Synthetic peptides of IL-1Ra and HSP70 have anti-inflammatory activity on human primary monocytes and macrophages: Potential treatments for inflammatory diseases.

Chronic inflammatory diseases are increasing in developed societies, thus new anti-inflammatory approaches are needed in the clinic. Synthetic peptides complexes can be designed to mimic the activity of anti-inflammatory mediators, in order to alleviate inflammation. Here, we evaluated the anti-inflammatory efficacy of tethered peptides mimicking the interleukin-1 receptor antagonist (IL-1Ra) and the heat-shock protein 70 (HSP70). We tested their biocompatibility and anti-inflammatory activity in vitro in primary human monocytes and differentiated macrophages activated with two different stimuli: the TLR agonists (LPS + IFN-γ) or Pam3CSK4. Our results demonstrate that IL-1Ra and HSP70 synthetic peptides present a satisfactory biocompatible profile and significantly inhibit the secretion of several pro-inflammatory cytokines (IL-6, IL-8, IL-1ß and TNFα). We further confirmed their anti-inflammatory activity when peptides were coated on a biocompatible material commonly employed in surgical implants. Overall, our findings support the potential use of IL-1Ra and HSP70 synthetic peptides for the treatment of inflammatory conditions.

Anti-angiogenic potential of VEGF blocker dendron loaded on to gellan gum hydrogels for tissue engineering applications.

Damage of non-vascularised tissues such as cartilage and cornea can result in healing processes accompanied by a non-physiological angiogenesis. Peptidic aptamers have recently been reported to block the vascular endothelial growth factor (VEGF). However, the therapeutic applications of these aptamers are limited due to their short half-life in vivo. In this work, an enhanced stability and bioavailability of a known VEGF blocker aptamer sequence (WHLPFKC) was pursued through its tethering of molecular scaffolds based on hyperbranched peptides, the poly(ɛ-lysine) dendrons, bearing three branching generations. The proposed design allowed simultaneous and orderly-spaced exposure of 16 aptamers per dendrimer to the surrounding biological microenvironent, as well as a relatively hydrophobic core based on di-phenylalanine aiming to promote an hydrophobic interaction with the hydrophobic moieties of ionically crosslinked methacrylated gellan gum (iGG-MA) hydrogels. The VEGF blocker dendrons were entrapped in iGG-MA hydrogels, and their capacity to prevent endothelial cell sprouting was assessed qualitatively and quantitatively using 3D in vitro models and the in vivo chick chorioallantoic membrane assay. The data demonstrate that at nanoscale concentrations, the dendronised structures were able to enhance control of the biological actvity of WHLPFKC at the material/tissue interface and hence the anti-angiogenic capacity of iGG-MA hydrogels not only preventing blood vessel invasion, but also inducing their regression at the tissue/iGG-MA interface. The in ovo study confirmed that iGG-MA functionalised with the dendron VEGF blockers do inhibit angiogenesis by controlling both size and ramifications of blood vessels in the proximity of the implanted gel surface. Copyright © 2016 John Wiley & Sons, Ltd.

Hyperbranched poly(ϵ-lysine) substrate presenting the laminin sequence YIGSR induces the formation of spheroids in adult bone marrow stem cells.

Unlike the fibroblast-like cells formed upon monolayer culture of human mesenchymal stem cells, the natural stem cell niche of the bone marrow and other types of tissues favours the formation of 3-dimensional (3D) cell clusters. The structuring and biological activity of these clusters are regulated by the contacts established by cells with both the basement membrane and neighbour cells and results in their asymmetric division and the consequent maintenance of both a stem population and a committed progeny. The present work demonstrates the potential of a synthetic substrate to mimic the stem cell niche in vitro. The side amino groups of a linear Poly-L-lysine were modified with hyperbranched poly-(ϵ-lysine) peptides, named as dendrons, tethered with the laminin-mimicking sequence, YIGSR. These dendrons presented the YIGSR sequence at the uppermost molecular branching ensuring a controlled spacing of the bioligand. When used to coat the surface of tissue culture plates in a serum-free in vitro cell culture system, the substrate was able to mimic the most relevant features of the basement membrane of the stem cell niche, i.e. the mesh structure of Collagen Type IV and the availability of laminin bioligands relevant to integrin biorecognition. The substrate biomimetic properties were tested for their ability to support the formation of human bone marrow mesenchymal stem cells (hMSCs) 3D spheroids similar to those observed in the natural stem cell niches and their ability to maintain stem cell pluripotency markers. These features were related to the substrate-specific expression and localisation of (i) cell adhesion receptors (i.e. ß-integrin and N-cadherin), (ii) transcription factors of pluripotency markers and cytoskeleton protein and (iii) regulators of cell migration throughout cell culture passages 2 to 4. The results clearly demonstrate the formation of 3D spheroids starting from the asymmetric division of substrate-adhering spread cells, the clustering of relevant integrins and the expression of specific intracellular pathways controlling cytoskeleton formation suggesting their potential use as a substrate for the handling of stem cells prior to transplantation procedures.

Carboxybetaine-modified succinylated chitosan-based beads encourage pancreatic ß-cells (Min-6) to form islet-like spheroids under in vitro conditions.

In vitro, pancreatic ß-cells tend to reduce their ability to aggregate into islets and lose insulin-producing ability, likely due to insufficient cell-cell and cell-matrix interactions that are essential for ß-cell retention, viability and functionality. In response to these needs, surfaces of succinylated chitosan-based beads (NSC) were modified with zwitterionic carboxy-betaine (CB) moieties, a compatible osmolyte known to regulate cellular hydration state, and used to promote the formation of ß-cell spheroids using a conventional 2D cell culture technique. The NSC were synthesised by ionic gelation and surface-functionalised with CB using carbodiimide chemistry. Scanning electron microscopy (SEM), dynamic laser scattering (DLS) and Fourier transform infrared spectroscopy (FTIR) were employed as characterisation tools to confirm the successful modification of the succinylated chitosan material into spherical beads with rough surfaces and a diameter of 0.4 µm. NSC with and without CB were re-suspended at concentrations of 0.1, 0.3 and 0.6 mg/mL in saline medium and tested in vitro with MIN6 murine pancreatic ß-cell line. Results showed that a concentration of 0.3 mg/mL, NSC-CB encouraged pancreatic MIN6 cells to proliferate and form spheroids via E-cadherin and Pdx-1 activation within 48 h in culture. These spheroids, with a size of approximately 80 µm, exhibited high cell viability and enhanced insulin protein expression and secretion when compared to cells organised by the non-modified beads.

Synthesis, characterisation and in vitro anti-angiogenic potential of dendron VEGF blockers.

To overcome the lack of in vivo stability of certain peptides used in cancer treatment and to increase their retention time in the extracellular matrix of the target tissue, the anti-angiogenic WHLPFKC sequence is synthesised at the uppermost branching generation of a poly(ε-lysine) dendron. The root of these dendrons is designed to interact preferentially with macromolecules of the extracellular matrix, whilst the uppermost branching generation of the dendron increased the exposed density of the bioactive peptide. Bioactivity testing of the blockers is performed on HUVECs. The results show that the dendron tethered with VEGF blockers was still able to inhibit proliferation and angiogenesis. Their relatively larger structure did not prevent the interaction with VEGF.

Substrate-induced phenotypic switches of human smooth muscle cells: an in vitro study of in-stent restenosis activation pathways.

In-stent restenosis is a clinical complication following coronary angioplasty caused by the implantation of the metal device in the atherosclerotic vessel. Histological examination has shown a clear contribution of both inflammatory and smooth muscle cells (SMCs) to the deposition of an excess of neointimal tissue. However, the sequence of events leading to clinically relevant restenosis is unknown. This paper aims to study the phenotype of SMCs when adhering on substrates and exposed to biochemical stimuli typical of the early phases of stent implantation. In particular, human SMC phenotype was studied when adhering on extracellular matrix-like material (collagen-rich gel), thrombus-like material (fibrin gel) and stent material (stainless steel) in the presence or absence of a platelet-derived growth factor (PDGF) stimulus. Cells on the collagen and fibrin-rich substrates maintained their contractile phenotype. By contrast, cells on stainless steel acquired a secretory phenotype with a proliferation rate 50 per cent higher than cells on the natural substrates. Cells on stainless steel also showed an increase in PDGF-BB receptor expression, thus explaining the increase in proliferation observed when cells were subject to PDGF-BB stimuli. The stainless steel substrate also promoted a different pattern of ß1-integrin localization and an altered expression of hyaluronan (HA) synthase isoforms where the synthesis of high-molecular-weight HA seemed to be favoured. These findings highlighted the induction of a phenotypic pattern in SMC by the stainless steel substrate whereby the formation of a HA-rich neointimal tissue is enhanced.

Direct comparison of the short-term clinical performance of Z Guidant and Taxus stents.

The recent introduction of drug-eluting stents in angioplasty of atherosclerotic blood vessels has significantly reduced the risks of in-stent restenosis (ISR) [1]. Indeed, it is known that in conventional stents ISR takes place in over 20% of the cases and up to 60% when implanted in diabetic patients. Conversely, clinical trials have shown that drug-eluting stents have significantly reduced ISR. Among the drug-eluting stents available on the market, Taxus stents (Tax, Boston Scientific, USA) are among the most used devices [2]. Tax are stainless-steel stents coated with Translute, a poly(styrene-b-isobutylene-b-styrene) polymer (PSIBS) eluting Placlitaxel, an anti-mitotic drug. Clinical trials on this type of drug-eluting stents have shown an incidence of restenosis of approximately 4%. The majority of these trials were randomized studies where conventional stents and drug-eluting devices have been implanted in separate patients' cohorts. Such a randomized design, although fundamental to collect statistically-relevant data, does not allow a direct histological comparison of different stent types when implanted in the same patient and do not show the individual susceptibility to the host response especially at short-term implantation times. Here, an interesting case study is presented where two chrome-cobalt stents (Z Guidant, ZG, Guidant Corp.) and a Tax have been simultaneously implanted in the same patient in three separate coronary arteries, retrieved after only 8 weeks and histologically analysed.

Stent material surface and glucose activate mononuclear cells of control, type 1 and type 2 diabetes subjects.

In stent restenosis (ISR) has been described as an unaccomplished tissue healing and its rate is particularly high in diabetic patients. Evidence has been collected which relates the formation of ISR proteoglycan-rich neointimal tissue to the accumulation and protracted activation of macrophages around the stent metal struts. Here, the in vitro activation of mononuclear cells adhering to stainless steel (a material of choice in stent manufacturing) from control and diabetic (types 1 and 2) subjects was assessed in the presence of different glucose levels. The results showed that cells from the control and type 1 diabetes groups produced significantly higher levels of TGF-beta1 when adhering on stainless steel (p = 0.04 and p = 0.01), but a significant PDGF-BB secretion was observed only in control subjects. When tested at physiological glucose concentration, the effect of the stainless steel on control cells was more pronounced. The present study shows that mononuclear cells adhering onto stainless steel secrete growth factors relevant to ISR. Cells from diabetic subjects seem to secrete relatively higher levels of PDGF under hyperglycaemic conditions regardless of the substrate exposed thus offering an explanation for the higher incidence of restenosis in these patients.