Clinical evidence for thrombospondin-1 as a relevant suppressor of liver regeneration
Authors: Patrick Starlinger, Dominic Schauer, Lejla Alidzanovic, Silvia Zikeli, Kristina Gebhardt, Florian Luf, Edith Fleischmann, Beata Perisanidis, Birgit Gruenberger, Thomas Gruenberger, Christine Brostjan
Journal: Journal of Hepatology (2012)

The most significant factor determining morbidity and mortality following hepatectomy is the ability of the remnant liver to regenerate. In this context, Hayashi et al. [[1]Hayashi H. Sakai K. Baba H. Sakai T. Thrombospondin-1 is a novel negative regulator of liver regeneration after partial hepatectomy through transforming growth factor-beta1 activation in mice.Hepatology. 2012; 55: 1562-1573Crossref PubMed Scopus (57) Google Scholar] recently reported on a previously unknown role of thrombospondin-1 (TSP-1) in liver regeneration (LR). During tissue remodeling, TSP-1 is released into the pericellular space to regulate cell survival and extracellular matrix remodeling [[2]Sweetwyne M.T. Pallero M.A. Lu A. Van Duyn Graham L. Murphy-Ullrich J.E. The calreticulin-binding sequence of thrombospondin 1 regulates collagen expression and organization during tissue remodeling.Am J Pathol. 2010; 177: 1710-1724Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar]. A major TSP-1 function is the conversion of latent TGF-β1 (transforming growth factor-beta 1) to its active form [[3]Murphy-Ullrich J.E. Poczatek M. Activation of latent TGF-beta by thrombospondin-1: mechanisms and physiology.Cytokine Growth Factor Rev. 2000; 11: 59-69Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar]. Based on a mouse model of partial hepatectomy, Hayashi et al. [[1]Hayashi H. Sakai K. Baba H. Sakai T. Thrombospondin-1 is a novel negative regulator of liver regeneration after partial hepatectomy through transforming growth factor-beta1 activation in mice.Hepatology. 2012; 55: 1562-1573Crossref PubMed Scopus (57) Google Scholar] demonstrated that TSP-1 was rapidly released in response to liver resection and triggered TGF-β1 activation and signal transduction in hepatocytes as a mechanism to inhibit the proliferative hepatic response [4McMahon J.B. Richards W.L. del Campo A.A. Song M.K. Thorgeirsson S.S. Differential effects of transforming growth factor-beta on proliferation of normal and malignant rat liver epithelial cells in culture.Cancer Res. 1986; 46: 4665-4671PubMed Google Scholar, 5Russell W.E. Coffey Jr., R.J. Ouellette A.J. Moses H.L. Type beta transforming growth factor reversibly inhibits the early proliferative response to partial hepatectomy in the rat.Proc Natl Acad Sci U S A. 1988; 85: 5126-5130Crossref PubMed Scopus (370) Google Scholar]. Accordingly, knockdown of TSP-1 expression resulted in a significant reduction of TGF-β1 signaling, which promoted the proliferation of hepatocytes and accelerated LR [[1]Hayashi H. Sakai K. Baba H. Sakai T. Thrombospondin-1 is a novel negative regulator of liver regeneration after partial hepatectomy through transforming growth factor-beta1 activation in mice.Hepatology. 2012; 55: 1562-1573Crossref PubMed Scopus (57) Google Scholar]. Thus, TSP-1 was proposed as an inhibitory element and a potential therapeutic target of LR. We are now able to present the first clinical data, which support the relevance of TSP-1 in LR based on a cohort of metastasized colorectal cancer (mCRC) patients undergoing hepatic resection of liver metastases. A total of 66 mCRC patients without preexisting liver disease were included. Blood was retrieved before surgery (pre OP) and on day 1 (POD1) or days 5–8 (POD5) after liver resection, and plasma was prepared with an optimized method [6Starlinger P. Alidzanovic L. Schauer D. Brugger P. Sommerfeldt S. Kuehrer I. et al.Platelet-stored angiogenesis factors: clinical monitoring is prone to artifacts.Dis Markers. 2011; 31: 55-65Crossref PubMed Google Scholar, 7Starlinger P. Moll H.P. Assinger A. Nemeth C. Hoetzenecker K. Gruenberger B. et al.Thrombospondin-1: a unique marker to identify in vitro platelet activation when monitoring in vivo processes.J Thromb Haemost. 2010; 8: 1809-1819Crossref PubMed Scopus (51) Google Scholar]. Samples were assayed by ELISA for human TSP-1 (R&D Systems, Minneapolis, MN, USA) and TGF-β1 (eBioscience, San Diego, CA, USA). For statistical analysis, non-parametric tests (Mann–Whitney U test, Wilcoxon test and Spearman correlation) and the Fisher exact test were applied. A significant increase in TSP-1 (Fig. 1A) and TGF-β1 was observed on POD1 (p = 0.007 and p = 0.006, respectively), which dropped moderately until POD5 (p = 0.016 and p = 0.397). Circulating TSP-1 and TGF-β1 showed a highly significant correlation (k = 0.736, p <0.001). To further investigate whether a high postoperative release of TSP-1 or TGF-β1 was associated with delayed LR, we assessed the incidence of postoperative liver dysfunction (LD), based on the previously defined "50–50 criteria" [[8]Balzan S. Belghiti J. Farges O. Ogata S. Sauvanet A. Delefosse D. et al.The "50–50 criteria" on postoperative day 5: an accurate predictor of liver failure and death after hepatectomy.Ann Surg. 2005; 242 ([discussion 828–829]): 824-828Crossref PubMed Scopus (831) Google Scholar], with a prothrombin time (PT) <50% and a serum bilirubin (SB) level >50 μmol/L (>2.9 mg/dl). Our classification referred to measurements of any day within the first postoperative week to identify patients with delayed postoperative hepatic recovery. TSP-1 concentrations were significantly elevated (Fig. 1B) in patients with LD (median TSP-1 for LD: 98.8 ng/ml; no LD: 44.6 ng/ml, p = 0.003). Comparably, circulating TGF-β1 tended to be increased in individuals with LD (median TGF-β1 for LD: 2266 pg/ml; no LD: 1726 pg/ml), but the difference was not statistically significant (p = 0.102). Of note, our analyses were restricted to total TGF-β1 in patient blood since activated TGF-β1 was below ELISA detection limit. This may explain the lack of association between TGF-β1 levels and postoperative LD. To further characterize the potential of TSP-1 to predict postoperative LD, a ROC analysis was performed (Fig. 1C), revealing a significant predictive value of circulating TSP-1 on POD1 (AUC: 0.870; p = 0.003). A cut-off level of 100 ng/ml TSP-1 on POD1 was deduced to determine the high-risk group for postoperative LD with 95% specificity. No statistically significant difference in age and sex distribution or the site of primary tumor, type of hepatic resection and preoperative liver function parameters was observed between the TSP-1high individuals (>100 ng/ml TSP-1) and patients with lower circulating TSP-1 levels on POD1. When biochemical markers of postoperative liver function were compared between these groups, the TSP-1high patients showed a significant elevation in SB (Fig. 1D) and γ-glutamyltransferase (p <0.001; p = 0.011). Prothrombin time (Fig. 1E) was found to be significantly decreased in TSP-1high patients (p <0.001). A trend towards elevated alanine and aspartate aminotransferase levels was found in TSP-1high patients but did not reach statistical significance (p = 0.125 and p = 0.092). We further assessed the clinical performance of TSP-1high individuals in terms of morbidity and hospital stay. Only 4 patients suffered from severe morbidity of grade III, IV or V according to Dindo et al. [[9]Dindo D. Demartines N. Clavien P.A. Classification of surgical complications: a new proposal with evaluation in a cohort of 6336 patients and results of a survey.Ann Surg. 2004; 240: 205-213Crossref PubMed Scopus (21510) Google Scholar], which limits the conclusions of the statistical analysis. However, TSP-1 levels were significantly elevated in patients with severe complications (p = 0.016) and we found a substantial increase of postoperative severe morbidity from 4% to 33% in the TSP-1high subgroup (p = 0.053). With respect to hospital stay, a highly significant correlation of TSP-1 levels on POD1 with days of postoperative hospitalization was observed (k = 0.488; p <0.001). In accordance, TSP-1high patients required a doubled median hospitalization time of 16.5 days as compared to 8 days for the remaining patients to recover from hepatectomy (p = 0.011). In conclusion, these are the first clinical data confirming that TSP-1 negatively correlates with liver regeneration in humans, which warrants validation in large-scale studies. TSP-1 may prove a helpful clinical marker to predict LD on the first postoperative day and to identify high-risk patients for potential complications and suitable interventions. This work was supported by the Medical Scientific Fund of the Mayor of the City of Vienna (grant 8064 issued to Thomas Gruenberger). The authors who have taken part in this study declare that they do not have anything to disclose regarding conflict of interest with respect to this manuscript.