Lysyl oxidase enzymes mediate TGF-β1-induced fibrotic phenotypes in human skin-like tissues

Abstract

Cutaneous fibrosis is a common complication seen in mixed connective tissue diseases. It often occurs as a result of TGF-β-induced deposition of excessive amounts of collagen in the skin. Lysyl oxidases (LOXs), a family of extracellular matrix (ECM)-modifying enzymes responsible for collagen cross-linking, are known to be increased in dermal fibroblasts from patients with fibrotic diseases, denoting a possible role of LOXs in fibrosis. To directly study this, we have developed two bioengineered, in vitro skin-like models: human skin equivalents (hSEs), and self-assembled stromal tissues (SASs) that contain either normal or systemic sclerosis (SSc; scleroderma) patient-derived fibroblasts. These tissues provide an organ-level structure that could be combined with non-invasive, label-free, multiphoton microscopy (SHG/TPEF) to reveal alterations in the organization and cross-linking levels of collagen fibers during the development of cutaneous fibrosis, which demonstrated increased stromal rigidity and activation of dermal fibroblasts in response to TGF-β1. Specifically, inhibition of specific LOXs isoforms, LOX and LOXL4, in foreskin fibroblasts (HFFs) resulted in antagonistic effects on TGF-β1-induced fibrogenic hallmarks in both hSEs and SASs. In addition, a translational relevance of these models was seen as similar antifibrogenic phenotypes were achieved upon knocking down LOXL4 in tissues containing SSc patient-derived-dermal fibroblasts (SScDFs). These findings point to a pivotal role of LOXs in TGF-β1-induced cutaneous fibrosis through impaired ECM homeostasis in skin-like tissues, and show the value of these tissue platforms in accelerating the discovery of antifibrosis therapeutics.