Benjamin D. Matthews

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.

A Human Disease Model of Drug Toxicity–Induced Pulmonary Edema in a Lung-on-a-Chip Microdevice

An in vitro model of human pulmonary edema predicts drug toxicity and efficacy previously observed in humans. Pulmonary Edema-on-a-Chip Drug testing in animal models is time-consuming, costly, and often does not accurately predict the adverse effects in humans. Toward a more reliable output, Huh and colleagues developed a “lung-on-a-chip” that models human lung function in both normal and disease states. The authors cultured two types of human lung cells in parallel microchannels, which were separated by a thin membrane. Much like the human lung, the upper “alveolar” channel was filled with air, whereas the lower “microvascular” channel was filled with liquid. Vacuum was cyclically applied to the sides of the channels to mimic the breathing motion of the lung. When Huh and colleagues added interleukin-2 (IL-2) to the microvascular channel, the fluid started to leak into the air compartment. This process reproduces what is seen in the clinic, where IL-2 induces pulmonary leakage, also known as “edema.” Cyclic mechanical strain introduced with IL-2 compromised the pulmonary barrier even further and led to a threefold increase in leakage. As expected, the addition of angiopoietin-1 stabilized the endothelial junctions and inhibited IL-2–induced vascular leakage. Lastly, the authors tested their pulmonary disease model against a new pharmacological agent, GSK2193874, which blocks certain ion channels activated by mechanical strain. This drug inhibited leakage, suggesting that it would be a viable treatment option for patients with pulmonary edema who are being mechanically ventilated. Huh et al. have recreated the human lung on a microfluidic chip and shown that it not only mimics lung function in response to IL-2 and mechanical strain but also successfully predicts the activity of a new drug for pulmonary edema. The beneficial effects of GSK2193874 still need to be confirmed in humans, but were preliminary validated in animals in a study by Thorneloe et al. (this issue). The next step is to hook this lung up to other chip-based organs—heart, liver, pancreas, etc.—with the goal of one day being able to rapidly screen many drugs and conditions that could affect patient health. Preclinical drug development studies currently rely on costly and time-consuming animal testing because existing cell culture models fail to recapitulate complex, organ-level disease processes in humans. We provide the proof of principle for using a biomimetic microdevice that reconstitutes organ-level lung functions to create a human disease model-on-a-chip that mimics pulmonary edema. The microfluidic device, which reconstitutes the alveolar-capillary interface of the human lung, consists of channels lined by closely apposed layers of human pulmonary epithelial and endothelial cells that experience air and fluid flow, as well as cyclic mechanical strain to mimic normal breathing motions. This device was used to reproduce drug toxicity–induced pulmonary edema observed in human cancer patients treated with interleukin-2 (IL-2) at similar doses and over the same time frame. Studies using this on-chip disease model revealed that mechanical forces associated with physiological breathing motions play a crucial role in the development of increased vascular leakage that leads to pulmonary edema, and that circulating immune cells are not required for the development of this disease. These studies also led to identification of potential new therapeutics, including angiopoietin-1 (Ang-1) and a new transient receptor potential vanilloid 4 (TRPV4) ion channel inhibitor (GSK2193874), which might prevent this life-threatening toxicity of IL-2 in the future.

Cellular adaptation to mechanical stress: role of integrins, Rho, cytoskeletal tension and mechanosensitive ion channels.

To understand how cells sense and adapt to mechanical stress, we applied tensional forces to magnetic microbeads bound to cell-surface integrin receptors and measured changes in bead displacement with sub-micrometer resolution using optical microscopy. Cells exhibited four types of mechanical responses: (1) an immediate viscoelastic response; (2) early adaptive behavior characterized by pulse-to-pulse attenuation in response to oscillatory forces; (3) later adaptive cell stiffening with sustained (>15 second) static stresses; and (4) a large-scale repositioning response with prolonged (>1 minute) stress. Importantly, these adaptation responses differed biochemically. The immediate and early responses were affected by chemically dissipating cytoskeletal prestress (isometric tension), whereas the later adaptive response was not. The repositioning response was prevented by inhibiting tension through interference with Rho signaling, similar to the case of the immediate and early responses, but it was also prevented by blocking mechanosensitive ion channels or by inhibiting Src tyrosine kinases. All adaptive responses were suppressed by cooling cells to 4°C to slow biochemical remodeling. Thus, cells use multiple mechanisms to sense and respond to static and dynamic changes in the level of mechanical stress applied to integrins.

Shear-Activated Nanotherapeutics for Drug Targeting to Obstructed Blood Vessels

Bio-Inspired Drug Delivery Noting that platelets naturally migrate to narrowed blood vessels characterized by high fluid shear stress, Korin et al. (p. 738, published online 5 July; see the Perspective by Lavik and Ustin) developed a nanoparticle-based therapeutic that uses a similar targeting mechanism to deliver a drug to vessels obstructed by blood clots. Aggregates of nanoparticles coated with the clot-dissolving drug tPA (tissue plasminogen activator) were designed to fall apart and release the drug only when encountering high fluid shear stress. In preclinical models, the bio-inspired therapeutic dissolved clots and restored normal blood flow at lower doses than free tPA, suggesting that this localized delivery system may help reduce the risk of side effects such as excessive bleeding. Nanoparticles carrying a drug that dissolves blood clots disintegrate at sites of stenosis. Obstruction of critical blood vessels due to thrombosis or embolism is a leading cause of death worldwide. Here, we describe a biomimetic strategy that uses high shear stress caused by vascular narrowing as a targeting mechanism—in the same way platelets do—to deliver drugs to obstructed blood vessels. Microscale aggregates of nanoparticles were fabricated to break up into nanoscale components when exposed to abnormally high fluid shear stress. When coated with tissue plasminogen activator and administered intravenously in mice, these shear-activated nanotherapeutics induce rapid clot dissolution in a mesenteric injury model, restore normal flow dynamics, and increase survival in an otherwise fatal mouse pulmonary embolism model. This biophysical strategy for drug targeting, which lowers required doses and minimizes side effects while maximizing drug efficacy, offers a potential new approach for treatment of life-threatening diseases that result from acute vascular occlusion.

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.

Ultra-rapid activation of TRPV4 ion channels by mechanical forces applied to cell surface β1 integrins

Integrins are ubiquitous transmembrane mechanoreceptors that elicit changes in intracellular biochemistry in response to mechanical force application, but these alterations generally proceed over seconds to minutes. Stress-sensitive ion channels represent another class of mechanoreceptors that are activated much more rapidly (within msec), and recent findings suggest that calcium influx through Transient Receptor Potential Vanilloid-4 (TRPV4) channels expressed in the plasma membrane of bovine capillary endothelial cells is required for mechanical strain-induced changes in focal adhesion assembly, cell orientation and directional migration. However, whether mechanically stretching a cells extracellular matrix (ECM) adhesions might directly activate cell surface ion channels remains unknown. Here we show that forces applied to beta1 integrins result in ultra-rapid (within 4 msec) activation of calcium influx through TRPV4 channels. The TRPV4 channels were specifically activated by mechanical strain in the cytoskeletal backbone of the focal adhesion, and not by deformation of the lipid bilayer or submembranous cortical cytoskeleton alone. This early-immediate calcium signaling response required the distal region of the beta1 integrin cytoplasmic tail that contains a binding site for the integrin-associated transmembrane CD98 protein, and external force application to CD98 within focal adhesions activated the same ultra-rapid calcium signaling response. Local direct strain-dependent activation of TRPV4 channels mediated by force transfer from integrins and CD98 may therefore enable compartmentalization of calcium signaling within focal adhesions that is critical for mechanical control of many cell behaviors that underlie cell and tissue development.

α-Actinin-4 Is Required for Normal Podocyte Adhesion

Mutations in the α-actinin-4 gene ACTN4 cause an autosomal dominant human kidney disease. Mice deficient in α-actinin-4 develop a recessive phenotype characterized by kidney failure, proteinuria, glomerulosclerosis, and retraction of glomerular podocyte foot processes. However, the mechanism by which α-actinin-4 deficiency leads to glomerular disease has not been defined. Here, we examined the effect of α-actinin-4 deficiency on the adhesive properties of podocytes in vivo and in a cell culture system. In α-actinin-4-deficient mice, we observed a decrease in the number of podocytes per glomerulus compared with wild-type mice as well as the presence of podocyte markers in the urine. Podocyte cell lines generated from α-actinin-4-deficient mice were less adherent than wild-type cells to glomerular basement membrane (GBM) components collagen IV and laminin 10 and 11. We also observed markedly reduced adhesion of α-actinin-4-deficient podocytes under increasing shear stresses. This adhesion deficit was restored by transfecting cells with α-actinin-4-GFP. We tested the strength of the integrin receptor-mediated linkages to the cytoskeleton by applying force to microbeads bound to integrin using magnetic pulling cytometry. Beads bound toα-actinin-4-deficient podocytes showed greater displacement in response to an applied force than those bound to wild-type cells. Consistent with integrin-dependent α-actinin-4-mediated adhesion, phosphorylation of β1-integrins on α-actinin-4-deficient podocytes is reduced. We rescued the phosphorylation deficit by transfecting α-actinin-4 into α-actinin-4-deficient podocytes. These results suggest that α-actinin-4 interacts with integrins and strengthens the podocyte-GBM interaction thereby stabilizing glomerular architecture and preventing disease.

α4β1-dependent adhesion strengthening under mechanical strain is regulated by paxillin association with the α4-cytoplasmic domain

The capacity of integrins to mediate adhesiveness is modulated by their cytoplasmic associations. In this study, we describe a novel mechanism by which α4-integrin adhesiveness is regulated by the cytoskeletal adaptor paxillin. A mutation of the α4 tail that disrupts paxillin binding, α4(Y991A), reduced talin association to the α4β1 heterodimer, impaired integrin anchorage to the cytoskeleton, and suppressed α4β1-dependent capture and adhesion strengthening of Jurkat T cells to VCAM-1 under shear stress. The mutant retained intrinsic avidity to soluble or bead-immobilized VCAM-1, supported normal cell spreading at short-lived contacts, had normal α4-microvillar distribution, and responded to inside-out signals. This is the first demonstration that cytoskeletal anchorage of an integrin enhances the mechanical stability of its adhesive bonds under strain and, thereby, promotes its ability to mediate leukocyte adhesion under physiological shear stress conditions.

Role of the actin cytoskeleton in insulin action.

Insulin has diverse effects on cells, including stimulation of glucose transport, gene expression, and alterations of cell morphology. The hormone mediates these effects by activation of signaling pathways which utilize, 1) adaptor molecules such as the insulin receptor substrates (IRS), the Src and collagen homologs (Shc), and the growth factor receptor binding protein 2 (Grb2); 2) lipid kinases such as phosphatidylinositol 3‐kinase (PI 3‐Kinase); 3) small G proteins; and 4) serine, threonine, and tyrosine kinases. The activation of such signaling molecules by insulin is now well established, but we do not yet fully understand the mechanisms integrating these seemingly diverse pathways. Here, we discuss the involvement of the actin cytoskeleton in the propagation and regulation of insulin signals. In muscle cells in culture, insulin induces a rapid actin filament reorganization that coincides with plasma membrane ruffling and intense accumulation of pinocytotic vesicles. Initiation of these effects of insulin requires an intact actin cytoskeleton and activation of PI 3‐kinase. We observed recruitment PI 3‐kinase subunits and glucose transporter proteins to regions of reorganized actin. In both muscle and adipose cells, actin disassembly inhibited early insulin‐induced events such as recruitment of glucose transporters to the cell surface and enhanced glucose transport. Additionally, actin disassembly inhibited more prolonged effects of insulin, including DNA synthesis and expression of immediate early genes such as c‐fos. Intact actin filaments appear to be essential for mediation of early events such as association of Shc with Grb2 in response to insulin, which leads to stimulation of gene expression. Preliminary observations support a role for focal adhesion signaling complexes in insulin action. These observations suggest that the actin cytoskeleton facilitates propagation of the morphological, metabolic, and nuclear effects of insulin by regulating proper subcellular distribution of signaling molecules that participate in the insulin signaling pathway. Microsc. Res. Tech. 47:79–92, 1999.

Rapamycin Suppresses Self-Renewal and Vasculogenic Potential of Stem Cells Isolated from Infantile Hemangioma

Infantile hemangioma (IH) is a common childhood vascular tumor. Although benign, some hemangiomas cause deformation and destruction of features or endanger life. The current treatments, corticosteroid or propranolol, are administered for several months and can have adverse effects for the infant. We designed a high-throughput screen to identify FDA-approved drugs that could be used to treat this tumor. Rapamycin, an mTOR inhibitor, was identified based on its ability to inhibit proliferation of a hemangioma-derived stem cell population, human vasculogenic cells we had previously discovered. In vitro and in vivo studies show that Rapamycin reduces the self-renewal capacity of the hemangioma stem cells, diminishes differentiation potential, and inhibits the vasculogenic activity of these cells in vivo. Longitudinal in vivo imaging of blood flow through vessels formed with hemangioma stem cells shows that Rapamycin also leads to regression of hemangioma blood vessels, consistent with its known anti-angiogenic activity. Finally, we demonstrate that Rapamycin-induced loss of stemness can work in concert with corticosteroid, the current standard therapy for problematic hemangioma, to block hemangioma formation in vivo. Our studies reveal that Rapamycin targets the self-renewal and vascular differentiation potential in patient-derived hemangioma stem cells and suggests a novel therapeutic strategy to prevent formation of this disfiguring and endangering childhood tumor.