Inhaled high molecular weight hyaluronan ameliorates respiratory failure in acute COPD exacerbation: a pilot study.

BACKGROUND: Acute exacerbations of chronic obstructive pulmonary disease (AECOPD) carry significant morbidity and mortality. AECOPD treatment remains limited. High molecular weight hyaluronan (HMW-HA) is a glycosaminoglycan sugar, which is a physiological constituent of the lung extracellular matrix and has notable anti-inflammatory and hydrating properties. RESEARCH QUESTION: We hypothesized that inhaled HMW-HA will improve outcomes in AECOPD. METHODS: We conducted a single center, randomized, placebo-controlled, double-blind study to investigate the effect of inhaled HMW-HA in patients with severe AECOPD necessitating non-invasive positive-pressure ventilation (NIPPV). Primary endpoint was time until liberation from NIPPV. RESULTS: Out of 44 screened patients, 41 were included in the study (21 for placebo and 20 for HMW-HA). Patients treated with HMW-HA had significantly shorter duration of NIPPV. HMW-HA treated patients also had lower measured peak airway pressures on the ventilator and lower systemic inflammation markers after liberation from NIPPV. In vitro testing showed that HMW-HA significantly improved mucociliary transport in air-liquid interface cultures of primary bronchial cells from COPD patients and healthy primary cells exposed to cigarette smoke extract. INTERPRETATION: Inhaled HMW-HA shortens the duration of respiratory failure and need for non-invasive ventilation in patients with AECOPD. Beneficial effects of HMW-HA on mucociliary clearance and inflammation may account for some of the effects (NCT02674880, www.clinicaltrials.gov ).

Multiwalled Carbon Nanotube Functionalization with High Molecular Weight Hyaluronan Significantly Reduces Pulmonary Injury.

Commercialization of multiwalled carbon nanotubes (MWCNT)-based applications has been hampered by concerns regarding their lung toxicity potential. Hyaluronic acid (HA) is a ubiquitously found polysaccharide, which is anti-inflammatory in its native high molecular weight form. HA-functionalized smart MWCNTs have shown promise as tumor-targeting drug delivery agents and can enhance bone repair and regeneration. However, it is unclear whether HA functionalization could reduce the pulmonary toxicity potential of MWCNTs. Using in vivo and in vitro approaches, we investigated the effectiveness of MWCNT functionalization with HA in increasing nanotube biocompatibility and reducing lung inflammatory and fibrotic effects. We utilized three-dimensional cultures of differentiated primary human bronchial epithelia to translate findings from rodent assays to humans. We found that HA functionalization increased stability and dispersion of MWCNTs and reduced postexposure lung inflammation, fibrosis, and mucus cell metaplasia compared with nonfunctionalized MWCNTs. Cocultures of fully differentiated bronchial epithelial cells (cultivated at air-liquid interface) and human lung fibroblasts (submerged) displayed significant reduction in injury, oxidative stress, as well as pro-inflammatory gene and protein expression after exposure to HA-functionalized MWCNTs compared with MWCNTs alone. In contrast, neither type of nanotubes stimulated cytokine production in primary human alveolar macrophages. In aggregate, our results demonstrate the effectiveness of HA functionalization as a safer design approach to eliminate MWCNT-induced lung injury and suggest that HA functionalization works by reducing MWCNT-induced epithelial injury.

Decreased Uptake and Enhanced Mitochondrial Protection Underlie Reduced Toxicity of Nanoceria in Human Monocyte-Derived Macrophages.

Cerium dioxide nanoparticles (nanoceria), currently used as catalysts including additives to diesel fuel, also present potential as a novel therapeutic agent for disorders involving oxidative stress. However, little is known about the effects of nanoceria on primary human cells involved in the innate immune response. Here, we evaluate nanoceria effects on monocyte derived macrophages (MDMs) from healthy human subjects. Peripheral blood monocytes were isolated from healthy human volunteers. MDMs were obtained by maturing monocytes over a five-day period. MDMs were exposed to well-characterized nanoceria suspensions (0, 5, 10, 20 µg/mL) for 24 or 48 hours. We evaluated particle uptake, ultrastructural changes, cytotoxicity, and mitochondrial damage in MDMs through transmission electron microscopy (TEM), confocal imaging, flow cytometry, spectrometry, western blots, and immunofluorescence techniques. The role that intracellular concentration of nanoceria plays in the toxicity of MDMs was evaluated by 3D image analysis and compared to monocytes as a nanoceria sensitive cell model. Nanoceria failed to induce cytotoxicity in MDMs at the tested doses. Nanoceria-exposed MDMs showed no mitochondrial damage and displayed significant accumulation of anti-apoptotic proteins (Mcl-1 and Bcl-2) during the maturation process. TEM and confocal analyses revealed efficient uptake of nanoceria by MDMs, however 3D image analyses revealed lower nanoceria accumulation per unit cell volume in MDMs compared to monocytes. Taken together, our results suggest that mitochondrial protection and reduced volume-corrected intracellular nanoparticle concentration account for the lower sensitivity of human MDMs to nanoceria.

p38 mitogen-activated protein kinase interacts with vinculin at focal adhesions during fatty acid-stimulated cell adhesion.

Arachidonic acid stimulates cell adhesion by activating α2ß1 integrins in a process that depends on protein kinases, including p38 mitogen activated protein kinase. Here, we describe the interaction of cytoskeletal components with key signaling molecules that contribute to the spreading of, and morphological changes in, arachidonic acid-treated MDA-MB-435 human breast carcinoma cells. Arachidonic acid-treated cells showed increased attachment and spreading on collagen type IV, as measured by electric cell-substrate impedance sensing. Fatty acid-treated cells displayed short cortical actin filaments associated with an increased number of ß1 integrin-containing pseudopodia, whereas untreated cells displayed elongated stress fibers and fewer clusters of ß1 integrins. Confocal microscopy of arachidonic acid-treated cells showed that vinculin and phospho-p38 both appeared enriched in pseudopodia and at the tips of actin filaments, and fluorescence ratio imaging indicated the increase was specific for the phospho-(active) form of p38. Immunoprecipitates of phospho-p38 from extracts of arachidonic acid-treated cells contained vinculin, and GST-vinculin fusion proteins carrying the central region of vinculin bound phospho-p38, whereas fusion proteins expressing the terminal portions of vinculin did not. These data suggest that phospho-p38 associates with particular domains on critical focal adhesion proteins that are involved in tumor cell adhesion and spreading, and that this association can be regulated by factors in the tumor microenvironment.

15-lipoxygenase-1 activates tumor suppressor p53 independent of enzymatic activity.

15-LOX-1 and its metabolites are involved in colorectal cancer. Recently, we reported that 15-LOX-1 overexpression in HCT-116 human colorectal cancer cells inhibited cell growth by induction of p53 phosphorylation (4). To determine whether the 15-LOX-1 protein or its metabolites are responsible for phosphorylation of p53 in HCT-116 cells, we used HCT-116 cells that expressed a mutant 15-LOX-1. The mutant 15-LOX-1 enzyme, with a substitution of Leu at residue His361, was devoid of enzymatic activity. HCT-116 cells transiently transfected with either native or mutant 15-LOX-1 showed an increase in p53 phosphorylation and an increase in the expression of downstream genes. Thus, 15-LOX-1 induces p53 phosphorylation independent of enzymatic activity. Treatment of A549 human lung carcinoma cells with IL-4 increased the expression of 15-LOX-1 and also increased the expression of downstream targets of p53. This confirmed that the activation of p53 was also observed in wild-type cells expressing physiological 15-LOX-1. Immunoprecipitation experiments revealed that 15-LOX-1 interacts with, and binds to, DNA-dependent protein kinase (DNA-PK). The binding of 15-LOX-1 to DNA-PK caused an approximate 3.0-fold enhancement in kinase activity, resulting in increased p53 phosphorylation at Ser15. Knockdown of DNA-PK by small interfering RNA (siRNA) significantly reduced p53 phosphorylation. Furthermore, confocal microscopy demonstrated a colocalization of 15-LOX and DNA-PK in the cells. We propose that the 15-LOX-1 protein binds to DNA-PK, increasing its kinase activity and results in downstream activation of the tumor suppressor p53, thus revealing a new mechanism by which lipoxygenases (LOX) may influence the phenotype of tumor cells.

P-selectin activates integrin-mediated colon carcinoma cell adhesion to fibronectin.

During hematogenous cancer metastasis, tumor cells separate from a primary mass, enter the bloodstream, disperse throughout the body, migrate across vessel walls, and generate distant colonies. The later steps of metastasis superficially resemble leukocyte extravasation, a process initiated by selectin-mediated cell tethering to the blood vessel wall followed by integrin-mediated arrest and transendothelial migration. Some cancer cells express P-selectin ligands and attach to immobilized P-selectin, suggesting that these cells can arrest in blood vessels using sequential selectin- and integrin-mediated adhesion, as do leukocytes. We hypothesize that selectin binding may regulate subsequent integrin-mediated steps in metastasis. Using a model system of cultured Colo 320 human colon adenocarcinoma cells incubated with soluble P-selectin-IgG chimeric protein, we have found that P-selectin can stimulate activation of the alpha(5)beta(1) integrin resulting in a specific increase of adhesion and spreading of these cells on fibronectin substrates. P-selectin binding also induced activation of p38 mitogen-activated protein kinase (p38 MAPK) and phosphatidylinositol 3-kinase (PI3-K). PI3-K inhibitors blocked P-selectin-mediated integrin activation, cell attachment, and cell spreading. Inhibition of p38 MAPK activation blocked cell spreading, but not cell attachment. P-selectin binding also resulted in formation of a signaling complex containing PI3-K and p38 MAPK. These results suggest that P-selectin binding to tumor cells can activate alpha(5)beta(1) integrin via PI3-K and p38 MAPK signaling pathways leading to increased cell adhesion. We propose that P-selectin ligands are important tumor cell signaling molecules that modulate integrin-mediated cell adhesion in the metastatic process.