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Department of Medicine, Institute of Clinical Endocrinology, Tokyo Women’s Medical College (D.S.W., H.D., K.S.), Shinjuku-ku; and Mitsubishi Kagaku BCL (M.M.), Itabashi-ku, Tokyo, Japan
Address all correspondence and requests for reprints to: Kanji Sato, M.D., Institute of Clinical Endocrinology, Tokyo Women’s Medical College, Kawada-cho 8–1, Shinjuku-ku, Tokyo 162, Japan.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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When HOB with high alkaline phosphatase (Al-P) activity and human umbilical vein endothelial cells (HUVEC) with little activity were cultured together, Al-P activity increased, accompanied by an increase in cell number. When HOB and HUVEC were cultured separately, 1,25-(OH)2D3 did not directly stimulate [3H]thymidine incorporation into HUVEC, but stimulated it in the presence of HOB. VEGF did not directly stimulate the Al-P activity of HOB but stimulated it in the presence of HUVEC. The conditioned medium of HOB stimulated the proliferation of HUVEC, and this was partially blocked by anti-VEGF antibody. Conversely, the conditioned medium of HUVEC increased Al-P activity and [3H]thymidine incorporation into HOB, and this was partially blocked by antiinsulin-like growth factor I antibody and BQ-123, a specific antagonist of the endothelin-1 (ET-1) receptor. 1,25-(OH)2D3 stimulated the release of VEGF and ET-1 from HOB and HUVEC, respectively. Furthermore, the 1,25-(OH)2D3-induced release of VEGF was enhanced in HOB cocultured with HUVEC. A quantitative reverse transcription-PCR study revealed that genes for VEGF receptors (Flt-1 and KDR) were expressed in HUVEC, but not in HOB, and that 1,25-(OH)2D3 increased the levels of expression of VEGF receptor genes in endothelial cells only when cocultured with HOB.
In summary, we demonstrated that 1,25-(OH)2D3 exerts an anabolic effect on osteoblasts by enhancing their production of VEGF, which stimulates its receptors on endothelial cells, followed by increased production of osteotropic growth factors, such as insulin-like growth factor I and ET-1. These in vitro findings suggest that the VEGF/VEGF receptor system may be involved in both bone formation and bone remodeling in vivo.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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VEGF, a homodimeric protein with a signal peptide, stimulates specifically endothelial cell proliferation by binding to VEGF receptors (Flt-1 and KDR) that are expressed exclusively on the cells (11, 12). In view of the recent findings that endothelial cells produce growth factors for osteoblasts, such as endothelin-1 (ET-1) and IGFs (13, 14), and that osteoblasts express receptors for ET-1 and IGF-I (15, 16), it is highly likely that a mutual communication system exists between them, as demonstrated in liver and thyroid (17, 18).
This hypothesis is substantiated by histological findings indicating that osteoblasts and osteoprogenitor cells are always located adjacent to endothelial cells in blood vessels at sites of new bone formation (19, 20, 21). In embryonic skeletal tissue, osteogenesis and angiogenesis are temporally related (22). Furthermore, older subjects and patients with osteoporosis have decreased blood vessels in their skeletal tissue, accompanied by a parallel decrease in osteoblasts (23, 24, 25). These in vivo findings also suggest that angiogenesis and osteogenesis are mutually interdependent (26, 27), and that endothelial cells may accelerate bone formation through angiogenesis as well as in bone remodeling.
Therefore, we employed a coculture system of human osteoblast-like cells (HOB) and human umbilical venous endothelial cells (HUVEC), and investigated 1) whether osteoblast function is enhanced by coculture with endothelial cells; 2) if so, what osteotropic growth factors are involved in the interaction between osteoblasts and endothelial cells; and 3) whether the anabolic effects of 1,25-(OH)2D3 on HOB are enhanced by coculture with HUVEC. Furthermore, using quantitative reverse transcription-PCR (RT-PCR) (28), we investigated the effects of 1,25-(OH)2D3 on expression of VEGF receptor genes on endothelial cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MEM) and reagents were supplied by
Life Technologies (Grand Island, NY). FCS was purchased from Filtron
(Brooklyn, Australia). 1,25-(OH)2D3 was
obtained from Wako Pure Chemical Industries (Tokyo, Japan). IGF-I was
purchased from Becton Dickinson Labware (Bedford, MA). Recombinant
human VEGF was obtained from Pepro Tech (Rocky Hill, NJ). Human ET-1,
ET-2, and ET-3 were obtained from Cosmo Bio Co. (Tokyo, Japan). IGF-I
and ET-1 were dissolved in PBS containing 0.2% BSA and stored in
aliquots at -80 as stock solution. Dilutions of the stock solution
were prepared immediately before use. ET-A receptor antagonist (BQ-123)
(26) was purchased from Research Biochemical International (Natick, MA)
(29). Anti-IGF-I monoclonal and anti-VEGF polyclonal antibodies were
obtained from Oncogene Science (Uniondale, NY) and Santa Cruz
Biotechnology (Santa Cruz, CA), respectively.
[Methyl-3H]thymidine was obtained from
Amersham Corp. (Arlington Heights, IL). [
-32P]ATP was
purchased from Amersham (Downers Grove, IL). Nylon filters were
purchased from Schleicher and Schuell (Tokyo, Japan). Reagents for DNA
synthesis and AmpliTaq DNA polymerase were obtained from Life
Technologies (Gaithersburg, MD). Reverse transcriptase and T4
polynucleotide kinase were purchased from Takara (Kyoto, Japan). All
chemicals were of reagent grade and were purchased from Sigma Chemical
Co. (St. Louis, MO).
Cell cultures
HOB were cultured from trabecular bone explants obtained at the
time of orthopedic procedures performed on patients who had no evidence
of metabolic bone disease. The bone fragments were washed extensively
and repeatedly with culture medium to remove adherent marrow cells and
to expose the trabecular surface of the bone. Small bone chips (1
x 1 x 1 mm) were then placed in culture flasks (75
cm2), each containing 15 ml MEM supplemented with 10%
heat-inactivated FCS, penicillin (100 U/ml), and streptomycin (50
µg/ml; MEM-10% FCS), and cultured at 37 C in a humidified
atmosphere with 5% CO2. Cell outgrowth from the trabecular
bone surfaces was apparent after 5 days, and the osteoblast-like cells
became confluent after 10–14 days of culture. Cell passages were
performed by incubating confluent cells in 0.25% trypsin diluted in
calcium- and magnesium-free PBS and replating the cells at a density of
1:3. Experiments were usually performed with HOB subcultured at the
fourth to eighth passage. Under the culture conditions employed, HOB
produced alkaline phosphatase (Al-P) activity for more than 10 passages
(30).
HUVEC were obtained from Kurabo (Osaka, Japan). The cells were cultured
in the manufacturer’s recommended medium (E-BM) supplemented with 2%
FBS, recombinant human EGF (10 ng/ml), hydrocortisone (1 µg/ml),
bovine brain extract (12 µg/ml), gentamicin (50 µg/ml), and
amphotericin-b (50 ng/ml) (supplemented E-BM). When cells
reached subconfluence or confluence, they were cultured in MEM-10%
FCS containing 1 nM VEGF at 37 C in 95% air-5%
CO2. Under the present culture conditions, the cells
continued to proliferate for more than 5 days. Preliminary experiments
revealed that HUVEC, when cultured in MEM-10% FCS without VEGF,
continued to incorporate [3H]thymidine for 24–36 h and
showed rapidly decreased uptake by 48 h. Experiments were
performed using endothelial cells at the third to sixth passage from
different donors.
Coculture of HOB and HUVEC with direct contact
HOB were plated in a 24-multiwell dishes at 2–4 104
x cells/well in 1 ml MEM-10% FCS. After 24 h, when the cell
reached 50% confluence, HUVEC were added to each well at 2–4 x
104 cells in 200 µl MEM-10% FCS without VEGF. In
control culture, the same volume of MEM-10% FCS was added. On the
following day, 1,25-(OH)2D3 (dissolved in 10%
ethanol) or VEGF was added to HUVEC, HOB, and HOB cultured with HUVEC.
The final ethanol concentration in the culture medium was below 0.1%.
After 2–4 days of culture, the cell number and Al-P activity in HUVEC,
HOB, and HOB cocultured with HUVEC were determined as described
previously (30).
In the same experiments, [3H]thymidine (2 µCi/ml) was added to each culture well, and the cells were cultured for an additional 5 h. Then, the cell monolayer was washed with Hanks’ solution (pH 7.4) and extracted with cold 5% trichloroacetic acid. The resulting precipitates were washed with ethanol-ether (volume ratio, 4:1) and solubilized with 1 N sodium hydroxide. The radioactivity was determined with a liquid scintillation counter (LSC-3500, Aloka, Tokyo, Japan). All determinations were performed in quadruplicate.
Coculture of HOB and HUVEC without direct contact
In several experiments, HUVEC and HOB were cultured in the same
well, but separately, by placing a 0.4-µm filter insert (12 mm in
diameter; Millipore, Nunclon). In this coculture system, each well was
composed of double chambers, consisting of an outer chamber
(24-multiwell plate) and an inner Millcell-CM chamber. In the inner
chamber, HUVEC (2–4 x 104) were seeded in 0.5 ml
MEM-10% FCS, and 1 ml MEM-10% FCS containing HOB was poured
into the outer chamber. At 50% confluence of HOB, the inserts
containing HUVEC were placed in wells of HOB. In control cultures, the
cell inserts without HOB were also placed in the control wells. After
an additional 2–4 days of culture, Al-P activity and cell number were
measured.
Effects of conditioned medium of HOB (HOB-CM) on
[3H]thymidine incorporation into HUVEC
HOB were cultured in MEM-10% FCS until they reached
confluence. Then, the medium was changed to fresh MEM-10% FCS, and
the cells were cultured for an additional 3 days. At the end of the
incubation period, the HOB-CM was centrifuged at 200 x
g for 5 min at room temperature, followed by filtering
through a 0.45-µm Millipore filter. The supernatants were used
immediately or frozen at -20 C until further assay. In a few
experiments, HOB-CM was obtained by culturing HOB in MEM-10% FCS
supplemented with 10 nM
1,25-(OH)2D3 for 3 days.
HUVEC were grown in 24-multiwell dishes containing 1 ml supplemented
E-BM until 70–80% confluence. Then, the medium was changed to
MEM-10% FCS containing various concentrations of HOB-CM. After an
additional 1–2 days of culture, [3H]thymidine
incorporation into HUVEC was determined.
To investigate which growth factors are involved in the anabolic
effects of HOB-CM on HUVEC, HUVEC were cultured in MEM-10% FCS
supplemented with 50% HOB-CM and various concentrations of
anti-VEGF-antibody (31). After 2 days of culture,
[3H]thymidine was added, and after an additional 5 h
of culture, [3H]thymidine incorporation was
determined.
Effects of HUVEC-conditioned medium on HOB
HUVEC were grown in the supplemented E-BM on 75-cm2
plastic dishes (Nunclon) until they reached confluence. The confluent
monolayers (
4 days after the dishes had been seeded) were washed
twice with Hanks’ solution (pH 7.4), and the medium was replaced with
MEM-10% FCS supplemented with 1 nM VEGF. The
conditioned medium (HUVEC-CM) was obtained as described above.
HOB were plated at a density of 4 x 104 cells/well in
24-multiwell dishes. After the cells had reached 50% confluence, the
medium was changed to MEM-10% FCS supplemented with various
concentrations of HUVEC-CM. Cultures were carried out for 1–4 days,
and then Al-P activity and cell number were determined.
To investigate which growth factors are involved in the anabolic
effects of HUVEC-CM on osteoblast-like cells, HOB were cultured in
MEM-10% FCS supplemented with 50% HUVEC-CM and various
concentrations of anti-IGF-I antibody and/or BQ-123, a specific
inhibitor of the ET-1 receptor. After 2 days of culture,
[3H]thymidine was added, and after an additional 5 h
of culture, [3H]thymidine incorporation was
determined.
Measurement of VEGF and ET-1 in conditioned medium
The VEGF concentration in HOB-CM was measured at Mitsubishi
Kagaku BCL (Tokyo, Japan) using a solid phase enzyme-linked
immunosorbent assay designed to measure levels in cell culture
supernatants, serum, and plasma (R&D Systems, Minneapolis, MN). This
assay contains insect cell Sf21-expressed recombinant human
VEGF165 and antibodies raised against the recombinant
protein, and its sensitivity is less than 15 pg/ml. The ET-1
concentration in HUVEC-CM was also measured by enzyme-linked
immunosorbent assay (Wako Jun-yaku, Tokyo, Japan). The minimal
sensitivity of the assay was less than 0.5 pg/ml.
Isolation of total RNA
HUVEC and HOB were cultured alone or cocultured in 6-cm diameter
dishes and then treated with hormones or growth factors as described
above. Total RNA was extracted by the method of Chomczynski and Sacchi
(32). Isolated RNAs were stored at -20 C until assayed.
Primers and probes
Oligodeoxyribonucleotide primers and probes for quantitative
RT-PCR were synthesized by Kurabo (Osaka, Japan). The primer sequences
were 5'-ACTATGGAAGATCTGATTTCTTACAGT-3' (nucleotides 3232–3258) and
5'-GGTATAAATACACATGTGCTTCTAG-3' (complement of nucleotides 4289–4314)
(33) for detecting fms-like tyrosine kinase 1
(flt-1) mRNA, and 5'-TATAGATGGTGTAACCCGGA-3' (nucleotides
873–892) and 5'-TTTGTCACTGAGACAGCTTGG-3' (complement of nucleotides
1406–1427) (34) for kinase insert domain-containing receptor
(kdr) mRNA.
The internal oligodeoxyribonucleotide probes were 5'-GAGCTGGAAAGGAAAATCGCGTGCTGCTCC-3' for detecting flt-1 complementary DNA (cDNA) and 5'-ATCCAGTGGGCTGATGACCAAGAAGAACAG-3' for kdr cDNA, corresponding to nucleotides 4186–4215 (33) and 921–950 (34), respectively. The sequences of the primers and probe for detecting ß-actin mRNA have been reported previously (35).
RT-PCR
PCR was performed in 25 µl reaction solution containing cDNA
derived from 1 µg total RNA, 1.25 UAmpliTaq polymerase (Life
Technologies), 200 µmol/liter of each deoxy-NTP, and 0.5 µmol/liter
sense and antisense primers. The reaction mixture was overlaid with 15
µl mineral oil and heated at 94 C for 3 min. Each PCR cycle included
1 min of denaturation at 94 C, 1 min of primer annealing at 55 C, and
1.5 min of extension/synthesis at 72 C. PCR was performed with a DNA
thermal cycler (Perkin-Elmer/Cetus, Norwalk, CT). After the last cycle,
all samples were incubated for an additional 5 min at 72 C.
Quantitative RT-PCR
Quantitative RT-PCR was performed as described previously (28).
A 10-µl aliquot of each RT-PCR reaction mixture was electrophoresed
on 2% agarose gel and transferred to nylon membranes (Nytran, Keene,
NH). The membranes were UV autocross-linked, prehybridized at 42 C for
4 h, and hybridized with specific oligodeoxyribonucleotide probes
that had been 32P end labeled with
[-32P]ATP and polynucleotide kinase, using a DNA 5'-
end labeling kit (Takara Shuzo Co., Shiga, Japan). The filters were
washed once for 30 min in 2 x SSC (standard saline citrate)-0.1%
Denhardt’s solution at 42 C, once for 30 min in 0.1 x SSC-1%
SDS at 42 C, and then twice for 30 min in 0.1 x SSC at room
temperature. Autoradiographic exposure of the washed membranes was
performed at -80 C for various time periods, and the radioactivities
of the hybridization bands were measured with a BioImagin analyzer
(Fuji Photo Film Co., Hamamatsu, Japan).
Statistical analysis
All values are expressed as the mean ± SD.
Means were compared by Student’s t test. Most experiments
were repeated at least three times. ANOVA with Bonferroni’s test was
employed to determine the significance of differences in multiple
comparisons. Differences at P < 0.05 were considered
statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The number of HUVEC increased continuously when cultured in the
supplemented E-BM. However, the cells ceased to grow in MEM-10% FCS
within 1 day and started to degenerate after 2 days of culture. After 4
days of culture, the number of HUVEC, which had been growing to more
than 10,000/well, decreased to 4,818 ± 2,933 cells/well
(mean ± SD of eight experiments). The number of HOB
more than doubled during 4 days of culture in MEM-10% FCS and
increased to 103,431 ± 48,866 cells/well. When both cell types
were cultured together, total cell number increased synergistically to
172,003 ± 76,023 cells/well (mean ± SD of eight
experiments; P < 0.01).
Effects of VEGF and
1,25-(OH)2D3 on HUVEC,
HOB, and HOB cocultured with HUVEC
As expected, VEGF significantly stimulated the proliferation of
HUVEC (Fig. 1A
), but had no effect on HOB (Fig. 1A).
VEGF did not stimulate Al-P activity in HOB per se, but
stimulated the enzyme activity significantly at 10 nM in
HOB cocultured with HUVEC (Fig. 1B).
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). At 10 nM, active vitamin D
stimulated enzyme activity by 116 ± 34% (mean ±
SD of eight experiments). When HOB and HUVEC were cultured
together, 1,25-(OH)2D3 stimulated Al-P activity
by 127 ± 24% (mean ± SD of eight experiments).
Although there was no significant difference between the increased
ratio of the enzyme activity (expressed in milliunits per mg protein),
the enzyme activity per Al-P-positive cell was increased to a greater
extent in HOB cocultured with HUVEC than in HOB alone. Consistent with
previous reports, 1,25-(OH)2D3 decreased the
number of HOB, and this was also the case in HOB cocultured with HUVEC
(Fig. 1A).
Effects of VEGF and
1,25-(OH)2D3 on HOB or
HUVEC cultured in nondirect contact with each other
To investigate whether the increase in Al-P activity is mediated
by cell to cell contact, HUVEC and HOB were cultured separately in the
same well using filter inserts. In the presence of HUVEC that had been
cultured on the inserts, the Al-P activity of HOB grown on the bottom
of culture flask increased significantly compared with that of HOB
cultured alone (without HUVEC, 235 ± 110; with HUVEC, 304 ±
132 mU/mg protein; mean ± SD of
quadruplicate samples; P < 0.01). The number of HOB
also significantly increased in the presence of HUVEC (91,910 ±
5,852 vs. 130,635 ± 25,688 cells/well; mean ±
SD of quadruplicate samples; P < 0.01).
1,25-(OH)2D3 significantly increased the Al-P
activity of HOB cultured alone (292 ± 20 mU/mg protein;
P < 0.01), and this was further enhanced in the
presence of HUVEC on the insert (564 ± 25 mU/mg protein;
P < 0.01). In contrast, VEGF elicited no effect on
Al-P activity of HOB alone, whereas the angiogenic factor slightly, but
significantly, increased the Al-P activity of HOB in the presence of
HUVEC on the insert (without VEGF, 279 ± 14 mU/mg protein; with
10-8 M VEGF, 347 ± 30 mU/mg protein;
P < 0.05).
Conversely, when HUVEC were cultured on the bottom of culture flask in the presence of HOB on the cell inserts, the number of HUVEC was greater than that of HUVEC cultured alone (without HOB, 8,344 ± 3,890; with HOB, 10,086 ± 4,203 cells/well; mean ± SD of quadruplicate samples; P < 0.01). 1,25-(OH)2D3 did not affect the growth of HUVEC directly (data not shown), but it increased the number of HUVEC slightly, but significantly, in the presence of HOB [without 1,25-(OH)2D3, 10,086 ± 4,203; with 1,25-(OH)2D3 (10-8 M), 11,846 ± 5,721 cells/well; P < 0.05].
Anabolic effects of HOB-CM on HUVEC
As reported previously (5), 1,25-(OH)2D3
stimulated the release of VEGF from HOB in a concentration-dependent
manner (Table 1). Consistent with this finding, HOB-CM
increased the number of HUVEC significantly, accompanied by a
dose-dependent increase in [3H]thymidine incorporation
into the cells (Fig. 2). The proliferative effect of
HOB-CM on endothelial cells was potentiated when HOB was cultured with
1,25-(OH)2D3 (Table 2D), whereas
the active vitamin D had no direct proliferative effect on HUVEC (Table 2B).
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). However, anti-VEGF antibody could not completely
inhibit HOB-CM-induced [3H]thymidine incorporation, even
at a final concentration of 1.6 x 10-8
M, which nearly completely inhibited the
[3H]thymidine incorporation induced by 10-10
M VEGF (data not shown), suggesting that an angiogenic
factor(s) other than VEGF is present in HOB-CM.
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).
Furthermore, HUVEC-CM significantly increased the Al-P activity of HOB
in a dose-dependent manner.
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). Interestingly,
1,25-(OH)2D3 significantly increased the level
of ET-1 in HUVEC-CM (Table 1B). This was also observed when HUVEC were
cultured in MEM-10% FCS supplemented with 1 nM VEGF
(data not shown). In contrast to HUVEC, a trace amount of ET-1 was
detected in HOB-CM.
Consistent with the above findings, 10-6 M
BQ-123 and 1.5 x 10-8 M anti-IGF-I
antibody inhibited HOB-CM-induced [3H]thymidine
incorporation by 24% and 35%, respectively (Fig. 5).
However, the simultaneous addition of BQ-123 and anti-IGF-I inhibited
it additively, but not completely (by 58%), suggesting that HUVEC
produce an angiogenic factor(s) other than VEGF.
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, ET-1 stimulated Al-P activity and
[3H]thymidine incorporation in a concentration-dependent
manner. However, ET-2 and ET-3 elicited no significant effect on
HOB.
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). VEGF (1 nM) increased the levels
of kdr and flt-1 gene expression in HUVEC (Fig. 7, lanes 3 and 4). In contrast, 1,25-(OH)2D3
elicited no significant effect on VEGF receptor expression in HUVEC
alone (data not shown) or in HUVEC in the presence of VEGF. However,
when HUVEC were cultured together with HOB,
1,25-(OH)2D3 increased the levels of VEGF
receptor gene expression (Fig. 7, lane 6).
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, lanes 1, 5, and 9), whereas
HUVEC cultured in MEM-10% FCS without VEGF showed a time-dependent
decrease in the level of VEGF receptor gene expression (Fig. 8, lanes
2, 6, and 10); the levels of KDR expression in HUVEC cultured for 24,
36, and 48 h without VEGF decreased to 90%, 56%, and 21% of
that in the control culture, respectively. However, HUVEC cocultured
with HOB maintained their level of VEGF receptor gene expression, which
was enhanced by 48% (kdr) and 60% (flt-1) by
1,25-(OH)2D3 (Fig. 8, lane 12).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Furthermore, 1,25-(OH)2D3
enhanced all of these phenomena by stimulating the level of VEGF
receptor gene expression on endothelial cells. Therefore, there is a
mutual communication system between osteoblasts and endothelial cells
that leads to enhancement of each cellular function by
1,25-(OH)2D3 (Fig. 9). A similar mutual
communicating system between endothelial cells and parenchymal cells
has been demonstrated in the liver involving hepatocyte growth factor
and in the thyroid involving TSH (17, 18).
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As reported by Guenther et al. (44), endothelial cells synthesize potent growth factors for osteoblasts, and these have recently been identified as IGF-I, ET-1, basic fibroblast growth factor, etc. (13, 14, 37, 38, 39, 40, 41). We also confirmed that HUVEC under our present experimental conditions produce IGF-I and ET-1. Receptors for 1,25-(OH)2D3 and IGF-I are present not only on osteoblasts, but also on endothelial cells (45). Kurose et al. (46) reported that 1,25-(OH)2D3 and IGF-I synergistically stimulate Al-P activity on osteoblast-like cells. As 1,25-(OH)2D3 is capable of increasing the number of IGF-I receptors on osteoblast-like cells (47), it is reasonable that HOB cultured in direct or indirect contact with HUVEC increased Al-P activity to a greater extent than in HOB cultured alone, and that the effect was further potentiated by 1,25-(OH)2D3.
The effects of ET-1 on bone metabolism are a matter of debate (13). The vasoactive peptide stimulates proliferation of osteoblast-like cells (MC3T3-E1) (48) and increases the steady state levels of expression of osteopontin and osteocalcin mRNA in rat osteosarcoma cells (49). We demonstrated that 1,25-(OH)2D3 increased ET-1 concentration in the HUVEC-CM. To the best of our knowledge, this is the first report of 1,25-(OH)2D3 on the vasoactive peptides produced by HUVEC. Furthermore, we demonstrated that ET-1 stimulated Al-P activity in human osteoblast-like cells, although the opposite effect was reported in neonatal osteoblasts or murine cell lines (13). ET-1 can also affect osteoclasts, either inhibiting osteoclastic bone resorption or stimulating bone resorption depending on the organ culture system employed. As endothelial cells are abundant in bone marrow and lie in close proximity to osteoblasts and osteoclasts (50), ETs may be added to the list of potential modulating factors in bone remodeling (51). The histochemical localization of ET-1 in endothelial cells, osteoblasts, and osteoclasts supports this hypothesis (52).
Under our present experimental conditions, endothelial cells expressed the VEGF receptor genes (flt-1 and kdr) constitutively. In addition, when HUVEC were cultured with HOB, the level of VEGF receptor expression was maintained for a longer period and was further augmented by 1,25-(OH)2D3. However, 1,25-(OH)2D3 had no apparent direct effect on VEGF receptors in the absence of HOB. These results raise the possibility that VEGF produced by HOB may act on adjacent endothelial cells as an angiogenic factor in a paracrine manner. These in vitro findings are compatible with the in vivo observation that bone formation was stimulated in a diffusion chamber containing both endothelial cells and osteoblasts, which were implanted into rats (53). Furthermore, this hypothesis is substantiated by the adjacent histological localization of endothelial cells and osteoblast-like cells in skeletal tissue, suggesting that VEGF produced by osteoblast-like cells and VEGF receptors expressed on endothelial cells are involved in both bone formation and bone remodeling.
There is increasing evidence to suggest that vitamin D is
effective for the treatment of patients with osteoporosis, mainly by
stimulating the absorption of calcium and phosphate in the intestine
and maintaining the serum levels of calcium and phosphate, so that bone
mineralization proceeds efficiently (54, 55). Furthermore, active
vitamin D elicits a number of anabolic effects on osteoblasts, such as
stimulation of the production of osteocalcin, osteopontin, and bone
matrix protein; stimulation of Al-P activity; and increase in the
number of IGF-I receptors and the release of IGF-binding proteins (3, 4, 47, 48) (Fig. 9). Our present in vitro findings suggest
that these anabolic effects of 1,25-(OH)2D3 are
intensified by endothelial cells, which are abundant in the vicinity of
osteoblasts in bone marrow.
In summary, we demonstrated that 1,25-(OH)2D3 stimulates Al-P activity in osteoblasts and increases the steady state level of VEGF mRNA, followed by increased secretion of VEGF. The secretable angiogenesis factor acts on KDR and Flt-1 receptors on endothelial cells in a paracrine manner, thereby causing the proliferation of endothelial cells.
The activated endothelial cells expressing a greater amount of VEGF
receptor genes, in turn, produce osteotropic growth factors, such as
ET-1 and IGF-I, which synergistically stimulate the proliferation of
HOB accompanied by an increase in Al-P activity (Fig. 9). Therefore,
osteogenesis and angiogenesis may be mutually dependent, and it is
reasonable to assume that the anabolic effects of
1,25-(OH)2D3 on skeletal tissue are mediated in
a paracrine manner and enhanced by the VEGF/VEGF receptor system
between osteoblasts and endothelial cells.
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Acknowledgments |
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Footnotes |
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Received January 3, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(OH)-vitamin D3. Bone Miner 3:47–52[Medline]
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 M. L. Yen, J. L. Su, C. L. Chien, K. W. Tseng, C. Y. Yang, W. F. Chen, C. C. Chang, and M. L. Kuo Diosgenin Induces Hypoxia-Inducible Factor-1 Activation and Angiogenesis through Estrogen Receptor-Related Phosphatidylinositol 3-kinase/Akt and p38 Mitogen-Activated Protein Kinase Pathways in Osteoblasts Mol. Pharmacol., October 1, 2005; 68(4): 1061 - 1073. [Abstract] [Full Text] [PDF]
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 A. Germeyer, A. E. Hamilton, L. S. Laughlin, B. L. Lasley, R. M. Brenner, L. C. Giudice, and N. R. Nayak Cellular Expression and Hormonal Regulation of Neuropilin-1 and -2 Messenger Ribonucleic Acid in the Human and Rhesus Macaque Endometrium J. Clin. Endocrinol. Metab., March 1, 2005; 90(3): 1783 - 1790. [Abstract] [Full Text] [PDF]
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 M. J. Levine and D. Teegarden 1{alpha},25-Dihydroxycholecalciferol Increases the Expression of Vascular Endothelial Growth Factor in C3H10T1/2 Mouse Embryo Fibroblasts J. Nutr., September 1, 2004; 134(9): 2244 - 2250. [Abstract] [Full Text] [PDF]
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L. Qin, P. Qiu, L. Wang, X. Li, J. T. Swarthout, P. Soteropoulos, P. Tolias, and N. C. Partridge Gene Expression Profiles and Transcription Factors Involved in Parathyroid Hormone Signaling in Osteoblasts Revealed by Microarray and Bioinformatics J. Biol. Chem., May 23, 2003; 278(22): 19723 - 19731. [Abstract] [Full Text] [PDF]
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 T. Maeda, T. Kawane, and N. Horiuchi Statins Augment Vascular Endothelial Growth Factor Expression in Osteoblastic Cells via Inhibition of Protein Prenylation Endocrinology, February 1, 2003; 144(2): 681 - 692. [Abstract] [Full Text] [PDF]
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M. M. L. Deckers, R. L. van Bezooijen, G. van der Horst, J. Hoogendam, C. van der Bent, S. E. Papapoulos, and C. W. G. M. Lowik Bone Morphogenetic Proteins Stimulate Angiogenesis through Osteoblast-Derived Vascular Endothelial Growth Factor A Endocrinology, April 1, 2002; 143(4): 1545 - 1553. [Abstract] [Full Text] [PDF]
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 F. Villars, B. Guillotin, T. Amedee, S. Dutoya, L. Bordenave, R. Bareille, and J. Amedee Effect of HUVEC on human osteoprogenitor cell differentiation needs heterotypic gap junction communication Am J Physiol Cell Physiol, April 1, 2002; 282(4): C775 - C785. [Abstract] [Full Text] [PDF]
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 N. Akutsu, R. Lin, Y. Bastien, A. Bestawros, D. J. Enepekides, M. J. Black, and J. H. White Regulation of Gene Expression by 1{{alpha}},25-Dihydroxyvitamin D3 and Its Analog EB1089 under Growth-Inhibitory Conditions in Squamous Carcinoma Cells Mol. Endocrinol., July 1, 2001; 15(7): 1127 - 1139. [Abstract] [Full Text] [PDF]
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 D. J. Mantell, P. E. Owens, N. J. Bundred, E. B. Mawer, and A. E. Canfield 1{alpha},25-Dihydroxyvitamin D3 Inhibits Angiogenesis In Vitro and In Vivo Circ. Res., August 4, 2000; 87(3): 214 - 220. [Abstract] [Full Text] [PDF]
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 P. ESBRIT, M. V. ALVAREZ-ARROYO, F. DE MIGUEL, O. MARTIN, M. E. MARTINEZ, and C. CARAMELO C-Terminal Parathyroid Hormone-Related Protein Increases Vascular Endothelial Growth Factor in Human Osteoblastic Cells J. Am. Soc. Nephrol., June 1, 2000; 11(6): 1085 - 1092. [Abstract] [Full Text]
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M. M. L. Deckers, M. Karperien, C. van der Bent, T. Yamashita, S. E. Papapoulos, and C. W. G. M. Lowik Expression of Vascular Endothelial Growth Factors and Their Receptors during Osteoblast Differentiation Endocrinology, May 1, 2000; 141(5): 1667 - 1674. [Abstract] [Full Text] [PDF]
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 M. Carlevaro, S Cermelli, R Cancedda, and F Descalzi Cancedda Vascular endothelial growth factor (VEGF) in cartilage neovascularization and chondrocyte differentiation: auto-paracrine role during endochondral bone formation J. Cell Sci., January 1, 2000; 113(1): 59 - 69. [Abstract] [PDF]
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 S. Niida, M. Kaku, H. Amano, H. Yoshida, H. Kataoka, S. Nishikawa, K. Tanne, N. Maeda, S.-I. Nishikawa, and H. Kodama Vascular Endothelial Growth Factor Can Substitute for Macrophage Colony-Stimulating Factor in the Support of Osteoclastic Bone Resorption J. Exp. Med., July 19, 1999; 190(2): 293 - 298. [Abstract] [Full Text] [PDF]
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 M. D. Mueller, J.-L. Vigne, A. Minchenko, D. I. Lebovic, D. C. Leitman, and R. N. Taylor Regulation of vascular endothelial growth factor (VEGF) gene transcription by estrogen receptors alpha and beta PNAS, September 26, 2000; 97(20): 10972 - 10977. [Abstract] [Full Text] [PDF]
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,
Without HOB-CM; 
; with HOB-CM. *, P < 0.05,
with vs. without the antibody.
, Al-P
activity. *, P < 0.05; **, P
< 0.01 (with vs. without HUVEC-CM).
