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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
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
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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 ETB receptor antagonist (BQ788; 10-6 mol/L) and ETA/B receptor antagonists (PD142893 and PD145065; 10-6 mol/L), but not the ETA 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 ETB receptor antagonist (BQ788; 10-8 to 10-6 mol/L), but not the ETA 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 ETB receptor.
Key Words: endothelin-1 • ETB receptor • cell survival • apoptosis • endothelial cells
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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 (ETA and ETB)30 31 by three endothelin isopeptides leads to the regulation of vascular tonus29 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 ETB 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.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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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.
ETA/B receptor antagonists (PD142893 and
PD145065) were supplied by Parke-Davis Pharmaceutical Research, and
TAK044 was supplied by Takeda Research Laboratory. ETA
receptor antagonist (BQ123), ETB receptor
antagonist (BQ788), and ETB 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 described44 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.
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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.
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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.
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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 ETB receptor
antagonist BQ788 (10-6
mol/L) and the nonselective ETA/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 ETA receptor
antagonist BQ123 (10-6
mol/L) did not show any significant effect on cell death (Fig 5). The ETB-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
ETB receptor.
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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 ETB receptor
antagonist (BQ788; 10-10 to
10-8 mol/L) and ETA/B
receptor antagonists (TAK044 and PD145065;
10-10 to 10-8
mol/L) significantly increased apoptosis, whereas the
ETA 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).
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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).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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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 ETB 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 ETA receptor and the non–isopeptide-selective ETB receptor, both of which are functionally coupled to phospholipase C to induce phosphoinositide breakdown. Vascular smooth muscles express mainly ETA receptors mediating contraction, whereas vascular endothelium has ETB receptors that are involved in vasodilation.30 31 Endothelial cells express ETB 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 ETB receptor, because ETB receptor antagonist (BQ788) but not ETA receptor antagonist (BQ123) reduced cell proliferation and blocked apoptosis protection by ET-1. Furthermore, ETB receptor agonist (BQ3020) suppressed apoptosis, whereas both ETB receptor antagonist (BQ788) and ETA/B receptor antagonists (TAK044 and PD145065) but not ETA 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 ETA 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 dexamethasone23 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 ETB 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.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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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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  D. S. McLeod, R. Grebe, I. Bhutto, C. Merges, T. Baba, and G. A. Lutty Relationship between RPE and Choriocapillaris in Age-Related Macular Degeneration Invest. Ophthalmol. Vis. Sci., October 1, 2009; 50(10): 4982 - 4991. [Abstract] [Full Text] [PDF]
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 M. Shichiri, N. Fukai, Y. Kono, and Y. Tanaka Rifampicin as an Oral Angiogenesis Inhibitor Targeting Hepatic Cancers Cancer Res., June 1, 2009; 69(11): 4760 - 4768. [Abstract] [Full Text] [PDF]
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 C. F. Opitz, R. Ewert, W. Kirch, and D. Pittrow Inhibition of endothelin receptors in the treatment of pulmonary arterial hypertension: does selectivity matter? Eur. Heart J., August 2, 2008; 29(16): 1936 - 1948. [Abstract] [Full Text] [PDF]
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 M. G. Neerhof, T. Jilling, S. Synowiec, S. Khan, and L. G. Thaete Altered Endothelin Receptor Binding in Response to Nitric Oxide Synthase Inhibition in the Pregnant Rat Reproductive Sciences, April 1, 2008; 15(4): 366 - 373. [Abstract] [PDF]
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 L. G Thaete, T. Jilling, S. Synowiec, S. Khan, and M. G Neerhof Expression of Endothelin 1 and Its Receptors in the Hypoxic Pregnant Rat Biol Reprod, September 1, 2007; 77(3): 526 - 532. [Abstract] [Full Text] [PDF]
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 R. Jain, P. W. Shaul, Z. Borok, and B. C. Willis Endothelin-1 Induces Alveolar Epithelial-Mesenchymal Transition through Endothelin Type A Receptor-Mediated Production of TGF-beta1 Am. J. Respir. Cell Mol. Biol., July 1, 2007; 37(1): 38 - 47. [Abstract] [Full Text] [PDF]
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 D. Ribatti, M. T. Conconi, and G. G. Nussdorfer Nonclassic Endogenous Novel Regulators of Angiogenesis Pharmacol. Rev., June 1, 2007; 59(2): 185 - 205. [Abstract] [Full Text] [PDF]
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 D. Montani, R. Souza, C. Binkert, W. Fischli, G. Simonneau, M. Clozel, and M. Humbert Endothelin-1/Endothelin-3 Ratio: A Potential Prognostic Factor of Pulmonary Arterial Hypertension Chest, January 1, 2007; 131(1): 101 - 108. [Abstract] [Full Text] [PDF]
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 M.-N. Raymond, C. Bole-Feysot, Y. Banno, Z. Tanfin, and P. Robin Endothelin-1 Inhibits Apoptosis through a Sphingosine Kinase 1-Dependent Mechanism in Uterine Leiomyoma ELT3 Cells Endocrinology, December 1, 2006; 147(12): 5873 - 5882. [Abstract] [Full Text] [PDF]
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 G. Muller, R. A. Catar, B. Niemann, M. Barton, L. Knels, M. Wendel, and H. Morawietz Upregulation of endothelin receptor B in human endothelial cells by low-density lipoproteins. Experimental Biology and Medicine, June 1, 2006; 231(6): 766 - 771. [Abstract] [Full Text] [PDF]
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 G. Guruli, B. R. Pflug, S. Pecher, V. Makarenkova, M. R. Shurin, and J. B. Nelson Function and survival of dendritic cells depend on endothelin-1 and endothelin receptor autocrine loops Blood, October 1, 2004; 104(7): 2107 - 2115. [Abstract] [Full Text] [PDF]
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 I. A. Mawji, G. B. Robb, S. C. Tai, and P. A. Marsden Role of the 3'-Untranslated Region of Human Endothelin-1 in Vascular Endothelial Cells: CONTRIBUTION TO TRANSCRIPT LABILITY AND THE CELLULAR HEAT SHOCK RESPONSE J. Biol. Chem., March 5, 2004; 279(10): 8655 - 8667. [Abstract] [Full Text] [PDF]
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 N. Galie, A. Manes, and A. Branzi The endothelin system in pulmonary arterial hypertension Cardiovasc Res, February 1, 2004; 61(2): 227 - 237. [Abstract] [Full Text] [PDF]
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D. C. Sullivan, L. Huminiecki, J. W. Moore, J. J. Boyle, R. Poulsom, D. Creamer, J. Barker, and R. Bicknell EndoPDI, a Novel Protein-disulfide Isomerase-like Protein That Is Preferentially Expressed in Endothelial Cells Acts as a Stress Survival Factor J. Biol. Chem., November 21, 2003; 278(47): 47079 - 47088. [Abstract] [Full Text] [PDF]
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 K.-H. Ding, Q. Zhong, and C. M. Isales Glucose-dependent insulinotropic peptide stimulates thymidine incorporation in endothelial cells: role of endothelin-1 Am J Physiol Endocrinol Metab, August 1, 2003; 285(2): E390 - E396. [Abstract] [Full Text] [PDF]
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 Y. Ogata, M. Takahashi, S. Ueno, K. Takeuchi, T. Okada, H. Mano, S. Ookawara, K. Ozawa, B. C. Berk, U. Ikeda, et al. Antiapoptotic Effect of Endothelin-1 in Rat Cardiomyocytes In Vitro Hypertension, May 1, 2003; 41(5): 1156 - 1163. [Abstract] [Full Text] [PDF]
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A. Ishaque, M. J. Dunn, and A. Sorokin Cyclooxygenase-2 Inhibits Tumor Necrosis Factor alpha -mediated Apoptosis in Renal Glomerular Mesangial Cells J. Biol. Chem., March 14, 2003; 278(12): 10629 - 10640. [Abstract] [Full Text] [PDF]
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N. Fukai, M. Shichiri, N. Ozawa, M. Matsushita, and Y. Hirata Coexpression of Calcitonin Receptor-Like Receptor and Receptor Activity-Modifying Protein 2 or 3 Mediates the Antimigratory Effect of Adrenomedullin Endocrinology, February 1, 2003; 144(2): 447 - 453. [Abstract] [Full Text] [PDF]
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 M. R. Dashwood and J. C. S. Tsui Endothelins and the 'French Paradox': Are Detrimental Effects of Red Wine Also Associated with an Action on Endothelin Synthesis? Angiology, November 1, 2002; 53(6): 749 - 751. [PDF]
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 D. Del Bufalo, V. Di Castro, A. Biroccio, M. Varmi, D. Salani, L. Rosano, D. Trisciuoglio, F. Spinella, and A. Bagnato Endothelin-1 Protects Ovarian Carcinoma Cells against Paclitaxel-Induced Apoptosis: Requirement for Akt Activation Mol. Pharmacol., March 1, 2002; 61(3): 524 - 532. [Abstract] [Full Text] [PDF]
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 G. S. Filippatos, N. Gangopadhyay, O. Lalude, N. Parameswaran, S. I. Said, W. Spielman, and B. D. Uhal Regulation of apoptosis by vasoactive peptides Am J Physiol Lung Cell Mol Physiol, October 1, 2001; 281(4): L749 - L761. [Abstract] [Full Text] [PDF]
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 M. SHICHIRI and Y. HIRATA Antiangiogenesis signals by endostatin FASEB J, April 1, 2001; 15(6): 1044 - 1053. [Abstract] [Full Text] [PDF]
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M. C. Y. HO, A. C. Y. LO, H. KURIHARA, A. C. H. YU, S. S. M. CHUNG, and S. K. CHUNG Endothelin-1 protects astrocytes from hypoxic/ischemic injury FASEB J, March 1, 2001; 15(3): 618 - 626. [Abstract] [Full Text] [PDF]
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 G. Egidy, L. Juillerat-Jeanneret, J.-F. Jeannin, P. Korth, F. T. Bosman, and F. Pinet Modulation of Human Colon Tumor-Stromal Interactions by the Endothelin System Am. J. Pathol., December 1, 2000; 157(6): 1863 - 1874. [Abstract] [Full Text] [PDF]
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 T. F. Luscher and M. Barton Endothelins and Endothelin Receptor Antagonists : Therapeutic Considerations for a Novel Class of Cardiovascular Drugs Circulation, November 7, 2000; 102(19): 2434 - 2440. [Abstract] [Full Text] [PDF]
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D. Salani, G. Taraboletti, L. Rosano, V. Di Castro, P. Borsotti, R. Giavazzi, and A. Bagnato Endothelin-1 Induces an Angiogenic Phenotype in Cultured Endothelial Cells and Stimulates Neovascularization In Vivo Am. J. Pathol., November 1, 2000; 157(5): 1703 - 1711. [Abstract] [Full Text] [PDF]
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 G. Egidy, L. Juillerat-Jeanneret, P. Korth, F. T. Bosman, and F. Pinet The endothelin system in normal human colon Am J Physiol Gastrointest Liver Physiol, July 1, 2000; 279(1): G211 - G222. [Abstract] [Full Text] [PDF]
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 H. Morawietz, R. Talanow, M. Szibor, U. Rueckschloss, A. Schubert, B. Bartling, D. Darmer, and J. Holtz Regulation of the endothelin system by shear stress in human endothelial cells J. Physiol., June 15, 2000; 525(3): 761 - 770. [Abstract] [Full Text] [PDF]
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H. J. Kwak, S. J. Lee, Y.-H. Lee, C. H. Ryu, K. N. Koh, H. Y. Choi, and G. Y. Koh Angiopoietin-1 Inhibits Irradiation- and Mannitol-Induced Apoptosis in Endothelial Cells Circulation, May 16, 2000; 101(19): 2317 - 2324. [Abstract] [Full Text] [PDF]
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 J. R. Wu-Wong, W. J. Chiou, and J. Wang Extracellular Signal-Regulated Kinases Are Involved in the Antiapoptotic Effect of Endothelin-1 J. Pharmacol. Exp. Ther., May 1, 2000; 293(2): 514 - 521. [Abstract] [Full Text]
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 M. Shichiri, M. Yokokura, F. Marumo, and Y. Hirata Endothelin-1 Inhibits Apoptosis of Vascular Smooth Muscle Cells Induced by Nitric Oxide and Serum Deprivation via MAP Kinase Pathway Arterioscler Thromb Vasc Biol, April 1, 2000; 20(4): 989 - 997. [Abstract] [Full Text] [PDF]
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T. Stefanec Endothelial Apoptosis: Could It Have a Role in the Pathogenesis and Treatment of Disease? Chest, March 1, 2000; 117(3): 841 - 854. [Abstract] [Full Text] [PDF]
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 I. Kim, H. G. Kim, J.-N. So, J. H. Kim, H. J. Kwak, and G. Y. Koh Angiopoietin-1 Regulates Endothelial Cell Survival Through the Phosphatidylinositol 3'-Kinase/Akt Signal Transduction Pathway Circ. Res., January 7, 2000; 86(1): 24 - 29. [Abstract] [Full Text] [PDF]
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Q. N. Diep, H. D. Intengan, and E. L. Schiffrin Endothelin-1 Attenuates {omega}3 Fatty Acid-Induced Apoptosis by Inhibition of Caspase 3 Hypertension, January 1, 2000; 35(1): 287 - 291. [Abstract] [Full Text] [PDF]
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P. J. M. Best, L. O. Lerman, J. C. Romero, D. Richardson, D. R. Holmes Jr, and A. Lerman Coronary Endothelial Function Is Preserved With Chronic Endothelin Receptor Antagonism in Experimental Hypercholesterolemia In Vitro Arterioscler Thromb Vasc Biol, November 1, 1999; 19(11): 2769 - 2775. [Abstract] [Full Text] [PDF]
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 M. Shichiri, H. Kato, M. Doi, F. Marumo, and Y. Hirata Induction of Max by Adrenomedullin and Calcitonin Gene-Related Peptide Antagonizes Endothelial Apoptosis Mol. Endocrinol., August 1, 1999; 13(8): 1353 - 1363. [Abstract] [Full Text]
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P. J. M. Best, D. Hasdai, G. Sangiorgi, R. S. Schwartz, D. R. Holmes Jr, R. D. Simari, and A. Lerman Apoptosis : Basic Concepts and Implications in Coronary Artery Disease Arterioscler Thromb Vasc Biol, January 1, 1999; 19(1): 14 - 22. [Abstract] [Full Text] [PDF]
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N. Suenobu, M. Shichiri, M. Iwashina, F. Marumo, and Y. Hirata Natriuretic Peptides and Nitric Oxide Induce Endothelial Apoptosis via a cGMP–Dependent Mechanism Arterioscler Thromb Vasc Biol, January 1, 1999; 19(1): 140 - 146. [Abstract] [Full Text] [PDF]
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M. Iwashina, M. Shichiri, F. Marumo, and Y. Hirata Transfection of Inducible Nitric Oxide Synthase Gene Causes Apoptosis in Vascular Smooth Muscle Cells Circulation, September 22, 1998; 98(12): 1212 - 1218. [Abstract] [Full Text] [PDF]
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P. Moreau Endothelin in hypertension: A role for receptor antagonists? Cardiovasc Res, September 1, 1998; 39(3): 534 - 542. [Abstract] [Full Text] [PDF]
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M. Shichiri, J. M. Sedivy, F. Marumo, and Y. Hirata Endothelin-1 Is a Potent Survival Factor for c-Myc-Dependent Apoptosis Mol. Endocrinol., February 1, 1998; 12(2): 172 - 180. [Abstract] [Full Text] [PDF]
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