Lijun Xia - US grants

intravital microscopy; biomedical facility; cell cell interaction; bioimaging /biomedical imaging; clinical research;

enzyme activity; galactosyltransferases; enzyme mechanism; clinical research; laboratory mouse;

Serine- or threonine-linked oligosaccharides (O-glycans) are commonly found on membrane and secreted proteins. The functions of this form of posttranslational modification are not well understood. Our experiments indicate that there is a fundamental requirement for core 1-derived O-glycans during angiogenesis. Because vessel development is essential to many physiologic and pathologic processes such as cancer, ischemic diseases, and chronic inflammation, elucidating the role of O-glycans in angiogenesis will potentially provide new insight into numerous disease processes and may lead to novel therapeutic approaches to many common human diseases. O-glycans with GalNAc in a1 linkage to serine or threonine have four main core structures. Among them, core 1 and 2 are widely expressed in many tissues. Formation of the core 1 structure (GalB1-3GalNAc-), the precursor for the core 2 structure and for many extended O-glycans (core 1-derived O-glycans), is catalyzed by the enzyme core 1 pi ,3-galactosyltransferase (T-synthase). We have engineered mice that are globally deficient for T-synthase (T-syn[-/-]). The T-syn[-/-] mice developed brain hemorrhage that was uniformly fatal by embryonic day 14. The T-syn[-/-] brains formed a disorganized microvascular network with distended endothelial cells and defective association of endothelial cells with pericytes, extracellular matrix and neural tissues. These data reveal a novel requirement for core 1-derived O-glycans during angiogenesis. We have developed mice with tissue-specific T-syn deficiencies using Cre/loxP technology. We propose to investigate the role of core 1-derived O-glycans in vascular development through three specific aims: Aim 1: Identify cell types requiring O-glycans for angiogenesis. Blood vessels are composed of endothelial cells and mural cells (pericytes and vascular smooth muscle cells) embedded in the extracelluar matrix and surrounding tissues. O-glycans associated with any of these cell types may contribute to the defective angiogenesis observed in the T-syn[-/-] mice. We will use mice with endothelial cell-specific, neural cell-specific, and mural cell-specific T-syn deficiencies to identify the cell type(s) that cause defective angiogenesis when lacking T-synthase. In addition, we have developed transgenic mouse lines expressing T-synthase specifically in endothelial cells. By breeding these mice with T-syn[-/-] mice, we will examine whether the endothelial cell specific expression of T-synthase can rescue the T-syn[-/-] embryonic lethality. Aim 2: Characterize the functional defect of T-syn[-/-] endothelial cells during angiogenesis. Endothelial cells are essential in angiogenesis. During angiogenesis, endothelial cells undergo multiple processes including migration, proliferation, apoptosis, and specialization. Our published and preliminary experiments suggest that O-glycans in endothelial cells play a key role during vessel formation. We will use in-vivo assays such as retina angiogenesis and tumor angiogenesis to identify specific defect(s) of T-syn[-/-] endothelial cells in vascular development. In addition, we have developed T-syn[+/+]and T-syn[-/-] endothelial cell lines to evaluate the function of T-syn[-/-] endothelial cells using in-vitro assays such as endothelial cell tube formation. Aim 3: Analyze the profiles and function of O-glycans in endothelial cells. We will combine lectins and mAb screening, as well as HPLC and mass spectrometry, to profile the O-glycan structures in T-syn[-/-] and T-syn[-/-] endothelial cells. We will investigate how differences in O-glycan expression affect endothelial cell function using in-vitro angiogenesis assay.

DESCRIPTION (provided by applicant): Mucin-type O-glycans are primary components of the colonic mucus gel layer. Altered intestinal O- glycosylation has been observed in patients with ulcerative colitis (UC), but whether this alteration is an etiological factor is unknown. O-glycans consist mainly of core 1- and core 3-derived O-glycans. The biosynthesis of these two types of O-glycans is controlled by core 11,3-galactosyltransferase (T-synthase) and Core 31,3-glucosaminyltransferase (C3GnT), respectively. T-synthase function requires a specific chaperone, Cosmc. Human Cosmc is on the X-chromosome, increasing the significance of somatic mutations in this gene. In preliminary studies, we detected somatic mutations in the Cosmc gene in DNA, isolated from colonic epithelial cells, expressing abnormal O-glycans from UC patients. We hypothesize that altered O-glycans impair mucus barrier function, which in turn allows intestinal microflora to interact abnormally with epithelium and mucosal immune cells, thus causing colitis. To test this hypothesis, we developed mice lacking either core 1- or combined core 1- and core 3-derived O-glycans (IEC T-syn-/- and DKO mice, respectively). In addition, we have also developed mice with tamoxifen (TM) inducible deletion of T-syn in intestinal epithelial cells (TM-IEC T-syn-/-). These mice develop spontaneous colitis, which is associated with a massive granulocyte infiltration and cryptic abscesses, closely resembling active human UC. Significantly, IEC T-syn-/- and DKO mice in the Rag1-/- background, who lack adaptive immunity, manifested similarly severe colitis, suggesting an essential role for innate immune cells such as granulocytes in colitis development. This supports an etiological role for O-glycans in colitis and provides a unique model system to test whether altered O-glycans is a potential molecular mechanism in the pathogenesis of human UC. We propose to 1) analyze how abnormal O-glycosylation impairs the expression of intestinal mucins and add additional patient samples to improve the statistical power of our preliminary observations that Cosmc mutations cause abnormal expression of colon epithelial O-glycans in UC patients;2) determine the role of O- glycans in intestinal barrier function, investigate changes in bacterial variety or density in O-glycan-deficient mice before and after disease onset by phylogenetic analysis, and test definitively the role of microbiota in colitis development by developing germ-free O-glycan-deficient mice;and 3) identify mechanisms initiating granulocyte infiltration and determine the role of granulocytes in colitis. Our proposed studies will reveal novel insights into the pathogenesis of colitis and may lead to new therapies. PUBLIC HEALTH RELEVANCE: Ulcerative colitis (UC) is a chronic inflammation of the large intestine with an unknown cause or cure that can last years to decades. UC often leads to physical as well as psychological discomfort and even disability. Altered expression of large sugar molecules called O-glycans in the large intestine is seen in UC patients. However, whether this alteration causes the disease is unknown. The proposed project is poised to provide novel insights into the role of intestinal mucin-type O-glycans in intestinal mucus barrier function and in pathogenesis of the common human disease. This work may lead to a novel therapy for patients with UC.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The lymphatic system originates from the blood vascular system. Lymphatic endothelial cells (LECs) differentiate from blood endothelial cells (BECs), and this lineage decision as well as the maintenance of the LEC phenotype are tightly controlled processes, which is essential for development and functions of the two discrete vascular compartments. The transcription factor Prox1 is a binary regulator that promotes LEC lineage identity and suppresses BEC phenotype. However, how it functions is not well understood. We observed that mice lacking podoplanin, which is highly expressed in LECs, exhibit blood-filled lymphatic vessels that closely resemble those seen in time-specific deletion of Prox1 (inducible Prox1[unreadable]/[unreadable]) mice. We hypothesize that podoplanin, like Prox1, is critical in regulating endothelial cell identity. To test this, we propose two aims. Aim 1 will determine the embryonic and postnatal stages at which podoplanin controls LEC identity using mice with time-specific podoplanin deficiency in endothelial cells. Aim 2 will determine whether forced expression of podoplanin suppresses BEC identity in vivo using a transgenic line that over-expresses podoplanin in BECs and LECs (TetO-PdpnEC). Increased expression of Prox1 and podoplanin is associated with malignant transformation of BEC-derived angiosarcoma with a mixed BEC and LEC identity. We will investigate whether over-expression of podoplanin in endothelial cell lines (EOMA and Py-4-1) induces formation of aggressive angiosarcoma in vivo.

DESCRIPTION (provided by applicant): This renewal proposal for our Program Project Grant continues to employ an interdisciplinary approach to study the functions of protein-glycan interactions in the vascular system. The three thematically related projects are led by investigators with complementary expertise and a strong published record of collaborative research. Project 1 uses gene-targeted mice to study the interplay of adhesion and signaling molecules in the vasculature. The emphasis is on processes that regulate the interactions of selectins with their glycoconjugate ligands, a critical early response during inflammation and thrombosis. Mice with altered expression of selectins, selectin ligands, or signaling proteins will be studied. Project 2 studies the interactions of galectins with leukocytes. Major themes include the defining the glycan structures recognized by each galectin, the signaling mechanisms for the novel exposure of phosphatidylserine on activated neutrophils without apoptosis, and the biological roles of galectins in regulating inflammation in vivo. Project 3 studies how the 0-glycoprotein podoplanin maintains separated blood and lymphatic vessels, how mixing of blood and lymphatic vessels contributes to disease, and how 0-glycosylation regulates the functions of podoplanin in vitro and in vivo. There is unusually high synergy among the projects that results from the intellectually overlapping themes and the sharing of reagents and methods. An administrative core cements the interactions, in particular through maintenance of a server for data exchange among the projects and through computer support for processing images and other complex data. The data obtained will enhance our understanding of the functions of lectins and glycoconjugates during inflammation, thrombosis, and vascular development. This information may suggest new approaches to treat heart attacks, strokes, and other cardiovascular disorders.

? DESCRIPTION (provided by applicant): During development, angiogenesis mediated by vascular endothelial growth factor (VEGF) signaling is extremely active in the central nervous system (CNS). The newly formed vessel is often immature and sensitive to bleeding and thus requires special mechanisms to maintain its endothelial barrier stability. However, known mechanisms for vascular integrity, such as blood-brain-barrier function, are not fully developed before birth. Additional mechanisms for vascular stability during CNS development remain elusive. We have recently identified a novel mechanism of platelet activation in which the O-glycoprotein podoplanin (PDPN) activates platelet C-type lectin-like 2 (CLEC-2) receptors. Our preliminary experiments show that PDPN is expressed specifically in neural cells surrounding vessels in the early developing CNS. Mice lacking PDPN or CLEC-2 (Pdpn-/- or Clec-2-/-) develop CNS-specific hemorrhages primarily during early embryonic development. Furthermore, mice with conditional deletion of PDPN in neural cells also exhibit spontaneous brain bleeding. These preliminary results support a novel hypothesis that neural cell PDPN-mediated platelet activation is essential for the stability of nascent vessels in the early developing CNS. To test this hypothesis, we will determine 1) whether PDPN-CLEC-2-mediated platelet activation protects integrity of newly formed vessels in the early developing CNS. We found that PDPN is expressed on neural cells closely associated with vessels in the early developing CNS and that platelets are present outside vessels. These data support a novel hypothesis that interactions between PDPN on perivascular neural cells and CLEC-2 on extravasated platelets are essential for vascular integrity of newly formed vessel during early CNS development. We will test this using the state-of-the-art two-photon confocal imaging microscopy and vascular permeability assays; we will determine 2) whether sphingoshine 1-phosphate (S1P) released from PDPN-CLEC-2-activated platelets balances VEGF action to maintain vascular stability in the developing CNS. We hypothesize that release of S1P from platelets after PDPN-CLEC-2-mediated activation is essential for vascular endothelial barrier stability by balancing the functin of VEGF, and will use mice lacking S1P functions and pharmacological approaches to address this question. If the proposed studies support our hypotheses, it will define a novel mechanism of tissue-specific platelet activation in regulation of vascular integrity in the developing CNS. Identification of such a new mechanism may provide new insights into brain bleeding disease, such as germinal matrix- intraventricular hemorrhage (GMH-IVH), which affects ~35% of premature human infants. In addition, Pdpn-/- or Clec-2-/- mice may be used as a valuable model for testing novel therapies promoting vascular integrity that target hemorrhage in the developing brain.

PROJECT SUMMARY: The Cardiovascular Phenotyping Core was added during Phase II of the OMRF ?Interdisciplinary Research in Vascular Biology? COBRE grant in response to growing demand from COBRE investigators for scientific and technical expertise in developing cardiovascular disease models and conducting specialized pathophysiological tests. The Phase III Cardiovascular Phenotyping Core objective is to expand resources, services, and training available to COBRE and non-COBRE investigators for establishing precise cardiovascular and metabolic phenotypes for their genetically-altered mice. All COBRE investigators use gene- targeted mice and mouse models of cardiovascular disease as major research approaches to elucidate the molecular mechanisms of disease and generate novel discoveries in cardiovascular biology. This year, we significantly strengthened the Core capabilities by purchasing a cutting-edge VisualSonics Vevo-2100 Ultrasound Imaging System. With institutional support, we upgraded our Hemavet analyzer for blood cell count and differentials, and purchased a new Catalyst Dx Chemistry Analyzer that permits automated analysis of a broad spectrum of clinical chemical parameters of mouse blood and urine samples. The Core will provide a centralized system for accessing this state-of-the-art technology for evaluating complex cardiovascular disease pathologies to COBRE and non-COBRE investigators and will be guided by 3 specific aims: 1) Provide services of echocardiographic and Doppler imaging of cardiovascular structures and functions of genetically modified mice using Vevo-2100 system; 2) Provide consultation, training, genetic resources, and special instrumentation for general phenotyping, including peripheral blood cell and chemistry analyses, and sophisticated mouse cardiovascular procedures/disease models. The Core will maintain colonies of valuable mouse genetic tools (e.g. inducible or tissue-specific Cre or reporter lines) for genetic analysis of cardiovascular phenotypes; 3) Develop Core into sustainable facility that continues to provide services to COBRE and external cardiovascular investigators after Phase III funding. To achieve the Aims, the Core will provide COBRE and external investigators: 1) direct ultrasound imaging services, 2) technical training to use validated cardiovascular phenotyping techniques and instruments, 3) service for procedures requiring highly specialized skills, and 4) mouse genetic tools. A Core Coordinator will be designated for service requests and conduct regular surveys of end users to evaluate quality, turn-around, service costs, and training effectivness: this will be evaluated quarterly by Core Directors and annually by the External Advisory Committee, providing accountability for the Core's success. The Aims will be implemented under highly qualified director/co-director leadership and dedicated expert technical staff. By offering unique training and collaborative opportunities, we expect to develop this Core into a sustainable facility and further nurture establishment of an internationally- recognized center of excellence.

? DESCRIPTION (provided by applicant) Principal signs of inflammation include edema and bleeding, indicators of impaired vascular integrity. Vascular leakage often leads to impaired wound healing, and delayed clearance of pathogens, even hypotension and organ failure during severe systemic inflammation. Platelets prevent microvessels from leakage during inflammation. However, how platelets safeguard vascular integrity remains unclear. Our preliminary studies show that mice lacking platelet C-type lectin-like receptor 2 (CLEC-2) and its ligand podoplanin have increased vascular leakage in a model of lipopolysaccharide (LPS)-induced endotoxemia. These data support the novel hypothesis that interaction of platelet CLEC-2 with podoplanin protects vascular integrity in systemic inflammation. In this proposal, we will test this hypothesi by determining: 1) whether and if so, how platelet CLEC-2 interacts with perivascular podoplanin to protect vascular integrity during inflammation. Our preliminary data on confocal imaging detected staining for platelets on the abluminal side of venules and staining for podoplanin on perivascular macrophages during inflammation. These data suggest that interactions of CLEC-2 on extravasated platelets with podoplanin on perivascular macrophages limit vascular leakage during inflammation. In addition, platelets interact with leukocytes during inflammation. We will use spinning-disk and two-photon confocal intravital microscopy to determine whether platelets emigrate with or follow emigrating leukocytes through venules to interact with perivascular podoplanin. Models of systemic inflammation (such as cecal ligation and puncture) will be used to determine whether platelet CLEC-2 and perivascular podoplanin protect vascular integrity in different organs. 2) How platelets activated by podoplanin maintain vascular integrity during inflammation. We hypothesize that podoplanin-CLEC-2 interactions induce local platelet secretion of S1P that activates S1PR1 on endothelial cells to stabilize endothelial adherens junctions during inflammation. We will examine whether deficient podoplanin-CLEC-2 interactions exacerbate or platelet S1P ameliorates vascular leakage and organ failure in systemic inflammation. We will determine whether platelet S1P protects vascular integrity by maintaining endothelial adherens junctions during inflammation. Our preliminary data show that platelets, after podoplanin-CLEC-2-mediated activation, also release angiopoietin 1 (Ang1), another mediator of vascular integrity. Therefore, we will determine whether platelet S1P and Ang1 function cooperatively to protect vascular integrity during inflammation. These studies will provide key mechanistic insights into how platelet CLEC-2 limits vascular permeability during inflammation. Our results could offer exciting translational applications, for example, S1P receptor agonists or related drugs to restore endothelial barrier function in inflammatory diseases such as sepsis.

PROJECT SUMMARY/ABSTRACT Arterial thrombotic diseases such as ischemic heart disease are the leading cause of disability and death in the United States. Platelet adhesion and formation of thrombotic platelet aggregates at the site of a ruptured atherosclerotic plaque or damaged endothelium under arterial blood flow is essential in the pathogenesis of arterial thrombosis. Under high or disturbed flow conditions, the initial interaction between platelets and the vessel wall is primarily mediated by von Willebrand factor (vWF) and platelet glycoprotein Iba (GPIba), which subsequently leads to platelet content release, aggregation, and activation of the coagulation. These mechanisms, which are critical for both hemostasis and thrombosis, are targets of current FDA-approved antiplatelet therapies. Although they are effective, all have the life-threatening side effect of causing bleeding, which significantly limits their clinical use. To address this unmet need, it is critical to further elucidate insights into mechanisms essential for thrombosis but dispensable for hemostasis. Recent published data from several independent labs show that platelet CLEC-2 (C-type lectin-like receptor 2) is important in arterial thrombosis. However, how CLEC-2 regulates arterial thrombosis is unknown. The lectin-domain of CLEC-2 is known to bind to sialylated O-glycans. Our preliminary data showed that CLEC-2 interacts with GPIba in a sialylation-dependent manner. Furthermore, our preliminary results reveal that CLEC-2 promotes GPIba- mediated activation of integrin ?IIb?3, which is critical for arterial thrombus growth and stability in vivo. Importantly, blocking CLEC-2 function does not prolong the bleeding time in vivo. Therefore, we hypothesize that CLEC-2 is critical for GPIba-mediated platelet activation that is required for arterial thrombus growth and stability. To test this, we will 1) test the hypothesis that CLEC-2 regulates GPIba-mediated platelet activation through interaction between its lectin-like domain and sialylated O-glycans of GPIba as GPIba is heavily modified by sialylated O-glycans; 2) determine if/how CLEC-2 stabilizes the arterial thrombus by facilitating GPIba-mediated integrin aIIbb3 activation using mouse and human arterial thrombosis models. CLEC-2 and GPIba are expressed at similar high levels on murine platelets, and both receptors are essential in arterial thrombosis. However, the mechanisms underlying their role in arterial thrombosis have been either elusive (GPIba) or unknown (CLEC-2). Our proposed study will provide new mechanistic insights into these outstanding questions in the field. It may lead to the development of a new effective and safe anti-thrombosis therapy.

The lymphatic vascular system is essential for transporting interstitial fluid, dietary fat, and immune cells. Defects  in these functions contribute to lymphedema, impaired lipid absorption, obesity, abnormal immune function, and  cancer metastasis. During embryonic development, lymphangiogenesis is robust, primarily driven by vascular  endothelial growth factor C (VEGF-­C)-­mediated activation of VEGFR-­3, a main VEGF-­C receptor on lymphatic  endothelial cells (LECs). Emerging evidence has shown the metabolism of endothelial cells is critical for vascular  development. Changes in EC metabolic pathways are found in pathologies such as cancer and diabetes as well. But  most research has been focused on blood endothelial metabolic pathways. Despite a few recent pioneering studies,  knowledge of LEC metabolism during lymphangiogenesis is limited. There is an unmet need to bridge the knowledge  gap between cellular metabolism and lymphatic vascular development. Site-­1 protease (S1P), encoded by  membrane-­bound transcription factor peptidase, site 1 (MBTPS1), is a serine protease in the Golgi apparatus. S1P is  a key regulator of cholesterol biosynthesis by proteolytic activation of a membrane-­bound latent transcription factor,  sterol-­regulatory element binding protein 2 (SREBP2). Recently, we found that mice with inducible endothelial cell-­ specific deficiency of S1P (iEC Mbtps1-­/-­, Mbtps1f/f;?Cdh5CreERT2) exhibited severe subcutaneous lymphedema and  defective lymphatic vasculature during development. Our pilot experiments also showed that mice with LEC-­specific  deficiency of SREBP2 (LEC Srebf2-­/-­, Srebf2f/f;?Lyve1Cre) had a similar lymphatic vascular defect during  development. These strong in vivo preliminary data support the central hypothesis that S1P/SREBP2-­mediated  cholesterol biosynthesis is required for lymphatic vascular development.  We will test the central hypothesis through two Aims: 1) determine whether lymphatic endothelial S1P/SREBP2-­ mediated cholesterol biosynthesis is required for lymphatic vascular development. We will characterize LEC cellular  defects, such as differentiation, migration, and proliferation, of S1P or SREBP2-­deficient mice at different stages of  embryonic development. These in vivo analyses will be complemented by in vitro assays using LECs isolated from  wild-­type (WT) or mutant mice as well as primary human LECs;? 2) determine mechanisms by which S1P/SREBP2-­ mediated cholesterol biosynthesis regulate lymphangiogenesis. Based on our preliminary results, we will primarily  test the hypothesis S1P/SREBP2-­mediated cholesterol biosynthesis is required for sustained VEGFR3 signaling  mainly by in vitro assays using WT or mutant LECs as well as human LECs with knockdown of S1P/SREBP2 or  functional inhibitors to S1P and SREBP2.  Based on strong preliminary data, our proposed study will reveal novel insights into roles of S1P-­mediated lipid  metabolism in lymphatic vascular development. Our study may lead to novel therapeutic opportunities for  pathologies with lymphatic vascular defects.    

The primary purpose of this COBRE Phase I application, ?Center for Cellular Metabolism Research in Oklahoma (CMRO),? is to establish a center of cellular metabolism research excellence at the Oklahoma Medical Research Foundation (OMRF). Cellular metabolism is one of the fastest moving areas of biomedical research; however, it has not been sufficiently integrated in Oklahoma. To address this unmet need, this COBRE will offer a critical opportunity to unify OMRF?s metabolism-related resources and expertise to support five newly recruited junior faculty and to foster increased cellular metabolism research and multidisciplinary scientific interactions overall in Oklahoma. Our goal is to establish a recognized center of excellence in cellular metabolism research. In support of these goals, the Administrative Core will provide centralized management and serve as the coordinating resource through five Aims: 1) Provide central management and logistical support for the COBRE; 2) Provide mentoring for the Project Leaders, and assist with their transition to stable independence; 3) Administer the Pilot Project program; 4) Promote multidisciplinary approaches to the research of and interactions among the COBRE investigators; and 5) Provide fiscal management, ensure compliance with regulatory issues, and promote data sharing strategies. The Administrative Core will specifically ensure easy access to outstanding Core facilities and a network of support that would be difficult to replicate elsewhere. This COBRE will have a long-lasting impact on biomedical research in Oklahoma.

PROJECT SUMMARY/ABSTRACT OMRF operates three mouse barrier facilities with the only germ-free mouse facility (GFM) and patient-derived xenograft core (PDX) in Oklahoma. The research programs of all five Research Project Leaders (RPLs) on this new COBRE use mouse models, which make up approximately 15% of the daily mouse population of the OMRF mouse facilities. As these investigators grow their research programs in the next five years, this percentage is expected to increase to ~22%. In addition, the laboratories of the CMRO COBRE mentors also utilize mouse models, and the mentors and RPLs combined currently constitute almost 60% of the census. Reliable sterilization of mouse caging and ancillary equipment is essential for mouse barrier facilities, especially those housing immunodeficient and germ-free mice. OMRF?s current sterilizer is 20 years old and is failing. To address this, OMRF plans to replace the failing sterilizer and add a second sterilizer for future expansion. This supplement requests partial support of the sterilizing equipment. OMRF will provide 50% of the total cost for this equipment. The requested equipment is critical for reproducible results for research of the CMRO COBRE RPLs and their mentors as well as animal health and well-being. Additionally, this equipment will also benefit other COBRE and COBRE-eligible investigators from additional institutions in Oklahoma (University of Oklahoma Health Sciences Center and Oklahoma State University), as the GFM and PDX facilities are not found elsewhere in the state.