Coagulation factor XIII is a critical driver of liver regeneration after partial hepatectomy.

BACKGROUND: Activation of coagulation and fibrin deposition in the regenerating liver appears to promote adequate liver regeneration in mice. In humans, perioperative hepatic fibrin deposition is reduced in patients who develop liver dysfunction after partial hepatectomy (PHx), but the mechanism underlying reduced fibrin deposition in these patients is unclear. METHODS AND RESULTS: Hepatic deposition of cross-linked (ie, stabilized) fibrin was evident in livers of mice after two-thirds PHx. Interestingly, hepatic fibrin cross-linking was dramatically reduced in mice after 90% PHx, an experimental setting of failed liver regeneration, despite similar activation of coagulation after two-thirds or 90% PHx. Likewise, intraoperative activation of coagulation was not reduced in patients who developed liver dysfunction after PHx. Preoperative fibrinogen plasma concentration was not connected to liver dysfunction after PHx in patients. Rather, preoperative and postoperative plasma activity of the transglutaminase coagulation factor (F)XIII, which cross-links fibrin, was lower in patients who developed liver dysfunction than in those who did not. PHx-induced hepatic fibrin cross-linking and hepatic platelet accumulation were significantly reduced in mice lacking the catalytic subunit of FXIII (FXIII-/- mice) after two-thirds PHx. This was coupled with a reduction in both hepatocyte proliferation and liver-to-body weight ratio as well as an apparent reduction in survival after two-thirds PHx in FXIII-/- mice. CONCLUSION: The results indicate that FXIII is a critical driver of liver regeneration after PHx and suggest that perioperative plasma FXIII activity may predict posthepatectomy liver dysfunction. The results may inform strategies to stabilize proregenerative fibrin during liver resection.

Intrahepatic neutrophil accumulation and extracellular trap formation are associated with posthepatectomy liver failure.

BACKGROUND: Posthepatectomy liver failure (PHLF) represents a life-threatening complication with limited therapeutic options. Neutrophils play a critical and dynamic role during regeneratory processes, but their role in human liver regeneration is incompletely understood, especially as underlying liver disease, detectable in the majority of patients, critically affects hepatic regeneration. Here we explored intrahepatic neutrophil accumulation and neutrophil extracellular traps (NETs) in patients with PHLF and validated the functional relevance of NETs in a murine partial hepatectomy (PHx) model. METHODS: We investigated the influx of neutrophils, macrophages, eosinophils, and mast cells and the presence of their respective extracellular traps in liver biopsies of 35 patients undergoing hepatectomy (10 patients with PHLF) before and after the initiation of liver regeneration by fluorescence microscopy. In addition, NET formation and neutrophil activation were confirmed by plasma analysis of 99 patients (24 patients with PHLF) before and up to 5 days after surgery. Furthermore, we inhibited NETs via DNase I in a murine PHx model of mice with metabolically induced liver disease. RESULTS: We detected rapid intrahepatic neutrophil accumulation, elevated levels of myeloperoxidase release, and NET formation in regenerating human livers, with a significantly higher increase of infiltrating neutrophils and NETs in patients with PHLF. Circulating markers of neutrophil activation, including elastase, myeloperoxidase, and citrullinated histone H3, correlated with markers of liver injury. In a murine PHx model, we showed that the inhibition of NET accelerated hepatocyte proliferation and liver regeneration. CONCLUSIONS: Patients with PHLF showed accelerated intrahepatic neutrophil infiltration and NET formation, which were associated with liver damage. Further, we identified postsurgical myeloperoxidase levels as predictive markers for adverse outcomes and observed that blocking NETs in a murine PHx model accelerated tissue regeneration.

Age-Dependent Surface Receptor Expression Patterns in Immature Versus Mature Platelets in Mouse Models of Regenerative Thrombocytopenia.

Aging is a multifaceted process that unfolds at both the individual and cellular levels, resulting in changes in platelet count and platelet reactivity. These alterations are influenced by shifts in platelet production, as well as by various environmental factors that affect circulating platelets. Aging also triggers functional changes in platelets, including a reduction in RNA content and protein production capacity. Older individuals and RNA-rich immature platelets often exhibit hyperactivity, contributing significantly to pathologic conditions such as cardiovascular diseases, sepsis, and thrombosis. However, the impact of aging on surface receptor expression of circulating platelets, particularly whether these effects vary between immature and mature platelets, remains largely unexplored. Thus, we investigated the expression of certain surface and activation receptors on platelets from young and old mice as well as on immature and mature platelets from mouse models of regenerative thrombocytopenia by flow cytometry. Our findings indicate that aged mice show an upregulated expression of the platelet endothelial cell adhesion molecule-1 (CD31), tetraspanin-29 (CD9), and Toll-like receptor 2 (TLR2) compared to their younger counterparts. Interestingly, when comparing immature and mature platelets in both young and old mice, no differences were observed in mature platelets. However, immature platelets from young mice displayed higher surface expression compared to immature platelets from old mice. Additionally, in mouse models of regenerative thrombocytopenia, the majority of receptors were upregulated in immature platelets. These results suggest that distinct surface receptor expressions are increased on platelets from old mice and immature platelets, which may partially explain their heightened activity and contribute to an increased thrombotic risk.

Transcriptomic landscapes of effective and failed liver regeneration in humans.

Background & Aims: Although extensive experimental evidence on the process of liver regeneration exists, in humans, validation is largely missing. However, liver regeneration is critically affected by underlying liver disease. Within this project, we aimed to systematically assess early transcriptional changes during liver regeneration in humans and further assess how these processes differ in people with dysfunctional liver regeneration. Methods: Blood samples of 154 patients and intraoperative tissue samples of 46 patients undergoing liver resection were collected and classified with regard to dysfunctional postoperative liver regeneration. Of those, a matched cohort of 21 patients were used for RNA sequencing. Samples were assessed for circulating cytokines, gene expression dynamics, intrahepatic neutrophil accumulation, and spatial transcriptomics. Results: Individuals with dysfunctional liver regeneration demonstrated an aggravated transcriptional inflammatory response with higher intracellular adhesion molecule-1 induction. Increased induction of this critical leukocyte adhesion molecule was associated with increased intrahepatic neutrophil accumulation and activation upon induction of liver regeneration in individuals with dysfunctional liver regeneration. Comparing baseline gene expression profiles in individuals with and without dysfunctional liver regeneration, we found that dual-specificity phosphatase 4 (DUSP4) expression, a known critical regulator of intracellular adhesion molecule-1 expression in endothelial cells, was markedly reduced in patients with dysfunctional liver regeneration. Mimicking clinical risk factors for dysfunctional liver regeneration, we found liver sinusoidal endothelial cells of two liver disease models to have significantly reduced baseline levels of DUSP4. Conclusions: Exploring the landscape of early transcriptional changes of human liver regeneration, we observed that people with dysfunctional regeneration experience overwhelming intrahepatic inflammation. Subclinical liver disease might account for DUSP4 reduction in liver sinusoidal endothelial cells, which ultimately primes the liver for an aggravated inflammatory response. Impact and implications: Using a unique human biorepository, focused on liver regeneration (LR), we explored the landscape of circulating and tissue-level alterations associated with both functional and dysfunctional LR. In contrast to experimental animal models, people with dysfunctional LR demonstrated an aggravated transcriptional inflammatory response, higher intracellular adhesion molecule-1 (ICAM-1) induction, intrahepatic neutrophil accumulation and activation upon induction of LR. Although inflammatory responses appear rapidly after liver resection, people with dysfunctional LR have exaggerated inflammatory responses that appear to be related to decreased levels of LSEC DUSP4, challenging existing concepts of post-resectional LR.

Tyrosine phosphorylation of YAP-1 in biliary epithelial cells mediates posthepatectomy liver regeneration and is affected by serotonin.

Experimental data suggested activation of yes-associated protein (YAP-1) as a critical regulator of liver regeneration (LR). Serotonin (5-HT) promotes LR in rodent models and has been proposed to act via YAP-1. How 5-HT affects LR is incompletely understood. A possible mechanism how 5-HT affects human LR was explored. Sixty-one patients were included. Tissue samples prior and 2 h after induction of LR were collected. Circulating levels of 5-HT and osteopontin (OPN) were assessed. YAP-1, its phosphorylation states, cytokeratin 19 (CK-19) and OPN were assessed using immunofluorescence. A mouse model of biliary epithelial cells (BECs) specific deletion of YAP/TAZ was developed. YAP-1 increased as early as 2 h after induction of LR (p = 0.025) predominantly in BECs. BEC specific deletion of YAP/TAZ reduced LR after 70% partial hepatectomy in mice (Ki67%, p < 0.001). SSRI treatment, depleting intra-platelet 5-HT, abolished YAP-1 and OPN induction upon LR. Portal vein 5-HT levels correlated with intrahepatic YAP-1 expression upon LR (R = 0.703, p = 0.035). OPN colocalized with YAP-1 in BECs and its circulating levels increased in the liver vein 2 h after induction of LR (p = 0.017). In the context of LR tyrosine-phosphorylated YAP-1 significantly increased (p = 0.042). Stimulating BECs with 5-HT resulted in increased YAP-1 activation via tyrosine-phosphorylation and subsequently increased OPN expression. BECs YAP-1 appears to be critical for LR in mice and humans. Our evidence suggests that 5-HT, at least in part, exerts its pro-regenerative effects via YAP-1 tyrosine-phosphorylation in BECs and subsequent OPN-dependent paracrine immunomodulation.

Platelet p110ß mediates platelet-leukocyte interaction and curtails bacterial dissemination in pneumococcal pneumonia.

Phosphatidylinositol 3-kinase catalytic subunit p110ß is involved in tumorigenesis and hemostasis. However, it remains unclear if p110ß also regulates platelet-mediated immune responses, which could have important consequences for immune modulation during anti-cancer treatment with p110ß inhibitors. Thus, we investigate how platelet p110ß affects inflammation and infection. Using a mouse model of Streptococcus pneumoniae-induced pneumonia, we find that both platelet-specific p110ß deficiency and pharmacologic inhibition of p110ß with TGX-221 exacerbate disease pathogenesis by preventing platelet-monocyte and neutrophil interactions, diminishing their infiltration and enhancing bacterial dissemination. Platelet p110ß mediates neutrophil phagocytosis of S. pneumoniae in vitro and curtails bacteremia in vivo. Genetic deficiency or inhibition of platelet p110ß also impairs macrophage recruitment in an independent model of sterile peritonitis. Our results demonstrate that platelet p110ß dysfunction exacerbates pulmonary infection by impeding leukocyte functions. Thereby, our findings provide important insights into the immunomodulatory potential of PI3K inhibitors in bacterial infection.

Hepatectomy-induced apoptotic extracellular vesicles stimulate neutrophils to secrete regenerative growth factors.

BACKGROUND & AIMS: Surgical resection of the cancerous tissue represents one of the few curative treatment options for neoplastic liver disease. Such partial hepatectomy (PHx) induces hepatocyte hyperplasia, which restores liver function. PHx is associated with bacterial translocation, leading to an immediate immune response involving neutrophils and macrophages, which are indispensable for the priming phase of liver regeneration. Additionally, PHx induces longer-lasting intrahepatic apoptosis. Herein, we investigated the effect of apoptotic extracellular vesicles (aEVs) on neutrophil function and their role in this later phase of liver regeneration. METHODS: A total of 124 patients undergoing PHx were included in this study. Blood levels of the apoptosis marker caspase-cleaved cytokeratin-18 (M30) and circulating aEVs were analyzed preoperatively and on the first and fifth postoperative days. Additionally, the in vitro effects of aEVs on the secretome, phenotype and functions of neutrophils were investigated. RESULTS: Circulating aEVs increased at the first postoperative day and were associated with higher concentrations of M30, which was only observed in patients with complete liver recovery. Efferocytosis of aEVs by neutrophils induced an activated phenotype (CD11bhighCD16highCD66bhighCD62Llow); however, classical inflammatory responses such as NETosis, respiratory burst, degranulation, or secretion of pro-inflammatory cytokines were not observed. Instead, efferocytosing neutrophils released various growth factors including fibroblast growth factor-2 and hepatocyte growth factor (HGF). Accordingly, we observed an increase of HGF-positive neutrophils after PHx and a correlation of plasma HGF with M30 levels. CONCLUSIONS: These data suggest that the clearance of PHx-induced aEVs leads to a population of non-inflammatory but regenerative neutrophils, which may support human liver regeneration. LAY SUMMARY: In this study, we show that the surgical removal of a diseased part of the liver triggers a specific type of programmed cell death in the residual liver tissue. This results in the release of vesicles from dying cells into the blood, where they are cleared by circulating immune cells. These respond by secreting hepatocyte growth factors that could potentially support the regeneration of the liver remnant.

Immunological Aspects of AXL/GAS-6 in the Context of Human Liver Regeneration.

AXL and its corresponding ligand growth arrest-specific 6 (GAS-6) are critically involved in hepatic immunomodulation and regenerative processes. Pleiotropic inhibitory effects on innate inflammatory responses might essentially involve the shift of macrophage phenotype from a pro-inflammatory M1 to an anti-inflammatory M2. We aimed to assess the relevance of the AXL/GAS-6-pathway in human liver regeneration and, consequently, its association with clinical outcome after hepatic resection. Soluble AXL (sAXL) and GAS-6 levels were analyzed at preoperative and postoperative stages in 154 patients undergoing partial hepatectomy and correlated with clinical outcome. Perioperative dynamics of interleukin (IL)-6, soluble tyrosine-protein kinase MER (sMerTK), soluble CD163 (sCD163), and cytokeratin (CK) 18 were assessed to reflect pathophysiological processes. Preoperatively elevated sAXL and GAS-6 levels predicted postoperative liver dysfunction (area under the curve = 0.721 and 0.722; P < 0.005) and worse clinical outcome. These patients failed to respond with an immediate increase of sAXL and GAS-6 upon induction of liver regeneration. Abolished AXL pathway response resulted in a restricted increase of sCD163, suggesting a disrupted phenotypical switch to regeneratory M2 macrophages. No association with sMerTK was observed. Concomitantly, a distinct association of IL-6 levels with an absent increase of AXL/GAS-6 signaling indicated pronounced postoperative inflammation. This was further supported by increased intrahepatic secondary necrosis as reflected by CK18M65. sAXL and GAS-6 represent not only potent and easily accessible preoperative biomarkers for the postoperative outcome but also AXL/GAS-6 signaling might be of critical relevance in human liver regeneration. Refractory AXL/GAS-6 signaling, due to chronic overactivation/stimulation in the context of underlying liver disease, appears to abolish their immediate release following induction of liver regeneration, causing overwhelming immune activation, presumably via intrahepatic immune regulation.

Adverse Outcome in COVID-19 Is Associated With an Aggravating Hypo-Responsive Platelet Phenotype.

Thromboembolic complications are frequently observed in Coronavirus disease 2019 (COVID-19). While COVID-19 is linked to platelet dysregulation, the association between disease outcome and platelet function is less clear. We prospectively monitored platelet activation and reactivity in 97 patients during the first week of hospitalization and determined plasma markers of platelet degranulation and inflammation. Adverse outcome in COVID-19 was associated with increased basal platelet activation and diminished platelet responses, which aggravated over time. Especially GPIIb/IIIa responses were abrogated, pointing toward impeded platelet aggregation. Moreover, platelet-leukocyte aggregate formation was diminished, pointing toward abrogated platelet-mediated immune responses in COVID-19. No general increase in plasma levels of platelet-derived granule components could be detected, arguing against platelet exhaustion. However, studies on platelets from healthy donors showed that plasma components in COVID-19 patients with unfavorable outcome were at least partly responsible for diminished platelet responses. Taken together this study shows that unfavorable outcome in COVID-19 is associated with a hypo-responsive platelet phenotype that aggravates with disease progression and may impact platelet-mediated immunoregulation.

Horizontal MicroRNA Transfer by Platelets - Evidence and Implications.

For decades, platelets have been known for their central role in hemostasis and their ability to release bioactive molecules, allowing inter-platelet communication and crosstalk with the immune system and vascular cells. However, with the detection of microRNAs in platelets and platelet-derived microvesicles (MVs), a new level of inter-cellular regulation was revealed. By shedding MVs from their plasma membrane, platelets are able to release functional microRNA complexes that are protected from plasma RNases. Upon contact with macrophages, endothelial cells and smooth muscle cells platelet microRNAs are rapidly internalized and fine-tune the functionality of the recipient cell by post-transcriptional reprogramming. Moreover, microRNA transfer by platelet MVs allows infiltration into tissues with limited cellular access such as solid tumors, thereby they not only modulate tumor progression but also provide a potential route for drug delivery. Understanding the precise mechanisms of horizontal transfer of platelet microRNAs under physiological and pathological conditions allows to design side-specific therapeutic (micro)RNA delivery systems. This review summarizes the current knowledge and the scientific evidence of horizontal microRNA transfer by platelets and platelet-derived MVs into vascular and non-vascular cells and its physiological consequences.

Till Death Do Us Part-The Multifaceted Role of Platelets in Liver Diseases.

Platelets are tightly connected with the liver, as both their production and their clearance are mediated by the liver. Platelets, in return, participate in a variety of liver diseases, ranging from non-alcoholic fatty liver diseases, (viral) hepatitis, liver fibrosis and hepatocellular carcinoma to liver regeneration. Due to their versatile functions, which include (1) regulation of hemostasis, (2) fine-tuning of immune responses and (3) release of growth factors and cellular mediators, platelets quickly adapt to environmental changes and modulate disease development, leading to different layers of complexity. Depending on the (patho)physiological context, platelets exert both beneficial and detrimental functions. Understanding the precise mechanisms through which platelet function is regulated at different stages of liver diseases and how platelets interact with various resident and non-resident liver cells helps to draw a clear picture of platelet-related therapeutic interventions. Therefore, this review summarizes the current knowledge on platelets in acute and chronic liver diseases and aims to shed light on how the smallest cells in the circulatory system account for changes in the (patho)physiology of the second largest organ in the human body.

Platelets and Antiplatelet Medication in COVID-19-Related Thrombotic Complications.

Coronavirus disease 2019 (COVID-19) induces a hypercoagulatory state that frequently leads to thromboembolic complications. Whereas anticoagulation is associated with reduced mortality, the role of antiplatelet therapy in COVID-19 is less clear. We retrospectively analyzed the effect of anticoagulation and antiplatelet therapy in 578 hospitalized patients with COVID-19 and prospectively monitored 110 patients for circulating microthrombi and plasma markers of coagulation in the first week of admission. Moreover, we determined platelet shape change and also thrombi in postmortem lung biopsies in a subset of patients with COVID-19. We observed no association of antiplatelet therapy with COVID-19 survival. Adverse outcome in COVID-19 was associated with increased activation of the coagulation cascade, whereas circulating microthrombi did not increase in aggravated disease. This was in line with analysis of postmortem lung biopsies of patients with COVID-19, which revealed generally fibrin(ogen)-rich and platelet-low thrombi. Platelet spreading was normal in severe COVID-19 cases; however, plasma from patients with COVID-19 mediated an outcome-dependent inhibitory effect on naïve platelets. Antiplatelet medication disproportionally exacerbated this platelet impairment in plasma of patients with fatal outcome. Taken together, this study shows that unfavorable outcome in COVID-19 is associated with a profound dysregulation of the coagulation system, whereas the contribution of platelets to thrombotic complications is less clear. Adverse outcome may be associated with impaired platelet function or platelet exhaustion. In line, antiplatelet therapy was not associated with beneficial outcome.