An easy-to-perform method for microvessel isolation and primary brain endothelial cell culture to study Alzheimer's disease.

Dysfunction of the blood-brain barrier (BBB) has been increasingly recognised as a critical early event in Alzheimer's disease (AD) pathophysiology. Central to this mechanism is the impaired function of brain endothelial cells (BECs), the primary structural constituents of the BBB, the study of which is imperative for understanding AD pathophysiology. However, the published methods to isolate BECs are time-consuming and have a low success rate. Here, we developed a rapid and streamlined protocol for BEC isolation without using transgenic reporters, flow cytometry, and magnetic beads, which are essential for existing methods. Using this novel protocol, we isolated high-purity BECs from cell clusters of cortical microvessels from wild-type and APPswe/PS1dE9 (APP/PS1, a classical AD model) mice at 2, 4 and 9 months of age. Reduced levels of tight junction proteins Claudin-5 and Zonula Occludens-1, as well as glucose transporter 1, were observed in the isolated cortical microvessels from APP/PS1 mice and amyloid-ß (Aß) oligomer-treated BECs from wild-type mice. Trans-well permeability assay showed increased FITC-dextran leakage in BECs treated with Aß, suggesting impaired BBB permeability. BECs obtained using our novel protocol can undergo various experimental analyses, including immunofluorescence staining, western blotting, real-time PCR, and trans-well permeability assay. In conclusion, our novel protocol represents a reliable and valuable tool for in vitro modelling BBB to study AD-related mechanisms and develop targeted therapeutic strategies.

Mitochondria-containing extracellular vesicles from mouse vs. human brain endothelial cells for ischemic stroke therapy.

Ischemic stroke-induced mitochondrial dysfunction in the blood-brain barrier-forming brain endothelial cells (BECs) results in long-term neurological dysfunction post-stroke. We previously reported data from a pilot study where intravenous administration of human BEC (hBEC)-derived mitochondria-containing extracellular vesicles (EVs) showed a potential efficacy signal in a mouse middle cerebral artery occlusion (MCAo) model of stroke. We hypothesized that EVs harvested from donor species homologous to the recipient species (e.g., mouse) may improve therapeutic efficacy, and therefore, use of mouse BEC (mBEC)-derived EVs may improve post-stroke outcomes in MCAo mice. We investigated potential differences in the mitochondria transfer of EVs derived from the same species as the recipient cell (mBEC-EVs and recipient mBECs or hBECs-EVs and recipient hBECs) vs. cross-species EVs and recipient cells (mBEC-EVs and recipient hBECs or vice versa). Our results showed that while both hBEC- and mBEC-EVs transferred EV mitochondria, mBEC-EVs outperformed hBEC-EVs in increasing ATP levels and improved recipient mBEC mitochondrial function via increasing oxygen consumption rates. mBEC-EVs significantly reduced brain infarct volume and neurological deficit scores compared to vehicle-injected MCAo mice. The superior therapeutic efficacy of mBEC-EVs in MCAo mice support the continued use of mBEC-EVs to optimize the therapeutic potential of mitochondria-containing EVs in preclinical mouse models.

Epigenetic regulation in liver regeneration.

The liver is considered unique in its enormous capacity for regeneration and self-repair. In contrast to other regenerative organs (i.e., skin, skeletal muscle, and intestine), whether the adult liver contains a defined department of stem cells is still controversial. In order to compensate for the massive loss of hepatocytes following liver injury, the liver processes a precisely controlled transcriptional reprogram that can trigger cell proliferation and cell-fate switch. Epigenetic events are thought to regulate the organization of chromatin architecture and gene transcription during the liver regenerative process. In this review, we will summarize how changes to the chromatin by epigenetic modifiers are translated into cell fate transitions to restore liver homeostasis during liver regeneration.

Spatially segregated defects and IGF1-responsiveness of hilar and peripheral biliary organoids from a model of Alagille syndrome.

BACKGROUND & AIMS: Alagille syndrome (ALGS) manifests with peripheral intrahepatic bile duct (IHBD) paucity, which can spontaneously resolve. In a model for ALGS, Jag1Ndr/Ndr mice, this occurs with distinct architectural mechanisms in hilar and peripheral IHBDs. Here, we investigated region-specific IHBD characteristics and addressed whether IGF1, a cholangiocyte mitogen that is downregulated in ALGS and in Jag1Ndr/Ndr mice, can improve biliary outcomes. METHODS: Intrahepatic cholangiocyte organoids (ICOs) were derived from hilar and peripheral adult Jag1+/+ and Jag1Ndr/Ndr livers (hICOs and pICOs, respectively). ICOs were grown in Matrigel or microwell arrays, and characterized using bulk RNA sequencing, immunofluorescence, and high throughput analyses of nuclear sizes. ICOs were treated with IGF1, followed by analyses of growth, proliferation, and death. CellProfiler and Python scripts were custom written for image analyses. Key results were validated in vivo by immunostaining. RESULTS: Cell growth assays and transcriptomics demonstrated that Jag1Ndr/Ndr ICOs were less proliferative than Jag1+/+ ICOs. IGF1 specifically rescued survival and growth of Jag1Ndr/Ndr pICOs. Jag1Ndr/Ndr hICOs were the least proliferative, with lower Notch signalling and an enrichment of hepatocyte signatures and IGF uptake/transport pathways. In vitro (Jag1Ndr/Ndr hICOs) and in vivo (Jag1Ndr/Ndr hilar portal tracts) analyses revealed ectopic HNF4a+ hepatocytes. CONCLUSIONS: Hilar and peripheral Jag1Ndr/Ndr ICOs exhibit differences in Notch signalling status, proliferation, and cholangiocyte commitment which may result in cholangiocyte-to-hepatocyte transdifferentiation. While Jag1Ndr/Ndr pICOs can be rescued by IGF1, hICOs are unresponsive, perhaps due to their hepatocyte-like state and/or expression of IGF transport components. IGF1 represents a potential therapeutic for peripheral bile ducts.

Relining of a Bridging Covered Stent to Treat a Rare Cause of Type IIIc Endoleak Following Fenestrated Endovascular Aneurysm Repair.

PURPOSE: To present a rare cause of type III endoleak via the left renal artery (LRA) fenestration following fenestrated endovascular aneurysm repair (FEVAR) and to describe a successful reintervention for treating this endoleak. TECHNIQUE: The patient presented with a type IIIc endoleak following FEVAR, due to inadvertent placement of the LRA bridging balloon expandable covered stent (BECS) via the superior mesenteric artery (SMA) fenestration, but deployed outside the SMA fenestration. The proximal part of the BECS was positioned outside of the main body. This caused a type IIIc endoleak via the open LRA fenestration. Reintervention was performed by relining the LRA with a new BECS. First, access to the lumen of the previously placed BECS was gained using a re-entry catheter, followed by placement of a new BECS via the LRA fenestration. Completion angiography, and computerized tomography angiography (CTA) at 3 months follow-up showed total obliteration of the endoleak and patency of the LRA. CONCLUSION: Placement of a bridging stent via an incorrect fenestration during FEVAR is a rare cause of type III endoleak. In certain cases, successful treatment of such an endoleak could be achieved by perforation and relining of the misplaced BECS via the correct fenestration of the target vessel. CLINICAL IMPACT: To our best knowledge, a type IIIc endoleak following fenestrated endovascular aneurysm repair, due to placement of a bridging covered stent through an incorrect fenestration and deployed short of the fenestration, has not been described before. Reintervention was performed with perforation of the previously placed covered stent and relining using a new bridging covered stent. The technique presented here was successful for treating the endoleak in this case and could help guide clinicians when dealing with this or similar complications.

Hepatic lipid overload triggers biliary epithelial cell activation via E2Fs.

During severe or chronic hepatic injury, biliary epithelial cells (BECs) undergo rapid activation into proliferating progenitors, a crucial step required to establish a regenerative process known as ductular reaction (DR). While DR is a hallmark of chronic liver diseases, including advanced stages of non-alcoholic fatty liver disease (NAFLD), the early events underlying BEC activation are largely unknown. Here, we demonstrate that BECs readily accumulate lipids during high-fat diet feeding in mice and upon fatty acid treatment in BEC-derived organoids. Lipid overload induces metabolic rewiring to support the conversion of adult cholangiocytes into reactive BECs. Mechanistically, we found that lipid overload activates the E2F transcription factors in BECs, which drive cell cycle progression while promoting glycolytic metabolism. These findings demonstrate that fat overload is sufficient to reprogram BECs into progenitor cells in the early stages of NAFLD and provide new insights into the mechanistic basis of this process, revealing unexpected connections between lipid metabolism, stemness, and regeneration.

Apoptotic biliary epithelial cells and gut dysbiosis in the induction of murine primary biliary cholangitis.

Primary biliary cholangitis (PBC) is a female-predominant liver autoimmune disease characterized by the specific immune-mediated destruction of the intrahepatic small bile duct. Although apoptosis of biliary epithelial cells (BECs) and alterations in gut microbiota are observed in patients with PBC, it is still unclear whether these events happen in the early stage and cause the breakdown of tolerance in PBC. In this study, we examined the early events in the loss of tolerance in our well-defined 2-OA-OVA-induced murine autoimmune cholangitis (AIC) model. We report herein that apoptosis of BECs was notable in the early stage of murine AIC. An altered gut microbiota, in particular, an increased percentage of gram-positive Firmicutes in AIC mice was also observed. BECs in AIC mice expressed adhesion molecule ICAM-1, cytokines/chemokines TNF-α, CCL2, CXCL9, CXCL10, and toll-like receptor (TLR) 2. Moreover, BECs treated with TLR2 ligand had elevated apoptosis and CXCL10 production. These data collectively suggest a new mechanism of tolerance breakdown in AIC. Altered gut microbiota induces apoptosis of BECs through TLR2 signaling. BECs secrete chemokines to recruit CD8 T cells to damage BECs further.

Clonorchis sinensis infection induces hepatobiliary injury via disturbing sphingolipid metabolism and activating sphingosine 1-phosphate receptor 2.

Clonorchis sinensis (C. sinensis) infection induces severe hepatobiliary injuries, which can cause inflammation, periductal fibrosis, and even cholangiocarcinoma. Sphingolipid metabolic pathways responsible for the generation of sphingosine-1-phosphate (S1P) and its receptor S1P receptors (S1PRs) have been implicated in many liver-related diseases. However, the role of S1PRs in C. sinensis-mediated biliary epithelial cells (BECs) proliferation and hepatobiliary injury has not been elucidated. In the present study, we found that C. sinensis infection resulted in alteration of bioactive lipids and sphingolipid metabolic pathways in mice liver. Furthermore, S1PR2 was predominantly activated among these S1PRs in BECs both in vivo and in vitro. Using JTE-013, a specific antagonist of S1PR2, we found that the hepatobiliary pathological injuries, inflammation, bile duct hyperplasia, and periductal fibrosis can be significantly inhibited in C. sinensis-infected mice. In addition, both C. sinensis excretory-secretory products (CsESPs)- and S1P-induced activation of AKT and ERK1/2 were inhibited by JTE-013 in BECs. Therefore, the sphingolipid metabolism pathway and S1PR2 play an important role, and may serve as potential therapeutic targets in hepatobiliary injury caused by C. sinensis-infection.

Functional Expression of Multidrug-Resistance (MDR) Transporters in Developing Human Fetal Brain Endothelial Cells.

There is little information about the functional expression of the multidrug resistance (MDR) transporters P-glycoprotein (P-gp, encoded by ABCB1) and breast cancer resistance protein (BCRP/ABCG2) in the developing blood−brain barrier (BBB). We isolated and cultured primary human fetal brain endothelial cells (hfBECs) from early and mid-gestation brains and assessed P-gp/ABCB1 and BCRP/ABCG2 expression and function, as well as tube formation capability. Immunolocalization of the von Willebrand factor (marker of endothelial cells), zonula occludens-1 and claudin-5 (tight junctions) was detected in early and mid-gestation-derived hfBECs, which also formed capillary-like tube structures, confirming their BEC phenotype. P-gp and BCRP immunostaining was detected in capillary-like tubes and in the cytoplasm and nucleus of hfBECs. P-gp protein levels in the plasma membrane and nuclear protein fractions, as well as P-gp protein/ABCB1 mRNA and BCRP protein levels decreased (p < 0.05) in hfBECs, from early to mid-gestation. No differences in P-gp or BCRP activity in hfBECs were observed between the two age groups. The hfBECs from early and mid-gestation express functionally competent P-gp and BCRP drug transporters and may thus contribute to the BBB protective phenotype in the conceptus from early stages of pregnancy.

Impact of chronic stress exposure on cognitive performance incorporating the active and healthy aging (AHA) concept within the cross-sectional Bern Cohort Study 2014 (BeCS-14).

PURPOSE: This study aims to verify that the mental-cognitive domain of the validated generic bio-functional status (BFS)/bio-functional age (BFA) assessment tool, incorporating the concept of Active and Healthy Ageing (AHA), reflects cognitive performance. In addition, the effects of chronic stress exposure on the mental-cognitive BFS/BFA should be investigated. METHODS: The study was carried out as a monocenter, cross-sectional, observational, non-interventional trial (Bern Cohort Study 2014, BeCS-14) with the participation of 147 non-pediatric, non-geriatric subjects. All participants followed a standardized battery of biopsychosocial assessments consisting of BFS/BFA, a validated cognitive performance test battery (Inventar zur Gedächtnisdiagnostik; IGD) and a validated questionnaire for the assessment of chronic stress (Trier Inventory for the assessment of Chronic Stress; TICS), respectively. RESULTS: Mean cognitive performance was average and higher in younger or better educated individuals. The BFA of the participants was 7.8 ± 7.8 year-equivalents below their chronological age. The mental-cognitive BFS/BFA assessment correlated well with the validated questionnaire for cognition assessment, the IGD. Further, three TICS subdomains (work overload (r = - 0.246, p = 0.003), work discontent (r = - 0.299, p = 0.006) and pressure to succeed (r = - 0.274, p < 0.001)), reflecting mainly work-related stress, showed a significant negative correlation with the mental-cognitive BFS/BFA. CONCLUSIONS: Our study shows that the BFS/BFA assessment tool follows European Innovation Partnership on Active and Healthy Ageing (EIP-AHA) requirements. Further, we could demonstrate that higher levels of chronic work-related stress may be associated with poorer mental-cognitive performance and a pro-aging state indicating that cognitive impairments can be reduced by stress management interventions.

Geo-epidemiology and environmental co-variate mapping of primary biliary cholangitis and primary sclerosing cholangitis.

BACKGROUND & AIMS: Autoimmune liver disease (AILD) is thought to result from a complex interplay between genetics and the environment. Studies to date have focussed on primary biliary cholangitis (PBC) and demonstrated higher disease prevalence in more urban, polluted, and socially deprived areas. This study utilises a large cohort of patients with PBC and primary sclerosing cholangitis (PSC) to investigate potential environmental contributors to disease and to explore whether the geo-epidemiology of PBC and PSC are disease-specific or pertain to cholestatic AILD in general. METHODS: All adult patients with PBC and PSC in a tightly defined geographical area within the UK were identified. Point- and area-based analyses and structural equation modelling (SEM) were used to investigate for disease clustering and examine for relationships between prevalence, distribution of environmental contaminants, and socio-economic status. RESULTS: We identified 2,150 patients with PBC and 472 with PSC. Significant spatial clustering was seen for each disease. A high prevalence of PBC was found in urban, post-industrial areas with a strong coal-mining heritage and increased environmental cadmium levels, whereas a high PSC prevalence was found in rural areas and inversely associated with social deprivation. CONCLUSIONS: This study demonstrates spatial clustering of PBC and PSC and adds to our understanding of potential environmental co-variates for both diseases. Disease clustering, within the same geographical area but over different scales, is confirmed for each disease with distinct risk profiles identified and associations with separate putative environmental factors and socio-economic status. This suggests that different triggers and alternative pathways determine phenotypic expression of autoimmunity in the affected population. Co-variate analysis points towards the existence of specific disease triggers. LAY SUMMARY: This study looked for potential environmental triggers in patients with primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) living in the north-east of England and north Cumbria. We found that PBC was more common in urban areas with a history of coal mining and high levels of cadmium whereas PSC was more common in rural areas with lower levels of social deprivation.

Non-histone nuclear protein HMGN2 differently regulates the urothelium barrier function by altering expression of antimicrobial peptides and tight junction protein genes in UPEC J96-infected bladder epithelial cell monolayer.

The urinary tract is vulnerable to frequent challenges from environmental microflora. Uropathogenic Escherichia coli (UPEC) makes a major contribution to urinary tract infection (UTI). Previous studies have characterized positive roles of non-histone nuclear protein HMGN2 in lung epithelial innate immune response. In the study presented here, we found HMGN2 expression was up-regulated in UPEC J96-infected urothelium. Surprisingly, over-expression of HMGN2 promoted disruption of BECs 5637 cells' intercellular junctions by down-regulating tight junction (TJs) components' expression and physical structure under J96 infection. Further investigation showed that BECs 5637 monolayer, in which HMGN2 was over-expressed, had significantly increased permeability to J96. Our study systemically explored the regulatory roles of HMGN2 in BECs barrier function during UPEC infection and suggested different modulations of intracellular and paracellular routes through which UPEC invades the bladder epithelium.

The multiple antibacterial activities of the bladder epithelium.

The urinary tract is subject to frequent challenges from the gut microflora. Indeed, up to 40% of women will experience at least one urinary tract infection (UTI) during their lifetime. Uropathogenic Escherichia coli (UPEC) contribute to an overwhelming majority of these cases and they typically initiate UTIs by invading the superficial epithelium that lines the bladder lumen. In addition to serving as an effective barrier to noxious agents found in urine, bladder epithelial cells (BECs) play a key physiological role in regulating bladder volume to accommodate urine flow. UPEC appear to coopt this latter property to circumvent this normally impregnable epithelial barrier. However, in spite of this shortcoming, recent studies suggest that BECs possess several immune mechanisms to combat bacterial invasion including expulsion of invading bacteria back into the bladder lumen following infection. These antibacterial activities of BECs are triggered and coordinated by sensory molecules located on the epithelial cell membrane and within the cells. Although, they are the primary targets of microbial attack, BECs appear to be equipped with a diverse repertoire of defense schemes to fend off many of these microbial challenges.

Alpha-naphthylisothiocyanate modulates hepatobiliary transporters in sandwich-cultured rat hepatocytes.

Alpha-naphthylisothiocyanate (ANIT) induces intra-hepatic cholestasis mixed with hepatocellular injury mainly by bile ductular damage. However, its direct effect on hepatic parenchymal cells (hepatocytes) is unclear. Sandwich-cultured rat hepatocytes (SCRH) were applied to clarify this question. Though cytotoxicity was not observed (0-180 µM) in ANIT-treated SCRH, metabonomics analysis of the hepatocytes revealed a shift in the metabolic pattern and a decrease in cellular cholesterol level, accompanied by an increase in total bile acids after 48 h ANIT (5-45 µM) treatment. To assess the function of major hepatic bile acid transporters, the accumulation and efflux of [D-Pen(2,5)]-enkephalin (DPDPE), 5 (and 6)-carboxy-2',7'-dichlorofluorescein (CDF) diacetate promoiety and deuterium-labeled sodium taurocholate (d8-TCA) were measured. ANIT incubation for either 30 min or 48 h led to dose-dependent decreases in the biliary excretion index (BEI) of DPDPE and CDF, as well as the intracellular accumulation of d8-TCA, CDF and DPDPE. The basolateral efflux of d8-TCA was also decreased with its BEI barely changed. mRNA expression of multiple uptake transporters and bile acid synthesizing enzymes was down-regulated after 48 h incubation. In conclusion, ANIT could directly induce retention of bile acids in hepatocytes by inhibiting the function of bile acid transporters, which might contribute to its cholestatic effect.

Vitamin D in autoimmune liver disease.

The development of autoimmune disease is based on the interaction of genetic susceptibility and environmental causes. Environmental factors include infectious and non-infectious agents, with some of these factors being implicated in several autoimmune diseases. Vitamin D is now believed to play a role in the development (or prevention) of several autoimmune diseases, based on its immunomodulatory properties. As well, the increasing incidence of autoimmune disease as one moves away from the equator, may be due to the lack of sunlight, which is crucial for the maintenance of normal vitamin D levels. A deficiency in vitamin D levels or vitamin D receptors is commonly indicated in autoimmune diseases, with multiple sclerosis (MS) being one of the best-studied and well-known examples. However, the role of vitamin D in other autoimmune diseases is not well defined, including autoimmune liver diseases such as primary biliary cirrhosis, autoimmune hepatitis, and primary sclerosing cholangitis. This review will examine the role of vitamin D as an immunomodulator, followed by a comparison of vitamin D in MS versus autoimmune liver disease. From this comparison, it will become clear that vitamin D likely plays a role in the development of autoimmune liver disease, but this area requires further investigation.