Annalisa La Gatta

The most widespread desmosomal cadherin, desmoglein 2, is a novel target of caspase 3-mediated apoptotic machinery

Apoptotic cells are known to regulate the ordered dismantling of intercellular contacts through caspase activity. Despite the important role of desmoglein (Dsg) 2 in epithelial cell–cell adhesion, the fate of this widespread desmosomal cadherin during apoptosis is yet poorly understood. Here, by means of pharmacological approaches, we investigated whether Dsg2 was targeted by caspases in HaCaT and HT‐29 cell lines undergoing staurosporine (STS)‐induced apoptosis. Results showed that STS induced a caspase‐dependent form of cell‐death in both keratinocytes (HaCaT) and enterocytes (HT‐29), that associated with progressive depletion of Dsg2 from cell lysates. The proteolytic processing of full‐length Dsg2 resulted in the appearance of a 70‐kDa fragment which was released into the cytosol. Consistently, immunofluorescence studies revealed that Dsg2 staining was abolished from cell surface whereas the cytoplasmic region of Dsg2 did localize intracellularly. Plakoglobin (Pg) also underwent cleavage and detached from Dsg2. Apoptotic changes paralleled with progressive loss of intercellular adhesion strength. All these biochemical, morphological, and functional changes were regulated by caspase 3. Indeed, in the presence of the caspase 3‐inhibitor z‐DEVD‐fmk, full‐length Dsg2 protein levels were preserved, whereas the amount of the 70‐kDa fragment was maintained on control levels. Furthermore, cells pretreated with z‐DEVD‐fmk retained the membrane labeling of Dsg2. Taken together, our data demonstrate that the apoptotic processing of Dsg2 is mediated by caspase 3 in epithelial cells. J. Cell. Biochem. 103: 598–606, 2008.

Structure–activity relationship study of arbidol derivatives as inhibitors of chikungunya virus replication

Chikungunya virus (CHIKV), a mosquito-borne arthrogenic Alphavirus, causes an acute febrile illness in humans, that is, accompanied by severe joint pains. In many cases, the infection leads to persistent arthralgia, which may last for weeks to several years. The re-emergence of this infection in the early 2000s was exemplified by numerous outbreaks in the eastern hemisphere. Since then, the virus is rapidly spreading. Currently, no drugs have been approved or are in development for the treatment of CHIKV, which makes this viral infection particularly interesting for academic medicinal chemistry efforts. Several molecules have already been identified that inhibit CHIKV replication in phenotypic virus-cell-based assays. One of these is arbidol, a molecule that already has been licensed for the treatment of influenza A and B virus infections. For structural optimization, a dedicated libraries of 43 indole-based derivatives were evaluated leading to more potent analogues (IIIe and IIIf) with anti-chikungunya virus (CHIKV) activities higher than those of the other derivatives, including the lead compound, and with a selective index of inhibition 13.2 and 14.6, respectively, higher than that of ARB (4.6).

Biotechnological Production and Application of Hyaluronan

Glycosaminoglycans (GAGs) are linear polysaccharides formed from repetitions of a disaccharide unit composed of one aminosugar and one uronic acid residue. Among these, hyaluronic acid (HA) ([D-glucuronic acid (1-β-3) N-acetyl-D-glucosamine (1-β-4)]n) (figure 1), that differs from the other for not presenting sulphate groups, is a biopolymer of broad scientific interest and largely applied in different biomedical fields. This macromolecule is most frequently referred to as hyaluronan, because of the many different forms the molecule can assume in physiological conditions (i.e. the acid form, HA, and the salts, such as sodium hyaluronate) (Balazs & Gibbs, 1970).

Semi-interpenetrated Hydrogels Composed of PVA and Hyaluronan or Chondroitin Sulphate: Chemico-Physical and Biological Characterization

Physical hydrogels based on poly (vinyl alcohol) (PVA) were synthesized in the presence of natural polysaccharides, either hyaluronan or Chondroitin sulphate, in order to obtain versatile biomaterials with enhanced performances. The physical network is obtained by the freeze thawing technique, a simple method widely used for structuring PVA blends. The chemico-physical characterization of resulting materials consisted of swelling studies and mechanical analysis. Furthermore the release of embedded polysaccharides from the network was evaluated to improve understanding of the strength of hydrogen bonding between the different polymeric chains, and the effect of the sulphate groups on the interaction promoting network formation and stability. Biological response in terms of cytotoxicity, adhesion and cell vitality of murine fibroblast and human keratinocytes showed that the addition of glycosaminoglycans to a PVA polymer leads to a biomaterial with potential applications in biomedical fields.

Properties of Newly-Synthesized Cationic Semi-Interpenetrating Hydrogels Containing Either Hyaluronan or Chondroitin Sulfate in a Methacrylic Matrix

Extracellular matrix components such as hyaluronan (HA) and chondroitin sulfate (CS) were combined with a synthetic matrix of p(HEMA-co-METAC) (poly(2-hydroxyethylmethacrylate-co-2-methacryloxyethyltrimethylammonium)) at 1% and 2% w/w concentration following a previously developed procedure. The resulting semi-interpenetrating hydrogels were able to extensively swell in water incrementing their dry weight up to 13 fold depending on the glycosamminoglycan content and nature. When swollen in physiological solution, materials water uptake significantly decreased, and the differences in swelling capability became negligible. In physiological conditions, HA was released from the materials up to 38%w/w while CS was found almost fully retained. Materials were not cytotoxic and a biological evaluation, performed using 3T3 fibroblasts and an original time lapse videomicroscopy station, revealed their appropriateness for cell adhesion and proliferation. Slight differences observed in the morphology of adherent cells suggested a better performance of CS containing hydrogels.

Hyaluronan hydrogels with a low degree of modification as scaffolds for cartilage engineering

In the field of cartilage engineering, continuing efforts have focused on fabricating scaffolds that favor maintenance of the chondrocytic phenotype and matrix formation, in addition to providing a permeable, hydrated, microporous structure and mechanical support. The potential of hyaluronan-based hydrogels has been well established, but the ideal matrix remains to be developed. This study describes the development of hyaluronan sponges-based scaffolds obtained by lysine methyl-ester crosslinking. The reaction conditions are optimized with minimal chemical modifications to obtain materials that closely resemble elements in physiological cellular environments. Three hydrogels with different amounts of crosslinkers were produced that show morphological, water-uptake, mechanical, and stability properties comparable or superior to those of currently available hyaluronan-scaffolds, but with significantly fewer hyaluronan modifications. Primary human chondrocytes cultured with the most promising hydrogel were viable and maintained lineage identity for 3 weeks. They also secreted cartilage-specific matrix proteins. These scaffolds represent promising candidates for cartilage engineering.

In Vitro Evaluation of Hybrid Cooperative Complexes of Hyaluronic Acid as a Potential New Ophthalmic Treatment

PURPOSE The purpose of this in vitro study was to assess the potential benefits of eye drops based on hybrid cooperative complexes (HCCs) obtained from high and low molecular weight hyaluronic acid (HA). METHODS Rheological measurements were performed to adjust the HCC concentration toward optimal resistance to drainage from the ocular surface. The viscosity and mucoadhesion profiles of the optimized preparation were derived. Primary porcine corneal epithelial cells were used for biological studies. Cells were exposed to dehydration after being pretreated with the HCC solution, and protection from desiccation was evaluated using cell viability assays. Time-lapse experiments were carried out to evaluate the ability of the HCC preparation to promote corneal wound healing. The characterization studies were performed in comparison with a control HA solution representative of commercial HA-based products. RESULTS The HCC formulation is able to deliver twice the amount of biopolymer compared with conventional products while avoiding discomfort due to excessive viscosity. The viscosity and mucoadhesion profiles allowed the authors to predict the longer in vivo retention and, therefore, an improved HCC formulation bioavailability. The new preparation also proved superior in protecting porcine corneal epithelial cells from desiccation and in hastening corneal cell wound repair in vitro. CONCLUSIONS The results suggest that the developed formulation may be a promising topical ophthalmic medical treatment.

A biophysically-defined hyaluronic acid-based compound accelerates migration and stimulates the production of keratinocyte-derived neuromodulators

ABSTRACT Hyaluronic acid (HA) preparations are widely used in clinical practice and recent data suggest that commercially available HA-based compounds promote ulcer re-epithelialization and induce pain relief. However, the pathophysiological basis of these effects remains poorly understood. In the present study, we investigated the biophysical, biomolecular and functional properties of a HA preparation combined with a pool of collagen precursor synthetic aminoacids, namely l-proline, l-leucine, l-lysine and glycine (Aminogam®). Hydrodynamic characterization of Aminogam® by size exclusion chromatography-triple detector array (SEC-TDA) revealed an average molecular weight in the range of 700–1700 kDa. Rheological measurements of the 1700kDa Mw lot showed a pseoudoplastic behaviour with a zero-shear viscosity (η0) equal to 90 ± 9 Pa∙s at 25°C and 55 ± 6 Pa∙s at 37°C. Automated time-lapse videomicroscopy studies in a fibroblast-free system demonstrated that 1% (v/v) Aminogam® significantly reduced the healing time of wounded keratinocyte monolayers. In AKGOS assays, Aminogam® stimulated cellular locomotion (chemokinesis) and directional migration (chemotaxis) of keratinocytes. Analysis of microarray data suggested that keratinocytes had a functional neuroendocrine machinery, and this was confirmed by testing the secretion of six neuroactive molecules by ELISA, namely α-MSH, β-endorphins, melatonin, substance P, cortisol, and neurotensin. Interestingly, Aminogam® regulated the production of several neuropeptides, including β-endorphins. In conclusion, our data shed light on the epithelial-dependent mechanisms that underlie the efficacy of Aminogam®, particularly in reference to wound healing and nociception.

Macroporous alginate foams crosslinked with strontium for bone tissue engineering

Nowadays, the need of novel strategies to repair and regenerate bone defects in the field of biomedical applications has increased. Novel approaches include the design of natural bioactive scaffolds mimicking bone tissue. These bioactive scaffolds have to possess biophysical properties suitable to address biological response towards newly bone tissue formation. In particular, scaffold porosity and pore size play a pivotal role in cell migration, adhesion and proliferation, thus increasing cell-material surface interaction and osteogenic signals transmission. Here we propose the development of macroporous alginate foams (MAFs) with porous and well interconnected structure, useful to enhance growth and osteogenic differentiation of human Mesenchymal Stem Cells (hMSCs). Moreover, in this study we report a new method for MAFs fabrication based on the combination of internal gelation technique with gas foaming. Strontium was employed in combination with calcium as cross-linking agent for the alginate chains and as enhancer of the osteogenic differentiation. The influence of strontium ions on the gelation kinetics, physical properties and degradation in physiological medium of MAFs was investigated. Our results suggest that the combination of internal gelation technique with gas foaming followed by freeze-drying is an easy and straightforward procedure to prepare alginate foams with high porosity and interconnectivity, able to support cell infiltration. Finally, biological assays showed how scaffolds with high strontium content are able to support cell growth and differentiation in long times by promoting osteogenic marker expression.