[show abstract][hide abstract] ABSTRACT: Photodynamic therapy (PDT) is an effective clinical treatment for a number of different cancers. PDT can induce hypoxia and inflammation, pro-angiogenic side effects, which may counteract its angio-occlusive mechanism. The combination of PDT with anti-angiogenic drugs offers a possibility for improved anti-tumour outcome. We used two tumour models to test the effects of the clinically approved angiostatic tyrosine kinase inhibitors sunitinib, sorafenib and axitinib in combination with PDT, and compared these results with the effects of bevacizumab, the anti-VEGF antibody, for the improvement of PDT. Best results were obtained from the combination of PDT and low-dose axitinib or sorafenib. Molecular analysis by PCR revealed that PDT in combination with axitinib suppressed VEGFR-2 expression in tumour vasculature. Treatment with bevacizumab, although effective as monotherapy, did not improve PDT outcome. In order to test for tumour vessel normalization effects, axitinib was also applied prior to PDT. The absence of improved PDT outcome in these experiments, as well as the lack of increased oxygenation in axitinib-treated tumours, suggests that vascular normalization did not occur. The current data imply that there is a future for certain anti-angiogenic agents to further improve the efficacy of photodynamic anti-cancer therapy.
Journal of Cellular and Molecular Medicine 01/2014; · 4.75 Impact Factor
[show abstract][hide abstract] ABSTRACT: Galectins are carbohydrate binding proteins with versatile functions in tumor progression. Galectin-9, encoded by LGALS9, has been associated with metastasis and immunosuppresion. We previously reported on regulation of LGALS9 expression during endothelial cell activation. Here, we show increased galectin-9 protein levels in the endothelium of different tumors, including carcinomas of lung, liver, breast and kidney. Endothelial cells were found to express five LGALS9 splice variants, two of which have not been reported before. Splicing was found to be confined to exons 5, 6 and 10. Transfection of human microvascular endothelial cells (HMEC) with galectin-9∆5, a specific LGALS9 splice variant, induced a small but significant increase of proliferation, while migration was not affected by any LGALS9 splice variant. Application of recombinant galectin-9∆5 protein dose-dependently reduced proliferation and migration of HMEC as well as human umbilical vein endothelial cells in vitro. Enhanced sprouting and migration of HUVEC towards a galectin-9∆5 gradient was observed. Interestingly, galectin-9∆5 was found to induce a small inhibitory effect on angiogenesis in vivo. Collectively, these data show that endothelial cells regulate the expression and splicing of LGALS9 during angiogenesis. The function of the dominant splice variant, i.e. galectin-9∆5, in endothelial cell biology depends on the concentration and environmental context in which it is presented to the cells.
Biochimica et Biophysica Acta 12/2013; · 4.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: Heparan sulfate (HS), a long linear polysaccharide, is implicated in various steps of tumorigenesis, including angiogenesis. We successfully interfered with HS biosynthesis using a peracetylated 4-deoxy analog of the HS constituent GlcNAc and studied the compound's metabolic fate and its effect on angiogenesis. The 4-deoxy analog was activated intracellularly into UDP-4-deoxy-GlcNAc and HS expression was inhibited up to ~96% (IC50 = 16 µM). HS chain size was reduced, without detectable incorporation of the 4-deoxy analog, likely due to reduced levels of UDP-GlcNAc. Comprehensive gene expression analysis revealed reduced expression of genes regulated by HS binding growth factors as FGF-2 and VEGF. Cellular binding and signaling of these angiogenic factors was inhibited. Micro-injection in zebrafish embryos strongly reduced HS biosynthesis, and angiogenesis was inhibited in both zebrafish and chicken model systems. All these data identify 4-deoxy-GlcNAc as a potent inhibitor of HS synthesis which hampers pro-angiogenic signaling and neo-vessel formation.
ACS Chemical Biology 08/2013; · 5.44 Impact Factor
[show abstract][hide abstract] ABSTRACT: Galectins are a family of carbohydrate binding proteins with a broad range of cytokine and growth factor-like functions in multiple steps of cancer progression. They contribute to tumor cell transformation, promote tumor angiogenesis, hamper the anti-tumor immune response, and facilitate tumor metastasis. Consequently, galectins are considered as multifunctional targets for cancer therapy. Interestingly, many of the functions related to tumor progression can be linked to galectins expressed by endothelial cells in the tumor vascular bed. Since the tumor vasculature is an easily accessible target for cancer therapy, understanding how galectins in the tumor endothelium influence cancer progression is important for the translational development of galectin-targeting therapies.
[show abstract][hide abstract] ABSTRACT: Galectin family members have been shown to exert multiple roles in the context of tumor biology. Several recent findings support a similar multi-faceted role for galectin-9. Galectin-9 expression is frequently altered in cancer as compared to normal tissues. In addition, an increasing amount of evidence suggests that galectin-9 is involved in several aspects of tumor progression, including tumor cell adhesion and survival, immune escape and angiogenesis. Also, galectin-9 shows potential as a prognostic marker and a therapeutic target for several malignancies. In this review we summarize both the established and the emerging roles of galectin-9 in tumor biology and discuss the potential application of galectin-9 in anti-cancer therapy.
Biochimica et Biophysica Acta 05/2013; · 4.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: CD63 is a member of the transmembrane-4 glycoprotein superfamily (tetraspanins) implicated in the regulation of membrane protein trafficking, leukocyte recruitment and adhesion processes. We have investigated the involvement of CD63 in endothelial cell (EC) signaling downstream of b1 integrin and vascular endothelial growth factor (VEGF). We report that silencing of CD63 in primary ECs arrested capillary sprouting and tube formation in vitro due to impaired adhesion and migration of ECs. Mechanistically, CD63 associated to both b1 integrin and the main VEGF receptor on ECs, VEGFR2. Our data suggest that CD63 serves to bridge between b1 integrin and VEGFR2 since CD63 silencing disrupted VEGFR2/b1 integrin complex formation identified using proximity ligation assays. Signaling downstream of b1 integrin and VEGFR2 was attenuated in CD63-silenced cells although their cell surface expression levels remained unaffected. CD63 was furthermore required for efficient internalization of VEGFR2 in response to VEGF. Importantly, systemic delivery of VEGF failed to potently induce VEGFR2 phosphorylation and downstream signaling in CD63-deficient mouse lungs. Taken together, our findings demonstrate a previously unrecognized role for CD63 in coordinated integrin and receptor tyrosine kinase signaling in vitro and in vivo.
Journal of Biological Chemistry 04/2013; · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Interleukin (IL)-10 is a major cancer-related immunosuppressive factor, exhibiting a unique ability to hamper the maturation of dendritic cells (DCs). We have previously reported that IL-10 induces the conversion of activated, migratory CD1a(+) DCs found in the human skin to CD14(+)CD141(+) macrophage-like cells. Here, as a model of tumor-conditioned DC maturation, we functionally assessed CD14(-) and CD14(+) DCs that matured in vitro upon exposure to IL-10. IL-10-induced CD14(+) DCs were phenotypically characterized by a low maturation state as well as by high levels of BDCA3 and DC-SIGN, and as such they closely resembled CD14(+) cells infiltrating melanoma metastases. Compared with DC matured under standard conditions, CD14(+) DCs were found to express high levels of B7-H1 on the cell surface, to secrete low levels of IL-12p70, to preferentially induce TH2 cells, to have a lower allogeneic TH cell and tumor antigen-specific CD8(+) T-cell priming capacity and to induce proliferative T-cell anergy. In contrast to their CD14(+) counterparts, CD14(-) monocyte-derived DCs retained allogeneic TH priming capacity but induced a functionally anergic state as they completely abolished the release of effector cytokines. Transcriptional and cytokine release profiling studies indicated a more profound angiogenic and pro-invasive signature of CD14(+) DCs as compared with DCs matured in standard conditions or CD14(-) DCs matured in the presence of IL-10. Importantly, signal transducer and activator of transcription 3 (STAT3) depletion by RNA interference prevented the development of the IL-10-associated CD14(+) phenotype, allowing for normal DC maturation and providing a potential means of therapeutic intervention.
[show abstract][hide abstract] ABSTRACT: New triruthenium-carbonyl clusters derivatized with glucose-modified bicyclophosphite ligands have been synthesized. These compounds were found to have cytostatic and cytotoxic activity and depending on the number of bicyclophosphite ligands, and could be tuned for either anti-cancer or specific anti-angiogenic activity. While some compounds had a broad cellular toxicity profile in several cell types others showed endothelial cell specific dose-dependent anti-proliferative and anti-migratory efficacy. A profound inhibition of angiogenesis was also observed in the in vivo chicken chorioallantoic membrane (CAM) model, and consequently, these new compounds have considerable potential in drug design, e.g. for the treatment of cancer.
[show abstract][hide abstract] ABSTRACT: One of the challenges of tailored antiangiogenic therapy is the ability to adequately monitor the angiogenic activity of a malignancy in response to treatment. The α(v)β(3) integrin, highly overexpressed on newly formed tumor vessels, has been successfully used as a target for Arg-Gly-Asp (RGD)-functionalized nanoparticle contrast agents. In the present study, an RGD-functionalized nanocarrier was used to image ongoing angiogenesis in two different xenograft tumor models with varying intensities of angiogenesis (LS174T > EW7). To that end, iron oxide nanocrystals were included in the core of the nanoparticles to provide contrast for T(2)*-weighted magnetic resonance imaging (MRI), whereas the fluorophore Cy7 was attached to the surface to enable near-infrared fluorescence (NIRF) imaging. The mouse tumor models were used to test the potential of the nanoparticle probe in combination with dual modality imaging for in vivo detection of tumor angiogenesis. Pre-contrast and post-contrast images (4 hours) were acquired at a 9.4-T MRI system and revealed significant differences in the nanoparticle accumulation patterns between the two tumor models. In the case of the highly vascularized LS174T tumors, the accumulation was more confined to the periphery of the tumors, where angiogenesis is predominantly occurring. NIRF imaging revealed significant differences in accumulation kinetics between the models. In conclusion, this technology can serve as an in vivo biomarker for antiangiogenesis treatment and angiogenesis phenotyping.
Neoplasia (New York, N.Y.) 10/2012; 14(10):964-73. · 5.48 Impact Factor
[show abstract][hide abstract] ABSTRACT: The pathogenesis of plasma leakage during dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) is largely unknown. Angiopoietins are key regulators of vascular integrity: Angiopoietin-1 is stored in platelets and maintains vascular integrity, and endothelium-derived angiopoietin-2 promotes vascular leakage. We determined Angiopoietin-1 and Angiopoietin-2 levels in a cohort of children in Indonesia with DHF/DSS and related them to plasma leakage markers. Patients with DHF/DSS had reduced Angiopoietin-1 and increased Angiopoietin-2 plasma levels on the day of admission when compared with levels at discharge and in healthy controls. There was an inverse correlation between angiopoietin-1 and markers of plasma leakage and a positive correlation between angiopoietin-2 and markers of plasma leakage. Angiopoietin-1 levels followed the same trend as the soluble platelet activation marker P-selectin and correlated with platelet counts. Dengue-associated thrombocytopenia and endothelial activation are associated with an imbalance in angiopoietin-2: angiopoietin-1 plasma levels. This imbalance may contribute to the transient plasma leakage in DHF/DSS.
The American journal of tropical medicine and hygiene 09/2012; · 2.53 Impact Factor
[show abstract][hide abstract] ABSTRACT: The use of omonasteine (Omo) in sequential peptide ligation strategies extends the scope of homocysteine (Hcy) ligation to longer, methionine-rich proteins. Hcy-to-Omo conversion can be performed on-resin, while the Omo-to-Hcy deprotection can be performed in situ after peptide ligation. This strategy was successfully applied in the synthesis of the BRD7 bromodomain.
Chemical Communications 08/2012; 48(75):9403-5. · 6.38 Impact Factor
[show abstract][hide abstract] ABSTRACT: To investigate the angiogenic changes in primary tumor tissue of renal cell carcinoma (RCC) patients treated with VEGF-targeted therapy.
Phase II trials of VEGF pathway-targeted therapy given before cytoreductive surgery were carried out with metastatic RCC patients with the primary tumor in situ to investigate the necessity of nephrectomy. Primary tumor tissues were obtained and assessed for angiogenesis parameters. Results were compared with similar analyses on untreated tumors.
Sunitinib or bevacizumab pretreatment resulted in a significant reduction of microvessel density in the primary tumor. Also, an increase in vascular pericyte coverage was found in sunitinib-pretreated tumors, consistent with efficient angiogenesis inhibition. Expression of several key regulators of angiogenesis was found to be suppressed in pretreated tissues, among which VEGFR-1 and VEGFR-2, angiopoietin-1 and angiopoietin-2 and platelet-derived growth factor-B. In addition, apoptosis in tumor and endothelial cells was induced. Interestingly, in sunitinib-pretreated tissues a dramatic increase of the number of proliferating endothelial cells was observed, which was not the case in bevacizumab-pretreated tumors. A positive correlation with the interval between halting the therapy and surgery was found, suggesting a compensatory angiogenic response caused by the discontinuation of sunitinib treatment.
This study describes, for the first time, the angiostatic response in human primary renal cancers at the tissue level upon treatment with VEGF-targeted therapy. Discontinuation of treatment with tyrosine kinase inhibitors leads to accelerated endothelial cell proliferation. The results of this study contribute important data to the ongoing discussion on the discontinuation of treatment with kinase inhibitors.
Clinical Cancer Research 05/2012; 18(14):3961-71. · 7.84 Impact Factor
[show abstract][hide abstract] ABSTRACT: A large number of patients that undergo radiotherapy develop local failure. To improve the efficacy of treatment, there is an increasing interest in combining radiotherapy with novel targeted therapies. Inhibiting the growth of new tumor blood vessels, i.e. tumor angiogenesis, is such a targeted therapy. Growing tumors induce angiogenesis to ensure an adequate delivery of oxygen and nutrients and several angiostatic drugs have been approved for the treatment of cancer patients. Both pre-clinical and clinical studies have shown that radiotherapy can influence tumor angiogenesis and that angiogenesis inhibition can potentiate the effect of radiotherapy. Therefore, the combination of angiogenesis inhibition and radiotherapy holds a promising future in cancer treatment. However, the radiosensitizing effects of angiogenesis inhibition are transient and recent findings indicate that the effects of irradiation on angiogenesis depend on the dose and treatment schedule. This raises questions regarding the scheduling of both treatment modalities in order to achieve the optimal treatment efficacy with minimal toxicity. In this review the opportunities and pitfalls of combining angiostatic agents with radiotherapy are discussed. The lessons learned from (pre)clinical studies are summarized with an emphasis on scheduling and dosing of the combination therapy. Finally, the opportunities of ongoing clinical studies are discussed and opportunities to improve the combination of angiostatic drugs with radiotherapy are presented.
Drug resistance updates: reviews and commentaries in antimicrobial and anticancer chemotherapy 05/2012; 15(3):173-82. · 12.58 Impact Factor
[show abstract][hide abstract] ABSTRACT: Photodynamic therapy (PDT) is a minimally invasive form of treatment, which is clinically approved for the treatment of angiogenic disorders, including certain forms of cancer and neovascular eye diseases. Although the concept of PDT has existed for a long time now, it has never made a solid entrance into the clinical management of cancer. This is likely due to secondary tissue reactions, such as inflammation and neoangiogenesis. The recent development of clinically effective angiogenesis inhibitors has lead to the initiation of research on the combination of PDT with such angiostatic targeted therapies. Preclinical studies in this research field have shown promising results, causing a revival in the field of PDT. This review reports on the current research efforts on PDT and vascular targeted combination therapies. Different combination strategies with angiogenesis inhibition and vascular targeting approaches are discussed. In addition, the concept of increasing PDT selectivity by targeted delivery of photosensitizers is presented. Furthermore, the current insights on sequencing the therapy arms of such combinations will be discussed in light of vascular normalization induced by angiogenesis inhibition.
Biochimica et Biophysica Acta 03/2012; 1826(1):53-70. · 4.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: The endothelium plays a pivotal role in the progression of solid tumors and is considered a highly relevant target for therapy. However, it emerges that current clinical angiogenesis inhibitors that act through inhibition of tumor-derived growth factors are prone to inducing drug resistance. Therefore, markers of tumor endothelial cells (ECs) themselves provide attractive novel therapeutic targets. In a screen for markers of tumor angiogenesis, we recently identified high-mobility group box 1 (HMGB1), known to act as proinflammatory cytokine and chromatin-binding molecule. Here we report on the role of HMGB1 in angiogenesis by showing that its overexpression is associated with an increased angiogenic potential of ECs. HMGB1 stimulates the expression of players in vascular endothelial growth factor and platelet-derived growth factor signaling, both in vitro and in vivo. Importantly, we show that HMGB1 triggers and helps to sustain this proangiogenic gene expression program in ECs, additionally characterized by increased activity of matrix metalloproteinases, integrins and nuclear factor-κB. Moreover, we found that HMGB1 is involved in several autocrine and/or paracrine feedback mechanisms resulting in positive enforcement of HMGB1 expression, and that of its receptors, RAGE (receptor for advanced glycation end products) and Toll-like receptor 4 (TLR4). Interference in HMGB1 expression and/or function using knockdown approaches and antibody-mediated targeting to break this vicious circle resulted in inhibited migration and sprouting of ECs. Using different in vivo models, therapeutic efficacy of HMGB1 targeting was confirmed. First, we demonstrated induction of HMGB1 expression in the chicken embryo chorioallantoic membrane (CAM) neovasculature following both photodynamic therapy and tumor challenge. We subsequently showed that anti-HMGB1 antibodies inhibited vessel density in both models, accompanied by a reduced vascular expression of angiogenic growth factor receptors. Collectively, these data identify HMGB1 as an important modulator of tumor angiogenesis and suggest the feasibility of targeting HMGB1 for multi-level cancer treatment.Oncogene advance online publication, 5 March 2012; doi:10.1038/onc.2012.49.
[show abstract][hide abstract] ABSTRACT: The functional shift of quiescent endothelial cells into tip cells that migrate and stalk cells that proliferate is a key event during sprouting angiogenesis. We previously showed that the sialomucin CD34 is expressed in a small subset of cultured endothelial cells and that these cells extend filopodia: a hallmark of tip cells in vivo. In the present study, we characterized endothelial cells expressing CD34 in endothelial monolayers in vitro. We found that CD34-positive human umbilical vein endothelial cells show low proliferation activity and increased mRNA expression of all known tip cell markers, as compared to CD34-negative cells. Genome-wide mRNA profiling analysis of CD34-positive endothelial cells demonstrated enrichment for biological functions related to angiogenesis and migration, whereas CD34-negative cells were enriched for functions related to proliferation. In addition, we found an increase or decrease of CD34-positive cells in vitro upon exposure to stimuli that enhance or limit the number of tip cells in vivo, respectively. Our findings suggest cells with virtually all known properties of tip cells are present in vascular endothelial cell cultures and that they can be isolated based on expression of CD34. This novel strategy may open alternative avenues for future studies of molecular processes and functions in tip cells in angiogenesis.