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From the Reid Rheumatology Laboratory, Division of Autoimmune Diseases and Transplantation, The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, Victoria, Australia
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-mediated cytotoxicity. These
findings may reveal a new aspect of vascular physiology, which
may also be relevant to formation and maintenance of the abnormal
vasculature in the rheumatoid synovium.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A number of receptor tyrosine kinase (RTK)-ligand interactions have been identified that regulate vascular development and angiogenesis.5 Gene knockout mice have been particularly informative in understanding the role of RTKs in the developmental biology of the vascular system. Vascular endothelial cell growth factor (VEGF) is one of the key regulators of vascular development and has two RTK receptors, VEGFR-1 and -2. Mice lacking VEGFR-2 die early in embryonic development due to lack of endothelial and hemopoietic cells,6 whereas VEGFR-1-null mice generate both these cell types, but die because of failure to form early vascular structures.7 Another RTK involved in vascular development is Tie-2. Tie-2-null mice generate endothelial cells and early vascular patterning, but cannot organize an appropriate lattice of supporting cells to stabilize the developing vascular network.8 With the exception of physiological processes such as those in the female reproductive cycle and wound healing, angiogenesis is usually a pathological process in the adult.9 VEGF is probably the major regulator of angiogenesis in the adult, but basic fibroblast growth factor, platelet-derived growth factor, and hepatocyte growth factor signaling through cognate RTKs, as well as a variety of inflammatory cytokines and their receptors, can also cause angiogenesis.3,4 Endothelial cells are normally long-lived, but much less is known about survival signals in this cell type, particularly in the presence of tissue inflammation.
Cell growth and survival are active and interconnected processes that depend on the integration of signals from the external environment and the intrinsic differentiation programs of particular cells.10 In the absence of appropriate signals, cells die by apoptosis. Many growth factor receptors belong to the RTK family. RTKs have unique extracellular domains that specifically bind growth factor ligands but share homologous intracellular kinase domains that have intrinsic kinase activity and also bind signal transduction molecules. The presence of conserved motifs within the catalytic domain of RTKs has been exploited in the search for new members of the TK family.11
As one approach to understanding the molecular basis of cellular
hyperplasia in RA, we used reverse transcriptase-polymerase chain
reaction (RT-PCR) to search for RTKs expressed in RA synovium. We
report the identification of the RTK Axl in RA synovium and the
expression of Axl in endothelial cells and vascular smooth muscle
cells. We have also found Gas6, a recently discovered ligand for Axl,
in synovial fluid. Exogenous Gas6 bound to human umbilical vein
endothelial cells (HUVECs) and protected these cells from apoptosis in
response to growth factor withdrawal and also from TNF-mediated
cytotoxicity. These findings may reveal a novel survival pathway for
endothelial cells, which may be relevant to the pathology of RA.
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Materials and Methods |
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Synovial fluid samples were obtained from three RA patients, one ballet dancer with a traumatic knee effusion, and one patient with psoriatic arthritis. Synovial tissue samples were obtained from eight patients with RA and six with osteoarthritis (OA) at the time of joint replacement surgery.
Cloning of Axl via RT-PCR from RA Synovial Tissue
A cloning technique based on RT-PCR using degenerate oligonucleotides was used to search for RTKs in RA synovium. RNA was extracted from the synovium of a patient with RA as described below and used to generate cDNA using a cDNA synthesis kit (Amersham, Buckinghamshire, UK). Primers corresponding to sequence motifs within the catalytic domains of protein tyrosine kinase (PTK) family members were used as described elsewhere.12 The PCR products obtained were gel purified and digested with BamHI and EcoRI before ligation to BamHI/EcoRI-digested pBluescript II plasmid DNA (Stratagene, La Jolla, CA). Following transformation, plasmid DNA was isolated from individual bacterial colonies and the sequence of the insert was determined using the PCR oligonucleotide primers, a Taq Dye Deoxy Terminator Cycle Sequencing Kit (Perkin Elmer, Norwalk, CT) and an ABI automated sequencer (Perkin Elmer).
RNA Analysis
Total RNA was extracted from synovium using guanidinium thiocyanate and electrophoresed in agarose containing formaldehyde. Poly A-+ mRNA was extracted from SV40-transformed synovial cells (SV40.SYN)13 using oligo(dT)-cellulose. Northern blots were performed by capillary transfer to nylon membranes (Hybond N+, Amersham) and hybridized to 32P-labeled, full length Axl cDNA (Mega Prime DNA labeling system, Amersham). Axl cDNA was provided by Dr. Johannes Janssen.
Immunohistochemistry
Synovial tissue specimens from patients with RA were fixed in paraformaldehyde and embedded in paraffin. Paraffin sections 5 µm thick were dewaxed, hydrated, and incubated in methanol containing 3% peroxidase. Sections were digested with pepsin for 10 minutes at 37°C (Digest-All Kit, Zymed Laboratories, San Francisco, CA), then washed in phosphate-buffered saline (PBS). Immunohistochemistry was performed using a streptavidin-horseradish peroxidase system with 3-amino-9-ethylcarbazole as the chromogen, according to the manufacturer's instructions (Histostain-Plus Kit, Zymed Laboratories). The primary antibody (anti-Axl) was diluted 1:20 in PBS and incubated on the sections overnight at 4°C in a humidified chamber. As a negative control, normal rabbit IgG (Sigma Chemical Co., Steinheim, Germany) was substituted for anti-Axl at a corresponding protein concentration. The sections were counterstained with hematoxylin and mounted (DAKO, Glostrup, Denmark).
Protein Analysis
Protein was extracted from synovial tissue specimens using 25 mmol/L Tris, pH 7.5, 150 mmol/L NaCl, 0.1% TritonX-100, and protease inhibitors (10 µmol/L E-64, 100 µmol/L leupeptin, 10 mmol/L EDTA, 1 µmol/L pepstatin, 1 mmol/L phenylmethylsulfonyl fluoride, 10 mmol/L 1,10-phenanthroline, and 10 µmol/L Z-Phe-Ala-CHN2, all from Sigma). Synovial tissue lysates were ultracentrifuged at 50,000 rpm for 1 hour at 4°C and frozen at -80°C. Synovial fluids were centrifuged at 2000 rpm for 10 minutes at 4°C to remove cellular components and treated with hyaluronidase at 37°C for 1 hour. For primary cells and cell lines, protein was extracted using 25 mmol/L Tris, pH 7.5, 150 mmol/L NaCl, and 1% 3–3-cholamidopropyl-dimethylamonnio-1-propanesulfonate containing protease inhibitors as above. Lysates were incubated on ice for 30 minutes and cell debris was removed by centrifugation in a microfuge at 13,000 rpm for 10 minutes at 4°C. The amount of protein was estimated using the Bio-Rad Protein Assay (Bio-Rad, Richmond, CA). Samples (75 µg of synovial fluid or 25 µg of synovial tissue lysate) were run on 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred to nitrocellulose (Hybond-C, Amersham). Membranes were blocked with 10% skim milk powder in Tris-buffered saline, 0.1% Tween 20 (TBS-T) and incubated with primary antibody diluted 1:1000 in 1% skim milk powder in TBS-T. In some experiments, Axl and Gas6 anti-sera were pre-incubated for 30 minutes at room temperature with 5 µg of the extracellular domain of Axl fused to human immunoglobulin (Axl-Ig) or recombinant human Gas6 (rhGas6) as specificity controls. After washing in TBS-T, blots were incubated with sheep anti-rabbit horseradish peroxidase (Silenus, Hawthorn, Australia) diluted in 1% skim milk powder in TBS-T and proteins were visualized using the ECL detection system (Amersham). Gas6, anti-Gas6 rabbit polyclonal antibody, and Axl-Ig peptide were provided by Dr. Brian Varnum and anti-Axl rabbit polyclonal antibody was provided by Dr. Edison Liu.
Cell Culture Conditions
For in vitro culture of synovial lining cells, synovial tissue from RA patients was dissected and minced into 2–3 mm pieces. The tissue was washed in RPMI and dissociated for 1.5 hours at 37°C with gentle agitation in RPMI containing 2.4 mg/ml dispase II (Boehringer Mannheim, Mannheim, Germany), 1 mg/ml collagenase Type II (Sigma), and 100µg/ml DNase I (Boehringer Mannheim) in RPMI. The tissue was then ground gently through a sieve and washed several times in RPMI containing 10% fetal bovine serum (FBS, Life Technologies, Auckland, New Zealand). Cells were cultured in RPMI containing 10% FBS at 37°C, 5% CO2, with medium changes at 24 hours to remove nonadhering cells and debris. Early passage HUVECs were cultured at 37°C in 5% CO2 in complete medium consisting of M199 (Earle's salts) medium for endothelial cells supplemented with 25% (v/v) conditioned medium, 20% (v/v) FBS, 50 ng/ml transferrin (Boehringer Mannheim), 10 ng/ml endothelial cell growth supplement (Sigma), 10 µg/ml insulin (Sigma), and 2 mmol/L glutamine (Life Technologies, Grand Island, NY). The cells were seeded in 24-well plates precoated in 2.5% (w/v) gelatin (BDH Chemicals, Poole, UK) in PBS at approximately 5 x 105 cells per well.
Gas6 Binding to HUVECs
HUVECs were detached using trypsin/EDTA and incubated in complete M199 medium either with or without 500 ng/ml Gas6 for 1 hour at 37°C. In some experiments, 10 µg/ml soluble Axl-Ig was added in addition to Gas6. After washing in PBS containing 2% FBS, the cells were stained with anti-Gas6 polyclonal antibody at 10 µg/ml or an irrelevant rabbit antibody, washed, and stained with fluorescein isothiocyanate-labeled sheep anti-rabbit Ig (Silenus). Stained cells were analyzed on a Becton Dickinson (San Jose, CA) FACScan.
Cell Survival Assay
Cells were grown in complete medium (containing serum and growth factors), M199 medium (to induce cell death by apoptosis), or M199 medium supplemented with 100 ng/ml Gas6. The medium was changed every 48 hours and cell death was monitored at days 1, 2, 5, and 8 after detachment of the cells using trypsin/EDTA. Viable cells were counted using trypan blue exclusion. There were three replicates for each time point and the experiment was performed three times.
TNF-Mediated Cytotoxicity
Cells were grown in M199 base medium supplemented with 0, 10, or
100 ng/ml Gas6. At day 1, 10-8 mol/L TNF
(Boehringer Ingelheim, Frankfurt, Germany) was added to half the
wells without changing the medium to induce cell death by apoptosis.
Fresh Gas6 was added at day 2 and cells were harvested by
trypsinization at day 5. Viable cells were counted using trypan blue
exclusion. There were four replicates for each condition and the
experiment was performed three times.
Cell Cycle Analysis by Flow Cytometry
HUVECs collected from a TNF-mediated cytotoxicity experiment
were washed in PBS, fixed in 70% ethanol, and stained with propidium
iodide as described previously.14
Measurement of propidium
iodide fluorescence and analysis of the cell cycle was performed on a
Becton Dickinson FACScan using CellFit SOBR computer software. The
combination of dead and apoptotic cells was measured by counting the
percentage of events to the immediate left of the G1 histogram peak.
Ten thousand events were collected for each sample.
Statistical Analysis
Student's t-test was used to measure the difference between group means.
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Results |
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To detect members of the PTK family we performed RT-PCR using degenerate oligonucleotide primers that correspond to sequences within the catalytic domain of PTK family members. RNA was extracted from the synovium of a patient with RA and, after synthesis of cDNA, tyrosine kinase sequences were amplified using PTK-I and PTK-II oligonucleotides as primers.12 A PCR product of approximately 200 bp was obtained, purified, and subcloned into pBluescript II. After transformation of competent E. coli, individual bacterial colonies were picked and plasmid DNA isolated for sequence determination. Members of the jak family of tyrosine kinases were the clones most frequently obtained, but DNA sequences from multiple distinct colonies revealed 100% homology with the RTK Axl,15 also known as UFO.16 We chose to study the expression and possible function of this RTK in RA in more detail.
Axl Expression in RA Synovium
Northern Blotting
Northern blot analysis was performed to assess the level of Axl
expression in synovial tissues. Human Axl mRNA occurs as two
transcripts of 4.9 and 3.4 kb. These gene products are generated by
alternative splicing of exon 10 and differential usage of two imperfect
polyadenylation sites.17
Figure 1
shows Axl mRNA in synovial tissues from
several patients with RA and one patient with OA. OA was used as a
control for rheumatoid joint disease. Both mRNAs are present in all
samples, as well as in the SV40.SYN cell line, showing that Axl
expression is not unique to RA.
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We next performed immunohistochemistry to determine which cell
types express Axl within RA synovial tissue (Figure 2)
. The most striking finding was of Axl
expression associated with blood vessels, in particular endothelial
cells. In subsynovial capillaries, Axl was expressed in endothelial
cells (Figure 2, D and E)
. However, in larger blood vessels, both
arterioles and veins, Axl expression was confined to smooth muscle
cells (Figure 2, B and C)
. Expression was also seen in some but not all
synovial lining cells in RA synovium (Figure 2F)
. Normal rabbit IgG
used at the same protein concentration as the Axl polyclonal antibody
gave widespread nonspecific staining (Figure 2A)
.
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To confirm our immunohistochemistry findings, protein lysates from
a number of relevant cell types were analyzed for Axl expression by
Western blotting (Figure 3A)
. Axl is a
140-kd glycosylated protein.14
Soluble Axl-Ig was used a
positive control (molecular weight, 110 kd). Primary cultured RA
synovial cells showed low level, but detectable Axl expression and this
was greater in the synovial cell line SV40.SYN. However, in accord with
the immunohistochemistry results, HUVEC protein lysates were strongly
positive for Axl. A number of lower molecular weight immunoreactive
bands were also detected in HUVECs, consistent with multiple
glycosylation sites in the extracellular domain of Axl.18
The specificity of the anti-Axl antibody was shown by pre-incubation
with the soluble Axl-Ig peptide (Figure 3B)
before addition to a
Western blot. The 110-kd band of Axl-Ig was almost completely
abolished, indicating antibody specificity for the extracellular domain
of Axl. Rabbit IgG showed no reactivity against HUVEC lysates or Axl-Ig
(data not shown).
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Gas6 has been identified as a ligand for Axl.19,20
Protein lysates from synovial tissue of patients with either RA or OA
were used in a Western blot to detect expression of Gas6 (Figure 4A)
. Using a polyclonal anti-Gas6
antibody, a major immunoreactive protein of approximately 75 kd
corresponding to recombinant human Gas6 could be identified in all
synovial tissue specimens, although the intensity was generally greater
in the RA synovial tissues. Equivalent amounts of protein, as estimated
by the BioRad protein assay, were loaded in each lane. Specificity of
the anti-Gas6 antibody was confirmed by competition with recombinant
human Gas6 (Figure 4B)
. Figure 4C
shows Western blot analysis of
synovial fluids from patients with RA, psoriatic arthritis, and a
noninflammatory joint effusion probed with the anti-Gas6 antibody. A
band of approximately 75 kd was identified in all synovial fluid
specimens, corresponding to the expected size of Gas6. A second band of
approximately 90 kd was also seen, possibly corresponding to a
previously described Gas6 splice variant.21
The
proteolytic products of this variant are thought to be approximately 36
and 50 kd,22
and immunoreactive bands of this size were
identified in the Western blot of synovial fluid (Figure 4C)
but not
synovial tissue.
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Gas6 has been shown to be a ligand for Axl and the related RTKs
Sky and Mer.19,23-25
To confirm that Gas6 is a
physiological ligand for Axl expressed by HUVECs, Gas6 was added to
HUVECs and the cells were stained with an anti-Gas6 antibody. As shown
in Figure 5
, HUVECs bound added Gas6
and this was competed out in the presence of soluble Axl-Ig.
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Gas6 is able to protect a variety of cells from apoptosis induced
by complete growth factor depletion.26-28
To determine
whether Gas6 has similar activity in HUVECs, we induced apoptosis by
complete growth factor depletion (Figure 6)
. In the growth factor-deprived
cultures, cell viability had decreased at 24 hours and by day 8 all
cells were dead. In contrast, when Gas6 was added to growth
factor-deprived HUVECs, there was an initial drop in viability at 24
hours, but thereafter cell viability was retained. Rescue from
apoptosis by Gas6 was statistically significant at days 5
(P < 0.001) or 8 (P <
0.002). HUVECs produced some endogenous. Gas6 under normal culture
conditions (data not shown), but cell-associated Gas6 was clearly
unable to rescue HUVECs to the same extent.
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-Induced Cell Death
TNF is known to induce apoptosis of some cell types, especially
upon withdrawal of growth factors.29
Gas6 has been found
to rescue TNF-treated NIH3T3 cells from apoptosis.27
We
therefore examined the ability of Gas6 to protect HUVECs against
TNF-mediated apoptosis. As shown in Figure 7
, TNF efficiently induced cell death
in growth factor-starved HUVECs (P < 0.001) and
100 ng/ml (but not 10 ng/ml) of Gas6 partially protected HUVECs
from TNF-induced cytotoxicity (P < 0.001).
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Flow cytometric analysis was used to demonstrate the survival
effects of Gas6. Figure 8
shows
representative cell cycle profiles of HUVECs under conditions of growth
factor deprivation (Figure 8A)
and after treatment with Gas6 (Figure 8B)
, TNF (Figure 8C)
, or both (Figure 8D)
. Dead or apoptotic cells
accumulate in the hypodiploid region to the left of the vertical
marker. Cells retained in the various phases of the cell cycle at the
time of sampling appear to the right of the vertical marker. The
percentage of apoptotic or dead cells was significantly higher in the
growth factor-starved cells compared to starved cells supplemented with
Gas6 (65 ± 4% compared with 49 ± 3%, P <
0.001). Rescue from TNF-induced apoptosis by Gas6 was incomplete but
statistically significant. This experiment was performed three times
with a total of seven replicates for each condition. Because most
TNF-treated cells were killed it was difficult to determine the true
hypodiploid region, but there was at least 85 ± 4% cell death
with TNF, compared with 76 ± 6% in the presence of Gas6
(P < 0.01). The G1 peak was obliterated in
cells treated with TNF (Figure 8C)
but remained obvious when Gas6
was added (Figure 8D)
, indicating retention of cells in the cell cycle.
The percentage of cells in the G1 phase of the cell cycle was
significantly higher in the serum-starved, Gas6-treated group compared
to cells without Gas6 (20 ± 3% compared with 13 ± 3%,
P < 0.001).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Axl expression has been found in myeloid, erythroid, and megakaryocytic leukemic cell lines,31 in myeloid leukemias,32 and in colonic33 and hepatocellular carcinomas.30 ARK (the murine counterpart of Axl) is expressed within mesenchymal elements by day 12.5 of murine embryonic development34 and is broadly expressed in adult mouse tissues.16 Less is known about the cellular distribution of Axl, but rat vascular smooth muscle cells,35,36 human chondrocytes,37 human CD34+ hemopoietic stem cells, and mature myeloid hemopoietic cells32 have been shown to express Axl. Our study is the first to show clear expression of Axl by human endothelial cells and vascular smooth muscle cells in situ and provides further support for the possibility that Axl may be involved in vascular structure or function. We found an intriguing pattern of Axl expression: in small capillaries, Axl was expressed by endothelial cells, whereas in larger arterioles and veins, surrounding smooth muscle cells were Axl-positive. The extracellular domain of Axl contains adjacent fibronectin type III and immunoglobulin-like repeats16,38 and homophilic binding between the extracellular domains of Axl has been demonstrated.39 This suggests a role in cell adhesion which could be relevant to tube formation in angiogenesis. Vascular smooth muscle cell expression has been previously noted in the rat and may suggest involvement of Axl in some other aspect of vascular function.35,36 Clearly, the phenotype of mice with a targeted deletion of Axl will be of great interest in this regard, both in the basal state and in response to inflammatory and angiogenic stimuli.
One ligand for Axl has been identified as Gas6.19,20
Gas6
was originally discovered and named due to its production by cells in
the quiescent phase of the cell cycle.40
Gas6 is a
multimodular protein with an N-terminal 
-carboxyglutamic acid (Gla)
domain, epidermal growth factor-like repeats, and a sex hormone-binding
globulin-like domain.41
The last feature may be sufficient
for receptor binding and activation.42,43
Gas6 requires
vitamin K-dependent -carboxylation and has homology to Protein S, a
key protease regulator of coagulation.41
The full spectrum
of Gas6 biological activity is currently under investigation, but it is
of interest that Protein S and several other serum proteases including
thrombin,44
urokinase-type plasminogen
activator,45
and factor Xa46
have also
been found to contribute to inflammatory pathways.
Gas6 has a number of properties that may be relevant to vascular biology. Gas6 expression has been documented in unstimulated endothelial cells 41,47 and conditioned media from a bovine endothelial cell line was used to stimulate Axl phosphorylation and subsequently to purify Gas6 as an Axl ligand.20 Gas6 was also found in conditioned media of rat vascular smooth muscle cells that had been treated with thrombin and endothelin.48 Gas6 can promote adhesion between Axl-expressing cells49 and can elicit chemotaxis of vascular smooth muscle cells.50 Both of these properties are reminiscent of the Tie-2 ligand angiopoietin-1 and Gas6 could be similarly involved in formation or modeling of the vasculature. Avanzi et al47 reported that Gas6 inhibited adhesion of neutrophils to stimulated, but not resting, HUVECs and speculated that Gas6 exerts a protective anti-inflammatory effect. Nakano et al51 showed that the Gla domain of Gas6 can specifically bind phosphatidylserine, a phospholipid normally positioned on the inner leaflet of the plasma membrane but thought to be exposed on dying cells, leading those investigators to propose a role for Gas6 in the clearance of apoptotic cells.
We found Gas6 in synovial tissue and fluid from patients with OA and RA. Endothelial cells,41,47 rat vascular smooth muscle cells,48 and cultured human chondrocytes37 have been found to produce Gas6 and these cell types are therefore potential sources of Gas6 in synovial fluid. However, to our knowledge Gas6 has not been detected in the serum, suggesting local production or an alteration of half-life within the joint. It is of interest that the levels of Gas6 were generally higher in RA synovial tissue, suggesting that Gas6 may be up-regulated or overproduced in the setting of joint inflammation.
Gas6 is now well characterized as a promiscuous ligand for the Axl
subfamily but, in contrast to most RTK ligands, Axl-Gas6 interaction
alone induces only modest mitogenic effects in some
cells.26,35,37,48,52-54
However, Gas6 has been shown to
protect a number of Axl-positive cells from stimuli that induce
apoptosis.26-28,37
Other nonmitogenic properties of Gas6
include chemotactic effects on vascular smooth muscle
cells50
and up-regulation of osteoclast
function.55
A number of effects of Gas6 on vascular smooth
muscle cells have been documented;28,35,48,50
however,
much less is known about Axl-Gas6 interaction in endothelial cells. We
chose HUVECs as a model system and have shown that these cells
express Axl and bind Gas6. Upon growth factor withdrawal, exogenous
Gas6 acted as a survival factor for HUVECs and protected them from
TNF-induced cytotoxicity. Little is known about regulation of
endothelial cell survival and how it changes in
inflammation.56
The synovial cavity is a relatively
hypoxic and acidotic environment and synovial effusions can result in
ischemia of the synovium.57
Inflammatory cytokines such as
TNF are produced in high local concentrations in RA and attract
leukocytes from the bloodstream. The major role of Axl-Gas6 interaction
may therefore be in survival of the vasculature under conditions of
cellular stress or injury.36
Within the normal synovial joint, Axl and Gas6 could function as a survival pathway for endothelial cells and perhaps for vascular smooth muscle cells, synovial cells, and chondrocytes. Our results raise the possibility that Gas6 may also promote survival of activated endothelial cells, and perhaps other Axl-positive cells, within the hostile environment of the inflamed rheumatoid joint. In this way, a survival mechanism normally involved in tissue homeostasis could also contribute to maintenance of a pathological vasculature in RA.
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Acknowledgements |
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Footnotes |
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Supported by the Reid Charitable Trusts and the Australian National Health and Medical Research Council.
Accepted for publication January 22, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-carboxyglutamic acid residues for the biological activity of Gas6: contribution of endogenous Gas6 to the proliferation of vascular smooth muscle cells. Biochem J 1997, 323:387-392
-carboxylation and a sex hormone-binding globulin-like domain in receptor-binding and in biological activities of Gas6. FEBS Lett 1997, 408:306-310[Medline]
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 S. L. Shankar, K. O'Guin, M. Cammer, F. A. McMorris, T. N. Stitt, R. S. Basch, B. Varnum, and B. Shafit-Zagardo The Growth Arrest-Specific Gene Product Gas6 Promotes the Survival of |
