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Hyaluronidase 1 Deficiency Preserves Endothelial Function and Glycocalyx

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Hyaluronidase 1 Deficiency Preserves Endothelial Function and Glycocalyx Page 1 of 32 Diabetes Hyaluronidase 1 deficiency preserves endothelial function and glycocalyx integrity in early streptozotocin-induced diabetes Sophie Dogné,1 Géraldine Rath,2 François Jouret,3 Nathalie Caron,1 Chantal Dessy,2* and Bruno Flamion,1* 1 Molecular Physiology Research Unit, NARILIS, University of Namur, Namur, Belgium; 2 Pole of Pharmacology and Therapeutics, IREC, Université catholique de Louvain, Brussels, Belgium; 3 Groupe Interdisciplinaire de Génoprotéomique Appliquée (GIGA), Cardiovascular Sciences, University of Liège, Liège, Belgium. * These authors contributed equally to the study Corresponding author: Sophie Dogné, Laboratory of Physiology & Pharmacology, University of Namur, 61 rue de Bruxelles, 5000 Namur, Belgium; Phone: +3281725660; Fax: +3281724329; [email protected] Short running title: HYAL1 deficiency protects the endothelium Word count of main text (max 4000 words): 4052 Number of Tables: 1 Number of Figures: 7 Diabetes Publish Ahead of Print, published online May 31, 2016 Diabetes Page 2 of 32 ABSTRACT (max. 200 words): 193 Hyaluronic acid (HA) is a major component of the glycocalyx involved in vascular wall and endothelial glomerular permeability barrier. Endocytosed hyaluronidase HYAL1 is known to degrade HA into small fragments in different cell types including endothelial cells. In diabetes, the size and permeability of the glycocalyx are altered. In addition, type 1 diabetic patients present increased plasma levels of both HA and HYAL1. To investigate the potential implication of HYAL1 in the development of diabetes-induced endothelium dysfunction, we measured endothelial markers, endothelium-dependent vasodilation, arteriolar glycocalyx size, and glomerular barrier properties in wild-type and HYAL1 knockout (KO) mice with or without streptozotocin-induced diabetes. We observed that, 4 weeks after streptozotocin injections, the lack of HYAL1: 1) prevents diabetes-induced increases in soluble P-selectin concentrations and limits the impact of the disease on endothelium-dependent hyperpolarization (EDH)-mediated vasorelaxation; 2) increases glycocalyx thickness and maintains glycocalyx structure and HA content during diabetes; 3) prevents diabetes-induced glomerular barrier dysfunction assessed using urinary albumin/creatinine ratio and urinary 70/40-kDa dextran ratio. Our findings suggest that HYAL1 contributes to endothelial and glycocalyx dysfunction induced by diabetes. HYAL1 inhibitors could be explored as a new therapeutic approach to prevent vascular complications in diabetes. INTRODUCTION The glycosaminoglycan hyaluronic acid (HA), or hyaluronan, is a major component of the extracellular matrix. HA mediates cell-cell and cell-matrix interactions and plays key roles in cell migration, tumor growth and progression, inflammation, and wound healing (1). HA is synthesized at the plasma membrane by different HA synthases and degraded by a family of Page 3 of 32 Diabetes endoglucosaminidases named hyaluronidases, mainly HYAL1 and HYAL2 in somatic tissues (2). HYAL1 is the only hyaluronidase present in human and mouse plasma (3). In all cell types, its enzymatic activity occurs at pH levels <4.0, which requires the enzyme to undergo endocytosis (4). HYAL1 deficiency in humans is a rare disease; it is associated with bone erosions, synovitis, and polyarthritis together with high plasma HA levels (5). A mouse model of HYAL1 deficiency showed HA accumulation in serum without gross abnormalities except for a loss of proteoglycans in knee joints (6). In the vascular network, HA is a major component of the endothelial glycocalyx alongside heparan sulfate and chondroitin sulfate-containing proteoglycans (7). In the glycocalyx, HA binds to its receptor CD44 but has no covalent linkage and may freely exchange with the bloodstream. The glycocalyx is recognized as a major factor in vascular physiology and pathology; it contributes to shear force sensing and transduces these forces into intracellular responses, such as NO release (7). The glycocalyx also acts as a regulator of vascular permeability, a reservoir for various antithrombotic factors, and an anti-adhesive barrier for leukocytes (8). Through in vivo perfusion of hyaluronidase, which removes all HA in the endothelial surface layer, HA has been found to be essential to maintain glycocalyx integrity and functional barrier (9). Hyaluronidase infusion also abolishes the NO-dependent response to increased shear stress in segments of pig iliac artery or dog coronary arteries but not the acetylcholine- induced NO production (10,11). In patients with Type 1 and Type 2 diabetes, endothelial dysfunction appears to be a consistent finding underlying the pathophysiology of macro- and microvascular complications, and therefore contributes to the increased mortality rates observed in the diabetic population. Glycocalyx defects may play a central role in diabetes pathogenesis by Diabetes Page 4 of 32 contributing to the pro-inflammatory state implicated in impaired skin wound healing and atherosclerosis (12). Indeed, the glycocalyx itself is disturbed during both acute (13) and chronic hyperglycemia in man (14,15). In addition, both Type 1 and Type 2 diabetic patients have increased plasma HA levels (15,16) and hyaluronidase activity (14,15). To date, the implication of elevated plasma HYAL1 and/or HA levels in the pathogenesis of diabetes remains unexplored. As plasma HYAL1 is endocytosed into endothelial cells and could therefore modulate their function possibly through glycocalyx regulation, we decided to investigate the potential role of HYAL1 in the development of diabetes-induced endothelial dysfunction. To this aim, diabetes was induced in wild-type and HYAL1 KO mice using streptozotocin (STZ) injections, and endothelial-dependent vasorelaxation, circulating endothelial markers, and the size and HA content of the glycocalyx were measured. RESEARCH DESIGN AND METHODS Animals. All experiments were performed on 7- to 9-week-old male C57Bl/6 (WT) mice and B6.129X1-Hyal1tm1Stn/Mmcd (Hyal1-/- or KO) mice obtained from MMRRC (Mutant Mouse Regional Resource Centers, USA) backcrossed onto a C57Bl/6 genetic background for 9 generations. The animals were fed regular chow and tap water ad libitum. All experiments were approved by the local animal ethics committees of the University of Namur and the Université catholique de Louvain (2012/UCL/MD/004). Type 1 diabetes was induced by 5 daily intraperitoneal injections of 55 mg/kg STZ in 10mM citrate buffer, pH 4.5. Control mice received buffer alone. Four weeks after treatment, glycemia was measured using One Touch Vita test strips (LifeScan Europe, Zug, Switzerland, limited to an upper value of 600 mg/dl). Animals with glycemia ≥300 mg/dl were assigned to the diabetic groups for the experiments. Mean arterial blood pressure was measured using a Page 5 of 32 Diabetes noninvasive computerized tail-cuff method (CODA, Kent Scientific, Torrington, CT) in non- anesthetized mice after acclimation (17). Chemical assays. Blood was collected through cardiac puncture into 0.2% EDTA tubes. Soluble intercellular cell adhesion molecule-1 (sICAM1), vascular cell adhesion molecule-1 (sVCAM1), and P-selectin (sP-selectin) were quantified using ELISA kits, and HA using an ELISA-like assay that allows detection of HA molecules ≥15 kDa (18), all obtained from R&D Systems, Minneapolis, MN. Syndecan-1 was measured using an ELISA kit from Diaclone (Besançon, France). Albumin and creatinine concentrations were measured in urine samples using Albuwell (Exocell, Philadelphia, PA) and Creatinine (Enzo Life Sciences, Lausen, Switzerland) kits, respectively. Preparation of aortic samples. Aortas were isolated, cleaned of fat on ice, frozen in liquid nitrogen, and stored at -80°C. They were then lyophilized during 16h and treated with Pronase (at 3mg/ml in 100 mM ammonia/formic acid buffer, pH 7-8) for 24h at 55°C. After relyophilization, the samples were resuspended in water to allow HA measurement. In some experiments, aortas were first flushed on ice with a strong injection of cold PBS (2ml) by holding their extremity on the needle using pliers. Hyaluronidase activity. Plasma HYAL1 activity was measured using two different approaches: a) zymography in renatured and native conditions, as described previously (4); b) gel electrophoresis (19) followed by quantification of oligosaccharide bands using image-J (public domain, NIH). Glycocalyx staining in myocardial arterioles. The method followed a procedure previously described (20). Briefly, the aorta of anesthetized mice was retrogradely cannulated and the vena cava transsected. The following solutions were infused at a flow rate of 0.4 ml/min and a pressure of 33±5 mmHg: a cardioplegic solution during 3 min; a phosphate buffered 4% paraformaldehyde – 1% glutaraldehyde (pH 7.4) fixative solution for 2 min, and finally, the Diabetes Page 6 of 32 same solution containing 0.05% Alcian Blue 8GX (Sigma-Aldrich) during 30 min. The left ventricular wall was cut in 2-mm segments, fixed for 1h in the fixative solution, and postfixed in 1% osmium tetroxide and 1% lanthanum nitrate for 1h, then processed for transmission electronic microscopy using a standard procedure. Sections were visualized with a FEI Tecnai microscope and photomicrographs analyzed using image-J. The glycocalyx thickness of cardiac arterioles was calculated by dividing the surface area by the underlying endothelium length. Dextran excretion. Anesthetized mice were intravenously injected with a mixture of 10 mg/ml Texas Red-40kDa
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