© 1996 American Heart Association, Inc.
Articles |
Evidence That Implicates the Parathyroid Hormone–Related Peptide in Vascular Stenosis
Increased Gene Expression in the Intima of Injured Rat Carotid Arteries and Human Restenotic Coronary Lesions
From the Third Department of Internal Medicine (S.O., S.S., S.T., K.Y.) and the Departments of Cell Physiology (A.O., H.Y., S.Y.) and Pathology (T.N., M.I.), Atomic Disease Institute, Nagasaki University School of Medicine, Nagasaki, and the Kokura Memorial Hospital, Kitakyushu (T.Y., M.N.), Japan; and the Endocrine Unit, Massachusetts General Hospital, Boston, Mass (G.V.S.).
Correspondence to Akira Ohtsuru, MD, Department of Cell Physiology, Atomic Disease Institute, Nagasaki University School of Medicine, 1-12-4 Sakamoto, Nagasaki 852, Japan.
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Abstract Proliferation of vascular smooth muscle cells (VSMCs) is considered to be one key event underlying the pathophysiology of restenosis after angioplasty. The parathyroid hormone–related peptide (PTHrP) and its receptor, a local autocrine and paracrine regulator of cellular growth in a variety of normal cell types, have been reported in the vicinity of VSMCs. To investigate how PTHrP might be involved in the process of neointimal formation after balloon angioplasty, we examined PTHrP expression in balloon-denuded rat carotid arteries and human coronary arteries that had been retrieved by directional atherectomy. In rat carotid arteries, the RNase protection assay and in situ hybridization demonstrated that PTHrP mRNA expression increased fourfold to sixfold 1 to 7 days after denudation and continued for 28 days, coincident with downregulation of PTH/PTHrP receptor mRNA expression. In situ hybridization and immunohistochemistry revealed that PTHrP expression in balloon-denuded carotid arteries was mainly localized to the neointima. To confirm the involvement of the PTHrP in human coronary artery restenotic lesions, immunohistochemical analysis of human coronary atherectomy specimens (23 primary and 10 restenotic lesions) was then performed. The number of intimal cells that expressed PTHrP protein was significantly higher in restenotic (407±53 cells/mm2; range, 143 to 739) than in stable angina (50±12 cells/mm2; range, 18 to 132; P<.05) or unstable angina (129±16 cells/mm2; range, 21 to 232; P<.05) specimens. These data demonstrate that PTHrP gene expression in VSMCs markedly increases during neointimal formation, supporting the hypothesis that PTHrP may play an important role in vascular stenosis as a regulator of VSMC proliferation.
Key Words: PTH/PTHrP receptor • restenosis • atherectomy • neointimal formation • PTHrP
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Introduction |
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The vascular injury that is associated with percutaneous revascularization therapy initiates a complex cascade of biologic events, such as thrombosis, VSMC proliferation and migration, and extracellular matrix production. Among these, proliferation and migration of VSMCs may represent key events that underlie the pathophysiology of restenosis,1 2 3 which occurs in as many as 50% of patients within 6 months after balloon angioplasty.4 5 During the last decade, several polypeptides have been identified as potential regulators of VSMC proliferation and migration. These include transforming growth factor–ß, epidermal growth factor, insulin-like growth factor–I, platelet-derived growth factor, basic fibroblast growth factor, endothelin I, and Ang II.6 7 8
Recently, PTHrP has also been reported to be a potential regulator of VSMCs.9 PTHrP was first isolated from human tumor cells and shown to be responsible for the hypercalcemia in patients with humoral hypercalcemia of malignancy.10 11 12 PTHrP has limited N-terminal sequence homology with PTH13 and has been demonstrated to share the same receptor with PTH (PTH/PTHrP receptor).14 15 In fact, the in vitro biologic activity of the N-terminal fragment of PTHrP is similar to that of PTH.13 Unlike PTH, which is produced extensively in the parathyroid gland and acts in a classic endocrine fashion to regulate mineral ion homeostasis through bone and kidney, PTHrP does not normally circulate in the bloodstream except in humoral hypercalcemia of malignancy. PTHrP gene expression is observed in a variety of normal tissues,16 17 which often also express the PTH/PTHrP receptor gene within or near such tissues,18 suggesting that PTHrP may act locally in a paracrine and autocrine fashion.
PTHrP and PTH/PTHrP receptors are also expressed in VSMCs,9 19 and PTHrP is considered to be involved in the control of vascular tone as a vasodilator.20 On the other hand, PTHrP expression is stimulated by at least some of the aforementioned growth factors (eg, endothelin I, epidermal growth factor, transforming growth factor–ß, and Ang II).21 22 23 24 These findings of regulated PTHrP production in VSMCs raise the possibility that locally produced PTHrP opposes not only the contractile but also the mitogenic effects of these growth factors. In addition, the promoter regions of the human PTHrP gene are GC rich and include a number of GC "boxes." Furthermore, transcript turnover is rapid, presumably due to the AUUUA motifs within the 3' untranslated region.25 26 27 These characteristics are commonly seen in the mRNA of cytokines that are involved in cell proliferation and differentiation. Accordingly, we speculated that PTHrP might play an important role as a cytokine in regulating VSMC proliferation and might be involved in the process of neointimal formation after balloon angioplasty. The present study was designed to investigate the extent to which PTHrP expression was increased during neointimal formation after balloon arterial injury. Furthermore, analysis of human atherosclerotic lesions that were retrieved at the time of coronary atherectomy from patients with angina pectoris was performed to understand how PTHrP expression might be involvement in restenotic and primary lesions.
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Methods |
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Arterial Injury Model and Tissue Preparation
We examined PTHrP gene expression in balloon-injured arteries in male Wistar rats (n=90) weighing 450 to 500 g (Charles River Japan). Protocols for this and all subsequent series of experiments were approved by the Nagasaki University Institutional Animal Care and Use Committee. The rats were anesthetized with pentobarbital sodium (50 mg/kg body wt IP). A Fogarty balloon catheter (2F, Baxter Healthcare) was introduced into the left external carotid artery through an arteriotomy. The catheter was advanced to the aortic arch and pulled back three times while the balloon was inflated with air. After removal of the catheter, the left external carotid artery was ligated and the wound closed. The right carotid artery served as a control and underwent identical manipulation except for balloon denudation. The rats were euthanized with an overdose of pentobarbital sodium at various times after arterial denudation (n
10 at
each time). Both common carotid arteries were dissected and processed
as described below.
RNase Protection Assay
Expression of PTHrP and PTH/PTHrP receptor mRNAs was evaluated
by RNase protection assay. At the times indicated, both denuded and
nondenuded common carotid arteries were excised; adherent perivascular
connective tissue and adventitia were carefully dissected away in
ice-cold PBS ([in mmol/L] KCl 2.7, KH2PO4
1.5, NaCl 137, and Na2HPO4 8.1; pH 7.0); and
the arteries were immediately frozen in liquid N2. Total
cellular RNA was isolated from arterial samples by the
guanidinium thiocyanate method according to the manufacturer's
instructions (RNAzol, Tel-Test). Hybridization probes were labeled with
[
-32P]CTP using T3 RNA polymerase according to the
supplier's guidelines (MAXI script kit, Ambion). The RNA probes were
used as follows: (1) Rat PTHrP, a 994-bp
EcoRI/Xba I fragment of rPLPm10 (courtesy of Drs
G.N. Hendy and D. Goltzman, McGill University, Montreal, Canada), was
subcloned into pBluescript SK+ (Stratagene) and linearized with
Kpn I.25 (2) Rat PTH/PTHrP receptor, a 569-bp
HindIII/Not I fragment of R15B, was subcloned
into pBluescript SK+ and linearized with
HindIII.28 (3) Rat GAPDH, a 318-bp
Sac I/BamHI fragment of pTRI-GAPDH
(Ambion).29 The total RNA (30 µg) from rat carotid
artery was hybridized with PTHrP, PTH/PTHrP receptor riboprobe
(1x105 cpm), and GAPDH (5x103 cpm) riboprobe
overnight. After hybridization, the remaining single-strand RNA
probe and unhybridized sample RNA were removed by digestion with a
mixture of RNase A and RNase T1 (RPAII ribonuclease protection kit,
Ambion). The reaction products were then separated on a 4%
polyacrylamide (19:1 wt/wt, acryl/bis) gel containing 8 mol/L
urea for detection of PTHrP mRNA and on a 5% polyacrylamide
gel containing 8 mol/L urea for detection of PTH/PTHrP receptor mRNA.
Blots were then exposed for 24 (PTHrP) or 72 (PTH/PTHrP receptor) hours
to Kodak AR film. The intensity of the hybridization signal was
quantitated by use of a Fuji BAS 2000 Bio-Image Analyzer (Fuji
Film Co Ltd). The ratios of PTHrP to GAPDH and of the PTH/PTHrP
receptor to GAPDH mRNA radioactivity were calculated for each
sample.
In Situ Hybridization
In situ hybridization was performed to identify the locale and
extent of cells that expressed PTHrP and PTH/PTHrP receptor mRNAs.
Arterial tissues for in situ hybridization analysis
were fixed in 4% p-formaldehyde for 12 hours, dehydrated,
embedded in paraffin, and serially sectioned at 4 µm. After removal
of the paraffin by application of a xylene and ethanol series, the
tissue sections were immersed in 0.2N HCl for 15 minutes. Membrane
protein digestion was performed with 20 µg/mL proteinase K (Sigma
Chemical Co) in PBS for 10 minutes at 37°C. Proteolysis was stopped
by immersing the slides in 4% p-formaldehyde for 10
minutes. The slides were then treated with 2 mg/mL Gly for 10 minutes
and dehydrated. PTHrP and PTH/PTHrP receptor mRNAs were detected by
using a 343-bp Pvu II/Bgl II fragment of rPLPm10
and a 569-bp HindIII/Not I fragment of R15B,
respectively, subcloned into pBluescript SK+ (Stratagene). Antisense
and sense single-strand cRNA probes were synthesized with
digoxigenin-labeled UTP (Boehringer Mannheim) and T3/T7 RNA
polymerase (Ambion). Sections were hybridized at 50°C for 16 hours
with the digoxigenin-labeled riboprobe (50x dilution) with a
solution containing 50% deionized formamide; 10 mmol/L Tris HCl, pH
7.6; 200 µg/mL yeast RNA (Boehringer Mannheim); 1x
Denhardrt's solution (Sigma); 10% dextran sulfate; 600 mmol/L
NaCl; 0.25% SDS; and 1 mmol/L EDTA, pH 8.0. After the slides
were washed in 2x SSC/50% formamide at 60°C for 20 minutes (1x SSC
is 150 mmol/L NaCl and 15 mmol/L sodium citrate), they were subjected
to RNase A treatment (10 µg/mL in 10 mmol/L Tris HCl, pH 8.0; 500
mmol/L NaCl; and 1 mmol/L EDTA) at 37°C for 30 minutes. Hybridized
digoxigenin-labeled riboprobe was detected with a DIG DNA detection
kit (Boehringer Mannheim) according to the supplier's
guidelines.
Immunohistochemistry
To identify those cells that expressed PTHrP protein,
immunohistochemical staining with a mouse monoclonal antibody to PTHrP
(Oncogene Science) was applied to deparaffinized sections, with minor
modifications of a method described previously.30
Arterial tissues were fixed in 4%
p-formaldehyde, dehydrated, embedded in paraffin, cut (4
µm), and then mounted on glass slides. Nonspecific antibody binding
was blocked by incubation with 2% normal goat serum in PBS for 15
minutes. Sections were then incubated with anti-PTHrP antibody (5
µg/mL) overnight at 4°C. The bound primary antibody was detected
using a streptavidin-biotin–alkaline phosphatase method.
Slides were incubated with alkaline phosphatase–conjugated goat
anti-mouse immunoglobin antibodies (American Qualex) for 30
minutes. A mixture of 5-bromo-4-chloro-3-indolyl phosphate and nitro
blue tetrazolium chloride (BRL) was used as a chromogenic
substrate for alkaline phosphatase. The slides were flooded with
Tris-EDTA buffer to stop the reaction, dehydrated, and mounted. As
negative controls, adjacent sections were incubated with nonimmunized
mouse serum in lieu of the primary antibody.
Human Atherectomy Specimens
A total of 33 atherosclerotic lesions were examined in this
study (Table
). All lesions were obtained from patients
with angina pectoris at the time of directional atherectomy
(Simpson Coronary Atherocath, Devices for Vascular
Intervention) at Kokura Memorial Hospital, Kitakyushu. The
atherectomy site was determined by angiography immediately before
atherectomy. A total of 22 lesions were removed by directional
atherectomy from patients who were undergoing
percutaneous revascularization for
the first time. These lesions were designated as primary. Of these 22
lesions, 9 were obtained from patients with stable angina pectoris
(patients 1 through 9, Table). Another 13 were obtained from patients
with unstable angina pectoris (patients 10 through 22, Table). Unstable
angina pectoris was classified as one of the following clinical
syndromes: angina pectoris at rest, recent-onset angina pectoris
(<2 months), and accelerated angina pectoris. In contrast, 11 lesions
from patients 23 through 33 were identified at the site of a previous
angioplasty (Table) and therefore designated as restenotic.
All 11 restenotic lesions were obtained from patients with
clinical evidence of recurrent myocardial ischemia. In these 11
patients, the period between the initial angioplasty and subsequent
atherectomy ranged from 45 to 180 days. Informed consent was obtained
from all patients.
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After atherectomy, all retrieved specimens were immediately immersed in
4% p-formaldehyde solution for 2 or 3 days. Tissues were
then embedded in paraffin, cut into 4-µm-thick serial sections,
and placed on glass slides. Immunohistochemical staining for PTHrP was
then performed to compare the expression of PTHrP protein between
primary and restenotic human atherosclerotic plaques. To
characterize cells that expressed PTHrP, VSMCs were identified with a
mouse monoclonal antibody to human –smooth muscle actin (Dako
Corp).
Immunohistochemical Analysis of Atherectomy
Specimens
Histological analysis was performed
without knowledge of the clinical data. Analysis of
hematoxylin-and-eosin–stained and
PTHrP-immunostained sections included counting the total
number of cells and PTHrP-positive cells within the intima and
measuring the total intimal area by using a computerized image
analysis system (MCID, Fuji film). To ensure that the results
were not influenced by differences in cellular density between primary
and restenotic lesions, the number of PTHrP-positive cells
was also expressed as a PTHrP labeling index (in percent). The latter
was defined as the number of PTHrP-positive cells divided by the total
number of cells in each specimen.
Statistics
Results were expressed as mean±SE. Statistical
significance was evaluated by an unpaired Student's t test
for comparisons between two groups or by ANOVA followed by
Scheffé's procedure for comparisons between more than two
groups. A value of P<.05 was considered statistically
significant.
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Results |
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RNase Protection Assay
Using the RNase protection assay, we examined PTHrP expression in rat carotid arteries after balloon denudation (Fig 1
). Normal (nondenuded) rat carotid arteries weakly
expressed PTHrP mRNA. A significant increase in PTHrP mRNA levels was
observed within 4 hours after balloon denudation, and these were
fourfold to sixfold higher than those in nondenuded carotid arteries 1
to 7 days after balloon denudation. This increased level of gene
expression was not transient but continued for 28 days. However,
increased expression of PTHrP almost returned to control level by 12
weeks.
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Because balloon denudation increased PTHrP mRNA levels, we examined
whether such an increase would also change the level of PTH/PTHrP
receptor mRNA expression. As shown in Fig 2, PTH/PTHrP
receptor mRNA also existed in normal rat carotid arteries. After
balloon denudation, PTH/PTHrP receptor mRNA content decreased gradually
and reached about 20% of the control level in carotid arteries that
were harvested 14 days after denudation. This result showed that
downregulation of PTH/PTHrP receptor mRNA was not transient but
continuous. At 28 days to 12 weeks after balloon denudation, however,
downregulation gradually recovered to near control levels.
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In Situ Hybridization
To identify the locale and extent of cells that expressed PTHrP,
in situ hybridization of PTHrP mRNA was performed. Using
digoxigenin-labeled cRNA sense and antisense probes for PTHrP, we
examined nondenuded and denuded carotid arteries for PTHrP mRNA
expression. Nondenuded arteries that had hybridized with the antisense
probe showed a very low hybridization signal in the medial layer (Fig 3A, arrowhead). In denuded arteries at day 3, a
hybridization signal was noted in the media, particularly the
inner layer (Fig 3B). At 7, 14, and 28 days after balloon denudation,
PTHrP mRNA was abundant in the developing neointima but low
in the underlying media (Fig 3C, 3D, and 3E). However, 12 weeks after
denudation, PTHrP mRNA decreased substantially, especially in the
neointima (Fig 3F).
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Next, we examined PTH/PTHrP receptor mRNA expression. In the nondenuded
carotid artery, hybridization signal was observed in the entire media
(Fig 4A). In the denuded artery at day 3, hybridization
signal was low or absent in the media (Fig 4B). In addition, 7 days
after balloon denudation the sections revealed a faint hybridization
signal in both the developed neointima and media (Fig 4C).
At 14 days after balloon denudation, however, PTH/PTHrP receptor mRNA
again appeared in the neointima, especially the inner layer
(Fig 4D). At 28 days after balloon denudation, hybridization signal had
increased further and was present in the entire
neointima (Fig 4E). By 12 weeks after balloon denudation, a
hybridization signal had became stronger not only in the
neointima but also in the media (Fig 4F). In contrast,
arteries that had been hybridized with the sense probe lacked a
positive hybridization signal in both nondenuded and denuded
arteries.
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Immunohistochemistry
To complement the RNase protection assay that was designed to
identify the time course of PTHrP gene expression and the in situ
hybridization analysis that was designed to identify the locale
and extent of cells that expressed PTHrP, we also performed
immunostaining for PTHrP protein. Normal (nondenuded)
rat carotid arteries showed very little staining for PTHrP protein in
the media (Fig 5A).On the first day after denudation,
the intensity of PTHrP staining was greater in the entire media,
particularly the inner layer (Fig 5B). At this time, no
neointimal cells were observed. On the seventh
postdenudation day, the intensity of PTHrP staining in the media
had increased further (Fig 5C) and a neointima with strong,
homogeneous staining for PTHrP protein was observed (Fig 5C). At 14 weeks after denudation, the intensity of PTHrP protein
staining had increased further in the neointima as well as
the media (Fig 5D). The distribution of PTHrP protein on day 14 was
similar to that of PTHrP mRNA on the same day, as demonstrated by in
situ hybridization. Strong staining for PTHrP protein was still
observed on day 28 in both the neointima and media (Fig 5E). However, the intensity of staining at this time was slightly lower
in both layers compared with that on day 14. After 12 weeks, the extent
of PTHrP immunostaining had decreased in both the
neointima and media (Fig 6F). We found no
positive staining in negative control specimens.
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PTHrP Expression in Human Atherosclerotic
Plaques
Finally, to investigate whether PTHrP might be involved in the
development of human restenotic coronary lesions
after balloon angioplasty, we performed immunohistochemical staining
for PTHrP protein in 33 coronary specimens that had been
retrieved by directional atherectomy (13 unstable angina, 9 stable
angina, and 11 restenotic specimens; Table
). Fig 6A and 6B
shows hematoxylin-and-eosin–stained and
PTHrP-immunostained restenotic specimens.
Hypercellularity and a high proportion of PTHrP-positive cells were
observed in the intima. Immunohistochemical staining for human
–smooth muscle actin showed that the majority of cells that
expressed PTHrP were VSMCs (Fig 6C). Fig 7 shows a
hematoxylin-and-eosin–stained and
PTHrP-immunostained specimen from a patient with stable
angina. In contrast to restenotic specimens,
hypercellularity was not observed and only a few PTHrP-positive cells
in the intima were noted. Mean cell density in the intima of
restenotic specimens was 632±73 cells/mm2
(range, 282 to 1050); of stable angina specimens, 376±63
cells/mm2 (range, 121 to 625); and of unstable angina
specimens, 559±54 cells/mm2 (range, 300 to 903). The
difference in intimal cell density between restenotic and
stable angina specimens was significant, but the difference between
restenotic and unstable angina specimens was not. The
density of PTHrP protein–positive cells was at least three times
higher in restenotic (407±53 cells/mm2; range,
143 to 739) than in stable angina (50±12 cells/mm2; range,
18 to 132; P<.05) and unstable angina (129±16
cells/mm2; range, 21 to 232; P<.05) specimens
(Fig 8A). We found no immunostaining in
negative control specimens (data not shown). To ensure that this
observation was not influenced by the difference in cell density
between restenotic and stable or unstable angina specimens,
we also examined the PTHrP index in each sample. The average
PTHrP index in restenotic specimens (66.2±7.1%; range,
30.0% to 91.2%) was significantly higher than that of stable angina
(16.6±3.7%; range, 2.9% to 36.8%; P<.05) and unstable
angina (25.6±3.2%; range, 3.4% to 42.2%; P<.05)
specimens (Fig 8B).
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Discussion |
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The main finding of the present study is that balloon denudation of rat carotid arteries causes marked and persistent stimulation of PTHrP mRNA and protein expression that lasts for 28 days. A small amount of PTHrP expression was detected in the outer layer of the tunica media in the normal (nondenuded) carotid artery. Although no neointimal formation was observed 1 and 3 days after denudation, the number of PTHrP-positive cells increased throughout the entire media, particularly the inner layer. From 7 to 28 days after balloon denudation, PTHrP mRNA and protein were observed mainly in the neointima. These serial changes in the numbers of PTHrP-positive cells after balloon denudation are similar to those of VSMCs, which are thought to migrate from the media to the intima and proliferate during the process of neointimal formation.3 In addition to our observations, induction of PTHrP expression by mechanical stretch has already been demonstrated in SMCs of the chick oviduct31 and the rat uterus,32 33 urinary bladder,34 and gastrointestinal tract.30 In VSMCs, increased expression of PTHrP has also been recently reported in a balloon-distended abdominal artery that did not exhibit neointimal formation but that did undergo mechanical stress.35 However, that study also showed that PTHrP expression lasted only 2 to 6 hours and returned to control levels after 24 hours. Our results also revealed increases in PTHrP mRNA at 4 hours, and this early expression of PTHrP might have resulted from mechanical stress. However, continuous PTHrP expression was still observed 28 days after balloon denudation and was localized to neointimal VSMCs rather than the media, suggesting that mechanical distention caused a temporary increase in PTHrP while continuous PTHrP induction was not due to mechanical stress but mainly to denudation of the endothelial layer and subsequent neointimal formation.
The present study also demonstrated overexpression of PTHrP in the neointima of human restenotic atherectomy specimens. Furthermore, the density and proportion of intimal PTHrP-positive cells in restenotic specimens were significantly higher than those in both stable and unstable angina specimens. The increased PTHrP expression in human restenotic atherectomy specimens corresponded with that observed in the neointima of balloon-denuded rat carotid arteries, suggesting that PTHrP may be involved in the process of neointimal formation in human restenotic coronary arteries. In addition, recent studies have demonstrated neointimal hypercellularity in atherectomy specimens not only from patients with restenosis but also from those with unstable angina.36 Although we detected no difference in intimal cell density between restenotic and unstable angina specimens, the density of PTHrP-positive cells in unstable angina specimens was twice that of stable angina specimens, suggesting that PTHrP-positive cells may be related to the induction of unstable angina.
Formation of a neointima is most commonly cited as the
dominant cellular event in the process leading to
restenosis and is known to consist of VSMCs and
macrophages.37 38 The most prominent cell type in
the neointima is thought to be VSMCs.39 40
Immunohistochemical staining for –smooth muscle actin in
restenotic atherectomy specimens in the present study
confirmed these earlier results and revealed that VSMCs were dominant
in the intimal layer. These results indicate that
neointimal VSMCs overexpresses PTHrP.
The PTH/PTHrP receptor has been recently cloned and sequenced and includes seven transmembrane domains that link guanyl nucleotide–binding proteins.15 Binding of PTHrP to its receptor stimulates several intracellular second messengers, including cAMP and [Ca2+]i.28 In VSMCs, PTHrP signals exclusively by activating cAMP and does not influence [Ca2+]i.41 Interestingly, increased levels of intracellular cAMP have been demonstrated to inhibit VSMC proliferation.42 In fact, exogenous addition of PTHrP has an inhibitory effect on Ang II–induced DNA synthesis24 and insulin-like growth factor–I–induced cell proliferation in primary cultures of VSMCs,43 pointing out that PTHrP has a suppressive effect on neointimal formation.
Accordingly, some question remains as to why overexpressed PTHrP in VSMCs after balloon denudation cannot inhibit neointimal formation. Our results demonstrated a change in the level of not only PTHrP but also its receptor after balloon denudation. The decrease in PTH/PTHrP receptor mRNA coincided with a reciprocal increase in PTHrP mRNA content, suggesting that increased PTHrP synthesis after angioplasty results in downregulation of the PTH/PTHrP receptor. Previous studies have demonstrated reduced levels of PTH/PTHrP receptor mRNA by Ang II and endothelin I treatment in VSMCs.19 Therefore, "escape" from the physiological regulation of VSMCs by PTHrP due to homologous or heterologous downregulation of the PTH/PTHrP receptor may allow VSMC proliferation despite overexpression of PTHrP in the neointima in vivo. After 28 days, however, PTH/PTHrP receptor mRNA content in the neointima tends to recover from downregulation. It may be that PTHrP has a suppressive effect on neointimal proliferation at this later time. Additionally, in rat bone cells, PTHrP stimulates collagen synthesis,44 suggesting that overexpressed PTHrP in VSMCs may promote extracellular matrix protein and result in neointimal formation.
In summary, our study has demonstrated that PTHrP mRNA and protein in the intima are induced during intimal formation in injured rat carotid and human restenotic coronary arteries. The characteristic distribution of PTHrP and the PTH/PTHrP receptor was revealed by immunohistochemical and in situ hybridization analyses. This increased PTHrP expression was not transient but continuous and was accompanied by downregulation of its receptor. Among the several cytokines that typically bind to tyrosine kinase receptors and that have been shown to initiate proliferation of VSMCs during neointimal formation after balloon injury, PTHrP may be unique, in that it binds to seven transmembrane receptors, upregulates intracellular cAMP, and thus may play an important role in the process of neointimal formation.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture, Japan (06670729, 06454340, 06807051). The authors are grateful to Yoshiko Tsunoo for secretarial work.
Received March 25, 1995; accepted December 20, 1995.
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