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First published online 8 March 2006
doi: 10.1242/dev.02315
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1 Center of Experimental Medicine, Institute of Anatomy I, University Hospital
Hamburg-Eppendorf, Hamburg, Germany.
2 Department of Cardiac Surgery, University Hospital Hamburg-Eppendorf, Hamburg,
Germany,
3 Department of Hematology and Oncology, University Hospital Hamburg-Eppendorf,
Hamburg, Germany.
4 Department of Cardiology, University Hospital Hamburg-Eppendorf, Hamburg,
Germany.
5 Department of Internal Medicine, University of Schleswig-Holstein, Campus
Lübeck, Germany.
Author for correspondence (e-mail:
erguen{at}uke.uni-hamburg.de)
Accepted 8 February 2006
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Rat, Vasculogenesis, VEGFR
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Folkman, 2003;
Jain, 2002). Main steps of
this process comprise endothelial cell migration, proliferation and tube
formation. Accumulating data indicate a role for circulating (C-EPC) and/or
bone marrow-derived endothelial precursor cells (BM-EPC) involved in the new
blood vessel formation, (Asahara et al.,
1997; Asahara and Isner,
2002; Gehling et al.,
2000; Grant et al.,
2002; Pelosi et al.,
2002), a process defined as postnatal vasculogenesis. Although
homing of C-EPCs at sites of new vessel formation has been shown, up to now
the exact role that BM-EPCs play in the tissue neovascularization has been a
matter of debate (Asahara et al.,
1999; Bagley et al.,
2003; Carmeliet and Luttun,
2001; Rajantie et al.,
2004). Recent data demonstrate that BM-EPC are recruited to the
vessel wall rather than to endothelium
(Rajantie et al., 2004) More
recent data suggest a role for tissue-bound precursor cells as source of
vascular cells and concomitantly accumulating macrophages during collateral
vessel growth (Khmelewski et al.,
2004). The existence of local tissue-bound adult precursor cells
has been reported for different organs
(Aarum et al., 2003;
Conboy et al., 2003) but not
yet for the wall of adult blood vessels
(Asahara and Kawamoto, 2004).
Our data suggest the existence of a `vasculogenic zone' in the adult human
vascular wall that is, to our knowledge, the first reported location for EPCs
outside the bone marrow. |
MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immunohistochemical analysis
Immunohistochemical staining for CD34, CD31, CD45 (final dilution: 1:50),
CD105, CD133, occludin (final dilution 1:25), CD68 (final dilution 1:250), von
Willebrand Factor, TIE2 (final dilution 1:200), VEGFR2, VE-cadherin (final
dilution 1:100), 
-smooth-muscle-actin (final dilution 1:400) and
CEACAM1 using the antibody 4D1/C2 (final dilution: 1:400) was performed on
paraffin wax sections obtained from 10 different human internal thoracic
arteries before and after the ring assay using antibodies recognizing
endothelial and precursor cell markers mentioned above. After
de-paraffinization and rehydration, the sections were subjected to
immunohistochemistry. The nickel-enhanced glucose oxidase technique was used
to develop the immunostaining as previously described
(Davidoff and Schulze, 1990;
Kilic and Ergun, 2001).
Negative controls were performed by omitting or replacing the primary
antibodies with Ig-matched isotypes or PBS. All sections were counterstained
with Calcium Red for 1-2 minutes to visualize the tissue structure.
In two cases, cells that sprouted into the collagen gel from HITA-wall during ring assay were isolated from the collagen gel, subsequently cultured in endothelial growth medium (MV Medium, PromoCell, Heidelberg, Germany) flasks and then transferred into chamber slides and used for immunocytochemistry to detect VEGF receptors, the angiopoietin receptor TIE2 and CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1). On some sections, double immunostaining was performed for VEGFR2 and CEACAM1 combining the glucose oxidase technique as described above and immune fluorescence method using the TRITC-conjugated secondary antibody.
Apoptotic rat macrophages were double stained with a PE-labeled anti-ED2 (Serotec) antibody and an anti-caspase 3 antibody (R&D Systems). For detection of immature endothelial cells, specimens were double stained with a polyclonal rabbit anti FLK1 antibody and a polyclonal goat anti VE-cadherin antibody (both from Santa Cruz, CA, USA). As secondary antibodies we used a biotinylated donkey anti-goat followed by rhodamine conjugated streptavidine (both from Dianova, Hamburg, Germany) for the VE-cadherin antibody and a Cy2-conjugated goat anti rabbit antibody (Dianova) for the FLK1 antibody. Negative controls were performed by omitting the first antibody.
Dispersion cell culture
Dispersion cell culture preparation was performed essentially according to
a technique described by Brewer (Brewer,
1997). Tissue pieces were minced and placed into PBS buffer. After
centrifugation for 10 minutes at 230 g, tissues were
subsequently resuspended in digestion solution containing trypsin/EDTA at
37°C for 5 minutes. Non-digested tissues were removed by filtration. The
suspension was centrifuged for 5 minutes at 230 g. The cell
suspension was placed into endothelial growth culture medium (MV Medium,
PromoCell), washed twice with endothelial growth culture medium, resuspended
in culture medium and plated on collagen- or fibronectin-coated wells using
six-well culture dishes (Nunc, Wiesbaden, Germany; 2 ml of ccll suspension per
well). After 24 hours of incubation at 37°C and 5% CO2, medium
was renewed and changed every second day.
Ring assay
Fragments of 10 different HITAs were cut into 1 mm thick rings and embedded
between two layers of collagen gel, consisting of collagen type 1, sodium
hydroxide, glutamine, sodium bicarbonate, vitrogen-gel (Invitrogen) and MEM
(10x) using 48-well cluster tissue culture plates. Embedded rings were
incubated at 37°C. The medium was changed every third day. Rings were
evaluated every day using a phase contrast microscope (Zeiss, Jena, Germany)
equipped with a digital camera (Zeiss, Jena Germany). In three experiments,
rings were fixed in Bouin's solution and embedded in paraffin wax for
immunohistochemistry. In two experiments, rings were fixed in glutaraldehyde
(5.5%), subsequently embedded in Epon 812 and used for electron microscopic
studies.
To monitor the cells of the HITA-wall during capillary sprouting within
collagen gel, we introduced 1 µl of an adenoviral construct (AdV5-GFP)
containing the reporter gene GFP into the outer layer of the HITA-wall under
observation with stereo microscope in two cases. Prior to this, the rings have
been placed into the first layer of collagen gel that was prepared in the
wells of a 48-well cluster culture dish. After addition of the second layer of
collagen gel the rings were observed twice daily using an inverse fluorescence
microscope (Zeiss, Jena, Germany) equipped with a digital camera (Zeiss). For
the preparation of adenoviral construct containing GFP gene we used the
homologous recombination technique of adenoviral vectors
(He et al., 1998) as we also
reported previously (Ergun et al.,
2000).
In vitro endothelial tube assay
Cells isolated from collagen gel after performing the ring assay were
cultured in endothelial growth medium (MV Medium, PromoCell) containing 5% FCS
until confluence. They were then trypsinized and seeded on the pre-prepared
polymerized collagen gel in 48-well cluster tissue culture dishes as described
previously (Ergun et al.,
2000). At different stages of confluence, the full MV medium was
replaced by hunger medium containing the 2% FCS without supplements. After 24
hours of culture, the cells in a part of the wells were stimulated with VEGF
(50 ng/ml) and FGF (10 ng/ml). They were then observed and photographed using
an inverse microscope (Zeiss) equipped with a digital camera.
Flow cytometry
Cells isolated from HITA wall and ring assays were incubated with
phycoerythrin (PE)-conjugated anti-CD34, anti-CD31, anti-VE-cadherin (all from
Pharmingen, Hamburg, Germany) or AC133 monoclonal antibodies (Miltenyi
Biotec). For two-color flow cytometry, FITC-conjugated anti-CD105 (Pharmingen)
or vWF (Serotec, Düsseldorf, Germany) MoAbs were used as described
(Gehling et al., 2000).
Isotype-matched mouse immunoglobulin served as controls. All incubations were
performed at 4°C. Cells were incubated with the MoAb for 30 minutes in the
presence of normal goat serum. After each incubation, cells were washed in PBS
containing 0.1% BSA. Single- and two-color flow cytometric analyses were
performed, using a FACS SCAN flow cytometer (Becton Dickinson, Heidelberg,
Germany) and Cell Quest software (Becton Dickinson). Each analysis included at
least 5000 events.
Depletion of CD105+ mature endothelial cells from HITA wall
Cells isolated from HITA walls were incubated with CD105-conjugated super
paramagnetic microbeads (Miltenyi Biotec), washed and processed using the
VarioMacs system as previously described. Purified cells were discarded,
except for an aliquot that was analyzed by flow cytometry.
Electron microscopy
The arteries used in the ring assay were perfused with 5.5%
phosphate-buffered glutaraldehyde under manual pressure after rinsing with
0.15 M phosphate-buffered saline. Small blocks of these arteries were excised
and immersed for 8 hours in the same fixative, followed by fixation in 1%
OsO4 for 2 hours. The blocks were then embedded in Epon 812. Serial
semithin sections (1 µm) were stained with Toluidine Blue/Pyronin. For
electron microscopy, ultrathin sections (about 80 nm thick) of perfused
arteries were obtained using a Porter MTB2 ultramicrotome contrasted with
uranyl acetate and lead citrate, and examined using a Philips EM 300
(Einthoven, Netherlands) transmission electron microscope.
In vivo studies
The right femoral artery was occluded in Spraque Dawley rats (400-500 g) as
previously described (Ito et al.,
1997). Bone marrow depletion was induced with intraperitoneal
injections of cyclophosphamide (80 mg/kg bodyweight 5 days prior to occlusion
followed by an additional injection of 40 mg/kg bodyweight 3 days prior to
occlusion), as previously described
(Khmelewski et al., 2004).
Isolation and cultivation of tissue resident macrophages
Tissue pieces of collateral arteries were minced in CBFHH buffer and
subjected to numerous rounds of trypsin and DNAse digestion as previously
described. After filtration (30 µm filter), cells were incubated with an
anti-ED2 antibody (Serotec), followed by incubation with IgG-conjugated
microbeads (Miltenyi Biotec). Magnetically labeled cells were purified using a
VarioMacs as previously described (Gehling
et al., 2000). Cells were plated in a chamber slide and cultured
in serum-free Macrophage Medium (Invitrogen, Karlsurhe, Germany).
Proliferation assay
Isolated ED2+ cells were grown in Macrophage SMF Medium
(Invitrogen) that was supplemented with macrophage-colony stimulating factor.
As positive control, CD133+ cells isolated from G-CSF mobilized
peripheral blood were cultured and expanded as previously described
(Loges et al., 2004).
Immunoselected CD133+ cells and ED2+ cells were
incubated with BrdU (0.3 mg/ml) for 3 days and were subsequently stained using
the BrdU Flow Kit (Pharmingen). Positive staining was evaluated using a Zeiss
fluorescence microscope (Zeiss, Jena, Germany).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-smooth muscle actin (Fig.
1E,F), indicating that they do not belong to the vascular smooth
muscle cells. In this mural CD34+ cell zone, KDR+
(VEGFR2+) and TIE2+ cells were also found, whereas no
staining was detected for CD105 (endoglin, a TGFß receptor) and
VE-cadherin (not shown). Both CD34 and CD31 marked endothelial cells lining
the lumen of HITA and the lumina of vasa vasorum as expected (not shown).
Similar results were obtained after immunostaining for von Willebrand Factor
(vWF) (not shown).
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). After 5-7 days of culture in the gel, we found
capillary-like outgrowth into the vessel lumen as well as into the periphery
of the rings (Fig. 2A,B).
Histological sections confirmed the capillary formation
(Fig. 2C). Finally, electron
microscopic studies revealed the establishment of inter-endothelial contacts
(Fig. 2D).
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Immunostaining for CEACAM1, a cell-cell adhesion molecule with angiogenic properties that is upregulated in endothelial cells of only angiogenicly activated or newly formed capillaries, and for CD34 using serial sections of HITA after the ring assay showed the presence of CEACAM1 in new endothelial sprouts within the vessel lumen (Fig. 4A) and in capillary sprouts into the outside of the rings (Fig. 4B). Immunostaining for CD34 on the serial section following the section presented in Fig. 4B confirms the formation of these capillaries by the VW-EPC (Fig. 4C). There was no CEACAM1 staining in the HITA sections without the ring assay, as expected except few granulocytes adherent to the endothelium lining vascular lumen (Fig. 4D).
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Corresponding to the immunohistochemical results above isolated cells from collagen gel after ring assay also exhibited strong staining for CEACAM1 (Fig. 5A) and the majority of them were positive for both CEACAM1 and VEGFR2 (Fig. 5B). They were also positive for TIE2 (Fig. 5C), although no specific staining was seen in the corresponding control section (Fig. 5D).
To test the capability of sprouting cells to form capillary-like tubes under in vitro conditions, the cells isolated from collagen gel after performing the ring assay were used in endothelial tube assay at different stage of cell confluence. In all cases, they formed a tight network of capillary-like tubes in response to VEGF (Fig. 6A,C) and to FGF2 (not shown), although no such tubes were observed in corresponding controls (Fig. 6B,D).
To determine whether hematopoietic and/or mesodermal cells are also present in the HITA wall, we performed immunostaining for CD14, CD133 and CD45. However, only few cells in the CD34+ cell zone were also positive for the pan-leukocyte marker CD45 (Fig. 7A,B), whereas no staining was found for AC133 and CD14, probably owing to the possible failure of the antibodies in paraffin tissue. In a further step, we wanted to explore whether macrophages are present in the HITA wall and may contribute to the capillary sprouting from the HITA rings as shown above. Immunostaining for the macrophage marker CD68 on paraffin sections of untreated HITA revealed only single and randomly distributed macrophages, mostly associated with the lumina or the wall of vasa vasorum (Fig. 7C) but not located in the CD34+ cell zone. Unexpectedly, the number of CD68+ cells within the HITA wall dramatically increased during culture of the HITA rings in collagen gel (Fig. 7D). To our surprise, this proliferation and accumulation of macrophages in the HITA rings was strongly localized in the CD34+ cell zone of the HITA wall, increasing with culture time and expanded to the entire adventitial layer. Additionally, we found that macrophages migrated from this area into the gel outside the HITA rings (Fig. 7E) and few of them were involved in the capillary-like tube formation within the collagen gel (Fig. 7F). To further characterize the cells migrating into the collagen gel, we extracted them from the gel and cultured them in culture dishes to achieve a sufficient amount for FACS analysis. These studies confirmed our immunohistochemical results shown above and demonstrated the existence of CD34+ cells (Fig. 7G), CD105+ cells (Fig. 7H), AC133+ cells (Fig. 7I) and CD45+ cells (Fig. 7J). The surface expression pattern of the cells studied here is summarized in Table S1 (see supplementary material).
|
).
Recently, it has been shown that tissue resident macrophages rather than
circulating and bone marrow-derived macrophages contribute to collateral
vessel growth (Khmelewski et al.,
2004). Using this in vivo model, we obtained findings that were
similar to those obtained from HITA rings studies above. Immunostaining
studies on sections of collateral arteries grown in rats after femoral artery
ligation and depletion of bone marrow cells by treatment with cyclophosphamide
revealed an accumulation of macrophages within the wall of these arteries
within 3 days after arterial occlusion
(Fig. 8C). Immunostaining of
these vessels for VEGFR2 marked endothelial cells lining the arterial lumen
(Fig. 8D). As no considerable
number of mature macrophages was found in the wall of the collateral arteries
prior to their activation through femoral artery ligation and because
accumulation of macrophages upon femoral artery occlusion did not differ
between monocyte-competent and monocyte-deficient animals, we hypothesize that
the accumulating mature macrophages in the wall of these arteries originate
from vascular wall resident precursor cells. Furthermore, immunoselected
tissue-resident macrophages themselves did not proliferate in vitro
(Fig. 8E), although we were
able to show that local macrophage accumulation during collateral growth
occurs because of pronounced cellular proliferation. CD133+ cells
isolated from human peripheral blood and used as positive control show an
extensive BrdU incorporation (Fig.
8F). Additional evidence for the existence of tissue-resident
macrophage precursors was gained in our HITA ring assays. In the absence of
circulating blood cells, there was a pronounced increase of CD68+
cells, representing mature macrophages within the wall during cultivation.
These cells did not only accumulate in the adventitial space but were also
integrated into vascular sprouts.
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; Gehling et al.,
2000; Ribatti et al.,
2003). We wanted to explore whether the CD34+ cells in
the wall of HITA can be induced to capillary outgrowth by tumor cells. To
reach this goal, we established a modified arterial ring assay, in which cells
of the prostate cancer cell line DU-145 were implanted into a collagen gel
containing the rings of HITA. Although capillary-like sprouting from untreated
HITA rings was initially observable after 3-4 days of culture, the presence of
DU-145 cells resulted in capillary sprouting at the outside of HITA rings, in
most experiments within 24 hours of culture
(Fig. 9A,B). No sprouts were
seen at this time in ring cultures without tumor cells
(Fig. 9C,D). The amount of
capillary outgrowth at the outside of HITA rings and the very short time in
which they developed suggest that they are derived from vascular wall resident
CD34+ EPCs rather than from sprouting of pre-existing mature
endothelial cells lining the HITA lumen and/or the lumina of vasa vasorum. Our
data clearly indicate that these cells can be acquired for tumor
vascularization. The general relevance of VW-EPCs for tumor vascularization is
that they are present not only in the wall of HITA but also in the wall of
proper blood vessels of organs where primary tumors develop. Indeed, detailed
immunohistochemical studies on sections of different human organs, including
urinary bladder, testis, prostate, kidney, lung, heart, liver and brain
disclosed the existence of vascular wall resident CD34+ but
CD31- cells in the same zone of the vessel as demonstrated
in HITA. CD34+ cells were found in mid-sized and large arteries and
veins, e.g. human prostate blood vessels (9E-H).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) but no
exact location of these cells within the vascular wall has been identified in
this study. The ex vivo marking of the cells of the vasculogenic zone by
AdV5-GFP-system demonstrates that this zone of vascular wall serves a niche
containing VW-EPC, which are capable of forming capillary sprouts. During this
process, the cells became positive for endothelial markers and cell-adhesion
molecules such as VEGF receptors, TIE2, VE-cadherin and CEACAM1, indicating
their predetermination for endothelial cells. This interpretation is also
supported by our findings showing the co-existence of CEACAM1 and CD34 in
capillary-like sprouts from HITA-wall, as CEACAM1 is detectable in endothelial
cells that are activated angiogenicly or form new endothelial tubes, and in
endothelial cells from AC133-positive progenitor cells
(Ergun et al., 2000;
Gehling et al., 2000).
|
A further interesting aspect of the VW-EPC is that they can be activated to formation of capillary sprouts by tumor cells as shown here in co-culture of the HITA rings with the prostate cancer cell line DU-145. Together with the findings here showing the presence of the CD34+ cells in the same location of vascular wall of arteries and veins of several organs, including prostate these results implicate a new view of local mechanisms providing tumor vascularization. Tumor cells can not only activate the pre-existing mature endothelial cells and C-EPCs but also EPCs residing in the wall of neighboring blood vessels that can apparently be easily acquired for the formation of new vessels. The better understanding of biology of these cells is necessary to understand the complex local processes that regulate tumor vascularization.
One of the cell populations we detected in the vasculogenic zone are the
CD45+ cells. However, at this time it is not possible to determine
exactly the source and the fate of the CD45+ cells residing in this
zone of the vascular wall. It has been shown that a part of differentiating
endothelial cells lining the embryonic aorta are capable of differentiating
into CD45+ cells and to serve as basis for hematopoiesis
(Jaffredo et al., 1998). As we
did not find a significant change in the number of CD45+ cells
within the HITA wall after ring assay, we postulate that this may be due to
their differentiation into macrophages or other hematopoietic cells. However,
although CD34+ cells, CD105+ cells,
VE-cadherin+ cells, occludin+ cells, KDR+
cells, TIE2+ cells and occasionally CD68+ cells were
found to be involved in capillary sprouts from HITA, apparently no
CD45+ cells participated in the capillary formation, although a few
of them had migrated into the collagen gel outside the rings. These data are
in line with previous findings showing that CD45+ cells originating
from embryonic aortic endothelium became negative for endothelial markers once
they expressed CD45 (Jaffredo et al.,
1998).
It has been shown that the migration and proliferation capacity of BM-EPCs
decreases at higher ages (Dimmeler and
Zeiher, 2004; Scheubel et al.,
2003). In contrast to these circulating EPCs, the vascular wall
resident EPCs of the vasculogenic zone are not directly exposed to shear
stress, as they are distant from the luminal forces acting on mature
endothelial and circulating EPCs. Moreover, this zone apparently contains
multipotent stem cells that are capable of differentiating into macrophages
but probably also into HPCs. These findings lead to the assumption that this
vasculogenic zone of the vascular wall has not only the capability to serve as
a source for vascular cells forming new blood vessels, but also to serve as a
reservoir for inflammatory cells important for local immune response. As blood
vessels containing this `vasculogenic zone' are apparently present in all
organs rather than being a unique phenomenon, we hypothesize that pre-existing
blood vessels serve as basis for tumor vascularization not only by
angiogenesis but also by postnatal vasculogenesis. Development of systems and
techniques manipulating the function of these cells may be of therapeutic
relevance for tissue vascularization as well as for anti-angiogenic tumor
therapy.
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ACKNOWLEDGMENTS |
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Footnotes |
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* These authors equally contributed to this work 
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42,2073
-2080.
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