Christopher S. Chen - US grants

DESCRIPTION: (Description adapted from applicant's abstract): While current understanding of growth factor biology has been incorporated into the design of tissue-engineered products, little is known about how the architecture and composition of the adhesive matrix regulates angiogenesis. It is proposed that changes in cell shape, as governed by the architecture of the extracellular matrix (ECM), act as a distinct signal to regulate capillary endothelial cell proliferation, apoptosis and differentiation in angiogenesis. To test this hypothesis, we will probe how cells respond to changes in cell shape and structure as controlled by the geometry of microfabricated islands of ECM. Preliminary findings suggest that the degree of cell spreading regulates a switch between cell proliferation and apoptosis. In Specific Aim 1, geometric parameters controlling this switch will be identified by using multiple series of geometric islands to specifically vary projected cell area, perimeter, length, aspect ratio, cell height, membrane surface area or volume. The P.I. will determine whether an island size exists where cells neither grow nor die, and therefore can be effectively maintained for long periods in vitro without passage to a new substrate. In Aim 2, he will explore the ability of integrins and growth factors to modulate the degree to which changes in cell shape regulate cell proliferation and apoptosis. In Aim 3, he will determine the geometric requirements for ECM-induced capillary differentiation and tube formation. In this manner, he will seek to determine the ability of matrix architecture to regulate capillary cell behavior and, thus, provide a rational basis for the design of the engineered matrices to promote tissue vascularization.

[unreadable] DESCRIPTION (provided by applicant): This project focuses on the mechanisms by which cadherins, integrins and the cytoskeleton cooperate to regulate proliferation in endothelial and smooth muscle (vascular) cells. Aberrant proliferation of vascular cells plays a major role in the pathophysiology of atherosclerosis and arteriosclerosis, and likely is triggered by changes in the local tissue microstructure that alter cell-cell and cell-extracellular matrix interactions. Understanding the molecular basis for how cues within the tissue microenvironment are integrated within cells to regulate proliferation is hence a priority in the development of rational strategies to interrupt progression of vascular disease. It is proposed that Rho-mediated tension generated in the actin cytoskeleton couples signals from cadherins and integrins in an integrated mechanochemical signaling system that regulates proliferation of both endothelial and smooth muscle cells. During the past grant period, the investigator developed a micropatterning tool to independently manipulate cell-cell contact and cell-substrate adhesion, and combined this tool with molecular approaches to demonstrate that cadherin-mediated cell-cell contact initiates a stimulatory signal for proliferation. This novel cadherin-mediated proliferative signal requires Rho-driven changes in cytoskeletal tension, and is associated with increased focal adhesion signaling. Specific Aim 1 will be to further characterize the mechanism by which cadherin engagement triggers proliferative signaling. In particular, the investigator will examine whether cadherin engagement is necessary or also sufficient for RhoA activity and proliferation. Specific Aim 2 will be to investigate the mechanism by which cadherins activate RhoA. We will examine the role of the cadherin-associated scaffolding protein, p120-catenin, in the induction of RhoA activity and cytoskeletal tension by cadherin engagement. Specific Aim 3 will be to examine the role of focal adhesions and FAK in the transduction of cadherin-induced proliferative signals. Specific Aim 4 will be to explore the involvement of vascular cadherins, RhoA, cytoskeletal tension, and FAK in atherosclerotic lesions. This project will lead to an integrated molecular understanding of how endothelial and smooth muscle cells coordinate signals from cadherins, integrins, and cytoskeletal tension into a proliferative response, and may suggest new therapeutic strategies to interrupt the progression of atherosclerosis and arteriosclerosis. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The vascularization of engineered tissues is critical to the ultimate success of tissue engineering as an organ replacement therapy. The formation of new capillary vessels in vivo, or angiogenesis, also is linked to the pathogenesis of numerous diseases including cancer, and is regulated by local cues within the tissue microenvironment. The general goal of this RENEWAL proposal is to understand the mechanism by which local extracellular matrix (ECM) properties regulate capillary endothelial cell proliferation, gene expression, and capillary tube morphogenesis required in angiogenesis. The investigator has found that adhesion to ECM cooperates with growth factors to generate not only biochemical, but also mechanical signals that are important in driving capillary endothelial cell function. Studies from the past grant period demonstrated that ECM stiffness and composition could be used to regulate proliferation, gene expression, and capillary tube formation by modulating contractile tension generated by the actin cytoskeleton. In this proposal, the investigator proposes to further investigate the role of these mechanical and adhesive cues in regulating angiogenic behaviors. Specific Aim 1 will be to investigate the role of ECM stiffness in regulating angiogenesis. Specific Aim 2 will be to investigate the role of ECM peptide ligands in regulating angiogenesis. Specific Aim 3 will be to investigate the role of spatial organization of the ECM in regulating angiogenesis. Together, these studies will define the mechanisms by which local structural and mechanical properties within ECM modulate endothelial cell function and capillary morphogenesis, and establish new strategies to promote angiogenesis in native ischemic tissues as well as in ex-vivo engineered tissues. PUBLIC HEALTH RELEVANCE: The formation of new capillary vessels in vivo, or angiogenesis, is a rate limiting challenge in the development of engineered implants for organ replacement. Angiogenesis is also critical to many disease processes, including tumor growth and the establishment of atherosclerosis. This project is designed to develop a better understanding of how angiogenesis is regulated by local adhesive and mechanical cues, such that we may better design future approaches to control the growth of blood vessels in tissues.

[unreadable] DESCRIPTION (provided by applicant): Human mesenchymal stem cells (MSCs) are multipotent stem cells that differentiate into many of the cells resident in musculoskeletal and stromal tissues of the human body, including fibroblasts, chondrocytes, osteoblasts, myocytes, and adipocytes. While differentiation of the MSCs into appropriate lineages may enhance healing of injured tissues, inappropriate lineage specification may be responsible for numerous pathophysiologic processes, including the decreased bone and increased fat in osteoporotic bones, and the calcification of atherosclerotic vessel walls. Regulation of the lineage commitment of MSCs by local microenvironmental cues therefore may be critical to our fundamental understanding of numerous degenerative as well as healing processes. The long term objective of this research is to characterize the cues within the local surrounding microenvironment that drive the lineage specification and differentiation of human mesenchymal stem cells (MSCs), and the molecular pathways involved. The investigator has recently discovered that adhesion of MSCs to fibronectin regulates a commitment switch in the MSCs between adipogenic and osteogenic lineage specification, through a mechanism involving RhoA signaling and cytoskeletal tension. In this proposal, the investigator proposes to further characterize how adhesive and mechanical cues regulate the commitment of MSCs to osteogenic and adipogenic lineages. Specific Aim 1 will be to investigate the role of integrin-mediated cell adhesion in modulating mesenchymal stem cell commitment by characterizing the binding interactions that drive the MSC lineage commitment switch between osteoblasts and adipocytes. Specific Aim 2 will be to investigate the coordination of RhoA and cytoskeletal tension in the regulation of stem cell commitment. Specific Aim 3 will be to investigate the ability of RhoA to regulate stem cell commitment and differentiation in an animal model. Together, these studies will define roles of cell adhesion, RhoA, and cytoskeletal tension in MSC lineage commitment, and establish a molecular basis for the regulation of MSC differentiation by microenvironmental cues. [unreadable] [unreadable]

Human mesenchymal stem cells (MSCs) are the precursor cells that form and heal nearly all of the[unreadable] mechanical tissues in the human body. MSCs are now being isolated from adults to understand the[unreadable] fundamental biology of how these cells are regulated as a population, and to explore whether these cells can[unreadable] be differentiated and re-implanted as a cellular therapy in order to arrest or even reverse degeneration and[unreadable] damage to specific tissues. In several disease processes such as osteoporosis, a major cause of[unreadable] progressive tissue degeneration and damage may involve a shift in lineage specification of the MSCs leading[unreadable] to an inadequate supply of healthy MSCs and their daughter cells. The long term objective of this research[unreadable] is to characterize the role of adhesive and soluble cues, and the downstream signaling pathways, that drive[unreadable] the lineage specification and differentiation of human mesenchymal stem cells, in order to identify novel[unreadable] mechanisms to treat these degenerative diseases.[unreadable] The mesenchymal stem cell (MSC) is a multipotent cell capable of differentiating into distinct[unreadable] connective tissue lineages depending on cues present in the surrounding tissue microenvironment. While[unreadable] much effort has focused on identifying soluble differentiation factors such as the bone morphogenic proteins[unreadable] (BMPs), little is known about the role of cell adhesion to extracellular matrix (ECM) in determining MSC fate.[unreadable] Understanding these cues may provide better handles to specifically direct stem cell fate in many settings[unreadable] involving stem cell therapy. We have recently discovered that integrin-mediated adhesion of MSCs triggers[unreadable] changes in cell morphology and RhoA activity, which in turn modulate a commitment switch in MSCs[unreadable] between adipogenic and osteogenic lineages. The working hypothesis underlying the present proposal is[unreadable] that cell adhesion cooperates with signals from soluble cues to regulate the commitment and differentiation[unreadable] of human mesenchymal stem cells, and that this cooperative signaling involves RhoA.[unreadable] The goal of this 2-year Pilot and Feasibility proposal therefore is to obtain additional preliminary data[unreadable] in three key studies in order to elaborate our working hypothesis, and then to pursue support for this[unreadable] research by the R01 mechanism. The specific aims are: 1. To investigate the cooperative role of BMP[unreadable] signaling and cell adhesion in MSC gene expression; 2. To investigate the cooperation between BMP[unreadable] signaling and cell adhesion in regulating RhoA activity; and 3. To investigate the effects of BMP and RhoA[unreadable] signaling on MSC commitment in an animal model.

DESCRIPTION (provided by applicant): This project focuses on how the spatial organization of cells and resultant cell-cell interactions regulate the development and maintenance of stable tissue function within a tissue engineered construct. In vivo, cell-to cell communication and cooperation mediated through juxtacrine and paracrine signals is a hallmark of multicellular life, and is thought to play a critical role in the establishment of native tissue functions. Because the spatial organization of cells within tissues defines which juxtapositions exist between which cell types, this architecture ultimately can determine whether a tissue engineered construct ultimate will fail or succeed. Unfortunately, few tools currently exist to manipulate multicellular spatial organization;thus little is known about the true impact of tissue architecture to tissue function. The long-term goal of this project is to develop such cellular patterning tools, to use them to investigate the role of multicellular organization in regulating tissue function, and to explore how such organization can be used to enhance the function of engineered tissues. While the tools to be developed can be considered generic, the investigators will focus as a case study on the development of a vascularized engineered liver. The investigators have recently developed several multicellular patterning tools, and used them to demonstrate the importance of both hepatocyte-stromal cell interactions in supporting hepatocyte function, and interactions between parenchymal and vascular compartments in driving angiogenesis. Interestingly, there appear to be relevant pair wise interactions that occur between several cell types in this setting, and involve a combination of soluble paracrine signals and direct effects through cadherin engagement. It is apparent from these early studies that careful mechanistic studies are necessary to deconvolute and understand how these multiple interactions will contribute to the vascularization and differentiated function of the liver construct, so that a rational strategy can be developed to ultimately construct a functional tissue. It is proposed that a multifaceted in vitro and in vivo effort will be required to develop the necessary tools and studies to meet these goals. Specific Aim 1 will be to investigate the role of cell-cell interactions between hepatocytes, fibroblasts, and endothelial cells in regulating liver and angiogenic functions using several novel two-dimensional patterning tools. Specific Aim 2 will be to investigate how the organization of cells in three-dimensional constructs affects tissue function. Specific Aim 3 will be to explore the involvement of multicellular organization in regulating tissue integration and vascularization in an in vivo setting. In addition to novel approaches to generate patterned multi-cell type constructs, the investigators will also develop nanoparticles for non-invasive monitoring of tissue vascularization. This project will lead to an integrated understanding of the role of multicellular organization and cell-cell communication in stabilizing tissue function, and provide new tools and strategies to engineer complex multicellular tissues. Public Health Relevance Statement: This project will develop tools to organize multiple cell types within an engineered liver construct to maximize tissue function and integration with the patient's blood supply. As such, these studies will address several major hurdles towards the engineering of tissues for treating diseases that are otherwise only cured by whole organ transplantation.

DESCRIPTION (provided by applicant): The Gordon Research Conference (GRC) on Signal Transduction By Engineered Extracellular Matrices was established in 2000 and has become the premier meeting at the intersection of engineered materials, cellular adhesion and signaling, tissue engineering, and stem cell biology. The 2010 meeting, to be held June 27 to July 2, 2010, at the University of New England in Biddeford, ME, adds another important dimension - linking stem cell/engineered matrix constructs to developmental models and human clinical therapy. The meeting Chair will be Dr. Chris Chen and the Vice-Chair will be Dr. Karen Hirschi. These investigators provide complimentary expertise in these emerging fields. The main objective of the conference will be to share the newest knowledge from research on: the establishment and regulation of the cellular microenvironment during development and in postnatal tissues;how the cellular microenvironment can be engineered to control cell function;how the fate of cells can be dynamically tracked in vivo;and how such insights can be applied to the development and optimization of human clinical therapies for tissue repair and regeneration. Thus, this meeting necessarily brings together researchers in diverse fields of biology (including stem cell and developmental biology), chemistry, bioimaging and engineering for both the understanding of cellular function and how this can be harnessed for the repair and replacement of tissues lost or damaged due to disease or injury, which serves to encourage cross-disciplinary thinking. Speakers will include well-established leaders in the field as well as young investigators of exceptional promise. In addition to the plenary talks, the meeting includes lively and highly interactive poster sessions. Importantly, 3 poster presenters will be invited to give short oral presentations in every a.m. session, for a total of 12 oral poster presenters. As an important new feature of the 2010 meeting, the main conference will be preceded by a weekend meeting planned by and for pre-doctoral and post-doctoral trainees in this growing interdisciplinary field. This pre-meeting, the Gordon- Kenan Graduate Research Seminar, will provide a forum for these future leaders in the field to present their work in talks and posters, in a collegial and interactive environment that will add outstanding value to their overall GRC experience. Overall, the meeting will provide a platform for the development of long term interdisciplinary interactions, and expose students and young professionals to the latest ideas and opportunities at the confluence of signal transduction, molecular and cellular biology, biomaterials and tissue engineering. 2 PUBLIC HEALTH RELEVANCE: The Gordon Research Conference on Signal Transduction By Engineered Extracellular Matrices is a biennial meeting of the top investigators in the fields of bioengineering and stem cell biology. The 2010 meeting will be held June 27 to July 2, 2010 at the University of New England in Biddeford, Maine. Nine seminar sessions will be held in which leading researchers will present recent data on a variety of topics relevant to the field. In addition to these invited talks, poster sessions will enable attendees to share new experimental findings in a lively and interactive setting. The meeting will include ample time for informal, collegial interactions that are so essential for carrying out science. A pre-conference organized by and for pre-doctoral and post-doctoral trainees will be held preceding the main meeting. This pre-conference will provide an exceptional forum to establish peer group interaction among future leaders of the field, just prior to the main meeting.

DESCRIPTION (provided by applicant): This project focuses on the mechanisms by which cadherins, integrins and the cytoskeleton cooperate to regulate proliferation in vascular smooth muscle cells (VSMCs). Aberrant dedifferentiation and proliferation of VSMCs plays a major role in vascular stenosis at sites of injury and atherosclerosis. The investigators have shown an aberrant increase in local tissue stiffness at these sites that changes integrin-mediated cell-ECM and cadherin-mediated cell-cell adhesion, both of which appear to be critical for the proliferative sequelae. Understanding how these changes in arterial mechanics regulate proliferation is not only a priority in the development of rational strategies to interrupt progression of vascular disease, but also provides an opportunity to more broadly understand how mechanical forces, cadherins, and integrins cooperate to influence biological functions. We propose that Rho-mediated tension generated in the actin cytoskeleton couples signals from cadherins and integrins in an integrated mechanochemical signaling system that regulates proliferation. In this proposal, the Chen and Assoian labs bring to bear their respective expertise in engineered microenvironments and adhesive regulation of the cell cycle to investigate how tissue stiffening initiates a stimulatory signal for proliferation in an integrated approach that combines in vitro, x vivo, and in vivo model systems. We show that tissue stiffening at sites of vascular injury results in a dramatic increase in N-cadherin expression and that this effect is required for VSMC proliferation and vascular stenosis. Specific Aim 1 will characterize the mechanisms underlying the stimulatory effect of ECM stiffness on N-cadherin and the stimulatory effect of N-cadherin on VSMC cycling. Specific Aim 2 will examine the role of RhoA and cytoskeletal tension in stiffness- and cadherin-induced cycling. To begin to explore the relevance of this novel proliferative pathway in vivo, Specific Aim 3 will examine the interplay between ECM stiffening, FAK and N-cadherin during VSMC cycling in vivo. This project will lead to an integrated molecular understanding of how vascular smooth muscle cells coordinate signals from cadherins, integrins, and cytoskeletal tension into a proliferative response, provide novel approaches to study these complex adhesive and mechanical effects both in vivo and in vitro, and may suggest new therapeutic strategies to interrupt the progression of restenosis and arteriosclerosis.

? DESCRIPTION: The goals of our proposal are to bring together an expert team of bioengineers and stem cell/developmental biologists to create a Human Islet Biomimetic that will facilitate (i) long term culture and manipulation of human islets and (ii) maturation of stem-cell derived or reprogrammed islets. Specifically, we will combine our expertise in cell and developmental biology with our experience molding three dimensional vascularized scaffolds in which cellular inputs, matrix composition, and microscale organization (including flow) can be varied with precision. Although much is known about islet function under homeostatic conditions in vivo, current methods for studying islet physiology and pathophysiology are severely limited. Studies that rely on model organisms - particularly mice - are hampered by cellular and molecular discrepancies between human and rodent islets. The use of human islets for studies of islet physiology is also problematic, as limited availability and exposure to non-physiological conditions during isolation impede the use of this cellular source. Most importantly, there is no system currently available which supports the full function of islets or b-cells for more than a fe days in culture. Thus, our understanding of islet function and dysfunction - particularly as it relates to type 1 diabetes (T1D) - has been constrained by the lack of tools for maintaining and studying human islets in vitro. Into this gap, we will take cadaveric human islets, pancreatic progenitors from human ES and iPS cells, and endocrine cells that are trans-differentiated from intestinal stem cells as starting material, and incorporate them into innovative scaffold devices that provide control over local structure, cellular content, and fluid dynamics to stabilize b-cell function. Overall, we plan to reconstitute human islet biomimetics that recapitulate the diverse cellular types and their organization within the natural human islet. In addition, we will use the system to explore the reasons why islets are prone to lose function when placed ex vivo and to model human islet diseases. This system will be critical for the success of other HIRN consortia, as well for the b-cell biology community at large by providing an accessory system for studying b-cell survival, immune interactions, and alternate sources of b-cells. Our Aims are as follows: Aim 1: To establish a human islet biomimetic for sustained islet viability and function in vitro. Aim 2: To optimize human islet biomimetic function with respect to glucose sensing, insulin release, and stable maintenance of islet phenotypes. Our study is designed to provide a deeper understanding of the molecular and cellular events that lead to islet dysfunction in T1D and related islet disorders and to help develop strategies to restore normal islet function in these disorders.

? DESCRIPTION: The goal of this project is to define multicellular interactions in engineered hepatic tissue that will enable its engraftment and expansion in a living host. In vivo, cell-to-cel communication and cooperation mediated through juxtacrine and paracrine signals is a hallmark of multicellular life, and is thought to play a critical role in the establishment of native tissue functions. Specifically in liver, such interactions appear to be critical for tissue function and regeneration. Unfortunately, few tools currently exist to manipulate multicellular spatial organization; thus little is known about the true impact of tissue architecture to tissue function. During the past 4 years of this collaborative project, the investigators have shown that biomaterials can be used to support the transplantation and peritoneal engraftment of human engineered artificial livers composed of randomly- organized human hepatocytes, endothelial cells and stromal cells. Then, by using novel microtechnology tools to control the organization of these cell types within a 3D context, the team has shown that architecture impacts both the differentiated state of the hepatocyte and the function of the transplanted graft. In addition, the investigators have developed bioprinting tools to build vascular networks in these 3D hydrogels and demonstrated that these improve the survival of co-embedded hepatocytes as well as methods to prevacularize hepatic tissues and thereby accelerate the peritoneal engraftment. In these model systems, we observe that there is a reciprocal interaction via paracrine signals- that is endothelial cells impact hepatocyte function and conversely that hepatocytes impact the endothelial network. Interestingly, many of the paracrine signals are interrelated with perfusion of the network as they are regulated either by shear stress, hypoxia or both. In the current application, the investigators seek to define the spatial dependence on paracrine signaling and perfusion within engineered livers that would efficiently allow them to engraft and expand upon stimulation. The specific aims of this competitive renewal are: (1) To define the role of 3D positioning on paracrine signaling between hepatocytes and endothelial cells in vitro and in vivo, (2) To understand the role of network perfusion on cell function in 3D constructs in vitro and in vivo, and (3) To assess the functional role of network architecture and perfusion on graft expansion in vivo. This project will lead to an integrated understanding of the role of multicellulr organization and cell-cell communication in stabilizing hepatic tissue vascularization and function, and provide new tools and strategies to the broader community to engineer complex multicellular tissues.

Cells in living organisms reside in a meshwork of protein fibers and other molecules, termed the extracellular matrix, which provides both structural support and chemical inputs that are critical to the functioning of the cells. The structural properties of the extracellular matrix are largely determined by how these protein fibers are aligned and organized, and changes in these structures and are associated with the progression of many human diseases, but in ways that in many cases are still not well understood. This award supports fundamental research that will provide needed insights into how the organization and resulting mechanical properties of the extracellular matrix influence and control both the normal and pathological behavior of cells. This research is relevant to ongoing efforts to develop methods to speed wound healing, and to engineer artificial tissues to repair damaged organs, and so the results from this work will have broad impact and provide critical resources to the biomedical community. This collaborative research program involves techniques and insights drawn from several fields, including bioengineering, physics, microfabrication, and cell biology, and provides interdisciplinary training needed to prepare the next generation of scientists and engineers.

The adhesion of cells to the extracellular matrix plays a major role in many critical cellular functions important to embryonic development, adult tissue homeostasis, and disease pathogenesis, including cell survival, migration, proliferation, and differentiation. Yet, there have been few systems that allow control and therefore study the specific effects of fibrillar extracellular matrix architecture on cell function. This project use engineered microtissues to address the key hypothesis that alterations in the alignment of matrix, contractile activity of resident cells, geometry of boundary constraints, and external mechanical forces are highly coupled in a mechanotransduction machinery that will drive changes in cellular phenotype. These studies will determine how these factors regulate the transition of fibroblasts to an activated, fibrotic phenotype that is critical in wound healing as well as chronic fibrosis and scarring, but will apply more broadly to many cell types that response to mechanoadhesive cues.

The Science and Technology Center for Engineering Mechano-Biology (CEMB) brings together leading researchers from a diverse group of disciplines and institutions to investigate, understand, and innovate at the intersection of biology, mechanics, and engineering. The mission of this Center is to discover the governing principles of molecular and cellular communication, provide the intellectual foundations and materials for engineering new and powerful cell-based devices, and to train students in the multilingual foundations of engineering mechano-biology preparing them to be innovative leaders able to explore and exploit these interconnections to impact society. This award supports an innovative network of researchers and educators to investigate the fundamental relationships between cells, their environment, and the forces that act upon them. The team will train a new generation of scientists and engineers in the emerging discipline of Mechano-biology, and will partner with industry to translate new scientific discoveries into products and solutions for the health and prosperity of the nation.

Engineering Mechano-biology, with its focus on the interactions between structure, mechanics, and function, will have a major impact on our ability to construct and repair tissues, organs, and implants; to adapt plants to changing environments; to treat inflammation and fibrosis; to understand the effects of exercise, activity, and trauma; and to engineer optimized synthetic and biomimetic materials. This interdisciplinary field will enable fundamental discoveries in biological function and spur the development of cutting edge technologies for interrogation and guidance of plant and animal structures on multiple scales. Projects will span the length and time scales over which forces operate: from single molecules to supramolecular complexes, cells, tissues and whole organisms, and from milliseconds to hours, weeks or months. Major research efforts will focus on three Integrated Research Thrusts (IRTs) requiring new interfaces across disciplines and organized following a cell's hierarchical perspective from mechano-responsive molecules to signaling pathways to the extracellular niche. Thrust 1: Mechano-biology of Biomolecules and Nanostructures will characterize and engineer proteins and molecules, enabling detection and manipulation of the pN and nm mechano-responsiveness of proteins, scaffolds, and cells to probe or generate increasingly complex engineered mechano-biological reagents, materials, and systems. Thrust 2: Mechano-biology of Cells and Signaling will elucidate how cells dynamically react to mechanical forces through feedback between the cytoskeleton, the nucleus and the surrounding matrix, uncover the ways mechano-signaling contributes to cell-cell communication, and discover how cells distinguish and integrate mechano-signals across length and time scales. Thrust 3: Mechano-biology of Tissues, Materials and Microenvironments will identify matrix-based mechanical cues in plants and animals and investigate the fabrication of bulk gels, fibrous networks, and engineered micro-devices. This will lead to the generation of materials with mechano-responsive properties as well as novel means for studying the structural and biophysical cues in mechano-transduction.

ABSTRACT Heart failure (HF), a leading cause for cardiac transplantation and premature death, is usually preceded by ventricular dilatation and diminished systolic performance or dilated cardiomyopathy (DCM). DCM has many etiologies, including damaging variants in genes with diverse functions in cardiac biology. During the prior funding period we showed that the most common genetic cause of DCM was truncating variants in titin (TTNtv). These account for 25% of familial and 12% of sporadic DCM and for ~10% of DCM that occurs with pregnancy, alcohol abuse, and after cancer therapies. In addition, ~0.2% of the general population carries a TTNtv; these individuals have substantially higher lifelong risks for developing DCM and heart failure. We also identified mechanisms by which TTNtv and other recently recognized DCM genes (FLNC and ALPK3) cause disease. With this competitive renewal we propose to focus on the discovery of genes and mechanisms that account for unexplained DCM, which remains an unmet need. We propose that some unexplained DCM is mechanistically related to established genetic causes and results from sequence variants that are not routinely interrogated, or that have unclear functional consequences. We will study the roles of somatic variants, non-coding regulatory variants, mitochondrial variants, and variants of unknown significance (VUS) in established and newly identified DCM genes. We will also define cell populations and transcriptional profiles of all cells in human hearts with unexplained DCM and DCM with established genetic etiologies, so as to identify shared or distinct pathways that may inform therapeutic opportunities. Our analyses will employ state-of-the art technologies. We will exploit whole genome sequencing (WGS) from blood- and cardiac tissue-derived DNAs obtained from unexplained DCM subjects. We will use single nuclear RNA sequencing (NucSeq) to define how cell populations and transcription change in DCM hearts in comparison to normal hearts, using our recently completed normal human heart NucSeq data. We will perturb new identified variants and mechanisms in iPSC-CMs and mouse models. These studies will improve knowledge of the molecules and pathways that enable normal heart function, the molecular causes and mechanisms of DCM, information that will improve diagnosis and inform precision therapies to prevent heart failure. Our analyses will also contribute functional insights into noncoding sequences. To accomplish these goals, we will: 1) Identify coding and non-coding, germline and somatic variants that contribute to unexplained DCM; 2) Define perturbed cell populations and associated transcriptional profiles in hearts from variant-positive and unexplained DCM; 3) Define DCM mechanisms using engineered iPSC-CMs and mouse models.

ABSTRACT The Gordon Research Conference (GRC) on Signaling by Adhesion Receptors was established in 2000 and has become the premier meeting at the intersection of cellular adhesion and signaling, mechanobiology, and its relevance to physiology, disease, and development. The 2018 meeting subtitled ?Connecting structure, mechanics, and function in cells and tissues,? to be held June 24 to June 29, 2018, at the University of New England in Biddeford, ME, adds another important dimension ? connecting the structure-function relationship of cellular adhesions during homeostasis and pathogenesis. The meeting Chair will be Dr. Christopher Chen and the Vice-Chair will be Dr. Ann Miller. These investigators provide complimentary expertise in these emerging fields. One new goal of this meeting will be to integrate critical bioengineering and clinical perspectives into this expanding and ever more interdisciplinary community. The main objective of the conference will be to share the newest knowledge from research on: (i) cell-cell adhesions and cell-matrix adhesions, (ii) the mechanical regulation of adhesions, and (iii) adhesion and growth factor receptor crosstalk. Furthermore, as the development of novel tools and techniques have advanced our understanding of adhesion signaling at the fundamental level, the program will examine the impact of this field on: (i) tissue remodeling in development and disease, and (ii) clinical applications of adhesion modulation. Lastly, the meeting will introduce emerging topics to promote new frontiers in adhesion signaling. This interdisciplinary meeting is paramount in bringing together scientists with diverse backgrounds in bioimaging, bioengineering, and cell and developmental biology, to not only understand how the structure-function relationship of adhesions is maintained during homeostasis and disrupted in pathologic settings such as cancer, fibrosis, and wound healing but also how to utilize advances in this knowledge to advance tissue engineering and develop targeted therapies. Speakers will include established leaders as well as young investigators of exceptional promise. In addition to plenary talks, the meeting includes lively and highly interactive poster sessions and unscheduled afternoons to foster informal engagement amongst trainees and investigators. Importantly, four junior scientists (trainees or newly appointed young investigators) will be invited to give oral presentations every day of the meeting, for a total of 16 oral presenters that will shape the mentoring culture of the meeting. As an important feature of this meeting, the main conference will be preceded by a two-day Gordon Research Seminar planned by and for pre-doctoral and post-doctoral trainees in this growing interdisciplinary field. The Seminar will not only provide a forum for future leaders in the field to present their work in oral and poster presentations in a collegial and interactive environment, but also expose these trainees to the latest ideas and opportunities at the interface of adhesion receptor signaling, molecular and cellular biology, and bioengineering, thereby preparing them to further the scientific field.

Project Description and Summary The goal of this proposal is to characterize a new mechanism by which the Notch receptor regulates changes in cell adhesion dynamics. Notch signaling is highly conserved across the animal kingdom to regulate cell fates during development, and its dysregulation has been implicated in a variety of vascular inflammatory diseases, developmental abnormalities, and cancers. Binding of ligand to Notch receptor leads to proteolytic cleavages that release the intracellular domain (ICD) as a transcriptional activator, and this mechanism has been the primary focus in describing the role of Notch in development and disease. The investigator has recently found that shear stress caused by blood flow activates Notch, which in turn leads to rapid assembly of endothelial cell-cell junctions and heightened barrier function. In this work, they demonstrated that the transmembrane domain (TMD) left behind after Notch proteolysis initiates the formation of a cortical signaling complex that is responsible for stimulating junction assembly. Here, the investigator will identify the components, underlying mechanisms, and cellular impact of this previously unappreciated non-transcriptional, cortical pathway for Notch and elucidate the biological contexts in which this pathway is engaged. These objectives will be achieved through an interdisciplinary program built around three Aims: Specific Aim 1 will be to define mechanisms underlying the non-canonical cortical Notch signaling pathway. Specific Aim 2 will examine crosstalk between adhesion, force, and the cortical Notch signaling pathway. Specific Aim 3 will be to explore the extent to which the cortical Notch pathway generalizes to broader biological contexts. Together, these studies will offer important insights into this new arm of Notch signaling, and provide a molecular basis for how transcriptional and adhesive programs might be coordinated by a single receptor.

Project Summary/Abstract This application seeks funding for a Biotechnology Predoctoral Training Program in Synthetic Biology at Boston University. Three predoctoral training slots per year are requested, to support trainees for their second and third years of residence. The Synthetic Biology and Biotechnology (SB2) Program aims to prepare top researchers for careers in synthetic biology. Synthetic biology uses engineering approaches to build complex biological systems for research and applications, ranging from industrial chemistry to medical breakthroughs. This fast-growing discipline has the potential to revolutionize many scientific fields and industries, including biotechnology, and our program will be the first in the nation to prepare students specifically in synthetic biology. An intrinsically interdisciplinary field, our program integrates (a) solid technical foundation in the principles and practices of synthetic biology, (b) hands-on exposure to biotech research and the principles of commercialization for translating new synthetic biology technologies, (c) and extensive professional skills development, including teamwork and communication, necessary to success in interdisciplinary teams. The curriculum includes a strong didactic foundation in molecular and cellular engineering, the theory and practice of synthetic biology, data analysis, and basic understanding of commercialization and industrial research goals, along with in-depth examination of a broad range of potential applications. Experiential learning is emphasized throughout the curriculum. Program features include (1) summer internship in a local biotech company, (2) Synthetic Biology Laboratory to introduce state-of-the-art techniques and data analysis methodologies, (3) rigorous dissection of literature focusing on critical thinking and experimental design and validation, (4) a 5-day intensive training course in Biotechnology Entrepreneurship, (5) an SB2 Annual Seminar and Poster Day at which trainees present their research. Emphasis is placed throughout in developing skills in reproducible and rigorous research, in communicating science, and in understanding how to apply synthetic biology to find new solutions to existing problems in academia and biotechnology. Numerous mechanisms are employed to assist trainees in preparing for diverse career paths with formal and informal opportunities to interact with scientists from a range of careers. A distinguished training faculty of 20 mentors is drawn from various departments and colleges at Boston University, with expertise in engineering, biology, chemistry, pharmacology, computer engineering, and physics. All students have a primary and secondary advisor, one focused on foundational tools and one on applications, to ensure their research is clearly motivated and far-reaching. Students play critical roles in defining their interdisciplinary research projects by developing a formal Individual Development Plan.