dothelial cells [16,17], and a common receptor for parathyroid hormone PTH and PTHrP exists in vas-
cular smooth muscle [18]. However, there have been few reports demonstrating the expression of PTHrP in
the thickened intima of atherosclerotic and restenotic lesions [19,20]. Moreover, the pathophysiological role
of PTHrP in the vascular wall, either in the normal or diseased state, has not been elucidated.
PTHrP has a potent vasodilator action [21 – 24] which is associated with increased production of cyclic AMP
and decreased intracellular free calcium concentration in vascular smooth muscle [24]. Also, PTHrP exhibits
an inhibitory effect on VSMC migration and prolifera- tion [25]. These actions of PTHrP and its expression in
a variety of tissues have led us to examine the expres- sion of PTHrP and its pathophysiological role in
atherosclerotic lesions. Here we report that PTHrP is expressed in human atherosclerotic lesions as well as
experimentally produced neointimal lesions. Further- more, we demonstrate that locally administered PTHrP
inhibits and PTHrP antagonist enhances intimal thick- ening induced by non-obstructive polyethylene cuff in
rats.
2. Materials and methods
2
.
1
. Materials Human h PTHrP-1-34 and hPTHrP-7-34 were
purchased from Peptide Institute Inc., Osaka, Japan. Rabbit anti-hPTHrP-1-34 polyclonal antibody was
purchased from Peninsula Laboratories Inc. Belmont, CA, USA [16]. This antibody showed no cross-reactiv-
ity with hPTH-1-34, rat PTH-1-34 or Asp6-hPTH- 1-84.
Anti-muscle actin
monoclonal antibody,
HHF-35 [26] Enzo Diagnostics Inc., New York, NY, USA, and anti-macrophage monoclonal antibody,
HAM-56 [27] Enzo Diagnostics Inc., were also used.
2
.
2
. Rat neointimal lesion Male Wistar rats 12 weeks old, purchased from
Nippon Bio-Supply Center Tokyo, Japan, were anes- thetized with ether. A 2F embolectomy balloon catheter
Baxter Healthcare Co., Santa Ana, CA, USA was passed into the aorta via the left femoral artery and
positioned at the distal end of the aortic arch. The balloon was then inflated with 1.0 ml saline and was
withdrawn slowly to the aortic bifurcation. This proce- dure was repeated twice. Thoracic aorta was removed 2
weeks after injury, fixed in 10 formalin neutral buffer solution, and embedded in paraffin.
Male Wistar rats 12 weeks old were anesthetized with ether. Through a left inguinal incision, the left
femoral artery was cleared of surrounding connective tissue and loosely sheathed with a PE-160 polyethylene
cuff 10 mm in length, 1.14 mm in inner diameter, 1.57 mm in outer diameter, Becton Dickinson and Com-
pany, NJ, USA and the incision was then closed [28,29]. The femoral artery was removed 2 weeks later,
fixed in 10 formalin neutral buffer solution, and embedded in paraffin.
2
.
3
. Human atherosclerotic lesion The atherosclerotic lesion of a human coronary
artery was obtained at directional coronary atherec- tomy DCA performed at Toho University Ohashi
Hospital, Tokyo, Japan. The patient was a 55-year-old male in whom restenosis occurred 3 months after percu-
taneous transluminal coronary angioplasty PTCA of the left anterior descending coronary artery. Another
human coronary artery with a primary atherosclerotic lesion was obtained at autopsy at The Social Health
Insurance Medical Center, Tokyo, Japan. The patient was a 72-year-old male. The specimens were fixed in
10 formalin neutral buffer solution Wako Pure Chemical Industries Ltd., Tokyo, Japan and were em-
bedded in paraffin blocks.
2
.
4
. Immunohistochemical staining of PTHrP The formalin-fixed and paraffin-embedded tissues
were sliced. The tissue sections were deparaffinized with xylene three times 3 min for each xylene treatment
and rehydrated with 100 ethanol twice and 95 etha- nol twice 3 min for each ethanol treatment. The
sections were then washed with phosphate-buffered sa- line PBS, pH 7.4 for 5 min. After blocking endoge-
nous peroxidase activity with 0.03 hydrogen peroxide in methanol, the sections were incubated with 10
normal goat serum in PBS for 30 min. After these procedures, the sections were incubated overnight at
4°C with rabbit polyclonal antibody raised against hPTHrP-1-34 diluted with PBS containing 10 fetal
bovine serum. The dilution of this antibody was opti- mized, and 1:500 dilution was used in this study. The
streptavidin-biotin-peroxidase SAB method was per- formed using Histofine SAB kit Nichirei Co., Tokyo,
Japan. Briefly, the tissue sections were incubated with biotinylated secondary antibody for 45 min at room
temperature, and were subsequently incubated with streptavidin conjugated with horseradish peroxidase for
30 min at room temperature. The reaction was termi- nated by washing three times with PBS 5 min for each
washing. Immunoreactive PTHrP was visualized by 3,3-diaminobenzidine substrate kit Vector Laborato-
ries Inc., Burlingame, CA, USA. Counterstaining of the cell nuclei was performed with Meyer’s hematoxylin
solution. Control staining was performed by substitut- ing the primary antibody for non-immune rabbit
serum. HHF-35 1:50 dilution or HAM-56 1:50 dilu- tion
was also
used to
identify VSMCs
and macrophages, respectively.
2
.
5
. Effect of PTHrP-
1
-
34
and PTHrP-
7
-
34
on cuff-induced intimal thickening
Male Wistar rats 12 weeks old were anesthetized with ether. Through a left inguinal incision, the left
femoral artery was cleared of the surrounding connec- tive tissue and loosely sheathed with a non-obstructive
polyethylene cuff as above described. PTHrP-1-34 andor PTHrP-7-34, a PTHPTHrP receptor antago-
nist [30] was dissolved in 25 wv pluronic F-127 gel solution [31,32] BASF Wyandotte Co., Wyandotte,
MI, USA. Immediately after this procedure, 200 ml of the gel solution containing PTHrP-1-34 andor
PTHrP-7-34 was applied to the femoral artery, and the wound was closed. The unique characteristic of the
gel is reverse thermal gelation. Briefly, 25 wv solu- tion of the gel is fluid at refrigerator temperature 4 –
5°C, but is soft gel at body temperature. Preliminary experiments showed that PTHrP molecule was slowly
and constantly released from the gel solution. The artery was treated with the gel solution alone in the
control study. Two weeks later, the rats were perfusion- fixed with 10 formalin neutral buffer solution. Then
the femoral artery was removed, postfixed in 10 for- malin neutral buffer solution, and embedded in
paraffin. The middle segment of the artery was cut into cross-sectional pieces with 5 mm thickness and stained
by Elastica van Gieson staining. Cross-sectional area of the intima and of the media of the femoral arteries were
measured with NIH Image software using a Macintosh computer, and the ratio of intimal area to medial area
IM ratio and the ratio of intimal area to the area within the internal elastic lamina stenosis were
calculated.
2
.
6
. Statistics Data were analyzed by one-way analysis of variance.
When statistically significant effects were found, Bon- ferroni test was performed to isolate the differences
between the groups. A P value of less than 0.05 was considered significant. All data are presented in the text
and figures as mean 9 S.E.M.
3. Results
