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

Interactions between endothelial cells (ECs) and mural pericytes (PCs) are critical in 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 of PCs in signaling microvascular dysfunction remains underexplored. We hypothesized that integrin-matrix interactions contribute to PC migration from the vascular wall and conversion into interstitial myofibroblasts. Herein, pro-inflammatory tumor necrosis factor α (TNFα) or a fibrotic growth factor [transforming growth factor ß1 (TGF-ß1)] were used to evaluate human PC inflammatory and fibrotic phenotypes 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.

Engineering multicellular living systems-a Keystone Symposia report.

The ability to engineer complex multicellular systems has enormous potential to inform our understanding of biological processes and disease and alter the drug development process. Engineering living systems to emulate natural processes or to incorporate new functions relies on a detailed understanding of the biochemical, mechanical, and other cues between cells and between cells and their environment that result in the coordinated action of multicellular systems. On April 3-6, 2022, experts in the field met at the Keystone symposium "Engineering Multicellular Living Systems" to discuss recent advances in understanding how cells cooperate within a multicellular system, as well as recent efforts to engineer systems like organ-on-a-chip models, biological robots, and organoids. Given the similarities and common themes, this meeting was held in conjunction with the symposium "Organoids as Tools for Fundamental Discovery and Translation".

Endothelial cell secreted VEGF-C enhances NSC VEGFR3 expression and promotes NSC survival.

Although delivery of neural stem cell (NSC) as a therapeutic treatment for intracerebral hemorrhage (ICH) provides promise, NSC delivery typically has extremely low survival rates. Here, we investigate endothelial cell (EC) and pericyte (PC) interactions with NSC, where our results demonstrate that EC, and not PC, promote NSC cell proliferation and reduce cytotoxicity under glucose deprivation (GD). Additionally, NSC proliferation was increased upon treatment with EC conditioned media, inhibited with antagonism of VEGFR3. In an NSC + EC co-culture we detected elevated levels of VEGF-C, not seen for NSC cultured alone. Exogenous VEGF-C induced NSC upregulation of VEGFR3, promoted proliferation, and reduced cytotoxicity. Finally, we delivered microbeads containing NSC + EC into a murine ICH cavity, where VEGF-C was increasingly present in the injury site, not seen upon delivery NSC encapsulated alone. These studies demonstrate that EC-secreted VEGF-C may promote NSC survival during injury, enhancing the potential for cell delivery therapies for stroke.

Endothelial cell secreted metalloproteinase-2 enhances neural stem cell N-cadherin expression, clustering, and migration.

Neuroblasts have a clustered phenotype critical for their unidirectional migration, which in part is dependent on signaling from microvascular endothelial cells (EC) and pericytes (PC). Diffusible signals secreted by vascular cells have been demonstrated to increase survival, proliferation, and differentiation of subventricular zone resident neural stem cells (NSC); however, the signals that promote the necessary initiating step of NSC clustering are undefined. To investigate the role of vascular cells in promoting NSC clustering and directing migration, we created a 3-D hydrogel that mimics the biomechanics, biochemistry, and architectural complexity of brain tissue. We demonstrate that EC, and not PC, have a crucial role in NSC clustering and migration, further verified through microfluidic chamber systems and traction force microscopy. Ablation of the extended NSC aggregate arm halts aggregate movement, suggesting that clustering is a prerequisite for migration. When cultured with EC, NSC clustering occurs and NSC coincidentally increase their expression of N-cadherin, as compared to NSC cultured alone. NSC-presented N-cadherin expression was increased following exposure to EC secreted metalloproteinase-2 (MMP2). We demonstrate that inhibition of MMP2 prevented NSC N-cadherin surface expression and subsequent NSC clustering, even when NSC were in direct contact with EC. Furthermore, with exogenous activation of EGFR, which serves as a downstream activator of N-cadherin cleavage, NSC form clusters. Our results suggest that EC secretion of MMP2 promotes NSC clustering through N-cadherin expression. The insight gained about the mechanisms by which EC promote NSC migration may enhance NSC therapeutic response to sites of injury.

ECM hydrogel improves the delivery of PEG microsphere-encapsulated neural stem cells and endothelial cells into tissue cavities caused by stroke.

Intracerebral implantation of neural stem cells (NSCs) to treat stroke remains an inefficient process with <5% of injected cells being retained. To improve the retention and distribution of NSCs after a stroke, we investigated the utility of NSCs' encapsulation in polyethylene glycol (PEG) microspheres. We first characterized the impact of the physical properties of different syringes and needles, as well as ejection speed, upon delivery of microspheres to the stroke injured rat brain. A 20 G needle size at a 10 µL/min flow rate achieved the most efficient microsphere ejection. Secondly, we optimized the delivery vehicles for in vivo implantation of PEG microspheres. The suspension of microspheres in extracellular matrix (ECM) hydrogel showed superior retention and distribution in a cortical stroke caused by photothrombosis, as well as in a striatal and cortical cavity ensuing middle cerebral artery occlusion (MCAo). Thirdly, NSCs or NSCs + endothelial cells (ECs) encapsulated into biodegradable microspheres were implanted into a large stroke cavity. Cells in microspheres exhibited a high viability, survived freezing and transport. Implantation of 110 cells/microsphere suspended in ECM hydrogel produced a highly efficient delivery that resulted in the widespread distribution of NSCs in the tissue cavity and damaged peri-infarct tissues. Co-delivery of ECs enhanced the in vivo survival and distribution of ∼1.1 million NSCs. The delivery of NSCs and ECs can be dramatically improved using microsphere encapsulation combined with suspension in ECM hydrogel. These biomaterial innovations are essential to advance clinical efforts to improve the treatment of stroke using intracerebral cell therapy.

Author Correction: Minimally Invasive Delivery of Microbeads with Encapsulated, Viable and Quiescent Neural Stem Cells to the Adult Subventricular Zone.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Minimally Invasive Delivery of Microbeads with Encapsulated, Viable and Quiescent Neural Stem Cells to the Adult Subventricular Zone.

Stem cell therapies demonstrate promising results as treatment for neurological disease and injury, owing to their innate ability to enhance endogenous neural tissue repair and promote functional recovery. However, delivery of undifferentiated and viable neuronal stem cells requires an engineered delivery system that promotes integration of transplanted cells into the inflamed and cytotoxic region of damaged tissue. Within the brain, endothelial cells (EC) of the subventricular zone play a critical role in neural stem cell (NSC) maintenance, quiescence and survival. Therefore, here, we describe the use of polyethylene glycol microbeads for the coincident delivery of EC and NSC as a means of enhancing appropriate NSC quiescence and survival during transplantation into the mouse brain. We demonstrate that EC and NSC co-encapsulation maintained NSC quiescence, enhanced NSC viability, and facilitated NSC extravasation in vitro, as compared to NSC encapsulated alone. In addition, co-encapsulated cells delivered to an in vivo non-injury model reduced inflammatory response compared to freely injected NSC. These results suggest the strong potential of a biomimetic engineered niche for NSC delivery into the brain following neurological injury.

Engineered Biomimetic Neural Stem Cell Niche.

PURPOSE OF REVIEW: Neural stem cells (NSCs) have the potential to proliferate and differentiate into functional neurons, heightening their potential use for therapeutic applications. This review explores bioengineered systems which recapitulate NSC niche cell-cell and cell-matrix interactions. RECENT FINDINGS: Delivery of NSCs to the cytotoxic injured brain is limited by low cell survival rates post-transplantation and poor maintenance of native niche bioactive components. The use of biomaterial platforms can mimic in vivo the environment of the two germinal areas of the adult brain in which NSCs thrive. An environmental mimic that includes extracellular proteins and moieties, along with appropriate biomechanical cues has recently demonstrated promising results in enhancing neurogenesis, aiding the production of a bioengineered niche. SUMMARY: Biocomposition, biomechanics, and biostructure can be manipulated through engineered platforms to re-create the biofunctionality of an NSC niche. Upon transplantation and delivery with biomimetic scaffolds, NSCs show potential to promote functional recovery and rebuild neural circuitry post neurological trauma.

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.

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.

Extracellular Mitochondrial DNA Is Generated by Fibroblasts and Predicts Death in Idiopathic Pulmonary Fibrosis.

RATIONALE: 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-ß1 (TGF-ß1) 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. OBJECTIVES: We aimed to define an association between mtDNA and fibroblast responses in IPF. METHODS: We evaluated the response of normal human lung fibroblasts (NHLFs) to stimulation with mtDNA and determined whether the glycolytic reprogramming that occurs in response to TGF-ß1 stimulation and direct contact with stiff substrates, and spontaneously in IPF fibroblasts, is associated with excessive levels of mtDNA. We measured mtDNA concentrations in bronchoalveolar lavage (BAL) from subjects with and without IPF, as well as in plasma samples from two longitudinal IPF cohorts and demographically matched control subjects. MEASUREMENTS AND MAIN RESULTS: Exposure to mtDNA augments α-smooth muscle actin expression in NHLFs. The metabolic changes in NHLFs that are induced by interactions with TGF-ß1 or stiff hydrogels are accompanied by the accumulation of extracellular mtDNA. These findings replicate the spontaneous phenotype of IPF fibroblasts. mtDNA concentrations are increased in IPF BAL and plasma, and in the latter compartment, they display robust associations with disease progression and reduced event-free survival. CONCLUSIONS: These findings demonstrate a previously unrecognized and highly novel connection between metabolic reprogramming, mtDNA, fibroblast activation, and clinical outcomes that provides new insight into IPF.

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.

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

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.

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.

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

OBJECTIVE: 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. METHODS: We performed biomechanical evaluation of decellularized scaffolds prepared from lung explants from healthy control subjects and patients with scleroderma, using tensile testing and biochemical and proteomic analysis. Cells obtained from healthy controls and patients with SSc-related ILD were cultured on these scaffolds, and CD45+pro-ColIα1+ cells meeting the criteria for fibrocytes were quantified. The contribution of the neuromolecule netrin-1 to fibrosis was assessed using neutralizing antibodies in this system and by administering bleomycin via inhalation to netrin-1(+/-) mice. RESULTS: Compared with control lung scaffolds, lung scaffolds from patients with SSc-related ILD showed aberrant anatomy, enhanced stiffness, and abnormal ECM composition. Culture of control cells in lung scaffolds from patients with SSc-related ILD increased production of pro-ColIα1+ cells, which was stimulated by enhanced stiffness and abnormal ECM composition. Cells from patients with SSc-related ILD demonstrated increased pro-ColIα1 responsiveness to lung scaffolds from scleroderma patients but not enhanced stiffness. Enhanced detection of netrin-1-expressing CD14(low) cells in patients with SSc-related ILD was observed, and antibody-mediated netrin-1 neutralization attenuated detection of CD45+pro-ColIα1+ cells in all settings. Netrin-1(+/-) mice were protected against bleomycin-induced lung fibrosis and fibrocyte accumulation. CONCLUSION: Factors present in the lung matrices of patients with scleroderma regulate fibrocyte accumulation via a netrin-1-dependent pathway. Netrin-1 regulates bleomycin-induced pulmonary fibrosis in mice. Netrin-1 might be a novel therapeutic target in SSc-related ILD.

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.

Human microvascular pericyte basement membrane remodeling regulates neutrophil recruitment.

OBJECTIVE: 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. METHODS: To elucidate the role of human pericyte-derived ECM in EC activation, we characterized PC-derived ECM following transforming growth factor-ß1, IL-1ß, CCL2, or bleomycin activation, and examined surface adhesion molecule expression and neutrophil recruitment by EC cultured on PC-ECM. RESULTS: Pro-inflammatory activation of PC-induced deposition of compositionally distinct ECM compared with non-activated control. Bleomycin activation induced fibronectin-rich and collagen-poor ECM remodeling by PC, facilitating increased neutrophil transendothelial migration when compared with non-activated pericyte ECM (49.9 ± 3.4% versus 29.7 ± 1.4%). Increases in fibronectin compared to collagen I, are largely responsible for ECM-regulated neutrophil recruitment, as EC cultured on fibronectin supported increased neutrophil transmigration compared to collagen I (51.6 ± 6.2% versus 28.0 ± 4.8%). We attribute this difference to increased expression of ICAM-1 and its redistribution to EC borders. CONCLUSIONS: This is the first demonstration of human pericyte sensitivity to inflammatory stimuli, inducing fibrotic matrix deposition that regulates EC adhesion molecule expression and neutrophil recruitment.

αVß3 Integrin Regulation of Respiratory Burst in Fibrinogen Adherent Human Neutrophils.

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 αVß3 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 ß2 (Mac-1, CD11b/CD18) and ß1 integrin response to matrix proteins. Here we investigated αVß3 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 αVß3-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 αVß3-mediated adhesion dependent downstream signaling of ROS regulation via a Mac-1 independent mechanism.

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

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.