Relaxin decreases the severity of established hepatic fibrosis in mice.

BACKGROUND & AIMS: Hepatic fibrosis is characterized by excess collagen deposition, decreased extracellular matrix degradation and activation of the hepatic stellate cells. The hormone relaxin has shown promise in the treatment of fibrosis in a number of tissues, but the effect of relaxin on established hepatic fibrosis is unknown. The aim of this study was to determine the effect of relaxin on an in vivo model after establishing hepatic fibrosis METHODS: Male mice were made fibrotic by carbon tetrachloride treatment for 4 weeks, followed by treatment with two doses of relaxin (25 or 75 µg/kg/day) or vehicle for 4 weeks, with continued administration of carbon tetrachloride. RESULTS: Relaxin significantly decreased total hepatic collagen and smooth muscle actin content at both doses, and suppressed collagen I expression at the higher dose. Relaxin increased the expression of the matrix metalloproteinases MMP13 and MMP3, decreased the expression of MMP2 and tissue inhibitor of metalloproteinase 2 (TIMP2) and increased the overall level of collagen-degrading activity. Relaxin decreased TGFß-induced Smad2 nuclear localization in mouse hepatic stellate cells. CONCLUSIONS: The results suggest that relaxin reduced collagen deposition and HSC activation in established hepatic fibrosis despite the presence of continued hepatic insult. This reduced fibrosis was associated with increased expression of the fibrillar collagen-degrading enzyme MMP13, decreased expression of TIMP2, and enhanced collagen-degrading activity, and impaired TGFß signalling, consistent with relaxin's effects on activated fibroblastic cells. The results suggest that relaxin may be an effective treatment for the treatment of established hepatic fibrosis.

Expression of green fluorescent protein in human foreskin fibroblasts for use in 2D and 3D culture models.

The availability of fibroblasts that express green fluorescent protein (GFP) would be of interest for the monitoring of cell growth, migration, contraction, and other processes within the fibroblast-populated collagen matrix and other culture systems. A plasmid lentiviral vector-GFP (pLV-GFP) was utilized for gene delivery to produce primary human foreskin fibroblasts (HFFs) that stably express GFP. Cell morphology, cell migration, and collagen contraction were compared between nontransduced HFFs and transduced GFP-HFFs; no differences were observed. Immunocytochemical staining showed no differences in cell morphology between nontransduced and GFP-HFFs in both two-dimensional and three-dimensional culture systems. Furthermore, there was no significant difference in cellular population growth within the collagen matrix populated with nontransduced vs. GFP-HFFs. Within the limits of our assays, we conclude that transduction of GFP into HFFs did not alter the observed properties of HFFs compared with nontransduced fibroblasts. The GFP-HFFs may represent a new tool for the convenient monitoring of living primary fibroblast processes in two-dimensional or three-dimensional culture.

Degradation of relaxin family peptides by insulin-degrading enzyme.

Insulin-degrading enzyme (IDE) is a ubiquitously expressed metalloproteinase responsible for the intracellular degradation of insulin. IDE also interacts with other members of the insulin superfamily, including relaxin, but no studies have been reported regarding the interaction of other relaxin-like peptides with IDE. In this study, we determined that relaxin, relaxin-3, and InsL3 all competitively inhibited the degradation of insulin by IDE to different degrees, and all inhibited covalent cross-linking of insulin to IDE. Each of the peptides was degraded by IDE to various degrees (insulin > relaxin > InsL3 = relaxin-3). In summary, relaxin, InsL3, and relaxin-3 all bound to IDE, competed for the binding and degradation of insulin, and were all substrates for the proteolytic activity of IDE. Therefore, it is possible that in addition to insulin, IDE may be important for the cellular proteolysis of relaxin, InsL3, and relaxin-3.

Relaxin reduces fibrosis in models of progressive and established hepatic fibrosis.

The effect of relaxin administration before (prevention) or after (treatment) the establishment of hepatic fibrosis in a mouse model was examined. In the prevention study, relaxin reduced collagen and smooth muscle actin content and significantly reduced serum levels of the liver enzymes alanine aminotransferase and aspartate aminotransferase. In the treatment study, administration of relaxin for 1 week reduced collagen and smooth muscle actin but not liver enzyme levels. Relaxin administered for 2 weeks had no significant effect. In conclusion, the data suggest that relaxin treatment before fibrosis can reduce collagen and improve liver function but that there is little effect of short-term relaxin treatment after fibrosis is established.