Kenneth Walsh - US grants

During the next year we shall continue to pursue the broad goals outlined in our specific aims, with a continuing emphasis on the determination of the amino acid sequences of certain proteins. We expect to continue or finish structural analyses of myosin light chain kinase, phosphodiesterase, a sex steroid binding protein, a protein accumulating in amyloidosis, the lectin binding and casein kinase II. In each case, the structural data is expected to provide either new information about the domain substructure and probable origin of the protein or new insights into the function of the protein in healthy or diseased tissue. We shall continue to examine the chemical nature of profilaggrin, the very large, multidomain, precursor of a keratin assembling protein in differentiating epidermis. Three of the proteins under study are regulated by calcium and calmodulin (phosphodiesterase, myosin light chain kinase, and the Gamma chain of phosphorylase kinase). We plan to identify common features of the calmodulin docking sites and attempt to understand how that interaction modulates the activity of the interacting protein. Finally, we plan to continue to study the covalent processing events associated with the maturation or regulation of proteins during or after translation--particularly myristylation and phosphorylation.

This research proposal is directed toward an understanding of the structure/function relationships of multifunctional proteins that participate in major regulatory systems. Details of the organization and specific binding properties of structural and functional domains of selected proteins will be examined by methods of sequence analysis, limited proteolysis and recombination of subfragments constituting functional domains. Evidence will be sought in relation to the thesis that various cyclic nucleotide phosphodiesterases that respond to different regulatory signals belong to one protein family of homologous but chimeric character, and that this family bears a mechanistic, if not evolutionary, relationship to an analogous family of protein kinases. Domain isolation and recombination experiments will be designed to explore the mechanisms of signal transduction among representative members of these two super-gene families. It is expected that this study will lead to a more comprehensive understanding of the diverse mechanisms of regulation of enzymes and will direct attention to featrues common to regulated enzymes, whether controlled by zymogen activation, allostery or covalent modification. The proposal also describes plans to examine the multi-domain organization of profilaggrin, a protein that requires multiple posttranslational modification events (phosphorylation/ dephosphorylation and proteolysis) for its maturation and physiological function. Details of the unusual repetitive domain organization of profilaggrin will be determined. The protease(s) involved in its posttranslational processing will be identified. The chimeric domain organization of these various proteins will be compared and contrasted in efforts to develop a more comprehensive theory of the domain arrangements within multifunctional proteins.

This is a proposal to undertake a detailed study of structural features of human von Willebrand factor, and unusually large oligomeric glycoprotein that plays an important role in the formation of the platelet plug in hemostasis and blood coagulation. The protein acts as an adhesive bridge between platelets normally circulating in blood plasma and macromolecules such as collagen in the subendothelial extracellular matrix. The protein is found in blood plasma as a set of multimers of two to eighty identical subunits held together by disulfide bonds. We have established for each subunit the sequence of 2050 amino acid residues as well as the location of 22 oligosaccharide attachment sites and 26 disulfide bonds. These data are leading to the formulation of a structural model that accounts for the multiple ligand interactions of this multivalent molecule. Collaborative studies in our laboratory and elsewhere have tentatively identified several substructural domains, and we propose to explore their locations, boundaries and their mutual interactions using techniques of limited proteolysis and disulfide identification. Each isolated domain will be tested for independent folding of its polypeptide chain(s) and for this specific ligand binding capacity. We expect to refine the model of the protein in such detail that peptides can be designed as competitive ligands that could ameliorate thromobotic conditions. We expect to provide a molecular basis for understanding a wide range of variations collectively described as von Willebrand disease.

This proposal requests funds for the purchase of a plasma desorption mass spectrometer (PDMS) with time-of-flight (TOF) analytical capability. It is planned that the instrument will serve primarily 12 user groups in 10 departments at the University of Washington. Virtually al of the projects are directed at analysis of peptides of biomedical interest. This instrumentation will provide these users with access for the first time to mass spectrometer analyses of peptides of mass greater than 3000 amu. This biomedical community will benefit from access to this rapid, sensitive, flexible technique for examining peptides of high mass, for detecting posttranslational modifications, and for application of chemical strategies of partial structural analysis of proteins.

Atherosclerosis results, at least in part from the "differentiation" of smooth muscle cells in that they proliferate, decrease the expression of contractile proteins, and accumulate lipid. To understand the modulation of smooth muscle cell differentiation the proposed research will isolate and characterize the gene that may have a key role in regulating smooth muscle cell development. This approach takes advantage of recent advances in studies of skeletal muscle development which have led to the isolation of master regulatory genes for this tissue. Three genes (MyoD1, myogenin and Myf-5) have been isolated that have the ability to convert non-muscle cell types to skeletal muscle when they are constitutively expressed with a viral promoter. Normally these genes are not expressed in smooth muscle or any other cell type and they appear to be specific for controlling the differentiation program of skeletal muscle. The three skeletal muscle determination genes share a region of homology that is also found in the myc and lyl oncogenes, tissue-specific transcription factors, and in genes that are important in Drosphila development. This motif, a basic region- helix-loop-helix structure, may be shared by a general family of molecules that are of critical importance in determining the developmental fates of many cell types. Probes to this highly conserved motif will be used to isolate homologous sequences from smooth muscle cDNA libraries. Clones of these cDNAs will be used to determine the tissue specificity of mRNA expression. Full length cDNAs for these smooth muscle factors will be isolated and subcloned into a eucaryotic activate the expression of smooth muscle-specific contractile proteins and to autoregulate the endogenous factor gene. Clones will also be tested for their ability to trans- activate the expression of the vascular a-smooth actin gene. These clones may provide powerful tools to analyze the control of smooth muscle differentiation.

It is becoming apparent that myogenesis is controlled, at least in part, by proteins that bind to DNA regulatory elements in muscle-specific genes. This research will investigate a minimal, muscle-specific DNA regulatory element to understand how gene expression is controlled by complex protein DNA-interactions. A 28 bp fragment of the skeletal actin promoter is sufficient for muscle-specific expression when it is placed upstream of a nonmuscle TATA element. This muscle regulatory element (MRE) contains the core sequence motif, CC(A/T)6GG, which is referred to as CArG. The MRE is functionally different from nonmuscle elements, such as the c-fos SRE, yet they share the CC(A/T)6GG motif and bind to the same subset of nuclear factors. Presumably sequence differences between these elements alter the factor binding properties and this contributes to the different expression properties of the cognate promoters. The proteins that bind to the skeletal actin MRE are MAPF1 (and 2), SRF, and a recently identified factor, MF3. We have purified the MF3 binding activity to homogeneity from muscle. Molecular clones for MF3 and MAPF1 will be isolated. This will be accomplished by screening expression libraries with binding site probes or by screening with degenerate oligonucleotides designed from peptide sequence data. The analysis of these clones will provide clues about the nature of the heterogeneity found with the MAPF1 and 2 and the MF3 polypeptides. Transcript level measurements will indicate whether the factors are themselves regulated by muscle-specific promoters. Molecular clones will enable the production of recombinant factors and allow a genetic analysis of factor function. Purified factors will also be used to initiate studies on the assembly of the nucleoprotein complex. Experiments will be performed to evaluate whether these factors form higher-order complexes or compete for binding to the MRE. We are constructing a series of chimeric DNA elements to elucidate the molecular basis for the functional differences between the MRE and the SRE. Analyses of expression and factor binding properties of the synthetic elements will provide insights about the structural requirements for muscle-specific expression.

This proposal is directed towards understanding the structural organization of a large receptor-like, transmembrane enzyme, CD45, that appears to be a prototype for tandem-domain protein tyrosine phosphatase molecules subject to, and participant in, elaborate control mechanisms in living cells. The details of the organization of the molecule will be assessed, largely with modern methods of protein chemistry, in a search for facets of the substructural domains that appear to account for their binding, regulatory and catalytic functions. Particular attention will be given to understanding the importance to regulation of interactions between domains, as well as the modulating effects of reversible phosphorylation on those interactions. This study is focussed on CD45, which provides a vital link in the response of T-cells to antigens in the normal immune response. This transmembrane enzyme, and its homologs in a large family of related enzymes, are also thought to have the potential to act in opposition to growth-promoting factors and certain oncogene products.

Atherosclerosis is a major cause of death in industrialized nations. Part of the pathophysiology of atherosclerosis involves the dedifferentiation and proliferation of vascular smooth muscle (VSM) in response to PDGF and other growth factors. Nothing is known about the involvement of homeobox genes in the control of VSM, but these transcription factors have been identified as key regulators of cellular differentiation and proliferation in a wide variety of tissues and organs. Thus, we reasoned that alterations in homeobox gene expression may have a role in vascular disease and normal blood vessel development, and we looked for their presence in VSM. We have isolated a novel, divergent homeobox cDNA, dubbed V-Hox, that maps to a new position on mouse chromosome 12. In adults V-Hox is predominantly expressed in VSM, is present at low levels in cardiomyocytes and lung tissue, and it is not detected in visceral smooth muscle, skeletal muscle, brain nor many other tissues. A unique feature of V-Hox is that its expression is rapidly and dramatically downregulated by the addition of PDGF and serum to quiescent VSM cells. The aims of this research are to analyze the involvement of V-Hox in the control of differentiation and proliferation in cultured VSM cells and in transgenic mice. The proposed experiments will determine the DNA-binding and gene regulatory properties of V-Hox and identify potential downstream targets for this transcription factor. Work will also focus on elucidating the molecular basis of the rapid PDGF-induced downregulation of V-Hox expression because this will provide clues about the early events of mitogen action on VSM cells. Other experiments will overexpress and underexpress V-Hox to assess the role of this factor in the control of VSM cell growth and differentiation. Genomic clones for V-Hox will be isolated. The characterization of clones will elucidate V-Hox gene structure, and the study of promoter and enhancer sequences may provide clues about the hierarchy of regulatory events that control the development of VSM from precursor cells. The genomic clone will be used to disrupt the V-Hox gene in embryonic stem cells by homologous recombination. Chimeric mice will be derived by injecting blastocysts with these modified embryonic stem cells, and the subsequent breeding of these founder mice will produce V-Hox homozygous mice. Analyses of these mice will provide clues about V-Hox function in the developing cardiovascular system, and VSM cultures derived from the null mice would provide an ideal model system to study the transcriptionaI, developmental, and growth regulatory properties of the V-Hox protein.

This proposal is directed towards understanding the structural organization of a large receptor-like, transmembrane enzyme, CD45, that appears to be a prototype for tandem-domain protein tyrosine phosphatase molecules subject to, and participant in, elaborate control mechanisms in living cells. The details of the organization of the molecule will be assessed, largely with modern methods of protein chemistry, in a search for facets of the substructural domains that appear to account for their binding, regulatory and catalytic functions. Particular attention will be given to understanding the importance to regulation of interactions between domains, as well as the modulating effects of reversible phosphorylation on those interactions. This study is focussed on CD45, which provides a vital link in the response of T-cells to antigens in the normal immune response. This transmembrane enzyme, and its homologs in a large family of related enzymes, are also thought to have the potential to act in opposition to growth-promoting factors and certain oncogene products.

DESCRIPTION (the applicant's description verbatim): An increase in skeletal muscle mass is accompanied by the proliferation of endothelial cells and growth of arterioles. This vessel growth is triggered by the production of endothelial cell trophic factors by differentiating myofibers, such as vascular endothelial growth factor (VEGF), leading to the induction of angiogenesis. Recent data in cell culture systems demonstrate that Akt signaling is both essential and sufficient for endothelial cell survival, NO production and migration in response to angiogenic growth factor stimulation. Since these cellular responses are believed to be features of the angiogenic process, Akt may be uniquely situated within the endothelial cell signaling cascade to function as a key regulator of blood vessel growth. The experiments proposed here will extend the in vitro observations by testing the role of Akt signaling in blood vessel growth and vascular homeostasis in animal models. The proposed gene transfer studies will provide a better understanding of the signaling pathways that mediate angiogenesis within skeletal muscle and may lead to the development of new molecular therapeutic strategies to control blood vessel growth. Therefore, we propose to: 1) Assess the role of Akt signaling in vascular permeability using the Miles assay, 2) Examine the role of Akt signaling on blood vessel formation in matrigel plugs in mice, 3) Assess the consequences of enhanced endothelial cell Akt signaling in transgenic mice, and 4) Determine the role of endothelial Akt signaling in the revascularization of skeletal muscle in ischemic rabbit hindlimb.

DESCRIPTION (Adapted from Investigator's Abstract): Part of the pathology of atherosclerosis and post-angioplasty restenosis involves the regression of vascular smooth muscle cells (VSMCs) to a less differentiated cell that is capable of proliferation and migration. To understand these processes at a molecular level, we have isolated and characterized genes encoding transcription factors that are differentially expressed in quiescent and proliferating VSMCs. Previous work in my laboratory has led to the isolation of a homeobox transcription factor gene dubbed gax from an adult aorta VSM cDNA library. In VSMCs, gax expression is rapidly repressed by growth factor stimulation, and more slowly upregulated under conditions that promote quiescence. Similarly, expression of gax is rapidly downregulated in rat carotid arteries following an injury that stimulates VSMC proliferation. Adenovirus-mediated gax overexpression markedly inhibits injury-induced intimal hyperplasia in both rat carotid artery and rabbit iliac artery models of balloon denudation. Constitutive gax expression in vitro blocks both mitogen-stimulated cell cycle activity and mitogen-directed VSMC migration. Under conditions of prolonged mitogen activation, forced gax expression ultimately leads to p53-independent apoptotic cell death. The goal of the proposed research is to use Gax as a molecular reagent to characterize mechanisms that coordinate VSMC differentiation, proliferation and death. Therefore, renewal of funding is requested to: 1) perform a mutational analysis of the Gax transcription factor to determine structure-function relationships; 2) isolate and characterize Gax-binding protein; 3) identify downstream genes regulated by Gax and determine mechanisms of transcriptional regulation; and 4) identify promoter/enhancer regulatory elements in the gax gene that confer tissue-specific expression during embryogenesis and mediate the downregulation of expression that occurs upon vascular injury.

DESCRIPTION (Adapted from Investigator's Abstract): Vascular injury has been shown to induce proliferation and apoptosis in vascular smooth muscle cells (VSMCs), and this balance between cell growth and cell death will ultimately influence the size of the injury-induced lesion. Apoptotic cell death has been documented in human atherectomy and endarterectomy specimens and in a number of animal models of vessel wall stenosis. Recently, they have shown that as early as 30 minutes following balloon injury VSMCs of rat carotid and rabbit iliac arteries undergo apoptotic cell death at a high frequency as demonstrated by TUNEL staining, and by the appearance of condensed chromatin and other morphological features characteristic of apoptosis in electron micrographs. This induction of apoptosis coincides with a marked downregulation of the bcl-X protein, a potential cell death antagonist. Their data suggest that VSMC apoptosis is a rapid and prominent cellular response to acute vascular wall injury, the extent of this apoptotic response may ultimately influence characteristics of the lesion that result from the insult. To more fully understand the regulation and the role of apoptosis in vessel wall lesion formation, it is proposed to: 1) determine the frequencies of VSMC apoptosis in single injury and double-injury model of angioplasty in rabbit external iliac arteries; 2) assess the effects of enhanced apoptosis on vessel lesion formation using a replication defective adenovirus encoding Fas ligand; 3) characterize apoptosis in a mouse model of arterial injury; and 4) study the mechanisms that coordinate cell cycle and apoptosis at a molecular level.

This proposal describes the need in ongoing biomedical research of a rapid, sensitive and accurate Matrix-Assisted Laser Desorption Time-Of- Flight mass spectrometer, (MALDI-TOF) with delayed ion extraction and an improved detector in a shared-used facility in the Department of Biochemistry at the University of Washington Medical School. The facility has leased and earlier model of the instrument for three years and determined that it has technical advantages for certain studies that complement the utility of the Electrospray triple quadrupole instrument that was purchased for the same laboratory by a SIG in 1991. The new MALDI-TOF will extend service to a diverse user group of 15-45 principle investigators (in more than 8 Departments) with more than 22 NIH- supported research programs. Targets of these studies range from large proteins and their post-translational modifications to smaller peptides, drugs and metabolites. No other MALDI-TOF is available at the University to serve these needs.

Proliferative myocytes precuursor cells migrate from the somite and fuse to form distinct groups of post-mitotic myotubes in the trunk and limbs. Myocyte differntiation is characterized by an ordered progression through discrete developmental steps. Cell cycle exit occurs early during the differntiation program and it it essential for normal expression of the contractile phenotype. In contrast to precursor cells, differentiated myocytes display a markedly lower frequency of apoptotic cell death. Previous studies have shown that the upregulation of the cdk inhibitor p21 and the dephosphorylation of pRb appear to be criitical regulatory events for the establishment of both the post-mitotic and apoptosis-resistant states in vitro and in vivo. However, the mechanisms that coordinate cell cycle activity and apoptosis are largely unknown. The purpose of this proposed research is totest the hypothesis that the Art kinases (Akt and Akt2) play important roles in regulating the survival of differentiating myocytes. Specifically, we will test whether Akt kinases are regulated by developmental cues during myogenesis with consequences on cell survival. We will also test whether the induction of Akt during mycyte differentiation is inhibited by cell cycle activity in a manner similar to contractile protein genes. Finally, we will examine the temporal and spatial patterns of Akt and Akt2 peptide expression in the developing mouse embryo and determine the mechanisms relating Akt and Akt2 induction during muscle differentiation. Collectively, thesee experiments will explore the hypothesis that myocyte survival and proliferation are coordinately regulated through the ability of cell cycle activity to modulate the myogenic induction of Akt and/or Akt2.

The goal of this research is to characterize the post-natal regulatory mechanisms that control heart growth and apply these findings to develop new approaches to treat heart failure. Trophic factors such as growth hormone (GH) and insulin-like growth factor (IGF) are required for normal heart development and have been shown to inhibit and reverse the progression of heart failure. The beneficial effects of GH/IGF on cardiac function are thought to originate, at least in part, from their ability to promote physiological cardiac growth. Furthermore, insulin administered with glucose and potassium (GIK therapy) can decrease the rate of ischemic cell death following acute myocardial infarction, thereby reducing patient mortality. These factors activate the phosphatidylinositol 3-kinase/Akt intracellular signaling pathway, and we have previously shown that the serine-threonine kinase Akt mediates IGF-induced survival signals in cardiac myocytes. Therefore, we hypothesize that Akt signaling is a component of cardiac hypertrophy (Aim 1). In addition, our preliminary data suggest that insulin is an important regulatory of normal post-natal cardiac growth. Together with the finding that insulin is a potent activator of Akt in cardiac myocytes, we hypothesize that insulin regulates cardiac growth during postnatal development through Akt-dependent pathways(Aim 2). We also hypothesize that activation of Akt signaling will improve heart function in models of heart failure by promoting physiological cardiomyocyte hypertrophy (Aim 3). Finally, we hypothesize that the growth-promoting and protective activities of Akt signaling are partly mediated through the regulation of a specific subset of Forkhead transcription factors (Aim 4). Akt-regulated Forkhead factors are implicated in the control of cell growth survival, but they have not been investigated with regard to their regulation and function in the heart. Collectively, the proposed study will contribute to our understanding of the mechanisms that regulate physiological and pathological cardiac growth, and will provide insight for the development of novel approaches to treat heart failure.

DESCRIPTION: The objective of this proposal is to determine the role that Akt1 plays in the phenomenon of vascular smooth muscle cell (VSMC) polyploidization. It is well known that pathological remodeling of vascular smooth muscle in capacitance arteries is responsible for arterial stiffness and contributes to the development of associated coronary and ventricular pathologies. Previous studies have shown that the hypertrophy of vascular smooth muscle cells (VSMCs) is the main factor responsible for the enlargement and rigidity of the aorta and that hypertrophied. VSMCs on capacitance arteries of hypertensive individuals and animals are frequently polyploid. Our laboratory has found that the aortic vascular smooth muscles of hypertensive animals have elevated levels and activity of PKB/Akt1, an enzyme that provides signals for cell growth and survival. We have also seen that ectopic expression of Akt1 induces VSMC polyploidization and up regulation of Cks1, a Cyclin B/Cdc2-associated protein that promotes the progression of mitosis. We hypothesize that Cks1 mediates some of the effects of Akt1 on VSMC mitosis. To test these hypotheses, we propose the following aims: Aim 1: To determine the effect of Akt1 gene transfer on the activity of the mitotic spindle and postmitotic cell cycle checkpoints in VSMCs. Aim 2: To determine the molecular bases for the up regulation of Cks 1 expression in VSMCs with activated Akt 1. Aim 3: To determine the requirement of Cks1 function for Akt1-induced VSMC polyploidization. Aim 4: To generate transgenic animals that over express Cks1 or Akt1 in vascular smooth muscle.

It is recognized that our infrastructure to a very large extent is founded on unsaturated soils. In fact, construction in unsaturated soils is typically preferred when practical, due to reduced costs and effort. However, the geotechnical research community has not yet solved all of the issues of implementation of unsaturated soil theory. One of the primary difficulties in bringing the relatively new fundamental theories of unsaturated soil mechanics into engineering design, analysis, and construction is the difficulty, time, and cost associated with unsaturated soil characterization. Two major elements are needed to bring unsaturated soil mechanics and fundamental analyses into practice: (1) estimates or measurements of soil suction, and (2) moduli or coefficients relating soil suction to performance parameters such as soil shear strength and compressibility. In this research, a range in sophistication of approach will be employed for unsaturated soil characterization. Hierarchical Level 1 will be developed for extremely high-level, large budget projects for which significant expenditures are likely. Hierarchical Level 4 will be developed for lower budget, routine projects for which very limited testing and analysis are common. This research is an important step in bringing unsaturated soil mechanics into present day geotechnical engineering and construction practice. The implementation of unsaturated soils theory would result in more fundamentally sound analyses and design and would lead to substantial savings in construction costs for many applications.

CORE ABSTRACT NOT AVAILABLE

0090559
Bashford

This award is to Arizona State University to support the activity described below for 36 months. The proposal was submitted in response to the Partnerships for Innovation Program Solicitation (NSF 0082).

Partners
The partners are Arizona State University; Partnership for Advancing Technology in Housing; Home Building Association in Central Arizona; Del Webb Corporation; Pulte Homes; Eagle Homes; Trend Homes; Maracay Homes; Gateway Community College.

Proposed Activities
The partners are doing the following: identification of challenges faced by home builders where technology could change the process significantly; identification of technologies to address the challenges; research on new materials, products, and processes (especially where manufacturing can lower cost and increase quality); modeling and simulation of home construction; develop prototypes and work techniques to apply them; education/training of a workforce to use the new building technology developed.

Proposed Innovation
Housing is the industry that missed the innovation revolution in America. The industry is mature and extremely fragmented, making innovation very difficult. The proposed innovation activities include research and a management plan to incorporate new materials, manufacturing practice with obvious economic and quality control benefits, training of skilled craftsmen, energy savings, and modification of building codes could modernize the house construction industry with enormous savings for the US economy.

Potential Economic Impact
The following economic impacts are likely: improve durability and reduce maintenance costs for new homes by 50% by 2010; reduce cost of new homes by 20% by 2010; reduce energy costs by 50%; increased safety of construction workers. The housing industry is a huge driver in the national economy.

Potential Societal Impact
Active recruitment of Hispanics through chamber of commerce activities and Gateway Community College will help provide opportunity for this group to participate in the program. Affordable and maintainable housing will provide obvious benefits to all Americans, especially Americans who can afford custom homes.

The overall goal of this research is to realize the vision of making appropriate information available anywhere, anytime in the homebuilding process through the use of innovative information technology tools. The project vision will be accomplished through the development of a Pervasive Production Space (PPS) for building code compliance results that uses the emerging field of pervasive computing. This research will focus upon the development and identification of pervasive computing paradigms, methodologies, design tools and technologies to meet this vision for the US homebuilding industry. The project vision can ultimately apply to all phases of the homebuilding process including land development, house design, pre-construction, building construction, and post-construction. While pervasive computing is being generally adapted to structured home and office environments, this research will expand fundamental underpinnings of such computing theory through its application in an "unstructured" production environment. The intellectual merit of the proposed research also relates to its confirmation of widely held, but insufficiently documented, hypotheses about the value of IT implementation in the residential construction industry. The broader impacts of the proposed research are dramatic. Housing is a basic human need. Current management methods have led to an increase in the cycle time for the construction of new housing units, and a concomitant increase in the cost. The proposers estimate that some $90B in excess capital is tied up in the industry due to communications-induced cycle time waste. This capital could be put to other uses in the economy, and the impact on housing prices would increase affordability nationwide. The proposed data collection strategies will provide significant material for classroom instruction in residential construction, improving education methods via conduct of the research. The proposers will make every effort to disseminate the results broadly; positive data vetted by industry partners could be a compelling tool to drive other builders to seek similar partnerships, which could in turn lead to wider partnerships. Revolving around the core research activity will be other important activities that will ensure integration of research and education, involvement of persons with diverse backgrounds and career aspirations, creation of an enabling infrastructure that will foster and sustain innovation in the long-term, and transformation of demand generated by diffusions of innovation into new businesses.

Description: This award supports the US-India cooperative research project entitled Investigation of Lightweight Materials and Construction Techniques for Cost-effective Housing in India and the U.S. Professors Anil Sawhney, Arizona State University (ASU), Kenneth Walsh, San Diago State University (SDSU), and Krishnamurthy Ramamurthy, Indian Institute of Technology Madras (IITM) will use the "whole house approach" to investigate lightweight materials and construction processes to build quality cost-effective homes. They will examine the potential for autoclaved aerated concrete (AAC) and other similar materials to become a major component in housing production in the U.S. and India. While wood has been a popular home building material in the U.S., brick and concrete are widely used in India. The investigators believe that the energy and thermal mass properties, durability, and cost effectiveness of AAC have advantages over both other systems.

Scope: The collaboration between ASU, SDSU, and the IITM is expected to be mutually beneficial through the intellectual exchange of ideas, results, and approaches to housing research. The manufacture, construction, and implications for residential production system design, which have been extensively studied in the US, will be integrated with the Indian researchers' extensive experience in materials characterization of AAC. The collaboration is expected to expand understanding of AAC use in residential production in both settings. It will create technical linkages among the institutions, researchers and students, and will contribute to the development of improved graduate and undergraduate courses. The Division of Civil and Mechanical Systems is co funding this research; and India's Department of Science & Technology (DST) supports this project under the NSF/DST joint program.

Recent studies have found that bone marrow-derived endothelial progenitor cells (EPCs) are present in[unreadable] the systemic circulation and home to sites of ischemic injury where they function to promote[unreadable] neovascularization. Cardiovascular diseases characterized by increased reactive oxygen species (ROS)[unreadable] stress and reduced NO bioavailability are associated with reductions in the number and functional activity[unreadable] of circulating EPC, and these reductions may diminish their angiogenic and reendothelialization capacity.[unreadable] EPCs share antigenic markers with hematopoietic stem cells and differentiate into the endothelial lineage[unreadable] in vitro. A number of cytokines, growth factors and adhesion molecules have been implicated in the[unreadable] mobilization of EPCs from the bone marrow stem cell niche and in their homing and differentiation at sites[unreadable] of vascular damage. However, the signaling and transcript!onal regulatory mechanisms that control EPC[unreadable] behavior are incompletely understood. The PI3K/Akt signaling pathway is an important regulator of growth[unreadable] responses in a number of systems, and some evidence has implicated this signaling pathway in promoting[unreadable] the proliferation and differentiation of EPCs under conditions of basal ROS-mediated signaling and[unreadable] compensated oxidant stress. PI3K/Akt signaling contributes to the regulation of the FOXO transcription[unreadable] factors that play a role in cellular resistance to oxidant stress. The proposed studies will test the[unreadable] hypothesis that FOXO transcription factors control EPC proliferation and differentiation and regulate their[unreadable] resistance to oxidant stress. To achieve these objectives, we will first characterize the FOXO isoforms in[unreadable] EPCs with respect to their expression, regulation by ROS and subcellular localization (Aim 1). We will then[unreadable] use gain-of-function and loss-of-function strategies to determine the role of FOXO isoforms in controlling[unreadable] EPC function in vitro including proliferation, differentiation, migration, apoptosis and resistance to oxidant[unreadable] stress (Aim 2). Finally, we will construct lines of transgenic mice that conditionally express constitutivelyactive[unreadable] and dominant-negative FOXO isoforms in EPC and examine the implications for EPC behavior and[unreadable] response to ischemic injury (Aim 3). These studies should provide mechanistic information on the FOXO-mediated[unreadable] regulatory pathways that control EPC phenotype and the relationships between redox stress and[unreadable] EPC function.

DESCRIPTION (provided by applicant): Several lines of evidence have suggested that growth hormone (GH), and its local effector insulin-like growth factor-l (IGF-I), promote physiological cardiac growth. GH and/or IGF-I treatment have also been demonstrated to increase myocardial contractility and cardiac output in experimental models of cardiac failure. Similarly, insulin administered with glucose and potassium (GIK therapy) can decrease the rate of ischemic cell death following acute myocardial infarction. However, the effects of GH therapy in human subjects is still inconclusive, and recent reports in animal models suggest that long-term IGF-I treatment may not be beneficial because chronic IGF-I stimulation can induce aspects of pathological hypertrophy. Therefore, the development of therapeutic strategies for maintaining cardiac function in the failing heart could benefit from a better understanding of the molecular distinction between normal cardiac growth and pathological cardiac hypertrophy. We have shown that IGF-1 and insulin activate the phosphatidylinositol 3-kinase/Akt signaling pathway in cardiac myocytes and vascular cells. We have also shown that activation of this pathway is an essential step in mediating the IGF- and insulin-induced survival and growth signals in cardiac myocytes. Therefore, this grant will focus on mechanisms of myocardial remodeling in mice that have gain-of-function or loss-of-function genetic alterations at this and subsequent signaling steps. We propose to analyze myocardial remodeling and failure in inducible Akt transgenic and knockout mouse models (Aim 1); assess coronary vasculature growth and regression during myocardial growth and regression (Aim 2); analyze the role of the Akt-regulated transcription factors in controlling heart growth and function (Aim 3); and perform genomic and proteomic analyses of Akt and forkhead-mediated remodeling in the heart (Aim 4). These studies should provide a better understanding of the mechanism that regulate adaptive and maladaptive remodeling, and provide new insights about the development and treatment of heart failure.

This award is to the University of New Orleans to support the activity described below for 24 months. The proposal was submitted in response to the Partnerships for Innovation Program Solicitation (NSF-04556).

Partners
The partners include University of New Orleans (Lead Institution), Tulane University, Department of the Navy-SPAWAR, Department of the Navy-Stennis, NASA-Stennis, Louisiana Department of Economic Development, City of New Orleans, Jefferson Parrish, Jefferson Parrish Economic Development Commission, Louisiana Technology Council, MetroVision, Idea Village, Lockheed Martin, Northrup Grumman, NVE, Parallel Synthesis Technologies, g.c.r.&Associates, Resurgent Technologies, Science & Engineering Associates, Solutient, Xavier University, Louisiana State University, and Louisiana Tech.

The primary objectives are: The University of New Orleans proposes the creation of a Center For Innovation (CFI) as an effective way to facilitate the transformation of research knowledge and ideas into
innovations that create new business opportunities in Southern Louisiana and the Gulf Coast region. The CFI will bring together scientists with representatives of private businesses, government agencies, and financiers to explore and develop business opportunities. CFI will meet the goals of the Partnership for Innovation by facilitating the transformation of knowledge emerging from researchers into innovations that create new wealth, build strong local, regional and national economies and improve the national well
being. The strength of this proposal rests primarily on the novel program. The center will proactively search out faculty with promising ideas for emerging technologies using a network of college representatives and the staff in the Office of Research and Sponsored Programs. Faculty with emerging technology ideas that are judged to have a high potential for commercialization will be partnered with an Entrepreneurial advisor who will take an active role in educating the faculty member on the importance of protecting intellectual property and working with the faculty member on assessing the potential marketability of their product. Faculty will then be connected with the appropriate partners in the
business community. Thus, the Center For Innovation will serve as the conduit for connecting the academic community with these public/private partners to help turn innovations into economically beneficial projects. Novel ideas in the proposal include combining reporting of IP disclosures across multiple universities, which will increase the likelihood of visibility of innovations.


Potential Economic Impact
Growing the businesses in the area through collaborative research will provide an economic benefit to the area.

The intellectual merit of the project is the recognition of a need for wealth creation and economic development. This solution, creating a center, is a long-term far-reaching plan to answer the need for new and different jobs. University of New Orleans seeks to create an atmosphere of learning, collaboration, and commercialization. The plan has a variety of partners with similar goals. This proposal provides a vehicle for not only moving university innovations to the market but also partnering with businesses to link appropriate faculty research personnel to business interests.

The broader impacts of the activity concentrate on a Gulf Coast Region initiative. There is a concern for fellow institutions in the area facing similar problems. This is important because in the future when other institutions want to start similar centers there will be a helpful voice from a cooperative university. University of New Orleans does seek to create a template to help other universities create similar organizations for their regions. Also the program goes out of its way to address the inclusion of minority students in the project, which will be easy because of their location.

DESCRIPTION (provided by applicant): Ischemic heart disease is the leading cause of morbidity and mortality in the United States. Obesity- linked diseases including type 2 diabetes largely contribute to the incidence, severity and outcome of ischemic heart disease. However, the link between obesity and the development of cardiovascular disease is poorly understood at a molecular level. Adipose tissues secrete adipokines that directly or indirectly affect obesity-linked disorders. Adiponectin is a circulating adipokine that is downregulated in patients with obesity-linked diseases including type 2 diabetes, the metabolic syndrome and ischemic heart disease. Studies with adiponectin-deficient mice show that adiponectin has important anti- atherogenic and anti-diabetic properties. Previously, we showed that adiponectin promotes angiogenic repair of ischemic hindlimbs. Recently, we also demonstrated that adiponectin protects against acute myocardial ischemia-reperfusion injury. These cardiac protective effects are mediated by the anti- apoptotic actions of AMP-activated kinase (AMPK) and the anti-inflammatory actions of cyclooxygenase- 2 (COX-2) in myocardial cells. In contrast to AMPK, the signaling mechanisms by which adiponectin activates the COX-2 pathway are unknown. In the proposed studies, we hypothesize that adiponectin accelerates angiogenic response to chronic myocardial ischemia and regulates cardiac remodeling after myocardial infarction. The proposed studies will also focus on the receptor-mediated signaling mechanisms that mediate the adiponectin-COX-2 and -AMPK regulatory axes. To achieve these objectives, we will: Aim 1) Analyze whether adiponectin has beneficial effects on cardiac remodeling after myocardial infarction. Aim 2) Analyze the role of adiponectin in EPC mobilization and function. Aim 3) Analyze the adiponectin-COX-2 and -AMPK regulatory axes in vitro. Collectively, the proposed study will contribute to our understanding of the mechanisms that regulate cardiac remodeling and angiogenesis in association with obesity-linked disorders, and may provide insight for the development of novel approaches to treat ischemic cardiovascular disease.

DESCRIPTION (provided by applicant): The endothelial cell is topologically poised in the vasculature to sense and respond to a host of environmental signals, including reactive oxygen species (ROS). These chemically active molecules serve important roles in normal, homeostatic signaling; and their potential for oxidative injury is ameliorated by an elaborate system of antioxidant defenses. As the flux of ROS increases, the endothelial cell responds by enhancing protective mechanisms, leading to a state of compensated stress; when the flux of ROS increases further, these protective mechanisms are overwhelmed, leading to a state of uncompensated oxidant stress. In this program project application, five project leaders have come together to investigate mechanisms that underlie oxidant signaling and adaptation to oxidant stress in the endothelial cell in health and disease. Project 1 focuses on the mitochondrion as an important component of endothelial redox signaling; Project 2 addresses the role of glucose-6- phosphate dehydrogenase and its enzymatic product, NADPH, as key determinants of the thiol redox state in the endothelial cell; Project 3 examines the role of Foxo transcription factors in promoting resistance to oxidant stress in endothelial progenitor cells; Project 4 considers the effect of ROS dependent oxidative modifications of Ras on insulin signaling in the endothelial cell; and Project 5 tests the hypothesis that mitochondrial dysfunction and resulting oxidant stress contribute to endothelial dysfunction in human atherosclerosis. This conceptually cohesive program brings together five well established project leaders who have a long history of productive collaboration to focus on a key theme in endothelial cell biology. Using contemporary methods of cell and molecular biology, as well as genetic animal models and human studies, mechanisms will be dissected at the molecular and cellular levels and applied to mammalian systems. With the work proposed in this proposal, we hope to be able to identify novel approaches to understanding the biomolecular basis of oxidant signaling and the adaptive and maladaptive consequences of oxidant stress in endothelial (patho) biology and vascular disease.

Infrastructure construction globally over the next several decades will occur primarily in the developing world. The resulting demand for construction materials, energy, and water, and the resulting global pollution load, must be addressed on the front end to seek sustainable approaches. We must come to understand how to design and build infrastructure so as to accomplish the lofty objectives of the infrastructure itself, while at the same time finding approaches that maximize the investment impact while preserving social and political acceptance and the needs of the environment. Developing countries present unique development challenges in the efficient, affordable, and sustainable delivery of infrastructure especially due to weak government institutions, low-income economies, lower productivity, negligible private sector participation, and higher unemployment. And, while it is true that the infrastructure itself, upon completion, fuels economic growth, the process of constructing that infrastructure can have powerful impacts on national capacity of the construction industry. In addition to the direct gains which can be obtained by solving these riddles, it is also true that US infrastructure construction will be a continuing significant national expense into the foreseeable future, so the lessons learned from US technological leadership in developing country settings can also be transferred to US infrastructure projects. The challenges created by these critical problems must be addressed in a systematic way, but at present there is no broadly understood research agenda.

A workshop will be conducted to provide this agenda. A Workshop Advisory Panel (WAP) has already been established for this purpose. The workshop will focus on two broad themes of research. The first research theme is focused upon broadening the global capacity for engineering design and construction to accelerate the pace of knowledge creation. The second research theme is focused upon redefining the role of the civil and construction engineering community in an interdisciplinary setting consisting of economists, public policy experts, social scientists, and financial experts. In general, educational systems in the United States have an excellent record of preparing engineers for the technical challenges they will face. The workshop is planned to take place in Washington DC in fall 2007. The participants will include industry professionals, university researchers, graduate and undergraduate students, lead experts from sponsoring agencies, international development agencies, and members from research organizations. Students will be engaged in the process as participants and assistants.

Intellectual Merit: Developing countries represent a significant opportunity for growth in the future and it is important to consider strategies both for building sustainable infrastructures and for broadening participation in the solutions identified. How to explore and establish the appropriate technological and economic level of infrastructure capacity is a challenge that will be addressed by this workshop. This workshop will also create a new body of knowledge specifically related to infrastructure development issues in developing countries.

Broader Impacts: This workshop is focused upon training and educating future scholars and leaders in successful infrastructure development and growth in developing countries. Because the problem itself is so critical, and because its solution utterly depends on expanding the diversity of participation in its solution, the impacts are inherently broader than the knowledge created itself. Practitioners, faculty, and students from different countries will participate to learn about this topic. Through the involvement of an industry expert as a Co-PI the workshop content will be further strengthened. A website will be developed for this workshop to include the specific details on the workshop activities. It will be used to act as a clearing house for all relevant research and literature related to building sustainable infrastructure in developing countries. Results will be published in appropriate journals, newsletters, and websites. The case study project data will be provided on the workshop website and participants will be encouraged to use it as a training and educational resource. This award was co-funded by the Office of International Science and Engineering.

[unreadable] DESCRIPTION (provided by applicant): Vascular endothelial cells respond to ischemic stress in tissues by undergoing an angiogenic repair process. We have previously shown that activation of Akt signaling is an important mediator of the angiogenic response in endothelial cells. More recently, we have shown that hypoxia and hypoxic-reoxygenation are powerful stimuli of AMP-activated kinase (AMPK) signaling within endothelial cells, and that AMPK signaling significantly contributes to biological responses in endothelial cells under conditions of ischemic stress including eNOS activation, cellular survival and migration. Based upon these data, it has been proposed that AMPK signaling promotes endothelial cell function and angiogenic responses in ischemic tissues. Recent studies have shown that LKB1 functions as a key regulator of both AMPK and Akt signaling in a number of tissues including liver, skeletal muscle, myocardium and various cancers. However, very little is known about the role of LKB1 in regulating endothelial cell function and angiogenic responses. Thus, we will investigate the role of LKB1 in endothelial cells biology in vitro utilizing adenoviral expression vectors and siRNA procedures to understand how this signaling step regulates AMPK and Akt pathways under conditions of normoxia and hypoxia. A series of related experiments will analyze the role of LKB1 in endothelial cell function in vitro including NO production, survival, migration and proliferation. To understand the role of vascular cell LKB1 signaling in vivo, endothelial LKB1-knockout mice will be created using a cre/lox approach, and their baseline phenotype will be assessed. Finally, endothelial LKB1-deficient mice will be subjected to ischemic stresses and vascular responses will be evaluated. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE Vascular endothelial cells respond to ischemic stress in tissues by undergoing an angiogenic repair process. A series of experiments will analyze the role of LKB1 in endothelial cell function. LKB1-knockout mice will be created and their baseline phenotype will be assessed. Endothelial LKB1-deficient mice will be subjected to ischemic stresses and vascular responses will be evaluated. [unreadable] [unreadable] [unreadable]

Assumptions of extremes of wetted state of unsaturated soils during infrastructure lifetime (saturated or dry properties) have significant implications for design, construction, functionality, safety, and structural longevity. Assessment of the degree of saturation that occurs in the subsurface requires understanding and quantifying actual surface flux, a complex function of soil surface conditions, including in particular soil suction, degree of cracking, and slope geometry. There are two major related research issues yet to be adequately addressed in geotechnical applications: (1) the effect of surface cracking in volume-change-sensitive clays on unsaturated flow property functions, and (2) the effect of drainage conditions (well-sloped surface versus poorly drained) on surface flux of cracked and intact clays. Seasonal cracking of soil results in poor estimates of runoff and infiltration due to the changing soil storage conditions (Arnold et al, 2005). Prediction of soil suction profiles requires substantial improvements in current capabilities, both from a soil property and numerical modeling perspective. Several conditions present numerical solution challenges: (i) strong nonlinearities in soil properties, (ii) abrupt changes of moisture conditions at the surface boundary and wetting front, and (iii) the presence of surface runoff conditions (Scanlon et. al., 2002). The behavior of unsaturated cracked soil is quite different from that of intact soil, further complicating evaluation of surface flux conditions for clays.

This study addresses key remaining questions rarely, or only superficially, discussed in the geotechnical literature, and is geared toward transformation of surface flux modeling capabilities for cracked and intact clays. The geotechnical research team will work with a co-investigator in Applied Math towards addressing these needs through development of: (1) data and models for unsaturated soil properties of cracked clays including volume change of both the cracks and matrix; (2) data and models for run-off for well-inclined (sloped) and level-grade cracked and intact (3) improved solution methods for surface flux, including run-off, and (4) evaluation of field damage related to drainage and cracking for consistency with modeling and data. A key element of this research is the collaboration and sharing of physical resources among partners (ASU?s advanced unsaturated soils testing equipment and SDSU?s unique tilt table).

A wide range of problems arise from unsaturated soils. Damage to infrastructure from expansive clays alone is estimated to be as much as 15 billion/yr (Nuhfer et al., 1993; Wray and Meyer 2004). Krohn and Slossom (1980) estimated that 20% of surface soils of the U.S. are subject to shrink-swell (and cracking). Concerns over movement of contaminants to great depth have heightened interest in understanding unsaturated flow and the complex interactions between climate, human surface activities and unsaturated soil subsurface conditions and processes. Researchers have demonstrated the role of unsaturated soil behavior in rainfall-induced slope failure (Toll, 1999; Yin, 1998). Surface flux and surface runoff are critical to the performance of slopes, and little is known about the influence of cracks on the flux or on slope stability itself. In California in 1982, over 18,000 slides swept down slopes with little warning, damaging homes and killing 14 residents (Ellen and Wieczorek, 1988). U.S. costs for landslide repairs exceed $2 billion/yr and landslides, nearly all rainfall-induced, result in 25-50 deaths/yr (Spike and Gori, 2003). This research will have impact on solutions to all of these problems through enhancing our understanding and modeling of surface flux and unsaturated flow.

Students will be trained and will be engaged in dissemination, including conferences and publications. Findings will be presented in CE classrooms at ASU and SDSU, and integrated into ASU?s Math program where students will perform numerical simulations and compare with existing codes and data. On-going ASU recruiting programs focused on teachers and underrepresented students will be used to bring aspects of this study into junior and high school classrooms. A set of lectures will be developed on the research process and presented at a San Diego high school having an 82% female and non-white male student body. Students will also tour labs. Working with pre-engineering teachers, Co-Is will develop a means of attracting students to engineering and research.

Vascular endothelial cells respond to ischemic stress in tissues by undergoing an angiogenic repair process. We have previously shown that activation of Akt signaling is an important mediator of the angiogenic response in endothelial cells. More recently, we have shown that hypoxia and hypoxic- reoxygenation are powerful stimuli of AMP-activated kinase (AMPK) signaling within endothelial cells, and that AMPK signaling significantly contributes to biological responses in endothelial cells under conditions of ischemic stress including eNOS activation, cellular survival and migration. Based upon these data, it has been proposed that AMPK signaling promotes endothelial cell function and angiogenic responses in ischemic tissues. Recent studies have shown that LKB1 functions as a key regulator of both AMPK and Akt signaling in a number of tissues including liver, skeletal muscle, myocardium and various cancers. However, very little is known about the role of LKB1 in regulating endothelial cell function and angiogenic responses. The proposed research will examine the role of endothelial cell LKB1 in regulating downstream signaling proteins, involving both AMPK and Akt signaling pathways. Related experiments will manipulate the expression of LKB1 and downstream proteins and assess the effects of these perturbations on cellular responses that are associated with angiogenesis, including migration, proliferation, survival and NO production. These observations will be extended in vivo through the construction and characterization of conditional knockout mice that are deficient for LKB1 in the vascular endothelium. LKB1 conditional knockout mice will then be subjected to ischemic surgeries and angiogenic responses will be analyzed. ]n the revised application, new experiments are proposed to examine the upstream regulators of LKB1. Specifically, experiments will focus on the role of the histone deacetvlate SIRT1. To accomplish these goals we will: 1. Dissect signaling pathways that function downstream from LKB1 in endothelial cells that are acutely exposed to ROS. 2. Assess the role of LKB1in regulating endothelial cell responses that contribute to angiogenesis. 3. Construct and characterize mice that are deficient for LKB1 in the vascular endothelium. I 4. Construct and characterize mice that are deficient for SIRT1 in the vascular endothelium.

DESCRIPTION (provided by applicant): Accumulating evidence suggests that there is a strong mitochondrial component in cardiac physiology and pathophysiology. Heart failure (HF) is a condition commonly seen in elderly;it can compromise the quality of a patient's life and it is associated with an increased rate of mortality. Currently, HF is recognized as a disease that occurs due to aberrant cardiac energy metabolism. The cardiac mitochondrion is considered the central integrator of myocyte metabolism and it may be possible to ameliorate HF progression through the direct stimulation of mitochondrial function. The molecular mechanisms that regulate mitochondrial function are gradually being uncovered but the critical link between mitochondrial morphology and mitochondrial function is poorly understood. Mitofusin (Mfn) 1 and 2 are two recently discovered mitochondria-shaping proteins that are found on the outer mitochondrial membrane. Research using a variety of experimental approaches shows that Mfn1 and Mfn2 are major regulators of mitochondrial architecture. Interestingly, Mfn1 and Mfn2 are robustly expressed in heart but so far there are no reports that describe their specific role in this tissue. In the proposed studies, we hypothesize that Mfn1 and Mfn2 are produced by the cardiac myocytes where they perform crucial roles in the maintenance of proper architecture and function of the mitochondrial compartment and that their perturbation will have profound and detrimental effects on heart performance. To address these possibilities we will generate Cardiomyocyte-specific Knockout mice for Mfn1 and Mfn2 (CKOMfn1, CKOMfn2). These mice will be used to assign the physiological function of Mfn1 and Mfn2 in the intact heart. Adult myocytes will be isolated from CKOMfn1, CKOMfn2 and control hearts to assess contractility and calcium handling. Adult myocytes from these strains will also be used to rigorously assess mitochondrial dynamics and function. Collectively, the proposed study will assess for the first time the importance of Mfn1 and Mfn2 in cardiac myocytes using powerful mouse genetic reagents. These experiments will provide a comprehensive top-down analysis of mitofusin function in the heart linking the intact heart phenotype with isolated cardiac myocyte function and mitochondrial function and dynamics. The outcome of this research will provide important information about mitochondrial fusion in the myocardium. PUBLIC HEALTH RELEVANCE In heart, organelles referred to as mitochondria have critical roles in energy production, calcium homeostasis and the generation of reactive oxygen species. It is now recognized that mitochondria are dynamic structures that undergo continuous fission and fusion. To understand the role of mitochondrial dynamics in heart function we will construct and analyze mouse models that lack proteins required for mitochondrial fusion.

DESCRIPTION (provided by applicant): Despite the prevalence and socioeconomic costs associated with conditions of muscle wasting, few treatment strategies for muscle atrophy have been identified. The Akt signaling pathway profoundly influences muscle growth, degradation and survival. In older animals, the activation of Akt and its downstream effectors is impaired in response to anabolic stimuli. We propose the direct activation of Akt may bypass age-related alterations in muscle that mediate this effect. Furthermore, we propose that proteins secreted from muscle participate in inter-tissue communication and that these regulatory mechanisms are diminished in elderly organisms where muscle mass is lost. Therefore, the proposed studies will test the hypothesis that acute genetic activation of Akt signaling in older (24 month) and middle-aged (12 month) mice will reverse, at least in part, age-associated decreases in strength, activity and metabolism to levels observed in young (4 month) mice. Related experiments will test whether the loss of Akt1 or Akt2 signaling in knockout mice will exacerbate the aging muscle phenotype in middle-aged and older mice. Other experiments will test the hypothesis that the age-dependent decline in the production of the muscle secreted protein follistatin-like 1 (Fstl1), i.e. a myokine, will contribute to the age-dependent abnormalities in muscle function and regeneration. This aim will examine the Akt-, injury- and age-dependent regulation of Fstl1 in muscle and characterize genetic gain- and loss-of- function models to understand the role of Fstl1 in basal muscle phenotype and regeneration in injury models.

This Partnerships for Innovation (PFI) project is a Type III (A:C) partnership between Arizona State University (ASU), an NSF PFI graduate 0090559, and two institutions that are new to the PFI Program (defined as ones that have never been PFI grantees), San Diego State University and Cleveland State University. Building upon a previous housing partnership effort, the project seeks to develop sustainability metrics related to construction operations which can be used by construction companies throughout the nation and beyond. While the transformation focused upon sustainability in the built environment has certainly touched the construction industry; paradoxically, the construction industry itself has been left out of efforts to encourage green practices. Metrics for construction operations do not currently exist in any form and construction companies are left to measure their sustainability in their own way. For the most part, they do no measuring at all. Through discovery and applied research, this project will provide a base upon which sustainable construction operations can be built, measured, weighed, and improved, thereby stimulating significant new research and activity.

Improvements in the environmental performance of construction operations are high on the agenda of many national and global organizations. The long-term goal of this project is to provide the construction industry with a standard protocol that will enable measurement and improvement of the environmental performance of onsite construction processes. Conserving material and energy resources will engender economic benefits to the U.S. The project will also contribute toward a fundamental understanding of just how our efforts to build the things we build affect the environment in which we live. Partnership products will enable participation in the discovery process for education groups at all levels, K-12 through university, and for industry groups.

Partners at the inception of the project are Academic Institutions: Arizona State University (lead institution), San Diego State University, and Cleveland State University; Private Sector Companies: Pulte Building Systems, SCP Concrete, LLC, DR Wastchak, LLC, and Baker Concrete; and Professional and Trade Organizations: American Concrete Institute (including, specifically, the Concrete Research Council, the ACI Committee 130 Sustainability in Concrete Construction, and the Strategic Development Council), American Coal Ash Association, American Concrete Pavement Association, American Society of Concrete Contractors, National Concrete Pavement Technology Center, and National Ready Mixed Concrete Association.

DESCRIPTION (provided by applicant): It is becoming increasingly appreciated that adipose tissue macrophage activation plays an important role in the development of chronic inflammation and metabolic dysfunction associated with obesity. The status of the vascular cells in adipose tissue has also received recent attention because it can contribute to fat pad dysfunction. Obese organisms display capillary rarefaction and diminished perfusion in their fat pads. Adipose tissue hypoxia is thought promote inflammation because this stress will favor adipocyte necrosis and lead to macrophage recruitment. In turn, the fat pad milieu of an obese organism will favor macrophage activation and lead to further degradation of the vascular bed. Thus, it is reasonable to speculate that obesity favors a vicious cycle of fat pad hypoxia and inflammation in the fat pad. Adiponectin is a fat-derived cytokine that has both anti-inflammatory and pro-angiogenic activities. Adipose tissues from lean organisms express high levels of adiponectin and there is a progressive decline in adiponectin expression as fat mass increases. Both inflammatory cytokines and hypoxia will lead to reductions in adiponectin expression by adipocytes. Published work from my lab has shown that adiponectin will promote endothelial cell function and angiogenesis in a variety of ischemia models, but the role of adiponectin in fat pad vascularization has never been examined. Furthermore, while adiponectin has recognized anti- inflammatory properties, its effect on adipose tissue macrophage polarization has never been systematically delineated. In this proposal, we will perform a series of experiments to investigate the role of adiponectin in fat pad biology to define the functional interrelationship between inflammation and hypoxia in obesity. The proposed experiments will test the hypotheses that adiponectin functions as a direct regulator of macrophage phenotype, favoring anti- inflammatory, M2-like polarization, and that it promotes fat pad perfusion. We propose that these activities of adiponectin control the microenvironment of the fat pad, and thereby influence systemic metabolism and cardiovascular function. Furthermore, gene ablation experiments in vitro and in mouse genetic models will be performed to determine the identities of the receptors that confer adiponectin's actions on fat pad perfusion and inflammation. PUBLIC HEALTH RELEVANCE: Clinical studies have documented that obese individuals differ markedly in their abilities to cope with excess fat tissue or develop a chronic low-grade inflammatory state and insulin resistance. This research will examine how the microenvironment of the fat pad, determined by the status of the vascular bed and composition of inflammatory cells, influences the overall metabolic health of the organism.

It is becoming increasingly appreciated that adipose tissue macrophage activation plays an important role in the development of chronic inflammation and metabolic dysfunction associated with obesity. Obese organisms also display capillary rarefaction and diminished perfusion in their fat pads. Adipose tissue hypoxia is thought to be linked to inflammation because this stress will favor adipocyte necrosis and lead to macrophage recruitment. In turn, the fat pad milieu of an obese organism will favor macrophage activation and lead to further degradation of the vascular bed. Thus, it is reasonable to speculate that obesity favors a vicious cycle of hypoxia and inflammation in adipose tissue. Adiponectin is a fat-derived cytokine that has both anti-inflammatory and pro-angiogenic activities. Adipose tissues from lean organisms express high levels of adiponectin and there is a progressive decline in adiponectin expression as fat mass increases. Both infiammatory cytokines and hypoxia will lead to reductions in adiponectin expression by adipocytes. Published work from my lab has shown that adiponectin will promote endothelial cell function and angiogenesis in a variety of ischemia models, but the role of adiponecfin in fat pad vascularization has never been examined. Furthermore, while adiponectin has recognized anti-inflammatory properties, its effect on adipose tissue macrophage polarization has never been systematically delineated. In this proposal, we will perform a series of experiments to investigate the role of adiponectin in fat pad biology to define the functional interrelationship between inflammation and hypoxia in obesity. The proposed experiments will test the hypotheses that adiponectin functions as a direct regulator of macrophage phenotype, favoring anti-inflammatory, M2-like polarization, and that it promotes fat pad perfusion. We propose that these activities of adiponectin control the microenvironment of the fat pad, and thereby infiuence systemic metabolism and cardiovascular function. Furthermore, gene ablation experiments in vitro and in mouse genetic models will be performed to determine the identifies of the receptors that confer adiponectin[unreadable]s actions on fat pad perfusion and inflammation.

A. Specific Aims The Administrative Core (AC) will provide support for planning, execution, and evaluation of the proposed research. The AC will support all administrative activities relating to the five projects and high throughput core enabling cohesion among the projects.

DESCRIPTION (provided by applicant): It is widely recognized that the endothelium is affected by the metabolic state of the organism, and endothelial function can also influence systemic metabolism. This program project renewal application brings together five productive project leaders who have a long history of collaboration to investigate mechanisms that underlie how the endothelium is both affected by and contributes to metabolic homeostasis. Project 1 will examine the mechanism by which endothelial cells switch from aerobic respiration to anaerobic glycolysis under conditions that stimulate vascular growth (i.e. hypoxia and pseudo-hypoxia) by focusing on a HIF1a-regulated microRNA that regulates the expression of mitochondrial respiratory complex proteins. Project 2 will examine mechanisms of redox regulation of cell signaling in vascular function and how these processes are perturbed by endothelial cell exposure to oxidants and reactive lipids that are associated with inflammation and metabolic disease. Project 3 will examine the functional interplay between endothelial function and inflammation in adipose tissue and assess how these processes influence systemic metabolism by focusing on mouse models that over- and under-express the adipocyte-derived cytokine adiponectin. Project 4 will also examine the interrelationship between the endothelium and inflammation in fat by measuring microvascular function and inflammatory markers in the fat of obese individuals before and after extensive weight loss resulting from bariatric surgery. Project 5 will examine the role of mitochondrial homeostasis in endothelial and inflammatory cells isolated from patients with Type 2 diabetes mellitus. This conceptually cohesive program focuses on an under-explored, yet clinically important, area of endothelial cell biology. With these proposed studies, we hope develop a better understanding of how endothelium functions at the interface of cardiovascular disease and metabolic dysfunction.

For the individual projects, gene expression analyses will be provided for specific cellular, animal, and clinical phenotypes. Importantly, this core lab will develop custom targeted gene expression and miRNA arrays that will provide the capacity to examine and directly compare related gene sets involved in endothelial function, mitochondrial biogenesis and function, reactive oxygen species, adipocytes, macrophages and hypoxia/angiogenesis. The Gene Expression Core has previously collaborated or published with Drs. Gokce and Vita and has begun studying miRNA and high-throughput gene expression with Drs. Cohen and Walsh. The Gene Expression Core Laboratory is an established facility with automated robotic pipeting using computerized programs, automated RNA isolation, storage, and custom chip capacity. The Laboratory is already involved in high-throughput analysis of over 10,000 subjects' gene expression, miRNA, and protein samples from various clinical projects. The laboratory has also assisted investigators in gene expression analysis from small volume tissue using murine and cell culture models. Importantly, there will be developed an established panel of genes and miRNA that will be compared across studies to determine the mechanistic overlap between specific inflammatory states and metabolic diseases and the role individual cell types play in endothelial and vascular disease (see Table 1).

It is becoming increasingly appreciated that adipose tissue macrophage activation plays an important role in the development of chronic inflammation and metabolic dysfunction associated with obesity. Obese organisms also display capillary rarefaction and diminished perfusion in their fat pads. Adipose tissue hypoxia is thought to be linked to inflammation because this stress will favor adipocyte necrosis and lead to macrophage recruitment. In turn, the fat pad milieu of an obese organism will favor macrophage activation and lead to further degradation of the vascular bed. Thus, it is reasonable to speculate that obesity favors a vicious cycle of hypoxia and inflammation in adipose tissue. Adiponectin is a fat-derived cytokine that has both anti-inflammatory and pro-angiogenic activities. Adipose tissues from lean organisms express high levels of adiponectin and there is a progressive decline in adiponectin expression as fat mass increases. Both infiammatory cytokines and hypoxia will lead to reductions in adiponectin expression by adipocytes. Published work from my lab has shown that adiponectin will promote endothelial cell function and angiogenesis in a variety of ischemia models, but the role of adiponecfin in fat pad vascularization has never been examined. Furthermore, while adiponectin has recognized anti-inflammatory properties, its effect on adipose tissue macrophage polarization has never been systematically delineated. In this proposal, we will perform a series of experiments to investigate the role of adiponectin in fat pad biology to define the functional interrelationship between inflammation and hypoxia in obesity. The proposed experiments will test the hypotheses that adiponectin functions as a direct regulator of macrophage phenotype, favoring anti-inflammatory, M2-like polarization, and that it promotes fat pad perfusion. We propose that these activities of adiponectin control the microenvironment of the fat pad, and thereby infiuence systemic metabolism and cardiovascular function. Furthermore, gene ablation experiments in vitro and in mouse genetic models will be performed to determine the identifies of the receptors that confer adiponectin¿s actions on fat pad perfusion and inflammation.

DESCRIPTION (provided by applicant): Mitochondria are the central regulators of myocardial metabolism and are responsible for maintaining metabolic homeostasis across a wide range of cardiac workloads. The molecular mechanisms that regulate mitochondrial function are gradually being uncovered, but the critical link between mitochondrial morphology and oxidative capacity is unknown. Mitofusin (Mfn) 1 and 2 are two recently-discovered mitochondrial-shaping proteins that are found on the outer membrane and are major regulators of mitochondrial architecture. Recent evidence from our laboratory suggests that cardiac myocyte-specific ablation of both Mfn1 and Mfn2 leads to a greater number of fragmented mitochondria, left ventricular remodeling and systolic dysfunction, and increased mortality during the transition from fetal to post-natal life. Concomitant with changes in mitochondrial and cardiac morphology was decreased expression of nuclear and mitochondrial transcription factors which collectively play a critical role in mitochondrial biogenesis. These findings suggest that mitofusins likely participate in the heart's adaptive metabolic response to increased energetic demand. To address this possibility, we will, for the first time, examine the role of mitofusins in the development of pathological and physiological cardiac hypertrophy and characterize the differential effects of Mfn1 and/or Mfn2 ablation in the adult cardiac myocyte. We will employ an extensive mouse genetic toolkit containing mitofusin conditional single and double knockouts, as well as mice with three of four mitofusin alleles deleted (monoallelics). These mice will be used to investigate mitofusins in post-natal cardiac growth and to explore mechanisms by which mitochondrial morphology affects mitochondrial content and respiratory function. These experiments will provide a comprehensive top-down analysis of mitofusin function in the adult mammalian myocardium, linking the intact heart phenotype, isolated cardiac myocyte physiology, and mitochondrial respiration and dynamics.

DESCRIPTION (provided by applicant): A growing body of evidence shows that the heart secretes factors to maintain its performance and coordinate cellular activities in response to stress/ or injury. Collectively, we refer to these cardiac-secreted factors as cardiokines. The identification and study of cardiokines is significant because it will provide information about intertissue communication within the heart, and these secreted factors may serve useful therapeutic or diagnostic functions. Via high throughput transcript analyses, we identified the secreted glycoprotein Follistatin-like 1 (Fstl1), also referred to as TSC36 and FRP, as a novel cardiokine. We demonstrated that Fstl1 is markedly upregulated by cardiac stress including ischemia-reperfusion injury, pressure overload and permanent left anterior descending coronary artery (LAD) ligation. We recently showed that acute overexpression of Fstl1 will protect the heart from ischemia-reperfusion injury and will promote revascularization of ischemic hind limbs in murine models. However, the functional role of Fstl1 in post- myocardial infarction (MI) cardiac remodeling is unknown, nor is it known how Fstl1 production by different cell types in the heart influence the remodeling process. In collaborative efforts with clinical laboratories we have found that Fstl1 can be detected in human serum, and these levels are prognostic for clinical outcomes in patients with either acute coronary syndrome or heart failure. Our pilot work in experimental models show that Fstl1 induction occurs both in cardiac myocytes and macrophages recruited to the infarct, suggesting that Fstl1 is involved in cardiac myocyte-macrophage crosstalk that coordinates the wound healing response in heart failure. Here we will evaluate the relative contributions of myocyte- versus macrophage-derived Fstl1 in post-MI remodeling. We will also explore the regulatory mechanisms and significance of Fstl1-mediated GDF15 regulation, whose importance is underscored by clinical data showing that these two factors participate in a biomarker network. Finally, we will examine the role of Dip2a, a recentl identified Fstl1 receptor molecule, in mediating the cardioprotective actions of Fstl1 on the heart To assess the role of Fstl1 in these processes, we will employ novel conditional knockout and overexpression models that have been created for these studies.

DESCRIPTION (provided by applicant): Obesity has emerged as one of the most critical health care problems in the US as 69% of the US population is currently overweight or obese. Adipose tissue dysfunction is an essential hallmark linking obesity to the pathogenesis of cardiometabolic disease, and prior work form our group has demonstrated that qualitative properties of adipose tissue shape systemic phenotypes. In particular, impaired adipose tissue angiogenesis in obesity has been associated with inflammation and metabolic dysfunction; however pathogenic mechanisms are incompletely understood. We describe a novel endogenous isoform of vascular endothelial growth factor (VEGF-A), VEGF-A165b that is selectively over-expressed in obesity and inhibits angiogenesis. Our preliminary data suggest that perturbations in the Wnt5a signaling system up-regulates VEGF-A165b under conditions of obesity that is modified by bariatric surgical weight loss. In aim 1, we will examine adipose depot-specific microvascular angiogenic responses in biopsied fat samples from 150 obese and 50 lean subjects. We will characterize VEGF-A isoforms in relation to angiogenic capacity and vascularization. We hypothesize that inhibitory isoform VEGF-A165b will be up- regulated in obesity, associated with anti-angiogenic actions in fat, and relate to whole body metabolic dysfunction. In aim 2, specific inhibitors of Wnt signaling will be employed using human adipose tissue samples secured from aim 1 to provide a molecular framework for understanding the regulation of VEGF-A 165b expression. In aim 3, we will re-examine adipose angiogenic capacity and VEGF isoform expression at 6 months following bariatric surgery in the same 150 obese subjects from aim 1. We will test whether relevant molecular pathways specifically identified in aim 2 are influenced by weight reduction. We seek to identify novel determinants of adipose tissue biology and angiogenesis in relation to metabolic changes which will develop in association with marked weight loss in obese individuals. Our proposal may identify the Wnt5a-VEGF-A 165b axis as a novel modulator of angiogenesis, adipose tissue biology, and consequently, systemic disease in clinical obesity and potentially lead to the identification of new therapeutic targets.

SUMMARY Non-alcoholic fatty liver disease (NAFLD) is a liver condition characterized by hepatic fat accumulation (hepatic steatosis) in the absence of excessive alcohol consumption. This disorder represents the most common form of chronic liver disease and is associated with obesity, aging and diabetes. An increasing body of evidence suggests a strong connection between reduced skeletal muscle mass/function and NAFLD, but the underlying mechanisms remain unknown. It has been suggested that skeletal muscle mediates some of the systemic benefits of physical exercise through the secretion of multiple bioactive proteins called myokines. Follistatin- like-1 (Fstl1) is an emerging myokine that is upregulated in a mouse model that mimics strength training- induced muscle growth, and has been shown to be upregulated in humans by either aerobic or strength training. Nothing is known about the potential metabolic actions of this myokine. The present project will test the hypothesis that skeletal muscle-derived Fstl1 exerts protective metabolic actions in the liver, preventing obesity-induced hepatic lipid accumulation and associated insulin resistance. This project has two specific aims: 1. To examine the effects of skeletal muscle-derived Fstl1 in the development of hepatic steatosis and insulin resistance in obese mice; 2. To investigate the role of AMPK signaling in the protective effects of Fstl1 against hepatic steatosis

? DESCRIPTION (provided by applicant): Obesity is the major worldwide epidemic of the 21st century. Cardiovascular disease (CVD) is the predominant cause of mortality in obese individuals. However, the mechanisms that link adipose tissue dysfunction to CVD remain incompletely understood. A growing body of evidence shows that adipose tissue secretes bioactive molecules called adipokines, and that obesity contributes to CVD due to unbalanced adipokine secretion, creating a chronic low-grade inflammatory state. Notably, we and others have shown that experimental manipulations of adipokine levels can have marked effects on cardiovascular disease processes in mouse genetic models fed a normal chow diet, documenting that changes in adipokine levels are sufficient to confer changes in cardiovascular function independent of its confounding metabolic actions. Our laboratory has identified Sfrp5 as a new anti-inflammatory adipokine, which antagonizes the pro-inflammatory activity of Wnt5a, a regulator of non-canonical Wnt signaling. While these studies showed that the Sfrp5/Wnt5a axis modulates inflammation in the microenvironment of adipose tissue and the peripheral vascular compartment, its actions in the heart remain unexplored. Here, we propose to investigate the role of the non-canonical Wnt5a regulatory system in the development post-myocardial infarction (post-MI) remodeling. We hypothesize that the aberrant regulation of Sfrp5/Wnt5a in the obese state is a highly significant, but previously unrecognized, mechanism by which metabolic dysfunction promotes ischemic heart disease.

? DESCRIPTION (provided by applicant): AAAs are age-associated localized dilations of the abdominal aorta that expand over the years and frequently lead to aortic rupture with a mortality rate as high as 90 %. Importantly, AAAs are one of the few cardiovascular disorders for which there is no pharmacological therapy. The only available treatment is surgical repair of the aortic wall, an expensive and risky procedure that is not a valid option for patients with small AAAs. Thus, the lack of a drug that prevents or slows down AAA growth represents an important unmet clinical need, which highlights the need for a precise knowledge of the molecular mechanisms underlying this vascular condition. It is widely accepted that the primary events in AAA development involve inflammation and proteolytic degradation of extracellular matrix in the vessel wall. We recently reported the existence of a new signaling axis that involves non-canonical Wnt signaling modulators and controls the inflammatory response in the adipose tissue micro-environment. Specifically, we identified Sfrp5 as a new anti-inflammatory adipokine, which antagonizes the pro-inflammatory activity of Wnt5a, a regulator of non-canonical Wnt signaling. Our recently published studies have combined the analysis of human fat specimens and the characterization of novel genetic mouse models to show that Sfrp5/Wnt5a signaling is a regulator of adipose tissue inflammation and systemic metabolic health. However, the endocrine actions of Sfrp5 and the direct contribution of this non- canonical Wnt signaling axis to inflammatory conditions independent of obesity remain to be demonstrated. In this regard, the expression patterns of Sfrp5 and Wnt5a suggest an important role of these molecules in vascular inflammatory disorders in general and AAAs in particular. Sfrp5 is highly expressed in adipose tissue, but severely downregulated by aging (as well as obesity). Conversely, Wnt5a is expressed at high levels in the abdominal aorta and markedly upregulated in human and mouse AAAs. Based on these findings, we propose to test the hypothesis that the aberrant regulation of the Sfrp5/Wnt5a axis represents a previously unrecognized insult to the aortic wall that contributes to vascular inflammation and AAA development. Additionally, given the urgent need for drugs that prevent AAA growth and rupture, we also aim to evaluate whether Wnt5a-inhibitory strategies are effective at preventing the growth of pre-established experimental AAAs. The specific aims of this project are: 1. To investigate the role of the Sfrp5/Wnt5a regulatory axis in experimental abdominal aortic aneurysm formation. 2. To investigate the role of the Wnt5a co-receptors Ror1 and Ror2 in experimental abdominal aortic aneurysm formation. 3. To evaluate the therapeutic potential of Wnt5a-inhibitory strategies in the setting of experimental abdominal aortic aneurysms

? DESCRIPTION (provided by applicant): Recent studies from our laboratory and others suggest the existence of a complex cross- regulation of energy substrate metabolism between heart and periphery, mediated by peptides/proteins that add to insulin, glucagon or other classical regulators. Among the potential candidate mediators, particular attention deserve muscle-released, hormone-like factors collectively named myokines. One of the emerging members is follistatin-like protein 1 (Fstl1), which is produced also by the heart, as first showed by the Walsh laboratory, and can be therefore ascribed to the myokine subgroup named cardiokines. High circulating Fstl1 is a reliable marker of heart failure (HF), while, if acutely overexpressed, Fstl1 can protect the heart against ischemia-reperfusion injury. However, while the molecular biology and even the therapeutic potential of Fstl1 are being progressively elucidated and defined, virtually nothing is known yet about the integrative physiology and pathophysiology of this hormone. In particular, no studies have explored the potential role of Fstl1 as a metabolic modulator. Preliminary studies from Recchia and Walsh labs indicate that Fstl1 overexpression or infusion alters cardiac and systemic metabolism in mice and dogs. Therefore the present project will test the hypothesis that the relative fractions of Fstl1 produce by heart and skeletal muscle vary in response to physiological and pathological conditions and contribute proportionally to the autocrine and the reciprocal endocrine regulation of cardiac and systemic energy metabolism. Three specific aims will be pursued combining studies in dog and mouse models. Specific aim 1 will determine overall turnover and relative rate of Fstl1 production by cardiac and skeletal muscle at baseline and in response to exercise or to moderate and severe HF. These studies will be performed in chronically instrumented dogs under resting conditions and during exercise or the development of tachypacing-induced HF. Specific aim 2 will test whether Fstl1 modulates cardiac and systemic oxygen and energy substrate consumption under physiological conditions and in HF. These studies will be performed in mouse models of cardiac or skeletal muscle- selective Fstl1 loss of function and in chronically instrumented dogs. The underlying molecular changes and mechanisms will be determined in tissue samples, ex vivo, as well as in cultured cardiomyocytes and skeletal myofibers exposed to Fstl1. Specific aim 3 will test whether sustained, selective enhancement of cardiac Fstl1 synthesis can attenuate myocardial and/or systemic metabolic alterations and exert therapeutic effects in HF. These studies will be performed in cardio-specific Fstl1 knockout mice subjected to aortic constriction and in dogs with tachypacing-induced HF and subjected to cardiac gene transfer of Fstl1 carried by adeno- associated viral vectors. Combined studies in mice and dogs may lead to the translation of novel findings into a new biological therapy for HF.

PI: Koley, Goutam
Proposal Number: 1606882

The goal is to develop bio-implantable sensors utilizing the unique material properties of graphene and boron nitride, which would offer much higher sensitivity for detecting concentration of various ions in blood and tissue in real time, enabling prediction of the onset of disease states before their clinical manifestation. The project activities would integrate research with education and training of high school, undergraduate and graduate students, with significant minority participation.

The overall goal of the proposed research is to develop novel graphene-based flexible ion-sensitive field effect transistor (ISFET) sensors for the measurement of K+, Ca2+ and Na+ ion concentrations and correlated ion fluxes in cardiomyocytes and glial cells, with the overall objective of developing a new approach for assaying cell membrane ion transport in primary cell culture. The detection of the aforementioned ions is clinically significant as they serve as important bio-markers for onset of myocardial ischemia and epilepsy. The proposed graphene ISFET sensor array will enable critical understanding of K+, Ca2+ and Na+ membrane transport in glial cells and cardiomyocytes. The approach of ISFET development utilizing the novel properties of boron nitride and graphene addresses several critical issues including: (i) the complicacy, low throughput and cell invasiveness issues of patch clamp and microelectrode array based techniques, and (ii) the low sensitivity and degradation of commercially available Si-based ISFETs. The experimental plan includes growing cells directly on graphene and performing continuous, real-time and label-free measurements of ion fluxes in cardiomyocytes and glial cells. The measurements acquired are expected to provide electrophysiological properties of primary cells in culture, especially regulatory and drug-induced changes in their electrical properties. Successful completion would result in the development of useful ISFET sensors for in vivo detection of the onset of myocardial ischemia and epilepsy. As part of the educational and outreach activities, the PIs would each involve at least one undergraduate and one high school student to work on this project every year focusing strongly on recruiting minority students. The PIs would also integrate research results in a graduate course, and disseminate them through conference participation as well as research websites.
 

Project Summary/Abstract: In this S10 proposal, we request funds for a QUANTSTUDIO 12K FLEX OPEN ARRAY REAL-TIME PCR SYSTEM manufactured by Thermo/Life Science/ABI that will enable BMC and Boston University users to carry out studies involving gene expression analysis, genotyping, microRNA profiling, and other RNA research projects. This innovative and versatile system, will replace the old and obsolete RT-PCR cyclers within the Analytical Instrumentation Core (AIC) that are no longer being supported by the manufacturer. Over the past eight years, more than 50 principal investigators and their laboratories have used the AIC's qPCR instruments. This new system will not only allow researchers to continue their on-going studies, but it will also create opportunities for new and original projects. In addition, having the latest software analysis tools and the additional capabilities of the new instrument will further promote the diversity and efficacy of nucleic acid research. It will allow the users to perform a wider-range of assays using different detection platforms at both the micro and nano- scales much more efficiently and will enhance data processing. Dr. Kenneth Walsh is the Principal Director and Principal Investigator (PD/PI) for this team project and is a leading scientist in the field of cardiovascular disease. His laboratory examines the molecular events that promote cardiac cell muscle growth, starting from differentiation and ending with cell death. Dr. Bryant will continue to use the AIC to determine the contribution of natural genetic variation on the mechanisms that confer susceptibility vs. resilience in substance abuse addictions. Dr. Myers will take advantage of the new qPCR system by conducting miRNA profiling to further examine the role of genes and gene regulation in both Huntington's (HD) and Parkinson's Diseases (PD). The above three scientists composed the Major User Group. The new instrument will enable other BU investigators (both major and minor users) to examine the molecular mechanisms involved in various areas of research that include: metabolic heart disease (Dr. Colucci), Alzheimer's Disease (AD) and Amyotrophic Lateral Sclerosis (ALS) (Dr. Wolozin and Dr. Qiu), diabetes (Dr. Ruderman and Dr. Rameh-Plant), ischemic limb vascularization (Dr. Matsui), obesity (Dr. Jing), calcium signaling (Dr. Bolotina), breast cancer and melanoma (Dr. Thiagalingam and Dr. Cui), dental disease (Dr. Gibson), sexually transmitted diseases (STDs) (Dr. Wetzler), virology (Dr. Connor) and hepatic disease (Dr. Bachschmid). The new instrument will also be used by AIC personnel to continue being able to provide the ?Gene Array Service? for all members of the BU research community. The new instrument will be housed within the AIC's qRT-PCR facility which is located on the 2nd floor at 670 Albany Street. The AIC has extensive experience when it comes to maintaining instruments and ensures that they are in excellent working condition with very little down time. Finally, the AIC has a long track record of having excellent equipment usage when it comes to RNA studies.  

ABSTRACT The accumulation of somatic DNA mutations over time is a hallmark of aging in many tissues. However, the causal role of somatic mutations in age-associated disorders other than cancer is a matter of debate, and remains unexplored in the setting of cardiovascular disease (CVD), the leading cause of death in elderly individuals. Recent large exome sequencing studies in humans have shown that aging is inevitably associated with an increased frequency of somatic mutations in the hematopoietic system, which provide a competitive growth advantage to the mutant cell and thus allow its clonal expansion (clonal hematopoiesis). Unexpectedly, these somatic mutations were associated with a higher rate of cardiovascular-related deaths, suggesting a previously unrecognized link between somatic mutations in bone marrow-derived cells and CVD . However, whether there is a causal connection between these somatic mutations and CVD remains unclear and the potential underlying mechanisms are completely unknown, and this is the scientific premise of the proposed research.

SUMMARY The accumulation of somatic DNA mutations over time is a hallmark of aging in many tissues. However, the causal role of somatic mutations in age-associated disorders other than cancer is a matter of debate, and remains unexplored in the setting of cardiovascular disease (CVD), the leading cause of death in elderly individuals. Recent large exome sequencing studies in humans have shown that aging is inevitably associated with an increased frequency of somatic mutations in the hematopoietic system, which provide a competitive growth advantage to the mutant cell and thus allow its clonal expansion (clonal hematopoiesis). Unexpectedly, these somatic mutations were associated with a higher rate of cardiovascular-related deaths, suggesting a previously unrecognized link between somatic mutations in bone marrow-derived cells and CVD. Recently, we reported that pre-cancerous driver mutations in Tet2 that occur in hematopoietic stem cells may be causally linked to cardiovascular disease. However, whether there is a causal connection between other clonal hematopoiesis genes and CVD remains unclear and the potential underlying mechanisms are completely unknown; and this is the scientific premise of the proposed research. Here, we will use a lentivirus/CRISPR gene editing approach to manipulate other hematopoietic stem cell driver genes and assess their impacts in a multi-faceted model of cardio-metabolic disease.

ABSTRACT The accumulation of somatic DNA mutations over time is a hallmark of aging in many tissues. However, the causal role of somatic mutations in age-associated disorders other than cancer is a matter of debate, and remains unexplored in the setting of metabolic disease. Recent large exome sequencing studies in humans have shown that aging is inevitably associated with an increased frequency of somatic mutations in the hematopoietic system, which provide a competitive growth advantage to the mutant cell and thus allow its clonal expansion (clonal hematopoiesis). Unexpectedly, these somatic mutations were associated with a higher rates of cardio-metabolic disease, suggesting a previously unrecognized link between somatic mutations in bone marrow-derived cells and these disease processes. However, whether there is a causal connection between these somatic mutations and metabolic dysfunction remains unclear and the potential underlying mechanisms are unknown, and this is the scientific premise of the proposed research.

Project Summary/Abstract The current proposal represents a combined clinical patient-oriented and experimental murine investigation which seeks to examine mechanisms of vascular dysfunction in obesity. Obesity has developed into one of our most critical health care problems as 69% of the US population is currently overweight or obese. Adipose tissue dysfunction, inflammation, and insulin resistance are essential hallmarks linking obesity to the pathogenesis of cardiovascular disease. Our preliminary data demonstrate marked up-regulation of a unique pro-inflammatory Wnt signaling pathway that may play a major role in mechanisms of vascular dysfunction in obesity. In this proposal, we will examine the role of Wnt signaling in the regulation of microvascular endothelial function in intact blood vessels and isolated endothelial cells acquired from living subjects. We will utilize a multidisciplinary approach and complementary expertise between clinical and basic scientists to characterize the pathophysiological role of dysfunctional Wnt5a signaling. In aim 1, we will characterize depot-specific mechanisms of vascular endothelial dysfunction in human adipose tissue arterioles using videomicroscopy of small vessels isolated from subcutaneous and visceral fat compartments during elective surgical procedures in 150 obese and 50 age- and gender-matched lean subjects. We will characterize vascular phenotypes in relation to Wnt signaling and test the hypothesis that over-activation of Wnt5a-mediated signaling is a dominant regulatory feature that leads to vascular dysfunction. In aims 2 and 3, specific pharmacological and biological inhibitors of the Wnt5a and TGF? pathways will be employed using arterioles and endothelial cells from aim 1 to test the hypothesis that antagonism of Wnt5a reverses vascular dysfunction, in part through its ability to modulate EndoMT in adipose tissue, and seek to identify novel regulators and therapeutic targets in obesity. To corroborate these findings in genetic models, we will explore EndoMT and the vascular and metabolic phenotypes of mice that are engineered to conditionally ablate or overexpress Wnt5a in myeloid cells. In aim 4, studies of endothelial phenotyping will be repeated 6-months after life-saving bariatric weight loss surgical intervention in the same 150 obese subjects from aim 1 to examine the effects of marked weight reduction on arteriolar responses and relevant Wnt molecular pathways identified in aims 2 and 3. The overall project combines studies of cellular signaling and whole vessel physiology using primary tissues from severely obese individuals where clinically very little vascular data currently exist. Our proposal may identify the Wnt5a-Sfrp5 axis as a novel modulator of vascular biology and potentially lead to the identification of new targets and approaches to combat obesity-induced cardiovascular disease.

SUMMARY COVID-19 disease has a diverse range of outcomes, and this individual-to-individual variability is poorly understood. Clonal hematopoiesis is a prevalent, age-associated condition that arises from the accumulation of various somatic mutations in hematopoietic cells and can lead to their clonal amplification. These mutant clones corrupt immune cell function and contribute to mortality and increased cardiovascular disease risk through cytokine dysregulation. The proposed research will investigate the hypothesis that clonal hematopoiesis is a hematologic host factor that predisposes persons to develop severe COVID-19 disease. Through a collaborative effort between Kenneth Walsh Ph.D. (UVA) and Christopher deFilippi M.D. (Inova) the proposed research will explore the possibility that clonal hematopoiesis-mediated alterations to the immune system are associated with clinical laboratory measures of a marked inflammatory response, biochemical evidence of cardiac injury and poor clinical outcomes in patients with COVID-19 infection. Patients will be consented and enrolled at the Inova hospital system in northern Virginia that delivers service to more than 2 million people per year in the Washington, D.C metro area with a large volume of hospitalized COVID-19 positive patients. Upon enrollment, biospecimens will be collected and banked. Clinical data will be extracted from the electronic medical record and stored in research form in Research Electronic Data Capture software. DNA will be sent to Dr. Walsh?s laboratory at UVA for analysis of clonal hematopoiesis. DNA from the Inova group will be processed at UVA to assess clonal hematopoiesis via targeted, error-corrected DNA sequencing. This analysis employs an enrichment panel to capture driver genes of interest and the construction of libraries with DNA barcodes. Following deep next generation DNA sequencing, a bioinformatic platform is employed to distinguish true variant calls from noise at a particular exonic location. These data on clonal hematopoiesis will then be shared with the team at Inova to test whether there are associations between somatic mutations, clinical outcome, and markers of inflammation and cardiac injury.

SUMMARY The accumulation of somatic DNA mutations in driver genes within the hematopoietic system can provide a fitness advantage to the mutant cell and thus allow for its clonal expansion. This process is referred to as clonal hematopoiesis and leads to a situation where a substantial fraction of an individual?s blood cells are replaced by clones with the driver gene mutation. Previous large exome sequencing studies in humans have shown that these somatic mutations accumulate during aging and are associated with a higher rate of cardiovascular-related deaths. Moreover, recent experimental studies support the idea that clonal hematopoiesis causally promotes cardiovascular disease (CVD). More recently, genotoxic stresses such as radiation or chemotherapy have also been shown to facilitate hematopoietic clonal expansion in cancer patients. Compared with age-related clonal hematopoiesis that is mediated primarily by mutations in epigenetic regulators such as DNMT3A and TET2, clonal hematopoiesis resulting from prior exposure to cytotoxic therapy is uniquely associated with high frequencies of mutations in TP53 and PPM1D. This clonal selection/expansion of TP53 and PPM1D mutant clones by cancer therapy may subsequently increase the risk of CVD, and addressing this clinically-relevant question represents the main objective of the current proposal. In fact, over the past decade, the number of cancer survivors has grown, thus there has been a paradigm shift in the approach to survivor care with a renewed focus on maximizing non-cancer-related outcomes, such as CVD. Therefore, there is an unmet need to understand the potential connection between cancer-therapy related clonal hematopoiesis and CVD. Thus, this study aims to examine whether a causal connection exists between therapy-related clonal hematopoiesis and chemotherapy-associated cardiomyopathy. As a proof-of-concept, I will test the hypothesis that TP53- and PPM1D-mediated clonal hematopoiesis contributes to anthracycline-induced cardiomyopathy using sophisticated animal models.