CARDIOVASCULAR JOURNAL OF AFRICA • Vol 23, No 6, July 2012
AFRICA
e5
Nx mice but scattered spots of black-stained material were
found in the arterial media layer (Fig. 2A, B) at 12 weeks. In
the 5/6 Nx
+
HP mice, diffuse or massive mineral deposition
was detected in the media layer (Fig. 2C, D) at 12 weeks, and
the calcification in p53–/– mice was much more severe than
in the p53
+
/
+
mice. The aorta in the 5/6 Nx mice had mild/
moderate calcification, whereas the aorta in the 5/6 Nx
+
HP
mice, particularly the p53–/– mice, had severe calcification.
The score showed that vascular calcification in the p53–/– mice
had increased significantly compared with that in the p53
+
/
+
mice. Vascular calcification in the 5/6 Nx
+
HP mice was also
increased compared with that in the 5/6 Nx only mice (Fig. 2).
In groups 1 and 2, almost no positive staining of p53, BMP-2,
RUNX2, Osx and ALP protein was found in the VSMCs of
the aorta, but expression of
α
-SMA was detected. In group 4
and particularly group 6, positive staining of BMP-2 (Fig. 3C,
D), RUNX2 (Fig. 3G, H), Osx (Fig. 3K, L) and ALP protein
(Fig. 4G, H) was always detected in the VSMCs. However, no
p53 (Fig. 4C, D) and only weak staining of
α
-SMA (Fig. 4K,
L) was detected in the same tissues. This pattern presented at
eight weeks after 5/6 Nx, and became significantly increased 12
weeks after 5/6 Nx. On the other hand, weak positive staining of
BMP-2 (Fig. 3B, A), RUNX2 (Fig. 3F, E), Osx (Fig. 3J, I) and
ALP proteins (Fig. 4F, E), and enhanced positive staining of p53
(Fig. 4B, A) and
α
-SMA (Fig. 4J, I) was detected in group 5, and
particularly in group 3. Quantitative analysis of these proteins
was performed using the Western blot analysis, and this showed
changes similar to those found by the immunohistochemistry and
indirect immunofluorescence analyses (Figs 5, 6).
Statistical analysis showed that changes in vascular
calcification were positively correlated with the levels of
expression of BMP-2, RUNX2, Osx and ALP, but negatively
correlated with the levels of expression of p53 and
α
-SMA.
Fig. 3. Indirect immunofluorescence staining analysis for BMP-2, RUNX2 and Osx in the aorta at 12 weeks after 5/6
Nx or 5/6 Nx
+
HP (original magnification,
×
200). Positive staining of BMP-2, RUNX2 and Osx protein was detected in
VSMCs, clumping or a green line demonstrated the expression of protein, but in p53
+
/
+
or 5/6 Nx mice, a weaker signal
was detected compared with those in p53–/– or 5/6 Nx
+
HP mice.
p53
+
/
+
mice
p53–/– mice
BMP–2
RUNX2
OsX
Nx
Nx
+
HP
Nx
Nx
+
HP
A
B
C
D
E
F
G
H
I
J
K
L
TABLE 2. LINEAR CORRELATION BETWEENVASCULAR
CALCIFICATIONAND CALCIFICATION-RELATED
PROTEINS INVSMCSAFTER 5/6 NEPHRECTOMY
P53(
+
/
+
) mice
p53(–/–) mice
Protein
R
p
-value
R
p
-value
p53
–0.725
<
0.001
BMP-2
0.569
<
0.001
0.677
<
0.001
RUNX2
0.457
<
0.001
0.594
<
0.001
Osx
0.510
<
0.001
0.618
<
0.001
ALP
0.670
<
0.001
0.773
<
0.001
α
-SMA –0.582
<
0.001
–0.755
<
0.001
