JOURNAL OF CELLULAR PHYSIOLOGY 148:152-156 (1991)

Bovine Aortic Endothelial Cells Release Hydrogen Peroxide T. SUNDQVIST Department of Medical Microbiology, University of linkoping, 5-58 1 85 Linkoping, Sweden Endothelial cells grown on microcarriers are able to release H,O, to the extracellular environment without any added stimulus. The extracellularly released H,O, can be detected by luminol-amplified chemiluminescence (CL) if horseradish peroxidase is added. The CL response can be reduced by catalase and could be a precursor for blocked by superoxide dismutase, indicating that 0,H,O,. The CL kinetics, i.e., a long lag time followed by a rapid shift to a new level, indicate activation of an 0,--producing enzyme. The cells are also able to protect themselves from H,O, stimulation by both catalase and the glutatione system. Bradykinin stimulates the H,O, release, but if the effect is directly stiniulatory or if it acts by reduction of the protective system is at present unclear. The extracellularly released H,O, could be a cause of injury to the endothelial cells or to the subendothelial matrix.

The microvascular endothelium is the primary physical barrier between the blood and the tissues, and it participates in the exchange of materials between these compartments. One of the manifestations of inflammation is edema, the loss of plasma fluid and protein into the tissues. This condition can be induced by a wide range of biochemical and mechanical insults to a microvessel. It is currently thought that oxidantinduced injury to endothelium is an important mechanism of vascular damage (Ward and Varani, 19901,and cytolysis of cultured endothelial cells by neutro hils is dependent on the generation of hydrogen peroxi e from the latter cells (Weiss et al., 1981; Shasby et al., 1983). Under pathological conditions several metabolic pathways undergo alterations and new ones may become operative. It is likely that a linkage exists between the release of oxygen products from activated phagocytic cells and the conversion of xantine dehydrogenase to xantine oxidase in endothelial cells (McCord, 1987). Endothelial cells are able t o generate and secrete superoxide anion in response to certain agents (Rosen and Freeman, 1984; Matsubara and Ziff, 1986a,b).This production by endothelial cells has also been demonstrated in anoxia-reoxygenation models (Schinetti et al., 1989; Ratych et al., 1987). The mechanism proposed is that, during ischemia, xantine dehydrogenase is converted to xantine oxidase, which generates superoxide oxide as reduction product. Recently, Holland et al. (1990) demonstrated that bradykinin induced superoxide anion release from endothelial cells and proposed that this may be an important component in the pathophysiological action of bradykinin on vascular function. (Holland et al. 1990). The production of oxidative metabolites (02-,H20,) by neutrophils can be measured as light emission, i.e., chemiluminescence (Allen et al., 1972). With the introduction of luminol to the system, the sensitivity in-

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creased 100-fold (Allen and Loose, 1976; Stevens et al., 1978). This amplification permits the use of fewer neutrophils or studies of other cell types producing smaller amounts of oxidative metabolites. The present study was undertaken to investigate whether intact endothelial cells release HzOz to the extracellular environment when grown on microcarrier, as assessed by chemiluminescence (CL) measurements in a luminol-amplified system. MATERIALS AND METHODS Fresh bovine aorta from a slaughterhouse was kept in phosphate-buffered saline (PBS) with penicillin (100 pg/ml), streptomycin (100 pg/ml), and fungizone (5 pgiml). Within 1hr endothelial cells were obtained in the following ways: 1)by filling the vessel with 0.2% collagenase (Sigma Chemical Co., St. Louis, MO) for 15 min (37°C)after ligating inter costals and cutting ends (Jaffe et al., 1973) or 2) a single gentle scraping of the exposed endothelial surface with a scalpel blade. The procedure depended on the size of the inter costals. The cells were rinsed in culture medium and seeded in 80 cm2 cell culture flasks (Nunc, Roskilde, Denmark) in a complete culture medium composed of RPMI 1640 with 2 mM L-glutamin, 100 pgiml penicillin, 100 pgiml streptomycin, and 20% fetal calf serum (Flow Laboratories, Irvine, Scotland). After 1 week in the incubator (type GF3; Kebo-ASSAB Medicine, Stockholm, Sweden) with 5% CO, and 95% air, the cells had reached a confluent layer and were detached from flasks using trypsin-EDTA. The cells were prepared for growth on Cytodex 3 microcarriers (Pharmacia LKB Biotechnology AB, Uppsala, Sweden) according to Busch et al. (1982)in a 125 ml microcarrier spinner flask (J.Bibby Science Products Ltd, Stone, Staffordshire, England). After about 1week the cells reached a confluent layer, Received October 2, 1990; accepted April 1, 1991.

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and after another week they were used for CL studies. The medium was changed three times per week, and cells from the culture flasks were used for the microcarrier attachment up to passage 20. Endothelial cell identity was verified by indirect immunofluorescence microscopic detection of factor VIII-related antigen in the cells (Dakopatts AIS, Glostrup, Denmark). The cells were sedimentated and washed twice with KrebsRinger phosphate buffer, supplemented with lucose (10 mmol/liter), Ca2+ (1 mmol/liter), and M$+ (1.5 mmoliliter) ( H 7.3) KRG. The cells were never stored more than 1 r at 4°C before use. CL was measured in a six-channel Biolumat LB9505 (Berthold Co., Wildbad, Germany) using disposable 4 ml polypropylene tubes. A 0.9 ml reaction mixture containing endothelial cells on microcarriers, 2 X lop5 moliliter luminol in KRG and horseradish peroxidase (HRP) (Sigma Chemical Co.). The reaction mixture without cells was placed in the Biolumat and allowed to equilibrate for 5 min at 37°C. Then, 5 x lo5 or 1 X lo6 cells were added and the light emission was recorded continuously. Superoxide dismutase (SOD; 200 U) and catalase (2,000 U) were obtained from Boeringer Mannheim (Mannheim, Germany) and bradykinin from Sigma.

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RESULTS The generation of luminol-enhanced luminescence is a H202-and peroxidase-dependent process. In the endothelial cells there is no CL without the addition of peroxidase (Fig. 1).Increasing concentrations of HRP resulted in increased CL (Fig. 2) up to saturation at 4 U in a mixture of 1 x lo6 cells. The CL was at least ten times lower compared with stimulated neutrophils (Lock et al., 1988). The light emission started slowly, and increased gradually, reaching a maximum after 5-25 min (Fig. 1).The onset and time at maximum light emission seemed to be dependent on the washing procedure. Increasing the number of cells in the suspension also delayed the chemiluminescence response (Fig. 3) up to the saturation point. The CL was blocked by SOD (200 U) independent of at which time it was added during the response (Fig. 4). The CL was also reduced by the addition of catalase (2,000 U)(Fig. 5). Addition of azide (100 FM) to block the catalase activity from the cells resulted in an even faster conversion to the new CL level (Fig. 6), but addition of azide during the high CL production had no effect. When bradykinin (50 nM) was added as stimulus, a transient CL peak was achieved, followed by a rapid increase up to a steady-state level (Fig. 7). DISCUSSION Although CL has become increasingly popular for determining the production of oxidative metabolites from neutrophils, the mechanisms underlying the reaction are not fully understood. It is well known that the addition of luminol to a CL system can amplify the response by acting as a “bystander” substrate for the oxidative metabolites generated during activation of the neutrophils (Allen and Loose, 1976). However, current information favors the role of a peroxidaseperoxide reaction in a luminol-dependent CL (Wymann

et al., 1987; Lock et al., 1988), so the luminol-HRP system has been suggested to be a sensitive assay for H202 (Cormier and Prichard, 1968; Wymann et al., 1987).The advantages of using CL compared with other methods for studyin H202production by microcarrierattached endothelia cells are that the cells are minimally disturbed as they maintain their contact with each other and the growth surface and that it is simple and rapid, permitting rapid kinetic studies of cell responses to different stimuli. Previous in vitro studies have described that endothelial cells release 02-in res onse to various stimuli (Matsubara and Ziff, 1986a,b; orog et al., 1987; Steinbrecher, 1988; Schinetti et al., 1989; Holland et al., 1990). However, continuous 02- dismutation should also result in a production of H202. The CL response from the endothelial cells demonstrates that H202 is released (Fig. 1) without any added stimulus. If mechanical interaction between the cells on different microcarriers or if the washing procedure is sufficient to stimulate an H20z production is hard to tell. The same CL response was obtained using human umbilical vein endothelial cells on microcarrier beads (unpublished). There was no CL without HRP. The facts that added HRP does not penetrate the cell membrane and that CL is reduced with catalase (Fig. 4) indicates an extracellular release of H202. The lack of CL in the presence of SOD (Fig. 4)could show either that 02- is a precursor for H202 or the existence of 02-from H202produced luminol radicals. Furthermore, since luminol-dependent CL detects H202 when luminol is oxidized (Vilim and Wilheim, 19891, the addition of SOD to the system might have reduced the 02- oxidizing luminol, thereby blocking the CL. Judging from the CL kinetics, there is a lag time between 5 and 25 min and then a rapid increase in the CL response. The sensitivity in the kinetics to the amount of microcarrier and a more rapid conversion after blocking catalase activity with azide suggest a

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Fig. 1. Time trace of chemiluminescence emitted from bovine endothelial cells (1 x 106cells) grown on microcarriers, in the presence of luminol (dotted line), HRP 4 U (dashed line), and luminol and HRP (solid line). Abscissa, time of study (rnin); ordinate, chemiluminescence (CPM x

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Fig. 2. Chemiluminescence from bovine endothelial cells in the presence of different amounts of HRP. Abscissa, amount of HRP (U) present in the reaction vials; ordinate, chemiluminescence in percent of maximum peak value. Each symbol represents the mean I SEM of three to eight determinations. Fig. 3. Time trace of chemiluminescence emitted from bovine endothelial cells grown on microcarriers, after addition of 50 ~1 (5 x lo5 cells) (a). 100 ul (1 x lo6 cells) (b). and 200 u.1 (2 X lo6 cells) (c) cell suspension. Abscissa, time of study (rnin); ordinate, chemiluminescence (CPM x 10-9. Fig. 4. Time trace of chemiluminescence emitted from bovine endothelial cells (1 x lo6 cells) grown on microcarriers, after addition of superoxide dismutase (SOD; 200 U) (solid line) at different times (arrows) compared with control (dotted line). Abscissa, time of study b i n ) ; ordinate, chemiluminescence (CPM x 10-9.

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Fig. 5 . Time trace of chemiluminescence emitted from bovine endothelial cells (1 x lo6 cells) grown on microcarriers, after addition of catalase (2000 U) (solid line) compared with control (dotted line). Abscissa, time of study (min); ordinate, chemiluminescence (CPM X 10-9. Fig. 6. Time trace of chemiluminescence emitted from bovine endothelial cells (1 x l o 6 cells) grown on microcarriers after addition of azide (100 (*MI at time (a) resulted in a faster conversion to the higher chemiluminescence (b) compared with control. Abscissa, time of study (min); ordinate, chemiluminescence (CPM x Fig. 7. Time trace of chemiluminescence emitted from bovine endothelial cells (1 x lo6 cells) grown on microcarriers, after addition of bradykinin (50 nM) (solid line) compared with control (dotted line). Abscissa, time of study (min); ordinate, chemiluminescence (CPM x 10- 6).

ENDOTHELIAL CELLS RELEASE HYDROGEN PEROXIDE

self-stimulatory effect by H202 but also a self-regulatory mechanism by which the cells are able to protect themselves from a limited concentration of released H202. Glutathione is one protective agent in the cells (Andreoli et al., 19861, but also serum albumin in the suspension may act as a scavenger. With addition of 1% serum albumin to the CL system with neutrophils a reduction of the extracellular CL is expected (Briheim et al., 1984). Since the endothelial cells are grown in 20% serum, there might be albumin in the cell suspension that could delay CL. This could explain the sensitivity to washing the cells and to the amount of cell suspension added (Fig. 3). Miirtensson and Meister (1989) mention that it is easy to wash out glutation from cells, and this should also reduce cell protection. Furthermore, it has been shown that the endothelial cells can protect themself from H202-inducedinjury in a defined range of H202 concentrations by actively degrading the peroxide (Dobrina and Patriarca, 1986). When the production exceeds this concentration, H202 might stimulate the cell to produce even more up to a maximal rate. This could explain the constant CL from 5 to 25 min, followed by a rapid increase, which possibly indicates a sudden release of H202 up to a constant production level. The proposed conversion of xantine dehydrogenase to xantine oxidase in the superoxidegenerating system (Schinetti et al., 1989) fits with this model provided that 02- or H202 converts xantine dehydrogenase to xantine oxidase. This may occur when exogenous or endogenous scavengers are consumed. The anoxiaireoxygenation model of cultured rat pulmonary artery endothelial cells has shown that the majority of xantine dehydrogenase is converted to xantine oxidase (Ratych et al., 1987),which should give a saturated CL response (Fig. 1) as long as the substrate exists. On the other hand, by indirect immunofluorescence microscopy, xantine oxidase has been detected mainly in the microvascular endothelium (Jarasch et al., 1986). Therefore, it cannot be stated that xantine oxidase is the source of the hydrogen peroxide. A feedback mechanism may explain the kinetics of the CL response. The xantine oxidase-induced injury to endothelial cells may indicate that H202 is the cytotoxic oxygen derived product (Kvietys et al., 1989). The release of hydrogen peroxide from the endothelial cells indicates a possible endogenous cause of injury to the cells or the subendothelial matrix. Bradykinin-induced H,02 release from the endothelial cells was rapid, with an initial peak in CL. The subsequent conversion to the steady-state level could, however, either be due to bradykinin or a result of the released radicals. The latter su ports the theory of a feedback mechanism that is bloc ed by scavengers. The 02-and H202production from endothelial cells can be stimulated by bradykinin (Holland et al., 1990; Fig. 6). Moreover, it is known that the production of endothelial-derived relaxing factor (EDRF) is stimulated by bradykinin (Ignarro et al., 1986). Superoxide anion inactivates EDRF (Gryglewski et al., 19861, indicating a vasoregulatory mechanism in the endothelial cells involvin free radicals. The spontaneous and SODmediateC f dismutation of 02-to H202could increase the

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amount of intact EDRF but also result in injury to the cells or the subendothelial matrix. In conclusion, endothelial cells release H202to the extracellular environment without any added stimulus. The production was detected as extracellular CL in a HRP-luminol-containing system. The extracellularly released H202could be noxious to the endothelial cells or to the subcellular matrix.

ACKNOWLEDGMENTS I thank Drs. K.-E. Magnusson, C. Dahlgren, and T. Skogh for valuable discussions. This research was supported by grants from the Swedish Medical Research Council (Project 9055), King Gustaf V80 Years Foundation, and Ostergotlands Lans Landstings Forskningsfond. LITERATURE CITED Allen, R.C., and Loose, L.D. (1976) Phagocytic activation of a luminoldependent chemiluminescence in rabbit alveolar and peritoneal macrophages. Biochem. Biophys. Res. Commun., 69:245-252. Allen, R.C., Stjernholm, R.L., and Steel, R.H. (1972) Evidence for the generation of electronic excitation state(s) in human polymorphonuclear leukocytes and its partition in bactericidal activity. Biochem. Biophys. Res. Commun., 47:679-684. Andreoli, S.P., Mallett, C.P., and Bergstein, J.M. (1986) Role of glutathione in protecting endothelial cells against hydrogen peroxide oxidant injury. J . Lab. Clin. Med., 108:190-198. Briheim, G., Stendahl, O., and Dahlgren, C. 11984) Intra- and extracellular events in Luminol-dependent chemiluminescence of polymorphonuclear leukocytes. Infect. Immun., 45:l-5. Busch, C., Cancilla, P.A., DeBault, L.E., Goldsmith, J.C., and Owen, G.W. (1982) Methods in laboratorv investigation. Use of endothelium cultured on microcarriers as model &r the microcirculation. Lab. Invest., 47:498-505. Cormier, M.J., and Prichard, P.M. (1968) An investigation of the mechanism of the luminescent aeroxidation of luminol bv- stouued flow techniques. J. Biol. Chem.: 243:4706%4714. Dobrina, A,, and Patriarca, P. (1986) Neutrophil-endothelial cell interaction: Evidence for and mechanisms of the self-protection of bovine microvascular endothelial cells from hydrogen peroxodeinduced oxidative stress. J. Clin. Invest., 78:462-471. Gorog, P., Pearson, J.D., and Kakkar, V.V. (1987) Generation of reactive oxygen metabolites by phagocytosing endothelial cells: Regulatory role of the glycocalyx. Thromb. Haemostas., 58:63 (abstract). Gryglewski, R.J., Palmer, R.M.J., and Moncada, S. (1986)Superoxide anion is involved in the breakdown of endothelium-derived vascular relaxing factor. Nature, 320:45&456. Holland, J.A., Pritchard, K.A., Pappolla, M.A., Wolin, M.S., Rogers, N.J., and Stemerman, M.B. (1990) Bradykinin induces superoxide anion release from human endothelial cells. J. Cell. Physiol., 143:2 1-25. Ignarro, L.J., Harbison, R.G., Wood, K.S., and Kadowitz, P.J. (1986) Activation of purified soluble guanylate cyclase by endothelium derived relaxing factor from intrapulmonary artery and vein: Stimulation bv acetvlcholine. bradvkinin. and adenosine triuhos~" phate. J. Pharmacol. Exp. Ther., 227:893-900. Jaffe, E.A., Hoyer, L.W., and Nachman, R.L. (1973) Synthesis of antihemoohilic factor antigen bv cultured human endothelial cells. J . Clin. Irbest., 52:2757-fi64. " Jarasch, E.-D., Bruder, G., and Heid, H.W. (1986) Significance of xanthine oxidase in capillary endothelial cells. Acta Physiol. Scand., 548(suppl):39-46. Kvietys, P.R., Inauen, W., Bacon, B.R., and Grisham, M.B. (1989) Xanthine oxidase-induced injury to endothelium: Role of intracellular iron and hydroxyl radical. Am. J . Physiol., 257:H1640-H1646. Lock, R., Johansson, A,, Orselius, K., and Dahlgren, C. (1988) Analysis of horseradish peroxidase-amplified chemiluminescence produced by human neutrophils reveals a role for the superoxide anion in the light emitting reaction. Anal. Biochem., 173:450-455. MBrtensson, J., and Meister, A. (1989) Mitochondria] damage in muscle occurs after marked depletion of glutathione and is pre-

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vented by giving glutathione monoester. Proc. Natl. Acad. Sci. USA, 86:471-475. Matsubara, T., and Ziff, M. (1986a) Superoxide anion release bynbhuman endothelial cells: Synergism between a phorbol ester and a calcium ionophore. J. Cell. Physiol., 128:207-210. Matsubara, T., and Ziff, M. (198613)Increased superoxide anion release from human endothelial cells in response to cytokines. J . Immunol., I37:3295-3298. McCord, J.M. (1987) Oxygen-derived radicals: A link between reperfusion injury and inflammation. Fed. Proc., 46:2402-2406. Ratych, R.A., Chuknyiska, R.S., and Bulkley, G.B. (1987)The primary localization of free radical generation after anoxiaireoxygenation in isolated endothelial cells. Surgery, 102:122-130. Rosen, G.M., and Freeman, B.A. (1984) Detection of superoxide generated by endothelial cells. Proc. Natl. Acad. Sci. USA, 81:72697273. Schinetti, M.L., Sbarbati, R., and Scarlattini, M. (1989) Superoxide production by human umbilical vein endothelial cells in a n anoxiareoxygenation model. Cardiovasc. Res., 23:76-80. Shasby, M.D., Shasby, S.S.,and Peach, M.J. (1983) Granulocytes and phorbol myristate acetate increase permeability to albumin of cultured endothelial monolayers and isolated perfused lung. Role of

oxygen radicals and granulocyte adherence. Am. Rev. Respir. Dis., 127:72-76. Steinbrecher, U.P. (1988) Role of superoxide in endothelial-cell modification of low-density lipoproteins. Biochim. Biophys. Acta, 959:20-30. Stevens, P., Winston, D., and van Dyke, K. (1978) In vitro evaluation of opsonic and cellular granulocyte function by luminol-dependent chemiluminescence.Utility in patients with severe neutropenia and cellular state. Infect. Immun., 22:41-51. Vilim, V., and Wilhelm, J. (1989) What do we measure by a luminoldependent chemiluminescence of phagoeytes? Free Rad. Biol. Med., 6:623-629. Ward, P.A., and Varani, J. (1990)Mechanisms of neutrophil-mediated killing of endothelial cells. J. Leukocyte Biol., 48:97-102. Weiss, S.J., Young, J., LoBuglio, A.F., and Slivka, A. (1981) Role of hydrogen peroxide in neutrophil-mediated destruction of cultured endothelial cells. J . Clin. Invest., 68:714-721. Wymann, M.P., von Tscharner, V., Deranleau, D.A., and Baggiolini, M. (1987) Chemiluminescence detection of H,O, produced by human neutrophils during the respiratory burst. Anal. Biochem., 165:371-378.

Bovine aortic endothelial cells release hydrogen peroxide.

Endothelial cells grown on microcarriers are able to release H2O2 to the extracellular environment without any added stimulus. The extracellularly rel...
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