Total RNA was extracted from cultured cells using Trizol (Gibco) according to the manufacturer’s protocol. The amount of RNA was determined using spectoscopy at 260 nm.
Five ug of RNA was reverse transcribed to cDNA using a standard RT protocol. One ul (0.4 ug) of cDNA was added to PCR-reaction premix (GenDEPOT) with 10 pM corresponding primer pairs. The following primers were used for polymerase chain reaction : GAPDH, 5’-GTG TAG TTC ACG CCC ACG TC-3’ (forward), 5’-5’-GTG ATG GCA TGG ACT 5’-GTG GT-3’
(reverse) ; neurocan, 5’-GCC ACA CTC TAC ACT CGT CCC-3’ (f), 5’TCT CCC CAG CAT AGC CCT GAT-3’(r) ; phosphacan, 5’-GCA AGT CCT GCC GTC CTT GCA 3’-(f), 5’-GGA ATA GGG ATT AGT AAC AGC-3’ (r) ; c-Met, 5’-TGT CTC TGA AAT CCA CCC GA 3’ (f), 5’ GTG TAG TTC ACG CCC ACG TC-3’ (r). Their specificity was verified using the basic local alignment search tool (BLAST) on the GenBank database. PCR amplification was performed with 35 cycles of 95 ℃ for 30 s, 55 ℃ ~ 60 ℃ (property Tm) for 30 s, 72 ℃ for 90 s. The PCR products were separated on a 1% agarose gel and stained by Ethidium Bromide (EtBr). The amount of each product was quantified by a gel document system (Bio-Rad). GAPDH expression was used as an internal reference to verify equal concentrations of cDNA in each sample. We have executed experimental number of each group as described
the table 1. And we quantified mRNA expression level of neurocan and phosphacan by band thickness using the image J program based on band thickness of non-TGFβ1 treatment.
H. Statistical Analysis
Statistical comparison of mean values was performed using one-way ANOVA followed by Tukey’s post hoc tests. All values are expressed as mean ± SD. Quantitation graphs were generated by GraphPad Prism version 4.00 (GraphPad Software, San Diego, CA, USA)
Ⅲ. RESULTS
A. HGF prevents cytokine-induced astrocytic activation in vitro.
To examine whether HGF affects formation of glial scars, we used primary cultured astrocytes isolated from 1 day pub rat’s neocortex to mimic glial scars (Fig. 1A). When more than 95% cultured astrocytes express glial fibrillary acidic protein (GFAP), we induced astrocyte activation by treatment of TGFβ1 at a concentration of 10 ng/ml (Baghdassarian et al., 1993; Reilly et al., 1998) (Fig. 1B, C). Immunocytochemistry revealed that TGFβ1-treated astrocytes upregulated GFAP positive intermediate filaments and showed hypertrophic morphology. HGF was added to the medium at three different concentrations: 5 ng/ml, 50 ng/ml, and 250 ng/ml. Fifty ng/ml and 250 ng/ml treatment of HGF obviously prevented morphological change, although 5 ng/ml was not effective (Fig. 1E-G). To quantify changes in astrocytic morphologies, we measured mean GFAP positive areas (Fig.
1G). We found that treatment of TGFβ1 clearly increased the mean size of astrocytes and co-treatment of HGF at 50 ng/ml and 250 ng/ml significantly reduced the extent of hypertrophic changes.
It has been previously reported that HGF stimulated neuronal cell survival and oligodendrocyte precursor cell proliferation (Yan and Rivkees, 2002; Ohya et al., 2007). To examine whether HGF affects proliferation of astrocytes, cells were stained with anti Ki-67, which is a cellular marker of proliferation and detects nuclei-during interphase. Ki-67 immunoreactivity was not different between different treatment groups (Fig. 2A-C).
Quantification of the number of Ki-67 positive cells showed that proliferation between TGFβ1 treatment and HGF treatment with TGFβ1 was not significantly different (Fig. 2E).
As shown in Fig 2D, DAPI counts in regular area made no difference between TGFβ1 and HGF treatment, including control.
We next examined expression of HGF receptor c-Met in primary astrocyte. RT-PCR showed that c-Met mRNA is present in primary astrocytes and its expression is greatly increased after TGFβ1 treatment (Fig. 3A). HGF treatment did not change expression of c-Met. By immunostaining with antibodies that detect the beta-chain of c-Met (Fig. 3B-D), we confirmed the expression of c-Met in HGF treated astrocytes.
Fig. 1. Astrocytic activation in vitro glial scars model A: Illustration of primary astrocyte cultures and experimental scheme. B: GFAP stained astrocytes in serum free medium. B: 10 ng/ml treatment of TGFβ1 induced astrocytic hypertrophy. C: 5ng/ml treatment of HGF. D-F:5 ng/ml, 50 ng/ml, and 250 ng/ml HGF was added to culture medium with TGFβ1 respectively. Cells were treated with the above conditions for 24 hours and were fixed for immunocytochemistry with GFAP. G: Quantification of GFAP positive areas. *p<0.05,
**p<0.01 and ***p<0.001 by one-way ANOVA followed by Tukey’s post hoc test for the comparison of mean GFAP areas between different groups.
Fig. 2. Absence of HGF effect on astrocyte proliferation A-C: Ki-67 immunocytochemistry and DAPI staining of primary astrocytes without TGFβ1 (A), with TGFβ1 (B), and TGFβ1 with HGF 50 ng/ml (C). D: DAPI counting results by groups. E:
Quantification of Ki-67 positive cells counts among the all strocytes in the one taken picture.
Fig. 3. Expression of HGF receptor, c-Met, in primary astrocytes A: RT-PCR analysis showed mRNA expression of c-Met in primary astrocytes with or without TGFβ1 and HGF contreatment condition. B-D: Images of immunocytochemistry with c-Met and DAPI in primary astrocytes treated with TGFβ1 and HGF 50 ng/ml. B: Immunoreactivity of DAPI. C. Immunoreactivity of c-Met. D: merge image of DAPI and c-Met immunoreactivities.
B. HGF decreases expression of chondroitin sulfate proteoglycans (CSPGs)
To examine whether HGF affects production of CSPGs that is a major component of glial scars, we measured mRNA expression of neurocan and phosphacan, which are two species of CSPGs upregulated following cytokine stimulation (McKeon et al., 1999; Asher et al., 2000) and spinal cord injury (Jones and Tuszynski, 2002). TGFβ1 induced astrocytic activation resulted in dramatic increases in neurocan and phosphacan mRNAs.
Quantification data showed that neurocan mRNA expression was elevated 8 times higher than control level. HGF treatment at a concentration of 50 ng/ml almost completely blocked the increase of neurocan mRNA expression (Fig. 4A, B). Phosphacan mRNA level was also increased 2 times with TGFβ1, and HGF co-treatment evidently reduce the expression level (Fig. 4C, D). These experiments confirmed that HGF obviously suppresses mRNA production of CSPGs.
Fig. 4. Modulation of chondroitin sulfate proteoglycans (CSPGs) mRNA expression by HGF A: Quantification of mRNA expression of neurocan. B: neurocan RNA analysis by RT-PCR. C: Quantification of mRNA expression of phosphacan D: phosphacan RNA analysis by RT-PCR. *p<0.05, **p<0.01 , and ***p<0.001 by one-way ANOVA followed by Tukey’s post hoc test for the comparison with hypertrophic effects in primary astrocytes.
Table 1. Experimental groups and treatment number in vitro
C. Effects of HGF on TGFβ secretion
Following spinal cord injury, TGFβ1 and TGFβ2 were secreted from reactive astrocytes around the lesion site. TGFβ1 induces inflammatory responses and is involved in initial formation of the glial scars, while TGFβ2 maintains glial scars at later time points (Buss et al., 2008). Inhibition of TGFβ1 and TGFβ2 functions by neutralizing antibodies reduced the extent of scar formation (Logan et al., 1999; Moon and Fawcett, 2001). Therefore, TGFβs are regarded as strong inducers for glial scars formation.
For these reasons, we determined whether reduction of astrocytic activation is associated with changes in secretion of TGFβs. To this end, we adopted a different activation scheme. Treatment of interleukin-1 beta 1 (IL-1β) and interferon-gamma (IFNγ) has been shown to strongly induce astrocytic activation (Hewett et al., 1993). Applying IL-1β and IFNγ together is known to increase GFAP expression by NO induction (Brahmachari et al., 2006).
Purified astrocytes were treated with 10 ng/ml of IL-1β and 10 ng/ml of IFNγ for 48 hours after starvation during 24 hours (Fig. 5A). Treatment of IL-1β and IFNγ together elicited dramatic changes in astrocytic morphology (Fig 5B-D). Astrocytes that were exposed to IL-1β and IFNγ showed highly elongated morphology and tended to aggregate together compared to untreated astrocytes (Fig 5C). HGF treatment partially restored astrocytic morphology (Fig 5E). To compare secretion level of TGFβ1 and TGFβ2 from astrocytes cultured supernatants were harvested and analyzed for TGFβ1 and TGFβ2 ELISA after 48 hours of cytokines addition (Fig 5A, F-G). TGFβ1 secretion was significantly increased by IL-1β and IFNγ, which was completely blocked by HGF co treatment (Fig 5D).
TGFβ2 secretion is not significantly increased by addition of IL-1β and IFNγ, but clearly decreased by HGFs (Fig 5E). Thus, HGF inhibited secretion of TGFβ1 and TGFβ2 in IL-1β / IFNγ induced activated astrocyte culture system.
Fig. 5. HGF inhibited secretion of TGFβ1 and TGFβ2 from activated astrocytes. A:
Schematic illustration of the experimental procedure. B-D: Representative images of GFAP Immunocytochemistry of primary astrocyte without cytokine treatment (B), with IL-1β and IFNγ at 10 ng/ml each for 48 hours (C), and addition of 50 ng/ml of HGF with IL-1β and IFNγ treatment (D). E-F: ELISA analysis of TGFβ1 (E) and TGFβ2 (F) in supernatants collected from cultures for 48 hours under designated experimental conditions. *p<0.05, and ***p<0.001 by one-way ANOVA followed by Tukey’s post hoc test for the comparison of TGFβ1 and TGFβ2 concentration between different groups.
Table 2. Experimental groups and treatment number of IL-1β and IFNγ-induced in vitro scar model
ELISA (TGFβ1 and TGFβ2)
groups number
Non IL-1β and IFNγ (control) 4
IL-1β 10 ng/ml + IFNγ 10 ng/ml 4
IL-1β 10 ng/ml + IFNγ 10 ng/ml + HGF 5 ng/ml 4
D. Ex vivo delivery of HGF using mesenchymal stem cells in hemisection spinal cord injury
In vitro data demonstrated that HGF prevents cytokine-induced astrocytic hypertrophy
and reduced CSPGs expression. We attempted to test whether HGF could regulate glial scars formation in vivo. To achieve stable and robust delivery of HGF, we transplanted HGF overexpressing-mesenchymal stem cells (MSCs) into the hemisection spinal cord injury site at T8 level (Fig. 6A). After hemisection spinal injury, we immediately implanted gelfoams soaked with PΒS (control group), human bone marrow derived MSCs (MSC alone), and MSCs transduced with HGF gene (HGF-MSC).
Immunohistochemistry was performed to detect surviving grafted cell at 2 weeks after surgery. GFP immunoreactivity was used to detect MSCs because enhanced GFPs were incorporated into MSCs. MSCs were also detected by human specific mitochondria antibodies. Spinal cord tissue of control group (PBS) did not show any immunoreactivity for eGFP or human mitochondria (Fig. 6B). In animals with MSCs alone or HGF-MSCs, eGFP and human mitochondria positive grafted cells were detected inside gelfoam as well as spinal cord parenchyme surrounding the lesion (Fig 6C-F). Thus, grafted MSCs were found to survive for at least 2 weeks post transplantation in both gelfoam around circumference of lesion.
We next examined if HGF overexpressing MSCs were capable of secreting HGF outside of cells in vitro. ELISA analysis of supernatants obtained from culture for different duration showed that the level of HGF secretion increased as the duration of culture was prolonged (Table 3). In contrast, the amount of HGF in supernatants from MSC alone cultures was
almost negligible. To verify adequate delivery of HGF using the ex vivo approach, animals were sacrificed 1 week after injury, and the level of HGF was measured in the spinal cord tissue around the lesion site. The level of HGF in the spinal cord with HGF-MSCs was 14 times higher than that of control or MSC group (Fig. 7A). This result indicated that the ex vivo delivery of HGF using MSCs successfully delivered HGF to the injured spinal cord
tissue. Immunohistochemistry using anti-HGF antibodies was also performed to detect presence of HGF in the injured spinal cord tissue (Fig 7C-E). Confocal image showed diffuse HGF immunoreactivity surrounding hemisected injury site in HGF-MSCs group (Fig 7E). Partial expression of PΒS and MSCs groups might be due to endogenous expression of HGFs (Fig. 7C, D).
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). To determine whether c-Met signaling was effectively induced by the ex vivo approach, phosphorylation of c-Met was measured using phospho c-Met specific antibodies can detect phosphorylation of Tyr 1230, 1234, and 1235 (Fig. 7B). Expression of c-Met receptor was detected to a similar extent in all three groups. Apparent phosphorylation of c-Met was observed in HGF-MSC group, and c-Met phosphorylation was also detected in MSC alone group to a much smaller extent (Fig.
7B).
Fig. 6. Ex vivo delivery of HGF gene using human mesenchymal stem cells as carriers A:
Schematic illustration of hemisection spinal cord injury and transplantation of gelfoams with PBS, MSCs alone, and HGF-MSCs. B-E: Results of immunohistochemistry analysis after 1 week post-surgery. B: No cells that were reactive to GFP or human mitochondrial antibodies were detected in animals with PBS soaked gelfoam. C: GFP positive MSCs are readily observed inside the gelfoam. D: GFP and human mitochondria immunofluorescence signals in spinal cord tissue of lesion boundary. E: High magnification fluorescence signal in square of D.
Fig. 7. Verification of effective HGF delivery by the ex vivo method A: Quantification of ELISA analysis of HGF level in injured spinal cord at 1 week. B: Western blot analysis result of phospho-c-Met and c-Met, HGF receptor after 1 week post-surgery. C-E:
Immunohistochemistry result of HGF in PΒS (C), MSC (D) and HGF-MSCs (E) implanted tissue. ***p<0.001 by one-way ANOVA followed by Tukey’s post hoc test.
Table 3. HGF concentration in cultured supernatant by culture period condition
Sample HGF concentration (ng/ml)
MSC 3day culture sup (10 MOI) 0.002
HGF 3day culture sup (10 MOI) 2.401
HGF 1day culture sup (10 MOI) 0.599
HGF 2day culture sup (10 MOI) 1.307
HGF 5day culture sup (10 MOI) 17.967
Table 4. Experimental groups and treatment number in vivo
Western blot (neurocan), ELISA (TGFβ1 and TGFβ2) - 1week
groups number
PΒS 5
MSC 6
HGF-MSC 6
Immunohistochemistry (GFAP, CS-56, HGF, eGFP, and Hu-Mito) – 2 weeks
groups number
PΒS 3
MSC 4
HGF-MSC 4
E. HGF inhibits astrocytic activation and CSPGs production after spinal cord injury.
We sought to investigate whether glial scars formation is influenced by HGF treatment and production of chondroitin sulfate proteoglycans is reduced by HGF treatment after spinal cord injury. First, we examined whether astrocytic hypertrophy in vivo was reduced by HGF as was shown in vitro. At 2 weeks post-injury, glial limitance was formed around the boundary of the hemisection lesion and GFAP expression was increased along the glial limitance (Fig. 8A). Intensity of GFAP immunoreactivity was dramatically suppressed in HGF-MSCs group compared to both PΒS (A) and MSCs (B) groups (Fig. 8A-C).
Quantification data showed that transplantation of HGF-MSCs reduced GFAP intensity by more than 50% (Fig. 8D).
In vitro results suggested that HGF prevented astrocytic activation by inhibiting
secretion of TGFβ1 and β2 from reactive astrocytes. To address this issue, we explore whether HGF treatment reduces production of TGFβs in vivo using ELISA analysis (Fig. 9A-B). We found that production of endogenous TGFβ1 was significantly inhibited by transplantation MSCs and HGF-MSCs (Fig. 9A) and the decrease was significantly only by HGF-MSCs transplantation (Fig. 9B).
We next examined the production and deposition of CSPGs. It is well known that CSPGs produced from reactive astrocytes is accumulated in perineuronal nets (PNN) near epicenter after spinal cord injury (Jones et al., 2003). Accumulation of CSPGs was detected by immunoreactivities against GAG sugar chains using CS-56 antibody (Fig. 10A-D). We found that CS-56 immunoreactivity was strongly upregulated in perineuronal net in PΒS implanted group (Fig. 10A-C), indicating that GAGs chains of CSPGs were densely
deposited around the lesion. Transplantation of HGF-MSC markedly inhibited accumulation of CSPGs (Fig. 10C). Transplantation of MSC alone also seemed to decrease CS-56 immunoreactivity (Fig. 10B). Quantification results from CSPG intensity at 3 points that are rostral, caudal and contra lateral was significantly reduced the CS-56 intensity per unit area by both HGF-MSCs and MSCs implantation (Fig. 10D). We also analyzed expression of core proteins of CSPGs by western blot. Neurocan, one of CSPG species, is expressed in injured spinal cord and reaches a peak level at 2 weeks after injury (Jones et al., 2003).
Protein homogenates were pretreated with chondroitinase ABC to remove GAGs chains from core protein. The removal of GAG chains allowed visualization of discrete neurocan band at the position expected from its expected molecular weight. We found that HGF-MSCs transplantation reduced intensity of neurocan signals compared to control and MSC alone groups at all the three expected molecular weights (130 kDa, 160 kDa and 240 kDa) (Fig 10E, F). Collectively, these results provide supporting evidence that HGF prevents astrocytic activation and production of CSPGs.
Fig. 8. Reduction of astrocytic scars by transplantation of HGF overexpressing mesenchymal stem cells A-C: Representative images of GFAP stained longitudinal spinal cord tissue sections from animals with PBS (A), MSCs alone (B), and HGFMSCs (C). D:
Quantification of GFAP intensity from regions of interests described in methods section.
**p<0.01, and ***p<0.001 by one-way ANOVA followed by Tukey’s post hoc test.
Fig. 9. Regulation of TGFβ1 and TGFβ2 level in glial scars by transplantation of HGF-MSCs A-B: Endogenous level of TGFβ1 (A) and TGFβ2 (B) by ELISA analysis.
*p<0.05, and ***p<0.001 by one-way ANOVA followed by Tukey’s post hoc test.
Fig. 10. Reduction of CSPGs production by HGF-MSCs in spinal cord injury model A-C: Representative images of CS-56 immunohistochemistry in longitudinal spinal cord sections at 2 weeks post injury in PΒS implantation group (A), MSCs alone implantation group (B), and HGF-MSCs implantation group (C). D: Quantification of CS-56 intensity. E:
Western blot analysis result of neurocan. Three bands corresponding to intact (240kDa), N-terminal (160kDa), and C-N-terminal neurocan (130kDa) are shown up. β-actin signal was used as a loading control of tissue lysates. F: Quantification of thickness from 3 bands of 3 sizes.
**p<0.01, and ***p<0.001 by one-way ANOVA followed by Tukey’s post hoc test.
Ⅳ. 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