FIBROGENIC PATHWAYS IN 3D TISSUE MODELS: FROM LYSYL OXIDASE ENZYMES TO SCLERODERMA
(SSc) is a heterogeneous, progressive disease characterized by collagen accumulation in
skin. The majority of the patients with this condition present with debilitating
cutaneous fibrosis and contractual changes that affect tendons and subcutaneous tissues.
In the past decade, tremendous progress has been achieved to understand this devastating
disease. Unfortunately, no ... read moreeffective therapy has been developed to treat fibrotic
progression in skin. Recent studies have identified that lysyl oxidases (LOXs), a family
of extracellular matrix (ECM)-modifying enzymes responsible for collagen cross-linking,
were upregulated in dermal fibroblasts from patients with fibrotic diseases including
SSc. To better understand mechanisms linked to fibrosis, we have adapted two
bioengineered, in vivo-like, 3D tissue models for human skin (hSE) and human dermis (SA)
to study the ECM expression, organization and remodeling that occurs in the presence of
fibroblasts derived from SSc (SScDF) in comparison to age-and-site matched, normal
donors (NDF). We validated multiphoton microscopy (SHG/TPEF) in 3D tissue models as a
non-invasive tool to evaluate human foreskin fibroblast (HFF) responses to TGF- in ECM
by assessing collagen fiber density, organization, and cross-linking level, that were
correlated with tissue stiffness measured by Atomic Force Microscopy (AFM). These
TGF--induced changes were detected in the dermis of 3D tissues (hSE) and found that
increasing collagen content and crosslinks were accompanied by elevated rigidity that
were inhibited by suppression of LOX using the inhibitor BAPN or by specific siRNA
knockdown of LOX and lysyl oxidase-like 4 (LOXL4), which abolished TGF--induced ECM
alteration and dermal stiffness. In the initial stage of SSc, a fibroblasts transition
to myofibroblasts can be activated by increasing circulating levels of TGF- in patient
serum. In SScDF, expression of alpha smooth muscle actin (SMA) and LOXL4 were increased
in both 2D and 3D. Knockdown LOXL4 in SScDF not only inhibited the rigidity and
contraction in hSE by comparing them to NDF, but also abolished TGF--induced ECM
deposition and organization in SA tissues. Thus, LOXs plays a pivotal role in the
pathogenesis of dermal fibrosis in 3D skin-like tissues that were directly monitored by
SHG/TPEF and AFM, which may serve as new prognostic and diagnostic platforms for
studying and treating fibrotic diseases.
Thesis (Ph.D.)--Tufts University, 2017.
Submitted to the Dept. of Cell, Molecular & Developmental Biology.
Advisors: Jonathan Garlick, and Lauren Black.
Committee: Lauren Black, Peter Brooks, and John Castellot.
Keyword: Cellular biology.read less