Stephen Young - US grants

During late fetal development, the lung must prepare for the transition to air breathing. Maintaining alveolar stability becomes vital for survival. Therapy with glucocorticoids to enhance lung maturity may have immediate and long term consequences for lung structure and function. This series of investigations is directed toward furthering our basic knowledge of lung secretory cell development. We will use structural and biochemical methods to test hypotheses of secretory cell function and to test the probabilities that glucocorticoids damage fetal and newborn lung structure. We intend to study biochemical and 3-dimensional morphologic changes during epithelial cell maturation in the last days of fetal development. We will test hypotheses of the assembly of lamellar bodies and assess the changes in type II cell maturation induced by glucocorticoids. This drug induced accelerated maturation is a relevant clinical concern as the effects are widespread within the lung and have not been shown to be entirely reversible during postnatal growth. A detailed 3-dimensional morphologic study of groups of alveolar septa and of terminal bronchiolar structure will be conducted at allow magnification level that will test the microanatomic basis for theories of cell-cell interactions.

As a cardiologist, I have been impressed with how frequently young patients are seen with coronary artery disease (CAD) but who lack traditional risk factors, such as elevated levels of low density lipoprotein (LDL). It is now clear that LDL particles are heterogeneous and differ in size, density, chemical composition and immunochemical properties. Work from this laboratory, and other, has demonstrated that such physical heterogeneity results in metabolic heterogeneity as well. Over the next five years I will determine if such heterogeneity is pathophysiologically relevant to the atherogenic process. My investigative work will take place in the productive environment of the La Jolla Atherosclerosis SCOR program, under the direction of my Sponsor, Dr. Josepth L. Witztum. My research will focus on several areas. First, I have documented that alterations in LDL occur in response to bile sequestrant therapy. Such therapy produces LDL particles that are smaller and more dense, consistent with the type of LDL particles found in many normocholesterolemic CAD patients. Since bile sequestrant therapy can produce such particles even in normals, a detailed study of the mechanisms, and the consequences of such changes will be made to determine if this is a model for similar alterations in LDL seen in CAD patients. We have also shown that probucol alters LDL so as to cause an increase in LDL receptor independent uptake of the altered particle. Understanding how probucol enhances clearance of LDL by LDL receptor independent mechanism is also highly relevant to understanding atherogenesis. Another major focus will be to define immunochemical heterogeneity of apo B. I have already defined one genetic polymorphism in apo B by use of a specific mouse monoclonal antibody. In addition I plan to develop new monoclonal antibodies against further allotypes of apo B by use of human-human hybridomas. This technique should facilitate the development of a panel of antibodies to define apo B genetic polymorphism. A detailed study will then be made of subjects with premature CAD to determine if allotypic patterns predict atherosclerosis prone LDL (as has been reported in an animal model). The definition of such apo B polymorphism should also be of considerable importance to those interested in the molecular biology of apo B as well.

An advanced interactive serial reconstruction system will be developed, and programs will be implemented for the derivation and visualization of data from thick section electron microscopy tilt series. This award will provide an initial basis for these efforts. The facilities of the San Diego Supercomputer Center will be utilized to implement and explore computer graphic methods for the three dimensional reconstruction and visualization of data represented as a series of planes or cross sections through a three dimensional medium. The computer software tools will be available not only for biologists but also for meteorologists, oceanographers and geologists who wish to explore three dimensional distributional aspects of data collected in the form of layers or strata. The programs are being implemented on an Ardent Titan and a Silicon Graphics IRIS workstation. The electron microscopic images are being analyzed to obtain substantial three dimensional information about the structure of biological specimens. The methods employ tomographic reconstruction of two dimensional images and exploit prior developments for registration of digitized tilt series and three dimensional energy distribution. The computer advances will be applied to three dimensional reconstruction of cellular images from both electron and light microscopy, to images of molecular and macromolecular assemblies, and to neuroanatomical mapping systems.

Extracellular matrix (ECM) molecules such as fibronectin (FN) and laminin (LN) are restricted in their spatial and temporal expression. They have important influences upon cellular proliferation, differentiation and mobility. Antibodies to LN will inhibit key lung developmental processes like branching morphogenesis. A more recently described ECM molecule, tenascin (TN), also has a restricted spatio-temporal expression in mammalian lung. It may modulate the binding of cells to ECM substrates like FN and it can induce fibroblast metalloproteinase genes such as collagenase. My hypothesis is that cell-extracellular matrix interactions regulate pulmonary branching morphogenesis and alveolarization; I propose that TN is an ECM molecule which has a major role as a regulator of lung development and that inhibition of TN expression may be important in the response of the developing lung to postnatal injury. Using in vitro and in vivo methods we will investigate two key periods of lung development: Aim 1. Branching morphogenesis a) Determine the spatio-temporal expression of tenascin, including its alternatively spliced forms, in lung by morphologic measures at light and electron microscopic resolutions. b) Measure lung TN gene and protein expression. c) Test the functional importance of TN upon branching morphogenesis with polyclonal anti-TN antiserum and with monoclonal domain-specific anti-TN antibodies and recombinant expression proteins. d) Test whether interactions of tenascin with laminin and fibronectin modify adherence and spreading of fetal lung fibroblasts and epithelial cells on plastic substrate. Aim 2. Alveolarization: We will use autoradiography to measure cell kinetics of the lung parenchyma. Alveolar septal cellular proliferation will be compared to the spatio- temporal expression of lung tenascin. Completion of these specific aims will extend our current work on cellular kinetics and ECM structural molecules of growing lung. Previously identified milestones of lung epithelial development will be correlated with epithelial-mesenchymal interactions to be studied with this support. Understanding of mechanisms leading to bronchopulmonary dysplasia will be advanced.

technology /technique development; microscopy; building /facility design /renovation; biotechnology; biomedical resource; biomedical equipment development; bioengineering /biomedical engineering;

We are developing a system to control the NCMIR Intermediate Voltage Electron Microscope (IVEM) remotely over the Internet and to easily distribute computationally intensive tasks such as those required for electron microscope tomography to high performance computers. A preliminary system was designed to test the feasibility of remote control and demonstrated at the SIGGRAPH and the Supercomputing conferences in 1992. As a result of the success of this demonstration and the interest and positive response from the NCRR we have been developing a more refined system. Our ultimate goal is to turn the IVEM and associated image processing and analysis software into a network resource available to researchers nationwide as part of the National Information Infrastructure. We expect that remote operation will increase access and use of the resource, facilitate more extensive data acquisition and analyses for collaborative projects, and allow more efficient use of the resource staff. To carry out this extensive project, we were encouraged by the NCRR to seek additional funding to support development of those aspects of the telemicroscopy project primarily related to remote operation and going beyond the development of enhancements for the local use of the microscope and associated computation. Accordingly, we submitted a proposal for a "Collaboratory for Microscopic Digital Anatomy (CMDA)" that was funded by the NSF as a National Challenge grant in September 1994. Mark Ellisman is PI and Sid Karin, Director of the San Diego Supercomputer Center (SDSC) and Don Greenberg, Director of Cornell University Program of Computer Graphics are Co-PIs. NCMIR and SDSC are primarily responsible for the overall system design of the CMDA including task management, network communication, enhancement of functions controlling the microscope, and the development of an improved user interface. Cornell is focusing on the development of advanced rendering methods that will in corporate error metrics. This NSF grant funds the major portion of the work on the telemicroscopy project. This funding provides additional benefits to the NCMIR beyond the develop of a remote control system since many of the CMDA activities, in particular those associated with microscope automation, are synergistic with the goals of the resource. In addition, the project supports and enhances a mutually beneficial interaction with the SDSC (and now the NPACI). As the result of a positive reviews by NSF site visit teams we were awarded an NSF Creativity Extension Award, adding two more years of support to the CMDA project which will carry it to September of 1999. An early version of the CMDA was demonstrated at the Supercomputing 95 conference in San Diego in December 1995 and is described in Young et al. (Int. J. Supercomputer Applications and High Performance Computing, 10: 170-181, 1996). The present CMDA (CMDA 1.0) incorporates routines developed by NCMIR to automate electron microscope focus and registration as well as the sequential operations required for acquisition of mosaics and tomographic tilt series. Initial testing with researchers at UC Berkeley, and the first intensive use began in March 1997 with collaborative researchers at the University of Oregon. Results of these tests have already indicated the value of remote interaction in conducting collaborative research. In addition, they have provided important feedback on the system leading to changes which are being incorporated into the next version, CMDA 2.0. The CMDA project has been recently recognized as a state-of-the-art next generation technology. NCMIR was i nvited to demonstrate the system at the Internet 2 conference held in October 1997 in Washington DC. The CMDA project has been recently selected as a finalist in the Next Generation category of the Global Information Infrastrucure competition, an industry-wide competition. The winner of this competition will be announced on April 20, 1998. After a second extensive design period focused on extending the functionality of the current system, the next version, CMDA II, is now undergoing testing and final development. As described in the CMDA II design document, the new CMDA II software architecture will incorporate numerous improvements into the current CMDA I system. These include the capability of collaboration involving an unlimited number of users, and enhanced system and data security. The CMDA II also incorporates image data compression for more rapid transmission of data acquired from the microsocpe during remote sessions. The new design incorporates a CORBA interface. This language and platform independent distributed processing interface will facilitate remote access to high performance computing for tomographic reconstruction, analysis, and visualization. In a related research project, partly supported by another NCRR-sponsored Resource grant to Sid Karin, the "National Biological Computing Resource" (NBCR), we have implemented both iterative and simple R-weighted tomographic reconstruction on the massively parallel 400 node Intel Paragon computer at the SDSC and have recently ported this program to the Cray T3E. We have also developed a remote interface these reconstruction platforms. A major emphasis in the current period is to bring on an increasing number of remote sites using the CMDA. To accomplish this, we have extended the initial design to incorporate web-based technology, including initial work with a web-based user interface using video images from the microscope to provide enhanced interactive control of the microscope to augment automated functions. The platform-independent web interface will enable use of the CMDA by a large number of researchers. As part of this design, we have written and are testing a data organization system, Gridset, based on the HDF file format. Tests of the web-based interactive microscope controller are currently underway with NCMIR collaborators in Oregon, Montana, and New York. Finally, during this year we have submitted a proposal to the NIH/NCRR call for supplementary grant to on collaboratory test-beds. This proposal will expand of the teleinstrumentation / collaboratory activities to include other NCRR resources and will benefit from resources provided by the newly established National Partnership for Advanced Computing Infrastructure.

The major goals for these software projects are to design, develop and implement programs for semiautomated segmentation and classification of 3D volumes, and to develop software for the visualization of three-dimensional structure. In the previous years of this project we have developed the program suites, Synu and Ducky, for performing sophisticated three-dimensional visualizations. These programs have been found to be extremely useful by biologists and have been distributed to many investigators throughout the US and internationally. During this year we have focused on improvements in software for segmentation and classification as well as programs for combining volume datasets for use in performing serial tomography and for merging volume correlated datasets acquired from the light and microscope (Perkins et al., 1997). Manual segmentation-tracing programs: In many light and electron microscopic studies, the objects of interest for three-dimensional reconstruction or analysis are delineated by membranes. These membranes are often extremely difficult to define using image processing techniques. Generally it has been found necessary to specify the location of the membranes using an interactive manual tracing method. The program XVOXTRACE developed at NCMIR is specifically designed for manually contouring features of structures in tomographic volumes by tracing with a mouse on planes of the volume along any one of the three major axes. Additional guides are provided to achieve accurate tracing. Thus, as the contours are traced, they can be viewed simultaneously superimposed on selected images from the original tilt series. Pairs of tilt-series images with the contours superimposed may also be displayed as anaglyphic or stereoscopic views. In addition, the accuracy of tracing can be evaluated by examining the contours superimposed on renderings of the volume viewed from an arbitrarily selected orientation. We have developing an improved version of this program which will include stereoscopic volume rendering as well as enhanced contour editing facilities such as automatic contour resplining. Capabilities for panning and zooming on a view of a selected region of the specimen in the volume slice, volume rendering, and tilt displays will also be provided. Semiautomated particle detection: Several on-going NCMIR research projects are interested in defining the three-dimensional distribution of particle-like structures such as synaptic vesicles. In collaboration with Dr. Ioana Martin (Dept. Computer Science, Univ. Indiana, South Bend) we have been developing semiautomated tool for detecting and defining particles. An initial tool was developed based on methods used by Dr. Martin to detect virus particles in two-dimensional electron micrographs. The tool was evaluated to detection of synaptic vesicles in tomographic reconstructions. Although partly successful, we expect that algorithms, currently under development, using three-dimensional information will be considerably more accurate. Measurements based on contours: XDEND is a graphics-based analysis tool developed at NCMIR to obtain measurements of the length, surface area and volume of objects defined as sets of contours such as those produced with XVOXTRACE. In addition, contours can also be derived directly from data volumes in those cases in which the structure can be segmented using simple built-in thresholding. Components of the structure may be resectioned into contours in the transverse, sagittal, or coronal axes to obtain the best orientation for length measurements. The length measurements are based on linear segments interconnecting the centroids of the contours. The program automatically detects branching segments. Objects may be viewed in perspective as contour outlines, centroids or skeletons. XDEND can also be used to reclassify and edit objects after recontouring. We are currently improving this program by providing resectioning along an arbitrary axis, and more accurate skeletonization methods. Software for serial tomography and volume merging We previously developed and implemented a procedure for combining serial section techniques with tomographic reconstruction (Soto et al., 1994). For a fixed tilt range and tilt increment, the resolution of the volume reconstructed by tomography decreases as the thickness of the object under reconstruction is increased. Thus, the resolution obtainable in reconstructions of very thick specimens is relatively poor. To overcome this limitation, we combined tomography with the serial section reconstruction technique to reconstruct large structures with adequate resolution. A series of consecutive sections is cut with a thickness sufficient to achieve the resolution desired in the reconstruction. Each section is reconstructed using tomography. Subsequently, the resulting volumes in the series are aligned and merged into a single volume. We recently developed two programs, MOG and TRANSMOG that are useful in the alignment of serial volumes. Fiducial marks are assigned to features in the raw or segmented sections from the consecutive volumes using the NCMIR fiducial marking tool, FIDO. MOG computes the polynomial function coefficients necessary to align the two sections, including a correction for in-plane warpage that may have occurred between the two volumes. The translations computed by MOG are then applied by TRANSMOG to the remaining sections in the volume. These have programs are also useful for aligning and scaling correlated three-dimensional light and electron microscope data and have been used for this purpose on a collaborative study examining alterations in cardiac muscle in a model of heart failure. Further, this software has also been found useful for assessing changes in structures due to shrinkage. The NCMIR program MOG can be used to obtain numeric estimates of changes in single sections resulting from beam exposure or specimen preparation.

DESCRIPTION (provided by applicant): The proteins of the nuclear lamina have generated enormous interest because of recent studies showing that mutations in the gene for lamin A/C (LMNA) develop a host of different diseases, including cardiomyopathy, muscular dystrophy, and partial lipodystrophy. The objectives of this proposal are to define the enzymes that are important in the posttranslational processing of the nuclear lamins and to understand the consequences of defective posttranslational processing at both the cellular and tissue levels. Prelamin A (664 amino acids) terminates with a "CAAX" sequence motif and undergoes a complicated series of posttranslational modifications. First, the cysteine (C) of the CAAX motif is farnesylated by protein farnesyltransferase. Second, the last three amino acids of the protein (i.e., the -AAX) are released by a prenylprotein-specific endoprotease (likely Rce1 or Zmpste24 or both). Third, the newly exposed farnesylcysteine is methylated by isoprenylcysteine carboxyl methyltransferase (Icmt), a membrane protein of the endoplasmic reticulum. Fourth, once the cell has gone to all of this effort, the carboxyl-terminal 15 residues of the protein (including the farnesylcysteine methyl ester) are clipped off and degraded, leaving mature lamin A (646 amino acids). Zmpste24 might carry out that final endoproteolytic-processing step. Lamin B1 and B2 have a CAAX sequence motif and undergo the first three processing steps, but do not undergo a second endoproteolysis step; thus, their sequences terminate with a methylated farnesylcysteine. During the past few years, the laboratory of Dr. Stephen Young has generated knockout alleles [as well as some conditional ("floxed") alleles] for many of the genes involved in CAAX protein processing (e.g., Fntb, Rce1, Zmpste24, and Icmt) for the purpose of analyzing the importance of the posttranslational processing steps. In mice lacking Zmpste24, the processing of prelamin A to lamin A was blocked. Of note, the Zmpste24-deficient mice exhibited reduced muscle strength (suggestive of a laminopathy), and also developed spontaneous bone fractures, a peculiar finding not generally observed in humans with lamin mutations. The first aim of this grant application is to define, biochemically, the precise role(s) of Zmpste24 in prelamin A processing. The second aim is to further define the cellular and tissue pathology of Zmpste24 mice and then to determine whether all of the pathologic findings are due to defective prelamin A processing. The third aim is to understand the posttranslational processing of lamin B1 and to define the consequences of lamin B1 deficiency in mammals.

DESCRIPTION (provided by applicant): Proteins that terminate with a "CAAX" sequence motif (e.g., the Ras proteins, Rho proteins, and nuclear lamins) undergo three sequential posttranslational modifications. First, the cysteine (C) is isoprenylated by enzymes in the cytosol (farnesyltransferase or geranylgeranyltransferase I). Second, the last three amino acids of the protein (i.e., the -AAX) are released by Rce1, an integral membrane endoprotease of the endoplasmic reticulum. Third, the newly exposed isoprenylcysteine is methylated by isoprenylcysteine carboxyl methyltransferase (Icmt), also an integral membrane protein of the endoplasmic reticulum. Each of these modifications is important for the targeting of CAAX proteins to membrane surfaces and for their proper function. For example, inhibitors of farnesyltransferase block the membrane targeting and downstream effects of mutationally activated Ras proteins and have shown promise in the treatment of certain cancers. A potential therapeutic drawback of the farnesyltransferase inhibitors is that K-Ras, the Ras isoform most commonly involved in human cancers, is geranylgeranylated by geranylgeranyltransferase I in the setting of farnesyltransferase inhibitors. The existence of this alternate prenylation pathway has raised interest in defining the physiologic effects of blocking the enzymes involved in the "post-isoprenylation" processing of CAAX proteins (Rce 1 and Icmt), as those enzymes act on both farnesylated and geranylgeranylated proteins. The goal of this project is to gain insights into the potential therapeutic utility of interfering with protein processing by Icmt and Reel, using cell culture systems and genetically modified mice. The effects of a deficiency in Fntb (the gene encoding the beta-subunit of protein farnesyltransferase) will also be assessed. Many useful reagents have already been generated for this project, including mice with knockout and conditional alleles of lcmt, Reel, and Ftnb. Preliminary studies have uncovered many exciting findings, including the fact that Icmt deficiency strikingly reduces oncogenic transformation of fibroblasts by K-Ras. Specific Aim 1 of this project is to assess the physiologic impact of Icmt deficiency, further defining its effects on Ras transforming activity. Specific Aim 2 is to generate cell culture and animal models for Fntb deficiency, and to compare the impacts of Fntb deficiency, Icmt deficiency, and Rcel deficiency. Specific Aim 3 is to determine if deficiencies in Icmt and Reel render cells more sensitive to farnesyltransferase inhibition. These studies should define the therapeutic potential of blocking the three different steps of CAAX protein processing.

BayGenomics has used gene-trap vectors to inactivate thousands of genes in mouse embryonic stem (ES) cells. More than 8000 ES cell lines with well-characterized insertional mutations have been generated and posted on our web site (http://baygenomics.ucsf.edu/). The ES cells have been made freely available to the scientific community, for the purpose of generating knockout mice. Thus far, we have responded to the requests for more than 930 different ES cell lines. The vast majority of our mutant ES cell lines yield germline-transmitting chimeras, and hundreds of knockout mice have been generated by our group and outside laboratories. Many of the knockout mice are highly relevant to cardiopulmonary disease and development. BayGenomics has also produced two widely used programs for analyzing microarray expression data according to biochemical pathways (GenMAPP and MAPPFinder). In addition, BayGenomics investigators have generated and characterized multiple lines of knockout mice for understanding cardiopulmonary development and disease. BayGenomics has an active, high-quality education program. BayGenomics involves two leading San Francisco Bay Area research institutions: The J. David Gladstone Institutes and the University of California, San Francisco. BayGenomics is organized into seven Components: (1) Gene Trapping in Embryonic Stem Cells, (2) Computational Methods and Genomics Education, (3) In Situ Hybridization, (4) Mouse Resource for Lipid Metabolism, (5) Mouse Resource for Pulmonary Disease, (6)Mouse Resource for Cardiopulmonary Development, and (7) Administration. A major objective of BayGenomics will be to use custom gene-trap vectors to generate 2,500 ES cell lines per year with well characterized insertional mutations (Component 1). Each "trapped" ES cell line will be posted on our web site and will be distributed freely to the research community. A second objective will be to improve the annotation of our web site and to provide relevant genomics education programs to our users (Component 2). A third objective is to use in situ hybridization studies to define gene-expression patterns for a subset of the trapped genes, making it easier for our group and others to make informed choices about which genes are likely to be relevant to cardiopulmonary development (Component 3). A fourth objective is to generate a limited number of genetically modified mice, for the purpose of defining the relevance of specific genes to cardiopulmonary development and disease (Components 4-6).

DESCRIPTION (provided by applicant): Many intracellular signaling proteins (e.g., the Ras and Rho proteins) and several nuclear lamins terminate with a carboxyl-terminal CAAX motif. CAAX proteins undergo three sequential posttranslational modifications. First, the cysteine (i.e., the C of the CAAX motif) is farnesylated or geranylgeranylated by a pair of cytosolic enzymes--farnesyltransferase (FTase) and geranylgeranyltransferase I (GGTase I). Second, the last three amino acids (i.e., the -AAX) are cleaved off by Ras and a-factor converting enzyme (Rcel), an integral membrane protease of the endoplasmic reticulum (ER). Third, the newly exposed carboxyl-terminal isoprenylcysteine is methylated by another ER protein, isoprenylcysteine carboxyl methyltransferase (Icmt). These posttranslational modifications render the C-terminus of CAAX proteins more hydrophobic, enhancing the attachment of the proteins to membrane surfaces and facilitating certain protein-protein interactions. Activating Ras mutations have been detected in 30% of all human cancers, and are common in leukemia and myeloproliferative diseases. Inhibitors of FTase have been used to treat cancers that harbor mutationally activated Ras proteins. Unfortunately, K-Ras and N-Ras--the Ras isoforms most often implicated in human cancers--are readily geranylgeranylated by GGTase I in the setting FTase inhibition. This alternate isoprenylation pathway has focused attention on other enzymes in the pathway, such as GGTase I, Rcel, and Icmt. Surprisingly, there are no data on the impact of inhibiting these other enzymes on the development of cancer in mice. In this project, this void will be addressed. In preliminary studies, mice harboring both a Cre-inducible latent oncogenic Kras2 allele (KrasLsL) and the inducible Mx1-Cre transgene have been generated. Induction of Cre in those mice activates the latent Ras allele and results in a full-fledged, lethal, myeloproliferative disease that is reminiscent of chronic myelogenous leukemia or juvenile myelomonocytic leukemia in humans. Recently, conditional "floxed" alleles for the posttranslational processing enzymes (FTase, GGTase I, Rcel, and Icmt) have been generated. Thus, it is now possible to breed mice in which Cre expression can be used to simultaneously activate the latent oncogenic K-Ras allele and inactivate the CAAX processing enzymes. Using these mice, we will define the impact of defective CAAX processing on the development, progression, and lethality of Ras-induced myeloproliferative disease.

DESCRIPTION (provided by applicant): Mutations in LMNA, the gene for lamin A/C, have been shown to cause several forms of muscular dystrophy, a cardiomyopathy, familial partial lipodystrophy, one form of Charcot Marie Tooth neuropathy, mandibuloacral dysplasia, and Hutchinson Gilford progeria syndrome (HGPS). HGPS patients have many aging-like phenotypes, including a wizened appearance, alopecia, osteoporosis, and coronary heart disease. HGPS is a merciless disease; death occurs on average at age 13, nearly always from atherosclerosis in the coronary and cerebral arteries. A recent NIH workshop on HGPS concluded that a thorough understanding of vascular disease in HGPS syndrome represents a key research priority. In most cases, HGPS is caused by a de novo single nucleotide substitution in exon 11. This mutation generates a cryptic splice site, resulting in an in-frame deletion of 50 amino acids near the carboxyl terminus of the lamin A. Interestingly, the deletion prevents the endoproteolytic processing of prelamin A. The objective of this grant application is to create mouse models of HGPS for the purpose of exploring mechanisms underlying the strikingly increased susceptibility to atherosclerosis in HGPS. The Principal Investigator (PI), Dr. Stephen G. Young, is well-positioned to pursue this objective. For the past 10 years, the PI has worked both on the posttranslational processing enzymes required for the biogenesis of lamin A and on the genetic underpinnings of atherosclerosis. For both projects, the PI has relied heavily on studies with gene targeted mice. Recently, the PI showed that the targeted inactivation of Zmpste24 prevents the proper endoproteolytic processing of lamin A, leading to the accumulation of prelamin A. Of note, Zmpste24-/- mice have a host of phenotypes similar to those in HGPS. The PI has also created mice lacking other enzymes involved in the posttranslational processing of the lamins (protein farnesyltransferase, Rcel, and Icmt). The first aim of this application is to create, with gene-targeting approaches, authentic mouse models of HGPS. The second specific aim is to compare, at both cellular and whole-animal levels, phenotypes in HGPS mouse models and the Zmpste24-deficient mice. The third specific aim is to understand mechanisms underlying the strikingly high susceptibility to atherosclerosis in HGPS. The PI's laboratory has extensive experience in analyzing atherogenesis in mice and is thrilled with the prospect of exploring mechanisms of atherosclerosis in HGPS.

[unreadable] DESCRIPTION (provided by applicant): Hyperlipidemia is a major public health problem. The objective of this proposal is to define the role of glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1) in plasma triglyceride metabolism. During the past few months, we have made a stunning observation-that GPIHBP1 plays a major role in the metabolism of triglyceride-rich lipoproteins in the plasma. Remarkably, the sole phenotype of male and female Gpihbpl-deficient mice on a chow diet is chylomicronemia, with plasma triglycerides of -10,000 mg/ dl. GPIHBP1 is expressed on the surface of cells, mainly in adipose tissue and heart, the principal tissue sites of lipolysis of triglyceride-rich lipoproteins. Because the expression pattern of GPIHBP1 faithfully mirrors that of lipoprotein lipase (LPL), it is overwhelmingly likely that the hypertriglyceridemia in Gpihbpl - deficient mice is due to impaired lipolysis of chylomicrons and very low density lipoproteins (VLDL). Only one description of GPIHBP1 exists in the literature. That study that showed that GPIHBP1 can bind to high density lipoproteins in cultured cells. However, no link was made to triglyceride-rich lipoproteins, and the in vivo physiologic importance of the molecule was not explored. We propose that GPIHBP1 plays a major role in the lipolysis of triglyceride-rich lipoproteins. We hypothesize that GPIHBP1 is involved in "capturing" lipoproteins as they flow through capillaries and recruiting LPL (from a reservoir on the surface of cells) so that lipolysis can begin. However, at this stage, there are more questions than answers about the functions of GPIHBPL in Specific Aim 1 of this proposal; we will further characterize the hyperlipidemia in Gpihbpl-deficient mice and to test the hypothesis that the severe chylomicronemia is related to defective LPL-mediated lipolysis along the capillary endothelium. In Specific Aim 2, we will define the pattern of Gpihbpl expression in various mouse tissues and determine if Gpihbpl expression is regulated according to different metabolic states (e.g., fasting, refeeding). Defining the Gpihbpl expression will be facilitated by the fact that the Gpihbpl knockout mice have a lacZ reporter. In Specific Aim 3, we will test the idea that GPIHBP1 binds directly to triglyceride-rich lipoproteins and/or to LPL, thereby facilitating lipolysis. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] The proposed Program Project Grant (PPG), "New Molecules in Triglyceride Metabolism and [unreadable] Adipogenesis," is guided by a common objective-to study mechanisms for triglyceride delivery to peripheral tissues and to define new mechanisms in adipogenesis. This PPG is highly relevant to obesity and hyperlipidemia, two public health problems that are central to the mission of the NHLBI. [unreadable] [unreadable] During the past year, the project and core leaders of this PPG have uncovered new molecules regulating plasma lipid metabolism, fuel delivery to cells, and adipogenesis. Although these novel molecules and targets were discovered by diverse experimental approaches, they all are directly connected to lipogenesis and to PPARy (peroxisome proliferator-activated receptor gamma), a key regulator of adipogenesis and triglyceride metabolism. The convergence of intellectual interests around a single topic, triglyceride metabolism and adipogenesis, has fueled our collaborative interactions, which in turn have led to entirely new discoveries. [unreadable] [unreadable] This PPG is organized into three projects and two cores. Project 1, "Function and Regulation of [unreadable] GPIHBP1 in Lipid Metabolism," will be led by Dr. Stephen G. Young. This project will deal with a novel endothelial cell protein, GPIHBP1 (glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1), which is critically important for the lipolytic processing of triglyceride-rich lipoproteins and for the delivery of lipid nutrients to adipose tissue, heart, and skeletal muscle. Project 2, "The Lipin Protein Family and Triglyceride Metabolism," will be led by Dr. Karen Reue. Dr. Reue's identification of the mutation causing fatty liver dystrophy led to the discovery of the lipin family of proteins, which have key roles in adipogenesis and triglyceride synthesis. The lipin proteins are intriguing because they function as triglyceride biosynthetic enzymes as well as transcriptional coactivators. Project 3, "Novel Pathways for Triglyceride Storage and Adipogenesis," will be led by Dr. Peter Tontonoz. Using a novel high-throughput screening approach, Dr. Tontonoz has identified entirely new cDNAs involved in adipogenesis, as well as small-molecule regulators of adipogenesis. Dr. Tontonoz is poised to elucidate these new players in the field of triglyceride metabolism and adipogenesis. A Mouse Model and Antibody Core (Core A) will create new genetically modified mice and polyclonal and monoclonal antibody reagents for the three projects. An Administration Core (Core B) will support the personnel of each project, organize advisory board meetings, and ensure compliance with institutional and NIH guidelines.

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The main objective of Project 1 ("Function and Regulation of GPIHBP1 in Lipid Metabolism") is to define the function of glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1) in plasma triglyceride metabolism and in the regulation of fuel delivery to adipose tissue and muscle. This objective is closely aligned with the overall objectives of this PPG, which are to elucidate the basic mechanisms of triglyceride metabolism and adipogenesis, focusing on newly discovered molecules and pathways that are regulated by PPARy. Gpihbpl-deficient mice have severe chylomicronemia, even on a low-fat diet, with plasma triglyceride levels as high as 5000 mg/dl. GPIHBP1 is located on the luminal surface of endothelial cells of heart, muscle, and fat, where lipolysis of plasma triglycerides occurs. Expression of GPIHBP1 in cultured cells confers the ability to bind both lipoprotein lipase (LpL) and chylomicrons, suggesting that GPIHBP1 is a key platform for the lipolytic processing of chylomicrons along the luminal face of capillaries. Although GPIHBP1 is clearly important for the lipolysis of triglyceride-rich lipoproteins, many issues regarding the biological function of GPIHBP1 have not been explored. We have not yet examined the impact of different diets on lipid and glucose metabolism in Gpihbpl-deficient mice, nor do we know why Gpihbpl - deficient mice are protected from diet-induced obesity. Also, the molecular basis for chylomicron and LpL binding to GPIHBP1 is unknown[unreadable]in particular whether the highly negatively charged domain in GPIHBP1 interacts with positively charged "heparin-binding" domains in LpL and various apolipoproteins within chylomicrons (e.g., apo-E, apo-B, and apo-AV). Finally, Gpihbpl is highly regulated by fasting and refeeding and by PPARy, but the molecular basis for this regulation has not been defined. In Specific Aim 1, we will further define the metabolic abnormalities in Gpihbpl-deficient mice. In Specific Aim 2, we will identify the structural domains of GPIHBP1 required for the binding of LpL and chylomicrons and define the apolipoproteins that mediate the binding of chylomicrons (and other lipoprotein fractions) to Gpihbpl-transfected cells. In Specific Aim 3, we will define the tissue pattern of Gpihbpl expression and changes in Gpihbpl expression with PPARy agonists and different metabolic conditions.

DESCRIPTION (provided by applicant): This application addresses Challenge Area (06): Enabling Technologies and Specific Challenge Topic 06- HL-102, which is to develop high affinity/high specificity targeted molecular probes for molecular imaging of cardiovascular and pulmonary disease targets. We will use antibody probes to better define the pathogenesis of hypertriglyceridemia. Hypertriglyceridemia is caused by inherited defects in lipoprotein lipase (LPL), but the etiology of most cases of hypertriglyceridemia remains mysterious. Fortunately, recent discoveries on the mechanism by which LPL enters capillaries may uncloak the mystery. By applying new discoveries in lipolysis and molecular imaging, we will better define the underpinnings of hypertriglyceridemia. Reduced lipolysis by LPL underlies many cases of hypertriglyceridemia, but the explanation for the defective lipolysis is mysterious. This mystery is compounded when one considers the fact that many patients with hypertriglyceridemia have normal levels of LPL both in tissues and in the "postheparin" plasma. Arguably, the mechanisms for hypertriglyceridemia constitute the most perplexing riddle in lipoprotein metabolism. We have fresh insights into this problem. We identified an endothelial cell protein, GPIHBP1, which binds LPL and serves as a "platform" for the lipolysis in capillaries. Also, we found that GPIHBP1 serves as the "LPL transporter." GPIHBP1 transports LPL from the basolateral to the apical (luminal) surface of endothelial cells, where it hydro-lyzes lipoprotein triglycerides. We hypothesize that many cases of hypertriglyceridemia are due to defective GPIHBP1-mediated transport of LPL into capillaries. Drs. Stephen Young, Loren Fong, and colleagues have developed monoclonal and polyclonal antibodies against GPIHBP1 and LPL, as well as new gene-targeted models for assessing GPIHBP1 and LPL function. Meanwhile, Drs. Anna Wu and Tove Olafsen are pioneers in immunodiagnostics and molecular imaging. Together, we have already taken the first step and performed positron-based molecular imaging studies with an 124I-labeled monoclonal antibody against GPIHBP1. Over the next two years, we will develop molecular imaging approaches to measure the intracapillary levels of both GPIHBP1 and LPL. This topic can only be approached with molecular imaging techniques. Our imaging studies will begin with mouse models, but we will simultaneously prepare the reagents required for molecular analysis of lipolysis in humans. We expect that our efforts will clarify the mechanisms of hypertriglyceridemia and establish molecular imaging as a critical tool in understanding hypertriglyceridemia. H PUBLIC HEALTH RELEVANCE: Defective lipolysis can lead to atherosclerosis as well as life-threatening episodes of atherosclerosis. Genetic studies have shown that accelerated rates of lipolysis lead to lower plasma lipid levels and a reduced prevalence of coronary disease. Molecular imaging to define intracapillary levels of GPIHBP1 and LPL will lead to a vastly improved understanding of the molecular underpinnings of hypertriglyceridemia.

DESCRIPTION (provided by applicant): The Ras family of proteins and several of the nuclear lamins contain a carboxyl-terminal "CaaX motif" that triggers protein isoprenylation and methylation. We have intensively studied these posttranslational modifications. We created gene-targeted mice to examine the role of all of the enzymes involved in the posttranslational processing of CaaX proteins [protein farnesyltransferase (FTase), protein geranylgeranyl- transferase-I (GGTase-I), ICMT, RCE1, ZMPSTE24]. We have examined the importance of each enzyme in the growth of Ras-induced tumors. We also discovered that ZMPSTE24 is a prelamin A protease and that its absence leads to an accumulation of farnesyl-prelamin A. This accumulation of farnesyl-prelamin A underlies certain progeroid disorders in humans, including Hutchinson-Gilford progeria syndrome. We created gene- targeted mice and investigated the importance of protein farnesylation in the pathogenesis and treatment of progeria. We also discovered that lamins B1 and B2 are crucial for neuronal migration in the brain. Our research program on CaaX proteins has been productive, resulting in 38 publications during the past 5 years. The objective of this renewal application is to continue our work on the in vivo importance of the modifications of CaaX proteins, focusing on specific topics relevant to human disease. Specific Aim 1 is to use genetically modified mice to examine the importance of lamins B1 and B2 in the brain and in other tissues. Our preliminary studies suggest that B-type lamins are essential for neuronal migration. We need to investigate this issue in depth. Also, we need to determine whether the individual proteins, lamin B1 and lamin B2, have intrinsically different roles in this process. Finally, we need to investigate the roles of lamin B1 and lamin B2 in adult tissues. The fact that lamin B1 and lamin B2 knockout mice die at birth means that no one has yet explored the physiologic importance of these proteins in adult tissues. Very recently, we have created conditional knockout alleles for both Lmnb1 and Lmnb2, and over the next few years, we intend to use these tools to address the importance of the B-type lamins in adult tissues. Specific Aim 2 is to define the importance of the posttranslational modifications of prelamin A in the assembly of the nuclear lamina, and to define the importance of protein farnesylation in the pathogenesis of progeroid syndromes. Over the past few years, our laboratory has generated knock-in mice that synthesize exclusively prelamin A and other knock-in mice that produce exclusively mature lamin A (where lamin A is produced directly, bypassing the posttranslational processing). Already, we have discovered differences in the assembly of the nuclear lamina in these mice. Further analyses of these mice should help us to define the physiologic importance of the posttranslational processing of prelamin A. Also, additional knock-in mice created by our group have strongly suggested that the farnesyl lipid modification may be critical for certain progeroid disorders, but less critical for others. We plan to explore the cell biology underlying this finding. PUBLIC HEALTH RELEVANCE: The proteins of the nuclear lamina have generated enormous interest because of recent studies showing that genetic defects in nuclear lamins cause multiple human diseases. The main objective of this renewal application is to continue to explore the physiologic and medical relevance of the posttranslational processing of the nuclear lamins, particularly as it relates to devastating neuronal migration defects and the pathogenesis and treatment of progeroid syndromes (precocious aging syndromes). Our studies will be focused on the physiologic importance of the B-type lamins in the developing brain and other tissues and the physiologic importance of the posttranslational processing of prelamin A.

DESCRIPTION (provided by applicant): The objectives of this proposal are to identify the structural features of lipoprotein lipase (LPL) required for binding to GPIHBP1 and to define mechanisms by which LPL is transported across endothelial cells. We discovered that GPIHBP1, an endothelial cell protein, binds LPL and is absolutely required for the lipolytic processing of triglyceride-rich lipoproteins. We showed that an absence of GPIHBP1 in mice causes severe hypertriglyceridemia (chylomicronemia) and that humans with GPIHBP1 mutations exhibit the same phenotype. Initially, we proposed that GPIHBP1 was a binding site for LPL in capillaries, but we recently found that GPIHBP1 has a second role-transporting LPL from the extracellular spaces into the capillary lumen. The identification of GPIHBP1 as the LPL transporter solved a longstanding mystery of plasma lipid metabolism, posing many new questions for investigation. Among these are: What amino acid sequences within LPL are important for binding to GPIHBP1? Do certain LPL mutations cause disease by abolishing LPL's ability to bind to GPIHBP1? Do other mutations enhance LPL transport? What is the efficiency of LPL transport across endothelial cells? Is LPL movement across capillaries bidirectional? Is LPL transported across capillaries in transcytotic vesicles? We have begun to investigate each of these issues. We have identified amino acid substitutions within the carboxyl terminus of LPL that abolish its ability to bind to GPIHBP1. These mutations have provided important clues regarding the location of LPL's GPIHBP1-binding domain, but many more studies are required to fully understand LPL-GPIHBP1 interactions. We have made great progress in understanding mechanisms of LPL transport, both in vitro and in vivo. Our studies have taken advantage of a host of new antibody reagents and expertise with both confocal and electron microscopy. We have two specific aims, each designed to better understand lipolysis in health and disease. In Aim 1, we will test the hypothesis that the carboxyl terminus of LPL is crucial for binding GPIHBP1 and examine LPL mutations that interfere with GPIHBP1 binding and transport across endothelial cells. In Aim 2, we will test the hypothesis that LPL is transported across endothelial cells by caveolar-mediated transcytosis. We are excited by these aims, and are poised-with all of the necessary expertise, reagents, and experimental techniques-to carry out the proposed studies.

The objective of Project 1 (Function of GPIHBPI in Triglyceride Metabolism) in our Program Project Grant (PPG) is to define the function of GPIHBPI, a member of the Ly6 family of proteins, in plasma triglyceride metabolism and in the delivery of lipid nutrients to adipose tissue and muscle. This objective is closely aligned with the theme of our PPG?to understand new molecules and mechanisms underlying triglyceride delivery to parenchymal tissues, triglyceride synthesis, and adipogenesis. Over the past 5 years, our PPG team showed that GPIHBPI transports lipoprotein lipase (LPL) from the interstitial spaces (where it is secreted by adipocytes and myocytes) to its site of action within the capillary lumen. This discovery solved a longstanding mystery in plasma lipid metabolism but simultaneously highlighted other lingering mysteries within the field. For example, no one has yet defined the mechanisms by which triglyceride-rich lipoproteins (TRLs) marginate within capillaries, so that lipolysis can proceed. Other mysteries include the mechanism by which TRL-derived lipids move across endothelial cells and the precise structures governing LPL-GPIHBP1 interactions and interactions of TRLs with the LPL-GPIHBP1 complex. Also, it is unclear whether other members of the Ly6 protein family, aside from GPIHBP1, contribute to metabolism and obesity. Recently, Project 1 investigators have shown that knockouts of several Ly6 genes near Gpihbp1 are associated with protection from obesity and lower plasma lipid levels. For the next 5 years. Project 1 investigators will pursue three Specific Aims. Specific Aim 1 is to define mechanisms for the margination of TRLs in capillaries. As part of this aim, we will investigate the functional relevance of a newly discovered endothelial cell organelle, nanovilli, in TRL margination and in the transport of LPL across endothelial cells. Specific Aim 2 is to better define interactions between GPIHBP1 and LPL and to further elucidate the features of the LPL-GPIHBP1 complex required for interactions with TRLs. Specific Aim 3 is to investigate how the inactivation of a cluster of Ly6 genes (Slurpl, Slurp2, Lypd2) near Gpihbp1 affects energy balance in mice and protects against adiposity.

All three projects rely on genetically modified mice and the production of purified proteins and polyclonal and monoclonal antibodies. Accordingly, all three projects will be supported by the Mouse Model and Protein Expression Core (Core A), led by Dr. Loren Fong. Dr. Fong is an expert in a wide variety of practical techniques in cellular and molecular biology, including growing and manipulating mouse embryonic stem cells and the use of mouse models in biomedical research. This expertise has paid huge dividends for this PPG. Dr. Fong, working with Drs. Reue, Young, and Tontonoz, has generated conventional and tissue- specific Lipin2, Lipin3, Idol, and Tle3 knockout mice. In addition, the Core houses and genotypes mice, backcrosses mice onto inbred strains, maintains animal protocols, and organizes histological studies on mouse models. The Core also produces purified proteins for biochemical studies and for generating polyclonal and monoclonal antibodies; this has been an essential function, since our PPG has focused on newly discovered molecules, for which few reagents exist. During the prior funding period, Drs. Bensadoun, Fong and Young produced multiple antibody reagents for this PPG. Their expertise and experience will be a boon for the new antibody projects proposed in this application. Aside from antibody reagents, the Core will also produce purified recombinant proteins for biochemical and structural biology studies. All three projects of the PPG will use Core A. RELEVANCE (See instructions): This Program Project Grant proposes to define the molecular mechanisms that regulate lipid metabolism and adiposity-two events that play centrol roles in the pathogenesis of metablic disesase (e.g., obeisity and atherosclerosis). The Mouse Model and Protein Expression Core will support the PPG's goals by providing new mouse models, antibodies, and recombinant proteins to all three projects.

? DESCRIPTION (provided by applicant): Our multi-institutional team will investigate ZMPSTE24 inhibition by HIV-protease inhibitors and the resulting accumulation of farnesyl-prelamin A as a mechanism for metabolic syndrome and atherosclerotic cardiovascular disease in HIV-PI-treated patients. This grant proposal is submitted in response to a Request for Applications (RFA-HL-14-024) on mechanisms of heart disease in HIV patients. Our team was enthusiastic about responding to this RFA because we have collective expertise in cardiology, HIV medicine, lipid metabolism, atherogenesis, and nuclear lamins. Also, this RFA provides an opportunity for us to pursue one of our own discoveries-that commonly used HIV-PIs (e.g., atazanavir, ritonavir, lopinavir) inhibit ZMPSTE24 and lead to an accumulation of farnesyl-prelamin A in cells and tissues. This discovery is noteworthy because genetic syndromes associated with an accumulation of prelamin A (e.g., Hutchinson-Gilford progeria syndrome) lead to severe atherosclerotic heart disease. We fully appreciate that heart disease in HIV patients is multifactorial and influenced by several classes of drugs. No single research group could possibly follow-up on all of the potential mechanisms. Our team has decided to focus and to dig deeper into the connection between HIV-PIs, reduced ZMPSTE24 activity, farnesyl-prelamin A accumulation, and heart disease. Over the past few years, several stumbling blocks have slowed progress in understanding the connection between HIV-PIs, ZMPSTE24 inhibition, and heart disease. First, a paucity of specific prelamin A antibodies has made it difficult to perform rigorous research in animal models or with the cells and tissues of HIV patients. Our laboratory's recent development of a panel of monoclonal antibodies against prelamin A will be very helpful in overcoming that obstacle. Second, the link between prelamin A accumulation and atherogenesis is poorly understood. The cells in the arterial wall that are susceptible to prelamin A toxicity-and the link to atherogenesis-are not understood. To overcome that obstacle, we have created a conditional knockout allele for Zmpste24. Third, the mechanisms by which HIV-PIs inhibit ZMPSTE24 are not known. However, overcoming that obstacle is now quite feasible because the structure for human ZMPSTE24, alone and complexed to a prelamin A peptide substrate, was recently solved by our collaborator, Dr. Elisabeth Carpenter. We have three Specific Aims. The first is to investigate the impact of HIV-PIs on prelamin A processing in cultured cell lines, in mouse models, and in the cells and tissues of HIV patients. The second is to determine, with tissue-specific Zmpste24 knockout mice, how prelamin A accumulation affects different cell types of the arterial wall and leads to atherogenesis. The third is to investigate, with Dr. Carpenter, mechanisms for ZMPSTE24 catalysis and how HIV-PIs block ZMPSTE24 activity. We are excited by all three Aims and are poised-with all of the expertise, reagents, techniques, and expert collaborators-to carry out our proposed studies. We expect that our studies will yield fresh insights into mechanisms of heart disease in HIV patients.

? DESCRIPTION (provided by applicant): Title of Project: Defining mechanisms for lipid transport across capillary endothelial cells Our objective is to understand how lipids from triglyceride-rich lipoproteins (TRLs) move across capillary endothelial cells towards parenchymal cells. Our interest in this topic-the least understood area within plasma triglyceride metabolism-arose from our efforts to understand molecular mechanisms for the intravascular processing of TRLs. During the past few years, we showed that GPIHBP1, a GPI-anchored protein of capillary endothelial cells, is solely responsible for shuttling lipoprotein lipase (LPL) from the interstitial spaces to its site of action in the capillary lumen. More recenty, we showed that the LPL-GPIHBP1 complex is critical for the margination of TRLs along capillaries (so that the LPL-mediated processing of TRLs can proceed). The movement of lipid nutrients across capillaries to parenchymal cells is crucial for delivering fuel to vital organs an lipids for storage in adipose tissue. Unfortunately, there are few insights into this process. No one understands: (1) whether the fatty acid products of lipolysis simply diffuse across endothelial cells; (2) whether lipids move across endothelial in the very same vesicles that shuttle GPIHBP1 and LPL; (3) whether intact TRLs move across endothelial cells to the subendothelial spaces; and (4) whether binding of fatty acids by CD36 on endothelial cells is required for lipid transport across capillaries. Also, no one understands how lipids move across capillaries in other vertebrates (e.g., birds, fish). In those organisms, GPIHBP1 is absent and LPL appears to be located largely, if not exclusively, in the extravascular spaces (i.e., it is not associated with capillaries). In other vertebrates, we suspect that the TRLs might be transported across capillaries to the subendothelial spaces-to where the LPL is located. An improved understanding of TRL metabolism in other vertebrates will likely yield insights into accessory mechanisms for TRL processing in mammals. One of the main reasons for the slow progress in understanding lipid transport across capillaries is that there has been no way to visualize lipid transport. Seeing how lipids move across capillaries is crucial for deciphering molecular mechanisms and designing testable hypotheses. Fortunately, we have overcome the imaging roadblock. During the past two years, we have used NanoSIMS and backscattered electron (BSE) imaging to create high-resolution images of TRLs as they marginate along capillaries and as the TRL lipids move across endothelial cells to parenchymal cells. These studies have demonstrated that some TRL lipids move across endothelial cells in vesicles, but additional high-resolution imaging studies are required to determine if diffusion of fatty acids along plasma membranes-or transport of lipids across the cytosol-is involved. The same methods can be used to define the role of specific proteins (e.g., CD36) in lipid transport across endothelial cels. For the next five years, we will use NanoSIMS and BSE imaging to define the cellular and molecular mechanisms for lipid transport across capillaries. We will also define the in vivo functional relevance of CD36 for the transport of TRL-derived lipids across capillary endothelial cells.

During the past 5 years, we have pursued our interest in lipoprotein lipase (LPL) and intravascular lipolysis, focusing on the specific aims in our original grant application. Our studies have been productive, yielding papers that describe insights into the LPL sequences that interact with GPIHBP1 and insights into the cellular mechanisms for GPIHBP1-mediated transport of LPL across endothelial cells. In this renewal application, we have proposed three new specific aims. The first aim relates to our discovery that some cases of human hypertriglyceridemia are caused by GPIHBP1 autoantibodies. We now need to better characterize this new disease syndrome and define its frequency. Our second aim relates to our discovery that GPIHBP1 is present in human plasma and that GPIHBP1 levels can be quantified with an ELISA. We now need to determine if GPIHBP1 levels in the plasma are a useful biomarker of metabolic or vascular disease. Our third aim deals with persistent and fundamental questions regarding the LPL?GPIHBP1 complex, including the stoichiometry of the complex and how its stability and activity are influenced by physiologic variables. We have accumulated substantial preliminary data to support the feasibility of our proposed studies. With regard to the first specific aim, we have already documented GPIHBP1 autoantibodies in plasma samples from six patients with hypertriglyceridemia. One of the patients became pregnant and, as expected, the autoantibodies crossed the placenta. The newborn infant?s plasma contained GPIHBP1 autoantibodies, resulting in extremely severe (but transient) hypertriglyceridemia. The discovery that GPIHBP1 autoantibodies cause hypertriglyceridemia is a translational discovery with major implications for medical diagnostics and therapeutics. We now need to better characterize GPIHBP1 autoantibodies and determine the frequency of the ?GPIHBP1 autoantibody syndrome.? For the second specific aim, we already developed and characterized monoclonal antibodies against human GPIHBP1 and used two of the antibodies to create an ELISA for human GPIHBP1. Our ELISA accurately quantifies GPIHBP1 in the plasma. We are poised to collaborate with leaders of genetic?epidemiology studies to test whether plasma GPIHBP1 levels are a useful biomarker of vascular or metabolic disease. With regard to the third specific aim, we have made substantial progress in understanding the LPL?GPIHBP1 complex and in developing experimental approaches to study that complex. Our studies suggest that there is only a single binding site for GPIHBP1 on newly secreted LPL (despite the fact that LPL is widely presumed to be a homodimer). Related studies suggest that GPIHBP1 binds only one molecule of LPL. We need to confirm these results with additional experimental platforms, including surface plasmon resonance studies. We also need to determine how LPL?GPIHBP1 interactions are affected by physiologic variables such as active triglyceride lipolysis or inhibitor proteins (e.g., ANGPTL4). These studies will add substantially to our understanding of LPL?GPIHBP1 interactions and the physiology of intravascular lipolysis.

Abstract Our laboratory focuses on lipoprotein lipase (LPL)?mediated processing of triglyceride-rich lipoproteins (TRLs) in capillaries. This process, intravascular lipolysis, is essential for delivering lipid nutrients to vital tissues (e.g., the heart) and is highly relevant to plasma lipid levels and coronary artery disease risk. We discovered a protein expressed exclusively in capillary endothelial cells, GPIHBP1, that is required for intravascular lipolysis. GPIHBP1 binds LPL within the interstitial spaces and shuttles it across endothelial cells to its site of action in the capillary lumen. GPIHBP1 is also required for the margination of TRLs in capillaries and for preserving the catalytic activity of LPL. These discoveries have already transformed textbook descriptions of lipolysis, but many challenges remain. One is to define the cellular and molecular mechanisms by which the fatty acid (FA) products of TRL processing traverse endothelial cells and move into parenchymal cells. No one understands this process, in part because there were no methods for visualizing FA movement into and across capillary endothelial cells. To formulate hypotheses about the mechanisms for FA movement within tissues and to test the roles of specific genes and metabolites in this process, we are now imaging tissues with NanoSIMS. NanoSIMS uses a Cs+ beam to bombard a tissue section, releasing secondary ions that can be collected, quantified, and used to create high-resolution images of cells and tissues based solely on their isotopic content. We routinely prepare fresh TRLs enriched in 13C- or 2H-labeled triglycerides, inject them intravenously into mice, and then use NanoSIMS to create high-resolution images of 13C- and 2H-FAs as they move into and across capillary endothelial cells. We obtain backscattered electron (BSE) images on the same section. Our correlative imaging approach, which is unique in the fields of lipid metabolism and vascular biology, allows us to match the chemical information from NanoSIMS to the ultrastructural morphology provided by the BSE images. We are now positioned to identify the cellular and molecular mechanisms for the movement of lipids to vital tissues. A second challenge has been to identify additional proteins in capillary endothelial cells that are relevant to lipid metabolism; a related issue is to determine if active TRL processing alters gene expression in capillary endothelial cells. Fortunately, our GPIHBP1-specific monoclonal antibodies have made it possible to purify capillary endothelial cells from complex mixtures of cells, facilitating analyses of gene expression in capillary endothelial cells. A third challenge?and one that is particularly relevant to clinical medicine?is to explore the importance of GPIHBP1 and capillary endothelial cells to human hypertriglyceridemia. We discovered autoantibodies against GPIHBP1 in the plasma of multiple patients with hypertriglyceridemia; these autoantibodies cause disease by blocking the binding of LPL to GPIHBP1. The GPIHBP1 autoantibodies now need characterization, and the frequency of this new autoimmune/metabolic disease syndrome needs to be defined. With our reagents and assays, we are uniquely positioned to address these issues.  

PPG Title: New Approaches for Understanding Lipid Movement in Health and Disease SUMMARY/ABSTRACT Our Program Project Grant (PPG) focuses on lipid metabolism and transport, with the goal of defining mechanisms for metabolic and cardiovascular disease. Our PPG team has made seminal discoveries in lipid metabolism and transport. We discovered an endothelial cell protein, GPIHBP1, that transports lipoprotein lipase (LPL) to the capillary lumen and stabilizes the structure of LPL. Recently, we defined the structure of the GPIHBP1?LPL complex, providing fresh insights into mutations causing hypertriglyceridemia and opening the door to understanding mechanisms that regulate intravascular lipolysis. In the realm of cholesterol metabolism, our PPG discovered that macrophages release, by plasma membrane (PM) budding, particles that are enriched in cholesterol. Our PPG uncovered a link between inflammatory signaling and cholesterol metabolism in macrophages, and we identified a new protein, Aster-B, that is critical for cholesterol movement between the PM and the endoplasmic reticulum (ER). A deficiency of Aster-B impairs cholesterol movement to the ER, causing a striking upregulation of lipid biosynthetic genes. These discoveries, all relevant to the pathogenesis of atherosclerosis, were utterly dependent on collaborations between our PPG leaders and the advanced molecular, biochemical, and imaging capabilities in their laboratories. As we look to the future, we will dig deeper into the molecules and mechanisms that we have uncovered. In project 1, Drs. Young and his PPG colleagues will use biochemical and biophysical tools to elucidate the functions of the LPL?GPIHBP1 complex, including the role of GPIHBP1?s acidic domain in stabilizing LPL activity and capturing LPL within the subendothelial spaces. They will also study, with electron microscopy and NanoSIMS imaging, the budding of cholesterol-rich particles from the macrophage PM. They will define the composition of the particles and explore their relevance to reverse cholesterol transport. In project 2, Dr. Bensinger and coworkers will determine how inflammatory signals modulate the lipidome of macrophages. They will also define mechanisms by which alterations in cholesterol homeostasis affect STING signaling and the impact of the STING pathway on dyslipidemia, inflammation, and atherogenesis. In Project 3, Dr. Tontonoz and his PPG coworkers will explore the role of Aster-B in cholesterol transport, efflux, and esterification and elucidate the function of Aster-B in sterol transport in vivo. They will also assess the contribution of the ?macrophage Aster pathway? to atherosclerosis and screen for additional proteins required for the nonvesicular transport of cholesterol within cells. The three component projects will be supported by a single scientific core, led by Dr. Loren Fong and colleagues. They will produce recombinant proteins, provide advanced microscopy services, including NanoSIMS imaging of lipids. They will also work with Dr. Keriann Backus to provide chemical proteomics for investigating lipid metabolism. Our PPG is confident in success because we have exciting hypotheses and we work as a team at both scientific and technical levels.

Core B (Administration Core) SUMMARY/ABSTRACT The Administration Core of this NHLBI Program Project Grant has four main functions: general administration, accounting, clerical support, and organizing the annual external advisory board meeting. The coordination of research activities and the handling of routine paperwork for the University of California and the National Institutes of Health necessitate a strong and efficient administrative component. General administration includes supervision (by the core leader, Dr. Stephen G. Young) of Ms. Lovelyn Edillo, assisting with clerical and accounting functions, scheduling weekly PPG meetings, providing assistance with travel arrangements, submitting paperwork for travel reimbursements, assisting with material transfer agreements, solving computer software and connectivity issues, preparing documents for domestic and international shipments, and notifying project and core leaders of due dates for the animal research applications, radiation safety applications, biosafety applications, and training courses. The clerical component includes final formatting of reports and manuscripts, preparing bibliographies, writing and distributing memos, sending emails to PPG staff, coordinating letters of collaboration, performing final formatting of the project and core reports for our external advisory board meeting and for the annual ?Application for Continuation Grant.? The accounting component includes ordering and receiving supplies purchased by the cores and projects, tracking and recording deliveries, returning damaged shipments or supplies that were delivered by mistake, maintaining laboratory equipment service contracts, managing consortium agreements, and maintaining financial ledgers in accordance with university procedures. In coordinating these functions, Dr. Young will work closely with both Ms. Edillo and Ms. Kayla Brown, a full-time fund manager in UCLA?s department of medicine. Planning the annual external advisory board meeting is an important duty of the Administration Core; preparing for the external advisory board meeting involves assembling the annual report, assisting with travel, housing and transportation for the external advisors, reserving a retreat location, and deciding the menu and dining arrangements (luncheons, coffee breaks, and evening dinners). The Administration Core will serve the three component projects and Core A equally.

Project 1: Deciphering Mechanisms for Triglyceride and Cholesterol Transport SUMMARY/ABSTRACT Project 1 investigators have devoted their careers to exploring basic mechanisms of lipoprotein metabolism in health and disease. They discovered that an endothelial cell protein, GPIHBP1, is responsible for transporting lipoprotein lipase (LPL) to the capillary lumen; that the LPL?GPIHBP1 complex is crucial for the margination of triglyceride-rich lipoproteins (TRLs) along capillaries; and that GPIHBP1 protects LPL from spontaneous and ANGPTL4-catalyzed unfolding/inactivation. Their efforts have resulted in >60 publications, many reflecting a commitment to understanding human disease. For example, they identified GPIHBP1 mutations causing chylomicronemia and uncovered a new human disease?chylomicronemia from GPIHBP1 autoantibodies. Recently, Project 1 investigators and coworkers determined the structure of the LPL?GPIHBP1 complex. During the next 5 years, Project 1 investigators will pursue two independent objectives. The first is to pursue ongoing studies of intravascular lipolysis, building on insights from the structure of the GPIHBP1?LPL complex. That structure, along with new reagents, new methodologies, and expert collaborators, have made intravascular lipolysis more exciting than ever. Key goals include defining amino acid residues required for GPIHBP1?LPL interactions, exploring mechanisms underlying specific ?chylomicronemia mutations,? understanding a gain-of- function polymorphism in LPL, defining the role of GPIHBP1?s acidic domain in stabilizing LPL from unfolding/inactivation, examining the function of GPIHBP1?s acidic domain in recruiting LPL from heparan sulfate proteoglycan binding sites in the subendothelial spaces, and investigating how ANGPTL4 initiates the unfolding and inactivation of LPL. We will also determine the structure of GPIHBP1 and LPL in association with an Fab fragment of the LPL?specific monoclonal antibody 5D2. Our second objective is to investigate the distribution of cholesterol in macrophages and the mechanisms by which macrophages dispose of cholesterol. In preliminary studies, Project 1 investigators found that macrophages release, by plasma membrane budding, numerous 30? 70-nm particles. By NanoSIMS imaging, these particles are highly enriched in cholesterol, including the metabolically active ?accessible cholesterol? detectable by bacterial cytolysins (e.g., ALO-D4). The finding that cholesterol-rich particles ?bud? from macrophages raises many questions. What is the function of particle budding? What is the composition of these particles? Is particle budding regulated? In collaboration with projects 2 and 3, project 1 will investigate the numbers and composition of macrophage particles in different settings (e.g., sterol starvation, cholesterol loading, LXR agonist treatment, and deficiencies of LXRs, ABCA1, or ABCG1). Preliminary NanoSIMS imaging studies showed that high-density lipoproteins are effective in unloading cholesterol from macrophage-derived particles, implying that macrophage particle budding could be relevant to reverse cholesterol transport and the emergence of cholesterol-laden cells in atherosclerotic plaques.

This award is funded in whole or in part under the American Rescue Plan Act of 2021 (Public Law 117-2).

This doctoral dissertation project examines the processes that result in concrete being a leading construction material in rapidly developing cities despite environmental impacts that may reduce resilience to extreme weather events. The research examines the financial investments that drive the growth of concrete residential buildings. The research also highlights the environmental impacts of urban infrastructure development, such as increased in flood risks, by studying the extraction of sand from riverbeds for subsequent use in concrete construction. The project provides data that can inform attempts to reduce the impacts of sand and stone extraction on weather-related disasters, including floods and landslides. In addition to providing funding for the training of a graduate student in the methods of scientific data collection and analysis, the project contributes to localized efforts to advance sustainable construction methods. The findings from this research have the potential to contribute to policy conversations that seek to improve working conditions for construction workers, thereby providing security and acceptable working conditions to laborers.

The researchers examine development of concrete construction in urban areas, particularly those that are impacted by extreme weather. In these settings, the widespread use of concrete is implemented amid concerns that it increases the risk of impacts from natural disasters. Drawing on theory from economic geography and urban political ecology, the researchers examine the financing of residential construction, the tradeoffs faced by laborers in the construction industry, and the processes by which the extraction of sand contributes to concrete production and related environmental impacts. The research includes a complementary set of ethnographic methods, such as participant observation, semi-structured interviews, and focus groups with diverse stakeholders in the construction industry. These stakeholders include real estate developers, architects, urban designers, building and labor contractors, government bureaucrats, and sand traders. Findings from this study will address the imbalance between rapid urban economic growth in developing contexts and the social and environmental impacts of concrete, the primary raw material for construction in many settings.

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

PPG Title: New Approaches for Understanding Lipid Movement in Health and Disease SUMMARY/ABSTRACT Our Program Project Grant (PPG) focuses on lipid metabolism and transport, with the goal of defining mechanisms for metabolic and cardiovascular disease. Our PPG team has made seminal discoveries in lipid metabolism and transport. We discovered an endothelial cell protein, GPIHBP1, that transports lipoprotein lipase (LPL) to the capillary lumen and stabilizes the structure of LPL. Recently, we defined the structure of the GPIHBP1?LPL complex, providing fresh insights into mutations causing hypertriglyceridemia and opening the door to understanding mechanisms that regulate intravascular lipolysis. In the realm of cholesterol metabolism, our PPG discovered that macrophages release, by plasma membrane (PM) budding, particles that are enriched in cholesterol. Our PPG uncovered a link between inflammatory signaling and cholesterol metabolism in macrophages, and we identified a new protein, Aster-B, that is critical for cholesterol movement between the PM and the endoplasmic reticulum (ER). A deficiency of Aster-B impairs cholesterol movement to the ER, causing a striking upregulation of lipid biosynthetic genes. These discoveries, all relevant to the pathogenesis of atherosclerosis, were utterly dependent on collaborations between our PPG leaders and the advanced molecular, biochemical, and imaging capabilities in their laboratories. As we look to the future, we will dig deeper into the molecules and mechanisms that we have uncovered. In project 1, Drs. Young and his PPG colleagues will use biochemical and biophysical tools to elucidate the functions of the LPL?GPIHBP1 complex, including the role of GPIHBP1?s acidic domain in stabilizing LPL activity and capturing LPL within the subendothelial spaces. They will also study, with electron microscopy and NanoSIMS imaging, the budding of cholesterol-rich particles from the macrophage PM. They will define the composition of the particles and explore their relevance to reverse cholesterol transport. In project 2, Dr. Bensinger and coworkers will determine how inflammatory signals modulate the lipidome of macrophages. They will also define mechanisms by which alterations in cholesterol homeostasis affect STING signaling and the impact of the STING pathway on dyslipidemia, inflammation, and atherogenesis. In Project 3, Dr. Tontonoz and his PPG coworkers will explore the role of Aster-B in cholesterol transport, efflux, and esterification and elucidate the function of Aster-B in sterol transport in vivo. They will also assess the contribution of the ?macrophage Aster pathway? to atherosclerosis and screen for additional proteins required for the nonvesicular transport of cholesterol within cells. The three component projects will be supported by a single scientific core, led by Dr. Loren Fong and colleagues. They will produce recombinant proteins, provide advanced microscopy services, including NanoSIMS imaging of lipids. They will also work with Dr. Keriann Backus to provide chemical proteomics for investigating lipid metabolism. Our PPG is confident in success because we have exciting hypotheses and we work as a team at both scientific and technical levels.