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.

Endothelial cell-secreted MIF reduces pericyte contractility and enhances neutrophil extravasation.

Dysregulated neutrophil extravasation contributes to the pathogenesis of many inflammatory disorders. Pericytes (PCs) have been implicated in the regulation of neutrophil transmigration, and previous work demonstrates that endothelial cell (EC)-derived signals reduce PC barrier function; however, the signaling mechanisms are unknown. Here, we demonstrate a novel role for EC-derived macrophage migration inhibitory factor (MIF) in inhibiting PC contractility and facilitating neutrophil transmigration. With the use of micro-ELISAs, RNA sequencing, quantitative PCR, and flow cytometry, we found that ECs secrete MIF, and PCs upregulate CD74 in response to TNF-α. We demonstrate that EC-derived MIF decreases PC contractility on 2-dimensional silicone substrates via reduction of phosphorylated myosin light chain. With the use of an in vitro microvascular model of the human EC-PC barrier, we demonstrate that MIF decreases the PC barrier to human neutrophil transmigration by increasing intercellular PC gap formation. For the first time, an EC-specific MIF knockout mouse was used to investigate the effects of selective deletion of EC MIF. In a model of acute lung injury, selective deletion of EC MIF decreases neutrophil infiltration to the bronchoalveolar lavage and tissue and simultaneously decreases PC relaxation by increasing myosin light-chain phosphorylation. We conclude that paracrine signals from EC via MIF decrease PC contraction and enhance PC-regulated neutrophil transmigration.-Pellowe, A. S., Sauler, M., Hou, Y., Merola, J., Liu, R., Calderon, B., Lauridsen, H. M., Harris, M. R., Leng, L., Zhang, Y., Tilstam, P. V., Pober, J. S., Bucala, R., Lee, P. J., Gonzalez, A. L. Endothelial cell-secreted MIF reduces pericyte contractility and enhances neutrophil extravasation.

Novel Nanoparticulate and Ionic Titanium Antigens for Hypersensitivity Testing.

Titanium is used in a wide variety of materials ranging from medical devices to materials used in everyday life. Adverse biological reactions that could occur in patients, consumers, and workers should be monitored and prevented. There is a lack of available agents to test and predict titanium-related hypersensitivity. The aim of this study was to develop two bioavailable titanium substances in ionic and nanoparticulate form to serve as antigens for hypersensitivity testing in vitro. Peripheral blood mononuclear cells from 20 test subjects were stimulated with the antigens and secretion of monocytic and lymphatic cytokines and chemokines were measured by a multiplex bead assay. Lymphocyte stimulation indices were also determined in a subset of test subjects by measuring CD69 and HLA-DR expression by flow cytometry. Cytokine profiling revealed that both antigens increased production of typical monocyte and macrophage secreted cytokines after 24 h, with significant increases in IL-1ß, IL-7, IL-10, IL-12, IL-2R, IL-6, GM-CSF, TNF-α, IL-1RA, MIP-1α, MIP-1ß, IFN-α, and IL-15. Lymphatic cytokines and chemokines were not significantly induced by activation. After seven days of stimulation, ionic-Ti (2.5 µg/mL) caused proliferation (stimulation index > 2) of CD4+ cells and CD8+ cells in all persons tested (N = 6), while titanium dioxide nanoparticles (50 µg/mL) only caused significant proliferation of CD4+ cells. Our preliminary results show that the experimental titanium antigens, especially the ionic form, induce a general inflammatory response in vitro. A relevant cohort of test subjects is required to further elucidate their potential for predictive hypersensitivity testing.

Tumor Necrosis Factor-α and IL-17A Activation Induces Pericyte-Mediated Basement Membrane Remodeling in Human Neutrophilic Dermatoses.

Sweet syndrome (SS) is a prototypical neutrophilic dermatosis, a class of inflammatory diseases marked by elevated levels of tumor necrosis factor (TNF)-α and IL-17A, pathologic neutrophil recruitment, and microvascular remodeling. Histologic analyses of four matrix proteins-collagen I and IV, laminin, and fibronectin-in skin biopsies of patients with SS reveal that the basement membrane of dermal postcapillary venules undergoes changes in structure and composition. Increased neutrophil recruitment in vivo was associated with increases in collagen IV, decreases in laminin, and varied changes in fibronectin. In vitro studies using TNF-α and IL-17A were conducted to dissect basement membrane remodeling. Prolonged dual activation of cultured human pericytes with TNF-α and IL-17A augmented collagen IV production, similar to in vivo remodeling. Co-activation of pericytes with TNF-α and IL-17A also elevated fibronectin levels with little direct effect on laminin. However, the expression of fibronectin- and laminin-specific matrix metalloproteinases (MMPs), particularly MMP-3, was significantly up-regulated. Interactions between pericytes and neutrophils in culture yielded even higher levels of active MMPs, facilitating fibronectin and laminin degradation, and likely contributing to the varied levels of detectable fibronectin and the decreases in laminin observed in vivo. These data indicate that pericyte-neutrophil interactions play a role in mediating microvascular changes in SS and suggest that targeting MMP-3 may be effective in protecting vascular wall integrity.

Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture.

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies of endothelial-epithelial bilayers have traditionally relied on permeable support models that enable bilayer culture, but permeable supports are limited in their ability to replicate the diversity of human basement membranes. In contrast, hydrogel models that require chemical synthesis are highly tunable and allow for modifications of both the material stiffness and the biochemical composition via incorporation of biomimetic peptides or proteins. However, traditional hydrogel models are limited in functionality because they lack pores for cell-cell contacts and functional in vitro migration studies. Additionally, due to the thickness of traditional hydrogels, incorporation of pores that span the entire thickness of hydrogels has been challenging. In the present study, we use poly-(ethylene-glycol) (PEG) hydrogels and a novel zinc oxide templating method to address the previous shortcomings of biomimetic hydrogels. As a result, we present an ultrathin, basement membrane-like hydrogel that permits the culture of confluent cellular bilayers on a customizable scaffold with variable pore architectures, mechanical properties, and biochemical composition.

IL-17 Promotes Neutrophil-Mediated Immunity by Activating Microvascular Pericytes and Not Endothelium.

A classical hallmark of acute inflammation is neutrophil infiltration of tissues, a multistep process that involves sequential cell-cell interactions of circulating leukocytes with IL-1- or TNF-activated microvascular endothelial cells (ECs) and pericytes (PCs) that form the wall of the postcapillary venules. The initial infiltrating cells accumulate perivascularly in close proximity to PCs. IL-17, a proinflammatory cytokine that acts on target cells via a heterodimeric receptor formed by IL-17RA and IL-17RC subunits, also promotes neutrophilic inflammation but its effects on vascular cells are less clear. We report that both cultured human ECs and PCs strongly express IL-17RC and, although neither cell type expresses much IL-17RA, PCs express significantly more than ECs. IL-17, alone or synergistically with TNF, significantly alters inflammatory gene expression in cultured human PCs but not ECs. RNA sequencing analysis identifies many IL-17-induced transcripts in PCs encoding proteins known to stimulate neutrophil-mediated immunity. Conditioned media from IL-17-activated PCs, but not ECs, induce pertussis toxin-sensitive neutrophil polarization, likely mediated by PC-secreted chemokines, and they also stimulate neutrophil production of proinflammatory molecules, including TNF, IL-1α, IL-1ß, and IL-8. Furthermore, IL-17-activated PCs, but not ECs, can prolong neutrophil survival by producing G-CSF and GM-CSF, delaying the mitochondrial outer membrane permeabilization and caspase-9 activation. Importantly, neutrophils exhibit enhanced phagocytic capacity after activation by conditioned media from IL-17-treated PCs. We conclude that PCs, not ECs, are the major target of IL-17 within the microvessel wall and that IL-17-activated PCs can modulate neutrophil functions within the perivascular tissue space.

Extracellular matrix biomimicry for the creation of investigational and therapeutic devices.

The extracellular matrix (ECM) is a web of fibrous proteins that serves as a scaffold for tissues and organs, and is important for maintaining homeostasis and facilitating cellular adhesion. Integrin transmembrane receptors are the primary adhesion molecules that anchor cells to the ECM, thus integrating cells with their microenvironments. Integrins play a critical role in facilitating cell-matrix interactions and promoting signal transduction, both from the cell to the ECM and vice versa, ultimately mediating cell behavior. For this reason, many advanced biomaterials employ biomimicry by replicating the form and function of fibrous ECM proteins. The ECM also acts as a reservoir for small molecules and growth factors, wherein fibrous proteins directly bind and present these bioactive moieties that facilitate cell activity. Therefore biomimicry can be enhanced by incorporating small molecules into ECM-like substrates. Biomimetic ECM materials have served as invaluable research tools for studying interactions between cells and the surrounding ECM, revealing that cell-matrix signaling is driven by mechanical forces, integrin engagement, and small molecules. Mimicking pathological ECMs has also elucidated disease specific cell behaviors. For example, biomimetic tumor microenvironments have been used to induce metastatic cell behaviors, and have thereby shown promise for in vitro cancer drug testing and targeting. Further, ECM-like substrates have been successfully employed for autologous cell recolonization for tissue engineering and wound healing. As we continue to learn more about the mechanical and biochemical characteristics of the ECM, these properties can be harnessed to develop new biomaterials, biomedical devices, and therapeutics.