Targeting immunomodulation following cardiac injury

Cardiovascular diseases cause more than a quarter of all deaths in the UK, (approximately 170,000 deaths each year - an average of 460 deaths each day or one every three minutes). Of these, coronary heart disease is the most common and is the leading cause of heart attack. Heart attack manifests as massive death of muscle cells, upwards of around one billion, accounting for around 25% loss of the total heart muscle. There is no means to replace these lost cells and consequently the injury is patched-up by the formation of a non-contractile, scar to prevent rupture of the wall of the heart. This, in turn, increases the burden on survived muscle and results in increased cell size, wall thinning, dilation of the chambers and ultimately progression to heart failure. Currently there are around 900,000 people living in the UK with heart failure; organ transplantation is the only current long-term solution, but is complicated by immune rejection and the fact that demand continually outstrips the availability of donor hearts. Alternative regenerative approaches have centred on the replacement of lost cells generated from a variety of sources (bone marrow, fat tissue and skeletal muscle), however, these have largely failed, with disappointing clinical trial results. One reason for this is that the local environment of the injured heart becomes highly inflamed and scarred due to the invasion of immune cells which in turn leads to further cell death and failure to support the integration of new cells into survived tissue.

Elsewhere in the body immune cells are cleared to draining lymph nodes by lymphatic vessels after injury (for eg. following skin excision injury). We recently discovered that the lymphatics of the heart respond to injury in mice by sprouting and further identified that these expanding vessels function to clear immune cells which, when increased by growth factor treatment, improved the outcome after a heart attack. In this proposal, we seek to determine whether timed stimulation of the growth of cardiac lymphatics can define the optimal window for intervention after injury and which types of immune cells are best retained in the heart versus cleared lymph nodes to optimise heart repair and function after a heart attack. Correlating timed clearance of precisely defined immune cells with outcome, will enable us to uniquely attribute function to different subsets of immune cells as an important insight. We will also model human lymphatic vessels and their interactions with immune cells and screen these models for drug compounds which might activate the lymphatics. This will represent the first stage of a drug-discovery pipeline targeting the local environment, to enable cell repopulation and tissue restoration, as part of a combined therapy to treat heart attack patients.

Regenerative medicine approaches to cardiovascular disease have largely centred on replacement of lost cardiovascular cells, with little attention paid to the local environment into which the new cells emerge: a cytotoxic mixture of inflammation and fibrosis which prevents engraftment and integration with survived heart tissue. We demonstrated previously that the cardiac lymphatic vasculature can respond to cardiac injury (Klotz et al., 2015. Nature) and more recently that increased lymphatic vessel sprouting (lymphangiogenesis) functions to clear immune cells to draining lymph nodes and constrain the inflammatory and reparative responses for improved outcome (Vieira et al., 2018. JCI). We now hypothesis that the cardiac lymphatics can be utilised to define functionally-relevant immune cell sub-types following cardiac injury. To test this, we will establish a time course of cardiac lymphatic vessel induction and analyses of lymphatic-compromised mouse models (Prox1-haploinsufficiency- a model of impaired lymphangiogenesis and Lyve-1 mutants- a model of impaired immune cell uptake) post-myocardial infarction (MI) to probe the temporal effects of immune cell clearance and resolution, focusing specifically on macrophages, dendritic cells and T-cells. Using combined single-cell transcriptomics and CYTOF imaging, we will identify precisely which sub-populations are cleared at which time points and how altered clearance correlates with outcome. To extrapolate to humans, we propose to study a model of human lymphatic vessels and their role in improving cardiac function post-MI. Finally, we will carry-out an unbiased chemical screen for novel activators of human lymphatics to establish the basis for promoting therapeutic lymphangiogenesis in human MI patients.

This MRC Programme application seeks to understand mechanistically how increased lymphangiogenesis and altered immune cell load in the heart results in improved prognosis following a heart attack and to develop ways to enhance this further and optimise tissue repair and regeneration. Our goals at the end of the 5-years are to identify lymphatic-based immune cell clearance as a determinant of improved cardiac repair; define functional subsets of immune cells as correlates with prognosis post-MI; to identify a window for optimal immunomodulation and induction of a "hospitable" environment for adjunct cell-based tissue repair and ultimately to seed strategies to promote therapeutic lymphangiogenesis in human AMI patients.

Realising the therapeutic potential of this programme is important because currently there are no successful strategies in the clinic for effective immunomodulation to improve prognosis in patients following a heart attack. To-date, relatively blunt approaches to target inflammation post-MI utilising corticosteroids, non-steroidal anti-inflammatories, or non-specific immunosuppression (cyclosporine, methotrexate), have largely failed due to broad ranging effects on the initial inflammatory response and repair phase. As such this programme will begin to address a genuine unmet clinical need which could significantly improve outcomes in patients suffering from ischaemic heart disease leading to acute myocardial infarction and congestive heart failure.

At this stage, the programme is has a significant discovery science-focus around the timing of manipulation of lymphatics and the functional phenotyping of cleared versus retained immune cells; consequently the major impact will be via novel insights into lymphatic-immune cell interactions which will benefit other groups and individuals working within the field of cardiovascular regenerative medicine, fostering further related studies.

The second aim, however, seeks to establish a high-throughput chemical screen to identify small molecule activators human lymphangiogenesis. This represents the initiation of a drug-repurposing and/or new drug-discovery pipeline for heart attack patients, which will be progressed through lead candidate testing and validation, medicinal chemistry to optimise structure-activity relationships and pre-clinical models. By the end of the 5-year programme we will have identified drug candidates which can be progressed through further funding or integration into existing programmes within Oxford-based spin-outs such as OxStem- https://www.oxstem.com/. Here there is potential for commercial exploitation and addition to the portfolio of a UK biotechnology company to potentially benefit the UK economy.

Staff working on the programme will benefit from comprehensive research training in state-of-the-art molecular techniques, comparative study of animal and human models and in the rigours and multi-disciplinarity associated with establishing and developing a robust phenotypic screen and successful drug-discovery pipeline.