1 Department of Pathology, University of Washington, Seattle, WA 98195,
USA
2 IFOM, Institute FIRC for Molecular Oncology, Milan 20139, Italy
3 Department of Engineered Biomaterials, University of Washington, Seattle, WA
98195, USA
4 Veterans Administration Puget Sound Health Care System, Seattle, WA 98108,
USA
* Author for correspondence (e-mail: roberto.nicosia{at}med.va.gov)
Accepted 22 April 2003
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Angiogenesis, Angiopoietins, Tie2, Mural cells, Pericytes
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The angiopoietins, a newly discovered family of angioregulatory
polypeptides, play a crucial role in endothelial sprouting, vessel wall
remodeling and mural cell recruitment. Angiopoietin-1 (Ang-1) binds to and
induces phosphorylation of Tie2 (Sato et
al., 1995), a tyrosine kinase receptor expressed by endothelial
cells (Sato et al., 1995
;
Davis et al., 1996
).
Angiopoietin-2 (Ang-2) has different effects on Tie2 phosphorylation depending
on the context in which it operates. It can function as an agonist or an
antagonist depending on its concentration and duration of treatment
(Teichert-Kuliszewska et al.,
2001
) or the extracellular matrix environment
(Kim et al., 2000a
;
Maisonpierre et al., 1997
).
Ang-2 binds to endothelial Tie2 without inducing its phosphorylation but can
also activate Tie2 in nonendothelial mesenchymal cells genetically engineered
to express this receptor (Maisonpierre et
al., 1997
). Ang-3 and Ang-4 bind to Tie2 and are believed to be
the mouse and the human counterparts, respectively, of the same gene. Ang-3
acts as an antagonist, whereas Ang-4 appears to function as an agonist
(Valenzuela et al., 1999
).
Ang-1 is produced predominantly by mural and perivascular mesenchymal cells
(Davis et al., 1996;
Kim et al., 2001
). Ang-2 is
produced by endothelial, mural and mesenchymal cells
(Holash et al., 1999
;
Mandriota et al., 1998). Genetic ablation of the Ang-1 or
Tie2 genes results in lethal defects in vascular remodeling,
maturation and stabilization (Sato et al.,
1995
; Suri et al.,
1996
). Vessels formed in the absence of a functioning Ang-1/Tie2
system do not acquire a properly assembled layer of mural cells and fail to
mature into an arborizing network of small and large vessels
(Sato et al., 1995
;
Suri et al., 1996
). Point
mutations of the gene encoding the Tie2 receptor resulting in constitutive
Tie2 phosphorylation cause arteriovenous malformations characterized by
abnormal layering of smooth muscle cells around the endothelium
(Vikkula et al., 1996
).
Overexpression of Ang-2 in transgenic mice causes abnormal vascular
development similar to that observed in Ang-1 and Tie2
knockout mice (Maisonpierre et al.,
1997
). Overexpression of Ang-1 in mice genetically engineered to
express this molecule in the skin induces formation of a dense vascular
network composed of vessels that are larger and more branched than those of
control animals (Suri et al.,
1998
).
Although there is compelling evidence that the angiopoietin/Tie2 system is
involved in mural cell recruitment, the mechanisms of this process are poorly
understood. To gain further insight into the mechanisms by which mural cells
are recruited by sprouting neovessels, we established a method to isolate
mural precursor cells (MPCs) from the rat aorta. MPCs were isolated from the
aortic intimal/subintimal layers, which have the capacity to generate mural
cell-coated neovessels in collagen gel culture. Isolated MPCs are polygonal in
shape, have a high proliferative rate and produce large amounts of laminin-
and type-IV collagen-rich extracellular matrix
(Villaschi et al., 1994). In
addition, they respond to endothelial cues by transforming into a dendritic
phenotype and becoming incorporated as pericytes in capillary networks
bioengineered from isolated endothelial cells in a collagen gel overlay assay
(Nicosia and Villaschi,
1995
).
Tie2 is reportedly expressed in endothelial cells but not in mural cells
(Sato et al., 1995;
Kim et al., 2001
;
Suri et al., 1996
). In this
paper, we report that aorta-derived MPCs express the Tie2 receptor, respond
chemotactically to Ang-1 and Ang-2, and produce matrix metalloproteinase-2
(MMP-2) when stimulated by these factors. These observations suggest that
mural cells of angiogenic neovessels might arise from a precursor
subpopulation of less differentiated mesenchymal cells capable of responding
directly to angiopoietins produced by the sprouting endothelium.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Rat aorta model of angiogenesis
Rings of rat aorta were prepared and cultured in floating collagen gels as
previously described (Nicosia and
Ottinetti, 1990). Each ring was embedded in approximately 300
µl of interstitial collagen purified from rat tail tendons as reported
(Elsdale and Bard, 1972
).
Collagen gel cultures of rat aorta were kept at 35.5°C in serum-free MCDB
131 culture medium (Clonetics, San Diego, CA) with or without Ang-1 and/or
Ang-2, in the presence or absence of soluble Tie2. The medium was changed
three times a week. Branching morphogenesis was evaluated by counting branch
points at the peak of angiogenic growth (day 7). Mural cell recruitment was
scored by counting the number of mural cells visible under phase contrast
microscopy in the ten longest microvessels of each culture, as previously
reported (Nicosia and Villaschi,
1995
).
Isolation of MPCs
MPCs were isolated nonenzymatically from everted rat aortas, as previously
described (Villaschi et al.,
1994) and grown at 37°C in MCDB 131 growth medium supplemented
with 10% fetal bovine serum (FBS) (Sigma, St Louis, MO) and 50 µg
ml-1 gentamicin (Sigma). MPCs were
-smooth muscle actin
(
-SMA) positive and factor-VIII-related antigen (FVIII-RA) negative by
immunocytochemistry. The capacity of MPCs to transform into pericytes was
evaluated in a collagen gel overlay coculture assay with rat aorta endothelial
cells (Nicosia and Villaschi,
1995
). MPC were periodically reisolated to insure a constant
supply of early passage cells (passages 7-12, at 1:3 split ratio).
MPCs were also isolated with an immunomagnetic method by incubating an
endothelial-depleted suspension of aortic outgrowth cells with
anti-Tie2-coated magnetic beads (Alessandri
et al., 2001). A mixed population of aorta-derived cells grown on
gelatin-coated dishes were trypsin treated and first incubated with magnetic
beads (anti-mouse-IgG-coated beads, cell:bead ratio 1:4; Dynal Biotech, Oslo,
Norway) coated with an anti-CD31 mouse monoclonal antibody (BD Biosciences
Pharmingen, San Diego, CA). Endothelial cells, which selectively express CD31,
bound to the beads coated with anti-CD31 antibody and were removed with a
magnetic particle concentrator (Dynal Biotech). The remaining CD31-negative
cells were then recovered by centrifugation and incubated with magnetic beads
coated with mouse monoclonal antibody to Tie2 (BD Biosciences Pharmingen;
cell:bead ratio 1:4). The CD31-/Tie2+ cells were
recovered with a magnetic particle concentrator, seeded on a gelatin-coated 60
mm dish and cultured at 37°C in MCDB 131 growth medium supplemented with
10% FBS and gentamicin. Detachment of beads from cells was facilitated by
gently trypsinizing the isolated cells after a few days in culture.
Endothelial cell lines
Rat aortic endothelial cells (RAECs) were isolated and subcultured as
reported (Nicosia et al.,
1994). The human-endothelium-derived permanent cell line EAhy 926
(Edgell et al., 1983
) was
kindly provided by C. J. S. Edgell (Department of Pathology, University of
North Carolina, Chapel Hill, NC). These cells were grown in Dulbecco's
Modified Eagle Medium (DMEM) (Life Technologies, Rockville, MD) containing 10%
FBS (Sigma) and HAT growth supplement (Life Technologies).
Immunocytochemistry and immunohistochemistry
For immunocytochemical studies, MPCs were cultured in 35 mm dishes in
endothelial basal medium (EBM) supplemented with 10% FBS and gentamicin. EAhy
926 cells and RAECs were used as positive controls of endothelial cell
differentiation. Subconfluent monolayers were fixed in cold methanol or
ethanol and stained, according to previously published procedures
(Villaschi and Nicosia, 1994),
with biotinylated Griffonia Simplicifolia isolectin-B4 (Vector Laboratories,
Burlingame, CA), rabbit polyclonal anti-FVIII-RA antibody (DAKO, Carpinteria,
CA) or mouse monoclonal antibodies against CD31 (BD Biosciences, Pharmingen),
-SMA (Sigma) or calponin (Sigma). Tie2 protein expression in isolated
cells was demonstrated in separate experiments with two rabbit polyclonal
anti-Tie2 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA; Alpha
Diagnostics International, San Antonio, TX). Biotinylated secondary antibodies
of the appropriate animal species were used after the primary antibody, as
described (Villaschi and Nicosia,
1994
). The cultures were overlaid with ABC solution (Vectastain
Elite Kit, Vector Laboratories) for 10 minutes, reacted with diaminobenzidine
(DAB), rinsed in distilled water and mounted in Gelvatol (Monsanto, St Louis,
MO). Negative controls included cultures incubated with non-immune IgG of the
same species as the primary antibody of interest and cultures reacted only
with secondary antibody. For the immunohistochemical localization of Tie2 in
the aortic wall, the native rat aorta was fixed in buffered formalin, embedded
in paraffin, cross-sectioned and mounted on Fisherbrand Superplus slides
(Fisher Scientific, Pittsburgh, PA). Histological sections were
deparaffinized, rehydrated and blocked according to standard protocols. They
were then immunostained by the ABC immunoperoxidase procedure with a rabbit
polyclonal anti-Tie2 antibody (Alpha Diagnostics International) and mounted in
Gelvatol (Monsanto).
Confocal microscopy and double immunofluorescence
Double immunofluorescence followed by confocal microscopy was used to
localize Tie2 and -SMA in MPCs. MPCs cultured in gelatin-coated Lab-Tek
chamber slides (Nalge Nunc, Naperville, IL) were fixed in formalin, washed
twice with PBS, permeabilized with 0.2% Triton X-100 solution and reacted for
1 hour with a cocktail of rabbit polyclonal anti-Tie2 antibody (Santa Cruz
Biotechnology, Santa Cruz, CA) and mouse antibody against
-smooth-muscle actin (Sigma). They were then rinsed in PBS, incubated
for 30 minutes with a cocktail of AlexaFluor-488-labeled goat anti-rabbit
antibody (green fluorochrome) and AlexaFluor-568-labeled goat anti-mouse
antibody (red fluorochrome) (Molecular Probes, Eugene, OR, USA), rinsed twice
with PBS, transferred onto histology glass slides, mounted in aqueous mounting
medium (Gelvatol) and examined by confocal microscopy using a Leica TCS-SP
laser scanning microscope. Image analysis and z-plane sectioning were
performed using proprietary Leica software.
Chemotaxis analysis
Chemotaxis assay was carried out in a 48-well Boyden chamber (Neuro Probe,
Cabin John, MD) as reported (Albini et al.,
1993). MPCs were harvested in trypsin 0.05% NaHCO3
(Life Technologies), incubated in MCDB 131 plus 10% FBS to neutralize trypsin,
centrifuged, resuspended in serum-free MCDB 131 (Clonetics) and seeded in the
upper compartment of the chemotaxis chamber (5x104 cells per
50 µl). Each experimental group comprised six wells, whose lower and upper
compartments were separated by a gelatin-coated polycarbonate filter of
3-µm-pore-size (Nucleopore/Costar, Cambridge, MA). The lower compartment of
the wells was filled with PDGF-BB, Ang-1, Ang-2, the Ang-1/Ang-2 combination,
the Ang-1/Tie2 combination, albumin or serum-free medium. To rule out
chemokinesis (random migration), the Ang-1 effect was further evaluated by
adding Ang-1 to both lower and upper compartments. After a 4-hour incubation
at 37°C in a humidified 5% CO2 incubator, the filters were
removed from the Boyden chamber. Nonmigrating cells were scraped off the upper
side of the filter with a rubber spatula. Filters were then stained with a
HEMA 3 kit (Biochemical Sciences, Swedesboro, NJ) to demonstrate cells that
had migrated toward the lower compartment of the chamber. Cell migration was
measured as optical density (OD) with a GS-710 densitometer (Bio-Rad
Laboratories, Hercules, CA).
Zymography and MMP gelatinase activity assay
MMP secretion in cultures of MPC treated with Ang-1, Ang-2, the Ang-1/Ang-2
combination or serum-free medium was evaluated by gelatin zymography
(Kleiner and Stetler-Stevenson,
1994). Equal volumes of medium conditioned by MPC (300,000 cells
per well) for 48 hours were electrophoresed onto a 7.5% SDS-polyacrylamide gel
containing 0.6 mg ml-1 type A gelatin from porcine skin (Sigma).
After electrophoresis, the gel was washed in 2.5% Triton X-100 for 2 hours to
remove SDS, incubated for 18 hours at 37°C and stained with 0.1% Coomassie
brilliant blue. Regions of gelatinolytic activity appeared as white bands
against a blue background. Gelatinase bands were quantitatively evaluated with
a Biorad GS710 imaging densitometer. The optical density value of each band
was normalized by the total protein content of each sample. Total protein was
measured with the Micro BCA assay (Pierce, Rockford, IL).
Proliferation assay
Thymidine incorporation was used to evaluate MPC proliferation in response
to stimulation by angiopoietins. Cells were trypsin treated, suspended in MCDB
131 growth medium supplemented with 10% FBS (Life Technologies) and seeded in
a 24 well plate (5x103 in 0.5 ml in each well). Two days
later, the cells were washed with serum-free MCDB 131 and incubated with or
without Ang-1, Ang-2 or the Ang-1/Ang-2 combination under serum-free
conditions. FBS (10%) was used as a positive control. After a 24-hour
treatment, triplicate cultures were pulsed with 2 µCi ml-1
[3H]-thymidine (NEN Life Science Products, Zaventum, Belgium) for 2
hours and treated with 10% trichloroacetic acid (TCA). The TCA precipitates
were solubilized in 0.5 N NaOH for 30 minutes and evaluated in a liquid
scintillation counter.
Western-blot analysis
Proteins were extracted from MPCs or EAhy 926 monolayers in RIPA buffer
containing protease inhibitors (Tris-HCl 50 mM, pH 7.4, 1% NP-40, 0.25% sodium
deoxycholate, 150 mM NaCl, 1 mM PMSF, 1 mM NaVD, 1 mM NaF). After
centrifugation and boiling, protein concentration was measured with the Micro
BCA assay (Pierce, Rockford, IL). 50 µg total protein per lane was
resuspended in Laemmli buffer and loaded onto 6% SDS-polyacrylamide gel. After
electrophoresis, the gel was transferred to a nitrocellulose membrane (Bio-Rad
Laboratories) and probed with monoclonal mouse anti-Tie2 antibody (Upstate
Biotechnology, Lake Placid, NY) or polyclonal rabbit anti-Tie2 antibody (Santa
Cruz Biotechnology). A peroxidase-conjugated donkey anti-mouse or goat
anti-rabbit antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) was
used as secondary antibody. Immunoreactive bands were visualized with the ECL
chemiluminescence detection system (Amersham, Piscataway, NJ). In some
experiments protein samples were immunoprecipitated with a polyclonal rabbit
anti-Tie2 antibody (Santa Cruz Biotechnology) prior to western-blot analysis,
as described below for the Tie2 phosphorylation study.
Tie2 phosphorylation study
MPCs or EAhy 926 were grown in gelatin-coated 100 mm dishes until
confluence, rinsed with PBS pH 7.4 and exposed for 2.5 minutes to Ang-1 or
Ang-2 in serum-free EBM. Treated cells and untreated controls were collected
with a cell scraper in RIPA buffer. Protein concentration was measured with
the Micro BCA assay (Pierce). 200 µg of each sample were then incubated on
ice with 100 µl protein-G-coated agarose beads (Sigma) for 10 minutes and
centrifuged at 10,000 g for 5 minutes. The supernatant was
collected and immunoprecipitated overnight with anti-Tie2 rabbit polyclonal
antibody (Santa Cruz Biotechnology) at 4°C. The immunoprecipitated samples
were washed three times in RIPA buffer, boiled for 3 minutes to release the
agarose beads, resuspended in Laemmli buffer and run onto 6%
SDS-polyacrylamide gel. The gel was then blotted onto a nitrocellulose
membrane (Bio-Rad Laboratories), which was probed with a mouse monoclonal
anti-phosphotyrosine antibody (Upstate Biotechnology, Lake Placid, NY). A
peroxidase-conjugated donkey polyclonal anti-mouse antibody (Jackson
ImmunoResearch Laboratories) was used as secondary antibody. Immunoreactive
bands were visualized with the ECL luminescence detection system (Amersham,
Piscataway, NJ). The membrane was incubated in stripping buffer according to
recommended protocol in the Amersham ECL manual and re-exposed to ECL
detection reagents to ensure absence of residual signals. It was then probed
with rabbit polyclonal anti-Tie2 antibody (Santa Cruz Biotechnology) followed
by a secondary peroxidase-conjugated goat anti-rabbit antibody to demonstrate
that equal amounts of receptor protein were present in each lane.
Immunoreactive protein bands were visualized by chemiluminescence as described
above.
Reverse transcriptase polymerase chain reaction
RNA was extracted from MPCs and EAhy 926 cells with the TRIZOL method (Life
Technologies), according to the manufacturer's protocol. 2 µg of total RNA
was reverse transcribed with random hexamer primers. The reaction mixture
included 100 pmoles random hexamers (Pharmacia, Peapack, NJ) previously
incubated at 70°C for 10 minutes, 20 units RNase inhibitor (Life
Technologies), 1 µM dNTPs (Pharmacia), 0.1 M DTT (Life Technologies),
5x first strand buffer (Life Technologies), 200 Units Superscript II
(Life Technologies) and the RNA of interest. After a 1-hour incubation at
42°C, the reaction was inactivated at 70°C for 15 minutes. For PCR,
the following reagents were mixed to a final volume of 5 µl
tube-1: 10x polymerase chain reaction buffer (Life
Technologies), 2.5 mM MgCl2 (Life Technologies), 0.2 µM dNTP
mix, 1 µM reverse (R) primer and 1 µM forward (F) primers, 2 µl cDNA
template and sterile distilled water. The reaction mixture was heated to
94°C for 5 minutes before adding 2.5 U Taq polymerase (Life Technologies).
35 cycles were performed under the following conditions: denaturation,
94°C for 45 seconds; primer annealing, 55°C for 1 minute; primer
extension, 72°C for 1 minute; final extension at 72°C for 2 minutes.
The Tie2 primers were designed from consensus sequences of human, mouse and
bovine Tie2/Tek to amplify a region of 307 bp. Tie2 F primer:
5'-GAGGACAGGCAATAAGGATACG-3'; Tie2 R primer:
5'-GGGTGAAGAGGTTTCCTCCTAT-3'. PCR products purified with a PCR
purification kit (Qiagen) were cloned into the TA vector PCR 2.1-TOPO (Life
Technologies) according to the manufacturer's recommendations. Two colonies
with the proper size insert were sequenced in both directions at the Veterans
Administration Puget Sound Health Care System (Seattle, WA). Complete
sequences of the amplified PCR products were compared to GenBank using the
BLAST program.
Enzyme-linked immunosorbent assay
The effect of Tie2 activation on endothelial PDGF-BB production was tested
by treating EAhy 926 cells with Ang-1. The EAhy 926 cell system was chosen
because the Tie2 of these cells is unphosphorylated under control conditions
and can be activated by exogenous Ang-1 (data not shown). PDGF-BB was measured
by performing ELISA (R&D Systems) on medium conditioned by EAhy 926 cells
in the presence or absence of stimulatory doses of Ang-1. The production of
PDGF-BB was measured in duplicate using a commercial ELISA kit.
Statistical analysis
Data were analysed with GraphPad Prism statistics software (GraphPad
Software, San Diego, CA). One-way ANOVA followed by Newman-Keuls multiple
comparison test was used to evaluate whether differences between groups were
significant. Statistical significance was set at P<0.05.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Isolation and characterization of MPCs from the rat aorta
We have previously reported that mural cells of rat aorta-derived
neovessels originate from the intimal/subintimal layers of the aortic wall
(Villaschi and Nicosia, 1994).
These cells can be isolated nonenzymatically from everted aortic tubes. When
co-cultured with branching capillaries formed by endothelial cells in collagen
gel overlay co-cultures, MPCs migrate towards the endothelium, becoming
incorporated as pericytes around the capillary tubes
(Nicosia and Villaschi, 1995
).
To evaluate the molecular mechanisms regulating the recruitment of MPCs by
endothelial cells, we isolated and characterized MPCs based on their
morphologic, immunohistochemical and behavioral characteristics using
published protocols (Villaschi and
Nicosia, 1994
; Nicosia and
Villaschi, 1995
). Confluent MPCs displayed a polygonal shape
(Fig. 2A), grew mainly as a
monolayer, had a high proliferative rate and produced large amounts of
laminin- and type-IV-collagen-rich extracellular matrix, as described
previously (Villaschi and Nicosia,
1994
). Immunostaining demonstrated that these cells were positive
for calponin (Fig. 2B) and
-SMA, (Fig. 2C) and
negative for the endothelial markers FVIII-RA
(Fig. 2D) and CD31 (data not
shown).
|
Ang-1 fails to induce the production of MPC chemoattractants by
endothelial cells
To evaluate whether the Ang-1/Tie2 system induced mural cell migration
indirectly through endothelial activation, cultures of EAhy 926 cells were
treated for 24 hours with Ang-1 under serum-free conditions.
Immunoprecipitation of the EAhy 926 Tie2 receptor followed by western-blot
analysis with an anti-phosphotyrosine antibody confirmed activation of the
endothelial Tie2 by Ang-1, as previously reported
(Davis et al., 1996) (data not
shown). Medium conditioned by endothelial cells in the presence or absence of
Ang-1 was tested in a modified 48-well Boyden chamber for its capacity to
promote MPC chemotaxis. Treatment of endothelial cells with Ang-1 failed to
stimulate MPC migration above control values. In addition, ELISA studies
showed no evidence that Ang-1 induces endothelial production of PDGF-BB (data
not shown).
Ang-1 and Ang-2 directly stimulate MPC chemotaxis
While performing the chemotaxis experiments with endothelium-conditioned
medium, we noticed that MPCs migrated in response to Ang-1-containing medium
not exposed to endothelial cells. This suggested that Ang-1 might function as
a direct chemoattractant for MPCs. Dose/response experiments confirmed this
observation, showing a dose-dependent and saturable chemotactic effect by
Ang-1 (data not shown). Interestingly, a similar chemotactic response was also
observed with Ang-2. Ang-2 had no antagonistic effects when administered
together with Ang-1 and the chemotactic effect of the Ang-1/Ang-2 combination
was actually higher than that obtained with the single molecules
(Fig. 3A). To rule out a
nonspecific chemokinetic effect of Ang-1, the experiment was repeated by
placing Ang-1 at the same concentration in both the lower and upper
compartments of the Boyden chamber, a condition that eliminates chemotactic
gradients in the system. MPCs exposed to Ang-1 under these conditions did not
migrate (Fig. 3B). The Ang-1
effect was also significantly reduced by preincubating Ang-1 with soluble Tie2
for 15 minutes prior to the chemotaxis assay
(Fig. 3B). This observation
supports the hypothesis that Ang-1 stimulates MPC migration through a
Tie2-mediated chemotactic mechanism.
|
Ang-1 and Ang-2 stimulate MMP-2 production by MPCs
It has recently been reported that Ang-1 stimulates production of MMPs by
endothelial cells (Kim et al.,
2000a). To evaluate whether angiopoietins have similar effects on
MPCs, these cells were exposed for 48 hours under serum-free conditions to
Ang-1, Ang-2 or the Ang-1/Ang-2 combination. Zymographic analysis of
MPC-conditioned medium showed that MMP-2, which was detectable at low levels
in the untreated control, was significantly increased in cultures treated with
Ang-1, Ang-2 or the Ang-1/Ang-2 combination
(Fig. 4). MPC-conditioned
medium showed no detectable MMP-9 activity in either controls or
angiopoietin-treated cultures.
|
Ang-1 has no effect on DNA synthesis by MPCs
DNA synthesis by MPCs cultured in the presence or absence of Ang-1, Ang-2
or the Ang-1/Ang-2 combination was studied by incubating the cultures with
[H3]-thymidine. Evaluation of thymidine uptake showed that
angiopoietins had no effect on MPC proliferation (data not shown), as
previously reported for endothelial cells
(Huang et al., 1999).
MPCs express Tie2 protein and Tie2 mRNA
The observation that MPCs respond directly to angiopoietin stimulation
suggested that these cells might express Tie2. Western-blot analysis of MPC
extracts immunoreacted for Tie2 demonstrated a 140-kDa band that migrated with
the corresponding band of an endothelial Tie2 control
(Fig. 5), consistent with the
reported molecular weight of Tie2 (Wong et
al., 1997). RT-PCR confirmed expression of Tie2 in MPCs
(Fig. 5). The PCR product was
of 282 bp, as expected from published human, mouse and bovine Tie2 sequences,
and was confirmed to be Tie2 by sequence analysis. The predicted amino acid
sequence from this region was 90%, 88% and 87% homologous to the mouse, human
and bovine Tie2 receptors, respectively. A control RT-PCR reaction using RNA
from the endothelial cell line EAhy 926 produced a product that was 100%
identical to the human sequence, as expected. Immunoprecipitation of MPC
extracts with anti-Tie2 antibody followed by western-blot analysis with an
anti-phosphotyrosine antibody demonstrated marked induction of Tie2
phosphorylation upon stimulation with both Ang-1 and Ang-2
(Fig. 5). Immunocytochemical
staining of MPCs showed peripheral membrane-type localization of Tie2
(Fig. 6A,B), as also
demonstrated in control endothelial cells
(Fig. 6F). Tie2 expression on
the surface of MPCs was further confirmed by isolating these cells with
anti-Tie2-antibody-coated magnetic beads from a mixed population of rat aortic
cells that had been previously depleted of endothelial cells with
anti-CD31-coated beads. Confocal microscopy showed co-localization of
-SMA and Tie2 in isolated MPCs (Fig.
6C-E).
|
|
Tie2 is expressed in intimal/subintimal nonendothelial cells of the
native rat aorta
To evaluate whether MPCs express Tie2 in their native environment (i.e. in
the intimal/subintimal layers of the aortic wall from which they originate),
we immunostained histological sections with an anti-Tie2 antibody. Tie2 was
found not only in endothelial cells but also in mesenchymal cells located in
the intimal/subintimal layers of the aortic wall
(Fig. 6G,H), as predicted by
the MPC isolation and characterization studies.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We previously reported that the rat aortic intima has the capacity to form
neovessels composed of endothelial and mural cells
(Nicosia et al., 1992).
Subsequent isolation experiments led to the identification of rat aortic
intima-derived
-SMA-positive and FVIII-RA-negative cells with mural
cell precursor properties (Villaschi et
al., 1994
). Collagen gel coculture experiments demonstrated that
these cells, which we termed MPCs, have a marked endothelial tropism and
become incorporated into microvascular networks formed by isolated endothelial
cells (Nicosia and Villaschi,
1995
). MPCs stabilize the microvessels, which are unable to
survive in the absence of a nonendothelial mesenchymal cell support.
Our observation that MPCs express Tie2 and respond directly to angiopoietin
stimulation provides the basis for a novel understanding of how mural cells
are recruited by developing neovessels during angiogenesis. Of particular
interest is the observation that MPCs respond not only to Ang-1 but also to
Ang-2. Initial reports indicated that Ang-1 was the main activating ligand of
Tie2, whereas Ang-2 (which bound to this receptor without inducing its
phosphorylation) was considered an antagonist
(Maisonpierre et al., 1997).
Recent studies, however, have shown that Ang-2 might act as an agonist in the
context of a three-dimensional matrix
(Teichert-Kuliszewska et al.,
2001
) and when used at high concentration
(Kim et al., 2000b
) or for
prolonged periods of time
(Teichert-Kuliszewska et al.,
2001
), conditions that might all occur at sites of angiogenesis
(Kim et al., 2001
;
Goede et al., 1998
). The
cellular context in which Tie2 is expressed has also a profound influence on
the response of the receptor to Ang-2. Maisonpierre et al.
(Maisonpierre et al., 1997
)
found that Ang-2 was able to induce Tie2 activation when the receptor was
transgenically expressed in NIH 3T3 fibroblasts. Our results confirm and
expand these observations demonstrating that Ang-2 functions as a Tie2 agonist
in MPC, which spontaneously express this receptor. Ang-2 promotes both MPC
migration and MMP-2 production and does not inhibit the stimulatory effect of
Ang-1 on these cells. In some experiments we actually observed greater MPC
migration and MMP2 production when the cells were exposed to both Ang-1 and
Ang-2. Ang-2 produced by the endothelium during the early stages of
angiogenesis might provide crucial migratory and proteolytic signals to
Tie2+ MPCs. Ang-1 produced by the mural cells themselves and
surrounding fibroblasts may further potentiate this process. According to this
model, Tie2+ MPCs would eventually differentiate into
Tie2- mural cells, a process suggested by the observation in our
laboratory that Tie2 expression is eventually lost in MPCs after repeated
passages in culture (M.I. and R.F.N., unpublished).
The finding of Tie2+ MPCs in the rat aorta confirms recent
observations that Tie2 expression is not restricted to the endothelium and can
also be demonstrated in nonendothelial mesenchymal cell types such as stromal
cells of pyogenic granulomas (Yuan et al., 2001), vascular smooth muscle cells
of breast cancer xenograft tumors (Tian et
al., 2002), vascular smooth muscle cells of reactive synovial
tissue (Shahrara et al.,
2002
), pericytes associated with venous malformations
(Calvert et al., 1999
) and
fibroblast-like cells of choroidal neovascular membranes
(Otani et al., 1999
). Tie2 is
also expressed by hematopoietic stem cells
(Hattori et al., 2001
;
Takakura et al., 1998
) and has
been reported in cytotrophoblastic cells
(Dunk et al., 2000
).
Establishing the origin of the MPCs might be crucial for understanding how
vessels develop and mature. One possibility is that MPCs originate through
transdifferentiation of endothelial cells into a mesenchymal cell type, which
maintains the capacity to express Tie2. An endotheliummesenchyme
transformation has been reported previously in other systems such as
endocardial cushion development (Nakajima
et al., 1997). Alternatively, Tie2+ MPCs might arise
from a vascular progenitor stem cell capable of both endothelial and mural
cell differentiation. Recent studies have identified a common Flk1+
progenitor cell from which both endothelial and mural cells originate in
response to vascular endothelial growth factor (VEGF) or PDGF-BB, respectively
(Yamashita et al., 2000
;
Carmeliet, 2000
). These
immature cells can be found in bone marrow, circulation and peripheral tissues
during angiogenic responses (Asahara et
al., 1997
; Asahara et al.,
1999
; Harraz et al.,
2001
). They have been identified in the human embryonic aorta
(Alessandri et al., 2001
),
raising the possibility that the ex vivo angiogenic response of the postnatal
aorta might be, at least in part, the result of de novo formation of
neovessels from mesenchyme, as previously suggested by others
(Akita et al., 1997
).
In conclusion, our results provide a novel mechanistic explanation for the angiopoietin/Tie2-mediated recruitment of mural cells during angiogenesis. Although more studies are needed to determine whether this model can be universally applied to angiogenic responses in different tissues, our observations raise the intriguing possibility that mural cell recruitment during angiogenesis can be genetically promoted or blocked by targeting the Tie2 receptor of MPCs. Possible applications of this approach include stimulation of neovessel survival and arteriogenesis in diseases characterized by inadequate vascularization, induction of neovessel regression in angiogenesis-dependent disorders such as cancer and bioengineering of vascularized tissue constructs for in vivo implantation. The implications of these observations go beyond the angiogenesis field because of the crucial role molecular mechanisms of mural cell recruitment play in the pathogenesis of a number of vascular disorders including atherosclerosis and restenosis of bypass grafts.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Akita, M., Murata, E., Merker, H. J. and Kaneko, K. (1997). Formation of new capillary-like tubes in a three-dimensional in vitro model (aorta/collagen gel). Anat. Anz. 179,137 -147.[Medline]
Albini, A., Melchiori, A., Parodi, S. and Kleinman, H. (1993). Chemotaxis and invasiveness. In Connective Tissue Disease of the Skin (eds Lapiere, C. M. and Krieg, T.), pp. 129-139. New York: Marcel Dekker.
Alessandri, G., Girelli, M., Taccagni, G., Colombo, A., Nicosia, R., Caruso, A., Baronio, M., Pagano, S., Cova, L. and Parati, E. (2001). Human vasculogenesis ex vivo: embryonal aorta as a tool for isolation of endothelial cell progenitors. Lab. Invest. 81,875 -885.[Medline]
Asahara, T., Murohara, T., Sullivan, A., Silver, M., van der
Zee, R., Li, T., Witzenbichler, B., Schatteman, G. and Isner, J. M.
(1997). Isolation of putative progenitor endothelial cells for
angiogenesis. Science
275,964
-967.
Asahara, T., Masuda, H., Takahashi, T., Kalka, C., Pastore, C.,
Silver, M., Kearne, M., Magner, M. and Isner, J. M. (1999).
Bone marrow origin of endothelial progenitor cells responsible for postnatal
vasculogenesis in physiological and pathological neovascularization.
Circ. Res. 85,221
-228.
Benjamin, L. E., Golijanin, D., Itin, A., Pode, D. and Keshet,
E. (1999). Selective ablation of immature blood vessels in
established human tumors follows vascular endothelial growth factor
withdrawal. J. Clin. Invest.
103,157
-158.
Calvert, J. T., Riney, T. J., Kontos, C. D., Cha, E. H., Prieto,
V. G., Shea, R. C., Berg, J. N., Nevin, N. C., Simpson, S. A, Pasyk, K. A. et
al. (1999). Allelic and locus heterogeneity in inherited
venous malformations. Hum. Mol. Genet.
8,1279
-1289.
Carmeliet, P. (2000). One cell, two fates. Nature 408,43 -44.[CrossRef][Medline]
Carmeliet, P. and Jain, R. K. (2000). Angiogenesis in cancer and other diseases. Nature 407,249 -257.[CrossRef][Medline]
Davis, S., Aldrich, T. H., Jones, P. F., Acheson, A, Compton, D. L., Jain, V., Ryan, T. E., Bruno, J., Radziejewski, C., Maisonpierre, P. C. and Yancopoulos, G. D. (1996). Isolation of angiopoietin-1, a ligand for the Tie2 receptor, by secretion-trap expression cloning. Cell 87,1153 -1155.[Medline]
Dunk, C., Shams, M., Nijjar, S., Rhamam, M., Qiu, Y., Bussolati,
B. and Ahmed, A. (2000). Angiopoietin-1 and angiopoietin-2
activate trophoblast Tie2 to promote growth and migration during placental
development. Am. J. Pathol.
156,2185
-2199.
Edgell, C. J. S., McDonald, C. C. and Graham, J. B. (1983). Permanent cell line expressing factor VIII related antigen established by hybridization. Proc. Natl. Acad. Sci. USA 80,3734 -3737.[Abstract]
Elsdale, T. and Bard, J. (1972). Collagen
substrate for studies on cell behaviour. J. Cell Biol.
54,626
-637.
Folkman, J. and D'Amore, P. A. (1996). Blood vessel formation: what is its molecular basis? Cell 87,1153 -1155.[Medline]
Goede, V., Schmidt, T., Kimmina, S., Kozian, D. and Augustin, H. G. (1998). Analysis of blood vessel maturation processes during cyclic ovarian angiogenesis. Lab. Invest. 11,1385 -1394.
Harraz, M., Jiao, C., Hanlon, H. D., Hartley, R. S. and
Schatteman, G. C. (2001). Cd34- blood-derived
human endothelial cell progenitors. Stem Cells
19,304
-312.
Hattori, K., Dias, S., Heissig, B., Hackett, N. R., Lyden, D.,
Tateno, M., Hicklin, D. J., Zhu, Z., Witte, L., Crystal, R. G., Moore M. A.
and Rafii, S. (2001). Vascular endothelial growth factor and
angiopoietin-1 stimulate postnatal hematopoiesis by recruitment of
vasculogenic and hematopoietic stem cells. J. Exp.
Med. 193,1005
-1014.
Hirschi, K. K. and D'Amore, P. A. (1996). Pericytes in the microvasculature. Cardiovasc. Res. 32,687 -698.[CrossRef][Medline]
Holash, J., Wiegand, S. J. and Yancopoulos, G. D. (1999). New model of tumor angiogenesis: dynamic balance between vessel regression and growth mediated by angiopoietins and VEGF. Oncogene 18,53 -56.
Huang, X. L., Takakura, N. and Suda, T. (1999). In vitro effects of angiopoietins and VEGF on hematopoietic and endothelial cells. Biochem. Biophys. Res. Commun. 264,133 -138.[CrossRef][Medline]
Kim, I., Kim, H. G., Moon, S. O., Chae, S. W., So, J. N., Koh,
K. N., Ahn, B. C. and Koh, G. Y. (2000a). Angiopoietin-1
induces endothelial cell sprouting through the activation of focal adhesion
kinase and plasmin secretion. Circ. Res.
86,952
-959.
Kim, I., Kim, J. H., Moon, S. O., Kwak, H. J., Kim, N. G. and Koh, G. Y. (2000b). Angiopoietin-2 at high concentration can enhance endothelial cell survival through the phosphatidylinositol 3'-kinase/Akt signal transduction pathway. Oncogene 19,4549 -4552.[CrossRef][Medline]
Kim, I., Moon, S. O., Han, C. Y., Pak, Y. K., Moon, S. K., Kim, J. J. and Koh, G. Y. (2001). The angiopoietin-Tie2 system in coronary artery endothelium prevents oxidized low-density lipoprotein-induced apoptosis. Cardiovasc. Res. 49,872 -881.[CrossRef][Medline]
Kleiner, D. E. and Stetler-Stevenson, W. G. (1994). Quantitative zymography: detection of picogram quantities of gelatinases. Anal. Biochem. 218,325 -329.[CrossRef][Medline]
Maisonpierre, P. C., Suri, C., Jones, P. F., Bartunkova, S.,
Wiegand, S. J., Radziejewski, C., Compton, D., McClain, J., Aldrich, T. H.,
Papadopoulos, N. et al. (1997). Angiopoietin-2, a natural
antagonist for Tie2 that disrupts in vivo angiogenesis.
Science 277,55
-60.
Mandriota, S. J. and Pepper, M. S. (1998).
Regulation of angiopoietin-2 mRNA levels in bovine microvascular endothelial
cells by cytokines and hypoxia. Circ. Res.
83,552
-559.
Nakajima, Y., Mironov, V., Yamagishi, T., Nakamura, H. and Markwald, R. R. (1997). Expression of smooth muscle alpha-actin in mesenchymal cells during formation of avian endocardial cushion tissue: a role for transforming growth factor beta3. Dev. Dyn. 209,296 -309.[CrossRef][Medline]
Nicosia, R. F. and Ottinetti, A. (1990). Growth of microvessels in serum-free matrix culture of rat aorta: a quantitative assay of angiogenesis in vitro. Lab. Invest. 63,115 -122.[Medline]
Nicosia, R. F. and Villaschi, S. (1995). Rat aortic smooth muscle cells become pericytes during angiogenesis in vitro. Lab. Invest. 73,658 -666.[Medline]
Nicosia, R. F. and Villaschi, S. (1999). Autoregulation of angiogenesis by cells of the vessel wall. Int. Rev. Cytol. 185,1 -43[Medline]
Nicosia, R. F., Bonanno, E. and Villaschi, S. (1992). Large-vessel endothelium switches to a microvascular phenotype during angiogenesis in collagen gel culture of rat aorta. Atherosclerosis 95,191 -199.[Medline]
Nicosia, R. F., Villaschi, S. and Smith, M. (1994). Isolation and characterization of vasoformative endothelial cells from the rat aorta. In vitro Cell. Dev. Biol. 30A,394 -399.
Otani, A., Takagi, H., Oh, H., Koyama, S., Matsumura, M. and
Honda, Y. (1999). Expressions of angiopoietins and Tie2 in
human choroidal neovascular membranes. Invest. Ophthalmol. Vis.
Sci. 40,1912
-1920.
Sato, T. N., Tozawa, Y., Deutsch, U., Wolbur-Bulchholz, K., Fujiwara, Y., Gendron-Maguire, M., Gridley, T., Wolburg, H., Risau, W. and Quin, Y. (1995). Distinct roles of the receptor tyrosine kinases Tie1 and Tie2 in blood vessel formation. Nature 376,70 -74.[CrossRef][Medline]
Shahrara, S., Volin, M. V., Connors, M. A., Haines, K. G. and Koch, A. E. (2002). Differential expression of the angiogenic Tie receptor family in arthritic and normal synovial tissue. Arthritis Res. 4,201 -208.[CrossRef][Medline]
Suri, C., Jones, P. F., Patan, S., Bartunkova, S., Maisonpierre, P. C., Davis, S., Sato, T. N. and Yancopoulos, G. (1996). Requisite role of angiopoietin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis. Cell 87,1171 -1180.[Medline]
Suri, C., McClain, J., Thurston, G., McDonald, D. M., Zhou, H.,
Oldmixon, E. H., Sato, T. N. and Yancopoulos, G. D. (1998).
Increased vascularization in mice overexpressing angiopoietin-1.
Science 282,468
-471.
Takakura, N., Huang, X. L., Naruse, T., Hamaguchi, T., Dumont, D. J., Yancopoulos, G. D. and Suda, T. (1998). Critical role of the Tie2 endothelial cell receptor in the development of definitive hematopoiesis. Immunity 9, 677-686.[Medline]
Teichert-Kuliszewska, K., Maisonpierre, P. C., Jones, N., Campbel, A. L., Master, Z., Bendeck, M. P., Alitalo, K., Dumont, D. J., Yancopoulos, G. D. and Stewart, D. J. (2001). Biological action of angiopoietin-2 in a fibrin matrix model of angiogenesis is associated with activation of Tie2. Cardiovasc. Res. 49,459 -470.
Tian, S., Hayes, A. J., Metheny-Barlow, L. J. and Li, L. Y. (2002). Stabilization of breast cancer xenograft tumour neovasculature by angiopoietin-1. Br. J. Cancer 86,645 -651.[CrossRef][Medline]
Valenzuela, D. M., Griffiths, J. A., Rojas, J., Aldrich, T. H.,
Jones, P. F., Zhou, H., McClain, J., Copeland, N. G., Gilbert, D. J., Jenkins,
N. A. et al. (1999). Angiopoietins 3 and 4: diverging gene
counterparts in mice and humans. Proc. Natl. Acad. Sci.
USA 96,1904
-1909.
Vikkula, M., Boon, L. M., Carraway, K. L., Calvert, J. T., Diamonti, A. J., Goumnerov, B., Pasyk, K. A., Marchuck, D. A., Warman, M. L., Cantley, L. C. et al. (1996). Vascular dysmorphogenesis cause by an activating mutation in the receptor tyrosine kinase TIE2. Cell 87,1181 -1190.[Medline]
Villaschi, S. and Nicosia, R. F. (1994). Paracrine interactions between fibroblasts and endothelial cells in a serum-free coculture model: modulation of angiogenesis and collagen gel contraction. Lab. Invest. 71,291 -299.[Medline]
Villaschi, S., Nicosia, R. F. and Smith, M. R. (1994). Isolation of a morphologically and functionally distinct smooth muscle cell type from the intimal aspect of the normal rat aorta. Evidence for smooth muscle cell heterogeneity. In vitro Cell Dev. Biol. 30A,589 -595.
Wong, A. L., Haroon, Z. A., Werner, S., Dewhirst, M. W.,
Greenberg, C. S. and Peters, K. G. (1997). Tie2 expression
and phosphorylation in angiogenic and quiescent adult tissues.
Circ. Res. 81,567
-574.
Yamashita, J., Itoh, H., Hirashima, M., Ogawa, M., Nishikawa, S., Yurugi, T., Nalto, M., Nakao, K. and Nishikawa, S. (2000). Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors. Nature 408, 92-96.[CrossRef][Medline]
Yuan, K., Jin, Y. T. and Lin, M. T. (2000). Expression of Tie-2, angiopoietin-1, angiopoietin-2, EphrinB2 and EphB4 in pyogenic granuloma of human gingival implicates their roles in inflammatory angiogenesis. J. Periodont. Res. 35,165 -171.[CrossRef][Medline]