Radiation therapy attenuates lymphatic vessel repair by reducing VEGFR-3 signalling

Introduction: Surgery and radiotherapy are key cancer treatments and the leading causes of damage to the lymphatics, a vascular network critical to fluid homeostasis and immunity. The clinical manifestation of this damage constitutes a devastating side-effect of cancer treatment, known as lymphoedema. Lymphoedema is a chronic condition evolving from the accumulation of interstitial fluid due to impaired drainage via the lymphatics and is recognised to contribute significant morbidity to patients who survive their cancer. Nevertheless, the molecular mechanisms underlying the damage inflicted on lymphatic vessels, and particularly the lymphatic endothelial cells (LEC) that constitute them, by these treatment modalities, remain poorly understood.Methods: We used a combination of cell based assays, biochemistry and animal models of lymphatic injury to examine the molecular mechanisms behind LEC injury and the subsequent effects on lymphatic vessels, particularly the role of the VEGF-C/VEGF-D/VEGFR-3 lymphangiogenic signalling pathway, in lymphatic injury underpinning the development of lymphoedema.Results: We demonstrate that radiotherapy selectively impairs key LEC functions needed for new lymphatic vessel growth (lymphangiogenesis). This effect is mediated by attenuation of VEGFR-3 signalling and downstream signalling cascades. VEGFR-3 protein levels were downregulated in LEC that were exposed to radiation, and LEC were therefore selectively less responsive to VEGF-C and VEGF-D. These findings were validated in our animal models of radiation and surgical injury.Discussion: Our data provide mechanistic insight into injury sustained by LEC and lymphatics during surgical and radiotherapy cancer treatments and underscore the need for alternative non-VEGF-C/VEGFR-3-based therapies to treat lymphoedema

Next Generation Cell Culture Tools Featuring Micro- and Nanotopographies for Biological Screening

Cells are able to perceive complex mechanical cues across both the micro- and nanoscale which can influence their development. Whilst causative effects between surface topography and cellular function can be demonstrated, the variability in materials used in this screening process makes it difficult to discern whether the observed phenotypic changes are indeed a result of topographical cues alone, or the inherent difference in material properties. A novel approach to directly imprint both micro- and nanoscaled topographical features into the base of conventional cell cultureware is thus developed, facilitating its compatibility with standard biological techniques and methods of analysis. The utility of this technology is demonstrated by performing high-throughput screening across five distinct cell types to interrogate the effects of 12 surface topographies, exemplifying unique cell specific responses to both behavior and cell morphological characteristics. The ability of this technology to underpin new insights into how surface topographies can regulate key image descriptors to drive cell fate determination is further demonstrated. These findings will inform the future development of advanced micro- and nanostructured cell culture substrates that can regulate cell behavior and fate determination across the life sciences, including fundamental cell biology, drug screening, and cell therapy

Screening of the 'Open Scaffolds' collection from Compounds Australia identifies a new chemical entity with anthelmintic activities against different developmental stages of the barber's pole worm and other parasitic nematodes

The discovery and development of novel anthelmintic classes is essential to sustain the control of socioeconomically important parasitic worms of humans and animals. With the aim of offering novel, lead-like scaffolds for drug discovery, Compounds Australia released the 'Open Scaffolds' collection containing 33,999 compounds, with extensive information available on the physicochemical properties of these chemicals. In the present study, we screened 14,464 prioritised compounds from the 'Open Scaffolds' collection against the exsheathed third-stage larvae (xL3s) of Haemonchus contortus using recently developed whole-organism screening assays. We identified a hit compound, called SN00797439, which was shown to reproducibly reduce xL3 motility by ≥ 70%; this compound induced a characteristic, "coiled" xL3 phenotype (IC50 = 3.46-5.93 μM), inhibited motility of fourth-stage larvae (L4s; IC50 = 0.31-12.5 μM) and caused considerable cuticular damage to L4s in vitro. When tested on other parasitic nematodes in vitro, SN00797439 was shown to inhibit (IC50 = 3-50 μM) adults of Ancylostoma ceylanicum (hookworm) and first-stage larvae of Trichuris muris (whipworm) and eventually kill (>90%) these stages. Furthermore, this compound completely inhibited the motility of female and male adults of Brugia malayi (50-100 μM) as well as microfilariae of both B. malayi and Dirofilaria immitis (heartworm). Overall, these results show that SN00797439 acts against genetically (evolutionarily) distant parasitic nematodes i.e. H. contortus and A. ceylanicum [strongyloids] vs. B. malayi and D. immitis [filarioids] vs. T. muris [enoplid], and, thus, might offer a novel, lead-like scaffold for the development of a relatively broad-spectrum anthelmintic. Our future work will focus on assessing the activity of SN00797439 against other pathogens that cause neglected tropical diseases, optimising analogs with improved biological activities and characterising their targets

Dual microRNA Screens Reveal That the Immune-Responsive miR-181 Promotes Henipavirus Entry and Cell-Cell Fusion

<div><p>Hendra and Nipah viruses (family <i>Paramyxoviridae</i>, genus <i>Henipavirus</i>) are bat-borne viruses that cause fatal disease in humans and a range of other mammalian species. Gaining a deeper understanding of host pathways exploited by henipaviruses for infection may identify targets for new anti-viral therapies. Here we have performed genome-wide high-throughput agonist and antagonist screens at biosafety level 4 to identify host-encoded microRNAs (miRNAs) impacting henipavirus infection in human cells. Members of the miR-181 and miR-17~93 families strongly promoted Hendra virus infection. miR-181 also promoted Nipah virus infection, but did not affect infection by paramyxoviruses from other genera, indicating specificity in the virus-host interaction. Infection promotion was primarily mediated via the ability of miR-181 to significantly enhance henipavirus-induced membrane fusion. Cell signalling receptors of ephrins, namely EphA5 and EphA7, were identified as novel negative regulators of henipavirus fusion. The expression of these receptors, as well as EphB4, were suppressed by miR-181 overexpression, suggesting that simultaneous inhibition of several Ephs by the miRNA contributes to enhanced infection and fusion. Immune-responsive miR-181 levels was also up-regulated in the biofluids of ferrets and horses infected with Hendra virus, suggesting that the host innate immune response may promote henipavirus spread and exacerbate disease severity. This study is the first genome-wide screen of miRNAs influencing infection by a clinically significant mononegavirus and nominates select miRNAs as targets for future anti-viral therapy development.</p></div

Select Eph receptors inhibit HeV infection and cell-cell-fusion and are miR-181 target genes.

<p>(A) Relative mRNA levels of indicated target genes in HeLa cells 72 h post-transfection with siRNAs (40 nM). ***p<0.001 compared to siNEG (B) Relative percentage of cells infected with HeV (24 h, MOI 0.5), after 72 h transfection with siRNAs targeting indicated molecules. *p<0.05, ***p<0.001 compared to siNEG. (C) Relative mRNA levels of Eph receptors A4, A5, A7 and B4 in HeLa cells, 72 h post transfection with miRNA agonists (25 nM). N.s. not significant; **p<0.01, ***p≤0.001, compared to control agonist. (D) Cell-cell fusion of HeV-F and–G expressing HEK-293T cells to HeLa cells treated with indicated siRNAs. Values are normalised as a percentage to siNEG or control agonist.</p

miR-181d promotes henipavirus infection specifically.

<p>Infectivity assays were applied to assess changes in virus production or virus infection of HeLa cells infected with NiV, MeV, MuV, RSV or influenza A/WSN/33 virus for 24 h. Cells were previously transfected with miR-181d or negative control agonists (miNEG) for 72 h. **p≤0.01; n.s. not significant.</p

miR-181 significantly enhances HeV RNA synthesis and F- and G-mediated cell-cell fusion.

<p>(A) qRT-PCR measurements of intracellular viral RNA copy number in HeLa cells infected with HeV (MOI 5). ***p≤0.001; *p≤0.05. HeV RNA values were normalised to cellular 18S levels. (B) Cell-to-cell fusion of HeV-F and HeV-G-expressing HEK-293T effector cells to HeLa cells treated with indicated siRNA or miRNA agonists. Syncytia were imaged using automated fluorescence microscopy. Nuclei are shown in blue (DAPI), effector cells in green (HeV-G staining) and target cells red (DiO lipid dye). (f1) and (f2) are images of cells transfected with miR-181d from two different microscopy fields. (C) Quantification of fusion events by counting all nuclei present in all syncytia. Values are normalised as a percentage to siNEG or miNEG. *p ≤ 0.05, ***p ≤0.001 compared to respective negative controls.</p

Expression levels of miR-181 in biofluids of animals infected with HeV are increased.

<p>Sixteen ferrets were infected with HeV at BSL-4. At predetermined time-points, ten different tissues were harvested and analysed for viral RNA loads by qRT-PCR (A). (B) qRT-PCR analysis of miR-181d levels in the serum samples of the ferrets. Values were normalized to the U6 RNA. *p≤0.05 compared to day 0. (C) qRT-PCR analysis of miR-181d in blood of horses from a published HeV infection study [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005974#ppat.1005974.ref052" target="_blank">52</a>].</p

miR-17 promotes henipavirus infection but does not enhance HeV F- and G-mediated cell-cell fusion.

<p>(A) Percentage of cells infected with HeV or RSV (24 h, MOI 1), after 72 h transfection with miR-17, miR-93 or control agonists. ***p≤0.001, **p≤0.01, *p≤0.05 compared to control agonist. (B) TCID₅₀ virus titres of supernatants derived from HeLa cells infected with HeV for 24 h (MOI 1), at 72 h post-transfection with agonists. *p≤0.05 compared to control agonist. (C) Cell-cell fusion of HeV-F and -G expressing HEK-293T cells to HeLa cells treated with indicated siRNA or miRNA agonists. Syncytia were imaged using automated fluorescence microscopy. Nuclei are shown in blue (DAPI) and effector cells in green (HeV-G staining). (D) Quantification of fusion events by counting of all nuclei present in all syncytia. Values are normalised as a percentage to siNEG or control agonist. n.s. not significant; *p≤0.05, **p≤0.01; ***p≤0.001 compared to respective negative controls.</p

Genome-wide complementary agonist and antagonist screens of host-encoded miRNAs impacting henipavirus infection at BSL-4.

<p>(A) Schematic of optimized protocol for performing functional RNAi screens with an infectious BSL-4 virus. Two sets of daughter plates were generated from library plates consisting of 1,239 miRNA agonists (grey plates) and 1,225 antagonists (black plates). HeLa cells were added to both plate sets for reverse transfection of the miRNA agonists and antagonists. 72 h post transfection, one set of plates were processed at BSL-2 for cell viability analysis by DAPI staining. The other set was then transferred into BSL-4 (red box), infected with luciferase-expressing HeV, and lysed for luminescence reading at 24 hours post-infection (h.p.i.) in BSL-4. (B and C) Results from the miRNA agonist (B) and antagonist (C) screens, with miRNAs ranked using a robust Z-score approach, from lowest (decreased virus infection) to highest (increased virus infection). Dotted horizontal lines represent the threshold of hit identification (Z ≥ 2 or ≤ -2). The number of miRNA hits above this threshold is shown. (D) Venn diagram identifying pro- and anti-viral miRNAs from both screens.</p