Role of calcium integrin-binding protein 1 in the mechanobiology of the liver endothelium.

Liver sinusoidal endothelial cells (LSECs) dysfunction is a key process in the development of chronic liver disease (CLD). Progressive scarring increases liver stiffness in a winch-like loop stimulating a dysfunctional liver cell phenotype. Cellular stretching is supported by biomechanically modulated molecular factors (BMMFs) that can translocate into the cytoplasm to support mechanotransduction through cytoskeleton remodeling and gene transcription. Currently, the molecular mechanisms of stiffness-induced LSECs dysfunction remain largely unclear. Here we propose calcium- and integrin-binding protein 1 (CIB1) as BMMF with crucial role in LSECs mechanobiology in CLD. CIB1 expression and translocation was characterized in healthy and cirrhotic human livers and in LSECs cultured on polyacrylamide gels with healthy and cirrhotic-like stiffnesses. Following the modulation of CIB1 with siRNA, the transcriptome was scrutinized to understand downstream effects of CIB1 downregulation. CIB1 expression is increased in LSECs in human cirrhosis. In vitro, CIB1 emerges as an endothelial BMMF. In human umbilical vein endothelial cells and LSECs, CIB1 expression and localization are modulated by stiffness-induced trafficking across the nuclear membrane. LSECs from cirrhotic liver tissue both in animal model and human disease exhibit an increased amount of CIB1 in cytoplasm. Knockdown of CIB1 in LSECs exposed to high stiffness improves LSECs phenotype by regulating the intracellular tension as well as the inflammatory response. Our results demonstrate that CIB1 is a key factor in sustaining cellular tension and stretching in response to high stiffness. CIB1 downregulation ameliorates LSECs dysfunction, enhancing their redifferentiation, and reducing the inflammatory response.

IRF3 regulates neuroinflammatory responses and the expression of genes associated with Alzheimer's disease.

The pathological role of interferon signaling is emerging in neuroinflammatory disorders, yet, the specific role of Interferon Regulatory Factor 3 (IRF3) in neuroinflammation remains poorly understood. Here, we show that global IRF3 deficiency delays TLR4-mediated signaling in microglia and attenuates the hallmark features of LPS-induced inflammation such as cytokine release, microglial reactivity, astrocyte activation, myeloid cell infiltration, and inflammasome activation. Moreover, expression of a constitutively active IRF3 (S388D/S390D: IRF3-2D) in microglia induces a transcriptional program reminiscent of the Activated Response Microglia and the expression of genes associated with Alzheimer's disease, notably apolipoprotein-e. Using bulk-RNAseq of IRF3-2D brain myeloid cells, we identified Z-DNA binding protein-1 (ZBP1) as a target of IRF3 that is relevant across various neuroinflammatory disorders. Lastly, we show IRF3 phosphorylation and IRF3-dependent ZBP1 induction in response to Aß in primary microglia cultures. Together, our results identify IRF3 as an important regulator of LPS and Aß -mediated neuroinflammatory responses and highlight IRF3 as a central regulator of disease-specific gene activation in different neuroinflammatory diseases.

4 in 1: Antibody-free protocol for isolating the main hepatic cells from healthy and cirrhotic single rat livers.

Liver cells isolated from pre-clinical models are essential tools for studying liver (patho)physiology, and also for screening new therapeutic options. We aimed at developing a new antibody-free isolation method able to obtain the four main hepatic cell types (hepatocytes, liver sinusoidal endothelial cells [LSEC], hepatic macrophages [HMΦ] and hepatic stellate cells [HSC]) from a single rat liver. Control and cirrhotic (CCl4 and TAA) rat livers (n = 6) were perfused, digested with collagenase and mechanically disaggregated obtaining a multicellular suspension. Hepatocytes were purified by low revolution centrifugations while non-parenchymal cells were subjected to differential centrifugation. Two different fractions were obtained: HSC and mixed LSEC + HMΦ. Further LSEC and HMΦ enrichment was achieved by selective adherence time to collagen-coated substrates. Isolated cells showed high viability (80%-95%) and purity (>95%) and were characterized as functional: hepatocytes synthetized albumin and urea, LSEC maintained endocytic capacity and in vivo fenestrae distribution, HMΦ increased expression of inflammatory markers in response to LPS and HSC were activated upon in vitro culture. The 4 in 1 protocol allows the simultaneous isolation of highly pure and functional hepatic cell sub-populations from control or cirrhotic single livers without antibody selection.

Impaired endothelial autophagy promotes liver fibrosis by aggravating the oxidative stress response during acute liver injury.

BACKGROUND & AIMS: Endothelial dysfunction plays an essential role in liver injury, yet the phenotypic regulation of liver sinusoidal endothelial cells (LSECs) remains unknown. Autophagy is an endogenous protective system whose loss could undermine LSEC integrity and phenotype. The aim of our study was to investigate the role of autophagy in the regulation of endothelial dysfunction and the impact of its manipulation during liver injury. METHODS: We analyzed primary isolated LSECs from Atg7control and Atg7endo mice as well as rats after CCl4 induced liver injury. Liver tissue and primary isolated stellate cells were used to analyze liver fibrosis. Autophagy flux, microvascular function, nitric oxide bioavailability, cellular superoxide content and the antioxidant response were evaluated in endothelial cells. RESULTS: Autophagy maintains LSEC homeostasis and is rapidly upregulated during capillarization in vitro and in vivo. Pharmacological and genetic downregulation of endothelial autophagy increases oxidative stress in vitro. During liver injury in vivo, the selective loss of endothelial autophagy leads to cellular dysfunction and reduced intrahepatic nitric oxide. The loss of autophagy also impairs LSECs ability to handle oxidative stress and aggravates fibrosis. CONCLUSIONS: Autophagy contributes to maintaining endothelial phenotype and protecting LSECs from oxidative stress during early phases of liver disease. Selectively potentiating autophagy in LSECs during early stages of liver disease may be an attractive approach to modify the disease course and prevent fibrosis progression. LAY SUMMARY: Liver endothelial cells are the first liver cell type affected after any kind of liver injury. The loss of their unique phenotype during injury amplifies liver damage by orchestrating the response of the liver microenvironment. Autophagy is a mechanism involved in the regulation of this initial response and its manipulation can modify the progression of liver damage.

Patients With Signs of Advanced Liver Disease and Clinically Significant Portal Hypertension Do Not Necessarily Have Cirrhosis.

BACKGROUND & AIMS: Patients with hepatic venous pressure gradients (HVPGs) of 10 mm Hg or greater and chronic liver disease often are assumed to have cirrhosis. We investigated the association between HVPGs and cirrhosis, using histologic findings as the reference standard. We also assessed the prevalence and characteristics of patients with HVPGs of 10 mm Hg or greater without cirrhosis. METHODS: We performed a retrospective analysis of 157 consecutive patients, 89 with suspected cirrhosis and hepatic hemodynamic data collected from 2015 through 2017. Biopsy specimens collected had 10 or more portal tracts from each patient and were analyzed for features of cirrhosis. Biopsy specimens with histologic features of cirrhosis were excluded and the remaining biopsy specimens were re-reviewed by an expert pathologist. The fibrosis area was calculated digitally by image analysis. RESULTS: HVPG identified patients with cirrhosis with an area under the receiver operating characteristic curve of 0.879: 14 of 89 patients with HVPG of 10 mm Hg or greater (16%) had no histologic features of cirrhosis (METAVIR scores <4 and Ishak scores <6). The median HVPG was 11 mm Hg (range, 10-22 mm Hg). Based on METAVIR scores, 7 patients had fibrosis stage F3, 4 patients had fibrosis stage F2, and 3 patients had fibrosis stages F0 or F1. The mean area of fibrosis in livers was 16.2% ± 6.5%. All 14 patients had perisinusoidal fibrosis and 8 patients had hepatocyte ballooning. The most common diagnoses were nonalcoholic steatohepatitis (n = 5) and nodular regenerative hyperplasia (n = 4). An HVPG cut-off value of 12 mm Hg identified patients with cirrhosis with 92% specificity, misclassifying 5 patients with different etiologies of liver disease. CONCLUSIONS: In a retrospective analysis of 89 consecutive patients with chronic liver disease and an HVPG of 10 mm Hg or greater, 16% were not found to have cirrhosis upon biopsy analysis. Most of these patients had nonalcoholic steatohepatitis or nodular regenerative hyperplasia. Perisinusoidal fibrosis and hepatocyte ballooning might increase sinusoidal pressure. An HVPG cut-off value of 12 mm Hg or greater identified patients with cirrhosis with 92% specificity.

Cross-talk between autophagy and KLF2 determines endothelial cell phenotype and microvascular function in acute liver injury.

BACKGROUND & AIMS: The transcription factor Krüppel-like factor 2 (KLF2), inducible by simvastatin, confers endothelial vasoprotection. Considering recent data suggesting activation of autophagy by statins, we aimed to: 1) characterize the relationship between autophagy and KLF2 in the endothelium, 2) assess this relationship in acute liver injury (cold ischemia/reperfusion) and 3) study the effects of modulating KLF2-autophagy in vitro and in vivo. METHODS: Autophagic flux, the vasoprotective KLF2 pathway, cell viability and microvascular function were assessed in endothelial cells and in various pre-clinical models of acute liver injury (cold storage and warm reperfusion). RESULTS: Positive feedback between autophagy and KLF2 was observed in the endothelium: KLF2 inducers, pharmacological (statins, resveratrol, GGTI-298), biomechanical (shear stress) or genetic (adenovirus containing KLF2), caused endothelial KLF2 overexpression through a Rac1-rab7-autophagy dependent mechanism, both in the specialized liver sinusoidal endothelial cells (LSEC) and in human umbilical vein endothelial cells. In turn, KLF2 induction promoted further activation of autophagy. Cold ischemia blunted autophagic flux. Upon reperfusion, LSEC stored in University of Wisconsin solution did not reactivate autophagy, which resulted in autophagosome accumulation probably due to impairment in autophagosome-lysosome fusion, ultimately leading to increased cell death and microvascular dysfunction. Simvastatin pretreatment maintained autophagy (through the upregulation of rab7), resulting in increased KLF2, improved cell viability, and ameliorated hepatic damage and microvascular function. CONCLUSIONS: We herein describe for the first time the complex autophagy-KLF2 relationship, modulating the phenotype and survival of the endothelium. These results help understanding the mechanisms of protection conferred by KLF2-inducers, such as simvastatin, in hepatic vascular disorders. LAY SUMMARY: Autophagy and the transcription factor KLF2 share a common activation pathway in the endothelium, being able to regulate each other. Statins maintain microvascular function through the inhibition of Rac1, which consequently liberates Rab7, activates autophagy and increments the expression of KLF2.

Resistin and visfatin in steatotic and non-steatotic livers in the setting of partial hepatectomy under ischemia-reperfusion.

BACKGROUND & AIMS: This study examined whether the regulation of resistin and visfatin could reduce damage and improve regeneration in both steatotic and non-steatotic livers undergoing partial hepatectomy under ischemia-reperfusion, a procedure commonly applied in clinical practice to reduce bleeding. METHODS: Resistin and visfatin were pharmacologically modulated in lean and obese animals undergoing partial hepatectomy under ischemia-reperfusion. RESULTS: No evident role for these adipocytokines was observed in non-steatotic livers. However, obese animals undergoing liver surgery showed increased resistin in liver and plasma, without changes in adipose tissue, together with visfatin downregulation in liver and increment in plasma and adipose tissue. Endogenous resistin maintains low levels of visfatin in the liver by blocking its hepatic uptake from the circulation, thus regulating the visfatin detrimental effects on hepatic damage and regenerative failure. Indeed, the administration of anti-resistin antibodies increased hepatic accumulation of adipocyte-derived visfatin, exacerbating damage and regenerative failure. Interestingly, treatment with anti-visfatin antibodies protected steatotic livers, and similar results were obtained with the concomitant inhibition of resistin and visfatin. Thus, when visfatin was inhibited, the injurious effects of anti-resistin antibodies disappeared. Herein we show that upregulation of visfatin increased NAD levels in the remnant steatotic liver, whereas visfatin inhibition decreased them. These later observations suggest that visfatin may favour synthesis of NAD instead of DNA and induces alterations in amino acid metabolism-urea cycle and NO production, overall negatively affecting liver viability. CONCLUSIONS: Our results indicate the clinical potential of visfatin blocking-based therapies in steatotic livers undergoing partial hepatectomy with ischemia-reperfusion.

Pathophysiology and therapeutic options for cirrhotic portal hypertension.

Portal hypertension represents the primary non-neoplastic complication of liver cirrhosis and has life-threatening consequences, such as oesophageal variceal bleeding, ascites, and hepatic encephalopathy. Portal hypertension occurs due to increased resistance of the cirrhotic liver vasculature to portal blood flow and is further aggravated by the hyperdynamic circulatory syndrome. Existing knowledge indicates that the profibrogenic phenotype acquired by sinusoidal cells is the initial factor leading to increased hepatic vascular tone and fibrosis, which cause increased vascular resistance and portal hypertension. Data also suggest that the phenotype of hepatic cells could be further impaired due to the altered mechanical properties of the cirrhotic liver itself, creating a deleterious cycle that worsens portal hypertension in the advanced stages of liver disease. In this Review, we discuss recent discoveries in the pathophysiology and treatment of cirrhotic portal hypertension, a condition with few pharmacological treatment options.

Sinusoidal communication in chronic liver disease.

The liver sinusoid, mainly composed of liver sinusoidal endothelial cells, hepatic macrophages and hepatic stellate cells, shapes the hepatic vasculature and is key maintaining liver homeostasis and function. During chronic liver disease (CLD), the function of sinusoidal cells is impaired, being directly involved in the progression of liver fibrosis, cirrhosis, and main clinical complications including portal hypertension and hepatocellular carcinoma. In addition to their roles in liver diseases pathobiology, sinusoidal cells' paracrine communication or cross-talk is being studied as a mechanism of disease but also as a remarkable target for treatment. The aim of this review is to gather current knowledge of intercellular signalling in the hepatic sinusoid during the progression of liver disease. We summarise studies developed in pre-clinical models of CLD, specially emphasizing those pathways characterized in human-based clinically relevant models. Finally, we describe pharmacological treatments targeting sinusoidal communication as promising options to treat CLD and its clinical complications.

IRF3 regulates neuroinflammatory responses and the expression of genes associated with Alzheimer's disease.

The pathological role of interferon signaling is emerging in neuroinflammatory disorders, yet, the specific role of Interferon Regulatory Factor 3 (IRF3) in neuroinflammation remains poorly understood. Here, we show that global IRF3 deficiency delays TLR4-mediated signaling in microglia and attenuates the hallmark features of LPS-induced inflammation such as cytokine release, microglial reactivity, astrocyte activation, myeloid cell infiltration, and inflammasome activation. Moreover, expression of a constitutively active IRF3 (S388D/S390D:IRF3-2D) in microglia induces a transcriptional program reminiscent of the Activated Response Microglia and the expression of genes associated with Alzheimer's Disease, notably apolipoprotein-e. Lastly, using bulk-RNAseq of IRF3-2D brain myeloid cells, we identified Z-DNA binding protein-1 as a target of IRF3 that is relevant across various neuroinflammatory disorders. Together, our results identify IRF3 as an important regulator of LPS-mediated neuroinflammatory responses and highlight IRF3 as a central regulator of disease-specific gene activation in different neuroinflammatory diseases.

Stiffness-induced modulation of ERG transcription factor in chronic liver disease.

Chronic liver disease (CLD) is characterised by liver sinusoidal endothelial cells (LSECs) dysfunction. Mechanical forces and inflammation are among the leading factors. ETS-related gene (ERG) is an endothelial-specific transcription factor, involved in maintaining cell quiescence and homeostasis. Our study aimed to understand the expression and modulation of ERG in CLD. ERG expression was characterised and correlated to clinical data in human liver cirrhosis at different disease stages. ERG dynamics in response to stiffness and inflammation were investigated in primary healthy and cirrhotic rat LSEC and in human umbilical vein endothelial cells (HUVECs). ERG is markedly downregulated in cirrhosis independently of disease stage or aetiology and its expression is modulated by substrate stiffness in ECs. Inflammation downregulates ERG in cells on physiological stiffness, but not on high stiffness, suggesting a complementary role of inflammation and stiffness in suppressing ERG. This study outlines ERG as an LSEC inflammation and stiffness-responsive transcription factor in cirrhosis.

Simvastatin maintains function and viability of steatotic rat livers procured for transplantation.

BACKGROUND & AIMS: Liver grafts obtained from healthy rat donors develop acute microcirculatory dysfunction due to cold-storage and warm-reperfusion injuries. These detrimental effects are avoided adding simvastatin to the cold-storage solution. Considering the importance of increasing organ donor pool for transplantation, we characterized whether simvastatin pretreatment can protect steatotic grafts from cold-storage and warm-reperfusion injuries. METHODS: Rats fed with high-fat diet received a single dose of simvastatin, or its vehicle, 30 min before liver procurement. Grafts were then cold stored for 0 h (control group) or 16 h and warm reperfused. At the end of the reperfusion period, hepatic vascular resistance, endothelial function, nitric oxide pathway, cell death, oxidative stress, autophagy, and liver injury were evaluated. Hepatic vascular resistance and endothelial function were determined in a group of simvastatin-treated livers in the presence of the nitric oxide synthase inhibitor L-NNA. RESULTS: Cold-stored rat steatotic livers exhibit increased hepatic vascular resistance and marked endothelial dysfunction, together with liver damage, oxidative stress, and low nitric oxide. Simvastatin markedly improved liver injury and prevented hepatic endothelial dysfunction. The beneficial effects of simvastatin were associated with cell death diminution, autophagy induction, and nitric oxide release. Statin-derived liver microcirculation protection was not observed when nitric oxide production was blunted. CONCLUSIONS: Pretreatment of steatotic liver donors with simvastatin shortly before procurement of the liver graft strongly protects both parenchymal and endothelial components of the liver after warm reperfusion. Our data reinforce the use of statins to protect liver grafts undergoing transplantation.

Mechanobiology of portal hypertension.

The interplay between mechanical stimuli and cellular mechanobiology orchestrates the physiology of tissues and organs in a dynamic balance characterized by constant remodelling and adaptative processes. Environmental mechanical properties can be interpreted as a complex set of information and instructions that cells read continuously, and to which they respond. In cirrhosis, chronic inflammation and injury drive liver cells dysfunction, leading to excessive extracellular matrix deposition, sinusoidal pseudocapillarization, vascular occlusion and parenchymal extinction. These pathological events result in marked remodelling of the liver microarchitecture, which is cause and result of abnormal environmental mechanical forces, triggering and sustaining the long-standing and progressive process of liver fibrosis. Multiple mechanical forces such as strain, shear stress, and hydrostatic pressure can converge at different stages of the disease until reaching a point of no return where the fibrosis is considered non-reversible. Thereafter, reciprocal communication between cells and their niches becomes the driving force for disease progression. Accumulating evidence supports the idea that, rather than being a passive consequence of fibrosis and portal hypertension (PH), mechanical force-mediated pathways could themselves represent strategic targets for novel therapeutic approaches. In this manuscript, we aim to provide a comprehensive review of the mechanobiology of PH, by furnishing an introduction on the most important mechanisms, integrating these concepts into a discussion on the pathogenesis of PH, and exploring potential therapeutic strategies.

Neuroblastoma RAS viral oncogene homolog (N-RAS) deficiency aggravates liver injury and fibrosis.

Progressive hepatic damage and fibrosis are major features of chronic liver diseases of different etiology, yet the underlying molecular mechanisms remain to be fully defined. N-RAS, a member of the RAS family of small guanine nucleotide-binding proteins also encompassing the highly homologous H-RAS and K-RAS isoforms, was previously reported to modulate cell death and renal fibrosis; however, its role in liver damage and fibrogenesis remains unknown. Here, we approached this question by using N-RAS deficient (N-RAS-/-) mice and two experimental models of liver injury and fibrosis, namely carbon tetrachloride (CCl4) intoxication and bile duct ligation (BDL). In wild-type (N-RAS+/+) mice both hepatotoxic procedures augmented N-RAS expression in the liver. Compared to N-RAS+/+ counterparts, N-RAS-/- mice subjected to either CCl4 or BDL showed exacerbated liver injury and fibrosis, which was associated with enhanced hepatic stellate cell (HSC) activation and leukocyte infiltration in the damaged liver. At the molecular level, after CCl4 or BDL, N-RAS-/- livers exhibited augmented expression of necroptotic death markers along with JNK1/2 hyperactivation. In line with this, N-RAS ablation in a human hepatocytic cell line resulted in enhanced activation of JNK and necroptosis mediators in response to cell death stimuli. Of note, loss of hepatic N-RAS expression was characteristic of chronic liver disease patients with fibrosis. Collectively, our study unveils a novel role for N-RAS as a negative controller of the progression of liver injury and fibrogenesis, by critically downregulating signaling pathways leading to hepatocyte necroptosis. Furthermore, it suggests that N-RAS may be of potential clinical value as prognostic biomarker of progressive fibrotic liver damage, or as a novel therapeutic target for the treatment of chronic liver disease.

Review article: vascular effects of PPARs in the context of NASH.

BACKGROUND: Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors known to regulate glucose and fatty acid metabolism, inflammation, endothelial function and fibrosis. PPAR isoforms have been extensively studied in metabolic diseases, including type 2 diabetes and cardiovascular diseases. Recent data extend the key role of PPARs to liver diseases coursing with vascular dysfunction, including nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). AIM: This review summarises and discusses the pathobiological role of PPARs in cardiovascular diseases with a special focus on their impact and therapeutic potential in NAFLD and NASH. RESULTS AND CONCLUSIONS: PPARs may be attractive for the treatment of NASH due to their liver-specific effects but also because of their efficacy in improving cardiovascular outcomes, which may later impact liver disease. Assessment of cardiovascular disease in the context of NASH trials is, therefore, of the utmost importance, both from a safety and efficacy perspective.

Regression of portal hypertension: underlying mechanisms and therapeutic strategies.

Portal hypertension is the main non-neoplastic complication of chronic liver disease, being the cause of important life-threatening events including the development of ascites or variceal bleeding. The primary factor in the development of portal hypertension is a pathological increase in the intrahepatic vascular resistance, due to liver microcirculatory dysfunction, which is subsequently aggravated by extra-hepatic vascular disturbances including elevation of portal blood inflow. Evidence from pre-clinical models of cirrhosis has demonstrated that portal hypertension and chronic liver disease can be reversible if the injurious etiological agent is removed and can be further promoted using pharmacological therapy. These important observations have been partially demonstrated in clinical studies. This paper aims at providing an updated review of the currently available data regarding spontaneous and drug-promoted regression of portal hypertension, paying special attention to the clinical evidence. It also considers pathophysiological caveats that highlight the need for caution in establishing a new dogma that human chronic liver disease and portal hypertension is reversible.

The Hepatic Sinusoid in Chronic Liver Disease: The Optimal Milieu for Cancer.

The liver sinusoids are a unique type of microvascular beds. The specialized phenotype of sinusoidal cells is essential for their communication, and for the function of all hepatic cell types, including hepatocytes. Liver sinusoidal endothelial cells (LSECs) conform the inner layer of the sinusoids, which is permeable due to the fenestrae across the cytoplasm; hepatic stellate cells (HSCs) surround LSECs, regulate the vascular tone, and synthetize the extracellular matrix, and Kupffer cells (KCs) are the liver-resident macrophages. Upon injury, the harmonic equilibrium in sinusoidal communication is disrupted, leading to phenotypic alterations that may affect the function of the whole liver if the damage persists. Understanding how the specialized sinusoidal cells work in coordination with each other in healthy livers and chronic liver disease is of the utmost importance for the discovery of new therapeutic targets and the design of novel pharmacological strategies. In this manuscript, we summarize the current knowledge on the role of sinusoidal cells and their communication both in health and chronic liver diseases, and their potential pharmacologic modulation. Finally, we discuss how alterations occurring during chronic injury may contribute to the development of hepatocellular carcinoma, which is usually developed in the background of chronic liver disease.

Novel therapeutics for portal hypertension and fibrosis in chronic liver disease.

Portal hypertension (PH) is the most common non-neoplastic complication of chronic liver disease, determining clinical complications that lead to death or liver transplantation. PH results from increased resistance to portal blood flow through the cirrhotic liver, which is due to hepatic fibrosis and microcirculatory dysfunction. The present review focuses on the pathophysiology of fibrosis and PH, describes currently used treatments, and critically discusses potential therapeutic options.