(Received for publication, March 28, 1996, and in revised form, July 30, 1996)
From the Department of Medicine, State University of New York, Stony Brook, New York 11794-8152 and Rensellaer Polytechnic Institute, Troy, New York 12180-3590
Endothelin (ET) synthesis is enhanced at sites of ischemia or in injured vessels. The purpose of this study was to explore the possibility of autocrine stimulation of endothelial cell migration by members of the endothelin family. Experiments with microvascular endothelial cell transmigration in a Boyden chemotactic apparatus showed that endothelins 1 and 3, as well as a selective agonist of ETB receptor IRL-1620, equipotently stimulated migration. Endothelial cell migration was unaffected by the blockade of ETA receptor, but it was inhibited by ETB receptor antagonism. Based on our previous demonstration of signaling from the occupied ETB receptor to constitutive nitric oxide (NO) synthase (Tsukahara, H., Ende, H., Magazine, H. I., Bahou, W. F., and Goligorsky, M. S. (1994) J. Biol. Chem. 269, 21778-21785), we next examined the contribution of ET-stimulated NO production to endothelial cell migration. In three independent cellular systems, 1) migration and wound healing by microvascular endothelial cells, 2) wound healing by Chinese hamster ovary cells stably expressing ETB receptor with or without endothelial NO synthase, and 3) application of antisense oligodeoxynucleotides targeting endothelial NO synthase in human umbilical vein endothelial cells, an absolute requirement for the functional NO synthase in cell migration has been demonstrated. These findings establish the permissive role of NO synthesis in endothelin-stimulated migration of endothelial cells.
Formation of new blood vessels, driven by the morphogenetic program and/or by the functional demand for increased blood supply, is initiated by the budding of endothelial cells off the microcirculatory bed. Tissue hypoxia represents a physiologic stimulus for angiogenesis, and several autocrine angiogenic mediators produced by endothelial cells have been recognized (1-3). Endothelin, one of such mediators, is constitutively produced by endothelial cells and its production is enhanced by hypoxia (4-6). Two investigative teams have recently demonstrated that ET-11 and ET-3, acting via the ETB receptor, stimulate endothelial cell migration and proliferation (7, 8). Since we and others have previously demonstrated that occupancy of ETB receptors in endothelial cells is accompanied by the activation of constitutive endothelial NO synthase (9, 10), we hypothesized that the observed motogenic effects of members of the ET family may in fact be mediated by the release of NO. Indeed, there is emerging evidence that NO release serves as a prerequisite for epithelial and endothelial cell motility (11-13). Leibovich et al. (13) have shown that production of angiogenic activity by activated monocytes (assayed by chemotaxis of endothelial cells and corneal angiogenesis) is absolutely dependent on L-arginine and NO synthase. These observations are in concert with findings reported by Ziche et al. (12) who detected the potentiation by sodium nitroprusside of the angiogenic effect of substance P in the rabbit cornea. Our own observations expand this function to the classical angiogenic signal, vascular endothelial growth factor. We demonstrated that endogenous NO production by the endothelial cells is a prerequisite for the motogenic and angiogenic effects of this factor.2 In the present study, we used three different approaches (migration and wound healing by endothelial cells, wound healing by Chinese hamster ovary cells expressing ETB receptor with or without eNOS, and application of antisense oligodeoxynucleotides targeting eNOS) to provide evidence that the effect of ET-1 on cell migration is mediated via ETB receptor and requires functional enzymatic machinery for NO generation.
Renal microvascular endothelial cells (RMVEC) were previously established and characterized (15). This cell line shows expression of receptors for acetylated low density lipoprotein, staining with an antibody to von Willebrand antigen, and capillary tube formation. Human umbilical vein endothelial cells (HUVEC) were isolated according to the previously established technique (9, 16). RMVEC were grown in gelatin-coated dishes in M199 culture medium (Mediatech, Washington, D. C.) supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT), 100 units/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Inc.) HUVEC were cultured in M199 supplemented with 100 µg/ml heparin, 30 µg/ml endothelial cell growth factor, and 10% fetal bovine serum. Endothelin-1 (ET-1) and endothelin-3 (ET-3) were purchased from Calbiochem. IRL-1620, BQ123, and BQ788 were generously provided by Dr. Magazine (Queens College, Flushing, NY). NG-Nitro L-arginine methyl ester (L-NAME) was obtained from Bachem California (Torrance, CA), and S-nitroso-N-acetyl-DL-penicillamine was purchased from Molecular Probes, Inc. (Eugene, OR).
Functional Expression of ETBR and eNOS in CHO CellsThe generation and characterization of individual cell lines used in this study have been detailed previously (9). Briefly, CHO-ETB cell cultures stably integrated copies of the ETBR cDNA cloned, and the amplified insert encompassing the ETB open reading frame was digested with PstI, gel-purified, and ligated into the eukaryotic expression plasmid pMT2. This plasmid is driven by the adenovirus major late promoter and contains the sequences encoding murine dihydrofolate reductase immediately downstream of the cDNA insert, thereby allowing for the establishment of stably integrated cell lines with greatly amplified copy numbers when propagated in the presence of methotrexate (17). Dihydrofolate reductase-deficient CHO cells (18) were propagated in nucleoside-free Iscove's modified Dulbecco's medium (Sigma), supplemented with 10% dialyzed fetal calf serum, and subsequently plated at a density of 5 × 105/100 mm2 dish. Cells were transfected with 30 µg of pMT2-ETB or pMT2-wild type using the calcium phosphate transfection method of Chen and Okayama (19). After 5 days, selection and amplification were initiated by supplementation of the media with 0.02 µM methotrexate and cells were cloned by limiting dilution. Resistant colonies were subsequently expanded and simultaneously screened for stable integration by genomic hybridization using the 32P-radiolabeled ETB cDNA as probe and for functional expression using fluorescence mapping with biotinylated highly specific ligand, IRL-1620, as previously detailed (9).
For double transfectants, CHO-ETB cells were retransfected with the cDNA for endothelial cell-derived NOS (20) (kindly supplied by Dr. K. D. Bloch) using the eukaryotic expression plasmid pMEP4 (Invitrogen, San Diego, CA), which contains an inherent hygromycin B resistance gene and an inducible metallothionein promoter upstream from the NOS cloning site. Stable transfectants (CHO-ETBNOS) were selected using culture medium supplemented with 300 µg/ml hygromycin B (1000 units/mg, Sigma). Resistant colonies were ring cloned, propagated, and functionally evaluated for NO expression using an NO-selective electrode, as detailed previously (9). NOS expression was maximized by supplementation of the medium with 50 µM ZnCl2 for 5 h prior to functional measurements.
Cell Migration AssayMigration assay was performed according to the previously described technique (22), with minor modifications, in a Boyden chemotactic apparatus (Neuroprobe, Cabin John, MD). RMVEC were lifted with 0.05% trypsin, 0.53 mM EDTA (Life Technologies, Inc.) and washed, and 106 cells/ml suspended in 25 µl of M199 with 0.1% bovine serum albumin were added to the lower chamber of a Boyden apparatus. Polycarbonate filters with 8-µm pores (Poretics Corporation, Livermore, CA) were coated with 10 µg/ml ProNectin F (Protein Polymer Technologies, San Diego, CA), washed twice with phosphate-buffered saline, and positioned above the wells of the lower chemotactic chamber which contained cells. The top half of the chamber was reattached, and the chamber was incubated in an inverted position at 37 °C in 95% air + 5% CO2 for 2 h to allow a uniform cell attachment to the filter. The test agents, as specified under "Results," or a vehicle suspended in 50 µl of M199 with 0.1% bovine serum albumin were added to upper chambers. The chambers were wrapped with Parafilm and incubated for an additional 6 h in an upright position. After incubation, the filter was removed from the apparatus, and cells were fixed with methanol and stained with Dff-Quick (Baxter, Miami, FL). The number of migrated cells on the upper surface of the filter was counted in six randomly chosen fields under × 400 magnification and averaged. All experiments were performed in quadruplicate, and each experiment was repeated at least three times.
Monitoring of NO ReleaseRMVEC or HUVEC grown in gelatin-coated 35-mm dishes were preincubated in Krebs-Ringer buffer of the following composition (in mM): NaCl 136, KCl 5.4, CaCl2 1.8, MgSO4 0.8, NaH2PO4 1.0, HEPES 10, and 1 g/liter glucose, adjusted to pH 7.4, which was supplemented with 7.5 units/ml superoxide dismutase (Sigma). NO release was monitored with an NO-selective microprobe (Inter Medical Co., Nagoya, Japan), as previously detailed (11). Tip diameter of the probe (25 µm) permitted the use of a micromanipulator (Zeiss-Eppendorff) attached to the stage of an inverted microscope (Nikon Diaphot) and enclosed in a Faraday's chamber to position the sensor approximately 5 µm above the cell surface. Calibration of the electrochemical sensor was performed using different concentrations of a nitrosothiol NO donor S-nitroso-N-acetyl-DL-penicillamine, as previously detailed (23).
Wound Healing AssaysElectrode fabrication and the design of electric cell-substrate impedance sensor, a device that detects with high precision the electrical resistance of cells cultured inside small wells on the surface of a miniature gold electrode, have been reported previously (24, 25). Electrodes were precoated with 10 µg/ml fibronectin (Collaborative Biomedical Products, Bedford, MA). Endothelial cells at the density of 2 × 105 were resuspended in Basal Medium (Life Technologies, Inc.) supplemented with 0.1% bovine serum albumin, 250 µM L-arginine (Sigma), and 100 units/ml penicillin 100 µg/ml streptomycin, and seeded on electrodes. To generate "wounds" on the surface of microelectrodes, confluent endothelial monolayers were electropermeabilized with direct current from a 6-V battery generating a local electrical current of 0.12 mA. This procedure resulted in a loss of cells from the surface of the microelectrode, while all surrounding cells remained intact. The rate of repopulation of the microelectrode with migrating cells was monitored for 20 h as changes in resistance and capacitance. Alternatively, uniform wounds were generated using a laser beam. CHO-ETB and CHO-ETBNOS cell monolayers grown on glass coverslips for 2 days were exposed to a wounding procedure as previously detailed (11). Wounds were produced using a single pulse generated by a YAG laser (Coherent 7900, Palo Alto, CA) with energy of 6.5-7.5 mJ. Cell monolayers were arranged perpendicularly to and in the focal plane of the beam (using a micromanipulator, these parameters were kept constant between different experiments). After wounding, the medium was immediately changed to Dulbecco's modified Eagle's medium containing 0.1% bovine serum albumin supplemented with 100 units/ml penicillin and 100 µg/ml streptomycin. After the wounding, images were recorded immediately and 3 and 6 h later and stored for analysis (Universal Imaging Corp., West Chester, PA). The surface area of wounds in the consecutive images was measured, the size of the initial wound was expressed as 100%, and the timed progression of wound healing was expressed as fractions of the initial size.
Antisense Oligodeoxynucleotide TreatmentsThe antisense
phosphorothioate derivatives of oligodeoxynucleotides (S-ODN) to the
human endothelial constitutive NO synthase (eNOS) 5-[AGT TGC CCA TGT
TAC TGT GCG TCC GTC]-3
, nucleotides 56-30 of human eNOS cDNA
(20), as well as the sense 5
-[GAC GGA CGC ACA GTA ACA TGG GCA
ACT]-3
and scrambled 5
-[CTG GGA CCT GTT CGT ACA GGT CTC TTC]-3
phosphorothioate sequences were synthesized using an automated
solid-phase DNA synthesizer (Applied Biosystems, Foster City, CA).
These sequences included the 5
-untranslated region of eNOS cDNA and
initiation codon. Thus designed sequences showed no homology with other
known mammalian sequences deposited in the GenBank data base, as
screened using a Blast program (21). All the S-ODNs were purified using
oligonucleotide purification cartridges, dried down, resuspended in
Tris-EDTA (10 mM Tris, pH 7.4, 1 mM EDTA, pH
8.0), and quantified spectrophotometrically.
Prior to migration assays, HUVEC were incubated with 10 µM S-ODNs for 12 h under a serum-free condition. During typical experiments in a Boyden chemotactic chamber (see above), S-ODNs were present in the medium. In parallel experiments, companion HUVEC treated with 10 µM S-ODNs in the serum-free medium were seeded on gelatin-coated coverslips, allowed to attach, and used to verify their responsiveness to ET-1 (1 nM) by monitoring NO release or were stained with monoclonal antibodies directed against eNOS (Transduction Laboratories, Lexington, KY), according to the previously described protocol (11).
With regard to NO
release and locomotive responses, RMVEC displayed sensitivity to ET-1
within the physiologic concentration range (Fig. 1).
ET-1 stimulated NO production by RMVEC, and L-NAME blunted
NO release in a dose-dependent manner, with 2 mM L-NAME resulting in complete inhibition of
NO release (Fig. 1A). Transwell migration of RMVEC, studied
in a modified Boyden chamber, showed the similar exquisite sensitivity
to ET-1. A statistically significant almost 50% increase in the number
of migrated cells was detected with ET-1 concentrations of 10 pM, and it almost doubled with further elevation of ET-1
concentration to 1 nM (Fig. 1B). Inhibition of
NO synthase with 2 mM L-NAME completely
abrogated the ET-1-induced cell migration. Under the conditions of NOS
inhibition, endothelial cell migration could be restored in the
presence of 8-bromo-cyclic GMP (100 µM), as shown in Fig.
2.
To elucidate the type of ET receptor(s) involved in the observed
stimulation of RMVEC migration, transwell migration experiments were
performed with different endothelins and in the presence of selective
inhibitors of their receptors. As summarized in Fig. 3,
ET-1 and ET-3 were equipotent in stimulating endothelial cell migration. A selective ETBR agonist, IRL-1620 exhibited a
similar stimulatory effect, suggesting that the observed phenomena were mediated via ETBR. This conclusion is further supported
by the observation that a selective ETA receptor antagonist
BQ123 did not affect ET-1-induced endothelial cell transmigration,
whereas a selective ETBR antagonist BQ788 completely
abrogated the stimulatory action of ET-1. These findings were in
concert with ET-1-induced NO release. While BQ123 did not interfere
with NO production by endothelial cells stimulated with 1 nM ET-1, pretreatment with BQ788 abrogated NO release in
response to ET-1 but not to bradykinin (Fig. 4). It is
therefore concluded that ET-1 induces endothelial cell migration via
the ETBR and that the previously demonstrated coupling of
this receptor to eNOS (9) is critical for NO production and cell
migration.
Effects of Endothelin on the Rate of Endothelial Wound Healing
The rate of endothelial wound healing was examined by
electroporation-induced RMVEC denudation from the surface of a gold miniature electrode, as previously detailed (25). ET-1 accelerated wound healing at 10 h by 66.0 ± 3.2% (n = 5) compared with the control medium (40.4 ± 2.0%
(n = 5), p < 0.05) (Fig.
5). The rate of restitution of endothelial integrity was
equally enhanced by IRL-1620 and by ET-3, and this effect was abolished
by co-application of ETB receptor antagonist BQ788, further
implicating ETB receptor in the observed phenomena (Fig. 5,
A and B). The addition of L-NAME to
ET-1- or ET-3-stimulated endothelial cells virtually abrogated this
response (Fig. 5, C and D).
Effects of Endothelin on Wound Healing in CHO Cells
To
further test the above premise that NO production is required for
ET-1-induced RMVEC migration, experiments were performed in a model
system of CHO cells expressing ETBR and eNOS, as verified and detailed previously (9). ET-1 was ineffective in stimulating wound
healing in CHO cells transfected with ETBR alone (Fig.
6). However, co-expression of both ETBR and
eNOS in CHO cells imparted on them the migratory response to ET-1 and
resulted in acceleration of wound healing. The data further confirm the
permissive role of NO production in ET-1-induced migration.
Effects of Endothelin in the Presence of Antisense Oligodeoxynucleotides Targeting eNOS
In the next series of
experiments, a selective knockdown of eNOS was performed using
antisense S-ODNs. To avoid any possible species-specific variations in
the sequence, experiments were performed in HUVEC using an antisense
construct directed against the initiation codon of human eNOS cDNA.
Cells were treated with either antisense, sense, or scrambled S-ODNs,
as detailed under "Materials and Methods." The expression of
immunodetectable eNOS in endothelial cells subjected to S-ODNs is
presented in Fig. 7, confirming the adequacy of
treatments. Functional analysis of endothelial cells subjected to
S-ODNs was accomplished by monitoring NO release. Fig.
8A depicts typical tracings of ET-1-induced
NO release. Both sense and scrambled S-ODN-treated cells responded to
ET-1 with increased NO release, similar to that observed in intact
control cells. In contrast, antisense S-ODN-treated HUVEC failed to
produce NO in response to ET-1. These data confirm the validity of the
utilized S-ODN constructs. To study the effects of ET-1 on motility of
S-ODN-treated HUVEC, experiments were performed in a modified Boyden
apparatus. As summarized in Fig. 8B, pretreatment of HUVEC
with antisense S-ODN resulted in a dramatic deceleration of
transmigration, as compared with intact control cells and cells exposed
to sense or scrambled constructs.
In this study, two different endothelial cell preparations, derived from rats and humans, showed a consistent acceleration of motility in response to members of the endothelin family. Through application of specific agonists and antagonists of known endothelin receptors, ETA and ETB, it was possible to implicate ETB receptor in the observed responses. However, these effects of ET were mediated by endogenous NO production, as confirmed by the results of three independent experimental approaches. In the first series of experiments, L-NAME prevented ET-1-induced endothelial cell transwell migration and wound healing. In the second series, CHO-ETBNOS cell, a model cell system devoid of complexity inherent to endothelial cells, showed NO dependence of migration induced by ET-1. Finally, the application of eNOS isoform-selective antisense S-ODNs, but not sense or scrambled constructs, to endothelial cells suppressed their migratory responsiveness to ET-1. From these observations, we derive a conclusion that stimulated NO production serves a permissive role in ET-induced acceleration of endothelial cell motility and wound healing.
ETB receptor in endothelial cells serves a unique purpose
for autocrine feedback regulation of several physiologic functions by
the produced ET-1. The application of anti-ET -globulin to cultured
endothelial cells inhibits the rate of proliferation (26, 27).
Moreover, this receptor is implicated in ET-induced vasodilation (10).
We have recently demonstrated that this non-isopeptide-selective receptor is functionally coupled to eNOS and coordinates the release of
NO from endothelial cells (9). Stably transfected
CHO-ETBNOS cells were successfully employed in those
studies to reveal, in a simplified model system, that receptor-enzyme
coupling involves the tyrosine kinase- and
calcium/calmodulin-dependent pathways (9). Hence, the
studies established a physiologically meaningful communication and
coordination between endothelium-derived vasoconstrictor and
vasodilator systems that control the vascular tone. Other investigators
have also viewed ET-1 generated by endothelial cells as an autocrine
motility factor (7, 8). However, results presented herein implicate for
the first time the integral complex system of endothelium-derived
vasoactive agonists, endothelin and NO, in endothelial cell motility
and implicate this system in vascular remodeling.
We have previously demonstrated that NO production is a prerequisite for epithelial cell migration guided by several motogens, including hepatocyte growth factor and epidermal growth factor (11). The establishment of head-to-tail gradients of NO synthase in locomoting, but not in stationary, epithelial cells was construed as an important regulator of locomotion driven by these guidance cues (11). More recently, we have established that NO interferes with processes of endothelial cell adhesion to matrix proteins and participates in locomotion initiated by vascular endothelium growth factor.2 These findings provided an experimental basis for a hypothesis ascribing to NO the function of a modulator of cell-matrix adhesion. In this vein, ET-induced NO production by endothelial cells can be considered as a particular example of a more generalized phenomenon of NO-regulated cell motility.
The described NO production serving as a prerequisite for endothelial cell locomotion in response to activation of ETB receptor may explain a host of pathophysiologic observations on inadequate angiogenesis despite enhanced generation of ET-1. In hypercholesterolemic pigs and in humans with atherosclerotic lesions, ET-1 generation is augmented (28-30). The reason for the insufficient angiogenesis toward ischemic sites could be conceptually explained by the fact that NO production by the endothelium is suppressed under these conditions (31-33). Similarly, endothelial wound healing after balloon angioplasty is retarded, neointimal formation by proliferating smooth muscle cells is enhanced, and endothelin receptor antagonist SB 209670 protects angioplastic vessels against neointimal formation (14). Interpretation of these observations may rest on the above presented findings on the permissive role of NO in endothelial cell migration and wound healing.