Genetic analysis of heparan sulfate in vascular biology

硫酸乙酰肝素在血管生物学中的遗传分析

基本信息

批准号：
7510385
负责人：
Jeffrey D Esko
金额：
$ 28.48万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7510385
关键词：
Acetylgalactosamine Adoptive Transfer Alleles Allergic Contact Dermatitis Alteplase Anabolism Antithrombins Arts Binding Binding Sites Biochemical Biological Assay Biology Bleeding time procedure Blood Chemical Analysis Blood Vessels Blood coagulation Bone Marrow Transplantation Breeding Cells Characteristics Chondroitin Sulfate A Chondroitin Sulfates Cutaneous Deacetylase Defect Depressed mood Dermal Dermatan Sulfate Development Disaccharides Endopeptidases Endothelial Cells Endothelium Enzyme Inhibitor Drugs Enzyme Inhibitors Enzymes Evaluation Event Exons Extracellular Matrix Extracellular Matrix Proteins Family Fibrinolysis Fibroblast Growth Factor Fibroblast Growth Factor 2 Fibroblasts Gene Deletion Genes Genetic Recombination Genomics Glucuronic Acid Glucuronic Acids Glycosaminoglycans Goals Growth Factor Hematology Hemostatic function Heparan Sulfate Proteoglycan Heparin Heparin Binding Heparitin Sulfate Histology Iduronic Acid In Vitro Infection Inflammation Inflammatory Response Inorganic Sulfates Intervention Invasive Isoenzymes Laboratories Leukocytes Libraries Ligands Modeling Modification Molecular Mus Mutant Strains Mice Mutation Numbers Oxazolone Penetrance Peptide Hydrolases Peritonitis Pharmacologic Substance Physiology Plasminogen Inactivators Play Principal Investigator Program Research Project Grants Protein C Protein-Carbohydrate Interaction Proteoglycan Purpose Range Reaction Relative (related person)Reporter Role Serpins Site Skin Staging Streptococcus pyogenes Structure System TFPI Techniques Technology Testing Thioglycolates Thrombus Time Tissues Transfection Transgenic Mice Unspecified or Sulfate Ion Sulfates Wound Healing angiogenesis blastocyst cDNA Library chemokine cytokine embryonic stem cell epimerase epimerization genetic analysis heparan sulfate L-iduronyl 2-O-sulfotransferase heparan sulfate-N-deacetylase-N-sulfotransferase in vivo keratinocyte growth factor mature animal member mutant neutrophil null mutation programs research study sulfation sulfotransferase tool vector

项目摘要

Endothelial cells express heparan sulfate proteoglycans that bind to plasma and extracellular matrix proteins through carbohydrate-protein interactions. These interactions have led to the hypothesis that endothelial cell proteoglycans play important roles in vascular biology and hemostasis. The objective of this project is to test this idea by genetically altering endothelial HS in mice. The specific genes that will be studied include two N-deacetylase/N-sulfotransferase isozymes (NDST1 and NDST2) that initiate the modification reactions, the uronosyl 2-O-sulfotransferase (HS2OST) and glucosaminyl 6-O-sulfotransferase-1 (HS6OST1), which generate the binding sites for ligands on the chains. Systemic inactivation of NDST1 and HS2OST in mice have shown that they are essential for early development, making it difficult to assess their role in physiology in adult animals. Therefore, our plan consists of examining mutant mice with selective endothelial-cell null mutations in these genes, using the Cre-loxP recombination system. Mutant alleles of NDST1 have already been made, and targeting of HS2OST and HS6OST1 are in different stages of development. Targeted deletion of these genes in endothelial cells and leukocytes will be accomplished by breeding to Tie2Cre mice. For each mutant, the degree of penetrance of the mutations will be assessed in isolated cells, and the proteoglycan and glycosaminoglycan composition will be analyzed. Bone marrow transplantation and adoptive transfer experiments will allow us to study heparan sulfate in both the endothelium and in leukocytes. To examine the role of heparan sulfate in hemostasis, blood coagulation, thrombus formation and fibrinolysis will be examined, with particular emphasis on heparin-binding proteinases (Protein C and tissue plasminogen activator) and serpins (antithrombin, tissue factor pathway inhibitor, and plasminogen activator inhibitor-I). Initial experiments indicate that the bleeding time is altered in NDST1 deficient mice, which may reflect alterations in one or more of these factors. Inflammatory responses appear to be depressed in the mutant as well, in models of thioglycollate-induced peritonitis, oxazolone-induced allergic contact dermatitis, and excisional wound healing. To study these systems further, the effect of altering heparan sulfate on P- and Lselectin and neutrophil chemokines KC and MIP-2 will be examined. The long range goal of these studies is to determine the role of heparan sulfate in vascular biology, with the purpose of defining potential targets for pharmaceutical intervention.

内皮细胞通过碳水化合物 - 蛋白质相互作用表达与血浆和细胞外基质蛋白结合的肝蛋白聚糖。这些相互作用导致了一个假设，即内皮细胞蛋白聚糖在血管生物学和止血中起重要作用。该项目的目的是通过基因改变内皮HS来测试这一想法在老鼠中。 The specific genes that will be studied include two N-deacetylase/N-sulfotransferase isozymes (NDST1 and NDST2) that initiate the modification reactions, the uronosyl 2-O-sulfotransferase (HS2OST) and glucosaminyl 6-O-sulfotransferase-1 (HS6OST1), which generate the binding sites for ligands on the链。 NDST1和HS2OST在小鼠中的全身失活表明，它们对于早期发育至关重要，因此很难评估它们在成年动物中的生理学作用。因此，我们的计划包括使用CRE-LoxP重组系统在这些基因中检查具有选择性内皮细胞无效突变的突变小鼠。 NDST1的突变等位基因已经制造，HS2OST和HS6OST1的靶向处于不同的发展阶段。这些基因的目标缺失内皮细胞和白细胞将通过繁殖到扎2切小鼠来完成。对于每个突变体，将在分离的细胞中评估突变的渗透率，并将分析蛋白聚糖和糖胺聚糖组成。骨髓移植和产卵实验将使我们能够在内皮和白细胞中研究硫酸盐。为了检查硫酸乙酰肝素在止血中的作用，将检查血液凝结，血栓形成和纤维蛋白溶解，特别强调肝素结合蛋白酶（蛋白C和组织纤溶酶原激活剂）和蛇毒蛋白酶（抗凝结物，抗凝结物，组织因子因子途径途径途径途径抑制剂，纤溶酶蛋白激活剂Inbimogentor-i Inbibintor-i）。最初的实验表明，在NDST1缺乏的小鼠中，出血时间发生了变化，这可能反映了其中一个或多个因素的改变。在突变体中，炎症反应似乎也抑制了硫代糖果诱导的腹膜炎，恶唑酮诱导的过敏性接触性皮炎和解除伤口愈合的模型。为了进一步研究这些系统，将研究改变硫酸乙酰肝素对P-和Lselectin和中性粒细胞趋化因子KC和MIP-2的影响。这些研究的远距离目标是确定硫酸乙酰肝素在血管生物学中的作用，目的是确定药物干预的潜在靶标。