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• W2557882611 abstract "Viral infection is an exacerbating factor contributing to chronic airway diseases, such as asthma, via mechanisms that are still unclear. Polyinosine-polycytidylic acid (poly(I:C)), a Toll-like receptor 3 (TLR3) agonist used as a mimetic to study viral infection, has been shown to elicit inflammatory responses in lungs and to exacerbate pulmonary allergic reactions in animal models. Previously, we have shown that poly(I:C) stimulates lung fibroblasts to accumulate an extracellular matrix (ECM), enriched in hyaluronan (HA) and its binding partner versican, which promotes monocyte adhesion. In the current study, we aimed to determine the in vivo role of versican in mediating inflammatory responses in poly(I:C)-induced lung inflammation using a tamoxifen-inducible versican-deficient mouse model (Vcan−/− mice). In C57Bl/6 mice, poly(I:C) instillation significantly increased accumulation of versican and HA, especially in the perivascular and peribronchial regions, which were enriched in infiltrating leukocytes. In contrast, versican-deficient (Vcan−/−) lungs did not exhibit increases in versican or HA in these regions and had strikingly reduced numbers of leukocytes in the bronchoalveolar lavage fluid and lower expression of inflammatory chemokines and cytokines. Poly(I:C) stimulation of lung fibroblasts isolated from control mice generated HA-enriched cable structures in the ECM, providing a substrate for monocytic cells in vitro, whereas lung fibroblasts from Vcan−/− mice did not. Moreover, increases in proinflammatory cytokine expression were also greatly attenuated in the Vcan−/− lung fibroblasts. These findings provide strong evidence that versican is a critical inflammatory mediator during poly(I:C)-induced acute lung injury and, in association with HA, generates an ECM that promotes leukocyte infiltration and adhesion. Viral infection is an exacerbating factor contributing to chronic airway diseases, such as asthma, via mechanisms that are still unclear. Polyinosine-polycytidylic acid (poly(I:C)), a Toll-like receptor 3 (TLR3) agonist used as a mimetic to study viral infection, has been shown to elicit inflammatory responses in lungs and to exacerbate pulmonary allergic reactions in animal models. Previously, we have shown that poly(I:C) stimulates lung fibroblasts to accumulate an extracellular matrix (ECM), enriched in hyaluronan (HA) and its binding partner versican, which promotes monocyte adhesion. In the current study, we aimed to determine the in vivo role of versican in mediating inflammatory responses in poly(I:C)-induced lung inflammation using a tamoxifen-inducible versican-deficient mouse model (Vcan−/− mice). In C57Bl/6 mice, poly(I:C) instillation significantly increased accumulation of versican and HA, especially in the perivascular and peribronchial regions, which were enriched in infiltrating leukocytes. In contrast, versican-deficient (Vcan−/−) lungs did not exhibit increases in versican or HA in these regions and had strikingly reduced numbers of leukocytes in the bronchoalveolar lavage fluid and lower expression of inflammatory chemokines and cytokines. Poly(I:C) stimulation of lung fibroblasts isolated from control mice generated HA-enriched cable structures in the ECM, providing a substrate for monocytic cells in vitro, whereas lung fibroblasts from Vcan−/− mice did not. Moreover, increases in proinflammatory cytokine expression were also greatly attenuated in the Vcan−/− lung fibroblasts. These findings provide strong evidence that versican is a critical inflammatory mediator during poly(I:C)-induced acute lung injury and, in association with HA, generates an ECM that promotes leukocyte infiltration and adhesion. Viral lung infection is one of the exacerbating factors contributing to chronic lung diseases, such as asthma (1Shibata T. Habiel D.M. Coelho A.L. Kunkel S.L. Lukacs N.W. Hogaboam C.M. Axl receptor blockade ameliorates pulmonary pathology resulting from primary viral infection and viral exacerbation of asthma.J. Immunol. 2014; 192: 3569-3581Crossref PubMed Scopus (44) Google Scholar2Tan W.C. Viruses in asthma exacerbations.Curr. Opin. Pulm. Med. 2005; 11: 21-26PubMed Google Scholar, 3Saraya T. Kurai D. Ishii H. Ito A. Sasaki Y. Niwa S. Kiyota N. Tsukagoshi H. Kozawa K. Goto H. Takizawa H. Epidemiology of virus-induced asthma exacerbations: with special reference to the role of human rhinovirus.Front. Microbiol. 2014; 5: 226Crossref PubMed Scopus (33) Google Scholar, 4Kurai D. Saraya T. Ishii H. Takizawa H. Virus-induced exacerbations in asthma and COPD.Front. Microbiol. 2013; 4: 293Crossref PubMed Scopus (117) Google Scholar, 5Papadopoulos N.G. Christodoulou I. Rohde G. Agache I. Almqvist C. Bruno A. Bonini S. Bont L. Bossios A. Bousquet J. Braido F. Brusselle G. Canonica G.W. Carlsen K.H. Chanez P. et al.Viruses and bacteria in acute asthma exacerbations–a GA(2) LEN-DARE systematic review.Allergy. 2011; 66: 458-468Crossref PubMed Scopus (223) Google Scholar6Clarke D.L. Davis N.H. Majithiya J.B. Piper S.C. Lewis A. Sleeman M.A. Corkill D.J. May R.D. Development of a mouse model mimicking key aspects of a viral asthma exacerbation.Clin. Sci. 2014; 126: 567-580Crossref PubMed Scopus (31) Google Scholar). During acute lung inflammation, extracellular matrix (ECM) 6The abbreviations used are: ECMextracellular matrixHAhyaluronanCSchondroitin sulfatePGproteoglycanTLRToll-like receptorpoly(I:C)polyinosine-polycytidylic acidVcanversicanVcan−/−versican-deficientHashyaluronan synthaseGAGglycosaminoglycanBALFbroncoalveolar lavage fluidBACbacterial artificial chromosomeESCembryonic stem cell. around blood vessels and airways remodels to allow for infiltration of leukocytes. This “provisional” ECM involves accumulation of the hygroscopic molecules hyaluronan (HA) and the chondroitin sulfate (CS) proteoglycan (PG) versican, which together create a loose and hydrated space necessary for leukocyte ingress and additionally for migration and expansion of resident stromal cells. Versican expression, which is high in lungs during embryonic development (7Shannon J.M. McCormick-Shannon K. Burhans M.S. Shangguan X. Srivastava K. Hyatt B.A. Chondroitin sulfate proteoglycans are required for lung growth and morphogenesis in vitro.Am. J. Physiol. Lung Cell Mol. Physiol. 2003; 285: L1323-L1336Crossref PubMed Scopus (35) Google Scholar, 8Faggian J. Fosang A.J. Zieba M. Wallace M.J. Hooper S.B. Changes in versican and chondroitin sulphate proteoglycans during structural development of the lung.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2007; 293: R784-R792Crossref PubMed Scopus (25) Google Scholar9Snyder J.M. Washington I.M. Birkland T. Chang M.Y. Frevert C.W. Correlation of versican expression, accumulation, and degradation during embryonic development by quantitative immunohistochemistry.J. Histochem. Cytochem. 2015; 63: 952-967Crossref PubMed Scopus (24) Google Scholar) but low in adult lungs, is reactivated in numerous lung diseases, including pulmonary fibrosis, chronic obstructive pulmonary disease, acute respiratory distress syndrome, and asthma (10Johnson P.R. Role of human airway smooth muscle in altered extracellular matrix production in asthma.Clin. Exp. Pharmacol. Physiol. 2001; 28: 233-236Crossref PubMed Scopus (84) Google Scholar11Roberts C.R. Is asthma a fibrotic disease?.Chest. 1995; 107: 111S-117SAbstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 12Huang J. Olivenstein R. Taha R. Hamid Q. Ludwig M. Enhanced proteoglycan deposition in the airway wall of atopic asthmatics.Am. J. Respir. Crit. Care Med. 1999; 160: 725-729Crossref PubMed Scopus (173) Google Scholar, 13de Medeiros Matsushita M. da Silva L.F. dos Santos M.A. Fernezlian S. Schrumpf J.A. Roughley P. Hiemstra P.S. Saldiva P.H. Mauad T. Dolhnikoff M. Airway proteoglycans are differentially altered in fatal asthma.J. Pathol. 2005; 207: 102-110Crossref PubMed Scopus (81) Google Scholar, 14Westergren-Thorsson G. Chakir J. Lafrenière-Allard M.J. Boulet L.P. Tremblay G.M. Correlation between airway responsiveness and proteoglycan production by bronchial fibroblasts from normal and asthmatic subjects.Int. J. Biochem. Cell Biol. 2002; 34: 1256-1267Crossref PubMed Scopus (84) Google Scholar, 15Weitoft M. Andersson C. Andersson-Sjöland A. Tufvesson E. Bjermer L. Erjefält J. Westergren-Thorsson G. Controlled and uncontrolled asthma display distinct alveolar tissue matrix compositions.Respir. Res. 2014; 15: 67Crossref PubMed Scopus (47) Google Scholar, 16Andersson-Sjöland A. Hallgren O. Rolandsson S. Weitoft M. Tykesson E. Larsson-Callerfelt A.K. Rydell-Törmänen K. Bjermer L. Malmström A. Karlsson J.C. Westergren-Thorsson G. Versican in inflammation and tissue remodeling: the impact on lung disorders.Glycobiology. 2015; 25: 243-251Crossref PubMed Scopus (60) Google Scholar, 17Zhu Z. Ma B. Zheng T. Homer R.J. Lee C.G. Charo I.F. Noble P. Elias J.A. IL-13-induced chemokine responses in the lung: role of CCR2 in the pathogenesis of IL-13-induced inflammation and remodeling.J. Immunol. 2002; 168: 2953-2962Crossref PubMed Scopus (174) Google Scholar18Lowry M.H. McAllister B.P. Jean J.C. Brown L.A. Hughey R.P. Cruikshank W.W. Amar S. Lucey E.C. Braun K. Johnson P. Wight T.N. Joyce-Brady M. Lung lining fluid glutathione attenuates IL-13-induced asthma.Am. J. Respir. Cell Mol. Biol. 2008; 38: 509-516Crossref PubMed Scopus (40) Google Scholar). Our published work has shown that versican and molecules that associate with versican, such as HA, are the principal ECM components that accumulate in inflamed lungs at early times following exposure to pathogens, such as LPS (19Chang M.Y. Tanino Y. Vidova V. Kinsella M.G. Chan C.K. Johnson P.Y. Wight T.N. Frevert C.W. A rapid increase in macrophage-derived versican and hyaluronan in infectious lung disease.Matrix Biol. 2014; 34: 1-12Crossref PubMed Scopus (51) Google Scholar). The accumulation of a versican-enriched ECM coincides with invasion and retention of leukocytes within different compartments of the lung during these early inflammatory responses. Previous studies have shown that bronchial fibroblasts cultured from subjects with asthma have elevated production of versican (14Westergren-Thorsson G. Chakir J. Lafrenière-Allard M.J. Boulet L.P. Tremblay G.M. Correlation between airway responsiveness and proteoglycan production by bronchial fibroblasts from normal and asthmatic subjects.Int. J. Biochem. Cell Biol. 2002; 34: 1256-1267Crossref PubMed Scopus (84) Google Scholar, 20Malmström J. Larsen K. Hansson L. Löfdahl C.G. Nörregard-Jensen O. Marko-Varga G. Westergren-Thorsson G. Proteoglycan and proteome profiling of central human pulmonary fibrotic tissue utilizing miniaturized sample preparation: a feasibility study.Proteomics. 2002; 2: 394-404Crossref PubMed Scopus (39) Google Scholar, 21Ludwig M.S. Ftouhi-Paquin N. Huang W. Pagé N. Chakir J. Hamid Q. Mechanical strain enhances proteoglycan message in fibroblasts from asthmatic subjects.Clin. Exp. Allergy. 2004; 34: 926-930Crossref PubMed Scopus (30) Google Scholar), and in a recent study in a cockroach antigen-induced mouse model of asthma, we showed that versican, produced by airway epithelial cells, consistently accumulates in the subepithelial space and precedes infiltration of leukocytes, suggesting a specific immunomodulatory role for versican (22Reeves S.R. Kaber G. Sheih A. Cheng G. Aronica M.A. Merrilees M.J. Debley J.S. Frevert C.W. Ziegler S.F. Wight T.N. Subepithelial accumulation of versican in a cockroach antigen-induced murine model of allerigic asthma.J. Histochem. Cytochem. 2016; 64: 364-380Crossref PubMed Scopus (21) Google Scholar). Whether the acute lung inflammation stimulated by viral infection worsens asthma by altering the ECM microenvironment to facilitate leukocyte infiltration and accumulation is not yet known. One of the major inflammatory signaling pathways activated by virus is the Toll-like receptor 3 (TLR3) pathway, which recognizes double-stranded RNA, such as polyinosine-polycytidylic acid (poly(I:C)), and thus is often used as a viral mimetic and a TLR3 agonist. It has been shown to generate an HA-enriched ECM in colon and in kidney, which promotes leukocyte accumulation (23de la Motte C. Hascall V.C. Drazba J.A. Strong S.A. Poly I:C induces mononuclear leukocyte-adhesive hyaluronan structures on colon smooth muscle cells: IaI and versican facilitate adhesion.in: Kennedy J.F. Phillips G.O. Williams P.A. Hascall V.C. Hyaluronan: Chemical, Biochemical and Biological Aspects. Woodhead Publishing Ltd., Cambridge, UK2002: 381-388Crossref Google Scholar24de la Motte C.A. Hascall V.C. Drazba J. Bandyopadhyay S.K. Strong S.A. Mononuclear leukocytes bind to specific hyaluronan structures on colon mucosal smooth muscle cells treated with polyinosinic acid:polycytidylic acid: inter-α-trypsin inhibitor is crucial to structure and function.Am. J. Pathol. 2003; 163: 121-133Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 25Lauer M.E. Fulop C. Mukhopadhyay D. Comhair S. Erzurum S.C. Hascall V.C. Airway smooth muscle cells synthesize hyaluronan cable structures independent of inter-α-inhibitor heavy chain attachment.J. Biol. Chem. 2009; 284: 5313-5323Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 26Lauer M.E. Mukhopadhyay D. Fulop C. de la Motte C.A. Majors A.K. Hascall V.C. Primary murine airway smooth muscle cells exposed to poly(I,C) or tunicamycin synthesize a leukocyte-adhesive hyaluronan matrix.J. Biol. Chem. 2009; 284: 5299-5312Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar27Wang A. Hascall V.C. Hyaluronan structures synthesized by rat mesangial cells in response to hyperglycemia induce monocyte adhesion.J. Biol. Chem. 2004; 279: 10279-10285Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), and has also been shown to elicit acute lung inflammation in vivo and further to exacerbate pulmonary allergic reactions (28Torres D. Dieudonné A. Ryffel B. Vilain E. Si-Tahar M. Pichavant M. Lassalle P. Trottein F. Gosset P. Double-stranded RNA exacerbates pulmonary allergic reaction through TLR3: implication of airway epithelium and dendritic cells.J. Immunol. 2010; 185: 451-459Crossref PubMed Scopus (73) Google Scholar, 29Stowell N.C. Seideman J. Raymond H.A. Smalley K.A. Lamb R.J. Egenolf D.D. Bugelski P.J. Murray L.A. Marsters P.A. Bunting R.A. Flavell R.A. Alexopoulou L. San Mateo L.R. Griswold D.E. Sarisky R.T. et al.Long-term activation of TLR3 by poly(I:C) induces inflammation and impairs lung function in mice.Respir. Res. 2009; 10: 43Crossref PubMed Scopus (128) Google Scholar). A number of studies by our group have demonstrated that lung fibroblasts synthesize and deposit HA- and versican-enriched ECM in response to poly(I:C). This ECM is strongly adhesive for monocytes and T lymphocytes and is hyaluronidase-sensitive, indicating that HA is a necessary component of this adhesive ECM (30Evanko S.P. Potter-Perigo S. Bollyky P.L. Nepom G.T. Wight T.N. Hyaluronan and versican in the control of human T-lymphocyte adhesion and migration.Matrix Biol. 2012; 31: 90-100Crossref PubMed Scopus (105) Google Scholar31Evanko S.P. Potter-Perigo S. Johnson P.Y. Wight T.N. Organization of hyaluronan and versican in the extracellular matrix of human fibroblasts treated with the viral mimetic poly I:C.J. Histochem. Cytochem. 2009; 57: 1041-1060Crossref PubMed Scopus (62) Google Scholar, 32Potter-Perigo S. Johnson P.Y. Evanko S.P. Chan C.K. Braun K.R. Wilkinson T.S. Altman L.C. Wight T.N. Polyinosine-polycytidylic acid stimulates versican accumulation in the extracellular matrix promoting monocyte adhesion.Am. J. Respir. Cell Mol. Biol. 2010; 43: 109-120Crossref PubMed Scopus (62) Google Scholar33Wight T.N. Kang I. Merrilees M.J. Versican and the control of inflammation.Matrix Biol. 2014; 35: 152-161Crossref PubMed Scopus (145) Google Scholar). Interfering with versican accumulation in this ECM also inhibits leukocyte adhesion in vitro, suggesting that versican and HA may form an immunomodulatory complex in response to viral lung infection (32Potter-Perigo S. Johnson P.Y. Evanko S.P. Chan C.K. Braun K.R. Wilkinson T.S. Altman L.C. Wight T.N. Polyinosine-polycytidylic acid stimulates versican accumulation in the extracellular matrix promoting monocyte adhesion.Am. J. Respir. Cell Mol. Biol. 2010; 43: 109-120Crossref PubMed Scopus (62) Google Scholar, 33Wight T.N. Kang I. Merrilees M.J. Versican and the control of inflammation.Matrix Biol. 2014; 35: 152-161Crossref PubMed Scopus (145) Google Scholar34Kang I. Yoon D.W. Braun K.R. Wight T.N. Expression of versican V3 by arterial smooth muscle cells alters TGFβ-, EGF-, and NFκB-dependent signaling pathways, creating a microenvironment that resists monocyte adhesion.J. Biol. Chem. 2014; 289: 15393-15404Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). However, specific roles for versican in the regulation of pulmonary inflammatory responses are not yet well defined due to lack of versican knock-out animals, which are embryonically lethal due to defective cardiac development (35Mjaatvedt C.H. Yamamura H. Capehart A.A. Turner D. Markwald R.R. The Cspg2 gene, disrupted in the hdf mutant, is required for right cardiac chamber and endocardial cushion formation.Dev. Biol. 1998; 202: 56-66Crossref PubMed Scopus (261) Google Scholar). In this study, we examine the formation of HA- and versican-enriched ECM in lungs of conditionally versican-deficient mice, developed recently in our laboratory, in response to poly(I:C) as a surrogate for viral infection. We report that global deficiency of versican perturbs both the accumulation of HA and the accumulation and infiltration of leukocytes, demonstrating that versican is a critical ECM component mediating HA-dependent leukocyte accumulation in the lungs and a potential therapeutic target. extracellular matrix hyaluronan chondroitin sulfate proteoglycan Toll-like receptor polyinosine-polycytidylic acid versican versican-deficient hyaluronan synthase glycosaminoglycan broncoalveolar lavage fluid bacterial artificial chromosome embryonic stem cell. The distribution of HA and versican was initially examined in unchallenged and poly(I:C)-instilled lungs of 8–10-week-old C57Bl/6 mice. In the unchallenged animals, moderate to strong HA staining was present in the stromal connective tissues of airways but not in alveolar sacs. In the pulmonary vasculature, moderate to strong HA staining was present mostly in the adventitial and peri-adventitial regions (Fig. 1, A–C). Versican levels were low throughout the lung with weak staining in the epithelium of bronchi and bronchioles (Fig. 1 (G–I) and Table 1). HA and versican accumulation associated with infiltrating leukocytes was prominent 48 h after the second poly(I:C) instillation in the perivascular and peribroncheal spaces as well as in the alveolar septa (Fig. 1 (D–H and J–L) and Table 1). These in vivo observations support our previously published in vitro findings that poly(I:C) treatment of lung fibroblasts promotes the formation of an HA- and versican-rich ECM, which enhances monocyte binding (30Evanko S.P. Potter-Perigo S. Bollyky P.L. Nepom G.T. Wight T.N. Hyaluronan and versican in the control of human T-lymphocyte adhesion and migration.Matrix Biol. 2012; 31: 90-100Crossref PubMed Scopus (105) Google Scholar, 31Evanko S.P. Potter-Perigo S. Johnson P.Y. Wight T.N. Organization of hyaluronan and versican in the extracellular matrix of human fibroblasts treated with the viral mimetic poly I:C.J. Histochem. Cytochem. 2009; 57: 1041-1060Crossref PubMed Scopus (62) Google Scholar32Potter-Perigo S. Johnson P.Y. Evanko S.P. Chan C.K. Braun K.R. Wilkinson T.S. Altman L.C. Wight T.N. Polyinosine-polycytidylic acid stimulates versican accumulation in the extracellular matrix promoting monocyte adhesion.Am. J. Respir. Cell Mol. Biol. 2010; 43: 109-120Crossref PubMed Scopus (62) Google Scholar). These findings led us to hypothesize that versican plays an intergral role in promoting leukocyte infiltration into lungs during poly(I:C)-induced pulmonary inflammation. To test this hypothesis, and because the homozygous hdf (heart defect) mouse that lacks Vcan expression (35Mjaatvedt C.H. Yamamura H. Capehart A.A. Turner D. Markwald R.R. The Cspg2 gene, disrupted in the hdf mutant, is required for right cardiac chamber and endocardial cushion formation.Dev. Biol. 1998; 202: 56-66Crossref PubMed Scopus (261) Google Scholar) is embryonic lethal, we developed a novel mouse strain with conditional global versican deficiency, which enabled us to study the contribution of versican to inflammation in adult animals.TABLE 1HA and versican staining in unchallenged and poly(I:C)-stimulated lungsControlPoly(I:C)HAVersicanHAVersicanAirways Bronchi Epithelium++++++ Stroma+++0/+++++++ Bronchioles Epithelium++++++ Stroma+++0++++++ Respiratory bronchioles Epithelium0+++++ Stroma++0++++++ Alveoli Alveolar ducts++0/+++++++ Alveolar sacs (rims)+0++++ Alveolar walls0/+0+++++Vessels Pulmonary arteries (bronchi) Endothelium0000/+ Media00/+0/+++ Adventitia++0+++++ Peri-adventitial+++0+++++++ Pulmonary arteries (bronchioles) Endothelium0000/+ Media00/+0/++++ Adventitia++0++++++ Peri-adventitial+++0+++++++ Pulmonary veins Endothelium0000 Media+0++++++ Adventitia++0++++++ Venules Endothelium0000 Wall+0+++++++ Open table in a new tab Conditional versican-deficient (Vcan−/−) mice were successfully generated by inserting LoxP sites flanking exon 4 of the Vcan gene on the C57Bl/6 genetic background (Fig. 2A). When these B6.Vcan-e4fl/fl mice were crossed to Rosa26-CreERT2 mice, deletion of exon 4 was successfully achieved after treating the mice with tamoxifen (Fig. 2B). Deletion of exon 4 results in the generation of a stop codon in exon 5, preventing versican expression. Vcan−/− animals and littermate controls lacking Cre were treated with poly(I:C) and observed after 48 h, when the inflammatory response was at its peak. Distribution of HA and versican accumulation in PBS-treated control and Vcan−/− animals were similar to our findings in wild type C57Bl/6 mice (Fig. 3, A and B and E and F). Poly(I:C) induced the accumulation of HA- and versican-enriched ECM in the perivascular and peribronchial spaces in the inflamed lungs of control animals (Fig. 3, C and G). This ECM accumulation was reduced in Vcan−/− mice. Both HA and versican accumulation were dramatically reduced in Vcan−/− lungs (Fig. 3, D and H). Messenger RNA levels of all isoforms of versican in unchallenged lungs were low in both control and Vcan−/− animals and not significantly different between genotypes. In response to poly(I:C) challenge, however, total versican mRNA levels significantly increased in lungs of control animals but not in Vcan−/− animals (Fig. 4A). Specifically, levels of V0, V1, and V2 versican isoforms were significantly increased in poly(I:C)-challenged lungs but showed no significant elevation in Vcan−/− animals (Fig. 4A). Hyaluronan synthase 2 (Has2) mRNA levels were also significantly increased in poly(I:C)-challenged control animals but not in Vcan−/− animals (Fig. 4B). Similarly, poly(I:C) challenge induced significant increase in protein accumulation of versican in the lungs in control animals but not in Vcan−/− animals (Fig. 5, A and B). When normalized to Vcan mRNA levels in PBS-treated control lungs, Vcan mRNA levels in poly(I:C)-treated control lungs increased to 310 ± 43%, whereas PBS- and poly(I:C)-treated Vcan−/− lungs were 20 ± 4 and 64 ± 13% of PBS-treated control lungs, respectively (supplemental Fig. 1). This represents a nearly 80% reduction in PBS- as well as poly(I:C)-induced gene expression in Vcan−/− lungs. Similarly, normalized Vcan protein levels in poly(I:C)-treated control lungs increased to 365 ± 57%, whereas PBS- and poly(I:C)-treated Vcan−/− lungs were at 100 ± 27 and 170 ± 28% of PBS-treated control lungs, respectively. This translates to no significant change in protein levels in PBS-treated lungs but a 53.4% reduction in poly(I:C)-stimulated Vcan protein in Vcan−/− lungs. When calculated as a percentage of induction above PBS-treated lungs, poly(I:C)-treated Vcan−/− lungs had 78.8% less mRNA and 73.6% less protein than poly(I:C)-treated controls.FIGURE 5A and B, protein levels of Vcan in whole lung extracts as measured by Western blot (A) were also significantly increased by poly(I:C) (pIC) stimulation in control mice (Cre−) but not in Vcan−/− (Cre+) mice, as quantified by densitometry (B). n = 6–15 mice/group. *, p < 0.05; **, p < 0.01. Error bars, S.D.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We observed that leukocyte accumulation associated with a versican- and HA-enriched ECM in poly(I:C)-challenged lungs was blunted in Vcan−/− mice (Fig. 6A). To confirm this finding, we examined whether the numbers of total cells in broncoalveolar lavage fluid (BALF) from poly(I:C)-challenged animals were affected by versican deficiency. Total cell counts in BALF increased in response to poly(I:C) challenge in control animals, which was significantly reduced in Vcan−/− mice (Fig. 6B). These BALF cells were further subjected to flow cytometry analysis to examine differential counts of leukocytes. The relative percentages of neutrophils, alveolar macrophages, dendritic cells, B and T lymphocytes, eosinophils, and interstitial macrophages in the BALF were not significantly affected by reduction in versican (supplemental Fig. 2, A–C). We further examined whether the presence of versican affects the inflammatory cytokines and chemokines induced by poly(I:C) challenge. In total lung lysates, poly(I:C) significantly increased expression of inflammatory cytokines and chemokines, including TNFα, IL1β, MIP2 (Cxcl2), IFNα, IFNγ, and IL10, in control mice. This response was attenuated or absent in Vcan−/− mice (Fig. 7). To determine whether there was a direct relationship between versican expression and cytokine levels, we performed linear regression analysis and found a significant positive relationship between versican and levels of inflammatory cytokines and chemokines (supplemental Fig. 3). We further examined whether versican deficiency affected the expression of cytokines and chemokines in lung stromal fibroblasts in vitro. Cultured primary lung fibroblasts, isolated from control and Vcan−/− mice, were treated with PBS or poly(I:C), and expression of cytokines and chemokines was measured. Poly(I:C) stimulation induced a significant increase in transcript levels of versican as well as IL1β expressed by control lung fibroblasts, consistent with the changes shown in the poly(I:C)-instilled lungs. In contrast, lung fibroblasts isolated from Vcan−/− mice showed significantly reduced levels of IL1β as well as versican (Fig. 8). To determine whether versican has an impact on leukocyte chemotaxis, we tested chemotactic migration of monocytic U937 cells toward CCL2 in the presence or absence of versican or CS chains. The addition of purified exogenous versican to the bottom chamber, along with CCL2, significantly enhanced migration of monocytic cells toward the chemokine (Fig. 9). Similarly, the addition of CS enhanced chemotaxis in a dose-dependent manner. In contrast, adding the purified versican to the monocytic cells in the filter well on the top chamber abolished this chemotactic migration, indicating that the interaction between the chemokine and versican, potentially via CS side chains, enhances leukocyte chemotaxis. Because we observed a relationship between HA, versican, and leukocyte accumulation in poly(I:C)-treated mouse lungs, we examined the formation of HA cables induced by poly(I:C) stimulation of lung fibroblasts in vitro, which promote leukocyte adhesion, as we have previously shown (30Evanko S.P. Potter-Perigo S. Bollyky P.L. Nepom G.T. Wight T.N. Hyaluronan and versican in the control of human T-lymphocyte adhesion and migration.Matrix Biol. 2012; 31: 90-100Crossref PubMed Scopus (105) Google Scholar, 31Evanko S.P. Potter-Perigo S. Johnson P.Y. Wight T.N. Organization of hyaluronan and versican in the extracellular matrix of human fibroblasts treated with the viral mimetic poly I:C.J. Histochem. Cytochem. 2009; 57: 1041-1060Crossref PubMed Scopus (62) Google Scholar32Potter-Perigo S. Johnson P.Y. Evanko S.P. Chan C.K. Braun K.R. Wilkinson T.S. Altman L.C. Wight T.N. Polyinosine-polycytidylic acid stimulates versican accumulation in the extracellular matrix promoting monocyte adhesion.Am. J. Respir. Cell Mol. Biol. 2010; 43: 109-120Crossref PubMed Scopus (62) Google Scholar, 34Kang I. Yoon D.W. Braun K.R. Wight T.N. Expression of versican V3 by arterial smooth muscle cells alters TGFβ-, EGF-, and NFκB-dependent signaling pathways, creating a microenvironment that resists monocyte adhesion.J. Biol. Chem. 2014; 289: 15393-15404Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). As anticipated, lung fibroblasts isolated from control animals generated HA cable structures in response to poly(I:C) (supplemental Fig. 4A). On the other hand, lung fibroblasts isolated from Vcan−/− animals did not generate these HA cable structures (supplemental Fig. 4B), suggesting that versican deficiency disrupts HA cable formation. We found that poly(I:C) treatment of control lung fibroblasts in cultures induces formation of HA cables, which allows U937 monocytic cells to adhere (Fig. 10, A and B). In contrast, fibroblasts from Vcan−/− mice showed a reduction in HA cable formation and associated adherent monocytes (Fig" @default.
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• W2557882611 date "2017-01-01" @default.
• W2557882611 modified "2023-10-17" @default.
• W2557882611 title "Versican Deficiency Significantly Reduces Lung Inflammatory Response Induced by Polyinosine-Polycytidylic Acid Stimulation" @default.
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• W2557882611 doi "https://doi.org/10.1074/jbc.m116.753186" @default.
• W2557882611 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/5217699" @default.
• W2557882611 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/27895126" @default.

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