Donald E. Ingber, MD, PhD - US grants

The general goal of this renewal proposal is to understand the biomechanical mechanism by which extracellular matrix (ECM) regulates angiogenesis during tumor development, with a specific focus on how physical interactions between capillary endothelial (CE) cells and their ECM adhesions control directional cell motility. During the last grant period, we showed that mechanical changes at the cell-ECM interface govern the direction in which cells move because local variations of physical force distributions dictate where cells will form focal adhesions (FAs) and extend new motile processes when stimulated with soluble motility factors. Analysis of this motility steering mechanism and the mechanism of FA repositioning revealed a central role for transfer of mechanical forces across transmembrane integrin receptors which elicit signaling responses that, in turn, activate additional p1 integrin receptors. Other signaling molecules, including the small GTPases, Rho and Rac, also contribute to the mechanism by which ECM influences FA location, and cells that lack the FA protein paxillin fail to exhibit spatial coupling between FA formation and lamellipodia extension. In separate studies, we discovered that an upstream regulator of Rho, p190RhoGAP, may link cytoskeletal signaling to cell motility and angiogenesis by another mechanism: this Rho inhibitor regulates the activity of the transcription factor TFII-I and thereby controls expression of the vascular endothelial growth factor (VEGF) receptor VEGFR2. Thus, the specific aims include: 1) To explore how stress- dependent activation of (31 integrin and Rho alter focal adhesion position, 2) To determine how focal adhesions govern lamellipodia positioning and directional cell migration, and 3) To analyze how cytoskeletal signaling through p190RhoGAP influences VEGFR2 gene expression. These studies will include in vitro mechanistic experiments as well as in vivo studies in a tumor angiogenesis model to determine the potential clinical relevance of our findings. Understanding the molecular basis of this mechanical signaling response that controls direction migration of capillary blood vessel cells could lead to identification of new molecular targets for therapeutic intervention in virtually all solid cancers that require continuous angiogenesis for their own growth and expansion.

The main goal of this proposal (Project 6 of 11 of a U54 Consortium grant entitled, "SysCODE: Systemsbased Consortium for Organ Design and Engineering") is to define engineering principles and microstructural design criteria that when combined with the molecular blueprint uncovered by this Consortium will permit us to fabricate biomimetic materials with appropriate mechanical and chemical signals necessary to induce organ regeneration. We will define how micromechanical forces generated by tissue cells and resisted by extracellular matrices (ECMs) with different mechanical compliance and internal microstructure contribute locally to the regional tissue shape transformations and progressive structural remodeling that mediate morphogenesis and hierarchical self assembly of complex organs. The long term goal is to use the physical design criteria identified in this effort to fabricate multifunctional biomimetic scaffolds that can reprogram stem cells to recapitulate organ formation. These scaffolds will mimic the micromechanical features of living ECMs that control cell fate switching locally, and will spatially orient chemical and adhesive signals that. trigger appropriate developmental cascades. To identify fundamental design principles, we will break down this hierarchical self assembly process into individual steps or critical "morphogenetic modules" ,(e.g., mesenchyme condensation, epithelial budding and folding, cell fate switching, and epithelial-mesenchymal transitions) that underlie epithelial-mesenchymal interactions during development of the tooth, as well as pancreatic islets and heart valves. Relevant molecular regulators and high throughput ECM fabrication strategies will be accessed through collaboration with other members of this Consortium. The new information, ECM materials and design criteria discovered in this proposal will then be integrated with the other projects to develop prototype materials for tissue and organ engineering. The specific aims include: 1) To analyze how cell-generated contractile forces and ECM micromechanics vary spatially during morphogenetic shape transformations in the developing tooth, 2) To determine the effects of altering endogenous cell-generated tensional forces or applying external mechanical loads on tooth development, and 3) To determine the effects of varying the mechanics, structure and chemistry of artificial ECMs on morphogenesis and cell fate switching in tooth, pancreatic islet and heart valve.

DESCRIPTION (provided by applicant): In this competitive renewal, five principal investigators, who have a strong track record in making novel contributions to angiogenesis research, propose to continue their collaborative efforts by studying novel activities of a set of endogenous molecules and structures that regulate angiogenesis by previously unknown mechanisms. J. Folkman (Project I) will elucidate new mechanisms by which specific endogenous angiogenesis regulatory proteins (e.g., thrombospondin-1 and endostatin) found in platelets, and in stromal cells and their extracellular matrix, suppress pathological angiogenesis, and oppose the tumorigenic activity of activated oncogenes. M. Klagsbrun (Project II) will study new angiogenesis regulatory functions of two proteins, neuropilin and semaphorin, that were previously thought to exclusively regulate neuronal growth and migration. D. Ingber (Project III) will investigate how physical interactions between cells and extracellular matrix regulate the small GTPases Rho and Rac, and thereby govern directional migration of capillary cells during tumor angiogenesis in vitro and in vivo. Patricia D'Amore (Project IV) will examine the potential antiadhesive function of endomucin, a down stream target of VEGF, in vascular lumen formation. Marsha Moses (Project V) will analyze the mechanism by which the metalloproteinase ADAM12 regulates angiogenesis, as well as tumor growth and metastasis. She will also determine whether ADAM 12 may be used as an early biomarker of recurrent cancer, disease status or efficacy of therapy in cancer patients. Together, the five research programs in this application cover a broad range of investigation that should significantly enhance our understanding of how angiogenesis is regulated. The results of these experiments are likely to provide a broader spectrum of targets for antiangiogenic therapy, and lead to the development of new diagnostic and prognostic biomarkers. Summary: The body has a variety of defenses against cancer. One of the strongest defenses consists of special proteins that prevent early tumors smaller than a pinhead from recruiting new blood vessels. These are called endogenous angiogenesis inhibitors. Many of them were first discovered by the five principal investigators who are working together on this Program Project. These investigators will now study the different mechanisms by which a set of these proteins operate to prevent tumor angiogenesis. The expected result from this body of work should be the possibility of new angiogenesis inhibitors for which there would be less risk of the development of resistance by a tumor, and the possibility of developing blood tests and urine tests that could detect recurrent cancer long before symptoms appear, or before a tumor can be seen by current methods.

DESCRIPTION (provided by applicant): One of the major problems slowing development and regulatory approval of new and safer medical products is the lack of experimental in vitro model systems that can replace costly and time-consuming animal studies by predicting drug efficacy, bioavailability and toxicity in man. Although considerable advances have been made in the development of cell culture models, these methods fail to reconstitute structural and mechanical features of whole living organs and integrated multi-organ system physiology that are central to their function. This project is based on recent breakthroughs in the laboratories ofthe PI and Co-PI that make it possible to engineer biomimetic microsystems technologies that use living human cells cultured within three- dimensional microfluidic systems to replicate the complex physiological functions and mechanical microenvironment ofthe breathing lung and beating heart. The long-term goal of this project is to intejgrate these 'organ-on-chip'microdevices to produce a 'Heart-Lung Micromachine'that can provide quantitative real-time measures of the efficacy, bioavailability and safety of aerosol-based drugs, nanotherapeutics and other medical products on integrated lung and heart function. The specific aims of this proposal include: 1) to demonstrate the ability ofthe breathing lung-on-a-chip device to measure pulmonary absorption, efficacy and toxicity of aerosol-based drugs and nanotherapeutics, 2) to demonstrate the ability ofthe beating heart microdevlce to detect cardiotoxicity by measuring changes in cardiac cell contractility, electrical conduction, and tissue inflammation, and 3) to create an integrated heart-lung microsystem technology that can assess the effects of drugs and nanotherapeutics delivered to the lung by aerosol on cardiac function and toxicity in vitro. In these studies, we will demonstrate proof-of-principle for a new biomimetic microsystem technology that can analyze efficacy and bioavailability, as well as detect adverse toxicities, associated with use of therapeutic agents before entering clinical trials. If successful, these organ-on-chip microdevices could greatly shorten the timeline and reduce costs associated with development of aerosolized drugs, nanotherapies and other medical products, as well as inform regulatory decision-making in the future. PUBLIC HEALTH RELEVANCE: We propose to build a 'Heart-Lung Micromachine'composed of microfluidic channels lined by living cells as a screening platform that could replace animal assays currently used for development and regulatory review of drugs and nanotherapies. This biomimetic technology could greatly shorten the time required to bring drugs to patients, increase their safety, decrease their costs, and improve clinical outcome.

DESCRIPTION (provided by applicant): The overall goal of this REVISED proposal is to design and fabricate biomimetic scaffolds that mimic how certain embryonic tissues induce whole organ formation in the embryo, and ultimately, to use these developmentally-inspired materials to engineer artificial tissues and organs in adult animals. As a proof-of- principle, we propose to fabricate multifunctional scaffolds that induce formation of differentiated tooth starting with adult mesenchymal stem cells (aMSCs) and adult oral epithelial cells (aOECs). Our approach is based our past work which shows that during embryogenesis, the dental epithelium transfers its inductive capabilities to undifferentiated dental mesenchyme by stimulating a mesenchymal condensation response, and that the resulting physical compaction of cells is sufficient to trigger this developmental switch in vitro as well as subsequent tooth differentiatio in vivo. In addition, we now include new preliminary results that show we can fabricate thermosensitive polymer scaffolds that shrink and artificially induce mesenchymal condensation when placed at body temperature, and that tooth differentiation can be stimulated both by warming in vitro and by combining these materials with embryonic dental mesenchymal cells and implanting them under the kidney capsule in mice in vivo. Based on analysis of the embryonic tooth induction process, we believe that we can enhance the efficiency of this artificial induction process by including other key extracellular matrix (ECM) molecules and morphogens in these scaffolds that contribute to normal tooth development; we also plan to optimize the design and fabrication of the mechano-inductive scaffolds. Thus, the long-term goal of this proposal is to fabricate biomimetic polymer scaffolds that can reprogram aMSCs to differentiate into inductive mesenchyme that instructs normal aOECs to form a differentiated tooth in vivo. The Specific Aims of this proposal include: 1) to fabricate a mechanically actuatable biomimetic polymer scaffold that induces aMSCs to undergo odontogenic differentiation in vitro by producing cell compaction, 2) to identify molecules that mediate tooth formation during embryogenesis that can enhance compaction-induced tooth differentiation in vitro, and 3) to bioengineer a differentiated tooth in vivo using an optimized biomimetic scaffold in combination with aMSCs and aOECs in a mouse model.

? DESCRIPTION: The overall goal of this application is to demonstrate the feasibility of using a microengineered `Lung-on-a-Chip microfluidic device to probe the molecular mechanism of mechano-chemical signaling in the human lung, and to use this knowledge to develop new and improved inhibitors of pulmonary edema development. One of the most rapid (< 5 msec) mechanical signaling events triggered by force transmission from the microenvironment to the cell via their extracellular matrix adhesions involves integrin-dependent activation of the stress-activated membrane ion channel TRPV4, which appears to be critical for the development of many disease processes, including pulmonary edema. The molecular mechanism by which forces applied to integrin mediate this `early- immediate' mechanical signaling response that activates TRPV4 and lead to pulmonary disease is not well understood. To study this process in vitro, we will use a recently developed human Lung-on-a-Chip microfluidic device that contains an artificial alveolar-capillary interface lined by living human lung alveolar and capillary cells hat experiences physiological breathing motions and regenerates a functional vascular permeability barrier in vitro. Importantly, we previously used this microengineered lung chip to show that a specific chemical inhibitor of TRPV4 activity can prevent pulmonary vascular leakage induced by both interleukin-2 and mechanical deformation (breathing motions). In addition, our preliminary results have revealed that the transmembrane protein CD98 binds to both ¿1-integrin and TRPV4, and that it is required for mechanical, but not chemical, activation of TRPV4. Thus, in this project, we propose to use our microengineered human Lung- on-a-Chip device to delineate the molecular mechanism by which forces applied to integrins activate TRPV4, and to develop new therapeutics for pulmonary edema that targets this molecular mechanism. The specific aims include: 1) to define the molecular mechanism by which CD98 mediates ¿1-integrin-dependent mechanical activation of TRPV4 in human microvascular endothelial cells, 2) to develop peptide modulators of mechanical signaling through TRPV4 that prevent vascular leakage in the lung-on-a-chip pulmonary edema model, and 3) to validate the peptide inhibitors by demonstrating their ability to prevent vascular leakage in an ex vivo mouse pulmonary edema model.

SUMMARY The goal of this proposal is to use Organs-on-Chips (Organ Chips) to develop clinically relevant in vitro models of influenza infection in humans that can be used to test efficacy of candidate therapeutics, explore variation in responses in different patient populations, and potentially develop anti-influenza drugs that target the host response to infection, rather than the virus itself. Our Organ Chips are 2-channel microfluidic culture devices that are lined by human organ-specific tissue cells and vascular endothelium grown in parallel microchannels separated by a porous extracellular matrix-coated membrane. We have previously created Lung Alveolus Chips as well as Small Airway Chips lined by bronchiolar epithelial cells from either normal donors or diseased patients, such as individuals with chronic obstructive pulmonary disease (COPD), and we showed that they faithfully recapitulate human pathophysiology observed in vivo, including lung inflammation and pulmonary edema In addition, we have created human Liver Chips that metabolize drugs in vitro, and engineered an instrument for automated culture and fluidic coupling of up to 10 human organ chips for up to 4 weeks, which can be used to link different Organ Chips in a physiological way. Importantly, in preliminary studies, we successfully infected these bronchiolar epithelium with H1N1 influenza A virus (IAV), identified molecular mediators of the host response to infection, and discovered a potential new antiviral therapeutic that targets these mediators. In the UG3 phase of this project, we will demonstrate that Lung Airway and Alveolus Chips lined by primary cells isolated from human healthy donors or COPD patients can be used to model clinical features of IAV infection and related lung disorders previously observed in human patients, including viral replication and shedding, release of characteristic inflammatory cytokines, recruitment of circulating immune cells, and pulmonary edema, all of which we will measured non-invasively. During the UH3 phase, we will conduct preclinical efficacy testing of existing antiviral drugs and use multi-omics analysis and bioinformatics approaches to define translatable biomarkers and identify new potential molecular targets. We also will leverage these insights to discover new potential therapeutics that target the host response to infection, rather than the virus itself. Our UG3 Specific Aims are 1) to develop models of influenza infection in human Lung Airway and Alveolus Chips lined by cells from healthy donors and COPD patients that recapitulate in vivo disease responses, and 2) to develop an integrated model for influenza drug testing by fluidically linking Lung Airway, Lung Alveolus Chips, and Liver Chips via their vascular channels. Our UH3 Aims include: 1) to use the integrated Organ Chip influenza model to measure efficacy and safety of known antiviral therapeutics, 2) to validate translatable biomarkers for influenza infection and therapeutic responses identified using the Organ Chip model by comparison with clinical measures in humans, and 3) to use the integrated Organ Chip influenza model to identify new antiviral therapeutics that target host responses to infection.

PROJECT SUMMARY This COMPETITIVE REVISION application is being submitted to expand the scope of our ongoing NIH grant UH3HL141797 in order to leverage our human organ-on-a-chip (Organ Chip) microfluidic culture devices for the rapid development and assessment of potential therapeutic agents for COVID-19. Our ongoing UH3 grant supports the development of human Lung Chips as in vitro preclinical tools for rapid discovery of new therapeutics for viral pandemics caused by influenza. In recent studies, we showed that highly differentiated human cells in our Lung Chips, as well as human intestinal cells within Intestine Chips we developed, express high levels of ACE2 and TMPRSS2 that mediate SARS-CoV-2 virus (CoV2) infection. We also were able to infect these Organ Chips with CoV2 spike protein-expressing viral pseudoparticles (CoV2pp) that closely mimic the effects of native CoV2 virus when tested against multiple FDA approved drugs in cell-based assays. Human Lung Chips were also shown to be more stringent models for assessing potential COVID19 inhibitory activity as only a subset of these drugs significantly inhibited entry of the CoV2pp when administered under flow on-chip at their maximum concentration (Cmax) in human blood reported in clinical studies. Here, we propose to use human Intestine and Lung Chips in combination with computational discovery and synthetic chemistry approaches to develop broad-spectrum coronavirus therapeutics that would both help infected COVID19 patients now, and allow us to be prepared to prevent infections by related pandemic viruses that emerge in the future. In preliminary studies, multiple novel compounds designed with our computational tools exhibited significant inhibitory activities when tested against both CoV2pp and native CoV2 virus in cell based assays. Thus, our Specific Aims include: 1) to use computational and synthetic chemistry approaches to create new compounds that are predicted to inhibit infection by CoV2 virus and related coronaviruses, 2) to prioritize active molecules by analyzing their structure-activity relationships in cell-based assays infected with native CoV2 and related coronaviruses, 3) to identify lead compounds and effective doses based on inhibition of infection and host inflammatory responses in human Organ Chips using native coronaviruses, and 4) to carry out pharmacokinetic studies in mice coupled with iterative chemical synthesis and testing in cell-based assays to optimize the pharmaceutical properties and safety of the lead compounds, while retaining efficacy. Through this effort, we will identify new compounds that demonstrate broad spectrum inhibiting activities against CoV2 as well as related coronaviruses, and generate pharmacokinetic data necessary to move these drugs into animal validation studies and, eventually, human clinical trials. This work will also further establish the value of human Organ Chips as preclinical tools for accelerating drug development.