Hyaluronan and Reactive Oxygen Species Signaling-Novel Cues from the Matrix?

Hyaluronan (HA) is a naturally occurring non-sulfated glycosaminoglycan (GAG) localized to the cell surface and the tissue extracellular matrix (ECM). It is composed of disaccharides containing glucuronic acid and N-acetylglucosamine, is synthesized by the HA synthase (HAS) enzymes and is degraded by hyaluronidase (HYAL) or reactive oxygen and nitrogen species (ROS/RNS) actions. HA is deposited as a high molecular weight (HMW) polymer and degraded to low molecular weight (LMW) fragments and oligosaccharides. HA affects biological functions by interacting with HA-binding proteins (hyaladherins). HMW HA is anti-inflammatory, immunosuppressive, and antiangiogenic, whereas LMW HA has pro-inflammatory, pro-angiogenetic, and oncogenic effects. ROS/RNS naturally degrade HMW HA, albeit at enhanced levels during tissue injury and inflammatory processes. Thus, the degradation of endothelial glycocalyx HA by increased ROS challenges vascular integrity and can initiate several disease progressions. Conversely, HA exerts a vital role in wound healing through ROS-mediated HA modifications, which affect the innate immune system. The normal turnover of HA protects against matrix rigidification. Insufficient turnover leads to increased tissue rigidity, leading to tissue dysfunction. Both endogenous and exogenous HMW HA have a scavenging capacity against ROS. The interactions of ROS/RNS with HA are more complex than presently perceived and present an important research topic.

The Role of IGF/IGF-IR-Signaling and Extracellular Matrix Effectors in Bone Sarcoma Pathogenesis.

Bone sarcomas, mesenchymal origin tumors, represent a substantial group of varying neoplasms of a distinct entity. Bone sarcoma patients show a limited response or do not respond to chemotherapy. Notably, developing efficient chemotherapy approaches, dealing with chemoresistance, and preventing metastasis pose unmet challenges in sarcoma therapy. Insulin-like growth factors 1 and 2 (IGF-1 and -2) and their respective receptors are a multifactorial system that significantly contributes to bone sarcoma pathogenesis. Whereas failures have been registered in creating novel targeted therapeutics aiming at the IGF pathway, new agent development should continue, evaluating combinatorial strategies for enhancing antitumor responses and better classifying the patients that could best benefit from these therapies. A plausible approach for developing a combinatorial strategy is to focus on the tumor microenvironment (TME) and processes executed therein. Herewith, we will discuss how the interplay between IGF-signaling and the TME constituents affects sarcomas' basal functions and their response to therapy. This review highlights key studies focusing on IGF signaling in bone sarcomas, specifically studies underscoring novel properties that make this system an attractive therapeutic target and identifies new relationships that may be exploited. Potential direct and adjunct therapeutical implications of the extracellular matrix (ECM) effectors will also be summarized.

Lumican mediates HTB94 chondrosarcoma cell growth via an IGF­IR/Erk1/2 axis.

Chondrosarcoma is a malignant bone tumor characterized by the production of a modified cartilage­type extracellular matrix (ECM). In the present study, the expression levels of the small leucine­rich proteoglycans (SLRPs), decorin, biglycan and lumican, were examined in the HTB94 human chondrosarcoma cell line. HTB94 cells were found to express and secrete the 3 SLRP members. RT­qPCR and western blot analysis demonstrated that lumican was the most abundantly secreted SLRP, whereas decorin and biglycan expression levels were low. The utilization of short interfering RNA specific for the decorin, biglycan, and lumican genes resulted in the efficient downregulation of the respective mRNA levels (P≤0.001). The growth of the HTB94 cells was stimulated by lumican (P≤0.001), whereas their migration and adhesion were not affected (P=NS). By contrast, these cellular functions were not sensitive to a decrease in low endogenous levels of decorin and biglycan. Lumicandeficiency significantly inhibited both basal and insulin­like growth factor I (IGF­I)­induced HTB94 cell growth (P≤0.001 andP≤0.01, respectively). These effects were executed through the insulin­like growth factor I receptor (IGF­IR), whose activation was markedly attenuated (P≤0.01) in lumican­deficient HTB94 cells. The downregulation of lumican induced the substantial inhibition of extracellular regulated kinase (ERK1/2) activation (P≤ 0.01), indicating that ERK1/2 is a necessary component of lumican/IGF­IR­mediated HTB94 cell proliferation. Moreover, the lumican­deficient cells exhibit increased mRNA levels of p53 (P≤0.05), suggesting that lumican facilitates HTB94 cell growth through an IGF­IR/ERK1/2/p53 signaling cascade. On the whole, the findings of the present study demonstrate that endogenous lumican is a novel regulator of HTB94 cell growth.

Contact allergen (PPD and DNCB)-induced keratinocyte sensitization is partly mediated through a low molecular weight hyaluronan (LMWHA)/TLR4/NF-κB signaling axis.

Allergic contact dermatitis (ACD) is caused by topical exposure to chemical allergens. Keratinocytes play a key role in innate immunity, as well as in ACD progression. The transmembrane Toll-like receptor 4 (TLR4), strongly implicated in skin inflammation, has the ability to bind Damage Associated Molecular Patterns (DAMPs), like Low Molecular Weight Hyaluronan (LMWHA). Previously, we had determined that p-phenylenediamine (PPD) and 2,4-dinitrochlorobenzene (DNCB) modulate keratinocyte HA deposition in a manner correlated to their sensitization. In the present study, we aimed to investigate putative co-operation of HA and TLR4 in the process of PPD and DNCB-induced keratinocyte activation. Contact sensitizers were shown to significantly increase the expression of Hyaluronan Synthases (HAS) and TLR4 in NCTC2544 human keratinocytes, as demonstrated by western blot and Real-Time PCR. These data, in correlation to earlier shown enhanced HA degradation suggest that the contact sensitizers facilitate HA turnover of keratinocytes and increase the release of pro-inflammatory, LMWHA fragments. Treatment with exogenous LMWHA enhanced TLR4, HAS levels and Nuclear factor-kappa beta (NF-κΒ) activation. PPD, DNCB and LMWHA-effects were shown to be partly executed through TLR4 downstream signaling as shown by Real-Time, western blot, siRNA and confocal microscopy approaches. Specifically, PPD and DNCB stimulated the activation of the TLR4 downstream mediator NF-κB. Therefore, the shown upregulation of TLR4 expression is suggested to further facilitate the release of endogenous, bioactive HA fragments and sustain keratinocyte activation. In conclusion, keratinocyte contact allergen-dependent sensitization is partly mediated through a LMWHA/TLR4/ NF-κB signaling axis.

Proteoglycans-Biomarkers and Targets in Cancer Therapy.

Proteoglycans (PGs), important constituents of the extracellular matrix, have been associated with cancer pathogenesis. Their unique structure consisting of a protein core and glycosaminoglycan chains endowed with fine modifications constitutes these molecules as capable cellular effectors important for homeostasis and contributing to disease progression. Indeed, differential expression of PGs and their interacting proteins has been characterized as specific for disease evolvement in various cancer types. Importantly, PGs to a large extent regulate the bioavailability of hormones, growth factors, and cytokines as well as the activation of their respective receptors which regulate phenotypic diversibility, gene expression and rates of recurrence in specific tumor types. Defining and targeting these effectors on an individual patient basis offers ground for the development of newer therapeutic approaches which may act as either supportive or a substitute treatment to the standard therapy protocols. This review discusses the roles of PGs in cancer progression, developing technologies utilized for the defining of the PG "signature" in disease, and how this may facilitate the generation of tailor-made cancer strategies.

Role of the extracellular matrix in cancer-associated epithelial to mesenchymal transition phenomenon.

The epithelial to mesenchymal transition (EMT) program is a crucial component in the processes of morphogenesis and embryonic development. The transition of epithelial to mesenchymal phenotype is associated with numerous structural and functional changes, including loss of cell polarity and tight cell-cell junctions, the acquisition of invasive abilities, and the expression of mesenchymal proteins. The switch between the two phenotypes is involved in human pathology and is crucial for cancer progression. Extracellular matrices (ECMs) are multi-component networks that surround cells in tissues. These networks are obligatory for cell survival, growth, and differentiation as well as tissue organization. Indeed, the ECM suprastructure, in addition to its supportive role, can process and deliver a plethora of signals to cells, which ultimately regulate their behavior. Importantly, the ECM derived signals are critically involved in the process of EMT during tumorigenesis. This review discusses the multilayer interaction between the ECM and the EMT process, focusing on contributions of discrete mediators, a strategy that may identify novel potential target molecules. Developmental Dynamics 247:368-381, 2018. © 2017 Wiley Periodicals, Inc.

HA metabolism in skin homeostasis and inflammatory disease.

Hyaluronan (HA), an unsulfated glycosaminoglycan, is an important component of the complex extracellular matrix network which surrounds and supports cells in tissues. HA is detected in all vertebrate tissues, but the bulk of HA is produced and deposited in the skin. In this review we focus on the role of HA in skin-associated inflammatory disease and wound healing. Properties of HA are directly dependent on its molecular weight. Thus, high molecular weight HA (HMWHA) is deposited in normal tissues during homeostasis and promotes their stability whereas low molecular weight HA fragments (LMWHA), on the other hand, may arise from enzymatic or chemical activities. The degradation of HMWHA to LMWHA fragments, often leads to the generation of biologically active oligosaccharides with different properties and postulated functions in wound scar formation and inflammation. More detailed studies of HA involvement in skin-associated inflammatory disease may result in novel treatment modalities.

Bacteriostatic and bactericidal activities of tigecycline against Coxiella burnetii and comparison with those of six other antibiotics.

The present article is a study of the in vitro susceptibility of eight Greek Coxiella burnetii isolates, derived from patients with acute Q fever, and two reference strains of Coxiella burnetii to tigecycline. The bacteriostatic activity of tigecycline was compared with those of six other antibiotics using a shell vial assay. The MICs of the examined antibiotics were as follows: tigecycline ranged from 0.25 to 0.5 microg/ml; doxycycline, trovafloxacin, and ofloxacin ranged from 1 to 2 microg/ml; linezolid and clarithromycin ranged from 2 to 4 microg/ml; and ciprofloxacin ranged from 4 to 8 microg/ml. Tigecycline was effective in inhibiting the infection of Vero cells by C. burnetii. No bactericidal activity was observed against C. burnetii at 4 microg/ml.

First isolation and identification of Rickettsia conorii from ticks collected in the region of Fokida in Central Greece.

Three different spotted-fever group rickettsiae-Rickettsia conorii, R. massiliae, and R. rhipicephali-were detected and identified by PCR-restriction fragment length polymorphism analysis in Rhipicephalus ticks collected from domestic animals in the Fokida region of Greece, where a high seroprevalence of antibodies to R. conorii was previously demonstrated. The infection rate of ticks was 1.6%. Moreover, R. conorii was isolated from one Rhipicephalus sanguineus tick.

Fourteen-year seroepidemiological study of zoonoses in a Greek village.

A seroepidemiological study carried out in a high-risk village in Crete in 1985-1987 and 1998 showed that although the awareness of the people concerning zoonoses had increased during this period, the situation did not improve: there was a significant increase of the spread of seroprevalence in time and space of Coxiella burnetii, Rickettsia typhi, Brucella sp., and Entamoeba histolytica. Toxoplasma gondii, Rickettsia conorii, Borrelia burgdorferi, Echinococcus granulosus, Leishmania sp., and Fasciola hepatica stayed at the same levels. This first study of Bartonella henselae in Crete showed that 15.9% of the children tested were seropositive. The results indicate that reservoirs and vectors of the pathogens studied are widespread in the environment, and the way of life of the people favors contact with them. Seven of 30 milk samples were positive for Brucella sp. by seminested polymerase chain reaction.

Isolation of Coxiella burnetii by a centrifugation shell-vial assay from ticks collected in Cyprus: detection by nested polymerase chain reaction (PCR) and by PCR-restriction fragment length polymorphism analyses.

Ticks are the principal vectors and reservoirs of Coxiella burnetii. The identification of isolates is necessary for understanding the clinical diversity of Q fever in different geographic areas. This is the first report of isolation of C. burnetii from ticks by the shell-vial assay and by nested polymerase chain reaction (PCR) assay for the detection of this pathogen in ticks. Of 141 ticks collected in Cyprus (Rhipicephalus sanguineus and Hyalloma spp.), 10% were found to be infected with C. burnetii. Three ticks were positive by hemolymph test, and 11 triturated ticks were positive by nested PCR. Three isolates were obtained by the centrifugation shell-vial technique. Analysis by PCR, then restriction fragment length polymorphism showed that the 3 Cyprus isolates had identical restriction profiles to reference strains Nine Mile and Q212. The methods described are useful in studying the epidemiology and ecology of C. burnetii.

Mechanisms of resistance to fluoroquinolones in Coxiella burnetii.

Mechanisms of resistance to quinolones were investigated in two in vitro DNA gyrase mutant Coxiella burnetii strains. Accumulation of pefloxacin was found to be lower in resistant strains than in susceptible strains. Pre-incubation with carbonyl cyanide m-chlorophenylhydrazone had no apparent effect on intracellular quinolone fluorescence, indicating that an active efflux mechanism is not responsible for the decrease of pefloxacin accumulation. No differences were detected between the strains after outer membrane protein analysis. Decreased accumulation was found in all strains at acidic pH. It is possible that, apart from the mutation-induced mechanism of resistance, a pH-related mechanism is involved in the drug accumulation process.