Titration of RAS alters senescent state and influences tumour initiation.

Oncogenic RAS-induced senescence (OIS) is an autonomous tumour suppressor mechanism associated with premalignancy1,2. Achieving this phenotype typically requires a high level of oncogenic stress, yet the phenotype provoked by lower oncogenic dosage remains unclear. Here we develop oncogenic RAS dose-escalation models in vitro and in vivo, revealing a RAS dose-driven non-linear continuum of downstream phenotypes. In a hepatocyte OIS model in vivo, ectopic expression of NRAS(G12V) does not induce tumours, in part owing to OIS-driven immune clearance3. Single-cell RNA sequencing analyses reveal distinct hepatocyte clusters with typical OIS or progenitor-like features, corresponding to high and intermediate levels of NRAS(G12V), respectively. When titred down, NRAS(G12V)-expressing hepatocytes become immune resistant and develop tumours. Time-series monitoring at single-cell resolution identifies two distinct tumour types: early-onset aggressive undifferentiated and late-onset differentiated hepatocellular carcinoma. The molecular signature of each mouse tumour type is associated with different progenitor features and enriched in distinct human hepatocellular carcinoma subclasses. Our results define the oncogenic dosage-driven OIS spectrum, reconciling the senescence and tumour initiation phenotypes in early tumorigenesis.

Senescence-induced endothelial phenotypes underpin immune-mediated senescence surveillance.

Senescence is a stress-responsive tumor suppressor mechanism associated with expression of the senescence-associated secretory phenotype (SASP). Through the SASP, senescent cells trigger their own immune-mediated elimination, which if evaded leads to tumorigenesis. Senescent parenchymal cells are separated from circulating immunocytes by the endothelium, which is targeted by microenvironmental signaling. Here we show that SASP induces endothelial cell NF-κB activity and that SASP-induced endothelial expression of the canonical NF-κB component Rela underpins senescence surveillance. Using human liver sinusoidal endothelial cells (LSECs), we show that SASP-induced endothelial NF-κB activity regulates a conserved transcriptional program supporting immunocyte recruitment. Furthermore, oncogenic hepatocyte senescence drives murine LSEC NF-κB activity in vivo. Critically, we show two distinct endothelial pathways in senescence surveillance. First, endothelial-specific loss of Rela prevents development of Stat1-expressing CD4+ T lymphocytes. Second, the SASP up-regulates ICOSLG on LSECs, with the ICOS-ICOSLG axis contributing to senescence cell clearance. Our results show that the endothelium is a nonautonomous SASP target and an organizing center for immune-mediated senescence surveillance.

Convergent somatic mutations in metabolism genes in chronic liver disease.

The progression of chronic liver disease to hepatocellular carcinoma is caused by the acquisition of somatic mutations that affect 20-30 cancer genes1-8. Burdens of somatic mutations are higher and clonal expansions larger in chronic liver disease9-13 than in normal liver13-16, which enables positive selection to shape the genomic landscape9-13. Here we analysed somatic mutations from 1,590 genomes across 34 liver samples, including healthy controls, alcohol-related liver disease and non-alcoholic fatty liver disease. Seven of the 29 patients with liver disease had mutations in FOXO1, the major transcription factor in insulin signalling. These mutations affected a single hotspot within the gene, impairing the insulin-mediated nuclear export of FOXO1. Notably, six of the seven patients with FOXO1S22W hotspot mutations showed convergent evolution, with variants acquired independently by up to nine distinct hepatocyte clones per patient. CIDEB, which regulates lipid droplet metabolism in hepatocytes17-19, and GPAM, which produces storage triacylglycerol from free fatty acids20,21, also had a significant excess of mutations. We again observed frequent convergent evolution: up to fourteen independent clones per patient with CIDEB mutations and up to seven clones per patient with GPAM mutations. Mutations in metabolism genes were distributed across multiple anatomical segments of the liver, increased clone size and were seen in both alcohol-related liver disease and non-alcoholic fatty liver disease, but rarely in hepatocellular carcinoma. Master regulators of metabolic pathways are a frequent target of convergent somatic mutation in alcohol-related and non-alcoholic fatty liver disease.

Somatic mutations and clonal dynamics in healthy and cirrhotic human liver.

The most common causes of chronic liver disease are excess alcohol intake, viral hepatitis and non-alcoholic fatty liver disease, with the clinical spectrum ranging in severity from hepatic inflammation to cirrhosis, liver failure or hepatocellular carcinoma (HCC). The genome of HCC exhibits diverse mutational signatures, resulting in recurrent mutations across more than 30 cancer genes1-7. Stem cells from normal livers have a low mutational burden and limited diversity of signatures8, which suggests that the complexity of HCC arises during the progression to chronic liver disease and subsequent malignant transformation. Here, by sequencing whole genomes of 482 microdissections of 100-500 hepatocytes from 5 normal and 9 cirrhotic livers, we show that cirrhotic liver has a higher mutational burden than normal liver. Although rare in normal hepatocytes, structural variants, including chromothripsis, were prominent in cirrhosis. Driver mutations, such as point mutations and structural variants, affected 1-5% of clones. Clonal expansions of millimetres in diameter occurred in cirrhosis, with clones sequestered by the bands of fibrosis that surround regenerative nodules. Some mutational signatures were universal and equally active in both non-malignant hepatocytes and HCCs; some were substantially more active in HCCs than chronic liver disease; and others-arising from exogenous exposures-were present in a subset of patients. The activity of exogenous signatures between adjacent cirrhotic nodules varied by up to tenfold within each patient, as a result of clone-specific and microenvironmental forces. Synchronous HCCs exhibited the same mutational signatures as background cirrhotic liver, but with higher burden. Somatic mutations chronicle the exposures, toxicity, regeneration and clonal structure of liver tissue as it progresses from health to disease.

Hepatocytes undergo punctuated expansion dynamics from a periportal stem cell niche in normal human liver.

BACKGROUND & AIMS: While normal human liver is thought to be generally quiescent, clonal hepatocyte expansions have been observed, though neither their cellular source nor their expansion dynamics have been determined. Knowing the hepatocyte cell of origin, and their subsequent dynamics and trajectory within the human liver will provide an important basis to understand disease-associated dysregulation. METHODS: Herein, we use in vivo lineage tracing and methylation sequence analysis to demonstrate normal human hepatocyte ancestry. We exploit next-generation mitochondrial sequencing to determine hepatocyte clonal expansion dynamics across spatially distinct areas of laser-captured, microdissected, clones, in tandem with computational modelling in morphologically normal human liver. RESULTS: Hepatocyte clones and rare SOX9+ hepatocyte progenitors commonly associate with portal tracts and we present evidence that clones can lineage-trace with cholangiocytes, indicating the presence of a bipotential common ancestor at this niche. Within clones, we demonstrate methylation CpG sequence diversity patterns indicative of periportal not pericentral ancestral origins, indicating a portal to central vein expansion trajectory. Using spatial analysis of mitochondrial DNA variants by next-generation sequencing coupled with mathematical modelling and Bayesian inference across the portal-central axis, we demonstrate that patterns of mitochondrial DNA variants reveal large numbers of spatially restricted mutations in conjunction with limited numbers of clonal mutations. CONCLUSIONS: These datasets support the existence of a periportal progenitor niche and indicate that clonal patches exhibit punctuated but slow growth, then quiesce, likely due to acute environmental stimuli. These findings crucially contribute to our understanding of hepatocyte dynamics in the normal human liver. IMPACT AND IMPLICATIONS: The liver is mainly composed of hepatocytes, but we know little regarding the source of these cells or how they multiply over time within the disease-free human liver. In this study, we determine a source of new hepatocytes by combining many different lab-based methods and computational predictions to show that hepatocytes share a common cell of origin with bile ducts. Both our experimental and computational data also demonstrate hepatocyte clones are likely to expand in slow waves across the liver in a specific trajectory, but often lie dormant for many years. These data show for the first time the expansion dynamics of hepatocytes in normal liver and their cell of origin enabling the accurate measurment of changes to their dynamics that may lead to liver disease. These findings are important for researchers determining cancer risk in human liver.

Lipid Remodeling in Hepatocyte Proliferation and Hepatocellular Carcinoma.

BACKGROUND AND AIMS: Hepatocytes undergo profound metabolic rewiring when primed to proliferate during compensatory regeneration and in hepatocellular carcinoma (HCC). However, the metabolic control of these processes is not fully understood. In order to capture the metabolic signature of proliferating hepatocytes, we applied state-of-the-art systems biology approaches to models of liver regeneration, pharmacologically and genetically activated cell proliferation, and HCC. APPROACH AND RESULTS: Integrating metabolomics, lipidomics, and transcriptomics, we link changes in the lipidome of proliferating hepatocytes to altered metabolic pathways including lipogenesis, fatty acid desaturation, and generation of phosphatidylcholine (PC). We confirm this altered lipid signature in human HCC and show a positive correlation of monounsaturated PC with hallmarks of cell proliferation and hepatic carcinogenesis. CONCLUSIONS: Overall, we demonstrate that specific lipid metabolic pathways are coherently altered when hepatocytes switch to proliferation. These represent a source of targets for the development of therapeutic strategies and prognostic biomarkers of HCC.

A minority of somatically mutated genes in pre-existing fatty liver disease have prognostic importance in the development of NAFLD.

BACKGROUND: Understanding the genetics of liver disease has the potential to facilitate clinical risk stratification. We recently identified acquired somatic mutations in six genes and one lncRNA in pre-existing fatty liver disease. We hypothesised that germline variation in these genes might be associated with the risk of developing steatosis and contribute to the prediction of disease severity. METHODS: Genome-wide association study (GWAS) summary statistics were extracted from seven studies (>1.7 million participants) for variants near ACVR2A, ALB, CIDEB, FOXO1, GPAM, NEAT1 and TNRC6B for: aminotransferases, liver fat, HbA1c, diagnosis of NAFLD, ARLD and cirrhosis. Findings were replicated using GWAS data from multiple independent cohorts. A phenome-wide association study was performed to examine for related metabolic traits, using both common and rare variants, including gene-burden testing. RESULTS: There was no evidence of association between rare germline variants or SNPs near five genes (ACVR2A, ALB, CIDEB, FOXO1 and TNRC6B) and risk or severity of liver disease. Variants in GPAM (proxies for p.Ile43Val) were associated with liver fat (p = 3.6 × 10-13 ), ALT (p = 2.8 × 10-39 ) and serum lipid concentrations. Variants in NEAT1 demonstrated borderline significant associations with ALT (p = 1.9 × 10-11 ) and HbA1c, but not with liver fat, as well as influencing waist-to-hip ratio, adjusted for BMI. CONCLUSIONS: Despite the acquisition of somatic mutations at these loci during progressive fatty liver disease, we did not find associations between germline variation and markers of liver disease, except in GPAM. In the future, larger sample sizes may identify associations. Currently, germline polygenic risk scores will not capture data from genes affected by somatic mutations.

Cellular senescence and its effector programs.

Cellular senescence is a stress response that accompanies stable exit from the cell cycle. Classically, senescence, particularly in human cells, involves the p53 and p16/Rb pathways, and often both of these tumor suppressor pathways need to be abrogated to bypass senescence. In parallel, a number of effector mechanisms of senescence have been identified and characterized. These studies suggest that senescence is a collective phenotype of these multiple effectors, and their intensity and combination can be different depending on triggers and cell types, conferring a complex and diverse nature to senescence. Series of studies on senescence-associated secretory phenotype (SASP) in particular have revealed various layers of functionality of senescent cells in vivo. Here we discuss some key features of senescence effectors and attempt to functionally link them when it is possible.

Sorafenib is associated with a reduced rate of tumour growth and liver function deterioration in HCV-induced hepatocellular carcinoma.

BACKGROUND & AIMS: Sorafenib has been the standard of care for patients with advanced hepatocellular carcinoma and although immunotherapeutic approaches are now challenging this position, it retains an advantage in HCV-seropositive patients. We aimed to quantify the rate of tumour progression in patients receiving sorafenib and relate this figure to survival, both overall, and according to viral status. METHODS: Using serial data from an international clinical trial we applied a joint model to combine survival and progression over time in order to estimate the rate of tumour growth as assessed by tumour burden and serum alpha-fetoprotein, and the impact of treatment on liver function. RESULTS: High tumour burden at baseline was associated with an increased risk of death. In patients still alive at the end of the study, the progression in relation to tumour burden was very low compared to those who died within the study. Overall, the change in mean tumour burden was 0.12 mm per day or an absolute growth rate of 3.6 mm/month. Median doubling time was 665 days. For those who progressed above 0.12 mm per day or the 12% rate, median survival was 234 days compared to 384 days if the rate was below 12%. Tumour growth rate and serum alpha-fetoprotein rise were significantly lower in those who were HCV seropositive as was the rate of decline in liver function. These results were replicated in 2 independent patient groups. CONCLUSION: Our analysis suggests that sorafenib treatment is associated with improved survival in patients with advanced hepatocellular carcinoma mainly by decreasing the rate of tumour growth and liver function deterioration among patients with HCV infection. LAY SUMMARY: Among patients receiving sorafenib for advanced hepatocellular carcinoma the rate of tumour growth (as assessed by changes in tumour size and the biomarker alpha-fetoprotein) and the deterioration of liver function is less in those who have the hepatitis C virus, than in those who do not.

Notch and Senescence.

Cellular senescence, previously thought of as an autonomous tumour suppressor mechanism, is emerging as a phenotype and effector present throughout the life of an organism from embryogenesis to senile decline. Senescent cells have powerful non-autonomous effects upon multiple players within their microenvironment mainly through their secretory phenotype. How senescent cells co-ordinate numerous, sometimes functionally contrasting outputs through their secretome had previously been unclear. The Notch pathway, originally identified for its involvement in Drosophila wing development, has more recently been found to underpin diverse effects in human cancer. Here we discuss recent findings that suggest that Notch is intimately involved in the development of senescence and how it acts to co-ordinate the composition and functional effects of the senescence secretome. We also highlight the complex physical and functional interplay between Notch and p53, critical to both senescence and cancer. Understanding the interplay between Notch, p53 and senescence could allow us develop the therapeutics of the future for cancer and ageing.

Selective insulin resistance in hepatocyte senescence.

Insulin resistance has been described in association with chronic liver disease for decades. Hepatocyte senescence has been demonstrated in chronic liver disease and as many as 80% of hepatocytes show a senescent phenotype in advanced liver disease. The aim of this study was to understand the role of hepatocyte senescence in the development of insulin resistance. Senescence was induced in HepG2 cells via oxidative stress. The insulin metabolic pathway was studied in control and senescent cells following insulin stimulation. GLUT2 and GLUT4 expressions were studied in HepG2 cells and human liver tissue. Further, GLUT2 and GLUT4 expressions were studied in three independent chronic liver disease cohorts. Signalling impairment distal to Akt in phosphorylation of AS160 and FoxO1 was evident in senescent HepG2 cells. Persistent nuclear localisation of FoxO1 was demonstrated in senescent cells despite insulin stimulation. Increased GLUT4 and decreased GLUT2 expressions were evident in senescent cells, human cirrhotic liver tissue and publically available liver disease datasets. Changes in GLUT expressions were associated with a poor clinical prognosis. In conclusion, selective insulin resistance is evident in senescent HepG2 cells and changes in GLUT expressions can be used as surrogate markers of hepatocyte senescence.

Replication-timing boundaries facilitate cell-type and species-specific regulation of a rearranged human chromosome in mouse.

In multicellular organisms, developmental changes to replication timing occur in 400-800 kb domains across half the genome. While examples of epigenetic control of replication timing have been described, a role for DNA sequence in mammalian replication-timing regulation has not been substantiated. To assess the role of DNA sequences in directing developmental changes to replication timing, we profiled replication timing in mice carrying a genetically rearranged Human Chromosome 21 (Hsa21). In two distinct mouse cell types, Hsa21 sequences maintained human-specific replication timing, except at points of Hsa21 rearrangement. Changes in replication timing at rearrangements extended up to 900 kb and consistently reconciled with the wild-type replication pattern at developmental boundaries of replication-timing domains. Our results are consistent with DNA sequence-driven regulation of Hsa21 replication timing during development and provide evidence that mammalian chromosomes consist of multiple independent units of replication-timing regulation.

The clinical value of the hepatic venous pressure gradient in patients undergoing hepatic resection for hepatocellular carcinoma with or without liver cirrhosis.

The role of hepatic venous pressure gradient (HVPG) measurement in risk stratification before liver resection is an ongoing area of debate. This study examines the impact of preoperative HVPG levels on overall survival (OS)/time to recurrence (TTR) and postoperative complications after hepatic resection of hepatocellular carcinoma (HCC). Thirty-eight HCC patients undergoing HVPG measurement before liver resection at Cambridge University Hospitals NHS Foundation Trust between January 2014 and April 2022 were retrospectively analysed. Statistical analysis comprised univariable/multivariable Cox/logistic regression to identify risk factors of reduced OS/TTR or 90-day post-resection complications and Kaplan-Meier estimator, log-rank, chi-squared, Fisher's exact, and Mann-Whitney U test, or Student's t-test for survival/subgroup analysis. The median HPVG was 6 (range: 0-14) mmHg. The HVPG was an independent risk factor for poorer TTR in the overall cohort (cut-off: ≥7.5 mmHg (17.18/43.81 months; P = 0.009)). In the subgroup analysis of cirrhotic patients (N = 29 (76%)), HVPG was additionally an independent risk factor for lower OS (cut-off: ≥8.5 mmHg [44.39/76.84 months; P = 0.012]). The HVPG had no impact on OS/TTR in non-cirrhotic patients (N = 9 (24%)), nor was it associated with postoperative complications in any cohort. In conclusion, preoperative HVPG levels are useful predictors for TTR and OS in cirrhotic HCC patients undergoing hepatic resection.

Cross-talk between ILC2 and Gata3high Tregs locally constrains adaptive type 2 immunity.

Regulatory T cells (Tregs) control adaptive immunity and restrain type 2 inflammation in allergic disease. Interleukin-33 promotes the expansion of tissue-resident Tregs and group 2 innate lymphoid cells (ILC2s); however, how Tregs locally coordinate their function within the inflammatory niche is not understood. Here, we show that ILC2s are critical orchestrators of Treg function. Using spatial, cellular, and molecular profiling of the type 2 inflamed niche, we found that ILC2s and Tregs engage in a direct (OX40L-OX40) and chemotaxis-dependent (CCL1-CCR8) cellular dialogue that enforces the local accumulation of Gata3high Tregs, which are transcriptionally and functionally adapted to the type 2 environment. Genetic interruption of ILC2-Treg communication resulted in uncontrolled type 2 lung inflammation after allergen exposure. Mechanistically, we found that Gata3high Tregs can modulate the local bioavailability of the costimulatory molecule OX40L, which subsequently controlled effector memory T helper 2 cell numbers. Hence, ILC2-Treg interactions represent a critical feedback mechanism to control adaptive type 2 immunity.

Novel immunotherapeutics against LGR5 to target multiple cancer types.

We have developed and validated a highly specific, versatile antibody to the extracellular domain of human LGR5 (α-LGR5). α-LGR5 detects LGR5 overexpression in >90% of colorectal cancer (CRC), hepatocellular carcinoma (HCC) and pre-B-ALL tumour cells and was used to generate an Antibody-Drug Conjugate (α-LGR5-ADC), Bispecific T-cell Engager (α-LGR5-BiTE) and Chimeric Antigen Receptor (α-LGR5-CAR). α-LGR5-ADC was the most effective modality for targeting LGR5+ cancer cells in vitro and demonstrated potent anti-tumour efficacy in a murine model of human NALM6 pre-B-ALL driving tumour attrition to less than 1% of control treatment. α-LGR5-BiTE treatment was less effective in the pre-B-ALL cancer model yet promoted a twofold reduction in tumour burden. α-LGR5-CAR-T cells also showed specific and potent LGR5+ cancer cell killing in vitro and effective tumour targeting with a fourfold decrease in pre-B-ALL tumour burden relative to controls. Taken together, we show that α-LGR5 can not only be used as a research tool and a biomarker but also provides a versatile building block for a highly effective immune therapeutic portfolio targeting a range of LGR5-expressing cancer cells.

γ-H2AX+CD8+ T lymphocytes cannot respond to IFN-α, IL-2 or IL-6 in chronic hepatitis C virus infection.

BACKGROUND & AIMS: Age is the dominant prognostic factor influencing the natural history of hepatitis C virus (HCV) infection and treatment response. Accelerated lymphocyte telomere shortening in HCV infection correlates with adverse clinical outcomes. Critical telomere shortening generates double-stranded DNA breaks (DSB) inducing the DNA damage response, leading to replicative senescence. The phenotype and function of CD8+ T lymphocytes and the in vitro response to IFN-α in relation to the DNA damage response were investigated in patients with chronic HCV infection. METHODS: CD8+ T lymphocytes with DSB were identified by expression of γ-H2AX (Ser-139) in 134 HCV-exposed subjects and 27 controls. Telomere length was determined by flow-FISH; cytokine expression by intracellular cytokine staining; in vitro responses to IFN-α, IL-2 or IL-6 by phospho-STAT1 (Y701) or phospho-STAT5 (Y694) expression. RESULTS: The proportion of circulating CD8+γ-H2AX+ T lymphocytes rose with increasing fibrosis stage (p=0.0023). CD8+γ-H2AX+ T lymphocytes were enriched in liver compared to blood (p=0.03). CD8+γ-H2AX+ T lymphocytes demonstrated increased IFN-γ (p=0.02) and reduced IL-2 expression (p=0.02). CD8+γ-H2AX+ T lymphocytes failed to phosphorylate STAT1 in response to IFN-α compared to unfractionated CD8+ T lymphocytes (p <0.0001). More widespread failure of Jak/Stat signalling in CD8+γ-H2AX+ T lymphocytes was suggested by impaired phosphorylation of STAT1 with IL-6 (p=0.002) and STAT5 with IL-2 (p=0.0039) compared to unfractionated CD8+ T-lymphocytes. CONCLUSIONS: In chronic HCV infection, CD8+γ-H2AX+ T lymphocytes are highly differentiated with shortened telomeres, are more frequent within the liver, are associated with severe fibrosis and fail to activate Jak/Stat pathways in response to IFN-α, IL-2 or IL-6, perhaps explaining treatment failure in those with severe fibrosis.

Autophagy in cancer: having your cake and eating it.

Autophagy, one of two major intracellular degradation pathways, plays a critical role in energy homeostasis and the quality control of macromolecules and intracellular organelles. Previous work has demonstrated the importance of autophagy in maintaining cellular fitness, both in healthy and stressful conditions, revealing the complex interplay between autophagy and other stress-responsive phenotypes. The complex outcomes of stress-responsive autophagy confer on it both pro- and anti-tumourigenic roles, depending on the cellular and environmental context. Furthermore, recent findings that functionally link autophagy to the tumour suppressor mechanism, cellular senescence, have revealed a new role of autophagy in cancer biology. In this review we summarise the current evidence on the relationship between autophagy and cancer, with a focus on its role in senescence.

Breath Biopsy® to Identify Exhaled Volatile Organic Compounds Biomarkers for Liver Cirrhosis Detection.

Background and Aims: The prevalence of chronic liver disease in adults exceeds 30% in some countries and there is significant interest in developing tests and treatments to help control disease progression and reduce healthcare burden. Breath is a rich sampling matrix that offers non-invasive solutions suitable for early-stage detection and disease monitoring. Having previously investigated targeted analysis of a single biomarker, here we investigated a multiparametric approach to breath testing that would provide more robust and reliable results for clinical use. Methods: To identify candidate biomarkers we compared 46 breath samples from cirrhosis patients and 42 from controls. Collection and analysis used Breath Biopsy OMNI™, maximizing signal and contrast to background to provide high confidence biomarker detection based upon gas chromatography mass spectrometry (GC-MS). Blank samples were also analyzed to provide detailed information on background volatile organic compounds (VOCs) levels. Results: A set of 29 breath VOCs differed significantly between cirrhosis and controls. A classification model based on these VOCs had an area under the curve (AUC) of 0.95±0.04 in cross-validated test sets. The seven best performing VOCs were sufficient to maximize classification performance. A subset of 11 VOCs was correlated with blood metrics of liver function (bilirubin, albumin, prothrombin time) and separated patients by cirrhosis severity using principal component analysis. Conclusions: A set of seven VOCs consisting of previously reported and novel candidates show promise as a panel for liver disease detection and monitoring, showing correlation to disease severity and serum biomarkers at late stage.

The senescent secretome drives PLVAP expression in cultured human hepatic endothelial cells to promote monocyte transmigration.

Liver sinusoidal endothelial cells (LSEC) undergo significant phenotypic change in chronic liver disease (CLD), and yet the factors that drive this process and the impact on their function as a vascular barrier and gatekeeper for immune cell recruitment are poorly understood. Plasmalemma-vesicle-associated protein (PLVAP) has been characterized as a marker of LSEC in CLD; notably we found that PLVAP upregulation strongly correlated with markers of tissue senescence. Furthermore, exposure of human LSEC to the senescence-associated secretory phenotype (SASP) led to a significant upregulation of PLVAP. Flow-based assays demonstrated that SASP-driven leukocyte recruitment was characterized by paracellular transmigration of monocytes while the majority of lymphocytes migrated transcellularly. Knockdown studies confirmed that PLVAP selectively supported monocyte transmigration mediated through PLVAP's impact on LSEC permeability by regulating phospho-VE-cadherin expression and endothelial gap formation. PLVAP may therefore represent an endothelial target that selectively shapes the senescence-mediated immune microenvironment in liver disease.