Title: Understanding the impact of ageing on ‘active Extracellular Vesicles’ from Mesenchymal Stromal Cells (MSC) and consequences on repair responses: towards novel therapies in wound healing.
Funder: Dunhill Medical Trust

Abstract: Poor wound healing is a challenge that is becoming more important with the advancing age of our population and incidence of important co-morbidities such as diabetes. Estimates suggest that each year 3.8 million patients are managed by the NHS for wounds, with over 130 million patient visits from healthcare staff, at an annual NHS cost of £8.3 billion, of which £5.6 billion is associated with managing non-healing wounds. Consequently, there is an urgent and unmet need to understand the immune control that becomes impaired with ageing and to develop novel approaches to promote wound healing. Chronic (non-healing) wounds are a great health burden, both financially and personally, and are a significant and increasing challenge within an ageing society. A number of approaches (e.g. debridement, topical growth factors and dressings and antibiotics) are taken to manage these hard-to-treat conditions and reduce healing times. However, some wounds fail to heal and become ‘stalled’ and this typically happens within the inflammatory phase of wound healing. This is the phase in which the innate immune system acts to both clear any challenge (e.g. microbial or damaged cells) before promoting a ‘repair’ response. This repair stage is heavily reliant on macrophages which switch from a pro-defence phenotype (‘classically-activated’, often called M1) to a pro-repair phenotype (‘alternatively-activated’, often called M2) to help drive wound healing. Mesenchymal Stromal Cells (MSC) have great potential for promoting repair responses and it has become increasingly clear over recent years that a major mechanism by which MSC may promote repair is through modulation of immune responses, particularly inflammatory responses and macrophage phenotype. Extracellular Vesicles (EV) released by MSC (MSC-derived EV: MSCdEV) also promote these beneficial macrophage phenotype changes towards a pro-repair phenotype. However, MSC from older individuals are much less competent to modulate macrophage phenotype and this may explain the profound defect in repair responses as we age. This seed-funding will support our programme of work to begin to define a molecular mechanism by which MSC and their EV control macrophage phenotype – a novel mechanism that we hypothesise becomes defective in ageing. We have recently shown that EV from dying, apoptotic cells (Apoptotic Cell-derived EV: ACdEV) carry a family of inflammation modulating enzymes (Lipoxygenase: LOX). Different LOX isoforms can promote and inhibit inflammation and the balance of these different isoforms is crucial to deciding the outcome of inflammatory control and wound healing. We have called these enzyme-carrying EV ‘active EV’ and have shown that these are responsible for promoting repair responses in macrophages through promoting macrophage phenotype change. Our recent preliminary data now reveals that MSCdEV also carry these same enzymes and, importantly, the activity is altered in MSC from older donors. This proposed programme of work will now characterise the presence, balance and activity of LOX isoforms within MSC and MSCdEV from older and younger donors. This will define the altered carriage of LOX isoforms in MSCdEV from older individuals, and crucially whether they are less able to drive macrophages from older and younger individuals towards a ‘pro-repair’ phenotype. Cellular senescence is also known to have profound effects on the inflammatory environment as we age, promoting a pro-inflammatory (non-repair) environment. Here, we will also consider the senescent MSCdEV to understand their LOX composition and activity to understand how this may also contribute to the senescence-associated secretome and its profound inflammatory consequences. This work will provide new insight in to the control of macrophage phenotype and repair responses and those defects that may occur with ageing. It will define the ageing-associated changes in active EV and defects in modulating macrophage repair responses. This has important implications for MSC-based therapies used clinically for both chronic inflammatory conditions and solid organ transplantation. Future work will then seek to mitigate these defects through the development of novel approaches to overcome the defect (e.g. through synthetic vesicles carrying the necessary enzymes or their encoding messenger RNA). Using this approach, we will enable the development of novel therapeutic agents to be targeted for important, debilitating, and hard-to-treat wounds associated with ageing.

Lay abstract: Non-healing (chronic) wounds such as abscesses and ulcers are a great health burden, both financially and personally, and are a significant and increasing challenge within an ageing society. Estimates suggest that each year 3.8 million patients are managed by the NHS for wounds at a total cost of £8.3 billion and 80+% of this is incurred in community care, including over 130 million patient visits from healthcare staff. Chronic wounds are difficult to treat. In a recent study, up to 55% of them failed to heal within the year of study. It is clear that there is a pressing need for new approaches to wound management and therapy. Wound healing involves the immune system promoting a defence response (to clear infection and damaged tissue) and subsequently, a repair response is initiated. This involves macrophages (an important immune cell) which switch their function from defence to repair (wound healing) responses. This switch is impaired in older people and leads to chronic wounds becoming ‘stalled’. However, we do not understand the mechanism that is affected by ageing. This programme of work will address this gap in our knowledge directly. Over recent years, stem cells and small membrane bags (Extracellular Vesicles : EV) that they release have been shown to promote repair responses and wound healing. However, stem cells and EV are less able to promote repair as we age and our work will define why this is the case. Our work reveals that EV from stem cells carry a family of crucial inflammation-controlling enzymes that can both promote and inhibit inflammation. Consequently, the precise balance of these family members within EV will decide the outcome: beneficial wound repair or detrimental chronic inflammation. We call these enzyme-carrying EV ‘active EV’. Our preliminary work reveals that EV from stem cells of older people carry more enzyme activity, and we hypothesise that the precise enzyme composition of EV changes during ageing, causing impaired wound healing. By studying stem cells and their vesicles from older and younger people, we will understand how EV changes alter macrophage repair responses. This work therefore aims to reveal the repair pathway from stem cells that becomes defective as we age so that therapies can be designed to overcome the defects. Consequently, this work will pave the way for novel therapeutic approaches for hard to treat and debilitating conditions associated with ageing such as chronic non-healing wounds.

Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFLAMMATORY AND IMMUNE SYSTEM