Vascular trauma due to metabolic, mechanical, or immunological
insult is characterized by changes in the vascular endothelium,
including migration and proliferation of smooth muscle cells (SMCs),
loss of expression of SMC phenotype, enhanced extracellular-matrix
synthesis, and cell death due to apoptosis and necrosis. These cellular
changes gradually result in intimal thickening of the blood vessel
and loss of elasticity in the vasculature
1
. Vasculature also plays an important role in endochondral ossification
2
, and vascularization is a key event in the development of normal
cartilage and bone.
A number of local and systemic factors participate in the maintenance
of vascular integrity
3,4
. Platelet-derived growth factor (PDGF), basic fibroblast growth
factor (bFGF), and insulin-like growth factor-1 (IGF-1) are known
to exert strong growth-promoting activity on SMCs
in vitro5
and play critical roles in the migration and proliferation of SMCs
in vivo6,7
. Notably, these growth factors are detected at high concentrations
within vascular proliferative lesions
8
. Conversely, systemic factors such as gamma-interferon and progesterone
have been shown to exhibit growth-inhibitory activity on SMCs
9,10
. TGF-ß, a bifunctional growth-regulatory molecule for vascular
SMCs, stimulates or inhibits cell growth, depending on the cell
culture conditions, the presence of other growth-regulatory molecules, and
the dose and frequency with which they are employed
11
. It has been shown that the injury site has high concentrations
of TGF-ß, which subsequently cause an enhanced extracellular-matrix
production leading to intimal thickening in blood vessels. Thus,
the regulation of SMC proliferation and expression of SMC phenotype
is a dynamic process, tightly governed by local and systemic factors
to maintain the vascular integrity and continuous blood flow
in vivo
.
Bone morphogenetic proteins (BMPs), also called osteogenic proteins
(OPs), are members of the TGF-ß superfamily, originally identified
by their ability to induce endochondral bone formation at an extraskeletal
site
12,13
. They have been shown to regulate the migration, proliferation,
and differentiation of pluripotent progenitor cells involved in
the development of organ systems during embryogenesis and in adult
tissue repair
14
. We have shown that systemic administration of recombinant human
BMP-7 is capable of protecting heart
15
, brain
16,17
, and in particular kidney function following ischemia-reperfusion
injury in rat models
18
. The tissue protective function of BMP-7 may be complex; however,
we demonstrated that BMP-7 maintains the expression of vascular
smooth muscle phenotype in peritubular capillaries after ischemic
acute renal failure (ARF) in the rat
18
. In addition, administration of BMP-7 in norepinephrine-induced
ARF in rats has been shown to improve renal function by increasing
the glomeruli filtration rate, renal blood flow, and urine flow
rate
19
.
In this study, we show that BMP-7 inhibits proliferation and
stimulates the expression of the SMC phenotype when it is added
to human aorta-derived SMCs in culture. This BMP-7-induced growth-inhibitory
effect was also observed when the SMC proliferation was stimulated
by PDGF-BB and TGF-ß, suggesting that in addition to its role in
the prevention of ischemic injury, BMP-7 may be a cytoprotective
agent for vascular proliferative disorders.
Cell Culture
Human primary aortic smooth muscle cells (HASM) were purchased
from Clonetics (San Diego, California). They were grown in growth medium
provided by the same company. Cells from passages 4-9 were used
for all experiments.
3
H-Thymidine Incorporation
HASM cells were plated into 24-well plates at a concentration
of 4
×
10
4
cells/well such that they were 60-70% confluent the next day. At
this time, they were rendered quiescent by changing the medium to
Dulbecco's modified Eagle medium (DMEM) (Gibco BRL, Gaithersburg,
Maryland) containing 0.1% serum for 60-72 hours. The medium was
then replaced with DMEM containing 1% serum with or without various
concentrations of a homogenous preparation of recombinant human
mature BMP-7
20
, PDGF-BB, or TGF-ß (Upstate Biologicals, Syracuse, New York) for
24 hours. During the last 3 hours of culture, cells were labeled
with [methyl-
3
H]-thymidine (Dupont/NEN, Boston, Massachusetts) at 1 Ci/ml/well
in thymidine-free medium. At the end of the incubation period, the
cells were fixed in 75% methanol/25% acetic acid (v/v) for at least
2 hours, followed by several washes with 80% methanol. Incorporated
3
H-thymidine was measured in a liquid scintillation counter. All
experiments were performed multiple times employing cells from different
passages (passages 4-9). The data presented are representative of one
such experiment.
Cell Counting
HASM cells were plated into a 24-well plate at a concentration
of 8
×
10
3
cells/well. After 24 hours, various concentrations of BMP-7, PDGF-BB,
or TGF-ß1 were added and the cells were incubated for a total of
72 hours. The total cell number was determined at 0, 24, 48, and 72
hours with use of a hemocytometer and trypan blue staining for viable
cells. Cells from four wells were combined for an average reading.
Long-Term Cultures of HASM Cells
Eight 100-mm dishes were plated with HASM cells at a concentration
of 1.2
×
10
6
cells/dish. After 24 hours, 100 ng/ml BMP-7 was added to four plates
and an equal amount of vehicle was added to the other four. The
day that the cultures reached confluence was noted and marked as day
0. The medium was changed that day and every other day thereafter,
and fresh BMP-7 was added. Beginning day 3 and every other day thereafter,
one plate from each group was harvested and total RNA was isolated.
Thus, total RNA was made from BMP-7-treated and untreated cultures
on day 3, 5, 7, and 9, respectively.
Total RNA Preparation and Semiquantitative Reverse
Transcriptase-Polymerase Chain Reaction (RT-PCR) Analysis
Total RNA from HASM cells was isolated with TRIzol as indicated
(Gibco BRL). Contaminating genomic DNA was removed with RNAase-free DNAase
following the manufacturer's protocol. Complementary DNA was synthesized
with Superscript reverse transcriptase (Gibco BRL) as described
in the same protocol. Four micrograms of DNAase-treated RNA was
incubated with 2 µloligo dT at 70°C for 10 minutes. Next, 14 µlof
53 buffer, 7 µlDTT (0.1
M
), 3.5 µlof each nucleotide (10 m
M
), 1.5 µlRNAase inhibitor, and 2 µlSuperscript reverse transcriptase
(Gibco BRL) were added to each reaction. The samples were incubated
for 10 minutes at room temperature, 50 minutes at 42°C, 10 minutes
at 50°C, and 5 minutes at 90°C. RNAase-H (1 µl) (Gibco BRL) was added
to the reactions, and samples were incubated for 20 minutes at 37°C.
PCR was performed as indicated by the manufacturer (Perkin-Elmer,
Norwalk, Connecticut) with the following primers: a-actin sense 5'GCT
CAC GGA GGC ACC CCT GAA, antisense 5'CTG ATA GGA CAT TGT TAG CAT; glyceraldehyde-3-phosphate
(GAPDH) senseACC ACA GTC CAT GCC ATC AC, antisense 5'TCC ACC
ACC CTG TTG CTG TA; collagen III sense 5'GGT GTG GAC GTT GGC CCT
GTT TGC, antisense 5'CTA AGT AAC CGT ACG GTG TCC C; collagen I sense
59TGA CTT GAG ACT CAG CCA CCC A, antisense 5'AGG TTG CCA GTC TCC
TCA TCC A; and intercellular adhesion molecule-1 (ICAM-1) sense
59GGG AAT CCA GCC CCT AAT CTG A, ICAM-1 antisense 5'GAC TGT CCC
AGC TTT CCC ATG T.
Reactions included 5 µlof 103 buffer, 3 µlof 25 m
M
MgCl2, 1 µlof 10 m
M
dNTP, 1 µlof 20
M
3' primer, 1 µlof 20
M
5' primer, 0.5 µlTaq polymerase (Perkin-Elmer, Foster City, California),
and 1 µlcDNA. Samples were incubated for 5 minutes at 94°C followed
by 25-40 cycles of 45 seconds at 94°C, 45 seconds at 59°C, 1 minute
at 72°C, and final extension at 72°C for 10 minutes in a Perkin-Elmer
DNA Thermal Cycler. To compare the relative quantity of the RT-PCR
reactions, the transcription level of GAPDH was used as a control.
GAPDH gene expression was found to be similar at all time points
examined, enabling analysis of the relative levels of gene expression
for the desired genes. PCR primer sets were optimized with use of
human placental and embryonic cDNA libraries purchased from Clonetech
(Palo Alto, California). The PCR product was verified at least twice.
Quantitative RT-PCR Analysis
cDNA was generated by reverse-transcribing 1 µg of total RNA in
a volume of 100 µlwith use of a kit provided by Promega (Madison,
Wisconsin). This cDNA (2 µl) was then used as a template for PCR reactions.
Quantitative PCR was performed with use of a PE Biosystems 5700
SDS with SYBR Green core reagents (PE Biosytems, Foster City, California).
Reactions were performed in triplicate and quantitated to that of
ß-actin, which remained unchanged by the addition of BMP-7. The
primers used were ß-actin sense 5'CTG GAA CGG TGA AGG TGA CA, antisense
59CGG CCA CAT TGT GAA CTT TG; p21 sense 5'CCC GTT TCT CCA CCT AGA
CTG T, antisense 5'TCA GCA TTG TGC GAG GAG CT; Smad6 sense 5'GCC
ACT GGA TCT GTC CGA TT, antisense 5'CAC CCG GAG CAG TGA TGA G; Smad7
sense 5'CTG GGA GGG ACA TGC TTA GC, antisense 5'TCA GCC TAG GAT
GGT ACC TTG G; a-actin sense 5'CCA GCA GAT GTG GAT CAG CA, antisense 5'AAG
CAT TTG CGG TGG ACA AT; Id-1 sense 5'AGA ACC GCA AGG TGA GCA A,
antisense 5'TCC ACC TGA AGG TCC CTG ATG; Id-2 sense 5'CCA CCC TCA
ACA CGG ATA TCA, antisense 5'ACA CAG TGC TTT GCT GTC ATT TG; Id-3
sense 5'GCG GCA GAG CTG GTC TTC, antisense 5'TCA GGG CAA CAG AAC CTT
TCT C; basic calponin sense 5'AGG AGC TGA GAG AGT GGA TCG A, antisense
59TCG CAA AGA ATG ATG CCA TCT; and H-chain myosin sense 5'TCA ACA
TGC AGG CGC TCA, antisense 5' CGT CTC ATA CTC GTG AAG CTG TCT.
Northern Blot Analysis
Five micrograms of total RNA was electrophoresed in a 1.2% formaldehyde-agarose
gel and blotted onto nitrocellulose filters that were hybridized
with randomly primed
32
P-labeled Smad6 cDNA probe as described
21
. The hybridized filters were washed in 30 m
M
NaCl, 3 m
M
sodium citrate, and 0.1% sodium dodecyl sulfate at 55°C and autoradiographed
on Kodak XAR film at -80°C on phosphor screen. To correct for differences
in RNA loading, the filters were washed at 80°C in 50% formamide
solution and rehybridized with a radiolabeled GAPDH probe. The GAPDH probe
was generated from RT-PCR product.
ICAM-1 Estimation by ELISA
HASM cells were plated at 5
×
10
4
cells/well in a 24-well plate, in growth medium containing 5% fetal
bovine serum (FBS). The next day, the medium was replaced with medium
containing 0.1% FBS. Then, 1 ng/ml of IL-1b (R&D Systems, Minneapolis,
Minnesota) and various concentrations of BMP-7 were added as indicated. Three
or five days later, the medium was removed and the cell layer was
washed twice with phosphate buffered saline (PBS) and lysed in 0.5
ml PBS by three freeze-thaw cycles. Finally, 20-100 µlof cell lysates
was analyzed for soluble human ICAM-1 with use of ELISA kits (R&D
Systems). Recombinant human ICAM-1 and IL-6 were provided by the manufacturer
and used as standards.
The results are indicated as units/mg of protein in cell lysates.
SMCs Express BMP-7 Receptors
Prior to evaluating the direct effect of BMP-7 on vascular SMCs,
we examined cultures of HASM cells for the presence of BMP-7 and
its type-I and II receptors by RT-PCR. Although there was no detectable
BMP-7, BMP type-I receptors ALK-3 (ActR-IA) and ALK-6 (BMPR-IB)
were abundant. The presence of several type-I receptors for BMPs [ALK-2
(ActR-I), ALK-3 (BMPR-IA), and ALK-6 (BMPR-IB)] in vascular SMC
also has been reported previously
22-24
. Although we were unable to detect BMP type-II receptors (BMPR-II),
we found abundant activin type-IIB receptor (ActR-IIB), which has
been shown to function as a type-II receptor for BMP-7
25
. Similarly, BMP-2 has been shown to bind to ActR-II and ActR-IIB
in the presence of the appropriate type-I receptors
26
.
BMP-7 Inhibits Serum and Growth Factor-Induced Proliferation
of SMCs
We examined the effect of exogenously added BMP-7 on cell growth
in HASM cell cultures by
3
H-thymidine incorporation into DNA. Prior to the addition of BMP-7,
cells were synchronized by serum starvation for 60-72 hours and
then induced to proliferate by providing the medium with 1% serum.
Various concentrations of BMP-7 with or without PDGF-BB or TGF-ß1
were added. BMP-7 exhibits a biphasic effect on the serum-induced
proliferation of HASM cells in culture (
Fig. 1-A
). At very low concentrations (0.1-1.0 ng/ml), BMP-7 had a small
but reproducible stimulatory effect (about 10%) on these cells,
whereas at higher concentrations (10-200 ng/ml) it inhibited the serum-induced
HASM cell proliferation in a dose-dependent manner. About 60% inhibition
of proliferation was obtained within 24 hours by the addition of
BMP-7 (200 ng/ml). Under identical experimental conditions, TGF-ß1
(1 ng/ml) and PDGF-BB (40 ng/ml) stimulated the proliferation of
HASM cells by 30% and 300%, respectively (
Fig. 1-A
). Interestingly, when BMP-7 was added in conjunction with these
growth factors, the same biphasic pattern was observed as seen with
BMP-7 alone (
Fig. 1-B
and
Fig. 1-C
). At very low concentrations (0.1-1 ng/ml), BMP-7 had either no
effect or a small but reproducible stimulatory effect, whereas at
higher concentrations (10-200 ng/ml), BMP-7 was able to inhibit
the stimulatory activity of TGF-ß1 and PDGF-BB in a dose-dependent
manner. In accordance with reduced
3
H-thymidine incorporation into DNA, the cell number was also decreased
when cells were treated with BMP-7 and PDGF as compared with PDGF
alone (
Fig. 1-D
).
Several cell-cycle proteins, including cyclin-dependent kinase-2
(cdk-2) and cdk-2 kinase inhibitor, p21 (cip), mediate SMC proliferation
27,29
, and BMP-7 inhibits the proliferative activity of PDGF in HASM
cells. Therefore, we examined the effect of BMP-7 on these cell-cycle-modulated genes
by quantitative RT-PCR. As shown in
Fig. 2
, 40 ng PDGF-BB per ml dramatically downregulated the expression
of cdk-2 kinase inhibitor, p21, which was completely neutralized
by BMP-7 in a dose-dependent manner. Correspondingly, the cdk-2
kinase gene expression was upregulated by PDGF and this effect was
inhibited by BMP-7.
BMP-7 Induces the Expression of Inhibitors
of Helix-Loop-Helix Transcription Factors and Inhibitory Smads
Id proteins control cell differentiation by interfering with
DNA binding of transcription factors. Previously, it was shown that
BMP-2 upregulated Id-1 in the process of osteoblast differentiation
of myoblast cell line C2C12
29
. Investigating the mechanism of BMP-7 action, we found that the
expression of inhibitors of helix-loop-helix transcription factors
Id-1 and Id-2 was dramatically upregulated by BMP-7 (
Fig. 3-A
). Greater than 25-fold induction of both Id-1 and Id-2 was obtained
by 100 ng/ml BMP-7. In contrast, TGF-ß and PDGF had little or no
effect on the expression of Ids. This induction was rapid and was detected
at the earliest time point we have examined: 15 minutes after the
addition of BMP-7. When BMP-7 was added in conjunction with TGF-ß
or PDGF, Id-1 and Id-2 were induced to a lesser extent.
The inhibitory Smads, Smad6 and Smad7, have been shown to act
as an inhibitor of Smad phosphorylation in the BMP and TGF-ß signaling
pathway and have been implicated in the control of these pathways
21,30,32
. Both Smad6 (35-fold) and Smad7 (15-fold) were induced by 100 ng/ml
BMP-7 (
Fig. 3-A
). Under identical culture conditions, PDGF had no effect on the
expression of these two genes and TGF-ß induced only Smad7. A dose-dependent induction
of Smad6 could be detected by Northern blot analysis of the same
RNA (
Fig. 3-B
).
BMP- 7 Downregulates Endogenous and Induced ICAM-1
One key feature of vascular proliferative disorders is inflammation.
IL-1b and other pro-inflammatory cytokines from aggregating macrophages
and monocytes induce in turn the expression of ICAM-1, MCP-1, and
other pro-inflammatory cytokines in SMC cells. IL-1b (1 ng/ml) dramatically
induced the expression of ICAM-1, which can be inhibited to as much
as 40% by 200 ng/ml BMP-7 (
Fig. 4-A
). Additionally, BMP-7 can dose-dependently downregulate the expression
of endogenous ICAM-1 (
Fig. 4-B
and
Fig. 4-C
) and IL-6 (data not shown) as examined by semiquantitative RT-PCR.
BMP-7 Maintains the Expression of Markers Characteristic
of SMC Phenotype
When primary HASM cells are maintained in culture for long periods,
they tend to adopt a more fibroblast-like morphology with a concomitant
loss of SMC markers such as a-actin and SMC-specific H-chain myosin.
We found that HASM cultures containing BMP-7 (100 ng/ml) maintained
the expression of a-actin beyond day 6 in confluent cultures, whereas
untreated cultures started to loose a-actin expression as early
as day 3 (
Fig. 5-A
). As it has been shown that in atherosclerotic lesions type-I collagen
synthesis is enhanced compared with that of type-III collagen, we
examined the ability of BMP-7 to maintain type-III collagen synthesis
in long-term cultures by semiquantitative RT-PCR. Although the expression
of collagen type I remained unaltered beyond day 3 following confluence
in treated and untreated cultures (
Fig. 5-A
), the expression of collagen type III was significantly decreased
over the same time period in untreated cultures (
Fig. 5-A
. However, its expression remained unaltered or even enhanced in
BMP-7-treated cultures. This is confirmed by densitometer scanning
of the gel followed by calculations of ratios (collagen III/GAPDH).
Furthermore, BMP-7 induced a dose-dependent increase in the expression
of a-actin, basic calponin, and H-chain myosin as examined by quantitative
and semiquantitative RT-PCR (
Fig. 5-B
and
Fig. 5-C
). Additionally, there was a discernable change in morphology (
Fig. 5-D
) from needle-shaped fibroblast-like cells that are stacked on top
of each other to more randomly organized smaller cells at the addition
of BMP-7 to these cultures. The true appearance of SMCs in culture
is unclear.
We show here that HASM cells when treated with recombinant human
BMP-7 (also called osteogenic protein-l [OP-1]) inhibit serum-stimulated
cell proliferation in culture over a wide range of physiologic concentrations
(10-200 ng/ml). Notably, this BMP-7-mediated inhibition of SMC proliferation was
more pronounced when cell growth had been stimulated with PDGF-BB
or TGF-ß, suggesting for the first time a potential role for BMPs
in the prevention of SMC proliferation following vascular injury.
The inhibitory effect of BMP-7 was due in part to an increased expression
of the inhibitor of cyclin-dependent kinase-2, p21, which was inhibited
during PDGF-BB-mediated cell proliferation. BMP-7 also induced the
expression of developmentally regulated genes such as Id-1 and Id-2,
which are required for several key cellular differentiation pathways
and inhibitory Smads. Smad6 and Smad7, which negate both the TGF-ß
and TGF-ß superfamily of proteins, mediated downstream signaling.
Concurrently, BMP-7-mediated inhibition of cell growth is accompanied
by enhanced expression of markers that are characteristic of the
SMC phenotype, a-actin, SMC-specific heavy-chain myosin, and maintenance
of the type III/I collagen ratio. Finally, BMP-7 downregulated endogenous and
IL-1b induced ICAM-1, a cell-surface molecule known to play a role
in neutrophil adhesion and activation during inflammation. Our findings suggest
that BMP-7 may provide a basis for developing therapeutic strategies
to effectively suppress SMC proliferation, delay the onset of inflammation,
and diminish the formation of connective tissue in and around the
vascular endothelium associated with vascular proliferative disorders
4
.
Several peptide growth factors (PDGF, FGF, IGF-1, and TGF-ß),
pro-inflammatory cytokines (interleukin-1 [IL-1] and tumor necrosis
factor-alpha [TNF-a]), and agents such as nitric oxide and lipids
are known to regulate migration, proliferation, and differentiation
of vascular SMCs
6,7,32-34
. Specifically, following injury, the migration and proliferation
of SMCs are stimulated by (1) the paracrine action of PDGF-BB, released
by platelets at their activation
4
, (2) the autocrine action of PDGF-AA induced by the pro-inflammatory
cytokines (IL-1 and TNF-a), and (3) TGF-ß
33,34
. In addition, FGF and IGF-1 are released locally by activated SMCs
4
that act on SMC growth. Studies attempting to antagonize the activities
of growth factors PDGF, FGF, and TGF-ß and their receptors using
specific antibodies and antisense oligonucleotides
35,36
have provided protection against SMC proliferation following balloon
angioplasty and thus help to reduce myo-intimal thickening. In addition,
therapeutic modalities such as the infusion of IFN-gamma
37
and anti-ICAM
38
antibodies have been reported to diminish intimal thickening in
various animal models. Since an ideal artery contains SMCs that
are both contractile and quiescent with respect to proliferation,
therapies that would decrease the likelihood of SMC hyperplasia
and consequent luminal occlusion may provide cyto-protection against
the vascular proliferative disorders and delayed dysfunction that occur
after organ transplantation.
Although BMPs were originally identified in bone, their morphogenic
roles have been widely documented in a wide variety of other nonskeletal
tissues
14
. BMPs are novel growth and differentiation factors constituting
a large subfamily in the TGF-ß superfamily of proteins. BMPs have
been shown to play inductive roles in migration and proliferation
of pluripotent stem cells
39
and have a subsequent differentiation into lineage-specific cell
types during morphogenesis. Furthermore, BMPs are capable of stimulating
the expression of phenotypes characteristic to a specific cell type
40
and serve as maintenance factors during the repair and regeneration
of adult tissue
18,41
. Therefore, when BMPs are made available in and around the vascular
endothelium, they may assist in maintaining SMCs in a contractile
and quiescent state rather than aid in the proliferation and loss
of phenotype at vascular injury and as observed in various proliferative
disorders. To support this notion, BMP-2, a related member of BMP-7,
has been shown to inhibit rat vascular smooth muscle proliferation
in vitro
and to inhibit injury-induced intimal hyperplasia when administered
locally by adenovirus-mediated gene transfer following balloon angioplasty
in rats
42
. Our present study supports and extends these observations. Potentially,
BMPs can be used to maintain the expression of SMC phenotype in
cultures during cell expansion in order to engineer functional vascular
grafts for clinical need. Since the effect of BMPs on cell growth
and differentiation is dependent on the developmental stage of the responding
cells, it is likely that the presence of the BMPs in the culture
medium will help to differentiate the expanded cells into the more
"mature" contractile quiescent SMCs.
Among TGF-ß superfamily proteins, TGF-ß has been studied in the
context of vascular integrity
in vitro
and
in vivo43
. This morphogen is a bifunctional growth regulator of SMCs that
was shown to stimulate or inhibit the growth of SMCs depending on
the conditions of cell culture, the presence of other regulatory
molecules, the sequence of their addition, and the concentration
of TGF-ß
44
. Moreover, several studies have shown that TGF-ß is potent in stimulating
the deposition of connective tissues by SMCs
43
. Under our culture conditions, TGF-ß had a proliferative effect
on serum-stimulated DNA synthesis of SMCs. Thus, the beneficial
effect of BMP-7 on SMCs does not appear comparable with that of TGF-ß.
Although the mode of action of the BMP-7 activity is currently unknown,
the results suggest that BMP-7 upregulates both Smad6 and Smad7, the
two inhibitory Smads in the TGF-ß superfamily. Under these conditions,
Smad7 but not Smad6 was upregulated by TGF-ß, whereas neither was induced
by PDGF. Additionally, it has been shown that BMP-7 upregulates
Smad7 expression in lung cancer cell lines
45
and in myoblast cell line C2C12 (unpublished observation).
In vivo
studies have shown that both Smad6 and Smad7 were induced in vascular
endothelium at mechanical stimulus
46
, suggesting potential protection against vascular injury. The fact
that Smad6 is important for the inhibition of BMP signaling
29
suggests that this molecule may in part be involved in the regulation
of the effects of BMP on SMCs.
The observed cytoprotective effect of BMPs on vascular SMC may
provide a basis for developing therapeutic agents for intervention
against vascular proliferative disorders.