Procoagulant phenotype of virus-infected pericytes is associated with portal thrombosis and intrapulmonary vascular dilations in fatal COVID-19.

BACKGROUND & AIMS: Mechanisms and clinical impact of portal microthrombosis featuring severe COVID-19 are unknown. Intrapulmonary vascular dilation (IPVD)-related hypoxia has been described in severe liver diseases. We hypothesized that portal microthrombosis is associated with IPVD and fatal respiratory failure in COVID-19. METHODS: Ninety-three patients who died from COVID-19, were analysed for portal microvascular damage (histology), IPVD (histology and chest-computed tomography, CT), and hypoxemia (arterial blood gas). Seventeen patients who died from COVID-19-unrelated pneumonia served as controls. Vascular lesions and microthrombi were phenotyped for endothelial (vWF) and pericyte (αSMA/PDGFR-ß) markers, tissue factor (TF), viral spike-protein and nucleoprotein (SP, NP), fibrinogen, platelets (CD41a). Viral particles in vascular cells were assessed by transmission electron microscopy (TEM). Cultured pericytes were infected with SARS-CoV-2 to measure TF expression and tubulisation of human pulmonary microvascular endothelial cells (HPMEC) was assessed upon vWF treatment. RESULTS: IPVD was present in 16/66 COVID-19 patients with both liver and lung histology, with a younger age (62 vs 78yo), longer illness (25 vs 14 days), worsening hypoxemia (PaO2/FiO2 from 209 to 89), and more ventilatory support (63 vs 22%) compared to COVID-19/Non-IPVD. IPVD, absent in controls, were confirmed by chest-CT. COVID-19/IPVD liver histology showed portal microthrombosis in >82.5% of portal areas, with a thicker wall of αSMA/PDGFR-ß+/ SP+/NP+ pericytes compared with COVID-19/Non-IPVD. Thrombosed portal venules correlated with αSMA+ area, whereas infected SP+/NP+ pericytes expressed TF. SARS-CoV-2 viral particles were observed in portal pericytes. In-vitro SARS-CoV-2 infection of pericytes up-regulated TF and induced endothelial cells to overexpress vWF, which expanded HPMEC tubules. CONCLUSIONS: SARS-CoV-2 infection of liver pericytes elicits a local procoagulant response associated with extensive portal microthrombosis, IPVD and worsening respiratory failure in fatal COVID-19. IMPACT AND IMPLICATIONS: Vascular involvement of the liver represents a serious complication of COVID-19 infection that must be considered in the work-up of patients with long-lasting and progressively worsening respiratory failure, as it may associate with the development of intrapulmonary vascular dilations. This clinical picture is associated with a pro-coagulant phenotype of portal venule pericytes, which is induced by SARS-CoV-2 infection of pericytes. Both observations provide a model that may apply, at least in part, to other vascular disorders of the liver, featuring obliterative portal venopathy, similarly characterized at the clinical level by development of hypoxemia and at the histological level, by phlebosclerosis and reduced caliber of the portal vein branches in the absence of cirrhosis. Moreover, our findings bring light to an as yet overlooked player of thrombosis pathophysiology, i.e. pericytes, which may provide novel therapeutic tools to halt prothrombotic mechanisms.

The Neglected Role of Bile Duct Epithelial Cells in NASH.

Nonalcoholic fatty liver disease (NAFLD) is the most prevalent liver disease worldwide, and affects 25% of the population in Western countries. NAFLD is the hepatic manifestation of the metabolic syndrome, linked to insulin resistance, which is the common pathogenetic mechanism. In approximately 40% of NAFLD patients, steatosis is associated with necro-inflammation and fibrosis, resulting in nonalcoholic steatohepatitis (NASH), a severe condition that may progress to cirrhosis and liver cancer. Although the hepatocyte represents the main target of the disease, involvement of the bile ducts occurs in a subset of patients with NASH, and is characterized by ductular reaction and activation of the progenitor cell compartment, which incites portal fibrosis and disease progression. We aim to dissect the multiple biological effects that adipokines and metabolic alterations exert on cholangiocytes to derive novel information on the mechanisms driven by insulin resistance, which promote fibro-inflammation and carcinogenesis in NASH.

NFkappaB, cytokines, TLR 3 and 7 expression in human end-stage HCV and alcoholic liver disease.

BACKGROUND/AIMS: Conflicting observations exist concerning the role of nuclear factor kappa B (NFjB) in alcoholic liver disease (ALD) in animal models. To date no studies have examined this aspect in human liver tissue. We here assessed cytokines and toll-like receptors (TLRs) expressions in conjunction with NFkappaB activation in non-active end-stage human ALD compared with normal livers and hepatitis C virus (HCV) related end-stage disease. METHODS: mRNA and protein expression were examined by quantitative PCR and Western blotting, DNA-binding by electrophoretic mobility shift assays and NFkappaB sub-cellular localization by immunofluorescent staining of livers. RESULTS: NFkappaB mRNA and protein expression as well as strong DNA-binding were preserved in ALD but significantly down-regulated in HCV compared with normal livers. P50 immunofluorescence was found in hepatocytes and bile ducts in ALD and normal livers, whereas a shift was observed in p65 staining from non-parenchymal cells in normal livers to hepatocytes in ALD. NFkappaB responsive genes mRNA levels IkBalpha and interleukin 6 were significantly higher in ALD compared with HCV. Tumour necrosis factor alpha (TNFalpha), TLRs 3 and 7 mRNA were up-regulated in ALD and HCV compared with normal liver with TNFalpha and TLR7 being the highest in HCV. Strong induction of interferon beta was found in HCV but not in ALD or normal liver tissue. CONCLUSIONS: Persistent NFkappaB activation together with high pro-inflammatory cytokine expression and upregulation of TLR3 and TLR7 is associated with end-stage ALD in humans and could contribute to disease progression even in absence of alcohol intake.

Human haematopoietic stem cells express Oct4 pseudogenes and lack the ability to initiate Oct4 promoter-driven gene expression.

The transcription factor Oct4 is well defined as a key regulator of embryonic stem (ES) cell pluripotency. In recent years, the role of Oct4 has purportedly extended to the self renewal and maintenance of multipotency in adult stem cell (ASC) populations. This profile has arisen mainly from reports utilising reverse transcription-polymerase chain reaction (RT-PCR) based methodologies and has since come under scrutiny following the discovery that many developmental genes have multiple pseudogenes associated with them. Six known pseudogenes exist for Oct4, all of which exhibit very high sequence homology (three >97%), and for this reason the generation of artefacts may have contributed to false identification of Oct4 in somatic cell populations. While ASC lack a molecular blueprint of transcription factors proposed to be involved with 'stemness' as described for ES cells, it is not unreasonable to assume that similar gene patterns may exist. The focus of this work was to corroborate reports that Oct4 is involved in the regulation of ASC self-renewal and differentiation, using a combination of methodologies to rule out pseudogene interference. Haematopoietic stem cells (HSC) derived from human umbilical cord blood (UCB) and various differentiated cell lines underwent RT-PCR, product sequencing and transfection studies using an Oct4 promoter-driven reporter. In summary, only the positive control expressed Oct4, with all other cell types expressing a variety of Oct4 pseudogenes. Somatic cells were incapable of utilising an exogenous Oct4 promoter construct, leading to the conclusion that Oct4 does not appear involved in the multipotency of human HSC from UCB.

Tissue engineering of human biliary epithelial cells on polyglycolic acid/polycaprolactone scaffolds maintains long-term phenotypic stability.

The biliary tree is the target of damage in a number of important liver diseases. Although human biliary epithelial cells (hBECs) can be maintained in vitro for up to 8 weeks, using double-collagen gels, which offer a substantial improvement compared with conventional tissue culture plastic, such gels are unstable and, being only semisolid, they do not lend themselves readily to routine analysis. In this study we have investigated the behavior of primary hBECs on polyglycolic acid (PGA) fiber mesh scaffolds. Experiments showed that PGA fiber mesh scaffolds collapsed after 3 or 4 weeks; hence, in order to improve the integrity of the construct, we also developed a polycaprolactone (PCL)-stabilized PGA scaffold. Cells formed spheroidal aggregates while continuing to proliferate long term and expressing phenotypic stability. Aggregates spontaneously detached from the fibers and could either be left to attach to tissue culture plastic, after which cells spread out and continued to proliferate, or they could be reseeded onto fresh constructs, which then became recolonized and the same pattern of tissue formation was repeated. This behavior was observed even after 6 months and is of major significance because this culture model could therefore be used as a longterm strategy for growing, expanding, and exploiting hBECs for subsequent studies of bile duct morphogenesis and tissue engineering of artificial bile ducts.

A novel cell-surface marker found on human embryonic hepatoblasts and a subpopulation of hepatic biliary epithelial cells.

The nature of the cells that contribute to the repopulation of the liver after hepatic necrosis or cirrhosis remains uncertain, in part because we lack specific markers to facilitate identification and prospective isolation of progenitor cells. The monoclonal antibody GCTM-5 reacts with a minority subpopulation of cells in spontaneously differentiating cultures of pluripotent human embryonal carcinoma or embryonic stem cells. The epitope recognized by GCTM-5 is found on a 50-kDa protein present on the surface of these cells. In tissue sections of first-trimester human embryos, GCTM-5 specifically stained hepatoblasts and no other cell type examined. In normal pediatric or adult liver, GCTM-5 reacted with a minority population of luminal bile duct cells. In diseased livers, the numbers of GCTM-5-positive cells were increased compared with normal liver; antibody staining was restricted to a subpopulation of ductular reactive cells, and among this subpopulation we observed GCTM-5-positive cells that did not express cytokeratin 19 or N-CAM, classical makers of ductular reactive cells. Live GCTM-5-positive cells could be isolated from diseased livers by immunomagnetic sorting. These results suggest that GCTM-5 will be a useful reagent for defining cell lineage relationships between putative progenitor populations in embryonic liver and in the biliary epithelium during tissue repair.

A bioartificial liver--state of the art.

End-stage liver disease is treated by liver transplantation, but donor organ shortages remain a serious problem. This has prompted the design of bioartificial liver devices to "bridge" patients until they either recover or receive a liver transplant. In these devices, patient plasma is circulated extracorporeally through a bioreactor that houses liver cells (hepatocytes) sandwiched between artificial plates or capillaries.

Expression and DNA-binding activity of signal transducer and activator of transcription 3 in alcoholic cirrhosis compared to normal liver and primary biliary cirrhosis in humans.

In rats, activation of the cytokine-inducible transcription factor signal transducer and activator of transcription 3 (Stat3) is impaired in the liver after ethanol administration. The aim was to examine Stat3 expression, localization, and activity in alcoholic liver disease (ALD) in humans. Explanted livers of ALD patients were compared to normal and primary biliary cirrhosis livers. Protein expression, DNA-binding, and subcellular localization of Stat3 was examined by Western blotting, electrophoretic mobility shift assays, and immunohistochemistry; and interleukin-6, Stat3, and suppressor of cytokine signaling (SOCS)-3 mRNA expression by quantitative polymerase chain reaction. Stat3 proteins increased markedly in ALD, mainly in hepatocyte and proliferating biliary epithelial cell nuclei. In contrast to normal and primary biliary cirrhosis livers where Stat3 DNA-binding occurred normally, no Stat3 DNA-binding complexes were observed in ALD, although the tyrosine and serine phosphorylation of Stat3 was not altered. Elevated interleukin-6 mRNA was found in ALD whereas Stat3 and suppressor of cytokine signaling-3 mRNA levels were decreased. Although end-stage ALD is characterized by up-regulation of Stat3 proteins, this transcription factor appears to be functionally inactive. Furthermore, decreased transcription of the Stat3 gene in ALD might also affect cytoplasmic reserves of inactivated Stat3 in the long term. Impaired activation and restoration of Stat3 might thus contribute to the development of cell damage leading to liver cirrhosis in ALD.

Altered Notch ligand expression in human liver disease: further evidence for a role of the Notch signaling pathway in hepatic neovascularization and biliary ductular defects.

The Jagged and Delta family of transmembrane proteins are ligands for Notch receptors, which control the proliferation and/or differentiation of many cell lineages. Expression and localization of these ligands in the adult human liver has not been fully elucidated, nor whether dysregulation of these proteins contributes to liver disease processes. We have examined expression of the five known Notch ligands in human liver. Expression of Jagged-1 and Delta-4 mRNA was seen in normal and diseased liver tissue, whereas Jagged-2, Delta-1, and Delta-3 mRNA was undetectable. In primary liver cell isolates, Jagged-1 expression was found in all cell types, whereas Delta-4 was present in biliary epithelial and liver endothelial cells, but absent in hepatocytes. Interestingly, Jagged-1 mRNA expression was significantly up-regulated in diseased liver tissue. By immunohistochemistry, Jagged-1 expression was present on most structures in normal tissue. However in disease, strikingly strong Jagged-1 immunoreactivity was observed on many small neovessels and bile ductules. The expression of downstream modulators and effectors of Notch signaling was also detectable in purified cell isolates. This, together with aberrant Jagged-1 expression suggests that the Notch signaling pathway may play a role in the neovascularization and biliary defects observed in the liver during the development of cirrhosis.

Human liver-derived stem cells.

The search for human oval cells or bi-potential stem cells in the human liver is the subject of intensive investigation. Fetal hepatocytes (hepatoblasts) have some proliferative and bipotential capacity, but access to sufficient numbers of cells remains limiting. Candidate stem cells in the adult normal and diseased human liver have been identified using markers such as OV6, CD34, c-kit and NCAM. Lack of stem cell marker specificity however, remains a problem and further, more specific markers are required. The molecular signaling molecules and transcription factors that control proliferation and cell differentiation pathways into hepatocyte or biliary phenotype are beginning to emerge. However, whether any of the current liver stem cell approaches will be converted into effective clinical cell transplantation or gene therapy treatments has yet to be proven.

Progenitor cells of the biliary epithelial cell lineage.

Stem-like cells have been identified in liver that are able to differentiate in vivo and in culture to biliary epithelial cells (BEC), hepatocytes and oval cells. The growth factors/cytokines and signal pathways required for the differentiation processes are beginning to be evaluated. There is increasing evidence to suggest that these stem-like cells may originate from both the bone marrow population or from a precursor remnant from liver embryogenesis, as they share many of the same markers (CD34, c-kit, CD45). Most recently, it has been shown that a population of progenitor cells can copurify with mesenchymal bone marrow cells and differentiate under specific culture conditions to form both hepatic epithelial and also endothelial cells. The interaction of haemopoietic and mesenchymal stem cells needs further evaluation. The close association of ductular reactive cells and neovessels in end-stage cholestatic liver diseases and the relation to Jagged/Notch signalling pathway may be important in the regulation of stem cells to form both biliary epithelial and endothelial cells.

The role of Notch receptor expression in bile duct development and disease.

Mutations in the Jagged1 gene, a ligand for the Notch signalling pathway, have been implicated in the pathogenesis of Alagille syndrome (AGS), resulting in bile duct paucity. Recently, a mouse model for AGS suggested that abnormalities of the Notch2 receptor, as well as of Jagged1, may be present. Expression patterns of Notch receptors have not been described in the developing human liver or in paediatric liver. The expression of Notch receptors and ligands was examined in fetal, paediatric normal, and diseased human liver by RT-PCR and immunohistochemistry. RT-PCR showed Notch1-4 mRNA expression to be present. In fetal liver, Notch3 protein was expressed on mesenchymal cells, closely adjacent to ductal plate cells that expressed Jagged1. In paediatric normal liver, Notch1 and Notch2 were present on mature bile duct cells. Notch expression was altered in disease, with distinct differences in AGS from extrahepatic biliary atresia (EHBA) and alpha1-anti-trypsin deficiency (alpha1AT). In AGS, where extensive ductular reaction was present, Jagged1 was expressed on ductular reactive cells (DRCs), along with marked Notch2 and Notch3 staining. Where there was ductular paucity, Notch2 and Notch3 were not expressed on remaining biliary epithelial cells. In EHBA and alpha1AT, Notch receptor expression was not seen on DRCs. Instead, Notch2 and Notch3 were expressed by stromal cells. In all diseases, Notch3 was expressed on neovessels in portal tracts and cirrhotic fibrous septa. In conclusion, Notch3 is expressed in close proximity to Jagged1 at the time of ductal plate formation, suggesting that Notch3 is important for bile duct development. The expression of both Notch2 and Notch3 in AGS on DRCs confirms that these receptors may be important in the pathogenesis of this disease. Further studies are required to investigate the presence of Notch2 and Notch3 at other periods in liver development and to clarify the role of Notch signalling in paediatric cholestases.

Nomenclature of the finer branches of the biliary tree: canals, ductules, and ductular reactions in human livers.

The work of liver stem cell biologists, largely carried out in rodent models, has now started to manifest in human investigations and applications. We can now recognize complex regenerative processes in tissue specimens that had only been suspected for decades, but we also struggle to describe what we see in human tissues in a way that takes into account the findings from the animal investigations, using a language derived from species not, in fact, so much like our own. This international group of liver pathologists and hepatologists, most of whom are actively engaged in both clinical work and scientific research, seeks to arrive at a consensus on nomenclature for normal human livers and human reactive lesions that can facilitate more rapid advancement of our field.