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. 2021 Sep 8;22(18):9729.
doi: 10.3390/ijms22189729.

CD146 Delineates an Interfascicular Cell Sub-Population in Tendon That Is Recruited during Injury through Its Ligand Laminin-α4

Neil Marr  1 , Richard Meeson  2 , Elizabeth F Kelly  2 , Yongxiang Fang  3 , Mandy J Peffers  4 , Andrew A Pitsillides  1 , Jayesh Dudhia  2 , Chavaunne T Thorpe  1
Affiliations

CD146 Delineates an Interfascicular Cell Sub-Population in Tendon That Is Recruited during Injury through Its Ligand Laminin-α4

Neil Marr et al. Int J Mol Sci. .

Abstract

The interfascicular matrix (IFM) binds tendon fascicles and contains a population of morphologically distinct cells. However, the role of IFM-localised cell populations in tendon repair remains to be determined. The basement membrane protein laminin-α4 also localises to the IFM. Laminin-α4 is a ligand for several cell surface receptors, including CD146, a marker of pericyte and progenitor cells. We used a needle injury model in the rat Achilles tendon to test the hypothesis that the IFM is a niche for CD146+ cells that are mobilised in response to tendon damage. We also aimed to establish how expression patterns of circulating non-coding RNAs alter with tendon injury and identify potential RNA-based markers of tendon disease. The results demonstrate the formation of a focal lesion at the injury site, which increased in size and cellularity for up to 21 days post injury. In healthy tendon, CD146+ cells localised to the IFM, compared with injury, where CD146+ cells migrated towards the lesion at days 4 and 7, and populated the lesion 21 days post injury. This was accompanied by increased laminin-α4, suggesting that laminin-α4 facilitates CD146+ cell recruitment at injury sites. We also identified a panel of circulating microRNAs that are dysregulated with tendon injury. We propose that the IFM cell niche mediates the intrinsic response to injury, whereby an injury stimulus induces CD146+ cell migration. Further work is required to fully characterise CD146+ subpopulations within the IFM and establish their precise roles during tendon healing.

Keywords: Achilles tendon; CD146; LAMA4; interfascicular matrix; tendon injury.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Effect of injury on tendon morphology and cellularity. H&E stained tendon sections visualised under bright and polarising microscopy demonstrate alterations in lesion appearance over time in injured tendons compared to contralateral controls. Lesions are outlined by dotted lines. Alterations in collagen organisation were also observed up to 500 µm proximal to the lesion. Scale bar = 100 µm (a). Scoring was based on the Bonar scoring system and demonstrated a significant increase in overall score in injured compared to control and sham samples (indicated by ***), but no difference between injury time points (b). Lesion area (c) and cellularity (d) were significantly greater at day 21 than at day 4 or day 7. Data are displayed as mean ± S.D; n = 4 per time point. Significance indicated by *: * = p < 0.05, ** = p < 0.01, *** = p < 0.001.
Figure 2
Figure 2
Injury response over time was assessed using immunohistochemistry for a panel of markers. Images show the lesion site in injured tendons and the corresponding region in uninjured tendons. Positive staining is brown, with cell nuclei counterstained blue. Inset shows the region of interest magnified 2.5 times. Scale bar = 50 µm.
Figure 3
Figure 3
Three-dimensional visualisation of uninjured tendons labelled for CD146. Reconstruction of Achilles tendon labelled for CD146 (red, a,b) and with nuclei counterstained (blue, b). Reconstructions have been clipped longitudinally to allow for visualisation of CD146 within the tendon core. Inset shows magnified region. Transverse (xz) views of reconstructions (c,d) show a complex network of CD146 in uninjured tendon, localised to the IFM (indicated by *) and epitenon.
Figure 4
Figure 4
Three-dimensional visualisation of lesion sites in injured tendons at days 4, 7 and 21 after injury. CD146 is labelled red, with nuclei counterstained blue. At day 4 post injury, the area surrounding the lesion was mainly acellular, with CD146 expression restricted to the epitenon. By day 7, some CD146 positive-labelling could be visualised surrounding the lesion, and by day 21 CD146 expression was present within the lesion site.
Figure 5
Figure 5
Three-dimensional visualisation of lesion site 21 days after injury. CD146 is labelled red, collagen is labelled green and nuclei labelled blue. Blue and green channels have been digitally removed in the centre of the tendon to allow for the visualisation of CD146-positive labelling within the lesion in sagittal (a) and transverse (b) views.
Figure 6
Figure 6
Injury response over time was assessed using immunohistochemistry for a panel of markers for progenitor cells, pericytes and macrophages. Images show the lesion site in injured tendons and the corresponding region in uninjured tendons. Positive staining is brown, with cell nuclei counterstained blue. Inset shows the region of interest magnified 2.5 times. Scale bar = 50 µm.
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