Activation of P2X7 and P2Y11 purinergic receptors inhibits migration and normalizes tumor-derived endothelial cells via cAMP signaling.

Purinergic signaling is involved in inflammation and cancer. Extracellular ATP accumulates in tumor interstitium, reaching hundreds micromolar concentrations, but its functional role on tumor vasculature and endothelium is unknown. Here we show that high ATP doses (>20 µM) strongly inhibit migration of endothelial cells from human breast carcinoma (BTEC), but not of normal human microvascular EC. Lower doses (1-10 µM result ineffective. The anti-migratory activity is associated with cytoskeleton remodeling and is significantly prevented by hypoxia. Pharmacological and molecular evidences suggest a major role for P2X7R and P2Y11R in ATP-mediated inhibition of TEC migration: selective activation of these purinergic receptors by BzATP mimics the anti-migratory effect of ATP, which is in turn impaired by their pharmacological or molecular silencing. Downstream pathway includes calcium-dependent Adenilyl Cyclase 10 (AC10) recruitment, cAMP release and EPAC-1 activation. Notably, high ATP enhances TEC-mediated attraction of human pericytes, leading to a decrease of endothelial permeability, a hallmark of vessel normalization. Finally, we provide the first evidence of in vivo P2X7R expression in blood vessels of murine and human breast carcinoma. In conclusion, we have identified a purinergic pathway selectively acting as an antiangiogenic and normalizing signal for human tumor-derived vascular endothelium.

Lenalidomide normalizes tumor vessels in colorectal cancer improving chemotherapy activity.

BACKGROUND: Angiogenesis inhibition is a promising approach for treating metastatic colorectal cancer (mCRC). Recent evidences support the seemingly counterintuitive ability of certain antiangiogenic drugs to promote normalization of residual tumor vessels with important clinical implications. Lenalidomide is an oral drug with immune-modulatory and anti-angiogenic activity against selected hematologic malignancies but as yet little is known regarding its effectiveness for solid tumors. The aim of this study was to determine whether lenalidomide can normalize colorectal cancer neo-vessels in vivo, thus reducing tumor hypoxia and improving the benefit of chemotherapy. METHODS: We set up a tumorgraft model with NOD/SCID mice implanted with a patient-derived colorectal cancer liver metastasis. The mice were treated with oral lenalidomide (50 mg/Kg/day for 28 days), intraperitoneal 5-fluorouracil (5FU) (20 mg/Kg twice weekly for 3 weeks), combination (combo) of lenalidomide and 5FU or irrelevant vehicle. We assessed tumor vessel density (CD146), pericyte coverage (NG2; alphaSMA), in vivo perfusion capability of residual vessels (lectin distribution essay), hypoxic areas (HP2-100 Hypoxyprobe) and antitumor activity in vivo and in vitro. RESULTS: Treatment with lenalidomide reduced tumor vessel density (p = 0.0001) and enhanced mature pericyte coverage of residual vessels (p = 0.002). Perfusion capability of tumor vessels was enhanced in mice treated with lenalidomide compared to controls (p = 0.004). Accordingly, lenalidomide reduced hypoxic tumor areas (p = 0.002) and enhanced the antitumor activity of 5FU in vivo. The combo treatment delayed tumor growth (p = 0.01) and significantly reduced the Ki67 index (p = 0.0002). Lenalidomide alone did not demonstrate antitumor activity compared to untreated controls in vivo or against 4 different mCRC cell lines in vitro. CONCLUSIONS: We provide the first evidence of tumor vessel normalization and hypoxia reduction induced by lenalidomide in mCRC in vivo. This effect, seemingly counterintuitive for an antiangiogenic compound, translates into indirect antitumor activity thus enhancing the therapeutic index of chemotherapy. Our findings suggest that further research should be carried out on synergism between lenalidomide and conventional therapies for treating solid tumors that might benefit from tumor vasculature normalization.

Class 3 semaphorins: physiological vascular normalizing agents for anti-cancer therapy.

Findings from preclinical and clinical studies show that vascular normalization represents a novel strategy to enhance the efficacy of and overcome the acquired resistance to anti-angiogenic therapies in cancer. Several mechanisms of tumour vessel normalization have been revealed. Amongst them, secreted class 3 semaphorins (Sema3), which regulate axon guidance and angiogenesis, have been recently identified as novel vascular normalizing agents that inhibit metastatic dissemination by restoring vascular function. Here, we discuss the different biological functions and mechanisms of action of Sema3 in the context of tumour vascular normalization, and their impact on the different cellular components of the tumour microenvironment.

Percolation, morphogenesis, and burgers dynamics in blood vessels formation.

Experiments of in vitro formation of blood vessels show that cells randomly spread on a gel matrix autonomously organize to form a connected vascular network. We propose a simple model which reproduces many features of the biological system. We show that both the model and the real system exhibit a fractal behavior at small scales, due to the process of migration and dynamical aggregation, followed at large scale by a random percolation behavior due to the coalescence of aggregates. The results are in good agreement with the analysis performed on the experimental data.

Tat-induced platelet-activating factor synthesis contributes to the angiogenic effect of HIV-1 Tat.

This study shows that human umbilical cord vein-derived endothelial cells (HUVEC) stimulated with HIV-1 Tat synthesized platelet-activating factor (PAF), a phospholipid mediator of inflammation that possesses angiogenic properties. The synthesis of PAF by HUVEC stimulated with Tat was dose and time dependent. Moreover, in vitro experiments were performed to evaluate whether migration of HUVEC induced by Tat was dependent on the synthesis of PAF. It was found that the cell motility induced by Tat was inhibited by WEB 2170, a specific PAF receptor antagonist. In vivo, the neoangiogenesis induced by Tat was also inhibited by WEB 2170 in a murine model, in which matrigel subcutaneously injected was used as substratum for angiogenesis. These results suggest that the synthesis of PAF by endothelial cells mediates, at least in part, the angiogenic activity of Tat by promoting the endothelial cell migration.

Src-mediated activation of alpha-diacylglycerol kinase is required for hepatocyte growth factor-induced cell motility.

Diacylglycerol kinases are involved in cell signaling, either as regulators of diacylglycerol levels or as intracellular signal-generating enzymes. However, neither their role in signal transduction nor their biochemical regulation has been elucidated. Hepatocyte growth factor (HGF), upon binding to its tyrosine kinase receptor, activates multiple signaling pathways stimulating cell motility, scattering, proliferation and branching morphogenesis. Herein we demonstrate that: (i) the enzymatic activity of alpha-diacylglycerol kinase (alphaDgk) is stimulated by HGF in epithelial, endothelial and alphaDgk-transfected COS cells; (ii) cellular expression of an alphaDgk kinase-defective mutant inhibits activation of endogenous alphaDgk acting as dominant negative; (iii) specific inhibition of alphaDgk prevents HGF-induced cell movement of endothelial cells; (iv) HGF induces the association of alphaDgk in a complex with Src, whose tyrosine kinase activity is required for alphaDgk activation by HGF; (v) Src wild type stimulates alphaDgk activity in vitro; and (vi) alphaDgk can be tyrosine phosphorylated in intact cells.

Tumor necrosis factor-alpha regulates expression of vascular endothelial growth factor receptor-2 and of its co-receptor neuropilin-1 in human vascular endothelial cells.

Tumor necrosis factor-alpha (TNF-alpha) modulates gene expression in endothelial cells and is angiogenic in vivo. TNF-alpha does not activate in vitro migration and proliferation of endothelium, and its angiogenic activity is elicited by synthesis of direct angiogenic inducers or of proteases. Here, we show that TNF-alpha up-regulates in a dose- and time-dependent manner the expression and the function of vascular endothelial growth factor receptor-2 (VEGFR-2) as well as the expression of its co-receptor neuropilin-1 in human endothelium. As inferred by nuclear run-on assay and transient expression of VEGFR-2 promoter-based reporter gene construct, the cytokine increased the transcription of the VEGFR-2 gene. Mithramycin, an inhibitor of binding of nuclear transcription factor Sp1 to the promoter consensus sequence, blocked activation of VEGFR-2, suggesting that the up-regulation of the receptor required Sp1 binding sites. TNF-alpha increased the cellular amounts of VEGFR-2 protein and tripled the high affinity 125I-VEGF-A165 capacity without affecting the Kd of ligand-receptor interaction. As a consequence, TNF-alpha enhanced the migration and the wound healing triggered by VEGF-A165. Since VEGFR-2 mediates angiogenic signals in endothelium, our data indicate that its up-regulation is another mechanism by which TNF-alpha is angiogenic and may provide insight into the mechanism of neovascularization as occurs in TNF-alpha-mediated pathological settings.

The angiogenesis induced by HIV-1 tat protein is mediated by the Flk-1/KDR receptor on vascular endothelial cells.

The HIV-1 Tat protein transactivates HIV, viral and some host cell genes. Tat can be released by infected cells and acts extracellularly in the microenvironment, regulating functions of immunocompetent and mesenchymal cells. One of the most striking effects of Tat is the induction of a functional program in vascular cells related to angiogenesis and inflammation (migration, proliferation and expression of plasminogen activator inhibitor-1 and E selectin). Tat induces growth of Kaposi's sarcoma (KS) spindle cells and is angiogenic in vivo and in transgenic mice10-12. We previously reported that Tat is a direct angiogenic factor and noted the Tat arginine- and lysine-rich sequence is similar to that of other potent angiogenic growth factors, such as vascular endothelial growth factor-A (VEGF-A). It is possible that Tat mimics one of these factors by interacting with its growth factor tyrosine kinase receptor. Here we demonstrate that Tat specifically binds and activates the Flk-1/kinase insert domain receptor (Flk-1/KDR), a VEGF-A tyrosine kinase receptor (for review see ref. 13), and that Tat-induced angiogenesis is blocked by agents blocking the Flk-1/KDR receptor. Endothelial cell stimulation by Tat occurs in the absence of activation of FLT-1, another VEGF-A tyrosine kinase receptor.

IL-6 is an in vitro and in vivo autocrine growth factor for middle T antigen-transformed endothelial cells.

Murine endothelial cells immortalized with the middle-size Ag of polyomavirus (PmT) cause vascular tumors in syngenic mice by recruitment of host normal endothelial cells. This pathogenic process is similar to that occurring in Kaposi's sarcoma, in which the core of the lesion is constituted by "spindle cells," which recruit normal vascular mesenchymal cells. In murine endothelial cells, PmT induces modification of the expression of genes, including that of IL-6. Since IL-6 is a pleiotrophic cytokine that also regulates endothelial cell functions related to angiogenesis, we studied the relevance of IL-6 in the tumorigenicity of PmT-endothelial cells. In vitro studies demonstrated that the spontaneous PmT-endothelial cell proliferation rate was slow during the first 6 days of culture and then increased rapidly and paralleled the IL-6 release. The addition of recombinant IL-6 during the first days of culture induced a marked proliferation in a dose-dependent manner. PmT-endothelial cells expressed on their surface a high-affinity binding site for IL-6 constituted by both IL-6Ralpha and gp130 transmembrane receptors. The growth-promoting effect of exogenous IL-6 or that released by PmT-endothelial cells was abrogated by mAbs anti-IL-6Ralpha, whereas a mAb recognizing the endothelial cell CD31 molecule was inactive. 15A7 mAb anti-murine IL-6Ralpha was also active in vivo, reducing the number of metastases forming after transplantation of PmT-endothelial cells in DBA/2 mice. 15A7 mAb also increased the survival of mice bearing vascular tumors. We conclude that IL-6 is involved in the progression of vascular tumors induced by PmT, and that the blockage of IL-6-mediated intercellular circuits could be useful in the management of human vascular tumors, including Kaposi's sarcoma.

In vivo activation of met tyrosine kinase by heterodimeric hepatocyte growth factor molecule promotes angiogenesis.

Hepatocyte growth factor (HGF) is a powerful motogen and mitogen for epithelial cells. The factor is a 90-kD heterodimer composed of an alpha chain containing four kringle motifs and a beta chain showing structural homologies with serine proteases. It is, however, devoid of enzymatic activity. Recently, it has been reported that HGF activates migration and proliferation of endothelial cells and is angiogenic. In this article we discuss (1) the molecular domains of HGF required to activate in vitro and in vivo endothelial cells, studied by use of molecular mutants, and (2) the characteristics of the angiogenic response to HGF in an experimental model system of implanted reconstituted basement membrane (Matrigel). Two groups of mutants were made and used in vitro and in vivo: one with deletions of kringle domains and one with substitution at the cleavage site of the HGF precursor. In vitro, HGF variants containing only the first two (HGF-NK2) or the first three kringles (HGF-NK3) of the alpha chain did not induce proliferation of endothelial cells even if used at concentration 160-fold higher than that optimal for HGF (0.05 nmol/L). High concentrations of these mutants (4 to 8 nmol/L) activated a little endothelial cell motogenic response that was 60% lower than that elicited by HGF. Substitution of Arg 489 with Gln 489 in the HGF precursor generated an uncleavable single-chain factor, unable to induce either endothelial cell migration or proliferation. In vivo, HGF induced a dose-dependent angiogenic response, which was enhanced by heparin.

A brain phantom for studying contrast recovery in emission computerized tomography.

A brain phantom is described that is characterized by a high anatomical definition and by the possibility of varying the internal contrast with the use of a single radioactive solution. The experimental work was done with a single-photon emission computerized tomographic (SPET) rotating camera. The phantom was used to study the contrast recovery of both the filtered back-projection and an iterative reconstruction algorithm. Moreover, it was also used to find a cross-calibration factor between activity concentrations in the SPET slices and an external reference.