This study aimed to explore the association between dietary fiber intake and the risk of metabolic dysfunction-associated steatotic liver disease (MASLD), as well as liver fat content, while considering genetic predispositions of MASLD, gut microbial abundance, and butyrate levels. This study analyzed data from 190,276 participants in the UK Biobank. Dietary fiber intake was assessed using 24-h dietary recall. MASLD cases were diagnosed through hospital admission records and death registries, and liver fat content was measured via magnetic resonance imaging. The genetic predispositions of MASLD, gut microbial abundance, and butyrate levels were evaluated using single nucleotide polymorphisms. Cox proportional hazards models were used to calculate hazard ratios (HRs) and 95 % confidence intervals (CIs). Over a median follow-up of 10.49 years, 1423 MASLD cases were recorded. Elevated dietary fiber intake was associated with a reduced risk of MASLD (HR: 0.72; 95 % CI: 0.58, 0.90) and a lower level of liver fat content (β: -0.97; 95 % CI: -1.21, -0.73) (all P for trend <0.05). Restricted cubic spline analyses further confirmed the linear inverse associations between fiber intake and the risk of MASLD. Notably, the negative associations between dietary fiber intake and both MASLD and liver fat content were consistent across different genetic predispositions of gut microbial abundance and butyrate levels. Moreover, the inverse association between dietary fiber intake and liver fat was strengthened by high genetic susceptibility of MASLD and elevated body mass index (both P for interaction <0.05). Overall, increased dietary fiber consumption was associated with a lower MASLD risk and decreased liver fat content regardless of genetic predispositions of gut microbial abundance and butyrate levels.

Growth differentiation factor 15 (GDF15) is a stress-induced hepatokine with emerging roles in liver injury. Estrogen-related receptor γ (ERRγ), a nuclear receptor regulating mitochondrial function and metabolic stress, has also been implicated in various liver injury conditions. However, the regulatory interplay between ERRγ and GDF15 remains unclear. This study investigates the molecular mechanisms underlying GDF15 expression and secretion in the liver, focusing on the role of ERRγ during acute and chronic liver injury.

Wild-type and hepatocyte-specific ERRγ knockout (ERRγ-LKO) mice were administered with a single dose of carbon tetrachloride (CCl4) or fed an alcohol-containing diet for 4 weeks to establish acute or chronic liver injury models, respectively. ERRγ was overexpressed through an adenoviral construct (Ad-ERRγ). The ERRγ-specific inverse agonist GSK5182 was employed to inhibit the transactivation of ERRγ. The luciferase reporter assays were used to assess the binding of ERRγ protein to the regulatory region of GDF15 gene.

Hepatic ERRγ and GDF15 gene expression, and GDF15 protein secretion were significantly elevated in both acute and chronic liver injury. Adenovirus-mediated overexpression of ERRγ is sufficient to substantially increase hepatic GDF15 expression and secretion. Genetic ablation of ERRγ expression or pharmacological inhibition of ERRγ transactivation substantially inhibited the upregulation of hepatic GDF15 expression and production in both acute and chronic liver injury. Furthermore, reporter assays showed that ERRγ, but not ERRα or ERRβ, directly binds to and activates the GDF15 gene promoter.

Our findings highlight the crucial role of ERRγ in transcriptional regulation of GDF15 gene expression and production in response to liver damage. Understanding the regulatory mechanisms of GDF15 expression could lead to new therapeutic targets for protecting the liver from various types of injuries and associated diseases.

Liver regeneration is a remarkable and unique biological process, orchestrated by an intricate cellular crosstalk of (non-)parenchymal, immune, and nerve cells. In this issue of Immunity, Modares et al. uncover the pivotal role of choline acetyltransferase (ChAT+)-expressing B cells as key orchestrators of liver regeneration.

The biotransformation of inhalational anesthetic agents can result in production of hepatotoxic metabolites, which may potentially impair liver regeneration. Thus, isoflurane and propofol may have differential impacts on liver regeneration after donor hepatectomy.

To compare the regeneration liver volume (RgLV) via computed tomography (CT) volumetry on post-operative day (POD) 14 in isoflurane and propofol groups.

Randomized controlled pilot trial.

Sixty donors, who underwent donor hepatectomy.

Patients were randomized into isoflurane and propofol group using computer generated random number tables. In isoflurane group, anesthesia was maintained with isoflurane 1-2 % in air‑oxygen mixture. In propofol group, anesthesia was maintained with the target-controlled infusion (TCI) of propofol using a TCI system (Perfusor® Space- B. Braun) at a plasma target concentration of 3-6 μg.ml-1. BIS was recorded in all patients.

Liver CT volumetry was assessed at POD 14.

RgLV on POD14 was comparable in two groups [449.3(170) & 437.8 (177.8) cm3, [Mean Difference (MD) -11.904 95 % Confidence Interval(CI) -101.83, 78.03 respectively; p = 0.79].

Administering propofol or isoflurane may not have differential effect on liver regeneration after donor hepatectomy.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a growing global health concern, with limited effective treatments. KCNMA1 potassium channel has been implicated in the pathogenesis of various metabolic diseases. However, whether and how KCNMA1 regulates MASLD have been elusive.

Global, hepatic stellate cells (HSCs)-specific, and hepatocyte-specific Kcnma1 knockout mice were fed either a standard chow or a high-fat diet (HFD). Serum and liver tissues were collected and analyzed by biochemical assay, histology, qPCR and western blotting. HSCs conditioned medium (CM) treatment hepatocytes experiment model and three-dimensional (3D) hepatocytes-HSCs spheroids were employed to study lipid accumulation in hepatocytes. A Cytokine Antibody Array was used to analyze the cytokine profile.

Our study demonstrated that global Kcnma1 deletion prevented diet-induced hepatic steatosis and improved insulin sensitivity. Further analyses using HSC-specific and hepatocyte-specific Kcnma1 knockout MASLD mouse models revealed that the protective effect against hepatic steatosis was predominantly mediated by Kcnma1 deletion in HSCs, rather than in hepatocytes. CM transfer experiment and 3D spheroid studies show Kcnma1 deletion effectively prevents lipid accumulation in hepatocytes. Mechanically, Kcnma1-deficient HSCs secrete Amphiregulin (AREG) to regulate lipid metabolism in hepatocytes via epidermal growth factor receptor (EGFR) signaling. Of clinical significance, AREG levels were notably reduced in the liver tissue of MASLD patients, while injection of recombinant AREG protein significantly ameliorated MASLD in mice.

Our study uncovers a novel mechanism in which Kcnma1 deletion in HSCs enhances AREG secretion, thereby reducing lipid accumulation in hepatocytes through the AREG/EGFR signaling, ultimately inhibiting the progression of MASLD.

Autophagy plays an important role in liver regeneration. However, most studies are limited to hepatocytes, and the function and mechanism of macrophage autophagy in liver regeneration remain unclear. This study investigated the role of the essential autophagy gene encoding autophagy-related 16-like 1 (ATG16L1), which regulates the macrophage phenotype in liver regeneration.

We generated FloxP-Atg16l1 (Atg16l1 FL/FL ), Lyz2-Cre Atg16l1 knockout (KO) (Atg16l1 M-KO ), and myeloid-specific Atg16l1-overexpression-knock-in (Atg16l1 OE ) mice. These mice were subjected to 70% partial hepatectomy to demonstrate the role of ATG16L1 in macrophages during liver regeneration.

ATG16L1 expression was significantly upregulated in macrophages during the early stages of liver regeneration. ATG16L1 deletion in macrophages substantially delayed liver regeneration in mice and caused a marked imbalance in Ly6Chi and Ly6Clo macrophage proportions in the liver. RNA-sequencing analysis revealed that, compared with macrophages isolated from Atg16l1 FL/FL mice, those from Atg16l1 M-KO mice exhibited significant downregulation of genes associated with oxidative phosphorylation and upregulation of proinflammatory gene expression. Mechanistically, ATG16L1 loss impaired mitophagy in macrophages, leading to the accumulation of mitochondrial damage and a metabolic shift that promoted proinflammatory macrophage polarization. ATG16L1 deficiency not only promoted macrophage mitochondrial (mt)DNA release and cyclic GMP-AMP synthase-stimulator of interferon genes (STING) activation, but also suppressed STING degradation. Sustained STING hyperactivation and subsequent increased release of downstream interferons further contributed to the inhibition of liver regeneration. Notably, pharmacological activation or genetic overexpression of ATG16L1 significantly enhanced liver regeneration in mice.

ATG16L1 has a pivotal role in liver regeneration by affecting the phenotype and function of macrophages. Thus, targeting ATG16L1 in macrophages could present a novel strategy for promoting liver regeneration.

The autophagy-related gene ATG16L1 mediates mitophagy, facilitating the clearance of damaged mitochondria in macrophages following partial hepatectomy and maintaining a reparative macrophage phenotype. ATG16L1 deficiency triggers excessive STING activation and inhibits its degradation, thereby suppressing liver regeneration. Thus, targeting ATG16L1 in macrophages could represent a novel strategy to promote liver regeneration.

The liver has a unique ability to regenerate to meet the body's metabolic needs, even following acute or chronic injuries. The cellular and molecular mechanisms underlying normal liver regeneration have been well investigated to improve organ transplantation outcomes. Once liver regeneration is impaired, pathological regeneration occurs, and the underlying cellular and molecular mechanisms require further investigations. Nevertheless, a plethora of cytokines and growth factor-mediated pathways have been reported to modulate physiological and pathological liver regeneration. Regenerative mitogens play an essential role in hepatocyte proliferation. Accelerator mitogens in synergism with regenerative ones promote liver regeneration following hepatectomy. Finally, terminator mitogens restore the proliferating status of hepatocytes to a differentiated and quiescent state upon completion of regeneration. Chronic loss of hepatocytes, which can manifest in chronic liver disorders of any etiology, often has undesired structural consequences, including fibrosis, cirrhosis, and liver neoplasia due to the unregulated proliferation of remaining hepatocytes. In fact, any impairment in the physiological function of the terminator mitogens results in the progression of pathological liver regeneration. In the current review, we intend to highlight the updated cellular and molecular mechanisms involved in liver regeneration and discuss the impairments in central regulating mechanisms responsible for pathological liver regeneration.

Surgical resection remains the gold standard for liver tumor treatment, yet the emergence of postoperative liver failure, known as the small-for-size syndrome (SFSS), poses a significant challenge. The activation of hypoxia sensors in an SFSS liver remnant initiated early angiogenesis, improving the vascular architecture, safeguarding against liver failure, and reducing mortality. The study aimed to elucidate vascular remodeling mechanisms in SFSS and their impact on hepatocyte function and subsequent liver failure.

Mice underwent extended partial hepatectomy to induce SFSS, with a subset exposed to hypoxia immediately after surgery. Hypoxia bolstered posthepatectomy survival rates. The early proliferation of liver sinusoidal cells, coupled with recruitment of putative endothelial progenitor cells, increased vascular density, improved lobular perfusion, and limited hemorrhagic events in the regenerating liver under hypoxia. Administration of granulocyte colony-stimulating factor in hepatectomized mice mimicked the effects of hypoxia on vascular remodeling and endothelial progenitor cell recruitment but failed to rescue survival. Compared to normoxia, hypoxia favored hepatocyte function over proliferation, promoting functional preservation in the regenerating remnant. Injection of Adeno-associated virus serotype 8-thyroxine-binding globulin-hepatocyte nuclear factor 4 alpha virus for hepatocyte-specific overexpression of hepatocyte nuclear factor 4 alpha, the master regulator of hepatocyte function, enforced functionality in proliferating hepatocytes but did not rescue survival. The combination of hepatocyte nuclear factor 4 alpha overexpression and granulocyte colony-stimulating factor treatment rescued survival after SFSS-setting hepatectomy.

In summary, SFSS arises from an imbalance and desynchronized interplay between functional regeneration and vascular restructuring. To improve survival following SFSS hepatectomy, it is essential to adopt a 2-pronged strategy aimed at preserving the function of proliferating parenchymal cells and simultaneously attenuating vascular damage.

The liver fulfils a plethora of vital functions and, due to their importance, liver dysfunction has life-threatening consequences. Liver disorders currently account for more than two million deaths annually worldwide and can be classified broadly into three groups, considering their onset and aetiology, as acute liver diseases, inherited metabolic disorders and chronic liver diseases. In the most advanced and severe forms leading to liver failure, liver transplantation is the only treatment available, which has many associated drawbacks, including a shortage of organ donors. Cell therapy via fully mature cell transplantation is an advantageous alternative that may be able to restore a damaged organ's functionality or serve as a bridge until regeneration can occur. Pioneering work has shown that transplanting adult hepatocytes can support liver recovery. However, primary hepatocytes cannot be grown extensively in vitro as they rapidly lose their metabolic activity. Therefore, different cell sources are currently being tested as alternatives to primary cells. Human pluripotent stem cell-derived cells, chemically induced liver progenitors, or 'liver' organoids, hold great promise for developing new cell therapies for acute and chronic liver diseases. This Review focuses on the advantages and drawbacks of distinct cell sources and the relative strategies to address different therapeutic needs in distinct liver diseases.

The plasticity of plant cells underlies their wide capacity to regenerate, with increasing evidence in plants and animals implicating cell-cycle dynamics in cellular reprogramming. To investigate the cell cycle during cellular reprogramming, we developed a comprehensive set of cell-cycle-phase markers in the Arabidopsis root. Using single-cell RNA sequencing profiles and live imaging during regeneration, we found that a subset of cells near an ablation injury dramatically increases division rate by truncating G1 phase. Cells in G1 undergo a transient nuclear peak of glutathione (GSH) prior to coordinated entry into S phase, followed by rapid divisions and cellular reprogramming. A symplastic block of the ground tissue impairs regeneration, which is rescued by exogenous GSH. We propose a model in which GSH from the outer tissues is released upon injury, licensing an exit from G1 near the wound to induce rapid cell division and reprogramming.

Posthepatectomy liver failure is a serious clinical issue with high mortality, similar in pathophysiology to small-for-size syndrome seen in liver transplantation. This study evaluates the efficacy of splenic artery ligation in reducing posthepatectomy liver failure in patients with portal venous pressure >15 mm Hg after hepatectomy.

This single-center, randomized controlled trial was conducted from May 2019 to November 2023. Eligible participants were patients scheduled for open hepatectomy for any indication. Patients with a portal venous pressure >15 mm Hg were randomized into splenic artery ligation and control groups in a 1:1 ratio. The primary outcomes were posthepatectomy liver failure grades B and C (International Study group of Liver Surgery criteria), and secondary outcomes included 90-day mortality, comprehensive complication index, and ascites volume.

The study was terminated early, before reaching the calculated sample size, because the primary outcome in the intervention group demonstrated statistically significant results. Of the 92 patients, 36 had elevated portal venous pressure, which was associated with greater rates of posthepatectomy liver failure grades B and C (41.67% vs 3.57%, P < .001), increased ascites volume (5,340 mL vs 1,055 mL, P < .001), and a greater comprehensive complication index (20.90 vs 8.70, P < .001). In the randomized subset, splenic artery ligation significantly reduced portal venous pressure and the portal venous pressure-central venous pressure gradient compared with both presplenic artery ligation values and the control group and significantly lowered the incidence of posthepatectomy liver failure grades B and C (16.67% vs 66.67%, P = .006), comprehensive complication index (8.70 vs 20.90, P = .034). Splenic artery ligation was identified as an independent factor in reducing posthepatectomy liver failure (adjusted relative risk, 0.29).

Splenic artery ligation is effective in reducing posthepatectomy liver failure in patients with high portal venous pressure after hepatectomy.

The ability of an organism to regrow tissues is regulated by various signaling pathways. One such pathway that has been studied widely both in the context of regeneration and development is the Notch signaling pathway. Notch is required for the development of the eye and regeneration of tissues in multiple organisms, but it is unknown if Notch plays a role in the regulation of Xenopus laevis embryonic eye regrowth. We found that Notch1 is required for eye regrowth and regulates retinal progenitor cell proliferation. Chemical and molecular inhibition of Notch1 significantly decreased eye regrowth by reducing retinal progenitor cell proliferation without affecting retinal differentiation. Temporal inhibition studies showed that Notch function is required during the first day of regrowth. Interestingly, Notch1 loss-of-function phenocopied the effects of the inhibition of the proton pump, vacuolar-type ATPase (V-ATPase), where retinal proliferation but not differentiation was blocked during eye regrowth. Overexpression of a form of activated Notch1, the Notch intracellular domain (NICD) rescued the loss of eye regrowth due to V-ATPase inhibition. These findings highlight the importance of the Notch signaling pathway in eye regeneration and its role in inducing retinal progenitor cell proliferation in response to injury.

Small-for-size syndrome is a clinical syndrome of early allograft dysfunction usually following living donor liver transplantation due to a mismatch between recipient metabolic and functional requirements and the graft's functional capacity. While graft size relative to the recipient size is the most commonly used parameter to predict risk, small-for-size syndrome is multifactorial and its development depends on a number of inter-dependant factors only some of which are modifiable. Intra-operative monitoring of portal haemodynamics and portal flow modulation is widely recommended though there is wide variation in clinical practice. Management of established small-for-size syndrome centres around meticulous patient care, infection prevention, fluid management and identifying correctable technical complications. However, retransplantation is the only treatment in severe cases. While small-for-size syndrome per se is associated with increased peri-operative mortality, the contribution of non-hepatic organ failure in determining patient outcomes needs further studies.

Fibrosis is a disease that negatively affects liver regeneration, resulting in severe complications after liver surgery. However, there is still no clinically effective treatment for promoting fibrotic liver regeneration because the underlying hepatocellular mechanism remains poorly understood. Through microRNA microarrays combined with the application of AAV6, we found that high expression of miR-181a-5p in activated hepatic stellate cells (HSCs) suppressed the expression of hepatic growth factor (HGF) and partially contributed to impaired regeneration potential in mice with hepatic fibrosis that had undergone two-thirds partial hepatectomy. As nanotherapeutics, mesenchymal stem-cell-derived extracellular vesicles (MSC-EVs) have been verified as effective treatments for liver regeneration. Here we observe that MSC-EVs can also promote fibrotic liver regeneration via enriched lncEEF1G, which acts as a competing endogenous RNA to directly sponge miR-181a-5p, leading to the upregulated expression of HGF in HSCs. Finally, engineered MSC-EVs with high expression of lncEEF1G (lncEEF1GOE-EVs) were constructed, suggesting greater potential for this model. In summary, our findings indicate that lncEEF1GOE-EVs have a nanotherapeutic effect on promoting regeneration of fibrotic livers by modulating the miR-181a-5p/HGF pathway in HSCs, which highlights the potential of extracellular vesicle engineering technology for patients with hepatic fibrosis who have undergone hepatic surgery. Engineered mesenchymal stem cells that overexpress lncEEF1G can secrete extracellular vesicles that are rich in lncEEF1G (lncEEF1GOE-EVs). Upon injection of lncEEF1GOE-EVs into a fibrotic 70% partial hepatectomy mouse model, lncEEF1G competitively binds to miR-181a-5p in hepatic stellate cells, preventing the interaction between miR-181a-5p and the messenger RNA of hepatocyte growth factor. This consequently leads to an increase in the secretion of hepatocyte growth factor and the promotion of hepatocyte proliferation.

Non-compressible hemostasis and promoting tissue healing are important in soft tissue trauma repair. Inorganic aerogels show superior performance in rapid hemostasis or promoting tissue healing, but simultaneously promoting non-compressive hemostasis and soft tissue healing still remains a challenge. Herein, SiO2-based inorganic nanofiber aerogels (M2+@SiO2, M=Ca, Mg, and Sr) were prepared by freeze-drying the mixture of bioactive silicates-deposited SiO2 nanofibers and SiO2 sol. These M2+@SiO2 aerogels have a three-dimensional highly-interconnected porous structure, remarkable flexibility, high absorption, good hydrophilicity, negative zeta potential, and bioactive ions releasing capacity. M2+@SiO2 aerogels not only exhibited satisfactory hemostasis activities in vitro, but also possessed high hemostatic efficacy in compressible rabbit femoral artery injury bleeding model and non-compressible rat liver puncture bleeding model compared to medical gauze and gelatin sponge. M2+@SiO2 aerogel had low blood clotting index of Ca. 10 % and short partial thromboplastin time of ca. 82 s in vitro, and could greatly short bleeding time by >50 % and decrease blood loss by about 80 % compared to medical gauze and gelatin sponge in non-compressible hemostasis. Sr2+@SiO2 aerogel showed optimal bioactivities on promoting cell proliferation, cell migration, and the expression of liver function and angiogenesis related genes and proteins in vitro. Importantly, Sr2+@SiO2 aerogel possessed a noteworthy function to promote liver soft tissue healing in vivo by releasing bioactive ions and providing a highly-interconnected porous structure to support vascular development and tissue regeneration. Overall, Sr2+@SiO2 aerogel has great potential for integrated rapid hemostasis and soft tissue healing, which is promising in soft tissue trauma therapy. STATEMENT OF SIGNIFICANCE: Non-compressible hemorrhage and soft tissue impairment are the main causes of mortality in emergency trauma. Inorganic aerogels with high porosity and outstanding flexibility can rapidly absorb blood to pro-coagulation and fill in irregular trauma without compression, but the low bioactivity limited the ability to promote soft tissue healing. Herein, SiO2-based inorganic nanofiber aerogels (M2+@SiO2, M=Ca, Mg, and Sr) were prepared by freeze-drying the mixture of bioactive silicates-deposited SiO2 nanofibers and SiO2 sol. M2+@SiO2 aerogels possessed high bioactivity and exhibited superior hemostatic performance in compressible and non-compressible bleeding model. Furthermore, Sr2+@SiO2 aerogel showed optimal bioactivities on cell responses and effectively promoted liver healing by releasing bioactive ions and providing highly-interconnected porous support structure for vascular development and tissue regeneration.

We have developed a single-cell assay that combines Cell Painting-a morphological profiling assay-with trajectory inference analysis. We have applied this morphological trajectory inference to the bi-potent HepaRG liver progenitor cell line allowing us to track liver cell fate and map small-molecule-induced changes using a morphological atlas of liver cell differentiation. Our overarching goal is to demonstrate the potential of Cell Painting to study biological processes as continuous trajectories at the single-cell level, enhancing resolution and biological understanding. This work has identified small-molecule Src family kinase inhibitors that promote the differentiation of HepaRG cells toward a hepatocyte-like lineage as well as primary human hepatic progenitor cells toward a hepatocyte-like phenotype in vitro. These findings could significantly advance research on liver cell regeneration mechanisms and facilitate the development of cell-based and small-molecule therapies.

Liver diseases pose significant health challenges, underscoring the importance of understanding liver regeneration mechanisms. Systemic adipose tissue is thought to be a primary source of lipids and energy during this process; however, empirical data on the effects of adipose tissue deficiency are limited. This study investigates the role of adipose tissue in liver regeneration, focusing on transient regeneration-associated steatosis (TRAS) and hepatocyte proliferation using a Seipin knockout mouse model that mimics severe human lipodystrophy. Additionally, the study explores therapeutic strategies through adipose tissue transplantation.

Male Seipin knockout (Seipin-/-) and wild-type (WT) mice underwent 2/3 partial hepatectomy (PHx). Liver and plasma samples were collected at various time points post-surgery. Histological assessments, lipid accumulation analyses and measurements of hepatocyte proliferation markers were conducted. Additionally, normal adipose tissue was transplanted into Seipin-/- mice to evaluate the restoration of liver regeneration.

Seipin-/- mice exhibited significantly reduced liver regeneration rates and impaired TRAS, as evidenced by histological and lipid measurements. While WT mice demonstrated extensive hepatocyte proliferation at 48 and 72 h post-PHx, characterised by increased mitotic cells, elevated proliferating cell nuclear antigen and Ki67 expression, Seipin-/- mice showed delayed hepatocyte proliferation. Notably, adipose tissue transplantation into Seipin-/- mice restored TRAS and improved liver regeneration and hepatocyte proliferation. Conversely, liver-specific overexpression of Seipin in Seipin-/- mice did not affect TRAS or liver regeneration, indicating that the observed effects are primarily due to adipose tissue deficiency rather than hepatic Seipin itself.

Systemic adipose tissue is essential for TRAS and effective liver regeneration following PHx. Its deficiency impairs these processes, while adipose tissue transplantation can restore normal liver function. These findings underscore the critical role of adipose tissue in liver recovery and suggest potential therapeutic strategies for liver diseases associated with lipodystrophies.

Seipin-/- mice, which lack adipose tissue, exhibit significantly impaired TRAS and delayed liver regeneration following partial hepatectomy. Transplantation of normal adipose tissue into Seipin-/- mice restores TRAS and enhances liver regeneration, highlighting the essential role of adipose tissue in these processes. Liver-specific overexpression of Seipin has no effect on TRAS and liver regeneration in Seipin-/- mice.

The liver is a vital organ responsible for numerous metabolic processes in the human body, including the metabolism of drugs and nutrients. After liver damage, the organ can rapidly return to its original size if the causative factor is promptly eliminated. However, when the harmful stimulus persists, the liver's regenerative capacity becomes compromised. Substantial theoretical feasibility has been demonstrated at the levels of gene expression, molecular interactions, and intercellular dynamics, complemented by numerous successful animal studies. However, a robust model and carrier that closely resemble human physiology are still lacking for translating these theories into practice. The potential for liver regeneration has been a central focus of ongoing research. Over the past decade, the advent of organoid technology has provided improved models and materials for advancing research efforts. Liver organoid technology represents a novel in vitro culture system. After several years of refinement, human liver organoids can now accurately replicate the liver's morphological structure, nutrient and drug metabolism, gene expression, and secretory functions, providing a robust model for liver disease research. Regenerative medicine aims to replicate human organ or tissue functions to repair or replace damaged tissues, restore their structure or function, or stimulate the regeneration of tissues or organs within the body. Liver organoids possess the same structure and function as liver tissue, offering the potential to serve as a viable replacement for the liver, aligning with the goals of regenerative medicine. This review examines the role of liver organoids in regenerative medicine.

Golgi protein 73 (GP73) is implicated in key pathogenic processes, particularly those related to inflammation and fibrogenesis. In the last years, its measurement has emerged as a promising biomarker for detection of liver fibrosis (LF), a common consequence of chronic liver disease that can progress to cirrhosis and eventually hepatocellular carcinoma. GP73 concentrations in blood appear significantly increased in LF patients, correlating with disease severity, making this biomarker a possible non-invasive alternative for detecting and monitoring this condition regardless of etiology. Understanding the molecular mechanisms involving GP73 expression could also lead to new therapeutic strategies aimed at modulating its synthesis or function to prevent or reverse LF. Despite its clinical potential, GP73 as a LF biomarker faces several challenges. The lack of demonstrated comparability among different assays as well as the lack of knowledge of individual variability can make difficult the result interpretation. Further research is therefore needed focusing on robust clinical validation of GP73 as a LF biomarker. Addressing analytical, biological, and clinical limitations will be critical to exploiting its potential for improving detection and monitoring of advanced LF.

While significant progress has been made in understanding various aspects of liver regeneration, the molecular mechanisms responsible for the initiation and termination of cell proliferation in the liver following massive tissue loss or injury of liver remain unknown. As it was previously shown, the loss of liver mass affects putative hepatocyte-specific mitogenic inhibitors in the blood. Although the presence of these putative inhibitors regulating precise liver regeneration has been described in numerous publications, they have never been identified. Extracellular vesicles (EVs) are nano-sized, membrane-limited structures secreted by cells into the extracellular space. Their proposed role is stable intercellular carriers of proteins and RNAs, predominantly micro-RNA, from secreted to recipient cells. Upon uptake by the recipient cells, EVs can significantly modulate their biological functions. In the present study, using in vivo and in vitro models, we demonstrate that hepatocyte proliferation and liver regeneration are regulated by EVs secreted by hepatocytes into the bloodstream. This regulation occurs through a negative feedback mechanism, which explains the precise regeneration of liver tissue after massive damage. We also demonstrate that an essential component of this mechanism is RNA carried by hepatocyte-derived EVs. Our findings open up a new and unexplored area of liver biology regarding the mechanisms involved in the precise regulation of liver regeneration after a massive tissue loss or injury. Further study of this mechanism will have a great influence on the development of new approaches to liver transplantation, various liver pathologies, and hepatic tumors.

The liver possesses an impressive capability to regenerate following various injuries. Given its profound implications for the treatment of liver diseases, which afflict millions globally, liver regeneration stands as a pivotal area of digestive organ research. Zebrafish (Danio rerio) has emerged as an ideal model organism in regenerative medicine, attributed to their remarkable ability to regenerate tissues and organs, including the liver. Many fantastic studies have been performed to explore the process of liver regeneration using zebrafish, especially the extreme hepatocyte injury model. Biliary-mediated liver regeneration was first discovered in the zebrafish model and then validated in mammalian models and human patients. Considering the notable expansion of biliary epithelial cells in many end-stage liver diseases, the promotion of biliary-mediated liver regeneration might be another way to treat these refractory liver diseases. To date, a comprehensive review discussing the current advancements in zebrafish liver regeneration models is lacking. Therefore, this review aims to investigate the utility of different zebrafish models in exploring liver regeneration, highlighting the genetic and cellular insights gained and discussing the potential translational impact on human health.

Even with advancement of medical technologies, liver transplantation still faces several major challenges. Hence, other treatment modalities are urgently needed for patients with end-stage liver disease. Stem cells from human exfoliated deciduous teeth (SHED) was discovered to have highly proliferative and pluripotent properties; including differentiation into hepatocyte-like cells. This study aims to investigate the capability of intrasplenic transplanted SHED and SHED-Hep cells in inducing proliferation of stem cells and native hepatocytes in order to accelerate liver regeneration in liver fibrosis mice models.

Three carbon tetrachloride (CCl4)-injured male mice groups were used in this study. Two of those groups were transplanted with either SHED or SHED-Hep, while the other did not undergo transplantation. One age- and sex- matched healthy mice group was used as control. All specimens were immunohistochemically stained with anti-Ki-67 antibodies and anti-proliferating cell nuclear antigen (PCNA) antibodies before counter stained with hematoxylin-eosin.

Anti-Ki-67 antibodies staining: at both 8 and 12 weeks, proliferating activity was predominantly seen on both SHED- and SHED-Hep-transplanted CCl4-injured mice groups, while control and non-transplanted CCl4-injured mice group showed little to no sign of proliferation activity. Anti-PCNA staining: at both 8 and 12 weeks, significant proliferating activity was detected by PCNA staining, mainly on stem cells population area on SHED- and SHED-Hep-treated group.

In conclusion, this study has provided the evidence that transplantation of SHED or SHED-Hep on liver-injured mice induced proliferation of both transplanted stem cells and native liver cells in order to accelerate liver regeneration.

Liver regeneration is an intricate pathophysiological process that has been a subject of great interest to the scientific community for many years. The capacity of liver regeneration is very critical for patients with liver diseases. Therefore, exploring the mechanisms of liver regeneration and finding good ways to improve it are very meaningful. Mesencephalic astrocyte-derived neurotrophic factor (MANF), a member of newly identified neurotrophic factors (NTFs) family, extensively expresses in the liver and has demonstrated cytoprotective effects during ER stress and inflammation. However, the role of MANF in liver regeneration remains unclear. Here, we used hepatocyte-specific MANF knockout (MANFHep-/-) mice to investigate the role of MANF in liver regeneration after 2/3 partial hepatectomy (PH). Our results showed that MANF expression was up-regulated in a time-dependent manner, and the peak level of mRNA and protein appeared at 24 h and 36 h after 2/3 PH, respectively. Notably, MANF knockout delayed hepatocyte proliferation, and the peak proliferation period was delayed by 24 h. Mechanistically, our in vitro results showed that MANF physically interacts with LRP5 and β-catenin, two essential components of Wnt/β-catenin pathway. Specifically, as a cofactor, MANF binds to the extracellular segment of LRP5 to activate Wnt/β-catenin signaling. On the other hand, MANF interacts with β-catenin to stabilize cytosolic β-catenin level and promote its nuclear translocation, which further enhance the Wnt/β-catenin signaling. We also found that MANF knockout does not affect the c-Met/β-catenin complex after 2/3 PH. In summary, our study confirms that MANF may serve as a novel hepatocyte factor that is closely linked to the activation of the Wnt/β-catenin pathway via intracellular and extracellular targets.

Hydrogel microfibers are hydrogel materials engineered into fiber structures. Techniques such as wet spinning, microfluidic spinning, and 3D bioprinting are often used to prepare microfibers due to their ability to precisely control the size, morphology, and structure of the microfibers. Microfibers with different structural morphologies have different functions; they provide a flow-through culture environment for cells to improve viability, and can also be used to induce the differentiation of cells such as skeletal muscle and cardiac muscle cells to eventually form functional organs in vitro through special morphologies. This Review introduces recent advances in microfluidics, 3D bioprinting, and wet spinning in the preparation of microfibers, focusing on the materials and fabrication methods. The applications of microfibers in tissue engineering are highlighted by summarizing their contributions in engineering biomimetic blood vessels, vascularized tissues, bone, heart, pancreas, kidney, liver, and fat. Furthermore, applications of engineered fibers in tissue repair and drug screening are also discussed.

Partial hepatectomy-induced liver regeneration causes the increase in relative blood flow rate within the liver, which dilates hepatic sinusoids and applies mechanical stretch on liver sinusoidal endothelial cells (LSECs). Heparin-binding EGF-like growth factor is a crucial growth factor during liver regeneration. We aimed to investigate whether this sinusoidal dilation-induced stretch promotes HB-EGF secretion in LSECs and what the related molecular mechanism is.

In vivo partial hepatectomy, ex vivo liver perfusion, and in vitro LSEC mechanical stretch were applied to detect HB-EGF expression in LSECs and hepatocyte proliferation. Knockdown or inhibition of mechanosensitive proteins was used to unravel the molecular mechanism in response to stretch. This stretch triggers amplitude-dependent and duration-dependent HB-EGF upregulation in LSECs, which is mediated by Yes-associated protein (YAP) nuclear translocation and binding to TEA domain family. This YAP translocation is achieved in 2 ways: On one hand, F-actin polymerization-mediated expansion of nuclear pores promotes YAP entry into nucleus passively. On the other hand, F-actin polymerization upregulates the expression of BAG family molecular chaperone regulator 3, which binds with YAP to enter the nucleus cooperatively. In this process, β1-integrin serves as a target mechanosensory in stretch-induced signaling pathways. This HB-EGF secretion-promoted liver regeneration after 2/3 partial hepatectomy is attenuated in endothelial cell-specific Yap1 -deficient mice.

Our findings indicate that mechanical stretch-induced HB-EGF upregulation in LSECs through YAP translocation can promote hepatocyte proliferation during liver regeneration through a mechanocrine manner, which deepens the understanding of the mechanical-biological coupling in liver regeneration.

Liver regeneration (LR) is a complex process encompassing three distinct phases: priming, proliferation phase and restoration, all influenced by various regulatory factors. After liver damage or partial resection, the liver tissue demonstrates remarkable restorative capacity, driven by cellular proliferation and repair mechanisms. The essential roles of non-coding RNAs (ncRNAs), predominantly microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNA (circRNA), in regulating LR have been vastly studied. Additionally, the impact of ncRNAs on LR and their abnormal expression profiles during this process have been extensively documented. Mechanistic investigations have revealed that ncRNAs interact with genes involved in proliferation to regulate hepatocyte proliferation, apoptosis and differentiation, along with liver progenitor cell proliferation and migration. Given the significant role of ncRNAs in LR, an in-depth exploration of their involvement in the liver's self-repair capacity can reveal promising therapeutic strategies for LR and liver-related diseases. Moreover, understanding the unique regenerative potential of the adult liver and the mechanisms and regulatory factors of ncRNAs in LR are crucial for improving current treatment strategies and exploring new therapeutic approaches for various liver-related diseases. This review provides a brief overview of the LR process and the ncRNA expression profiles during this process. Furthermore, we also elaborate on the specific molecular mechanisms through which multiple key ncRNAs regulate the LR process. Finally, based on the expression characteristics of ncRNAs and their interactions with proliferation-associated genes, we explore their potential clinical application, such as developing predictive indicators reflecting liver regenerative activity and manipulating LR processes for therapeutic purposes.

The normal liver has an extraordinary capacity of regeneration. However, this capacity is significantly impaired in steatotic livers. Emerging evidence indicates that metabolic dysfunction associated steatotic liver disease (MASLD) and liver regeneration share several key mechanisms. Some classical liver regeneration pathways, such as HGF/c-Met, EGFR, Wnt/β-catenin and Hippo/YAP-TAZ are affected in MASLD. Some recently established therapeutic targets for MASH such as the Thyroid Hormone (TH) receptors, Glucagon-like protein 1 (GLP1), Farnesoid X receptor (FXR), Peroxisome Proliferator-Activated Receptors (PPARs) as well as Fibroblast Growth Factor 21 (FGF21) are also reported to affect hepatocyte proliferation. With this review we aim to provide insight into common molecular pathways, that may ultimately enable therapeutic strategies that synergistically ameliorate steatohepatitis and improve the regenerating capacity of steatotic livers. With the recent rise of prolonged ex-vivo normothermic liver perfusion prior to organ transplantation such treatment is no longer restricted to patients undergoing major liver resection or transplantation, but may eventually include perfused (steatotic) donor livers or even liver segments, opening hitherto unexplored therapeutic avenues.

The liver is a complex organ that performs vital functions in the body. Despite its extraordinary regenerative capacity compared to other organs, exposure to chemical, infectious, metabolic and immunologic insults and toxins renders the liver vulnerable to inflammation, degeneration and fibrosis. Abnormal wound healing response mediated by aberrant signaling pathways causes chronic activation of hepatic stellate cells (HSCs) and excessive accumulation of extracellular matrix (ECM), leading to hepatic fibrosis and cirrhosis. Fibrosis plays a key role in liver carcinogenesis. Once thought to be irreversible, recent clinical studies show that hepatic fibrosis can be reversed, even in the advanced stage. Experimental evidence shows that removal of the insult or injury can inactivate HSCs and reduce the inflammatory response, eventually leading to activation of fibrolysis and degradation of ECM. Thus, it is critical to understand the role of gene-environment interactions in the context of liver fibrosis progression and regression in order to identify specific therapeutic targets for optimized treatment to induce fibrosis regression, prevent HCC development and, ultimately, improve the clinical outcome.

The phosphatidylinositol-4,5-bisphosphate 3-kinase delta isoform (Pik3cd), usually considered immune-specific, was unexpectedly identified as a gene potentially related to either regeneration and/or differentiation in animals lacking hepatocellular Integrin Linked Kinase (ILK). Since a specific inhibitor (Idelalisib, or CAL101) for the catalytic subunit encoded by Pik3cd (p110δ) has reported hepatotoxicity when used for treating chronic lymphocytic leukemia and other lymphomas, the authors aimed to elucidate whether there is a role for p110δ in normal liver function. To determine the effect on normal liver regeneration, partial hepatectomy (PHx) was performed using mice in which p110δ was first inhibited using CAL101. Inhibition led to over a 50% decrease in proliferating hepatocytes in the first 2 days after PHx. This difference correlated with phosphorylation changes in the HGF and EGF receptors (MET and EGFR, respectively) and NF-κB signaling. Ingenuity Pathway Analyses implicated C/EBPβ, HGF, and the EGFR heterodimeric partner, ERBB2, as three of the top 20 regulators downstream of p110δ signaling because their pathways were suppressed in the presence of CAL101 at 1 day post-PHx. A regulatory role for p110δ signaling in mouse and rat hepatocytes through MET and EGFR was further verified using hepatocyte primary cultures, in the presence or absence of CAL101. Combined, these data support a role for p110δ as a downstream regulator of normal hepatocytes when stimulated to proliferate.

Endothelial cells (ECs) form the inner lining of blood vessels and play crucial roles in angiogenesis. While it has been known for a long time that there are considerable differences among ECs from lymphatic and blood vessels, as well as among arteries, veins and capillaries, the full repertoire of endothelial diversity is only beginning to be elucidated. It has become apparent that the role of ECs is not just limited to their exchange functions. Indeed, a multitude of organ-specific functions, including release of growth factors, regulation of immune functions, have been linked to ECs. Recent years have seen a surge into the identification of spatiotemporal molecular and functional heterogeneity of ECs, supported by technologies such as single-cell RNA sequencing (scRNA-seq), lineage tracing and intersectional genetics. Together, these techniques have spurred the generation of epigenomic, transcriptomic and proteomic signatures of ECs. It is now clear that ECs across organs and in different vascular beds, but even within the same vessel, have unique molecular identities and employ specialized molecular mechanisms to fulfil highly specialized needs. Here, we focus on the molecular heterogeneity of the endothelium in different organs and pathological conditions.

The liver is a remarkable organ that can regenerate in response to injury. Depending on the extent of injury, the liver can undergo compensatory hyperplasia or fibrosis. Despite decades of research, the molecular mechanisms underlying these processes are poorly understood. Here, we developed a new model to study liver regeneration based on cryoinjury. To visualise liver regeneration at cellular resolution, we adapted the CUBIC tissue-clearing approach. Hepatic cryoinjury induced a localised necrotic and apoptotic lesion characterised by inflammation and infiltration of innate immune cells. After this initial phase, we observed fibrosis, which resolved as regeneration re-established homeostasis in 30 days. Importantly, this approach enables the comparison of healthy and injured parenchyma within an individual animal, providing unique advantages to previous models. In summary, the hepatic cryoinjury model provides a fast and reproducible method for studying the cellular and molecular pathways underpinning fibrosis and liver regeneration.

The liver has the remarkable capacity to regenerate. In the clinic, regeneration is induced by portal vein embolization, which redirects portal blood flow, resulting in liver hypertrophy in locations with increased blood supply, and atrophy of embolized segments. Here, we apply single-cell and single-nucleus transcriptomics on healthy, hypertrophied, and atrophied patient-derived liver samples to explore cell states in the regenerating liver. Our data unveils pervasive upregulation of genes associated with developmental processes, cellular adhesion, and inflammation in post-portal vein embolization liver, disrupted portal-central hepatocyte zonation, and altered cell subtype composition of endothelial and immune cells. Interlineage crosstalk analysis reveals mesenchymal cells as an interaction hub between immune and endothelial cells, and highlights the importance of extracellular matrix proteins in liver regeneration. Moreover, we establish tissue-scale iterative indirect immunofluorescence imaging for high-dimensional spatial analysis of perivascular microenvironments, uncovering changes to tissue architecture in regenerating liver lobules. Altogether, our data is a rich resource revealing cellular and histological changes in human liver regeneration.

Chronic liver injury induces liver cirrhosis and facilitates hepatocarcinogenesis. However, the effects of this condition on hepatocyte proliferation and differentiation are unclear. We showed that rodent hepatocytes display a ductular phenotype when they are cultured within a collagenous matrix. This process involves transdifferentiation without the emergence of hepatoblastic features and is at least partially reversible. During the ductular reaction in chronic liver diseases with progressive fibrosis, some hepatocytes, especially those adjacent to ectopic ductules, demonstrate ductular transdifferentiation, but the majority of increased ductules originate from the existing bile ductular system that undergoes extensive remodeling. In chronic injury, hepatocyte proliferation is weak but sustained, and most regenerative nodules in liver cirrhosis are composed of clonally proliferating hepatocytes, suggesting that a small fraction of hepatocytes maintain their proliferative capacity in chronic injury. In mouse hepatocarcinogenesis models, hepatocytes activate the expression of various fetal/neonatal genes, indicating that these cells undergo dedifferentiation. Hepatocyte-specific somatic integration of various oncogenes in mice demonstrated that hepatocytes may be the cells of origin for a broad spectrum of liver tumors through transdifferentiation and dedifferentiation. In conclusion, the phenotypic plasticity and heterogeneity of mature hepatocytes are important for understanding the pathogenesis of chronic liver diseases and liver tumors.

Liver regeneration induced by partial hepatectomy (PHx) has attracted intensive research interests due to the great significance for liver resection and transplantation. The zebrafish (Danio rerio) is an excellent model to study liver regeneration. In the fish subjected to PHx (the tip of the ventral lobe was resected), the lost liver mass could be fully regenerated in seven days. However, the regulatory mechanisms underlying the liver regeneration remain largely unknown. In this study, gene expression profiles during the regeneration of PHx-treated liver were explored by RNA sequencing (RNA-seq). The genes responsive to the injury of PHx treatment were identified and classified into different clusters based on the expression profiles. Representative gene ontology (GO) enrichments for the early responsive genes included hormone activity, ribosome biogenesis and rRNA processing, etc., while the late responsive genes were enriched in biological processes such as glutathione metabolic process, antioxidant activity and cellular detoxification. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichments were also identified for the differentially expressed genes (DEGs) between the time-series samples and the sham controls. The proteasome was overrepresented by the up-regulated genes at all of the sampling time points. Inhibiting proteasome activity by the application of MG132 to the fish enhanced the expression of Pcna (proliferating cell nuclear antigen), an indicator of hepatocyte proliferation after PHx. Our data provide novel insights into the molecular mechanisms underlying the regeneration of PHx-treated liver.

Liver regeneration is a complex process involving the crosstalk between parenchymal and non-parenchymal cells, especially macrophages. However, the underlying mechanisms remain incompletely understood. Here, we identify the E3 ubiquitin ligase TRIM26 as a crucial regulator of liver regeneration. Following partial hepatectomy or acute liver injury induced by carbon tetrachloride, Trim26 knockout mice exhibit enhanced hepatocyte proliferation compared to wild-type controls, while adeno-associated virus (AAV)-mediated overexpression of Trim26 reverses the promotional effects. Mechanistically, Trim26 deficiency promotes the recruitment of macrophages to the liver and their polarization towards pro-inflammatory M1 phenotype. These M1 macrophages secrete Wnts, including Wnt2, which subsequently stimulate hepatocyte proliferation through the activation of Wnt/β-catenin signaling. In hepatocytes, Trim26 knockdown reduces the ubiquitination and degradation of β-catenin, thereby further enhancing Wnt/β-catenin signaling. Pharmacological inhibition of Wnt/β-catenin pathway by ICG-001 or depletion of macrophages by clodronate liposomes diminishes the pro-regenerative effects of Trim26 deficiency. Moreover, bone marrow transplantation experiments provide evidence that Trim26 knockout in myeloid cells alone can also promote liver regeneration, highlighting the critical role of macrophage Trim26 in this process. Taken together, our study uncovers TRIM26 as a negative regulator of liver regeneration by modulating macrophage polarization and Wnt/β-catenin signaling in hepatocytes, providing a potential therapeutic target for promoting liver regeneration in clinical settings.

While significant progress has been made in understanding different aspects of liver regeneration, the molecular mechanisms responsible for the initiation and termination of cell proliferation in the liver after massive loss or injury of liver tissue remain unknown. The loss of liver mass affects tissue-specific mitogenic inhibitors in the blood, which in turn regulate the proliferation of remaining hepatocytes and liver regeneration. Although well described in a number of publications, which inhibitory substances or "sensor molecules" control the regeneration mechanisms to properly maintain liver size remain unknown. Extracellular vesicles (EVs) are nano-sized, membrane-limited structures secreted by cells into the extracellular space. Their proposed role is stable intercellular carriers of proteins and RNAs, mostly micro-RNA, from secreted to recipient cells. Taken up by the recipient cells, EVs can significantly modulate their biological functions. In the present study, using in vivo and in vitro models, we demonstrate that hepatocyte proliferation and liver regeneration are regulated by EVs secreted by hepatocytes into the bloodstream. This regulation is carried out through a negative feedback mechanism, which explains the very precise regeneration of liver tissue after massive damage. We also demonstrate that an essential component of this mechanism is RNA carried by hepatocyte-derived EVs. These findings open up a new and unexplored area of biology regarding the mechanisms involved in the homeostasis regulation of various constantly renewing tissues by maintaining the optimal size and correct ratio between differentiating and proliferating cells.

Hepatic stellate cells (HSCs) perform a significant function in liver regeneration (LR) by becoming active. We propose to investigate if activated HSCs enhance glycolysis via PFKFB3, an essential glycolytic regulator, and whether targeting this pathway could be beneficial for LR. The liver and isolated HSCs of mice subjected to 2/3 partial hepatectomy (PHx) exhibited a significant rise in PFKFB3 expression, as indicated by quantitative RT-PCR analyses and Western blotting. Also, the primary HSCs of mice subjected to PHx have a significant elevation of the glycolysis level. Knocking down PFKFB3 significantly diminished the enhancement of glycolysis by PDGF in human LX2 cells. The hepatocyte proliferation in mice treated with PHx was almost completely prevented when the PFKFB3 inhibitor 3PO was administered, emerging that PFKFB3 is essential in LR. Furthermore, there was a decline in mRNA expression of immediate early genes and proinflammatory cytokines. In terms of mechanism, both the p38 MAP kinase and ERK1/2 phosphorylation in LO2 cells and LO2 proliferation were significantly reduced by the conditioned medium (CM) obtained from LX2 cells with either PFKFB3 knockdown or inhibition. Compared to the control group, isolated hepatocytes from 3PO-treated mice showed decreased p38 MAP kinase and ERK1/2 phosphorylation and proliferation. Thus, LR after PHx involves the activation of PFKFB3 in HSCs, which enhances glycolysis and promotes lactate production, thereby facilitating hepatocyte proliferation via the p38/ERK MAPK signaling pathway.