Cellular and Molecular Mechanisms of Novel Therapies to Ameliorate Liver Sinusoidal Dysfunction in Cirrhotic Portal Hypertension

[spa] El aumento de la resistencia vascular intrahepática, debido principalmente a un aumento en el tono vascular junto a un cambio en la función endotelial hepática y a la fibrosis, es el factor principal en el desarrollo de la hipertensión portal en la cirrosis. La presente tesis doctoral se dirigió a revelar los mecanismos celulares y moleculares necesarios para la identificación de nuevas dianas terapéuticas así como investigar sobre el cross-talk entre las células hepáticas en condiciones estáticas y fisiológicas (shear stress). En el estudio 1 describimos como el bloqueo de la vía de señalización de la leptina en la cirrosis reduce significativamente la presión portal, probablemente por una mejora del tono vascular hepático debido a una mayor disponibilidad de óxido nítrico. Con el estudios 2 y 3 demostramos que las estatinas (simvastatina in primis) mejoran la función endotelial hepática, inhiben el desarrollo de fibrosis hepática e inducen también su regresión en modelos experimentales de cirrosis, a través del factor de transcripción Kruppel-like factor 2 (KLF2). KLF2 media los efectos de la simvastatina pero es también capaz de orquestar una respuesta vasoprotectora y anti-fibrótica eficiente por sí mismo, mejorando la cirrosis y disminuyendo la hipertensión portal. De hecho, la sobre-expresión de KLF2 (mediante el tratamiento con adenovirus o simvastatina) mejora la cirrosis en modelos experimentales in vitro e in vivo. Además vimos que la sobre- expresión de KLF2 mejora no solo el fenotipo de las células tratadas sino también el de las células cercanas, que no sobre-expresan KLF2, gracias a mecanismos paracrinos, bajo condiciones estáticas o mejor en condiciones fisiológicas. Globalmente, los estudios incluidos en la presente tesis doctoral proponen nuevos tratamientos para la hipertensión portal cirrótica e intentan explicar algunos de los mecanismos moleculares subyacentes los beneficios de las estatinas, proponiendo la regulación al alza de KLF2 como una nueva y altamente efectiva terapia para mejorar la microcirculación y promover la regresión de la cirrosis hepática. Por lo tanto, una acción sobre la vía de señalización de la leptina o el utilizo de las estatinas podrían ser una buena opción terapéutica para los pacientes con hipertensión portal y cirrosis.[eng] Increased intrahepatic vascular resistance (IHVR), mainly due to elevated vascular tone together with the maturation of hepatic fibrosis and the drop of the hepatic endothelial function, is the main factor in the development of portal hypertension (PH) in cirrhosis. This PhD thesis investigates the cellular and molecular mechanisms necessary for the identification of new therapeutic targets and evaluates the possible cross- talk between the hepatic cells in static and physiological conditions (shear stress). Study 1: Leptin is a pro-oxidant and pro-fibrotic hormone increased in patients with cirrhosis. Therefore, we evaluated whether leptin could influence the increased IHVR in PH. Cirrhotic animals with portal hypertension received the leptin receptor blocking antibody (OBR-Ab), or its vehicle, every other day for 1 week. In cirrhotic rats, leptin-receptor blockade significantly reduces portal pressure without modifying portal blood flow, suggesting a reduction in the intrahepatic resistance. Portal pressure reduction is associated with increased nitric oxide bioavailability and with decreased O2 levels and nitro-tyrosinated proteins. Thus, the blockade of the leptin signaling pathway in cirrhosis significantly reduces portal pressure, probably due to a nitric oxide-mediated reduction in the hepatic vascular tone. Study 2&3: Statins improve hepatic endothelial function and liver fibrosis in experimental models of cirrhosis, thus they have been proposed as therapeutic options to ameliorate portal hypertension syndrome. In cirrhosis, the transcription factor Kruppel-like factor 2 (KLF2) is early over-expressed during the progression of the disease, nevertheless it is not enough to slow down the development of vascular dysfunction. For this reason, we aimed to explore the effects, and the underlying mechanisms, of hepatic KLF2 over-expression in in vitro and in vivo models of liver cirrhosis. These studies demonstrate that KLF2 is induced by statins (simvastatin in primis) in both normal and cirrhotic liver sinusoidal endothelial cells (LSEC), orchestrating an efficient vasoprotective response, and in cirrhotic hepatic stellate cells (HSC), inducing their apoptosis and de-activating their phenotype partly via the activation of the nuclear factor Nrf2. Simvastatin vasoprotection and its anti-fibrotic properties are attenuated or even inhibited in the presence of isoprenoids or specific siRNA for KLF2, and are magnified in cells cultured (using bi and tri- dimensional systems) under physiological shear stress conditions. Indeed, LSEC over-expressing KLF2 induce quiescence of HSC through a KLF2–nitric oxide–guanylate cyclase-mediated paracrine mechanism as well as activated HSC over-expressing KLF2 reverse their phenotype and induce an amelioration of LSEC probably through a VEGF-mediated mechanism, but no paracrine interactions between hepatocytes and HSC are observed. All these effects are amplified in cells co-cultured in a sinusoidal-like environment. Pharmacological or adenoviral up-regulation of hepatic KLF2 expression provokes a profound amelioration in portal hypertension and cirrhosis, mainly due to hepatic stellate cells inactivation and apoptosis, together with reduction in hepatic oxidative stress and improvement in endothelial function. Specific induction of hepatic KLF2 within the liver represents an easy and highly effective strategy to promote liver cirrhosis regression and portal hypertension amelioration. Overall, the studies included in this PhD thesis propose new treatments for cirrhotic portal hypertension and try to explain some of the molecular mechanisms underlying the benefits of statins, suggesting the up- regulation of KLF2 as a new and highly effective therapy to improve hepatic microcirculation and promote regression of liver cirrhosis. Therefore, acting on leptin signaling pathway or using statins could be a good therapeutic option for patients with portal hypertension and cirrhosis

WIF1, A WNT PATHWAY INHIBITOR, IS SILENCED IN SYSTEMIC SCLEROSIS BY DNA DAMAGE: A MECHANISM LINKING DNA DAMAGE TO WNT AND FIBROSIS

Dysregulation of Wnt signaling is common in a variety of human malignancies, carcinogenesis, aging and fibrosis. Wnt signaling is tightly controlled by several negative regulators, such as WIF1 (Wnt inhibitor factor 1). Activation of canonical Wnt signaling has been recently found in fibrotic diseases included Systemic Sclerosis (SSc). Objectives The objective of the present work is to identify the mechanism responsible for the silencing of WIF1 in SSc. Methods Skin fibroblasts from SSc patients and normal controls were treated with bleomycin or ATM-HDAC inhibitors. Cells were transiently transfected with the siRNA against c-jun and ATF-3 with Lipofectamine (Invitrogen). Total RNA was isolated and reverse-transcribed, according to the manufacturer’s instructions (Bio-Rad). Quantitative real-time PCR reactions were performed using SYBR-Green PCR Master Mix (Bio-Rad). The relative expression levels were calculated using the 2-ΔΔCT method. To analyzed protein expression, cells were lysed with RIPA buffer and subjected to western blot with specific antibodies. Results Our data indicate that WIF1 is silenced by DNA damage and the check point kinase, ATM. Cell derived from SSc patients reactivate WIF1 expression if exposed to ATM or HDACI-III inhibitors. ROS and SSc immunoglobulins silence WIF1 expression via PDGF receptor, stimulate b-catenin accumulation by inducing ROS-dependent DNA damage. Bleomycin, a drug widely used to induce local skin fibrosis in vivo, silences WIF1 and stimulates Wnt signaling and its effects are suppressed by ATM or HDAC inhibitors. Silencing of WIF1 in normal cells amplifies Wnt signaling and increases collagen expression. As molecular actors that silence WIF1 in DNA damaged cells, we report that the knocking down of the expression of transcription factors ATF-3 and c-jun relieves WIF1 inhibition and dowregulates collagen expression in SSc cells. Bleomycin profibrotic phenotype is caused by activation of ATF-3 which silences WIF1 and amplifies Wnt signaling and by c-jun which cooperates with Wnt and stimulates collagen expression. Conclusions These results explain Wnt signaling hypertrophy in fibrotic disease, unveil a direct link between DNA damage and Wnt, and pave a novel route to treat fibrosis

Comparison between two packages for pectoral muscle removal on mammographic images

Pectoral muscle removal is a fundamental preliminary step in computer-aided diagnosis systems for full-field digital mammography (FFDM). Currently, two open-source publicly available packages (LIBRA and OpenBreast) provide algorithms for pectoral muscle removal within Matlab environment

Oxidative DNA damage induces the ATM-mediated transcriptional suppression of the Wnt inhibitor WIF-1 in systemic sclerosis and fibrosis

Systemic sclerosis (SSc) is an autoimmune disease characterized by extensive visceral organ and skin fibrosis. SSc patients have increased production of autoreactive antibodies and Wnt signaling activity. We found that expression of the gene encoding Wnt inhibitor factor 1 (WIF-1) was decreased in fibroblasts from SSc patient biopsies. WIF-1 deficiency in SSc patient cells correlated with increased abundance of the Wnt effector β-catenin and the production of collagen. Knocking down WIF-1 in normal fibroblasts increased Wnt signaling and collagen production. WIF-1 loss and DNA damage were induced in normal fibroblasts by either SSc patient immunoglobulins or oxidative DNA-damaging agents, such as ultraviolet light, hydrogen peroxide, or bleomycin. The DNA damage checkpoint kinase ataxia telangiectasia mutated (ATM) mediated WIF-1 silencing through the phosphorylation of the transcription factor c-Jun, which in turn activated the expression of the gene encoding activating transcription factor 3 (ATF3). ATF3 and c-Jun were recruited together with histone deacetylase 3 (HDAC3) to the WIF-1 promoter and inhibited WIF-1 expression. Preventing the accumulation of reactive oxygen species or inhibiting the activation of ATM, c-Jun, or HDACs restored WIF-1 expression in cultured SSc patient cells. Trichostatin A, an HDAC inhibitor, prevented WIF-1 loss, β-catenin induction, and collagen accumulation in an experimental fibrosis model. Our findings suggest that oxidative DNA damage induced by SSc autoreactive antibodies enables Wnt activation that contributes to fibrosis

Hepatic microcirculation and mechanisms of portal hypertension.

The liver microcirculatory milieu, mainly composed of liver sinusoidal endothelial cells (LSECs), hepatic stellate cells (HSCs) and hepatic macrophages, has an essential role in liver homeostasis, including in preserving hepatocyte function, regulating the vascular tone and controlling inflammation. Liver microcirculatory dysfunction is one of the key mechanisms that promotes the progression of chronic liver disease (also termed cirrhosis) and the development of its major clinical complication, portal hypertension. In the present Review, we describe the current knowledge of liver microcirculatory dysfunction in cirrhotic portal hypertension and appraise the preclinical models used to study the liver circulation. We also provide a comprehensive summary of the promising therapeutic options to target the liver microvasculature in cirrhosis