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
3
.
1
. Immunohistochemical detection of PTHrP in experimentally produced neointima and human
atherosclerotic lesion As shown in Fig. 1, a marked intimal thickening
was observed in the rat thoracic aorta 2 weeks after balloon injury. Immunoreactivity of PTHrP in the
thickened intima of the balloon-injured rat thoracic aorta was detected by immunohistochemical staining
with anti-hPTHrP-1-34 polyclonal antibody Fig. 1A. Negative control prepared with non-immune rab-
bit serum showed no significant staining Fig. 1B. In non-injured rat aorta, weak immunoreactivity of
PTHrP was detected at medial smooth muscle layer Fig. 1E.
As shown in Fig. 2, non-obstructive cuff-induced intimal thickening was observed after 2 weeks in the
rat femoral artery [29]. Immunoreactivity of PTHrP was also detected in the cuff-induced thickened intima
of the rat femoral artery as well as in the media Fig. 2A.
In human coronary arteries, immunoreactivity of PTHrP was detected in the restenotic lesion after
PTCA obtained at DCA and a primary atheroscle- rotic lesion obtained at autopsy Fig. 3A and Fig.
4A. Negative control prepared with non-immune rab- bit serum showed no significant staining in these spec-
imens Fig. 3B and Fig. 4B.
In the thickened intimal lesion of balloon-injured rat aorta, in the cuff-induced intimal thickening of rat
femoral artery and in the restenotic lesion of human coronary artery after PTCA obtained at DCA, the
immunoreactivity of PTHrP was observed in the re- gions where smooth muscle cells were predominant
Fig. 1C and Fig. 2C and Fig. 3C. In contrast, in the primary atherosclerotic lesion of human coronary
artery obtained at autopsy, the immunoreactivity of PTHrP
was observed
in the
regions where
macrophages were predominant Fig. 4D although weak staining for muscle actin was also detected in
the same regions Fig. 4C.
3
.
2
. Effect of PTHrP-
1
-
34
and PTHrP-
7
-
34
on cuff-induced intimal thickening
As shown in Fig. 5, moderate intimal thickening was induced by a non-obstructive cuff in the rat
femoral artery. Quantitative analysis of intimal thick- ening revealed that the IM ratio was 0.27 9 0.04 and
stenosis was 19.6 9 3.3 in the control group. Lo- cally applied PTHrP-1-34 dissolved in pluronic F-
127
gel decreased
intimal thickening
whereas PTHrP-7-34 increased that Fig. 5. Morphometric
analysis revealed that the decreases in the IM ratio and stenosis by PTHrP-1-34 were dose-de-
pendent Fig. 6. In contrast, PTHrP-7-34 dose-de- pendently
enhanced stenosis.
Furthermore, PTHrP-1-34 and PTHrP-7-34 overcame their ef-
fects each other when both reagents were applied simultaneously Fig. 6.
Fig. 1. Photomicrographs showing immunohistochemical staining of balloon-injured rat thoracic aorta A, B, C and D and uninjured rat thoracic aorta E and F. Rabbit anti-human parathyroid hormone-related protein PTHrP-1-34 polyclonal antibody was used as the first antibody A
and E. Control study was performed using non-immune rabbit serum B and F. HHF-35, a mouse anti-muscle actin monoclonal antibody C, or HAM-56, a mouse anti-macrophage monoclonal antibody D, was used as the first antibody. Original magnification × 100.
Fig. 2. Photomicrographs showing immunohistochemical staining of thickened intima induced by non-obstructive polyethylene cuff. Rabbit anti-human parathyroid hormone-related protein PTHrP-1-34 polyclonal antibody was used as the first antibody A; control study was
performed using non-immune rabbit serum B; HHF-35, a mouse anti-muscle actin monoclonal antibody C, or HAM-56, a mouse anti-macrophage monoclonal antibody D, was used as the first antibody. Original magnification × 200.
Fig. 3. Photomicrographs showing immunohistochemical staining of a restenotic lesion of human coronary artery after percutaneous transluminal coronary angioplasty obtained at directional coronary atherectomy. Rabbit anti-human parathyroid hormone-related protein PTHrP-1-34
polyclonal antibody was used as the first antibody A; The control study was performed using non-immune rabbit serum B; HHF-35, a mouse anti-muscle actin monoclonal antibody C; or HAM-56, a mouse anti-macrophage monoclonal antibody D, was used as the first antibody.
Original magnification × 400. Fig. 4. Photomicrographs showing immunohistochemical staining of an atherosclerotic lesion of human coronary artery obtained at autopsy.
Rabbit anti-human parathyroid hormone-related protein PTHrP-1-34 polyclonal antibody was used as the first antibody A; control study was performed using non-immune rabbit serum B; HHF-35, a mouse anti-muscle actin monoclonal antibody C; or HAM-56, a mouse
anti-macrophage monoclonal antibody D, was used as the first antibody. Original magnification × 40.
4. Discussion
