miR-1, miR-133a, and miR-29b and Skeletal Muscle Fibrosis in Chronic Limb-Threatening Ischaemia.

Abstract

Background. Chronic limb-threatening ischaemia (CLTI) is the most severe manifestation of peripheral arterial disease (PAD), and is associated with poor prognosis and high amputation rates. Novel therapeutic approaches are currently being investigated, but to date, they have not demonstrated significant benefits in promoting CLTI limb salvage. Understanding the molecular pathways of skeletal muscle dysfunction in CLTI is imperative for a rational design of successful therapeutic approaches. The full spectrum of dysregulated microRNAs (miRNAs) associated with PAD disease stages is currently poorly understood. The purpose of this study was to identify dysregulated miRNA transcripts in gastrocnemius muscle biopsies that were differentially expressed in different PAD cohorts. Methods. We performed an analysis of a publicly available RNA-sequencing database of PAD cohorts using MIcroRNA ENrichment TURned NETwork(MIENTURNET), a web tool for miRNA-target enrichment and network-based analysis. Following this, we validated the dysregulation of miRNAs and their targets in mice with hindlimb ischaemia (HLI). Results. Our miRNA-target interaction enrichment analysis identified a list of miRNAs over-represented amongst upregulated differentially expressed genes (DEGs) in CLTI. Upon validation in the HLI model, our results showed a significant and marked ischaemia-induced decrease of miR-1, miR-133a, and miR-29b in the ischaemic limbs vs the contralateral non-ischaemic limbs. A miR-1, miR-133a, and miR-29b target protein-protein interaction network identified many extracellular matrix components including collagen-1a1, -3a1 and -4a1, fibronectin-1, fibrin-1, matrix metalloproteinase-2 and -14, and the secreted protein acidic and rich in cysteine (Sparc), which were upregulated in the ischaemic muscle in mice. Functional enrichment analysis performed on this subset of DEGs identified pathways related to fibrosis and vascular pathology. Conclusions. We have identified, for the first time, a CLTI-specific miRNA signature. Our results indicate miR-1, miR-133a, and miR-29b as potential contributors to the fibrosis and vascular pathology in CLTI muscle, which supports its investigation as potential novel therapeutics.

Mesenchymal Stromal Cell Transplantation Ameliorates Fibrosis and Dysregulated microRNA Signature Associated with Ischaemic Skeletal Muscle Damage.

“…Results from these analyses identified a number of extracellular matrix components including a family of collagens, fibronectin and fibrin, which were targeted by these miRNAs. Concurrent with the downregulation of the three miRNAs, their gene targets were found to be upregulated 7 days after ischaemia 34 . Similarly, in this study, we observed a sustained upregulation of pro-fibrotic markers at 28 days post-ischaemia, including as Col1a1, Fn1 and Acta2, and a downregulation of these genes after cell treatment.…”

“…Previously, bioinformatics-based analyses were performed on the combined gene targets of these three miRNAs, which were also upregulated in CLTI muscle, and which interaction was validated in the literature 34 . Results from these analyses identified a number of extracellular matrix components including a family of collagens, fibronectin and fibrin, which were targeted by these miRNAs.…”

“…Our group have previously identified a dysregulated miRNA signature on human CLTI skeletal muscle using publicly available transcriptomic data from Ryan et al 67 . We previously predicted a downregulation of miR-1, miR-133a and miR-29b in gastrocnemius biopsies from patients with CLTI, which was then validated in an HLI mouse model at 7 days postischaemia 34 . While cardiac and skeletal muscle-specific miR-1 and miR-133a are considered canonical 'myomiRs' which play an essential role in regulating myogenesis and muscle development [68][69][70][71] , miR-29b is well-known for its anti-fibrotic properties [72][73][74] .…”

“…The presence of CD68+ macrophages was significantly higher in the control group compared to the hUC-MSCs treated group Previous work from our group identified several miRNAs including miR-1, miR-133a and miR-29b, which were predicted to be downregulated in humans with CLTI. Downregulation of these miRNAs was validated in ischaemic mouse muscle at 7 days post-ischaemic injury 34 . Here, we investigated the effect of ischaemia and cell delivery on the level of expression of these miRNAs at 28 days post-ischaemia.…”

“…Our results have shown an amelioration of ischaemia-induced skeletal muscle inflammation, fibrosis and muscle wasting features after hUC-MSC transplantation. We have previously identified a miRNA dysregulated signature in ischaemic muscles associated with fibrosis 34 . Here, we investigated whether miRNA levels were restored after cell treatment.…”