CLUSTERING OF SPECIFIC MOLECULES IN SHED VESICLES.

In several tumor cell lines serum addition causes release of vesicles that bud from the cell surface and can be purified from cell conditionated media. These vesicles are known to be involved in cell migration and tumor progression. We recently demonstrated that FGF-2, a growth factor devoid of the classical signaling sequence, is secreted as a component of these vesicles. In order to analyze how molecules are clustered in shed vesicles we followed their intracellular movements by immunofluorescence techniques. The role of cytoskeletal components was analyzed using molecules such as paclitaxol, nocodazole, colchicin and cytochalasin which destabilize their organization. In the absence of serum, no clear localization of FGF-2 was observed. After serum addition, FGF-2 was localized partially in the nucleus and nucleolus, and partially in granules near the plasma membrane. Nocodazole and paclitaxol, which interfere with microtubular organization, inhibit FGF-2 nuclear localization but do not appear to modify FGF-2 movements toward the plasma membrane. Cytocalasine, which interferes with actin polymerization, decreases FGF-2 clustering in granules localized near the cell membrane. In summary, microtubular organization seems to be required for FGF-2 nuclear localization while actin filaments appear to be needed for FGF-2 translocation toward the plasma membrane. In a different set of experiments, we analyzed localization of neutral ceramidase (ncDase) and of Sphingosine Kinase (SphK). Ceramidase catalyzes ceramide hydrolysis giving rise to sphingosine, which in turn can be phosphorilated to S1P by SphK. Sp1P is an important signaling molecule involved in induction of cell migration and apoptosis. SphK-1 was known to be shed into the extracellular medium by an unconventional mechanism, we hypothesized that shed vesicles could vehicle its release. We therefore analyzed the localization of membrane-bound isoforms of ceramidase (ncDase) and of SphK (SphK-1 and SphK-2) by western blotting and Immunofluorescence techniques. Immunolocalization showed that ncDase is located into the plasma membrane and in cellular extensions. The concentration of ncDase was found to be higher in extracts of shed vesicles than in cell extracts. SphK-1 was found to be localized in plasma membrane and in vesicles, which appear to be enriched in this enzyme. SphK-2 was preferentially located in the nucleus and it was not detected in vesicles. In conclusion, ncDase and SphK were found to be clustered in shed vesicles. In order to analyze the role of SphK-1, either in the shedding phenomenon or in vesicle functions, we used transiently transfected SK-Hep1 cells, which overexpress SphK or express a non-functional mutant of this enzyme

Shed vesicles are involved in the release of some leaderless proteins.

Most proteins destined for secretion in the extracellular matrix are characterized by the presence of N-terminal signal peptides which direct their translocation into the endoplasmic reticulum, they are subsequently transferred to the Golgi apparatus and then secreted in the extracellular space. A growing number of secreted proteins, are being identified which, however, lack signal peptides allowing their entrance into the endoplasmic reticulum. They include the inflammatory cytokine interleukin 1b, galactins, macrophage migration inhibitory factor (MIF), acid and basic fibroblast growth factors (FGF-1, FGF-2) and Sphingosine kinase1(SphK-1). These proteins are secreted from the cell by unconventional processes which are the subject of numerous studies. Several types of normal and tumor cells can release in the extracellular medium microvesicles, called esovesicles, which result from budding of their plasma membranes. The vesicle diameter ranges between 100nm and 1000nm, the vesicle composition and function depends on the kind of the cell from which they have been produced. We already reported that FGF-2, a secreted lectin that transmits proangiogenic signals, and which is recognized as a potential oncoprotein able to modulate tumour growth and malignancy (Sorensen et al 2006), is released from SkHep1 cells, and from transfected NIH 3T3 cells through vesicle shedding (Taverna et al.2003). Now we are trying to elucidate the intracellular route followed by the growth factor from the site of synthesis to vesicles budding from the cell membrane. Actin filaments appear to be a binary for this intracellular trafficking. After 6h of treatment with cytocalasine, a drug that interferes with actin polymerization, the amount of vesicles was in fact decreased and FGF-2 clustering in granules localized near the cell surface was avoided. On the contrary no effects were observed when cells were treated with drugs which interfere with microtubule polymerization or de-polymerization. We also observed that FGF-2 granules are not included in lipid-coated vesicles. We are also analyzing the possibility that esovesicles are involved in the secretion of another leader-less signalling protein: Sphingosine kinase1 (SphK1). SphK1 has been shown to regulate a wide variety of cellular processes, including promotion of cell proliferation, survival and motility (Spiegel et al. 2003). SphK1 is primarily localized in the cytosol; when a signal induces the phosphorylation of Ser 225 of SphK1 through the activation of MAPK and ERK1/2, the molecule is translocated in plasma membranes and the involvement of actin filaments in its targeting has been reported (Pitson et. al. 2003). Three SphK1 isoforms having a different number of amino acids (384, 398 and 470) have been identified, we found that extracellular vesicles are enriched in the 47kDa isoform. SphK assays with TLC confirm that the enzyme is present in shed vesicles and that it has enzymatic activity. The substrate Sphingosine is also present in esovesicles therefore shed vesicles are likely to be a site of Sphingosine 1 Phosphate production

3D polylactide-based scaffolds for studying human hepatocarcinoma processes in vitro

We evaluated the combination of leaching techniques and melt blending of polymers and particles for the preparation of highly interconnected three-dimensional polymeric porous scaffolds for in vitro studies of human hepatocarcinoma processes. More specifically, sodium chloride and poly(ethylene glycol) (PEG) were used as water-soluble porogens to form porous and solvent-free poly(L,D-lactide) (PLA)-based scaffolds. Several characterization techniques, including porosimetry, image analysis and thermogravimetry, were combined to improve the reliability of measurements and mapping of the size, distribution and microarchitecture of pores. We also investigated the effect of processing, in PLA-based blends, on the simultaneous bulk/surface modifications and pore architectures in the scaffolds, and assessed the effects on human hepatocarcinoma viability and cell adhesion. The influence of PEG molecular weight on the scaffold morphology and cell viability and adhesion were also investigated. Morphological studies indicated that it was possible to obtain scaffolds with well-interconnected pores of assorted sizes. The analysis confirmed that SK-Hep1 cells adhered well to the polymeric support and emitted surface protrusions necessary to grow and differentiate three-dimensional systems. PEGs with higher molecular weight showed the best results in terms of cell adhesion and viability

Water-borne Polymeric Nanoparticles for Glutathione-Mediated Intracellular Delivery of Anticancer Drugs.

A new family of water-borne, biocompatible and carboxyl- functionalized nanogels was developed for glutathione- mediated delivery of anticancer drugs. Poly(N-vinyl- pyrrolidone)-co-acrylic acid nanogels were generated by e- beam irradiation of aqueous solutions of a crosslinkable polymer, using industrial-type linear accelerators and set- ups. Nanogels physico-chemical properties and colloidal stability, in a wide pH range, were investigated. In vitro cell studies proved that the nanogels are fully biocompatible and able to quantitatively bypass cellular membrane. An anticancer drug, doxorubicin (DOX), was linked to the carboxyl groups of NGs through a spacer containing a disulphide cleavable linkage. In vitro release studies showed that glutathione is able to trigger the release of DOX through the reduction of the S-S linkage at a concentration comparable to its levels in the cytosol

An Active Form of Sphingosine Kinase-1 Is Released in the Extracellular Medium as Component of Membrane Vesicles Shed by Two Human Tumor Cell Lines

Expression of sphingosine kinase-1 (SphK-1) correlates with a poor survival rate of tumor patients. This effect is probably due to the ability of SphK-1 to be released into the extracellular medium where it catalyzes the biosynthesis of sphingosine-1-phosphate (S1P), a signaling molecule endowed with profound proangiogenic effects. SphK-1 is a leaderless protein which is secreted by an unconventional mechanism. In this paper, we will show that in human hepatocarcinoma Sk-Hep1 cells, extracellular signaling is followed by targeting the enzyme to the cell surface and parallels targeting of FGF-2 to the budding vesicles. We will also show that SphK-1 is present in a catalitycally active form in vesicles shed by SK-Hep1 and human breast carcinoma 8701-BC cells. The enzyme substrate sphingosine is present in shed vesicles where it is produced by neutral ceramidase. Shed vesicles are therefore a site for S1P production in the extracellular medium and conceivably also within host cell following vesicle endocytosis

In vivo angiogenic activity induction by collagen- soaked Poly-L-lactic acid scaffolds

Angiogenesis is essential in tissue integration and it is involved in the biological response to biomaterials. Poly-L-lactic acid (PLLA), a synthetic polymer, is utilized as scaffolding to regenerate new tissues. This study investigated the short and long term degradation and the induction of neovascularization of both native PLLA (n-PLLA) and collagen type I soaked PLLA (c-PLLA) porous scaffolds, implanted subcutane- ously in balb/c mice.The comparative analysis by phase contrast, optical, and scanning electron micros- copy (SEM) of scaffolds 7 and 21 days after implantation showed a mild inflam- matory response at the implant site of c-PLLA scaffold. No significant difference in systemic immune response was detected by hematology analyzer, and by histologi- cal evaluation of lymph node and spleen features. On the contrary, immune reaction was moderate in n-PLLA. Pores of both PLLA networks laying on the muscle fibers were partially infiltrated by appositional collagen/elastin tissue, phagocytic cells, and fibroblast, with respect to the inner side. The presence of numerous and large blood vessels into pores of c-PLLA scaffolds showed an enhancing vascularization rate. These characteristics appeared to be less conspicuous in n-PLLA.At longer time points (42 and 84 days), there was low difference in inflammato- ry cell presence into scaffold pores and the number of cells infiltrating each implant was significantly decreased. In fact, we did not observe difference in the migration of inflammatory cells into PLLA scaffolds. Polymer degradation was detected in both PLLA networks, but there are no considerable differences, as confirmed by the SEM analysis.Our results suggest that tissue integration of PLLA is enhanced when it is soaked with collagen, as well as the angiogenic activity on c-PLLA. Furthermore, the colla- gen soaking makes PLLA polymer more suitable for supporting cell attachment, pro- liferation, and function by mimicking the natural extra cellular matrix.

Use of Modified 3D Scaffolds to Improve Cell Adhesion and Drive Desired Cell Responses.

In the most common approach of tissue engineering, a polymeric scaffold with a well-defined architecture has emerged as a promising platform for cells adhesion and guide their proliferation and differentiation into the desired tissue or organ. An ideal model for the regeneration should mimic clinical conditions of tissue injury, create a permissive microenvironment for diffusion of nutrients, gases and growth factors and permit angiogenesis. In this work, we used a 3D support made of synthetic resorbable polylactic acid (PLLA), which has considerable potential because of its well-known biocompatibility and biodegradability. One of the factors that influence cell adhesion to the scaffold is its porosity degree, but surface properties represent the main driving forces that influence the composition and orientation of proteins that will be absorbed onto material surfaces. We used scaffolds in which it was possible to control pore size and that had undergone on type-I collagen treatment, to mimic the extra cellular matrix, or plasma enhanced chemical vapor deposition (PE-CVD) combined with plasma treatment, in order to modify surface chemistry of the material. Our results show different cell affinity in non-treated scaffolds compared to type-I collagen or plasma modified ones. These surface changes are of considerable interest for tissue engineering and other areas of biomaterials science, where it can be useful to improve the surface of biomedical polymers to facilitate the colonization of the structure by the cells and obtain a more rapid regeneration or tissue replacement.In the most common approach of tissue engineering, a polymeric scaffold with a well-defined architecture has emerged as a promising platform for cells adhesion and guide their proliferation and differentiation into the desired tissue or organ. An ideal model for the regeneration should mimic clinical conditions of tissue injury, create a permissive microenvironment for diffusion of nutrients, gases and growth factors and permit angiogenesis. In this work, we used a 3D support made of synthetic resorbable polylactic acid (PLLA), which has considerable potential because of its well-known biocompatibility and biodegradability. One of the factors that influence cell adhesion to the scaffold is its porosity degree, but surface properties represent the main driving forces that influence the composition and orientation of proteins that will be absorbed onto material surfaces. We used scaffolds in which it was possible to control pore size and that had undergone on type-I collagen treatment, to mimic the extra cellular matrix, or plasma enhanced chemical vapor deposition (PE-CVD) combined with plasma treatment, in order to modify surface chemistry of the material. Our results show different cell affinity in non-treated scaffolds compared to type-I collagen or plasma modified ones. These surface changes are of considerable interest for tissue engineering and other areas of biomaterials science, where it can be useful to improve the surface of biomedical polymers to facilitate the colonization of the structure by the cells and obtain a more rapid regeneration or tissue replacement. Copyright © 2012, AIDIC Servizi S.r.l

Minimalism in Radiation Synthesis of Biomedical Functional Nanogels

A scalable, single-step, synthetic approach for the manufacture of biocompatible, functionalized micro- and nanogels is presented. In particular, poly(N-vinyl pyrrolidone)-grafted-(aminopropyl)methacrylamide microgels and nanogels were generated through e-beam irradiation of PVP aqueous solutions in the presence of a primary amino-group-carrying monomer. Particles with different hydrodynamic diameters and surface charge densities were obtained at the variance of the irradiation conditions. Chemical structure was investigated by different spectroscopic techniques. Fluorescent variants were generated through fluorescein isothiocyanate attachment to the primary amino groups grafted to PVP, to both quantify the available functional groups for bioconjugation and follow nanogels localization in cell cultures. Finally, a model protein, bovine serum albumin, was conjugated to the nanogels to demonstrate the attachment of biologically relevant molecules for targeting purposes in drug delivery. The described approach provides a novel strategy to fabricate biohybrid nanogels with a very promising potential in nanomedicine

Production of Injectable Marine Collagen-Based Hydrogel for the Maintenance of Differentiated Chondrocytes in Tissue Engineering Applications

Cartilage is an avascular tissue with limited ability of self-repair. The use of autologous chondrocyte transplants represent an effective strategy for cell regeneration; however, preserving the differentiated state, which ensures the ability to regenerate damaged cartilage, represents the main challenge during in vitro culturing. For this purpose, we produced an injectable marine collagen-based hydrogel, by mixing native collagen from the jellyfish Rhizostoma pulmo with hydroxy-phenyl-propionic acid (HPA)-functionalized marine gelatin. This biocompatible hydrogel formulation, due to the ability of enzymatically reticulate using horseradish peroxidase (HPR) and H2O2, gives the possibility of trap cells inside, in the absence of cytotoxic effects, during the cross-linking process. Moreover, it enables the modulation of the hydrogel stiffness merely varying the concentration of H2O2 without changes in the concentration of polymer precursors. The maintenance of differentiated chondrocytes in culture was then evaluated via morphological analysis of cell phenotype, GAG production and cytoskeleton organization. Additionally, gene expression profiling of differentiation/dedifferentiation markers provided evidence for the promotion of the chondrogenic gene expression program. This, combined with the biochemical properties of marine collagen, represents a promising strategy for maintaining in vitro the cellular phenotype in the aim of the use of autologous chondrocytes in regenerative medicine practices