Engineering Advanced In Vitro Models of Systemic Sclerosis for Drug Discovery and Development

Abstract

Systemic sclerosis (SSc) is a complex multisystem disease with the highest case-specific mortality among all autoimmune rheumatic diseases, yet without any available curative therapy. Therefore, the development of novel therapeutic antifibrotic strategies that effectively decrease skin and organ fibrosis is needed. Existing animal models are cost-intensive, laborious and do not recapitulate the full spectrum of the disease and thus commonly fail to predict human efficacy. Advanced in vitro models, which closely mimic critical aspects of the pathology, have emerged as valuable platforms to investigate novel pharmaceutical therapies for the treatment of SSc. This review focuses on recent advancements in the development of SSc in vitro models, sheds light onto biological (e.g., growth factors, cytokines, coculture systems), biochemical (e.g., hypoxia, reactive oxygen species) and biophysical (e.g., stiffness, topography, dimensionality) cues that have been utilized for the in vitro recapitulation of the SSc microenvironment, and highlights future perspectives for effective drug discovery and validation.

Keywords: 3D in vitro models; animal models; fibrosis; in vitro microenvironment; scleroderma; tissue engineering.

Figure 1.

Pathophysiology of SSc. Genetic and environmental factors trigger the onset of SSc. SSc is characterized by vascular alterations, inflammation and autoimmunity, and multisystemic excessive fibrosis, which ultimately lead to severe and life-threatening organ complications. Created with BioRender.com.

Figure 2.

Molecular mechanisms of SSc. 1) Preclinical stage. Vascular injury is the earliest event in SSc which leads to endothelial cell activation and entrapment of peripheral blood mononuclear cells. 2) Inflammatory stage. Progressive vascular damage causes endothelial cell apoptosis, which in turn secrete ET-1 and PDGF that stimulate smooth muscle cell proliferation, leading to luminal narrowing, and inflammatory cells recruitment. Plasma cells secrete autoantibodies (anti-Scl-70, anticentromere, anti-RNA-polymerase III) and IL-6. Type 2 T helper (T_H2) cells secrete TGF-β and IL-13. Polarized M2 macrophages secrete TGF-β. These soluble mediators contribute to fibroblasts activation and increase ECM deposition. 3) Late stage. Progressive endothelial cells apoptosis, smooth muscle cells proliferation and vessel narrowing lead to tissue hypoxia and oxidative stress which contribute to the maintenance of fibrosis. Fibroblasts undergo complete myofibroblasts differentiation and increase ECM deposition leading to mechanical stress and perpetuating the fibrotic process. M2 polarized macrophages infiltration further increases TGF-β secretion. Created with BioRender.com.

Figure 3.

Overview of biological, biochemical, and biophysical cues used in vitro to recapitulate the SSc microenvironment. Created with BioRender.com.

Figure 4.

Matrix stiffness and mechanotransduction in fibrosis. Mechanotransduction pathways mediate matrix stiffness-induced myofibroblast activation. Stiffness-mediated traction forces are transmitted across integrins, which induce actomyosin cell contractility mediated by focal adhesion kinase (FAK) and RHO-associated kinase (ROCK). These signals activate the downstream effectors YAP (Yes-associated protein), TAZ (transcriptional coactivator with PDZ-binding motif) and myocardin-related transcription factor (MRTF), which increase the expression of profibrotic markers such as α-SMA and collagen type I. Increased collagen deposition and crosslinking further increases ECM stiffening, creating a profibrotic positive feedback loop between matrix stiffness and myofibroblast activation. Created with BioRender.com.

Figure 5.

Advantages and limitations of in silico, in vitro, and in vivo models utilized to replicate complex pathophysiologies. Created with BioRender.com.