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.