Search

Background Continuous positive airway pressure (CPAP) is a primary mode of respiratory support for preterm infants. Animal studies have shown long-term detrimental effects on lung/airway development, particularly airway (AW) hyper-reactivity, as an unfortunate consequence of neonatal CPAP. Since the hyaluronan (HA) synthesizing enzyme hyaluronan synthase-3 (HAS3) is involved in various adult pulmonary disorders, the present study used a neonatal mouse model to investigate the role of HAS3 in CPAP-induced AW hyper-reactivity. Methods Male and female neonatal mice were fitted with a custom-made mask for delivery of daily CPAP 3 h/day for 7 days. At postnatal day 21 (2 weeks after CPAP ended), airway (AW) hyper-reactivity and HAS3 expression were assessed with and without in vitro HAS3 siRNA treatment. Results MRIs of 3-day-old mice confirmed that CPAP increased lung volume with incrementing inflation pressures. CPAP increased AW reactivity in both male and female mice, which was associated with increased airway smooth muscle and epithelial HAS3 immunoreactivity. CPAP did not affect HA accumulation, but HAS3 siRNA reversed CPAP-induced AW hyper-reactivity and reduced HAS3 expression. Conclusions These data in mice implicate a role for HAS3 in long-term effects of CPAP in the developing airway in the context of preterm birth and CPAP therapy. Impact Neonatal CPAP increases airway smooth muscle and epithelial HAS3 expression in mice. CPAP-induced airway hyper-reactivity is modulated by HAS3. These data enhance our understanding of the role mechanical forces play on lung development. These data are a significance step toward understanding CPAP effects on developing airway. These data may impact clinical recognition of the ways that CPAP may contribute to wheezing disorders of former preterm infants.

Infants born with neonatal opioid withdrawal syndrome (NOWS) can display abnormal cardiorespiratory patterns including tachypnea, tachycardia, and impaired ventilatory responses to hypoxia (HVR) and hypercapnia (HCVR). Chronic morphine exposure is associated with increased midbrain microglial expression. Using a rat model of pre- and post-natal morphine exposure, we assessed cardiorespiratory features of NOWS (resting tachycardia and tachypnea) including the attenuated HVR and HCVR and whether they are associated with increased brainstem microglia expression. Pregnant rats (dams) received twice-daily subcutaneous injections of morphine (5 mg/kg) during the third (last) week of pregnancy to simulate 3rd trimester in utero opioid exposure. Offspring then received once-daily subcutaneous injections of morphine (0.5 mg/kg) until postnatal (P) day P10 days of age to simulate postnatal morphine therapy. Cardiorespiratory responses were assessed 24 h later (P11 days) following spontaneous withdrawal. Compared to saline-treated pups, morphine-exposed offspring exhibited tachycardia and tachypnea as well as an attenuated HVR and HCVR. Microglial cell counts were increased in the nucleus tractus solitarius (nTS), dorsal motor nucleus of the vagus (DMNV) and nucleus ambiguous (NAamb), but not the retrapezoid nucleus (RTN) or the non-cardiorespriatory region, the cuneate nucleus (CN). These data suggest that the cardiorespiratory features and autonomic dysregulation in NOWS infants may be associated with altered microglial function in specific brainstem cardiorespiratory control regions.

Chronic hypoxia leads to pathologic remodeling of the pulmonary vasculature and pulmonary hypertension (PH). The antioxidant enzyme extracellular superoxide dismutase (SOD3) protects against hypoxia-induced PH. Hyaluronan (HA), a ubiquitous glycosaminoglycan of the lung extracellular matrix, is rapidly recycled at sites of vessel injury and repair. We investigated the hypothesis that SOD3 preserves HA homeostasis by inhibiting oxidative and enzymatic hyaluronidase-mediated HA breakdown. In SOD3-deficient mice, hypoxia increased lung hyaluronidase expression and activity, hyaluronan fragmentation, and effacement of HA from the vessel wall of small pulmonary arteries. Hyaluronan fragmentation corresponded to hypoxic induction of the cell surface hyaluronidase-2 (Hyal2), which was localized in the vascular media. Human pulmonary artery smooth muscle cells (HPASMCs) demonstrated hypoxic induction of Hyal2 and SOD-suppressible hyaluronidase activity, congruent to our observations in vivo. Fragmentation of homeostatic high molecular weight HA promoted HPASMC proliferation in vitro, whereas pharmacologic inhibition of hyaluronidase activity prevented hypoxia- and oxidant-induced proliferation. Hypoxia initiates SOD3-dependent alterations in the structure and regulation of hyaluronan in the pulmonary vascular extracellular matrix. These changes occurred soon after hypoxia exposure, prior to appearance of PH, and may contribute to the early pathogenesis of this disease.

The deposition of aggrecan/hyaluronan (HA)-rich matrix within the tendon body and surrounding peritenon impede tendon healing and result in compromised biomechanical properties. Hence, the development of novel strategies to achieve targeted removal of the aggrecan-HA pericellular matrix may be effective in treating tendinopathy. The current study examined the therapeutic potential of a recombinant human hyaluronidase, rHuPH20 (FDA approved for reducing HA accumulation in tumors) for treating murine Achilles tendinopathy. The 12-week-old C57Bl/6 male mice were injected with two doses of rHuTGF-β1 into the retrocalcaneal bursa (RCB) to induce a combined bursitis and tendinopathy. Twenty-four hours following induction of injury, treatment groups were administered rHuPH20 Hyaluronidase (rHuPH20; Halozyme Therapeutics) into the RCB. At either 6 h (acute), 9 days, or 25 days following hyaluronidase treatment, Achilles tendons were analyzed for gene expression, histology and immunohistochemistry, fluorophore-assisted carbohydrate electrophoresis, and biomechanical properties. The rHuPH20 treatment was effective, particularly at the acute and 9-day time points, in (a) removing HA deposits from the Achilles tendon and surrounding tissues, (b) improving biomechanical properties of the healing tendon, and (c) eliciting targeted increases in expression of specific cell fate, extracellular matrix metabolism, and inflammatory genes. The potential of rHuPH20 to effectively clear the pro-inflammatory, HA-rich matrix within the RCB and tendon strongly supports the future refinement of injectable glycosidase preparations as potential treatments to protect or regenerate tendon tissue by reducing inflammation and scarring in the presence of bursitis or other inducers of damage such as mechanical overuse. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:59-69, 2020.

Continuous positive airway pressure (CPAP) is a primary mode of respiratory support for preterm infants. Animal studies have shown long-term detrimental effects on lung/airway development, particularly airway (AW) hyper-reactivity, as an unfortunate consequence of neonatal CPAP. Since the hyaluronan (HA) synthesizing enzyme hyaluronan synthase-3 (HAS3) is involved in various adult pulmonary disorders, the present study used a neonatal mouse model to investigate the role of HAS3 in CPAP-induced AW hyper-reactivity.Male and female neonatal mice were fitted with a custom-made mask for delivery of daily CPAP 3 h/day for 7 days. At postnatal day 21 (2 weeks after CPAP ended), airway (AW) hyper-reactivity and HAS3 expression were assessed with and without in vitro HAS3 siRNA treatment.MRIs of 3-day-old mice confirmed that CPAP increased lung volume with incrementing inflation pressures. CPAP increased AW reactivity in both male and female mice, which was associated with increased airway smooth muscle and epithelial HAS3 immunoreactivity. CPAP did not affect HA accumulation, but HAS3 siRNA reversed CPAP-induced AW hyper-reactivity and reduced HAS3 expression.These data in mice implicate a role for HAS3 in long-term effects of CPAP in the developing airway in the context of preterm birth and CPAP therapy.Neonatal CPAP increases airway smooth muscle and epithelial HAS3 expression in mice. CPAP-induced airway hyper-reactivity is modulated by HAS3. These data enhance our understanding of the role mechanical forces play on lung development. These data are a significance step toward understanding CPAP effects on developing airway. These data may impact clinical recognition of the ways that CPAP may contribute to wheezing disorders of former preterm infants.

Hyaluronan (HA), a high molecular weight non-sulfated glycosaminoglycan, is an integral component of the extracellular matrix of developing and mature connective tissues including tendon. There are few published reports quantifying HA content during tendon growth and maturation, or detailing its effects on the mechanical properties of the tendon extracellular matrix. Therefore, the goal of the current study was to examine the role of HA synthesis during post-natal skeletal growth and maturation, and its influence on tendon structure and biomechanical function. For this purpose, the morphological, biochemical, and mechanical properties of Achilles tendons from wild type (WT) and hyaluronan synthase 1 and 3 deficient mouse strains (Has1-/- (Has1KO), Has3-/- (Has3KO), and Has1-/- 3-/- (Has1/3KO)) were determined at 4, 8, and 12 weeks of age. Overall, HAS-deficient mice did not show any marked differences from WT mice in Achilles tendon morphology or in the HA and chondroitin/dermatan sulfate (CS/DS) contents. However, HAS1-deficiency (in the single or Has1/3 double KO) impeded post-natal formation of the retrocalcaneal bursa, implicating HAS1 in regulating HA metabolism by cells lining the bursal cavity. Together, these data suggest that HA metabolism via HAS1 and HAS3 does not markedly influence the extracellular matrix structure or function of the tendon body, but plays a role in the formation/maintenance of peritendinous bursa. Additional studies are warranted to elucidate the relationship of HA and CS/DS metabolism to tendon healing and repair in vivo. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2622-2632, 2018.

Exposure to pollutants, such as ozone, exacerbates airway inflammation and hyperresponsiveness (AHR). TNF-stimulated gene 6 (TSG-6) is required to transfer inter-α-inhibitor heavy chains (HC) to hyaluronan (HA), facilitating HA receptor binding. TSG-6 is necessary for AHR in allergic asthma, because it facilitates the development of a pathological HA–HC matrix. However, the role of TSG-6 in acute airway inflammation is not well understood. Here, we hypothesized that TSG-6 is essential for the development of HA- and ozone-induced AHR. TSG-6−/− and TSG-6+/+ mice were exposed to ozone or short-fragment HA (sHA), and AHR was assayed via flexiVent. The AHR response to sHA was evaluated in the isolated tracheal ring assay in tracheal rings from TSG-6−/− or TSG-6+/+, with or without the addition of exogenous TSG-6, and with or without inhibitors of Rho-associated, coiled-coil–containing protein kinase (ROCK), ERK, or PI3K. Smooth-muscle cells from mouse tracheas were assayed in vitro for signaling pathways. We found that TSG-6 deficiency protects against AHR after ozone (in vivo) or sHA (in vitro and in vivo) exposure. Moreover, TSG-6−/− tracheal ring non-responsiveness to sHA was reversed by exogenous TSG-6 addition. sHA rapidly activated RhoA, ERK, and Akt in airway smooth-muscle cells, but only in the presence of TSG-6. Inhibition of ROCK, ERK, or PI3K/Akt blocked sHA/TSG-6–mediated AHR. In conclusion, TSG-6 is necessary for AHR in response to ozone or sHA, in part because it facilitates rapid formation of HA–HC complexes. The sHA/TSG-6 effect is mediated by RhoA, ERK, and PI3K/Akt signaling.

Allergic asthma is a major cause of morbidity in both pediatric and adult patients. Recent research has highlighted the role of hyaluronan (HA), an extracellular matrix glycosaminoglycan, in asthma pathogenesis. Experimental allergic airway inflammation and clinical asthma are associated with an increase of shorter fragments of HA (sHA), which complex with inter-α-inhibitor heavy chains (HCs) and induce inflammation and airway hyperresponsiveness (AHR). Importantly, the effects of sHA can be antagonized by the physiological counterpart high molecular weight HA (HMWHA). We used a mouse model of house dust mite-induced allergic airway inflammation and demonstrated that instilled HMWHA ameliorated allergic airway inflammation and AHR, even when given after the establishment of allergic sensitization and after challenge exposures. Furthermore, instilled HMWHA reduced the development of HA-HC complexes and the activation of Rho-associated, coiled-coil containing protein kinase 2. We conclude that airway application of HMWHA is a potential treatment for allergic airway inflammation.

Hyaluronan (HA) exhibits numerous important roles in physiology and pathologies, and these facts necessitate an ability to accurately and reproducibly measure its quantities in tissues and cell cultures. Our group previously reported a rigorous and analytical procedure to quantify HA (and chondroitin sulfate, CS) using a reductive amination chemistry and separation of the fluorophore-conjugated, unsaturated disaccharides unique to HA and CS on high concentration acrylamide gels. This procedure is known as fluorophore-assisted carbohydrate electrophoresis (FACE) and has been adapted for the detection and quantification of all glycosaminoglycan types. While this previous FACE procedure is relatively straightforward to implement by carbohydrate research investigators, many nonglycoscience laboratories now studying HA biology might have difficulties establishing this prior FACE procedure as a routine assay for HA. To address this need, we have greatly simplified our prior FACE procedure for accurate and reproducible assessment of HA in tissues and cell cultures. This chapter describes in detail this simplified FACE procedure and, because it uses an enzyme that degrades both HA and CS, investigators will also gain additional insight into the quantities of CS in the same samples dedicated for HA analysis.

Hyaluronan (HA) exhibits numerous important roles in physiology and pathologies, and these facts necessitate an ability to accurately and reproducibly measure its quantities in tissues and cell cultures. Our group previously reported a rigorous and analytical procedure to quantify HA (and chondroitin sulfate, CS) using a reductive amination chemistry and separation of the fluorophore-conjugated, unsaturated disaccharides unique to HA and CS on high concentration acrylamide gels. This procedure is known as fluorophore-assisted carbohydrate electrophoresis (FACE) and has been adapted for the detection and quantification of all glycosaminoglycan types. While this previous FACE procedure is relatively straightforward to implement by carbohydrate research investigators, many nonglycoscience laboratories now studying HA biology might have difficulties establishing this prior FACE procedure as a routine assay for HA. To address this need, we have greatly simplified our prior FACE procedure for accurate and reproducible assessment of HA in tissues and cell cultures. This chapter describes in detail this simplified FACE procedure and, because it uses an enzyme that degrades both HA and CS, investigators will also gain additional insight into the quantities of CS in the same samples dedicated for HA analysis.

The extracellular matrix glycosaminoglycan, hyaluronan, has been described as a regulator of tissue inflammation, with hyaluronan fragments reported to stimulate innate immune cells. High molecular mass hyaluronan is normally present in tissues, but upon inflammation lower molecular mass fragments are generated. It is unclear if these hyaluronan fragments induce an inflammatory response or are a consequence of inflammation. In this study, mouse bone marrow derived macrophages and dendritic cells (DCs) were stimulated with various sizes of hyaluronan from different sources, fragmented hyaluronan, hyaluronidases and heavy chain modified-hyaluronan (HA-HC). Key pro-inflammatory molecules, tumour necrosis factor alpha, interleukin-1 beta, interleukin-12, CCL3, and the co-stimulatory molecules, CD40 and CD86 were measured. Only human umbilical cord hyaluronan, bovine testes and Streptomyces hyaluronlyticus hyaluronidase stimulated macrophages and DCs, however, these reagents were found to be contaminated with endotoxin, which was not fully removed by polymyxin B treatment. In contrast, pharmaceutical grade hyaluronan and hyaluronan fragments failed to stimulate in vitro-derived or ex vivo macrophages and DCs, and did not induce leukocyte recruitment after intratracheal instillation into mouse lungs. Hence, endotoxin-free pharmaceutical grade hyaluronan does not stimulate macrophages and DCs in our inflammatory models. These results emphasize the importance of ensuring hyaluronan preparations are endotoxin free.

Formation of cell surface protrusions has long been associated with tumor cell migration, metastasis & bone marrow homing & retention. It was shown that increased expression of endogenous or exogenous hyaluronic acid synthesizing enzyme 3 (HAS3) induces growth of microvillus-like cell surface protrusions. Some of the biological signals triggered by HA are dependent on its recognition by the cell surface receptor CD44. In our studies we observe that overexpression of the eukaryotic translation initiation factor 4E (eIF4E) increases formation of cell surface protrusions resembling those produced by HAS3 upregulation. eIF4E regulates mRNA export & translation through binding to the m7G cap of mRNA in the nucleus & the cytoplasm, respectively. Strikingly, analysis of our two complimentary screens (immunoprecipitation of mRNAs bound to eIF4E in the nucleus, and mRNA export assay with ribavirin, an established inhibitor of eIF4E) that we carried out to identify candidate eIF4E mRNA export targets, showed that nearly all the enzymes involved in HA synthesis as well as CD44. Here, we hypothesize that eIF4E regulates HA synthesis & CD44 overexpression to reshape the surface of cancer cells producing HA-rich microvillus-like pseudopods. Following validation of the positive hits, we used immunofluorescence staining with biotinylated HA binding protein (HABP) to determine the effect of eIF4E expression on protrusion formation. Our data revealed that eIF4E overexpression increased formation of HA-rich cell surface protrusions & was correlated with increased invasiveness of eIF4E overexpressing cells in in vitro matrigel assay, compared to respective controls. Colocalization studies using confocal microscopy indicated that protrusions positively stained for CD44. Further, pharmacological inhibition with ribavirin and genetic knockdown of eIF4E (or HAS3/CD44 in the context of eIF4E overexpression) abrogated protrusion formation and decreased the invasion capacity of eIF4E overexpressing cells. Together, these data indicate that eIF4E modulates protrusion formation & tumor invasion through coordinately regulating the export of RNAs in the CD44-HA regulon. To establish the clinical relevance of our findings in AML, we examined primary AML specimens with high eIF4E levels for expression of HA synthesizing enzymes, CD44 & we directly measured HA using confocal methods. This comparison showed that these pathways are dysregulated in AML patients & that their inhibition correlates with response to eIF4E inhibition by ribavirin. While HA has long been considered as a component of the tumor microenvironment required for the migration of cancer cells though interaction with surface CD44, our results provide a novel role for eIF4E in the motility & bone marrow homing. These findings offer a rationale for potentially inhibiting tumor cell metastasis through the combinatorial inhibition of eIF4E and CD44 or HAS3. Citation Format: Hiba Zahreddine, Biljana Kraljacic-Culjkovic, Valbona Cali, Mark Lauer, Lucy Skrabanek, Nabilah Khan, Ronald Midura, Leandro Cerchietti, Vincent Hascall, Craig Jordan. eIF4E reshapes the surface of migrating AML cells through regulating Hyaluronic Acid synthesis & CD44 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4898. doi:10.1158/1538-7445.AM2017-4898