4 | DISCUSSION
In the present study, we confirmed for the first time that mechanical
stress induced WISP-1 expression in LF cells obtained from LF tissues of
LSCS patients. Moreover, we demonstrated that WISP-1 induced LF
hypertrophy and fibrosis by activating the Hedgehog-Gli1 pathway and
aggravating ECM deposition by promoting LF cell proliferation,
inhibiting cell apoptosis and inducing the transition of fibroblasts
into myofibroblasts. Most importantly, inhibition of Hedgehog signaling
suppressed mechanical stress-induced LF fibrosis in rabbit model,
indicating that targeting Hedgehog signaling may be a novel strategy for
the prevention and treatment of LF fibrosis.
Lumbar spinal canal stenosis (LSCS) is a very common disease in the
elderly population often with symptoms of pain, limb numbness, or
intermittent claudication, which is a serious threat to human health and
life (Schizas et al., 2010; Yang et al., 2017; Yan et al., 2018).
Currently, it is clear that LF hypertrophy is considered to be a major
pathogenic factor for the occurrence and development of LSCS(Sakai et
al., 2017). Similarly, the present study also demonstrated that the LF
in LSCS group was significantly thicker as compared with LDH group.
Histologically, the hypertrophic LF showed decrease of elastic fibers
and collagen accumulation, suggesting fibrotic changes, which were
consistent with those reported by previous studies (Yan et al., 2018;
Sun et al., 2020).
Emerging evidence has suggested that WISP-1, a secreted matricellular
protein belonging to the CCN family, is involved in the initiation and
progression of fibrosis in various organs (Berman et al., 2003; Berman
et al., 2002; Königshoff et al., 2009). Our previous studies showed that
WISP-1 expression increased in hypertrophic LF and was highly associated
with LF fibrosis, suggesting that it is a critical fibrotic effector in
LF fibrosis (Sun et al., 2017). However, the upstream mechanism of the
abnormal expression of WISP-1 in LF tissue is not yet understood. Of
note, it has been suggested that excessive mechanical stress commonly
contributes to various pathological diseases (Wu et al., 2020; Yuan et
al., 2018). As such, mechanical stress owing to segmental instability is
usually occurred in patients with LSCS, and this increasing instability
is considered to have a vital role in the development of LF fibrosis,
finally leading to LF hypertrophy (Yoshiiwa et al., 2016; Fukui et al.,
2015; Sairyo et al., 2007). Histologically, the degeneration of elastic
fibers becomes accelerated and the accumulation of collagen is markedly
increased because of the mechanical stress (Hur et al., 2015).
Therefore, it would be of interest to examine whether mechanical stress
induces WISP-1 expression during fibrogenesis. As expected, in the
present study, we confirmed for the first time that mechanical
stretching stress could directly induce WISP-1 expression in LF
fibroblasts obtained from LSCS patient and our animal model of rabbit
also demonstrated that mechanical stress induced LF fibrosis and
activated WISP-1 expression in LF tissues. To explore the subsequent
responses, we next focused on investigating the potential role of WISP-1
in LF fibrosis and the molecular mechanism probably included.
Previous research has clearly showed that Hedgehog signaling has been
characterised as a critical mechanism underlying the persistent
fibroblast activation in fibrotic diseases (Horn et al., 2012; Zerr et
al., 2012; Liang et al., 2012). As is known, Gli family includes three
members: namely Gli1, Gli2 and Gli3, which is a downstream transcription
factor of Hedgehog pathway (Omenetti et al., 2011). It is noteworthy
that Gli1 is the main transcription factor downstream of the Hedgehog
pathway and can be viewed as readouts of pathway activity (Bariwal et
al., 2019). In general, Gli1 is present in the cytoplasm and is in an
inactive state. Once activated, Gli1 is cleaved and transported to the
nucleus resulting in the transcription of Hedgehog target genes (Hegde
et al., 2008). Consistently, our results also demonstrated that the
Hedgehog signaling was activated during LF fibrosis. Briefly, we found
that Gli1 and Shh were highly expressed in hypertrophic LF and Gli1
expression was highly related to LF fibrosis. The in vitro experiment
demonstrated that activated Hedgehog-Gli1 signaling promoted LF
fibroblast proliferation and collagen accumulation, and inhibited cell
apoptosis. Furthermore, we found that WISP-1 over-expression promoted
the Gli1 expression and nucleus translocation. Meanwhile, silencing
WISP-1 suppressed the fibrotic effect of activated Hedgehog-Gli1
signaling. In addition, a growing body of evidence suggests that
Hedgehog inhibitor cyclopamine plays a crucial role in treating liver
fibrosis by inhibiting several fibrotic genes (Kumar et al., 2016;
Pratap et al., 2010). Similar to those studies, we demonstrated that
cyclopamine inhibited WISP-1-induced fibrogenesis in vitro and in vivo.
These results suggest that Hedgehog-Gli1 signaling was one of the
targets of WISP-1 and mediated the fibrotic effect of WISP-1.
Myofibroblasts are mainly originated from the fibroblasts acting in
producing a great number of ECM adhesive and structural proteins (Li &
Kuemmerle, 2014). It is noteworthy that the trans-differentiation of
fibroblasts into myofibroblasts is a key cellular event that drives the
fibrosis response in a number of tissues and organs (Yoshiiwa et al.,
2016; Fukui et al., 2015; Ono et al., 2018). The myofibroblasts are
characterized by the expression of a-SMA(Zhao et al., 2018; Pinchuk et
al., 2010). It has been reported that activated Hedgehog signaling
promotes fibroblast-to-myofibroblast transition and fibrosis, whereas
targeted inhibition of Hedgehog signalling ameliorates fibrosis in
several diseases (Horn et al., 2012; Zerr et al., 2012; Liang et al.,
2012). Similarly, in the present study, we demonstrated that a-SMA was
upregulated in hyperthrophic LF, indicating that
fibroblast-to-myofibroblast transition was also the important mechanism
of LF fibrosis. Furthermore, it was found that WISP-1 promoted a-SMA
expression through Hedgehog signaling as inhibition of Hedgehog
signaling suppressed WISP-1-induced a-SMA expression in vitro.
Consistently, in vivo inhibition of Hedgehog signaling by cyclopamine
ameliorated the fibrotic effect induced by mechanical stress in rabbit.
Taken together, it is concluded that mechanical
stress/WISP-1/Hedgehog/a-SMA signaling is a new pro-fibrotic axis and
provides a theoretical basis for the research and development of new
drugs for LF fibrosis and hypertrophy.
It is generally accepted that integrins are key cell surface receptors
for the CCN family (Sharma et al., 2010; Scotton et al., 2009; Ono et
al., 2018). Our ongoing study is to find the corresponding receptor that
are responsible for determining the selectivity of WISP-1 on Sonic
Hedgehog pathway. Overall, the present data revealed the important role
of the mechanical stress-WISP-1-Hedgehog axis in LF fibrosis and
provided a better understanding for LF hypertrophy. The results from in
vitro and in vivo experiments indicated that interfering with Hedgehog
signaling may be a novel strategy for the prevention and treatment of LF
fibrosis and hypertrophy.