George A. Truskey - US grants

Interactions between hemodynamic forces and vascular permeability may be important in atherogenesis. The focal nature of atherosclerosis suggests that the fluid shear stress at the endothelial surface can influence the development of atherosclerotic lesions. The exact relationship between shear stress and atherogenesis and the effect of hemodynamic forces on endothelial permeability are poorly understood. The long term goals of this project are to develop an understanding of 1) the distribution of shear stress and permeability in arterial vessels susceptible to atherosclerosis; and 2) the mechanisms by which hemodynamic forces influence the vascular permeability and cellular metabolism of low density lipoprotein (LDL). The proposed research will examine: a) the rates of entry of LDL into the rabbit abdominal aorta at sites prone to the development of atherosclerotic lesions; b) the dependence of permeability upon the local hemodynamic environment; c) receptor-mediated and receptor-independent LDL accumulation and degradation in the arterial wall; and d) the relative importance of the various transport and metabolic processes. Permeability and metabolism will be examined by measuring concentration profiles of 125I-and 131I-tyramine-cellobiose-labeled LDL and methylated LDL using the technique of quantitative autoradiography. This technique permits resolution of areas as small as 100-500 mum2. Samples will be taken from sites around the renal and superior mesenteric arteries and the iliac bifurcation. Sites exposed to low and high shear will be examined. Vascular casts will be used to construct models of arteries. Flow studies will be conducted in vitro using these model vessels and shear stresses will be measured under physiological conditions using flush mounted hot film anemometry. Electron microscopy autoradiography will be used to localize the sites (intra-v. extracellular) of LDL and mLDL accumulation in the vessel wall. Results from these studies will permit integration of our understanding of the effect of hemodynamic forces on vascular permeability and metabolism of LDL. Mathematical model will incorporate two dimensional diffusion in the media and permit determination of the spatial variability of permeability and metabolism. In vitro studies will yield information on the distribution of shear stress in the same vessels in which transport studies will be performed. These results will be compared to determine the effect of shear stress on permeability. Finally, the results of this study will be correlated with the distribution of lesions in the rabbit abdominal aorta to examine the relationship between LDL permeability, shear stress and atherosclerosis.

A FOURIER TRANSFORM INFRARED (FT-IR) Spectrometer with Attenuated Total Reflectance (ATR) capabilities is being purchased for the dedicated use of faculty within the Center for Biochemical Engineering. No such instrument exists within the School of Engineering. The nearest FT-IR spectrometer resides in the Chemistry Department at Duke and its use is dedicated to non-biological samples. On-going and future projects of the principal investigators currently requiring an ATR/FT-IR spectrometer include: In-Situ ATR/FT-IR SPECTROSCOPY OF BACTERIAL ADHESION AND BIOFILM FORMATION 1. Molecular Chemistry of Bacterial Adhesion and Immobilized Cell Physiology 2. Macromolecule Mass Transport Through Bacterial Biofilms 3. Mechanisms Governing Bacterial Biofilm Sloughing ATR/FT-IR SPECTROSCOPY OF ADSORBED PROTEIN FILMS 1. Protein Denaturation Upon Adsorption 2. Effects of Physiochemical Nature of Substratum on Adsorbed Protein Films 3. Mechanisms of Ligand binding to Adsorbed Proteins ATR/FT-IR SPECTROSCOPY OF ANCHORAGE-DEPENDENT MAMMALIAN CELL PROCESSES 1. Substratum Pretreatment Effects on Anchorage-Dependent Mammalian Cell Adhesion 2. Shear Stress and Growth Factor Effects on Anchorage- Dependent Mammalian Cell Physiology

9421425 Truskey This project is on research to evaluate the hypothesis that the size and number of focal contacts regulate cytoskeleton organization which, in turn, controls cell shape and growth. A major problem in cell culture and tissue engineering is retaining morphology and function in vitro. This research will study rat hepatocytes because they are difficult to maintain in a differentiated state in vitro, and spreading has been correlated with changes in cell replication and protein synthesis. The specific aims of this research are to: (1) develop micropatterned surfaces with immobilized peptides and sugar groups; (2) study the effect of pattern dimensions and ligand density upon focal contacts, cytoskeleton organization, cell shape, and growth; and (3) study the effect of perturbations in focal contact area and cytoskeleton organization upon hepatocyte spreading and function. ***

DESCRIPTION: (Adapted from the Investigator's Abstract) Characteristic features of early atherosclerotic lesions are lipid-filled macrophages, extracellular lipid, and localization of lesions to arterial branches or highly curved arteries. At lesion-prone sites, endothelial permeability to low density lipoprotein (LDL) is enhanced and intimal white blood cells are present. The objective of the proposed research is to evaluate the effect of LDL oxidation and fluid dynamics upon the initial steps of atherogenesis. The proposed research tests two hypotheses: 1) local oxidation of LDL within the arterial wall exacerbates endothelial cell injury which increases monocyte adhesion and LDL transport into the arterial wall, and 2) low and oscillating shear stresses localize injury a) by a direct effect upon endothelial cell function and b) by promoting increased monocyte adhesion. To address the first hypothesis, the investigators will determine the effect of the antioxidants probucol and beta-carotene on LDL transport and endothelial cell expression of adhesion proteins and chemotactic proteins for monocytes in normal and hypercholesterolemic rabbits. Quantitative autoradiography will be used to determine local concentrations of degraded and undegraded LDL around intercostal arteries and major branches of the abdominal aorta. Theoretical models will be used to characterize changes to transport and metabolic properties due to hypercholesterolemia. A panel of monoclonal antibodies will be used to examine endothelial cell expression of adhesion proteins and chemotactic agents, macrophage activation, and the cellular components of early lesions. To address the site of action of the antioxidants, the investigators will measure tissue and LDL levels of antioxidants and determine the effect of antioxidants upon conjugated diene formation by LDL. The second hypothesis will be addressed by correlating fluid dynamics at sites susceptible to lesion development with specific biological changes induced by hypercholesterolemia. Flow visualization will be used to characterize the general features of the flow field, and flush-mounted hot film anemometry will be used to assess wall shear stresses at selected sites of early lesion development. In vitro experiments will be performed to examine the effect of laminar shear stress on the susceptibility of LDL to oxidation. A numerical model of three-dimensional flow and mass transfer around arterial branches will be used to obtain detailed wall shear stress distributions under a variety of flow conditions. Model and experimental wall shear stresses, shear stress gradients, and oscillatory shear indices will be compared with experimentally measured distributions of sites of altered LDL permeability and metabolism and endothelial cell activation. Numerical models of monocyte contact with endothelium will be compared with the spatial distribution of monocyte adhesion protein expression, adherent monocytes and intimal macrophages to assess the extent to which fluid dynamics influences monocyte attachment to the endothelium.

The Center for Cellular and Biosurface Engineering at Duke University requests funding to purchase atomic force microscopy (AFM) instrumentation for the shared use of faculty within the Center. No such instrumentation currently exists within the Center for shared use. A significant number of the core investigators have used atomic force microscopes at other institutions. The requested Digital Instruments MultiMode/BioscopeTM AFM allows simultaneous optical microscopy and AFM imaging. We propose to further extend the capabilities of the BioscopeTM AFM by incorporating total internal reflection fluorescence microscopy (TIRFM) into its optics. This instrument will enhance the research of six members in the interdisciplinary Center for Cellular and Biosurface Engineering (from four departments) in the following research projects: (1) characterization of crystalline, two-dimensional bacterial s-layer protein arrays and nanopatterned protein arrays directed by the s-layer nanotemplates; (2) elucidation of the mechanism of mechanical signal transduction in musculoskeletal cells; (3) characterization of photopatterned antibody arrays designed for a multianalyte integrated optical waveguide sensor; (4) quantitative imaging of endothelial cell attachment and spreading processes on novel, micropatterned cell-adhesive ligand substrates; (5) characterization of a DNA chip: multiplexed, spatially-localized oligonucleotide arrays on silicon substrates; (6) analysis of motor proteins by AFM; and (7) development of a biophysical model to relate protein-ligand recognition energetics to AFM-measured intermolecular forces. The acquisition of an AFM will also significantly enhance the educational mission of the Center for Cellular and Biosurface Engineering at Duke University. The Center was the recent recipient of a special opportunity award from the Whitaker Foundation for educational enhancements to the program at both the undergraduate and graduate levels. The Center is committed to developing innovative laboratory classes that utilize contemporary experimental techniques to understand, quantify, and visualize the fundamental forces, stresses, and recognition processes at the molecular and cellular levels that are central to biological phenomena. The ability of the atomic force microscope to investigate these phenomena in a biologically-relevant milieu in real time combined with its relative ease of use makes it an ideal experimental technique for a number of the laboratory components of a new graduate course under development.

DESCRIPTION: Two of the earliest events in atherogenesis are the accumulation of low density lipoprotein (LDL) and macrophages in the arterial intima. Firm adhesion of monocytes to endothelium is necessary for transmigration of monocytes into the vessel wall. Monocyte adhesion depends upon the level of expression of adhesion proteins by endothelial cells and monocytes as well as the fluid forces acting on monocytes. Localization of early lesions to vessel branches may result from a hemodynamic environment which does not cause cell detachment and alters endothelial cell function to promote monocyte adhesion. The applicants will test the following two hypotheses: 1) arterial fluid dynamics and local oxidation of LDL activate the endothelium to express receptors for monocytes, and 2) localization of monocyte adhesion to vessel branch points represents a balance between hydrodynamic and adhesive interactions. Hydrodynamic interactions include cell transport to the endothelium and drag forces and torque acting on the attached or rolling cell. Objectives of the proposed research are to 1) identify conditions which promote monocyte rolling and arrest on endothelial cells at shear stresses encountered in arteries; 2) characterize the kinetics of these receptor-ligand interactions; and 3) determine the effect of the flow field upon localization of monocyte attachment. Specific aims are to 1) characterize the biophysics of monocyte interactions with endothelial cells activated with minimally modified LDL (mm LDL); 2) determine the effect of the flow field upon monocyte interactions with endothelial cells; and 3) examine monocyte adhesion to and rolling on activated endothelial cells in a region of flow recirculation with and without red blood cells. Experiments will be performed with U937 cells and human umbilical vein endothelial cells. Endothelial cells will be activated by mm LDL. Flow chamber studies will be used to study rolling and adhesion under conditions which simulate shear stresses and flows in the arterial system. Micropipet measurements will be performed to quantify bond lifetimes and rate constants for binding and dissociation. The applicants will use a sudden expansion flow chamber to create a region of flow recirculation. They will examine the effect of flow recirculation on the expression of adhesion molecules by endothelium and the transport and adhesion of monocytes to endothelium. These studies will provide important quantitative data on monocyte adhesion to endothelium and permit the investigators to evaluate how monocytes can adhere to activated endothelium in the high stress environment of the arterial system.

blood lipoprotein transport; low density lipoprotein; pathologic process; atherosclerosis; vascular endothelium; oxidation; gene expression; macrophage; monocyte; hemodynamics; cell adhesion; clinical research; tissue /cell culture; human subject;

Stimulated by advances in molecular and cellular biology, a new and evolving research activity within biomedical engineering is the application of engineering at the bimolecular, cellular and tissue levels. The overall goal of this research is to produce natural or modified therapeutic biomolecules and the design of hybrid tissues and organs. As a result of the importance of this developing activity, the objective of this proposal is to integrate biomolecular, cellular and tissue engineering into the undergraduate curriculum. We propose to achieve this objective by modifying and expanding the laboratory component of three required courses in biomedical engineering and two electives and to provide increased opportunities for independent study coursework in this area. Laboratory projects will complement class material by directly demonstrating fundamental concepts and illustrating relevant biomedical examples and applications of the material using modern instrumentation and techniques. This equipment will also be available for use in undergraduate independent study research. Matching funds will be provided by the School of Engineering and the Department of Biomedical Engineering at Duke University. Additionally, one of the Co-investigators will work to increase the presence of underrepresented groups in independent study research. Co-investigators on this project teach the courses which will be revised and supervise independent study research by undergraduates in bimolecular, cellular and tissue engineering.

DESCRIPTION: (Adapted from the Investigator's Abstract): This investigation concerns the sequence of events leading to the formation and localization of early atherosclerotic lesions. The long-term objectives are to determine the inter-relationship among lipoprotein accumulation and oxidation, intimal accumulation of macrophages, and arterial fluid mechanics during the initial stages of atherosclerosis. Three hypotheses will be tested: 1) Antioxidants reduce the rate of monocyte entry into the vessel wall but do not affect LDL oxidation and monocyte and LDL residence times; (2) Antioxidants alter the number of adhesion molecules present on the endothelium in lesion-prone areas; (3) The rate of monocyte entry into the vessel wall represents the interaction among cells in the luminal flow, the presence of secondary flows which transport monocytes to the endothelium, adhesion molecule expression, and local mass transport of chemotactic agents released by the endothelium and intimal macrophages. Three approaches will be used: fluorescently labeled monocytes to study monocyte transport into, and macrophage accumulation within, the intima; quantification of adhesion molecule densities; and computational fluid dynamics. These approaches will allow examination of the dynamics of macrophage accumulation, the relationship between monocyte adhesion and adhesion molecule density, the role of normal and oxidized LDL upon adhesion molecule expression and macrophage accumulation, and the influence of hemodynamics on adhesion molecule expression and monocyte adhesion. Specific Aims are as follows: 1) Determine the effect of antioxidants on macrophage dynamics and LDL residence times at lesion-prone sites during early atherogenesis, (2) evaluate the role of oxidized LDL upon adhesion molecule density and macrophage entry into the vessel wall, (3) examine the relationship between three-dimensional numerical simulations and measurements of adhesion molecule density and monocyte adhesion and accumulation. Results from these experiments will more clearly define the sequence of events occurring during atherogenesis and the contribution of normal and modified LDL to lesion initiation.

DESCRIPTION (provided by applicant): This proposal seeks support for the 2001 Annual Fall Meeting of the BMES. BMES is the full service professional society for biomedical engineers and BME. The meeting will be held from October 4 to 7, 2001, at the Sheraton Imperial Hotel in Durham, NC. Estimated number of participants at the meeting is 1200. Based on previous annual meetings, 55 percent of attendees hold doctorates and 45 percent are students. Approximately 90 percent of the participants are from academic institutions and 10 percent are from industry. About 15 percent of attendees are international. The goal of the meeting is to present the latest research and development advances in the field of BME and to serve as focal point for discussions about the future of the field. The field is growing rapidly and there is a need for annual meetings of the Society to provide an opportunity for exchange of scientific and engineering ideas. The meeting is divided into the following twelve tracks: (1)Artificial Organs and Prosthetics; (2) Bioinformatics and Computational Biology; (3) Biomechanics of Tissue and Cells; (4) Cardiac Electrophysiology and Mapping; (5) Cardiovascular Systems and Engineering; (6) Molecular and Cellular Bioengineering; (7) Drug and Gene Delivery; (8) Education, Industrial Relations and History; (9) Imaging and Biomedical Optics; (10) Neural Systems and Engineering; (11) Respiratory Systems Engineering; and (12) Tissue Engineering. The meeting will consist of approximately 930 presentations of which two-thirds are oral presentations and one-third are poster presentations. Graduate student participation in presentation is encouraged. Graduate student research awards are presented for outstanding research projects. A young investigator award is presented to stimulate careers in BME. BME continues to be a dynamic and growing field that plays an important role in the improvement of human health. The Annual Fall Meeting of the BMES serves an important function in the advancement of knowledge in BME and bioengineering.

DESCRIPTION (provided by applicant): Tissue engineering represents a promising approach to treat a number of cardiovascular problems including atherosclerosis, damaged valves and heart failure. Cultured blood vessels can be made from extracellular matrix alone or with smooth muscle cells embedded in polymer or collagen gel. Development of a functional, adherent endothelium is one of the major factors limiting the successful development of tissue-engineered grafts. Endothelial cells attachment and function on cultured blood vessels is limited and the adherent endothelium function in a procoagulant manner. The objective of the proposed research is to test the following four hypotheses: (1) endothelial cells cultured in direct contact with partially differentiated smooth muscle cells leads to improved adhesion and differentiation of endothelial cells, (2) exposure of cocultured endothelial cells and smooth muscle cells to flow and mechanical stretch regulates differentiation of both cell types; (3) coculture and flow-mediated effects on endothelial cell differentiation are due, in part, to nitric oxide generated by the endothelium in response to flow and mechanical stretch; and (4) enhanced endothelial cell adhesion in coculture can be used to improve endothelial cell adhesion and function on implanted tissue-engineered blood vessels. In order to test these hypotheses, we will develop a novel coculture system in which endothelial cells are cultured directly on partially differentiated smooth muscle cells. In contrast, current coculture systems do not use differentiated smooth muscle cells and separate the two cell types. Such a system is now feasible due to advances in our understanding of smooth muscle cell differentiation in vitro. Close apposition of the cells will facilitate cell-cell communication. Specific aims of the project are to: (1) develop an endothelial cell-smooth muscle coculture system, (2) determine the effect of flow and mechanical stretch on endothelial cell and smooth muscle cell function, and (3) apply coculture methodology to seed endothelial cells onto tissue-engineered vascular grafts. Results from this research will provide a new method to culture endothelium that more closely mimics the physical interaction found in vivo. This system is applicable to development of tissue-engineered vascular grafts.

DESCRIPTION (provided by applicant): Macrophage accumulation is one of the earliest events in atherosclerosis. Macrophages are derived from blood-borne monocytes that adhere to endothelium and transmigrate across the endothelium. In flowing blood monocytes are transported along streamlines, Contact is more likely at regions where curvature brings streamlines close to the vessel wall and flow reversal first occurs. In order for cells to adhere and activate signaling pathways that promote transmigration, the adhesive force arising from bond formation must resist the hydrodynamic force causing cells to detach. Firm adhesion in regions of flow reversal must still occur rapidly in order to resist higher shears tresses arising during systole. Recently we observed that adhesion of monocytic cells is enhanced by forces acting normal to the surface between the monocyte and endothelium. Bond formation increased leading to more stable adhesion. Normal forces arising from secondary flows may thus play an important role in creating adhesive forces able to resist detachment of monocytes from endothelium in the relatively high shear stress environment found in the arteries. These normal forces may be further enhanced by red cell interactions with monocytes about to contact or adhering to endothelium. Cell rolling and arrest involves bond and tether formation. The objective of the proposed research is to test the hypothesis that normal forces arising in regions of flow recirculation enhance monocyte adhesion by compression of the microvitli and increased bond formation between monocytes and endothelium. A combination of micropipette aspiration experiments, flow chamber adhesion measurements and total internal reflection fluorescence microscopy (TIRF) will be used to test this hypothesis in vitro through the following specific aims: (1) characterize the viscoelastic behavior of monocytic cells, endothelium and tether formation (2) Investigate the effect of normal forces on the biophysics of bond and tether formation; (3) characterize the receptors involved in adhesion mediated by normal forces;(4) Determine the effect of normal; and shear forces on the contact area of rnonocytes; and (5) Examine the role of normal forces in enhanced monocyte adhesion near flow reattachment. Experimental studies will be aided by application of theoretical studies to model the adhesion of monocytes to endothelium. Results from this research will provide new information about the mechanism by which monocytes adhere to arterial endothelium in atherosclerosis-prone regions.

DESCRIPTION (provided by applicant): Skeletal myoblasts are a population of progenitor cells in adult skeletal muscle that are capable of differentiating intovarious types of skeletal muscle. This unique capability of myoblasts makes them suitable candidates for tissue engineering applications such from cellular cardiomyoplasty and patches to replace damaged skeletal or cardiac tissue. Control of the differentiation and mechanical behavior of the cells is crucial to using these cells to repair damaged tissues. The use of a patch in which cells are grown in three-dimensional culture within a support material offers several advantages over direct injection of cells. The three-dimensional culture can be preconditioned to permit adaptation of cells to a specific mechanical environment and reducing apoptosis. Long-term objectives of our research are to determine the effect of mechanical stimulation upon skeletal muscle differentiation and function in order to design engineering replacements for damaged muscle tissue. In this project we propose to examine the hypotheses that (1) static and dynamic stimulation of myoblasts regulates the differentiated state of the skeletal muscle and (2) that mechanical stimulation regulates differentiation by coordinated interaction between nitric oxide synthase activity and cytoskeletal interactions with focal contacts. Our preliminary data show that nitric oxide release by mechanical stimulation accelerates differentiation and alters the mechanical properties of the cells. In order to address these hypotheses we will use the mouse myoblast cell line C2C12 which are mechanically stimulated in two- and three dimensional cultures. Two-dimensional cultures permit a detailed study of mechanical behavior and protein expression of the differentiating cells. Three-dimensional culture studies enable us to extrapolate the results to conditions in a realistic tissue engineering application. Specific aims of the proposed work are: (1) Evaluate the effect of static and dynamic mechanical stimulation on murine myoblast differentiation; (2) Determine the effect of type of mechanical stimulation upon the expression of nitric oxide synthase and NO release; (3) Examine the role of nitric oxide upon focal contact formation and interaction with the cytoskeleton; and (4) Evaluate the response of three-dimensional myoblast cultures to mechanical stimulation and the importance of nitric oxide delivery. Cells will be exposed to static and oscillatory stretch conditions that mimic conditions to which skeletal and cardiac muscle cells are exposed. We will examine the elastic and viscous behavior of the cells and the extent to which the behavior is influenced by nitric oxide release and nitric oxide synthase expression. Focal contact formation and actin filament organization will be assessed under different stretch conditions and the influence of focal contact formation on mechanical properties determined. These studies will provide important new data on the role of mechanical preconditioning on the differentiation of skeletal muscle myoblasts.

DESCRIPTION (provided by applicant): In order to establish a functional endothelium for tissue engineered arteries, we developed a novel co-culture model in which human endothelial cells (EC) form a confluent monolayer on quiescent smooth muscle cells (SMC). The co-culture model is a simplified representation of a blood vessel that permits rapid and efficient examination of a large number of experimental variables. Strong adhesion develops between EC and SMC, SMC are more differentiated in co-culture, and human co-cultures can be maintained for as long as 30 days. Integrins play an important role in endothelial force transduction. Our preliminary data suggest that EC cultured on extracellular matrix produced by SMC produce fibrillar adhesions rather than focal adhesions observed when ECs adhere to rigid substrates. Further, EC in co-culture show a reduced oxidative and inflammatory state relative to EC cultured on plastic suggesting a shift in the type of adhesion and integrins involved during co-culture may affect the function of endothelium. The shift in the type of adhesion can influence integrins involved in adhesion during co- culture and the subsequent function of ECs. To properly design tissue engineered vessels that produce appropriate EC function, it is necessary to understand the effect of EC adhesion to the matrix overlying SMC upon EC function following exposure to flow. Thus, we will test the hypotheses that (1) in co-culture, fibrillar adhesion formation and the elastic modulus of SMCs influence specific extracellular matrix proteins and integrins involved in EC adhesion;(2) fibrillar adhesions promote an anti-inflammatory and anti- thrombotic EC phenotype under static and flow conditions by regulating the level of the transcription factor KLF2, and (3) a combination of co-culture and pulsatile shear stresses lowers the permeability of endothelium by reducing the formation of actin stress fibers with fibrillar adhesions. Specific aims of the project are to: (1) identify integrins and adhesion molecules involved in fibrillar and focal adhesion formation between EC and SMC in co-culture;(2) determine the importance of SMC elasticity and fibrillar adhesions upon EC adhesion and function;(3) determine the effect of fibrillar adhesions upon the response of co-cultured EC to flow;and (4) determine the effect of long-term flow upon EC permeability in co-culture. These studies will provide important new information about EC and SMC function interactions that can influence the design of tissue-engineered blood vessels. Endothelial Cell Adhesion &Function on Smooth Muscle There is considerable need for new sources of blood vessels to repair or replace vessels damaged by atherosclerosis. Tissue engineering represents one such opportunity. The proposed research will examine the manner in which the cells that line arteries and veins (endothelial cells) attach and function on surfaces produced by smooth muscle cells. The studies will provide new insights into the manner in which these two cells of the vessel wall interact and provide a cell culture system to facilitate development of tissue- engineered arteries.

DESCRIPTION (provided by applicant): There is considerable interest in using skeletal muscle progenitor cells, or satellite cells, for cellular therapies and tissue engineering applications. These mononuclear satellite cells, known in the context of skeletal muscle as myoblasts, must fuse into multinucleated myotubes in a highly coordinated differentiation process. Like native muscle, an engineered construct should produce an aligned bundle of myotubes to produce mature muscle ultrastructure that functions properly. Different stimuli, such as mechanical stretch, have been shown to enhance this process. Although mechanical stimulation can promote differentiation, mature muscle fibers have not yet been produced in vitro. Recently, microRNAs (miRNAs) have been shown to regulate gene function to influence proliferation or differentiation in skeletal muscle. As such, miRNA delivery to mechanically stimulated myoblasts may represent an important and novel way to enhance differentiation. The proposed research will examine miRNA expression in skeletal myoblast proliferation and differentiation in response to different mechanical stimuli. In our preliminary data we have identified some novel stretch-sensitive microRNAs and show that the specific stretch regimen produce statistically significant changes in miRNA levels. We will test the following hypotheses: (1) myoblast proliferation and differentiation are modulated by mechanical stimulation via different sets of miRNAs that are temporally regulated, (2) stretch-sensitive miRNAs affect key proteins involved in myoblast differentiation and (3) differentiation can be enhanced by a combination of mechanical stimulation and regulation of miRNA levels. The specific aims of the proposed research are: (1) identify stretch-induced miRNAs by assessing the temporal effect of different levels of stretch, frequency and rest period on miRNA expression in myoblasts, (2) identify key downstream targets and functional effects of miRNAs during the response to mechanical stimulation, and (3) modulate miRNA levels to enhance the effect of applied stretch on myotube formation and differentiation in two-dimensional skeletal muscle cell cultures. Completion of these aims will provide important insights into the mechanism by which stretch mediates differentiation and new tools to modulate skeletal muscle function for applications in regenerative medicine. PUBLIC HEALTH RELEVANCE: This research involves a novel approach to promote the differentiation of cells into tissues using microRNAs, which have been recently identified as regulators of tissue development, and differentiation. While the study focuses upon the demonstration of proof-of-principle in skeletal myoblasts, the results can be extended to other tissues. The work, if successful, could have wide applicability both in the development of engineered tissues and cellular therapies.

DESCRIPTION (provided by applicant): Abstract Design and project-based learning are critical components of the undergraduate Biomedical Engineering program at Duke University. The design experience culminates in a semester-long design course taken during the senior year. Students select from a set of five one-semester design courses. These courses are divided topically. Each class requires the student to design, build, and test a biomedical device, or evaluate a therapy or process. Each course requires that the student design, build, and test a biomedical device or process. Students provide oral presentations and written reports to document progress and milestones. The semester culminates with a BME Design Fair, at which projects are presented to faculty and students from the entire School. All design courses provide each student the opportunity to be creative, to use the knowledge they have accumulated, and to develop an instrument, tool, or device that is of value to the Biomedical Engineering community. Common content in each course addresses standards, ethics, and intellectual property. Our goal is to create an integrated experience of BME design and translation so that students have the opportunity to be involved in all aspects of the design process including needs finding, project identification, design, prototyping and early stage translation. To enhance the design experience of biomedical engineering students at Duke we will (1) create a course on Medical Device Innovation, through case studies and invited guest lectures, students will learn to identify the key features of successful biomedical devices and the ways in which clinical needs affect device design;(2) establish a clinical needs finding practicum in which rising seniors and Master of Engineering students in BME, who have taken the medical device innovation course, engage in clinical needs finding and project identification at Duke Hospital and develop design project topics;(3) create a formal advanced lab-based design course in which teams fully validate their design, provide detailed product specifications, and develop an initial market assessment and provisional patent application;and (4) integrate the BME design experience with curricular and co-curricular activities in technology translation and entrepreneurship activities at Duke. Students enrolled in this program will be eligible for a certificate recognizing the detailed design experience. These changes will provide a wider range of projects for students;more closely link the projects to clinical applications, and allow students greater involvement in all stages of the design process. A faculty team from BME and clinical departments will organize the program, identify clinical needs finding experiences and provide mentoring. We will assess the program annually with an advisory board that will review student projects as well as surveys of students who complete the program. PUBLIC HEALTH RELEVANCE: This project will provide advanced training for BME seniors and first year graduate students on various aspects of biomedical device needs identification, design and development. As such, the training is expected to better prepare students to be professional engineers and facilitate the development of new devices for clinically important problems.

DESCRIPTION (provided by applicant): Skeletal muscle is important for drug and toxicity testing given the relative size of the muscle mass and cardiac output that passes through muscle beds, the key role of muscle in energy substrate metabolism and diabetes, its role in mediating the severity of peripheral arterial disease and heart failure, and the need for therapies for muscle diseases such as muscular dystrophy and sarcopenia. To develop a system for functional and drug testing under physiological conditions, we will incorporate three-dimensional skeletal muscle cultures in a circulatory system that consists of a high-pressure arterial system that carries media to various tissue microcirculatory organ beds and returned via a low-pressure venous system. Arterial vessels will consist of an inner layer of endothelium and layers of differentiated vascular smooth muscle cells or mesenchymal stem cells. A computer controlled pump and valve system will pump small volumes of fluid to mimic arterial flow. Measurement of O2, CO2 and pressure will be used to control flow to the various beds. The modular design of the microcirculatory organ beds facilitates integration with a broad array of other organ and tissue mimics as part of the UH3 phase of the Cooperative Agreement. All experiments will use primary human cells. To generalize the applicability of the test bed, we will develop mature smooth muscle cells and skeletal muscle from iPS cells. In Aim 1, we will fabricate and test a branching network of small caliber blood vessels consisting of several layers of contractile human smooth muscle cells or mesenchymal stem cells and a confluent layer of endothelium. Flow rates, vessel distension and contraction, and the resistance of the microfluidic microcirculatory beds lined with endothelium will control the flow distribution to the different microcirculatory beds. In Aim 2, we will develop three-dimensional constructs of skeletal muscle and fibroblasts under tension. Different levels of oxygen partial pressure in the arterial and venous inflow lines will be used to produce a range of oxygen gradients. The muscle will be connected to posts containing a ferrogel that contracts under electromagnetic stimulation and thereby loads the muscle fibers. Endothelium will cover the outside of the three-dimensional muscle cultures, serving as an interface between the perfusion medium and skeletal muscle fibers. Oxygen gradients across the muscle layer will be controlled by cell density and thickness of the cell layer. We will fabricate an electrode system to electrically stimulate the fibers and measure force production. In Aim 3, we will combine the vascular and muscle units and run the unit for four weeks. Measurement of O2, CO2 and pressure will be used to control overall flow and regulate flow to the different beds. We will assess vessel dilation and muscle function. In Aim 4, the completed system will be used to test the effect of local release of vasodilators and vasoconstrictors on flow distribution, glucose metabolism and oxygen uptake by muscle. We will examine the response of blood vessels and muscle to an inflammatory stimulus. Metabolic profiling will be performed to simulate different physiological conditions and response to drugs and toxins.

DESCRIPTION (provided by applicant): Skeletal muscle is important for drug and toxicity testing given the relative size of the muscle mass and cardiac output that passes through muscle beds, the key role of muscle in energy substrate metabolism and diabetes, its role in mediating the severity of peripheral arterial disease and heart failure, and the need for therapies for muscle diseases such as muscular dystrophy and sarcopenia. To develop a system for functional and drug testing under physiological conditions, we will incorporate three-dimensional skeletal muscle cultures in a circulatory system that consists of a high-pressure arterial system that carries media to various tissue microcirculatory organ beds and returned via a low-pressure venous system. Arterial vessels will consist of an inner layer of endothelium and layers of differentiated vascular smooth muscle cells or mesenchymal stem cells. A computer controlled pump and valve system will pump small volumes of fluid to mimic arterial flow. Measurement of O2, CO2 and pressure will be used to control flow to the various beds. The modular design of the microcirculatory organ beds facilitates integration with a broad array of other organ and tissue mimics as part of the UH3 phase of the Cooperative Agreement. All experiments will use primary human cells. To generalize the applicability of the test bed, we will develop mature smooth muscle cells and skeletal muscle from iPS cells. In Aim 1, we will fabricate and test a branching network of small caliber blood vessels consisting of several layers of contractile human smooth muscle cells or mesenchymal stem cells and a confluent layer of endothelium. Flow rates, vessel distension and contraction, and the resistance of the microfluidic microcirculatory beds lined with endothelium will control the flow distribution to the different microcirculatory beds. In Aim 2, we will develop three-dimensional constructs of skeletal muscle and fibroblasts under tension. Different levels of oxygen partial pressure in the arterial and venous inflow lines will be used to produce a range of oxygen gradients. The muscle will be connected to posts containing a ferrogel that contracts under electromagnetic stimulation and thereby loads the muscle fibers. Endothelium will cover the outside of the three-dimensional muscle cultures, serving as an interface between the perfusion medium and skeletal muscle fibers. Oxygen gradients across the muscle layer will be controlled by cell density and thickness of the cell layer. We will fabricate an electrode system to electrically stimulate the fibers and measure force production. In Aim 3, we will combine the vascular and muscle units and run the unit for four weeks. Measurement of O2, CO2 and pressure will be used to control overall flow and regulate flow to the different beds. We will assess vessel dilation and muscle function. In Aim 4, the completed system will be used to test the effect of local release of vasodilators and vasoconstrictors on flow distribution, glucose metabolism and oxygen uptake by muscle. We will examine the response of blood vessels and muscle to an inflammatory stimulus. Metabolic profiling will be performed to simulate different physiological conditions and response to drugs and toxins.

DESCRIPTION (provided by applicant): Skeletal muscle is important for drug and toxicity testing given the relative size of the muscle mass and cardiac output that passes through muscle beds, the key role of muscle in energy substrate metabolism and diabetes, its role in mediating the severity of peripheral arterial disease and heart failure, and the need for therapies for muscle diseases such as muscular dystrophy and sarcopenia. To develop a system for functional and drug testing under physiological conditions, we will incorporate three-dimensional skeletal muscle cultures in a circulatory system that consists of a high-pressure arterial system that carries media to various tissue microcirculatory organ beds and returned via a low-pressure venous system. Arterial vessels will consist of an inner layer of endothelium and layers of differentiated vascular smooth muscle cells or mesenchymal stem cells. A computer controlled pump and valve system will pump small volumes of fluid to mimic arterial flow. Measurement of O2, CO2 and pressure will be used to control flow to the various beds. The modular design of the microcirculatory organ beds facilitates integration with a broad array of other organ and tissue mimics as part of the UH3 phase of the Cooperative Agreement. All experiments will use primary human cells. To generalize the applicability of the test bed, we will develop mature smooth muscle cells and skeletal muscle from iPS cells. In Aim 1, we will fabricate and test a branching network of small caliber blood vessels consisting of several layers of contractile human smooth muscle cells or mesenchymal stem cells and a confluent layer of endothelium. Flow rates, vessel distension and contraction, and the resistance of the microfluidic microcirculatory beds lined with endothelium will control the flow distribution to the different microcirculatory beds. In Aim 2, we will develop three-dimensional constructs of skeletal muscle and fibroblasts under tension. Different levels of oxygen partial pressure in the arterial and venous inflow lines will be used to produce a range of oxygen gradients. The muscle will be connected to posts containing a ferrogel that contracts under electromagnetic stimulation and thereby loads the muscle fibers. Endothelium will cover the outside of the three-dimensional muscle cultures, serving as an interface between the perfusion medium and skeletal muscle fibers. Oxygen gradients across the muscle layer will be controlled by cell density and thickness of the cell layer. We will fabricate an electrode system to electrically stimulate the fibers and measure force production. In Aim 3, we will combine the vascular and muscle units and run the unit for four weeks. Measurement of O2, CO2 and pressure will be used to control overall flow and regulate flow to the different beds. We will assess vessel dilation and muscle function. In Aim 4, the completed system will be used to test the effect of local release of vasodilators and vasoconstrictors on flow distribution, glucose metabolism and oxygen uptake by muscle. We will examine the response of blood vessels and muscle to an inflammatory stimulus. Metabolic profiling will be performed to simulate different physiological conditions and response to drugs and toxins.

Abstract Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare autosomal dominant disease of accelerated aging which leads to death between 7 and 20 years of age. The disease arises from point mutations that produce an alternately spliced form of the nuclear protein lamin A, known as progerin, that accumulates in the cell nucleus. Mouse models of HGPS exhibit many phenotypical similarities with the HGPS lamin gene mutation, but atherosclerosis does not develop, suggesting a limit to the suitability of animal models. Since cardiovascular disease represents the primary cause of death among those with HGPS, we propose to use a novel tissue engineered blood vessel microphysiological system to develop biomarkers for the disease and assess the effectiveness of treatment against relevant physiological measurements. We have developed arteriolar-scale endothelialized tissue-engineered blood vessels (TEBVs) using smooth muscle cells (SMCs) derived from induced pluripotent stem cells (iPSCs) using healthy and HGPS cells. The TEBVs can be produced and perfused at physiological flow conditions within a few hours of preparation and exhibit endothelial-mediated vasoactivity and respond to inflammatory mediators. We can perform standard functional tests and examine the effects of inflammatory signals, thus tracking the progression of the disease in the same vessel. The HGPS-TEBVs provide a more realistic in vitro environment than cells cultured on plastic and can help advance the process of discovering novel therapeutics and identification of biomarkers. In this project, we will test the hypotheses that tissue-engineered blood vessels made with cells derived from individuals with HGPS recapitulate in vitro the structure and activity found in vivo and can aid in assessing the effectiveness and mode of action suitable drug candidates for clinical studies. In Aim 1, we will test the hypothesis that TEBVs with cells derived from HGPS patients have the same phenotype as a mouse model of HGPS. We will assess (1) the relative contribution of reduced cell number and oxidative stress on the altered function of HGPS- TEBVs, (2) the effect of flow on EC NRF2 activity and oxidative genes it regulates, and (3) compare TEBV structure and function with the mouse model for HGPS. Control conditions will consist of TEBVs prepared with cells derived from a parent of one of the HGPS patients. In Aim 2, we will modify our system to run multiple TEBVs simultaneously and test the hypothesis that combination therapies have been ineffective because they have not restored SMC number, differentiation, and vasoactivity. In Aim 3, we will assess the suitability of novel treatments for progeria to alter the HGPS phenotype in the TEBVs. We will examine the effect of agents which improve mitochondrial function and or protein degradation, alone or in combination with lonafarnib and anti-sense oligonucleotides that inhibit progerin production. Corresponding studies in mice will be performed to assess whether the HGPS-TEBV model reproduces changes to vessels found in mouse model of HGPS.

Abstract Rheumatoid arthritis (RA) is a chronic inflammatory disease that primarily affects the joints. Autoreactive immune cells enter articular joints as well as skeletal muscle, and inflammation of the synovial membrane damages the cartilage causing pain and discomfort. Disability rates from RA reach 40% from 5 to 10 years after diagnosis. In addition to disease activity and pain, muscle strength has a major impact on disability in RA patients. Skeletal muscle clearly contributes to RA disability, but the mechanisms by which this occurs are not well understood. Since animal models and cell culture systems are imperfect replicates of human disease and do not recapitulate biologic and physiologic features of human skeletal muscle, we propose to use a recently developed engineered electrically responsive, contractile human muscle tissues (myobundles) to model RA in vitro and identify features of the disease phenotype. These human myobundles exhibit structural hallmarks of native skeletal muscle including aligned architecture, multinucleated and striated myofibers, and a satellite cell pool. Over four weeks in culture, they contract spontaneously and respond to single or high frequency electrical stimuli with twitch and tetanic contractions; myobundles maintain functional acetylcholine and ?-adrenergic receptors and undergo structural and functional maturation. Preliminary data with myoblasts derived from RA patients indicate that RA reduces the ability of myofibers to form and produce significant contractile force, even after removal of excess fibroblasts. To establish the in vitro system as a model to test therapies for the disease in skeletal muscle, we will extend our preliminary results and test the hypotheses hypotheses that (1) engineered skeletal muscle myobundles derived from cells of rheumatoid arthritis patients show reduced capacity for repair and differentiation and reduced force production; (2) the decrement in differentiation and function is due to select myokine and cytokine production; and (3) addition cytokines and myokines secreted by RA skeletal muscle myofibers can induce the RA phenotype in healthy myoblasts. In Aim 1, we will extend our preliminary results to show that the engineered skeletal muscle myobundles reproduce the disease phenotype and exhibit reduced maturation and capacity for force generation. Myobundles prepared with myoblasts from healthy aged-matched individuals will serve as controls. Results will be compared to immunohistochemical staining of biopsy samples to establish cellular changes in RA muscle. In Aim 2, we will use an unbiased approach and measure proteins and cytokines released from RA myobundles and apply statistical model to determine the relationship between features of RA phenotype identified in Aim 1 and the levels of myokines and cytokines. In Aim 3, we will that the hypothesis that the abnormal secretion of myokines and cytokines by RA muscle is responsible for the disease phenotype.

ABSTRACT The initiation and progression of atherosclerosis is influenced by systemic inflammation and individuals suffering from autoimmune diseases, such as rheumatoid arthritis, have increased risk of developing cardiovascular diseases. Likewise, chronic and systemic inflammation in rheumatoid arthritis induces muscle wasting and loss of function. Therapies that reduce inflammation effectively treat rheumatoid arthritis and have the potential to reduce the severity of cardiovascular disease. To overcome limitations of animal models replicating some key disease phenotypes, but not the underlying mechanisms, we established functional human microphysiological systems (hMPS) for healthy human skeletal and cardiac muscle and endothelialized tissue-engineered blood vessels (eTBEVs) using primary and iPS-derived cells and assessed the response to drugs and pro-inflammatory cytokines. These models replicate the structure and key functions of the native tissue and maintain their structure and function for at least 4 weeks. These in vitro tissue systems accurately model the response to drugs. Our goal in this project is to develop clinically relevant hMPS disease models to examine rheumatoid arthritis (RA) risk for muscle dysfunction and atherosclerosis and the role of exercise in attenuating disease-associated inflammation. To meet this goal, we will expand our preliminary results to develop and validate an early atherosclerosis model that uses flow conditions promoting endothelial dysfunction, macrophage accumulation, foam cell formation, and altered vasoactivity. We will reproduce the RA phenotype in skeletal and cardiac muscle through addition of macrophages and cytokines present in RA, and demonstrate that simulated exercise conditions on muscle produce myokines that reduce inflammation in this RA model. Then, we will develop an integrated perfusion system for eTEBVs, skeletal and cardiac muscle and show that the RA model can increase macrophage accumulation in eTEBVs and cardiac bundles, and assess the response to exercise and drugs to treat atherosclerosis and inflammation. We will use CRISPR gene editing technology to generate mutations to proprotein convertase subtilisin/kexin type 9 (PCSK9) and genes that affect IL-6 shedding to assess their impact on endothelial dysfunction and foam cell formation in eTEBVs, and inflammation in skeletal and cardiac muscle bundles. We will profile cytokines and metabolites in the models with and without RA, and demonstrate that disease progression and biomarkers are reduced in the presence of common anti-inflammatory therapeutic interventions for atherosclerosis, and assess the effect of exercise. Likewise, in the RA muscle model, we will examine whether gene variants produce alterations in cytokine profiles impacting muscle function and response to exercise; these may point toward new disease-associated biomarkers and therapeutic targets. Results of this project will provide a general framework for in vitro modeling of atherosclerosis and autoimmune diseases and the role of gene variants in disease severity and drug development.

This study will test the hypothesis that awarding of tenure represents an opportunity to nurture innovation as well as non-incremental and novel research pursuits. Development of faculty as highly successful researchers and educators is a critical goal of all universities. Considerable time and effort are invested into recruiting and mentoring exceptionally promising junior faculty. Tenure recognizes the most promising faculty and provides long term security and stability to develop their research ideas. However, less attention is focused upon post-tenure faculty development and the formulaic tenure process in most universities discourages risk associated with pursuing innovative research. This project will test a program to effect a refocusing of research direction in newly tenured faculty, with the goal of increasing their desire and ability to engage with significant problems.

This study seeks to leverage achievement of tenure as a pivot point for inducing reflection, with the goal of stimulating newly tenured faculty to engage with novel research directions with increased potential for societal impact. The study will involve development of a unique, interdisciplinary program, the Faculty Springboard, targeting recently tenured associate professors drawn from the sciences and engineering at Duke University to facilitate the exploration of new and potentially groundbreaking research initiatives. The program will consist of: 1) an annual innovation workshop, focusing on community building, networking and brainstorming; 2) professional coaching and mentoring to further develop faculty?s novel research ideas; 3) a follow-up workshop to cement project development. Program delivery and effectiveness will be assessed through a comprehensive evaluation plan. Best practices will be institutionalized to ameliorate societal impact achieved through faculty research.

The anticipated Broader Impact of this research is the expansion of research programs focused on addressing societal challenges. Through developing a faculty network and skillsets in support of creative risk-taking for innovative research, this professional development model has the potential to enhance researchers? abilities to address "wicked problems". By establishing proof of principle at Duke through targeted iterative evaluation, core essential components critical to success of this model program will be identified and disseminated for adaptation by other universities.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

Abstract: Genome editing technologies have significant potential to treat a variety of devastating human diseases and disorders. However, there are a number of challenges that genome editing therapies must overcome to reach their full promise. Specifically, there are many possible adverse consequences that are unique to genome editing tools, such as genome integrity, immune responses, and loss of therapeutic efficacy due to cell turnover, for which there are currently are no optimal systems for rigorous assessment. Moreover, these consequences are unique to human physiology, genome sequence, and immune systems, and therefore typical animal models are not completely informative. To address this unmet need, we have assembled a team of collaborative investigators that have developed advanced genome editing strategies and methods for engineering human microphysiological tissue systems that recapitulate human physiology and function, with an emphasis on skeletal and cardiac muscle. We will combine these technologies in this proposal to systemically evaluate tissue physiology, genomic alterations, tissue regeneration, and immune response in response to various genome editing strategies and delivery methods. Specifically, this will include comprehensive and unbiased mapping of unintended modifications to human genome sequences, including at on-target and off-target sites. We will also determine the role of resident tissue stem cells, cell turnover, and tissue injury and regeneration in the stability of genome editing. Finally, we can incorporate immune cells into these microphysiological tissues to understand the consequences of immunity to bacteria-derived genome editing components. Collectively, this proposal will develop a platform to systematically address the most significant challenges to realizing the transformative potential of genome editing therapies in human tissues.

Abstract The appearance of SARS CVO2 in early 2020 has spurred efforts to limit the disease spread and develop effective treatments. The most promising long-term approach is a vaccine. While some vaccines are entering accelerated clinical trials, it may take 12 or more months before an effective vaccine is available. Even if successful, it may not be possible to treat everyone with a vaccine or the effectiveness of the vaccine may be limited. Given the severity of the disease among a number of those patients, alternative approaches to limit infection should be developed. The goal of this proposal is to use human cardiac, vascular, and lung alveolar microphysiological systems (MPS) to identify possible compounds that block SARS COV2 entry into cells and tissues. While cell binding assays can be used to screen drug candidates, human MPS offer the advantage of testing promising drug candidates under conditions encountered in the body. We propose a tiered approach in which primary cells and cells overexpressing angiotensin converting enzyme (ACE2) are used to identify promising candidates that block SARS COV2 virus entry into cells, and vascular, cardiac, and lung alveolar MPS are used to provide a robust evaluation of drugs that block SARS COV2 binding. The first tier with individual cell types enables a rapid screen and the screen with the microphysiological systems enables testing of the most promising candidates with the tissues most likely to be infected. We will develop the screening assays using a pseudovirus with the SARS COV2 spike protein. In Aim 1, we will develop an assay for pseudovirus binding to ACE2 expressing cells by verifying binding and fusion to cells that express ACE2. We will whether the binding specifically involves the spike protein and determine the levels of binding sites on the cell types used in subsequent aims. In Aim 2, we will screen individual cells types for molecules that block entry into the cell of pseudovirus expressing the spike proteins. Potential drug candidates include those that potentially block spike protein binding (e.g. spike proteins, Captopril, Lisinopril, human recombinant soluble ACE2, and antibodies to the spike protein or ACE2) and those inhibiting Transmembrane Serine Protease 2 (TMPRSS2), activity (e.g. camostat mesylate, nafamostat mesylate). In Aim 3, we will test most promising compounds in vascular, cardiac and lung microphysiological systems and compare against results from 2D studies. We will also examine the relationship between drug blocking and factors that affect ACE2 expression.