Cellular senescence – the culprit of MMVD?

13 August 2024

Michal Tkacz, a final year vet student at the Royal (Dick) School of Veterinary Studies, University of Edinburgh, and Professor Brendan Corcoran, Personal Chair of Veterinary Cardiopulmonary Medicine at the University of Edinburgh, have been awarded a PetSavers-funded student research project grant to investigate myxomatous mitral valve disease.

Myxomatous mitral valve disease (MMVD) is the single most important acquired cardiovascular disease of the dog. It involves the formation of myxomatous nodules on mitral valve leaflets, which prevents appropriate apposition of the valve, leading to mitral regurgitation, eventual cardiac remodelling and, in many cases, heart failure. Although the therapeutic management of heart failure has improved significantly in recent years, there is still no way to prevent, arrest or reverse valve pathology. Surgical options have also been described, but these do not address the root cause of the pathology, are expensive, and inaccessible to most, being performed in only a handful of centres worldwide.

Despite the high prevalence of MMVD amongst dogs, the aetiology has remained unclear. However, links to polygenic inheritance in more commonly affected breeds such as the Cavalier King Charles Spaniel have been shown. In recent years, there has been increased interest in the role of cellular senescence in a wide range of age-dependent degenerative diseases in human medicine, and work at the Corcoran Laboratory in the Roslin Institute at the University of Edinburgh has shown this likely also applies to canine MMVD.

The cells that are affected by senescence in this case are valve interstitial cells (VICs), which are specialized fibroblasts located in the mitral valve matrix. They are normally found in a quiescent state and become activated (aVICs) in response to damage to the mitral valve, contributing to life-long valve extracellular matrix (ECM) remodelling. They lay down collagen and other connective tissue substances as needed and subsequently return to a quiescent phenotype or are removed from the valve by programmed cell death (apoptosis). However, this does not occur in diseased valves, where they remain in an activated phenotype and enter the arrested cell cycle in a state known as senescence. Targeting of this cellular senescence has the potential to be a novel medical therapeutic approach to controlling the development of valve pathology.

FIGURE 1: (A) Effect of quercetin (Q) and quercetin with dasatinib (Q+D) on the expression of proteins associated with senescence (p53, p21 and p16) and cell apoptosis (caspase-3) in valve interstitial cells derived from diseased valves (C), indicating the reversal of senescence. The reduction in alpha-SMA expression indicates the cells have returned to a normal quiescent phenotype. (B) Cultured cells stained with the senescence marker ß-galactosidase show quercetin treatment is more effective than fisetin at reversing senescence.

What are senescent cells?

Senescent cells are a major topic of interest in human medicine, and their importance in veterinary disease is now being recognized and explored. They are involved in a number of conditions including chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, inflammatory bowel disease, diabetes, osteoarthritis and neoplasia. Senescence involves a number of controlling mechanisms, including damage-induced and developmentally-programmed pathways which lead to the upregulation of cell cycle inhibitory proteins such as p53, p21 and p16, and others such as TGF-ß, mTOR and AKT, which drive these effects. The process can be started by several triggers including proliferative exhaustion (an excessive number of cell divisions), genotoxic stress (DNA damage), mitochondrial dysfunction, inflammation, oncogene activation and other non-specific stressors. MMVD-induced senescence might be triggered by valve edge trauma from repeated closure, although the exact triggering mechanism is unknown. While senescent cells can no longer divide, they remain metabolically active and release a cocktail of damaging proteins including cytokines, chemokines and ECM products into the valve matrix. This is known as senescence-associated secretory phenotype (SASP) and also induces senescence in other nearby cells.

Senolytics – a novel idea

A very large class of medications has been produced or identified that target senescent cells. Of particular interest are the food-based flavonoids found in fruits, nuts and vegetables, mainly in those of a dark colour (e.g. red grapes, red apples and pomegranate seeds). This PetSavers-funded student research project examined the effect of senolytic candidate drugs (which eliminate senescent cells) including the flavonoids quercetin and fisetin, as well as dasatinib which has been shown to improve flavonoid effects. Each of these drugs targets different points of the senescent signalling pathway; quercetin inhibits the PI3K-AKT pathway, fisetin is a topoisomerase inhibitor and dasatinib is a tyrosine kinase inhibitor.

Our study showed that all three can inhibit senescence in aVICs, but that quercetin is more potent than fisetin, and the effect is more pronounced when quercetin and dasatinib are used together (Figure 1). While this was only examined in cell culture, it is a proof of principle that MMVD-associated cell senescence can be countered, and these findings will inform future studies in clinically affected patients.

The future of MMVD management

Traditionally, MMVD has been medically managed with a combination of drugs to postpone the development of left-sided congestive heart failure or treat it once it has occurred. These therapies manage the consequences of MMVD but have no effect on valve degeneration. Clinical trials examining the effects of senolytics on a range of human diseases are on-going, and our new data suggests that we can now envisage the same for MMVD. The data have also informed exciting on-going research projects at the Roslin Institute examining other methods to control cell senescence. This will allow us to identify the most promising agents for future drug trials in affected dogs.

About the authors

Michal is a current final year veterinary student at the Royal (Dick) School of Veterinary Studies, University of Edinburgh, due to graduate in Summer 2024. He undertook an elective project in his 2nd year about myxomatous mitral valve disease, which led to him undertaking his PetSavers-funded student research project in the same field. He has an interest in all aspects of small animal medicine with the intention of going into small animal practice once graduated.

 

 

 

 

Professor Corcoran graduated from the Veterinary College of Ireland in 1981 and joined the Royal (Dick) School of Veterinary Studies (R(D)SVS). He has subsequently held posts as a Wellcome Research Leave Fellow, Director of the Hospital for Small Animals, Head of Companion Animal Sciences, Director of International Affairs, and Deputy Head of School at the R(D)SVS, and Dean International for North America for the University of Edinburgh. He was awarded a Personal Chair in Veterinary Cardiopulmonary Medicine in 2006. His clinical and research interests are in respiratory medicine and the cell and molecular biology of myxomatous mitral valve disease.