American Journal of Pathology, Vol. 139, No. 4, October 1991 Copyright © American Association of Pathologists

Hyperthermia Stimulates Plasminogen Activator Inhibitor Type 1 Expression in Human Umbilical Vein Endothelial Cells In Vitro Johann Wojta,* Michael Holzer,* Peter Hufnagl,* Gunther Christ,* Richard L. Hoover,t and Berud R. Binder* From the Laboratoiy for Clinical Experimental Physiology,* University of Vienna, Austria; and the Department of Pathology, Vanderbilt University, Nashville, Tennessee

The effect of exposure to hyperthermia on the fibrinolytic potential of human umbilical vein endothelial cells (HUVEC) in culture was studied. HUVEC responded to exposure to 42°C with a time-dependent increase in plasminogen activator inhibitor type 1 (PAI-1) activity and antigen accompanied by afourto fivefold increase in PAI-i specific m-RNA and a decrease in tissue-type plasminogen activator (t-PA) antigen. The effect of 8 hours exposure to hyperthermia on PAI-1 activity and antigen could not be reversed by reexposure of the cells to 3 7Cfor 24 hours as evidenced by continuously increased amounts of PAI-i released into the conditioned media t-PA release, however, decreased during the 24-hour period at37°C after exposure to hyperthernmia No difference in PAI-I antigen present in the extracellular matrix of heat treated HUVEC as compared to HUVEC kept at 3 7C could be found Our data supports the idea that hyperthermia is one stress factor that influences the fibrinolytic potential of endothelial cells. (Am J Pathol 1991, 139:911-919)

The fibrinolytic capacity of human endothelial cells is maintained by the balance between plasminogen activators (PAs) and plasminogen activator inhibitor type 1 (PAI-1). This balance is regulated by a wide variety of stimuli (for a review see references 1 and 2).1 2 The fibrinolytic system is not only important for dissolving blood clots but also for extravascular processes like vascularization and plays a role in the body's defense system. Therefore, it is not surprising that physiologic and pathologic forms of "stress" like sheer stress or hypoxia, inflammation, and septicemia have been shown to regulate the

expression of PAs and PAI-1 by endothelial cells.3 6Exposure to hyperthermia is one form among stressful conditions and has been identified to regulate the expression of so-called "stress" or "heat-shock proteins" in both prokaryotic and eukaryotic cells.7'8 Endothelial cells have been shown to respond to heat shock with an increased production of thrombospondin.9 Therefore, this study investigated whether hyperthermia might also influence the fibrinolytic potential of human endothelial cells.

Materials and Methods

Cell Culture Human umbilical vein endothelial cells (HUVEC) were isolated by mild collagenase treatment as described.10 Cells were seeded into Petri dishes coated with gelatine (BioRad, Richmond, CA) and grown to confluency in Medium 199 (Sigma, St. Louis, MO) containing 20% supplemented calf serum (SCS) (Hyclone, Logan, UT), 50 ,ug/ml endothelial cell growth supplement (ECGS), prepared as described11 and 5 U/mI heparin (LyphoMed, Rosemont, IL). Cells were confirmed to be endothelial by their cobblestone morphology,12 by positive immunofluorescence using an anti von Willebrand factor VIII antibody13 and by uptake of acetylated LDL.14 Such characterized HUVEC were seeded into gelatine-coated six well-cluster dishes and grown to confluency as described earlier. All HUVEC used in this study were between passage 2 and 4.

Heat Treatment HUVEC grown to confluency in six well dishes were rinsed twice with Hank's balanced salt solution (HBSS) Supported by grants No. HL-36526 from the NIH and No. 6832 and 7617 from the Austrian Funds for the Promotion of Scientific Research Projects. Accepted for publication June 10, 1991. Address reprint requests to Dr. Johann Wojta, Laboratory for Clinical Expermental Physiology, University of Vienna, Schwarzspanierstr. 17, A-1090 Vienna, Austria.

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containing 10 mM HEPES. Thereafter, 1 ml of Medium 199 containing 1 mg/ml bovine serum albumine (BSA) was added to each well and cells were incubated for 4, 8, 12, and 24 hours at 370C or 420C, respectively, in a humidified atmosphere of 95% air and 5% C02. At time points indicated conditioned media of such treated cells were collected, centrifuged at 300xg and stored at -700C. Aliquots of these samples were taken before freezing and dialyzed against 6 M guanidinehydrochloride for 4 hours at 40C to activate the latent PAI-1. Such treated samples were then dialyzed against PBS to eliminate guanidinehydrochloride and stored at -700C. At the same time points, the HUVEC monolayers were rinsed with HBSS and lysed with PBS containing 0.1% Triton X100. Lysates were processed as described earlier. In an additional set of experiments, HUVEC were preincubated for 4, 8, and 12 hours at 420C or 37°C, respectively, and washed; fresh serum-free media was added and such treated cells were incubated for additional 24 hours at 370C. Conditioned media harvested at the end of the experiments were processed as described earlier.

Assay for Tissue Type PA (t-PA) Activity and Antigen t-PA activity and antigen in conditioned media and cell lysates were measured by a combined assay system employing monoclonal antibodies as described previ-

ously.15

Assay for Urokinase-type PA (u-PA) Antigen u-PA antigen in conditioned media and cell lysates was determined by an ELISA as described recently.16

Assay for PAI-1 Antigen PAI-1 antigen in the samples was quantified by an ELISA. Briefly, a monoclonal anti-PAI-1 antibody (5PA112) that recognizes active PAI-1, latent PAI-1 and PAI-1 in complex with t-PA immobilized to a micro-ELISA plate was

ng/ 105 cells

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12 4 8 24 hours Figure 1. Effect of byperthermia on PAI-I antigen released into conditioned media of HWVEC. The inset shows the effect ofhyperthermia on PAI-1 antigen in cell lysates ofHUVEC PAI-1 antigen after incubation at 37°C (full circles) and 42C (open circles) is given in ng/105 cells Values represent mean values + SD of three experments each done in triplicate. *P < 0.001, **P < 0.05

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used to bind the PAI-1 contained in the sample. A second peroxidase-labeled monoclonal anti-PAI-1 antibody (3PA15) that recognizes also active and latent PAI-1 as well as PAI-1 in complex with t-PA was used to quantify the amount of bound PAI-1. Purified melanoma PAI-117 was used as a calibration standard.18

Assay for PAI-1 Activity PAI-1 activity in the samples was measured by a modified ELISA. Briefly, an excess of t-PA was bound to microELISA plates by a monoclonal anti-t-PA antibody (MPW3VPA;15). Thereafter, the samples were added. Only the active PAI-1 present would bind to the immobilized t-PA by forming a complex. PAI-1 bound to t-PA was then quantified by a peroxidase-labeled monoclonal antiPAI-1 antibody (3PA15). The reference reagent for PAI-1 (87-512) agreed to have a unitage of 25 U per ampoule was used as a standard.18

Determination of PAI-1 Antigen in the ECM of HUVEC Extracellular matrix (ECM) of HUVEC, which had been exposed to 3TC or 420C for 4, 12, and 24 hours, respec-

tively, was prepared after the protocol of Levin and Santell.19 Confluent monolayers of HUVEC grown in six well plates were washed twice with 2 ml per well of 0.1% EDTA in Ca' +- and Mg' '-free PBS and then treated with 1 ml per well of 0.2 M urea, 0.1% EDTA in Ca+ +and Mg + +-free PBS to detach the cells. Remaining ECM was washed three times with Ca++ - and Mg ++-free PBS and incubated for 2 hours at 37°C with peroxidase labelled monoclonal anti-PAI-1 antibody (3PA15) at a final concentration of 1 ug/rml. Thereafter, ECM was washed three times with 2 ml per well of Ca+ +- and Mg + +-free PBS and 1 ml per well of a solution of 1 g/l 0-phenylendiaminedihydrochloride in 0.11 M sodium phosphate, 0.05 M citrate buffer, pH = 5.85, made 0.03% (v/v) in hydrogenperoxide was added. The color reaction was terminated by addition of 1 ml per well of 1.5 M sulfuric acid. As a control, gelatine-coated six well plates were processed as described earlier. Absorbances were read at 490 nm (630 nm reference wave length). Absorbances obtained with the gelatine-coated wells were deducted from the absorbance values obtained with the ECM to correct for unspecific binding of the monoclonal antibody 3PA15. Unspecific binding was less than 10%. Absorbances measured with ECM from cells exposed to 370C for the respective time points were set as 100%. Absorbances determined for the ECM from cells exposed to 420C for the respective timepoints were expressed as percent of the respective control exposed to 3°C.

t-PA inh. U/105 cells

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Figure 2. Effect of hyperthtnia on PAI-1 activity released into conditioned media of HLWEC. PAM-1 activity before (triangles) and after (circles) activation of latent PAI-1 with 6M guanidinebydrocbloride is given in t-PA inhibiting units/105 cellsfor HWVEC incubated at 37°C (full smbols) and 42C (open swmbols). Values represent mean values + SD of three experiments each done in triplicate. *P < 0.001, **P < 0.05

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minutes) with 0.1% SDS, 1.0 x SSC, 0.1 mM EDTA and 0.01 M sodium phosphate, pH 7.0, followed by one wash at room temperature (15 minutes) with 0.1% SDS, 0.2 x SSC, 0.1 mM EDTA and 0.01 M sodium phosphate, pH 7.0, air dried, and exposed to XAR film (Kodak) for 18 hours. Films were scanned using a densitometer (Hirschmann, Germany) to quantify differences in mRNA expression.

Northern Analysis Total RNA was recovered from cells cultured and incubated as indicated in figure legends by acid guanidine thiocyanate-phenol-chloroform extraction as described.20 Samples of total RNA were electrophoretically separated on 1.2% agarose gels containing 6% formaldehyde, transferred to nitrocellulose, and hybridized for 16 hours at 42°C with 106 cpm/ml hybridization solution (Cerenkov) random primed ax-[32P]dCTP-labeled probes21,22 for either human PAI-1 (a w1.3kb cDNAfragment) or human t-PA (a w1 .6kb cDNA-fragment, both provided by Dr. Pannekoek, Amsterdam)23' 24 or rat GAPDH (glyceraldehyde-3-phosphate dehydrogenase, a w1.3kb cDNA-fragment provided by Dr. Meinrad Busslinger, Vienna). 50 [lI hybridization buffer containing 50% v/v formamide 5.0 x SSC, 5.0 x Denhardt's solution, 0.01 M sodium phosphate, pH 7.0, 1 mM EDTA, 0.1% SDS, and 50 ,ug/ml sonicated and heat-denaturated salmon sperm DNA, were used per cm2 of nitrocellulose filter. Blots were washed once at room temperature (15

Determination of the Viability of the

Cultured HUVEC To determine the effect of hyperthermia on viability, confluent HUVEC monolayers prelabeled with 51Cr (1 ,uCi/ well for 2 hours at 37C) were incubated for 24 hours at 370C or 42°C. Thereafter, amounts of 51Cr released into the media and 51Cr bound to the cells were determined by gamma-counting. There was no difference in 51Cr release between heat-treated and control cultures, indicating that hyperthermia had no significant effect on cell

ng/105 cells 2.0

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Figure 3. Effect of hypertermia on t-PA antigen released into conditioned media ofHUVEC. The inset shows the effect of byperthermia on t-PA antigen in cell lysates ofHUVEC t-PA antigen after incubation at 37°C (full circles) and 42°C (open circles) is expressed in ng/105 cells. Values represent mean values + SD of three experiments each done in triplicate. *p < 0.001, **p < 0.05

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viability. Furthermore, no morphologic change of the respective endothelial cells at different duration of 420C or 370C incubation was observed.

Statistical Analysis Data were compared statistically by using a Student's t-test for paired observations. The P values calculated in all figures were based on differences between values at 420C and the corresponding control (37C) values at the respective time points indicated in the figures.

Results As can be seen in Figure 1, under hyperthermic conditions PAI-1 antigen in conditioned media was significantly increased over all time points tested. The value after 24 hours for cells at 420C was 349.8 ± 33.5 ng/10 cells (n = 3) compared with 227.9 ± 27.8 ng/1 05 cells (n = 3) for control (37°C). Also in cell lysates, PAI-1 antigen inng/ 10 5cells

creased significantly under hyperthermia (inset Figure 1). PAI-1 activity decreased in untreated conditioned media of hyperthermically treated HUVEC; however, when PAI-1 activity was measured in aliquots of these same samples activated with guanidinehydrochloride, an increase was found for HUVEC kept under hyperthermic conditions (Figure 2). The value after 24 hours at 420C was 55.4 ± 5.3 t-PA inhibiting U/105 cells (n = 3) compared with 38.2 ± 4.9 t-PA inhibiting U/105 cells (n = 3) for control. In contrast to PAI-1 antigen, t-PA antigen significantly decreased upon heat treatment (Figure 3). After 24 hours of incubation at 420C or 370C, the values were 1.2 ± 0.1 ng/105 and 1.6 ± 0.2 ng/105 cells (n = 3), respectively. Neither t-PA activity nor u-PA antigen could be detected in either conditioned media or cell lysates (data not shown). To determine whether the effect of heat treatment of HUVEC upon PAI-1 and t-PA antigen levels was reversible, HUVEC were incubated at 420C for the time periods indicated in the respective figures and then washed, supplemented with fresh serum-free media, and incubated at 370C for additional 24 hours. Values of t-PA antigen t-PA inh. U/ 105 cells

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24h 370 24h 370 24h 370 24h 370 Figure 4. Effect of hypertermnia followed by reexposure to 37°C on PAI-l antigen (A) and PAI-i activity (B) released into conditioned media of HUVEC. PAI-1 antigen is given in ng/105 cells, PAI-1 activity before (batched bars) and after activation with 6M guanidinebydrochloride (open bars) is exressed in t-PA inhibiting units/105 cells. Gray bars represent control conditions, uhite bars represent experimental conditions. Values represent mean values + SD of three experiments each done in triplicate. *P < 0.001, **P