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(Hypertension. 1997;30:1198-1203.)
© 1997 American Heart Association, Inc.

Articles

Endothelin-1 as an Autocrine/Paracrine Apoptosis Survival Factor for Endothelial Cells

Masayoshi Shichiri; Hiroki Kato; Fumiaki Marumo; Yukio Hirata

From the Endocrine-Hypertension Division, Second Department of Internal Medicine, Tokyo Medical and Dental University (Japan).

Correspondence to Masayoshi Shichiri, MD, Endocrine-Hypertension Division, Second Department of Internal Medicine, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113, Japan. E-mail mshichiri.med2{at}med.tmd.ac.jp

	Abstract

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Abstract Endothelin-1 (ET-1), an endothelium-derived vasoactive peptide, functions as a potent vasoconstrictor as well as mitogen. We show here a novel role for ET-1 as an apoptosis survival factor for cultured rat endothelial cells. When we rendered endothelial cells obtained from rat aorta quiescent by serum starvation, significant portions of cultured cells underwent apoptotic death as demonstrated by nucleosomal laddering on agarose gel electrophoresis, flow cytometry analysis with FACS, and the TdT-mediated dUTP biotin nick-end labeling (TUNEL) method. ET-1 dose-dependently (10^-12 to 10^-6 mol/L) suppressed the apoptosis induced by serum starvation. The ET_B receptor antagonist (BQ788; 10^-6 mol/L) and ET_A/B receptor antagonists (PD142893 and PD145065; 10^-6 mol/L), but not the ET_A receptor antagonist (BQ123; 10^-6 mol/L), blocked the apoptosis protective effect of 10^-7 mol/L ET-1. Nonimmune rabbit serum reduced the apoptotic event induced by serum deprivation, whereas neutralization of endogenous ET-1 by polyclonal anti–ET-1 antiserum abrogated this protective effect. The ET_B receptor antagonist (BQ788; 10^-8 to 10^-6 mol/L), but not the ET_A receptor antagonist (BQ123; 10^-8 to 10^-6 mol/L), significantly inhibited proliferation of endothelial cells. These data suggest that ET-1, as well as mitogen, functions as an apoptosis survival factor for endothelial cells in an autocrine/paracrine manner via the ET_B receptor.

Key Words: endothelin-1 • ET_B receptor • cell survival • apoptosis • endothelial cells

	Introduction

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Vascular endothelium, a monolayer of cells lining the intima of the blood vessels, is involved in a variety of functions, including coagulation, vascular permeability, vascular tonus, and remodeling.¹ Endothelial cells, which are in direct contact with plasma and cellular components of blood, are the target of many cytokines and growth factors,^{2 3} and they alter the metabolic status of their own cells.⁴ Abnormalities of endothelial function may contribute to certain diseases of blood vessel walls, such as thrombosis, atherosclerosis, and vasculitis.^{5 6} Endothelial cell survival under pathological conditions, such as hypertension, atherosclerosis, vascular prosthesis,⁷ endoarterectomies,⁸ and angioplasties,⁹ is assumed to be a consequence of the proliferative and apoptotic status of cells, which may be particularly important in preventing the development of vascular diseases.⁶

Apoptosis, a strategic biological process of eliminating unwanted cells, is involved in the regulation of cell number under physiological and certain pathological conditions.¹⁰ Apoptosis is associated with distinctive morphological and biological events, such as cellular shrinkage, nuclear condensation, and fragmentation of chromatin caused by cleavage by endonucleases at the internucleosomal linker region, yielding mono- and polynucleosomal DNA fragments. Apoptosis has been studied extensively for its involvement in the regulation of tumor growth, cell-mediated immunity, embryogenesis, and metamorphosis.^{11 12 13 14 15} The genetic program of apoptotic cell death in hematopoietic cells, fibroblasts, and thymocytes is under the control of environmental signals, such as growth factors and cytokines.^{16 17 18 19 20 21} However, little is known about its role in the cardiovascular system. Although it has been demonstrated that endothelial cells,^{22 23 24} vascular smooth muscle cells,^{25 26 27} and cardiomyocytes²⁸ undergo apoptosis, the regulation of apoptosis in normal cellular physiology as well as in pathophysiological conditions remains largely unexplored.

ET-1 is a potent 21-residue vasoconstrictor peptide originally isolated from vascular endothelium.²⁹ The activation of two distinct subtypes of G protein-coupled heptahelical receptors (ET_A and ET_B)^{30 31} by three endothelin isopeptides leads to the regulation of vascular tonus^{29 32} and remodeling.^{33 34 35} ET-1, produced and released mainly from endothelial cells, acts on the same and/or adjacent endothelial cells via ET_B receptor in an autocrine/paracrine manner.^{36 37} Gene targeting experiments have revealed the importance of ET-1 signaling in embryonic development and organogenesis.^{38 39 40} Despite extensive studies that characterize ET-1 as the most potent vasoconstrictor as well as a potent growth factor for many cell types, its role as a modulator of cell death remains undescribed thus far. In this report, we demonstrate a novel role of ET-1 as an apoptosis survival factor for cultured rat endothelial cells.

	Methods

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Cell Culture
Rat endothelial cells were prepared from 15-week-old male Wistar rat aorta by collagenase and elastase digestion, as described.⁴¹ The endothelial origin of the cultures was confirmed by the presence of factor VIII by immunohistochemical detection. All cells were cultured in DMEM in a 5% CO₂ atmosphere at 37°C, supplemented with 10% FBS.

Reagents
Synthetic ET-1 was purchased from Peptide Institute, Inc. ET_A/B receptor antagonists (PD142893 and PD145065) were supplied by Parke-Davis Pharmaceutical Research, and TAK044 was supplied by Takeda Research Laboratory. ET_A receptor antagonist (BQ123), ET_B receptor antagonist (BQ788), and ET_B receptor agonist (BQ3020) were generously provided by Banyu Research.

DNA Fragmentation
To demonstrate nucleosome laddering, cellular fragmented DNA extraction and fractionation on 1.2% agarose gels were performed using the NP-40 lysis method, which eliminates intact chromatin.^{42 43} After serum starvation for 4 hours, both floating and adherent endothelial cells were collected and apoptotic DNA fragments were extracted.

Flow Cytometry Analysis
Floating endothelial cells and/or trypsinized adherent cells were washed with PBS and stained with 0.1% Triton X-100 and 0.1% sodium citrate (pH 7.0) containing 50 µg/mL propidium iodide by incubation in subdued light (30 minutes at 4°C). Stained cells were then analyzed with an FACS Calibur flow cytometer (Becton Dickinson).

In Situ Detection of Apoptosis
Apoptotic cells were detected in situ by the TdT-mediated dUTP-biotin nick-end labeling (TUNEL) method using an in situ cell death detection kit (Takara Biomedicals). In brief, endothelial cells grown on a LAB-TEK Chamber Slide (Nalge Nunc) were placed in serum-free DMEM, incubated with or without ET-1, and fixed for 15 minutes in 4% paraformaldehyde in PBS. After fixation, cells were blocked for 15 minutes with 0.3% H₂O₂ in methanol, washed, and permeabilized for 2 minutes with 0.1% sodium citrate in PBS; cells were then sequentially exposed to the enzymatic reaction mixture (TdT enzyme plus Labeling Safe Buffer) for 60 minutes at 37°C, anti-FITC HRP conjugate for 30 minutes at 37°C, and 0.05% diaminobenzidine in 1% nickel sulfate and 0.01% H₂O₂.

Measurement of Viable and Apoptotic Cells
Rat endothelial cells were plated in 12-well dishes in serum-containing medium at a density of 10⁵ cells per well and incubated for 24 hours. The cells were extensively washed with PBS, placed in serum-free DMEM, and incubated with or without ET-1. After 24 hours, all floating cells were collected after two washes with PBS. All adherent cells were also collected after trypsinization for a quantitative analysis of total apoptotic events in a given cell population. The number of floating and adherent cells was determined with a Coulter Counter model ZM (Coulter Electronics).

Radioimmunoassay and Northern Hybridization
ET-1–like immunoreactivity was determined by the double-antibody radioimmunoassay as described^{44 45} ; the antibody used was directed toward the C-terminal Trp²¹ residue of ET-1 without any cross-reactivity with bigET-1, ET-2, or ET-3. Rat aortic endothelial cells under serum-free conditions secreted ET-1–like immunoreactivity as a function of time (9.65±2.27 fmol/24 h per 10⁶ cells; n=4). Northern blot analysis of total RNA from the cells using cDNA from rat preproET-1 as a probe demonstrated a single hybridization band corresponding to the size (2.3 kb) of mRNA coding for preproET-1.

Determination of Growth Rates
Trypsinized cells were seeded at a density of 500 cells/cm² in 10-cm dishes and incubated in DMEM containing 10% FBS. Culture dishes with and without various doses of ET-receptor antagonists were marked to allow successive photographs to be taken of the same area, and 30 discrete cohorts of cells for each culture condition were followed at 24-hour intervals for 3 days. Cell numbers were counted on photographic enlargements.

	Results

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When rendered quiescent by serum deprivation for 24 hours, rat endothelial cell cultures always contain a significant fraction (40%) of floating cells which, even after being replated in fresh medium containing 10% FBS, did not reattach to the dish or regrow. Floating cells displayed characteristic features of apoptosis such as membrane blebbing, cellular shrinkage, condensed chromatin, and subnuclear bodies. DNA samples, after extraction from total cell culture (4 hours of serum deprivation) by the NP-40 lysis method and separation by electrophoresis, showed a nucleosomal ladder (Fig 1, left lane). Flow cytometric analysis revealed that the number of cells with subdiploid DNA content increased after serum deprivation (Fig 2): 1.4% before serum withdrawal (Fig 2A), 13.6% after 4 hours (Fig 2B), and more than 40% after 24 hours (data not shown). When floating and adherent cells were separately analyzed with flow cytometry 4 hours after serum deprivation, adherent culture contained very few subdiploid cells (<2%), whereas most floating cells were subdiploid (Fig 2C). DNA strand breaks caused by endonuclease were detected in situ by the TUNEL method (Fig 3). In contrast with negative staining in control cells cultured with 10% FBS (Fig 3A), positive staining was visible in many nuclei of cells deprived of serum for 4 hours (Fig 3B). These results indicate that serum deprivation induces apoptosis of rat endothelial cells, resulting in apoptotic floating cells.

View larger version (95K): [in this window] [in a new window]   Figure 1. Demonstration of a nucleosomal ladder from serum-starved endothelial cells and its suppression by ET-1. Confluent cultures of rat endothelial cells in 10-cm dishes were deprived of serum and incubated for 4 hours in the absence and presence of the indicated concentrations of ET-1. After collection of both floating and adherent cells, fragmented DNA was extracted using the NP-40 lysis method and separated by electrophoresis in 1.2% agarose gel. Positions of molecular weight markers (base pairs) are shown on the left.

View larger version (28K): [in this window] [in a new window]   Figure 2. Flow cytometric DNA analysis of endothelial cells. Rat endothelial cells were serum-deprived for 4 hours, stained with propidium iodide, and subjected to flow cytometric analysis: total endothelial cells cultured in 10% FBS (A), total cultured cells after 4-hour serum starvation (B), and floating cells (C) (4-hour serum starvation) were collected and analyzed. Apoptotic cells with subdiploid DNA staining are shown in the region marked with the bar, and the percentage of such cells is indicated. Arrowheads indicate the positions of peak G0/G1 cells (left) and G2 cells (right).

View larger version (98K): [in this window] [in a new window]   Figure 3. In situ detection of serum deprivation-induced apoptosis in endothelial cells. Rat endothelial cells, before (A) and after serum deprivation for 4 hours without (B) or with (C) 10-7 mol/L ET-1, were stained in situ by the TdT-mediated dUTP-biotin nick-end labeling (TUNEL) method.

ET-1 dose-dependently (10^-9 to 10^-6 mol/L) inhibited the fragmented DNA induced by 4 hours of serum starvation as demonstrated by nucleosomal ladders (Fig 1). ET-1 also decreased the number of serum deprivation-induced floating cells in a dose-dependent manner (10^-12 to 10^-6 mol/L) (Fig 4); a significant apoptotic inhibition was induced with as low as 10^-12 mol/L ET-1, and a maximal antiapoptotic effect (60.6±4.0% survival of control apoptotic cells) was induced with 10^-6 mol/L ET-1. These data are complemented by TUNEL staining, which revealed that ET-1 also markedly reduced the number of adherent apoptotic cells (Fig 3C). These data demonstrate that ET-1 antagonizes apoptosis of endothelial cells induced by serum deprivation.

View larger version (13K): [in this window] [in a new window]   Figure 4. ET-1 acts as an apoptosis survival factor for endothelial cells. Exponentially growing rat endothelial cells were changed into serum-free medium containing the indicated concentrations of ET-1. After 24 hours, all floating cells were collected and counted with a Coulter counter. Each point represents mean±SEM (n=6); values were calculated by the percentage of floating cells in the absence of ET-1. *P<.05, **P<.01 for treated versus untreated cells, respectively.

To determine via which ET receptor subtype apoptosis inhibition is mediated, we examined the effects of several ET receptor antagonists on the protective effect of ET-1 in endothelial cells. The ET_B receptor antagonist BQ788 (10^-6 mol/L) and the nonselective ET_A/B receptor antagonists PD142893 and PD145065 (10^-6 mol/L) completely blocked the protective effect induced by ET-1 (10^-7 mol/L), whereas the ET_A receptor antagonist BQ123 (10^-6 mol/L) did not show any significant effect on cell death (Fig 5). The ET_B-selective receptor agonist BQ3020 dose-dependently suppressed apoptosis (10^-12 to 10^-9 mol/L), although the magnitude of maximal inhibition (20%) was less than that of ET-1 (data not shown). These results suggest that the cell protective effect provoked by ET-1 is mediated by the ET_B receptor.

View larger version (45K): [in this window] [in a new window]   Figure 5. ET-1-induced apoptosis inhibition is mediated by the ETB receptor. Cells were treated with or without ET-1 (10-7 mol/L) in the absence and presence of BQ123 (10-6 mol/L), BQ788(10-6 mol/L), PD142893 (10-6 mol/L), or PD145065 (10-6 mol/L) during the 24-hour serum deprivation. Apoptosis assay using exponentially growing rat endothelial cells was performed as described in Fig 4. Each column represents mean±SEM (n=6). **P<.01 for treated versus untreated cells.

To determine whether endogenous ET-1 affects apoptotic death of endothelial cells as a survival factor in an autocrine/paracrine manner, the effect of anti–ET-1 antibody was examined. Addition of preimmune rabbit serum to the serum-deprived cells led to a dose-dependent suppression of apoptosis. Polyclonal antibody at dilutions of both 1:10 000 and 1:1000, but not 1:100 000, completely abolished the protective effect of normal rabbit serum (Fig 6). These data are complemented by experiments using ET receptor antagonists. The ET_B receptor antagonist (BQ788; 10^-10 to 10^-8 mol/L) and ET_A/B receptor antagonists (TAK044 and PD145065; 10^-10 to 10^-8 mol/L) significantly increased apoptosis, whereas the ET_A receptor antagonist (BQ123; 10^-10 to 10^-8 mol/L) was without effect (data not shown). In control experiments, BQ123, BQ788, and PD145065 failed to affect apoptosis of fibroblast cell lines that do not express ET-1 (data not shown).

View larger version (21K): [in this window] [in a new window]   Figure 6. Effects of anti-ET-1 antiserum and preimmune serum on apoptosis. During the 24-hour serum deprivation period, cells were exposed to either rabbit preimmune serum (open bars) or anti-ET-1 antiserum (solid bars) at various dilutions. Apoptosis assay with exponentially growing rat endothelial cell cultures was performed as described in Fig 4. Each column represents mean±SEM (n=6); values were calculated by the percentage of floating cells in the absence of ET-1.

To investigate whether endogenous ET-1 is involved in the proliferation of rat endothelial cells, the effects of ET receptor antagonists on cell number were examined in subconfluent cultures growing in 10% FBS. BQ788 dose-dependently (10^-8 to 10^-7 mol/L) inhibited cell growth (Fig 7), whereas BQ123 was without effect (10^-9 to 10^-6 mol/L; data not shown). In control experiments, neither BQ123 nor BQ788 affected the growth of fibroblasts that do not express ET-1 (data not shown).

View larger version (18K): [in this window] [in a new window]   Figure 7. Effect of ETB receptor antagonist (BQ788) on the proliferation of rat endothelial cells. Subconfluent cells were incubated in DMEM containing 10% FBS in the absence (squares) or presence of 10-8 mol/L (diamonds) and 10-7 mol/L (circles) BQ788. For each growth curve, 30 discrete cohorts were selected 10 hours after trypsinization, and their cell numbers were determined at successive 24-hour intervals. Cell numbers were determined photographically (see Methods). Each point represents mean±SEM (n=30); values were calculated by the percentage of cells 10 hours after trypsinization (10±4 cells, mean±SEM).

	Discussion

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Increased cell number in culture could be a combined result of enhanced proliferative status of cells and suppressed apoptosis. Major growth factors, such as PDGF, IGF, and EGF, not only stimulate cell proliferation rate, but also suppress apoptosis, thereby maintaining the surviving cell number.¹⁸ Many types of cells undergo apoptosis when growth factors are deprived, whereas addition of one or more of such factors to serum-free culture media inhibits apoptosis. ET-1 is a novel vasoconstrictor peptide derived from vascular endothelial cells. ET-1 has been shown to have a potent mitogenic property in a variety of cells, including endothelial cells.^{33 34 35 37} However, its role as a regulator of apoptosis has not been described to date. A novel and intriguing finding of the present work is that ET-1 acts as a survival factor from apoptosis via the ET_B receptor in rat endothelial cells in an autocrine/paracrine manner.

The present study confirmed that a fraction of rat endothelial cells rapidly started to float and to undergo apoptosis after serum deprivation, as assessed by nucleosomal laddering on agarose gel electrophoresis and positive staining with the TUNEL method. Floating cells showed morphological characteristics of apoptosis, whereas adherent cells were mostly alive. These observations were also corroborated by the results of flow cytometric analysis. Because of chromatin condensation and DNA cleavage, apoptotic cells show less propidium iodide fluorescence than viable cells. Such subdiploid cell population corresponding to the floating dead cells started to increase as a function of time after serum deprivation. Therefore, subsequent quantitations of apoptotic events were performed by counting floating apoptotic cells with the Coulter Counter.

A marked inhibition by ET-1 of serum deprivation-induced apoptosis of rat endothelial cells is demonstrated by three lines of evidence: (1) the inhibition of nucleosomal ladder formation from total cell culture; (2) the morphology of adherent cells negative for apoptosis by the TUNEL method; and (3) the decrease in floating apoptotic cell number. The minimum effective concentrations that induced apoptotic protection by ET-1 (10^-12 mol/L) are comparable with those (10^-12 mol/L) of circulating ET-1 and lower than those (10^-11 mol/L) of ET-1 secreted into serum-free cultured media during 24 hours from endothelial cells. These levels are also far lower than those reported with other growth factors and cytokines to block apoptosis.¹⁸ Moreover, abrogation of serum-induced apoptotic protection by anti–ET-1 antiserum suggests that ET-1 produced by the endothelial cells acts on its own cells to protect them from apoptotic death. Because endothelial cells synthesize and secrete ET-1, endogenous ET-1 could function as "self-protection" by an autocrine/paracrine mechanism. These results demonstrate the physiological significance of ET-1 as a potent survival factor from apoptosis.

ET receptors consist of at least two subtypes: the ET-1-selective ET_A receptor and the non–isopeptide-selective ET_B receptor, both of which are functionally coupled to phospholipase C to induce phosphoinositide breakdown. Vascular smooth muscles express mainly ET_A receptors mediating contraction, whereas vascular endothelium has ET_B receptors that are involved in vasodilation.^{30 31} Endothelial cells express ET_B receptors, which mediate synthesis of nitric oxide³⁶ and cell proliferation and migration.³⁷ The present study shows that the mitogenic and apoptotic survival activities of ET-1 in endothelial cells are mediated by the ET_B receptor, because ET_B receptor antagonist (BQ788) but not ET_A receptor antagonist (BQ123) reduced cell proliferation and blocked apoptosis protection by ET-1. Furthermore, ET_B receptor agonist (BQ3020) suppressed apoptosis, whereas both ET_B receptor antagonist (BQ788) and ET_A/B receptor antagonists (TAK044 and PD145065) but not ET_A receptor antagonist (BQ123) increased apoptosis. These observations are in contrast to those of rat fibroblasts in which ET-1 antagonized c-Myc–dependent apoptosis via the ET_A receptor (M. Shichiri, J.M. Sedivy, F. Marumo, Y. Hirata, unpublished observations, 1997).

It has been shown that apoptosis of vascular endothelial cells is prevented by fibroblast growth factor and dexamethasone^{23 24} but aggravated by tumor necrosis factor-²² and transforming growth factor-ß.⁴⁶ Possible involvement of protein kinase C has been suggested in mediation of basic fibroblast growth factor protection of endothelial cells against apoptosis induced by serum starvation and radiation.^{24 47} Because ET-1 induces phosphoinositide breakdown to activate protein kinase C resulting from generation of diacylglycerol in endothelial cells,³⁶ it is possible that protein kinase C may be involved in the antiapoptotic effect by ET-1. However, the exact molecular mechanism responsible for the antiapoptotic effect by ET-1 remains to be elucidated.

In conclusion, we demonstrate a novel role of ET-1 as an autocrine/paracrine survival factor from apoptosis for rat endothelial cells via the ET_B receptor. The pathophysiological significance of ET-1 as a survival factor from apoptosis of endothelial cells of diseased blood vessels, such as in hypertension and atherosclerosis, remains to be determined.

	Selected Abbreviations and Acronyms

 

[Ca2+]i = intracellular free calcium concentration DMEM = Dulbecco's modified Eagle's medium ET-1 = endothelin-1 FBS = fetal bovine serum PBS = phosphate-buffered saline

	Acknowledgments

 
This work was supported in part by grants-in-aid from the Ministry of Education, Science, and Culture of Japan and from the Ministry of Health and Welfare of Japan; by the Chiiki-Igaku Research Fund; and by the Tanabe Medical Frontier Conference. We are grateful to Banyu Pharmaceutical Co, Ltd for providing BQ123, BQ788, and BQ3020 and to Takeda Pharmaceutical Co, Ltd for providing TAK044.

Received February 12, 1997; first decision March 25, 1997; accepted March 25, 1997.

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