Portal hypertension is a primary cause of complications leading to significant morbidity and mortality in patients with cirrhosis. Transjugular intrahepatic portosystemic shunt (TIPS) insertion has improved survival in well-selected patients with refractory ascites and high-risk variceal bleeding. We investigated the prognostic role of circulating extracellular vesicles (EVs), which are known for their role in immunomodulation and intercellular communication, in patients undergoing TIPS. 141 patients undergoing TIPS placement were included in this retrospective analysis. Median EVs size (X50) and total serum concentration were determined by nanoparticle tracking analysis (NTA) prior to TIPS placement, and transplant-free 1-year survival was assessed using time-to event analysis and Cox regression. EVs size but not their concentration moderately correlated with MELD and Child-Pugh scores based on its correlation with bilirubin and international normalized ratio. In addition, a significant correlation of EVs concentration with platelet count and the immune activation marker soluble urokinase plasminogen activator receptor was observed. In univariate analysis, larger EVs size (> 243 nm) was associated with 1-year transplant-free survival after TIPS placement (p = 0.012; HR: 2.539), which remained significant after adjusting for MELD scores in multivariable Cox-regression analysis (p = 0.033; HR: 2.204). Larger EVs size indicates advanced stages of chronic liver disease and served as an independent predictor of transplantation-free survival after TIPS placement. Larger prospective studies are needed to confirm these findings and to identify patients at particularly high-risk following TIPS placement.
Portal hypertension (PH) is the primary factor contributing to complications that cause morbidity and mortality in cirrhotic patients. It leads to the development of portal-systemic collaterals, as well as gastric and oesophageal varices, and is associated with a poorer prognosis. Several complications may occur, including bleeding from varices, ascites, and hepatic encephalopathy. In addition, complications related to circulatory dysfunction may occur, such as hepatorenal syndrome, portopulmonary syndrome, and hepatopulmonary syndrome. The introduction of the transjugular intrahepatic portosystemic shunt (TIPS) into clinical practice has been one of the most significant advances in the management of complications of PH. In cases of recurrent or refractory ascites, patients treated with TIPS have better survival than those who receive large volume paracentesis and albumin infusion. In patients with high risk variceal bleeding, preemptive TIPS placement is superior to conservative management and improves survival. However, PH is not the only cause of complications; increased systemic inflammation is also associated with decompensation, particularly in cases of acute-on-chronic liver failure (ACLF). Even when PH is effectively managed after TIPS, liver-derived inflammation appears to be the primary factor leading to organ failure and decompensation in cirrhotic patients. Various methods have been proposed to assess hepatic and systemic inflammation, but individual markers do not appear to reliably predict outcomes in acute decompensation.
Extracellular vesicles (EVs) are small lipid membrane structures released into the surrounding extracellular environment by almost all cell types. They have attracted worldwide interest due to their ability to carry specific cargoes, including proteins and nucleic acids. EVs are important mediators of intercellular communication and can play a critical role in modulatory immune responses. However, their potential prognostic value in patients undergoing TIPS placements has not been systematically analyzed.
The aim of this study was to evaluate the potential predictive and prognostic significance of circulating extracellular vesicles as a novel biomarker in patients undergoing TIPS placement for portal hypertension.
This is a retrospective analysis of 141 patients with cirrhosis and severe portal hypertension scheduled for TIPS insertion using ePTFE-covered stents were enrolled at the Jena University Hospital (Germany) between October 2013 and September 2022 and at the University Hospital RWTH Aachen (Germany) between August 2019 and May 2023. This study involves the validation cohort of the previously publishes study; Because serum from 9 patients was unavailable, we included 141 instead of 150 patients. The inclusion criteria were as follows: (1) age between 18 and 85 years, and (2) decompensated cirrhosis (ascites or variceal bleeding) with an indication for TIPS. Exclusion criteria included clinically assessed contraindications for TIPS placement, including severe heart failure, severe pulmonary hypertension, active systemic infection, spontaneous bacterial peritonitis, overt hepatic encephalopathy, or other medical conditions that would make the procedure technically unfeasible. Patients were followed until death or liver transplantation. The present study was performed in accordance with the Declaration of Helsinki. All protocols were approved by the ethics committee of the Jena University Hospital (No. 3683-02/3, 2019-1510, 2018-1080-BO) and the University Hospital RWTH Aachen (No. EK023-19). Written informed consent was obtained from each of the participating patients prior to intervention.
The TIPS procedure (8-10 mm VIATORR, W.L. Gore, Newark, Delaware, US) was carried out following local standard operating procedures as clinically indicated and previously described. Serum samples from the cubital vein were allowed to clot at room temperature for 30 min, then centrifuged at 1000×g for 10 min, and stored at - 80°C until further analysis. The median interval between blood collection and TIPS insertion was 0 days (IQR 0-1).
The size distribution and concentration of EVs were measured using the ZetaView multi-parameter Particle Tracking Analyzer (ParticleMetrix, Germany), as previously described. This technique, based on Brownian motion, allows for the analysis of nanometer-sized particles. To ensure accuracy, the ZetaView was auto-aligned before each measurement using a standard calibration nanoparticle solution (110 nm diameter) provided by ParticleMetrix. The camera focus was adjusted to make the particles appear as sharp dots prior to analysis. The sample with the highest anticipated vesicle concentration was used to set the camera sensitivity, which remained constant throughout subsequent measurements. Samples were diluted in particle-free PBS to achieve a particle concentration between 1 and 9 × 10^7 particles/mL (approximately 200 particles per visual field). For each sample, three 30-s videos were recorded using the script control function, with a 5-s pause between recordings and a sample advance between each video.
Statistical analyses were performed as previously described. The Shapiro-Wilk test was used to assess normality. Non-parametric data were analyzed using the Kruskal-Wallis ANOVA test. Box plots represent the median, quartiles, and ranges. For the correlation of two variables Spearman´s rank correlation coefficient was applied. The prognostic significance of EVs characteristics and the MELD score was further evaluated through univariate and multivariate Cox regression analyses, including parameters with a p value < 0.20 from univariate analysis in the multivariate model. Hazard ratios (HR) with 95% confidence intervals are reported. Kaplan-Meier curves were used to illustrate the effect of EVs parameters on transplantation (TX)-free survival and statistical differences were assessed using the log-rank test. The optimal cut-off value for identifying patients with reduced TX-free survival was determined by fitting Cox proportional hazards models to dichotomized survival status and survival time, defining the cut-off as the point with the most significant log-rank test split. All statistical analyses were performed using SPSS 23 (SPSS, Chicago, IL, USA) and RStudio 1.2.5033 (RStudio Inc., Boston, MA, USA). Differences were considered statistically significant at p < 0.05 (* p < 0.05; ** p < 0.01; *** p < 0.001).