu-Val-Asp fluoromethyl ketone were from R&D Systems (Minneapolis, MN). CHX, 4',6'-diamidino-2-phenylindole (DAPI), and pyrrolidine dithiolcarbamate (PDTC) were from Sigma-Aldrich (St. Louis, MO). A cell death detection DNA fragment ELISA kit and an annexin V-FITC staining kit were purchased from Roche (Indianapolis, IN) and BD PharMingen (San Diego, CA), respectively.
The following Abs were used: rabbit anti-IB and anti-TRAIL-R1 (Santa Cruz Biotechnology, Santa Cruz, CA); mouse anti-TRAIL-R2 mAb (eBioscience, San Diego, CA); rabbit anti-TRAIL-R4 and anti-FADD (Chemicon, Temecula, CA); mouse anti-human 
-actin (Novus Biologicals, Littleton, CO), mouse anti-human E-selectin (ascites preparations of H4/18) (26); mouse anti-human ICAM-1 (Immunotech, Marseille, France); isotype control Ab K16/16 (26); goat anti-rabbit IgG HRP-conjugated or anti-mouse IgG FITC-conjugated (Jackson ImmunoResearch, West Grove, PA).
HUVEC and HDMEC were isolated and cultured as described (26) and were used at passage levels 2 to 4. Such cultures are uniformly positive for von Willlebrand factor and CD31 and lack detectable contamination by CD45-expressing leukocytes. HEK293, HeLa, and HL-60 cells were obtained from the American Type Culture Collection (Manassas, VA) and cultured in DMEM medium containing 10% FBS (Life Technologies, Grand Island, NY).
Construction and transduction with recombinant retroviral vector
Plasmid pcDNA3 AU1-FADDmt (human FADD dominant negative (FADD.DN) with AU1 epitope tag) (27), provided by Dr. V. Dixit (Genentech, South San Francisco, CA), was subcloned into the pFB retroviral vector (Stratagene, La Jolla, CA) using PCR with primers 5'-CGACTCACTATAGGGAGACCCA-3' and 5'-GCGGCCGCTCAGGACGCTTCGGAGGTA-3', and topoisomerase cloning techniques (Invitrogen, Carlsbad, CA). The pFB vector was transfected into packaging cell line PA317 provided by Dr. G. Nolan (Stanford University, Palo Alto, CA) and G418 (Life Technologies)-resistant cells were derived as a source of retrovirus stock. Transduction and G418 selection of HUVEC was accomplished as described (28). In brief, this involved three serial transduction of primary cultures with retrovirus-containing supernatants followed by selection with G418. Approximately 50% of transduced HUVEC survived selection with 0.5 mg/ml G418. Multiple FADD.DN-transduced HUVEC clones were pooled to avoid clonal variability.
Similarly, human TRAIL cDNA was amplified using PCR with human T blast cDNA and two primers, 5'-GGATCATGGCTATGATGGAG-3' and 5'-GCGGCCGCAGTTAGCCAACTAAAAAGGC-3'. The amplified TRAIL cDNA fragment was confirmed by DNA sequencing and was subcloned into the pFB retroviral vector. The pFB vector was transfected into packaging cell PA317. G418 selection, retrovirus stock preparation, and HEK293 transduction were performed as described as for HUVEC. TRAIL expression by the transduced HEK293 was determined by TRAIL-R2-Fc staining and FACS analysis.
Assessment of TRAIL-mediated apoptosis
We treated 30,000 HUVEC or HDMEC plated in C6 plates with medium alone or with TRAIL for 3–15 h; CHX or caspase inhibitors were included during the entire treatment period. The doses of TRAIL were varied somewhat among experiments, because different HUVEC and HDMEC stocks showed some variability in sensitivity. To assess cell viability, EC were harvested, stained with 25 µg/ml propidium iodide (PI; Molecular Probes, Eugene, OR) for 5 min at 37°C and then subjected to analytic flow cytometry using a FACSort (BD Biosciences, Mountain View, CA). Apoptois of TRAIL-treated EC was assessed by an annexin V-FITC staining kit (BD PharMingen) following the manufacturer’s protocol. Nuclear morphology during EC apoptosis was assessed by DAPI staining and fluorescence microscopy, as described previously (28). DNA fragmentation was measured using a cell death detection ELISA kit (Roche) according to the manufacturer’s instructions.
Quantitation of protein expression
We measured protein expression by immunoblotting or flow cytometric analysis as described previously (28). Immunoblots were analyzed by chemiluminescence and -actin was used as protein loading control. For flow cytometry, Ab-stained cells were analyzed using FACSort cytometer and CellQuest software. We calculated corrected mean fluorescence intensities by subtracting the mean values of cells stained with the irrelevant control mAb from that of cells stained with specific primary mAb.
RT-PCR analysis
We performed total RNA extraction, reverse transcription, and PCR amplification as described previously (28, 29). The primers used for TRAIL-Rs were as follows: TRAIL-R1, 5'-ATGGCGCCACCACCAGCTAGA-3' and 5'-TGAGCAACGCAGACTCGCTGT-3'; TRAIL-R2, 5'-CAGGTGTGATTCAGGTGAAGT-3' and 5'-GGACATGGCAGAGTCTGCAT-3'; TRAIL-R4 5'-CTCCCTTCTCATGGGACTTTGG-3' and 5'-CCACCAGTTGGTCCTGAATTG-3'. We separated and visualized the RT-PCR products by electrophoresis in 1.2% agarose gel using ethidium bromide and UV illumination; identification of amplified bands was verified by DNA sequencing.
We performed real-time RT-PCR using a Multicolor Real-Time PCR detection system (Bio-Rad, Hercules, CA) using the SYBR Green PCR core reagents (4304886), according to the recommended protocol, to determine the threshold cycle (Tc). The primer sequences used were as follows: E-selectin, 5'-GAATGTGTAGAGACCATCATAATAAT-3' and 5'-AGGAAGAATTGRAGCTGAAGTTT-3'; ICAM-1, 5'-CTGTTCCCAGGACCTGGCAAT-3' and 5'-AGGCAGGAGCAACTCCTTTTTA-3'; IL-8, 5'-CTCTTGGCAGCCTTCCTGATT-3' and 5'-ACTCTCAATCACTCTCAGTTCT-3; and -actin, 5'-TGCACCACACCTTCTACAATGA-3' and 5'-CAGCCTGGATAGCAACGTACAT-3'. In TRAIL-treated HUVEC or HDMEC, up-regulation of E-selectin, ICAM-1, and IL-8 were expressed as fold induction (FI), calculated as follows: FI = 2- 7-[TRAIL (Tc target - Tc -actin) - control (Tc target - Tc -actin)].
NF-B promoter reporter assay
We transiently transfected HUVEC cells with NF-B promoter-firefly luciferase and -actin promoter-Renilla luciferase reporter plasmids using a DEAE-dextran protocol (30). The transfected HUVEC were incubated with 50 ng/ml TRAIL or TNF for 7 h, and their luciferase activity was determined using a Promega (Madison, WI) Dual-Luciferase reporter assay system according to the manufacturer’s instruction, and the activity of the NF-B promoter-reporter was normalized to that of the -actin promoter-reporter.
HL-60 cell adherence assay
We seeded HUVEC and HDMEC in 24-well plates, incubated cells with TRAIL or TNF for 5 h, and then added HL-60 cells loaded with calcein-AM (Molecular Probes) at a ratio of 5:1 of leukocytes to EC. After 30 min, unbound HL-60 cells were removed by washing gently three times with 1 ml of PBS, and the retained calcein per well was determined by fluorometry (Cytofluor2; PerSpective Biosystems, Framingham, MA). Binding was expressed by fluorescence and calculated as follows: (fluorescence associated with treated EC) - (fluorescence associated with untreated EC).
Human skin mouse model
We engrafted human skin on C.B-17 SCID/beige mice (Taconic Farms, Germantown, NY) as described (31), and the grafts were healed in 4–6 wk before use. We then intradermally injected 20 µl of saline vehicle, TRAIL (0.6 µg), or TNF (0.1 µg), and harvested the grafts 6 h later. The doses used were determined as optimal from preliminary experiments. Grafts were paraffin embedded, sectioned, and stained with H&E (31). The degreeof graft microvascular injury was evaluated from the H&E-stained section by a dermatopathologist (J. McNiff) blinded to the treatment protocols, assessing EC morphology, thrombosis, and leukocyte infiltration.
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Results |
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The goal of this study was to determine the effects of TRAIL on normal human EC in vitro and in vivo. We first examined whether cultured HUVEC and HDMEC express signaling TRAIL-Rs (i.e., TRAIL-R1, -R2, and -R4). Using a sensitive but nonquantitative RT-PCR method, we could readily detect TRAIL-R1 and -R2 mRNA in both cell types, as well as control cell lines HeLa or HEK 293 (Fig. 1a). TRAIL-R4 mRNA was detectable in HUVEC, HeLa, and HEK293, but not in HDMEC (Fig. 1a). We also measured TRAIL-R2 cell surface protein expression by indirect immunofluorescence and flow cytometry. As shown in Fig. 1b, HDMEC, HUVEC, HeLa, and HEK293 all express TRAIL-R2. Expression in HUVEC is somewhat greater than in HDMEC. Available Abs that react with TRAIL-R1 and -R4 do not work in flow cytometry, so we examined protein levels of these receptors by immunoblotting. TRAIL-R1 and -R4 proteins were detected in HUVEC, HeLa, and HEK293 (Fig. 1c), but HDMEC expressed only TRAIL-R1, consistent with the RT-PCR data (a).
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Next, we examined whether rTRAIL caused death of cultured HUVEC or HDMEC using PI exclusion and flow cytometry (28). As shown in Fig. 2a, 30% of HUVEC lose membrane integrity after treatment with 20 ng/ml TRAIL for 15 h, compared with 7.8% of cells in replicate control cultures not treated with TRAIL. Fifteen hours was determined in preliminary experiments to display maximal cell death, and longer incubations lead to interference with the assay by cell-derived debris. The dose-response curve varied somewhat in different HUVEC isolates, and cells showed progressive loss of sensitivity to TRAIL with serial passage (data not shown). HDMEC showed similar susceptibility to TRAIL (Fig. 2c). Cell death of both cell types was increased in the presence of the protein synthesis inhibitor CHX (Fig. 2, b and c). PI exclusion is highly quantitative but does not distinguish late apoptotic from necrotic cell death. To determine whether TRAIL-triggered HUVEC death was apoptotic or necrotic, we incubated HUVEC with 50 ng/ml TRAIL for 2.5, 5, or 10 h, and stained cells with Annexin V-FITC. At 5 or 10 h, 15% of HUVEC showed evidence of an early apoptotic phase which is Annexin V positive (Fig. 3a). In addition, TRAIL-induced HUVEC death is characterized by nuclear condensation and fragmentation, a more specific test of apoptosis (Fig. 3b), as shown by DAPI staining and fluorescence microscopy. The results of a DNA fragment ELISA also supported the conclusion that death caused by TRAIL with or without CHX, is apoptosis (Fig. 3c). Although HDMEC do not express TRAIL-R4, both HUVEC and HDMEC showed similar sensitivity to TRAIL (Fig. 2, b and c), which suggests that TRAIL-R4 expressed on HUVEC, but not HDMEC, is not a major inhibitor of TRAIL-mediated EC apoptosis. TNF does not induce a significant degree of apoptosis in either HUVEC or HDMEC unless CHX is added (data not shown).
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TRAIL is normally a membrane-bound ligand expressed by monocytes, T lymphocytes, dendritic cells, and NK cells (15). To address whether EC could be killed by membrane-bound TRAIL, HUVEC were cocultured with HEK293-TRAIL, or with control HEK293-pFB at 1:1 ratios for 15 h. The viability of the mixed population was then determined by PI exclusion. Coculture of HUVEC with HEK293-TRAIL resulted in a reproducible increase of PI-stained cells (15%), compared with HUVEC culture alone or with mock HEK293-pFB (5%) (Fig. 2e). Moreover, the HEK293-TRAIL-mediated cytotoxicity was markedly inhibited by 1 µg/ml TRAIL-R2-FC, but not by human Fas-Fc (Fig. 2e). Because only 50% of the cultures are HUVEC, this increase in HUVEC death is probably double that measured for the whole populations, similar to the sensitivity of HUVEC to the soluble rTRAIL used in our experiments.
TRAIL-triggered inflammatory gene expression in EC
Because in the absence of CHX, 70% of EC are not killed by TRAIL, we also examined the activation response of these cells. After treatment with TRAIL for 6–7 h, we observed an increase of E-selectin expression on HUVEC and HDMEC demonstrated by FACS (the time point was selected because of preliminary experiments for the optimal level of E-selectin protein expression) (Fig. 4a). This TRAIL response is consistently smaller than that induced by TNF (Fig. 4a). TRAIL was also effective at inducing E-selectin expression in HDMEC (Fig. 4b). Although our preparations of TRAIL contained only very low levels of endotoxin contaminant (tested by the manufacturer), we excluded the possibility that LPS contamination in the rTRAIL reagent was responsible for the E-selectin induction in EC by heating TRAIL at 80°C for 10 min before addition to EC. This treatment, which does not inactivate LPS actions on EC(data not shown), abolished induction of E-selectin expression in EC by TRAIL (Fig. 4a). The response to TRAIL was not confined to induction of E-selectin. ICAM-1 was also up-regulated in HUVEC surviving TRAIL treatment (Fig. 4c). We also examined the mRNA levels of E-selectin, ICAM-1, and IL-8 in TRAIL-stimulated human EC using real-time quantitative RT-PCR. Incubation with 50 ng/ml TRAIL for 3 h increased expression of E-selectin, ICAM-1, and IL-8 mRNAs in both HUVEC and HDMEC (Fig. 4d).
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TNF-mediated induction of proinflammatory genes in EC is dependent upon the activation of NF-B via degradation of IB. As demonstrated by immunoblot, TRAIL causes IB degradation in HUVEC, HDMEC, and HEK 293 cells (Fig. 5a). TRAIL-induced NF-B activation in HUVEC was more directly examined using an NF-B promoter reporter gene assay. Stimulation with 50 ng/ml TRAIL for 7 h caused an increase of luciferase activity driven by NF-B, although this response was smaller than that caused by 50 ng/ml TNF (Fig. 5b). We also find that the NF-B inhibitor PDTC prevented TRAIL-induced E-selectin expression in HUVEC (Fig. 5c), confirming a link between NF-B activation and the proinflammatory response of TRAIL in these cells.
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To examine the effects of TRAIL on human EC in vivo, we intradermally injected TRAIL into stable human skin xenografts on SCID/beige mice. Histological analysis confirmed that both TNF and TRAIL resulted in epidermal and vessel injury (Fig. 6, b–d). Vascular damage was seen as either loss of EC or formation of microthrombi (Fig. 6, b–d). This was accompanied by local infiltration of neutrophils and mononuclear cells (Fig. 6, b–d). The extent of leukocyte recruitment was much greater in TNF-injected skin than in TRAIL-injected skin. Heat-treated TRAIL did not induce inflammation or endothelial injury, excluding the possibility of an LPS contaminant producing these responses (data not shown).
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Discussion |
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30% of cultured EC lose membrane integrity (Fig. 2, a–c). EC death is increased by cotreatment with CHX but significantly reduced by selective caspase inhibitors or transduced FADD.DN (Fig. 2, b–d). Moreover, experiments with Annexin V staining, DAPI nuclear staining, and a DNA fragment ELISA demonstrate that TRAIL-triggered HUVEC death is predominantly apoptotic (Fig. 3). Most EC do not die in response to TRAIL in the absence of CHX, prompting us to examine other related responses. We found that TRAIL-treated EC also show NF-B-dependent activation responses and inflammatory gene expression that results in adhesion of leukocytes (Figs. 4 and 5). Most importantly, TRAIL induces EC damage and tissue injury in normal human skin in vivo. These responses are qualitatively similar to those induced by TNF (Fig. 6). However, compared with TNF, TRAIL is a stronger inducer of apoptosis in vitro (Fig. 2, a–c, e) but a weaker activator of NF-B (b). In vivo, the TRAIL response also caused less EC activation and inflammation than TNF but resulted in comparable levels of EC injury (Fig. 6). The dose responses of both HUVEC and HDMEC to TRAIL varied somewhat among isolates, similar to the previously noted variability of HUVEC cultures to TNF or IL-1 (33). In addition, apoptotic responses diminished somewhat with passage level. This is also true for the apoptosis response to TNF (J. Li and J. Pober, unpublished observation). The explanation for these changes is unknown. Previous investigations have reported the toxic effects of TRAIL on normal human cells, including hepatocytes (34), brain cells (35), and erythropoietic cells (36). In this regard, the effects on EC are not unique. However, the effects of injury to EC may be more biologically significant, because this can be amplified in vivo through secondary thrombosis and consequent tissue ischemia (Fig. 6, c and d). Our observations that HUVEC and HDMEC are susceptible to TRAIL death signals differ from those reported previously by others (17, 18). The basis of this difference is unclear but could result from the methods used to assess cell death, from the state of the EC cultures or from the potency of the TRAIL preparations used. We detected death by loss of membrane integrity, by Annexin V binding, by nuclear condensation, and by DNA fragmentation, which may be more sensitive than crystal violet staining used by other investigators (17, 18). We also do not know the characterization of EC cultures used in the previous published study. It may be relevant that HUVEC become increasingly resistant to apoptotic signals provided by TNF, CD95L, as well as (or) TRAIL after several passages in vitro. The different recombinant versions of TRAIL may also vary widely in biochemical properties and potential for cellular and whole animal toxicity (37). Soluble TRAIL could also be a weaker apoptosis inducer compared with membrane-bound TRAIL, the physiological form of this ligand on activated leukocytes. In one study, apoptosis through TRAIL-R2 was only activated by membrane-bound TRAIL or soluble TRAIL secondarily cross-linked by Abs (38). However, our results with TRAIL-transduced HEK293 cells do not support a major difference in the susceptibility of HUVEC to membrane-bound vs soluble ligand used in this study.
The system of cell receptors for TRAIL is complex. We have examined the expression of cultured HUVEC and HDMEC for those TRAIL-Rs involved in signaling, namely TRAIL-R1, -R2, and -R4. HUVEC express all three receptors, whereas HDMEC express only TRAIL-R1 and -R2. As shown in Fig. 1b, the expression level of TRAIL-R2 in HDMEC is somewhat less than that in HUVEC. We do not know the significance of these differences, because both cell types showed very similar dose-apoptotic responses to TRAIL addition. We do not know whether the death responses we have observed are mediated by TRAIL-R1 or -R2. We observed TRAIL-induced human EC injury in vivo as well as in vitro. We do not know which receptors are expressed by mouse EC or whether human TRAIL can act through all mouse receptors. Such information could shed light on the species difference of tissue injury produced by human TRAIL.
The TRAIL pathway represents a potentially promising target for anticancer therapy (17, 18, 19, 39), and the finding reported in this study that TRAIL induces apoptosis and inflammatory gene expression in EC does not exclude this possibility. TNF exerts its primary antitumor action in vivo via damage of tumor vessels. Our finding suggests TRAIL may also have indirect antitumor effects in addition to direct cytotoxicity toward tumor cells. TNF appears more active on tumor vessels than normal vessels, and it will be interesting to test whether TRAIL also shows such different sensitivity. A proinflammatory effect of a cytokine is also not necessarily a problem for tumor therapy. The inflammatory response provides signals that stimulate APCs, such as dendritic cells to express molecules that promote binding, stimulation, and activation of lymphocytes (40). In other words, the EC injury response we have observed in human skin after TRAIL treatment (Fig. 6c) could facilitate host immune responses by acting as an adjuvant.
In summary, we have found that recombinant human TRAIL can both injure and activate human EC in vitro and in vivo. Compared with TNF, TRAIL is potent at causing injury but less effective at stimulating inflammation. These findings need to be considered in planning and interpreting human therapeutic trials with rTRAIL as a treatment for cancer and for evaluating the physiological role of endogenous TRAIL in immune and inflammatory reactions.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jordan S. Pober, Boyer Center for Molecular Medicine, Yale University School of Medicine, 295 Congress Avenue, New Haven, CT 06510. E-mail address: jordan.pober{at}yale.edu
3 Abbreviations used in this paper: FADD, Fas-associated death domain protein; FADD.DN, FADD dominant negative; EC, endothelial cell; HUVEC, human umbilical vein EC; HDVEC, human dermal microvascular EC; CHX, cycloheximide; DAPI, 4',6'-diamidino-2-phenylindole; PDTC, pyrrolidine dithiolcarbamate; PI, propidium iodide; Tc, threshold cycle.
Received for publication December 31, 2002. Accepted for publication May 22, 2003.
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P. Secchiero, F. Corallini, M. G. di Iasio, A. Gonelli, E. Barbarotto, and G. Zauli TRAIL counteracts the proadhesive activity of inflammatory cytokines in endothelial cells by down-modulating CCL8 and CXCL10 chemokine expression and release Blood, May 1, 2005; 105(9): 3413 - 3419. [Abstract] [Full Text] [PDF]
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 E. Bremer, D. F. Samplonius, M. Peipp, L. van Genne, B.-J. Kroesen, G. H. Fey, M. Gramatzki, L. F.M.H. de Leij, and W. Helfrich Target Cell-Restricted Apoptosis Induction of Acute Leukemic T Cells by a Recombinant Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Fusion Protein with Specificity for Human CD7 Cancer Res., April 15, 2005; 65(8): 3380 - 3388. [Abstract] [Full Text] [PDF]
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J. Shi, D. Zheng, Y. Liu, M. H. Sham, P. Tam, F. Farzaneh, and R. Xu Overexpression of Soluble TRAIL Induces Apoptosis in Human Lung Adenocarcinoma and Inhibits Growth of Tumor Xenografts in Nude Mice Cancer Res., March 1, 2005; 65(5): 1687 - 1692. [Abstract] [Full Text] [PDF]
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 T. Wachter, M. Sprick, D. Hausmann, A. Kerstan, K. McPherson, G. Stassi, E.-B. Brocker, H. Walczak, and M. Leverkus cFLIPL Inhibits Tumor Necrosis Factor-related Apoptosis-inducing Ligand-mediated NF-{kappa}B Activation at the Death-inducing Signaling Complex in Human Keratinocytes J. Biol. Chem., December 17, 2004; 279(51): 52824 - 52834. [Abstract] [Full Text] [PDF]
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 L. B. Pritzker, M. Scatena, and C. M. Giachelli The Role of Osteoprotegerin and Tumor Necrosis Factor-related Apoptosis-inducing Ligand in Human Microvascular Endothelial Cell Survival Mol. Biol. Cell, June 1, 2004; 15(6): 2834 - 2841. [Abstract] [Full Text] [PDF]
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 C. Daly, V. Wong, E. Burova, Y. Wei, S. Zabski, J. Griffiths, K.-M. Lai, H. C. Lin, E. Ioffe, G. D. Yancopoulos, et al. Angiopoietin-1 modulates endothelial cell function and gene expression via the transcription factor FKHR (FOXO1) Genes & Dev., May 1, 2004; 18(9): 1060 - 1071. [Abstract] [Full Text] [PDF]
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