Telocytes in human liver fibrosis

Liver fibrosis is a wound-healing response which engages a variety of cell types to encapsulate injury. Telocyte (TC), a novel type of interstitial cell, has been identified in a variety of tissues and organs including liver. TCs have been reported to be reduced in fibrotic areas after myocardial infarction, human interstitial wall's fibrotic remodelling caused either by ulcerative colitis or Crohn's disease, and skin of systemic sclerosis. However, the role of TCs in human liver fibrosis remains unclear. Liver samples from human liver biopsy were collected. All samples were stained with Masson's trichrome to determine fibrosis. TCs were identified by several immunofluorescence stainings including double labelling for CD34 and c-kit/CD117, or vimentin, or PDGF Receptor-α, or β. We found that hepatic TCs were significantly decreased by 27%–60% in human liver fibrosis, suggesting that loss of TCs might lead to the altered organization of extracellular matrix and loss the control of fibroblast/myofibroblast activity and favour the genesis of fibrosis. Adding TCs might help to develop effective and targeted antifibrotic therapies for human liver fibrosis.


Introduction
Liver fibrosis, primarily as a result of chronic damages to the liver such as chronic viral hepatitis and fatty liver diseases associated with obesity, is a worldwide burden [1][2][3]. Liver fibrosis can further progress to cirrhosis, an advanced stage of liver fibrosis, leading to a major cause of morbidity and mortality worldwide [4][5][6]. This fibrotic response causes all the complications of end-stage liver disease, including portal hypertension, impaired metabolic capacity, synthetic dysfunction and ascites [1]. Unfortunately, currently no standard treatment for liver fibrosis is available and no appropriate antifibrotic drugs has emerged to be effective in human [6][7][8].
Telocytes have been reported to be reduced in myocardial infarction, especially in fibrotic areas [27]. In addition, TCs were progressively lost from the skin, gastric wall, the myocardium and the lung of systemic sclerosis patients [28,29]. Moreover, the number of TCs is decreased resulting in bowel architectural dysmotility and derangement during human intestinal wall's fibrotic remodelling affected either by ulcerative colitis or Crohn's disease [30,31]. We recently reported the existence of hepatic TCs in the Disse space with a similar density in the four hepatic lobes and their potential role in liver regeneration was emphasized in a mouse model of partial hepatectomy [11,32]. However, the role of TCs in liver fibrosis remains unclear.
In this study, based on immunofluorescence methods including double labelling for CD34 and c-kit/CD117, or vimentin, or PDGF Receptor-a, or b, we showed that in human liver fibrosis, hepatic TCs were significantly decreased, suggesting that loss of TCs might lead to the altered organization of extracellular matrix and loss the control of fibroblast/myofibroblast activity and favour the genesis of fibrosis.

Samples
Human liver fibrosis samples were obtained from liver biopsy examination in Division of Gastroenterology and Hepatology, Digestive Disease Institute, Fig. 1 Haematoxylin-eosin and Masson's trichrome stained sections of human liver. The liver section stained with Masson's trichrome from a control, compared with a liver fibrosis patient showed significant deposition of extracellular matrix proteins. Scale bar = 100 lM.

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Tongji Hospital. As healthy control tissues, samples were from patients who underwent surgery because of neoplastic pathologies. Specimens were collected at least 10 cm from the margin of the tumour. Healthy specimens were carefully chosen to be sure no inflammatory or neoplastic infiltration occur. All samples were divided to healthy or fibrosis specimens based on histopathological examination. All the patients had signed a written informed consent form and the Institutional ethical committees approved this study. The investigation conforms to the principles that are outlined in the Declaration of Helsinki regarding the use of human tissues.

Haematoxylin-eosin and Masson's trichrome staining
Liver samples were fixed in 4% paraformaldehyde in PBS, and were embedded in paraffin, and sectioned at 5-lm thick. Haematoxy-lin-eosin and Masson's trichrome staining were performed with standard procedures to investigate liver histological and fibrotic changes.

Immunofluorescent staining
For investigate the changes in TCs in human liver fibrosis, double immunofluorescent staining for CD34/PDGFR-a or CD34/PDGFR-b or CD34/Vimentin or CD34/c-Kit was used as our previously reported [32]. In brief, 6-lm-thick frozen sections were mounted on Superfrost Plus slides (Shitai, China) and were fixed in 4% paraformaldehyde containing 0.05% Triton-X-100 for 20 min. After three times wash with PBS, sections were pre-incubated for 1 hr in 5% Bovine serum albumin (BSA). After that, sections were incubated overnight at 4°C with mouse monoclonal anti-CD34 (1:100, ab54208; Abcam Cambridge, UK)

Statistical analysis
All analyses were evaluated using SPSS 19.0 International Business Machines Corporation, Armonk, NY, USA. Data are expressed as mean AE SD and were analysed by using the Student's t-test for independent samples. P-values that were less than 0.05 were considered to be statistically significant.

Results
The histopathological changes in all liver samples in this study were determined by haematoxylin-eosin and Masson's trichrome staining. In healthy liver stage group, hepatocytes had a radial array with clear central veins without occurrence of inflammation, necrosis and fibrosis (Fig. 1). In fibrosis samples, obvious collagen deposition, necro-  sis and inflammation cells' infiltration could be identified in liver interstitial (Fig. 1). The information for all participants is shown in Table 1. Based on the microscopic examination results obtained from haematoxylin-eosin and Masson's trichrome staining, liver specimens were categorized in a healthy stage (n = 4) or a fibrosis phase (n = 9).
Although transmission electron microscopy (TEM) examination is a golden standard for the identification of TCs, immunolabelling, especially double immunolabelling, remains a useful tool for discrimination between TCs and other cells, as well as for semi-quantitative data analysis. As we and many others have reported, here we used four different double labelling immunofluorescence methods to analyse the changes in TCs in human liver fibrosis, including double labelling for CD34 and PDGFR-a, CD34 and PDGFR-b, CD34 and vimentin, and CD34 and c-kit/CD117. Quantitative analysis of Figure 2 based on CD34 and PDGFR-a double labelling showed a significant reduction in the number of TCs in the fibrotic liver. In agreement with the results based on CD34 and PDGFR-a double labelling, Figures 3-5 also indicated that the number of TCs were severely reduced in liver fibrosis determined by CD34 and PDGFR-b, CD34 and vimentin, and CD34 and c-kit/ CD117 double labelling.

Discussion
This study reports a novel finding that TCs are reduced in human liver fibrosis, mirroring the recent findings as described in the colonic wall in ulcerative colitis, the terminal ileum of patients affected by small bowel Crohn's disease, and skins, gastric wall, lung and myocardium in systemic sclerosis [28][29][30][31]. Our data indicate that TCs might be involved in the pathological processes of liver fibrosis. Fibrosis is a wound-healing response which engages a variety of cell types to encapsulate injury [1,[5][6][7][8]. Hepatic stellate cells (HSCs) are considered as the primary source of the fibrogenic population in the liver [33]. In addition, other contributions are also appreciated including portal fibroblasts, bone marrow-derived cells, circulating fibrocytes and fibroblasts from epithelial mesenchymal transition [5-8, 33, 34]. There is now unequivocal and mounting evidence that TCs are definitely distinguished from other interstitial cells (mainly Kupffer cells and hepatic stellate cells) in liver by their location, morphology and immunophenotypes [11]. The microRNA signature, differential gene expression and proteomic profiles have been established between TCs and fibroblast [21][22][23]35], although these signatures between TCs and HSCs still deserve explored. We here showed that TCs were decreased in liver fibrosis, further supporting that TCs are different from HSCs which is well-known to be increased in liver fibrosis. Our data based on a series of double immunoreactions clearly indicate a potential role of TCs in the genesis of human liver fibrosis. Unfortunately, because of the lack of appropriate liver samples, we could not determine the ultrastructural features of TCs by TEM and this should be acknowledged as a limitation of this study.
It remains to be determined whether the reduction in TCs precede the process of fibrosis or just a consequence of the fibrotic process. However, based on the literature, several relevant and potential roles could be proposed. First, the reduction in TCs in liver could participate in the abnormal 3D organization of the extracellular matrix as they are involved in intercellular signalling via  either cell-to-cell contacts or shedding microvesicles or exosomes or paracrine secretion including microRNAs [36]. By this, TCs might be able to control the activity of HSCs. Second, the disappearance of TCs might impair hepatocytes and stem cells mediated liver regeneration. Indeed, in recent studies, we have shown that TCs had a close spatial relationships with hepatic putative stem (progenitor) cells and TCs might influence proliferation of hepatocytes and/or the activation of hepatic stem (progenitor) cells [32]. The reduction in TCs might contribute to the depletion of stem cell niches or proliferation of hepatocytes and impair liver regeneration and repair in human liver fibrosis. Further studies will be needed to reveal the functional relationship between TCs and hepatocytes/ hepatic stem cells. In addition, the therapeutic utility of TCs transplantation for the treatment of liver fibrosis requires further investigation.
In conclusion, this study firstly demonstrated the decrease in TCs in human liver fibrosis based on a series of double immunoreactions. Considering the fact that intramyocardial transplantation of cardiac TCs could decreases MI and improves post-infarcted cardiac function [37], adding TCs might help develop effective and targeted antifibrotic therapies that will modify the natural history of chronic fibrosing disease.