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Vessel Formation Is Induced Prior to the Appearance of Cartilage in BMP-2-Mediated Heterotopic Ossification

Article · October 2009with33 Reads
DOI: 10.1359/jbmr.091031 · Source: PubMed
Abstract
Heterotopic ossification (HO), or endochondral bone formation at nonskeletal sites, often results from traumatic injury and can lead to devastating consequences. Alternatively, the ability to harness this phenomenon would greatly enhance current orthopedic tools for treating segmental bone defects. Thus, understanding the earliest events in this process potentially would allow us to design more targeted therapies to either block or enhance this process. Using a murine model of HO induced by delivery of adenovirus-transduced cells expressing bone morphogenetic protein 2 (BMP-2), we show here that one of the earliest stages in this process is the establishment of new vessels prior to the appearance of cartilage. As early as 48 hours after induction of HO, we observed the appearance of brown adipocytes expressing vascular endothelial growth factors (VEGFs) simultaneous with endothelial progenitor replication. This was determined by using a murine model that possesses the VEGF receptor 2 (Flk1) promoter containing an endothelial cell enhancer driving the expression of nuclear-localized yellow fluorescent protein (YFP). Expression of this marker has been shown previously to correlate with the establishment of new vasculature, and the nuclear localization of YFP expression allowed us to quantify changes in endothelial cell numbers. We found a significant increase in Flk1-H2B::YFP cells in BMP-2-treated animals compared with controls. The increase in endothelial progenitors occurred 3 days prior to the appearance of early cartilage. The data collectively suggest that vascular remodeling and growth may be essential to modify the microenvironment and enable engraftment of the necessary progenitors to form endochondral bone.
Figures
Vessel Formation Is Induced Prior to the Appearance of
Cartilage in BMP-2-Mediated Heterotopic Ossification
Christine Fouletier Dilling ,
1
Aya M Wada,
2
Zawaunyka W Lazard,
1
Elizabeth A Salisbury ,
1
Francis H Gannon,
3
Tegy J Vadakkan,
2
Liang Gao,
2
Karen Hirschi,
1,4
Mary E Dickinson,
2
Alan R Davis,
1,5
and Elizabeth A Olmsted-Davis
1,5
1
Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX, USA
2
Departments of Molecular Physiology and Biophysics and Medicine, Baylor College of Medicine, Houston, TX, USA
3
Department of Pathology, Baylor College of Medicine, Houston, TX, USA
4
Department of Pediatrics, Pediatrics-Nutrition, Baylor College of Medicine, Houston, TX, USA
5
Department of Pediatrics, Hematology-Oncology, Baylor College of Medicine, Houston, TX, USA
ABSTRACT
Heterotopic ossification (HO), or endochondral bone formation at nonskeletal sites, often results from traumatic injury and can lead to
devastating consequences. Alternatively, the ability to harness this phenomenon would greatly enhance current orthopedic tools for
treating segmental bone defects. Thus, understanding the earliest events in this process potentially would allow us to design more
targeted therapies to either block or enhance this process. Using a murine model of HO induced by delivery of adenovirus-transduced
cells expressing bone morphogenetic protein 2 (BMP-2), we show here that one of the earliest stages in this process is the establishment
of new vessels prior to the appearance of cartilage. As early as 48 hours after induction of HO, we observed the appearance of brown
adipocytes expressing vascular endothelial growth factors (VEGFs) simultaneous with endothelial progenitor replication. This was
determined by using a murine model that possesses the VEGF receptor 2 (Flk1) promoter containing an endothelial cell enhancer driving
the expression of nuclear-localized yellow fluorescent protein (YFP). Expression of this marker has been shown previously to correlate
with the establishment of new vasculature, and the nuclear localization of YFP expression allowed us to quantify changes in endothelial
cell numbers. We found a significant increase in Flk1-H2B::YFP cells in BMP-2-treated animals compared with controls. The increase in
endothelial progenitors occurred 3 days prior to the appearance of early cartilage. The data collectively suggest that vascular remodeling
and growth may be essential to modify the microenvironment and enable engraftment of the necessary progenitors to form
endochondral bone. ß2010 American Society for Bone and Mineral Research.
KEY WORDS: BONE MORPHOGENTIC PROTEIN TYPE 2; HETEROTOPIC OSSIFICATION; VESSEL FORMATION
Introduction
Endochondral bone formation is thought to proceed through
an ordered series of events starting with the proliferation and
‘‘condensation’’ of presumptive mesenchymal cells to form
avascular cartilage. Hence it is presumed that the lack of
vasculature and associated cellular replication creates the hypoxic
environment necessary for chondrogenic differentiation. Using a
murine model that possesses the VEGF receptor 2 (Flk1) promoter
containing an endothelial cell enhancer driving the expression of
YFP,
(1)
we confirmed recent data from our laboratory using a
model of heterotopic ossification that suggested that vessels may
play an essential role in the induction of chondrogenesis.
(2)
It has been well established that vessel formation plays a key
role in late events during the process of bone formation. Vessels
invade the perichondrium and hypertrophic zone and are
required for the replacement of cartilage by bone.
(3)
The
angiogenic factor vascular endothelial growth factor (VEGF)
promotes vascular invasion via specific receptors, including Flk1
(VEGF receptor 2) expressed in endothelial cells, in the
perichondrium or surrounding tissue.
(4,5)
These events of cartilage
matrix remodeling and vascular invasion are necessary for
the migration and differentiation of osteoblasts and osteoclasts,
which remove mineralized cartilage matrix and replace it with
bone. However, much less is known about the role of vessel
formation prior to the appearance of the precartilage tissue.
During normal wound repair, a series of cell signaling events is
induced by the hypoxic state of the tissues, resulting in
upregulation of hypoxia inducible factor (HIF1), which, in turn,
upregulates a series of factors including several VEGFs (A, B, and
ORIGINAL ARTICLE J
J
BMR
Received in original form March 29, 2009; revised form September 30, 2009; accepted October 15, 2009. Published online October 17, 2009.
Address correspondence to: Alan R Davis, Center for Cell and Gene Therapy, One Baylor Plaza, Houston, TX 77030, USA. E-mail: ardavis@bcm.tmc.edu
C Fouletier Dilling and AM Wada contributed equally to the work.
Journal of Bone and Mineral Research, Vol. 25, No. 5, May 2010, pp 1147–1156
DOI: 10.1359/jbmr.091031
ß2010 American Society for Bone and Mineral Research
1147
D), leading to vessel formation. Hypoxia-induced angiogenesis
has been proposed to be necessary for creating specialized
vessels that facilitate progenitor homing and engraftment into
damaged tissues.
(6)
Little is known about whether such a process
plays a key role in the repair of bone.
Using a model of de novo bone formation to identify the
earliest events in this process, we have demonstrated that
myelomesenchymal stem cells are recruited to the tissues to
form the early cartilage.
(7)
One of the earliest events in this model
is the appearance of brown adipocytes. These cells are capable of
using their uncoupled aerobic respiration to reduce localized
oxygen tension and effectively pattern the newly forming
cartilage condensations.
(8)
This is consistent with in vitro data
showing that bone marrow–derived mesenchymal stem cells
can undergo chondrogenesis in the presence of bone
morphogenetic protein 2 (BMP-2) and low oxygen.
(9)
We also
observed the appearance of vessels lining the edges of the
perichondrial region, separated only by brown adipose tissue,
suggesting that perhaps the reduction in oxygen tension
coordinately activates new vessel formation in the region.
(8)
Thus these progenitors may indeed be recruited to the site of
new bone formation through the vasculature. In this study we
focused on defining this tentative early vessel formation.
To determine this, we chose to employ a transgenic mouse
model that expresses the fusion protein human histone H2B
with enhanced yellow fluorescent protein (EYFP) (H2B:YFP) in
endothelial cells under the regulation of a Flk1 promoter/
enhancer fragment (Flk1-H2B::YFP).
(1)
Recent improvements in
genetically encoded fluorescent protein expression in animal
models, along with advances in optical imaging and image
analysis software, have enabled the analysis of many aspects of
tissue development at a cellular level.
(10)
Previous studies using
this transgenic animal indicates that Flk1-H2B::YFP expression is
restricted to endothelial cells of smaller and/or newly forming
vessels,
(8)
thus providing a mechanism for quantification of new
vessels.
Here we demonstrate new vessel formation within the tissues
prior to the appearance of the presumptive cartilage. Quantifica-
tion of the number of endothelial cells shows that one of the
first steps of bone formation is to induce additional endothelial cell
proliferation. Histologic analysis shows that increases in endothelial
cell numbers are evident just prior to the influx of chondrocytic
progenitors. Immunohistochemical analysis of the tissues prior to
the mesenchymal condensations revealed a rapid and transient
expression of VEGF-A and -D from the brown adipocytes. The data
collectively suggest that the brown adipocytes may play a key role
in establishing patterning of the cartilage via regulation of oxygen
tension within the tissues through induction of both aerobic
respiration and early angiogenesis.
Materials and Methods
Cell culture
A murine C57BL/6-derived cell line (MC3T3-E1) was obtained
from American Type Culture Collection (Manassas, VA), propa-
gated in amodified essential medium (a-MEM) supplemented
with 10% FBS (Hyclone, Logan, UT, USA), 100 U/mL penicillin,
100 mg/mL streptomycin, and 0.25 mg/mL amphotericin B (Life
Technologies, Inc., Gaithersburg, MD, USA). Briefly, the cells were
grown in DMEM supplemented as described earlier and cultured
at a subconfluent density to maintain the phenotype. All cell
types were grown at 378C and 5% CO
2
in humidified air.
Transduction of cells with adenovirus in the presence of
GeneJammer adenoviruses
Replication defective first-generation human type 5 adeno-
virus (Ad5) deleted in regions E1 and E3 was constructed to
contain the cDNA for BMP-2 in the E1 region of the viral
genome.
(11)
The virus particle (vp) to plaque-forming unit (pfu)
ratios were 55 and 200 for Ad5-BMP-2 and Ad5-empty,
respectively, and all viruses were shown to be negative for
replication-competent adenovirus.
The C57BL/6 cell line, or MC3T3-E1 (1 10
6
), was transduced
with Ad5-BMP-2 or Ad5-empty cassette control virus at a
concentration of 5000 vp/cell with 1.2% GeneJammer, as
described previously.
(12)
Heterotopic bone assay
The transduced cells were resuspended at a concentration of
510
6
cells/100 mL of PBS and then delivered through
intramuscular injection into the hind limb quadriceps muscle
of Flk1 mice. Animals were euthanized at daily intervals, and hind
limbs were harvested, embedded, and stored at 808C. All
animal studies were performed in accordance with standards of
the Baylor College of Medicine, Department of Comparative
Medicine, after review and approval of the protocol by the
Institutional Animal Care and Use Committee (IACUC).
Histologic analysis and staining analysis
Soft tissues encompassing the site of new bone formation were
isolated from the rear hind limbs of the mice. Both the skin and
skeletal bone were removed from the tissues prior to freezing.
Serial sections (15 mm) were prepared that encompassed the
entire tissue (approximately 50 sections per tissue specimen). We
then performed hematoxylin and eosin staining on every fifth
slide, which allowed us to locate the region containing either our
delivery cells or the newly forming endochondral bone. Serial
unstained slides were used for immunohistochemical staining
(either single- or double-antibody labeling). For double-antibody
labeling, samples were treated with both primary antibodies
simultaneously, followed by washing and incubation with
respective secondary antibodies, used at 1:500 dilution, to
which Alexa Fluor 488, 594, or 647 was conjugated. Primary
antibodies were used as follows: SMA mouse monoclonal used at
1:200 dilution (Sigma Chemical Company, St Louis, MO, USA),
CD31 rat monoclonal used at 1:75 dilution (BD Pharmingen, San
Diego, CA, USA), Flk1 goat polyclonal used at 1:100 dilution (R&D
Systems, Minneapolis, MN, USA), Ki67 rat monoclonal used at
1:100 (Dako, Carpinteria, CA, UDA), and VEGF-D goat polyclonal
used at 1:100 dilution (Santa Cruz Biotechnology, Inc., Santa Cruz,
CA, USA). Stained tissue sections were examined by confocal
microscopy (LSM 510 META, Zeiss, Inc., Thornwood, NY, USA)
using a 20/0.75NA objective lens.
1148 Journal of Bone and Mineral Research DILLING ET AL.
Flk1-positive cell quantification in BMP-induced tissues
To quantify the increase in YFP-positive cells in the BMP-induced
tissues, frozen sections across these tissues were counterstained
with 4,6-diamidino-2-phenylindole (DAPI), and the YFP expression
was compared with that obtained in the control tissues. First, a
series of low-magnification (5.4and 12) bright-field images of
a tissue section was taken and overlapped to reconstruct the
tissue section using Adobe Photoshop CS3 (San Jose, CA, USA).
The reconstructed montage image was used to measure the area
of the tissue section using a manual contour-tracing method
(Zeiss Axiovision). The area of each of the frozen sections was
calculated in a similar manner. Area measurements are used to
determine the density of labeled cells, as indicated below.
High-resolution (10/NA0.45, 1024 1024 pixels) dual-chan-
nel images of tissue sections nuclear stained with DAPI were
taken using a confocal microscope (Zeiss LSM 510 META). In each
image, the number of nuclei in the DAPI and YFP channels was
counted using a modified watershed segmentation algorithm
(FARSIGHT, Farsight Image Segmentation Software, courtsey of
Badri Roysam, RPI, Troy, NY), which makes use of both intensity
and volume thresholds to distinguish two nuclei as separate. All
the nuclei counted using the software were DAPI
þ
. The fraction
of DAPI-stained nuclei marked by YFP was counted as YFP
þ
. The
density of YFP
þ
cells in a tissue section was defined as the ratio of
the number of YFP
þ
nuclei in the tissue section measured from
the high-magnification images to the area of the tissue section
measured from the low-magnification images. The density of the
YFP
þ
nuclei was calculated for a number of control and BMP-
treated tissues at 2 and 4 days after injection. The ratios then
were averaged over the various control and BMP-2-treated
tissues. The pvalues were calculated using a Student’s ttest.
Flk1-YFP
þ
cell association analysis
To characterize the cell type(s) that express YFP in the adult
muscle tissue, we performed immunoflourescence studies using
endothelial cell marker CD31 and Flk1 antibodies; for vascular
smooth muscle cells, we used smooth aactin (SMA). Association
of cells expressing YFP with immunolabeled cells was analyzed
using (FARSIGHT, RPI) and a custom program written in MATLAB
(MathWorks, Natick, MA, USA). After identification of each
nucleus by DAPI staining, YFP
þ
and YFP
cells then were
analyzed for association with the fluorescent signals of each
antibody. An intensity threshold was applied to the red channel
in each image to identify a cell positive or negative for the
immunofluorescent signal. Each identified nucleus and over-
lapping red channel were counted as CD31
þ
, Flk1
þ
, or SMA
þ
and
then as either YFP
and YFP
þ
. Colocalization percentages are
shown in the supplemental data section (Table S-1) and
described in detail the YFP
þ
cell types. The number of YFP
þ
/
Ki67
þ
nuclei in an area of the tissue was calculated by adding the
YFP
þ
/Ki67
þ
in each of the confocal images taken within the area.
The area fraction of YFP
þ
/Ki67
þ
was defined as the total number
of YFP
þ
/Ki67
þ
in the images taken within the area divided by the
number of images. The area fraction was measured for five
different areas, and the average area fraction was calculated for
control and BMP-treated tissues for every fifth slide sectioned
throughout the entire hind limb. The area fractions of YFP
þ
/
Ki67
þ
nuclei in the control and the BMP-treated tissues on day 2
were 3.97 2.96 and 6.11 1.76, respectively. The area fractions
for day 4 were 5.04 0.72 and 6.41 1.41 in the control and the
BMP-treated tissues. Based on the Student’s ttest, the pvalue for
the day 2 data was .21, and that for the day 4 data was .10. Taken
together, the data support the trend that the YFP
þ
/Ki67
þ
population increases on day 2 and day 4 after the BMP treatment.
qRT-PCR
Nonskeletal tissues (n¼4 per group) surrounding the site of
injection of the Ad5-BMP-2 or Ad5-control transduced cells were
isolated at daily intervals for 7 days and prepared as total RNA
using a Trizol reagent (Life Technologies, Carlsbad, CA, USA) in
accordance with the manufacturer’s specifications. The two
groups of RNAs were subjected to qRT-PCR analysis in parallel,
and the C
t
values obtained normalized to both internal 18S
ribosomal RNA used in multiplexing and to each other to remove
changes in gene expression common to both the BMP-2 and
control tissues by using the method of DDC
t
along with Taqman
primers and probes (Applied Biosystems, Carlsbad, CA, USA) as
described previously.
(8)
Results
Upregulation of vessel markers prior to the onset of
chondrogenesis
We have previously described a model of rapid endochondral
bone formation
(13)
in which mineralized bone is observed 7 days
after the initial induction with BMP-2. Observation of vessels
lining the newly forming perichondrium suggests that vessels
may undergo replication prior to chondrogenesis. To confirm this
hypothesis, we examined tissues, at 24-hour intervals over the
period leading up to chondrogenesis (day 5), for the presence or
absence of endothelial cell replication. Figure 1 shows the
coexpression of the endothelial cell–specific factor von Willibrand
factor (vWF) (red) and Ki67 (green), a marker of cellular
replication,
(14)
in the vessels from tissues that received Ad5-
BMP-2-transduced cells starting at 24 hours and going to 5 days
(panels A–E, respectively). As can be seen in Fig. 1B, we did observe
overlap of these two markers (yellow) in tissues receiving the Ad5-
BMP-2-transduced cells, whereas no replicating endothelial cells
were observed in the control tissues (Fig. 1F). We did not attempt
to quantify the amount and apparent timing of replication using
this method because vWF is an extracellular matrix protein. Instead,
we employed the Flk1-H2B::YFP model for quantifying endothelial
progenitor replication over the course of early bone formation.
Flk1-H2B::YFP in vessels
We next determined if there was a significant increase in the
number of Flk1
þ
endothelial progenitors during bone induction,
consistent with new vessel formation, prior to chondrogenesis.
We chose to use the Flk1-H2B::YFP mouse model,
(1)
in which new
vessel formation could be readily quantified within the muscle
tissues. Flk1 is a VEGF receptor transiently expressed on
endothelial cells and is thought to contribute to VEGF-induced
endothelial cell replication.
(15)
Therefore, quantification of the
nuclear YFP expression within tissues from animals receiving
VESSELS FORM PRIOR TO CARTILAGE Journal of Bone and Mineral Research 1149
either Ad5-BMP-2- or Ad5-empty-transduced cells allowed us to
to quantify increases in the number of endothelial progenitors
within the muscle prior to cartilage formation. We previously
quantified the association of Flk1-H2B::YFP mouse model with
other endothelial cells markers such as CD31 and found them to
be 95% overlapping (see Supplemental Data below). Frozen
sections were prepared by serial sectioning from Flk1-H2B::YFP
adult hind limb soft tissue (n¼4 per group) consisting of three
groups, those receiving (1) cells transduced with Ad5-BMP-2, (2)
cells transduced with Ad5-empty cassette control virus, and (3)
normal mouse muscle. To ensure uniform quantification and
adequate sampling, the entire region of soft tissues in the hind
limb was sectioned, and approximately every fifth section was
analyzed for YFP expression.
To quantify differences in the number of endothelial
progenitors, the number of YFP
þ
cells per the total number of
DAPI
þ
cells was determined using automated segmentation
methods (see Materials and Methods). The total YFP
þ
cells also
was quantified per total area of the tissue section to ensure that
there was no bias in the fields of view chosen for image analysis
(see Materials and Methods). The total area of each tissue section
was determined using a montage of images that were collected
using wide-field microscopy (Fig. 2A,F). As can be seen in Fig. 2,
we found Flk1-H2B::YFP
þ
cells in both tissues receiving Ad5-
empty-transduced cells (Fig. 2B–E) and Ad5-BMP-2-transduced
cells (Fig. 2G–O). These panels are higher-magnification confocal
images of the region within the corresponding white box on the
lower-magnification high-resolution wide-field montage of the
entire tissues (Fig. 2A, control; Fig. 2F, BMP-2). The results of
the quantification (Fig. 3) shows the average number of Flk1-
H2B::YFP
þ
cells on days 2 and 4. Analysis of the entire soft tissue
within several mice showed a significant elevation in tissues
receiving the Ad5-BMP-2-transduced cells ( p¼.017, day 2,
Fig. 3Aand p¼.006, day 4, Fig. 3B) compared with control on
both days 2 and 4. The peak was approximately 2 days after
induction of bone formation, with no statistically significant
difference between these results and those obtained in tissue
sections isolated 4 days after induction.
Endothelial progenitors undergo replication in tissues
receiving Ad5-BMP-2-transduced cells
In the tissues receiving cells transduced with a control adenoviral
vector we observed randomly scattered YFP
þ
cells along the
vessel structures, whereas in the tissues receiving Ad5-BMP-2-
transduced cells we saw clustering of the YFP
þ
cells (Fig. 2). This
prompted us to question whether the Flk1-H2B::YFP progenitors
could be replicating, so we next quantified the number of Flk-
H2B::YFP
þ
cells in these tissues. Representative images used for
quantification of YFP
þ
cell proliferation activity are shown in
Fig. 4. Replicating endothelial progenitors were defined as nuclei
positive for both Flk1-H2B::YFP (yellow) and the cell proliferation
marker Ki67 (red; Fig. 4). In both control and treated animals, we
also observed proliferating cells positive for Ki67 that did not
overlap with Flk1-H2B::YFP. Quantification of cells positive for
both Flk1-H2B::YFP and Ki67 (Fig. 4) indicates a large number of
replicating endothelial progenitors in both control and BMP-2-
treated tissues at 2 days after induction. Therefore, the
percentage of dual-positive cells was not significant at this
early time point compared with control. However, by 4 days after
induction with BMP-2, we observed much fewer replicating Flk1-
H2B::YFP cells in the control group and significantly more in the
experimental group (Fig. 4). This difference was found to be
statistically signifcant.
Vascular endothelial growth factor (VEGF) mRNA
expression
Endothelial progenitor replication appeared to start within 48
hours of induction with BMP-2. This correlated with a significant
elevation in VEGF-D [also termed fos-induced growth factor (FIGF)]
and VEGF-A RNA expression (Fig. 5). Figure 5 shows the changes
in VEGF mRNA expression from day 1 after injection of Ad5-BMP-
2-transduced cells until day 6, as determined by real-time RT-PCR
(qRT-PCR). Both VEGF-A and VEGF-D mRNA expression was
significantly increased on days 2 and 4 after induction of bone
formation. VEFG-B and VEGF-C RNA, however, remained on the
same level throughout the time course. Although the data
cannot differentiate between expansion of cells expressing
VEGF-A and -D and elevated transcription within cells residing in
the area, the results suggest that these potent endothelial
growth factors are rapidly and transiently increased within the
site of new bone formation prior to the onset of cartilage.
Role of brown adipose in vessel formation
The data collectively suggest that vessel replication is occurring
simultaneously with elevated expression of VEGFs within the
tissues. Since one of the earliest events observed in our model is
the recruitment and expansion of brown adipocytes,
(8)
we next
chose to determine if these cells might be expressing the VEGFs.
Immunohistochemical analysis of Flk1-H2B::YFP tissues that
received either Ad5-BMP-2- or Ad5-empty-transduced cells
Fig. 1. Immunohistochemical analysis of endothelial cell replication in
tissues isolated at daily intervals after induction of bone formation with
cells expressing BMP-2. (A–E) On days 1, 2, 3, 4, and 5, respectively, after
injection of BMP-2-producing cells, paraffin sections were prepared and
stained with an antibody against Ki67, followed by a secondary antibody
conjugated to Alexa fluor 488 (green) mixed with an anti–von Willibrand
Factor (vWF) antibody, followed by a secondary antibody conjugated to
Alexa fluor 547 (red). ( F) A representative image, similar staining, taken
from tissues isolated from mice injected with cells transduced with a
control vector (Ad5-empty).
1150 Journal of Bone and Mineral Research DILLING ET AL.
showed colocalization of VEGF-D (green, Fig. 6c) and the brown
adipocyte-specific marker uncoupling protein 1 (UCP1; red,
Fig. 6d) (day 2). As can be seen in Fig. 6e, expression of UCP1
overlaps expression of VEGF-D in cells that are adjacent to the
Flk1-H2B::YFP
þ
endothelial progenitors, suggesting that the
brown adipocytes may be contributing to the new vessel
formation. We observed additional fluorescence within the
surrounding muscle that appears to be punctate and not cell-
associated. This staining may represent VEGF-D protein
secreted in the tissues. To confirm the cell-specific expression
Fig. 2. Wide-field and confocal images of whole tissue sections and quantification of Flk1-H2B::YFP cells. (A,F) Representative montages of low-
magnification gray-scale images (1 pixel ¼0.003 mm) used for calculating total area for tissue sections. A single representative tissue section is depicted
after the entire hind limb muscles that encompassed the injection site were isolated 2 days after receiving an intramuscular injection of cells transduced
with either Ad5-empty control vector (A) or Ad5-BMP-2 (B) and sectioned at 15 mm thickness. Although every fifth section across the entire tissue was
analyzed, we show only a single representative image of each type. The corresponding regions with positive YFP signal, shown by the boxed areas, were
imaged by confocal microscopy (B–E,G–O) for counting the YFP
þ
cell numbers.
VESSELS FORM PRIOR TO CARTILAGE Journal of Bone and Mineral Research 1151
of VEGF-D in the brown adipocytes, we performed additional
immunostaining (Fig. 6f). Positive expression of VEGF-D (brown
staining) was observed only in the brown adipocytes, again
suggesting that these cells may play a role in the regulation of
new vessels.
Discussion
Similar physiologic steps lead to bone formation during
embryonic development and in adult organisms, for instance,
in fracture repair or heterotopic ossification. In both cases, bone
formation begins with mesenchymal condensation and ends
with maturation of the growth plate, recruitment of osteoblasts,
and the production of bone. Vascularization has been shown to
play a critical role in this process through infiltration into
cartilage to form vascularized bone.
(16)
Here we present data that
show that vessels play a much earlier role in patterning of the
cartilage and bone. The results show the presence of new vessel
formation prior to the onset of mesenchymal condensation and
cartilage.
We have previously reported the presence of brown
adipocytes within the tissues 2 days after the initial induction.
We also have shown that these cells regulate localized oxygen
tension through their unique metabolism.
(8)
In this study we
extend our knowledge of the functional role of brown adipocytes
to include their rapid and transient expression of the potent
angiogenic factors VEGF-A and -D. Interestingly, a similar rapid
and transient expression of VEGF-D also has been demonstrated
in limb development and has been shown to be critical for
patterning.
(17)
We observed a biphasic expression pattern for
VEGF-A and -D, suggesting multiple roles for this factor in bone
formation. The second peak of expression correlates nicely with
the transition of cartilage to bone formation, which has been
clearly documented.
(16,18)
However, the first phase is less well
studied, and in our model it appears to correlate with the
establishment of new vessels just prior to the onset of
chondrogenesis. Zelzer and colleagues also reported a similar
biphasic expression of VEGF-A during embryonic bone forma-
tion.
(19)
In these studies, the authors showed two functional roles
for VEGF-A, one prior to cartilage and one during the transition of
cartilage to bone, similar to our own observation in our model.
The data collectively suggest that the brown adipocytes may
induce the synthesis of new vessels as a component for
patterning the newly forming cartilage. In the proposed model,
the brown adipocytes induce new vessels, facilitating the
recruitment of chondrogenic precursors, while at the same time
lowering localized oxygen tension to allow for chondrogenic
differentiation. In support of this mechanism, we show in this
study the presence of brown adipocytes expressing VEGF-D only
in areas adjacent to our newly expanding vessels, as marked by
Flk1-H2B::YFP.
Using a model of rapid endochondral bone formation, we
show the immediate expansion of vessels within the tissues in
response to delivery of BMP-2. Although BMP-2 and -4 play a
critical role in the patterning of cartilage and bone in the
embryo,
(20)
much evidence now links the BMPs to a host of other
earlier physiologic functions, including vascularization of the
early embryo.
(21)
Thus it may not be surprising that the earliest
stage of bone formation in our model is the induction of new
vessel formation.
On BMP-2 stimulation, the Flk-1-H2B::YFP endothelial pro-
genitors expand as the total number of positive cells per tissue
area increases. The Flk1-H2B::YFP
þ
cells are clustered along
individual vessels, suggesting that these vessels are extending or
remodeling in response to BMP-2. At this point, we cannot
determine whether this increase occurs via replication of tissue-
resident endothelial progenitors or the recruitment of progeni-
tors to the site of new bone formation. Our data suggest that the
expansion of these progenitors, at least in part, is due to
replication because we observed an increase in the area of
replicating endothelial cells within the tissues receiving Ad5-
BMP-2-transduced cells on day 4 compared with control tissues.
However, we cannot rule out the possibility that at least some of
these cells are recruited from either the circulation or
surrounding tissues. Interestingly, their were significant clusters
of replicating Flk1-H2B::YFP cells on day 2 in tissues receiving
both the Ad5-BMP-2- and the Ad5-empty-transduced cells,
suggesting that perhaps the initial inflammatory reponse may be
somewhat masking the significance of the replication at this
early time point. Alternatively, the increase in replication of the
Flk1-H2B::YFP cell population at 4 days after induction of bone
formation may represent the need for vascularization to recruit
Fig. 3. Increase in Flk-H2B::YFP
þ
cells in BMP-2-induced tissue on days 2
and 4. Quantification of Flk1-H2B::YFP
þ
cells within the tissues 2 and
4 days after induction with Ad5-BMP-2-transduced or control cells. YFP
þ
nuclei were counted and reported as a ratio of the total area of the tissue
section determined using the wide-field montage. Flk-H2B::YFP
þ
cells
were significantly elevated in the tissues receiving BMP-2 compared with
controls. The graph depicts the average number of Flk-H2B::YFP
þ
cells in
five sections for day 2 control, 7 sections for day 2 BMP, 8 sections for
day 4 control, and 6 sections for day 4 BMP. The number of images taken
in each section ranged from 4 to 22.
Denotes a significant difference as
determined by the Student’s ttest.
1152 Journal of Bone and Mineral Research DILLING ET AL.
new chondro-osseous progenitors because this coincides with
the appearance of these cells within the tissues.
(22)
However,
recruitment from the surrounding tissue is equally likely
because recently Kaplan and colleagues
(34)
showed local stem
and progenitor cell contribution to heterotopic bone formation
in a murine model of stem cell transplantation, and this process
may require new vessel formation for establishment of these
cells.
VEGFs have been shown to be essential to expansion of both
endothelial cells and vascular smooth muscle cells that assemble
to form the vessel structure. Although VEGF-A most commonly
has been shown to be responsible for angiogenesis in most
systems, recent studies in murine muscle have found VEGF-D to
be an extremely potent angiogenic factor.
(23)
This family
member is better known for its critical role in the expansion
of lymphatic vasculature.
(23)
In our model we see both factors
highly expressed in the tissues receiving the Ad5-BMP-2-
transduced cells compared with those receiving control cells.
Again, the rapid but transient elevation in VEGF expression
suggests that these factors may be driving the endothelial cell
replication. Knockout studies have confirmed that BMPs regulate
vasculogenesis during embryonic development.
(24)
Functional
deletion of BMP-4 and the BMP I receptor in mice leads to
impaired mesoderm precursors required for vascular develop-
ment.
(25,26)
It also has been shown that addition of BMP-
neutralizing antibodies or noggin suppresses endothelial cell
formation during development, whereas addition of rhBMP-4
promotes it.
(27)
We and others have recently shown the chondrocyte to be of
myeloid origin, and it circulates to the site of new bone
formation.
(22,28)
These cells then must recruit and pass from the
vessels into the tissues, through a process known as extravasa-
tion.
(29)
This process has been shown to require small vasculature
that has a reduced blood flow.
(29)
Thus it is conceivable that
Fig. 4. Quantification of YFP
þ
cell proliferation. Representative images of Flk1-H2B::YFP and the cell proliferation marker Ki67. Colocalization of Flk1-
H2B::YFP (yellow) and Ki67 (red) was detected in BMP-2-treated and control tissues. Graphs show the total number of YFP
þ
/Ki67
þ
cells in the images taken
within the area divided by the number of images analyzed. The area fraction was measured for nine at day 2 and five at day 4 BMP and eight at day 2 and
four at day 4 control different areas, and the average area fraction was calculated for control and BMP-treated tissues. The area fractions of YFP
þ
/Ki67
þ
nuclei in the control and the BMP-treated tissues on day 2 were 7.32 3.26 and 10.20 6.95, respectively. The area fractions for day 4 were 6.97 2.32 and
11.26 2.58 in the control and the BMP-treated tissues. Based on the Student’s ttest, the pvalue for the day 2 data was .29 and that for the day 4 data was
.035. Taken together, the data showed significant YFP
þ
/Ki67
þ
population increases by day 4 after the BMP treatment, but on day 2 there were no
significant differences in dividing YFP cell population between control and BMP-treated tissues.
VESSELS FORM PRIOR TO CARTILAGE Journal of Bone and Mineral Research 1153
Fig. 5. Expression of VEGF-D during the early stages of endochondral bone formation. Results of qRT-PCR analysis of VEGF-A,-B,-C, and -D mRNA levels in
tissues surrounding the lesional site that received either the Ad5-BMP-2- or Ad5-empty-transduced cells isolated at daily intervals for up to 7 days after
initial injection. Four biologic replicates were run in triplicate, and the averages were normalized against an internal standard (ribosomal RNA). The
samples receiving Ad5-BMP-2-transduced cells then were compared with those obtained from the tissues receiving cells transduced with Ad5-empty
cassette virus. Therefore, the graph depicts the fold changes in VEGF RNAs in the BMP-2 samples over time compared with control tissues. Error bars
depict 1 SD unit.
Denotes samples that had a statistically significant ( p<.05) difference from all other samples by the ANOVA test.
Fig. 6. Immunohistochemical staining for brown adipocytes expressing VEGF-D (green, c) in tissues isolated from the Flk1-H2B::YFP mice 4 days after
receiving MC3T3 cells transduced with Ad5-BMP-2. Brown adipocytes were identified as cells expressing uncoupling protein 1 (UCP 1; red, d) and yellow (b)
represents the Flk-yfp
þ
endothelial cells within the muscle. The tissues also were stained with VEGF-D antibodies (c) and counterstained with DAPI (blue,
a), which stains the nucleus of cells. A merger of these stains (UCP-1, VEGF-D, and YFP) is shown in panel e. In panel f, a paraffin section taken 4 days after
injection of BMP-2-producing cells was stained with an antibody against UCP1, and staining was visualized using 3,3’-diaminobenzidine (DAB) as described
previously.
(8)
No staining was observed on a paraffin section taken 4 days after injection of cells transduced with the empty control vector Ad5-HM4 (data
not shown).
1154 Journal of Bone and Mineral Research DILLING ET AL.
brown adipocytes express the VEGFs to form new vessels that are
capable of permitting recruitment of chondrocytic progenitors
to the correct location for endochondral bone formation. Since
vascular invasion of the growth plate has been well documented
to precede the recruitment of osteoblast progenitors to form the
new bone,
(16,18,29,30)
it would not be surprising to have an earlier
phase of this process that recruited the chondrocytic progeni-
tors. We have shown previously that the brown adipocytes are
capable of inducing hypoxia in the local environment, which in
the presence of BMP-2 has been shown to induce chondrogen-
esis.
(8)
Thus we propose that the brown adipocytes are capable of
patterning the newly forming cartilage by inducing new vessel
formation while simultaneously removing oxygen through
uncoupled aerobic respiration. Once the progenitors differenti-
ate into chondrocytes, they then express a number of anti
angiogenic proteins to prevent in the growth of new vessels,
thus momentarily attenuating this early wave of angiogen-
esis.
(31–33,35)
Thus the results presented in this study extend our
knowledge about the critical role vascularization plays not only
in bone formation but also in cartilage formation as well. The
data collectively show a novel process for patterning of new
endochondral bone in adult organisms. Further, this is one of the
first studies that attempts to understand the biology of tissue
engineering of cartilage. Surprisingly, one of the critical
components we have identified is contradictory to our current
dogma that cartilage does not require vessels. This study
suggests that brown adipose may play a pivotal role in
establishing new vessels, essential for recruitment of chondro-
genic progenitors and patterning of the tissues. These findings
ultimately may play an important role in our efforts to replace
damaged cartilage through tissue engineering.
Disclosures
All the authors state that they have no conflicts of interest.
Acknowledgments
This study was funded in part by Grants RO1EB005173-01,
USMRMC 06135010, USMRMC 06136005 (DOD), and IW911NF-
09-1-0040 (DARPA).
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