Akiko Mammoto

Reconstituting Organ-Level Lung Functions on a Chip

Just Breathe Design of artificial systems that mimic in vivo organs could provide a better alternative for understanding mechanisms underlying physiological responses than current cell-based models or animal tests. Huh et al. (p. 1662) have created a tissue-tissue interface of human-cultured epithelial cells and endothelial cells together, with extracellular matrix in a device that models the alveolar-capillary interface of the human lung. The device mimicked physiological organ-level functions, including pathogen-induced inflammatory responses and responses to cytokine exposure. Breathing-type movements affected acute pulmonary cell toxicity and proinflammatory activity of widely used nanoparticulates. Endothelial and epithelial cells grown in a microfluidics apparatus mimic the alveolar-capillary interface of the lung. Here, we describe a biomimetic microsystem that reconstitutes the critical functional alveolar-capillary interface of the human lung. This bioinspired microdevice reproduces complex integrated organ-level responses to bacteria and inflammatory cytokines introduced into the alveolar space. In nanotoxicology studies, this lung mimic revealed that cyclic mechanical strain accentuates toxic and inflammatory responses of the lung to silica nanoparticles. Mechanical strain also enhances epithelial and endothelial uptake of nanoparticulates and stimulates their transport into the underlying microvascular channel. Similar effects of physiological breathing on nanoparticle absorption are observed in whole mouse lung. Mechanically active “organ-on-a-chip” microdevices that reconstitute tissue-tissue interfaces critical to organ function may therefore expand the capabilities of cell culture models and provide low-cost alternatives to animal and clinical studies for drug screening and toxicology applications.

Direct interaction of the Rho GDP dissociation inhibitor with ezrin/radixin/moesin initiates the activation of the Rho small G protein.

The Rho GDP dissociation inhibitor (GDI) forms a complex with the GDP-bound form of the Rho family small G proteins and inhibits their activation. The GDP-bound form complexed with Rho GDI is not activated by the GDP/GTP exchange factor for the Rho family members, suggesting the presence of another factor necessary for this activation. We have reported that the Rho subfamily members regulate the ezrin/radixin/moesin (ERM)-CD44 system, implicated in reorganization of actin filaments. Here we report that Rho GDI directly interacts with ERM, initiating the activation of the Rho subfamily members by reducing the Rho GDI activity. These results suggest that ERM as well as Rho GDI and the Rho GDP/GTP exchange factor are involved in the activation of the Rho subfamily members, which then regulate reorganization of actin filaments through the ERM system.

A mechanosensitive transcriptional mechanism that controls angiogenesis

Angiogenesis is controlled by physical interactions between cells and extracellular matrix as well as soluble angiogenic factors, such as VEGF. However, the mechanism by which mechanical signals integrate with other microenvironmental cues to regulate neovascularization remains unknown. Here we show that the Rho inhibitor, p190RhoGAP (also known as GRLF1), controls capillary network formation in vitro in human microvascular endothelial cells and retinal angiogenesis in vivo by modulating the balance of activities between two antagonistic transcription factors, TFII-I (also known as GTF2I) and GATA2, that govern gene expression of the VEGF receptor VEGFR2 (also known as KDR). Moreover, this new angiogenesis signalling pathway is sensitive to extracellular matrix elasticity as well as soluble VEGF. This is, to our knowledge, the first known functional cross-antagonism between transcription factors that controls tissue morphogenesis, and that responds to both mechanical and chemical cues.

Paper-supported 3D cell culture for tissue-based bioassays

Fundamental investigations of human biology, and the development of therapeutics, commonly rely on 2D cell-culture systems that do not accurately recapitulate the structure, function, or physiology of living tissues. Systems for 3D cultures exist but do not replicate the spatial distributions of oxygen, metabolites, and signaling molecules found in tissues. Microfabrication can create architecturally complex scaffolds for 3D cell cultures that circumvent some of these limitations; unfortunately, these approaches require instrumentation not commonly available in biology laboratories. Here we report that stacking and destacking layers of paper impregnated with suspensions of cells in extracellular matrix hydrogel makes it possible to control oxygen and nutrient gradients in 3D and to analyze molecular and genetic responses. Stacking assembles the “tissue”, whereas destacking disassembles it, and allows its analysis. Breast cancer cells cultured within stacks of layered paper recapitulate behaviors observed both in 3D tumor spheroids in vitro and in tumors in vivo: Proliferating cells in the stacks localize in an outer layer a few hundreds of microns thick, and growth-arrested, apoptotic, and necrotic cells concentrate in the hypoxic core where hypoxia-sensitive genes are overexpressed. Altering gas permeability at the ends of stacks controlled the gradient in the concentration of the O2 and was sufficient by itself to determine the distribution of viable cells in 3D. Cell cultures in stacked, paper-supported gels offer a uniquely flexible approach to study cell responses to 3D molecular gradients and to mimic tissue- and organ-level functions.

Mechanosensitive mechanisms in transcriptional regulation.

Summary Transcriptional regulation contributes to the maintenance of pluripotency, self-renewal and differentiation in embryonic cells and in stem cells. Therefore, control of gene expression at the level of transcription is crucial for embryonic development, as well as for organogenesis, functional adaptation, and regeneration in adult tissues and organs. In the past, most work has focused on how transcriptional regulation results from the complex interplay between chemical cues, adhesion signals, transcription factors and their co-regulators during development. However, chemical signaling alone is not sufficient to explain how three-dimensional (3D) tissues and organs are constructed and maintained through the spatiotemporal control of transcriptional activities. Accumulated evidence indicates that mechanical cues, which include physical forces (e.g. tension, compression or shear stress), alterations in extracellular matrix (ECM) mechanics and changes in cell shape, are transmitted to the nucleus directly or indirectly to orchestrate transcriptional activities that are crucial for embryogenesis and organogenesis. In this Commentary, we review how the mechanical control of gene transcription contributes to the maintenance of pluripotency, determination of cell fate, pattern formation and organogenesis, as well as how it is involved in the control of cell and tissue function throughout embryogenesis and adult life. A deeper understanding of these mechanosensitive transcriptional control mechanisms should lead to new approaches to tissue engineering and regenerative medicine.

Role of RhoA, mDia, and ROCK in Cell Shape-dependent Control of the Skp2-p27kip1 Pathway and the G1/S Transition

Cell shape-dependent control of cell-cycle progression underlies the spatial differentials of growth that drive tissue morphogenesis, yet little is known about how cell distortion impacts the biochemical signaling machinery that is responsible for growth control. Here we show that the Rho family GTPase, RhoA, conveys the “cell shape signal” to the cell-cycle machinery in human capillary endothelial cells. Cells accumulating p27kip1 and arrested in mid G1 phase when spreading were inhibited by restricted extracellular matrix adhesion, whereas constitutively active RhoA increased expression of the F-box protein Skp2 required for ubiquitination-dependent degradation of p27kip1 and restored G1 progression in these cells. Studies with dominant-negative and constitutively active forms of mDia1, a downstream effector of RhoA, and with a pharmacological inhibitor of ROCK, another RhoA target, revealed that RhoA promoted G1 progression by altering the balance of activities between these two downstream effectors. These data indicate that signaling proteins such as mDia1 and ROCK, which are thought to be involved primarily in cytoskeletal remodeling, also mediate cell growth regulation by coupling cell shape to the cell-cycle machinery at the level of signal transduction.

TRPV4 Channels Mediate Cyclic Strain–Induced Endothelial Cell Reorientation Through Integrin-to-Integrin Signaling

Cyclic mechanical strain produced by pulsatile blood flow regulates the orientation of endothelial cells lining blood vessels and influences critical processes such as angiogenesis. Mechanical stimulation of stretch-activated calcium channels is known to mediate this reorientation response; however, the molecular basis remains unknown. Here, we show that cyclically stretching capillary endothelial cells adherent to flexible extracellular matrix substrates activates mechanosensitive TRPV4 (transient receptor potential vanilloid 4) ion channels that, in turn, stimulate phosphatidylinositol 3-kinase–dependent activation and binding of additional β1 integrin receptors, which promotes cytoskeletal remodeling and cell reorientation. Inhibition of integrin activation using blocking antibodies and knock down of TRPV4 channels using specific small interfering RNA suppress strain-induced capillary cell reorientation. Thus, mechanical forces that physically deform extracellular matrix may guide capillary cell reorientation through a strain-dependent “integrin-to-integrin” signaling mechanism mediated by force-induced activation of mechanically gated TRPV4 ion channels on the cell surface.

Mechanobiology and Developmental Control

Morphogenesis is the remarkable process by which cells self-assemble into complex tissues and organs that exhibit specialized form and function during embryological development. Many of the genes and chemical cues that mediate tissue and organ formation have been identified; however, these signals alone are not sufficient to explain how tissues and organs are constructed that exhibit their unique material properties and three-dimensional forms. Here, we review work that has revealed the central role that physical forces and extracellular matrix mechanics play in the control of cell fate switching, pattern formation, and tissue development in the embryo and how these same mechanical signals contribute to tissue homeostasis and developmental control throughout adult life.

Cytoskeletal control of growth and cell fate switching.

Living cells must be able to switch between growth and differentiation with high fidelity and precise spatial control to ensure that normal tissue patterns are formed in the embryo and maintained throughout adult life. Most work on cell proliferation focuses on elucidating the signaling pathways responsible for control of cell cycle progression that are triggered by cell binding to soluble growth factors and insoluble extracellular matrix. Here we review recent work, which has revealed that mechanical tension-dependent changes in cell shape and cytoskeletal structure are equally critical for control of growth, as well as cell fate switching. This interplay between soluble, insoluble, and mechanical cues helps to explain how cell behaviors responsible for tissue formation are controlled in the physical context of living tissues.

Proteolytic release of the carboxy-terminal fragment of proHB-EGF causes nuclear export of PLZF

Cleavage of membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF) via metalloprotease activation yields amino- and carboxy-terminal regions (HB-EGF and HB-EGF-C, respectively), with HB-EGF widely recognized as a key element of epidermal growth factor receptor transactivation in G protein–coupled receptor signaling. Here, we show a biological role of HB-EGF-C in cells. Subsequent to proteolytic cleavage of proHB-EGF, HB-EGF-C translocated from the plasma membrane into the nucleus. This translocation triggered nuclear export of the transcriptional repressor, promyelocytic leukemia zinc finger (PLZF), which we identify as an HB-EGF-C binding protein. Suppression of cyclin A and delayed entry of S-phase in cells expressing PLZF were reversed by the production of HB-EGF-C. These results indicate that released HB-EGF-C functions as an intracellular signal and coordinates cell cycle progression with HB-EGF.