FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2009253716> ?p ?o ?g. }

• W2009253716 endingPage "528" @default.
• W2009253716 startingPage "520" @default.
• W2009253716 abstract "Hyaluronidase (HYAL) 2 is a membrane-anchored protein that is proposed to hydrolyze hyaluronan (HA) to smaller fragments that are internalized for breakdown. Initial studies of a Hyal2 knock-out (KO) mouse revealed a mild phenotype with high serum HA, supporting a role for HYAL2 in HA breakdown. We now describe a severe cardiac phenotype, deemed acute, in 54% of Hyal2 KO mice on an outbred background; Hyal2 KO mice without the severe cardiac phenotype were designated non-acute. Histological studies of the heart revealed that the valves of all Hyal2 KO mice were expanded and the extracellular matrix was disorganized. HA was detected throughout the expanded valves, and electron microscopy confirmed that the accumulating material, presumed to be HA, was extracellular. Both acute and non-acute Hyal2 KO mice also exhibited increased HA in the interstitial extracellular matrix of atrial cardiomyocytes compared with control mice. Consistent with the changes in heart structure, upper ventricular cardiomyocytes in acute Hyal2 KO mice demonstrated significant hypertrophy compared with non-acute KO and control mice. When the lungs were examined, evidence of severe fibrosis was detected in acute Hyal2 KO mice but not in non-acute Hyal2 KO or control mice. Total serum and heart HA levels, as well as size, were increased in acute and non-acute Hyal2 KO mice compared with control mice. These findings indicate that HYAL2 is essential for the breakdown of extracellular HA. In its absence, extracellular HA accumulates and, in some cases, can lead to cardiopulmonary dysfunction. Alterations in HYAL2 function should be considered as a potential contributor to cardiac pathologies in humans. Hyaluronidase (HYAL) 2 is a membrane-anchored protein that is proposed to hydrolyze hyaluronan (HA) to smaller fragments that are internalized for breakdown. Initial studies of a Hyal2 knock-out (KO) mouse revealed a mild phenotype with high serum HA, supporting a role for HYAL2 in HA breakdown. We now describe a severe cardiac phenotype, deemed acute, in 54% of Hyal2 KO mice on an outbred background; Hyal2 KO mice without the severe cardiac phenotype were designated non-acute. Histological studies of the heart revealed that the valves of all Hyal2 KO mice were expanded and the extracellular matrix was disorganized. HA was detected throughout the expanded valves, and electron microscopy confirmed that the accumulating material, presumed to be HA, was extracellular. Both acute and non-acute Hyal2 KO mice also exhibited increased HA in the interstitial extracellular matrix of atrial cardiomyocytes compared with control mice. Consistent with the changes in heart structure, upper ventricular cardiomyocytes in acute Hyal2 KO mice demonstrated significant hypertrophy compared with non-acute KO and control mice. When the lungs were examined, evidence of severe fibrosis was detected in acute Hyal2 KO mice but not in non-acute Hyal2 KO or control mice. Total serum and heart HA levels, as well as size, were increased in acute and non-acute Hyal2 KO mice compared with control mice. These findings indicate that HYAL2 is essential for the breakdown of extracellular HA. In its absence, extracellular HA accumulates and, in some cases, can lead to cardiopulmonary dysfunction. Alterations in HYAL2 function should be considered as a potential contributor to cardiac pathologies in humans. Hyaluronidase 2 is a glycophosphatidylinositol-linked protein and a member of the hyaluronoglucosaminidase family (1Lepperdinger G. Müllegger J. Kreil G. Hyal2–less active, but more versatile?.Matrix Biol. 2001; 20: 509-514Crossref PubMed Scopus (140) Google Scholar, 2Andre B. Duterme C. Van Moer K. Mertens-Strijthagen J. Jadot M. Flamion B. Hyal2 is a glycosylphosphatidylinositol-anchored, lipid raft-associated hyaluronidase.Biochem. Biophys. Res. Commun. 2011; 411: 175-179Crossref PubMed Scopus (43) Google Scholar). It is proposed to initiate the degradation of hyaluronan (HA), 3The abbreviations used are: HAhyaluronanGAGglycosaminoglycanECMextracellular matrixHYALhyaluronidaseMPSmucopolysaccharidosisKOknock-outHABPHA-binding proteinα-SMAα-smooth muscle actin. an abundant glycosaminoglycan (GAG) in the extracellular matrix (ECM) of many vertebrate tissues (including heart valves), vitreous of the eye, and synovial fluid. The viscoelastic properties of HA influence the properties of the ECM and are important for cell proliferation, differentiation, and migration. A role for HA in embryogenesis has been established using HA synthase 2-deficient mice, which die at embryonic day 9.5 due to loss of endocardial cushion swelling and a lack of epithelial-to-mesenchymal transition (3Camenisch T.D. Spicer A.P. Brehm-Gibson T. Biesterfeldt J. Augustine M.L. Calabro Jr., A. Kubalak S. Klewer S.E. McDonald J.A. Disruption of hyaluronan synthase-2 abrogates normal cardiac morphogenesis and hyaluronan-mediated transformation of epithelium to mesenchyme.J. Clin. Invest. 2000; 106: 349-360Crossref PubMed Scopus (715) Google Scholar). hyaluronan glycosaminoglycan extracellular matrix hyaluronidase mucopolysaccharidosis knock-out HA-binding protein α-smooth muscle actin. Regulation of HA levels is required for normal development and to maintain normal tissue homeostasis. Degradation of HA by hyaluronidases (HYALs) is important for maintaining these levels. Six HYAL-encoding genes have been identified, which are grouped into two tightly linked triplets, one on human chromosome 3p21.3 (HYAL1, HYAL2, and HYAL3) and one on human chromosome 7q31.3 (HYALP1, HYAL4, and SPAM1) (4Csóka A.B. Scherer S.W. Stern R. Expression analysis of six paralogous human hyaluronidase genes clustered on chromosomes 3p21 and 7q31.Genomics. 1999; 60: 356-361Crossref PubMed Scopus (207) Google Scholar). With the exception of HYAL4 and HYALP1, all of the HYALs are thought to be capable of degrading HA. A role for HYAL1 and HYAL2 in HA degradation in somatic cells has been proposed. In this model, HYAL2 initiates HA degradation into small fragments that are endocytosed and degraded in lysosomes by HYAL1 and exoglycosidases (5Hascall V. Sandy J.D. Handley C.J. Caterson B. Archer C.W. Benjamin M. Ralphs J. Biology of the Synovial Joint. Harwood Academic Publishers, The Netherlands1999: 101-120Google Scholar, 6Stern R. Devising a pathway for hyaluronan catabolism: are we there yet?.Glycobiology. 2003; 13: 105R-115RCrossref PubMed Scopus (288) Google Scholar). HYAL2 is a glycosylphosphatidylinositol-anchored protein (2Andre B. Duterme C. Van Moer K. Mertens-Strijthagen J. Jadot M. Flamion B. Hyal2 is a glycosylphosphatidylinositol-anchored, lipid raft-associated hyaluronidase.Biochem. Biophys. Res. Commun. 2011; 411: 175-179Crossref PubMed Scopus (43) Google Scholar) that could act at low pH at the cell surface (7Bourguignon L.Y. Singleton P.A. Diedrich F. Stern R. Gilad E. CD44 interaction with Na+-H+ exchanger (NHE1) creates acidic microenvironments leading to hyaluronidase-2 and cathepsin B activation and breast tumor cell invasion.J. Biol. Chem. 2004; 279: 26991-27007Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar). It has been suggested that HYAL2 resides in lysosomes in some cell types (8Chow G. Knudson C.B. Knudson W. Expression and cellular localization of human hyaluronidase-2 in articular chondrocytes and cultured cell lines.Osteoarthritis Cartilage. 2006; 14: 849-858Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Despite the proposed role for HYAL2 in HA degradation, only a deficiency of HYAL1 has been found to cause a human disorder. It causes mucopolysaccharidosis (MPS) IX, a lysosomal storage disorder characterized by joint abnormalities due to HA accumulation (9Natowicz M.R. Short M.P. Wang Y. Dickersin G.R. Gebhardt M.C. Rosenthal D.I. Sims K.B. Rosenberg A.E. Clinical and biochemical manifestations of hyaluronidase deficiency.N. Engl. J. Med. 1996; 335: 1029-1033Crossref PubMed Scopus (145) Google Scholar, 10Triggs-Raine B. Salo T.J. Zhang H. Wicklow B.A. Natowicz M.R. Mutations in HYAL1, a member of a tandemly distributed multigene family encoding disparate hyaluronidase activities, cause a newly described lysosomal disorder, mucopolysaccharidosis IX.Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 6296-6300Crossref PubMed Scopus (167) Google Scholar). In the heart valves, GAGs are abundant and play important roles in both developing and mature valves (11Armstrong E.J. Bischoff J. Heart valve development: endothelial cell signaling and differentiation.Circ. Res. 2004; 95: 459-470Crossref PubMed Scopus (523) Google Scholar, 12Hinton R.B. Yutzey K.E. Heart valve structure and function in development and disease.Annu. Rev. Physiol. 2011; 73: 29-46Crossref PubMed Scopus (307) Google Scholar). It is not surprising that cardiovascular manifestations are a prominent feature of many forms of MPS that result from GAG accumulation (13Braunlin E.A. Harmatz P.R. Scarpa M. Furlanetto B. Kampmann C. Loehr J.P. Ponder K.P. Roberts W.C. Rosenfeld H.M. Giugliani R. Cardiac disease in patients with mucopolysaccharidosis: presentation, diagnosis and management.J. Inherit. Metab. Dis. 2011; 34: 1183-1197Crossref PubMed Scopus (187) Google Scholar). For example, mitral valve thickening and stenosis are found in Hurler and Scheie diseases, and these findings are also reflected in the corresponding mouse model (14Jordan M.C. Zheng Y. Ryazantsev S. Rozengurt N. Roos K.P. Neufeld E.F. Cardiac manifestations in the mouse model of mucopolysaccharidosis I.Mol. Genet. Metab. 2005; 86: 233-243Crossref PubMed Scopus (31) Google Scholar). Therefore, defects in ECM-modifying enzymes are among the many causes of cardiovascular disease. HA is one of the major GAGs in heart valves. Surprisingly, no broad-spectrum HA accumulation, including in the heart, was identified during the characterization of Hyal1-, Hyal2-, or Hyal3-deficient mice (15Atmuri V. Martin D.C. Hemming R. Gutsol A. Byers S. Sahebjam S. Thliveris J.A. Mort J.S. Carmona E. Anderson J.E. Dakshinamurti S. Triggs-Raine B. Hyaluronidase 3 (HYAL3) knockout mice do not display evidence of hyaluronan accumulation.Matrix Biol. 2008; 27: 653-660Crossref PubMed Scopus (55) Google Scholar, 16Jadin L. Wu X. Ding H. Frost G.I. Onclinx C. Triggs-Raine B. Flamion B. Skeletal and hematological anomalies in HYAL2-deficient mice: a second type of mucopolysaccharidosis IX?.FASEB J. 2008; 22: 4316-4326Crossref PubMed Scopus (81) Google Scholar, 17Martin D.C. Atmuri V. Hemming R.J. Farley J. Mort J.S. Byers S. Hombach-Klonisch S. Csoka A.B. Stern R. Triggs-Raine B.L. A mouse model of human mucopolysaccharidosis IX exhibits osteoarthritis.Hum. Mol. Genet. 2008; 17: 1904-1915Crossref PubMed Scopus (77) Google Scholar). Previous studies of Hyal2−/− (knock-out (KO)) mice revealed craniofacial abnormalities and chronic anemia, as well as unexplained preweaning lethality (16Jadin L. Wu X. Ding H. Frost G.I. Onclinx C. Triggs-Raine B. Flamion B. Skeletal and hematological anomalies in HYAL2-deficient mice: a second type of mucopolysaccharidosis IX?.FASEB J. 2008; 22: 4316-4326Crossref PubMed Scopus (81) Google Scholar). However, in addition to these features, on an outbred background (C129;CD1;C57BL/6), we have found a gross enlargement of either the left or right atrium in more than half of the Hyal2 KO mice that survived to weaning. Because HA synthesis is known to be essential for normal heart development, we hypothesized that a failure to degrade HA in these mice had resulted in HA accumulation, leading to cardiopulmonary dysfunction and premature death. Hyal2 KO mice were generated as part of a previous study (16Jadin L. Wu X. Ding H. Frost G.I. Onclinx C. Triggs-Raine B. Flamion B. Skeletal and hematological anomalies in HYAL2-deficient mice: a second type of mucopolysaccharidosis IX?.FASEB J. 2008; 22: 4316-4326Crossref PubMed Scopus (81) Google Scholar). For this study, Hyal2 KO mice and control littermates, either wild type (+/+) or heterozygous (+/−), on an outbred background (C129;CD1;C57BL/6) were derived through breeding of Hyal2 heterozygotes. Mice were genotyped using PCR-based strategies on DNA samples isolated from ear punches. For amplification of wild type Hyal2 and the Neo-targeted Hyal2 allele, we used forward and reverse primers (5′-actcagctgctggttcccta-3′ and 5′-atagcactggcagcgaaagt-3′; 5′-aaggaacatcagggaagatcat-3′ and 5′-cggtgcccgagactaagtc-3′, respectively). All animal procedures were conducted following protocols approved by the University of Manitoba Animal Care Committee and following the guidelines of the Canadian Council on Animal Care. Mice were killed by carbon dioxide inhalation, and tissues for light microscopy were immediately collected and fixed overnight in either formalin or 10% buffered formalin (PROTOCOL) containing 1% hexadecylpyridinium chloride monohydrate. Tissues for subsequent biochemical studies were stored at −80 °C. Fixed tissues were embedded in paraffin, and 5-μm sections were stained for morphological analysis using established methods for hematoxylin and eosin (18Allen T.C. Prophet E.B. Mills B. Arrington J.B. Sobin L.H. Laboratory Methods in Histotechnology. American Registry of Pathology, Washington, D.C.1994: 53-58Google Scholar) or for GAG detection using Alcian blue (19Gaffney E. Prophet E.B. Mills B. Arrington J.B. Sobin L.H. Laboratory Methods in Histotechnology. American Registry of Pathology, Washington, D.C.1994: 149-174Google Scholar), with minor modifications in the time of incubation with the stain. Counterstaining for Alcian blue was performed with Nuclear Fast Red (ScyTek Laboratories) for 2.5 min. Heart and lung ECM was stained using Masson's trichrome stain (Sigma-Aldrich) according to the manufacturer's protocol. Slides were dehydrated, mounted, and visualized using bright-field microscopy. For analysis of hypertrophy in cardiomyocytes, slides were incubated for 30 min with 4 μg/ml wheat germ agglutinin (Alexa Fluor® 488 conjugate), mounted using Prolong Gold (Invitrogen), and visualized by fluorescence microscopy. The area of the cardiomyocytes, as defined by binding to wheat germ agglutinin, was measured using AxioVision 4.5 software. A minimum of seven fields, containing an average of eight cells per field, was examined for each animal. The valve and surrounding myocardium were collected by punch biopsy from a whole heart that was sliced using a rodent heart slicer matrix (Zivic Instruments). Tissues were fixed for 4 h at room temperature in 2% glutaraldehyde and 2% paraformaldehyde prepared in 100 mm sodium cacodylate buffer (pH 7.2) containing 10 mm CaCl2 and 0.7% Ruthenium red. After fixation, tissues were washed with buffer containing 100 mm sodium cacodylate, 10 mm CaCl2, 0.7% Ruthenium red, and 0.7% sucrose (pH 7.2) for 5 min, 30 min, and 1 h at room temperature. Tissues were post-fixed for 1 h in 100 mm sodium cacodylate containing 1% osmium tetroxide and 0.7% Ruthenium red, followed by three 10-min washes with double-distilled H2O. Tissues were dehydrated for 10 min each in 50% ethanol, 75% ethanol, 95% ethanol, two changes in 100% ethanol followed by 100% methanol, and three changes in propylene oxide. After dehydration, tissues were infiltrated by incubation in a mixture of propylene oxide and Araldite 502 (58.9:46:2% (w/v) mixture of Araldite, dodecenyl succinic anhydride, and 2,4,6-Tris (dimethylaminomethyl) phenol-30; Ted Pella, Inc.). The infiltration solutions were prepared with propylene oxide and Araldite (3:1, 1:1, and 1:3) and incubated for 1 h each. Slow hardening of the Araldite was performed through 1-day incubations at room temperature, 45 °C, and 60 °C. HA was detected with biotinylated HA-binding protein (HABP; Calbiochem) as described previously (17Martin D.C. Atmuri V. Hemming R.J. Farley J. Mort J.S. Byers S. Hombach-Klonisch S. Csoka A.B. Stern R. Triggs-Raine B.L. A mouse model of human mucopolysaccharidosis IX exhibits osteoarthritis.Hum. Mol. Genet. 2008; 17: 1904-1915Crossref PubMed Scopus (77) Google Scholar), except without enzyme retrieval and using HABP (1.67 μg/ml for heart and 6.68 μg/ml for lungs) in Tris-buffered saline (pH 7.5) overnight at 4 °C. Sections were incubated with avidin-conjugated horseradish peroxidase, detected with diaminobenzidine, and counterstained with Nuclear Fast Red. To verify the specificity of HABP, sections were incubated overnight with 25 units/ml HYAL from Streptomyces hyalurolyticus (Sigma) before HABP detection. For detection of α-smooth muscle actin (α-SMA), F4/80, and CD31, the sections were incubated overnight at 4 °C with mouse monoclonal anti-human smooth muscle actin (1:300; Dako), rat monoclonal anti-mouse F4/80 (1:100; AbD Serotec), or rabbit polyclonal anti-CD31 (1:50; Abcam) antibody, respectively. Secondary antibodies (biotinylated rabbit anti-mouse (1:1000; Dako) or goat anti-rat (1:500; Invitrogen)) were used to detect the primary complexes, and complex detection was as described above for HA. For F4/80 and CD31, antigen retrieval was performed by incubating the slides for 20 min at 95 °C in 10 mm sodium citrate (pH 6). Blood was collected from mice immediately following euthanasia or from the saphenous vein of living mice at 6 and 12 weeks. Serum was collected from clotted blood by centrifugation at 3000 rpm for 10 min and stored at −80 °C. HA levels were quantified using an ELISA-like HA test plate (R&D Systems) according to the manufacturer's directions. All samples were analyzed in duplicate, and the average value was used for subsequent comparisons. Fluorophore-assisted carbohydrate electrophoresis was performed for quantification of HA and chondroitin sulfate in tissues (20Gushulak L. Hemming R. Martin D. Seyrantepe V. Pshezhetsky A. Triggs-Raine B. Hyaluronidase 1 and β-hexosaminidase have redundant functions in hyaluronan and chondroitin sulfate degradation.J. Biol. Chem. 2012; 287: 16689-16697Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Briefly, GAGs isolated from frozen heart tissue by 50 μg/μl proteinase K digestion and ethanol precipitation were cleaved to disaccharide units by overnight incubation with hyaluronidase SD (Seikagaku catalog no. 100741-1) or chondroitinase ABC (Sigma-Aldrich) in 200 mm ammonium acetate. The disaccharides were isolated, labeled with 2-aminoacridone, and quantified in comparison with known disaccharide standards as described previously (21Plaas A.H. West L. Midura R.J. Hascall V.C. Disaccharide composition of hyaluronan and chondroitin/dermatan sulfate. Analysis with fluorophore-assisted carbohydrate electrophoresis.Methods Mol. Biol. 2001; 171: 117-128PubMed Google Scholar). Fluorescence was detected using the Fluor-S Max MultiImager (Bio-Rad) and quantified using Quantity One 4.6.9 software (Bio-Rad). For sizing of HA, GAGs were prepared from tissue (10–15 mg) or serum (200–400 μl) as for fluorophore-assisted carbohydrate electrophoresis, except 5 mm deferoxamine was included in the proteinase K digestion, and a 4-h incubation at 37 °C with 10 units of DNase I and 50 μg of RNase A was incorporated after destroying the proteinase K by boiling. Instead of digesting the GAGs to disaccharides, they were enriched by precipitation with 1% hexadecylpyridinium chloride monohydrate as described previously (22Gordon L.B. Harten I.A. Calabro A. Sugumaran G. Csoka A.B. Brown W.T. Hascall V. Toole B.P. Hyaluronan is not elevated in urine or serum in Hutchinson-Gilford progeria syndrome.Hum. Genet. 2003; 113: 178-187Crossref PubMed Google Scholar). The pellet was then resuspended in 100 μl of 20 mm Tris (pH 8.0), and Pronase was added to 0.2 mg/ml. Following overnight incubation, the GAGs were precipitated with 900 μl of 100% ethanol. The final pellet was resuspended in 15 μl of water, 3 μl of Orange G tracking dye was added, and the sample was separated by electrophoresis on a 0.8% agarose gel prepared in Tris acetate buffer (pH 8.3). The gel was stained overnight with 0.005% Stains-all prepared in 50% ethanol, destained with 10% ethanol until the bands were clearly visible, and photographed. Negative control samples to verify the identity of the HA were prepared by incubating the GAGs overnight with 2.5 units of hyaluronidase SD before they were analyzed by electrophoresis. The genotype distributions were compared with the expected Mendelian ratios using the χ2 test. For all other statistical analysis, data are presented as means ± S.E. Means were compared by Student's t test. p ≤ 0.05 was considered statistically significant. We obtained viable Hyal2 KO mice by intercrossing Hyal2 heterozygotes (+/−); although as reported previously (16Jadin L. Wu X. Ding H. Frost G.I. Onclinx C. Triggs-Raine B. Flamion B. Skeletal and hematological anomalies in HYAL2-deficient mice: a second type of mucopolysaccharidosis IX?.FASEB J. 2008; 22: 4316-4326Crossref PubMed Scopus (81) Google Scholar), preweaning lethality was observed. In fact, only ∼9% (70 of 789) of the offspring were Hyal2 KOs at weaning instead of the expected Mendelian 25% (χ22 = 109.86; p < 0.001). Of the viable Hyal2 KO mice, 54% were smaller in size than their littermates and exhibited rapid onset lethargy, weight loss, dull coat, and shortness of breath requiring euthanasia at an average of 3.2 months of age. Upon dissection, the left or right atrium of these mice was grossly dilated (Fig. 1). The remaining 46% of Hyal2 KO mice developed a slower onset lethargy, loss of weight, and poor grooming that required euthanasia at an average of 5.8 months of age. Severe atrial dilation was not found in these mice or in control littermates (Hyal2+/+/Hyal2+/−). However, one kidney was found to be missing in 1% of control mice and 43% (n = 29) of Hyal2 KO mice, suggesting that this phenotype is increased by Hyal2 deficiency. The kidney phenotype was not further investigated, as it was found to be independent of the atrial dilation. For the studies described herein, the Hyal2 KO mice were divided into two categories: mice with atrial dilation were defined as “acute,” and those without atrial dilation were defined as “non-acute.” The atrial dilation in the acute Hyal2 KO mice, together with the knowledge that HA is abundant in the heart valve, led us to look for a valve abnormality in the Hyal2 KO mice. H&E staining revealed a profound expansion of the pulmonary, mitral, aortic, and tricuspid valves in all mice that were examined (n = 9) compared with control littermates (n = 7). Representative sections of the pulmonary and mitral valves are shown in Fig. 2 (A–D). To determine whether there was any difference in the expansion of the valves in the acute and non-acute Hyal2 KO mice, we compared the expansion/length of the valve leaflet/cups by determining the number of serial sections that included each valve. The valve thickness was significantly greater in Hyal2 KO mice than in control mice, but there was no significant difference between acute and non-acute Hyal2 KO mice (Fig. 2E). To determine whether HA was accumulating in the Hyal2 KO mice, we used HABP to detect HA. Abundant HA was found throughout the valves (Fig. 3, A–J), and given the expanded size of the valves in the Hyal2 KO mice, this indicates that HA was accumulating in the Hyal2 KO valves. To determine whether the accumulating HA was altering the organization of the ECM of the valve, we examined ECM components using Masson's trichrome stain. Compared with normal valves (Fig. 4B), those from Hyal2 KO mice were disorganized, with GAGs (white material) deposited between the collagen fibers in the fibrosa and ventricularis/atrialis layers, respectively, and resulting in an expanded spongiosa layer (Fig. 4A). Endothelial cells in the valve were identified by staining for the endothelial marker CD31. The CD31-positive cells (brown) appeared less frequently in the Hyal2 KO valve, probably because these cells were stretched over the extended surface area of the expanded valve (Fig. 4, C and D). These findings indicate that the accumulated HA in the ECM of Hyal2 KO mice leads to the expansion, thickening, and disorganization of the valve leaflet/cups. We also examined the structure of the atrial and ventricular myocardium of acute and non-acute Hyal2 KO mice and control mice for additional pathologies. Staining for total GAG with Alcian blue revealed accumulation of interstitial GAGs between the atrial cardiac myocytes (Fig. 5, A, C, and E). This staining was more prominent in the acute Hyal2 KO mice than in the non-acute Hyal2 KO mice and was always stronger in Hyal2 KO mice than in control mice, indicating that GAGs were accumulating in the KO mice. In adjacent serial sections, HA was found to be abundant in the ECM of the acute mice compared with both the non-acute and control mice (Fig. 5, B, D, and F), providing further evidence that HA was accumulating in the Hyal2 KO mice. To determine whether there were additional abnormalities in the cardiac myocytes of Hyal2 KO mice, sections were stained with fluorescently labeled wheat germ agglutinin, and the sizes of the cardiomyocytes were determined. Significant cardiac hypertrophy was found in the upper ventricular region, close to the base of the heart, of acute Hyal2 KO mice compared with control mice (Fig. 6A), but this hypertrophy did not reach significance in the lower ventricular region of acute Hyal2 KO mice (Fig. 6B) or non-acute Hyal2 KO mice (Fig. 6, C and D). To further characterize the changes in the Hyal2 KO hearts, we performed transmission electron microscopy on the valves and the ventricular and atrial myocardium. In the control valves (Fig. 7E), the ECM was densely packed with collagen fibrils, whereas in the Hyal2 KO valves (Fig. 7A), the ECM separated the collagen fibrils. The accumulated material in the Hyal2 KO valves was clearly extracellular and was presumed to be HA. The atrial myocardium of Hyal2 KO mice (Fig. 7, B and C) contained cardiomyocytes, which showed a disorganized arrangement of myofibrils and mitochondria and a substantial reduction in myofibrils. In contrast, the atrial cardiomyocytes of control mice showed a sarcoplasm with tightly packed myofibrils and mitochondria (Fig. 7, F and G). Within the ventricular myocardium, we observed cardiomyocytes with a disorganized arrangement of myofibrils and mitochondria and a more loosely packed sarcoplasm (Fig. 7D) compared with controls (Fig. 7H). To look for additional differences in acute, non-acute, and control mice that might be related to atrial dilation, we examined the lungs. A dramatic difference between the acute and non-acute Hyal2 KO lungs was identified. The alveoli were reduced, and the alveolar septa were thickened in the acute Hyal2 KO mice compared with the non-acute and control mice (Fig. 8, A–C). Further, Masson's trichrome staining revealed increased collagen in the alveolar septa of the acute mice that was absent in both the non-acute and control mice (Fig. 8, D–F). Further investigation of the thickened alveolar septa using anti-α-SMA immunostaining revealed a strong signal for α-SMA in the alveolar interstitium of acute mice that was not detected in the lungs of non-acute and control mice (Fig. 8, G–I). Increased levels of HA were also detected in the lungs of acute and non-acute Hyal2 KO mice compared with control mice (Fig. 8, J–L), although the levels were much higher in the acutely affected animals. Therefore, pathology typical of severe pulmonary fibrosis was present only in the acute Hyal2 KO mice. We observed numerous alveolar macrophages (foam cells) in the inter- and intra-alveolar spaces of acute and, to a lesser extent, non-acute Hyal2 KO mice (Fig. 8, A and B) compared with control mice, suggesting that the accumulation of HA led to the recruitment of macrophages to the lung parenchyma. To confirm that these infiltrating cells were macrophages, we used an antibody against F4/80 (Fig. 8, M–O). A deficiency of HYAL2 had already been demonstrated to lead to increased serum HA (16Jadin L. Wu X. Ding H. Frost G.I. Onclinx C. Triggs-Raine B. Flamion B. Skeletal and hematological anomalies in HYAL2-deficient mice: a second type of mucopolysaccharidosis IX?.FASEB J. 2008; 22: 4316-4326Crossref PubMed Scopus (81) Google Scholar). As expected, we found the levels of HA to be ∼19-fold higher in Hyal2 KO mice compared with control mice at 12 weeks (Fig. 9A). The levels of HA increased with age in the Hyal2 KO mice, reaching an average 27-fold increase at the time of euthanasia (Fig. 9A). Interestingly, the levels of serum HA were elevated in both acute and non-acute Hyal2 KO animals, indicating that differences in the levels of circulating HA are unlikely to be the cause of the more severe phenotype in acute Hyal2 KO animals. We also quantified the levels of HA in the hearts and lungs of Hyal2 KO mice using fluorophore-assisted carbohydrate electrophoresis. Consistent with the detection of increased HA by immunohistochemistry, elevated levels of HA were found in Hyal2 KO hearts and lungs compared with control mice (Fig. 9, B and C). No significant difference between the levels of HA in non-acute and acute Hyal2 KO mice was detected in the heart (Fig. 7B). The HA levels also appeared similar in the lungs of acute and non-acute Hyal2 KO mice, but our sample size was not large enough for statistical analysis. We also examined the chondroitin sulfate levels in the heart by fluorophore-assisted carbohydrate electrophoresis, and no significant difference between Hyal2 KO (0.91 ± 0.17 ng/mg) and control (0.77 ± 0.12 ng/mg) mice was found (n = 4; p = 0.51). Given the predicted role of HYAL2 in initiating the degradation of HA by cleavage of high molecular mass HA to smaller fragments for internalization, we expected that the HA found in the tissues of Hyal2 KO mice would have a larger molecular mass than that in control animals. To assess this, HA was partially purified from the sera and hearts" @default.
• W2009253716 created "2016-06-24" @default.
• W2009253716 creator A5010625257 @default.
• W2009253716 creator A5050522824 @default.
• W2009253716 creator A5063398884 @default.
• W2009253716 creator A5067919633 @default.
• W2009253716 creator A5071342393 @default.
• W2009253716 date "2013-01-01" @default.
• W2009253716 modified "2023-10-01" @default.
• W2009253716 title "Murine Hyaluronidase 2 Deficiency Results in Extracellular Hyaluronan Accumulation and Severe Cardiopulmonary Dysfunction" @default.
• W2009253716 cites W1964541789 @default.
• W2009253716 cites W1964632662 @default.
• W2009253716 cites W1966422114 @default.
• W2009253716 cites W1969108243 @default.
• W2009253716 cites W1970139580 @default.
• W2009253716 cites W1970399260 @default.
• W2009253716 cites W1980194211 @default.
• W2009253716 cites W1985044730 @default.
• W2009253716 cites W1988424812 @default.
• W2009253716 cites W1989102849 @default.
• W2009253716 cites W1993962364 @default.
• W2009253716 cites W1995580605 @default.
• W2009253716 cites W2005318950 @default.
• W2009253716 cites W2005457203 @default.
• W2009253716 cites W2012856015 @default.
• W2009253716 cites W2044299317 @default.
• W2009253716 cites W2050413447 @default.
• W2009253716 cites W2050557934 @default.
• W2009253716 cites W2059041221 @default.
• W2009253716 cites W2060798250 @default.
• W2009253716 cites W2074536214 @default.
• W2009253716 cites W2099327671 @default.
• W2009253716 cites W2102103202 @default.
• W2009253716 cites W2114825542 @default.
• W2009253716 cites W2120499448 @default.
• W2009253716 cites W2128466784 @default.
• W2009253716 cites W2132284796 @default.
• W2009253716 cites W2148133781 @default.
• W2009253716 cites W2150058785 @default.
• W2009253716 cites W2160251803 @default.
• W2009253716 cites W2164021363 @default.
• W2009253716 cites W2166644552 @default.
• W2009253716 doi "https://doi.org/10.1074/jbc.m112.393629" @default.
• W2009253716 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3537049" @default.
• W2009253716 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/23172227" @default.
• W2009253716 hasPublicationYear "2013" @default.
• W2009253716 type Work @default.
• W2009253716 sameAs 2009253716 @default.
• W2009253716 citedByCount "58" @default.
• W2009253716 countsByYear W20092537162013 @default.
• W2009253716 countsByYear W20092537162014 @default.
• W2009253716 countsByYear W20092537162015 @default.
• W2009253716 countsByYear W20092537162016 @default.
• W2009253716 countsByYear W20092537162017 @default.
• W2009253716 countsByYear W20092537162018 @default.
• W2009253716 countsByYear W20092537162019 @default.
• W2009253716 countsByYear W20092537162020 @default.
• W2009253716 countsByYear W20092537162021 @default.
• W2009253716 countsByYear W20092537162022 @default.
• W2009253716 countsByYear W20092537162023 @default.
• W2009253716 crossrefType "journal-article" @default.
• W2009253716 hasAuthorship W2009253716A5010625257 @default.
• W2009253716 hasAuthorship W2009253716A5050522824 @default.
• W2009253716 hasAuthorship W2009253716A5063398884 @default.
• W2009253716 hasAuthorship W2009253716A5067919633 @default.
• W2009253716 hasAuthorship W2009253716A5071342393 @default.
• W2009253716 hasBestOaLocation W20092537161 @default.
• W2009253716 hasConcept C181199279 @default.
• W2009253716 hasConcept C185592680 @default.
• W2009253716 hasConcept C2780375986 @default.
• W2009253716 hasConcept C28406088 @default.
• W2009253716 hasConcept C55493867 @default.
• W2009253716 hasConcept C71924100 @default.
• W2009253716 hasConceptScore W2009253716C181199279 @default.
• W2009253716 hasConceptScore W2009253716C185592680 @default.
• W2009253716 hasConceptScore W2009253716C2780375986 @default.
• W2009253716 hasConceptScore W2009253716C28406088 @default.
• W2009253716 hasConceptScore W2009253716C55493867 @default.
• W2009253716 hasConceptScore W2009253716C71924100 @default.
• W2009253716 hasIssue "1" @default.
• W2009253716 hasLocation W20092537161 @default.
• W2009253716 hasLocation W20092537162 @default.
• W2009253716 hasLocation W20092537163 @default.
• W2009253716 hasLocation W20092537164 @default.
• W2009253716 hasOpenAccess W2009253716 @default.
• W2009253716 hasPrimaryLocation W20092537161 @default.
• W2009253716 hasRelatedWork W1965618318 @default.
• W2009253716 hasRelatedWork W1969467097 @default.
• W2009253716 hasRelatedWork W2041654773 @default.
• W2009253716 hasRelatedWork W2072090357 @default.
• W2009253716 hasRelatedWork W2098109427 @default.
• W2009253716 hasRelatedWork W2147785780 @default.
• W2009253716 hasRelatedWork W2293294922 @default.
• W2009253716 hasRelatedWork W2402022922 @default.
• W2009253716 hasRelatedWork W2748952813 @default.
• W2009253716 hasRelatedWork W2899084033 @default.
• W2009253716 hasVolume "288" @default.
• W2009253716 isParatext "false" @default.

• next