S100A9 is a Biliary Protein Marker of Disease Activity in Primary Sclerosing Cholangitis

<div><h3>Background and Aims</h3><p>Bile analysis has the potential to serve as a surrogate marker for inflammatory and neoplastic disorders of the biliary epithelium and may provide insight into biliary pathophysiology and possible diagnostic markers. We aimed to identify biliary protein markers of patients with primary sclerosing cholangitis (PSC) by a proteomic approach.</p> <h3>Methods</h3><p>Bile duct-derived bile samples were collected from PSC patients (n = 45) or patients with choledocholithiasis (n = 24, the control group). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed to analyse the proteins, 2-D-gel patterns were compared by densitometry, and brush cytology specimens were analysed by RT-PCR.</p> <h3>Results</h3><p>A reference bile-duct bile proteome was established in the control group without signs of inflammation or maligancy comprising a total of 379 non-redundant biliary proteins; 21% were of unknown function and 24% had been previously described in serum. In PSC patients, the biliary S100A9 expression was elevated 95-fold (p<0.005), serum protein expression was decreased, and pancreatic enzyme expression was unchanged compared to controls. The S100A9 expression was 2-fold higher in PSC patients with high disease activity than in those with low activity (p<0.05). The brush cytology specimens from the PSC patients with high disease activity showed marked inflammatory activity and leukocyte infiltration compared to the patients with low activity, which correlated with S100A9 mRNA expression (p<0.05).</p> <h3>Conclusions</h3><p>The bile-duct bile proteome is complex and its analysis might enhance the understanding of cholestatic liver disease. Biliary S100A9 levels may be a useful marker for PSC activity, and its implication in inflammation and carcinogenesis warrants further investigation.</p> </div

Correlation between biliary proteins and disease activity in PSC patients.

<p>Bile from PSC patients showed a marked increase in biliary protein content. (<b>A</b>) Box plots of the protein concentration per volume show a marked increase for the PSC patients. If the PSC patients were classified based on disease activity, either by endoscopic findings (<b>C</b>) or by Mayo Risk score (<b>E</b>), the corresponding biliary protein concentrations showed significantly greater concentrations in the patients with high PSC activity than in those with low activity. If the bile samples were normalised by the bile-acid content, the results were similar (<b>B,</b> controls versus all the PSC patients; <b>D,</b> PSC patients classified by endoscopic findings; <b>F,</b> PSC patients classified by Mayo Risk Score). Box plots of the protein concentration normalised by the BA concentration. (control = 24, PSC = 45, low MRS = 25, intermediate MRS = 20; low endoscopic = 26; high endoscopy activity = 19; ns = not significant; *p<0.05; **p<0.005).</p

Effects of Increased Von Willebrand Factor Levels on Primary Hemostasis in Thrombocytopenic Patients with Liver Cirrhosis

<div><p>In patients with liver cirrhosis procoagulant and anticoagulant changes occur simultaneously. During primary hemostasis, platelets adhere to subendothelial structures, via von Willebrand factor (vWF). We aimed to investigate the influence of vWF on primary hemostasis in patients with liver cirrhosis. Therefore we assessed in-vitro bleeding time as marker of primary hemostasis in cirrhotic patients, measuring the Platelet Function Analyzer (PFA-100) closure times with collagen and epinephrine (Col-Epi, upper limit of normal ≤165 s) or collagen and ADP (Col-ADP, upper limit of normal ≤118 s). If Col-Epi and Col-ADP were prolonged, the PFA-100 was considered to be pathological. Effects of vWF on primary hemostasis in thrombocytopenic patients were analyzed and plasma vWF levels were modified by adding recombinant vWF or anti-vWF antibody. Of the 72 included cirrhotic patients, 32 (44.4%) showed a pathological result for the PFA-100. They had mean closure times (± SD) of 180±62 s with Col-Epi and 160±70 s with Col-ADP. Multivariate analysis revealed that hematocrit (<i>P</i> = 0.027) and vWF-antigen levels (<i>P</i> = 0.010) are the predictors of a pathological PFA-100 test in cirrhotic patients. In 21.4% of cirrhotic patients with platelet count ≥150/nL and hematocrit ≥27.0%, pathological PFA-100 results were found. In thrombocytopenic (<150/nL) patients with cirrhosis, normal PFA-100 results were associated with higher vWF-antigen levels (462.3±235.9% vs. 338.7±151.6%, <i>P</i> = 0.021). These results were confirmed by multivariate analysis in these patients as well as by adding recombinant vWF or polyclonal anti-vWF antibody that significantly shortened or prolonged closure times, respectively. In conclusion, primary hemostasis is impaired in cirrhotic patients. The effect of reduced platelet count in cirrhotic patients can at least be partly compensated by increased vWF levels. Recombinant vWF could be an alternative to platelet transfusions in the future.</p></div

Identification of S100A9 as a marker of disease activity.

<p>The protein patterns among the PSC and choledocholithiasis (control) patients were compared. Intensities of 25 spots uniquely identified by mass spectrometry were measured using densitometry. The spot resembling S100A9 was markedly upregulated in the PSC patients with high activity (<b>B</b>) compared to those with low activity (<b>A</b>). Circled spots: haemoglobin (red), S100A9 (yellow), serum proteins (black), pancreatic enzymes (green), and others (blue). An evaluation of the relative amount of S100A9 in the spots demonstrated a clear upregulation of S100A9 in the PSC patients (<b>C</b>). Subgroup analyses of the PSC patients performed according to disease activity revealed a distinct increase in S100A9 in the PSC patients with high disease activity relative to those with low disease activity (<b>D</b>). The PSC patients were also stratified by their Mayo risk scores. (n: control = 6; PSC = 18; low MRS = 13; intermediate MRS = 5; low endoscopic = 12; high endoscopic = 6). The total S100 levels were significantly elevated in the PSC patients PSC (<b>F</b>) (*p<0.05; **p<0.005). The RT-PCR analyses of the brush cytology samples demonstrated marked upregulation of S100A9 expression (n = 8, 4 in the low activity group and 4 in the high activity group; p<0.05) (<b>G</b>).The brush cytology from the patients with unclear strictures demonstrated corresponding cytological findings. The cytology from a patient with low disease activity showed regular epithelial cells (arrowheads) (<b>H</b>), whereas the cytology from a patient with high disease activity showed leukocyte infiltrate (arrows) and altered epithelial cells (<b>I</b>).</p

Patient characteristics.

<p>MRS Mayo risk score; PSC low and high activities refer to the endoscopic finding.</p

Bile purification and identification of biliary proteins.

<p>(<b>A</b>) Comparison of serum (S), plasma (P), and bile duct-derived bile proteins (B) from two choledocholithiasis patients demonstrates many similar expressed proteins; however, the protein composition differs between the serum and bile samples. (<b>B</b>) Albumin and IgG depletion and purification for the crude bile duct-derived bile samples. The purification of the bile samples was monitored using 1-D silver stained gels, and the crude bile samples (Lane 1) were compared to the precipitated samples depleted of albumin and IgG. The arrow indicates the marked reduction in albumin. (<b>C</b>) A mixture of bile samples was separated by 1-D Gel electrophoresis, cut into the depicted slices and analysed by mass spectrometry. The 301 identified proteins with a Mascot score >100 were classified according to reported subcellular localisation (<b>D</b>), the biological processes in which they are involved (<b>E</b>) and the pathways the majority of proteins are involved (<b>F</b>).</p

2-D SDS-PAGE analysis of purified bile.

<p>2-D gels of biliary proteins. pH range of the first-dimension isoelectric focusing gel is shown at the bottom, and the sizes of the molecular mass standards (in kDa) for the second dimension are indicated on the left. The proteins corresponding to the spots on the gels (circled in blue) were identified by mass spectrometry. (<b>A</b>) 500 µg of protein was separated on a gradient from pH 3–11 (non-linear). To identify less abundant proteins, (<b>B</b>), 1 mg of protein was separated; more prominent spots were excluded, and the less prominent spots were identified by mass spectrometry.</p

Selection of the study population.

<p>The enrolment details of the study population are shown, including the number of patients excluded because of the use of antiplatelet drugs or because there was no information on use of these drugs. For all subgroups, the number of available test results for PFA-100 with Col-Epi and Col-ADP is given.</p

Analysis of the effect of vWF on results of PFA-100 in thrombocytopenic patients.

<p>Comparison of vWF-antigen and vWF-activity in thrombocytopenic (<150/nL) patients with liver cirrhosis and the effect of vWF on results of PFA-100 measured with Col-Epi and Col-ADP (norm.  =  normal test result, path.  =  pathological test result).</p><p>Analysis of the effect of vWF on results of PFA-100 in thrombocytopenic patients.</p

Characteristics of patients with liver cirrhosis.

<p>Description of baseline parameters such as age, sex, and etiology of patients with liver cirrhosis and labMELD-score; grouped by Child-Turcotte-Pugh score.</p>a<p>Including primary sclerosing cholangitis and biliary atresia.</p>b<p>Including Wilson disease and polycystic liver disease.</p><p>Characteristics of patients with liver cirrhosis.</p