Myofibroblast-specific inhibition of the Rho kinase-MRTF-SRF pathway using nanotechnology for the prevention of pulmonary fibrosis

Abstract

Pulmonary fibrosis is characterized by the accumulation of myofibroblasts in the lung and progressive tissue scarring. Fibroblasts exist across a spectrum of states, from quiescence in health to activated myofibroblasts in the setting of injury. Highly activated myofibroblasts have a critical role in the establishment of fibrosis as the predominant source of type 1 collagen and profibrotic mediators. Myofibroblasts are also highly contractile cells and can alter lung biomechanical properties through tissue contraction. Inhibiting signaling pathways involved in myofibroblast activation could therefore have significant therapeutic value. One of the ways myofibroblast activation occurs is through activation of the Rho/myocardin-related transcription factor (MRTF)/serum response factor (SRF) pathway, which signals through intracellular actin polymerization. However, concerns surrounding the pleiotropic and ubiquitous nature of these signaling pathways have limited the translation of inhibitory drugs. Herein, we demonstrate a novel therapeutic antifibrotic strategy using myofibroblast-targeted nanoparticles containing a MTRF/SRF pathway inhibitor (CCG-1423), which has been shown to block myofibroblast activation in vitro. Myofibroblasts were preferentially targeted via the angiotensin 2 receptor, which has been shown to be selectively upregulated in animal and human studies. These nanoparticles were nontoxic and accumulated in lung myofibroblasts in the bleomycin-induced mouse model of pulmonary fibrosis, reducing the number of these activated cells and their production of profibrotic mediators. Ultimately, in a murine model of lung fibrosis, a single injection of these drugs containing targeted nanoagents reduced fibrosis as compared with control mice. This approach has the potential to deliver personalized therapy by precisely targeting signaling pathways in a cell-specific manner, allowing increased efficacy with reduced deleterious off-target effects.

Keywords: cell-specific targeting; fibrosis; myofibroblast; nanoparticles; nanotechnology.

Graphical abstract

Figure 1.

Characterization of myofibroblast-targeted nanoparticles. Schematics depicting the PEG-PLGA core (green) of each of the three nanoparticles generated for these experiments. In the targeted particles with drug (T + D), the core is coated with AGTR2-targeted peptides (purple) and contain CCG-1423 drug inside the shell (blue). In the targeted particles without drug (T − D), there is no CCG-1423 within the shell but the targeting peptide is seen on the outer shell. In the nontargeting particle with drug (NT+D), CCG-1423 is present within the shell but no targeting peptide is visible. Size distribution of the particles is shown below.

Figure 2.

Ex vivo characterization of myofibroblast targeted nanoparticles after IT bleomycin. A: schematic of experimental design. C57BL/6 mice were injected with IT bleomycin (0.8 U/kg) on day 0, IV nanoparticles were injected day 10, and mouse lungs were harvested on day 14. B: ex vivo biodistribution demonstrated uptake of targeted nanoparticles in the lungs, kidney, and liver. C: quantification was performed of fluorescence uptake of organs ex vivo at day 14. n = 3 animals per group. NT + D, nontargeting nanoparticle with drug; T + D, targeting nanoparticle with drug; T − D, targeting nanoparticle without drug. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 3.

Myofibroblast targeted nanoparticles are specifically taken up by taken up by lung fibroblasts after lung injury with bleomycin. A: flow cytometry was performed on single cell suspension of lung tissue generated at day 14 after IT bleomycin. Hematopoietic cells (CD45⁺), epithelial cells (Epcam⁺), endothelial cells (CD31⁺), and fibroblasts (CD45⁻Epcam⁻CD31⁻) were identified through the gating strategy shown. B: nanoparticles were not well visualized in the lung fibroblasts by flow cytometry in mice who received IV nontargeting nanoparticles. In contrast, nanoparticles were well visualized by flow cytometry in the mice who received targeting nanoparticles without CCG-1423 (C) and in those who received targeting nanoparticles containing CCG-1423 (D). Representative images of n = 3 mice/group. E: nanoparticle containing cells were quantified for hematopoietic cells, endothelial cells, epithelial cells, and fibroblasts. n = 3 samples/group. NT + D, nontargeting nanoparticle with drug; T + D, targeting nanoparticle with drug; T − D, targeting nanoparticle without drug. **P < 0.01, ***P < 0.001.

Figure 4.

Myofibroblast targeted nanoparticles containing CCG-1423 attenuate bleomycin-induced pulmonary fibrosis. A: schematic of experimental design. C57BL/6 mice were injected with IT bleomycin (0.8 U/kg) on day 0, IV nanoparticles were injected on days 10, 13, 16, 20, 24 and mice were followed for 28 days. B: mouse body weight was measured at multiple time points over 28 days. C: survival was monitored over 28 days after IT bleomycin challenge. D: H&E staining was performed on mouse lung samples harvested on day 14 after IT bleomycin challenge. E: lung fibrosis was quantified by hydroxyproline assay on day 14 after IT bleomycin. n = 8–11 mice/group. *P < 0.05. NT + D, nontargeting nanoparticle with drug; T + D, targeting nanoparticle with drug; T − D, targeting nanoparticle without drug. *P < 0.05.

Figure 5.

Myofibroblast-targeted nanoparticles containing CCG-1423 reduce myofibroblast activation. A: immunofluorescent staining was performed on mouse lung samples harvested on day 14 after IT bleomycin challenge. Lung sections were stained for αSMA (green) and nanoparticles (red). Representative images shown. B: RNA was isolated from mouse lung fibroblasts containing nanoparticles and isolated by FACS at day 14 after IT bleomycin. qPCR was performed to compare CTGF expression in fibroblasts containing targeted nanoparticles containing CCG-1423 to fibroblasts from mice containing targeted nanoparticles without CCG-1423. C: immunoblotting was performed on lung homogenates from mice treated with IV nanoparticles on day 10 after IT bleomycin and probed for CTGF protein expression. n = 4 mice/group. NT + D, nontargeting nanoparticle with drug; T + D: targeting nanoparticle with drug; T − D, targeting nanoparticle without drug. *P < 0.05.