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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From Institut National de la Santé et de la
Recherche Médicale (INSERM) EMI 0105, Department of Responses and
Cellular Dynamics, Commissariat à l'Energie (CEA)-Grenoble,
France.
Activin receptor-like kinase 1 (ALK-1) is an orphan type I
receptor of the transforming growth factor beta (TGF- Angiogenesis, the formation of new blood
capillaries from a preexisting capillary network, is a multistep
process that can be roughly divided into 2 phases. In the activation
phase, endothelial cells degrade the perivascular basement membrane,
migrate into the extracellular space, proliferate, and form capillary
sprouts and tubular structures. In the maturation phase, endothelial
cells cease migration and proliferation, reconstitute a basement
membrane, and recruit smooth muscle cells permitting the maintenance of vessel wall integrity. A balance of proangiogenic and antiangiogenic factors very tightly regulates angiogenesis. Important angiogenic factors include basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and
transforming growth factor beta 1 (TGF- Activin receptor-like kinase 1 (ALK-1) is an orphan type I receptor of
the TGF- The physiologic ligand for ALK-1 is not known and the type II
receptor(s) it interacts with is not clearly identified. In experiments
where type II TGF- The aim of our study was to determine the biologic functions and
molecular targets of ALK-1 in capillary endothelial cells. Since the
natural ligand for ALK-1 has not been conclusively identified, we used
a constitutively active form of ALK-1 (ALK-1QD), containing a Gln to
Asp mutation in the penultimate residue of the regulatory domain (GS
domain), which results in constitutive ligand-independent receptor
activation. This study was performed in human microvascular endothelial
cells from the dermis (HMVEC-d's) since in HHT, numerous telangiectases can be observed in the skin. We observed that ALK-1QD inhibited proliferation of HMVEC-d's by stopping the cells in the
G1 phase of the cell cycle. ALK-1QD expression also inhibited migration, and decreased readhesion and spreading of HMVEC-d's to
different matrices. This led us to examine the dynamic formation of
adhesion complexes. We observed that ALK-1QD-expressing cells were not
able to reorganize these focal complexes at the edge of a wound.
Finally, a microarray analysis performed 15 hours after
infection revealed 13 genes that were either positively or negatively
regulated by ALK-1QD. Among the genes, 2 were related to the cell cycle
(p21/waf1 and c-myc), 3 to focal adhesion plaque and the cytoskeleton
( Cell culture, recombinant adenoviruses, and infection
Western blot analysis
Cell number At the indicated time of culture, the number of viable cells was determined using either a particle counter (Z1; Beckman Coulter, Roissy, France) or the WST-1 colorimetric assay (Roche).Cell cycle analysis At 15 hours after infection, the cells were rinsed and incubated for another 9 hours. Cells were then trypsinized and resuspended in phosphate buffered saline (PBS) containing 1 g/L glucose, 4% paraformaldehyde, 1% Triton X-100, and 2 µg/mL Hoechst no. 33258 (Sigma). We collected 20 000 events on a flow cytometer (FACScan; Becton Dickinson, Pont de Claix, France) and the percentage of cells in the G1, S, and G2/M phases of the cell cycle was determined.Apoptosis assay At 15, 24, and 48 hours after infection, the cells were trypsinized, fixed for 1 hour with 2% paraformaldehyde, and permeabilized in 0.1% triton X-100, 0.1% sodium citrate for 2 minutes on ice. Apoptotic cells were measured with TdT-mediated dUTP nick end labeling (TUNEL) in presence of fluorescein dUTP (Roche). The number of fluorescent apoptotic cells was determined by flow cytometry (FACScalibur; Becton Dickinson).Cell migration in the wound assay At 15 hours after infection, confluent HMVEC-d, HMEC-1, HMVEC-l, and HUVEC monolayers were wounded with a plastic pipet tip, placed back at 37°C in a CO2 incubator, and photographed at regular intervals (0, 24, 32, and 48 hours). Closure of the wound was followed by time-lapse microscopy with pictures taken every 30 minutes for 48 hours. Video images were collected with MetaVue software (Universal Imaging Corporation, Downingtown, PA) and analyzed using Track Image Bio processing software (Orme, Toulouse, France).Cell migration in the transwell assay Confluent monolayers of HMVEC-d's were infected with Ad -gal
or AdALK-1QD (MOI = 10) and labeled with the fluorophore DiI (5 µg/mL, Molecular Probes) for 15 hours. Cells were then trypsinized and suspended at a final concentration of 5 × 105/mL in
EGM-2-MV with 0.5% FBS. A 100 µL aliquot of the cell suspension was
added to transwell inserts (8 µm; Falcon HTSF Fluoroblok inserts; Becton Dickinson). The inserts were held in 24-well companion plates
(Becton Dickinson) containing 500 µL EGM-2-MV with either 0.5% or
5% FBS and incubated for 24 hours at 37°C. Migration of cells
through the optically opaque insert membrane to the lower culture plate
was assessed by measurement of DiI fluorescence on a fluorimeter (BMG
LabTechnologies, Offenburg, Germany).
Adhesion measurement At 15 hours after infection, the cells were trypsinized and seeded onto uncoated (culture-treated plastic; Falcon, Becton Dickinson) or coated (Greiner Bio-One, Frickenhausen, Germany) multiwells. Coating was performed with either 0.2 mg/mL fibronectin (Sigma), 1 mg/mL gelatin (Sigma), 0.1 mg/mL collagen I (Becton Dickinson), or 0.1 mg/mL collagen IV (Sigma). Cells were photographed 3 hours after readhesion. The number of attached cells was determined after trypsinization with a particle counter (Z1; Beckman Coulter).Cell spreading measurement At 15 hours after infection, the cells were trypsinized and seeded onto fibronectin-coated glass slides (10 µg/mL) for 30 minutes. Cells were then fixed and stained with Coomassie blue. The numbers of fully spread (> 1000 µm2), partially spread (between 100 and 1000 µm2), and round (< 100 µm2) cells were determined. Cell area was determined using National Institutes of Health Image software.Immunofluorescence For immunofluorescence staining, cells were cultured on vitronectin-coated (10 µg/mL vitronectin for 2 hours at 37°C) Lab-Tek 4-well chamber slides (NUNC; Polylabo, Strasbourg, France) and infected with adenovirus for 15 hours. Then, the monolayer was wounded as described above or left unwounded. The monolayers were fixed and permeabilized (4% paraformaldehyde, 0.2% Triton X-100) 3 hours after wounding. Cells were stained with mouse monoclonal antibody against paxillin (Upstate Biotechnology, Lake Placid, NY) or with phalloidin-fluorescein isothiocyanate (FITC; Sigma) to visualize actin microfilaments, and counterstained with Hoechst no. 33258 (Sigma).Differential hybridization of Atlas human cDNA expression arrays Total RNA from HMVEC-d's infected for 15 hours with Ad-gal,
AdALK-1QD, or AdALK-5TD were isolated (Rneasy; Qiagen, Courtaboeuf, France) and treated with Dnase I. 33P-radiolabeled cDNA
synthesis was carried out by RT-PCR as described in the Atlas cDNA
expression arrays user manual (Clontech, Palo Alto, CA). Microarray
analysis was performed in 2 independent experiments. Radiolabeled cDNAs
were hybridized to nylon membranes carrying 1176 cancer-related cDNAs
(Atlas Human Cancer 1.2 Arrays; Clontech, 7851-1). Hybridization
signals were quantitated with a -imager (FujifilmBAS-5000, Fuji)
using Image Reader and Image Gauge softwares (Fuji). A 1.5-fold change
in gene expression was arbitrarily deemed biologically significant.
Quantitative reverse transcriptase-real time PCR (RT-rtPCR) Quantitative RT-rtPCR analysis of ALK-1QD-controlled gene expression of-actin, paxillin, zyxin, c-myc, MIC-1/GDF15, and p21/waf1 was performed using the LightCycler apparatus (Roche) with
sequence-specific primer pairs. The FastStart DNA Master SYBR Green I
system (Roche) was used for real-time monitoring of amplification.
Quantitation was performed using the LightCycler software (Roche).
Statistics Statistical analysis was performed using Student t test. (*P < .05; **P < .01.)
Infection of HMVEC-d's HMVEC-d's were infected for 15 hours with either AdALK-1QD or Ad-gal, as a control, at an MOI of 10. Under these conditions, more
than 95% of Ad-gal-infected HMVEC-d's were -gal-positive. Expression of HA-tagged ALK-1QD was detected by Western blotting with
an anti-HA antibody (Figure 1A). This
antibody detected 2 specific bands, which probably represent
differentially N-glycosylated forms of ALK-1, as previously
noted.17 Constitutive ALK-1 activity was verified using a
specific anti-phosphoSmad1/5 antibody (Figure 1B). ALK-1QD
appeared to specifically induce the phosphorylation of Smad1/5 since no
signal was detected with an anti-phosphoSmad2/3 antibody (data
not shown).
ALK-1QD inhibits HMVEC-d proliferation To determine the potential role of ALK-1 in proliferation, we measured the number of viable cells after AdALK-1QD infection in comparison to Ad-gal infection. Figure
2A shows that ALK-1QD expression
significantly decreased the number of viable HMVEC-d's as soon as 15 hours after infection. This effect was dependent on the concentration
of adenovirus, being significative at an MOI of 5 (Figure 2B). This
decrease may have resulted from an inhibition of either cell
proliferation or cell survival. We therefore measured the number of
apoptotic cells following infection with the TUNEL method. We found
that ALK-1QD expression did not induce apoptosis of HMVEC-d's at the
times studied (15 hours: 0.08% ± 0.01% versus 0.06% ± 0.01%;
24 hours: 1.72% ± 0.04% versus 0.71% ± 0.435%; 48 hours: 0.74% ± 0.08% versus 0.65% ± 0.22%, Ad-gal and
AdALK-1QD, respectively). We then analyzed the effect of ALK-1QD expression on the distribution of cells in the cell cycle by flow cytometry. AdALK-1QD treatment led to an accumulation of cells in the
G1 phase (77.7% versus 63.1%, AdALK-1QD and Ad-gal) and a decrease
in S phase (6.9% versus 17.7%, AdALK-1QD and Ad-gal; Figure
2C). Again, no apoptosis could be detected as visualized by levels of
DNA lower than that seen in G1 phase.
ALK-1QD decreases HMVEC-d migration Endothelial cell migration is another important step of angiogenesis. Therefore, we examined the effect of ALK-1QD expression versus-gal expression on migration of endothelial cells using the
monolayer wound assay. At 15 hours after infection, the monolayers were
wounded with a pipet tip and the closing of the wound was photographed
at different time intervals and analyzed by time-lapse microscopy.
Videos depicting this may be found on the Blood website; see
the Supplemental Videos link at the top of the online article. In
Ad-gal-infected cells, the wound was half-closed after 24 hours and
completely closed by 48 hours (Figure
3A,C; -gal video). In
contrast, in ALK-1QD-expressing cells, the wound was still open after
48 hours and there was a decrease in cell density (Figure 3A,C; ALK-1
video). Cellular movements were recorded and measured using
time-lapse video microscopy. Comparison of the paths of migration of
ALK-1QD-expressing cells and -gal-infected cells showed that while
-gal-infected cells have a relatively direct trajectory toward the
center of the wound, ALK-1QD-expressing cells move in an unorientated
manner and many of them detach shortly after wounding. We also measured
ALK-1QD effect on migration using transwell chambers and we found a
similar effect. ALK-1QD expression resulted in a 2-fold decrease in
motility (no chemoattractant) and in migration (5% FBS in the lower
chamber; Figure 3D).
ALK-1QD inhibits migration of HMEC-1's, HMVEC-l's, and HUVECs We next wanted to know if ALK-1QD inhibition of migration could also be observed in other endothelial cell types. We therefore performed a similar experiment with HMEC-1's, HMVEC-l's, and HUVECs. Expression and constitutive activity of ALK-1QD were verified by Western blot analysis in these 3 different cell types (data not shown). These different endothelial cells were infected for 15 hours with either AdALK-1QD or Ad-gal (MOI = 50) and then a wound was
created. We observed that ALK-1QD expression inhibited the closing of
the wound of these 3 different endothelial cell types (Figure
4).
ALK-1QD decreases HMVEC-d readhesion and spreading Migration involves discrete phases of adhesion and disadhesion. Therefore, we also determined whether ALK-1QD induced changes in this aspect of cellular behavior. To this end, HMVEC-d's were infected with either AdALK-1QD or Ad-gal. At 15 hours after infection, the cells
were trypsinized and seeded back onto plastic. Less adherent and less
spread cells were observed in ALK-1QD-expressing cells (Figure
5A). The number of adherent cells was
determined 30 minutes after reseeding (Figure 5B). We found that
ALK-1QD expression significantly decreased the number of adherent cells on plastic (28%). We also observed a decrease in cell reattachment when the cells were seeded on plates coated with different
extracellular matrix proteins (fibronectin, gelatin, collagen I, and
collagen IV; Figure 5B). We also looked at the effect of ALK-1QD on
spreading. The numbers of fully spread versus partially spread and
round cells were determined 30 minutes after reseeding. ALK-1QD
expression decreased the number of fully spread cells (65% versus
36%, Ad-gal and AdALK-1QD, Figure 5C). The average cell area was
also significantly reduced by ALK-1QD expression (1012 µm2 versus 1362 µm2, AdALK-1 and
Ad-gal; P < .01).
ALK-1QD inhibits the relocalization of focal complexes Since ALK-1QD inhibited migration and readhesion, we postulated that ALK-1 might have an effect on the formation of focal adhesion. We therefore examined the effect of AdALK-1QD on these complexes in migrating cells and in confluent monolayers. To do this, at 15 hours after infection, some adherent cells were wounded and fixed 3 hours later. Staining of the control confluent monolayer of AdALK-1QD-infected HMVEC-d's for paxillin, a good marker of focal adhesions, did not reveal any significant difference with the staining of Ad-gal-infected cells (Figure 6,
top). Staining for paxillin in wounded
monolayers of Ad-gal-infected cells showed discrete focal adhesions
at the extreme leading edge of the wound and at the base of
lamellipodia (Figure 6, bottom). In contrast, in ALK-1QD-infected
cells, the staining remained in focal adhesions within the body of the
cells (Figure 6, bottom), suggesting a defect in dynamic formation of
focal adhesions in ALK-1QD-expressing cells. Phalloidin labeling of
the actin fibers also indicated a defect in their formation near the
wound in ALK-1QD-infected cells (Figure 6, bottom).
ALK-1QD-regulated genes in HMVEC-d's To further explore the molecular mechanisms behind ALK-1-induced changes in HMVEC-d behavior, we examined the changes in cellular gene expression, which occurred in response to ALK-1QD, by microarray analysis using a membrane spotted with 1176 cancer-related cDNAs. This experiment was performed twice and yielded similar results. As shown in Table 1, we found 13 genes that were either positively regulated (8 genes) or negatively regulated (5 genes) 15 hours after AdALK-1QD infection. Among these genes, 2 encoded proteins related to the cell cycle; the Cdk inhibitor p21/WAF1, which was up-regulated, and the product of the proto-oncogene c-myc, which was down-regulated. There were 3 genes that encode proteins that are related to the cytoskeleton and more precisely to focal adhesion plaques:-actin, paxillin, and zyxin, which were down-regulated. The
regulation of these 5 genes was confirmed by quantitative RT-rtPCR
(Table 1). There were 2 other genes that encoded proteins that belong
to the TGF- family, the growth and differentiation factor 15 (GDF-15) and the BMP type II receptor (BMPRII), which were
up-regulated. A similar experiment was performed with the constitutive
active form of ALK-5 (ALK-5TD). Out of the 13 genes found to be
regulated by ALK-1QD, only BMPRII and GRP78/BiP were also found to be
up-regulated by ALK-5TD (Table 1).
ALK-1 is an orphan endothelial-specific type I receptor of the
TGF- The most profound effect of ALK-1QD expression that we could observe in
HMVEC-d's was the complete absence of migration in the wound assay.
This inhibition of migration was also observed in the transwell assay.
Time-lapse recording of the wound assay experiment in HMVEC-d's
(pictures taken every 30 minutes for 48 hours) showed that
ALK-1QD-treated cells start to move in response to wounding in an
unorientated manner and many rapidly detached from the surface (Figure
3; ALK-1 video). To eliminate the possibility of toxic effects
of ALK-1QD expression, we measured apoptosis by the TUNEL technique. If
ALK-1QD was to directly elicit apoptosis directly some evidence of
TUNEL staining might be observed in adherent cells prior to detachment.
This was not the case. We could only visualize apoptotic cells in the
population of nonadherent detached cells. Therefore, it is probable
that cells detach when they are stimulated to move toward the center of
the wound, possibly due to a defect in cytoskeletal reorganization, and
then undergo anoïkis. Indeed, we observed a defect in the
formation of focal adhesion complexes, as visualized by paxillin and
actin staining at the edge of the wound. Further, we found that ALK-1QD
expression inhibited HMVEC-d readhesion to different matrices
(fibronectin, gelatin, collagen I, and collagen IV) and decreased
spreading to fibronectin. Interestingly, 3 genes that were found to be
down-regulated by ALK-1 are related to the cytoskeleton and more
precisely to focal adhesion complexes and stress fibers: The molecular control of actin filament assembly and disassembly
implicates the Rho families of small GTPases. Each has been ascribed to
a particular function: Rac1 is essential for the protrusion of
lamellipodia and for forward movement; Cdc42 is required to maintain
cell polarity and direction of the movement; and Rho is required to
maintain cell adhesion during movement.19 Therefore our
results suggest that ALK-1 might modify the balance between GTPases.
This is in accordance with previous studies that demonstrated that
ligands of the TGF- In the present work we also show that activated ALK-1 inhibits HMVEC-d
proliferation through an arrest in the G1 phase of the cell cycle.
Interestingly, we demonstrate that ALK-1QD regulates the expression of
2 genes that are implicated in cell cycle regulation: the
proto-oncogene c-myc and the Cdk inhibitor p21/waf1. These genes
represent 2 well-characterized targets in the pathway of TGF- There are 2 other genes regulated by ALK-1 that belong to the TGF- The cDNA array analysis also enabled us to identify genes that are not
regulated by ALK-1 at the time studied. Specifically, we found that the
expression of several genes encoding extracellular matrix components
was not altered by ALK-1QD (fibronectin, PAI-1, and SPARC). These genes
have been previously shown to be regulated by TGF- The results presented here provide the first evidence implicating ALK-1
in angiogenesis at the cellular level and provide several important
clues regarding the cellular signaling mechanisms behind the
development of HHT. Using a constitutively active ALK-1 receptor
(ALK-1QD), we could clearly demonstrate that ALK-1 inhibits proliferation and migration of HMVEC-d's. These in vitro experiments represent the conceptual opposite of the natural genetic disease HHT,
in which mutations in the ALK-1 gene result in haploinsufficiency and
reduced levels of ALK-1 receptor at the surface of endothelial cells.3 HHT patients present vessel enlargement and direct arteriovenous connections without intervening capillary beds. This
phenotype probably results from an increase in proliferation and
migration of capillary endothelial cells that is completely consistent
with the "inverse" phenotype observed in ALK-1QD-expressing cells.
In further support of these observations, homozygous ALK-1 knockout
mice die at midgestation, exhibiting severe vascular abnormalities
characterized by excessive fusion of capillaries and hyperdilation of
large vessels,4,5 and the zebrafish mutant ALK-1 has
an abnormal circulation pattern attributed to an increase in
endothelial cell number.6 These findings from HHT patients
and ALK-1
We thank Dr J. LaMarre (University of Guelph, ON, Canada) for his critical review of the manuscript and for his provocative suggestions. We thank Dr S. Souchelnitskiy (Ludwig Cancer Institute for Cancer Research, Uppsala, Sweden) for helpful discussions. We are grateful to Dr M. Fujii and Dr P. ten Dijke for providing us with ALK-1QD adenovirus and specific anti-phosphoSmad antibodies, respectively. We thank Dr D. Gulino and Dr F. Candal for providing us with HUVECs and HMEC-1's, respectively. We thank M. Keramidas and Dr B. Vailhé (INSERM EMI105, Grenoble, France) for introducing us to endothelial cell migration assays. We acknowledge C. Rosello and X. Ronot (Institut A Boniot, Grenoble, France) for helping us in video tracking analysis of migration in the wound assay. We thank Véronique Collin-Faure and Gwenaëlle LeMoigne for their skillful technical help. We thank the Gene Production Network (GVPN) and the Gene Therapy Laboratory of the CHU in Nantes who amplified the adenoviruses.
Submitted November 13, 2001; accepted August 2, 2002.
Supported by INSERM (EMI 0105), CEA (Direction Sciences du Vivant, Department of Responses and Cellular Dynamics (DRDC)/Angio0105), and a European Union grant (no. QLRT-2000-01302).
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Sabine Bailly, INSERM EMI 0105 DRDC/Angio, CEA-Grenoble, 17 rue des Martyrs, 38054 Grenoble, France; e-mail: sbailly{at}cea.fr.
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Abstract
Introduction
/