Leucocyte and Platelet-rich Fibrin: a carrier of autologous multipotent cells for regenerative medicine.

The wound healing is a complex process wherein inflammation, proliferation and regeneration evolve according to a spatio-temporal pattern from the activation of coagulation cascade to the formation of a plug clot including fibrin matrix, blood-borne cells and cytokines/growth factors. Creating environments conducive to tissue repair, the haemoderivatives are commonly proposed for the treatment of hard-to-heal wounds. Here, we explored in vitro the intrinsic regenerative potentialities of a leucocyte- and platelet-rich fibrin product, known as CPL-MB, defining the stemness grade of cells sprouting from the haemoderivative. Using highly concentrated serum-based medium to simulate wound conditions, we isolated fibroblast-like cells (CPL-CMCs) adhering to plastic and showing stable in vitro propagation, heterogeneous stem cell expression pattern, endothelial adhesive properties and immunomodulatory profile. Due to their blood derivation and expression of CXCR4, CPL-CMCs have been suggested to be immature cells circulating in peripheral blood at quiescent state until activation by both coagulation event and inflammatory stimuli such as stromal-derived factor 1/SDF1. Expressing integrins (CD49f, CD103), vascular adhesion molecules (CD106, CD166), endoglin (CD105) and remodelling matrix enzymes (MMP2, MMP9, MMP13), they showed a transendothelial migratory potential besides multipotency. Taken together, our data suggested that a standardized, reliable and economically feasible blood product such as CPL-MB functions as an artificial stem cell niche that, under permissive conditions, originate ex vivo immature cells that could be useful for autologous stem cell-based therapies.

Sex-dependent differences in inflammatory responses during liver regeneration in a murine model of acute liver injury.

A sexual dimorphism in liver inflammation and repair was previously demonstrated. Its cellular dissection in the course of acute liver injury (ALI) was explored. BALB/c mice were treated with carbon tetrachloride (CCl4) by intraperitoneal injection and killed after 3, 5, and 8 days. Histological and hepatic cell population analyses were performed. The correlation between androgen receptor (AR) expression and liver recruited inflammatory cells was investigated by treatment with the AR antagonist flutamide. Additionally, patients with a diagnosis of drug induced liver injury (DILI) were included in the study, with a particular focus on gender dimorphism in circulating monocytes. A delayed resolution of necrotic damage and a higher expression of proinflammatory cytokines were apparent in male mice along with a slower recruitment of inflammatory monocytes. F4/80+CD11b+ macrophages and CD11bhighGr-1high monocytes expressed AR and were recruited later in male compared with female livers after CCl4 treatment. Moreover, CD11bhighAR+Gr-1high recruitment was negatively modulated by flutamide in males. Analysis of DILI patients showed overall a significant reduction in circulating mature monocytes compared with healthy subjects. More interestingly, male patients had higher numbers of immature monocytes compared with female patients.A stronger cytotoxic tissue response was correlated with an impaired recruitment of CD11bhighAR+Gr-1high cells and F4/80+CD11b+ macrophages in the early inflammatory phase under AR signaling. During DILI, a dimorphic immune response was apparent, characterized by a massive recruitment of monocytes to the liver both in males and females, but only in males was this recruitment sustained by a turnover of immature monocytes.

Antiproliferative activity of 8-methoxypsoralen on DU145 prostate cancer cells under UVA and blue light.

The use of photoactivatable 8-methoxypsoralen (8-MOP) as potential focal treatment towards prostate cancer cells is proposed here. Our results, obtained on isolated DNA and DU145 cells, indicate that blue light, besides UVA, is able to activate 8-MOP. When compared to UVA, blue light irradiation led to a modulation of the extent and the types of 8-MOP-DNA damage, specially cross-links, coupled to a still valuable antiproliferative effect. Our data suggest that the proapototic activity of 8-MOP is related not only to DNA damage and reactive oxygen species generation but also to the modulation of cell signalling pathways. In particular, a different activation of p38 and p44/42 mitogen-activated protein kinases was detected depending on the light wavelengths.

Fluorescence-based gene reporter plasmid to track canonical Wnt signaling in ENS inflammation.

In several gut inflammatory or cancer diseases, cell-cell interactions are compromised, and an increased cytoplasmic expression of ß-catenin is observed. Over the last decade, numerous studies provided compelling experimental evidence that the loss of cadherin-mediated cell adhesion can promote ß-catenin release and signaling without any specific activation of the canonical Wnt pathway. In the present work, we took advantage of the ability of lipofectamine-like reagent to cause a synchronous dissociation of adherent junctions in cells isolated from the rat enteric nervous system (ENS) for obtaining an in vitro model of deregulated ß-catenin signaling. Under these experimental conditions, a green fluorescent protein Wnt reporter plasmid called ΔTop_EGFP3a was successfully tested to screen ß-catenin stabilization at resting and primed conditions with exogenous Wnt3a or lipopolysaccharide (LPS). ΔTop_EGFP3a provided a reliable and strong fluorescent signal that was easily measurable and at the same time highly sensitive to modulations of Wnt signaling following Wnt3a and LPS stimulation. The reporter gene was useful to demonstrate that Wnt3a exerts a protective activity in the ENS from overstimulated Wnt signaling by promoting a downregulation of the total ß-catenin level. Based on this evidence, the use of ΔTop_EGFP3a reporter plasmid could represent a more reliable tool for the investigation of Wnt and cross-talking pathways in ENS inflammation.

Ligand engagement of Toll-like receptors regulates their expression in cortical microglia and astrocytes.

BACKGROUND: Toll-like receptor (TLR) activation on microglia and astrocytes are key elements in neuroinflammation which accompanies a number of neurological disorders. While TLR activation on glia is well-established to up-regulate pro-inflammatory mediator expression, much less is known about how ligand engagement of one TLR may affect expression of other TLRs on microglia and astrocytes. METHODS: In the present study, we evaluated the effects of agonists for TLR2 (zymosan), TLR3 (polyinosinic-polycytidylic acid (poly(I:C)), a synthetic analogue of double-stranded RNA) and TLR4 (lipopolysaccaride (LPS)) in influencing expression of their cognate receptor as well as that of the other TLRs in cultures of rat cortical purified microglia (>99.5 %) and nominally microglia-free astrocytes. Elimination of residual microglia (a common contaminant of astrocyte cultures) was achieved by incubation with the lysosomotropic agent L-leucyl-L-leucine methyl ester (L-LME). RESULTS: Flow cytometric analysis confirmed the purity (essentially 100 %) of the obtained microglia, and up to 5 % microglia contamination of astrocytes. L-LME treatment effectively removed microglia from the latter (real-time polymerase chain reaction). The three TLR ligands robustly up-regulated gene expression for pro-inflammatory markers (interleukin-1 and interleukin-6, tumor necrosis factor) in microglia and enriched, but not purified, astrocytes, confirming cellular functionality. LPS, zymosan and poly(I:C) all down-regulated TLR4 messenger RNA (mRNA) and up-regulated TLR2 mRNA at 6 and 24 h. In spite of their inability to elaborate pro-inflammatory mediator output, the nominally microglia-free astrocytes (>99 % purity) also showed similar behaviours to those of microglia, as well as changes in TLR3 gene expression. LPS interaction with TLR4 activates downstream mitogen-activated protein kinase and nuclear factor-κB signalling pathways and subsequently causes inflammatory mediator production. The effects of LPS on TLR2 mRNA in both cell populations were antagonized by a nuclear factor-κB inhibitor. CONCLUSIONS: TLR2 and TLR4 activation in particular, in concert with microglia and astrocytes, comprise key elements in the initiation and maintenance of neuropathic pain. The finding that both homologous (zymosan) and heterologous (LPS, poly(I:C)) TLR ligands are capable of regulating TLR2 gene expression, in particular, may have important implications in understanding the relative contributions of different TLRs in neurological disorders associated with neuroinflammation.

Anti-inflammatory activity of Wnt signaling in enteric nervous system: in vitro preliminary evidences in rat primary cultures.

BACKGROUND: In the last years, Wnt signaling was demonstrated to regulate inflammatory processes. In particular, an increased expression of Wnts and Frizzled receptors was reported in inflammatory bowel disease (IBD) and ulcerative colitis to exert both anti- and pro-inflammatory functions regulating the intestinal activated nuclear factor κB (NF-кB), TNFa release, and IL10 expression. METHODS: To investigate the role of Wnt pathway in the response of the enteric nervous system (ENS) to inflammation, neurons and glial cells from rat myenteric plexus were treated with exogenous Wnt3a and/or LPS with or without supporting neurotrophic factors such as basic fibroblast growth factor (bFGF), epithelial growth factor (EGF), and glial cell-derived neurotrophic factor (GDNF). The immunophenotypical characterization by flow cytometry and the protein and gene expression analysis by qPCR and Western blotting were carried out. RESULTS: Flow cytometry and immunofluorescence staining evidenced that enteric neurons coexpressed Frizzled 9 and toll-like receptor 4 (TLR4) while glial cells were immunoreactive to TLR4 and Wnt3a suggesting that canonical Wnt signaling is active in ENS. Under in vitro LPS treatment, Western blot analysis demonstrated an active cross talk between canonical Wnt signaling and NF-кB pathway that is essential to negatively control enteric neuronal response to inflammatory stimuli. Upon costimulation with LPS and Wnt3a, a significant anti-inflammatory activity was detected by RT-PCR based on an increased IL10 expression and a downregulation of pro-inflammatory cytokines TNFa, IL1B, and interleukin 6 (IL6). When the availability of neurotrophic factors in ENS cultures was abolished, a changed cell reactivity by Wnt signaling was observed at basal conditions and after LPS treatment. CONCLUSIONS: The results of this study suggested the existence of neuronal surveillance through FZD9 and Wnt3a in enteric myenteric plexus. Moreover, experimental evidences were provided to clarify the correlation among soluble trophic factors, Wnt signaling, and anti-inflammatory protection of ENS.

Facile and selective covalent grafting of an RGD-peptide to electrospun scaffolds improves HUVEC adhesion.

The development of a biomimetic surface able to promote endothelialization is fundamental in the search for blood vessel substitutes that prevent the formation of thrombi or hyperplasia. This study aims at investigating the effect of functionalization of poly-ε-caprolactone or poly(L-lactic acid-co-ɛ-caprolactone) electrospun scaffolds with a photoreactive adhesive peptide. The designed peptide sequence contains four Gly-Arg-Gly-Asp-Ser-Pro motifs per chain and a p-azido-Phe residue at each terminus. Different peptide densities on the scaffold surface were obtained by simply modifying the peptide concentration used in pretreatment of the scaffold before UV irradiation. Scaffolds of poly-ε-caprolactone embedded with adhesive peptides were produced to assess the importance of peptide covalent grafting. Our results show that the scaffolds functionalized with photoreactive peptides enhance adhesion at 24 h with a dose-dependent effect and control the proliferation of human umbilical vein endothelial cells, whereas the inclusion of adhesive peptide in the electrospun matrices by embedding does not give satisfactory results.

Maintenance of the enteric stem cell niche by bacterial lipopolysaccharides? Evidence and perspectives.

The enteric nervous system (ENS) has to respond to continuously changing microenvironmental challenges within the gut and is therefore dependent on a neural stem cell niche to keep the ENS functional throughout life. In this study, we hypothesize that this stem cell niche is also affected during inflammation and therefore investigated lipopolysaccharides (LPS) effects on enteric neural stem/progenitor cells (NSPCs). NSPCs were derived from the ENS and cultured under the influence of different LPS concentrations. LPS effects upon proliferation and differentiation of enteric NSPC cultures were assessed using immunochemistry, flow cytometry, western blot, Multiplex ELISA and real-time PCR. LPS enhances the proliferation of enteric NSPCs in a dose-dependent manner. It delays and modifies the differentiation of these cells. The expression of the LPS receptor toll-like receptor 4 on NSPCs could be demonstrated. Moreover, LPS induces the secretion of several cytokines. Flow cytometry data gives evidence for individual subgroups within the NSPC population. ENS-derived NSPCs respond to LPS in maintaining at least partially their stem cell character. In the case of inflammatory disease or trauma where the liberation and exposure to LPS will be increased, the expansion of NSPCs could be a first step towards regeneration of the ENS. The reduced and altered differentiation, as well as the induction of cytokine signalling, demonstrates that the stem cell niche may take part in the LPS-transmitted inflammatory processes in a direct and defined way.

Amniotic fluid stem cells prevent ß-cell injury.

BACKGROUND AIMS: The contribution of amniotic fluid stem cells (AFSC) to tissue protection and regeneration in models of acute and chronic kidney injuries and lung failure has been shown in recent years. In the present study, we used a chemically induced mouse model of type 1 diabetes to determine whether AFSC could play a role in modulating ß-cell injury and restoring ß-cell function. METHODS: Streptozotocin-induced diabetic mice were given intracardial injection of AFSC; morphological and physiological parameters and gene expression profile for the insulin pathway were evaluated after cell transplantation. RESULTS: AFSC injection resulted in protection from ß-cell damage and increased ß-cell regeneration in a subset of mice as indicated by glucose and insulin levels, increased islet mass and preservation of islet structure. Moreover, ß-cell preservation/regeneration correlated with activation of the insulin receptor/Pi3K/Akt signaling pathway and vascular endothelial growth factor-A expression involved in maintaining ß-cell mass and function. CONCLUSIONS: Our results suggest a therapeutic role for AFSC in preserving and promoting endogenous ß-cell functionality and proliferation. The protective role of AFSC is evident when stem cell transplantation is performed before severe hyperglycemia occurs, which suggests the importance of early intervention. The present study demonstrates the possible benefits of the application of a non-genetically engineered stem cell population derived from amniotic fluid for the treatment of type 1 diabetes mellitus and gives new insight on the mechanism by which the beneficial effect is achieved.

Development of 3D melanoma cultures on a hyaluronic acid-based scaffold with synthetic self-assembling peptides: Electroporation enhancement.

Electrochemotherapy (ECT) with bleomycin is an effective antitumor treatment. Still, researchers are investigating new drugs and electroporation conditions to improve its efficacy. To this aim, in vivo assays are accurate but expensive and ethically questionable. Conversely, in vitro assays, although cheaper and straightforward, do not reflect the architecture of the biological tissue because they lack a tridimensional (3D) structure (as in the case of two-dimensional [2D] in vitro assays) or do not include all the extracellular matrix components (as in the case of 3D in vitro scaffolds). To address this issue, 3D in vitro models have been proposed, including spheroids and hydrogel-based cultures, which require a suitable low-conductive medium to allow cell membrane electroporation. In this study, a synthetic scaffold based on hyaluronic acid (HA) and self-assembling peptides (SAPs; EAbuK), condensed with a Laminin-derived adhesive sequence (IKVAV), is proposed as a reliable alternative. We compare SKMEL28 cells cultured in the HA-EAbuK-IKVAV scaffold to the control (HA only scaffold). Three days after seeding, the culture on the HA-EAbuK-IKVAV scaffold showed collagen production. SKMEL28 cells cultured on the HA-EAbuK-IKVAV scaffold started to be electroporated at 400 V/cm, whereas, at the same electric field intensity, those cultured on HA were not. As a reference, 2D experiments showed that electroporation of SKMEL28 cells starts at 600 V/cm using an electroporation buffer and at 800 V/cm in a culture medium, but with very low efficiency (<50 % of cells electroporated). 3D cultures on HA-EAbuK-IKVAV allowed the simulation of a more reliable microenvironment and may represent a valuable tool for studying electroporation conditions. Using Finite Element Analysis (FEA) to compute the transmembrane potential, we detected the influence of inhomogeneity of the extracellular matrix on electroporation effect. Our 3D cell culture electroporation simulations showed that the transmembrane potential increased when collagen surrounded the cells. Of note, in the collagen-enriched HA-EAbuK-IKVAV scaffold, EP was already improved at lower electric field intensities. This study shows the influence of the extracellular matrix on electric conductivity and electric field distribution on cell membrane electroporation and supports the adoption of more reliable 3D scaffolds in experimental electroporation studies.

Finite Element Evaluation of the Electric Field Distribution in a Non-Homogeneous Environment.

Finite element analysis is used in this study to investigate the effect of media inhomogeneity on the electric field distribution in a sample composed of cells and their extracellular matrix. The sample is supposed to be subjected to very high pulsed electric field. Numerically computed electric field distribution and transmembrane potential at the cell membrane in electroporation conditions are considered in order to study cell behavior at different degrees of inhomogeneity. The different inhomogeneity grade is locally obtained using a representative model of fixed volume with cell-cell distance varying in the range of 1-283 um. The conductivity of the extracellular medium was varied between plain collagen and a gel-like myxoid matrix through combinations of the two, i.e., collagen and myxoid. An increase in the transmembrane potential was shown in the case of higher aggregate. The results obtained in this study show the effect of the presence of the cell aggregates and collagen on the transmembrane potential. In particular, by increasing the cell aggregation in the two cases, the transmembrane potential increased. Finally, the simulation results were compared to experimental data obtained by culturing HCC1954 cells in a hyaluronic acid-based scaffold. The experimental validation confirmed the behavior of the transmembrane potential in presence of the collagen: an increase in electroporation at a lower electric field intensity was found for the cells cultured in the scaffolds where there is the formation of collagen areas.

Preclinical and clinical orthotopic transplantation of decellularized/engineered tracheal scaffolds: A systematic literature review.

Severe tracheal injuries that cannot be managed by mobilization and end-to-end anastomosis represent an unmet clinical need and an urgent challenge to face in surgical practice; within this scenario, decellularized scaffolds (eventually bioengineered) are currently a tempting option among tissue engineered substitutes. The success of a decellularized trachea is expression of a balanced approach in cells removal while preserving the extracellular matrix (ECM) architecture/mechanical properties. Revising the literature, many Authors report about different methods for acellular tracheal ECMs development; however, only few of them verified the devices effectiveness by an orthotopic implant in animal models of disease. To support translational medicine in this field, here we provide a systematic review on studies recurring to decellularized/bioengineered tracheas implantation. After describing the specific methodological aspects, orthotopic implant results are verified. Furtherly, the only three clinical cases of compassionate use of tissue engineered tracheas are reported with a focus on outcomes.

Development and In Vitro/In Vivo Comparative Characterization of Cryopreserved and Decellularized Tracheal Grafts.

Tracheal reconstruction represents a challenge when primary anastomosis is not feasible. Within this scenario, the study aim was to develop a new pig-derived decellularized trachea (DecellT) to be compared with the cryopreserved counterpart (CryoT) for a close predictive analysis. Tracheal segments underwent decellularization by a physical + enzymatic + chemical method (12 cycles); in parallel, cryopreserved samples were also prepared. Once decellularized (histology/DNA quantification), the two groups were characterized for Alpha-Gal epitopes/structural proteins (immunohistochemistry/histology/biochemical assays/second harmonic generation microscopy)/ultrastructure (Scanning Electron Microscopy (SEM))/mechanical behaviour. Cytotoxicity absence was assessed in vitro (extract-test assay/direct seeding, HM1SV40 cell line) while biocompatibility was verified in BALB/c mice, followed by histological/immunohistochemical analyses and SEM (14 days). Decellularization effectively removed Alpha-Gal epitopes; cartilage histoarchitecture was retained in both groups, showing chondrocytes only in the CryoT. Cryopreservation maintained few respiratory epithelium sparse cilia, not detectable in DecellT. Focusing on ECM, preserved structural/ultrastructural organization and collagen content were observed in the cartilage of both; conversely, the GAGs were significantly reduced in DecellT, as confirmed by mechanical study results. No cytotoxicity was highlighted by CryoT/DecellT in vitro, as they were also corroborated by a biocompatibility assay. Despite some limitations (cells presence/GAGs reduction), CryoT/DecellT are both appealing options, which warrant further investigation in comparative in vivo studies.

New vandetanib analogs: fused tricyclic quinazolines with antiangiogenic potential.

The antiangiogenic effects of three novel anilinoquinazoline derivatives were studied with the aim to find new multi-kinase inhibitors as anticancer agents. The compounds are characterized by dioxolane, dioxane and dioxepine rings and bear the same aniline substituent in 4 position as vandetanib, known antiangiogenic agent. The in vitro assays were carried out on human umbilical vein endothelial cells (HUVECs), whereas in vivo angiogenesis was evaluated by means of Matrigel plug assay. The results showed that these compounds exert, even though to different extents, antiangiogenic activity affecting the various step of the process that leads to the formation of new blood vessels. At high concentrations they induced antiproliferative effects, whereas at non-cytotoxic concentrations they inhibited cell migration and the formation of tubular structures in Matrigel. In in vitro assays the dioxolane derivative 1 was more effective than vandetanib. Indeed, it inhibited the effects induced by exogenous VEGF and FGF-2 on both cell proliferation and morphogenesis, whereas vandetanib was completely ineffective. Moreover, all the compounds, as vandetanib, counteracted the FGF-2-induced increase in the hemoglobin content in the Matrigel plugs. Our results showed that all the three novel derivatives possess both in vitro and in vivo antiangiogenic activity, with compound 1 more effective than vandetanib to inhibit in vitro angiogenesis induced by exogenous cytokines.

Ratiometric detection of Cr3+ and Hg2+ by a naphthalimide-rhodamine based fluorescent probe.

Newly synthesized rhodamine derivatives, L(1) and L(2), are found to bind specifically to Hg(2+) or Cr(3+) in presence of large excess of other competing ions with associated changes in their optical and fluorescence spectral behavior. These spectral changes are significant enough in the visible region of the spectrum and thus, allow the visual detection. For L(1), the detection limit is even lower than the permissible [Cr(3+)] or [Hg(2+)] in drinking water as per standard U.S. EPA norms; while the receptor, L2 could be used as a ratiometric sensor for detection of Cr(3+) and Hg(2+) based on the resonance energy transfer (RET) process involving the donor naphthalimide and the acceptor Cr(3+)/Hg(2+)-bound xanthene fragment. Studies reveal that these two reagents could be used for recognition and sensing of Hg(2+)/Cr(3+). Further, confocal laser microscopic studies confirmed that the reagent L(2) could also be used as an imaging probe for detection of uptake of these ions in A431 cells.

Systemic administration of a novel human umbilical cord mesenchymal stem cells population accelerates the resolution of acute liver injury.

BACKGROUND: Hepatocytes and stem cells transplantation may be an alternative to liver transplantation in acute or chronic liver disease. We aimed to evaluate the therapeutic potential of mesenchymal stem cells from human umbilical cord (UCMSCs), a readily available source of mesenchymal stem cells, in the CCl4-induced acute liver injury model. METHODS: Mesenchymal stem cells profile was analyzed by flow cytometry. In order to evaluate the capability of our UCMSCs to differentiate in hepatocytes, cells were seeded on three different supports, untreated plastic support, MatrigelTM and human liver acellular matrix. Cells were analyzed by immunocitochemistry for alpha-fetoprotein and albumin expression, qPCR for hepatocyte markers gene expression, Periodic Acid-Schiff staining for glycogen storage, ELISA for albumin detection and colorimetric assay for urea secretion.To assess the effects of undifferentiated UCMSCs in hepatic regeneration after an acute liver injury, we transplanted them via tail vein in mice injected intraperitoneally with a single dose of CCl4. Livers were analyzed by histological evaluation for damage quantification, immunostaining for Kupffer and stellate cells/liver myofibroblasts activation and for UCMSCs homing. Pro- and anti-inflammatory cytokines gene expression was evaluated by qPCR analysis and antioxidant enzyme activity was measured by catalase quantification.Data were analyzed by Mann-Whitney U-test, Kruskal-Wallis test and Cuzick's test followed by Bonferroni correction for multiple comparisons. RESULTS: We have standardized the isolation procedure to obtain a cell population with hepatogenic properties prior to in vivo transplantation. When subjected to hepatogenic differentiation on untreated plastic support, UCMSCs differentiated in hepatocyte-like cells as demonstrated by their morphology, progressive up-regulation of mature hepatocyte markers, glycogen storage, albumin and urea secretion. However, cells seeded on 3D-supports showed a minor or negligible differentiation capacity.UCMSCs-transplanted mice showed a more rapid damage resolution, as shown by histological analysis, with a lower inflammation level and an increased catalase activity compared to CCl4-treated mice. CONCLUSIONS: Our findings show that UCMSCs can be reliably isolated, have hepatogenic properties and following systemic administration are able to accelerate the resolution of an acute liver injury without any differentiation and manipulation. These features make UCMSCs strong candidates for future application in regenerative medicine for human acute liver disease.

Preliminary Discovery of Small Molecule Inhibitors of Epidermal Growth Factor Receptor (EGFR) That Bind to the Extracellular Domain.

The Epidermal Growth Factor Receptor (EGFR) is a transmembrane glycoprotein belonging to the protein kinase superfamily. It is composed of an extracellular domain, a transmembrane anchoring region and a cytoplasmic region endowed with tyrosine kinase activity. Genetic mutations of EGFR kinase cause higher activity thereby stimulating downstream signaling pathways that, in turn, impact transcription and cell cycle progression. Due to the involvement of mutant EGFR in tumors and inflammatory diseases, in the past decade, several EGFR inhibitory strategies have been extensively studied, either targeting the extracellular domain (through monoclonal antibodies) or the intracellular kinase domain (through ATP-mimic small molecules). Monoclonal antibodies impair the binding to growth factor, the receptor dimerization, and its activation, whereas small molecules block the intracellular catalytic activity. Herein, we describe the development of a novel small molecule, called DSF-102, that interacts with the extracellular domain of EGFR. When tested in vitro in KRAS mutant A549 cells, it impairs EGFR activity by exerting (i) dose-dependent toxicity effects; (ii) a negative regulation of ERK, MAPK p38 and AKT; and (iii) a modulation of the intracellular trafficking and lysosomal degradation of EGFR. Interestingly, DSF-102 exerts its EGFR inhibitory activity without showing interaction with the intracellular kinase domain. Taken together, these findings suggest that DSF-102 is a promising hit compound for the development of a novel class of anti-EGFR compounds, i.e., small molecules able to interact with the extracellular domain of EGFR and useful for overcoming the KRAS-driven resistance to TKI treatment.

In vitro and in vivo pro-angiogenic effects of thymosin-ß4-derived peptides.

Thymosin-ß4 (Tß4) is a G-actin sequestering peptide involved in regeneration and remodeling of injured tissues. In this work, we have designed and synthesized three peptide sequences containing the N-terminus (TYB4-n), the central part (TYB4-i) or the C-terminus (TYB4-c) of Tß4. All fragments are overlapping on the main central binding actin site. After a structural characterization, we have evaluated in vitro and in vivo their pro-angiogenic effects. The results of this study have shown that: (i) each fragment reproduces the native conformation; (ii) Tß4-derived peptides exert both in vitro and in vivo pro-angiogenic effects; (iii) their in vitro effect seem to be related to the activation of several signaling pathways and is positively modulated by the N-terminus of Tß4.

Synthesis, in vitro and in vivo preliminary evaluation of anti-angiogenic properties of some pyrroloazaflavones.

This work investigated the in vitro and in vivo anti-angiogenic activity of some pyrroloazaflavones, exactly 2-phenyl-1H-pyrrolo[2,3-h]quinolin-4(7H)ones, with vinblastine as reference compound. Growth inhibitory activity, migration, and capillary-like structures formation were determined in human umbilical vein endothelial cell cultures, and Matrigel plug assay was carried out to evaluate in vivo effects on angiogenesis. Collectively, our results indicate that some pyrroloazaflavone derivatives, at non-cytotoxic concentrations and like vinblastine are able: (i) to exert in vitro anti-angiogenic activity and (ii) to counteract in vitro and in vivo the pro-angiogenic effects of fibroblast growth factor-2 (FGF-2).

Enzymatic cross-linking of human recombinant elastin (HELP) as biomimetic approach in vascular tissue engineering.

The use of polymers naturally occurring in the extracellular matrix (ECM) is a promising strategy in regenerative medicine. If compared to natural ECM proteins, proteins obtained by recombinant DNA technology have intrinsic advantages including reproducible macromolecular composition, sequence and molecular mass, and overcoming the potential pathogens transmission related to polymers of animal origin. Among ECM-mimicking materials, the family of recombinant elastin-like polymers is proposed for drug delivery applications and for the repair of damaged elastic tissues. This work aims to evaluate the potentiality of a recombinant human elastin-like polypeptide (HELP) as a base material of cross-linked matrices for regenerative medicine. The cross-linking of HELP was accomplished by the insertion of cross-linking sites, glutamine and lysine, in the recombinant polymer and generating ε-(γ-glutamyl) lysine links through the enzyme transglutaminase. The cross-linking efficacy was estimated by infrared spectroscopy. Freeze-dried cross-linked matrices showed swelling ratios in deionized water (≈2500%) with good structural stability up to 24 h. Mechanical compression tests, performed at 37°C in wet conditions, in a frequency sweep mode, indicated a storage modulus of 2/3 kPa, with no significant changes when increasing number of cycles or frequency. These results demonstrate the possibility to obtain mechanically resistant hydrogels via enzymatic crosslinking of HELP. Cytotoxicity tests of cross-linked HELP were performed with human umbilical vein endothelial cells, by use of transwell filter chambers for 1-7 days, or with its extracts in the opportune culture medium for 24 h. In both cases no cytotoxic effects were observed in comparison with the control cultures. On the whole, the results suggest the potentiality of this genetically engineered HELP for regenerative medicine applications, particularly for vascular tissue regeneration.