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

Abstract

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.

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.