TGF-β-mediated Endothelial to Mesenchymal Transition (EndMT) and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

Abstract

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like phenotype, a process that is termed endothelial to mesenchymal transition (EndMT). Emerging results have suggested that EndMT is causally linked to multiple human diseases, such as fibrosis and cancer. In addition, endothelial-derived mesenchymal cells may be applied in tissue regeneration procedures, as they can be further differentiated into various cell types (e.g., osteoblasts and chondrocytes). Thus, the selective manipulation of EndMT may have clinical potential. Like epithelial-mesenchymal transition (EMT),EndMT can be strongly induced by the secreted cytokine transforming growth factorbeta (TGF-β), which stimulates the expression of so-called EndMT transcription factors (EndMT-TFs), including Snail and Slug. These EndMT-TFs then up-and downregulate the levels of mesenchymal and endothelial proteins, respectively. Here, we describe methods to investigate TGF-β-induced EndMT in vitro, including a protocol to study the role of particular TFs in TGF-β-induced EndMT. Using these techniques, we provide evidence that TGF-β2 stimulates EndMT in murine pancreatic microvascular endothelial cells (MS-1 cells), and that the genetic depletion of Snail using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPRassociated protein 9 (Cas9)-mediated gene editing, abrogates this phenomenon. This approach may serve as a model to interrogate potential modulators of endothelial biology, and can be used to perform genetic or pharmacological screens in order to identify novel regulators of EndMT, with potential application in human disease.

Single‐Cell Transcriptomic Census of Endothelial Changes Induced by Matrix Stiffness and the Association with Atherosclerosis

“…This finding suggests that SNAI1 gene upregulation likely occurred at time points earlier than 6 days, since SNAI1 regulates the early steps of EndMT. [ 27 ] Nevertheless, we observed distinct post‐transcriptional modification of snail‐1 protein expression at subcellular level (Figure 1A). Additionally, transcriptional analysis at an intermediate time‐point of 6 days demonstrated many similarities in gene expression patterns to that of day 14 (Figure S5, Supporting Information).…”

Targeting Inflammation to Control Tissue Fibrosis

“…Inhibitors targeting PI3K/Akt [23] and MAPK [24] have also been shown to inhibit fibrosis in preclinical studies and clinical trials. TGF-β' s role in EndMT has also been extensively investigated and was shown to regulate EndMT through the canonical Smad signalling pathway [25,26]. Connective Tissue Growth Factor (CTGF) was reported to mediate, at least partially, the profibrotic effects of TGF-β.…”

Cytoskeleton Reorganization in EndMT—The Role in Cancer and Fibrotic Diseases

“…To confirm the occurrence of the EndMT, the most often detected mesenchymal markers include α-smooth muscle actin (α-SMA), N-cadherin, calponin, fibroblast-specific protein-1 (FSP-1), vimentin, fibronectin (FN), collagen types I and III, and matrix metalloproteinase 2 and 9 (MMP-2 and MMP-9, respectively). It should be mentioned that other frequently used typical EMT markers, such as E-cadherin, claudin, occludin, and cytokeratin, are not usually used in studies of EndMT [ 46 , 47 ].…”