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The Roles of Glycans in Hemostasis, Inflammation and Vascular Biology

Executive Summary

Purpose

The Division of Blood Diseases and Resources of the National Heart, Lung, and Blood Institute (NHLBI) convened a Working Group of scientific investigators on February 25-26, 2008, in Bethesda, Maryland, to identify scientific opportunities and priorities, emerging from the recent explosion of technological and biological advances in the glycosciences. Aligned with the NHLBI Strategic Plan Goals 1, 2 and 3 (NHLBI Strategic Plan), the focus of this group was on "The Roles of Glycans in Hemostasis, Inflammation and Vascular Biology".

Background

As noted during the discussion, the first major collaborative efforts on glycomics began with the formation of the Consortium for Functional Glycomics funded by NIGMS in 2001. The program has been successful and has been awarded funding through 2011. Nonetheless, the consortium has focused on cataloging structures of glycans in selected cell types and organs using profiling techniques of mass spectrometry, developing core facilities to characterize phenotypes of mice engineered for deficiencies in glyco-related genes, and creating specialized microarrays for studies of gene expression.

While all of the above developments are important and address interesting topics of basic scientific importance, none of them are directed towards gaining a fundamental understanding of the role of glycans in human diseases particularly disorders of heart, lung and blood. Further, there has been no program focus to capitalize the information for diagnostics and developments of new treatments. The working group was charged to identify key research areas as well as to prioritize the most fruitful areas for basic, pre-clinical, and clinical research.

Discussion Overview

Glycosylation is the most common and abundant post-translational modification of proteins and lipids. Glycans affect most major developmental, biological and pathological processes, in multitudinous ways. Members of the working group first gave scientific presentations, which highlighted some unexpected and provocative new discoveries on the biological roles of Glycans. These stimulating presentations were followed by intense and thought-provoking discussions as detailed below.

The committee extensively discussed the delay in our ability to capitalize on opportunities in the field due to technological challenges that are inherent to glycan studies, and the dearth of investigators with expertise in the area. They emphasized that such challenges can be overcome and that the time is ripe for NHLBI, to exploit newly identified opportunities by applying an interdisciplinary approach to glycan research incorporating cross-cutting research themes that address blood and vascular diseases. The committee also stated that recent advances and breakthroughs in the technologies and knowledge base for studying the structure and function of glycans now allow "Glycobiology" to become an integral part of the genomic and post-genomic revolution. Indeed, as emphasized by the welcoming message to the group from NHLBI Deputy Director Dr. Susan Shurin, the full potential of the genomic and proteomic tools and knowledge bases recently generated by NHLBI funding will not be realized without an equally strong emphasis on glycomics.

Research Opportunities and Priorities

The committee identified many research opportunities and priorities regarding the impact of glycans in hemostasis, inflammation and vascular biology, and recommended developing new initiatives to catalyze the field of glycosciences. They emphasized that multi-PI R01 collaborative research projects between investigators having expertise in glycans and those who address biological questions in blood and vascular diseases will be the key to success in translational studies.

It was suggested that initiatives in one or more areas should catalyze advances in our knowledge far beyond where the field is currently. Some of the more remarkable areas along with specific examples are listed below:

  • Thrombotic disorders, e.g. effects of glycosylation on coagulation factor and platelet half-life and clearance.
  • Reperfusion injury, e.g. potential therapeutic value of non-anticoagulant glycosaminoglycans.
  • Sickle cell disease, e.g. role of P-selectin in mediating erythrocyte adhesion in vasoocclusive crises.
  • Hematopoietic disorders, e.g. the deficiency of glycophospholipid anchors in paroxysmal nocturnal hemoglobinuria.
  • Hematopoietic stem cells, e.g. use of in vitro glycan modification to optimize homing to the bone marrow.
  • Transfusion medicine, e.g. the key role of glycans as blood group antigens; the use of glycosidases to generate "universal donor" cells; and, the use of galactosyltransferase to increase the shelf-life of donor platelets.
  • Chronic inflammation, e.g. lectin-dependent activation of macrophages and dendritic cells.
  • Cancer associated coagulopathies, e.g. role of mucins and heparin in Trousseau's disease.
  • Glycan-based targeting, e.g. use of glycan-coated particles for gene therapy, drug delivery and imaging.
  • Vascular remodeling and angiogenesis, e.g. the critical role of O-glycosylation in endothelial cell development.
  • Tissue engineering, e.g. use of glycan-based biodegradable scaffolds.
  • Glycan-related therapeutics, e.g. PSGL-1 to improve graft recovery following transplantation.
  • Glycans as potential biomarkers, e.g. serum levels of mannose-binding protein as predictors of autoimmune vasculitis.
  • Atherosclerosis e.g. multiple roles of Glycans in the initiation and progression of coronary artery disease, stroke and peripheral vascular disease.

It is important to emphasize that opportunities for major breakthroughs are not limited to the examples given above. Many areas of potential importance remain completely unexplored e.g. effects of chronic hypertension on glycans that could affect vascular wall resistance; and potential polymorphisms in the enzymes, critical glycosylation sites or glycan-binding proteins that might correlate with propensity to disease.

Publication Plans

NHLBI website; publication in a scientific journal

Participating Institutes

In addition to NHLBI program officials from DBDR and DCVD, the meeting was well attended by NHLBI and NCI Frederick intramural scientists, CSR Scientific Review Officers, and extramural program officials from NCI, NIGMS, and NIAID. While, some invitees could not attend the working group meeting, these individuals were subsequently sent a copy of this report and asked to serve as 'Consulting Advisors". Their verbatim responses are appended at the end of this report.

Committee Report and Commentaries

The working group met at the request of Dr. Rita Sarkar, NHLBI to discuss the importance of glycans to the mission of the Institute, with a specific emphasis on their roles in hemostasis, inflammation and vascular biology. The members of the working group initially gave scientific presentations that included:

  • Chronic inflammation due to evolutionary loss of Siglec expression on human leukocytes
  • Incorporation of the non-human sialic acid Neu5Gc into human endothelia from dietary sources, with the potential for vascular inflammation caused by anti-Neu5Gc antibodies
  • Identification of heparin sulfate as the long-sought receptor for plasma lipoprotein clearance
  • Heparan sulfate mediated chemokine transcytosis and presentation during inflammation and leukocyte trafficking
  • Heparan sulfate dependence for angiogenesis
  • Chemoenzymatic synthesis of pharmaceutical heparins
  • Carbohydrate-based nanotechnology
  • Non-thrombogenic heparinized biomaterials
  • Biochemical and genetic regulation of protein glycosylation in blood leukocytes and endothelial cells
  • Roles of glycosylation in hematopoiesis, and in leukocyte trafficking during inflammation and thrombosis
  • Roles of Cosmc molecular chaperone in regulating O-glycosylation, and the role in disorders such as Tn polyagglutinability syndrome, IgA nephropathy, etc.
  • Recognition and signaling in blood cells through glycan recognition by the galectin, siglec and selectin families of glycan-binding proteins
  • Roles of the O-GlcNAc nuclear-cytosolic protein modification in diabetes-associated vascular disease and glucose toxicity
  • Role of O-GlcNAc in myocardial protection following ischemic injury
  • Control of T cell development by O-linked fucosylation of Notch
  • Modulation of myelopoiesis by O-linked fucosylation of Notch
  • Control of leukocyte trafficking by sialylated, fucosylated Glycans
  • Regulation of monocyte integrin function by variant sialylation
  • Role of BACE1 secretase in macrophage adhesion
  • Galectin regulation of dendritic cell migration into sites of inflammation
  • Galectins as "alarmins" to initiate sterile inflammation
  • Glycans as receptors for viral vectors for gene transfer in hematopoietic stem cells
  • Role of glycans in hematopoietic stem cell homing and engraftment

These scientific presentations exemplify a recent dramatic enhancement of our understanding of the cellular and molecular biology of glycans in nature, and their roles in evolution, development, physiology and pathology. Despite these exciting and unanticipated developments, there remains a gulf between this new knowledge and its applications to the biomedical sciences. One major reason for this anomaly is that the methodologies for glycan analysis have been more difficult to develop than those for DNA, RNA and proteins. A second reason is that there are currently a very small number of NIH-funded investigators studying glycans, resulting in slow pace of discovery relative to other molecules of biomedical importance, such as DNA, RNA and proteins.

Despite recent advances in understanding the role of glycosylation in themes relevant to the NHLBI; most active biomedical scientists have a limited knowledge and expertise in glycosciences. Thus, although the great majority of current NHLBI-funded projects involve molecules that contain and/or recognize glycans, most investigators typically do not address this key aspect of their systems. Consequently, at the very moment when major advances in understanding the roles of glycans in blood and vascular diseases are possible, there is a lack of investigators pursuing these opportunities.

Clearly, there is a need for funding mechanisms that will bring together the few existing experts in the study of glycans with investigators in blood and vascular diseases, to foster fruitful collaborations that will bridge the existing gulf. This frontier area is also ripe for translational research and drug development. The situation is different in many European and Asian countries e.g. Sweden, Australia and Japan, where there are major nationwide commitments to, and interest in the study of glycans, thus, the U.S. lags behind the rest of the world in taking advantage of these opportunities.

The study of glycans in blood and vascular diseases fits the recently defined three strategic goals of the NHLBI namely, Goal 1: To increase understanding of the molecular and physiological basis of health and disease and use that understanding to improve diagnosis, treatment, and prevention. Goal 2: To improve understanding of the clinical mechanisms of disease and thereby enable better prevention, diagnosis, and treatment. Goal 3: To translate research into practice for the benefit of personal and public health.

In this report, we focus on three specific areas of relevance to these NHLBI goals: the roles of glycans in hemostasis, inflammation and vascular biology. Some classic examples are the interactions of antithrombin with the anticoagulant heparin, and the roles of selectins in initiating leukocyte trafficking. In addition to the short-term goal of fostering collaborative interdisciplinary research in such areas of special interest to NHLBI, this report addresses the long-term need to educate the next generation of investigators, who must effectively integrate the study of glycans into their explorations of physiology and disease. Such measures will undoubtedly facilitate the overall NHLBI goal of improving diagnosis, treatment and prevention of diseases.

Existing Challenges

As discussed above, there are numerous exciting opportunities, but there are also challenges. The current generation of established researchers is largely unfamiliar with glycan structure and biology, and the related methodologies and terminology. Consequently, many opportunities for discovery are missed, and there is limited potential for younger generation of biomedical scientist to become exposed to this field. There is also a paucity of study section reviewers with expertise in glycans and awareness of their important biological roles. Given these issues, the group feels that the most acute need is to expand the pool of NHLBI-supported investigators who routinely incorporate the study of glycans into their research programs.

Suggested Funding Mechanisms

Some NIH institutes, namely NIGMS, NCI and NCRR, have already established consortia, and centers for technology and reagent development, and for core resources to perform glycan research analysis. Given the availability of these resources, the NHLBI is poised to take advantage of them to address novel and important questions regarding the roles of glycans in hemostasis, inflammation and vascular biology. The committee recommends an RFA supporting multi-PI R01-style collaborative grants, which would originate from 2-3 investigators with complementary expertise in glycoscience and in blood and vascular diseases. The objective is to promote synergistic interactions that could lead to groundbreaking discoveries. Examples of complementary fields of expertise include bioengineering, biophysics, synthetic chemistry, structural biology, hematology, vascular biology, hematopathology, etc. The mechanisms for review and evaluation of proposals should involve experts in both the glycosciences, as well as those in the relevant complementary field. The immediate outcome in implementing such an initiative is that it will amplify the pool of investigators at the interface between glycosciences and hemostasis, inflammation and vascular biology. An added benefit will be the education of trainees that work between the collaborating laboratories. Overall, this approach will have a major impact on American public health by opening new frontiers in diagnosis, treatment and prevention of numerous diseases involving hemostasis, inflammation and vascular biology.

Additional Comments from Consulting Advisors - Verbatim Responses

Comments from David Ginsburg, University of Michigan, Ann Arbor

This report summarizes an exciting meeting, bringing together an outstanding group of leading investigators in the field. The discussions identified limited expertise in glycan biology a deficiency of training opportunities in this area and offered recommendations for future initiatives from NHLBI to attract additional investigators and collaborative efforts into this field. I am happy to add my support to these proposals.

Comments from Richard Hynes, MIT, Boston

I agree that glycosylation is important in many biological phenomena, not least in vascular biology - both normal and pathological. The technologies for analyzing and manipulating these complex post-translational modifications have improved greatly and there is a definite opportunity in bringing together those who know how to do this (chemists, biochemist, mass spec etc) with the biologist and clinicians who understand the relevant biological systems. The likely outcomes from such interactions would include both improved understanding of the functions of glycans as well as insight into therapeutic application of this understanding to diseases of the vasculature. I think P01 (or clustered R01) grants to foster collaborations at this interface is a very sound one.

Comments from Stuart Kornfeld, Washington University School of Medicine, St. Louis

I believe the report of the NHLBI working group does a good job in pointing out the involvement and importance of glycans and glycan-recognizing molecules in many aspects of hemostasis, inflammation and vascular biology and in articulating the reasons why studies in this area lag other areas of research. Bringing together glycoscientist with experts in blood and vascular diseases has the potential to generate major advances in these fields. The idea of starting with appropriate RFA is asound one as it should be an effective way to stimulate work in this area. I congratulate the working group on their constructive report.

Comments from Thomas Stossel, Brigham & Women's Hospital, Boston

My recent brief fling with glycobiology now taken over by my colleague Dr. Karin Hoffmeister concerning how glycans appear to influence platelet survival with exquisite specificity and daunting complexity persuades me that the opportunities summarized in this excellent meeting report are absolutely valid and that the suggestions for capitalizing on the opportunities make sense. I also agree with the commentaries that better standardization of nomenclature and a focus on quantitative methodologies are important priorities for the field.

Comments from Erkki Ruoslahti, The Burnham Institute, La Jolla

I recruited a glycobiology group to the Burham Institute 25 years ago, and my assessment of the potential of this field has not changed in the intervening years. As stated in this report, glycan-directed studies may well be underrepresented in the current mix of academic research. The reason may be that glycans are primarily modulators of functions of proteins, DNA, lipids, and cellular structures, such as membranes, rather than the primary players. Nonetheless, the fine tuning glycans provide is critical, particularly in many aspects of vascular physiology and disease. The parallel with proteomics is compelling. The RFA mechanism proposed in the report would be a good way of making the vascular disease research community aware of the possibilities offered by current glycan research.

Comments from Michael Gimbrone, Harvard Medical School, Boston

As is well illustrated by this Working Group's report, the field of glycobiology clearly represents an exciting, and as yet under-realized, opportunity to cross-cut multiple areas of importance to the NHLBI. It provides a whole new context in which to view the post-translational modification of the transcriptome and its myriad implications for health and disease. The proposed RFA structure, which would combine investigators with complementary expertise in glycosciences with those in blood and vascular diseases, would help overcome the somewhat isolated nature of the field, and hopefully lead to translationally useful insights.

Comments from Ulrich von Andrian, Harvard Medical School, Boston

The working group report lucidly presents the current state and future challenges of the "Glycomics" field in the United States. More structured emphasis on this important area through the ROI mechanism, backed by adequate funding, could go a long way to broaden the scope and depth of researchers in this field. The working group report has identified several major areas of emphasis in the context of hemostasis, inflammation and vascular biology. One additional aspect that could be considered, especially in the context of transfusion medicine and chronic inflammatory diseases, are the mechanisms by which carbohydrates and glycoconjugates induce and/or modify adaptive immune responses. Such mechanisms may involve direct cognate recognition by lymphocytes or indirect interactions with antigen-presenting cells.

NHLBI Contacts

Rita Sarkar, Ph.D., NHLBI, NIH
Email: sarkarr@nhlbi.nih.gov
Telephone: 301-435-1324

Working Group Members

Chair: Ajit Varki, M.D., University of California, San Diego

Co-Chairs: Linda Baum, M.D., Ph.D., University of California, Los Angeles

Susan Bellis, Ph.D, University of Alabama, Birmingham

Members:

Richard D. Cummings, Ph.D., Emory University, Atlanta
Robert J. Linhardt, Ph.D., Rensselaer Polytechnic Institute
Jeffrey D. Esko, Ph.D., University of California, San Diego
Gerald W. Hart, Ph.D., Johns Hopkins University, Baltimore
Arun Srivastava, Ph.D., University of Florida, Gainesville
Rodger McEver, M.D., Ph.D., Oklahoma Med Res Foundation, Oklahoma
Thomas Stossel, M.D., Brigham & Women's Hospital, Boston
John Lowe, M.D., Ph.D., Case Western Reserve University, Cleveland

Consulting Advisors

David Ginsburg, M.D., University of Michigan, Ann Arbor
Richard Hynes, Ph.D., MIT, Boston
Stuart Kornfeld, M.D., Washington University School of Medicine, St. Louis
Thomas Stossel, M.D., Brigham & Women's Hospital, Boston
Erkki Ruoslahti, M.D., The Burnham Institute, La Jolla
Michael Gimbrone, M.D., Harvard Medical School, Boston
Ulrich von Andrian, M.D., Ph.D., Harvard Medical School, Boston

Last updated: March 19, 2008

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