DISCUSSION

In this study, we showed that HGF can prevent astrocytic activation and suppress production of chondroitin sulfate proteoglycans. We further showed that HGF could regulate astrocytic activation via decreasing the level of TGFβs. These effects of HGF on regulation of glial scars were demonstrated both in cultured primary astrocytes and in vivo spinal cord injury model. Our data showed that HGF treatment dramatically reduced morphological changes of astrocytes induced by TGF treatment (hypertrophy) or IFNγ and IL-1beta (elongated morphology) (Fig. 1, 5). As shown at figure 5A-C, we can observe that IL-1β and IFNγ induced activation is modulated the astrocyte active migration and association and HGF could partially prevent these effects, which is involved in cell dissociation and scatter function of HGF (Gherardi et al., 1993). However, HGF didn’t influence astrocyte proliferation in cultured astrocytes (Fig. 2). A previous study is consistent with our result that HGF does not stimulate proliferation of culture astrocytes (Machide et al., 2000).

Intriguingly, HGF also completely blocked mRNA expression of neurocan and phosphacan (Fig. 4) that are differently regulated following spinal cord injury (McKeon et al., 1999;

Jones et al., 2003). Immunoblotting and immunostaining results indicated that HGF evidently reduced not only depositions of GAG sugar chains (Fig. 10A-D) but also productions of core protein (Fig. 10E-F), which are actively upregulated following injury (Busch and Silver, 2007). Moreover, mRNA expression of c-Met, HGF receptor, is expressed in all condition using RT-PCR in vitro (Fig 3), especially induced at TGFβ1 treatment including HGF co treatment. Previous studies provided the evidence that c-Met is normally

expressed in astrocytes and increases expression level in response to cytokine treatment (TGFβ1, FGF etc) (Shimazaki et al., 2003). Binding of HGF to the c-Met receptor tyrosine kinase (RTK) triggers receptor dimerization, which is originally disulfide linked α-β heterodimeric RTK, and phosphorylation on multiple residues (Ma et al., 2003). We can detect phosphorylated c-Met expression in HGF-MSC transplantation as well as MSC transplantation (Fig. 7B). We thought that weak expression of phospho-c-Met in MSCs group is phosphorylated by other intracellular signaling proteins. TGFβ are well known factor by glial scars inducer, so we suggested that HGF might prevent secretion of TGFβ and then finally HGF can affect blockage of astrocyte activation. ELISA analysis results are supported our hypothesis that HGF treatment has significantly inhibited the secretion of TGFβ1 and TGFβ2 in vitro (Fig. 5E, F) and in vivo (Fig. 9). These results are indicated that hepatocyte growth factor is a potent factor of glial scars prevention and is may be induced the axonal outgrowth around the lesion site and this is first showed that HGF can prevent glial scars formation.

Glial scars are formed around the lesion after spinal cord injury and act as a barrier to regenerating axonal growth. Glial scars mainly consist of reactive astrocytes, which become hypertrophic and greatly increase their expression of intermediate filament proteins such as vimentin, and glial fibrillary acidic proteins (Rhodes and Fawcett, 2004; Busch and Silver, 2007). For in vitro studies, we used two different astrocytes activation schemes: 1) TGFβ1 (Baghdassarian et al., 1993) and 2) IL-1β and IFNγ (Hewett et al., 1993). HGF treatment was effective in preventing GFAP upregulation in TGFβ conditions (Fig. 1, 5). We demonstrated these effects also in in vivo hemisection spinal cord injury model. Thus, these results indicate

that HGFs are effective suppression of GFAP upregulation in response to cytokines or in vivo injury conditions.

Reactive astrocyte inhibits axonal regeneration by producing growth-inhibitory components such as CSPGs (McKeon et al., 1991; Jones et al., 2003). An important implication of our finding is that HGF could block or reduce the production of chondroitin sulfate proteoglycans. CSPGs, such as neurocan, brevican, phosphacan, and NG2, are major elements of the glial scars because secreted CSPGs from reactive astrocyte are accumulated in extracellular matrix (ECM). During the past few years, it has been shown that elimination CSPGs by chondroitinase is effective to improve functional recovery after SCI. In this study, we first found that HGFs decreased the RNA expression of neurocan and phosphacan (Fig.

4), and the deposition of CSPG around the lesion site (Fig. 10) was markedly reduced by transplantation of HGF overexpressing MSCs.

Based on these results, we propose that HGF can modulation of GFAP expression and CSPG production in astrocytes. These effects might be mediated by regulation of TGFβ1 level (Bradbury et al., 2002; Tester and Howland, 2008), which was demonstrated in internal organs such the live and kidney. TGFβ1 is secreted from reactive astrocyte, reactive microglia, and injured neurons, and plays a very important role in constructing the glial scars (Liu and Yang, 2006). Previous studies attempted to reduce glial scars using neutralizing antibodies against TGFβ1 and TGFβ2 (Logan et al., 1994; Buss et al., 2008). Moreover TGFβ1 regulated the production of CSPGs (Logan et al., 1999; Moon and Fawcett, 2001).

As expected from our hypothesis, reduction of TGFβ1 and β2 secretion was observed following HGF treatment in vivo and in vitro (Fig. 5, 9). Interestingly, MSC transplantation

is significantly inhibited the secretion of TGFβ1 in vivo (Fig. 9). Zhao et al (2008). already showed that TGFβ1 is decreased following MSC treatment (Asher et al., 2000; Smith and Strunz, 2005). Recently, reactive astrocytes were dramatically inhibited by rapamycin (Zhao et al., 2008a), which is antagonist of mTOR signaling that is serine/threonine kinase and a key regulator of cell size and proliferation downstream of growth factor receptors (Hara et al., 2002). This finding suggests that intracellular signaling of HGF-c-Met system might be somehow related with mTOR signaling. In addition, HGF-c-Met induced delayed STAT3 phosphorylation (Sabatini, 2006; Wullschleger et al., 2006; Bai et al., 2007) that is involved in TGFβ1 intracellular singling (Yin et al., 2008; Lee et al., 2009). Further studies will be required to clarify detailed mechanism by which HGF arrests the secretion TGFβ.

Recent studies have shown that HGF can be therapeutically utilized for various neurological disease conditions. As in our study, HGF overexpressing MSCs were transplanted in stroke animal model to repair the ischemia (Zhao et al., 2008b). They described that the HGF-MSCS were more powerful than MSC cell transplantation alone.

Furthermore, a recent study by Kitamura et al. (2007) demonstrated that HGF expressing replication-incompetent herpes simplex virous-1 (HSV-1) vector promoted endogenous repair and functional recovery after spinal cord injury (Zhao et al., 2006), although they did not look into the effects of HGF on glial scars formation. Versatile effects of HGF were actively studied in anti-tumor therapy (Kitamura et al., 2007) and anti-inflammatory inhibition effects (Matsumoto and Nakamura, 2003). HGF was also tested in transgenic mouse ALS model (Kadoyama et al., 2007). In this model, HGF played a role of preventing motoneuronal death and reducing microglia migration following inhibition of pro-apoptotic

protein activation. This study also suggested that secreted HGFs from motoneurons were uptaken by astrocytes and then astrocytes influenced suppression of gliosis via downregulated IL-1β secretion. Thus, HGF and its downstream signaling pathway seem to be a promising target for various neurological disorders.