Gretchen J. Mahler

Characterization of Caco-2 and HT29-MTX cocultures in an in vitro digestion/cell culture model used to predict iron bioavailability.

Cocultures of two human cell lines, Caco-2 and HT29-MTX mucus-producing cells, have been incorporated into an in vitro digestion/cell culture model used to predict iron bioavailability. A range of different foods were subjected to in vitro digestion, and iron bioavailability from digests was assessed with Caco-2, Caco-2 overlaid with porcine mucin, HT29-MTX or cocultures of Caco-2 and HT29-MTX at varying ratios. It was found that increasing the ratio of HT29-MTX cells decreased the amount of ferritin formed and resulted in an overall decline in the ability of the model to detect differences in iron bioavailability. At the physiologically relevant ratios of 90% Caco-2/10% HT29-MTX and 75% Caco-2/25% HT29-MTX, however, a mucus layer completely covered the cell monolayer and the in vitro digestion model was nearly as responsive to changes in sample iron bioavailability as pure Caco-2 cultures. The in vitro digestion/Caco-2 cell culture model correlates well with human iron bioavailability studies, but, as mucus appears to play a role in iron absorption, the addition of a physiologically realistic mucus layer and goblet-type cells to this model may give more accurate iron bioavailability predictions.

Characterization of a gastrointestinal tract microscale cell culture analog used to predict drug toxicity

The lining of the gastrointestinal (GI) tract is the largest surface exposed to the external environment in the human body. One of the main functions of the small intestine is absorption, and intestinal absorption is a route used by essential nutrients, chemicals, and pharmaceuticals to enter the systemic circulation. Understanding the effects of digestion on a drug or chemical, how compounds interact with and are absorbed through the small intestinal epithelium, and how these compounds affect the rest of the body is critical for toxicological evaluation. Our goal is to create physiologically realistic in vitro models of the human GI tract that provide rapid, inexpensive, and accurate predictions of the bodys response to orally delivered drugs and chemicals. Our group has developed an in vitro microscale cell culture analog (µCCA) of the GI tract that includes digestion, a mucus layer, and physiologically realistic cell populations. The GI tract µCCA, coupled with a multi‐chamber silicon µCCA representing the systemic circulation, is described and challenged with acetaminophen. Proof of concept experiments showed that acetaminophen passes through and is metabolized by the in vitro intestinal epithelium and is further metabolized by liver cells, resulting in liver cell toxicity in a dose‐dependent manner. The µCCA response is also consistent with in vivo measurements in mice. The system should be broadly useful for studies on orally delivered drugs or ingestion of chemicals with potential toxicity. Biotechnol. Bioeng. 2009; 104: 193–205

Aortic valve disease and treatment: The need for naturally engineered solutions☆

The aortic valve regulates unidirectional flow of oxygenated blood to the myocardium and arterial system. The natural anatomical geometry and microstructural complexity ensures biomechanically and hemodynamically efficient function. The compliant cusps are populated with unique cell phenotypes that continually remodel tissue for long-term durability within an extremely demanding mechanical environment. Alteration from normal valve homeostasis arises from genetic and microenvironmental (mechanical) sources, which lead to congenital and/or premature structural degeneration. Aortic valve stenosis pathobiology shares some features of atherosclerosis, but its final calcification endpoint is distinct. Despite its broad and significant clinical significance, very little is known about the mechanisms of normal valve mechanobiology and mechanisms of disease. This is reflected in the paucity of predictive diagnostic tools, early stage interventional strategies, and stagnation in regenerative medicine innovation. Tissue engineering has unique potential for aortic valve disease therapy, but overcoming current design pitfalls will require even more multidisciplinary effort. This review summarizes the latest advancements in aortic valve research and highlights important future directions.

Inflammatory Cytokines Promote Mesenchymal Transformation in Embryonic and Adult Valve Endothelial Cells

Objective—Inflammatory activation of valve endothelium is an early phase of aortic valve disease pathogenesis, but subsequent mechanisms are poorly understood. Adult valve endothelial cells retain the developmental ability to undergo endothelial-to-mesenchymal transformation (EndMT), but a biological role has not been established. Here, we test whether and how inflammatory cytokines (tumor necrosis factor-&agr; and interleukin-6) regulate EndMT in embryonic and adult valve endothelium. Methods and Results—Using in vitro 3-dimensional collagen gel culture assays with primary cells, we determined that interleukin-6 and tumor necrosis factor-&agr; induce EndMT and cell invasion in dose-dependent manners. Inflammatory-EndMT occurred through an Akt/nuclear factor-&kgr;B–dependent pathway in both adult and embryonic stages. In embryonic valves, inflammatory-EndMT required canonical transforming growth factor-&bgr; signaling through activin receptor-like kinases 2 and 5 to drive EndMT. In adult valve endothelium, however, inflammatory-induced EndMT still occurred when activin receptor-like kinases 2 and 5 signaling was blocked. Inflammatory receptor gene expression was significantly upregulated in vivo during embryonic valve maturation. Endothelial-derived mesenchymal cells expressing activated nuclear factor-&kgr;B were found distal to calcific lesions in diseased human aortic valves. Conclusion—Inflammatory cytokine–induced EndMT in valve endothelium is present in both embryonic and adult stages, acting through Akt/nuclear factor-&kgr;B, but differently using transforming growth factor-&bgr; signaling. Molecular signatures of valve EndMT may be important diagnostic and therapeutic targets in early valve disease.

Effects of shear stress pattern and magnitude on mesenchymal transformation and invasion of aortic valve endothelial cells.

Understanding the role of mechanical forces on cell behavior is critical for tissue engineering, regenerative medicine, and disease initiation studies. Current hemodynamic bioreactors are largely limited to 2D substrates or the application of general flow conditions at a tissue level, which eliminates the investigation of some essential physiological and pathological responses. One example is the mesenchymal transformation of endothelial cells in response to shear stress. Endothelial to mesenchymal transformation (EndMT) is a valve morphogenic mechanism associated with aortic valve disease initiation. The aortic valve experiences oscillatory shear on the disease‐susceptible fibrosa, and the role of hemodynamics on adult EndMT is unknown. The goal of this work was to develop and characterize a microfluidic bioreactor that applies physiologically relevant laminar or oscillatory shear stresses to endothelial cells and permits the quantitative analysis of 3D cell–extracellular matrix (ECM) interactions. In this study, porcine aortic valve endothelial cells were seeded onto 3D collagen I gels and exposed to different magnitudes of steady or oscillatory shear stress for 48 h. Cells elongated and aligned perpendicular to laminar, but not oscillatory shear. Low steady shear stress (2 dyne/cm2) and oscillatory shear stress upregulated EndMT (ACTA2, Snail, TGFB1) and inflammation (ICAM1, NFKB1) related gene expression, EndMT‐related (αSMA) protein expression, and matrix invasion when compared with static controls or cells exposed to high steady shear (10 and 20 dyne/cm2). Our system enables direct testing of the role of shear stress on endothelial cell mesenchymal transformation in a dynamic, 3D environment and shows that hemodynamics regulate EndMT in adult valve endothelial cells. Biotechnol. Bioeng. 2014;111: 2326–2337.

Transforming Growth Factor β, Bone Morphogenetic Protein, and Vascular Endothelial Growth Factor Mediate Phenotype Maturation and Tissue Remodeling by Embryonic Valve Progenitor Cells: Relevance for Heart Valve Tissue Engineering

Despite years of research, limited understanding of heart valve cell and tissue biology remains a key impediment to valvular tissue engineering progress. Heart valves rapidly evolve structural and cellular composition naturally during embryonic development, which suggests that mimicking these signaling events could advance engineered valve tissue research. Many inductive factors participate in the initial endocardial to mesenchymal transformation event necessary to form the prevalvular cushion, but far less is known about the regulation of cushion remodeling into fibrous leaflets and the associated maturation of valvular progenitors into fibroblasts. In this study, we combine in vitro three-dimensional tissue-engineered models of embryonic valvular remodeling with in vivo analysis to determine the roles of three prominent growth factors during avian mitral valvulogenesis. We show that transforming growth factor-β3 (TGFβ3), bone morphogenetic protein 2 (BMP2), and vascular endothelial growth factor A (VEGFA) are expressed in spatiotemporally distinct patterns and at significantly different levels within remodeling embryonic valves in vivo. We then establish dose-dependent functional roles for each growth factor in 3D cultured embryonic valve progenitor cells. TGFβ3 induced cell migration, invasion, and matrix condensation; BMP2 induced invasion. VEGFA inhibited invasion but increased migration. Finally, we determine that TGFβ3 induced myofibroblastic differentiation in a dose-dependent manner, whereas VEGFA and BMP2 did not. Collectively, these findings frame a naturally derived blueprint for controlling valvulogenic remodeling and phenotype maturation, which can be integrated into clinically needed regenerative strategies for heart valve disease and to accelerate the development of engineered tissue valves.

Modeling Barrier Tissues In Vitro: Methods, Achievements, and Challenges

Organ-on-a-chip devices have gained attention in the field of in vitro modeling due to their superior ability in recapitulating tissue environments compared to traditional multiwell methods. These constructed growth environments support tissue differentiation and mimic tissue–tissue, tissue–liquid, and tissue–air interfaces in a variety of conditions. By closely simulating the in vivo biochemical and biomechanical environment, it is possible to study human physiology in an organ-specific context and create more accurate models of healthy and diseased tissues, allowing for observations in disease progression and treatment. These chip devices have the ability to help direct, and perhaps in the distant future even replace animal-based drug efficacy and toxicity studies, which have questionable relevance to human physiology. Here, we review recent developments in the in vitro modeling of barrier tissue interfaces with a focus on the use of novel and complex microfluidic device platforms.

Titanium dioxide nanoparticle ingestion alters nutrient absorption in an in vitro model of the small intestine

Ingestion of titanium dioxide (TiO2) nanoparticles from products such as agricultural chemicals, processed food, and nutritional supplements is nearly unavoidable. The gastrointestinal tract serves as a critical interface between the body and the external environment, and is the site of essential nutrient absorption. The goal of this study was to examine the effects of ingesting the 30 nm TiO2 nanoparticles with an in vitro cell culture model of the small intestinal epithelium, and to determine how acute or chronic exposure to nano-TiO2 influences intestinal barrier function, reactive oxygen species generation, proinflammatory signaling, nutrient absorption (iron, zinc, fatty acids), and brush border membrane enzyme function (intestinal alkaline phosphatase). A Caco-2/HT29-MTX cell culture model was exposed to physiologically relevant doses of TiO2 nanoparticles for acute (four hours) or chronic (five days) time periods. Exposure to TiO2 nanoparticles significantly decreased intestinal barrier function following chronic exposure. Reactive oxygen species (ROS) generation, proinflammatory signaling, and intestinal alkaline phosphatase activity all showed increases in response to nano-TiO2. Iron, zinc, and fatty acid transport were significantly decreased following exposure to TiO2 nanoparticles. This is because nanoparticle exposure induced a decrease in absorptive microvilli in the intestinal epithelial cells. Nutrient transporter protein gene expression was also altered, suggesting that cells are working to regulate the transport mechanisms disturbed by nanoparticle ingestion. Overall, these results show that intestinal epithelial cells are affected at a functional level by physiologically relevant exposure to nanoparticles commonly ingested from food.

Inflammatory regulation of valvular remodeling: the good(?), the bad, and the ugly.

Heart valve disease is unique in that it affects both the very young and very old, and does not discriminate by financial affluence, social stratus, or global location. Research over the past decade has transformed our understanding of heart valve cell biology, yet still more remains unclear regarding how these cells respond and adapt to their local microenvironment. Recent studies have identified inflammatory signaling at nearly every point in the life cycle of heart valves, yet its role at each stage is unclear. While the vast majority of evidence points to inflammation as mediating pathological valve remodeling and eventual destruction, some studies suggest inflammation may provide key signals guiding transient adaptive remodeling. Though the mechanisms are far from clear, inflammatory signaling may be a previously unrecognized ally in the quest for controlled rapid tissue remodeling, a key requirement for regenerative medicine approaches for heart valve disease. This paper summarizes the current state of knowledge regarding inflammatory mediation of heart valve remodeling and suggests key questions moving forward.

Cardiac developmental toxicity.

Congenital heart disease (CHD) is a highly prevalent problem with mostly unknown origins. Many cases of CHD likely involve an environmental exposure coupled with genetic susceptibility, but practical and ethical considerations make nongenetic causes of CHD difficult to assess in humans. The development of the heart is highly conserved across all vertebrate species, making animal models an excellent option for screening potential cardiac teratogens. This review will discuss exposures known to cause cardiac defects, stages of heart development that are most sensitive to teratogen exposure, benefits and limitations of animal models of cardiac development, and future considerations for cardiac developmental toxicity research.