Publications by authors named "Thomas N Wight"

192 Publications

Modulation of hyaluronan synthases and involvement of T cell-derived hyaluronan in autoimmune responses to transplanted islets.

Matrix Biol Plus 2021 Feb 30;9:100052. Epub 2020 Dec 30.

Center for Fundamental Immunology, Matrix Biology Program, Benaroya Research Institute at Virginia Mason, Seattle, WA, USA.

The extracellular matrix glycosaminoglycan hyaluronan (HA) accumulates in human and mouse islets during the onset of autoimmune type 1 diabetes (T1D). HA plays a critical role in T1D pathogenesis, as spontaneous disease is blocked in mice fed the HA synthesis inhibitor 4-methylumbelliferone (4MU). The present study demonstrates the involvement of HA in T cell-mediated autoimmune responses to transplanted islets and in in vivo and in vitro T cell activation. Scaffolded islet implants (SIs) loaded with RIP-mOVA mouse islets expressing chicken ovalbumin (OVA) on their β cells were grafted into T and B cell-deficient RIP-mOVA mice, which subsequently received CD4 T cells from DO11.10 transgenic mice bearing OVA peptide-specific T cell receptors (TcRs), followed by injection of OVA peptide to induce an immune response to the OVA-expressing islets. By affinity histochemistry (AHC), HA was greatly increased in grafted islets with T cell infiltrates (compared to islets grafted into mice lacking T cells) and a portion of this HA co-localized with the infiltrating T cells. Transferred T cells underwent HA synthase (HAS) isoform switching - T cells isolated from the SI grafts strongly upregulated HAS1 and HAS2 mRNAs and downregulated HAS3 mRNA, in contrast to T cells from graft-draining mesenteric lymph nodes, which expressed HAS3 mRNA only. Expression of HAS1 and HAS2 proteins by T cells in SI infiltrates was confirmed by immunohistochemistry (IHC). DO11.10 mice fed 4MU had suppressed in vivo T cell immune priming (measured as a reduced recall response to OVA peptide) compared to T cells from control mice fed a normal diet. In co-cultures of naïve DO11.10 T cells and OVA peptide-loaded antigen-presenting cells (APCs), pre-exposure of the T cells (but not pre-exposure of APCs) to 4MU inhibited early T cell activation (CD69 expression). In addition, T cells exposed to 4MU during activation in vitro with anti-CD3/CD28 antibodies had inhibited phosphorylation of the CD3ζ subunit of the TcR, a very early event in TcR signaling. Collectively, our results demonstrate that T cell-derived HA plays a significant role in T cell immune responses, and that expression of T cell HAS isoforms changes in a locale-specific manner during in vivo priming and functional phases of the T cell response.
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http://dx.doi.org/10.1016/j.mbplus.2020.100052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7930869PMC
February 2021

The extracellular matrix molecules versican and hyaluronan in urethral and vaginal tissues in stress urinary incontinence.

Neurourol Urodyn 2021 Mar 1;40(3):771-782. Epub 2021 Mar 1.

Section of Urology and Renal Transplantation, Virginia Mason Medical Center, Seattle, Washington, USA.

Purpose: Abnormal extracellular matrix (ECM) changes are correlated with stress urinary incontinence (SUI). The ECM components versican (Vcan) and hyaluronan (HA) play key roles in regulating tissue inflammation and maintaining connective tissue homeostasis. We analyzed the localization and expression of these ECM components in urethral and vaginal tissues from a rat model of urinary incontinence and from human clinical specimens.

Methods: Nulliparous rats underwent vaginal distension (VD), a rodent model of SUI, or a sham procedure. Tissues were harvested from six rats per group at days 1, 4, and 21 for immunohistochemistry and RNA expression analysis of ECM components. Periurethral vaginal samples from female patients with SUI were also examined.

Results: High-intensity staining for Vcan was observed 1 day after procedure in both control and VD animals. This level of abundance persisted at day 4 in VD compared to control, with concurrent reduced messenger RNA (mRNA) expression of the Vcan-degrading enzymes ADAMTS5 and ADAMTS9 and reduced staining for the Vcan cleavage epitope DPEAAE. Abundance of HA was not different between VD and control, however mRNA expression of the HA synthase Has2 was significantly reduced in VD tissues at day 4. Abundant Vcan staining was observed in 60% of SUI patient samples, which was strongest in regions of disrupted elastin.

Conclusion: Reduction of Vcan-degrading enzymes and HA synthases at day 4 postsurgery indicates a potential delay in ECM turnover associated with SUI. Abundant Vcan is associated with inflammation and elastin fiber network disruption, warranting further investigation to determine its role in SUI pathogenesis.
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http://dx.doi.org/10.1002/nau.24635DOI Listing
March 2021

Hyaluronan synthesis inhibition impairs antigen presentation and delays transplantation rejection.

Matrix Biol 2021 02 5;96:69-86. Epub 2020 Dec 5.

Division of Infectious Diseases and Geographic Medicine, Dept. of Medicine, Stanford University School of Medicine, Beckman Center, 279 Campus Drive, Stanford, CA 94305, United States. Electronic address:

A coat of pericellular hyaluronan surrounds mature dendritic cells (DC) and contributes to cell-cell interactions. We asked whether 4-methylumbelliferone (4MU), an oral inhibitor of HA synthesis, could inhibit antigen presentation. We find that 4MU treatment reduces pericellular hyaluronan, destabilizes interactions between DC and T-cells, and prevents T-cell proliferation in vitro and in vivo. These effects were observed only when 4MU was added prior to initial antigen presentation but not later, consistent with 4MU-mediated inhibition of de novo antigenic responses. Building on these findings, we find that 4MU delays rejection of allogeneic pancreatic islet transplant and allogeneic cardiac transplants in mice and suppresses allogeneic T-cell activation in human mixed lymphocyte reactions. We conclude that 4MU, an approved drug, may have benefit as an adjunctive agent to delay transplantation rejection.
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http://dx.doi.org/10.1016/j.matbio.2020.12.001DOI Listing
February 2021

Loss of versican and production of hyaluronan in lung epithelial cells are associated with airway inflammation during RSV infection.

J Biol Chem 2020 Nov 13. Epub 2020 Nov 13.

Center for Immunity and Immunotherapies, Seattle Children's Research Institute, United States.

Airway inflammation is a critical feature of lower respiratory tract infections caused by viruses such as respiratory syncytial virus (RSV). A growing body of literature has demonstrated the importance of extracellular matrix (ECM) changes such as the accumulation of hyaluronan (HA) and versican in the subepithelial space in promoting airway inflammation; however, whether these factors contribute to airway inflammation during RSV infection remains unknown. To test the hypothesis that RSV infection promotes inflammation via altered HA and versican production, we studied an ex vivo human bronchial epithelial cell (BEC)/human lung fibroblast (HLF) co-culture model. RSV infection of BEC/HLF co-cultures led to decreased hyaluronidase expression by HLFs, increased accumulation of HA, and enhanced adhesion of U937 cells as would be expected with increased HA. HLF production of versican was not altered following RSV infection; however, BEC production of versican was significantly downregulated following RSV infection. In vivo studies with epithelial-specific versican-deficient mice [SPC-Cre(+) Vcan-/-] demonstrated that RSV infection led to increased HA accumulation compared to control mice which also coincided with decreased hyaluronidase expression in the lung. SPC-Cre(+) Vcan-/- mice demonstrated enhanced recruitment of monocytes and neutrophils in bronchoalveolar lavage fluid and increased neutrophils in the lung compared to SPC-Cre(-) RSV-infected littermates. Taken together, these data demonstrate that altered ECM accumulation of HA occurs following RSV infection and may contribute to airway inflammation. Additionally, loss of epithelial expression of versican promotes airway inflammation during RSV infection further demonstrating that versican's role in inflammatory regulation is complex and dependent on the microenvironment.
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http://dx.doi.org/10.1074/jbc.RA120.016196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7949086PMC
November 2020

Exuberant fibroblast activity compromises lung function via ADAMTS4.

Nature 2020 11 28;587(7834):466-471. Epub 2020 Oct 28.

Department of Immunology, St Jude Children's Research Hospital, Memphis, TN, USA.

Severe respiratory infections can result in acute respiratory distress syndrome (ARDS). There are no effective pharmacological therapies that have been shown to improve outcomes for patients with ARDS. Although the host inflammatory response limits spread of and eventually clears the pathogen, immunopathology is a major contributor to tissue damage and ARDS. Here we demonstrate that respiratory viral infection induces distinct fibroblast activation states, which we term extracellular matrix (ECM)-synthesizing, damage-responsive and interferon-responsive states. We provide evidence that excess activity of damage-responsive lung fibroblasts drives lethal immunopathology during severe influenza virus infection. By producing ECM-remodelling enzymes-in particular the ECM protease ADAMTS4-and inflammatory cytokines, damage-responsive fibroblasts modify the lung microenvironment to promote robust immune cell infiltration at the expense of lung function. In three cohorts of human participants, the levels of ADAMTS4 in the lower respiratory tract were associated with the severity of infection with seasonal or avian influenza virus. A therapeutic agent that targets the ECM protease activity of damage-responsive lung fibroblasts could provide a promising approach to preserving lung function and improving clinical outcomes following severe respiratory infections.
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http://dx.doi.org/10.1038/s41586-020-2877-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7883627PMC
November 2020

Juvenile, but Not Adult, Mice Display Increased Myeloid Recruitment and Extracellular Matrix Remodeling during Respiratory Syncytial Virus Infection.

J Immunol 2020 12 23;205(11):3050-3057. Epub 2020 Oct 23.

Benaroya Research Institute, Seattle, WA 98101;

Early life respiratory syncytial virus (RSV) infection has been linked to the onset of asthma. Despite this association, our knowledge of the progression of the initial viral infection is limited, and no safe or effective vaccine currently exists. Bronchioalveolar lavage, whole-lung cellular isolation, and gene expression analysis were performed on 3-wk- (juvenile) and 8-wk-old (adult) RSV-infected C57BL/6 mice to investigate age-related differences in immunologic responses; juvenile mice displayed a sustained myeloid infiltrate (including monocytes and neutrophils) with increased RNA expression of , , and , when compared with adult mice, at 72 h postinfection. Juvenile mice demonstrated αSma expression (indicative of myofibroblast activity), increased hyaluronan deposition in the lung parenchyma (attributed to asthma progression), and a lack of CD64 upregulation on the surface of monocytes (which, in conjunction with serum amyloid P, is responsible for clearing residual hyaluronan and cellular debris). RSV infection of human airway epithelial cell, human lung fibroblast, and U937 monocyte cocultures (at air-liquid interface) displayed similar CCL expression and suggested matrix metalloproteinase-7 and MMP9 as possible extracellular matrix modifiers. These mouse data, in conjunction with our findings in human monocytes, suggest that the sustained influx of myeloid cells in the lungs of juvenile mice during acute RSV infection could potentiate extracellular matrix remodeling, facilitating conditions that support the development of asthma.
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http://dx.doi.org/10.4049/jimmunol.2000683DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7747670PMC
December 2020

A Role for HAPLN1 During Phenotypic Modulation of Human Lung Fibroblasts In Vitro.

J Histochem Cytochem 2020 11 16;68(11):797-811. Epub 2020 Oct 16.

Matrix Biology Program, Benaroya Research Institute at Virginia Mason, Seattle, WA, USA.

Hyaluronan and proteoglycan link protein 1 (HAPLN1) stabilizes interactions between two important extracellular matrix (ECM) macromolecules, versican and hyaluronan, which facilitate proliferation of fibroblasts and their conversion to myofibroblasts. However, the role of HAPLN1 in these events has not been studied. Using immunocytochemistry, cellular and ECM locations of HAPLN1 were evaluated in cultured human lung fibroblasts during proliferation and conversion to myofibroblasts. HAPLN1 localized to pericellular matrices, associating with both versican and hyaluronan in the ECM and on the cell surface. Nuclear and total HAPLN1 immunostaining increased after myofibroblast induction. Confocal microscopy showed HAPLN1 predominant in the ECM under cells while versican predominated above cells. Versican and HAPLN1 were also juxtaposed in columnar inclusions in the cytoplasm and nucleus. Nuclear HAPLN1 staining in interphase cells redistributed to the cytosol during mitosis. In the absence of TGF-β1, addition of exogenous bovine HAPLN1 (together with aggrecan G1) facilitated myofibroblast formation, as seen by significant upregulation of α-smooth muscle actin (SMA) staining, while adding full-length bovine versican had no effect. Increased compaction of hyaluronan-rich ECM suggests that HAPLN1 plus G1 addition affects hyaluronan networks and myofibroblast formation. These observations demonstrate changes in both extracellular and intracellular localization of HAPLN1 during fibroblast proliferation and myofibroblast conversion suggesting a possible role in fibrotic remodeling.
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http://dx.doi.org/10.1369/0022155420966663DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7649966PMC
November 2020

Serum amyloid A-containing HDL binds adipocyte-derived versican and macrophage-derived biglycan, reducing its antiinflammatory properties.

JCI Insight 2020 09 24;5(20). Epub 2020 Sep 24.

Division of Metabolism, Endocrinology, and Nutrition, Department of Medicine, University of Washington, Seattle, Washington, USA.

The ability of HDL to inhibit inflammation in adipocytes and adipose tissue is reduced when HDL contains serum amyloid A (SAA) that is trapped by proteoglycans at the adipocyte surface. Because we recently found that the major extracellular matrix proteoglycan produced by hypertrophic adipocytes is versican, whereas activated adipose tissue macrophages produce mainly biglycan, we further investigated the role of proteoglycans in determining the antiinflammatory properties of HDL. The distributions of versican, biglycan, apolipoprotein A1 (the major apolipoprotein of HDL), and SAA were similar in adipose tissue from obese mice and obese human subjects. Colocalization of SAA-enriched HDL with versican and biglycan at the cell surface of adipocyte and peritoneal macrophages, respectively, was blocked by silencing these proteoglycans, which also restored the antiinflammatory property of SAA-enriched HDL despite the presence of SAA. Similar to adipocytes, normal HDL exerted its antiinflammatory function in macrophages by reducing lipid rafts, reactive oxygen species generation, and translocation of Toll-like receptor 4 and NADPH oxidase 2 into lipid rafts, effects that were not observed with SAA-enriched HDL. These findings imply that SAA present in HDL can be trapped by adipocyte-derived versican and macrophage-derived biglycan, thereby blunting HDL's antiinflammatory properties.
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http://dx.doi.org/10.1172/jci.insight.142635DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7605543PMC
September 2020

Hypothalamic perineuronal net assembly is required for sustained diabetes remission induced by fibroblast growth factor 1 in rats.

Nat Metab 2020 10 7;2(10):1025-1033. Epub 2020 Sep 7.

University of Washington Medicine Diabetes Institute, University of Washington, Seattle, WA, USA.

We recently showed that perineuronal nets (PNNs) enmesh glucoregulatory neurons in the arcuate nucleus (Arc) of the mediobasal hypothalamus (MBH), but whether these PNNs play a role in either the pathogenesis of type 2 diabetes (T2D) or its treatment remains unclear. Here we show that PNN abundance within the Arc is markedly reduced in the Zucker diabetic fatty (ZDF) rat model of T2D, compared with normoglycaemic rats, correlating with altered PNN-associated sulfation patterns of chondroitin sulfate glycosaminoglycans in the MBH. Each of these PNN-associated changes is reversed following a single intracerebroventricular (icv) injection of fibroblast growth factor 1 (FGF1) at a dose that induces sustained diabetes remission in male ZDF rats. Combined with previous work localizing this FGF1 effect to the Arc area, our finding that enzymatic digestion of Arc PNNs markedly shortens the duration of diabetes remission following icv FGF1 injection in these animals identifies these extracellular matrix structures as previously unrecognized participants in the mechanism underlying diabetes remission induced by the central action of FGF1.
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http://dx.doi.org/10.1038/s42255-020-00275-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7572652PMC
October 2020

Adipocyte-Derived Versican and Macrophage-Derived Biglycan Control Adipose Tissue Inflammation in Obesity.

Cell Rep 2020 06;31(13):107818

Department of Medicine, Division of Metabolism, Endocrinology, and Nutrition, University of Washington, Seattle, WA, USA. Electronic address:

Obesity is characterized by adipose tissue inflammation. Because proteoglycans regulate inflammation, here we investigate their role in adipose tissue inflammation in obesity. We find that adipose tissue versican and biglycan increase in obesity. Versican is produced mainly by adipocytes and biglycan by adipose tissue macrophages. Both proteoglycans are also present in adipose tissue from obese human subjects undergoing gastric bypass surgery. Deletion of adipocyte-specific versican or macrophage-specific biglycan in mice reduces macrophage accumulation and chemokine and cytokine expression, although only adipocyte-specific versican deletion leads to sustained improvement in glucose tolerance. Macrophage-derived biglycan activates inflammatory genes in adipocytes. Versican expression increases in cultured adipocytes exposed to excess glucose, and adipocyte-conditioned medium stimulates inflammation in resident peritoneal macrophages, in part because of a versican breakdown product, versikine. These findings provide insights into the role of adipocyte- and macrophage-derived proteoglycans in adipose tissue inflammation in obesity.
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http://dx.doi.org/10.1016/j.celrep.2020.107818DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7384517PMC
June 2020

High-molecular weight hyaluronan attenuates tubulointerstitial scarring in kidney injury.

JCI Insight 2020 06 18;5(12). Epub 2020 Jun 18.

Laboratory for Regenerative Tissue Repair, Division of Pediatric Surgery, Department of Surgery, Texas Children's Hospital/Baylor College of Medicine, Houston, Texas, USA.

Renal fibrosis features exaggerated inflammation, extracellular matrix (ECM) deposition, and peritubular capillary loss. We previously showed that IL-10 stimulates high-molecular weight hyaluronan (HMW-HA) expression by fibroblasts, and we hypothesize that HMW-HA attenuates renal fibrosis by reducing inflammation and ECM remodeling. We studied the effects of IL-10 overexpression on HA production and scarring in mouse models of unilateral ureteral obstruction (UUO) and ischemia/reperfusion (I/R) to investigate whether IL-10 antifibrotic effects are HA dependent. C57BL/6J mice were fed with the HA synthesis inhibitor, 4-methylumbelliferone (4-MU), before UUO. We observed that in vivo injury increased intratubular spaces, ECM deposition, and HA expression at day 7 and onward. IL-10 overexpression reduced renal fibrosis in both models, promoted HMW-HA synthesis and stability in UUO, and regulated cell proliferation in I/R. 4-MU inhibited IL-10-driven antifibrotic effects, indicating that HMW-HA is necessary for cytokine-mediated reduction of fibrosis. We also found that IL-10 induces in vitro HMW-HA production by renal fibroblasts via STAT3-dependent upregulation of HA synthase 2. We propose that IL-10-induced HMW-HA synthesis plays cytoprotective and antifibrotic roles in kidney injury, thereby revealing an effective strategy to attenuate renal fibrosis in obstructive and ischemic pathologies.
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http://dx.doi.org/10.1172/jci.insight.136345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7406305PMC
June 2020

Inhibitory Effects of PRG4 on Migration and Proliferation of Human Venous Cells.

J Surg Res 2020 09 19;253:53-62. Epub 2020 Apr 19.

Department of Surgery, University of Washington, Seattle, Washington; Center for Cardiovascular Biology and Institute for Stem Cells and Regenerative Medicine, University of Washington, Seattle, Washington. Electronic address:

Background: Proteoglycan 4 (PRG4; lubricin) is a member of two gene co-expression network modules associated with human vein graft failure. However, little is known about PRG4 and the vascular system. Therefore, we have investigated the effects of recombinant human PRG4 (rhPRG4) on cell migration and proliferation in human veins.

Methods: Effects of rhPRG4 on cell migration, proliferation, and neointima formation were determined in human venous tissue and cultured venous smooth muscle cells (SMCs), adventitial cells, and endothelial cells. Expression of PRG4 by cultured human saphenous veins, failed vein grafts, and varicose veins was determined by immunostaining or Western blotting.

Results: Limited expression of PRG4 in fresh saphenous veins was dramatically increased around medial SMCs after culture ex vivo. rhPRG4 inhibited the migration of cultured SMCs, adventitial cells, and endothelial cells, as well as the proliferation of endothelial cells. rhPRG4 also inhibited the migration of SMCs and adventitial cells from tissue explants, but there was no effect on cell proliferation or neointima formation in ex vivo whole veins. Finally, PRG4 was largely absent in two examples of venous pathology, that is, failed human vein grafts and varicose veins.

Conclusions: Although rhPRG4 can inhibit the migration of venous SMCs, endothelial cells, and adventitial cells, and the proliferation of endothelial cells, PRG4 was only increased around medial SMCs in veins after ex vivo culture. PRG4 was not observed around medial SMCs in failed human vein grafts and varicose veins, suggesting the possibility that a failure of PRG4 upregulation may promote these pathologies.
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http://dx.doi.org/10.1016/j.jss.2020.03.028DOI Listing
September 2020

Versican-A Critical Extracellular Matrix Regulator of Immunity and Inflammation.

Front Immunol 2020 24;11:512. Epub 2020 Mar 24.

Division of Pulmonary/Critical Care Medicine, Center for Lung Biology, University of Washington School of Medicine, Seattle, WA, United States.

The extracellular matrix (ECM) proteoglycan, versican increases along with other ECM versican binding molecules such as hyaluronan, tumor necrosis factor stimulated gene-6 (TSG-6), and inter alpha trypsin inhibitor (IαI) during inflammation in a number of different diseases such as cardiovascular and lung disease, autoimmune diseases, and several different cancers. These interactions form stable scaffolds which can act as "landing strips" for inflammatory cells as they invade tissue from the circulation. The increase in versican is often coincident with the invasion of leukocytes early in the inflammatory process. Versican interacts with inflammatory cells either indirectly via hyaluronan or directly via receptors such as CD44, P-selectin glycoprotein ligand-1 (PSGL-1), and toll-like receptors (TLRs) present on the surface of immune and non-immune cells. These interactions activate signaling pathways that promote the synthesis and secretion of inflammatory cytokines such as TNFα, IL-6, and NFκB. Versican also influences inflammation by interacting with a variety of growth factors and cytokines involved in regulating inflammation thereby influencing their bioavailability and bioactivity. Versican is produced by multiple cell types involved in the inflammatory process. Conditional total knockout of versican in a mouse model of lung inflammation demonstrated significant reduction in leukocyte invasion into the lung and reduced inflammatory cytokine expression. While versican produced by stromal cells tends to be pro-inflammatory, versican expressed by myeloid cells can create anti-inflammatory and immunosuppressive microenvironments. Inflammation in the tumor microenvironment often contains elevated levels of versican. Perturbing the accumulation of versican in tumors can inhibit inflammation and tumor progression in some cancers. Thus versican, as a component of the ECM impacts immunity and inflammation through regulating immune cell trafficking and activation. Versican is emerging as a potential target in the control of inflammation in a number of different diseases.
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http://dx.doi.org/10.3389/fimmu.2020.00512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7105702PMC
March 2021

Respiratory Syncytial Virus Infection of Human Lung Fibroblasts Induces a Hyaluronan-Enriched Extracellular Matrix That Binds Mast Cells and Enhances Expression of Mast Cell Proteases.

Front Immunol 2019 28;10:3159. Epub 2020 Jan 28.

Division of Pulmonary and Sleep Medicine, Seattle Children's Hospital, Seattle, WA, United States.

Human lung fibroblasts (HLFs) treated with the viral mimetic polyinosine-polycytidylic acid (poly I:C) form an extracellular matrix (ECM) enriched in hyaluronan (HA) that avidly binds monocytes and lymphocytes. Mast cells are important innate immune cells in both asthma and acute respiratory infections including respiratory syncytial virus (RSV); however, the effect of RSV on HA dependent mast cell adhesion and/or function is unknown. To determine if RSV infection of HLFs leads to the formation of a HA-enriched ECM that binds and enhances mast cell activity primary HLFs were infected with RSV for 48 h prior to leukocyte binding studies using a fluorescently labeled human mast cell line (LUVA). Parallel HLFs were harvested for characterization of HA production by ELISA and size exclusion chromatography. In separate experiments, HLFs were infected as above for 48 h prior to adding LUVA cells to HLF wells. Co-cultures were incubated for 48 h at which point media and cell pellets were collected for analysis. The role of the hyaladherin tumor necrosis factor-stimulated gene 6 (TSG-6) was also assessed using siRNA knockdown. RSV infection of primary HLFs for 48 h enhanced HA-dependent LUVA binding assessed by quantitative fluorescent microscopy. This coincided with increased HLF HA synthase (HAS) 2 and HAS3 expression and decreased hyaluronidase (HYAL) 2 expression leading to increased HA accumulation in the HLF cell layer and the presence of larger HA fragments. Separately, LUVAs co-cultured with RSV-infected HLFs for 48 h displayed enhanced production of the mast cell proteases, chymase, and tryptase. Pre-treatment with the HA inhibitor 4-methylumbelliferone (4-MU) and neutralizing antibodies to CD44 (HA receptor) decreased mast cell protease expression in co-cultured LUVAs implicating a direct role for HA. TSG-6 expression was increased over the 48-h infection. Inhibition of HLF TSG-6 expression by siRNA knockdown led to decreased LUVA binding suggesting an important role for this hyaladherin for LUVA adhesion in the setting of RSV infection. In summary, RSV infection of HLFs contributes to inflammation via HA-dependent mechanisms that enhance mast cell binding as well as mast cell protease expression via direct interactions with the ECM.
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http://dx.doi.org/10.3389/fimmu.2019.03159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6997473PMC
November 2020

The synthesis and secretion of versican isoform V3 by mammalian cells: A role for N-linked glycosylation.

Matrix Biol 2020 07 27;89:27-42. Epub 2020 Jan 27.

Matrix Biology Program, Benaroya Research Institute, Seattle, WA, USA. Electronic address:

Versican is a large extracellular matrix (ECM) chondroitin sulfate (CS) proteoglycan found in most soft tissues, which is encoded by the VCAN gene. At least four major isoforms (V0, V1, V2, and V3) are generated via alternative splicing. The isoforms of versican are expressed and accumulate in various tissues during development and disease, where they contribute to ECM structure, cell growth and migration, and immune regulation, among their many functions. While several studies have identified the mRNA transcript for the V3 isoform in a number of tissues, little is known about the synthesis, secretion, and targeting of the V3 protein. In this study, we used lentiviral generation of doxycycline-inducible rat V3 with a C-terminal tag in stable NIH 3T3 cell lines and demonstrated that V3 is processed through the classical secretory pathway. We further show that N-linked glycosylation is required for efficient secretion and solubility of the protein. By site-directed mutagenesis, we identified amino acids 57 and 330 as the active N-linked glycosylation sites on V3 when expressed in this cell type. Furthermore, exon deletion constructs of V3 revealed that exons 11-13, which code for portions of the carboxy region of the protein (G3 domain), are essential for V3 processing and secretion. Once secreted, the V3 protein associates with hyaluronan along the cell surface and within the surrounding ECM. These results establish critical parameters for the processing, solubility, and targeting of the V3 isoform by mammalian cells and establishes a role for V3 in the organization of hyaluronan.
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http://dx.doi.org/10.1016/j.matbio.2020.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7282976PMC
July 2020

Hyaluronan deposition in islets may precede and direct the location of islet immune-cell infiltrates.

Diabetologia 2020 03 6;63(3):549-560. Epub 2020 Jan 6.

Diabetes Research Program and Clinical Research Center, Benaroya Research Institute at Virginia Mason, Seattle, WA, USA.

Aims/hypothesis: Substantial deposition of the extracellular matrix component hyaluronan (HA) is characteristic of insulitis in overt type 1 diabetes. We investigated whether HA accumulation is detectable in islets early in disease pathogenesis and how this affects the development of insulitis and beta cell mass.

Methods: Pancreas tissue from 15 non-diabetic organ donors who were positive for islet autoantibodies (aAbs) and from 14 similarly aged aAb control donors were examined for the amount of islet HA staining and the presence of insulitis. The kinetics of HA deposition in islets, along with the onset and progression of insulitis and changes in beta cell mass, were investigated in BioBreeding DRLyp/Lyp rats (a model of spontaneous autoimmune diabetes) from 40 days of age until diabetes onset.

Results: Abundant islet HA deposits were observed in pancreas tissues from n = 3 single- and n = 4 double-aAb donors (aAbHA). In these seven tissues, the HA-stained areas in islets measured 1000 ± 240 μm (mean ± SEM) and were fourfold larger than those from aAb control tissues. The aAbHA tissues also had a greater prevalence of islets that were highly rich in HA (21% of the islets in these tissues contained the largest HA-stained areas [>2000 μm] vs less than 1% in tissues from aAb control donors). The amount of HA staining in islets was associated with the number of aAbs (i.e. single- or double-aAb positivity) but not with HLA genotype or changes in beta cell mass. Among the seven aAbHA tissues, three from single- and one from double-aAb donors did not show any islet immune-cell infiltrates, indicating that HA accumulates in aAb donors independently of insulitis. The three aAbHA tissues that exhibited insulitis had the largest HA-stained areas and, in these tissues, islet-infiltrating immune cells co-localised with the most prominent HA deposits (i.e. with HA-stained areas >2000 μm). Accumulation of HA in islets was evident prior to insulitis in 7-8-week-old presymptomatic DRLyp/Lyp rats, in which the islet HA-stained area measured 2370 ± 170 μm (mean ± SEM), which was threefold larger than in 6-week-old rats. This initial islet HA deposition was not concurrent with beta cell loss. Insulitis was first detected in 9-10-week-old rats, in which the HA-stained areas were 4980 ± 500 μm. At this age, the rats also exhibited a 44% reduction in beta cell mass. Further enlargement of the HA-positive areas (mean ± SEM: 7220 ± 880 μm) was associated with invasive insulitis. HA deposits remained abundant in the islets of rats with destructive insulitis, which had lost 85% of their beta cells.

Conclusions/interpretation: This study indicates that HA deposition in islets occurs early in type 1 diabetes and prior to insulitis, and points to a potential role of HA in triggering islet immune-cell infiltration and the promotion of insulitis.
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http://dx.doi.org/10.1007/s00125-019-05066-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7002022PMC
March 2020

Targeting the Extracellular Matrix Promotes Healing Following Myocardial Infarction.

Circ Res 2019 10 10;125(9):802-804. Epub 2019 Oct 10.

From the Matrix Biology Program, Benaroya Research Institute, Seattle, WA.

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http://dx.doi.org/10.1161/CIRCRESAHA.119.315902DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6788771PMC
October 2019

Cardiac Hyaluronan Synthesis Is Critically Involved in the Cardiac Macrophage Response and Promotes Healing After Ischemia Reperfusion Injury.

Circ Res 2019 05;124(10):1433-1447

From the Institut für Pharmakologie und Klinische Pharmakologie (A.P., M.G., D.J.G., M.A., M.P., R.S., S.H., J.M., S.G., J.W.F.), University Hospital, Heinrich-Heine-University Düsseldorf, Germany.

Rationale: Immediate changes in the ECM (extracellular matrix) microenvironment occur after myocardial ischemia and reperfusion (I/R) injury.

Objective: Aim of this study was to unravel the role of the early hyaluronan (HA)-rich ECM after I/R.

Methods And Results: Genetic deletion of Has2 and Has1 was used in a murine model of cardiac I/R. Chemical exchange saturation transfer imaging was adapted to image cardiac ECM post-I/R. Of note, the cardiac chemical exchange saturation transfer signal was severely suppressed by Has2 deletion and pharmacological inhibition of HA synthesis 24 hours after I/R. Has2 KO ( Has2 deficient) mice showed impaired hemodynamic function suggesting a protective role for endogenous HA synthesis. In contrast to Has2 deficiency, Has1-deficient mice developed no specific phenotype compared with control post-I/R. Importantly, in Has2 KO mice, cardiac macrophages were diminished after I/R as detected by F MRI (magnetic resonance imaging) of perfluorcarbon-labeled immune cells, Mac-2/Galectin-3 immunostaining, and FACS (fluorescence-activated cell sorting) analysis (CD45CD11bLy6GCD64F4/80cells). In contrast to macrophages, cardiac Ly6C and Ly6C monocytes were unaffected post-I/R compared with control mice. Mechanistically, inhibition of HA synthesis led to increased macrophage apoptosis in vivo and in vitro. In addition, α-SMA (α-smooth muscle actin)-positive cells were reduced in the infarcted myocardium and in the border zone. In vitro, the myofibroblast response as measured by Acta2 mRNA expression was reduced by inhibition of HA synthesis and of CD44 signaling. Furthermore, Has2 KO fibroblasts were less able to contract collagen gels in vitro. The effects of HA/CD44 on fibroblasts and macrophages post-I/R might also affect intercellular cross talk because cardiac fibroblasts were activated by monocyte/macrophages and, in turn, protected macrophages from apoptosis.

Conclusions: Increased HA synthesis contributes to postinfarct healing by supporting macrophage survival and by promoting the myofibroblast response. Additionally, imaging of cardiac HA by chemical exchange saturation transfer post-I/R might have translational value.
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http://dx.doi.org/10.1161/CIRCRESAHA.118.313285DOI Listing
May 2019

Increased Hyaluronan and TSG-6 in Association with Neuropathologic Changes of Alzheimer's Disease.

J Alzheimers Dis 2019 ;67(1):91-102

Department of Pathology, Division of Neuropathology, University of Washington, Seattle, WA, USA.

Little is known about the extracellular matrix (ECM) during progression of AD pathology. Brain ECM is abundant in hyaluronan (HA), a non-sulfated glycosaminoglycan synthesized by HA synthases (HAS) 1-3 in a high molecular weight (MW) form that is degraded into lower MW fragments. We hypothesized that pathologic severity of AD is associated with increases in HA and HA-associated ECM molecules. To test this hypothesis, we assessed HA accumulation and size; HA synthases (HAS) 1-3; and the HA-stabilizing hyaladherin, TSG-6 in parietal cortex samples from autopsied research subjects with not AD (CERAD = 0, Braak = 0- II, n = 12-21), intermediate AD (CERAD = 2, Braak = III-IV, n = 13-18), and high AD (CERAD = 3, Braak = V-VI, n = 32-40) neuropathologic change. By histochemistry, HA was associated with deposits of amyloid and tau, and was also found diffusely in brain parenchyma, with overall HA quantity (measured by ELSA) significantly greater in brains with high AD neuropathology. Mean HA MW was similar among the samples. HAS2 and TSG-6 mRNA expression, and TSG-6 protein levels were significantly increased in high AD and both molecules were present in vasculature, NeuN-positive neurons, and Iba1-positive microglia. These results did not change when accounting for gender, advanced age (≥ 90 years versus <90 years), or the clinical diagnosis of dementia. Collectively, our results indicate a positive correlation between HA accumulation and AD neuropathology, and suggest a possible role for HA synthesis and metabolism in AD progression.
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http://dx.doi.org/10.3233/JAD-180797DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6398602PMC
March 2020

Fibroblast gene expression following asthmatic bronchial epithelial cell conditioning correlates with epithelial donor lung function and exacerbation history.

Sci Rep 2018 10 25;8(1):15768. Epub 2018 Oct 25.

Division of Pulmonary and Sleep Medicine, Seattle Children's Hospital, Seattle, WA, USA.

Airway remodeling may contribute to decreased lung function in asthmatic children. Bronchial epithelial cells (BECs) may regulate fibroblast expression of extracellular matrix (ECM) constituents and fibroblast-to-myofibroblast transition (FMT). Our objective was to determine if human lung fibroblast (HLF) expression of collagen I (COL1A1), hyaluronan synthase 2 (HAS2), and the FMT marker alpha-smooth muscle actin (α-SMA) by HLFs conditioned by BECs from asthmatic and healthy children correlate with lung function measures and exacerbation history among BEC donors. BECs from asthmatic (n = 23) and healthy children (n = 15) were differentiated at an air-liquid interface (ALI) and then co-cultured with HLFs for 96 hours. Expression of COL1A1, HAS2, and α-SMA by HLFs was determined by quantitative polymerase chain reaction (qPCR). FMT was quantified by measuring HLF cytoskeletal α-SMA by flow cytometry. Pro-collagen Iα1, hyaluronan (HA), and PGE were measured in BEC-HLF supernatant. Correlations between lung function measures of BEC donors, and COL1A1, HAS2, and α-SMA gene expression, as well as supernatant concentrations of HA, pro-collagen Iα1, hyaluronan (HA), and PGE were assessed. We observed that expression of α-SMA and COL1A1 by HLFs co-cultured with asthmatic BECs was negatively correlated with BEC donor lung function. BEC-HLF supernatant concentrations of pro-collagen Iα1 were negatively correlated, and PGE concentrations positively correlated, with asthmatic BEC donor lung function. Expression of HAS2, but not α-SMA or COL1A1, was greater by HLFs co-cultured with asthmatic BECs from donors with a history of severe exacerbations than by HLFs co-cultured with BECs from donors who lacked a history of severe exacerbations. In conclusion, α-SMA and COL1A1 expression by HLFs co-cultured with BECs from asthmatic children were negatively correlated with lung function measures, supporting our hypothesis that epithelial regulation of HLFs and airway deposition of ECM constituents by HLFs contributes to lung function deficits among asthmatic children. Furthermore, epithelial regulation of airway HAS2 may influence the susceptibility of children with asthma to experience severe exacerbations. Finally, epithelial-derived PGE is a potential regulator of airway FMT and HLF production of collagen I that should be investigated further in future studies.
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http://dx.doi.org/10.1038/s41598-018-34021-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6202408PMC
October 2018

Versican is differentially regulated in the adventitial and medial layers of human vein grafts.

PLoS One 2018 28;13(9):e0204045. Epub 2018 Sep 28.

Matrix Biology Program, Benaroya Research Institute, Seattle, WA, United States of America.

Changes in extracellular matrix proteins may contribute significantly to the adaptation of vein grafts to the arterial circulation. We examined the production and distribution of versican and hyaluronan in intact human vein rings cultured ex vivo, veins perfused ex vivo, and cultured venous adventitial and smooth muscle cells. Immunohistochemistry revealed higher levels of versican in the intima/media compared to the adventitia, and no differences in hyaluronan. In the vasa vasorum, versican and hyaluronan associated with CD34+ progenitor cells. Culturing the vein rings for 14 days revealed increased versican immunostaining of 30-40% in all layers, with no changes in hyaluronan. Changes in versican accumulation appear to result from increased synthesis in the intima/media and decreased degradation in the adventitia as versican transcripts were increased in the intima/media, but unchanged in the adventitia, and versikine (the ADAMTS-mediated cleavage product of versican) was increased in the intima/media, but decreased in the adventitia. In perfused human veins, versican was specifically increased in the intima/media in the presence of venous pressure, but not with arterial pressure. Unexpectedly, cultured adventitial cells express and accumulate more versican and hyaluronan than smooth muscle cells. These data demonstrate a differential regulation of versican and hyaluronan in human venous adventitia vs. intima/media and suggest distinct functions for these extracellular matrix macromolecules in these venous wall compartments during the adaptive response of vein grafts to the arterial circulation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0204045PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6161854PMC
March 2019

A Role for Extracellular Matrix in Atherosclerotic Plaque Erosion.

Authors:
Thomas N Wight

J Am Coll Cardiol 2018 09;72(13):1504-1505

Matrix Biology Program, Benaroya Research Institute, Seattle, Washington. Electronic address:

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http://dx.doi.org/10.1016/j.jacc.2018.07.031DOI Listing
September 2018

Hyaluronan levels are increased systemically in human type 2 but not type 1 diabetes independently of glycemic control.

Matrix Biol 2019 07 6;80:46-58. Epub 2018 Sep 6.

Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Beckman Center, 279 Campus Drive, Stanford, CA 94305, USA.

Hyaluronan (HA), an extracellular matrix glycosaminoglycan, is implicated in the pathogenesis of both type 1 diabetes (T1D) as well as type 2 diabetes (T2D) and has been postulated to be increased in these diseases due to hyperglycemia. We have examined the serum and tissue distribution of HA in human subjects with T1D and T2D and in mouse models of these diseases and evaluated the relationship between HA levels and glycemic control. We found that serum HA levels are increased in T2D but not T1D independently of hemoglobin-A1c, C-peptide, body mass index, or time since diabetes diagnosis. HA is likewise increased in skeletal muscle in T2D subjects relative to non-diabetic controls. Analogous increases in serum and muscle HA are seen in diabetic db/db mice (T2D), but not in diabetic DORmO mice (T1D). Diabetes induced by the β-cell toxin streptozotozin (STZ) lead to an increase in blood glucose but not to an increase in serum HA. These data indicate that HA levels are increased in multiple tissue compartments in T2D but not T1D independently of glycemic control. Given that T2D but not T1D is associated with systemic inflammation, these patterns are consistent with inflammatory factors and not hyperglycemia driving increased HA. Serum HA may have value as a biomarker of systemic inflammation in T2D.
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http://dx.doi.org/10.1016/j.matbio.2018.09.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6401354PMC
July 2019

Immune Checkpoint Ligand PD-L1 Is Upregulated in Pulmonary Lymphangioleiomyomatosis.

Am J Respir Cell Mol Biol 2018 12;59(6):723-732

3 Penn Center for Pulmonary Biology, Pulmonary, Allergy and Critical Care Division, and.

Pulmonary lymphangioleiomyomatosis (LAM) is a slow-progressing metastatic disease that is driven by mutations in the tumor suppressor tuberous sclerosis complex 1/2 (TSC1/2). Rapamycin inhibits LAM cell proliferation and is the only approved treatment, but it cannot cause the regression of existing lesions and can only stabilize the disease. However, in other cancers, immunotherapies such as checkpoint blockade against PD-1 and its ligand PD-L1 have shown promise in causing tumor regression and even curing some patients. Thus, we asked whether PD-L1 has a role in LAM progression. In vitro, PD-L1 expression in murine Tsc2-null cells is unaffected by mTOR inhibition with torin but can be upregulated by IFN-γ. Using immunohistochemistry and single-cell flow cytometry, we found increased PD-L1 expression both in human lung tissue from patients with LAM and in Tsc2-null lesions in a murine model of LAM. In this model, PD-L1 is highly expressed in the lung by antigen-presenting and stromal cells, and activated T cells expressing PD-1 infiltrate the affected lung. In vivo treatment with anti-PD-1 antibody significantly prolongs mouse survival in the model of LAM. Together, these data demonstrate that PD-1/PD-L1-mediated immunosuppression may occur in LAM, and suggest new opportunities for therapeutic targeting that may provide benefits beyond those of rapamycin.
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http://dx.doi.org/10.1165/rcmb.2018-0123OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6293078PMC
December 2018

Asthmatic bronchial epithelial cells promote the establishment of a Hyaluronan-enriched, leukocyte-adhesive extracellular matrix by lung fibroblasts.

Respir Res 2018 08 2;19(1):146. Epub 2018 Aug 2.

Division of Pulmonary and Sleep Medicine, Seattle Children's Hospital, 4800 Sand Point Way NE, Seattle, WA, 98105, USA.

Background: Airway inflammation is a hallmark of asthma. Alterations in extracellular matrix (ECM) hyaluronan (HA) content have been shown to modulate the recruitment and retention of inflammatory cells. Bronchial epithelial cells (BECs) regulate the activity of human lung fibroblasts (HLFs); however, their contribution in regulating HLF production of HA in asthma is unknown. In this study, we tested the hypothesis that BECs from asthmatic children promote the generation of a pro-inflammatory, HA-enriched ECM by HLFs, which promotes the retention of leukocytes.

Methods: BECs were obtained from well-characterized asthmatic and healthy children ages 6-18 years. HLFs were co-cultured with BECs for 96 h and samples were harvested for analysis of gene expression, synthesis and accumulation of HA, and subjected to a leukocyte adhesion assay with U937 monocytes.

Results: We observed increased expression of HA synthases HAS2 and HAS3 in HLFs co-cultured with asthmatic BECs. Furthermore, we demonstrated greater total accumulation and increased synthesis of HA by HLFs co-cultured with asthmatic BECs compared to healthy BEC/HLF co-cultures. ECM generated by HLFs co-cultured with asthmatic BECs displayed increased HA-dependent adhesion of leukocytes in a separate in vitro binding assay.

Conclusions: Our findings demonstrate that BEC regulation of HA production by HLFs is altered in asthma, which may in turn promote the establishment of a more leukocyte-permissive ECM promoting airway inflammation in this disease.
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http://dx.doi.org/10.1186/s12931-018-0849-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6090698PMC
August 2018

Smooth muscle glucose metabolism promotes monocyte recruitment and atherosclerosis in a mouse model of metabolic syndrome.

JCI Insight 2018 06 7;3(11). Epub 2018 Jun 7.

Department of Medicine, University of Washington Medicine Diabetes Institute, University of Washington School of Medicine, Seattle, Washington, USA.

Metabolic syndrome contributes to cardiovascular disease partly through systemic risk factors. However, local processes in the artery wall are becoming increasingly recognized to exacerbate atherosclerosis both in mice and humans. We show that arterial smooth muscle cell (SMC) glucose metabolism markedly synergizes with metabolic syndrome in accelerating atherosclerosis progression, using a low-density lipoprotein receptor-deficient mouse model. SMCs in proximity to atherosclerotic lesions express increased levels of the glucose transporter GLUT1. Cytokines, such as TNF-α produced by lesioned arteries, promote GLUT1 expression in SMCs, which in turn increases expression of the chemokine CCL2 through increased glycolysis and the polyol pathway. Furthermore, overexpression of GLUT1 in SMCs, but not in myeloid cells, accelerates development of larger, more advanced lesions in a mouse model of metabolic syndrome, which also exhibits elevated levels of circulating Ly6Chi monocytes expressing the CCL2 receptor CCR2. Accordingly, monocyte tracing experiments demonstrate that targeted SMC GLUT1 overexpression promotes Ly6Chi monocyte recruitment to lesions. Strikingly, SMC-targeted GLUT1 overexpression fails to accelerate atherosclerosis in mice that do not exhibit the metabolic syndrome phenotype or monocytosis. These results reveal a potentially novel mechanism whereby arterial smooth muscle glucose metabolism synergizes with metabolic syndrome to accelerate monocyte recruitment and atherosclerosis progression.
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http://dx.doi.org/10.1172/jci.insight.96544DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6124428PMC
June 2018

A role for proteoglycans in vascular disease.

Authors:
Thomas N Wight

Matrix Biol 2018 10 27;71-72:396-420. Epub 2018 Feb 27.

Matrix Biology Program, Benaroya Research Institute at Virginia Mason, Seattle, WA 98101, United States. Electronic address:

The content of proteoglycans (PGs) is low in the extracellular matrix (ECM) of vascular tissue, but increases dramatically in all phases of vascular disease. Early studies demonstrated that glycosaminoglycans (GAGs) including chondroitin sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS) and heparan sulfate (HS) accumulate in vascular lesions in both humans and in animal models in areas of the vasculature that are susceptible to disease initiation (such as at branch points) and are frequently coincident with lipid deposits. Later studies showed the GAGs were covalently attached to specific types of core proteins that accumulate in vascular lesions. These molecules include versican (CSPG), biglycan and decorin (DS/CSPGs), lumican and fibromodulin (KSPGs) and perlecan (HSPG), although other types of PGs are present, but in lesser quantities. While the overall molecular design of these macromolecules is similar, there is tremendous structural diversity among the different PG families creating multiple forms that have selective roles in critical events that form the basis of vascular disease. PGs interact with a variety of different molecules involved in disease pathogenesis. For example, PGs bind and trap serum components that accumulate in vascular lesions such as lipoproteins, amyloid, calcium, and clotting factors. PGs interact with other ECM components and regulate, in part, ECM assembly and turnover. PGs interact with cells within the lesion and alter the phenotypes of both resident cells and cells that invade the lesion from the circulation. A number of therapeutic strategies have been developed to target specific PGs involved in key pathways that promote vascular disease. This review will provide a historical perspective of this field of research and then highlight some of the evidence that defines the involvement of PGs and their roles in the pathogenesis of vascular disease.
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http://dx.doi.org/10.1016/j.matbio.2018.02.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6110991PMC
October 2018

Proteoglycans as Immunomodulators of the Innate Immune Response to Lung Infection.

J Histochem Cytochem 2018 04 12;66(4):241-259. Epub 2018 Jan 12.

Center for Lung Biology, Division of Pulmonary/Critical Care Medicine, University of Washington School of Medicine, Seattle, Washington.

Proteoglycans (PGs) are complex, multifaceted molecules that participate in diverse interactions vital for physiological and pathological processes. As structural components, they provide a scaffold for cells and structural organization that helps define tissue architecture. Through interactions with water, PGs enable molecular and cellular movement through tissues. Through selective ionic interactions with growth factors, chemokines, cytokines, and proteases, PGs facilitate the ability of these soluble ligands to regulate intracellular signaling events and to influence the inflammatory response. In addition, recent findings now demonstrate that PGs can activate danger-associated molecular patterns (DAMPs) and other signaling pathways to influence production of many of these soluble ligands, indicating a more direct role for PGs in influencing the immune response and tissue inflammation. This review will focus on PGs that are selectively expressed during lung inflammation and will examine the novel emerging concept of PGs as immunomodulatory regulators of the innate immune responses in lungs.
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http://dx.doi.org/10.1369/0022155417751880DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5958380PMC
April 2018

The biochemistry and immunohistochemistry of versican.

Methods Cell Biol 2018 26;143:261-279. Epub 2017 Nov 26.

Matrix Biology Program, Benaroya Research Institute, Seattle, WA, United States. Electronic address:

Versican is a chondroitin sulfate proteoglycan found in the extracellular matrix that is important for changes in cell phenotype associated with development and disease. Versican has been shown to be involved in cardiovascular disorders, as well as lung disease and fibrosis, inflammatory bowel disease, cancer, and several other diseases that have an inflammatory component. Versican was first identified as a fibroblast proteoglycan and forms large multimolecular complexes with hyaluronan and other components of the provisional matrix during wound healing and inflammation. The biology of versican has been well studied. Versican plays a major role in embryogenesis, particularly heart formation, where versican deletion proves lethal. The ability to purify versican to characterize and to use in experimental systems is vital to defining its role in development and disease. Protein expression systems have proven challenging to obtain milligram quantities of full-length versican. Here, we describe proteoglycan biochemical purification techniques that have been developed by others, but which we have adapted to use with our source tissues and cells. We also include methods for immunohistochemical localization and quantitation of versican in tissue sections.
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http://dx.doi.org/10.1016/bs.mcb.2017.08.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6419768PMC
November 2018