Basic science232. Certolizumab pegol prevents pro-inflammatory alterations in endothelial cell function

Background: Cardiovascular disease is a major comorbidity of rheumatoid arthritis (RA) and a leading cause of death. Chronic systemic inflammation involving tumour necrosis factor alpha (TNF) could contribute to endothelial activation and atherogenesis. A number of anti-TNF therapies are in current use for the treatment of RA, including certolizumab pegol (CZP), (Cimzia ®; UCB, Belgium). Anti-TNF therapy has been associated with reduced clinical cardiovascular disease risk and ameliorated vascular function in RA patients. However, the specific effects of TNF inhibitors on endothelial cell function are largely unknown. Our aim was to investigate the mechanisms underpinning CZP effects on TNF-activated human endothelial cells. Methods: Human aortic endothelial cells (HAoECs) were cultured in vitro and exposed to a) TNF alone, b) TNF plus CZP, or c) neither agent. Microarray analysis was used to examine the transcriptional profile of cells treated for 6 hrs and quantitative polymerase chain reaction (qPCR) analysed gene expression at 1, 3, 6 and 24 hrs. NF-κB localization and IκB degradation were investigated using immunocytochemistry, high content analysis and western blotting. Flow cytometry was conducted to detect microparticle release from HAoECs. Results: Transcriptional profiling revealed that while TNF alone had strong effects on endothelial gene expression, TNF and CZP in combination produced a global gene expression pattern similar to untreated control. The two most highly up-regulated genes in response to TNF treatment were adhesion molecules E-selectin and VCAM-1 (q 0.2 compared to control; p > 0.05 compared to TNF alone). The NF-κB pathway was confirmed as a downstream target of TNF-induced HAoEC activation, via nuclear translocation of NF-κB and degradation of IκB, effects which were abolished by treatment with CZP. In addition, flow cytometry detected an increased production of endothelial microparticles in TNF-activated HAoECs, which was prevented by treatment with CZP. Conclusions: We have found at a cellular level that a clinically available TNF inhibitor, CZP reduces the expression of adhesion molecule expression, and prevents TNF-induced activation of the NF-κB pathway. Furthermore, CZP prevents the production of microparticles by activated endothelial cells. This could be central to the prevention of inflammatory environments underlying these conditions and measurement of microparticles has potential as a novel prognostic marker for future cardiovascular events in this patient group. Disclosure statement: Y.A. received a research grant from UCB. I.B. received a research grant from UCB. S.H. received a research grant from UCB. All other authors have declared no conflicts of interes

Utilising high throughput screening techniques to identify small molecule activators of the epicardium

The epicardium, the mesothelial layer covering the surface of the heart, plays an essential role during heart development. Epicardium derived cells (EPDCs) contribute essential cardiovascular cell types including vascular smooth muscle, interstitial fibroblasts and cardiomyocytes via the process of epithelial to mesenchymal transition (EMT) and EPDC paracrine signalling is critical for proper organ formation. Whilst dormant in the adult heart, the epicardium is reactivated in response to injury in both mouse and zebrafish. Activation is characterised by epicardial expansion, EMT, EPDC migration and re-expression of embryonic transcription factors including WT1. Moreover, priming the mouse heart with thymosin β4 (Tβ4) increases the number of Wt1+ EPDCs in vivo following myocardial infarction. Subsequently, small numbers of Tβ4 activated Wt1+ cells migrate into the wound forming functional cardiomyocytes. Unfortunately, the number of functional EPDC-derived cardiomyocytes is suboptimal to restore the lost heart muscle; therefore, the aim of this project was to augment the process using chemical or genetic modulation. To realise this aim, a high throughput phenotypic screen utilising primary human epicardium derived cells (hEPDCs) was developed in collaboration with the Target Discovery Institute. Recombinant transforming growth factor beta (TGFβ) was used to induce EMT in hEPDC cultures; morphological changes indicative of EMT were used as a surrogate read out for hEPDC activation. A rigorous analysis protocol was successfully developed, however, ongoing issues with access to primary human tissue led to investigation of alternate in vitro models of epicardial EMT. Preliminary investigations into an EPDC line derived from rat proved problematic and due to numerous inconsistencies in the model an alternate cell line was sought. Success was found with an immortalised EPDC line derived from mouse (mEPDC), which reproducibly underwent EMT in response to TGFβ treatment. However, mEPDC EMT was not accompanied by morphological changes as pronounced as in hEPDC cultures, thus the mouse line was deemed unsuitable for the previously described phenotypic screen. Moving forward, a high throughput scratch assay was developed which utilised migration as a surrogate read out for mEPDC activation. Upon screening of a small candidate library, two Bromodomain and Extra-Terminal motif (BET) inhibitors were found to reproducibly increase the speed of mEPDC wound closure. Screening a wider panel of BET inhibitors yielded several candidates that increased the rate of closure in a dose-responsive manner. To determine whether increased migration was correlated with a change in mEPDC phenotype the cells were cultured with an 'effective' dose of inhibitor and probed for a panel of differentiation markers. Several BET inhibitors were found to significantly upregulate expression of the EMT associated transcription factor Snai2 after 24 hours of treatment compared with the control. Together, the observed increase in cell migration and expression of the Snai2 transcription factor suggest a role for the BET bromodomains in epicardial activation and EMT.</p

Utilising high throughput screening techniques to identify small molecule activators of the epicardium

The epicardium, the mesothelial layer covering the surface of the heart, plays an essential role during heart development. Epicardium derived cells (EPDCs) contribute essential cardiovascular cell types including vascular smooth muscle, interstitial fibroblasts and cardiomyocytes via the process of epithelial to mesenchymal transition (EMT) and EPDC paracrine signalling is critical for proper organ formation. Whilst dormant in the adult heart, the epicardium is reactivated in response to injury in both mouse and zebrafish. Activation is characterised by epicardial expansion, EMT, EPDC migration and re-expression of embryonic transcription factors including WT1. Moreover, priming the mouse heart with thymosin &beta;4 (T&beta;4) increases the number of Wt1+ EPDCs in vivo following myocardial infarction. Subsequently, small numbers of T&beta;4 activated Wt1+ cells migrate into the wound forming functional cardiomyocytes. Unfortunately, the number of functional EPDC-derived cardiomyocytes is suboptimal to restore the lost heart muscle; therefore, the aim of this project was to augment the process using chemical or genetic modulation. To realise this aim, a high throughput phenotypic screen utilising primary human epicardium derived cells (hEPDCs) was developed in collaboration with the Target Discovery Institute. Recombinant transforming growth factor beta (TGF&beta;) was used to induce EMT in hEPDC cultures; morphological changes indicative of EMT were used as a surrogate read out for hEPDC activation. A rigorous analysis protocol was successfully developed, however, ongoing issues with access to primary human tissue led to investigation of alternate in vitro models of epicardial EMT. Preliminary investigations into an EPDC line derived from rat proved problematic and due to numerous inconsistencies in the model an alternate cell line was sought. Success was found with an immortalised EPDC line derived from mouse (mEPDC), which reproducibly underwent EMT in response to TGF&beta; treatment. However, mEPDC EMT was not accompanied by morphological changes as pronounced as in hEPDC cultures, thus the mouse line was deemed unsuitable for the previously described phenotypic screen. Moving forward, a high throughput scratch assay was developed which utilised migration as a surrogate read out for mEPDC activation. Upon screening of a small candidate library, two Bromodomain and Extra-Terminal motif (BET) inhibitors were found to reproducibly increase the speed of mEPDC wound closure. Screening a wider panel of BET inhibitors yielded several candidates that increased the rate of closure in a dose-responsive manner. To determine whether increased migration was correlated with a change in mEPDC phenotype the cells were cultured with an 'effective' dose of inhibitor and probed for a panel of differentiation markers. Several BET inhibitors were found to significantly upregulate expression of the EMT associated transcription factor Snai2 after 24 hours of treatment compared with the control. Together, the observed increase in cell migration and expression of the Snai2 transcription factor suggest a role for the BET bromodomains in epicardial activation and EMT.</p

Structure-based design of selective fat mass and obesity associated protein (FTO) inhibitors

FTO catalyzes the Fe(II) and 2-oxoglutarate (2OG)-dependent modification of nucleic acids, including the demethylation of N6-methyladenosine (m6A) in mRNA. FTO is a proposed target for anti-cancer therapy. Using information from crystal structures of FTO in complex with 2OG and substrate mimics, we designed and synthesized two series of FTO inhibitors, which were characterized by turnover and binding assays, and by X-ray crystallography with FTO and the related bacterial enzyme AlkB. A potent inhibitor employing binding interactions spanning the FTO 2OG and substrate binding sites was identified. Selectivity over other clinically targeted 2OG oxygenases was demonstrated, including with respect to the hypoxia-inducible factor prolyl and asparaginyl hydroxylases (PHD2 and FIH) and selected JmjC histone demethylases (KDMs). The results illustrate how structure-based design can enable the identification of potent and selective 2OG oxygenase inhibitors and will be useful for the development of FTO inhibitors for use in vivo

Structure-based design of selective fat mass and obesity associated protein (FTO) inhibitors

FTO catalyzes the Fe(II) and 2-oxoglutarate (2OG)-dependent modification of nucleic acids, including the demethylation of N6-methyladenosine (m6A) in mRNA. FTO is a proposed target for anti-cancer therapy. Using information from crystal structures of FTO in complex with 2OG and substrate mimics, we designed and synthesized two series of FTO inhibitors, which were characterized by turnover and binding assays, and by X-ray crystallography with FTO and the related bacterial enzyme AlkB. A potent inhibitor employing binding interactions spanning the FTO 2OG and substrate binding sites was identified. Selectivity over other clinically targeted 2OG oxygenases was demonstrated, including with respect to the hypoxia-inducible factor prolyl and asparaginyl hydroxylases (PHD2 and FIH) and selected JmjC histone demethylases (KDMs). The results illustrate how structure-based design can enable the identification of potent and selective 2OG oxygenase inhibitors and will be useful for the development of FTO inhibitors for use in vivo

Genetic profiling and surface proteome analysis of human atrial stromal cells and rat ventricular epicardium-derived cells reveals novel insights into their cardiogenic potential

Epicardium-derived cells (EPDC) and atrial stromal cells (ASC) display cardio-regenerative potential, but the molecular details are still unexplored. Signals which induce activation, migration and differentiation of these cells are largely unknown. Here we have isolated rat ventricular EPDC and rat/human ASC and performed genetic and proteomic profiling. EPDC and ASC expressed epicardial/mesenchymal markers (WT-1, Tbx18, CD73, CD90, CD44, CD105), cardiac markers (Gata4, Tbx5, troponin T) and also contained phosphocreatine. We used cell surface biotinylation to isolate plasma membrane proteins of rEPDC and hASC, Nano-liquid chromatography with subsequent mass spectrometry and bioinformatics analysis identified 396 rat and 239 human plasma membrane proteins with 149 overlapping proteins. Functional GO-term analysis revealed several significantly enriched categories related to extracellular matrix (ECM), cell migration/differentiation, immunology or angiogenesis. We identified receptors for ephrin and growth factors (IGF, PDGF, EGF, anthrax toxin) known to be involved in cardiac repair and regeneration. Functional category enrichment identified clusters around integrins, PI3K/Akt-signaling and various cardiomyopathies. Our study indicates that EPDC and ASC have a similar molecular phenotype related to cardiac healing/regeneration. The cell surface proteome repository will help to further unravel the molecular details of their cardio-regenerative potential and their role in cardiac diseases

Fish assemblage response to rehabilitation of a sand-slugged lowland river

The impact of excessive sediment supply on river channels has been&nbsp; described in many areas of the world. Sediment deposition disturbance alters habitat&nbsp; structure by decreasing channel depth, changing substrate composition and burying woody debris. River rehabilitation is occurring worldwide, but information is scant on fish assemblage responses to rehabilitation in sedimentdisturbed lowland rivers. Sediment removal and large woody debris (LWD) replacement&nbsp; were used to experimentally rehabilitate habitat along a 1500m stretch of the Glenelg River in western Victoria, Australia. Using an asymmetrical before-after control-impact (BACI) design, fish were captured before and after the reach was rehabilitated, from two control reaches and from a &lsquo;higher quality&rsquo; reference reach. After two years post-rehabilitation monitoring, the fish assemblage at the rehabilitated reach did not differ from control reaches. Temporal changes in taxa richness and the abundance of Philypnodon grandiceps, Nannoperca spp. and three angling taxa occurred after rehabilitation (winter 2003) compared with the before period (winter 2002), but these effects did not differ between rehabilitated and control locations. Highest taxa richness and abundances occurred at the reference location. High salinity coincided with the timing of rehabilitation works, associated with low river discharges due to drought. The negative effects of other large-scale disturbances may have impaired the effectiveness of reachscale rehabilitation or the effects of rehabilitation may take longer than two years to develop in a lowland river subjected to multiple environmental disturbances