ultrathin-porated-elastic-hydrogels-as-biomimetic-basement-membrane

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 ...

Yale University

Polymorphoneuclear leukocytes or neutrophils, a major component of white blood cells, contribute to the innate immune response in humans. Upon sensing changes in the microenvironment, neutrophils adhere to the vascular wall, migrate through the endothelial cell (EC)-pericyte bilayer, and subsequently through the extracellular matrix to reach the site of inflammation. These cells are capable of destroying microbes, cell debris, and foreign proteins by oxidative and non-oxidative processes. While primarily mediators of tissue homeostasis, there are an increasing number of studies indicating that neutrophil recruitment and transmigration can also lead to host-tissue injury and subsequently inflammation-related diseases. Neutrophil-induced tissue injury is highly regulated by the microenvironment of the infiltrated tissue, which includes cytokines, chemokines, and the provisional extracellular matrix, remodeled through increased vascular permeability and other cellular infiltrates. Thus, investigation of the effects of matrix proteins on neutrophil-EC interaction and neutrophil transmigration may help identify the proteins that induce pro- or anti-inflammatory responses. This area of research presents an opportunity to identify therapeutic targets in inflammation-related diseases. This review will summarize recent literature on the role of neutrophils and the effects of matrix proteins on neutrophil-EC interactions, with focus on three different disease models: 1) atherosclerosis, 2) COPD, and 3) tumor growth and progression. For each disease model, inflammatory molecules released by neutrophils, important regulatory matrix proteins, current anti-inflammatory treatments, and the scope for further research will be summarized.

The effects of extracellular matrix proteins on neutrophil-endothelial interaction--a roadway to multiple therapeutic opportunities.

During acute inflammation, neutrophil recruitment into extravascular tissue requires neutrophil tethering and rolling on cytokine-activated endothelial cells (ECs), tight adhesion, crawling towards EC junctions and transendothelial migration (TEM). Following TEM, neutrophils must still traverse the subendothelial basement membrane and network of pericytes (PCs). Until recently, the contribution of the PC layer to neutrophil recruitment was largely ignored. Here we analyze human neutrophil interactions with interleukin (IL)-1β-activated human EC monolayers, PC monolayers and EC/PC bilayers in vitro. Compared to EC, PC support much lower levels of neutrophil binding (54.6% vs. 7.1%, respectively) and transmigration (63.7 vs. 8.8%, respectively) despite comparable levels of IL-8 (CXCL8) synthesis and display. Remarkably, EC/PC bilayers support intermediate levels of transmigration (37.7%). Neutrophil adhesion to both cell types is Mac-1-dependent and while ICAM-1 transduction of PCs increases neutrophil adhesion to (41.4%), it does not increase transmigration through PC monolayers. TEM, which increases neutrophil Mac-1 surface expression, concomitantly increases the ability of neutrophils to traverse PCs (19.2%). These data indicate that contributions from both PCs and ECs must be considered in evaluation of microvasculature function in acute inflammation.

Transendothelial migration enables subsequent transmigration of neutrophils through underlying pericytes.

Neutrophil extravasation occurs across postcapillary venules, structures composed of endothelial cells (ECs), pericytes (PCs), and basement membrane (BM). We constructed composite models of the human postcapillary venule, combining ECs with PCs or PC-deposited BM, to better study this process. Quiescent and tumor necrosis factor α (TNF-α)-activated composites demonstrated in situ-like expression of cadherins, E-selectin, intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), platelet-endothelial cell adhesion molecule 1 (PECAM-1), CD99, and interleukin 8 (IL-8). After TNF-α activation, the ECs supported greater neutrophil adhesion (66.1 vs. 23.7% of input cells) and transmigration (35.1 vs. 7.20% of input cells) than did the PCs, but the composites behaved comparably (no significant difference) to ECs in both assays. TNF-α-activated EC-conditioned medium (CM) increased transmigration across the PCs, whereas TNF-α-activated PC-CM decreased transmigration across the ECs, and culturing on PC-derived BM decreased both adhesion to and transmigration across the ECs. Anti-very late antigen 4 (VLA-4; on neutrophils) inhibited adhesion to TNF-α-activated composites, but not to ECs alone. Anti-CD99 (expressed on all 3 cell types) inhibited transmigration across the composites (14.5% of control) more than across the ECs (39.0% of control), and venular shear stress reduced transmigration across the ECs (17.3% of static) more than across the composites (36.7% of static). These results provide proof of concept that our composite human EC/PC/BM venular construct can reveal new interactions in the inflammatory cascade.

A composite model of the human postcapillary venule for investigation of microvascular leukocyte recruitment.

Fibrosis is characterized by excessive extracellular matrix deposition and is the pathological outcome of repetitive tissue injury in many disorders. The accumulation of matrix disrupts the structure and function of the native tissue and can affect multiple organs including the lungs, heart, liver, and skin. Unfortunately, current therapies against the deadliest and most common fibrosis are ineffective. The pathogenesis of fibrosis is the result of aberrant wound healing, therefore, the microvasculature plays an important role, contributing through regulation of leukocyte recruitment, inflammation, and angiogenesis. Further exacerbating the condition, microvascular endothelial cells and pericytes can transdifferentiate into matrix depositing myofibroblasts. The contribution of the microvasculature to fibrotic progression makes its cellular components and acellular products attractive therapeutic targets. In this review, we examine many of the cytokine, matrix, and cellular microvascular components involved in fibrosis and discuss their potential as targets for fibrotic therapies with a particular focus on developing nanotechnologies.

Microvascular targets for anti-fibrotic therapeutics.

Neutrophil extravasation at post-capillary venules, consisting of EC, PC, and the shared ECM, increases following fibrotic remodeling in the lung, liver, and skin. The role of fibrotic pericyte-derived ECM in regulating EC activation and neutrophil recruitment remains unexplored.

Human microvascular pericyte basement membrane remodeling regulates neutrophil recruitment.

In response to inflammatory stimuli, microvascular endothelial cells become activated, initiating the capture and exit of neutrophils from the blood vessel and into the extravascular extracellular matrix (ECM). In the extravascular space, neutrophils bind to ECM proteins, regulating cellular functions via signaling through adhesion molecules known as integrins. The αVβ3 integrin is an important mediator of neutrophil adhesion to ECM proteins containing the Arg-Gly-Asp (RGD) peptide sequence, including fibrinogen and fibronectin. Despite the abundance of RGD sequence in the ECM, adhesion molecule-mediated neutrophil activity has been focused on the β2 (Mac-1, CD11b/CD18) and β1 integrin response to matrix proteins. Here we investigated αVβ3 integrin-mediated reactive oxidant suppression as a consequence of human neutrophil adhesion to RGD containing proteins. Using integrin ligand-modified (poly)ethylene glycol hydrogels and reactive oxygen species (ROS) sensitive fluorescent probes (dihydrotetramethylrhosamine, H2TMRos), we evaluated integrin-peptide interactions that effectively regulate ROS generation. This study demonstrates that neutrophil adhesion suppresses ROS production in an αVβ3-dependent manner. Additionally, we determine that p38 mitogen-activated protein kinase in the respiratory burst signaling pathway is interrupted by integrin-mediated adhesion. These data indicate that ECM/integrin interactions can induce αVβ3-mediated adhesion dependent downstream signaling of ROS regulation via a Mac-1 independent mechanism.

αVβ3 Integrin Regulation of Respiratory Burst in Fibrinogen Adherent Human Neutrophils.

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.

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

Fibrocytes are collagen-producing leukocytes that accumulate in patients with systemic sclerosis (SSc; scleroderma)-related interstitial lung disease (ILD) via unknown mechanisms that have been associated with altered expression of neuroimmune proteins. The extracellular matrix (ECM) influences cellular phenotypes. However, a relationship between the lung ECM and fibrocytes in SSc has not been explored. The aim of this study was to use a novel translational platform based on decellularized human lungs to determine whether the lung ECM of patients with scleroderma controls the development of fibrocytes from peripheral blood mononuclear cells.

Netrin-1 Regulates Fibrocyte Accumulation in the Decellularized Fibrotic Sclerodermatous Lung Microenvironment and in Bleomycin-Induced Pulmonary Fibrosis.

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-1β, 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.

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

The vascular basement membrane-a thin, elastic layer of extracellular matrix separating and encasing vascular cells-provides biological and mechanical cues to endothelial cells, pericytes, and migrating leukocytes. In contrast, experimental scaffolds typically used to replicate basement membranes are stiff and bio-inert. Here, we present thin, porated polyethylene glycol hydrogels to replicate human vascular basement membranes. Like commercial transwells, our hydrogels are approximately 10μm thick, but like basement membranes, the hydrogels presented here are elastic (E: 50-80kPa) and contain a dense network of small pores. Moreover, the inclusion of bioactive domains introduces receptor-mediated biochemical signaling. We compare elastic hydrogels to common culture substrates (E: >2GPa) for human endothelial cell and pericyte monolayers and bilayers to replicate postcapillary venules in vitro. Our data demonstrate that substrate elasticity facilitates differences in vascular phenotype, supporting expression of vascular markers that are increasingly replicative of venules. Endothelial cells differentially express vascular markers, like EphB4, and leukocyte adhesion molecules, such as ICAM-1, with decreased mechanical stiffness. With porated PEG hydrogels we demonstrate the ability to evaluate and observe leukocyte recruitment across endothelial cell and pericyte monolayers and bilayers, reporting that basement membrane scaffolds can significantly alter the rate of vascular migration in experimental systems. Overall, this study demonstrates the creation and utility of a new and accessible method to recapture the mechanical and biological complexity of human basement membranes in vitro.

Biomimetic, ultrathin and elastic hydrogels regulate human neutrophil extravasation across endothelial-pericyte bilayers.

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.

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

Idiopathic pulmonary fibrosis (IPF) involves the accumulation of α-smooth muscle actin-expressing myofibroblasts arising from interactions with soluble mediators such as transforming growth factor-β1 (TGF-β1) and mechanical influences such as local tissue stiffness. Whereas IPF fibroblasts are enriched for aerobic glycolysis and innate immune receptor activation, innate immune ligands related to mitochondrial injury, such as extracellular mitochondrial DNA (mtDNA), have not been identified in IPF.

Article	Year
The effects of extracellular matrix proteins on neutrophil-endothelial interaction--a roadway to multiple therapeutic opportunities. The Yale journal of biology and medicine\| PubMed ID: 22737047	2012
Transendothelial migration enables subsequent transmigration of neutrophils through underlying pericytes. PloS one\| PubMed ID: 23555870	2013
A composite model of the human postcapillary venule for investigation of microvascular leukocyte recruitment. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 24297702	2014
Microvascular targets for anti-fibrotic therapeutics. The Yale journal of biology and medicine\| PubMed ID: 24348218	2013
Human microvascular pericyte basement membrane remodeling regulates neutrophil recruitment. Microcirculation (New York, N.Y. : 1994)\| PubMed ID: 25214363	2015
αVβ3 Integrin Regulation of Respiratory Burst in Fibrinogen Adherent Human Neutrophils. Cellular and molecular bioengineering\| PubMed ID: 25632307	2014
Extracellular matrix biomimicry for the creation of investigational and therapeutic devices. Wiley interdisciplinary reviews. Nanomedicine and nanobiotechnology\| PubMed ID: 26053111	2016
Netrin-1 Regulates Fibrocyte Accumulation in the Decellularized Fibrotic Sclerodermatous Lung Microenvironment and in Bleomycin-Induced Pulmonary Fibrosis. Arthritis & rheumatology (Hoboken, N.J.)\| PubMed ID: 26749424	2016
IL-17 Promotes Neutrophil-Mediated Immunity by Activating Microvascular Pericytes and Not Endothelium. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 27534549	2016
Biomimetic, ultrathin and elastic hydrogels regulate human neutrophil extravasation across endothelial-pericyte bilayers. PloS one\| PubMed ID: 28234918	2017
Tumor Necrosis Factor-α and IL-17A Activation Induces Pericyte-Mediated Basement Membrane Remodeling in Human Neutrophilic Dermatoses. The American journal of pathology\| PubMed ID: 28609645	2017
Extracellular Mitochondrial DNA Is Generated by Fibroblasts and Predicts Death in Idiopathic Pulmonary Fibrosis. American journal of respiratory and critical care medicine\| PubMed ID: 28783377	2017

Anjelica L. Gonzalez

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