Local and Circulating Endothelial Cells Undergo Endothelial to Mesenchymal Transition (EndMT) in Response to Musculoskeletal Injury

Abstract

Endothelial-to-mesenchymal transition (EndMT) has been implicated in a variety of aberrant wound healing conditions. However, unambiguous evidence of EndMT has been elusive due to limitations of in vitro experimental designs and animal models. In vitro experiments cannot account for the myriad ligands and cells which regulate differentiation, and in vivo tissue injury models may induce lineage-independent endothelial marker expression in mesenchymal cells. By using an inducible Cre model to mark mesenchymal cells (Scx-creERT/tdTomato + ) prior to injury, we demonstrate that musculoskeletal injury induces expression of CD31, VeCadherin, or Tie2 in mesenchymal cells. VeCadherin and Tie2 were expressed in non-endothelial cells (CD31−) present in marrow from uninjured adult mice, thereby limiting the specificity of these markers in inducible models (e.g. VeCadherin- or Tie2-creERT). However, cell transplantation assays confirmed that endothelial cells (ΔVeCadherin/CD31+/CD45−) isolated from uninjured hindlimb muscle tissue undergo in vivo EndMT when transplanted directly into the wound without intervening cell culture using PDGFRα, Osterix (OSX), SOX9, and Aggrecan (ACAN) as mesenchymal markers. These in vivo findings support EndMT in the presence of myriad ligands and cell types, using cell transplantation assays which can be applied for other pathologies implicated in EndMT including tissue fibrosis and atherosclerosis. Additionally, endothelial cell recruitment and trafficking are potential therapeutic targets to prevent EndMT.

Heterotopic ossification and the elucidation of pathologic differentiation

“…In a recent study of ours, we demonstrated that musculoskeletal injury induces expression of CD31, VE-cadherin, and Tie2 in mesenchymal cells. Using cell transplantation assays, we also confirmed that endothelial cells isolated from uninjured muscle tissue undergoes in vivo endothelial mesenchymal transition when transplanted directly into the wound [65]. More recently, Olmsted-Davis et al found that the endoneurium played a key functional role in HO formation using Wnt1-CreERT for lineage tracing [39].…”

Stem cells and heterotopic ossification: Lessons from animal models

“…Interestingly, using a VE-Cadherin-Cre driver, a recent report described the contribution of endothelial cells to HO following combined burn injury and tendonectomy[78]. Transplantation of endothelial cells isolated from the aorta or hindlimb musculature into the tendon injury site further suggested a potential, albeit minor, contribution of endothelium to tendon HO.…”

“…There are two commercially available VE-Cadherin-Cre strains (B6;129-Tg(Cdh5-cre)1Spe/J[79] and B6.Cg-Tg(Cdh5-cre)7Mlia/J[77]), each exhibiting highly efficient labeling of endothelium, but differential labeling of hematopoietic and other lineages. In comparing the studies of Wosczyna et al[65] and Agarwal et al[78], it is possible that different outcomes resulted from the use of different Cre drivers (not reported in ref. [78]).…”

“…In comparing the studies of Wosczyna et al[65] and Agarwal et al[78], it is possible that different outcomes resulted from the use of different Cre drivers (not reported in ref. [78]). Alternatively, mechanisms and progenitor cells involved in directing HO following tendonectomy (discussed in Section 3.4) may be distinct from those governing intramuscular HO.…”

Function of cAMP scaffolds in obstructive lung disease: Focus on epithelial‐to‐mesenchymal transition and oxidative stress

“…Mesenchymal cell phenotype is characterized by α-smooth muscle actin (α-SMA), N-cadherin, vimentin, type I collagen, fibronectin, and β-catenin. For further details, see text confined to epithelial cells but also occurs in endothelial cells, indicating that this process is crucial for tissue repair (Agarwal et al, 2016).…”