Supplementary Figures, Table and Movie Legend from Blood Vessel Maturation and Response to Vascular-Disrupting Therapy in Single Vascular Endothelial Growth Factor-A Isoform–Producing Tumors
Background: Combretastatin A4 phosphate (CA4P) is a tumour vascular disrupting agent (VDA) that targets endothelial microtubules, triggering remodelling of the actin cytoskeleton, contractility and disruption of VE-cadherin junctions through RhoGTPase/ROCK-dependent pathways. These events lead to a rise in endothelial monolayer permeability. A rise in permeability is considered crucial for vascular shutdown elicited by CA4P in vivo. CA4P also inhibits endothelial migration and induces mitotic arrest and apoptosis, so potentially it could also target tumour angiogenesis.
Method: In this study, the nature of signalling interactions between Rho/ROCK and cAMP/cGMP and their influence on cytoskeletal and functional responses of endothelial cells to CA4P were investigated.
Results: Several cAMP/cGMP analogues inhibited CA4P-induced Rho/ROCK activation and prevented actin remodeling, disruption of cell-to-cell junctions and permeability rise in endothelial monolayers. cAMP inhibits Rho by either protein kinase A (PKA)-dependent mechanisms or via activation of Epac1/Rap1. O-Me-cAMP, an analogue that selectively activates Epac1/Rap1 abolished activation of Rho/ROCK by CA4P while selective PKA activator 6-Bnz-cAMP only partially inhibited Rho/ROCK activation and actin remodelling by CA4P. Inhibitors of PKA did not alter endothelial responses to CA4P in the presence of cAMP analogues suggesting that cAMP acts primarily via Epac1/Rap1 to inhibit Rho/CA4P interactions. CA-4-P also inhibited endothelial migration and abolished lamellipodia at the leading edge of migrating cells in injured monolayers. Rho inhibitor C3 exoenzyme and ROCK inhibitor Y27632 as well as cAMP analogues re-established cell movement and formation of lamellipodia in wounded monolayers exposed to CA-4-P, suggesting that inhibitory effects on migration were mediated via Rho/ROCK.
Conclusion: Deciphering molecular pathways that modulate endothelial responses to VDAs is important for further targeting. Our data demonstrate that interactions between cGMP/cAMP and Rho influence both the vascular disrupting and anti-angiogenic activities of CA-4-P and point to cAMP/cGMP as potential targets for improving VDA activity. Acknowledgements Funded by Cancer Research UK
Supplementary Figures, Table and Movie Legend from Blood Vessel Maturation and Response to Vascular-Disrupting Therapy in Single Vascular Endothelial Growth Factor-A Isoform–Producing Tumors
Supplementary Figure 3 from Blood Vessel Maturation and Response to Vascular-Disrupting Therapy in Single Vascular Endothelial Growth Factor-A Isoform–Producing Tumors
Supplementary Figure 3 from Blood Vessel Maturation and Response to Vascular-Disrupting Therapy in Single Vascular Endothelial Growth Factor-A Isoform–Producing Tumors
The combretastatins are an important class of tubulin-binding agents. Of this family, a number of compounds are potent tumor vascular disrupting agents (VDAs) and have shown promise in the clinic for cancer therapy. We have developed a modular synthetic route to combretastatin analogs based on a pyrazole core through highly regioselective alkyne cycloaddition reactions of sydnones. These compounds show modest to high potency against human umbilical vein endothelial cell proliferation. Moreover, evidence is presented that these novel VDAs have the same mode of action as CA4P and bind reversibly to β-tubulin, believed to be a key feature in avoiding toxicity. The most active compound from in vitro studies was taken forward to an in vivo model and instigated an increase in tumor cell necrosis.
Abstract Tubulin-binding vascular-disrupting agents (VDA) are currently in clinical trials for cancer therapy but the factors that influence tumor susceptibility to these agents are poorly understood. We evaluated the consequences of modifying tumor vascular morphology and function on vascular and therapeutic response to combretastatin-A4 3-O-phosphate (CA-4-P), which was chosen as a model VDA. Mouse fibrosarcoma cell lines that are capable of expressing all vascular endothelial growth factor (VEGF) isoforms (control) or only single isoforms of VEGF (VEGF120, VEGF164, or VEGF188) were developed under endogenous VEGF promoter control. Once tumors were established, VEGF isoform expression did not affect growth or blood flow rate. However, VEGF188 was uniquely associated with tumor vascular maturity, resistance to hemorrhage, and resistance to CA-4-P. Pericyte staining was much greater in VEGF188 and control tumors than in VEGF120 and VEGF164 tumors. Vascular volume was highest in VEGF120 and control tumors (CD31 staining) but total vascular length was highest in VEGF188 tumors, reflecting very narrow vessels forming complex vascular networks. I.v. administered 40 kDa FITC-dextran leaked slowly from the vasculature of VEGF188 tumors compared with VEGF120 tumors. Intravital microscopy measurements of vascular length and RBC velocity showed that CA-4-P produced significantly more vascular damage in VEGF120 and VEGF164 tumors than in VEGF188 and control tumors. Importantly, this translated into a similar differential in therapeutic response, as determined by tumor growth delay. Results imply differences in signaling pathways between VEGF isoforms and suggest that VEGF isoforms might be useful in vascular-disrupting cancer therapy to predict tumor susceptibility to VDAs. [Cancer Res 2008;68(7):2301–11]
The heart is a critical dose-limiting organ during radiotherapy. In patients treated for breast cancer or Hodgkin's disease, regions of the heart can receive radiation doses in excess of 40 Gy. More recently, epidemiological and clinical studies suggested that even moderate to low radiation doses increase the risk of cardiovascular disease that can present years after the initial exposure. Radiation can cause cardiac microvascular damage but the mechanisms of pathogenesis of radiation-induced heart disease have not been clearly defined. Cardiac injury triggers repair processes in the myocardium requiring neovascularisation. Here, we investigated latent effects of ionizing radiation on angiogenic responses in the mouse heart. We also assessed the effects of radiation on cardiac endothelial responses and angiogenesis in vitro. The hearts of C57BL/6 and ApoE-/- mice were locally irradiated with 0.2 to 16 Gy and animals were sacrificed at 20, 40 and 60 weeks post-irradiation. Formation of angiogenic sprouts was assessed in ventricular heart explants embedded in fibrin gels. The angiogenic capacity of irradiated hearts was also assessed through a novel fibroblast-endothelial assay we developed. Mouse hearts were enzymatically digested into a single cell suspension and cultured for 10 days. Within 5 to 7 days, endothelial cells began to form capillary-like structures surrounded by fibroblasts and pericytes, modeling the angiogenic process. Cardiac endothelial cells were also irradiated in vitro and effects on migration and capillary-tube formation on a fibroblast bed were established. Radiation caused a dose-dependent inhibition of endothelial sprout formation in explants from both mouse models. Significant changes were observed at 8 and 16 Gy after 20 weeks and at ≥2 Gy after 40 or 60 weeks post irradiation. Irradiation also significantly reduced the adherence and growth of extracted cardiac cells and inhibited the quantity and quality of the capillary-like structures formed in the self-assembling model. In vitro, irradiation (≥0.2 Gy) inhibited the migration of cardiac endothelial cells in scratch wound assays and capillary-like formation on a fibroblast bed. Our data show that radiation causes persistent and progressive vascular damage and suppresses angiogenic activity in the heart. Angiogenesis was inhibited at doses likely to be delivered during thoracic radiation in cancer patients. Impairment of the angiogenic response could lead to further ischemia and degeneration in the myocardium and contribute to the development of heart disease.
Abstract Vascular endothelial growth factor A (VEGF), a potent regulator of tumour angiogenesis, is differentially spliced to give rise to the predominant pro-angiogenic isoforms, VEGF121/120, 165/164 and 189/188 in human and mouse respectively. All VEGF isoforms bind to two tyrosine kinase receptors, VEGFR-1 and VEGFR-2, but elicit different signalling outcomes. However, this is further complicated by more recent work describing the existence of human anti-angiogenic VEGF isoforms (VEGFxxxb), which are thought to counteract pro-angiogenic isoforms, inferring that it is the balance between these two families of VEGF isoforms that determines tumour angiogenic outcome. We have shown previously that mouse fibrosarcoma cells expressing all VEGF isoforms (wt) or VEGF188 only, give rise to tumours which establish more slowly, exhibit higher rates of apoptosis, and exhibit more mature vasculature invested with pericytes, compared to VEGF164 or VEGF120 expressing tumours. Importantly this translated into resistance to the vascular disrupting agent (VDA) combretastatin A-4 phosphate (CA4P) (Cancer Res 2008: 68:2301). To address the hypothesis that differences in signalling pathways between VEGF isoforms contributes to the tumour vascular characteristics described above, we performed proteomic profiling of wt and VEGF isoform-specific tumour cell lines and solid tumour lysates using commercial angiogenesis and phospho-receptor tyrosine kinase arrays (R&D) as well as iTRAQ technology to identify differentially expressed proteins. In addition, using an RT-PCR based approach we determined whether or not the potential existence of mouse anti-angiogenic isoforms could be contributing to our data. Although able to infrequently detect mouse VEGFxxxb isoforms, we show that these must be PCR artefacts since they were detected in lysates derived from cell lines obtained from transgenic mice whose genetic construction precludes VEGFxxxb splicing from occurring. In contrast, protein profiling has revealed a number of VEGF isoform-specific differentially expressed proteins. In particular, we found that fibroblast growth factor (FGF-2), neuropilin 1 (NRP1) and platelet-derived growth factor receptor ≤ (PDGFRα) were up-regulated in VEGF188-specific tumour cells, whilst Neuropilin 2 (NRP2) was down-regulated in the same tumour extracts relative to VEGF164 and VEGF120. Our data infer VEGF188-specific differentially expressed candidate proteins in tumours, which may impact on the efficacy of VDA treatment therapies. Further experiments aim to determine whether manipulating the expression of these candidate proteins in VEGF188-specific tumours reverses their resistance to CA4P, thus identifying additional targets for anti-angiogenic therapy approaches. This work, was funded by a Program Grant from Cancer Research UK Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1371. doi:1538-7445.AM2012-1371
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