Ageing is associated with a dysregulated immune system that contributes to vulnerability in older adults to infection, malignancies, autoimmune diseases, and inflammatory disorders. This immune dysfunction can be categorised into two processes: progressive decline in immune responsiveness (immunosenescence) and chronic low-grade systemic inflammation (inflammaging). These processes perpetuate a cycle wherein persistent inflammation accelerates immune cell exhaustion and senescence, while diminished immune surveillance heightens inflammation, together promoting tissue damage and age-related disease. The liver, a crucial immune organ pivotal for maintaining systemic immune tolerance, assumes an increasingly prominent role in regulating peripheral immune tolerance as age-related thymic involution diminishes central tolerance. Ageing alters the liver's immune landscape, with diverse patterns of infiltration and structural remodelling marked by the emergence of ageing-related tertiary lymphoid-associated structures (ATLAS), enriched with focal clusters of inflammatory cells. These structures and associated fibrotic niches function as hubs for pro-inflammatory and pro-fibrotic signalling. Transcriptomic studies reveal consistent upregulation of inflammatory immune pathways and pro-inflammatory cytokines across the aged liver. Immune cells are dysregulated with liver macrophages shifting toward pro-inflammatory phenotypes, NK cells showing exhaustion with reduction in frequency and impaired senescent cell clearance. T and B cells accumulate exhausted phenotypes with expanding populations of senescence-associated T cells (SATs) and age-associated B cells (ABCs), respectively. Liver sinusoidal endothelial cells (LSECs) undergo pseudo-capillarization and defenestration, creating a physical barrier that impairs clearance of tissue-adjacent T cells by hepatocytes. Taken together, age-related immune changes in liver immune cells indicate that the liver plays a central role in systemic inflammation in old age.

Clozapine is the most effective antipsychotic drug, while also causing the most severe metabolic side effects. The underlying mechanisms of these side effects appear multifactorial and are not entirely understood. We hypothesized that liver sinusoidal endothelial cells (LSEC) dysfunction is linked to the metabolic side effects of clozapine, based on known association between reduced LSEC porosity, hypertriglyceridemia and insulin resistance. Accordingly, we conducted a translational study by assessing the porosity of murine LSECs following clozapine exposure in vitro and correlating serum clozapine concentrations with metabolic parameters in routine human blood samples. The patient data showed a correlation for clozapine serum concentrations with both triglycerides (Spearman’s ρ = 0.47, P = 0.00024) and glucose levels (ρ = 0.24, P = 0.034), but no correlation with LDL cholesterol (ρ = −0.04, P = 0.82) or HDL cholesterol (ρ = −0.22, P = 0.088). The in vitro experiments showed a concentration-dependent reduction in murine LSEC porosity (P for trend = 0.00036). Together, these findings support a link between clozapine exposure and reduced LSEC porosity. This represents a promising point of intervention that potentially could expand safe access to clozapine as a highly effective antipsychotic treatment for a broader population, also in individuals at high risk for metabolic and cardiovascular side effects.

As the global population ages, research on the biology of ageing and its role in chronic disease is expanding, alongside a growing clinical focus on the unique needs of older adults. In the past, the liver was not thought to undergo substantial age-related changes, nor was there thought to be any liver disease characteristic of older adults. Current studies challenge this perspective, revealing that ageing substantially influences liver pathophysiology at the organ level and within each of the liver cell types. These observations have implications for understanding the pathogenesis of liver diseases common in older adults, including hepatocellular carcinoma, hypoxic hepatitis and metabolic dysfunction-associated steatotic liver disease. Previously, managing older patients with liver disease mostly addressed age-related changes in drug metabolism and liver function tests. However, current clinical practice increasingly emphasizes age-specific issues such as frailty, sarcopenia, multimorbidity and polypharmacy. Given the liver’s pivotal role in systemic metabolism, immunity and detoxification, ageing of the liver can contribute to systemic diseases. In the future, interventions that target ageing biology might offer new treatment options for liver diseases. Here, we review those age-related changes in the liver that have substantial biological and clinical consequences for older adults. Older adults can be affected by multiple chronic medical conditions, including liver disease. This Review provides a comprehensive overview of age-related pathophysiological changes in the liver and discusses interventions and treatment options for older patients.

Ageing is established as the most significant risk factor for disease. About 75% of people over 75 years have diabetes or pre-diabetes and/or hyperlipidaemia which are established risk factors for cardiovascular outcomes, and risk factors for age-related conditions such as dementia, sarcopenia, frailty and osteoporosis. Age-related changes in the liver microcirculation, in particular relating to the cells lining the blood vessels, the liver sinusoidal endothelial cells (LSEC), are a potential cause for dyslipidaemia and insulin resistance in old age. There is also loss of LSEC mediated waste clearance functions essential for homeostasis. Finding ways to reverse these age-related changes in the LSEC will fill a significant gap in therapeutic options available for the treatment of ageing disorders. Such therapies may also benefit patients with fibrotic livers, since LSEC changes in this disease resemble those seen in the ageing LSEC in many aspects. Nanoparticles that access systemic circulation frequently accumulate in the liver. This could be utilized as a promising strategy for targeted drug delivery to the liver. The present study assessed if poly(alkyl-cyanoacrylate) nanoparticles (PACA NPs) are a suitable vector for the targeted transport of such therapeutics to LSEC, to reverse age-related changes such as fenestration/porosity loss. Mice were co-injected with PACA NPs and formaldehyde denatured serum albumin (FSA) and their livers were then examined by microscopy. PACA and FSA co-localised to LSEC, including at the sub-cellular level in endocytic vesicles. Isolated LSEC were challenged with Nile Red (NR668) labelled PACA NPs, which were rapidly internalized. HEK293 cells overexpressing stabilin-2 internalized PACA NPs, suggesting that stabilin-2 mediates PACA uptake on LSEC. Cultured LSEC from aged mice were challenged with PACA NPs containing sildenafil and examined with scanning electron microscopy to determine effects on fenestrations. Sildenafil PACA reversed age-related changes LSEC fenestration frequency and porosity at 3-fold lower sildenafil concentrations than sildenafil alone. If sildenafil PACA induces similar changes in vivo, age-related reduction of LSEC porosity could be reversed by the targeted delivery of sildenafil via PACA NPs.

BACKGROUND:During chronic liver diseases, LSECs undergo a dedifferentiation process contributing to the development of hepatic microvascular dysfunction. Although microRNAs (miRNAs) have been associated with chronic liver disease, their role as modulators of liver endothelial phenotype is mostly unknown. Therefore, the aim of this study was to analyze miRNAs as regulators of hepatic sinusoidal endothelial dysfunction in chronic liver disease to suggest novel and translatable therapeutic options for cirrhosis. METHODS:Global expression of miRNAs was determined in primary LSECs from healthy and cirrhotic patients (alcohol abuse) and rats (CCl4 inhalation). LSECs were transfected with the mimetic or inhibitor of dysregulated miRNAs or with quantum dot nano-complexes containing miR-27b-3p or negative control, and endothelial phenotype was analyzed by RNA sequencing, quantitative PCR, and western blot. Endothelial or mesenchymal phenotypes were analyzed in LSEC by RNA sequencing, followed by pathway analyses and gene deconvolution. RESULTS:In all, 30 and 69 dysregulated miRNAs were identified in human and rat cirrhosis, respectively, of which 6 miRNAs were commonly dysregulated. Specific exogenous downregulation of miR-27b-3p was associated with the upregulation of target genes, suggesting a correlation between loss of miR-27b-3p and LSEC dedifferentiation. Finally, the expression of miR-27b-3p was efficiently and physiologically re-established in cirrhotic LSECs using nano-miR-27b-3p, leading to modulation of 1055 genes compared with the negative control, ultimately leading to inhibition of the endothelial-to-mesenchymal transition process observed in cirrhosis. CONCLUSIONS:Loss of miR-27b-3p expression contributes to LSECs dedifferentiation in cirrhosis. The use of nano-miR-27b-3p represents a new therapeutic option for hepatic diseases coursing with endothelial dysfunction.

The clearance of peripheral beta amyloid (Aβ) is a potential target for the treatment of Alzheimer’s disease (AD). The liver has been implicated in the elimination of Aβ from the peripheral circulation. Here, the single-pass uptake of Aβ40 in perfused livers from young and old rats (6 to 10 rats per group) was investigated with the multiple indicator dilution technique. Aβ40 had volumes of distribution between those of the vascular marker Evans Blue and the extracellular marker sucrose. The hepatic extraction of Aβ40 was negligible, explained in part by the small permeability surface area products consistent with a high endothelial barrier to liver uptake. There were no substantial effects of age on any of these results. In vitro experiments with isolated hepatocytes and liver sinusoidal endothelial cells showed only very small amounts of Aβ uptake consistent with low intrinsic clearance. These results indicate that the hepatic clearance of Aβ is capacity-limited, explained by the low-permeability surface area products and hepatocyte uptake. However, this does not preclude an effect of aging in longer-term in vivo studies where age-related changes in liver blood flow and protein binding influence liver clearance.

Atherogenesis is associated with elevated plasma levels of oxidized low-density lipoproteins (oxLDL). In vivo, oxLDL causes liver endothelial swelling, and disrupts liver sinusoidal endothelial cell (LSECs) fenestrations. We mapped the nanoscale kinetics of these changes in vitro in isolated rat LSECs challenged with oxLDL and monitored viability with endocytosis and cytotoxicity assays. OxLDL disrupted LSEC ultrastructure – increasing oxLDL concentrations and oxidation levels caused sieve plate loss, fenestration fusion, and gap formation. Importantly, these effects were not uniform across all LSECs. LSECs retained the ability to endocytose ligands irrespective of the presence of oxLDL. However, increasing oxidation levels and concentrations of oxLDL inhibited LSEC mediated degradation of endocytosed ligands. Viability was unaffected by any oxLDL challenge. In conclusion, oxLDL disrupts LSEC ultrastructural morphology in vitro but LSECs remain viable and mostly maintain the scavenging function during oxLDL challenge.

Background & AimsLiver sinusoidal endothelial cells (LSECs) are important in liver development, regeneration and pathophysiology, but the differentiation process underlying their tissue-specific phenotype is poorly understood and difficult to study as primary human cells are scarce. The aim of this study was to use human induced pluripotent stem cell (hiPSC)-derived LSEC-like cells to investigate the differentiation process of LSECs.MethodshiPSC-derived endothelial cells were transplanted into the livers of Fah−/−/Rag2−/−/Il2rg−/− mice and assessed over a 12-week period. Lineage tracing, immunofluorescence, flow cytometry, plasma human factor VIII measurement and bulk and single cell transcriptomic analysis were used to assess the molecular and functional changes that occur with transplantation.ResultsProgressive and long-term repopulation of the liver vasculature occurred as iECs expanded along the sinusoids between hepatocytes and increasingly produced human factor VIII, indicating differentiation into LSEC-like cells. To chart the developmental profile associated with LSEC specification, the bulk transcriptome of transplanted cells between 1- and 12-weeks post-transplantation were compared against primary human adult LSECs. This demonstrated a chronological increase in LSEC markers, LSEC differentiation pathways, and zonation. Bulk transcriptome analysis suggested that the transcription factors NOTCH1, GATA4, and FOS play a central role in LSEC specification, interacting with a network of 27 transcription factors. Novel markers associated with this process include EMCN and CLEC14A. Additionally, single cell transcriptomic analysis demonstrated that transplanted iECs at 4-weeks contain zonal subpopulations with a region-specific phenotype.ConclusionsCollectively, this study confirms that hiPSC can adopt LSEC-like features and provides insight into LSEC specification. This humanised xenograft system can be applied to further interrogate LSEC developmental biology and pathophysiology, bypassing current logistical obstacles associated with primary human LSECs.Impact And ImplicationsLiver sinusoidal endothelial cells (LSECs) are important cells for liver biology, but better model systems are required to study them. We present a pluripotent stem cell xenografting model which produces human LSEC-like cells. A detailed and longitudinal transcriptomic analysis of the development of LSEC-like cells is included, which will guide future studies to interrogate LSEC biology and produce LSEC-like cells which could be used for regenerative medicine.

Unraveling the complex interplay between nutrients and drugs via their effects on "omics" features could revolutionize our fundamental understanding of nutritional physiology, personalized nutrition, and, ultimately, human health span. Experimental studies in nutrition are starting to use large-scale "omics" experiments to pick apart the effects of such interacting factors. However, the high dimensionality of the omics features, coupled with complex fully factorial experimental designs, poses a challenge to the analysis. Current strategies for analyzing such types of data are based on between-feature correlations. However, these techniques risk overlooking important signals that arise from the experimental design and produce clusters that are hard to interpret. We present a novel approach for analyzing high-dimensional outcomes in nutriomics experiments, termed experiment-guided NutriOmics DatA cLustering ('eNODAL'). This three-step hybrid framework takes advantage of both Analysis of Variance (ANOVA)-type analyses and unsupervised learning methods to extract maximum information from experimental nutriomics studies. First, eNODAL categorizes the omics features into interpretable groups based on the significance of response to the different experimental variables using an ANOVA-like test. Such groups may include the main effects of a nutritional intervention and drug exposure or their interaction. Second, consensus clustering is performed within each interpretable group to further identify subclusters of features with similar response profiles to these experimental factors. Third, eNODAL annotates these subclusters based on their experimental responses and biological pathways enriched within the subcluster. We validate eNODAL using data from a mouse experiment to test for the interaction effects of macronutrient intake and drugs that target aging mechanisms in mice.

Background and Aim: Orengedokuto is a traditional herbal medicine used in Japan to treat conditions such as dermatitis, gastric ulcers, and gastritis. Previously, orengedokuto given in combination with chemotherapy to patients with leukemia reduced the incidence of chemotherapy-induced diarrhea, highlighting a potential preventive effect on intestinal mucositis. This study sought to determine if orengedokuto could reduce the severity of mucositis in a mouse model induced by the commonly prescribed antimetab-olite chemotherapy drug 5-fl uorouracil (5-FU). Methods: Female Balb/c mice ( n = 60) were injected (intraperitoneal; Day 0) with either saline or 5-FU (200 mg/kg). Mice ( n = 10/group) were gavaged daily with water (160 μ L) or orengedokuto (0.5 mg/kg or 1 g/kg) for 4 days. Disease activity was monitored daily via the disease activity index (DAI; comprising stool consistency, rectal bleeding, weight loss, and general condition) and, on alternate days, by behavioral analyses (facial grimace and burrowing activity). Visceral organ weights and lengths were determined after euthanasia on Day 4. Small intestinal mucosal architecture (villus height and crypt depth

Figure S1 Dose response of CD5-2 in B16F10 tumor model Figure S2 CD5-2 penetrates into endothelial cells but has no effect on proliferation, senescence and migration in vitro. Figure S3 CD5-2 does not affect tumor cell proliferation, necrosis, senescence and number of leukocytes infiltrating into the B16F10 tumor. Figure S4 CD5-2 has effects on CD8+ T cells but no effect on CD4+ T cells or NKp46+ NK cells in the MC38 tumor. Figure S5 CD5-2 increases VE-cadherin expression and regulates tight junctions pathway. Figure S6 schematic diagram of proposed effects of CD5-2 on tumor microenvironment.

In vitro models of liver (patho)physiology, new technologies, and experimental approaches are progressing rapidly. Based on cell lines, induced pluripotent stem cells or primary cells derived from mouse or human liver as well as whole tissue (slices), such in vitro single- and multicellular models, including complex microfluidic organ-on-a-chip systems, provide tools to functionally understand mechanisms of liver health and disease. The International Society of Hepatic Sinusoidal Research (ISHSR) commissioned this working group to review the currently available in vitro liver models and describe the advantages and disadvantages of each in the context of evaluating their use for the study of liver functionality, disease modeling, therapeutic discovery, and clinical applicability.

Xanthines such as caffeine and theobromine are among the most consumed psychoactive stimulants in the world, either as natural components of coffee, tea and chocolate, or as added ingredients. The present study assessed if xanthines affect liver sinusoidal endothelial cells (LSEC). Cultured primary rat LSEC were challenged with xanthines at concentrations typically obtained from normal consumption of xanthine-containing beverages, food or medicines; and at higher concentrations below the in vitro toxic limit. The fenestrated morphology of LSEC were examined with scanning electron and structured illumination microscopy. All xanthine challenges had no toxic effects on LSEC ultrastructure as judged by LSEC fenestration morphology, or function as determined by endocytosis studies. All xanthines in high concentrations (150 μg/mL) increased fenestration frequency but at physiologically relevant concentrations, only theobromine (8 μg/mL) showed an effect. LSEC porosity was influenced only by high caffeine doses which also shifted the fenestration distribution towards smaller pores. Moreover, a dose-dependent increase in fenestration number was observed after caffeine treatment. If these compounds induce similar changes in vivo, age-related reduction of LSEC porosity can be reversed by oral treatment with theobromine or with other xanthines using targeted delivery.

Figure S1 Dose response of CD5-2 in B16F10 tumor model Figure S2 CD5-2 penetrates into endothelial cells but has no effect on proliferation, senescence and migration in vitro. Figure S3 CD5-2 does not affect tumor cell proliferation, necrosis, senescence and number of leukocytes infiltrating into the B16F10 tumor. Figure S4 CD5-2 has effects on CD8+ T cells but no effect on CD4+ T cells or NKp46+ NK cells in the MC38 tumor. Figure S5 CD5-2 increases VE-cadherin expression and regulates tight junctions pathway. Figure S6 schematic diagram of proposed effects of CD5-2 on tumor microenvironment.

Supplemental- Material and Methods