KNOCKDOWN OF SPARC (SECRETED PROTEIN ACIDIC AND RICH IN CYSTEINE) ATTENUATES THE PROFIBROGENIC RESPONSE INDUCED BY TGF-B1 AND PDGF-BB IN HEPATIC STELLATE CELLS

CATALINA ATORRASAGASTI; LEONARDO HOFMAN; JORGE B. AQUINO; LAURA ALANIZ; MARIANA MALVICINI; FLAVIA PICCIONI; JUAN BAYO; MARIANA G. GARCIA; OSVALDO PODHAJCER; MARCELO SILVA; GUILLERMO MAZZOLINI

Congreso; The Liver Meeting 2010--AASLD's 61st Annual Meeting; 2010

SPARC is an extracellular matrix (ECM) proteininvolved in many biological processes and over-expressed incirrhosis. We demonstrated that in vivo down-regulation ofSPARC ameliorates fibrogenic response to chronic thioacetamideintoxication in rats (Camino et al., 2008). Neverthelesscellular mechanisms involved remain largely unknown Aim:to assess if down-regulation of SPARC could affect TGF-β1 andPDGF-mediated profibrogenic activity of hepatic stellate cells(HSC) M&M: Experiments were carried out in CFSC-2G andLX2 cells, an immortalized HSC line from cirrhotic rats and aspontaneous immortalized human HSC line, respectively.SPARC inhibition was assessed by a synthetic specific smallinterfering RNA constructs siRNA in CFSC-2G cells and by aplasmid contained a specific siRNA for LX2 cells. The efficacyof SPARC knock-down was assessed by real time PCR (qPCR).For transwell cell migration assays, non-modified or SPARCdown-regulated HSC were seeded on the upper chambers of48-well chamber plates; in the lower chamber, TGF-β1 or PDGFwere added as the chemoattractants. Adhesion to plastic wasalso performed. TGF-β1 production was quantified by ELISA oncultured HSC supernatants. Comparative analyses of rat ECMand adhesion molecules by qPCR arrays of non-modified HSCversus SPARC down-regulated HSC were performed. Differentiallyexpressed genes were confirmed by qPCR Results: BothCFSC-2G and LX2 cells showed a significant inhibition SPARCmRNA expression at 72 h post-transfection (60% and 80%,respectively). Down-regulation of SPARC by specific siRNA didnot affect proliferation and have minor effects on HSC apoptosis.However, SPARC knockdown increased the adhesion ofHSC to fibronectin and prevents HSC chemotaxis in response toFBS, PDFG and TGF-β1. In addition, SPARC siRNA transfectedcells were found to secrete significantly lower TGF-β1 levelsthan controls. Chemotaxis deficiency in SPARC siRNA-treatedHSC was partially reversed by exogenous application of TGF-β1 suggesting its involvement downstream of SPARC effects.Comparative analyses by qPCR arrays of non-modified HSCversus SPARC down-regulated HSC revealed an important numberof genes (26) whose expression levels were modified morethan 1.5-fold. Importantly, SPARC knockdown increased E-cadherinexpression and a concomitant decrease in N-cadherinexpression Conclusions: These data indicate that a reduction inSPARC levels alters the adhesion/detachment balance in HSCand/or increases their adhesion and this likely impairs the abilityof HSC to move and migrate in response to PDGF-BB. Ourresults further suggest SPARC as a novel target for the treatmentof liver fibrosis