EC plasticity potential in EndMT: fate of EndMT-derived cells
The multi-step cell fate changes observed in EndMT can be described as a continuum rather than a binary event.87,88 As such, this transition can eventually lead to mesenchymal-like cells where the EC transcriptional signature is no longer present. However, intermediate stages are characterized by coexpression of both endothelial and mesenchymal signatures and can potentially shift between lineages.81,83,116 Conceivably, transitioning ECs can re-establish a homeostatic balance, or mesenchymal-like cells can further differentiate into other mature cell types.23,108 Considering process dynamics and EC heterogeneity within the backdrop of distinct EndMT stages, it is tempting to speculate that partial or complete activation of the EndMT programme can prime or induce ex novo formation of specialized EC subpopulations having a central role in human disease. Additional studies are warranted to better characterize the plasticity of EndMT-responsive EC subsets and define whether a common ‘transitioning signature’ is present and then modulated by local cues or if cells are already primed to follow lineage-specific changes. Additional investigation is also needed to define whether partial EndMT is the main manifestation of this process in the adult (as opposed to complete EndMT). Studying specific local cellular environments is crucial, not only because of the impact it may play on EndMT-responsive cells but also to understand how EndMT may reshape the biology of bystander cells and how it fits into an EndMT continuum model.
4.1 Identification and characterization of mesenchymal-derived cells of endothelial origin
The balance between competing cell fates is responsible for determining and maintaining cellular identity.44,81,128 For ECs to lose their identity and acquire that of another cell type, they must overcome signalling cues that maintain EC fate.81 This can be achieved when mesenchymal signals are strong enough to overcome those of the endothelial phenotype. The balance between these two opposing genetic programmes may guide partial vs. complete EndMT, with the latter representing a disruption of such equilibrium.
It is reported that the response of ECs to EndMT stimuli is highly heterogeneous and dependent on multiple factors, such as length of EndMT induction and local cues.3,43,45 Furthermore, different patterns of gene expression have been reported when inducing EndMT in differing EC populations, for example, in human microvascular ECs (HMECs) compared with macrovascular human umbilical vein ECs (HUVECs).129 A recent study stemming from our collaborative work also demonstrated that treatment of HUVECs and human pulmonary arterial ECs (HPAECs) with TGF-β2 (10 ng/mL) and IL-1β (1 ng/mL) for 7 days induced EndMT while TGF-β2 or IL-1β alone did not.35 Deep RNA sequencing of these cells showed that the effect of EndMT induction was stronger in HUVECs compared with HPAECs. As for the contribution of IL-1β to EndMT, a study showed that IL-1β (10 ng/mL) alone is sufficient to induce EndMT in human coronary arterial cells (HCAECs), even after 24 h of stimulation.99 However, previous work suggested that IL-1β supports EndMT initiation, without being essential for its progression and maintenance.130 Moreover, it was recently reported that stimulating HMECs with TNF-α induces EndMT in a dose-dependent manner (20–100 ng/mL) after 96 h,54 whereas a different study showed no effect of TNF-α on human intestinal microvascular cells even after 6 days of EndMT induction.131 Additionally, in PAECs, the combination of TGF-β1 with TNF-α and IL-1β was found to be more powerful for EndMT induction than any of these mediators alone.40 Overall, this heterogeneity is mainly attributed to different cell lines and stimulating cytokines in vitro.
The response of ECs becomes increasingly complicated when EndMT is induced via different stimulating factors, such as hypoxia, disturbed blood flow, and oxidative stress. Hypoxia is a well-known inducer of tissue fibrosis in various pathological processes, and its involvement with EndMT has been previously reported. Exposure of HCAECs to hypoxia (1% O2) for 5 days was shown to decrease smooth muscle marker α-SMA while increasing SM22α levels.61 When looking at HPAECs however, oxygen levels were shown to have no effects on α-SMA, compared with baseline levels. Furthermore, in both cell types, the endothelial marker CD31 was up-regulated. Other studies have also reported similar findings in terms of changes in EC markers.132,133 Of note, the expression of EC markers may not all occur simultaneously and may also be specific to individual transcripts/proteins. Additionally, it is likely that during partial EndMT, there is minimal initial loss of the EC phenotype, which only occurs once the transition is completed.132
When looking at the effects of disturbed blood flow in differing contexts, multiple changes arise such as a transition from atheroprotective phenotypes to pro-inflammatory cells, mesenchymal (EndMT-derived) cells, haematopoietic stem cells, endothelial stem/progenitor cells, and unexpected immune cell-like phenotypes.74 In a report studying the effects of oxidative stress on EC phenotype, it was shown that HUVECs exposed to hydrogen peroxide (H2O2) are converted to myofibroblasts via EndMT. This was confirmed based on reduced levels of CD31 and VE-cadherin and the increased protein levels of fibrotic and ECM markers, such as α-SMA, fibronectin, FSP-1, and COL3A1.134 The list of the above studies is in no way exhaustive; however, it accurately conveys that multiple factors, such as EC subtype, as well as the dose and nature of the inducing stimulus, result in heterogeneous EC responses. The lack of a standardized protocol for inducing EndMT in vitro to date remains a challenge, and a deeper characterization is required in order to understand the properties of these distinct EC populations and determine the fate of the resulting mesenchymal cells.
Overall, this body of evidence suggests that important cellular populations may reside at specific stages of the endothelial-to-mesenchymal continuum, ranging from cells with dual identity (both endothelial and mesenchymal) to those derived from a directed maturation of mesenchymal-like cells or in virtue of EndMT reversal. Hence, additional work in this direction could open a new realm of therapeutic strategies.
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