Synthesis, antiangiogenesis evaluation and molecular docking studies of 1-Aryl-3-[(thieno[3,2-b]pyridin-7-ylthio)phenyl]ureas: Discovery of a new substitution pattern for type II VEGFR-2 Tyr kinase inhibitors

The synthesis and biological evaluation of novel 1-aryl-3-[2-, 3- or 4-(thieno[3,2-b]pyridin-7-ylthio)phenyl]ureas 3, 4 and 5 as VEGFR-2 tyrosine kinase inhibitors, are reported. The 1-aryl-3-[3-(thieno[3,2-b]pyridin-7-ylthio)phenyl]ureas 4a-4h, with the arylurea in the meta position to the thioether, showed the lowest IC50 values in enzymatic assays (10-206 nM), the most potent compounds 4d-4h (IC50 10-28 nM) bearing hydrophobic groups (Me, F, CF3 and Cl) in the terminal phenyl ring. A convincing rationalization was achieved for the highest potent compounds 4 as type II VEGFR-2 inhibitors, based on the simultaneous presence of: (1) the thioether linker and (2) the arylurea moiety in the meta position. For compounds 4, significant inhibition of Human Umbilical Vein Endothelial Cells (HUVECs) proliferation (BrdU assay), migration (wound-healing assay) and tube formation were observed at low concentrations. These compounds have also shown to increase apoptosis using the TUNEL assay. Immunostaining for total and phosphorylated (active) VEGFR-2 was performed by Western blotting. The phosphorylation of the receptor was significantly inhibited at 1.0 and 2.5 microM for the most promising compounds. Altogether, these findings point to an antiangiogenic effect in HUVECs.

Gellan gum-hyaluronate spongy-like hydrogels promote angiogenesis in hindlimb ischemia

"Tissue engineering: part A", vol. 21, suppl. 1 (2015)

Closed hybrid hierarchical reservoirs based on the deconstruction of the cellular native microenvironment

The stem cell niche organization and dynamics provide valuable cues for the development of mimetic environments that could have potential to stimulate the regenerative process. We propose the use of biodegradable biomaterials to produce closed miniaturised structures able to encapsulate different cell types or bioactive molecules. In particular, capsules are fabricated using the so-called layer-by-layer technology, where the consecutive (nano-sized) layers are well stabilized by electrostatic interactions or other weak forces. Using alginate-based spherical templates containing cells or other elements (e.g. proteins, magnetic nanoparticles, microparticles) it is possible to produce liquefied capsules that may entrap the entire cargo under mild conditions. The inclusion of liquefied micropcapsules may be used to produce hierarchical compartmentalised systems for the delivery of bioactive agents. The presence of solid microparticles inside such capsules offers adequate surface area for adherent cell attachment increasing the biological performance of these hierarchical systems, while maintain both permeability and injectability. We demonstrated that the encapsulation of distinct cell types (including mesenchymal stem cells and endothelial cells) enhances the osteogenic capability of this system, that could be useful in bone tissue engineering applications.

Vascular Response of Ruthenium Tetraamines in Aortic Ring from Normotensive Rats

Background: Ruthenium (Ru) tetraamines are being increasingly used as nitric oxide (NO) carriers. In this context, pharmacological studies have become highly relevant to better understand the mechanism of action involved. Objective: To evaluate the vascular response of the tetraamines trans-[RuII(NH3)4(Py)(NO)]3+, trans-[RuII(Cl)(NO) (cyclan)](PF6)2, and trans-[RuII(NH3)4(4-acPy)(NO)]3+. Methods: Aortic rings were contracted with noradrenaline (10−6 M). After voltage stabilization, a single concentration (10−6 M) of the compounds was added to the assay medium. The responses were recorded during 120 min. Vascular integrity was assessed functionally using acetylcholine at 10−6 M and sodium nitroprusside at 10−6 M as well as by histological examination. Results: Histological analysis confirmed the presence or absence of endothelial cells in those tissues. All tetraamine complexes altered the contractile response induced by norepinephrine, resulting in increased tone followed by relaxation. In rings with endothelium, the inhibition of endothelial NO caused a reduction of the contractile effect caused by pyridine NO. No significant responses were observed in rings with endothelium after treatment with cyclan NO. In contrast, in rings without endothelium, the inhibition of guanylate cyclase significantly reduced the contractile response caused by the pyridine NO and cyclan NO complexes, and both complexes caused a relaxing effect. Conclusion: The results indicate that the vascular effect of the evaluated complexes involved a decrease in the vascular tone induced by norepinephrine (10−6 M) at the end of the incubation period in aortic rings with and without endothelium, indicating the slow release of NO from these complexes and suggesting that the ligands promoted chemical stability to the molecule. Moreover, we demonstrated that the association of Ru with NO is more stable when the ligands pyridine and cyclan are used in the formulation of the compound.

Lesões microscopicas do baço na purpura trombocitopenica idiopatica

Histopathological changes strikingly similar were found in the spleen of four cases (young female subjects) of idiopathic thrombocytopenic purpura hemorrhagica in which splenectomy was performed. The chief changes reported are enlargement of the marginal zone of the malpighian corpuscles, proliferation and mobilization of the reticulo-endothelial cells, myeloid metaplasia, local (tissue) eosinophilia, and stoppage of the circulation or stasis of platelets from which results a filling of the spelenic sinuses by such elements. The latter phenomenon will possibly present some bearing with thrombocytopenia which is such a characteristic feature in this disease and will perhaps account for the rapid increase in blood platelets which usually follows splenectomy and or the finding of increased megakaryocytes in the bone marrow.

Liprin (beta)1 is highly expressed in lymphatic vasculature and is important for lymphatic vessel integrity.

The lymphatic vasculature is important for the regulation of tissue fluid homeostasis, immune response, and lipid absorption, and the development of in vitro models should allow for a better understanding of the mechanisms regulating lymphatic vascular growth, repair, and function. Here we report isolation and characterization of lymphatic endothelial cells from human intestine and show that intestinal lymphatic endothelial cells have a related but distinct gene expression profile from human dermal lymphatic endothelial cells. Furthermore, we identify liprin beta1, a member of the family of LAR transmembrane tyrosine phosphatase-interacting proteins, as highly expressed in intestinal lymphatic endothelial cells in vitro and lymphatic vasculature in vivo, and show that it plays an important role in the maintenance of lymphatic vessel integrity in Xenopus tadpoles.

Pkd1 Regulates Lymphatic Vascular Morphogenesis during Development.

Lymphatic vessels arise during development through sprouting of precursor cells from veins, which is regulated by known signaling and transcriptional mechanisms. The ongoing elaboration of vessels to form a network is less well understood. This involves cell polarization, coordinated migration, adhesion, mixing, regression, and shape rearrangements. We identified a zebrafish mutant, lymphatic and cardiac defects 1 (lyc1), with reduced lymphatic vessel development. A mutation in polycystic kidney disease 1a was responsible for the phenotype. PKD1 is the most frequently mutated gene in autosomal dominant polycystic kidney disease (ADPKD). Initial lymphatic precursor sprouting is normal in lyc1 mutants, but ongoing migration fails. Loss of Pkd1 in mice has no effect on precursor sprouting but leads to failed morphogenesis of the subcutaneous lymphatic network. Individual lymphatic endothelial cells display defective polarity, elongation, and adherens junctions. This work identifies a highly selective and unexpected role for Pkd1 in lymphatic vessel morphogenesis during development.

VEGF and TNF up-regulate, NSAID down-regulate SOX18 protein level in HUVEC

Angiogenesis, the process of generating new blood vessels, is essential to embryonic development, organ formation, tissue regeneration and remodeling, reproduction and wound healing. Also, it plays an important role in many pathological conditions, including chronic inflammation and cancer. Angiogenesis is regulated by a complex interplay of growth factors, inflammatory mediators, adhesion molecules, morphogens and guidance molecules. Transcription factor SOX18 is transiently expressed in nascent endothelial cells during embryonic development and postnatal angiogenesis, but little is known about signaling pathways controlling its expression. The aim of this study was to investigate whether pro-angiogenic molecules and pharmacological inhibitors of angiogenesis modulate SOX18 expression in endothelial cells. Therefore, we treated human umbilical vein endothelial cells (HUVEC) with angiogenic factors, extracellular matrix proteins, inflammatory cytokines and nonsteroidal anti-inflammatory drugs (NSAID) and monitored SOX18 expression. We have observed that the angiogenic factor VEGF and the inflammatory cytokine TNF increase, while the NSAID ibuprofen and NS398 decrease the SOX18 protein level. These results for the first time demonstrate that SOX18 expression is modulated by factors and drugs known to positively or negatively regulate angiogenesis. This opens the possibility of pharmacological manipulation of SOX18 gene expression in endothelial cells to stimulate or inhibit angiogenesis.

A study on the pathogenesis of human cerebral malaria and cerebral babesiosis

Cerebral complications are important, but poorly understood pathological features of infections caused by some species of Plasmodium and Babesia. Patients dying from P. falciparum were classified as cerebral or non-cerebral cases according to the cerebral malaria coma scale. Light microscopy revealed that cerebral microvessels of cerebral malaria patients were field with a mixture of parazited and unparazited erythrocytes, with 94% of the vessels showing parasitized red blood cell (PRBC) sequestration. Some degree of PRBC sequestration was also found in non-cerebral malaria patients, but the percentage of microvessls with sequestered PRBC was only 13% Electron microscopy demonstrated knobs on the membrane of PRBC that formed focal junctions with the capillary endothelium. A number of host cell molecules such as CD36, thrombospondim (TSP) and intracellular adhesion molecule I (ICAM-1) may function as endothelial cell surfacereports for P. falciparum-infected erythrocytes. Affinity labeling of CD36 and TSP to the PRBC surface showed these molecules specifically bind to the knobs. Babesia bovis infected erythrocytes procedure projections of the erythrocyte membrane that are similar to knobs. When brain tissue from B. bovis-infected cattle was examined, cerebral capillaries were packed with PRBC. Infected erythrocytes formed focal attachments with cerebral endothelial cells at the site of these knob-like projections. These findings indicate that cerebral pathology caused by B. bovis is similar to human cerebral malaria. A search for cytoadherence proteins in the endothelial cells may lead to a better understanding of the pathogenisis of cerebral babesiosis.

Optimization and inhibition of the adherent ability of Plasmodium falciparum-infected erythrocytes

The vast majority of the 1-2 million malaria associated deaths that occur each year are due to anemia and cerebral malaria (the attachment of erythrocytes containing mature forms of Plasmodium falciparum to the endothelial cells that line the vascular beds of the brain). A "model" system"for the study of cerebral malaria employs amelanotic melanoma cells as the "target"cells in an vitro cytoadherence assay. Using this model system we determined that the optimum pH for adherence is 6.6 to 6.8, that high concentrations of Ca²* (50mM) result in increased levels of binding, and that the type of buffer used influences adherence (Bis Tris > MOPS > HEPES > PIPES). We also observed that the ability of infected erythrocytes to cytoadhere varied from (erythrocyte) donor to donor. We have produced murine monoclonal antibodies against P. falciparum-infected red cells which recognized modified forms of human band 3; these inhibit the adherence of infected erythrocytes to melanoma cells in a doso responsive fashion. Antimalarials (chloroquine, quinacrine, mefloquine, artemisinin), on the other hand, affected adherence in an indirect fashion i.e. since cytoadherence is due, in part to the presence of knobs on the surface of the infected erythrocyte, and knob formation is dependent on intracellular parasite growth, when plasmodial development is inhibited so is knob production, and consequently adherence is ablated.

Variation in the cytoadherence characteristics of malaria parasites: is this a true virulence factor?

The sequestration of Plasmodium falciparum-infected erythrocytes to the endothelial cells of brain capillaries is believed to represent one of the determining factors in the pathogenesis of cerebral malaria. In vitro studies of cytoadherence provide an experimental approach to understand the mechanism of sequestration and the respective roles played by parasite and host components in this interaction. This paper critically reviews current studies on cytoadherence, with particular emphasis on the nature of the information provided by such studies and their limitations. The paper also describes how cytoadherence studies using the patient's own monocytes can provide original information on the level of receptor up-regulation in the course of malarial infection.

Plasmodium coatneyi-infected rhesus monkeys: a primate modelfor human cerebral malaria

Although several animal models for human cerebral malaria have been proposed in the past, name have shown pathological findings that are similar to those seen in humans. In order to develop an animal model for human cerebral malaria, we studied the pathology of brains of Plasmodium coatneyi (primate malaria parasite)-infected rhesus monkeys. Our study demonstrated parazitized erythrocyte (PRBC) sequestration and cytoadherence of knobs on PRBC to endothelial cells in cerebral microvessels of these monkeys. This similar to the findings een in human cerebral malaria. Crebral microvessels with sequestred PRBC were shown by immunohistochemistry to possess CD36, TSP and ICAM-1. These proteins were not evident in cerebral microvessels of uninfected control monkeys. Our study indicates, for the first time, that rhesus monkeys infected with P. coatneyi can be used as a primate model to study human cerebral malaria.

Virulence and the immune response in malaria

Many factors determine the virulence of a malaria infection. These include host innate resistance mechanisms and, with Plasmodium falciparum, the ability to cytoadhere to endothelial cells, form rosetts, and induce release of cytokines. The effect on virulence of acquired immune responses can be determined by Class I and Class II MHC-antigens; levels of immunological responsiveness may be determined too in other ways. The structure of parasite surface antigens and their great diversity modulate the immune response and influence parasite survival and hence virulence, and transmission to the vector.

FOXC2 controls formation and maturation of lymphatic collecting vessels through cooperation with NFATc1.

The mechanisms of blood vessel maturation into distinct parts of the blood vasculature such as arteries, veins, and capillaries have been the subject of intense investigation over recent years. In contrast, our knowledge of lymphatic vessel maturation is still fragmentary. In this study, we provide a molecular and morphological characterization of the major steps in the maturation of the primary lymphatic capillary plexus into collecting lymphatic vessels during development and show that forkhead transcription factor Foxc2 controls this process. We further identify transcription factor NFATc1 as a novel regulator of lymphatic development and describe a previously unsuspected link between NFATc1 and Foxc2 in the regulation of lymphatic maturation. We also provide a genome-wide map of FOXC2-binding sites in lymphatic endothelial cells, identify a novel consensus FOXC2 sequence, and show that NFATc1 physically interacts with FOXC2-binding enhancers. As damage to collecting vessels is a major cause of lymphatic dysfunction in humans, our results suggest that FOXC2 and NFATc1 are potential targets for therapeutic intervention.

Propietats protectores de la lipoproteïna de alta densitat (HDL) sobre l'acció inflamatòria de la lipoproteïna de baixa densitat electronegativa (LDL(-)) en monòcits humans

Electronegative low-density lipoprotein (LDL(-)) is a modified fraction of LDL present in peripheral blood whose proportion is elevated in subjects with increased cardiovascular risk. LDL(-) has been shown to have an inflammatory effect on human endothelial cells and mononuclear blood cells. On the other hand, high-density lipoprotein (HDL) is known to have a protective effect against cardiovascular disease, partly mediated by its anti-inflammatory properties. The objective of the current work is to study the putative protective properties of HDL towards the inflammatory effect of LDL(-) in human monocytes, in order to elucidate the mechanisms behind their interaction. Total LDL and HDL were isolated by ultracentrifugation and LDL(-) was obtained from total LDL by anion exchange chromatography. HDL and LDL(-) were incubated together and then re-isolated, and their characteristics were compared to those of untreated lipoproteins. The inflammatory activity of the lipoproteins was determined by incubating monocytes with lipoproteins and measuring cytokine release from the cultured monocytes. The biochemical composition and electrophoretic mobility of the lipoproteins were also determined before and after their interaction. Incubation of HDL with LDL(-) reduced the inflammatory effect of LDL(-) and, in turn, HDL gained inflammatory properties. This indicates a transfer of inflammatory potential taking place during the interaction of LDL(-) and HDL. Additionally, LDL(-) lost non-esterified fatty acids (NEFAs) while HDL gained the same. We conclude that a transfer of NEFAs takes place between LDL(-) and HDL. These observations suggest that NEFAs play a role in the inflammatory effect mediated by LDL(-).