3. In clinical syudy
3.2. Secondary outcome
3.2.2. Gene expression associated with fibrosis
After stem cell administration, the relative expressions of transforming growth factor β1 (TGF-1), type 1 collagen (collagen-1), and α-smooth muscle actin (α-SMA) in real-time PCR significantly decreased from 1 ± 0.36 to 0.69 ± 0.32 (P<0.01), from 1 ± 0.58 to 0.70 ± 0.62 (P<0.01), and from 1 ± 0.44 to 0.55 ± 0.37 (P<0.01), respectively (Fig. 15).
６９ A B
C
Figure 15. Gene expression analysis associated with fibrosis.
After administration of BM-MSCs therapy, the relative expressions of (A) TGF-1, (B) collagen-1, and (C) α-SMA by real-time PCR decreased significantly from 1 ± 0.36 to 0.69 ± 0.32 (P<0.01), from 1 ± 0.58 to 0.70 ± 0.62 (P<0.01), and from 1 ± 0.44 to 0.55 ± 0.37 (P<0.01), respectively. Values are expressed as means ± standard deviation. *P<0.01.
７０
3.3. Safety
All 11 patients tolerated the therapy. They were monitored regularly for any sign of possible side effects of the stem cell therapy such as fever, hypersensitivity reaction, and acute rejection fever. There was no evidence of stem cell therapy related tumor developing during the follow-up period.
７１
IV. Discussion
At the overt cirrhotic stage, end-stage liver disease often necessitates liver transplantation 35. However, it has several limitations, such as shortage of organ donors, high medical cost, a widening donor recipient gap, and life long dependence on immunological suppression. Hence, stem cell transplantation has been suggested as an effective alternate therapy for hepatic disease 10, 12, 13
.
Stem cell therapies have shown promising benefits for hepatic fibrosis in experimental and clinical studies 13, 14, 19, 22
. BM comprises two main populations of stem cells, hematopoietic stem cells and MSCs, of which the latter have been considered as alternative cell sources for liver or hepatocyte transplantation 24. In liver damage, MSCs are able to differentiate into hepatocytes, stimulate the regeneration of endogenous parenchymal cells, migrate to damaged sites, and enhance fibrous matrix degradation (antifibrotic effects). Furthermore, several clinical studies have demonstrated favorable effects of BM-MSCs treatment such as improving the liver function in patients with hepatitis B or C virus-related cirrhosis 22, 36. However, no previous study has examined the effect of autologous BM-MCs on hepatic fibrosis in patients with alcoholic cirrhosis. We aimed to determine the safety and antifibrosis effect of MSCs on alcohol-related hepatic fibrosis in pre-clinical and clinical study.
In pre-clinical studies, we investigated whether BM-MSCs can attenuate hepatic fibrosis in a thioacetamide induced cirrhotic rat model. Thioacetamide (TAA)-induced cirrhosis is well-known classical experimental cirrhosis model. In present study, TAA administration results in deficiencies in liver functions and a progress
７２
increase in collagen accumulation in the liver with periportal cirrhotic characterized by portal-portal fibrous septa surrounding the hepatic lobules. These results were in agreement with 37 who indicated that TAA-induced cirrhosis characterized by cellular necrosis, fibrosis, micronodular reorganization of the parenchyma, duct proliferation, transaminase increase, peroxidation of lipid membranes, and macronodular cirrhosis
38, 39
.
Previous studies have suggested that MSC have antifibrotic effects on injured liver in animal models of liver fibrosis 40. Consistent with their result, we found that BM-MSCs treated group showed histologically significant improvement of hepatic fibrosis compared to untreated cirrhotic group. These results were further confirmed by immunohistochemical staining revealing α-SMA expression and Picrosirius Red staining. Additionally, the percentage of collagen proportionate area and content of hepatic hydroxyproline significantly decreased after BM-MSCs treatment. There results suggest that BM-MSCs improved liver structure in TAA-induced cirrhotic rats.
In present study, we investigated the effect of BM-MSCs on TGF-1, collagen-1,and α-SMA in TAA-induced cirrhotic rats. TGF-1 is a mediator particularly in liver fibrogenesis 41. TGF-1 promotes HSC to transit into MFB, stimulates the synthesis of ECM such as type I collagen and inhibits its degradation 42. And the expression of α-SMA in the liver is an indicator of HSC activation, which is recognized as the key player in hepatic fibrosis and cirrhosis 43. Likewise, this study showed that BM-MSCs recovered liver function, as indicated by decreasing gene expression of TGF-1, collagen-1, and α-SMA. Furthermore, the protein expression of α-SMA significantly decreased after BM-MSCs treatment. The results of our study are in agreement with
７３
those of Campbell JS et al 44. The finding a reduction in HSC activation during regeneration of the fibrotic liver in the rat after BM-MSCs treatment.
The signaling pathway of TGF-β1 was activated and catalyse Smad2/Smad3 phosphorylation, entering into the nucleus, binding to transcriptional factors and regulating the expression of target genes (e.g.,collagen, tissue inhibitor of matrix metallproteinases). Indeed, activation of TGF-β/Smad signaling is a key mechanism of liver fibrosis in both experimental and human chronic liver diseases 45–47. Similarly, the protein level of TGF-β1, p-Smad2, and p-Smad3 in the untreated cirrhotic group were increased compared to the sham, indicating that the TGF-β1/Smad signaling pathway was activated during the hepatic fibrosis. BM-MSCs in treated group significantly reduced the protein level of TGF-β1, p-Smad2, and p-Smad3 consistent to its inhibitory effect on hepatic fibrosis. There results suggest that main antifibrotic process by BM-MSCs treatment could be related to the inhibition of TGF-β/Smad signaling pathway.
In vitro study, we investigated the effects of paracrine factors secreted by
BM-MSCs on activated HSCs, the primary extracellular matrix-producing cell type in the liver. Bone marrow derived MSCs have the ability to repair damaged liver. In vivo activation of HSCs is divided into a fibrogenic and hyperplastic response 48 that is mediated by many autocrine and paracrine signals. And strategies targeting removal of activated HSCs have shown to reduce fibrosis and augment liver function 49. In the liver, IL-10 is produced by several types of cells including hepatocytes, sinusoidal cells, kupffer cells, stellate cells and liver-associated lymphocytes 50. It was reported that endogenous IL-10 in the liver increased for downregulating inflammatory
７４
response and improving liver fibrosis 51. IL-10 is a potent anti-inflammatory cytokine, which can protect hepatocytes, promote ECM degradation and modulate hepatic fibrogenesis by downregulating the inflammatory response, inhibiting the synthesis of pro-inflammatory cytokines and activating nuclear factor kB 52, 53.
HGF was identified as a mitogen for mature hepatocytes 54, and is considered to be important for the development and regeneration of liver 55. HGF showed the anti-apoptotic activity in hepatocytes and suppressed liver fibrosis 56, 57.
Previous studies have shown that the underlying mechanisms in the modulation of HSC activity by MSC were attributed to paracrine mediators, IL-10, TNF-α and HGF.
Blockade of MSC-derived IL-10 and TNF-α abolished the inhibitory effects of MSC on HSC proliferation and collagen synthesis; MSC-derived HGF was responsible for the marked induction of HSC apoptosis as determined by antibody neutralization studies. IL-6 secretion from activated HSCs induced IL-10 secretion from MSC, suggesting a dynamic response of MSC to HSCs in the microenvironment 25, 58. Consistent with their result, our data found that indirect coculture of activated HSCs with MSCs decreased the production of TGF-1 (24.7%) and IL-6 (8.4%).
Conversely, coculture of activated HSCs with MSCs increased the production of HGF (33.5%) and IL-10 (31.6%). HSC viability was decreased by 66% and apoptosis was increased by 43% with direct coculture of activated HSCs with MSCs. These results suggest that BM-MSCs inhibit the proliferative and fibrogenic function of activated HSCs in a paracrine manner and as a function of BM-MSC number. This inhibition could be caused by BM-MSC derived IL-10 which acted synergistically. The secretion of IL-10 by MSCs was found to be a dynamic response to IL-6 secretion by
７５
activated HSCs. Furthermore, BM-MSCs induced apoptosis in activated HSCs that is, in part, mediated by HGF. These results support the hypothesis that the therapeutic effect of BM-MSCs may be due to paracrine factors that modulate the proliferation, viability, and function of resident HSCs. Immunomodulation of HSCs by BM-MSC soluble factors provides the mechanistic evidence that BM-MSCs can exert a protective role through paracrine signaling to liver HSCs.
Our pre-clinical study has confirmed the effects of BM-MSCs on hepatic fibrosis, which probably are related with the inhibition of hepatic HSC activation and TGF-β1/Smad signaling pathways.
Furthermore, we investigated the antifibrotic effect of BM-MSCs on alcoholic cirrhosis as a phase II in clinical study. In clinical study, after autologous BM-MSCs injection, histological improvements were observed in 6 of 11 patients (54.5%). The Child-Pugh score improved in ten patients (90.9%), and the expressions of TGF-1, collagen-1 and α-SMA were significantly decreased (p<0.05). Importantly, no significant complications or side effects were observed during this study. These results indicate that BM-MSCs therapy has a potential as an antifibrotic treatment in cirrhosis and a bridging therapy for liver transplantation in advanced cirrhosis with hepatic insufficiency. Current antiviral therapies can improve the hepatic fibrosis in patients with B or C virus-related cirrhosis, but the only possible treatment for alcoholic cirrhosis is alcohol abstinence. Therefore, our results suggest a novel strategy for the treatment of alcoholic cirrhosis.
There is a clear histological variability of severity within cirrhosis classified as F4 by the METAVIR system. Cirrhosis is currently considered to be potentially
７６
reversible if the cause of the injury is removed. The lack of subclassification within cirrhosis can be problematic when assessing the antifibrotic effect of agents such as antiviral drugs. For instance, even though antifibrotic therapy leads to improvement of hepatic fibrosis from F4C to F4A in the Laennec system, the lack of change under the conventional METAVIR system will lead to the false conclusion that treatment is ineffective. Hence, further histological subclassification of cirrhosis is required 4, 30. In the present study, we applied the new Laennec fibrosis scoring system to provide a more detailed classification of F4 cirrhosis.
The quality of interventional studies of alcoholic liver disease patients is crucially dependent on monitoring the alcohol intake 59, 60. In the present study we did our best to monitor the subjects’ alcohol reintake and alcohol abstinence, using serologic liver function tests including serum AST/ALT ratio and γ-glutamyl transpeptidase, and mean corpus volume in complete blood counts. Furthermore, alcohol intake was monitored every week by phone calls and in monthly face to face interviews with patients and their family members. This was effective in preventing alcohol reintake in most of the participants due to the formation of a good rapport.
The present study was subject to some limitations. First, this pilot study was designed and performed as an open-label single arm study without control. Indeed, lack of a control group is certainly a major limitation of the present study. However, repeating biopsies in control group would be ethically difficult if no active treatment is being offered. Nevertheless, future prospective controlled studies would be necessary to validate the beneficial effect of MSCs on hepatic fibrosis shown in the present study. Although being an open-label study, the pathologist was blinded to the
７７
clinical data and the kind of therapy used, which enabled us to be confident about the accuracy of our histological data.
Second, like all biopsy studies there were potential limitations regarding liver biopsy sampling, and in particular sampling error. In the present study, our criteria of excluding biopsy specimens smaller than 15 mm should have minimized the magnitude of any sampling error. In addition, we incorporated other methods to assess hepatic fibrosis in this study to improve the validity of our morphometric analysis and measurement of TGF-1, collagen-1, and α-SMA by quantitative real-time PCR of the biopsied liver tissues 31.
This is the first study to determine the effect of BM-MSCs on hepatic fibrosis in patients with AC. The obtained results support the approval of this class of agents as a therapy for hepatic fibrosis.
７８
Ⅴ. CONCLUSION
This study has confirmed the significant inhibitory effect of bone marrow-derived mesenchymal stem cells on TAA-induced hepatic fibrosis in rats, which probably correlates with the inhibition of hepatic HSC activation and TGF-β1/Smad sign aling pathways. Furthermore, BM-MSCs therapy in alcoholic cirrhosis induces a histological and quantitative improvement of hepatic fibrosis. Therefore, the obtained results support the approval of this class of agents as a therapy for hepatic fibrosis.
In conclusion, BM-MSC therapy leads to the improvement of hepatic fibrosis, and so may provide a new strategy for antifibrosis therapy in near future.
７９
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