Antiangiogenic therapy of transgenic mice impairs de novo tumor growth.

Angiogenesis is activated during multistage tumorigenesis prior to the emergence of solid tumors. Using a transgenic mouse model, we have tested the proposition that treatment with angiogenesis inhibitors can inhibit the progression of tumorigenesis after the switch to the angiogenic phenotype. In this model, islet cell carcinomas develop from multifocal, hyperproliferative nodules that show the histological hallmarks of human carcinoma in situ. Mice were treated with a combination of the angiogenesis inhibitor AGM-1470 (TNP-470), the antibiotic minocycline, and interferon alpha/beta. The treatment regimen markedly attenuated tumor growth but did not prevent tumor formation; tumor volume was reduced to 11% and capillary density to 40% of controls. The proliferation index of tumor cells in treated and control mice was similar, whereas the apoptotic index was doubled in treated tumors. This study shows that de novo tumor progression can be restricted solely by antiangiogenic therapy. The results suggest that angiogenesis inhibitors represent a valid component of anticancer strategies aimed at progression from discrete stages of tumorigenesis and demonstrate that transgenic mouse models can be used to evaluate efficacy of candidate antiangiogenic agents.

Sequence-selective carbohydrate-DNA interaction: dimeric and monomeric forms of the calicheamicin oligosaccharide interfere with transcription factor function.

The synthetic oligosaccharide moiety of the antibiotic calicheamicin and the head-to-head dimer of this oligosaccharide are known to bind to the minor groove of DNA in a sequence-selective manner preferring distinct target sequences. We tested these carbohydrates for their ability to interfere with transcription factor function. The oligosaccharides inhibit binding of transcription factors to DNA in a sequence-selective manner, probably by inducing a conformational change in DNA structure. They also interfere with transcription by polymerase II in vitro. The effective concentrations of the oligosaccharides for inhibition of transcription factor binding and for transcriptional inhibition are in the micromolar range. The dimer is a significantly more active inhibitor than is the monomer.

Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric proteins.

The majority of severe visual loss in the United States results from complications associated with retinal neovascularization in patients with ischemic ocular diseases such as diabetic retinopathy, retinal vein occlusion, and retinopathy of prematurity. Intraocular expression of the angiogenic protein vascular endothelial growth factor (VEGF) is closely correlated with neovascularization in these human disorders and with ischemia-induced retinal neovascularization in mice. In this study, we evaluated whether in vivo inhibition of VEGF action could suppress retinal neovascularization in a murine model of ischemic retinopathy. VEGF-neutralizing chimeric proteins were constructed by joining the extracellular domain of either human (Flt) or mouse (Flk) high-affinity VEGF receptors with IgG. Control chimeric proteins that did not bind VEGF were also used. VEGF-receptor chimeric proteins eliminated in vitro retinal endothelial cell growth stimulation by either VEGF (P < 0.006) or hypoxic conditioned medium (P < 0.005) without affecting growth under nonstimulated conditions. Control proteins had no effect. To assess in vivo response, animals with bilateral retinal ischemia received intravitreal injections of VEGF antagonist in one eye and control protein in the contralateral eye. Retinal neovascularization was quantitated histologically by a masked protocol. Retinal neovascularization in the eye injected with human Flt or murine Flk chimeric protein was reduced in 100% (25/25; P < 0.0001) and 95% (21/22; P < 0.0001) 0.0001) of animals, respectively, compared to the control treated eye. This response was evident after only a single intravitreal injection and was dose dependent with suppression of neovascularization noted after total delivery of 200 ng of protein (P < 0.002). Reduction of histologically evident neovascular nuclei per 6-microns section averaged 47% +/- 4% (P < 0.001) and 37% +/- 2% (P < 0.001) for Flt and Flk chimeric proteins with maximal inhibitory effects of 77% and 66%, respectively. No retinal toxicity was observed by light microscopy. These data demonstrate VEGF's causal role in retinal angiogenesis and prove the potential of VEGF inhibition as a specific therapy for ischemic retinal disease.

Suppression of lung metastasis of B16 mouse melanoma by N-acetylglucosaminyltransferase III gene transfection.

The beta 1-6 structure of N-linked oligosaccharides, formed by beta-1,6-N-acetylglucosaminyltransferase (GnT-V), is associated with metastatic potential. We established a highly metastatic subclone, B16-hm, from low metastatic B16-F1 murine melanoma cells. The gene encoding beta-1,4-N-acetylglucosaminyltransferase (GnT-III) was introduced into the B16-hm cells, and three clones that stably expressed high GnT-III activity were obtained. In these transfectants, the affinity to leukoagglutinating phytohemagglutinin was reduced, whereas the binding to erythroagglutinating phytohemagglutinin was increased, indicating that the level of beta 1-6 structure was decreased due to competition for substrate between intrinsic GnT-V and ectopically expressed GnT-III. Lung metastasis after intravenous injection of the transfectants into syngeneic and nude mice was significantly suppressed, suggesting that the decrease in beta 1-6 structure suppressed metastasis via a mechanism independent of the murine system. These transfectants also displayed decreased invasiveness into Matrigel and inhibited cell attachment to collagen and laminin. Cell growth was not affected. Our results demonstrate a causative role for beta 1-6 branches in invasion and cell attachment in the extravasation stage of metastasis.

Cotranslational folding and calnexin binding during glycoprotein synthesis.

To analyze cotranslational folding of influenza hemagglutinin in the endoplasmic reticulum of live cells, we used short pulses of radiolabeling followed by immunoprecipitation and analysis with a two-dimensional SDS/polyacrylamide gel system which was nonreducing in the first dimension and reducing in the second. It separated nascent glycopolypeptides of different length and oxidation state. Evidence was obtained for cotranslational disulfide formation, generation of conformational epitopes, N-linked glycosylation, and oligosaccharide-dependent binding of calnexin, a membrane-bound chaperone that binds to incompletely folded glycoproteins via partially glucose-trimmed oligosaccharides. When glycosylation or oligosaccharide trimming was inhibited, the folding pathway was perturbed, suggesting a role for N-linked oligosaccharides and calnexin during translation of hemagglutinin.

Activation of mitogen-activated protein kinases by vascular endothelial growth factor and basic fibroblast growth factor in capillary endothelial cells is inhibited by the antiangiogenic factor 16-kDa N-terminal fragment of prolactin.

A number of factors both stimulating and inhibiting angiogenesis have been described. In the current work, we demonstrate that the angiogenic factor vascular endothelial growth factor (VEGF) activates mitogen-activated protein kinase (MAPK) as has been previously shown for basic fibroblast growth factor. The antiagiogenic factor 16-kDa N-terminal fragment of human prolactin inhibits activation of MAPK distal to autophosphorylation of the putative VEGF receptor, Flk-1, and phospholipase C-gamma. These data show that activation and inhibition of MAPK may play a central role in the control of angiogenesis.

T lymphocytes that infiltrate tumors and atherosclerotic plaques produce heparin-binding epidermal growth factor-like growth factor and basic fibroblast growth factor: a potential pathologic role.

Despite significant infiltration into tumors and atherosclerotic plaques, the role of T lymphocytes in these pathological conditions is still unclear. We have demonstrated that tumor-infiltrating lymphocytes (TILs) and plaque-infiltrating lymphocytes (PILs) produce heparin-binding epidermal growth factor-like growth factor (HB-EGF) and basic fibroblast growth factor (bFGF) in vitro under nonspecific conditions and in vivo in tumors by immunohistochemical staining. HB-EGF and bFGF derived from TILs and PILs directly stimulated tumor cells and vascular smooth muscle cells (SMCs) in vitro, respectively, while bFGF displayed angiogenic properties. Therefore, T cells may play a critical role in the SMC hyperplasia of atherosclerosis and support tumor progression by direct stimulation and angiogenesis.

Platelet-derived growth factor BB induces functional vascular anastomoses in vivo.

Neovascularization that generates collateral blood flow can limit the extent of tissue damage after acute ischemia caused by occlusion of the primary blood supply. The neovascular response stimulated by the BB homodimeric form of recombinant platelet-derived growth factor (PDGF-BB) was evaluated for its capacity to protect tissue from necrosis in a rat skin flap model of acutely induced ischemia. Complete survival of the tissue ensued, when the original nutritive blood supply was occluded, as early as 5 days after local PDGF-BB application, and the presence of a patent vasculature was evident compared to control flaps. To further evaluate the vascular regenerative response, PDGF-BB was injected into the muscle/connective tissue bed between the separated ends of a divided femoral artery in rats. A patent new vessel that functionally reconnected the ends of the divided artery within the original 3- to 4-mm gap was regenerated 3 weeks later in all PDGF-BB-treated limbs. In contrast, none of the paired control limbs, which received vehicle with an inactive variant of PDGF-BB, had vessel regrowth (P < 0.001). The absence of a sustained inflammatory response and granulation tissue suggests locally delivered PDGF-BB may directly stimulate the angiogenic phenotype in endothelial cells. These findings indicate that PDGF-BB can generate functional new blood vessels and nonsurgically anastomose severed vessels in vivo. This study supports the possibility of a therapeutic modality for the salvage of ischemic tissue through exogenous cytokine-induced vascular reconnection.

Oligogalacturonides and chitosan activate plant defensive genes through the octadecanoid pathway.

Jasmonic acid, synthesized from linolenic acid (the octadecanoid pathway), has been proposed to be part of a signal transduction pathway that mediates the induction of defensive genes in plants in response to oligouronide and polypeptide signals generated by insect and pathogen attacks. We report here that the induction of proteinase inhibitor accumulation in tomato leaves by plant-derived oligogalacturonides and fungal-derived chitosan oligosaccharides is severely reduced by two inhibitors (salicylic acid and diethyldi-thiocarbamic acid) of the octadecanoid pathway, supporting a role for the pathway in signaling by oligosaccharides. Jasmonic acid levels in leaves of tomato plants increased several fold within 2 hr after supplying the polypeptide systemin, oligogalacturonides, or chitosan to the plants through their cut stems, as expected if they utilize the octadecanoid pathway. The time course of jasmonic acid accumulation in tomato leaves in response to wounding was consistent with its proposed role in signaling proteinase inhibitor mRNA and protein synthesis. The cumulative evidence supports a model for the activation of defensive genes in plants in response to insect and pathogen attacks in which various elicitors generated at the attack sites activate the octadecanoid pathway via different recognition events to induce the expression of defensive genes in local and distal tissues of the plants.

Regulation of CD45 engagement by the B-cell receptor CD22.

The B-cell receptor CD22 binds sialic acid linked alpha-2-6 to terminal galactose residues on N-linked oligosaccharides associated with several cell-surface glycoproteins. The first of these sialoglycoproteins to be identified was the receptor-linked phosphotyrosine phosphatase CD45, which is required for antigen/CD3-induced T-cell activation. In the present work, we examine the effect of interaction between the extracellular domain of CD45 and CD22 on T-cell activation. Using soluble CD22-immunoglobulin fusion proteins and T cells expressing wild-type and chimeric CD45 forms, we show that engagement of CD45 by soluble CD22 can modulate early T-cell signals in antigen receptor/CD3-mediated stimulation. We also show that addition of sialic acid by beta-galactoside alpha-2,6-sialyltransferase to the CD22 molecule abrogates interactions between CD22 and its ligands. Together, these observations provide direct evidence for a functional role of the interaction between the extracellular domain of CD45 and a natural ligand and suggest another regulatory mechanism for CD22-mediated ligand engagement.

Disaccharide uptake and priming in animal cells: inhibition of sialyl Lewis X by acetylated Gal beta 1-->4GlcNAc beta-O-naphthalenemethanol.

Inhibitors of glycosylation provide a tool for studying the biology of glycoconjugates. One class of inhibitors consists of glycosides that block glycoconjugate synthesis by acting as primers of free oligosaccharide chains. A typical primer contains one sugar linked to a hydrophobic aglycone. In this report, we describe a way to use disaccharides as primers. Chinese hamster ovary cells readily take up glycosides containing a pentose linked to naphthol, but they take up hexosides less efficiently and disaccharides not at all. Linking phenanthrol to a hexose improves its uptake dramatically but has no effect on disaccharides. To circumvent this problem, analogs of Xyl beta 1-->6Gal beta-O-2-naphthol were tested as primers of glycosaminoglycan chains. The unmodified disaccharide did not prime, but methylated derivatives had activity in the order Xyl beta 1-->6Gal(Me)3-beta-O-2-naphthol > Xyl beta 1-->6Gal (Me)2 beta-O-2-naphthol >> Xyl beta 1-->6Gal(Me)beta-O-2-naphthol. Acetylated Xyl beta 1-->6Gal beta-O-2-naphthol also primed glycosaminoglycans efficiently, suggesting that the terminal xylose residue was exposed by removing the acetyl groups. The general utility of using acetyl groups to create disaccharide primers was shown by the priming of oligosaccharides on peracetylated Gal beta 1-->4GlcNAc beta-O-naphthalenemethanol. This disaccharide inhibited sialyl Lewis X expression on HL-60 cells.

Comparison of the defensive elicitation activity of cerato-populin and cerato-platanin: a structural model

Plants can defend themselves from potential pathogenic microorganisms relying on a complex interplay of signaling pathways: activation of the MAPK cascade, transcription of defense related genes, production of reactive oxygen species, nitric oxide and synthesis of other defensive compounds such as phytoalexins. These events are triggered by the recognition of pathogen’s effectors (effector-triggered immunity) or PAMPs (PAMP-triggered immunity). The Cerato Platanin Family (CPF) members are Cys-rich proteins secreted and localized on fungal cell walls, involved in several aspects of fungal development and pathogen-host interactions. Although more than hundred genes of the CPF have been identified and analyzed, the structural and functional characterization of the expressed proteins has been restricted only to few members of the family. Interestingly, those proteins have been shown to bind chitin with diverse affinity and after foliar treatment they elicit defensive mechanisms in host and non-host plants. This property turns cerato platanins into interesting candidates, worth to be studied to develop new fungal elicitors with applications in sustainable agriculture. This study focus on cerato-platanin (CP), core member of the family and on the orthologous cerato-populin (Pop1). The latter shows an identity of 62% and an overall homology of 73% with respect to CP. Both proteins are able to induce MAPKs phosphorylation, production of reactive oxygen species and nitric oxide, overexpression of defense’s related genes, programmed cell death and synthesis of phytoalexins. CP, however, when compared to Pop1, induces a faster response and, in some cases, a stronger activity on plane leaves. Aim of the present research is to verify if the dissimilarities observed in the defense elicitation activity of these proteins can be associated to their structural and dynamic features. Taking advantage of the available CP NMR structure, Pop1’s 3D one was obtained by homology modeling. Experimental residual dipolar couplings and 1H, 15N, 13C resonance assignments were used to validate the model. Previous works on CPF members, addressed the highly conserved random coil regions (loops b1-b2 and b2-b3) as sufficient and necessary to induce necrosis in plants’ leaves: that region was investigated in both Pop1 and CP. In the two proteins the loops differ, in their primary sequence, for few mutations and an insertion with a consequent diversification of the proteins’ electrostatic surface. A set of 2D and 3D NMR experiments was performed to characterize both the spatial arrangement and the dynamic features of the loops. NOE data revealed a more extended network of interactions between the loops in Pop1 than in CP. In addition, in Pop1 we identified a salt bridge Lys25/Asp52 and a strong hydrophobic interaction between Phe26/Trp53. These structural features were expected not only to affect the loops’ spatial arrangement, but also to reduce the degree of their conformational freedom. Relaxation data and the order parameter S2 indeed highlighted reduced flexibility, in particular for loop b1-b2 of Pop1. In vitro NMR experiments, where Pop1 and CP were titrated with oligosaccharides, supported the hypothesis that the loops structural and dynamic differences may be responsible for the different chitin-binding properties of the two proteins: CP selectively binds tetramers of chitin in a shallow groove on one side of the barrel defined by loops b1-b2, b2-b3 and b4-b5, Pop1, instead, interacts in a non-specific fashion with oligosaccharides. Because the region involved in chitin-binding is also responsible for the defense elicitation activity, possibly being recognized by plant's receptors, it is reasonable to expect that those structural and dynamic modifications may also justify the different extent of defense elicitation. To test that hypothesis, the initial steps of a protocol aimed to the identify a receptor for CP, in silico, are presented.

Microenvironment and microvascular wall modulate cancer cell progression

Tumour progression is a complex process that frequently brings to cancer metastasis, the first cause of poor prognosis of cancer affected patients. Metastasis are generated by cells escaped from a primary mass and able to enter in the circulation, survive and proliferate in a new, distant site of the organism. To reach all these goal, many different phenomena had occur within both the cancer cells and the surrounding microenvironment. In the first part of this thesis, the focus was pointed on the metastatic potential of a leiomyosarcoma cell model. The studied cancer cells demonstrated a strong invasive capacity of the ECM in vitro, principally by production of matrix metalloproteinases 2 and 9, and robust pro-angiogenic activity in the chick CAM model, that facilitate its dissemination through same chick embryo internal organs. This study, with the title “MMPs and angiogenesis affect the metastatic potential of a human vulvar leiomyosarcoma cell line”, is presented in the published form. In the second part of this work, the emphasis was given to the microvascular element of the tumour microenvironment and specifically to the perivascular pericytes. These are intriguing cells due to their uncertain involvement in the biology of cancer. It is not clear how pericytes change within the tumour microenvironment and which is their contribute during the tumour dissemination. After the characterization of the chosen pericytic cell model, an in vitro study of the interaction between pericytes and different cancer cell lines where performed. Indirect and direct cell-cell interaction as well as movement of cancer cells in presence of pericytes conditioned media was analysed, in order to investigate the reciprocal influence of pericytes and tumour cells in the context of cancer progression.

Well posedness of an integrodifferential kinetic model of Fokker-Planck type for angiogenesis.

Tumor induced angiogenesis processes including the effect of stochastic motion and branching of blood vessels can be described coupling a (nonlocal in time) integrodifferential kinetic equation of Fokker–Planck type with a diffusion equation for the tumor induced ingiogenic factor. The chemotactic force field depends on the flux of blood vessels through the angiogenic factor. We develop an existence and uniqueness theory for this system under natural assumptions on the initial data. The proof combines the construction of fundamental solutions for associated linearized problems with comparison principles, sharp estimates of the velocity integrals and compactness results for this type of kinetic and parabolic operators

Expressão gênica de metaloproteinases e de seus reguladores em neoplasias mieloproliferativas: associação com biomarcadores de angiogênese e status mutacional

As neoplasias mieloproliferativas (NMPs) BCR-ABL1 negativas compreendem a mielofibrose primária (PMF), trombocitemia essencial (TE) e a policitemia vera (PV). A patogênese e progressão dessas NMPs não estão completamente elucidadas. As metaloproteinases de matriz (MMPs) degradam a matriz extracelular, ativando citocinas e fatores de crescimento que, por sua vez, participam da tumorigênese e angiogênese. O objetivo deste estudo foi avaliar a relação da expressão gênica das MMPs, TIMPs, HIF1-α e SPARC com os marcadores angiogênicos bFGF e VEGFA em pacientes com MF e TE, considerando o status mutacional; bem como avaliar a regulação desses genes em camundongos submetidos à hipóxia, e em modelos HIF1-α(-/-) e VHL(-/-). Foram incluídos 21 pacientes com MF, 21 com MF pós-TE, 6 com MF pós-PV, 23 com TE e 78 indivíduos controle. As análises realizadas foram: dosagem sérica e expressão de RNAm de MMP2, MMP9, TIMP1, TIMP2 e SPARC, hemograma, determinação da proteína C reativa ultrassensível, determinação das concentrações de VEGFA e bFGF e avaliação das mutações nos genes JAK2, cMPL e CALR. A avaliação da densidade microvascular da medula óssea foi feita em 30 dos pacientes incluídos. Os pacientes com MFP, MFPTE e TE apresentaram maior expressão de MMP2, SPARC, TIMP1, TIMP2 e bFGF quando comparados aos seus controles (P<0,05), enquanto MMP9 foi mais expressa nos pacientes com MFPTE e TE (P= 0,011 e P=0,047, respectivamente). Os pacientes com TE apresentaram maior expressão de HIF1-α e VEGFA em relação ao grupo controle (P<0,05). Pacientes com MF JAK2V617F positivos apresentaram maiores concentrações de MMP9, TIMP2, bFGF e VEGFA quando comparados aos pacientes portadores de mutações na CALR (P<0,05). Os pacientes com TE JAK2V617F positivos apresentaram maiores concentrações de MMP2 e TIMP2 (P=0,049 e P=0,020, respectivamente). As concentrações das proteínas estudadas não apresentaram correlação com a carga alélica de JAK2V617F e nem com a densidade microvascular da medula óssea. Células de medula óssea de camundongos submetidos à hipóxia apresentaram maior expressão de MMP2 e TIMP1 comparados aos camundongos em normóxia. Camundongos VHL(-/-) apresentaram aumento na expressão dos genes MMP2, MMP9, TIMP1, TIMP2 e VEGFA. Diferentemente, embriões HIF1-α(-/-) não foram considerados um bom modelo para este estudo devido ao envolvimento das MMPs na embriogênese/organogênese. Frente aos resultados encontrados, pode-se sugerir que a maior expressão de MMP2, SPARC e de bFGF estão associadas às NMPs. A mutação JAK2V617F foi associada a maiores concentrações de MMPs, TIMP2 VEGFA e bFGF. HIF1-α foi mais expresso na PV e na TE, sugerindo uma possível regulação da expressão das MMPs e TIMPs nessas doenças.