CTGF is a central mediator of tissue remodeling and fibrosis and its inhibition can reverse the process of fibrosis

Abstract

CTGF is a secreted matricellular protein with very complex biology. It has been shown to modulate many signaling pathways leading to cell adhesion and migration, angiogenesis, myofibroblast activation, and extracellular matrix deposition and remodeling, which together lead to tissue remodeling and fibrosis. It has been reported in the literature that inhibition of CTGF expression by siRNA prevents CCl4-induced liver fibrosis and can reverse fibrosis when administered after significant collagen deposition is observed. A monoclonal antibody to CTGF that is currently in clinical development (FG-3019) has demonstrated the ability to reverse vascular stiffening and improve cardiac function in a rat model of diabetic complications. FG-3019 has also exhibited activity in a murine radiation-induced pulmonary fibrosis model. When FG-3019 was administered to mice after a significant radiation-induced increase in lung density could be observed by CT imaging, the density of the lungs was observed to decrease over the period during which the antibody was administered and to remain stable after therapy had ceased. When considered together, these data indicate that inhibition of CTGF can prevent and reverse the process of fibrosis.

Figure 1

CTGF affects multiple signaling pathways and processes important in pathophysiology. CTGF interacts with a variety of molecules, including cytokines and growth factors, receptors and matrix proteins. These interactions alter signal transduction pathways, either positively or negatively, which results in changes in cellular responses.

Figure 2

CTGF is a central mediator of tissue remodeling and fibrosis. Many different stimuli can induce expression of CTGF, which then promotes formation of myofibroblasts by modulating differentiation of other cells, including epithelial cells (EMT, epithelial to mesenchymal transition), resident fibroblasts or recruited fibrocytes (bone-marrow-derived, circulating mesenchymal stem cells). CTGF also promotes activation of the myofibroblasts and stimulates extracellular matrix (ECM) deposition and tissue remodeling. Remodeling in the vasculature can produce local hypertension that induces the expression of more CTGF, resulting in a positive feedback loop. Other positive feedback loops result from cytokines whose expression may be stimulated by CTGF, that in turn induce the expression of CTGF.

Figure 3

CTGF is essential for persistent fibrosis. One day old neonatal mice were injected SC daily for 7 days with 800 ng TGFβ2 alone or with 400 ng CTGF. FG-3019 was also administered to one group that received CTGF. The injections were then stopped for 4 days and the mice were sacrificed to examine the deposition of ECM components and cellular invasion. Panel A: the experimental design. Panel B: A cross section of the skin, SC space and underlying muscle from representative mice is shown. The arrows point out fibrotic response to the various treatments.

Figure 4

CTGF knock-down with shRNA inhibits serum-dependent cell proliferation of cardiac fibroblasts. Primary cardiac fibroblasts (P) were infected with lentiviruses encoding 2 different CTGF shRNAs (h2 and h5 from OpenBiosystems) or with a lentivirus generated from an empty vector (V) as control. Panel A: CTGF protein expression was measured by ELISA in culture supernatants. Panel B: 96 hrs after infection, cells were re-plated in quadruplicate wells of a 96-well plate in medium containing serum and defined growth supplements and incubated for up to 10 days. Cell proliferation was measured by Cyquant Fluorescense Intensity at the indicated timepoints. Panel C: Parallel cultures of cells were stained with crystal violet at day 10 and doubling time (DT) was determined by linear regression analysis on the data shown in B. * p < 0.001 vs. vector by ANOVA.

Figure 5

CTGF knock-down with shRNA inhibits TGFb-dependent cell proliferation of cardiac fibroblasts. Primary cardiac fibroblasts (P) were infected with lentiviruses encoding CTGF shRNAs (h2 or h5) or with a lentivirus expressing a scrambled shRNA as control. Cells were serum starved for 24 hrs and replated in medium containing growth supplements and the indicated concentrations of TGFb (TGFb). Panel A: CTGF protein expression was measured after 7 days. Panel B: Cell proliferation was measured by Cyquant at the indicated timepoints.