MesenmiR: Towards a microRNA-modified mesenchymal stromal cell therapeutic for chronic limb-threatening ischaemia

Chronic limb-threatening ischaemia is the most severe manifestation of peripheral artery disease and is defined by the presence of ischaemic rest pain, ulceration, or gangrene of the lower limb. Despite current treatment options, many patients have no option but to undergo amputation. In chronic limb-threatening ischaemia, vascular occlusion causes distal tissue ischaemia, which results in a severe skeletal muscle pathology which is characterised by muscle atrophy, impaired satellite cell function, fibrosis, myosteatosis, mitochondrial dysfunction, and other features. There has been significant interest in novel therapeutics, such as cell and gene therapies, to improve outcomes in these patients. However, despite early promising data, translational success has been limited. These novel therapeutics have largely focussed on "therapeutic angiogenesis"; however, more recently, there has been increasing interest in targeting the skeletal muscle. MicroRNAs are small, non-coding RNAs that have also gained attention as pathological mediators and potential therapeutics in chronic limb-threatening ischaemia. In this thesis, I aimed to investigate skeletal muscle pathology, with a particular focus on its associated microRNA dysregulation, to inform the rational design of novel microRNA- and cell-based therapeutics. In Chapter 3, I utilised publicly available RNA sequencing data from gastrocnemius biopsies from chronic limb-threatening ischaemia patients to perform microRNA-Target Enrichment Analyses and identified a panel of microRNAs with a potential regulatory role in chronic limb-threatening ischaemia-associated skeletal muscle pathology. In a preclinical murine hindlimb ischaemia model, I showed that three of these microRNAs, miR-1, miR-133a, and miR-29b, were downregulated in skeletal muscle ischaemia on day 7 post-hindlimb ischaemia. Through functional enrichment analysis, I showed a role of this three-microRNA panel in regulating targets associated with fibrosis in the ischaemic limb. In chapter 4, I optimised a pre-existing protocol to isolate RNA from formalin-fixed paraffin-embedded skeletal muscle sections to profile the expression of microRNAs and mRNAs. I investigated the three-microRNA panel in human umbilical cord mesenchymal stromal cell-treated skeletal muscle in the hindlimb ischaemia model and further validated their downregulation at day 28 post-hindlimb ischaemia. Further, human umbilical cord mesenchymal stromal cell treatment was associated with a partial but significant restoration of miR-133a levels towards non-ischaemic levels. miR-133a levels were strongly correlated with decreased ischaemic damage of skeletal muscle in the hindlimb. In chapter 5, I established a 2D in vitro NIH-3T3 fibroblast and C2C12 myoblast coculture model with markedly enhanced myogenesis. In this model, induction of simulated ischaemia partially but inconsistently recapitulated features of skeletal muscle pathology seen in human chronic limb-threatening ischaemia and the murine hindlimb ischaemia model, such as myotube atrophy. However, further optimisation of this model will be required to enhance its applicability to chronic limb-threatening ischaemia research. Overall, the results in this thesis highlight the role of miR-1, miR-133a, and miR-29b in chronic limb-threatening ischaemia-associated skeletal muscle fibrosis and further the potential therapeutic relevance of restoration of miR-133a by human umbilical cord mesenchymal stromal cell-treatment in skeletal muscle ischaemia. It is hoped that further work on the lines of investigation opened in this thesis will facilitate the development of novel microRNA- and cell-based therapeutics aimed at restoring homeostasis in the ischaemic limb and, ultimately, improving outcomes in patients living with chronic limb-threatening ischaemia.

Funder

Hardiman PhD Scholarship and Government of Ireland Postgraduate Scholarship (GOIPG/2020/1235)

Publisher

University of Galway

Rights

Attribution-NonCommercial-NoDerivatives 4.0 International

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Collections

University of Galway Theses (PhD Theses)