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Age related decline in cytokine induced nitric oxide synthase activation and apoptosis in cultured endothelial cells: minimal involvement of nitric oxide in the apoptosis
mEha&msofageing aMibvebpmellt
Mechanisms of Ageing and Development 81 (1995) 27-36
ELSEVIER
Age related decline in cytokine induced nitric oxide synthase activation and apoptosis in cultured endothelial cells: minimal involvement of nitric oxide in the apoptosis Ikuko
Satoa’b, Kazuhiko
KajP, Sei-itsu Murota*a
“Department of Physiological Chemistry, Graduate School, bDepartment of Geriatric Dentistry, Faculty of Dentistry, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo 113, Japan ‘Lbepartment of Biochemistry Isotopes, Tokyo Metropolitan Institute of Gerontology, Sakaecho, Itabashi-ku, Tokyo 173, Japan
Received 5 December 1994; revision received 23 March 1995; accepted 3 April 1995
Abstract
Nitric oxide synthase (NOS) activity was enhanced in human umbilical vein endothelial cells (HUVECs) by the combined stimulation with IFN-7 plus IL-lp, TNF-a and LPS which was accompanied by cell death. DNA analysis of the NOS induced dead HUVECs showed that intemucleosomal DNA fragmentation had occurred, suggesting that apoptosis was taken place. The enhanced NO production seemed to be associated with the death of HUVIECs, however, both NC-methyl-L-arginine (LNMMA) and nitro-L-arginine (N-arg), inhibitors of NOS, recovered the death of HUVECs by only 16%, suggesting that NO production was minimally involved in the cytokine induced apoptosis of HUVECs. Additional results demonstrated that both the induction of NOS activity and apoptosis in HUVlECs declined with in vitro aging, i.e. declined with increasing PDLs of HUVECs, which may explain the decreased immunity during inflammation in aged people. Keywords:
endothelial
Nitric oxide; Nitric oxide synthase; Cytokine; Apoptosis; Human umbilical vein cells (HUVECs); In vitro ageing
* Corresponding 0047-6374/95/$09.50
Author,
Fax:
+ 81 3 5684 3929.
0 1995 Elsevier
SSDI 13047-6374(94)01579-O
Science Ireland
Ltd. All rights
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28
I. Sate et al. 1 Mech. Ageing Dev. 81 (199.5) 27-36
1. Introduction
Endothelium-derived NO is a labile, highly reactive substance which is considered to have a role in vasodilation [l-4]. Numerous cell types are capable of NO synthesis including vascular smooth muscle cells, macrophages, neutrophils, neurons and astrocytes in addition to vascular endothelial cells [5-81. NO is synthesized as a result of the reaction of NO synthase (NOS) with the guanidino nitrogen of L-arginine. Characterization of NOS from a variety of tissues suggests the existence of two distinct enzyme isotypes. One is the constitutive type and the other is the cytokine-inducible. The vascular endothelial cells are known to possess both the constitutive and inducible NOS [9]. In the macrophage, cytokine-inducible NO contributes to the cytotoxic activity not only against tumor cells, bacteria and protozoa but also against the macrophages themselves [6,10]. Previous studies revealed that this NO dependent death of the macrophage was mediated through apoptosis, an active process of cellular self-destruction defined by unique morphologic and molecular characteristics [lo-121. While knowledge of the role of cytokine-inducible NO in human endothelial cells is limited, an autocytotoxic effect of NO generated by the cytokineinducible NOS has been reported in endothelial cells [9,13]. We reported the age-related reduction in NO release by constitutive NOS in human umbilical vein endothelial cells (HUVECs) [14], which may be utilized as an in vitro model system for human cellular ageing [15,16]. Therefore, we chose to investigate the mechanism of cell death in the cytokine-activated endothelial cells in relation to NO by analysis of internucleosomal DNA in HUVECs of known ages.
2. Materials and methods
2.1. Preparation of chemicals The following chemicals were used: Endothelial cell growth factor (ECGF) was prepared by heparin affinity chromatography as described previously [15,17]; Fetal bovine serum (FBS) (Hyclone Sterile Systems Inc., Logan, UT); Medium MCDB104, trypsin (GIBCO, Grand Island, NY); Epidermal growth factor (EGF) (Collaborative Res. Inc., Lexington, MA); L-[2,3,4,5-3H]arginine (spec. act. 62 Ci/mmol) (Amersham, Buckinghamshire, UK); [‘4C]citrulline (spec. act. 58 Ci/mmol) (NEN, Boston, MA); Nitro-L-arginine (N-arg) (Aldrich, Milwaukee, WC); NADPH (Boehringer, Mannheim, Germany); Tissue culture plasticware (Falcon, Becton Dickinson Labware, Oxnard, CA); Recombinant human tumor necrosis factor c( (TNF-a), recombinant human interleukin-lg (IL-l/3) (Endogen, Boston, MA); Human interferon y (IFN-y, 1 x lo6 JRU) was a generous gift of Shionogi Inc. (Osaka, Japan). Heparin, gelatin, NG-methyl-L-arginine (L-NMMA), lipopolysaccharide salmonella typhimurium (LPS), proteinase K, ethidium bromide were purchased from Sigma Chemical Co. (St. Louis, MO). All other chemicals were purchased from Wako Chemical Co. (Osaka, Japan).
I. &to et af. / Me&. Ageing Dev. 81 (1995) 27-36
29
2.2. HUVECs culture HUVECs were isolated from the inner lumen by the modified method of Jaffe et al. [18]. Cells were grown in 0.1% gelatin-coated dishes with a medium of MCDB104 supplemented with 10% FBS, 100 ng/ml ECGF, 10 ng/ml EGF, and 100 ,ug/ml heparin at 37°C in a humidified 5% CO* 95% air incubator and passaged weekly at a density of 1 x lo5 cells per lo-cm dish. During each passage, an aliquot of the cell suspension was counted by a Coulter counter ZBI (Coulter Electronics Inc., Hialeah, FL) to determine population doubling levels (PDLs) and another aliquot was frozen for later analysis of NOS activity. Cells were identified as endothelial cells by their positive response to a specific antibody to factor VIII-related antigen and their uptake of acetylated-LDL. The HUVECs used in the series of experiments in the present paper were free from mycoplasma contamination [19]. 2.3. NOS activity NO:3 activity was measured by the formation of [3H]citrulline from E3H]arginine, as previously described [20]. Briefly, confluent cultures of HUVECs were activated with several cytokine combinations for 24 h, then harvested from lo-cm dishes, sonicated in a 50 mM HEPES (pH 7.4) buffer and centrifuged for 10 min at 3000 rev./min. A 20 ~1 aliquot of the supernatant was removed for protein assay (Bio-Rad, Richmond, CA). A 200-~1 aliquot of the supernatant was incubated in a buffer containing 2 Ci/ml [3H]arginine, 2 mM NADPH and 0.45 mM calcium with or without 1 PM N-arg for 40 min at 37°C. The reaction was terminated by the addition of 10 ~1 6% HClO, and then 200 ~1 of the reaction solution was analyzed by high-performance liquid chromatography (HPLC). The radioactivity of [3H]cit rulline was determined by liquid scintillation counting. 2.4. Analysis of DNA fragmentation Floating cells which died during the 24-h stimulation of cytokines were collected by centrifugation and their DNA fragmentation was analyzed. The cell pellets were incubated for 2 h at 50°C in a digestion buffer containing 0.2 mg/ml proteinase K. The cellular DNA was extracted once with a 1:1 phenol/chloroform mixture and twice with chloroform. The residual RNA in the DNA sample was digested by the addition of RNase (100 pg/ml). The samples were electrophoresed on a 1.5% agarose gel in Tris-athetate buffer. Following electrophoresis, the gel was stained with et.hidium bromide and photographed on an UV transilluminator. 2.5. Statistical analysis All values in the figures and text are expressed as the means + S.E.M. The data were a.nalyzed either by two-tailed Student’s t-test or by one-way analysis of variance (ANOVA) followed by Fisher’s test. 3. Results 3.1. Induction of NOS activity in HUVECs by cytokines Fig. 1 shows a typical line graph of NOS activity in HUVECs (PDL 8). [3H]Citrulline, generated from [3H]arginine by NOS, was separated from
I. Sate et al. / Mech. Ageing Dev. 81 (1995) 27-36
30
[3H]arginine and other decqmposed products. The peak of [3H]citrulline was confirmed by the same retention time with [14C]citrulline, an authentic standard. The combinations of cytokines induced a high NOS activity in HUVECs. The peak was completely blocked in the presence of 1 PM of N-arg, one of the specific inhibitors of NOS. 3.2. Correlation of the induction of NOS activity and the autocytotoxicity to HUVECs Fig. 2A shows NOS activities in HUVECs (PDL 16) induced by several combinations of cytokines for 24 h. Treatment of HUVECs with IFN-y in the combination of IL-la, TNF-c( and LPS showed the maximal effects on the induction of NOS activity. The optimal combination of cytokines inducing NOS activity simultaneously caused cell death in the cultures (Fig. 2B). 3.3. Minimal protective efSect of the inhibitors of NOS on the autocytotoxicity induced by cytokines HUVECs (PDL 16) were plated onto 16-mm (2-cm2) wells at a density of 4 x lo4 cells/cm2. When the cells became confluent, the medium was replaced with a fresh one. Cells of quadruplicate cultures were activated with IFN-7 (100 U/ml) plus IL- l/3 (100 U/ml), TNF-a (50 U/ml) and LPS (10 pug/ml), in the presence or
v * -.--*------o---
01 8
’
I
10
.
1
1
12
14
Time
Control Cytokine treatment Control + N-arg Cytokine treatment + N-arg
1
’
16
16
(min)
Fig. 1. Line graph showing NOS activity in HUVECs at a young stage. The enzyme activity was assayed as described in the text with (dotted line) or without (solid line) 1 ,uM of N-arg. The peak indicated by C is that of citrulline. HUVECs showed high levels of NOS activity when they were activated with IFN-y (100 U/ml) plus IL-Ij? (10 U/ml), TNF-a (50 U/ml) and LPS (10 fig/ml) for 24 h.
I. Sara et al. 1 Mech. Ageing Dev. 81 (1995) 27-36
1
2
3
4
31
5
Fig. 2. Induction of NOS activity (A) and the associated autocytotoxicity (B) in HUVECs by various stimulatory conditions. Lane 1: Control; Lane 2: IFN-;r (100 U/ml) plus LPS (10 pg/ml); Lane 3: IFN-y (100 U/ml) plus IL-l/I (200 U/ml) and TNF-ol (50 U/ml); Lane 4: IFN-y (50 U/ml) plus IL-lb (200 U/ml) and TNF-c( (500 U/ml); Lane 5: IFN-y (100 U/ml) plus IL-l/I (100 U/ml); TNF-a (50 U/ml) and LPS (10 pg/ml). Values represent the means + S.E.M. from a few experiments run in duplicate; *P < 0.05; **I’ < 0.01.
absence of 1 mM of L-NMMA or N-arg. After 24 h, cells were removed from the wells by trypsin treatment, resuspended in the medium and counted by a Coulter counter. HUVECs activated with cytokines significantly reduced the cell viability (Fig. 3, P < 0.01). Addition of 1 mM of L-NMMA or N-arg to the cytokine containing medium minimally blocked the cell death (16%) induced by cytokines (Fig. 3: P < 0.01).
I. Sato et al. 1 Mech. Ageing Dev. 81 (1995) 27-36
80-
I
60 -
40-
20 -
OTControl
Cytokine
L-NMMA
N-arg
Fig. 3. Minimal inhibition of L-NMMA and N-arg on the autocytotoxicity induced by cytokine treatment in HUVECs. HUVECs were activated with IFN-y (100 U/ml) plus IL-IS (100 U/ml),‘IXF-/a/ (SO U/ml) and LPS (IO pg/ml) for 24 h. Values represent the means f S.E.M. from four cultures; **P < 0.01.
3.4. Cytokines induce apoptosis of HUVECs HUVECs (PDL 16) were activated with IFN-y (100 U/ml) plus IL-lp (100 U/ml), TNF-a (50 U/ml) and LPS (10 fig/ml) for 24 h in the presence or absence of ECGF. Fig. 4 shows the presence of low molecular weight DNA fragments in the cell pellets of the activated HUVECs supplied with ECGF (lane 4) or without ECGF (lane 2). No DNA fragmentation was detected at all in the sample from ECGF-treated inactivated control culture (lane 3) or in the sample from the remaining attached cells in ECGF-treated inactivated control culture (data not shown). Simultaneous experiments showed that the induction of NOS activity in the cytokine activated HUVECs occurred regardless of the presence of ECGF. 3.5, Age related decline in the cytokine inducible NO production and apoptosis HUVECs at different PDLs (PDL 10, 41, 64 and 68) were passaged every week until enough cells were obtained to measure the NOS activity. Cells of quadruplicate cultures were activated with IFN-)I (100 U/ml) plus IL-la (100 U/ml), TNF-a (50 U/ml) and LPS (10 pg/ml) for 24 h. Fig. 5 shows the NOS induction and the associated autocytotoxicity in HUVECs during in vitro ageing. Both the induction
I. Sara et al. / Mech. Ageing Dev. 81 (1995) 27-36
of NOS activity and the autocytotoxicity of HUVECs (P < 0.01).
33
significantly declined with advancing age
4. Discussion Our results confirm that treatment of cultured endothelial cells from human umbilical cord with IFN-y plus IL-lp, TNF-a and LPS induces NOS activity, as demonstrated by the conversion of L-arginine to L-citrulline in the cytosol extracts. The precise mechanism by which cytokines induces NOS activity is unclear, however, recent studies have demonstrated that cytokines increase NOS activity by increasing tetrahydrobiopterin levels [21,22].
1
2345
Fig. 4. Apoptosis of HUVECs activated with IFN-1 (100 U/ml) plus IL-l/? (100 U/ml), TNF-a (50 U/ml) and LPS (10 pg/ml) for 24 h. Untreated HUVECs in the absence (lane I) or presence (lane 3) of ECGF. ‘Treated cells in the absence (lane 2) or presence (lane 4) of ECGF. A standard DNA ladder is shown in lane 5.
I. Sato et al. 1 Mech. Ageing Dev. 81 (1995) 27-36
34
NOS activity ____*__._ Cellular protein
”
200l-
e 8
180I-
0
5
.z > *_ g
80
160I-
140 I_
H 50
I_
.% 2 $
-r
I
Young
I
Middle
I
Old
I
40
Very old
HUVECs (age) Fig. 5. Effect of in vitro ageing on the induction of NOS activity and the apoptosis of HUVECs. HUVECs were activated with IFN-y (100 U/ml) plus IL-IB (100 U/ml), TNF-a (50 U/ml) and LPS (10 pg/ml) for 24 h.
An autocytotoxic action of NO was demonstrated to be inversely proportional to the amount of NO produced in response to different stimuli (Fig. 2). Our results are consistent with earlier studies that demonstrated that the autocytotoxic mechanism of NO can be activated in endothelial cells from other species [9,13]. Thus it is envisaged that the normal physiological response of NO production by endothelial cells which acts as a cytotoxic compound against tumor cells [23] and parasites [24] may under conditions of pathology, also damage the endothelial cells themselves, thereby contributing to the evolution of novel pathologies in humans. The preventive effects of inhibitors of NOS on both porcine and bovine endothelial cells autocytolysis have confirmed that the cell death was mediated by NO [9,13]. Although NOS inhibitors completely prevented the cell death in other reports [9-131, the prevention of the cell death in HUVECs was extremely slight (16%), indicating that NO induction is not a determinant of cytokine-mediated cell death in HUVECs. We speculate that the cytokine-mediated cell death in activated endothelial cells occurs not by the direct necrosis of cells but rather by a process
I. Sato et al. 1 Mech. Ageing Dev. 81 (1995) 27-36
35
that involves the fragmentation of DNA, which is a change characteristic of apoptosis [25]. That NO-mediated cell death minimally shares this mechanism of apoptosis was confirmed by the concomitant induction of NOS through cytokine treatment. Although the relevance of NO is uncertain, our speculation that NO may partially induce apoptosis in endothelial cells is consistent with reports that demonstrate NO-mediated apoptosis in macrophages [lo- 121 and in pancreatic p-cells [26]. A more direct mechanism for NO-mediated apoptosis has been suggested by Nguyen et al. [27] who recently demonstrated that NO can deaminate purine and pyrimidine bases in DNA and result in DNA strand breaks. Alternatively, since apoptosis occurs via the activation of an endogenous endonuclease [28] and since NO can modulate the activity of certain enzymes through reaction with cofactors such as Fe and thiols [29], NO may activate the endonuclease involved in the cleavage of DNA [l 11. NOlS induction and autocytotoxicity of HUVECs significantly declined during ageing in vitro. This result is consistent with a report in our laboratory that the basal IV0 release is reduced during ageing in HUVECs [14], which may be reflected in the increase of vascular disorders expressed in aged people. In summary, activation of HUVECs with cytokines induced an increase in NOS activity and apoptosis. The increase in NOS activity was minimally associated with the apoptosis. The induction of NO was impaired during ageing which may be one of the mechanisms responsible for the reduction of immunity in aged people. Acknowledgments
The authors thank Professor Dentistry, Faculty of Dentistry, suggestions and critical reading by a Grant-in-Aid for Scientific and a grant from the Ministry
Masanori Nagao of the Department of Geriatric Tokyo Medical and Dental University, for helpful of the manuscript. This work was partly supported Research from the Ministry of Health and Welfare, of Education, Science and Culture of Japan.
References [I] RF. Furchgott and J.V. Zawardzki, The obligatory role of endothelial cells in the relaxation of art&al smooth muscle by acetylcholine. Nature, 288 (1980) 373-376. [2] R.M.J. Palmar, A.G. Ferrige and S. Moncada, Nitric oxide release accounts for the biological activity of endothelium-derived releasing factor. Nature, 327 (1987) 524-526. [3] M:W. Radomski, R.M.J. Palmar and S. Moncada, Glucocorticoids inhibit the expression of an inducible, but not the constitutive, nitric oxide synthase in vascular endothelial cells. Proc. Natl. Anrd. Sci. USA, 87 (1990) 10043-10047. [4] C. Suschek, H. Rothe, K. Fehsel, J. Enczmann and V. Kolb-Bachofen,
Induction of a macrophagelike nitric oxide synthase in cultured rat aortic endothelial cells. J. Immunol., 151 (1993) 3283-3291. [5] D. Beasley and M. Eldridge, Interleukin-l/? and tumor necrosis factor-a synergistically induce NO synthase in rat vascular smooth muscle cells. Am. J. Physiol., 266 (1994) RI 197-1203. [6] J.B. Hibbs, Z. Vavrin and R.R. Taintor, L-arginine is required for expression of the activated macrophage effector mechanism causing selective metabolic inhibition in target cells. J. Zmmunol., 13X(1987) 550-565. [7] D. Bredt and S. Snyder, Isolation of nitric oxide synthase, a calmodulin-requiring NatI. Acad. Sci. USA, 87 (1990) 682-685.
enzyme. Proc.
I. Sato et al. / Mech. Ageing Deu. 81 (1995) 27-36
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PI L. Ma, 1. Morita, and S. Murota, Presence of constitutive type nitric oxide synthase in cultured astrocytes isolated from rat cerebra. Neurosci. Lett., 174 (1994) 123- 126. 191R.M.J. Palmar, L. Bridge, N.A. Foxwell and S. Moncada, The role of nitric oxide in endothelial cell damage and its inhibition by glucocorticoids. Br. J. Pharmacol., 105 (1992) 1I-12. 1101J.E. Albina, S. Cui, R.B. Mateo and J.S. Reichner, Nitric oxide-mediated apoptosis in murine peritoneal macrophages. J. Immunol., 150 (1993) 5080-5085. [Ill M. Sarih, V. Souvannavong and A. Adam, Nitric oxide synthase induces macrophage death by apoptosis. Biochem. Biophys. Res. Commun., 191 (1993) 503-508. WI S. Cui, J.S. Reichner, R.B. Mateo and J.E. Albina, Activated murine macrophages induce apoptosis in tumor cells through nitric oxide-dependent or -independent mechanisms. Cancer Res., 54 (1994) 2462-2467.
[I31 C. Estrada, C. GBmez, C. Martin, S. Moncada and C. Gonzllez, Nitric oxide mediates tumor necrosis factor-cc cytotoxicity in endothelial cells. Biochem. Biophys. Rex Commun., 186 (1992) 475-482. [I41 I. Sato, I. Morita, K. Kaji, M. Ikeda, M. Nagao and S. Murota, Reduction of nitric oxide producing activity associated with in vitro aging in cultured human umbilical vein endothelial cell. Biochem. Biophys. Rex Commun., 195 (1993) 1070- 1076. iI51 I. Sato, K. Kaji, I. Morita, M. Nagao and S. Murota, Augmentation of endothelin-1, prostacyclin and thromboxane A, secretion associated with in vitro ageing in cultured human umbilical vein endothelial cells. Mech. Ageing Dee., 71 (1993) 73-84. [16] H. Hoshi and W.L. McKeehan, Isolation, growth requirements, cloning, prostacyclin production and life-span of human adult endothelial cells in low serum culture medium. In Vitro, 22 (1986) 51-56. [17] R.R. Lobb and J.W. Fett, Purification of two distinct growth factors from bovine neural tissue by heparin affinity chromatography. Biochemistry, 23 (1984) 6295-6299. [18] E.A. JalTe, R.L. Nachman, C.G. Becker and CR. Minick, Culture of human endothelial cells derived from umbilical veins: identification by morphologic and immunologic criteria. J. Clin. Invest., 52 (1973) 31-38. [19] K. Kihara, S. Ishida and H. Okumura, Detection of mycoplasmal contaminations in sera. J. Biol. Stand., 9 (1981) 243-351. [20] M. Ikeda, 1. Morita, S. Murota, F. Sekiguchi, T. Yuasa and T. Miyatake, Cerebellar nitric oxide synthase activity is reduced in nervous and purkinje cell degeneration mutants but not in climbing fiber-lesioned mice. Neurosci. Let?., 155 (1993) 148- 150. [21] G. Schoedon, M. Schneemann, N. Blau, C.-J..% Edge11 and A. Schaffner, Modulation of human endothelial cell tetrahydrobiopterin synthesis by activating and deactivating cytokines: new perspectives on endothelium-derived relaxing factor. Bioehem. Biophys. Res. Commun., J96(1993) 1343- 1348. P21 P. Rosenkranz-Weiss, W.C. Sessa, S. Milstien, S. Kaufman, C.A. Watson and J.S. Pober, Regulation of nitric oxide synthesis by proinflammatory cytokines in human umbilical vein endothelial cells. J. Clin. Invest., 93 (1994) 2236-2243.
~231L. Li, R.G. Kilbourn, J. Adams and I.J. Fidler, Role of nitric oxide in lysis of tumor cells by cytokine-activated endothelial cells. Cancer Res., 51 (1991) 2531-2535. 1241I.P. Oswald, I. Eltoum, T.A. Wynn, B. Schwartz, P. Caspar, D. Paulin, A. Sher and S.L. James, Endothelial cells are activated by cytokine treatment to kill an intravascular parasite, Schistosoma mansoni, through the production of nitric oxide. Proc. Natl. Acud. Sci. USA, 91 (1994) 999- 1003. 1251B. Robaye, R. Mosselmans, W. Fiers, J.E. Dumont and P. Galand, Tumor necrosis factor induces apoptosis (programmed cell death) in normal endothelial cells in vitro. Am. .I. Puthal., 138 (1991) 447-453. [26] M. Ankarcrona,
J.M. Dypbukt, B. Briine and P. Nicotera, lnterleukin-lp-induced nitric oxide production activates apoptosis in pancreatic RINm5F cells. Exp. Cell Res., 213 (1994) 172-177. [27] T. Nguyen, D. Brunson, CL. Crespi, B.W. Penman, J.S. Wishnok and S.R. Tannenbaum, DNA damage and mutation in human cells exposed to nitric oxide in vitro. Proc. Nat/. Aead. Sci. USA,
89 (1992) 3030-3034. [28] A.H. Wyllie, Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature, 284 (1980) 555-556. [29] J.S. Stamler, D.J. Singe1 and J. Loscalzo, Biochemistry of nitric oxide and its redox-activated forms.
Science, 258 (1992) 1898-1902.
Mechanisms of Ageing and Development 81 (1995) 27-36
ELSEVIER
Age related decline in cytokine induced nitric oxide synthase activation and apoptosis in cultured endothelial cells: minimal involvement of nitric oxide in the apoptosis Ikuko
Satoa’b, Kazuhiko
KajP, Sei-itsu Murota*a
“Department of Physiological Chemistry, Graduate School, bDepartment of Geriatric Dentistry, Faculty of Dentistry, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo 113, Japan ‘Lbepartment of Biochemistry Isotopes, Tokyo Metropolitan Institute of Gerontology, Sakaecho, Itabashi-ku, Tokyo 173, Japan
Received 5 December 1994; revision received 23 March 1995; accepted 3 April 1995
Abstract
Nitric oxide synthase (NOS) activity was enhanced in human umbilical vein endothelial cells (HUVECs) by the combined stimulation with IFN-7 plus IL-lp, TNF-a and LPS which was accompanied by cell death. DNA analysis of the NOS induced dead HUVECs showed that intemucleosomal DNA fragmentation had occurred, suggesting that apoptosis was taken place. The enhanced NO production seemed to be associated with the death of HUVIECs, however, both NC-methyl-L-arginine (LNMMA) and nitro-L-arginine (N-arg), inhibitors of NOS, recovered the death of HUVECs by only 16%, suggesting that NO production was minimally involved in the cytokine induced apoptosis of HUVECs. Additional results demonstrated that both the induction of NOS activity and apoptosis in HUVlECs declined with in vitro aging, i.e. declined with increasing PDLs of HUVECs, which may explain the decreased immunity during inflammation in aged people. Keywords:
endothelial
Nitric oxide; Nitric oxide synthase; Cytokine; Apoptosis; Human umbilical vein cells (HUVECs); In vitro ageing
* Corresponding 0047-6374/95/$09.50
Author,
Fax:
+ 81 3 5684 3929.
0 1995 Elsevier
SSDI 13047-6374(94)01579-O
Science Ireland
Ltd. All rights
reserved
28
I. Sate et al. 1 Mech. Ageing Dev. 81 (199.5) 27-36
1. Introduction
Endothelium-derived NO is a labile, highly reactive substance which is considered to have a role in vasodilation [l-4]. Numerous cell types are capable of NO synthesis including vascular smooth muscle cells, macrophages, neutrophils, neurons and astrocytes in addition to vascular endothelial cells [5-81. NO is synthesized as a result of the reaction of NO synthase (NOS) with the guanidino nitrogen of L-arginine. Characterization of NOS from a variety of tissues suggests the existence of two distinct enzyme isotypes. One is the constitutive type and the other is the cytokine-inducible. The vascular endothelial cells are known to possess both the constitutive and inducible NOS [9]. In the macrophage, cytokine-inducible NO contributes to the cytotoxic activity not only against tumor cells, bacteria and protozoa but also against the macrophages themselves [6,10]. Previous studies revealed that this NO dependent death of the macrophage was mediated through apoptosis, an active process of cellular self-destruction defined by unique morphologic and molecular characteristics [lo-121. While knowledge of the role of cytokine-inducible NO in human endothelial cells is limited, an autocytotoxic effect of NO generated by the cytokineinducible NOS has been reported in endothelial cells [9,13]. We reported the age-related reduction in NO release by constitutive NOS in human umbilical vein endothelial cells (HUVECs) [14], which may be utilized as an in vitro model system for human cellular ageing [15,16]. Therefore, we chose to investigate the mechanism of cell death in the cytokine-activated endothelial cells in relation to NO by analysis of internucleosomal DNA in HUVECs of known ages.
2. Materials and methods
2.1. Preparation of chemicals The following chemicals were used: Endothelial cell growth factor (ECGF) was prepared by heparin affinity chromatography as described previously [15,17]; Fetal bovine serum (FBS) (Hyclone Sterile Systems Inc., Logan, UT); Medium MCDB104, trypsin (GIBCO, Grand Island, NY); Epidermal growth factor (EGF) (Collaborative Res. Inc., Lexington, MA); L-[2,3,4,5-3H]arginine (spec. act. 62 Ci/mmol) (Amersham, Buckinghamshire, UK); [‘4C]citrulline (spec. act. 58 Ci/mmol) (NEN, Boston, MA); Nitro-L-arginine (N-arg) (Aldrich, Milwaukee, WC); NADPH (Boehringer, Mannheim, Germany); Tissue culture plasticware (Falcon, Becton Dickinson Labware, Oxnard, CA); Recombinant human tumor necrosis factor c( (TNF-a), recombinant human interleukin-lg (IL-l/3) (Endogen, Boston, MA); Human interferon y (IFN-y, 1 x lo6 JRU) was a generous gift of Shionogi Inc. (Osaka, Japan). Heparin, gelatin, NG-methyl-L-arginine (L-NMMA), lipopolysaccharide salmonella typhimurium (LPS), proteinase K, ethidium bromide were purchased from Sigma Chemical Co. (St. Louis, MO). All other chemicals were purchased from Wako Chemical Co. (Osaka, Japan).
I. &to et af. / Me&. Ageing Dev. 81 (1995) 27-36
29
2.2. HUVECs culture HUVECs were isolated from the inner lumen by the modified method of Jaffe et al. [18]. Cells were grown in 0.1% gelatin-coated dishes with a medium of MCDB104 supplemented with 10% FBS, 100 ng/ml ECGF, 10 ng/ml EGF, and 100 ,ug/ml heparin at 37°C in a humidified 5% CO* 95% air incubator and passaged weekly at a density of 1 x lo5 cells per lo-cm dish. During each passage, an aliquot of the cell suspension was counted by a Coulter counter ZBI (Coulter Electronics Inc., Hialeah, FL) to determine population doubling levels (PDLs) and another aliquot was frozen for later analysis of NOS activity. Cells were identified as endothelial cells by their positive response to a specific antibody to factor VIII-related antigen and their uptake of acetylated-LDL. The HUVECs used in the series of experiments in the present paper were free from mycoplasma contamination [19]. 2.3. NOS activity NO:3 activity was measured by the formation of [3H]citrulline from E3H]arginine, as previously described [20]. Briefly, confluent cultures of HUVECs were activated with several cytokine combinations for 24 h, then harvested from lo-cm dishes, sonicated in a 50 mM HEPES (pH 7.4) buffer and centrifuged for 10 min at 3000 rev./min. A 20 ~1 aliquot of the supernatant was removed for protein assay (Bio-Rad, Richmond, CA). A 200-~1 aliquot of the supernatant was incubated in a buffer containing 2 Ci/ml [3H]arginine, 2 mM NADPH and 0.45 mM calcium with or without 1 PM N-arg for 40 min at 37°C. The reaction was terminated by the addition of 10 ~1 6% HClO, and then 200 ~1 of the reaction solution was analyzed by high-performance liquid chromatography (HPLC). The radioactivity of [3H]cit rulline was determined by liquid scintillation counting. 2.4. Analysis of DNA fragmentation Floating cells which died during the 24-h stimulation of cytokines were collected by centrifugation and their DNA fragmentation was analyzed. The cell pellets were incubated for 2 h at 50°C in a digestion buffer containing 0.2 mg/ml proteinase K. The cellular DNA was extracted once with a 1:1 phenol/chloroform mixture and twice with chloroform. The residual RNA in the DNA sample was digested by the addition of RNase (100 pg/ml). The samples were electrophoresed on a 1.5% agarose gel in Tris-athetate buffer. Following electrophoresis, the gel was stained with et.hidium bromide and photographed on an UV transilluminator. 2.5. Statistical analysis All values in the figures and text are expressed as the means + S.E.M. The data were a.nalyzed either by two-tailed Student’s t-test or by one-way analysis of variance (ANOVA) followed by Fisher’s test. 3. Results 3.1. Induction of NOS activity in HUVECs by cytokines Fig. 1 shows a typical line graph of NOS activity in HUVECs (PDL 8). [3H]Citrulline, generated from [3H]arginine by NOS, was separated from
I. Sate et al. / Mech. Ageing Dev. 81 (1995) 27-36
30
[3H]arginine and other decqmposed products. The peak of [3H]citrulline was confirmed by the same retention time with [14C]citrulline, an authentic standard. The combinations of cytokines induced a high NOS activity in HUVECs. The peak was completely blocked in the presence of 1 PM of N-arg, one of the specific inhibitors of NOS. 3.2. Correlation of the induction of NOS activity and the autocytotoxicity to HUVECs Fig. 2A shows NOS activities in HUVECs (PDL 16) induced by several combinations of cytokines for 24 h. Treatment of HUVECs with IFN-y in the combination of IL-la, TNF-c( and LPS showed the maximal effects on the induction of NOS activity. The optimal combination of cytokines inducing NOS activity simultaneously caused cell death in the cultures (Fig. 2B). 3.3. Minimal protective efSect of the inhibitors of NOS on the autocytotoxicity induced by cytokines HUVECs (PDL 16) were plated onto 16-mm (2-cm2) wells at a density of 4 x lo4 cells/cm2. When the cells became confluent, the medium was replaced with a fresh one. Cells of quadruplicate cultures were activated with IFN-7 (100 U/ml) plus IL- l/3 (100 U/ml), TNF-a (50 U/ml) and LPS (10 pug/ml), in the presence or
v * -.--*------o---
01 8
’
I
10
.
1
1
12
14
Time
Control Cytokine treatment Control + N-arg Cytokine treatment + N-arg
1
’
16
16
(min)
Fig. 1. Line graph showing NOS activity in HUVECs at a young stage. The enzyme activity was assayed as described in the text with (dotted line) or without (solid line) 1 ,uM of N-arg. The peak indicated by C is that of citrulline. HUVECs showed high levels of NOS activity when they were activated with IFN-y (100 U/ml) plus IL-Ij? (10 U/ml), TNF-a (50 U/ml) and LPS (10 fig/ml) for 24 h.
I. Sara et al. 1 Mech. Ageing Dev. 81 (1995) 27-36
1
2
3
4
31
5
Fig. 2. Induction of NOS activity (A) and the associated autocytotoxicity (B) in HUVECs by various stimulatory conditions. Lane 1: Control; Lane 2: IFN-;r (100 U/ml) plus LPS (10 pg/ml); Lane 3: IFN-y (100 U/ml) plus IL-l/I (200 U/ml) and TNF-ol (50 U/ml); Lane 4: IFN-y (50 U/ml) plus IL-lb (200 U/ml) and TNF-c( (500 U/ml); Lane 5: IFN-y (100 U/ml) plus IL-l/I (100 U/ml); TNF-a (50 U/ml) and LPS (10 pg/ml). Values represent the means + S.E.M. from a few experiments run in duplicate; *P < 0.05; **I’ < 0.01.
absence of 1 mM of L-NMMA or N-arg. After 24 h, cells were removed from the wells by trypsin treatment, resuspended in the medium and counted by a Coulter counter. HUVECs activated with cytokines significantly reduced the cell viability (Fig. 3, P < 0.01). Addition of 1 mM of L-NMMA or N-arg to the cytokine containing medium minimally blocked the cell death (16%) induced by cytokines (Fig. 3: P < 0.01).
I. Sato et al. 1 Mech. Ageing Dev. 81 (1995) 27-36
80-
I
60 -
40-
20 -
OTControl
Cytokine
L-NMMA
N-arg
Fig. 3. Minimal inhibition of L-NMMA and N-arg on the autocytotoxicity induced by cytokine treatment in HUVECs. HUVECs were activated with IFN-y (100 U/ml) plus IL-IS (100 U/ml),‘IXF-/a/ (SO U/ml) and LPS (IO pg/ml) for 24 h. Values represent the means f S.E.M. from four cultures; **P < 0.01.
3.4. Cytokines induce apoptosis of HUVECs HUVECs (PDL 16) were activated with IFN-y (100 U/ml) plus IL-lp (100 U/ml), TNF-a (50 U/ml) and LPS (10 fig/ml) for 24 h in the presence or absence of ECGF. Fig. 4 shows the presence of low molecular weight DNA fragments in the cell pellets of the activated HUVECs supplied with ECGF (lane 4) or without ECGF (lane 2). No DNA fragmentation was detected at all in the sample from ECGF-treated inactivated control culture (lane 3) or in the sample from the remaining attached cells in ECGF-treated inactivated control culture (data not shown). Simultaneous experiments showed that the induction of NOS activity in the cytokine activated HUVECs occurred regardless of the presence of ECGF. 3.5, Age related decline in the cytokine inducible NO production and apoptosis HUVECs at different PDLs (PDL 10, 41, 64 and 68) were passaged every week until enough cells were obtained to measure the NOS activity. Cells of quadruplicate cultures were activated with IFN-)I (100 U/ml) plus IL-la (100 U/ml), TNF-a (50 U/ml) and LPS (10 pg/ml) for 24 h. Fig. 5 shows the NOS induction and the associated autocytotoxicity in HUVECs during in vitro ageing. Both the induction
I. Sara et al. / Mech. Ageing Dev. 81 (1995) 27-36
of NOS activity and the autocytotoxicity of HUVECs (P < 0.01).
33
significantly declined with advancing age
4. Discussion Our results confirm that treatment of cultured endothelial cells from human umbilical cord with IFN-y plus IL-lp, TNF-a and LPS induces NOS activity, as demonstrated by the conversion of L-arginine to L-citrulline in the cytosol extracts. The precise mechanism by which cytokines induces NOS activity is unclear, however, recent studies have demonstrated that cytokines increase NOS activity by increasing tetrahydrobiopterin levels [21,22].
1
2345
Fig. 4. Apoptosis of HUVECs activated with IFN-1 (100 U/ml) plus IL-l/? (100 U/ml), TNF-a (50 U/ml) and LPS (10 pg/ml) for 24 h. Untreated HUVECs in the absence (lane I) or presence (lane 3) of ECGF. ‘Treated cells in the absence (lane 2) or presence (lane 4) of ECGF. A standard DNA ladder is shown in lane 5.
I. Sato et al. 1 Mech. Ageing Dev. 81 (1995) 27-36
34
NOS activity ____*__._ Cellular protein
”
200l-
e 8
180I-
0
5
.z > *_ g
80
160I-
140 I_
H 50
I_
.% 2 $
-r
I
Young
I
Middle
I
Old
I
40
Very old
HUVECs (age) Fig. 5. Effect of in vitro ageing on the induction of NOS activity and the apoptosis of HUVECs. HUVECs were activated with IFN-y (100 U/ml) plus IL-IB (100 U/ml), TNF-a (50 U/ml) and LPS (10 pg/ml) for 24 h.
An autocytotoxic action of NO was demonstrated to be inversely proportional to the amount of NO produced in response to different stimuli (Fig. 2). Our results are consistent with earlier studies that demonstrated that the autocytotoxic mechanism of NO can be activated in endothelial cells from other species [9,13]. Thus it is envisaged that the normal physiological response of NO production by endothelial cells which acts as a cytotoxic compound against tumor cells [23] and parasites [24] may under conditions of pathology, also damage the endothelial cells themselves, thereby contributing to the evolution of novel pathologies in humans. The preventive effects of inhibitors of NOS on both porcine and bovine endothelial cells autocytolysis have confirmed that the cell death was mediated by NO [9,13]. Although NOS inhibitors completely prevented the cell death in other reports [9-131, the prevention of the cell death in HUVECs was extremely slight (16%), indicating that NO induction is not a determinant of cytokine-mediated cell death in HUVECs. We speculate that the cytokine-mediated cell death in activated endothelial cells occurs not by the direct necrosis of cells but rather by a process
I. Sato et al. 1 Mech. Ageing Dev. 81 (1995) 27-36
35
that involves the fragmentation of DNA, which is a change characteristic of apoptosis [25]. That NO-mediated cell death minimally shares this mechanism of apoptosis was confirmed by the concomitant induction of NOS through cytokine treatment. Although the relevance of NO is uncertain, our speculation that NO may partially induce apoptosis in endothelial cells is consistent with reports that demonstrate NO-mediated apoptosis in macrophages [lo- 121 and in pancreatic p-cells [26]. A more direct mechanism for NO-mediated apoptosis has been suggested by Nguyen et al. [27] who recently demonstrated that NO can deaminate purine and pyrimidine bases in DNA and result in DNA strand breaks. Alternatively, since apoptosis occurs via the activation of an endogenous endonuclease [28] and since NO can modulate the activity of certain enzymes through reaction with cofactors such as Fe and thiols [29], NO may activate the endonuclease involved in the cleavage of DNA [l 11. NOlS induction and autocytotoxicity of HUVECs significantly declined during ageing in vitro. This result is consistent with a report in our laboratory that the basal IV0 release is reduced during ageing in HUVECs [14], which may be reflected in the increase of vascular disorders expressed in aged people. In summary, activation of HUVECs with cytokines induced an increase in NOS activity and apoptosis. The increase in NOS activity was minimally associated with the apoptosis. The induction of NO was impaired during ageing which may be one of the mechanisms responsible for the reduction of immunity in aged people. Acknowledgments
The authors thank Professor Dentistry, Faculty of Dentistry, suggestions and critical reading by a Grant-in-Aid for Scientific and a grant from the Ministry
Masanori Nagao of the Department of Geriatric Tokyo Medical and Dental University, for helpful of the manuscript. This work was partly supported Research from the Ministry of Health and Welfare, of Education, Science and Culture of Japan.
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