Antagonizing the irreversible thrombomodulin-initiated proteolytic signaling alleviates age-related liver fibrosis via senescent cell killing.

Cellular senescence is a stress-induced, stable cell cycle arrest phenotype which generates a pro-inflammatory microenvironment, leading to chronic inflammation and age-associated diseases. Determining the fundamental molecular pathways driving senescence instead of apoptosis could enable the identification of senolytic agents to restore tissue homeostasis. Here, we identify thrombomodulin (THBD) signaling as a key molecular determinant of the senescent cell fate. Although normally restricted to endothelial cells, THBD is rapidly upregulated and maintained throughout all phases of the senescence program in aged mammalian tissues and in senescent cell models. Mechanistically, THBD activates a proteolytic feed-forward signaling pathway by stabilizing a multi-protein complex in early endosomes, thus forming a molecular basis for the irreversibility of the senescence program and ensuring senescent cell viability. Therapeutically, THBD signaling depletion or inhibition using vorapaxar, an FDA-approved drug, effectively ablates senescent cells and restores tissue homeostasis in liver fibrosis models. Collectively, these results uncover proteolytic THBD signaling as a conserved pro-survival pathway essential for senescent cell viability, thus providing a pharmacologically exploitable senolytic target for senescence-associated diseases.

Targeting senescent hepatocytes using the thrombomodulin-PAR1 inhibitor vorapaxar ameliorates NAFLD progression.

BACKGROUND AND AIMS: Senescent hepatocytes accumulate in parallel with fibrosis progression during NASH. The mechanisms that enable progressive expansion of nonreplicating cell populations and the significance of that process in determining NASH outcomes are unclear. Senescing cells upregulate thrombomodulin-protease-activated receptor-1 (THBD-PAR1) signaling to remain viable. Vorapaxar blocks the activity of that pathway. We used vorapaxar to determine if and how THBD-PAR1 signaling promotes fibrosis progression in NASH. APPROACH AND RESULTS: We evaluated the THBD-PAR1 pathway in liver biopsies from patients with NAFLD. Chow-fed mice were treated with viral vectors to overexpress p16 in hepatocytes and induce replicative senescence. Effects on the THBD-PAR1 axis and regenerative capacity were assessed; the transcriptome of p16-overexpressing hepatocytes was characterized, and we examined how conditioned medium from senescent but viable (dubbed "undead") hepatocytes reprograms HSCs. Mouse models of NASH caused by genetic obesity or Western diet/CCl 4 were treated with vorapaxar to determine effects on hepatocyte senescence and liver damage. Inducing senescence upregulates the THBD-PAR1 signaling axis in hepatocytes and induces their expression of fibrogenic factors, including hedgehog ligands. Hepatocyte THBD-PAR1 signaling increases in NAFLD and supports sustained hepatocyte senescence that limits effective liver regeneration and promotes maladaptive repair. Inhibiting PAR1 signaling with vorapaxar interrupts this process, reduces the burden of 'undead' senescent cells, and safely improves NASH and fibrosis despite ongoing lipotoxic stress. CONCLUSION: The THBD-PAR1 signaling axis is a novel therapeutic target for NASH because blocking this pathway prevents accumulation of senescing but viable hepatocytes that generate factors that promote maladaptive liver repair.

Rescue of glutaric aciduria type I in mice by liver-directed therapies.

Glutaric aciduria type I (GA-1) is an inborn error of metabolism with a severe neurological phenotype caused by the deficiency of glutaryl-coenzyme A dehydrogenase (GCDH), the last enzyme of lysine catabolism. Current literature suggests that toxic catabolites in the brain are produced locally and do not cross the blood-brain barrier. In a series of experiments using knockout mice of the lysine catabolic pathway and liver cell transplantation, we uncovered that toxic GA-1 catabolites in the brain originated from the liver. Moreover, the characteristic brain and lethal phenotype of the GA-1 mouse model was rescued by two different liver-directed gene therapy approaches: Using an adeno-associated virus, we replaced the defective Gcdh gene or we prevented flux through the lysine degradation pathway by CRISPR deletion of the aminoadipate-semialdehyde synthase (Aass) gene. Our findings question the current pathophysiological understanding of GA-1 and reveal a targeted therapy for this devastating disorder.

Targeting YAP-mediated HSC death susceptibility and senescence for treatment of liver fibrosis.

BACKGROUND AND AIMS: Liver fibrosis results from the accumulation of myofibroblasts (MFs) derived from quiescent HSCs, and yes-associated protein (YAP) controls this state transition. Although fibrosis is also influenced by HSC death and senescence, whether YAP regulates these processes and whether this could be leveraged to treat liver fibrosis are unknown. APPROACH AND RESULTS: YAP activity was manipulated in MF-HSCs to determine how YAP impacts susceptibility to pro-apoptotic senolytic agents or ferroptosis. Effects of senescence on YAP activity and susceptibility to apoptosis versus ferroptosis were also examined. CCl 4 -treated mice were treated with a ferroptosis inducer or pro-apoptotic senolytic to determine the effects on liver fibrosis. YAP was conditionally disrupted in MFs to determine how YAP activity in MF-HSC affects liver fibrosis in mouse models. Silencing YAP in cultured MF-HSCs induced HSC senescence and vulnerability to senolytics, and promoted ferroptosis resistance. Conversely, inducing HSC senescence suppressed YAP activity, increased sensitivity to senolytics, and decreased sensitivity to ferroptosis. Single-cell analysis of HSCs from fibrotic livers revealed heterogeneous sensitivity to ferroptosis, apoptosis, and senescence. In mice with chronic liver injury, neither the ferroptosis inducer nor senolytic improved fibrosis. However, selectively depleting YAP in MF-HSCs induced senescence and decreased liver injury and fibrosis. CONCLUSION: YAP determines whether MF-HSCs remain activated or become senescent. By regulating this state transition, Yap controls both HSC fibrogenic activity and susceptibility to distinct mechanisms for cell death. MF-HSC-specific YAP depletion induces senescence and protects injured livers from fibrosis. Clarifying determinants of HSC YAP activity may facilitate the development of novel anti-fibrotic therapies.

Hepatocyte Smoothened Activity Controls Susceptibility to Insulin Resistance and Nonalcoholic Fatty Liver Disease.

BACKGROUND & AIMS: Nonalcoholic steatohepatitis (NASH), a leading cause of cirrhosis, strongly associates with the metabolic syndrome, an insulin-resistant proinflammatory state that disrupts energy balance and promotes progressive liver degeneration. We aimed to define the role of Smoothened (Smo), an obligatory component of the Hedgehog signaling pathway, in controlling hepatocyte metabolic homeostasis and, thereby, susceptibility to NASH. METHODS: We conditionally deleted Smo in hepatocytes of healthy chow-fed mice and performed metabolic phenotyping, coupled with single-cell RNA sequencing (RNA-seq), to characterize the role of hepatocyte Smo in regulating basal hepatic and systemic metabolic homeostasis. Liver RNA-seq datasets from 2 large human cohorts were also analyzed to define the relationship between Smo and NASH susceptibility in people. RESULTS: Hepatocyte Smo deletion inhibited the Hedgehog pathway and promoted fatty liver, hyperinsulinemia, and insulin resistance. We identified a plausible mechanism whereby inactivation of Smo stimulated the mTORC1-SREBP1c signaling axis, which promoted lipogenesis while inhibiting the hepatic insulin cascade. Transcriptomics of bulk and single Smo-deficient hepatocytes supported suppression of insulin signaling and also revealed molecular abnormalities associated with oxidative stress and mitochondrial dysfunction. Analysis of human bulk RNA-seq data revealed that Smo expression was (1) highest in healthy livers, (2) lower in livers with NASH than in those with simple steatosis, (3) negatively correlated with markers of insulin resistance and liver injury, and (4) declined progressively as fibrosis severity worsened. CONCLUSIONS: The Hedgehog pathway controls insulin sensitivity and energy homeostasis in adult livers. Loss of hepatocyte Hedgehog activity induces hepatic and systemic metabolic stress and enhances susceptibility to NASH by promoting hepatic lipoxicity and insulin resistance.

Aging reduces liver resiliency by dysregulating Hedgehog signaling.

Older age is a major risk factor for damage to many tissues, including liver. Aging undermines resiliency and impairs liver regeneration. The mechanisms whereby aging reduces resiliency are poorly understood. Hedgehog is a signaling pathway with critical mitogenic and morphogenic functions during development. Recent studies indicate that Hedgehog regulates metabolic homeostasis in adult liver. The present study evaluates the hypothesis that Hedgehog signaling becomes dysregulated in hepatocytes during aging, resulting in decreased resiliency and therefore, impaired regeneration and enhanced vulnerability to damage. Partial hepatectomy (PH) was performed on young and old wild-type mice and Smoothened (Smo)-floxed mice treated with viral vectors to conditionally delete Smo and disrupt Hedgehog signaling specifically in hepatocytes. Changes in signaling were correlated with changes in regenerative responses and compared among groups. Old livers had fewer hepatocytes proliferating after PH. RNA sequencing identified Hedgehog as a top downregulated pathway in old hepatocytes before and after the regenerative challenge. Deleting Smo in young hepatocytes before PH prevented Hedgehog pathway activation after PH and inhibited regeneration. Gene Ontogeny analysis demonstrated that both old and Smo-deleted young hepatocytes had activation of pathways involved in innate immune responses and suppression of several signaling pathways that control liver growth and metabolism. Hedgehog inhibition promoted telomere shortening and mitochondrial dysfunction in hepatocytes, consequences of aging that promote inflammation and impair tissue growth and metabolic homeostasis. Hedgehog signaling is dysregulated in old hepatocytes. This accelerates aging, resulting in decreased resiliency and therefore, impaired liver regeneration and enhanced vulnerability to damage.

Dysregulation of the ESRP2-NF2-YAP/TAZ axis promotes hepatobiliary carcinogenesis in non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD), the hepatic correlate of the metabolic syndrome, is a major risk factor for hepatobiliary cancer (HBC). Although chronic inflammation is thought to be the root cause of all these diseases, the mechanism whereby it promotes HBC in NAFLD remains poorly understood. Herein, we aim to evaluate the hypothesis that inflammation-related dysregulation of the ESRP2-NF2-YAP/TAZ axis promotes HB carcinogenesis. METHODS: We use murine NAFLD models, liver biopsies from patients with NAFLD, human liver cancer registry data, and studies in liver cancer cell lines. RESULTS: Our results confirm the hypothesis that inflammation-related dysregulation of the ESRP2-NF2-YAP/TAZ axis promotes HB carcinogenesis, supporting a model whereby chronic inflammation suppresses hepatocyte expression of ESRP2, an RNA splicing factor that directly targets and activates NF2, a tumor suppressor that is necessary to constrain YAP/TAZ activation. The resultant loss of NF2 function permits sustained YAP/TAZ activity that drives hepatocyte proliferation and de-differentiation. CONCLUSION: Herein, we report on a novel mechanism by which chronic inflammation leads to sustained activation of YAP/TAZ activity; this imposes a selection pressure that favors liver cells with mutations enabling survival during chronic oncogenic stress. LAY SUMMARY: Non-alcoholic fatty liver disease (NAFLD) increases the risk of hepatobiliary carcinogenesis. However, the underlying mechanism remains unknown. Our study demonstrates that chronic inflammation suppresses hepatocyte expression of ESRP2, an adult RNA splicing factor that activates NF2. Thus, inactive (fetal) NF2 loses the ability to activate Hippo kinases, leading to the increased activity of downstream YAP/TAZ and promoting hepatobiliary carcinogenesis in chronically injured livers.

Aging and Chronic Liver Disease.

Aging increases the incidence of chronic liver disease (CLD), worsens its prognosis, and represents the predominant risk factor for its development at all different stages. The hepatic sinusoid, which is fundamental for maintaining liver homeostasis, is composed by hepatocytes, liver sinusoidal endothelial cells, hepatic stellate cells, and hepatic macrophages. During CLD progression, hepatic cells suffer deregulations in their phenotype, which ultimately lead to disease development. The effects of aging on the hepatic sinusoid phenotype and function are not well understood, nevertheless, studies performed in experimental models of liver diseases and aging demonstrate alterations in all hepatic sinusoidal cells. This review provides an updated description of age-related changes in the hepatic sinusoid and discusses the implications for CLD development and treatment. Lastly, we propose aging as a novel therapeutic target to treat liver diseases and summarize the most promising therapies to prevent or improve CLD and extend healthspan.

Ischemia/Reperfusion Injury in the Aged Liver: The Importance of the Sinusoidal Endothelium in Developing Therapeutic Strategies for the Elderly.

The liver endothelium plays a key role in the progression and resolution of liver diseases in young and adult individuals. However, its role in older people remains unknown. We have herein evaluated the importance of the sinusoidal endothelium in the pathophysiology of acute liver injury, and investigated the applicability of simvastatin, in aged animals. Eighteen-months-old male Wistar rats underwent 60 minutes of partial warm ischemia followed by 2 hours of reperfusion (WIR). A group of aged rats received simvastatin for 3 days before WIR. Endothelial phenotype, parenchymal injury, oxidative and nitrosative stress, and fenestrae dynamics were analyzed. The effects of WIR and simvastatin were investigated in primary LSEC from aged animals. The results of this study demonstrated that WIR significantly damages the liver endothelium and its effects are markedly worse in old animals. WIR-aged livers exhibited reduced vasodilation and sinusoidal capillarization, associated with liver damage and cellular stress. Simvastatin prevented the detrimental effects of WIR in aged livers. In conclusion, the liver sinusoidal endothelium of old animals is highly vulnerable to acute insult, thus targeted protection is especially relevant in preventing liver damage. Simvastatin represents a useful therapeutic strategy in aging.

New Rat Model of Advanced NASH Mimicking Pathophysiological Features and Transcriptomic Signature of The Human Disease.

Non-alcoholic steatohepatitis (NASH) is a major cause of chronic liver disease. However, most available animal models fail to reflect the whole spectrum of the disease. Liver fibrosis and portal hypertension are the strongest prognostic markers in advanced NASH. We herein aimed at developing a new model of NASH in male rats, obtained using a multi-hit protocol that combines the administration of a high fat and high-cholesterol diet with CCl4 and phenobarbital. Following this protocol, rats showed the full characteristics of advanced human NASH after 10 weeks and NASH with cirrhosis by 24 weeks. Specifically, our NASH rats exhibited: steatosis and metabolic syndrome, lipotoxicity, hepatocellular ballooning necrosis, inflammation and importantly, marked hepatic fibrosis and significant portal hypertension. Furthermore, a whole transcriptomic analysis of liver tissue from our rat model using next generation sequencing was compared with human NASH and illustrated the similarity of this pre-clinical model with the human disease. Pathway enrichment analysis showed that NASH animals shared a relevant number of central pathways involved in NASH pathophysiology, such as those related with cell death, as well as inflammatory or matrix remodeling. The present study defines a pre-clinical model of moderate and advanced NASH that mimics the human disease, including pathophysiologic characteristics and transcriptomic signature.

Aging Influences Hepatic Microvascular Biology and Liver Fibrosis in Advanced Chronic Liver Disease.

Advanced chronic liver disease (aCLD) represents a major public health concern. aCLD is more prevalent and severe in the elderly, carrying a higher risk of decompensation. We aimed at understanding how aging may impact on the pathophysiology of aCLD in aged rats and humans and secondly, at evaluating simvastatin as a therapeutic option in aged animals. aCLD was induced in young (1 month) and old (16 months) rats. A subgroup of aCLD-old animals received simvastatin (5 mg/kg) or vehicle (PBS) for 15 days. Hepatic and systemic hemodynamic, liver cells phenotype and hepatic fibrosis were evaluated. Additionally, the gene expression signature of cirrhosis was evaluated in a cohort of young and aged cirrhotic patients. Aged animals developed a more severe form of aCLD. Portal hypertension and liver fibrosis were exacerbated as a consequence of profound deregulations in the phenotype of the main hepatic cells: hepatocytes presented more extensive cell-death and poorer function, LSEC were further capillarized, HSC over-activated and macrophage infiltration was significantly increased. The gene expression signature of cirrhosis significantly differed comparing young and aged patients, indicating alterations in sinusoidal-protective pathways and confirming the pre-clinical observations. Simvastatin administration for 15-day to aged cirrhotic rats improved the hepatic sinusoidal milieu, leading to significant amelioration in portal hypertension. This study provides evidence that aCLD pathobiology is different in aged individuals. As the median age of patients with aCLD is increasing, we propose a real-life pre-clinical model to develop more reliable therapeutic strategies. Simvastatin effects in this model further demonstrate its translational potential.

Emricasan Ameliorates Portal Hypertension and Liver Fibrosis in Cirrhotic Rats Through a Hepatocyte-Mediated Paracrine Mechanism.

In cirrhosis, liver microvascular dysfunction is a key factor increasing hepatic vascular resistance to portal blood flow, which leads to portal hypertension. De-regulated inflammatory and pro-apoptotic processes due to chronic injury play important roles in the dysfunction of liver sinusoidal cells. The present study aimed at characterizing the effects of the pan-caspase inhibitor emricasan on systemic and hepatic hemodynamics, hepatic cells phenotype, and underlying mechanisms in preclinical models of advanced chronic liver disease. We investigated the effects of 7-day emricasan on hepatic and systemic hemodynamics, liver function, hepatic microcirculatory function, inflammation, fibrosis, hepatic cells phenotype, and paracrine interactions in rats with advanced cirrhosis due to chronic CCl4 administration. The hepato-protective effects of emricasan were additionally investigated in cells isolated from human cirrhotic livers. Cirrhotic rats receiving emricasan showed significantly lower portal pressure than vehicle-treated animals with no changes in portal blood flow, indicating improved vascular resistance. Hemodynamic improvement was associated with significantly better liver function, reduced hepatic inflammation, improved phenotype of hepatocytes, liver sinusoidal endothelial cells, hepatic stellate cells and macrophages, and reduced fibrosis. In vitro experiments demonstrated that emricasan exerted its benefits directly improving hepatocytes' expression of specific markers and synthetic capacity, and ameliorated nonparenchymal cells through a paracrine mechanism mediated by small extracellular vesicles released by hepatocytes. Conclusion: This study demonstrates that emricasan improves liver sinusoidal microvascular dysfunction in cirrhosis, which leads to marked amelioration in fibrosis, portal hypertension and liver function, and therefore encourages its clinical evaluation in the treatment of advanced chronic liver disease.

Effects of aging on liver microcirculatory function and sinusoidal phenotype.

The socioeconomic and medical improvements of the last decades have led to a relevant increase in the median age of worldwide population. Although numerous studies described the impact of aging in different organs and the systemic vasculature, relatively little is known about liver function and hepatic microcirculatory status in the elderly. In this study, we aimed at characterizing the phenotype of the aged liver in a rat model of healthy aging, particularly focusing on the microcirculatory function and the molecular status of each hepatic cell type in the sinusoid. Moreover, major findings of the study were validated in young and aged human livers. Our results demonstrate that healthy aging is associated with hepatic and sinusoidal dysfunction, with elevated hepatic vascular resistance and increased portal pressure. Underlying mechanisms of such hemodynamic disturbances included typical molecular changes in the cells of the hepatic sinusoid and deterioration in hepatocyte function. In a specific manner, liver sinusoidal endothelial cells presented a dysfunctional phenotype with diminished vasodilators synthesis, hepatic macrophages exhibited a proinflammatory state, while hepatic stellate cells spontaneously displayed an activated profile. In an important way, major changes in sinusoidal markers were confirmed in livers from aged humans. In conclusion, our study demonstrates for the first time that aging is accompanied by significant liver sinusoidal deregulation suggesting enhanced sinusoidal vulnerability to chronic or acute injuries.

Resemblance of the human liver sinusoid in a fluidic device with biomedical and pharmaceutical applications.

Maintenance of the complex phenotype of primary hepatocytes in vitro represents a limitation for developing liver support systems and reliable tools for biomedical research and drug screening. We herein aimed at developing a biosystem able to preserve human and rodent hepatocytes phenotype in vitro based on the main characteristics of the liver sinusoid: unique cellular architecture, endothelial biodynamic stimulation, and parenchymal zonation. Primary hepatocytes and liver sinusoidal endothelial cells (LSEC) were isolated from control and cirrhotic human or control rat livers and cultured in conventional in vitro platforms or within our liver-resembling device. Hepatocytes phenotype, function, and response to hepatotoxic drugs were analyzed. Results evidenced that mimicking the in vivo sinusoidal environment within our biosystem, primary human and rat hepatocytes cocultured with functional LSEC maintained morphology and showed high albumin and urea production, enhanced cytochrome P450 family 3 subfamily A member 4 (CYP3A4) activity, and maintained expression of hepatocyte nuclear factor 4 alpha (hnf4α) and transporters, showing delayed hepatocyte dedifferentiation. In addition, differentiated hepatocytes cultured within this liver-resembling device responded to acute treatment with known hepatotoxic drugs significantly different from those seen in conventional culture platforms. In conclusion, this study describes a new bioengineered device that mimics the human sinusoid in vitro, representing a novel method to study liver diseases and toxicology.

Liraglutide improves liver microvascular dysfunction in cirrhosis: Evidence from translational studies.

Hepatic stellate cells (HSC) play a key role in the development of chronic liver disease (CLD). Liraglutide, well-established in type 2 diabetes, showed anti-inflammatory and anti-oxidant properties. We evaluated the effects of liraglutide on HSC phenotype and hepatic microvascular function using diverse pre-clinical models of CLD. Human and rat HSC were in vitro treated with liraglutide, or vehicle, and their phenotype, viability and proliferation were evaluated. In addition, liraglutide or vehicle was administered to rats with CLD. Liver microvascular function, fibrosis, HSC phenotype and sinusoidal endothelial phenotype were determined. Additionally, the effects of liraglutide on HSC phenotype were analysed in human precision-cut liver slices. Liraglutide markedly improved HSC phenotype and diminished cell proliferation. Cirrhotic rats receiving liraglutide exhibited significantly improved liver microvascular function, as evidenced by lower portal pressure, improved intrahepatic vascular resistance, and marked ameliorations in fibrosis, HSC phenotype and endothelial function. The anti-fibrotic effects of liraglutide were confirmed in human liver tissue and, although requiring further investigation, its underlying molecular mechanisms suggested a GLP1-R-independent and NF-κB-Sox9-dependent one. This study demonstrates for the first time that liraglutide improves the liver sinusoidal milieu in pre-clinical models of cirrhosis, encouraging its clinical evaluation in the treatment of chronic liver disease.

Pathophysiology and a Rational Basis of Therapy.

Portal hypertension is a common complication of chronic liver disease. Its relevance comes from the fact that it determines most complications leading to death or liver transplantation in patients with cirrhosis of the liver: bleeding from esophageal or gastric varices, ascites and renal dysfunction, sepsis and hepatic encephalopathy. Portal hypertension results from increased resistance to portal blood flow through the cirrhotic liver. This is caused by two mechanisms: (1) distortion of the liver vascular architecture due to the liver disease causing structural abnormalities (nodule formation, remodeling of liver sinusoids, fibrosis, angiogenesis and vascular occlusion), and (2) increased hepatic vascular tone due to sinusoidal endothelial dysfunction, which results in a defective production of endogenous vasodilators, mainly nitric oxide (NO), and increased production of vasoconstrictors (thromboxane A2, cysteinyl leukotrienes, angiotensin II, endothelins and an activated adrenergic system). Hepatic endothelial dysfunction occurs early in the course of chronic liver disease as a consequence of inflammation and oxidative stress, and determines loss of the normal phenotype of liver sinusoidal endothelial cells (LSECs) that become proliferative, prothrombotic, proinflammatory and vasoconstrictor. The cross-talk between LSECs and hepatic stellate cells (HSCs) induces activation of the latter, which in turn proliferate, migrate and increase collagen deposition around the sinusoids, contributing to fibrogenesis, architectural disruption and angiogenesis, which further increase the hepatic vascular resistance and worsen liver failure by interfering with the blood perfusion of the liver parenchyma. An additional factor further worsening portal hypertension is an increased blood flow through the portal system due to splanchnic vasodilatation. This is an adaptive response to decreased effective hepatocyte perfusion, and is maximal once portal pressure has increased sufficiently to promote the development of intrahepatic shunts and portal-systemic collaterals, including varices, through which portal blood flow bypasses the liver. In human portal hypertension collateralization and hyperdynamic circulation start at a portal pressure gradient >10 mm Hg. Rational therapy for portal hypertension aims at correcting these pathophysiological abnormalities: liver injury, fibrogenesis, increased hepatic vascular tone and splanchnic vasodilatation. Continuing liver injury may be counteracted specifically by etiological treatments (the best example being the direct-acting antivirals for hepatitis C viral infection), while architectural disruption and fibrosis can be ameliorated by a variety of antifibrotic drugs and antiangiogenic strategies. Several drugs in this category are currently under investigation in phase II-III randomized controlled trials. Sinusoidal endothelial dysfunction is ameliorated by statins as well as by other drugs increasing NO availability. It is of note that simvastatin has already been proven to be clinically effective in two randomized controlled trials. Splanchnic hyperemia can be counteracted by nonselective ß-blockers (NSBBs), vasopressin analogs and somatostatin analogs, drugs that until recently were the only available treatments for portal hypertension, but that are not very effective in the initial stages of cirrhosis. There is experimental and clinical evidence indicating that a more effective reduction of portal pressure is obtained by combining agents acting on these different pathways. It is likely that the treatment of portal hypertension will evolve to use etiological treatments together with antifibrotic agents and/or drugs improving sinusoidal endothelial function in the initial stages of cirrhosis (preprimary prophylaxis), while NSBBs will be added in advanced stages of the disease.

New cellular and molecular targets for the treatment of portal hypertension.

Portal hypertension (PH) is a common complication of chronic liver disease, and it determines most complications leading to death or liver transplantation in patients with liver cirrhosis. PH results from increased resistance to portal blood flow through the cirrhotic liver. This is caused by two mechanisms: (a) distortion of the liver vascular architecture and (b) hepatic microvascular dysfunction. Increment in hepatic resistance is latterly accompanied by splanchnic vasodilation, which further aggravates PH. Hepatic microvascular dysfunction occurs early in the course of chronic liver disease as a consequence of inflammation and oxidative stress and determines loss of the normal phenotype of liver sinusoidal endothelial cells (LSEC). The cross-talk between LSEC and hepatic stellate cells induces activation of the latter, which in turn proliferate, migrate and increase collagen deposition around the sinusoids, contributing to fibrogenesis, architectural disruption and angiogenesis. Therapy for PH aims at correcting these pathophysiological abnormalities: liver injury, fibrogenesis, increased hepatic vascular tone and splanchnic vasodilatation. Continuing liver injury may be counteracted specifically by etiological treatments, while architectural disruption and fibrosis can be ameliorated by a variety of anti-fibrogenic drugs and anti-angiogenic strategies. Sinusoidal endothelial dysfunction is ameliorated by statins and other drugs increasing NO availability. Splanchnic hyperemia can be counteracted by non-selective beta-blockers (NSBBs), vasopressin analogs and somatostatin analogs. Future treatment of portal hypertension will evolve to use etiological treatments together with anti-fibrotic agents and/or drugs improving microvascular function in initial stages of cirrhosis (pre-primary prophylaxis), while NSBBs will be added in advanced stages of the disease.

KLF2 exerts antifibrotic and vasoprotective effects in cirrhotic rat livers: behind the molecular mechanisms of statins.

OBJECTIVE: In the liver, the transcription factor, Kruppel-like factor 2 (KLF2), is induced early during progression of cirrhosis to lessen the development of vascular dysfunction; nevertheless, its endogenous expression results insufficient to attenuate establishment of portal hypertension and aggravation of cirrhosis. Herein, we aimed to explore the effects and the underlying mechanisms of hepatic KLF2 overexpression in in vitro and in vivo models of liver cirrhosis. DESIGN: Activation phenotype was evaluated in human and rat cirrhotic hepatic stellate cells (HSC) treated with the pharmacological inductor of KLF2 simvastatin, with adenovirus codifying for this transcription factor (Ad-KLF2), or vehicle, in presence/absence of inhibitors of KLF2. Possible paracrine interactions between parenchymal and non-parenchymal cells overexpressing KLF2 were studied. Effects of in vivo hepatic KLF2 overexpression on liver fibrosis and systemic and hepatic haemodynamics were assessed in cirrhotic rats. RESULTS: KLF2 upregulation profoundly ameliorated HSC phenotype (reduced α-smooth muscle actin, procollagen I and oxidative stress) partly via the activation of the nuclear factor (NF)-E2-related factor 2 (Nrf2). Coculture experiments showed that improvement in HSC phenotype paracrinally ameliorated liver sinusoidal endothelial cells probably through a vascular endothelial growth factor-mediated mechanism. No paracrine interactions between hepatocytes and HSC were observed. Cirrhotic rats treated with simvastatin or Ad-KLF2 showed hepatic upregulation in the KLF2-Nrf2 pathway, deactivation of HSC and prominent reduction in liver fibrosis. Hepatic KLF2 overexpression was associated with lower portal pressure (-15%) due to both attenuations in the increased portal blood flow and hepatic vascular resistance, together with a significant improvement in hepatic endothelial dysfunction. CONCLUSIONS: Exogenous hepatic KLF2 upregulation improves liver fibrosis, endothelial dysfunction and portal hypertension in cirrhosis.