CARDIOVASCULAR JOURNAL OF AFRICA • Vol 23, No 6, July 2012
AFRICA
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implies that p53–/– VSMCs have the potential to transform to
osteoblast-like cells, and that p53 may inhibit the transformation
of VSMCs to osteoblast-like cells. We therefore propose that the
inhibition of expression of p53 in VSMCs may be involved in the
pathogenesis of the osteogenic differentiation of VSMCs in CKD.
To test this hypothesis, we used the experimental model of
CKD-associated vascular calcification in p53
+
/
+
and p53–/–
mice with 5/6 Nx and HP diet. At eight and 12 weeks
after 5/6 Nx, aortic calcification, and markers of osteogenic
differentiation, smooth muscle-specific protein and p53 proteins
in the aortic tissue were studied, and the effects of p53 on
osteogenic differentiation of VSMCs were assessed.
We found that changes in kidney histopathology and plasma
BUN levels showed no significant difference between the p53
+
/
+
and p53–/– mice at eight and 12 weeks following 5/6 Nx, which
indicates that both p53
+
/
+
and p53–/– mice developed to the
same stage of CKD at the same time following 5/6 Nx. There
were no obvious differences in deposition of serum calcium
phosphate between groups 1 and 2, groups 3 and 4, and groups
5 and 6, which indicated that the disruption by p53 had no effect
on the calcium–phosphate homeostasis.
In sham-operated mice, mineral deposition was not assessed.
In p53
+
/
+
mice with 5/6 Nx, and particularly with 5/6 Nx
+
HP,
scattered mineral deposition was found in the arterial media
layer. In p53–/– mice with 5/6 Nx, and particularly with 5/6 Nx
+
HP, diffuse or large-scale mineral deposition was detected in
the media layer. Therefore the aortas of p53
+
/
+
and p53–/– mice
with 5/6 Nx had mild/moderate calcification, and the aortas of
p53–/– mice with 5/6 Nx
+
HP had severe calcification.
The vascular calcification score showed that in p53–/– mice
and in mice with 5/6 Nx
+
HP, vascular calcification was
significantly increased compared with that in p53
+
/
+
mice and in
mice with 5/6 Nx only. This result suggests that (1) the vascular
calcification may be inhibited by p53 in CKD mice, and (2) the
5/6 Nx plays a vital role in vascular calcification, and the HP diet
aggravates vascular calcification.
Our further investigation showed that almost no positive
staining of p53, RUNX2, Osx and ALP proteins was found in
the VSMCs from sham-operated mice, but expression of
α
-SMA
was detected. In p53–/– mice, positive-staining of RUNX2,
Osx and ALP proteins was detected in VSMCs, but no p53 and
only weak staining of
α
-SMA was detected in the same tissue.
This pattern presented at eight weeks after 5/6 Nx, and became
more apparent at 12 weeks after 5/6 Nx. On the other hand,
weak positive staining of RUNX2, Osx and ALP proteins, and
enhanced positive staining of p53 and
α
-SMA was detected in
p53
+
/
+
mice.
Statistical analysis indicated that changes in vascular
calcification were positively correlated with levels of expression
of RUNX2, Osx andALP, but negatively correlated with levels of
expression of p53 and
α
-SMA. Levels of expression of RUNX2,
Osx and ALP were significantly negatively correlated with
levels of expression of p53 and
α
-SMA. These results indicate
Fig. 5. Western blot analysis for p53, BMP-2 and RUNX2
in the aorta at 12 weeks after 5/6 Nx or 5/6 Nx
+
HP. A
densitometry graph shows the expression levels of each
calcification-related protein identified by Western blot
analysis in the aorta. The graph indicates the relative
band density when levels of GAPDH protein expression
in each sample were calculated as 100%. Values are
means
±
SE;
n
=
5 per group.
a
p
<
0.001 vs p53–/– mice,
NS
=
no significant difference vs 5/6 Nx mice with the
same genetype.
p53
+
/
+
Mice
p53–/–
Mice
p53
53 kd
GAPDH
36 kd
BMP-2
16 kd
GAPDH
36 kd
RUNX2
55 kd
GAPDH
36 kd
Nx
p53
+
/
+
Mice
p53–/–
Mice
p53
53 kd
GAPDH
36 kd
BMP-2
16 kd
GAPDH
36 kd
RUNX2
55 kd
GAPDH
36 kd
Nx
+
HP
2.5
2
1.5
1
0.5
0
Calcification-related protein/GAPDH
Nx Nx
+
HP Nx Nx
+
HP Nx Nx
+
HP
p53+/+
p53–/–
NS
a
a
p53
BMP-2
RUNX2
NS
NS
aNS
aNS
Fig. 6. Western blot analysis for Osx, ALP and
α
-SMA
in the aorta at 12 weeks after 5/6 Nx or 5/6 Nx
+
HP. A
densitometry graph shows the expression levels of each
calcification-related protein identified by Western blot
analysis in the aorta. The graph indicates the relative
band density when levels of GAPDH protein expression
in each sample were calculated as 100%. Values are
means
±
SE;
n
=
5 per group.
a
p
<
0.001 vs p53–/– mice,
b
p
<
0.05,
c
p
<
0.01 vs 5/6 Nx mice with the same genetype.
p53
+
/
+
Mice
p53–/–
Mice
Osx
45 kd
GAPDH
36 kd
ALP
12 kd
GAPDH
36 kd
a
-SMA
42 kd
GAPDH
36 kd
Nx
p53
+
/
+
Mice
p53–/–
Mice
Osx
45 kd
GAPDH
36 kd
ALP
12 kd
GAPDH
36 kd
a
-SMA
42 kd
GAPDH
36 kd
Nx
+
HP
2.5
2
1.5
1
0.5
0
Calcification-related protein/GAPDH
Nx Nx
+
HP Nx Nx
+
HP Nx Nx
+
HP
p53+/+
p53–/–
a
a
p53
BMP-2
RUNX2
c
c
ac
ac
a
b
ab
