3.10 | Cyclopamine prevents mechanical stress-induced
LF fibrosis and hypertrophy in vivo
As stated previously, we built a rabbit model in which mechanical stress
was concentrated on the level of L3-4 segment with fixation of adjacent
segments (L2-3 and L4-5, Fig. 10A). We found that the L3-4 level LF in
group B was more thicker than that in group A (Fig. 10B). In addition,
it was shown that the structure of the hypertrophic LF in rabbits was
almost similar to that of the LF in humans (Fig. 10C, D) (Hayashi et
al., 2017). Those results demonstrated the degeneration and hypertrophy
of LF under the condition with continuously mechanical stress. Briefly,
the L3-4 level LF in Group B showed a significant decrease in the
density of elastic fibres, the disruption of elastic fibres and an
increase in collagen fibres (Fig. 10C, D). The levels of mRNA expression
in each group were evaluated by RT-PCR. The expression of
fibrosis-related genes on the L3-4 level LF in Group B, including
WISP-1, Gli1, Col1a2, Col2a1, and Col3a1 was significantly elevated as
compared with that on the same level in group A, whereas elastin
expression was significantly decreased (p<0.01) (Fig. 10 E,
F). These data indicated the activation of WISP-1/Hedgehog-Gli1
signaling in hypertrophic LF induced by mechanical stress in rabbit
model, which were similar to the results observed in hypertrophic LF in
patients with LSCS.
Moreover, our in vitro experiment suggested that cyclopamine suppressed
WISP-1-induced fibrogenesis. Therefore, we proposed that Hedgehog
signaling may serve as a therapeutic target for the prevention of LF
fibrosis and hypertrophy. To confirm this hypothesis, the in vivo effect
of cyclopamine was also analyzed in a rat LF hypertrophy model. Rat LF
sections were evaluated using immunohistochemistry and RT-PCR. The
results showed that LF thickness was significantly lower in
cyclopamine-treated LF group (Group C) compared to the Group B, whereas
the Group C showed approximately the same thickness as the group A (Fig.
10B). Cyclopamine treatment of LF in Group C attenuated the upregulation
of fibrosis-related genes such as collagen, Gli1 and a-SMA, and the
downregulation of elastin compared to the Group B (Fig. 10E, F). In
addition to these changes of ECM component, we detected the cellular
changes using our rabbit samples. The number of BrDu-positive
proliferating cells was significantly higher in the Group B as compared
with Group A, whereas the number of proliferating cells was reduced by
cyclopamine-treated LF in Group C as compared with Group B (Fig. 10G).
These data suggest that LF thickness is positively correlated with
mechanical stress/WISP-1/Hedgehog-Gli1/a-SMA axis and Hedgehog-Gli1
signaling may serve as a therapeutic target for the prevention and
treatment of LF fibrosis and hypertrophy.