Peter Francis Davies - US grants

Endocytosis, the capture and internalization of extracellular macromolecules in vesicles derived from invagination of the cell surface, occurs in all mammalian cells. The intracellular fate of the endocytosed substances appears mainly to be degradation in the lysosomal system of the cell. In some cases however, a large proportion of vesicles do not fuse with lysosomes but pass out of the cell by reverse endocytosis. This occurs in the arterial endothelium which therefore functions as a dynamic barrier to the passage of macromolecules from the blood into the artery wall. Thus transendothelial flux of macromolecules such as lipoproteins is regulated by endocytosis. Focal proliferation of arterial smooth muscle cells (SMC) and lipoprotein-lipid accumulation is a characteristic feature of atherogenesis. Therefore, the control of endocytosis assumes importance in transendothelial transport of lipoproteins and their subsequent internalization in SMC. Little is known concerning normal control of endocytosis. Because atherogenesis is associated with altered growth properties of endothelium and SMC, the project is designed to investigate the relationships between growth and quantitative lipoprotein endocytosis in cultured vascular endothelium and SMC. The rates of endocytosis of low density lopoproteins (LDL) via several pathways will be measured in quiescent and growing vascular cells. Growth status will be manipulated in part by specific growth factors. The studies will be extended to include investigations of the effects of LDL charge upon selective entry and degradation in vescular cells. Quantitative data on control of endocytosis will be related to various postulated mechanisms of atherogenesis.

Atherosclerosis, the underlying cause of most human heart disease, results from a focal imbalance of the normal equilibria of the arterial wall. While metabolic cooperation between vascular cells is essential for the maintenance of normal vascular homeostasis, little is known about the nature of these interactions or whether disturbance of their equilibria will precipitate irreversible pathological changes in arterial tissue. We propose to investigate mechanisms of cellular interactions between vascular endothelium, smooth muscle cells (SMC) and monocyte-derived macrophages in vitro and in intact vascular tissue. Two separate general mechanisms of cell cooperativity will be studied: a) cell contact-mediated communication via gap junctional channels connecting the cytoplasm of adjacent cells, and b) humoral communication in which diffusible substances secreted by one cell type pass via the interstitial fluid to specific receptors on the surface of the other cell population. The effects of cell biological pertubations associated with hypercholesterolemia (cellular cholesterol, lipoprotein metabolism), a major risk factor for atherogenesis, will be investigated. New techniques to probe cellular communication in intact normal and atherosclerotic (fibrofatty lesion) vascular tissue will be tested and developed to integrate the in vitro findings with vessel wall biology and pathology. Gap junctional-mediated cell interactions will be investigated biochemically in vitro (transfer of 3H-nucleotides, fluorescent dyes and putative second messengers such as cyclic nucleotides and Ca++ and electrophysiological measurements) using cocultures of endothelial cells, SMC and macrophages. The effects of various mediators of gap junctional transfer, particularly cellular cholesterol composition, will be evaluated. The effects of heterocellular communication upon receptor-mediated lipoprotein metabolism in endothelial cells will be measured. Gap junctional communication will also be investigated in normal and atherosclerotic intact arterial tissues both at the ultrastructural level and functionally using new methods to deliver tracers to the vascular tissue. The regulation of platelet-derived growth factor-like mitogens (c-sis related) synthesized and secreted by endothelial cells will be investigated in the context of the interactions of these cells with SMC and macrophages using molecular biology techniques.

This research is the renewal of a special Creativity Extension Award. The prior grant was highly productive resulting in more than twenty publications in refereed scientific journals with more than five addition being prepared. A summary of the significant accomplishments includes determination that the defect structure in barium titanate dielectrics was very dependent upon the processing of the material with little difference in stoichiometry noted; the defect structure of beta alumina solid electrolytes has both long range order and long range disorder which depends upon the stoichiometry and the thermal history; the mixed ion effect in beta alumina was explained and shown to be related to the defect structure (conductivity varies by as much as several thousand times for the same material); solid electrolytes with more control of the structure and properties were synthesized using ion exchange techniques. The renewal grant will emphasize research on increased understanding of the critical chemical factors affecting the structure and properties of oxide ceramics and on synthesis of new oxides using this understanding. Materials being investigated include layered transition metal oxides, copper oxides, and lanthanide beta alumina. Analytical techniques used include high resolution electron microscopy, x- ray diffraction, Raman spectroscopy, infrared spectroscopy, thermal analysis, and electronic characterization (conductivity, dielectric loss, magnetic susceptibility). The materials being investigated have technological importance because of their potential application in thermal batteries, lasers, high temperature superconductors, dielectrics for communications etc.

Atherosclerosis, the underlying cause of most human heart disease, results from a focal imbalance of the normal equilibria of the arterial wall. While metabolic cooperation between vascular cells is essential for the maintenance of normal vascular homeostasis, little is known about the nature of these interactions or whether disturbance of their equilibria will precipitate irreversible pathological changes in arterial tissue. We propose to investigate mechanisms of cellular interactions between vascular endothelium, smooth muscle cells (SMC) and monocyte-derived macrophages in vitro and in intact vascular tissue. Two separate general mechanisms of cell cooperativity will be studied: a) cell contact-mediated communication via gap junctional channels connecting the cytoplasm of adjacent cells, and b) humoral communication in which diffusible substances secreted by one cell type pass via the interstitial fluid to specific receptors on the surface of the other cell population. The effects of cell biological pertubations associated with hypercholesterolemia (cellular cholesterol, lipoprotein metabolism), a major risk factor for atherogenesis, will be investigated. New techniques to probe cellular communication in intact normal and atherosclerotic (fibrofatty lesion) vascular tissue will be tested and developed to integrate the in vitro findings with vessel wall biology and pathology. Gap junctional-mediated cell interactions will be investigated biochemically in vitro (transfer of 3H-nucleotides, fluorescent dyes and putative second messengers such as cyclic nucleotides and Ca++ and electrophysiological measurements) using cocultures of endothelial cells, SMC and macrophages. The effects of various mediators of gap junctional transfer, particularly cellular cholesterol composition, will be evaluated. The effects of heterocellular communication upon receptor-mediated lipoprotein metabolism in endothelial cells will be measured. Gap junctional communication will also be investigated in normal and atherosclerotic intact arterial tissues both at the ultrastructural level and functionally using new methods to deliver tracers to the vascular tissue. The regulation of platelet-derived growth factor-like mitogens (c-sis related) synthesized and secreted by endothelial cells will be investigated in the context of the interactions of these cells with SMC and macrophages using molecular biology techniques.

The application is for partial support of the 1991 Gordon Research Conference on Atherosclerosis to be held at Kimball Union Academy, New Hampshire, June 17-21, 1991. This international conference, which is held every two years, will comprise an integrated view of the most interesting and exciting developments in lipoprotein biology and vascular wall pathobiology as they relate to atherogenesis. 1991 represents the 10th Gordon conference on Atherosclerosis. Nine sessions are planned as follows: 1.Flow, Permeability and Genetic Mechanisms which Determine Lesion Localization 2.Regulation of Cell Proliferation in the Vascular Wall 3.Gene Activation in Atherogenesis 4.Leukocyte-Vascular Cell Interactions in Atherogenesis and Inflammation. 5.Monocyte Macrophage Receptors 6.Lipoproteins that Induce Foam Cell Formation 7.Genes of Lipoprotein Metabolism and their Regulation 8.Transplant Atherosclerosis 9.Genes Regulating Matrix Interactions with Vascular Cells and Lipoproteins.

An Atomic absorption spectrometer is acquired. The instrument will be used for research on catalysis, structures of zeolites and other molecular sieves, and novel oxides formed by soft chemistry.

Molecular mechanisms associated with the initiation and progression of atherosclerosis are the focus of this SCOR renewal. Particular emphasis will be upon integration of the molecular and cellular responses of the vessel wall, ranging from the effects of flow on the endothelium, circulating monocytes and subendothelial tissue, lipoprotein interactions with each other, the extracellular matrix and vascular cells, including postprandial lipoproteins in a clinical project, and the structure and regulation of apolipoprotein E. The center comprises 8 projects and 4 core units including one clinical project. Dr. Davies will seek a mechanosensor in endothelial cells, for which a potassium channel is a prime candidate. He will also study flow dependent mass transport, and the way the endothelial cells signal their flow responses to underlying smooth muscle cells. Dr. Giddens will extend his modeling studies of the flow profiles in vascular beds to characterize the spatial relationships between flow, pressure and vessel wall responses assayed by morphology, histochemistry and molecular probes to determine the hemodynamic mechanisms of lesion localization. Dr. Meredith will study LDL, modified by oxidation or hyperlipidemia in its interaction with itself (homogeneous fusion) and with matrix molecules (heterogeneous fusion). Dr. Mazzone will study the early response of the LDL receptor gene to growth activation and the mechanisms involved. Drs. Getz and Reardon will extend studies of apoprotein E structure and function (association with lipoproteins) and the regulation of apoprotein E production by transcriptional and post-transcriptional mechanisms. Dr. Schreiber will continue his experiments on apoprotein E in gonad steroidogenic cells with the emphasis on the role of the apoprotein in providing autocrine or paracrine communication between cells. Drs. Polonsky and Getz will study postprandial lipoproteins (chylomicron remnants, VLDL and HDL in obese and diabetic subjects (type II), emphasizing the function of post heparin lipases and the interaction of these particles with cells of the vessel wall. The four core units are administrative core, antigen/antibody core, lipid and lipoprotein analytical core, and molecular biology core.

This is a renewal proposal with a two-pronged approach. The first approach involves the development of new methodologies for the synthesis of metastable oxides. The proposed methodologies involve the chemical modification of materials at low temperatures. The oxides to be synthesized are the hexagonal form of MoO3 and its modified oxides. The second approach involves the development of new phases, and the intended oxides are the oxides related to a-PbO2. The objective of this proposal is to develop an understanding of the critical chemical factors affecting the synthesis of certain types of oxides. %%%% This proposal outlined a very good quality basic solid state chemistry research program dealing with specific types of oxides. It aims to provide information about the critical factors which affect the synthesis of the oxides chemically.

Atherosclerosis, the underlying cause of most morbidity and mortality in the Western world, results from a focal imbalance of the normal equilibria of the artery wall. While metabolic cooperation between vascular cells is essential for the maintenance of normal artery, very little is known about the nature of cellular interactions or how they are disturbed during the recruitment of monocyte-macrophages during atherogenesis. Failure of endothelial-mediated arterial relaxation is a prominent example of communication dysfunction in hypercholesterolemia and atherosclerosis. We will examine the hypothesis that compromised gap junctional (Gj) communication in hypercholesterolemia is responsible for inhibition of vasoregulation in atherogenesis. Direct cell-cell communication occurs via Gj communicating channels through which small metabolites and ions can pass between the cytoplasmic compartments of adjacent cells. Northern blot and riboprobe hybridization analyses have demonstrated that both vascular endothelial and smooth muscle cells in tissue culture express mRNA for the Gj protein connexin 43 (cxn43). In this competing renewal, we will investigate vascular endothelial and smooth muscle cell expression of Gj proteins in situ and in vitro as a function of atherogenic processes. Lesions induced during experimental hypercholesterolemia in rabbit and baboon arteries as well as fully developed atherosclerosis in human endarterectomy tissue will be probed for Gj protein transcription and translation by in situ hybridization and immunocytochemical techniques. The spatial and temporal relationships between expression in resident endothelial and smooth muscle cells, infiltrating plasma cells, and lipid filled foam cells will be studied as a function of hypercholesterolemia. In parallel in vitro studies, the influence of altered lipid environment upon vascular cells will be evaluated in terms of RNA, protein expression and functional communication (dye transfer and electrical coupling). The effects of atherogenesis and associated changes in Gj expression and upon direct electrical coupling of vascular cells will be evaluated . A recent novel finding is that lipid-filled macrophage foam cells in human atherosclerotic lesions express mRNA for cxn43 whereas the monocytes from which they are derived do not. The proposed research will delineate the mechanisms associated with vascular cells and macrophage induction of cxn43 with particular emphasis upon the cytokine environment, the role of intracellular cholesterol accumulation, and the transcriptional, translational and signal transduction mechanisms involved. These studies will address a poorly understood mechanism of vascular cell communication at the tissue, cell and molecular level as a function of atherogenesis.

Funds fron the Academic Research Infrastructure Program will support the acquisition of a Field Emission Transmission Electron Microscope (FE-TEM), an instrument that is specifically designed for high-resolution chemical analysis. The FE-TEM will include basic specimen stages, an x-ray flourescence spectrometer, a GATAN imaging filter, and a charge- coupled-device (CCD) camera and associated hardware. The FE-TEM will be employed in the study of: 1) structure and properties of alloy and model composite interfaces; 2) the effect of impurity segregation on the fracture mechanisms of metal/ceramic interfaces; 3) structural and compositional inhomogeneities in microwave ceramics; 4) the effects of concentration gradients near grain boundaries on the properties of electronic ceramics; 5) local structure and composition in catalytic materials; 6) morphological development of nanocrystalline ceramic composites from polymeric precursors; 7) chemistry/property relations at the interphase region in polymer/ceramic composites; 8) polymer interfaces and capillary wave formation. A field emission transmission electron microscope with associated peripheral hardware will be employed in the study of materials. A diverse range of topics from composite interfaces, fracture of metal/ceramic interfaces, microwave and electronic ceramics, catalytic materials, polymer/ceramic composities, and capillary wave formation at polymer interfaces will be studied.

Davies 9421184 The studies in this proposal focus on the relation between the structures and loss properties of a series of perovskite dielectrics. While these tantalate-based oxides have the lowest losses of all the microwave ceramics, optimization of their properties requires careful control of their chemistry, processing, and degree of structural order. Preliminary investigations made on one of these systems have indicated that the dielectric losses associated with internal domain boundaries can be reduced by introducing selected chemical substituents. The proposal describes a series of experimental studies designed to understand and explain this effect, and outlines new approaches for improving the loss properties of other petrovskite dielectrics. Many aspects of this work are being conducted in close collaboration with scientists from industry, and several aspects of this study are expected to have a direct impact on the processing and production commercial ceramic resonator systems. %%% The goals of this work are to understand what aspects of the structures of the existing microwave dielectric materials are critical in realizing an optimized electronic performance, and to use these crystal chemical insights to enhance the performance of other oxide systems. ***

9703550 Davies Preliminary experiments that have been conducted on a member of the PMN-relaxor family, PMT Pb(Mg1/3Ta2/3)03 indicate that the annealing temperatures utilized in all the previous attempts to promote domain growth lie in a range where the perovskite is kinetically inert. By taking special precautions that inhibit the volatilization of PbO, higher temperature heat treatments reveal that the size of the domains and degree of cation ordering can be increased by more than an order of magnitude. These observations unambiguously show that the currently accepted "space charge" models for the PMN relaxors are invalid. In this model the fine-scale inhomogeneities in the arrangements of the metal cations in the "B-sites" of the perovskite structure are interpreted in terms of the formation of negatively charged ordered nano-domains dispersed in a positively charged disordered matrix. The primary experimental support for this interpretation comes from the apparent absence of any growth of the domains or change in the degree of ordering with extended thermal treatment. This project will explore several new avenues of research that emanate from these results and focuses on the structure and properties of selected niobate and tantalate relaxor systems. The goals of these studies are: (1) to establish how the structural state of PMN-type relaxors respond to different high temperature treatments; (2) to develop new crystal chemical models for the cation ordering that are consistent with these observations; (3) to characterize how the new variable of degree of order and domain size affects the dielectric response of the PMN relaxers and; (4) to exploit these models to develop new relaxor ceramics. %%% Since their discovery over 35 years ago, relaxor ferroelectrics have attracted widespread interest for applications in multi-layer capacitors, transducers, electro-optic devices, and thin film memories. It is now well known that the dielectric properties of relaxor systems are intimately linked to the localized disorder in their crystal structures. However, a full understanding of the origins of the structural inhomogeneities and their relationship to the physics of the relaxor response does not yet exist. This is particularly true for the lead-based PMN (lead-magnesium-niobium oxide) complex perovskite relaxors, the most well known and widely studied family of relaxors, which are the focus of this exploratory study. The extensive studies that have been conducted on the PMN systems have led to the establishment and acceptance of one model as a basis for explaining their structures and properties. Preliminary work by the Principal Investigator has shown that this model may not be correct. The project will perform experiments to confirm the PI's model that will then be used to develop new relaxor materials. ***

Atherosclerosis, the underlying cause of most morbidity and mortality in the Western world, results from a focal imbalance of the normal equilibria of the artery wall. While metabolic cooperation between vascular cells is essential for the maintenance of normal artery, very little is known about the nature of cellular interactions or how they are disturbed during the recruitment of monocyte-macrophages during atherogenesis. Failure of endothelial-mediated arterial relaxation is a prominent example of communication dysfunction in hypercholesterolemia and atherosclerosis. We will examine the hypothesis that compromised gap junctional (Gj) communication in hypercholesterolemia is responsible for inhibition of vasoregulation in atherogenesis. Direct cell-cell communication occurs via Gj communicating channels through which small metabolites and ions can pass between the cytoplasmic compartments of adjacent cells. Northern blot and riboprobe hybridization analyses have demonstrated that both vascular endothelial and smooth muscle cells in tissue culture express mRNA for the Gj protein connexin 43 (cxn43). In this competing renewal, we will investigate vascular endothelial and smooth muscle cell expression of Gj proteins in situ and in vitro as a function of atherogenic processes. Lesions induced during experimental hypercholesterolemia in rabbit and baboon arteries as well as fully developed atherosclerosis in human endarterectomy tissue will be probed for Gj protein transcription and translation by in situ hybridization and immunocytochemical techniques. The spatial and temporal relationships between expression in resident endothelial and smooth muscle cells, infiltrating plasma cells, and lipid filled foam cells will be studied as a function of hypercholesterolemia. In parallel in vitro studies, the influence of altered lipid environment upon vascular cells will be evaluated in terms of RNA, protein expression and functional communication (dye transfer and electrical coupling). The effects of atherogenesis and associated changes in Gj expression and upon direct electrical coupling of vascular cells will be evaluated . A recent novel finding is that lipid-filled macrophage foam cells in human atherosclerotic lesions express mRNA for cxn43 whereas the monocytes from which they are derived do not. The proposed research will delineate the mechanisms associated with vascular cells and macrophage induction of cxn43 with particular emphasis upon the cytokine environment, the role of intracellular cholesterol accumulation, and the transcriptional, translational and signal transduction mechanisms involved. These studies will address a poorly understood mechanism of vascular cell communication at the tissue, cell and molecular level as a function of atherogenesis.

INT 9811609
Davies


This U.S.-Slovene research project involving Peter Davies of the University of Pennsylvania and Danilo Suvorov of the Josef Stefan Institute, University of Ljubljana, examines a set of microwave dielectric ceramics. Collaborative efforts will feature a range of steps including preparation, crystal chemistry and structure of new powder compositions as well as processing and dielectric characterization of the ceramic materials. Results should produce a detailed analysis of the microwave response for candidate oxides that include systems with pyrochlore and perovskite related crystal structures. The intent is to 1) improve our understanding of the critical chemical and structural requirements for a successful ceramic resonator and 2) identify new areas for microwave materials development where potential exists for creating novel ceramics with higher dielectric constants and lower dielectric losses. If successful, such materials may have applicability in microwave-based communication technologies.

This materials research project fulfills the program objective of advancing scientific knowledge by enabling leading experts in the United States and Central Europe to combine complementary talents and pool research resources in areas of strong mutual interest and competence.
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Davies
9809035

Following their discovery over thirty five years ago, relaxor ferroelectrics based on Pb(Mg1/3Nb2/3)03 (PMN) have attracted widespread interest for applications in multi-layer capacitors, transducers, electro-optic devices, and thin film memories. However, advances in the development of new PMN-type relaxors have been severely impeded by the apparent "frozen" nature of their chemical order, which has been assumed to be inert and unresponsive to thermal treatment. The inability to modify the nano-level cation order has also complicated attempts to characterize the local atomic structure or understand and model their relaxor response. Through experiments conducted on tantalate members of the PMN family of relaxors, Pb(Mg1/3Ta2/3)03 and its solid solutions with PbZrO3, Professor Davies has identified thermal conditions that can induce more than two orders of magnitude changes in the size of the chemically ordered domains. In contrast to other classes of perovskite relaxors, he has also found that the electronic properties of these large domain PMT systems retain a relaxor type response. These new observations cannot be reconciled in terms of the currently accepted "space charge" models for the structures of the PMN-type systems, and instead support a "random site" model for the cation order. Experiments using TEM and X-ray and neutron diffraction will be used to characterize the local chemistry and structure of the large domain systems. The dielectric response and ferroelectric properties will be measured. Several of the experiments will be carried out in collaboration with Professor Clive Randall of the Pennsylvania State University.
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Professor Davies' discovery of methods to modify the cation order opens a new avenue of research for the PMN family of relaxor ferroelectrics. In this renewal project, the findings from the previous project will be utilized to develop a new class of large domain, chemically ordered niobate, tantalate, and tungstate relaxors that will have improved properties over those currently available. Collaboration with Professor Clive Randall of the Pennsylvania State University will enhance the experimental capability of the PI's group.
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The initiation and progression of focal atherosclerotic lesions has long been associated with regions of disturbed blood flow, yet the relationships linking hemodynamics to vessel wall biology are poorly understood. Recent in vitro and in vivo studies from this laboratory have demonstrated major variations in the lumenal 3-dimensional surface geometry from cell to cell within the nominally homogeneous endothelial monolayer. Thus the detailed distribution of hemodynamic shear stresses over the lumenal cell surface varies significantly. The predicted consequences of these differences is the hypothesis to be addressed in this proposal: that throughout the arterial circulation there is significant heterogeneity of endothelial gene expression regulated by differential shear stress as a consequence of topographic differences from cell to cell. Both single cell and regional heterogeneities will be addressed. The hypothesis will be tested by analysis of gene expression in single cells and groups of cells, removed from the precise hemodynamic locations in vitro and in vivo and analyzed by single cell amplified antisense RNA technology. Disturbed flow regions will be identified in the mouse arterial circulation and in cultures of human endothelial cells in vitro. Topographic measurements of luminal endothelial surfaces will be performed by atomic force microscopy enabling force concentrations to be mapped for individual cells. Micro- manipulation/dissection will isolate single cells (and small groups of cells) for mRNA amplification. Microarray hybridization technology will be used to determine the expression profile of known stress-responsive genes and other known and unknown human and mouse genes resulting in cell-specific mRNA expression "fingerprints" related to the local hemodynamic forces. The arteries of atherosclerosis-susceptible LDL Receptor-Edit transgenic mice will be probed by in situ hybridization and immunocytochemistry (after antibody generation) to evaluate protein expression. Genes uniquely and prominently associated with specific hemodynamic stress profiles will be identified, a selective number of which will be studied by the generation of transgenic mice. In vitro flow systems will be manipulated to study the complex spatial and temporal characteristics of gene expression in controlled conditions of disturbed laminar flow. In a larger sense, expression profiles from disturbed flow regions in vivo will integrate gene discovery, hemodynamics, and focal atherosclerosis at the sites at which lesions develop.

Funds are requested for the purchase of a multi-photon microscope to be conveniently located near two of the major Penn Institutes whose faculty will share the equipment. Some 14 users from the School of Engineering and Applied Science, the School of Medicine, and several campus institutes will use the equipment for biomedical and biomaterial research investigations. The new equipment offers significant advantages over existing equipment in imaging penetration depth, sensitivity, photodamage, and the ability to observe multiple fluorophores simultaneously. Specific studies include the effect of environmental stimuli on cells, the microscopic basis of biomaterial behavior, polymer behavior at interfaces, etc. The facility will be made available to researchers at nearby Universities.

This proposal from an interdisciplinary (and interschool) Institute for Medicine and Engineering (IME) at the University of Pennsylvania is to purchase an atomic force microscope to become an integral part of microforce measurement and imaging laboratory. The PI and major users, who are members of 5 departments in 2 schools, represent an experienced group of investigators in molecular biomechanics and membrane biophysics. By placement in the IME, the instrumentation is formally committed to interdisciplinary research. No AFM suitable for biological research is available in Philadelphia area universities. This facility will provide: 1. Precise measurements of surface topography and force-related events in biological systems ranging from single molecules to intact cells, simultaneous with direct observation and quantitation of molecular dynamics through fluorophore reporter molecules. 2. Interdisciplinary research training opportunities for graduate students and postdoctoral fellows. The requested instrument is a Digital Instruments Nanoscope IIIa atomic force microscope system with Bioscope and Multimode tapping (in fluid) capabilities and fluorescence optical pathways. A recent modification devised by Yanagida for subpiconewton intermolecular force microscopy will be build into the system. House in a dedicated microscope room in the IME, the system will be used for: a. Topographic imaging of biological surfaces ranging from intact cell surfaces to isolated membranes at angstrom-nanometer resolution. b. The investigation of force interactions with cell surfaces and transmission to cell cytoskeleton. c. Intermolecular force measurements at the subpiconewton level in molecular motors; actin-myosin and kinesins. d. Cell-adhesion protein interactions. e. Investigation of cell surface deformation coincident with mechanotransduction (eg transmembrane ion flux). Optical pathways will permit simultaneous collection of quantitative fluorescence measurements during intact cell and single molecule manipulations, and the Yanagida refinement will permit subpiconewton force sensitivity.

DESCRIPTION (Adapted from applicant's abstract) This new interdisciplinary proposal for a Bioengineering Training Grant in Cardiovascular Pathophysiology is center in the recently established Institute for Medicine and Engineering (IME), an interschool initiative at the University of Pennsylvania. The proposal recognizes the need to train research and clinical fellows at the pre- and postdoctoral levels in quantitative cardiovascular pathophysiology particularly as it pertains to biomedical engineering at the molecular, cellular, and tissue levels. Hypothesis-driven and design-driven approaches to knowledge will be addressed. Fourteen interdisciplinary basic and clinical scientists in bioengineering and biomedical research interact within the IME with collaborative outreach to an additional 45 Members of the Institute. The core training investigators are recipient of approximately $10m (principally NIH and NSF) research grant support, with an additional $9m pending grant support. Eight of the training faculty are Full or Associate Professors together with 6 outstanding Assistant Professors. The IME with its state-of- the-art research labs and core laboratory facilities is adjacent to both the School of Medicine and the School of Engineering. A well-attended benchmark Interdisciplinary Seminar Series (46 lectures/year) will be developed to a full course, and a new graduate course in mechanotransduction that will complement current interdisciplinary graduate courses is proposed together with intensive short-courses, and a specialized Practicing Responsible Science course. Three pre- and 3 postdoctoral trainees are proposed for the first year increasing by one each year up to the 4th year (steady state 6/6). Two years of support will normally be provided. Trainees will be selected following competitive review by a Steering Committee of 5 faculty that will be carefully balanced to represent the interdisciplinary nature of the grant. Trainee selections will be made on the basis of academic record, quality of research abilities, and commitment to research in interdisciplinary cardiovascular biology. Postdoctoral M.D. and/or Ph.D. trainees will be expected to develop significant independence during their training period. Predoctoral trainees will be registered in one of the graduate programs of the Biomedical Graduate Group (Basic science, MSTP) or the Bioengineering, Chemical Engineering, and Mechanical Engineering Graduate Groups of the School of Engineering. Predoctoral trainees will be required or encourage to complete a 42 hour course in mechanotransduction in cardiovascular biology; all trainees are required to complete the Seminars/Journal Club/Discussion course. The IME interacts extensively with all clinical and basic departments of both schools. Responsiveness to 1) minority recruitment efforts, and 2) ethical conduct of research, are documented. An External Advisory Committee is named.

DESCRIPTION (provided by applicant): This BRP proposal from the University of Pennnsylvania is a partnership of interdisciplinary scientists in bioengineering and medical research focused on the biomechanics of cardiovascular cells, membranes, and tissues in the context of site-specific therapy and tissue engineering. Complementary design-driven and hypothesis-driven approaches to vascular cell physiology and pathology are proposed. The center for the program is the Institute for Medicine and Engineering located centrally on the Penn campus. There is a subcontract to Childrens Hospital of Philadelphia (CHOP) and a collaborative partnership with N.I.S.T. The Partnership is composed of two interactive components: (i) fundamental cell and molecular investigations of cardiovascular mechanotransduction. and (ii) preclinical studies of engineered arteries, heart valve calcification, and microcoil treatment of intracranial aneurysms. The basic studies focus on the continuum of force-membrane-cytoskeleton-adhesion and extracellular matrix. The experimental approaches include geometric constraints, spatial analyses, protein conformational changes, deformation properties, and mass transport characteristics that regulate vascular cell structure, gene expression, function, and maladaptation to hemodynamic forces that may lead to pathological change. Also proposed are the development of new materials to regulate cell adhesion (and hence phenotype), and for the delivery of therapeutics in situ. Parallel, complementary preclinical studies focus on tissue engineered arteries ex vivo, heart valve pathology (both ex vivo and in vivo), and the delivery of therapeutic factors to correct intracranial aneurysms in vivo. Specific first year objectives include quantitative spatial analyses of cell deformation, localization of mechanically-induced protein conformational changes, analyses of protein phosphorylation, and the first alterations of cell mechanics through manipulations of substrate chemistry. The preclinical short-term objectives are sustained retention of structure and function of arteries maintained ex vivo and their reintroduction in vivo, the elucidation of heart valve gene expression, and both in vitro and in vivo evaluation of the release of potential therapeutic agents from coated platinum microcoils. The proposal addresses the objectives of the BRP program by integrating physical, chemical, and engineering sciences into fundamental and preclinical studies of vascular mechanotransduction, and by developing innovative materials and devices for both basic research and clinical therapy.

With their ability to sustain an outstanding dielectric response in the microwave region and accommodate extensive chemical substitution, oxide perovskites (ABO3) are the most widely studied and technologically
important family of microwave dielectrics. For mixed-metal perovskites, where the A or B (or both) positions in the structure contain mixtures of different cations, a correlation between a high quality factor (low dielectric loss) and improved cation order has been clearly established. However, only a limited sub-set of ordered perovskite-forming stoichiometries has been examined, and many new systems are still to be explored. This proposal uses new approaches based on "non-integer" valence A-site chemistries to stabilize several completely new families of B-site ordered perovskites suitable for application as low-loss dielectrics in microwave communications. Many of the new materials lie in systems known to exhibit low melting points and high reactivity at low temperature, which also make them potentially useful for applications in low temperature co-fired ceramic (LTCC) technology.

The unique electrical properties of ceramic microwave dielectric oxides have revolutionized microwave-based communications by reducing the size and cost of resonators, filters, and oscillators in systems ranging from cellular telephones to global positioning technologies. Although the existing materials are adequate for current applications the next generation of systems requires new materials with an enhanced dielectric response. The research in this proposal is aimed toward identifying these materials by using a novel design strategy to prepare new families of ceramic microwave dielectrics based on the perovskite structure.

The endothelium is well recognized as a mechanotranduction interface influencing vessel physiology and pathology. The prevalence of atherogenesis in regions associated with complex disturbed flows in vivo has long been recognized yet the factors that predispose such sites and potentiate preferential atherosclerosis there are poorly understood. We propose that hemodynamic forces determine the susceptibility to atherosclerosis by regulation of the regional phenotype of endothelium at such sites. Preliminary profiling studies in the normal adult porcine aorta using microarray have shown differences in endothelial gene expression between areas prone to develop atherosclerotic lesions (disturbed flow) and those areas that are typically spared. Project 5 will address the hypothesis that at lesion-susceptible locations, the endothelial phenotype is in an equilibrium state that is primed to develop atherosclerosis by expression of pro-inflammatory genes but is protected by the up-regulation of anti-oxidant mechanisms and other genes that prevent the initiation/early development of atherosclerosis. Exposure to one or more risk factors (additional to hemodynamics) is proposed to be necessary for imbalances that lead to the development of vascular pathology. In a collaboration with the Center for Devices and Radiological Health animal facility at the FDA Laurel, MD, we will conduct the first multi-gene expresssion studies of endothelium in vivo in defined hemodynamic locations. Regional endothelial phenotypes will be defined in normal adult porcine arteries, and the effects of the important risk factors hypercholesterolemia, gender, and hormonal manipulations upon atherosclerosis-primed regions will be determined. The critical role of hemodynamics in regional susceptibility will be tested by creating flow disturbances in the common carotid artery, a location that is normally exposed to undisturbed laminar flow and which is resistant to atherogenesis; we propose that a susceptible phenotype will result from such intervention. The effects of hemodynamic forces upon anti-oxidant, coagulation, inflammatory and other gene classifications will be measured in vitro where transcription profiles of porcine endothelial cells exposed to oscillating and unidirectional flow will be analyzed. Using small interfering RNA, specific gene 'knock-downs' will be created (gene silencing) to determine the effect upon hemodynamic responses in vitro including perturbations of the phenotype as evaluated by microarray profiling with follow-up protein and proteomic studies.

DESCRIPTION (provided by applicant): This competing renewal HL07954 Multidisciplinary Training in Cardiovascular Biology is the principal NIH training grant integrating research between Penn's Schools of Medicine (SOM), Engineering (SEAS) and Arts and Sciences (SAS). We request continuing support of 6 pre- and 6 postdoctoral trainees in multidisciplinary cardiovascular pathophysiology with emphasis on integrated biomedicine - physical/ quantitative/ engineering sciences. Thirty two faculty mentors are distributed across Penn's SOM, Childrens Hospital of Philadelphia, SEAS, and SAS (Physics; Math) departments, Institutes and Centers. The proposal addresses NHLBI multidisciplinary training priorities in 5 key areas by ensuring (i) training breadth and depth through multidisciplinary cardiovascular research integrated across a full spectrum of scales from the molecular to in vivo, (ii) Addition o faculty to ensure training in new and evolving research areas including the development of new courses, (iii) A formalized individual mentorship plan required of each Mentor/ Trainee pairing, (iv) Workforce retention to academic or science-based private-sector positions (for Postdocs), and postdoctoral positions (for Predocs) with attention to diversity, and (v) A translational infrastructure that embraces basic translational potential scientists and Clinical Fellow representation. To sustain a vigorous program, Predocs and Postdocs will receive two and three years support respectively. Trainees are selected in competitive review by an inter-School Steering Committee representative of the interdisciplinary nature of the program. Trainees are selected on the basis of academic record, research potential, and commitment to interdisciplinary cardiovascular biology. There is internal and external advisory oversight and annual review of the program. PhD and/or MD Postdoctoral trainees develop significant independence during their training period. Both group and individual mentorship in grant-writing and career development are provided. Predoctoral trainees (including MD-PhD students) are registered in one of the graduate groups of the Biomedical Graduate Group in the SOM or in select Graduate Groups of SEAS (Bioengineering, Chemical/Biomolecular Engineering, or Materials Science Engineering) or SAS (Physics, Math). They complete courses in cardiovascular-related engineering as part of, or in addition to, individual graduate group requirements. Participation in an Interdisciplinary Seminar Series and a biweekly Interdisciplinary Chalk Talk series is required, as is continuous RCR training. Research Symposia tailored to the training program provide additional presentation opportunities. 10-year program metrics: (36 predocs; 34 postdocs). Of 26 Predocs who have graduated (mean 5.2 yrs) 10 are in postdoctoral training, and 16 are in faculty positions (8) or science jobs (8). The balance (10) are still in training. Of 24 Postdocs who have completed support, 17 (71%) were recruited to academic positions, 6 (25%) to private sector science-related jobs, and one is on family leave. The balance (10) are still in training. Trainees authored 323 peer-reviewed papers (14,560 citations to date). (End of Abstract)

NON-TECHNICAL DESCRIPTION: Advances in the control of chemistry, structure and functionality at the nanoscale are critical in enabling the engineering of new materials for application in nanotechnology. This research project focuses on a novel family of inorganic materials that spontaneously assemble into ordered "checkerboard" nanostructures with unprecedented levels of perfection. The proposed work will study the formation and atomic structure of these new materials to understand the forces responsible for their stabilization and investigate methods for tailoring the length scale of their assembly. The remarkable level of control over the arrangement of the building blocks within these systems provides an opportunity to use their surface structure as a template to organize nano-sized objects placed on their surface. The achievement of this aim could represent a new paradigm for self-assembly in nanoscience and nanotechnology, areas that are vital to US technology. The research will train undergraduate and graduate students in the synthesis and structure characterization of nanomaterials and expose them to the many advances being made in nanotechnology. The PI and students will be involved in the development of new laboratory modules for high school students and teachers to illustrate the use of electron microscopy in revealing and understanding structure and chemistry at the nano-level.

TECHNICAL DETAILS: The project focuses on a family of Li-based A-site perovskites where spontaneous nanoscale phase separation into ordered Li-rich domains and a Li-deficient matrix produces mesoscopic ordered checkerboard structures with extraordinary levels of perfection. The formation of the checkerboard structure appears to proceed via spinodal decomposition; the mechanistic studies will focus why the bulk chemistry and bonding of these Li systems promote such a remarkable level of control over the nanoscale periodicity. This understanding will be used to design new bulk checkerboard chemistries where magnetic and electronic functionality will be incorporated into the building blocks. The surfaces of the checkerboard systems present a unique modulated nanostructure; these will be patterned using self-assembled monolayers, which subsequently will be utilized to spontaneously assemble nanometer-sized objects placed on the surface. The experimental parts of the project will enable the students involved to develop expertise in state-of-the-art techniques in electron microscopy and use national facilities to study their samples by neutron scattering.

DESCRIPTION (provided by applicant): Drug-eluting stents (DES) are deployed to physically reopen stenotic regions of coronary arteries to restore blood flow to the heart and to inhibit restenosis by release of anti-proliferative drugs over an extended period. However, significant incidences of localized delayed inflammation and late stent thrombosis (LST) leading to morbidities and deaths several months-to-years after deployment were reported in 2006. These alerted the FDA to reconsider the safety of DES and to issue a safety warning. Because DES inhibits restenosis, the stent struts remain at the arterial surface in indefinite contact with the flowing blood instead of being rapidly overgrown by the neointima. Although averaging only 1005m in height, the struts significantly change the local flow characteristics to create flow separation zones containing unsteady vortices in the regions adjacent to the stent strut. These vortices are characterized by significantly lower blood flow velocities than the bulk flow and prolonged particle residence time. We propose that the flow separation regions represent micro-reaction chambers where pro-coagulant and pro-inflammatory elements from the blood and vessel wall accumulate. Furthermore, re-endothelialization of the stented region is inhibited by low shear stress of the separation zones thereby contributing to a pro-pathological environment. Learning from numerical simulations of coronary blood flow and our extensive hemodynamic studies of arterial geometries where natural blood flow disturbances such as unsteady vortices induce pro-pathological vascular cell phenotypes, we hypothesize that the stent strut geometry leads to a local pro-thrombotic and pro-inflammatory environment. This R21 research grant proposes topographic solutions to mitigate or eliminate these consequences and tests them by experiment under controlled conditions in vitro, which is a necessary set of proof-of-principle exploratory studies that precede experiments in vivo. Guided by fundamental fluid dynamic principles, CFD numerical simulations identified a range of streamlined stent strut geometries that minimize or eliminate flow separation. Aim 1 will use Particle Image Velocimetry to characterize the flow field about different manufactured strut stent geometries in a cell culture flow chamber modeling coronary arterial flow and in a flow tube scaled to manageable quantities of whole blood. Aim 2 will test the effects of the respective strut designs on deposition of activated platelets and characteristics of thrombus growth in a chemical and substrate milieu conducive to thrombosis. Finally, Aim 3 will evaluate the effects of the redesigned stents on re-endothelialization and the expression of coagulation-related molecular phenotypes of endothelium. The proposal addresses the mechanisms of an important clinical problem by exploring the potential high utility of stent redesign and is built upon our extensive experience in hemodynamics, biomedical engineering and vascular cell and molecular pathology. The same principles of streamlined strut design are also applicable to BMS where thrombosis linked to the physical presence of the stent at the vessel surface occurs earlier, before a neointima develops. PUBLIC HEALTH RELEVANCE: Coronary artery stents are a common and effective treatment for angina and heart attacks particularly when the metal stent is coated with a slow-release drug that inhibits tissue response-driven reclosure (restenosis) of the artery (drug eluting stents;DES). Recently however, late stent thrombosis has been reported in a significant number of DES patients after anti-coagulant therapy has ended months after stent deployment. This project proposes that the physical shape of currently used stent struts creates a flow environment that promotes inflammation and thrombosis, and that a streamlined stent strut geometry will reduce or eliminate flow disturbances with a predicted decrease in thrombosis risk. Before taking the streamlined designs into an animal model, it is important to optimize the geometry by conducting numerical simulations and proving it with experimental fluid flow measurements while demonstrating proof of biological principles through controlled experiments using blood and vascular cells. The project tests an important hemodynamics hypothesis related to stent function and is an essential exploratory bridge to preclinical testing in an animal model. Successful implementation of effective stent redesign will have an important impact on the use and efficacy of stents.

DESCRIPTION (provided by applicant): The training of quantitative basic scientists in clinically-related imaging science is increasingly important. Excellent imaging sciences are well represented at Penn in multiple schools, but no formal integration of efforts in graduate training existed, nor was there a formal clinical component to the training until the creation of the Training Program in Biomedical Imaging and Informational Sciences. Established in 2006 under the auspices of the HHMI-NIBIB Interfaces Initiative, the program represents a partnership led by the Institute for Medicine and Engineering and the Department of Radiology in collaboration with many other Departments across multiple Schools. Our premise is that the most successful research and technologies in quantitative imaging science are those that integrate clinical relevance, mathematical rigor, and engineering finesse. Accordingly, the program embraces strong clinical exposure alongside analytical imaging science. The objective is to develop a new kind of interdisciplinary training by ensuring that students attain a level of integration that woud allow them to become the next generation of leaders in hypothesis-driven, clinically focused biomedical imaging research. Program outcomes to date are strong across all impact measures, indicating successful progress toward training objectives: publications (107); numerous research awards and distinctions; and recruitment of 6 URM and disadvantaged trainees. A formalized curriculum, the doctoral foundation, developed for the program provides 18 months of vertical integration of the core didactic elements of biomedicine and basic science education in biomedical imaging through 4 components, two of them Foundational, followed by Integrative and Professional components. In the first, Foundations in Biomedical Science (2 courses), students participate in modified modules 1 and 2 of the medical student curriculum that teaches the Core Principles of Medicine (including Gross Anatomy) and a 12- month sequence of organ systems medicine, Integrative Systems and Diseases. This is complemented by 4 courses in Foundations of Imaging Science: Molecular Imaging, Biomedical Image Analysis, Fundamental Techniques of Imaging, and Mathematics of Medical Imaging & Measurements. The third component is an Integrative Module: Advanced Biomedical Imaging Applications and Biomedical Image Sciences Seminars. The fourth component is Professional Training: Responsible Conduct of Research, Teaching Practicum, Patient-Oriented Research Training, Research 'Survival' Skills, and Career Development Skills. The core curriculum is complemented by many elective courses offered through the program faculty and tailored to Biomedical Imaging. Obligatory Laboratory Rotations will be offered through the laboratories of the participating faculty. To ensure that the thesis research is directed to translational medicine through the solution of discrete clinical problems, trainees will be co-advised by members of the clinical and basic science faculty.