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Targeting novel lipid pathways for treatment of cardiovascular disease

Final Report Summary - ATHERO-FLUX (Targeting novel lipid pathways for treatment of cardiovascular disease)

Executive Summary:
Cardiovascular disease (CVD) causes much of the disease burden in Europe, claiming each year 4.3 million lives in Europe, 2.0 million in the EU (European Heart Network; www.ehnheart.org). The main forms of CVD are coronary artery disease (CAD) and stroke. CAD by itself is the single most common cause of death in Europe: accounting for 1.8 million deaths in Europe and 681,000 deaths in the EU per year (European Heart Network; www.ehnheart.org). Lipid lowering is the only therapeutic approach targeting the root cause of CVD, with statins achieving an impressive event reduction compared to other lipid lowering agents. Yet patients on high doses of statins still have a high residual CV risk, sparking attempts to mitigate this risk. However, alternatives and adjuncts to statins are difficult to develop, and complex problems were revealed whilst targeting the cholesteryl ester transfer protein (CEPT) pathway. New therapeutic targets in CVD are thus urgently required.
Athero-Flux was built on the lipidomics findings that a class of lipids, that is currently untargeted, is associated with significant CVD risk. Data generated by the “AtheroRemo” FP7 Consortium (http://www.atheroremo.org/) have revealed that specific sphingolipids (SLs) are associated with CV risk while others appear to be protective. Remarkably, their levels are a better predictor of clinical outcome than traditional risk factors such as low-density lipoprotein-cholesterol.
SLs are implicated in significant biological activities including cell survival, inflammation, and metabolic diseases. Moreover, their levels in metabolic diseases are modulated by previously unrecognized factors such as the gut microflora. Thus, we hypothesized that by controlling SL metabolism a better primary and secondary prevention of CVD events than with statins alone can be achieved.

Project Context and Objectives:
The Athero-Flux Consortium was aimed to generate new therapeutic targets and tools to address a hitherto unrecognised imbalance in lipid metabolism importantly linked to CV risk. It was built on the strengths of leading European SMEs with know-how in lipid metabolism and RNAi while giving them access to state-of-the-art models of disease and biological readouts and a whole new pipeline of therapeutic targets aligned with their priority areas. The Academic beneficiaries have taken advantage from collaboration with these companies with strong regulatory experience to accelerate the translation of their results into clinical applications. The Consortium has created a translational opportunity to turn lipidomics findings in large-scale clinical studies into new therapeutics for CVD. It also has elucidated the complex interaction between dyslipidaemia, atherosclerosis and inflammation essential for designing new therapeutic strategies for patients at risk or suffering from CVD.
Athero-Flux aimed to identify, characterise and validate novel therapeutic targets for CVD by dissecting the biosynthesis and pro-atherogenic potential of specific molecular species of SLs identified by lipidomics as strong predictors of CV risk in large-scale clinical studies.
Specific project objectives were:
1. To identify the steps regulating the generation and signalling of specific SLs relevant to CV risk
2. To validate therapeutic targets in ceramide metabolism and downstream signalling using atherosclerosis models in vitro and in vivo
3. To develop clinically applicable LNA-based therapeutics for the treatment of CVD and its acute complications

Project Results:
Over the 66 months, the Athero-Flux partners have developed LNAs against major AtheroFlux targets and they have succeeded in the modelling of ceramide production by the derivation of induced pluripotent stem cell (iPSC) derived hepatocytes that revealed differences in fatty acid turnover. Then the partners have built-up of a large scale siRNA library for the dissection of ceramide production and upstream inflammatory signalling pathways. The main S&T results are summarised below for each RTD WPs:

WP1. Discovery of regulatory nodes within SL biosynthesis in coronary artery disease via flux lipidomics. We have been able to successfully differentiate iPSCs into hepatocytes that closely resemble liver cells by both functionality and by lipid composition. Additionally, the efficient labelling of these cell lines with stable isotopes, targeted for sphingolipid metabolism aiming to facilitate tracer experiments, has been described by Viiri et al., 2019 in Science Report. This manuscript published in a peer-reviewed journal highlights the reprogramming of the transcriptome during the differentiation of iPSC into hepatocytes. Moreover, unique lipidomics platforms developed by Zora enabled us to absolutely quantify SL and ceramide species with distinct structural motifs. Thus, the turnover of 84 sphingolipids has been successfully monitored highlighting differences due to the length of the hydrophobic carbon chain. The analyses of the whole set of Flux lipidomic data combined with the gene assay performed in various cell lines highlighted differences in fatty acid turnover especially in ceramides.

WP2. Genome-scale RNAi screening to reveal rate-limiting pathways in ceramide metabolism and signalling. We have generated a large scale siRNA library targeting 569 genes coding namely protein kinases, ceramide synthases and genes involved in the inflammatory signaling cascades. The Upcyte immortalised primary human hepatocytes have been selected as the liver cell model for running the large scale RNAi screening. Thus in these cells, a screening of a library of siPOOLs targeting genes involved in ceramide and lipid biosynthesis combined to the use of quantitative proteomic analysis revealed approximately 5600 proteins related to the remodelling of IPCs from different patient groups during differentiation to iPSC-derived hepatocytes.
Moreover, the use of lipidomics platforms developed by Zora revealed the major role of 2 specific enzymes, a ceramide synthase and an elongase, in modulating the balance of very-long chain fatty acyl-containing sphingolipid species and long-chain fatty acyl-containing sphingolipids.

WP3. Study of the effect of prospective therapeutic targets within SL pathway on inflammatory signalling. Within the different reporting periods we have shown the crucial role of SL production in TNF signalling highlighting the important role of ceramide production in inflammatory signalling. Indeed, we have reported that ceramide synthase 2 (CerS2) null mice are unable to synthesize very-long acyl chain ceramides. Moreover, we have shown that CerS2 null mice are hypersensitive to LPS-mediated sepsis due to elevated TNFα secretion. This is due to elevated TNFα-converting enzyme (TACE) activity in both hepatocytes and macrophages. Consequently, this result highlights the major role of ceramide production in inflammatory signaling
Additionally, we have aimed to unravel the mechanisms responsible for the ceramide-induced inflammatory signalling. We have highlighted that the long-chain ceramides have been shown to be essential to elicit the proper signalling of some Pattern Recognition Receptors (PRRs). Moreover a specific ceramide synthase has been identified as pro-atherogenic trough its action on plaque formation.

WP4. Evaluation of biodistribution, bioactivity and pro-atherogenic properties of SLs. Significant results obtained in vivo in our CerS2 null mice model revealed the crucial role of CerS2 in the development of atherosclerosis, in insulin resistance and in lipid metabolism.
Moreover, we have succeeded in the identification of the ceramide species that accumulate in the aortic wall of both wild-type and hyperlipidaemic mice with atherosclerosis. Work on the effect of disruptions in ceramide synthesis in metabolism and atherosclerosis has highlighted a drastic impact on serum cholesterol and on the development of insulin resistance in mice. Innovative in vitro assays have been developed to aid the characterisation of the effects of gut microflora on sphingolipid metabolism and atherosclerosis. Investigations into whether specific components of the microbiome affect the secretion of ceramide species have successfully identified two bacteria of the genus Lactobacillus and Escherichia as major regulators. The underpinning molecular mechanisms of this modulation have been resolved and are intrinsic to the properties of each bacteria. Moreover, we have shown that pathways of inflammasome activation that sense danger and microbial stimuli are relevant to atherogenesis.

WP5 Development of LNA targeting SL metabolism and its associated signalling. In WP5, LNAs against various targets involved in sphingolipid metabolism and in PRR inflammatory signalling pathways have been synthesised. A dozen of LNAs that display powerful knockdown of the target in vitro and in vivo, two of which have safe pharmacology in mouse, have been generated and are ready for use by the beneficiaries of the consortium.
As a major target of the AtheroFlux consortium, CerS2 has been used in vivo in combination to lipidomics to decipher the dynamics of SL production in the presence and absence of CerS2 functionality. The analysis first demonstrated that the knockdown operated by the different GalNac conjugated LNA compounds is efficient ranging from 60% to 75% in primary hepathocytes. This efficient knockdown led to a drastic reduction of the long-chain ceramide C24 after only 2-3 weeks of treatments.

Potential Impact:
Potential Impact
The Athero-Flux project has addressed the challenge of the call, i.e. “HEALTH.2013.2.4.2-1: Discovery research to reveal novel targets for cardiovascular disease treatment.” by further characterisation and validation of therapeutic targets emerging from the use of lipidomics in large-scale clinical studies in CVD. The Consortium has pioneered research into the role of SL metabolism in metabolic disease and CV risk that has shown for the first time that ceramides with specific chain lengths are implicated in CVD. All Athero-Flux project participants promote translational medicine, providing access to clinical material and the necessary environment to nurture basic and clinical science with necessary multidisciplinary infrastructure that is required to conduct top-rate research at an international level. Athero-Flux has contributed to National and European Health by putting into practice new knowledge into hitherto untargeted lipids with strong prognostic predictive value in large scale clinical studies and ultimately generating new therapeutic tools with the latest oligonucleotide technology for gene silencing.
The most important impact of this project was the discovery of new therapeutic targets within SL metabolism that may abate the risk of previously unrecognized imbalances leading to the formation of structurally distinct ceramides. By defining in detail which sphingolipids, ceramides and their synthetic enzymes are really important in pathogenesis of cardiovascular disease we have identified new potential therapeutic targets and validated these as targets to define new therapeutics for a major disease. With help of RICC (formerly Santaris) we were able to test blockade of targets both in vitro and in animals leading to a portfolio of 3rd generation antisense drugs that can enter a pipeline of clinical development and prelude to the generation of further therapeutics.
A second area of impact was the boost of the competitiveness of EU-based SMEs by strengthening the SME pipeline with opportunities to capitalize on unique research platforms (human samples, iPSCs and mouse models) and scientific discoveries in need of effective translation to the clinic. The planned workflow enabled a two-way integration between the Academic and SME capabilities. The planned Academic and SME partnership has feed a significant number of potential targets into the SME pipeline.
The novelty of this project was the unique and multidisciplinary blend of academic and technical skills. Athero-Flux was buildt on established collaborations in previous EU Consortia but has enriched their interaction by bringing in experts in SL metabolism, high-throughput RNAi screening, LNA platforms and gut microflora in a true multidisciplinary effort to achieve better treatment for CVD beyond hypolipidemic drugs. Technological advance also took center stage in Athero-Flux: novel lipidomics platforms have defined the kinetic of the many existing ceramides and shed light on their production, while regulatory check-points were validated with innovative LNA platforms and further defined with high-resolution cell phenotyping and signaling analysis with CyTOF. This injection of expertise and technological advances have fuel significant progress towards solving existing challenges in CVD, such as the deep relationship between lipid metabolism and signaling and inflammation that is fueling an increase of metabolic diseases in EU and worldwide.
The validation capabilities of the Consortium were extensive and included a wealth of accredited in vitro human (i.e. iPSC-derived hepatocytes and macrophages from patients with CAD, atheroma and tissue cell cultures) and in vivo murine models of atherosclerosis and CVD (i.e.atherosclerosis via hyperlipidemia; arterial injury; stable vs. vulnerable plaque development under shear stress modulation) that are well suited to preclinical validation. Academic beneficiaries have established readouts with prognostic meaning in clinical cohorts that have correspondence in murine models.

Dissemination activities
Since the beginning of the project, the dissemination activities were aimed at enhancing visibility of the consortium and of the project, mainly for informing the scientific community and stakeholders and for communicating the value of the research funded by the European Community through FP7.
The main dissemination approaches from the project start included:

• Development of a Project website
The project website was created with its own domain: www.atheroflux.eu. The homepage summarizes the main focus and structure of Athero-Flux. Using the menu of the home page it is possible to access the main sessions of the public area:
The partners page describes the composition of the Athero-Flux consortium and contains email address of the scientists involved and a link to their organization.
The publications page list all the papers derived from the Athero-Flux project.
The reserved area is intended as a tool of information and updating for scientists; the access is restricted with a password in order to protect and dedicate to internal project communication. The project website has been updated periodically.

• Development of Project information material
A Brochure of the project was prepared by beneficiary 10-ALTA, that was distributed to partners in the occasion of project meetings.
ALTA has also designed the logo that visually identifies the project and that has been used in occasion of meetings, posters, and communication activities.

• Organisation of joint events
A joint AtheroSymposium was organized on September 22nd, 2014 in Siena (Italy) for Athero-Flux and Athero B Cell projects both focused on cardiovascular research and coordinated by the University of Oxford. Several lectures were organized on subjects related to cardiovascular diseases such as the development of new therapeutics, and the role of sphingolipids and microflora on these pathologies.
A final joint Cardiovascular Inflammation/Immunometabolism Workshop coordinated by Athero-Flux and Athero B Cell was organised in Evia (Greece) from 16th to 18th June 2018 to discuss the recent working theories and advancement in the field with excellent speakers both from within the consortium and externally.
• Participation at scientific events
see list in A2.

• Publications/chapter of books/reviews
See A1 for publications.

Exploitation activities
Several of Athero-Flux deliverables lend themselves clearly to successful exploitation. Namely these include:
1. Validation and standardization of high throughput screening assay in human hepatocytes and iPSC-derived hepatocytes and macrophages
2. Identification and validation of novel therapeutically relevant targets for CVD pertaining to SL metabolism and signalling
3. Identification of SLs as NKT cell antigens that could be suitable for generation of innovative vaccines against atherosclerosis development
4. Proof of efficacy and safety on CVD experimental models of new LNAs against selected targets
5. Generation and validation of an innovative CVD experimental model (ApoE-/- CerS2-/- mice) that could suitable for identification of new drugs for CVD targeting SL metabolism signalling
6. Identification of gut mutant bacteria as target and/or modulator of metabolic factors and SL synthesis in CVD.

We have obtained important results (particularly in our WP5 with the validation in vivo of the LNA against CerS2, one of the major target of the AtheroFlux consortium because of its crucial role in the development of atherosclerosis, in insulin resistance and in lipid metabolism as revealed in our WP4), which we anticipate could find applications in patients affected by CVD and populations at high-risk of developing CVD.

7 patents (already granted or filing ongoing) have been produced within Athero-Flux (see section 4.2 “Use and dissemination of foreground).

List of Websites:
http://www.atheroflux.eu/

Contractors involved:

UNITED KINGDOM
The Chancellor, Masters and Scholars of the University of Oxford- Marc Feldmann, Claudia Monaco

DENMARK
Roche Innovation Center Copenhagen A/S- Steffen Schmidt
Syddansk Universitet- Ole N. Jensen, Christer Ejsing

FINLAND
Pikkanmaa Hospital District- Katriina Aalto-Setälä, Leena Viiri
Zora Biosciences Ltd- Rejio Laaksonen
FRANCE
Institut Pasteur- Philippe J. Sansonetti
GERMANY
siTools Biotech GMBH- Michael Hannus
GREECE
Biomedical Reserach Foundation Academy of Athens- Evangelos Andreakos
ISRAEL
Weizmann Institute of Science- Tony Futerman
ITALY
ALTA Ricerca e Sviluppo in Biotecnologie Srlu- Paola Cesaroni
SWEDEN
Karolinska Institutet- Goran K. Hansson, Zhong-Qun Yan

Coordinator contact details
The Chancellor, Masters and Scholars of the University of Oxford
Prof Marc Feldmann
Head, Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford,
Roosevelt Drive
Headington Oxford, OX3 7FY