Steven C. George

Exhaled nitric oxide in pulmonary diseases: a comprehensive review

The upregulation of nitric oxide (NO) by inflammatory cytokines and mediators in central and peripheral airway sites can be monitored easily in exhaled air. It is now possible to estimate the predominant site of increased fraction of exhaled NO (FeNO) and its potential pathologic and physiologic role in various pulmonary diseases. In asthma, increased FeNO reflects eosinophilic-mediated inflammatory pathways moderately well in central and/or peripheral airway sites and implies increased inhaled and systemic corticosteroid responsiveness. Recently, five randomized controlled algorithm asthma trials reported only equivocal benefits of adding measurements of FeNO to usual clinical guideline management including spirometry; however, significant design issues may exist. Overall, FeNO measurement at a single expiratory flow rate of 50 mL/s may be an important adjunct for diagnosis and management in selected cases of asthma. This may supplement standard clinical asthma care guidelines, including spirometry, providing a noninvasive window into predominantly large-airway-presumed eosinophilic inflammation. In COPD, large/central airway maximal NO flux and peripheral/small airway/alveolar NO concentration may be normal and the role of FeNO monitoring is less clear and therefore less established than in asthma. Furthermore, concurrent smoking reduces FeNO. Monitoring FeNO in pulmonary hypertension and cystic fibrosis has opened up a window to the role NO may play in their pathogenesis and possible clinical benefits in the management of these diseases.

Prevascularization of a Fibrin-Based Tissue Construct Accelerates the Formation of Functional Anastomosis with Host Vasculature

One critical obstacle facing tissue engineering is the formation of functional vascular networks that can support tissue survival in vivo. We hypothesized that prevascularizing a tissue construct with networks of well-formed capillaries would accelerate functional anastomosis with the host upon implantation. Fibrin-based tissues were prevascularized with capillary networks by coculturing human umbilical vein endothelial cells (HUVECs) and fibroblasts in fibrin gels for 1 week. The prevascularized tissue and nonprevascularized controls were implanted subcutaneously onto the dorsal surface of immune-deficient mice and retrieved at days 3, 5, 7 and 14. HUVEC-lined vessels containing red blood cells were evident in the prevascularized tissue by day 5, significantly earlier than nonprevascularized tissues (14 days). Analysis of the HUVEC-lined vessels demonstrated that the number and area of perfused lumens in the prevascularized tissue were significantly larger compared to controls. In addition, collagen deposition and a larger number of proliferating cells were evident in the prevascularized tissue at day 14. Our results demonstrate that prevascularizing a fibrin-based tissue with well-formed capillaries accelerates anastomosis with the host vasculature, and promotes cellular activity consistent with tissue remodeling. Our prevascularization strategy may be useful to design large three-dimensional engineered tissues.

Concise review: maturation phases of human pluripotent stem cell-derived cardiomyocytes.

Human pluripotent stem cell‐derived cardiomyocytes (hPS‐CM) may offer a number of advantages over previous cardiac models, however, questions of their immaturity complicate their adoption as a new in vitro model. hPS‐CM differ from adult cardiomyocytes with respect to structure, proliferation, metabolism and electrophysiology, better approximating fetal cardiomyocytes. Time in culture appears to significantly impact phenotype, leading to what can be referred to as early and late hPS‐CM. This work surveys the phenotype of hPS‐CM, including structure, bioenergetics, sensitivity to damage, gene expression, and electrophysiology, including action potential, ion channels, and intracellular calcium stores, while contrasting fetal and adult CM with hPS‐CM at early and late time points after onset of differentiation. STEM CELLS 2013;31:829–837

Mesenchymal cells stimulate capillary morphogenesis via distinct proteolytic mechanisms

During angiogenesis, endothelial cells (ECs) degrade their surrounding extracellular matrix (ECM) to facilitate invasion. How interactions between ECs and other cells within their microenvironment facilitate this process is only partially understood. We have utilized a tractable 3D co-culture model to investigate the proteolytic mechanisms by which pre-committed or more highly committed mesenchymal cells stimulate capillary formation. On their own, ECs invade their surrounding matrix, but do not form capillaries. However, in the presence of either mesenchymal stem cells (MSCs) or fibroblasts, ECs form polarized, tubular structures that are intimately associated with mesenchymal cells. Further, ECs up-regulate gene expression of several extracellular proteases upon co-culture with either mesenchymal cell type. The administration of both broad spectrum and specific protease inhibitors demonstrated that MSC-stimulated capillary formation relied solely on membrane-type matrix metalloproteinases (MT-MMPs) while fibroblast-mediated sprouting proceeded independent of MMP inhibition unless the plasminogen activator/plasmin axis was inhibited in concert. While other studies have established a role for the ECM itself in dictating proteolysis and matrix degradation during capillary morphogenesis, the present study illustrates that heterotypic cellular interactions within the microenvironment can direct the proteolytic mechanisms required for capillary formation.

A microfluidic platform for generating large-scale nearly identical human microphysiological vascularized tissue arrays

This paper reports a polydimethylsiloxane microfluidic model system that can develop an array of nearly identical human microtissues with interconnected vascular networks. The microfluidic system design is based on an analogy with an electric circuit, applying resistive circuit concepts to design pressure dividers in serially-connected microtissue chambers. A long microchannel (550, 620 and 775 mm) creates a resistive circuit with a large hydraulic resistance. Two media reservoirs with a large cross-sectional area and of different heights are connected to the entrance and exit of the long microchannel to serve as a pressure source, and create a near constant pressure drop along the long microchannel. Microtissue chambers (0.12 μl) serve as a two-terminal resistive component with an input impedance >50-fold larger than the long microchannel. Connecting each microtissue chamber to two different positions along the long microchannel creates a series of pressure dividers. Each microtissue chamber enables a controlled pressure drop of a segment of the microchannel without altering the hydrodynamic behaviour of the microchannel. The result is a controlled and predictable microphysiological environment within the microchamber. Interstitial flow, a mechanical cue for stimulating vasculogenesis, was verified by finite element simulation and experiments. The simplicity of this design enabled the development of multiple microtissue arrays (5, 12, and 30 microtissues) by co-culturing endothelial cells, stromal cells, and fibrin within the microchambers over two and three week periods. This methodology enables the culturing of a large array of microtissues with interconnected vascular networks for biological studies and applications such as drug development.

A three-dimensional in vitro model of tumor cell intravasation

Metastasis is the cause of over 90% of all human cancer deaths. Early steps in the metastatic process include: the formation of new blood vessels, the initiation of epithelial-mesenchymal transition (EMT), and the mobilization of tumor cells into the circulation. There are ongoing efforts to replicate the physiological landscape of human tumor tissue using three-dimensional in vitro culture models; however, few systems are able to capture the full range of authentic, complex in vivo events such as neovascularization and intravasation. Here we introduce the Prevascularized Tumor (PVT) model to investigate early events of solid tumor progression. PVT spheroids are composed of endothelial and tumor cells, and are embedded in a fibrin matrix containing fibroblasts. The PVT model facilitates two mechanisms of vessel formation: robust sprouting angiogenesis into the matrix, and contiguous vascularization within the spheroid. Furthermore, the PVT model enables the intravasation of tumor cells that is enhanced under low oxygen conditions and is also dependent on the key EMT transcription factor Slug. The PVT model provides a significant advance in the mimicry of human tumors in vitro, and may improve investigation and targeting of events in the metastatic process.

Predicting bulk mechanical properties of cellularized collagen gels using multiphoton microscopy

Cellularized collagen gels are a common model in tissue engineering, but the relationship between the microstructure and bulk mechanical properties is only partially understood. Multiphoton microscopy (MPM) is an ideal non-invasive tool for examining collagen microstructure, cellularity and crosslink content in these gels. In order to identify robust image parameters that characterize microstructural determinants of the bulk elastic modulus, we performed serial MPM and mechanical tests on acellular and cellularized (normal human lung fibroblasts) collagen hydrogels, before and after glutaraldehyde crosslinking. Following gel contraction over 16 days, cellularized collagen gel content approached that of native connective tissues (∼200 mg ml⁻¹). Youngs modulus (E) measurements from acellular collagen gels (range 0.5-12 kPa) exhibited a power-law concentration dependence (range 3-9 mg ml⁻¹) with exponents from 2.1 to 2.2, similar to other semiflexible biopolymer networks such as fibrin and actin. In contrast, cellularized collagen gel stiffness (range 0.5-27 kPa) produced concentration-dependent exponents of 0.7 uncrosslinked and 1.1 crosslinked (range ∼5-200 mg ml⁻¹). The variation in E of cellularized collagen hydrogels can be explained by a power-law dependence on robust image parameters: either the second harmonic generation (SHG) and two-photon fluorescence (TPF) (matrix component) skewness (R²=0.75, exponents of -1.0 and -0.6, respectively); or alternatively the SHG and TPF (matrix component) speckle contrast (R²=0.83, exponents of -0.7 and -1.8, respectively). Image parameters based on the cellular component of TPF signal did not improve the fits. The concentration dependence of E suggests enhanced stress relaxation in cellularized vs. acellular gels. SHG and TPF image skewness and speckle contrast from cellularized collagen gels can predict E by capturing mechanically relevant information on collagen fiber, cell and crosslink density.

Two-photon laser scanning microscopy of epithelial cell-modulated collagen density in engineered human lung tissue.

Tissue remodeling is a complex process that can occur in response to a wound or injury. In lung tissue, abnormal remodeling can lead to permanent structural changes that are characteristic of important lung diseases such as interstitial pulmonary fibrosis and bronchial asthma. Fibroblast-mediated contraction of three-dimensional collagen gels is considered an in vitro model of tissue contraction and remodeling, and the epithelium is one factor thought to modulate this process. We studied the effects of epithelium on collagen density and contraction using two-photon laser scanning microscopy (TPLSM). TPLSM was used to image autofluorescence of collagen fibers in an engineered tissue model of the human respiratory mucosa -- a three-dimensional co-culture of human lung fibroblasts (CCD-18 lu), denatured type I collagen, and a monolayer of human alveolar epithelial cell line (A549) or human bronchial epithelial cell line (16HBE14o(-)). Tissues were imaged at days 1, 8, and 15 at 10 depths within the tissue. Gel contraction was measured concurrently with TPLSM imaging. Image analysis shows that gels without an epithelium had the fastest rate of decay of fluorescent signal, corresponding to highest collagen density. Results of the gel contraction assay show that gels without an epithelium also had the highest degree of contraction (19.8% +/- 4.0%). We conclude that epithelial cells modulate collagen density and contraction of engineered human lung tissue, and TPLSM is an effective tool to investigate this phenomenon.

Nitric oxide gas phase release in human small airway epithelial cells

BackgroundAsthma is a chronic airway inflammatory disease characterized by an imbalance in both Th1 and Th2 cytokines. Exhaled nitric oxide (NO) is elevated in asthma, and is a potentially useful non-invasive marker of airway inflammation. However, the origin and underlying mechanisms of intersubject variability of exhaled NO are not yet fully understood. We have previously described NO gas phase release from normal human bronchial epithelial cells (NHBEs, tracheal origin). However, smaller airways are the major site of morbidity in asthma. We hypothesized that IL-13 or cytomix (IL-1β, TNF-α, and IFN-γ) stimulation of differentiated small airway epithelial cells (SAECs, generation 10–12) and A549 cells (model cell line of alveolar type II cells) in culture would enhance NO gas phase release.MethodsConfluent monolayers of SAECs and A549 cells were cultured in Transwell plates and SAECs were allowed to differentiate into ciliated and mucus producing cells at an air-liquid interface. The cells were then stimulated with IL-13 (10 ng/mL) or cytomix (10 ng/mL for each cytokine). Gas phase NO release in the headspace air over the cells was measured for 48 hours using a chemiluminescence analyzer.ResultsIn contrast to our previous result in NHBE, baseline NO release from SAECs and A549 is negligible. However, NO release is significantly increased by cytomix (0.51 ± 0.18 and 0.29 ± 0.20 pl.s-1.cm-2, respectively) reaching a peak at approximately 10 hours. iNOS protein expression increases in a consistent pattern both temporally and in magnitude. In contrast, IL-13 only modestly increases NO release in SAECs reaching a peak (0.06 ± 0.03 pl.s-1.cm-2) more slowly (30 to 48 hours), and does not alter NO release in A549 cells.ConclusionWe conclude that the airway epithelium is a probable source of NO in the exhaled breath, and intersubject variability may be due, in part, to variability in the type (Th1 vs Th2) and location (large vs small airway) of inflammation.

3D microtumors in vitro supported by perfused vascular networks.

There is a growing interest in developing microphysiological systems that can be used to model both normal and pathological human organs in vitro. This “organs-on-chips” approach aims to capture key structural and physiological characteristics of the target tissue. Here we describe in vitro vascularized microtumors (VMTs). This “tumor-on-a-chip” platform incorporates human tumor and stromal cells that grow in a 3D extracellular matrix and that depend for survival on nutrient delivery through living, perfused microvessels. Both colorectal and breast cancer cells grow vigorously in the platform and respond to standard-of-care therapies, showing reduced growth and/or regression. Vascular-targeting agents with different mechanisms of action can also be distinguished, and we find that drugs targeting only VEGFRs (Apatinib and Vandetanib) are not effective, whereas drugs that target VEGFRs, PDGFR and Tie2 (Linifanib and Cabozantinib) do regress the vasculature. Tumors in the VMT show strong metabolic heterogeneity when imaged using NADH Fluorescent Lifetime Imaging Microscopy and, compared to their surrounding stroma, many show a higher free/bound NADH ratio consistent with their known preference for aerobic glycolysis. The VMT platform provides a unique model for studying vascularized solid tumors in vitro.