Analysis of capillary-like structures formed by endothelial cells in a novel organotypic assay developed from heart tissue

In this work, we present an algorithm to perform automatic segmentation, feature extraction and dosage classification of images derived from a novel organotypic angiogenesis assay developed to assess the effects of ionising radiation in the mouse heart. The images presented very different conditions of illumination and conditions of density and shapes of cells. The algorithm consists of a pipeline of several steps, which were validated against hand-segmented images. The algorithm provided satisfactory results, as all images were correctly dose-classified. The cells exposed to the lowest radiation dose were observed to have the greatest relative feature variability

Signaling interactions between RhoGTPase and cAMP/cGMP influence endothelial responses to the vascular disrupting agent combretastatin A4 phosphate

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 U

Sydnone Cycloaddition Route to Pyrazole-Based Analogs of Combretastatin A4.

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

Measuring cellular migration with image processing

An image-processing algorithm for the analysis of migration of vascular endothelial cells in culture is presented. The algorithm correctly detected the cellular regions on either side of an artificial ‘wound’ made by dragging a sterile pipette tip across the monolayer of cells (scratch wound assay). Frequency filtering and mathematical morphology were used to approximate the boundaries of cellular regions. This allowed the measurement of the distance between the regions, and therefore the migration rates, regardless of the orientation of the wound and even in cases where the cells were sparse and not tightly packed

A critical role for RhoA-GTPase signaling in the tumour vascular disrupting action of combretastatin A4-phosphate in vivo

Background: Tubulin binding microtubule depolymerising agents form a growing group of tumour vascular disrupting agents (VDAs) in clinical trial, with combretastatin A-4-phosphate (CA4P) the lead compound. Signalling through RhoGTPase/ROCK-dependent pathways is central to CA4P-induced effects on endothelial cells in vitro (Kanthou C. & Tozer GM, 2002, Blood 99: 2060-2069) . Here, we tested the hypothesis that RhoGTPase/ROCK signalling is also important in vivo. Method: SW1222 human colorectal carcinoma cells were grown as solid sub-cutaneous tumours in SCID mice. The Rho kinase (ROCK) inhibitor, Y-27632 (50 mg/kg) or saline control, was administered intraperitoneally (i.p.), 5 minutes prior to 100 mg/kg CA4P or saline i.p.. Laser Doppler flowmetry was used to assess tumour vascular response from 0 – 2h post-treatment. Intravenous administration of fluorescent tomato lectin was used for assessing tumour perfusion at 1, 3, 6 and 24 hours post-treatment. Necrosis (H&E) and leukocyte infiltration (immunohistochemistry) were assessed at 24h. Results: Y-27632 alone did not significantly increase necrosis at 24 hours (17±4% versus 10±3% of tumour sectional area). However, prior administration of Y-27632 significantly reduced CA4P-induced tumour necrosis from 61±5% to 35±7%, accompanied by a decrease in staining for the myeloid markers, myeloperoxidase and GR-1. Y-27632 pre-treatment did not affect laser Doppler and perfused vascular volume measurements in the first few hours after CA4P but significantly reduced the effect of CA4P on perfused vascular volume measured at 6 and 24 hours. Conclusion: Our data indicate that RhoGTPase/ROCK-dependent signalling is a critical factor in determining extent of tumour necrosis induction by CA4P and suggest that ROCK inhibition is acting downstream from initial vascular shut-down, potentially via modulation of myeloid cell recruitment. These mechanisms also have significance for similar VDAs in development. Funded by Cancer Research U

Wearable Insulin Biosensors for Diabetes Management: Advances and Challenges

We present a critical review of the current progress in wearable insulin biosensors. For over 40 years, glucose biosensors have been used for diabetes management. Measurement of blood glucose is an indirect method for calculating the insulin administration dosage, which is critical for insulin-dependent diabetic patients. Research and development efforts aiming towards continuous-insulin-monitoring biosensors in combination with existing glucose biosensors are expected to offer a more accurate estimation of insulin sensitivity, regulate insulin dosage and facilitate progress towards development of a reliable artificial pancreas, as an ultimate goal in diabetes management and personalised medicine. Conventional laboratory analytical techniques for insulin detection are expensive and time-consuming and lack a real-time monitoring capability. On the other hand, biosensors offer point-of-care testing, continuous monitoring, miniaturisation, high specificity and sensitivity, rapid response time, ease of use and low costs. Current research, future developments and challenges in insulin biosensor technology are reviewed and assessed. Different insulin biosensor categories such as aptamer-based, molecularly imprinted polymer (MIP)-based, label-free and other types are presented among the latest developments in the field. This multidisciplinary field requires engagement between scientists, engineers, clinicians and industry for addressing the challenges for a commercial, reliable, real-time-monitoring wearable insulin biosensor

Endogenous VEGF isoform expression regulates tumour cell motility

Background: Vascular endothelial growth factor-A (VEGF) is produced by most cancer cells as multiple alternatively spliced isoforms, which display distinct receptor and matrix binding characteristics. In addition to being a major inducer of tumour angiogenesis, VEGF also has complex functions in angiogenesis-independent aspects of tumour growth,but the role of individual VEGF isoforms remains poorly understood. Here we investigated the effects of endogenous VEGF isoform expression on tumour cell migration and invasion. Method: We used a panel of mouse fibrosarcoma cells we developed (fs188, fs164 and fs120) that express single VEGF isoforms (188, 164 or 120 respectively), under endogenous promoter control. We investigated adhesion to different matrices, 2D migration and invasion through 3D collagen. Results: Fs188 cells, are typically mesenchymal, form ruffles, display strong integrin-dependent adhesion and express high levels of pERK1/2 and pAKT. In contrast, fs164 and fs120 cells are not typically mesenchymal in morphology; they display weak binding to collagen, lack ruffles and align longitudinally forming long multicellular chains and abundant cell-cell contacts. On 3D collagen, fs188 cells remain mesenchymal while fs164 and fs120 cells adopt a rounded/amoeboid and a mix of rounded/mesenchymal morphologies respectively. Cell morphology and migration are dependent on the cytoskeleton and actinomyosin contractility, to provide traction force in mesenchymal movement, and cortical contraction for rounded amoeboid motility. Consistent with their mesenchymal characteristics, fs188 cells migrated faster in 2D and invaded 3D collagen more efficiently than fs164 or fs120 cells. Contractility inhibitors caused fs164/fs120 cells to switch to a mesenchymal morphology and accelerated their migration but not that of fs188 cells. Conclusion: VEGF isoforms are emerging as potential biomarkers for anti-VEGF therapies. Our results suggest that individual VEGF isoforms influence the migration and invasion strategies of tumour cells thus adding to the complexity of VEGF signaling within the tumor microenvironment. Acknowledgements This work was funded by Cancer Research UK

Segmentation and morphological analysis of microvessels in immunostained histological tumour sections

Segmentation and morphological analysis of microvessels in immunostained histological tumour sectionsA fully automatic segmentation and morphological analysis algorithm for the analy- sis of microvessels from CD31 immunostained histological tumour sections is presented. The algorithm exploited the distinctive hues of stained vascular endothelial cells, cell nuclei and background, which provided the seeds for a region-growing algorithm in the 3D Hue, Saturation, Value (HSV) colour model. The segmented objects, identified as microvessels by CD31 immunostaining, were post-processed with three morphological tasks: joining separate objects that were likely to belong to a single vessel, closing ob- jects that had a narrow gap around their periphery, and splitting objects with multiple lumina into individual vessels

The influence of hypoxia and energy depletion on the response of endothelial cells to the vascular disrupting agent combretastatin A-4-phosphate

Combretastatin A-4 phosphate (CA4P) is a microtubule-disrupting tumour-selective vascular disrupting agent (VDA). CA4P activates the actin-regulating RhoA-GTPase/ ROCK pathway, which is required for full vascular disruption. While hypoxia renders tumours resistant to many conventional therapies, little is known about its influence on VDA activity. Here, we found that active RhoA and ROCK effector phospho-myosin light chain (pMLC) were downregulated in endothelial cells by severe hypoxia. CA4P failed to activate RhoA/ROCK/pMLC but its activity was restored upon reoxygenation. Hypoxia also inhibited CA4P-mediated actinomyosin contractility, VE-cadherin junction disruption and permeability rise. Glucose withdrawal downregulated pMLC, and coupled with hypoxia, reduced pMLC faster and more profoundly than hypoxia alone. Concurrent inhibition of glycolysis (2-deoxy-D-glucose, 2DG) and mitochondrial respiration (rotenone) caused profound actin filament loss, blocked RhoA/ROCK signalling and rendered microtubules CA4P-resistant. Withdrawal of the metabolism inhibitors restored the cytoskeleton and CA4P activity. The AMP-activated kinase AMPK was investigated as a potential mediator of pMLC downregulation. Pharmacological AMPK activators that generate AMP, unlike allosteric activators, downregulated pMLC but only when combined with 2DG and/or rotenone. Altogether, our results suggest that Rho/ROCK and actinomyosin contractility are regulated by AMP/ATP levels independently of AMPK, and point to hypoxia/energy depletion as potential modifiers of CA4P response

Do Anti-Angiogenic VEGF (VEGFxxxb) Isoforms Exist? A Cautionary Tale

Splicing of the human vascular endothelial growth factor-A (VEGF-A) gene has been reported to generate angiogenic (VEGFxxx) and anti-angiogenic (VEGFxxxb) isoforms. Corresponding VEGFxxxb isoforms have also been reported in rat and mouse. We examined VEGFxxxb expression in mouse fibrosarcoma cell lines expressing all or individual VEGF isoforms (VEGF120, 164 or 188), grown in vitro and in vivo, and compared results with those from normal mouse and human tissues. Importantly, genetic construction of VEGF164 and VEGF188 expressing fibrosarcomas, in which exon 7 is fused to the conventional exon 8, precludes VEGFxxxb splicing from occurring. Thus, these two fibrosarcoma cell lines provided endogenous negative controls. Using RT-PCR we show that primers designed to simultaneously amplify VEGFxxx and VEGFxxxb isoforms amplified only VEGFxxx variants in both species. Moreover, only VEGFxxx species were generated when mouse podocytes were treated with TGFβ-1, a reported activator of VEGFxxxb splice selection in human podocytes. A VEGF164/120 heteroduplex species was identified as a PCR artefact, specifically in mouse. VEGFxxxb isoform-specific PCR did amplify putative VEGFxxxb species in mouse and human tissues, but unexpectedly also in VEGF188 and VEGF164 fibrosarcoma cells and tumours, where splicing to produce true VEGFxxxb isoforms cannot occur. Moreover, these products were only consistently generated using reverse primers spanning more than 5 bases across the 8b/7 or 8b/5 splice junctions. Primer annealing to VEGFxxx transcripts and amplification of exon 8b primer ‘tails’ explained the artefactual generation of VEGFxxxb products, since the same products were generated when the PCR reactions were performed with cDNA from VEGF164/VEGF188 ‘knock-in’ vectors used in the generation of single VEGF isoform-expressing transgenic mice from which the fibrosarcoma lines were developed. Collectively, our results highlight important pitfalls in data interpretation associated with detecting VEGFxxxb isoforms using current methods, and demonstrate that anti-angiogenic isoforms are not commonly expressed in mouse or human tissues