Hepatic stellate cell‐expressed endosialin balances fibrogenesis and hepatocyte proliferation during liver damage

Corresponding to the reported expression of EN by activated mesenchymal cells including pericytes and (myo)fibroblasts (Christian et al, 2008), weak expression restricted to HSC and portal fibroblasts was detected in healthy adult murine and human livers (Fig 1A–C and E). EN was strongly upregulated in human liver fibrosis (FL) and active cirrhosis (CL+) showing immunhistochemical overlap with well‐established pericyte and myofibroblast markers (such as PDGFR beta or α‐smooth muscle actin) but never with endothelial markers (Fig 1D, F and G; Supplementary Figs S1 and S2). Downregulated EN expression during late‐stage cirrhosis (Fig 1D and H) identified EN as an early HSC activation marker. Comparative analysis of EN expression with the established HSC activation marker α‐smooth muscle actin (αSMA) identified a substantially stronger differential of EN expression in diseased vs. healthy tissue (Fig 1I and M versus J and N). Correspondingly, qRT–PCR analyses determined a significant change in the ratio of EN‐to‐αSMA mRNA expression comparing normal with fibrotic or cirrhotic liver (Fig 1O). EN expression was also more pronounced in early stages of fibrogenesis compared to collagen 1a, the main product of HSC in fibrotic tissue (Fig 1P).

To study the consequences of acute liver damage in vivo, hepatotoxicity was induced in mice by injection of a single dose of carbon tetrachloride (CCl₄). Acute liver toxicity led to rapid upregulation of EN and αSMA (Fig 2A and B), followed by upregulated collagen 1a expression (Supplementary Fig S3). Analysis of isolated cell populations identified the vitamin A+, early activated HSC population (8.26% density gradient layer) as the primary EN‐positive cell population (Fig 2C).

Based on the observed prominent regulation of EN in a mouse model of acute liver damage, we next performed comparative hepatotoxicity experiments in wild‐type (WT) and EN‐deficient mice (EN^KO). EN^KO mice are viable and fertile and display no overt phenotype unless they are pathologically challenged (Nanda et al, 2006). Likewise, comparative analyses of WT and EN^KO livers from adult mice showed no overt morphological and liver‐related clinical chemistry alterations (Supplementary Fig S4). A single high‐dose injection of CCl₄ led to an equal extent of liver damage with acute zonal necrosis and increase in serum liver enzymes in both genotypes (Supplementary Fig S5). Twice weekly chronic CCl₄ administration resulted in a continuous increase in EN expression in WT mice over a period of 6 weeks (Fig 2D). Overall liver damage (assessed by cleaved caspase‐3 activity) was similar in WT and EN^KO mice (Supplementary Fig S6; Fig 2F and G). Likewise, the initiation of liver fibrosis was comparable in WT and EN^KO mice as indicated by similar levels of αSMA and desmin expression (Supplementary Fig S7). Yet, fibrosis progression was delayed in EN^KO mice as evidenced by significantly ameliorated (necro‐)inflammatory activity (assessed by METAVIR Score), reduced levels of collagen 1a, PDGFR beta, αSMA, desmin, and TIMP1 expression as well as lower liver serum enzymes after 6 weeks of repeated CCl₄ administration (Fig 2E–M; Supplementary Figs S8, S9 and S10). No differences in epithelial‐to‐mesenchymal transition were observed (Supplementary Fig S11). Fully activated hepatic stellate cells isolated from either wild‐type or EN^KO mice showed similar cell morphology after 8 days in culture (Supplementary Fig S12). However, lentiviral‐mediated endosialin knockdown in human immortalized hepatic stellate cells (LX‐2 cells) revealed dramatically reduced levels of αSMA and collagens 1 and 6 (Fig 2N) as well as a reduced myofibroblastic phenotype in the shEN HSC compared to control‐transfected cells (Fig 2O). While the initiation of liver fibrosis was not altered in EN^KO mice, hepatocyte proliferation was significantly increased in EN^KO mice compared to WT mice, most notably at the early time points (Fig 3A and B, Supplementary Fig S13), resulting in an increased mean bodyweight after 6 weeks of CCl₄ treatment (Supplementary Fig S14). These findings suggested that the observed ameliorated fibrosis phenotype after 6 weeks might not just have resulted from reduced fibrogenic activity, but rather a shift from a profibrogenic to a proregenerative phenotype in EN^KO mice as has previously been observed in 5‐hydroxtryptamine 2B receptor (5‐HT_2B)‐deficient mice (Ebrahimkhani et al, 2011).

We next performed comparative partial hepatectomy (PHx) experiments in WT and EN^KO mice in order to examine the role of HSC‐expressed EN in a model of rapid hepatocyte proliferation. PHx resulted in a characteristic biphasically upregulated expression of EN (Fig 3C) in HSC with maximum EN expression 1 day after PHx (coinciding with the initiation of hepatocyte proliferation) and 3 days after PHx (coinciding with the initiation of stromal cell proliferation) (Michalopoulos & DeFrances, 1997; Miyaoka et al, 2012). Hepatocyte proliferation was significantly increased in EN^KO mice after 2/3 and 1/3 PHx (Fig 3D and E; Supplementary Figs S15 and S16) showing more mitotic figures (Fig 3F), but equal numbers of double nuclei (Supplementary Fig S17). EN^KO livers presented with smaller hepatocytes 2 days after hepatectomy, resulting in only slight differences in the liver‐to‐bodyweight ratio (Supplementary Fig S17). A candidate‐based screen of known hepatocyte mitogens identified significantly upregulated levels of IGF2 in livers of EN^KO mice (Fig 3G). IGF2 has been proposed to act as a regulator of hepatocyte proliferation (Kimura & Ogihara, 1998), which corresponds to earlier work showing that IGF signaling during liver regeneration affected hepatocyte turnover but not necessarily cell volume (Leu et al, 2003). IGF receptor 1 and IRS1 were activated in EN^KO mice. Likewise, phosphorylation of downstream signaling pathways such as ERK and AKT was upregulated in EN^KO mice (Fig 3H and I). In turn, the expression of other hepatocyte mitogens such as IGF1, HGF, TGF‐β1, TGF‐β2, TGF‐β3, HB‐EGF, FGF21, IL‐1β, TIMP, or Cdkn1b was not altered (Supplementary Figs S18 and S19). The elevated levels of IGF2 identified in whole liver lysates could not be attributed to a particular cell type in the liver since liver sinusoidal endothelial cells (Fig 3J), hepatocytes (Fig 3K), and HSC (Supplementary Fig S20) were all identified as source of IGF2. Endosialin did not appear to directly affect IGF2 production in HSC as EdU uptake of cultured hepatocytes stimulated with conditioned medium collected from variably activated wild‐type or EN^KO HSC was increased to the same extent (Fig 3L). To examine whether IGF2 was able to directly affect hepatocyte proliferation, isolated hepatocytes from both genotypes were stimulated with recombinant IGF2, leading to a significantly increased EdU uptake after 24 h of stimulation (Fig 3M). Together, the selective regulation of IGF2 in livers from EN^KO mice suggests a possible role in the observed hepatocyte proliferation phenotype. Yet, further work will be needed to unravel the molecular details of the presumed endosialin–IGF2 axis.

In conclusion, this study has identified the hepatic stellate cell (HSC) marker endosialin (EN) as a critical balance of liver fibrogenesis and regenerative hepatocyte proliferation. Based on three independent pathology models (acute and chronic CCl₄‐induced liver damage, fibrosis, and partial hepatectomy) and validated by corresponding human pathology specimen expression profiling analyses, EN serves as positive regulator of the fibrogenic stromal cell compartment and as negative regulator of the parenchymal cell hepatocytic compartment (Supplementary Fig S21). Future work will need to unravel the molecular details of the paracrine endosialin‐mediated cross talk between HSC and hepatocytes, most notably the role of IGF2 as a regulator of hepatocyte proliferation. Yet, the identification of an HSC‐specifically expressed targetable and druggable cell surface receptor that controls the balance between liver fibrosis and liver regeneration may pave the way to fibrosis targeting and at the same time liver regeneration enhancing therapies: The dual compartment functions of endosialin further establish the molecular interdependency of HSC and hepatocyte contribution to liver function in health and disease (Ebrahimkhani et al, 2011). Moreover, EN targeting is presently pursued in early clinical tumor trials. Thus, the data identify EN as an attractive non‐oncologic target for liver fibrosis, whose therapeutic inhibition could negatively impact fibrosis, while at the same time stimulate hepatocyte proliferation.

Tissue samples were provided by the tissue bank of the National Center for Tumor Diseases (NCT, Heidelberg, Germany) in accordance with the regulations of the tissue bank and the approval of the ethics committee of the University of Heidelberg. This study was performed according to the Declaration of Helsinki; written informed consent was obtained from all patients. All patient specimen and corresponding clinical information were exclusively provided in a pseudonymized form according to the standard operating procedures of the NCT, approved by the ethic committee of the University of Heidelberg (Ethikvotes #206/207, 2005). Primary human hepatocytes were kindly provided by PD Dr. K. Breuhahn (Institute of Pathology, Heidelberg).

All animal experiments were performed according to the guidelines of the local animal use and care committees and approved by the Regierungspräsidium in Karlsruhe, including animal permits G195/10 (CCl₄ experiments) and G220/11 (partial hepatectomy). Animals were housed in barriers at the animal facility of the DKFZ with free admission to food and water. For sacrificing, mice were anesthetized by intraperitoneal injections of ketamine (100 mg/kg) and xylazine (10 mg/kg) diluted in isotonic 0.9% NaCl. Deeply anesthetized mice were heart punctured to obtain blood followed by rapid dislocation of the cervical spine. Whole blood was centrifuged at 8,000 g for 10 min, and subsequently, serum was collected from the supernatant. For determining clinically relevant serum parameters, 300 μl of serum (if necessary diluted with isotonic 0.9% NaCl) was analyzed by the Diagnostic Center of the Heidelberg University Hospital.

To induce acute liver damage, C57/Bl6 WT mice at an age of 10 weeks were injected intraperitoneally with a single high dose of CCl₄ (1.6 g/kg CCl₄, diluted in mineral oil). Mice were sacrificed after 3, 6, 24, 48, 72 h, or 6 days after injection. CCl₄‐induced liver fibrosis experiments were performed as previously described (Constandinou et al, 2005). Five to seven male C57/Bl6 endosialin WT and KO mice group at an age between 10 and 15 weeks were used for these experiments. Mice were injected intraperitoneally with CCl₄ (1 ml/kg bodyweight, 1:7 dilution in olive oil) twice a week for 2, 4, or 6 weeks. Control mice received the same amount of olive oil as CCl₄‐treated animals during the experimental time. Mice were sacrificed 3 days after the last CCl₄ injection. Partial hepatectomy was performed as previously described (Mitchell & Willenbring, 2008). In case of performing 1/3 partial hepatectomy, only left lobe was removed. Five to ten male C57BL/6 endosialin WT and KO mice group at an age between 10 and 15 weeks were used for partial hepatectomy experiments. Mice were anaesthetized by intraperitoneal injections of ketamine (100 mg/kg) and xylazine (10 mg/kg) diluted in isotonic 0.9% NaCl. As control, mice were sham‐operated. Mice were sacrificed after 1, 1.5, 2, 4, or 8 days posthepatectomy.

All results are expressed as mean ± s.e.m. or s.d., as indicated. The statistical differences between WT and EN^KO mice groups were analyzed using the two‐tailed unpaired Student's t‐test. The statistical difference between different time points in WT mice was analyzed using the two‐tailed paired or unpaired t‐test. Correlations were analyzed using the Pearson (bivariate) correlation. Differences P ≤ 0.05 were considered statistically significant.

Detailed information on additional materials and methods related to cells and cell culture experiments, molecular techniques, biochemical protocols and reagents, and histological and immunohistological techniques are summarized in the Supplementary Information.