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.

An in vitro model mimics the contact of biomaterials to blood components and the reaction of surrounding soft tissue.

The therapeutic efficacy of a medical product after implantation depends strongly on the host-initiated fibrotic response (foreign body reaction). For novel biomaterials, it is of high relevance to understand this fibrotic process. As an alternative to in vivo studies, in vitro models mimic parts of the whole foreign body reaction. Aim of this study was to develop a wound model with key cells and matrix proteins in coculture. This approach combined blood components such as primary macrophages in a plasma-derived fibrin hydrogel, directly exposed to reference biomaterials (PTFE, glass, titanium). The soft tissue reaction is resembled by integrating fibroblasts in a collagen or a fibrin matrix. Those two experimental setups were conducted to show whether a long-term in vitro culture of 13 days is feasible. The response to reference biomaterials was assessed by multi-parametric analyses, comprising molecular profiling (cytokines, collagen I and ß-actin) and tissue remodeling (cell adherence, histological structure, tissue deposition). Polytetrafluorethylene (PTFE) and titanium were tested as references to correlate the in vitro evaluation to previous in vivo studies. Most striking, both model setups evaluated references' fibrotic characteristics as previously reported by in vivo studies. STATEMENT OF SIGNIFICANCE: We present a test platform applied for assessments on the foreign body reaction to biomaterials. This test system consists of blood components - macrophages and plasma-derived fibrin - as well as fibroblasts and collagen, generating a three-dimensional wound microenvironment. By this modular approach, we achieved a suitable test for long-term studies and overcame the limited short-term stability of whole blood tests. In contrast to previous models, macrophages' viability is maintained during the extended culture period and excels the quality of the model. The potential to evaluate a foreign body reaction in vitro was demonstrated with defined reference materials. This model system might be of high potential as a screening platform to identify novel biomaterial candidates.

Human barrier models for the in vitro assessment of drug delivery.

In vitro test systems gain increasing importance in preclinical studies to increase the predictivity and reduce animal testing. Of special interest herein are barrier tissues that guard into the human body. These barriers are formed by highly specialized tissues such as the skin, the airways, and the intestine. However, to recapitulate these tissues, researchers are currently restricted by a lack of suitable supporting scaffolds. In this study, we present biological scaffolds based on decellularized porcine gut segments that offer a natural environment for cell growth and differentiation. Employing these scaffolds, human barrier models of the skin, the airways, and the intestine that mimic the natural histological architecture of the respective tissue are generated. These models show tissue specific barrier properties, such as the stratification of the skin, the mucociliary phenotype of the airways, and polarization of the intestinal epithelium. To investigate the transport characteristics of the intestinal test system, we incubated the tissue models with fluorescein (P app <1 × 106 cm/s), propranolol (P app >7 × 106 cm/s), and rhodamin123 (ratio 2.45). The here presented biological scaffolds facilitate the in vitro generation of human barrier models that might represent useful tools for drug delivery studies.

Skin tissue engineering--in vivo and in vitro applications.

Significant progress has been made over the years in the development of in vitro-engineered substitutes that mimic human skin, either to be used as grafts for the replacement of lost skin or for the establishment of human-based in vitro skin models. This review summarizes these advances in in vivo and in vitro applications of tissue-engineered skin. We further highlight novel efforts in the design of complex disease-in-a-dish models for studies ranging from disease etiology to drug development and screening.