Is there an effective therapy available for non-alcoholic fatty liver disease?

Non-alcoholic fatty liver disease (NAFLD) is defined as fat accumulation in the liver, ranging from simple steatosis to non-alcoholic steatohepatitis (NASH). Although it used to be considered a benign condition, nowadays it is known to be associated with liver injury and the development of end-stage liver disease. NAFLD is the hepatic manifestation of metabolic syndrome (MS) with an incidence rising in accordance with the increased prevalence of MS, the latter being considered the most common cause of liver enzyme elevation in Western countries. To date, no medications or surgical procedures have been approved for effective treatment of NAFLD, and all of the therapies tested so far must still be regarded as experimental. It is expected that, based on the large amount of data produced in the last few years and the ongoing large multicenter clinical trials, the effective treatment(s) for NASH will soon be defined. Meanwhile, lifestyle interventions and behavior therapy, the only treatments shown to be effective, must be introduced in daily clinical practice and, if possible, supported by public health programs

Transcriptional up-regulation of APE1/Ref-1 in hepatic tumor: Role in hepatocytes resistance to oxidative stress and apoptosis

OBJECTIVE: Human Hepatocellular Carcinoma (HCC) is the fifth most frequent neoplasm worldwide and the most serious complication of long-standing chronic liver diseases (CLD). Its development is associated with chronic inflammation and sustained oxidative stress. Deregulation of apurinic apyrimidinic endonuclease 1/redox effector factor 1 (APE1/Ref-1), a master regulator of cellular response to oxidative stress, has been associated with poor prognosis in several cancers including HCC. DESIGN: In the present study we investigated the APE1/Ref-1 mRNA levels in cirrhotic and HCC tissues obtained during HCC resection. The possible protective role of APE1/Ref-1 against oxidative stress and apoptosis was evaluated in vitro in immortalized human hepatocytes (IHH) over-expressing APE1/Ref-1. RESULTS: APE1/Ref-1 was up-regulated in HCC, regulation occurring at the transcriptional level. APE1/Ref-1 mRNA content increased with the progression of liver disease with the transcriptional up-regulation present in cirrhosis significantly increased in HCC. The up-regulation was higher in the less differentiated cancers. In vitro, over-expression of APE1/Ref-1 in normal hepatocytes conferred cell protection against oxidative stress and it was associated with BAX inhibition and escape from apoptosis. CONCLUSION: APE1/Ref-1 is up-regulated in HCC and this over-expression correlates with cancer aggressiveness. The up-regulation occurs at the transcriptional level and it is present in the earliest phases of hepatocarcinogenesis. The APE-1/Ref-1 over-expression is associated with hepatocyte survival and inhibits BAX activation and apoptosis. These data suggest a possible role of APE1/Ref-1 over-expression both in hepatocyte survival and HCC development calling attention to this molecule as a promising marker for HCC diagnosis and treatment

Studio della espressione di APE1/Ref-1 nel carcinoma epatocellulare. Valutazione del suo significato prognostico.

2007/2008ABSTRACT: Human Hepatocellular Carcinoma (HCC) is the fifth most frequent neoplasm worldwide and its incidence is rising in Western countries. HCC is frequently diagnosed at advanced stage and, until today, there are no effective treatments of this late phase. Therefore, research focused on finding new markers of progression and new molecular targets for therapy for HCC is of the utmost importance. Unquestionably, HCC is the most serious complication of long-standing chronic disease representing generally the final event of a liver disease usually originated decades earlier. Any chronic liver disease that causes cirrhosis is considered a risk factor for HCC development and this disease is characterized by chronic inflammation involving repeated rounds of necrosis and regeneration associated with sustained oxidative stress. Apurinic apyrimidinic endonuclease/redox effector factor 1 (APE1/Ref-1) is a multifunctional protein involved in redox regulation of transcription factors and acting as an endonuclease of the base excision repair (BER) pathway of DNA lesions. APE1/Ref-1 has been found to be up-regulated and abnormally localized in the cytoplasmic compartment of several cancer cells. Of notice is the finding that cytoplasmic localization is associated with a poor prognosis. Since at the time I started my thesis no data were available about the possible involvement of APE1/Ref-1 in HCC, the aim of this work was to investigate the expression and sub-cellular localization of APE1/Ref-1 in HCC. In addition, my aim was also to analyze the correlation of these parameters with the progression of the disease. To accomplish these points I analyzed human samples of HCC and cirrhosis (in vivo model) and an in vitro model constituted by HCC cell lines at different degree of differentiation. I also assessed the protective role of APE1/Ref-1 to oxidative damage as a function of its sub-cellular localization over-expressing, in normal hepatocytes, wild type and mutated forms of the protein that force its distribution to different cellular compartments. Using these approaches, we found that HCC is characterized by increased APE1/Ref-1 mRNA and protein levels and, that this up-regulation increases accordingly with tumor differentiation grading showing an association between tumor progression and APE1/Ref-1 levels. The increased amount of APE1/Ref-1 induces its nuclear and cytoplasmic accumulation in HCC. The cytoplasmic localization is peculiar of HCC and more frequently observed in poorly-differentiated cancers. The fact that APE1/Ref-1 accumulates in the cytoplasm of poorly-differentiated tumors and correlates with a shorter survival time after HCC resection points to a possible role of the APE1/Ref-1 sub-cellular localization as a prognostic marker of HCC aggressiveness. Thereafter, I demonstrated that APE1/Ref-1 over-expression in the normal hepatocyte cell line (IHH) enhances survival after oxidative damage induced by exposure to H2O2 and UV radiation although the different forms of the protein behave in a different ways. Wild type and truncated forms of APE1/Ref-1, that localize mainly in the cytoplasm and mitochondria, confer protection to H2O2 damage while the non-cleavable form, localized in the nucleus, does not. Conversely, all the mutants protect IHH from UV induced apoptosis. These data demonstrate for the first time that APE1/Ref-1 expression is deregulated in HCC, that its over-expression has a protective role in vitro and that APE1/Ref-1 cytoplasmic localization has a prognostic significance in vivo. We favor the conclusion that APE1/Ref-1 may be considered as a new prognostic marker for HCC aggressiveness. Its possible involvement in HCC development offer a new molecular key to elucidate the role of this protein in oxidative stress related hepatocarcinogenesis.RIASSUNTO: Il carcinoma primitivo del fegato è la quinta neoplasia più frequente al mondo e rappresenta la terza causa di morte per cancro. La sua incidenza negli ultimi anni è significativamente aumentata nei paesi occidentali e, nonostante i miglioramenti delle metodiche diagnostiche, la diagnosi di epatocarcinoma è spesso effettuata solo nella fase avanzata di malattia quando, le terapie al momento disponibili sono esclusivamente palliative. L’incremento di nuovi casi e la mancanza di terapia medica efficace, sottolineano la necessità di individuare nuovi marcatori prognostici e nuovi target terapeutici. Dal punto di vista fisiopatologico, il carcinoma epatocellulare rappresenta la complicanza più grave di una malattia cronica di fegato, generalmente iniziata molti anni prima della diagnosi di HCC, evoluta in cirrosi. La cirrosi epatica si sviluppa come conseguenza di un flogosi cronica che causa, cicli di necrosi e rigenerazione con un danno epatocitario sostenuto dallo stress ossidativo ed è considerata il fattore di rischio principale per lo sviluppo di HCC. APE1/Ref-1 (Apurinic apyrimidinic endonuclease/redox effector factor 1) è una proteina coinvolta nella regolazione dello stato redox cellular e agisce come co-attivatore di fattori trascrizionali e nel meccanismo BER (base excission repair) di riparo dei danni ossidativi al DNA. In molti tumori è stato dimostrata una up-regolazione di APE1/Ref-1 e in diversi casi la localizzazione citoplasmatica della proteina si è dimostrata correlare con una cattiva prognosi. Nel momento in cui ho cominciato il lavoro di tesi non era disponibile alcun dato sul possibile coinvolgimento di una alterata espressione di APE1/Ref-1 nel carcinoma primitivo del fegato. Gli obbiettivi di questa tesi sono stati, pertanto, la valutazione dell’espressione e del pattern di localizzazione cellulare di APE1/Ref-1 nel HCC in correlazione alla progressione della malattia e alla prognosi. In questa ottica ho valutato sia campioni di pazienti, diagnosticati affetti da HCC su cirrosi e trattati chirurgicamente, che un modello in vitro di epatocarcinoma a diversi gradi di differenziazione. Un ulteriore obbiettivo è stato quello di valutare l’eventuale effetto protettivo della overespressione di APE1/Ref-1 in una linea cellulare derivata da epatociti umani da fegato sano. In questo caso sono state overespresse differenti forme della proteina in modo da valutare il ruolo protettivo in relazione alle diverse localizzazioni intracellulari. Sulla base di questi modelli noi abbiamo osservato che l’epatocarcinoma è caratterizzato da un incremento dei livelli di mRNA e proteina di APE1/Ref-1 che cresce in modo inversamente proporzionale rispetto al grado di differenziazione del cancro. L’aumentata produzione della proteina si associa ad un accumulo della stessa sia nei nuclei che nei citoplasmi di HCC con livelli maggiori rispetto alla SLC, la localizzazione citoplasmatica nel tessuto tumorale si è dimostrata peculiare del cancro e più frequente negli HCC poco differenziati. L’osservazione che associa la maggiore frequenza della localizzazione citoplasmatica nei tumori poco differenziati e la correlazione di tale localizzazione con una ridotta sopravvivenza individua in APE1/Ref-1 un marker di aggressività di HCC. I dati ottenuti hanno inoltre dimostrato che la overespressione di APE1/Ref-1, seppur con peculiarità legate alle diverse forme, conferisce alle IHH una resistenza allo stress ossidativo indotto da H2O2 e radiazioni UV. La forma wild type e la forma tronca della proteina, che localizzano nel citoplasma, proteggono le cellule dal danno da H2O2, mentre la forma non clivabile, a localizzazione nucleare, non conferisce la stessa protezione. Per quanto riguarda invece il danno indotto da UV tutte le forme della proteina sono in grado di proteggere le cellule epatiche dall’apoptosi. Tutti questi dati dimostrano per la prima volta che l’espressione di APE1/Ref-1 è deregolata nel carcinoma epatocelluare e che la sua overespressione ha un ruolo protettivo in vitro. La localizzazione citoplasmatica nel tessuto tumorale della proteina ha significato prognostico. Pertanto, APE1/Ref-1 può essere considerato come un possibile nuovo marker prognostico di aggressività dell’epatocarcinoma. Il suo possibile coinvolgimento nello sviluppo di HCC apre nuove possibili strade nella comprensione del ruolo di questa proteina nei meccanismi che associano lo stress ossidativo all’epatocarcinogenesi

Small molecule membrane transporters in the mammalian podocyte: a pathogenic and therapeutic target

The intriguingly complex glomerular podocyte has been a recent object of intense study. Researchers have sought to understand its role in the pathogenesis of common proteinuric diseases such as minimal change disease and focal segmental glomerular sclerosis. In particular, considerable effort has been directed towards the anatomic and functional barrier to macromolecular filtration provided by the secondary foot processes, but little attention has been paid to the potential of podocytes to handle plasma proteins beyond the specialization of the slit diaphragm. Renal membrane transporters in the proximal tubule have been extensively studied for decades, particularly in relation to drug metabolism and elimination. Recently, uptake and efflux transporters for small organic molecules have also been found in the glomerular podocyte, and we and others have found that these transporters can engage not only common pharmaceuticals but also injurious endogenous and exogenous agents. We have also found that the activity of podocyte transporters can be manipulated to inhibit pathogen uptake and efflux. It is conceivable that podocyte transporters may play a role in disease pathogenesis and may be a target for future drug development

Warfarin-related nephropathy: possible role for the warfarin pharmacogenetic profile

Warfarin-related nephropathy (WRN) is a renal complication of warfarin treatment associated with over-anticoagulation. We describe a case of a 73-year-old man affected by chronic kidney disease, essential hypertension and atrial fibrillation treated with warfarin. The patient presented a rapid course of kidney failure after many episodes of over-anticoagulation, and renal biopsy demonstrated WRN. Interestingly, the patient's warfarin pharmacogenetic profile showed that he was warfarin sensitive. This is the first report describing the presence of gene polymorphisms affecting warfarin metabolism in a subject with a biopsy-proven WRN. The patient was treated with corticosteroids obtaining a partial clinical respons

Coupled Plasma Filtration Adsorption for Treatment of Capillary Leak Syndrome Superimposed to Acute Generalized Exanthematous Pustolosis: A Case Report

Coupled plasma filtration adsorption (CPFA) is an extracorporeal supportive therapy based on nonspecific adsorption of pro- and anti-inflammatory mediators combined with continuous renal replacement therapy. The main field of CPFA application is septic shock, and there are limited data about its efficacy in the treatment of other acute conditions characterized by a dysregulation in immune homeostasis. Capillary leak syndrome (CLS) defines a life-threatening condition sustained by hypercytokinemia and characterized by abrupt onset of increased capillary permeability leading to severe generalized edema and hypovolemic shock refractory to fluid administration. Therapy for CLS is not specific and, at present time, it consists in the use of steroids or intravenous immunoglobulins. We present the case of a 34-year-old woman who developed CLS superimposed to acute generalized exanthematous pustulosis after initiating therapy with hydroxychloroquine for undifferentiated connective tissue disease. CLS did not respond to steroids and intravenous immunoglobulins, while it was successfully treated with CPFA. This observation supports the possible role of CPFA in restoring a proper immunologic homeostasis not only in sepsis but also in other devastating conditions sustained by hypercytokinemia

Podocyte expression of membrane transporters involved in puromycin aminonucleoside-mediated injury.

Several complex mechanisms contribute to the maintenance of the intricate ramified morphology of glomerular podocytes and to interactions with neighboring cells and the underlying basement membrane. Recently, components of small molecule transporter families have been found in the podocyte membrane, but expression and function of membrane transporters in podocytes is largely unexplored. To investigate this complex field of investigation, we used two molecules which are known substrates of membrane transporters, namely Penicillin G and Puromycin Aminonucleoside (PA). We observed that Penicillin G pre-administration prevented both in vitro and in vivo podocyte damage caused by PA, suggesting the engagement of the same membrane transporters by the two molecules. Indeed, we found that podocytes express a series of transporters which are known to be used by Penicillin G, such as members of the Organic Anion Transporter Polypeptides (OATP/Oatp) family of influx transporters, and P-glycoprotein, a member of the MultiDrug Resistance (MDR) efflux transporter family. Expression of OATP/Oatp transporters was modified by PA treatment. Similarly, in vitro PA treatment increased mRNA and protein expression of P-glycoprotein, as well as its activity, confirming the engagement of the molecule upon PA administration. In summary, we have characterized some of the small molecule transporters present at the podocyte membrane, focusing on those used by PA to enter and exit the cell. Further investigation will be needed to understand precisely the role of these transporter families in maintaining podocyte homeostasis and in the pathogenesis of podocyte injury

Adsorption therapy and septic shock: a retrospective study comparing Coupled Plasma Filtration-Adsorption and CytoSorb

Background and Aims: Septic shock is a life-threatening organ dysfunction caused by a dysregulated host response to infection. Excessive release of cytokines and other inflammatory mediators is a key mechanism and cytokines adsorption therapies are applied in this context to restore a balanced immune homeostasis. Different adsorption techniques are available but there are no studies comparing these different adsorption approaches. The aim of this retrospective observational study was to compare Coupled Plasma Filtration Adsorption (CPFA) and CytoSorbTM in terms of hemodynamic and clinical response, mortality and renal function recover