Strategies for blocking the fibrogenic actions of connective tissue growth factor (CCN2): From pharmacological inhibition in vitro to targeted siRNA therapy in vivo

Abstract

Connective tissue growth factor (CCN2) is a major pro-fibrotic factor that frequently acts downstream of transforming growth factor beta (TGF-beta)-mediated fibrogenic pathways. Much of our knowledge of CCN2 in fibrosis has come from studies in which its production or activity have been experimentally attenuated. These studies, performed both in vitro and in animal models, have demonstrated the utility of pharmacological inhibitors (e.g. tumor necrosis factor alpha (TNF-alpha), prostaglandins, peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonists, statins, kinase inhibitors), neutralizing antibodies, antisense oligonucleotides, or small interfering RNA (siRNA) to probe the role of CCN2 in fibrogenic pathways. These investigations have allowed the mechanisms regulating CCN2 production to be more clearly defined, have shown that CCN2 is a rational anti-fibrotic target, and have established a framework for developing effective modalities of therapeutic intervention in vivo.

Fig. 1

a Production of targeted liposomes. A synthetic cyclic C*GRGDSPC* peptide was modified at its free N-terminus with the bifunctional linker succinimidyl-S-acetylthioacetate (SATA), the thiol group of which allowed for subsequent coupling to a maleimide linker in a modified lipid formulation (1,2-dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide]; 18:1 PE-MCC). Targeted liposomes were loaded with mouse CCN2 siRNA (sense: 5’-CCGCAAGAUUGGCGUGUGCtt; antisense: 5’-GCACACGCCAAUCUUGCGGtt; nucleotides 546-566). b CCN2 siRNA in targeted liposomes attenuates TGF-β-stimulated CCN2 production in mouse HSC in vitro. Day 2 primary mouse HSC were transfected for 6 hours with either 2 µM CCN2 siRNA or scrambled siRNA (SsiRNA) using targeted lipsomes. On the next day, the cells were stimulated with 20 ng/ml TGF-β for 24 hours and then analyzed for CCN2 protein or mRNA by, respectively, flow cytometry or RT-PCR (inset) as described (Leask et al.; Lawrencia et al.2009). CCN2 siRNA, but not SsiRNA, was effective in blocking TGF-β-induced CCN2 mRNA and protein

Fig. 2

Targeted CCN2 siRNA suppresses accumulation of αSMA or collagen deposition in preventative fibrosis model. Livers were sectioned and processed to detect αSMA (upper panel; brown stain) or collagen deposition (lower panel; blue stain) after administration of oil a or CCl₄ for 3 weeks b-e, the latter 2 weeks of which were given concurrent with either no therapy b, scrambled CCN2 siRNA in targeted liposomes c, CCN2 siRNA in non-targeted liposomes d, or CCN2 siRNA in targeted liposomes (E). CCl₄ was given i.m. as four 30 μl doses (1:1 CCl₄ : olive oil) per week. siRNA treatments (0.1 mg/kg) were given 4 times a week by i.p. injection. The staining intensity was assessed by image analysis (50 sections; 10 sections from each of 5 mice) and is shown in the bar graph. Data shown are indicative of the data from 3 separate experiments

Fig. 3

Targeted CCN2 siRNA prevents accumulation of αSMA or collagen deposition in curative fibrosis model Livers were sectioned and processed to detect αSMA (upper panel; brown stain) or collagen deposition (lower panel; blue stain) after administration of oil a or CCl₄ for 5 weeks b-e, the latter 2 weeks of which were given concurrent with either no therapy b, scrambled CCN2 siRNA in targeted liposomes c, CCN2 siRNA in non-targeted liposomes d, or CCN2 siRNA in targeted liposomes e. CCl₄ was given i.m. as four 30 μl doses (1:1 CCl₄:olive oil) per week. siRNA treatments (0.1 mg/kg) were given 4 times a week by i.p. injection. The staining intensity was assessed by image analysis (50 sections; 10 sections from each of 5 mice) and is shown in the bar graph. Data shown are indicative of the data from 3 separate experiments

Fig. 4

Expression of fibrosis-related genes. Livers were removed from mice after administration of oil a or CCl₄b-e for 3 weeks (preventative model; upper panel) or 5 weeks (curative model; lower panel). For the last 2 weeks in each model mice received no therapy b, scrambled CCN2 siRNA in targeted liposomes c, CCN2 siRNA in non-targeted liposomes d or CCN2 siRNA in targeted liposomes e. RNA was isolated and subjected to RT-PCR to establish the expression level of the indicated fibrotic marker genes as described (Lawrencia et al. 2009). Data are plotted relative to the expression of β-actin