In normal airways, hyaluronan (HA) matrices are primarily located within the airway submucosa, pulmonary vasculature walls, and, to a lesser extent, the alveoli. Following pulmonary injury, elevated ...levels of HA matrices accumulate in these regions, and in respiratory secretions, correlating with the extent of injury. Animal models have provided important insight into the role of HA in the onset of pulmonary injury and repair, generally indicating that the induction of HA synthesis is an early event typically preceding fibrosis. The HA that accumulates in inflamed airways is of a high molecular weight (>1600 kDa) but can be broken down into smaller fragments (<150 kDa) by inflammatory and disease-related mechanisms that have profound effects on HA pathobiology. During inflammation in the airways, HA is often covalently modified with heavy chains from inter-alpha-inhibitor via the enzyme tumor-necrosis-factor-stimulated-gene-6 (TSG-6) and this modification promotes the interaction of leukocytes with HA matrices at sites of inflammation. The clearance of HA and its return to normal levels is essential for the proper resolution of inflammation. These data portray HA matrices as an important component of normal airway physiology and illustrate its integral roles during tissue injury and repair among a variety of respiratory diseases.
The cell and its glycosaminoglycan-rich pericellular matrix (PCM) comprise a functional unit. Because modification of PCM influences cell behavior, we investigated molecular mechanisms that regulate ...PCM volume and composition. In fibroblasts and other cells, aggregates of hyaluronan and versican are found in the PCM. Dermal fibroblasts from Adamts5(-/-) mice, which lack a versican-degrading protease, ADAMTS5, had reduced versican proteolysis, increased PCM, altered cell shape, enhanced α-smooth muscle actin (SMA) expression and increased contractility within three-dimensional collagen gels. The myofibroblast-like phenotype was associated with activation of TGFβ signaling. We tested the hypothesis that fibroblast-myofibroblast transition in Adamts5(-/-) cells resulted from versican accumulation in PCM. First, we noted that versican overexpression in human dermal fibroblasts led to increased SMA expression, enhanced contractility, and increased Smad2 phosphorylation. In contrast, dermal fibroblasts from Vcan haploinsufficient (Vcan(hdf/+)) mice had reduced contractility relative to wild type fibroblasts. Using a genetic approach to directly test if myofibroblast transition in Adamts5(-/-) cells resulted from increased PCM versican content, we generated Adamts5(-/-);Vcan(hdf/+) mice and isolated their dermal fibroblasts for comparison with dermal fibroblasts from Adamts5(-/-) mice. In Adamts5(-/-) fibroblasts, Vcan haploinsufficiency or exogenous ADAMTS5 restored normal fibroblast contractility. These findings demonstrate that altering PCM versican content through proteolytic activity of ADAMTS5 profoundly influenced the dermal fibroblast phenotype and may regulate a phenotypic continuum between the fibroblast and its alter ego, the myofibroblast. We propose that a physiological function of ADAMTS5 in dermal fibroblasts is to maintain optimal versican content and PCM volume by continually trimming versican in hyaluronan-versican aggregates.
We propose a modification of a vacuum-insulator-electrode geometry that is commonly found in particle accelerators to reduce the electric field near the anode triple junction and reduce dielectric ...flashover.
Hyaluronan (HA) is a large (>1500 kDa) polysaccharide of the extracellular matrix that has been linked to severity and inflammation in asthma. During inflammation, HA becomes covalently modified with ...heavy chains (HC-HA) from inter-α-inhibitor (IαI), which functions to increase its avidity for leukocytes. Our murine model of allergic pulmonary inflammation suggested that HC-HA may contribute to inflammation, adversely effecting lower airway remodeling and asthma severity. Our objective was to characterize the levels of HA and HC-HA in asthmatic subjects and to correlate these levels with asthma severity. We determined the levels and distribution of HA and HC-HA (i) from asthmatic and control lung tissue, (ii) in bronchoalveolar lavage fluid obtained from non-severe and severe asthmatics and controls, and (iii) in serum and urine from atopic asthmatics after an experimental asthma exacerbation. HC-HA distribution was observed (i) in the thickened basement membrane of asthmatic lower airways, (ii) around smooth muscle cells of the asthmatic submucosa, and (iii) around reserve cells of the asthmatic epithelium. Patients with severe asthma had increased HA levels in bronchoalveolar lavage fluid that correlated with pulmonary function and nitric oxide levels, whereas HC-HA was only observed in a patient with non-severe asthma. After an experimental asthma exacerbation, serum HA was increased within 4 h after challenge and remained elevated through 5 days after challenge. Urine HA and HC-HA were not significantly different. These data implicate HA and HC-HA in the pathogenesis of asthma severity that may occur in part due to repetitive asthma exacerbations over the course of the disease.
Background: Hyaluronan has been linked to asthma severity and inflammation.
Results: We characterized the hyaluronan levels and its heavy chain modification in an experimental model of human asthma exacerbation.
Conclusion: These data implicate hyaluronan and its heavy chain modification in human asthma severity.
Significance: Repetitive asthma exacerbations exacerbate hyaluronan pathobiology, which contribute to the chronic inflammation associated with this disease.
Previous studies have demonstrated that ovalbumin sensitization promotes chronic asthma phenotype in murine asthma model. Human mesenchymal stem cells (hMSCs) are multipotent cells in vitro that have ...been shown to decrease inflammation and can reverse airway remodeling when infused into an in vivo chronic asthma model. However, the mechanism by which hMSCs reverse remodeling is still unclear. In this study, we hypothesized that hMSCs influence remodeling by decreasing extracellular matrix (ECM) deposition, more specifically by decreasing collagen I, collagen III, and hyaluronan synthesis.
Chronic asthma phenotype was produced in an in vitro model with 3 T3 murine airway fibroblast cells by stimulating with GM-CSF. Collagen I and collagen III gene expression was investigated using RT-PCR and Taqman techniques. Hyaluronan was evaluated using FACE and Western Blots. The chronic asthma phenotype was produced in vivo in murine model using sensitization with ovalbumin with and without hMSC infusion therapy. ECM deposition (specifically trichrome staining, soluble and insoluble collagen deposition, and hyaluronan production) was evaluated. Image quantification was used to monitor trichrome staining changes.
GM-CSF which induced collagen I and collagen III production was down-regulated with hMSC using co-culture. In the in vivo model, Ovalbumin induced enhanced ECM deposition, soluble and insoluble collagen production, and lung elastance. hMSC infusions decreased ECM deposition as evidenced by decreases in soluble and insoluble collagen production.
hMSCs participate in improved outcomes of remodeling by reversing excess collagen deposition and changing hyaluronan levels.
Asthma is a chronic inflammatory disease of the airways characterized by airway remodeling, which includes changes in the extracellular matrix (ECM). However the role of the ECM in mediating these ...changes is poorly understood. Hyaluronan (HA), a major component of the ECM, has been implicated in asthma as well as in many other biological processes. Our study investigates the processes involved in HA synthesis, deposition, localization and degradation during an acute and chronic murine model of ovalbumin (OVA)-induced allergic pulmonary inflammation. Mice were sensitized, challenged to OVA and sacrificed at various time points during an 8-week challenge protocol. Bronchoalveolar lavage (BAL) fluids, blood, and lung tissue were collected for study. RNA, HA, protein and histopathology were analyzed. Analyses of lung sections and BAL fluids revealed an early deposition and an increase in HA levels within 24
h of antigen exposure. HA levels peaked at day 8 in BAL, while inflammatory cell recovery peaked at day 6. Hyaluronan synthase (HAS)1 and HAS2 on RNA levels peaked within 2
h of antigen exposure, while hyaluronidase (HYAL)1 and HYAL2 on RNA levels decreased. Both inflammatory cell infiltrates and collagen deposition co-localized with HA deposition within the lungs. These data support a role for HA in the pathogenesis of inflammation and airway remodeling in a murine model of asthma. HA deposition appears largely due to up regulation of HAS1 and HAS2. In addition, HA appears to provide the scaffolding for inflammatory cell accumulation as well as for new collagen synthesis and deposition.
The covalent transfer of heavy chains (HCs) from inter-α-inhibitor (IαI) to hyaluronan (HA) via the protein product of tumor necrosis factor-stimulated gene-6 (TSG-6) forms the HC-HA complex, a ...pathological form of HA that promotes the adhesion of leukocytes to HA matrices. The transfer of HCs to high molecular weight (HMW) HA is a reversible event whereby TSG-6 can shuffle HCs from one HA molecule to another. Therefore, HMW HA can serve as both an HC acceptor and an HC donor. In the present study, we show that transfer of HCs to low molecular weight HA oligosaccharides is an irreversible event where subsequent shuffling does not occur, i.e. HA oligosaccharides from 8 to 21 monosaccharide units in length can serve as HC acceptors, but are unable to function as HC donors. We show that the HC-HA complex is present in the synovial fluid of mice subjected to systemic and monoarticular mouse models of rheumatoid arthritis. Furthermore, we demonstrate that HA oligosaccharides can be used, with TSG-6, to irreversibly shuffle HCs from pathological, HMW HC-HA to HA oligosaccharides, thereby restoring HC-HA matrices from the inflamed joint to their normal state, unmodified with HCs. This process was also effective for HC-HA in the synovial fluid of human rheumatoid arthritis patients (in vitro).
Background: TSG-6 transfers heavy chains from inter-α-inhibitor to hyaluronan.
Results: Heavy chain transfer to hyaluronan by TSG-6 is reversible for high molecular weight hyaluronan but irreversible for hyaluronan oligosaccharides.
Conclusion: High molecular weight hyaluronan functions as both a heavy chain acceptor and a heavy chain donor, whereas hyaluronan oligosaccharides function only as heavy chain acceptors.
Significance: Hyaluronan oligosaccharides have potential to remove heavy chains from pathological hyaluronan.
We previously reported an altered hyaluronan (HA) metabolism in idiopathic pulmonary arterial hypertension (IPAH) lung tissue and cultured smooth muscle cells. Hyaluronan was present in the smooth ...muscle cell layer surrounding the pulmonary vasculature and in plexigenic lesions. Additionally, cultured pulmonary artery smooth muscle cells produced spontaneous HA “cable” structures, without additional stimuli, that were leukocyte-adhesive. We now present evidence that the HA that accumulates in IPAH plexigenic lesions is a pathological form of HA in which heavy chains (HCs) from the serum-derived proteoglycan inter-α-inhibitor are covalently attached to the HA backbone to form a pathological HC-HA complex. CD45-positive leukocytes were identified within these HC-HA matrices. Elevated mRNA levels of the enzyme that transfers HCs to HA, known as tumor necrosis factor-stimulated gene 6, were detected in IPAH lung tissue.
Background: Hyaluronan metabolism is altered in idiopathic pulmonary arterial hypertension lung tissue.
Results: This hyaluronan is of a pathological form covalently modified by heavy chains from inter-α-inhibitor.
Conclusion: The distribution of this matrix in regions of abnormal vascular remodeling implies a mechanistic role.
Significance: Pathological hyaluronan matrices may direct inflammatory events and vascular changes in this disease.
Asthma is a chronic inflammatory disease that exhibits airway remodeling with changes in the extracellular matrix (ECM). The role of the ECM in mediating these changes is poorly understood. ...Hyaluronan (HA), a major component of the ECM, has been implicated in many biological processes in diseases. This study investigates the processes involved in HA synthesis, deposition and localization during the propagation of cockroach-induced asthma. Mice were sensitized and challenged with cockroach antigen, and sacrificed at various time points during an 8-week challenge protocol. Analysis of bronchoalveolar lavage (BAL) fluid revealed an increase in total nucleated cells as early as 6h, which peaked at 6 days. Histopathologic analysis of the lung tissue revealed an influx of inflammatory cells at the peribronchial and perivascular regions starting at 12 h, which peaked at 6 days and persisted to 8 weeks. Eosinophils predominated in the early time points while lymphocytes predominated during the late time points. Quantitative polymerase chain reaction (PCR) data showed that hyaluronan synthase 1 (HAS1) mRNA peaked within 6 h and then declined. HAS2 mRNA also peaked within 6 h but remained elevated throughout the 8-week exposure course. HA levels in lung tissue and BAL increased at 12 h and peaked by 6 and 8 days, respectively. Inflammatory cells and new collagen formation localized in areas of HA deposition. Taken together, these data support a role for HA in the pathogenesis in asthma.
Asthmatic attacks often follow viral infections with subsequent airway smooth muscle cell proliferation and the formation of an abnormal hyaluronan extracellular matrix with infiltrated leukocytes. ...In this study, we show that murine airway smooth muscle cells (MASM) treated with polyinosinic acid-polycytidylic acid (poly(I,C)), a double-stranded RNA that simulates a viral infection, synthesize an abnormal hyaluronan matrix that binds leukocytes (U937 cells). Synthesis of this matrix is initiated rapidly and accumulates linearly for ∼10 h, reaching a plateau level ∼7-fold higher than control cultures. MASM cells treated with tunicamycin, to induce endoplasmic reticulum stress, also rapidly initiate synthesis of the abnormal hyaluronan matrix with linear accumulation for ∼10 h, but only reach a plateau level ∼2-fold higher than control cultures. In contrast to poly(I,C), the response to tunicamycin depends on cell density, with pre-confluent cells producing more abnormal matrix per cell. Furthermore, U937 cell adhesion per hyaluronan content is higher in the sparse matrix produced in response to tunicamycin, suggesting that the structure in the poly(I,C)-induced matrix masks potential binding sites. When MASM cells were exposed to tunicamycin and poly(I,C) at the same time, U937 cell adhesion was partially additive, implying that these two toxins stimulate hyaluronan synthesis through two different pathways. We also characterized the size of hyaluronan produced by MASM cells, in response to poly(I,C) and tunicamycin, and we found that it ranges from 1500 to 4000 kDa, the majority of which was ∼4000 kDa and not different in size than hyaluronan made by untreated cells.
PDF
