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The Modulation of Tissue Factor by Endothelial Cells during Heat Shock

2003; Elsevier BV; Volume: 278; Issue: 13 Linguagem: Inglês

10.1074/jbc.m211540200

1083-351X

David L. Basi, Karen F. Ross, James S. Hodges, Mark C. Herzberg,

Hemoglobin structure and function

Tissue factor (TF) initiates the extrinsic coagulation cascade on the surface of macrophages and endothelial cells. In septic patients, the extrinsic coagulation cascade is activated. When septic patients are febrile, mortality is decreased. The purpose of this study was to investigate the role of elevated temperatures on TF expression by endothelial cells during a sepsis-like challenge. Human endothelial vein cells (HUVECs) were incubated with lipopolysaccharide (LPS) or interleukin-1β (IL-1β) for 0, 2, 4, 6, or 8 h. At the 0-h time point, some HUVECs were heat shocked at 43 °C for 2 h and then recovered at 37 °C for 0, 2, 4, or 6 h. Heat-shocked and non-heat-shocked LPS-stimulated HUVECs were analyzed for TF-specific mRNA expression by ribonuclease protection assay (RPA), surface TF expression by flow cytometry, and TF activity by a two-stage clotting assay. Heat shocked LPS-stimulated HUVECs expressed significantly reduced TF-specific mRNA, TF surface protein levels, and TF surface activity when compared with non-heat-shocked, LPS-stimulated HUVECs (p < 0.0125, p < 0.0125, andp< 0.0001, respectively; repeated measures analysis of variance, ANOVA). If heat shock models elevated core temperature, these results suggest that fever may protect the host during sepsis by reducing TF activity on the surface of endothelial cells. Tissue factor (TF) initiates the extrinsic coagulation cascade on the surface of macrophages and endothelial cells. In septic patients, the extrinsic coagulation cascade is activated. When septic patients are febrile, mortality is decreased. The purpose of this study was to investigate the role of elevated temperatures on TF expression by endothelial cells during a sepsis-like challenge. Human endothelial vein cells (HUVECs) were incubated with lipopolysaccharide (LPS) or interleukin-1β (IL-1β) for 0, 2, 4, 6, or 8 h. At the 0-h time point, some HUVECs were heat shocked at 43 °C for 2 h and then recovered at 37 °C for 0, 2, 4, or 6 h. Heat-shocked and non-heat-shocked LPS-stimulated HUVECs were analyzed for TF-specific mRNA expression by ribonuclease protection assay (RPA), surface TF expression by flow cytometry, and TF activity by a two-stage clotting assay. Heat shocked LPS-stimulated HUVECs expressed significantly reduced TF-specific mRNA, TF surface protein levels, and TF surface activity when compared with non-heat-shocked, LPS-stimulated HUVECs (p < 0.0125, p < 0.0125, andp< 0.0001, respectively; repeated measures analysis of variance, ANOVA). If heat shock models elevated core temperature, these results suggest that fever may protect the host during sepsis by reducing TF activity on the surface of endothelial cells. disseminated intravascular coagulation tissue factor immunoreactive TF lipopolysaccharide human endothelial vein cell interleukin-1β ribonuclease protection assay glyceraldehyde-3-phosphate dehydrogenase GAPDH short product heat shock protein 72 phosphate-buffered saline fetal bovine serum analysis of variance Disseminated intravascular coagulation (DIC)1 is a pathological condition precipitated by sepsis, trauma, or certain cancers (1Aderka D. Isr. J. Med. Sci. 1991; 27: 52-60Google Scholar, 2Esmon C.T. Fukudome K. Mather T. Bode W. Regan L.M. Stearns-Kurosawa D.J. Kurosawa S. Haematologica. 1999; 84: 254-259Google Scholar, 3Gando S. Nanzaki S. Sasaki S. Kemmotsu O. Thromb. Haemostasis. 1998; 76: 1111-1115Google Scholar, 4Bauer K.A. Conway E.M. Bach R. Konigsberg W.H. Griffin J.D. Demetri G. Thromb. Res. 1989; 56: 425-430Google Scholar). In DIC the coagulation system activates, promoting fibrin deposition in the microvasculature leading to thrombosis, organ failure, depletion of coagulation factors, and uncontrolled bleeding (5Regoeczi E. Brain M.C. Br. J. Haematol. 1969; 17: 73-81Google Scholar). Tissue factor (TF) is involved in the development and progression of DIC during sepsis; however, its precise role is unknown (3Gando S. Nanzaki S. Sasaki S. Kemmotsu O. Thromb. Haemostasis. 1998; 76: 1111-1115Google Scholar, 6Gando S. Nanzaki S. Sasaki S. Aoi K. Kemmotsu O. Crit. Care Med. 1998; 26: 2005-2009Google Scholar, 7Drake T.A. Cheng J. Chang A. Taylor Jr., F.B. Am. J. Pathol. 1993; 142: 1458-1470Google Scholar, 8Levi M. van der Poll T. ten Cate H. van Deventer S.J. Eur. J. Clin. Invest. 1997; 27: 3-9Google Scholar, 9Weiss D.J. Rashid J. J. Vet. Intern. Med. 1998; 12: 317-324Google Scholar). Fever is a physiological response that benefits the host during experimental infections and is correlated with improved patient survival (10Bryant R.E. Hood A.F. Hood C.E. Koenig M.G. Arch. Intern. Med. 1971; 127: 120-128Google Scholar, 11Mackowiak P.A. Browne R.H. Southern Jr., P.M. Smith J.W. Am. J. Med. Sci. 1980; 280: 73-80Google Scholar). Fever is characterized by the generation of acute phase proteins, activation of the immune response, and cytokine-mediated core temperature rise (12Mackowiak P.A. Arch. Intern. Med. 1998; 158: 1870-1881Google Scholar). The effects of elevated core temperature were modeled in rodents given a lethal dose of LPS (14Chu E.K. Ribeiro S.P. Slutsky A.S. Crit. Care Med. 1997; 25: 1727-1732Google Scholar). Heat-stressed rodents challenged with endotoxin express heat shock proteins, which positively correlate with survival (13Yang R.C. Wang C.I. Chen H.W. Chou F.P. Lue S.I. Hwang K.P. Kaohsiung J. Med. Sci. 1998; 14: 664-672Google Scholar, 14Chu E.K. Ribeiro S.P. Slutsky A.S. Crit. Care Med. 1997; 25: 1727-1732Google Scholar). The data suggest that heat stress and/or heat shock protein induction are important in survival during sepsis. Little is understood, however, about the molecular mechanism(s) that may explain protection. Heat shock inhibits cytokine- and endotoxin-mediated NF-κB nuclear translocation and I-κB degradation in cultured cells (15Wong H.R. Ryan M. Wispe J.R. Biochem. Biophys. Res. Commun. 1997; 231: 257-263Google Scholar, 16Curry H.A. Clemens R.A. Shah S. Bradbury C.M. Botero A. Goswami P. Gius D. J. Biol. Chem. 1999; 274: 23061-23067Google Scholar) andin vivo (17Pritts T.A. Wang Q. Sun X. Moon M.R. Fischer D.R. Fischer J.E. Wong H.R. Hasselgren P.O. Arch. Surg. 2000; 135: 860-866Google Scholar). Thus, the heat shock response may protect the host during infection by modulation of proinflammatory genes during sepsis. TF expression in response to LPS and cytokines is also partially regulated by NF-κB. We hypothesized, therefore, that heat shock modulates tissue factor expression by endothelial cells during LPS challenge. In this study we show that heat shock significantly reduces expression of TF-specific mRNA, surface protein, and activity by LPS-stimulated endothelial cells. HUVECs were isolated as described previously (18Balla G. Vercellotti G. Eaton J.W. Jacob H.S. Trans. Assoc. Am. Physicians. 1990; 103: 174-179Google Scholar, 19Platt J.L. Vercellotti G.M. Lindman B.J. Oegema Jr., T.R. Bach F.H. Dalmasso A.P. J. Exp. Med. 1990; 171: 1363-1368Google Scholar). HUVECs, which were provided by Dr. Gregory Vercellotti (Dept. of Medicine, University of Minnesota), were grown in modified Eagle's media 199 containing 10% fetal bovine serum (Invitrogen), 4.7 mm l-glutamine, 1 mm sodium pyruvate, 100 μg/ml penicillin/streptomycin, 25 μg/ml ampicillin (Invitrogen), 5 units/ml heparin sulfate (Sigma), and 50 μg/ml ENDOGRO™ (VecTechnologies, Rensselaer, NY) at 37 °C in 5% CO2. HUVECs from passages 1 to 4 were used in all experiments. To induce heat shock, HUVEC-containing flasks were immersed in a water bath equilibrated in a 43, 41.5, or 40 °C incubator. Some HUVEC cultures were stimulated with Escherichia coli LPS serotype 55:B5 (Sigma) or IL-1β (R & D Systems, St. Paul, MN). Total RNA was isolated from HUVEC monolayers by the guanidium-phenol extraction method with TRIzol™ reagent (Invitrogen) according to the manufacturer's protocol (20Chomczynski P.S.N. Anal. Biochem. 1987; 162: 156-159Google Scholar). Cell suspension/TRIzol was stored at −20 °C until RNA purification. The total amount of RNA isolated from each sample was quantified by absorbance at 260 nm. Purified RNA was stored at −20 or −80 °C until analyzed by ribonuclease protection assay (RPA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), GAPDH short product (GAPDHs), heat shock protein 72 (HSP72), and TF-purified polymerase chain reaction products were ligated into linearized plasmids that contained T3 and T7 RNA polymerase promoters (PCR-Script™; Amp Cloning Kit, Stratagene). The primers used to generate PCR products were as follows: GAPDH (5′-CGGAGTCAACGGATTTGGTCGTAT-3′, 5′-AGCCTTCTCCATGGTGGTGAAGAC-3′, PCR product length 307 bp); GAPDHs (5′-GACCCCTTCATTGACCTCAACTAC-3′, 5′-AGCCTTCTCCATGGTGGTGAAGAC-3′, PCR product length 222 bp); TF (5′-GACAATTTTGGAGTGGGAACCC-3′, 5′-CACTTTTGTTCCCACCTG-3′, PCR product length 310 bp); HSP72 (5′-CTCCAGCATCCGACAAGAAGC-3′, 5′-ACGGTGTTGTGGGGGTTCAG-3′, PCR product length 234 bp). Authenticity and orientation were confirmed by nucleotide sequencing. Using a combination of primers specific for each gene product and phage promoter flanking region, PCR products were generated containing T7 or T3 promoters. The flanking region primers were as follows: T7F, 5′-GGTAACGCCAGGGTTTTCCCAG-3′; and T3R, 5′-TCCGGCTCGTATGTTGTGTGGA-3′. Amplified PCR product size and purity were confirmed by gel electrophoresis. [α-32P]UTP-labeled single-stranded RNA target gene probes were transcribed with T3 or T7 RNA polymerase (Stratagene). Transcription products were purified by 5% acrylamide and 8m urea gel electrophoresis (45 min at constant 200 V) and exposed to radiographic film. Full-length, radioactively labeled RNA probes were excised and eluted from acrylamide by overnight incubation in elution buffer from Ambion RPA II kit (Ambion, Austin, TX) at 37 °C. To determine target probe specific activity, a liquid scintillation counter (LKB Wallac 1214 Rackbeta) was used to obtain counts per minute from aliquots of the transcription reaction mixture and target probe eluent. Probes were stored at −20 °C until use. Probe sizes were as follows: GAPDH (unprotected 393 bp, protected 307 bp); GAPDHs (unprotected 308 bp, protected 222 bp); TF (unprotected 396 bp, protected 310 bp); and HSP72 (unprotected 280 bp, protected 234 bp). RPA was performed using the Ambion RPA II kit (Ambion) according to the manufacturer's instructions. Total RNA was mixed with 32P-labeled antisense TF or HSP72 and GAPDH or GAPDHs probes. The RNA/probe mixture was hybridized overnight at 42 to 45 °C and then incubated with 1:50 RNase solution (T1/A) for 30 min at 37 °C. Protected RNA products were precipitated and then separated by electrophoresis on 5% acrylamide gel containing 8 m urea (60 min at 200 V). Gels were exposed to a PhosphorImager (Amersham Biosciences) for 20–24 h. To quantify mRNA, the phosphorimage of an exposed gel was scanned with a Storm System 840 scanner; ImageQuaNT™ analysis software (version 4.2a) was used to circumscribe the "protected" mRNA fragment and analyze the density of each pixel within each "box." After correction for background, relative abundance of TF- and HSP72-specific mRNAs was quantified as the ratio to GAPDH or GAPDHs (internal controls) within each sample. HUVECs were analyzed for surface TF expression by flow cytometry (BD Biosciences). Trypsin/EDTA solution was used to disrupt cell monolayers. Preliminary experiments were preformed to assess the effect of trypsin on the detection of surface TF. Two monolayer disruption protocols were compared. The first used EDTA to disrupt HUVEC monolayers, and the second protocol used a trypsin/EDTA combination. The trypsin/EDTA protocol produced no detectable differences in TF surface expression compared with EDTA treatment when assessed by flow cytometry (data not shown). Because TF was unaffected and the protocol disrupted HUVEC monolayers more efficiently, the trypsin/EDTA protocol was used for the flow cytometry experiments. HUVEC monolayers were washed with PBS containing 0.5 mm EDTA for 2 min. The supernatant wash was decanted, and HUVEC monolayers were dispersed by incubation with 0.5 mmEDTA/0.017% trypsin for 1 min. Dispersed HUVECs were washed with cold PBS containing 10% (v/v) FBS, centrifuged at 400 rpm for 5 min at 4 °C, and counted in a hemocytometer after resuspension in 250 μl of PBS containing 2% (v/v) FBS. HUVECs (5 × 105) were incubated with 8 μg/ml anti-TF or IgG isotype control antibodies for 30 min at 4 °C, washed with 25× the incubation volume with PBS containing 2% FBS, and incubated with an anti-isotype secondary antibody labeled with fluorescein isothiocyanate (FITC, Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 min at 4 °C. To assess nonspecific binding, some HUVECs were washed and resuspended in 300 μl of PBS containing 2% FBS and incubated with the secondary antibody only. To assess HUVEC viability, propidium iodine (1 μg/ml final concentration) was added to each cell suspension prior to flow cytometry. HUVEC monolayers were washed with PBS containing 0.5 mm EDTA for 2 min. Supernatants were removed, and the monolayers were disrupted by incubation with 0.5 mm EDTA/0.017% trypsin for 1 min. To inhibit trypsin activity, HUVEC suspensions were washed with cold Tris-HCl, pH 7.4, containing 10% (v/v) FBS. HUVECs (6 × 105) were pelleted, washed with buffer, and then incubated with 15 nmfactor VIIa (Clinical Enzyme Laboratories, South Bend, IN) and transferred to 96-well microtiter plates. Factor X (Clinical Enzyme Laboratories, South Bend, IN) and S-2222 (DiaPharma Group Inc, West Chester, OH) were added to each cell suspension at final concentrations of 300 μm and 1.4 μg/ml, respectively. Cleavage of S-2222 by factor Xa was detected by a change in absorbance at λ = 405 nm (Bio-Rad microplate reader model 3550). Repeated measures analysis of variance (ANOVAs) was applied in which HUVECs were "subjects," and the within-subject fixed effects were heat shock (present or absent), time (treated as categories), and ionomycin (present or absent). The one exception is the analysis supporting Fig. 3 I, which included the between-subject factors heat shock and temperature. Allpost hoc tests used the Bonferroni correction to maintain an alpha (type I error rate) of 0.05. Confluent HUVEC monolayers were incubated with 0.1 μg/ml LPS for various times. At each time point, cells were harvested, and total RNA was isolated and analyzed for TF-, HSP72-, and GAPDH-specific mRNA expression by an RPA. Fig.1 A is a representative phosphorimage from a time course experiment. The right panel shows ribonuclease digestion of the unbound RNA target probe. In the left panel, TF-specific mRNA expression by HUVECs appeared to maximize at 2 h of LPS-stimulation and return to near baseline levels at 6 h. The increase in the TF-specific message was significant at 1, 2, and 4 h when compared with non-stimulated HUVECs and maximized at 2 h (Fig. 1 B). To determine the LPS dose that would maximally stimulate TF-specific mRNA expression, confluent HUVEC monolayers were incubated for 2 h with each LPS concentration. LPS-stimulated HUVECs did not express detectable quantities of HSP72-specific mRNA at any time or concentration tested (data not shown). LPS concentrations from 0.01 to 10 μg/ml induced TF-specific mRNA expression, which was maximal at 0.1 μg/ml (Fig. 1, C andD). HUVEC cultures were heat shocked at 43 °C over time. At the times indicated, total RNA was isolated and analyzed for expression of HSP72-, TF-, and GAPDH-specific mRNA. HUVEC cells expressed HSP72-specific mRNA, which appeared to peak at 2 h (Fig. 2 A). Heat-shocked HUVECs did not express detectable quantities of TF-specific mRNA (data not shown). Based upon the optimization experiments, a standard protocol was used unless noted (Fig. 2 B). LPS (0.1 μg/ml) was added, and HUVEC cultures were heat shocked at 43 °C for 2 h and then re-equilibrated at 37 °C for up to 4 h (Fig.2 B). To determine the effect of heat shock on TF mRNA expression, LPS was incubated with HUVEC monolayers in the presence or absence of heat shock. LPS induced TF-specific mRNA in heat-shocked and non-heat-shocked HUVECs (Fig. 2 C). Heat-shocked HUVECs express HSP72-specific mRNA (Fig. 2 C, right panel). At 2 h, heat shock significantly reduced expression of TF-specific mRNA by LPS-stimulated HUVECs when compared with non-heat shocked, LPS-stimulated HUVECs (Fig.2 D). After 2 h of incubation with LPS, cells were allowed to re-equilibrate at 37 °C. The reduction in the expression of TF-specific mRNA caused by heat shock was not apparent (Fig.2 D). To determine whether the surface iTF expression of heat shocked, LPS-stimulated HUVECs paralleled the decrease in TF-specific mRNA expression, monolayers were stimulated with LPS or IL-1β and heat shocked. Monolayers were dispersed and analyzed by flow cytometry for iTF expression. Non-heat shocked, LPS-stimulated HUVECs were also analyzed for comparison. After 4 h of LPS stimulation, 31.7% of HUVECs were surface iTF-positive in a representative histogram (Fig.3 A, shaded area). After heat shock, only 22.2% of LPS-stimulated HUVECs were surface iTF positive (Fig. 3 B, shaded area) (reactions with isotype control antibodies are shown as unshaded histograms). Virtually no iTF was detected on HUVECs that were unstimulated (Fig.3 C) and heat shocked (2 h), followed by recovery for 2 h at 37 °C (Fig. 3 D), or stimulated with LPS for 4 h and incubated with secondary antibody only (Fig. 3 E). Surface expression of iTF by HUVECs stimulated with LPS (Fig.3 F) or IL-1β (Fig. 3 G) was significantly reduced by heat shock at all time points. Because reduced HUVEC viability caused by heat shock could explain the reduction of surface iTF, monolayers were assessed by propidium iodine dye exclusion. Heat-shocked and non-heat-shocked LPS-stimulated HUVECs maintained similar viability over time (Fig. 3 H). To determine whether a temperature between 37 and 43 °C depressed TF expression, LPS-stimulated HUVECs were heat shocked at 40 and 41.5 °C. At temperatures between 37 and 43 °C, smaller reductions in iTF surface expression were detected (Fig. 3 I). To determine whether the reduction in iTF surface expression by heat shock was functional, TF/factor VIIa activity was estimated as factor Xa generation by a serum-free, two-stage clotting assay. LPS-stimulated HUVECs were either maintained at 37 °C or heat shocked for 2 h and then allowed to recover for 2 h at 37 °C. Other cultures were heat shocked without LPS. Equal numbers of HUVECs were analyzed for TF activity. Factor Xa generation was largely inhibited by heat shock (Fig. 4 A). To show that the generation of factor Xa required iTF, anti-TF antibodies or isotype controls were added to the LPS-stimulated HUVECs before the addition of factor X and S-2222 (Fig. 4 B). Inhibition by anti-TF antibodies confirmed that factor Xa generation was TF-dependent. To determine whether the heat shock-induced reduction in surface activity might be due to the encryption of TF, ionomycin was added to LPS-stimulated heat-shocked and non-heat-shocked HUVECs. TF surface activity decreased significantly in heat-shocked, LPS-stimulated HUVECs independent of the treatment with ionomycin (Fig. 4 C). Although total TF activity increased, ionomycin did not substantially affect the ratio of TF surface activity in heat-shocked and non-heat-shocked LPS-stimulated HUVECs. In this study, we hypothesized that LPS-stimulated endothelial cells modulate TF when heat shocked. During LPS stimulation at 37 °C, TF-specific mRNA expression maximized in HUVECs at 2 h and returned to near baseline by 6 h (Fig. 1, A andB). For the first time, we showed that heat shock significantly reduced TF-specific mRNA expression during 2 h of LPS stimulation when compared with non-heat-shocked, LPS-stimulated cells (Fig. 2, C and D). To determine whether TF surface protein levels paralleled the decrease TF mRNA, we performed flow cytometry experiments. The largest number of surface iTF (+) HUVECs were detected at 4 h of LPS-stimulation (Fig.3 F). Heat shock significantly reduces surface iTF (+) HUVECs after stimulation with LPS for 2, 4, 6, or 8 h (Fig.3 F). Regulation of the expression of TF by heat shock may be under the control of the nuclear transcription factor NF-κB. NF-κB regulates cytokine- and LPS-mediated TF expression (21Pendurthi U.R. Williams J.T. Rao L.V. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 3406-3413Google Scholar, 22Zhang F.X. Kirschning C.J. Mancinelli R. Xu X.P. Jin Y. Faure E. Mantovani A. Rothe M. Muzio M. Arditi M. J. Biol. Chem. 1999; 274: 7611-7614Google Scholar). Heat shock attenuates NF-κB nuclear translocation and the induction of NF-κB-dependent nitric oxide synthase in murine epithelial cells (15Wong H.R. Ryan M. Wispe J.R. Biochem. Biophys. Res. Commun. 1997; 231: 257-263Google Scholar). Future studies will determine whether NF-κB nuclear translocation is reduced in heat shocked, LPS-stimulated HUVECs. 2D. L. Basi, K. F. Ross, J. S. Hodges, and M. C. Herzberg, manuscript in preparation. Heat shock regulation of TF expression was not specific to LPS. Flow cytometry experiments were repeated using IL-1β to stimulate TF expression. In response to IL-1β, heat shock significantly reduced surface iTF (+) HUVECs at all times (Fig. 3 G). Although the magnitude of the heat shock inhibition of iTF expression was temperature dependent (Fig. 3 I), the extent of reduction appeared to be independent of the strength of the procoagulant signal. Interleukin-1β or LPS stimulation induced similar surface iTF expression, with peak expression appearing to occur at 4 h. At 4 h, however, IL-1β induced expression on more HUVECs (≅ 60%) compared with LPS (≅ 36%) (Fig. 3, F and G). Heat shock also caused a greater reduction in the iTF (+) HUVEC population when IL-1β was used as the stimulating agent. In contrast, heat shock of LPS- and IL-1β-stimulated HUVECs reduced iTF (+) to 19.5 and 18% of the population, respectively. Heat shock appeared to down-regulate TF expression to this minimum level despite differences in stimulus potency, which may be an important control of coagulation if the anti-coagulant effect of heat shock occurs in vivo. TF surface expression was reduced on heat-shocked LPS- or IL-1β-stimulated HUVECs, but it was expressed. Therefore, we assessed surface TF procoagulant activity by a two-stage clotting assay. Heat shock significantly reduced surface TF activity by LPS-stimulated HUVECs (Fig. 4, A and C). Because TF-specific antibodies inhibited the two-stage clotting assay (Fig. 4 B), the procoagulant activity expressed on HUVECs was produced by TF. The reduction in TF surface activity was proportional to decreases in TF protein and TF-specific mRNA expression (Fig.5), which strongly suggests that heat shock mediated the modulation of transcription without detectable post-translational modification. Encrypted TF is the non-functional proportion of the total quantity of surface TF (23Bach R. Rifkin D.B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6995-6999Google Scholar, 24Maynard J.R. Heckman C.A. Pitlick F.A. Nemerson Y. J. Clin. Invest. 1975; 55: 814-824Google Scholar). Heat shock may have altered the proportion of encrypted TF and, therefore, could account for the decreased TF activity. To assess the total functional TF activity, we added ionomycin to HUVECs prior to incubation with factor VIIa. Ionomycin treatment increased surface TF activity, suggesting that a fraction of the TF was encrypted. Heat-shocked, LPS-stimulated HUVECs treated with ionomycin, however, showed significantly reduced surface TF activity (Fig. 4 C). Sixty-one percent of TF activity was encrypted on LPS-stimulated HUVECS compared with 83% on heat shocked cells. The data suggest that heat shock reduced the absolute quantity and functional activity of surface TF. We considered several other explanations for the results. The reduction in TF expression may have reflected the loss of LPS or IL-1β activity due to heat denaturation during heat shock. Therefore, LPS and IL-1β were pre-heated at 43 °C for 2 h and then added to HUVEC cultures. The surface iTF (+) HUVEC population was virtually identical when stimulated with pre-heated LPS or IL-1β (data not shown), indicating that heat denaturation or degradation did not alter activity. Perhaps heat shock itself contributed to cell injury or death and thus reduced TF expression. To test this possibility, cell viability was compared in heat stressed and unstressed LPS-stimulated HUVECs. Although HSP72-specific mRNA was up-regulated by heat shock and served as a positive control, viability was unaffected by heat shock when assessed by propidium iodine exclusion staining (Figs.2 C and 3 H). To rule out cellular injury as a cause of reduced TF expression, cellular respiration and gross morphological changes were compared in heat-stressed and -unstressed LPS-stimulated HUVECs. Cellular respiration and morphologic changes were evaluated by AlamarBlue™ reduction and light microscopy, respectively. Heat shock did not produce detectable changes in HUVEC respiration or morphology (data not shown). Heat shock modulates the expression of certain genes without apparent effect on other cell functions. For example, heat shock reduced the expression of TNF-α mRNA and protein by LPS-stimulated macrophages, which retained the ability to ingest antibody-coated erythrocytes like non-heat-shocked macrophages (25Snyder Y.M. Guthrie L. Evans G.F. Zuckerman S.H. J. Leukoc. Biol. 1992; 51: 181-187Google Scholar). Collectively, these experiments suggest that heat shock can modulate TF gene expression without an effect on specific cell functions, injury, or death. Reduction of TF expression by heat shock in LPS-stimulated HUVECs was therefore not due to cellular injury or death. Based on our study, we hypothesize that fever may protect septic hosts because of the reduced activation of extrinsic coagulation. Attenuated expression of TF would be expected to decrease development of DIC and improve the clinical prognosis. Fever may also play a role in other disease processes such as atherosclerosis. Viral and bacterial infections have been suggested as being implicated in the pathogenesis of atherosclerosis (26Gabrielli M. Santarelli L. Gasbarrini A. Circulation. 2002; 106: e32Google Scholar, 27Rugonfalvi-Kiss S. Endresz V. Madsen H.O. Burian K. Duba J. Prohaszka Z. Karadi I. Romics L. Gonczol E. Fust G. Garred P. Circulation. 2002; 106: 1071-1076Google Scholar, 28Shi Y. Tokunaga O. Pathol. Int. 2002; 52: 31-39Google Scholar). For example, specific pathogen-free chickens infected with Marek's disease virus and fed cholesterol-supplemented diets develop arterial fatty-fibro lesions similar to atheromas (29Minick C.R. Fabricant C.G. Fabricant J. Litrenta M.M. Am. J. Pathol. 1979; 96: 673-706Google Scholar). In humans, the presence of cytomegalovirus antibodies is an independent risk factor for the development of atherosclerosis (30Nieto F.J. Adam E. Sorlie P. Farzadegan H. Melnick J.L. Comstock G.W. Szklo M. Circulation. 1996; 94: 922-927Google Scholar). Chlamydia antigens are detected in ∼80% of coronary atherectomy sites compared with 4% in non-diseased vessels (31Muhlestein J.B. Hammond E.H. Carlquist J.F. Radicke E. Thomson M.J. Karagounis L.A. Woods M.L. Anderson J.L. J. Am. Coll. Cardiol. 1996; 27: 1555-1561Google Scholar). In view of our data, it is noteworthy that the host responds to chlamydia and cytomegalovirus infections with antibody production, but clinical symptoms such as fever are usually absent. We hypothesize that the absence of fever generation during acute chronic vascular infections may increase the risk of thrombosis, contributing to the development of atheromas and acute coronary events such as myocardial ischemia or infarction. We thank Julia Nguyen for isolation and characterization of the HUVECs used in our study.