Recombinant Adenosine Deaminase Ameliorates Inflammation, Vascular Disease, and Fibrosis in Preclinical Models of Systemic Sclerosis.

OBJECTIVE: Systemic sclerosis (SSc) is characterized by fibrosis, vascular disease, and inflammation. Adenosine signaling plays a central role in fibroblast activation. We undertook this study to evaluate the therapeutic effects of adenosine depletion with PEGylated adenosine deaminase (PEG-ADA) in preclinical models of SSc. METHODS: The effects of PEG-ADA on inflammation, vascular remodeling, and tissue fibrosis were analyzed in Fra-2 mice and in a B10.D2→BALB/c (H-2d ) model of sclerodermatous chronic graft-versus-host disease (GVHD). The effects of PEG-ADA were confirmed in vitro in a human full-thickness skin model. RESULTS: PEG-ADA effectively inhibited myofibroblast differentiation and reduced pulmonary fibrosis by 34.3% (with decreased collagen expression) (P = 0.0079; n = 6), dermal fibrosis by 51.8% (P = 0.0006; n = 6), and intestinal fibrosis by 17.7% (P = 0.0228; n = 6) in Fra-2 mice. Antifibrotic effects of PEG-ADA were also demonstrated in sclerodermatous chronic GVHD (reduced by 38.4%) (P = 0.0063; n = 8), and in a human full-thickness skin model. PEG-ADA treatment decreased inflammation and corrected the M2/Th2/group 2 innate lymphoid cell 2 bias. Moreover, PEG-ADA inhibited proliferation of pulmonary vascular smooth muscle cells (reduced by 40.5%) (P < 0.0001; n = 6), and prevented thickening of the vessel walls (reduced by 39.6%) (P = 0.0028; n = 6) and occlusions of pulmonary arteries (reduced by 63.9%) (P = 0.0147; n = 6). Treatment with PEG-ADA inhibited apoptosis of microvascular endothelial cells (reduced by 65.4%) (P = 0.0001; n = 6) and blunted the capillary rarefication (reduced by 32.5%) (P = 0.0199; n = 6). RNA sequencing demonstrated that treatment with PEG-ADA normalized multiple pathways related to fibrosis, vasculopathy, and inflammation in Fra-2 mice. CONCLUSION: Treatment with PEG-ADA ameliorates the 3 cardinal features of SSc in pharmacologically relevant and well-tolerated doses. These findings may have direct translational implications, as PEG-ADA has already been approved by the Food and Drug Administration for the treatment of patients with ADA-deficient severe combined immunodeficiency disease.

Dipeptidylpeptidase 4 as a Marker of Activated Fibroblasts and a Potential Target for the Treatment of Fibrosis in Systemic Sclerosis.

OBJECTIVE: Expression of dipeptidylpeptidase 4 (DPP-4) identifies a dermal fibroblast lineage involved in scarring during wound healing. The role of DDP-4 in tissue fibrosis is, however, unknown. The aim of the present study was to evaluate DPP-4 as a potential target for the treatment of fibrosis in patients with systemic sclerosis (SSc). METHODS: Expression of DPP-4 in skin biopsy samples and dermal fibroblasts was analyzed by real-time polymerase chain reaction, immunofluorescence, and Western blot analyses. The activity of DPP-4 was modulated by overexpression, knockdown, and pharmacologic inhibition of DPP4 using sitagliptin and vildagliptin. The effects of DPP4 inhibition were analyzed in human dermal fibroblasts and in different mouse models of SSc (each n = 6). RESULTS: The expression of DPP-4 and the number of DPP-4-positive fibroblasts were increased in the fibrotic skin of SSc patients, in a transforming growth factor ß (TGFß)-dependent manner. DPP-4-positive fibroblasts expressed higher levels of myofibroblast markers and collagen (each P < 0.001 versus healthy controls). Overexpression of DPP4 promoted fibroblast activation, whereas pharmacologic inhibition or genetic inactivation of DPP4 reduced the proliferation, migration, and expression of contractile proteins and release of collagen (each P < 0.001 versus control mice) by interfering with TGFß-induced ERK signaling. DPP4-knockout mice were less sensitive to bleomycin-induced dermal and pulmonary fibrosis (P < 0.0001 versus wild-type controls). Treatment with DPP4 inhibitors promoted regression of fibrosis in mice that had received bleomycin challenge and mice with chronic graft-versus-host disease, and ameliorated fibrosis in TSK1 mice (each P < 0.001 versus untreated controls). These antifibrotic effects were associated with a reduction in inflammation. CONCLUSION: DPP-4 characterizes a population of activated fibroblasts and shows that DPP-4 regulates TGFß-induced fibroblast activation in the fibrotic skin of SSc patients. Inhibition of DPP4 exerts potent antifibrotic effects when administered in well-tolerated doses. As DPP4 inhibitors are already in clinical use for diabetes, these results may have direct translational implications for the treatment of fibrosis in patients with SSc.

PU.1 controls fibroblast polarization and tissue fibrosis.

Fibroblasts are polymorphic cells with pleiotropic roles in organ morphogenesis, tissue homeostasis and immune responses. In fibrotic diseases, fibroblasts synthesize abundant amounts of extracellular matrix, which induces scarring and organ failure. By contrast, a hallmark feature of fibroblasts in arthritis is degradation of the extracellular matrix because of the release of metalloproteinases and degrading enzymes, and subsequent tissue destruction. The mechanisms that drive these functionally opposing pro-fibrotic and pro-inflammatory phenotypes of fibroblasts remain unknown. Here we identify the transcription factor PU.1 as an essential regulator of the pro-fibrotic gene expression program. The interplay between transcriptional and post-transcriptional mechanisms that normally control the expression of PU.1 expression is perturbed in various fibrotic diseases, resulting in the upregulation of PU.1, induction of fibrosis-associated gene sets and a phenotypic switch in extracellular matrix-producing pro-fibrotic fibroblasts. By contrast, pharmacological and genetic inactivation of PU.1 disrupts the fibrotic network and enables reprogramming of fibrotic fibroblasts into resting fibroblasts, leading to regression of fibrosis in several organs.