The high molecular weight of these compounds and the lectin binding properties suggest that they likely act predominantly on extracellular galectins. The potential mechanisms by which these galectin-3 binding drugs might have the demonstrated Temozolomide Autophagy inhibitor effect on fibrosis are not yet clear. Henderson, et al. showed that galectin-3 appeared to be required for activation of hepatic stellate cells to myofibroblasts. A reduction in activated stellate cells would clearly be important as they represent the primary cell for synthesis of extracellular collagen in liver fibrosis. In our experiments, there was a decrease in a-SMA protein in those treatment groups with the greatest anti-fibrotic effect, consistent with a reduction in stellate cell activation in response to the therapy. However this does not necessarily reflect a direct effect on stellate cells rather than an indirect effect by modulating the nature or extent of inflammation. Indeed, the effect of the drugs on isolated stellate cells and the LX-2 stellate cell line was extremely modest and therefore unlikely to account entirely for the significant efficacy in vivo. Future studies will need to determine whether the primary effect of these compounds in liver is via inhibition of galectin-3 on stellate cells, or through an indirect effect of changes in the cytokine and/or inflammatory milieu. Prior studies assessed the effect of galectin-1 and galectin-3 on proliferation and activation of cultured stellate cells and the effect of galectin-3 on phagocytosis-dependent activation of stellate cells. In our experiments there was no effect in LX-2 cells on proliferation, apoptosis, or the expression of most fibrogenesis-related genes and proteins. There was a reduction in the expression of TGF-b receptor-1 gene expression following treatment with both GRMD- 02 and GM-CT-01. TGF-b is an important cytokine in fibrogenesis and for the activation of stellate cells. Additionally, there is evidence that the activity of the TGF-b receptor in lung fibrosis is dependent on galectin-3 protein and that inhibition of galectin-3 is inhibits receptor activity. Therefore, inhibition of TGF-b-dependent stellate cell activation may be one mechanism that drug inhibition of galectin-3 could provide some of the effect seen in this animal model. The macrophage is another potential target by which galectin-3 binding drugs might affect fibrosis. Macrophages are pivotal to the development and resolution of collagen deposition in organs and are clearly important in liver fibrosis. Moreover, it is now clear that activated macrophages Pazopanib differentiate into a number of different subtypes, referred to as macrophage polarization, which have distinct functions along the continuum from inflammation and fibrogenesis to resolution of fibrosis.