TGF-ß1 Drives Integrin-Dependent Pericyte Migration and Microvascular Destabilization in Fibrotic Disease.

Interactions between endothelial cells (ECs) and mural pericytes (PCs) are critical to maintaining the stability and function of the microvascular wall. Abnormal interactions between these two cell types are a hallmark of progressive fibrotic diseases such as systemic sclerosis (also known as scleroderma). However, the role that PCs play in signaling microvascular dysfunction remains underexplored. It is hypothesized that integrin-matrix interactions contribute to PC migration from the vascular wall and conversion into interstitial myofibroblasts. Using pro-inflammatory tumor necrosis factor α (TNFα) or a fibrotic growth factor [transforming growth factor ß1 (TGF-ß1)], human PC inflammatory and fibrotic phenotypes were evaluated by assessing their migration, matrix deposition, integrin expression, and subsequent effects on endothelial dysfunction. Both TNFα and TGF-ß1 treatment altered integrin expression and matrix protein deposition, but only fibrotic TGF-ß1 drove PC migration in an integrin-dependent manner. In addition, integrin-dependent PC migration was correlated to changes in EC angiopoietin-2 levels, a marker of vascular instability. Finally, there was evidence of changes in vascular stability corresponding to disease state in human systemic sclerosis skin. This work shows that TNFα and TGF-ß1 induce changes in PC integrin expression and matrix deposition that facilitate migration and reduce vascular stability, providing evidence that microvascular destabilization can be an early indicator of tissue fibrosis.

Exploring the anomalous cytotoxicity of commercially-available poly(N-isopropyl acrylamide) substrates.

Poly(N-isopropyl acrylamide) (pNIPAM) is a stimulus-responsive polymer that has been of great interest to the bioengineering community. When the temperature is lowered below its lower critical solution temperature (∼32 °C), pNIPAM rapidly hydrates, and adherent cells detach as intact cell sheets. This cell-releasing behavior in a physiologically relevant temperature range has led to NIPAM's use for engineered tissues and other devices. In a previous study, however, the authors found that although most techniques used to polymerize NIPAM yield biocompatible films, some formulations from commercially-available NIPAM (cpNIPAM) can be cytotoxic. In this work, the authors investigate the reasons underlying this anomaly. The authors evaluated the response of a variety of cell types (e.g., bovine aortic endothelial cells, BAECs; monkey kidney epithelial cells, Vero cells; and mouse embryonic fibroblasts, 3T3s) after culture on substrates spin-coated with sol-gel (spNIPAM) and commercially-prepared (cpNIPAM). The relative biocompatibility of each cell type was evaluated using observations of its cell morphology and function (e.g., XTT and Live/Dead assays) after 48 and 96 h in culture. In addition, the substrates themselves were analyzed using NMR, goniometry, and XPS. The authors find that all the cell types were compromised by 96 h in culture with cpNIPAM, although the manner in which the cells are compromised differs; in particular, while Vero and 3T3 cells appear to be undergoing cytotoxic death, BAECs undergo apoptic death. The authors believe that this result is due to a combination of factors, including the presence of short chain oligomers of NIPAM in the commercially-available preparation. This work will provide valuable insights into the cytotoxicity of commercially-prepared polymer substrates for this type of bioengineering work and therefore into the applicability of cells grown on such surfaces for human subjects.

Human pericytes adopt myofibroblast properties in the microenvironment of the IPF lung.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease of unknown etiology characterized by a compositionally and mechanically altered extracellular matrix. Poor understanding of the origin of α-smooth muscle actin (α-SMA) expressing myofibroblasts has hindered curative therapies. Though proposed as a source of myofibroblasts in mammalian tissues, identification of microvascular pericytes (PC) as contributors to α-SMA-expressing populations in human IPF and the mechanisms driving this accumulation remain unexplored. Here, we demonstrate enhanced detection of α-SMA+ cells coexpressing the PC marker neural/glial antigen 2 in the human IPF lung. Isolated human PC cultured on decellularized IPF lung matrices adopt expression of α-SMA, demonstrating that these cells undergo phenotypic transition in response to direct contact with the extracellular matrix (ECM) of the fibrotic human lung. Using potentially novel human lung-conjugated hydrogels with tunable mechanical properties, we decoupled PC responses to matrix composition and stiffness to show that α-SMA+ PC accumulate in a mechanosensitive manner independent of matrix composition. PC activated with TGF-ß1 remodel the normal lung matrix, increasing tissue stiffness to facilitate the emergence of α-SMA+ PC via MKL-1/MTRFA mechanotranduction. Nintedanib, a tyrosine-kinase inhibitor approved for IPF treatment, restores the elastic modulus of fibrotic lung matrices to reverse the α-SMA+ phenotype. This work furthers our understanding of the role that microvascular PC play in the evolution of IPF, describes the creation of an ex vivo platform that advances the study of fibrosis, and presents a potentially novel mode of action for a commonly used antifibrotic therapy that has great relevance for human disease.