Increased sinusoidal pressure impairs liver endothelial mechanosensing pathways uncovering specific plasma biomarkers of portal hypertension.

Abstract: Background and aims: Portal hypertension (PH) is the most frequent and severe clinical syndrome associated to chronic liver disease (CLD), defined by a pathological increase in the hepatic venous pressure gradient (HVPG). Considering the known mechanobiological effects of hydrostatic pressure and shear stress on endothelial cells, we hypothesized that PH could not only be a consequence of, but significantly influence the phenotype of liver sinusoidal endothelial cells (LSECs) during disease progression. The aim of this study was to investigate the effects of pathological hydrodynamic pressure on LSECs and to identify endothelial-derived biomarkers of PH. Methods: Primary LSECs were cultured under normal or increased hydrodynamic pressure within a pathophysiological range (1 vs 12 mmHg) using a microfluidic liver-on-a-chip device. RNAseq was used to identify pressure-sensitive genes, which were validated in liver biopsies from two independent cohorts of CLD patients with PH (n=73) vs subjects without PH (n=23). Biomarker discovery was performed in plasma from a third independent cohort of 64 patients (46 with PH vs 18 w/o). Results: Transcriptomic analysis revealed a marked deleterious effect of pathological pressure in LSECs and identified chromobox 7 (CBX7) as a key transcription factor diminished by pressure. Hepatic CBX7 downregulation was validated in patients with PH and significantly correlated with HVPG. MicroRNA 181a-5p was identified as pressure-induced upstream regulator of CBX7. Analysis of two downstream targets of CBX7, ECAD and SPINK1, were found increased in the bloodstream of patients with PH and were highly predictive of PH and clinically significant PH, with a sensitivity of 91.3% and 91.4% respectively. Conclusions: We describe the detrimental effects of increased hydrodynamic pressure on the sinusoidal endothelium, identify CBX7 as a pressure-sensitive transcription factor, and propose that the combination of two of its reported products could be used as plasma biomarkers of PH.摘要：背景与目的：门静脉高压症（Portal Hypertension, PH）是慢性肝病（Chronic Liver Disease, CLD）最常见且严重的临床综合征，以肝静脉压力梯度（Hepatic Venous Pressure Gradient, HVPG）病理性升高为核心定义。鉴于静水压力与切应力对内皮细胞已知的机械生物学效应，本研究提出假说：门静脉高压症不仅是慢性肝病进展的继发结果，还可在疾病进程中显著影响肝窦内皮细胞（Liver Sinusoidal Endothelial Cells, LSECs）的表型。本研究旨在探究病理性流体动力压力对肝窦内皮细胞的作用，并筛选门静脉高压症的内皮源性生物标志物。 方法：采用微流控肝脏器官芯片（microfluidic liver-on-a-chip）装置，将原代肝窦内皮细胞置于生理病理范围内的两种流体动力压力条件下培养：正常压力（1 mmHg）与升高压力（12 mmHg）。通过RNA测序（RNAseq）鉴定压力敏感基因，并在两个独立慢性肝病患者队列的肝活检样本中进行验证：伴门静脉高压症患者73例，无门静脉高压症患者23例。此外，在第三个独立队列的64例患者血浆样本中开展生物标志物筛选：其中伴门静脉高压症患者46例，无门静脉高压症患者18例。 结果：转录组学分析显示，病理性压力对肝窦内皮细胞具有显著损伤效应，并鉴定出染色盒蛋白7（Chromobox 7, CBX7）为受压力下调的关键转录因子。门静脉高压症患者肝脏组织中CBX7表达下调得到验证，且其表达水平与肝静脉压力梯度显著相关。微小RNA 181a-5p（MicroRNA 181a-5p）被鉴定为CBX7的压力诱导型上游调控因子。对CBX7的两个下游靶标ECAD与SPINK1的分析发现，二者在门静脉高压症患者血液循环中表达升高，且对门静脉高压症及临床显著性门静脉高压症具有极高预测价值，灵敏度分别达91.3%与91.4%。 结论：本研究阐明了流体动力压力升高对窦状隙内皮细胞的损伤效应，鉴定出CBX7为压力敏感型转录因子，并提出将其两种已知下游产物联合应用，作为门静脉高压症的血浆生物标志物。