Biological Effects of Secreted Protein, Acidic and Rich in Cysteine (SPARC) Inhibition on Rat Hepatic Stellate Cells In Vitro

CATALINA ATORRASAGASTI; ALEJANDRA CAMINO; LAURA ALANIZ; DANIELA MACCIO; MARIANA MALVICINI; MIGUEL RIZZO,; PABLO MATAR; OSVALDO PODHAJCER; MARCELO SILVA; GUILLERMO MAZZOLINI

Boston, Massachusetts. USA

Congreso; The American Society of Gene Therapy (ASGT) 11th Annual Meeting.; 2008

The American Society of Gene Therapy (ASGT)

Introduction: Liver cirrhosis is characterized by the accumulation of extracellular matrix (ECM) that leads to disorganization of the parenchymal architecture and the subsequent liver failure. Activated hepatic stellate cells (HSC) are the primary cell responsible for the abnormal synthesis and deposition of ECM in hepatic fibrosis. SPARC is a 43-kDa ECM glycoprotein involved in many biological processes including cell adhesion, wound healing, differentiation and proliferation. It is upregulated in cirrhosis, mainly in HSC, although its role in the development of fibrosis remains unknown. We have previously observed that the administration of an adenovirus that encodes a SPARC antisense mRNA (AdasSPARC) in a rat model (Camino et al 2006) results in attenuation of liver fibrosis and decrease in number of activated HSC. Aim: To explore the mechanisms through which liver fibrosis is ameliorated upon SPARC inhibition treatment. Methods & Results: The in vitro experiments were carried out in CFSC-2G cells, an immortalized HSC line derived from cirrhotic rats. CFSC-2G cells expressed SPARC detected by RT-PCR and immunofluorescence. To evaluate the efficacy of SPARC knock-down, we measured SPARC mRNA by real time PCR (qPCR). AdasSPARC treatment showed >50% decrease in expression levels of SPARC at 5 days after infection when compared to controls. Since TGF-â1 is a key cytokine in liver fibrosis, we investigated whether SPARC modulations influence TGF-â1 release by HSC. TGF-â1 was determined in supernatants from non-infected and infected CFSC-2G cells by ELISA. Of note, inhibition of SPARC expression significantly decreased the amount of secreted TGF-â1 (MOI 62.5: 465pg/ml at day 3, and 1044pg/ml at day 5; MOI 125: 166pg/ml at day 3, and 926pg/ml at day 5) compared to Adâgal (MOI 62.5: 1327pg/ml at day 3, and 1777pg/ml at day 5; MOI 125: 1042pg/ml at day 3 and 2190pg/ml at day 5). In order to improve SPARC inhibition we tested specific small interfering RNAs (siRNA) constructs (siGENOME, Dharmacon). siRNA transfection efficiency was between 40% and 60%. Specific-siRNA strongly decreased the expression of SPARC at 72 h (80% reduction in the amount of SPARC mRNA vs. control). SPARC inhibition caused an increase in CFSC-2G proliferation assessed by 3H-Thymidine incorporation (2 fold compared to control siRNA). We next examined migration activity of SPARC siRNA transfected CFSC-2G cells (Transwell assay). Migration of CFSC-2G cells to FCS was significantly decreased after SPARC knock-down (p0,003). In addition, release of TGF-â1 from SPARC siRNA transfected cells was significantly reduced when compared to control siRNA-treated cells (1297pg/ml vs 3507pg/ml at day 3). Conclusions: Our results suggest that the downregulation of SPARC expression levels in vitro results in a decreased HSC proliferative activity, migration capability and production of TGF-â1. Therefore, SPARC could be considered as a novel target for the treatment of liver fibrosis.Keywords: Gene Correction/Modification; Adenovirus; RNAi and shRNA