title: Loss of function in heparan sulfate elongation genes EXT1 and EXT 2 results in improved nitric oxide bioavailability and endothelial function.
abstract: BACKGROUND
  Heparanase is the major enzyme involved in degradation of endothelial heparan sulfates, which is associated with impaired endothelial nitric oxide synthesis. However, the effect of heparan sulfate chain length in relation to endothelial function and nitric oxide availability has never been investigated. We studied the effect of heterozygous mutations in heparan sulfate elongation genes EXT1 and EXT2 on endothelial function in vitro as well as in vivo.
  
  
  RESULTS
  Flow-mediated dilation, a marker of nitric oxide bioavailability, was studied in Ext1(+/-) and Ext2(+/-) mice versus controls (n=7 per group), as well as in human subjects with heterozygous loss of function mutations in EXT1 and EXT2 (n=13 hereditary multiple exostoses and n=13 controls). Endothelial function was measured in microvascular endothelial cells under laminar flow with or without siRNA targeting EXT1 or EXT2. Endothelial glycocalyx and maximal arteriolar dilatation were significantly altered in Ext1(+/-) and Ext2(+/-) mice compared to wild-type littermates (glycocalyx: wild-type 0.67±0.1 μm, Ext1(+/-) 0.28±0.1 μm and Ext2(+/-) 0.25±0.1 μm, P<0.01, maximal arteriolar dilation during reperfusion: wild-type 11.3±1.0%), Ext1(+/-) 15.2±1.4% and Ext2(+/-) 13.8±1.6% P<0.05). In humans, brachial artery flow-mediated dilation was significantly increased in hereditary multiple exostoses patients (hereditary multiple exostoses 8.1±0.8% versus control 5.6±0.7%, P<0.05). In line, silencing of microvascular endothelial cell EXT1 and EXT2 under flow led to significant upregulation of endothelial nitric oxide synthesis and phospho-endothelial nitric oxide synthesis protein expression.
  
  
  CONCLUSIONS
  Our data implicate that heparan sulfate elongation genes EXT1 and EXT2 are involved in maintaining endothelial homeostasis, presumably via increased nitric oxide bioavailability.
authors.affiliation: Department of Vascular Medicine, AMC-UvA, Amsterdam, The Netherlands (M., B.M., U., G., K., D.T., S., N.).
authors.forename: H L
authors.initials: HL
authors.lastname: Mooij
authors.affiliation: Department of Bioengineering, UC San Diego, CA (C., T., I.).
authors.forename: P
authors.initials: P
authors.lastname: Cabrales
authors.affiliation: Department of Vascular Medicine, AMC-UvA, Amsterdam, The Netherlands (M., B.M., U., G., K., D.T., S., N.).
authors.forename: S J
authors.initials: SJ
authors.lastname: Bernelot Moens
authors.affiliation: Department of Cellular and Molecular Medicine, UC San Diego, CA (X., E.).
authors.forename: D
authors.initials: D
authors.lastname: Xu
authors.affiliation: Department of Vascular Medicine, AMC-UvA, Amsterdam, The Netherlands (M., B.M., U., G., K., D.T., S., N.).
authors.forename: S D
authors.initials: SD
authors.lastname: Udayappan
authors.affiliation: Department of Bioengineering, UC San Diego, CA (C., T., I.).
authors.forename: A G
authors.initials: AG
authors.lastname: Tsai
authors.affiliation: Department of Orthopedics, Leiden University Medical Center, Leiden, The Netherlands (S.).
authors.forename: M A J
authors.initials: MA
authors.lastname: van der Sande
authors.affiliation: Department of Vascular Medicine, AMC-UvA, Amsterdam, The Netherlands (M., B.M., U., G., K., D.T., S., N.).
authors.forename: E
authors.initials: E
authors.lastname: de Groot
authors.affiliation: Department of Bioengineering, UC San Diego, CA (C., T., I.).
authors.forename: M
authors.initials: M
authors.lastname: Intaglietta
authors.affiliation: Department of Vascular Medicine, AMC-UvA, Amsterdam, The Netherlands (M., B.M., U., G., K., D.T., S., N.).
authors.forename: J J P
authors.initials: JJ
authors.lastname: Kastelein
authors.affiliation: Department of Vascular Medicine, AMC-UvA, Amsterdam, The Netherlands (M., B.M., U., G., K., D.T., S., N.).
authors.forename: G M
authors.initials: GM
authors.lastname: Dallinga-Thie
authors.affiliation: Department of Cellular and Molecular Medicine, UC San Diego, CA (X., E.).
authors.forename: J D
authors.initials: JD
authors.lastname: Esko
authors.affiliation: Department of Vascular Medicine, AMC-UvA, Amsterdam, The Netherlands (M., B.M., U., G., K., D.T., S., N.).
authors.forename: E S
authors.initials: ES
authors.lastname: Stroes
authors.affiliation: Department of Vascular Medicine, AMC-UvA, Amsterdam, The Netherlands (M., B.M., U., G., K., D.T., S., N.).
authors.forename: M
authors.initials: M
authors.lastname: Nieuwdorp
mesh_terms: D000328:Adult; D000818:Animals; D001916:Brachial Artery / Q000201:enzymology* / Q000503:physiopathology; D016022:Case-Control Studies; D002460:Cell Line; D004730:Endothelium, Vascular / Q000201:enzymology* / Q000503:physiopathology; D005097:Exostoses, Multiple Hereditary / Q000175:diagnosis / Q000201:enzymology* / Q000235:genetics* / Q000503:physiopathology; D005260:Female; D020022:Genetic Predisposition to Disease; D019276:Glycocalyx / Q000201:enzymology; D006579:Heterozygote; D006801:Humans; D008297:Male; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008875:Middle Aged; D009154:Mutation; D017351:N-Acetylglucosaminyltransferases / Q000172:deficiency / Q000235:genetics*; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D010766:Phosphorylation; D014162:Transfection; D014664:Vasodilation; D000099065:Exostosin 2; D000099058:Exostosin 1
chemical_list: D009569:Nitric Oxide; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D017351:N-Acetylglucosaminyltransferases; D000099058:Exostosin 1; D000099065:Exostosin 2
keywords: EXT; endothelial function; heparan sulfate; nitric oxide