Finding the junction between claudins and endometrial carcinoma

: Endometrial carcinoma (EC) defines a heterogeneous group of neoplastic diseases originating from the transformation of endometrial cells that constitute the internal lining of the uterus. To date several molecular targets have been analysed to describe the natural course of the disease, claudins being among these. Claudins are the main components of tight junctions (TJs), and their main functions are ascribed to the compartmentalization of tissues and cell-cell communication by means of intracellular ions diffusion: these features are typical of epithelial cells. Their overexpression, mis-localization or loss contribute to the malignancy of EC cells. This review collected all available data regarding the expression, regulation and claudin-related signaling pathways to provide a comprehensive view on the influence of claudin in EC progression. Further, the translational potential of claudin differential expression was explored, indicating that their role in personalized medicine could also contribute to EC therapy besides their employment for diagnosis and prognosis

A study on the VEGFR2-ligand multi-physics interactions in Angiogenesis.

Tumorgrowthissustainedbyangiogenesis,i.e. theformationofnewbloodvesselsfrompre-existing ones. Angiogenesis is modulated by the interaction between tyrosine kinase receptors (TKRs), expressed by endothelial cells (ECs), and extracellular ligands, produced by tumor cells. This interaction triggers the activation of intracellular signaling cascades and kinetic processes, including cell deformationandadhesion,whicheventuallycausecelldivisionandproliferation. VascularEndothelial Growth Factor Receptor-2 (VEGFR2) is a pro-angiogenic receptor expressed on ECs. Ligand stimulation induces the polarization of ECs and the relocation of VEGFR2 in cell protrusion or in the basal aspect in cells plated on ligand enriched extracellular matrix (ECM) [1]. EC response to angiogenic growth factors is regulated by distinct sets of inputs conveyed by TRKs and different co-receptors including integrins, membrane proteins that are responsible of stress fibers formation and cell contractility [2]. Although biochemical pathways following VEGFR2 activation are well established, knowledge about the receptor dynamics on the plasma membrane remains limited. A multi-physics model has been developed [3] to describe: i) the diffusion of VEGFR2 on the cellularmembrane;ii)thechemicalkineticsoftheligand-receptorbindingreaction;iii)themechanical adhesion and spreading of the cell onto a ligand-rich extracellular substrate, in finite strain. The identification of the multi-physics interactions that regulate receptor polarization could open new perspectives to develop innovative anti-angiogenic strategies through the modulation of EC activation

Multi-physics interactions drive VEGFR2 relocation on endothelial cells.

Vascular Endothelial Growth Factor Receptor-2 (VEGFR2) is a pro-angiogenic receptor, expressed on endothelial cells (ECs). Although biochemical pathways that follow the VEGFR2 activation are well established, knowledge about the dynamics of receptors on the plasma membrane remains limited. Ligand stimulation induces the polarization of ECs and the relocation of VEGFR2, either in cell protrusions or in the basal aspect in cells plated on ligand-enriched extracellular matrix (ECM). We develop a mathematical model in order to simulate the relocation of VEGFR2 on the cell membrane during the mechanical adhesion of cells onto a ligand-enriched substrate. Co-designing the in vitro experiments with the simulations allows identifying three phases of the receptor dynamics, which are controlled respectively by the high chemical reaction rate, by the mechanical deformation rate, and by the diffusion of free receptors on the membrane. The identification of the laws that regulate receptor polarization opens new perspectives toward developing innovative anti-angiogenic strategies through the modulation of EC activatio

A tool for the quantification of radial neo-vessels in chick chorioallantoic membrane angiogenic assays

Angiogenesis, the process of new blood vessels formation, plays a key role in different physiological and pathological conditions and it is considered a promising target for the development of new anti-inflammatory and anti-tumor therapies. Several assays have been developed to mimic the angiogenic process in vitro and in vivo. Here we propose a technique for the quantification of the pro-angiogenic or anti-angiogenic responses induced by different molecules when implanted in vivo on the chick embryo chorioallantoic membrane (CAM). At day 11 of development CAM is completely vascularized and neo-vessels induced by exogenous molecules converge radially to the implant. Our algorithm is an effective and rapid tool to characterize molecules endowed with proor anti-angiogenic effects by means of the quantification of the vessels present in the CAM macroscopic images. Based on conventional and dedicated image morphology tools, the proposed technique is able to discriminate radial from non-radial vessels, excluding the last ones from the count

Tumor angiogenesis revisited: Regulators and clinical implications

Since Judah Folkman hypothesized in 1971 that angiogenesis is required for solid tumor growth, numerous studies have been conducted to unravel the angiogenesis process, analyze its role in primary tumor growth, metastasis and angiogenic diseases, and to develop inhibitors of proangiogenic factors. These studies have led in 2004 to the approval of the first antiangiogenic agent (bevacizumab, a humanized antibody targeting vascular endothelial growth factor) for the treatment of patients with metastatic colorectal cancer. This approval launched great expectations for the use of antiangiogenic therapy for malignant diseases. However, these expectations have not been met and, as knowledge of blood vessel formation accumulates, many of the original paradigms no longer hold. Therefore, the regulators and clinical implications of angiogenesis need to be revisited. In this review, we discuss recently identified angiogenesis mediators and pathways, new concepts that have emerged over the past 10 years, tumor resistance and toxicity associated with the use of currently available antiangiogenic treatment and potentially new targets and/or approaches for malignant and nonmalignant neovascular diseases

β3 Integrin promotes long-lasting activation and polarization of vascular endothelial growth factor receptor 2 by immobilized ligand

OBJECTIVE: During neovessel formation, angiogenic growth factors associate with the extracellular matrix. These immobilized factors represent a persistent stimulus for the otherwise quiescent endothelial cells (ECs), driving directional EC migration and proliferation and leading to new blood vessel growth. Vascular endothelial growth factor receptor 2 (VEGFR2) is the main mediator of angiogenesis. Although VEGFR2 signaling has been deeply characterized, little is known about its subcellular localization during neovessel formation. Aim of this study was the characterization and molecular determinants of activated VEGFR2 localization in ECs during neovessel formation in response to matrix-immobilized ligand. APPROACH AND RESULTS: Here we demonstrate that ECs stimulated by extracellular matrix-associated gremlin, a noncanonical VEGFR2 ligand, are polarized and relocate the receptor in close contact with the angiogenic factor-enriched matrix both in vitro and in vivo. GM1 (monosialotetrahexosylganglioside)-positive planar lipid rafts, β3 integrin receptors, and the intracellular signaling transducers focal adhesion kinase and RhoA (Ras homolog gene family, member A) cooperate to promote VEGFR2 long-term polarization and activation. CONCLUSIONS: A ligand anchored to the extracellular matrix induces VEGFR2 polarization in ECs. Long-lasting VEGFR2 relocation is closely dependent on lipid raft integrity and activation of β3 integrin pathway. The study of the endothelial responses to immobilized growth factors may offer insights into the angiogenic process in physiological and pathological conditions, including cancer, and for a better engineering of synthetic tissue scaffolds to blend with the host vasculature

The Novel Antitubulin Agent TR-764 Strongly Reduces Tumor Vasculature and Inhibits HIF-1α Activation

Tubulin binding agents (TBAs) are commonly used in cancer therapy as antimitotics. It has been described that TBAs, like combretastatin A-4 (CA-4), present also antivascular activity and among its derivatives we identified TR-764 as a new inhibitor of tubulin polymerization, based on the 2-(alkoxycarbonyl)-3-(3',4',5'-trimethoxyanilino)benzo[b]thiophene molecular skeleton. The antiangiogenic activity of TR-764 (1-10 nM) was tested in vitro on human umbilical endothelial cells (HUVECs), and in vivo, on the chick embryo chorioallantoic membrane (CAM) and two murine tumor models. TR-764 binding to tubulin triggers cytoskeleton rearrangement without affecting cell cycle and viability. It leads to capillary tube disruption, increased cell permeability, and cell motility reduction. Moreover it disrupts adherens junctions and focal adhesions, through mechanisms involving VE-cadherin/β-catenin and FAK/Src. Importantly, TR-764 is active in hypoxic conditions significantly reducing HIF-1α. In vivo TR-764 (1-100 pmol/egg) remarkably blocks the bFGF proangiogenic activity on CAM and shows a stronger reduction of tumor mass and microvascular density both in murine syngeneic and xenograft tumor models, compared to the lead compound CA-4P. Altogether, our results indicate that TR-764 is a novel TBA with strong potential as both antivascular and antitumor molecule that could improve the common anticancer therapies, by overcoming hypoxia-induced resistance mechanisms

Usefulness of melatonin as complementary to chemotherapeutic agents at different stages of the angiogenic process

Chemotherapeutics are sometimes administered with drugs, like antiangiogenic compounds, to increase their effectiveness. Melatonin exerts antitumoral actions through antiangiogenic actions. We studied if melatonin regulates the response of HUVECs to chemotherapeutics (docetaxel and vinorelbine). The inhibition that these agents exert on some of the processes involved in angiogenesis, such as, cell proliferation, migratory capacity or vessel formation, was enhanced by melatonin. Regarding to estrogen biosynthesis, melatonin impeded the negative effect of vinorelbine, by decreasing the activity and expression of aromatase and sulfatase. Docetaxel and vinorelbine increased the expression of VEGF-A, VEGF-B, VEGF-C, VEGFR-1, VEGFR-3, ANG1 and/or ANG-2 and melatonin inhibited these actions. Besides, melatonin prevented the positive actions that docetaxel exerts on the expression of other factors related to angiogenesis like JAG1, ANPEP, IGF-1, CXCL6, AKT1, ERK1, ERK2, MMP14 and NOS3 and neutralized the stimulating actions of vinorelbine on the expression of FIGF, FGFR3, CXCL6, CCL2, ERK1, ERK2, AKT1, NOS3 and MMP14. In CAM assay melatonin inhibited new vascularization in combination with chemotherapeutics. Melatonin further enhanced the chemotherapeutics-induced inhibition of p-AKT and p-ERK and neutralized the chemotherapeuticscaused stimulatory effect on HUVECs permeability by modifying the distribution of VE cadherin. Our results confirm that melatonin blocks proangiogenic and potentiates antiangiogenic effects induced by docetaxel and vinorelbine enhancing their antitumor effectiveness.This work was supported by grants from the Spanish Economy and Competitiveness Ministry (SAF2016-77103-P) and from Instituto de Investigación Sanitaria Valdecilla (IDIVAL) (APG/12)

Preliminary observations on scleral ossicles in performing functionalized 3D vascularized scaffolds for “critical-size” bone defect healing

The problem of “critical-size” bone defects occurs when a severe lesion is difficult to be self-recovered. Many strategies of regenerative medicine were used in the last decade, with translational approaches, to mimic both structure and function of the native bone tissue, making use of synthetic materials, nanotechnologies, bio/synthetic constructs or some of their combination. The main obstacle to engineering strategies is mostly due to the lack of a proper vascularization of the construct used. In this feasibility study, our attention is directed towards the main tissue engineering items: scaffolds, cells and conditioning factors. We propose the use of scleral ossicles of lower vertebrates (1), as natural scaffolds which will be functionalized to allow the best adhesion of endothelial cells along a geometrically controlled pattern on the bony surface of the construct; successively, on the functionalized scaffold, osteogenic cell lines will be cultured. In the preliminary phases of the study, the ossicles were scratched to remove soft tissue residues, variously flattened with different methods to reach a regular morphology on both sides, and finally autoclaved to eliminate cellular remnants and to annul antigenic properties. Ossicles were observed under SEM and subjected to micro-assay, to establish the best scaffold preparation and to characterize morphological properties more suitable for engineering phases. Functionalization will be made by immobilizing on the engineered ossicles specific growth factors for endothelial cells; later, mouse primary lung endothelial cells (ECs) and immortalized osteogenic cells (IDG-SW3) will be used. As expected results ECs should adhere to the ossicle surface and organize to form lumenized microvascular-like structures; later, supported by the vascular-like network, the osteogenic lineage should produce bone matrix on the construct. The production of newly-formed bone around vascular-like buds will be verify. 3D tissue constructs generated in vitro will be used in successive in vivo study for the healing of “critical-size” bone defects experimentally induced in mice