Published: February 26th, 2021
We describe methods to investigate TGF-β2-induced EndMT in endothelial cells by observing cell morphology changes and examining the expression EndMT-related marker changes using immunofluorescence staining. CRISPR/Cas9 gene editing was described and used to deplete the gene encoding Snail to investigate its role in TGF-β2-induced EndMT.
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 factor-beta (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)/CRISPR-associated 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.
Endothelial to mesenchymal transition (EndMT) is a multistep and dynamic biological phenomenon that has been linked to diverse physiological and pathological processes1,2. Upon EndMT endothelial cells gradually lose their endothelial traits, while acquiring mesenchymal properties3; thus, tightly compacted and well organized endothelial cells differentiate into elongated mesenchymal-like cells. Morphological changes in EndMT coincide with alterations in the expression of certain genes and proteins. In general, the expression of proteins that maintain endothelial characteristics, including vascular endothelial (VE)-cadherin, platelet/EC adhesion molecule-1 (CD31/Pecam-1) declines. Simultaneously, proteins related to mesenchymal functions, such as α-smooth muscle actin (α-Sma) and smooth muscle protein 22α (Sm22α) accumulate. Emerging results have demonstrated that postnatal EndMT contributes to the development of human diseases, such as cancer, cardiac fibrosis, pulmonary arterial hypertension (PAH), atherosclerosis (AS), organ fibrosis, etc2,4,5,6,7. A deeper understanding of the underlying mechanisms of EndMT and how to direct the EndMT process will provide novel therapeutic methods for EndMT-related diseases and regenerative medicine.
TGF-β is one of the main EndMT inducers, and other known involved factors include Wnt/β-catenin, Notch, and some inflammatory cytokines1. As the cellular context is key for responses triggered by TGF-β, the interplay of TGF-β with other EndMT promoting signals is relevant for TGF-β to elicit an EndMT response. Upon the activation of TGF-β cell surface type I and type II serine/threonine kinase receptors, the intracellular canonical Smad pathway is activated. TGF-β receptor-mediated phosphorylated Smad2/3 form heteromeric complexes with Smad4 that translocate into the nucleus, where they upregulate the expression of EndMT-related transcription factors. Similar to epithelial-mesenchymal transition (EMT), transcription factors such as Snail, Slug, Twist, Zeb1 and Zeb2 are induced by TGF-β signaling and contribute to gene reprogramming in EndMT8.
Snail has been frequently identified as a key factor in EndMT. Snail binds to the promoter of genes encoding cell-cell adhesion proteins and suppresses their transcription, which is counterbalanced by the enhancement of the expression of mesenchymal proteins9. Endothelial cells comprise a very heterogeneous population and the relative influence of diverse extracellular stimuli on EndMT may differ among endothelial cellular contexts or cell types10. Due to its similarities with EMT, some methodologies are useful to investigate both mechanisms EMT and EndMT8. In this regard, the EMT International Association (TEMTIA) strongly emphasizes the need of complementary techniques to ultimately demonstrate the occurrence of EMT/EndMT11.
Here we describe a method to monitor and visualize the TGF-β-induced EndMT process. Immunofluorescence staining provides the basic information about expression changes in targeted proteins/markers, which are used as indicators of whether the EndMT process occurs. Additionally, the immunofluorescence staining can visualize the localization of proteins/markers and cell morphology. To study the potential activity of specific TFs (or other upstream or downstream regulators) involved in TGF-β mediated EndMT, we describe a protocol using clustered regularly interspaced short palindromic repeats (CRISPR) /CRISPR-associated protein 9 (Cas9) gene editing to deplete specific genes from cells, using the TF Snail as an example. Cas9 is a dual RNA-guided DNA endonuclease that recognizes and cleaves sequences complementary to CRISPR sequences in bacteria12. The CRISPR/Cas9 system is currently extensively utilized because it facilitates genetic engineering in vitro and in vivo13. Directed by a single guide RNA (sgRNA), ectopically expressed Cas9 generates a double strand break at a preselected targeting sequence in a specific gene locus. Non-homologous end joining (NHEJ) takes place to repair Cas9-induced strand breaks, via random nucleotide insertions or deletions thereby leading to the disruption and inactivation of the targeted gene. We describe in detail methods for designing selective sgRNAs and generating lentiviral-compatible vectors containing the designed sgRNAs. As a result, stable gene-depleted endothelial cells can be generated in an efficient and reliable manner.
In this study, we used murine pancreatic microvascular endothelial cells (MS-1)14 as a model system to examine the TGF-β2-induced EndMT process. Our previous study demonstrated that Snail is the main transcription factor increased by TGF-β2, by which EndMT is induced in MS-1 cells15. Upon CRISPR/Cas9 gene editing to abrogate Snail expression in MS-1 cells, TGF-β2 failed to mediate EndMT. This workflow can be applied to study other (suspected) EndMT-related genes.
1. Induction of EndMT by TGF-β2
2. Immunofluorescence staining
3. Knock out of Snail using CRISPR/Cas9 editing
4. Generate Snail knockout MS-1 cells
TGF-β2 induces EndMT and stimulates Snail expression in MS-1 endothelial cells
TGF-β is one of the cytokines with greatest potential to induce EndMT. After treating MS-1 cells with TGF-β2 (1 ng/mL) for 3 days, endothelial MS-1 cells lose their cobblestone-like structure and differentiate into spindle-shaped mesenchymal-like cells (Figure 1A)15. To further verify the role of TGF-β2 in inducing cell phenotypic changes, we pre-treated the cells with the small molecule activin receptor-like kinase (ALK)4/ALK5/ALK7 inhibitor SB431542 before TGF-β2 stimulation19. SB431542 completely abrogated TGF-β2-induced cell morphology changes (Figure 1A). The TGF-β2 induced EndMT process was further investigated by studying changes in the expression of EndMT-related markers. As shown in Figure 1B, the endothelial protein Pecam-1 was potently decreased after TGF-β2 stimulation, while the mesenchymal factor Sm22α was profoundly upregulated by TGF-β215. These data are consistent with the notion that TGF-β2 triggered EndMT in MS-1 cells. Next, we investigated the effects of TGF-β2 on Snail and Slug expression. As shown in Figure 1C, Snail was markedly upregulated by TGF-β2, while Slug expression was not influenced by TGF-β2 in MS-1 cells15. The quantification of Snail expression from three independent experiments is shown in Figure 1D.
Depletion of Snail by CRISPR/Cas9 in MS-1 endothelial cells
As Snail was induced by TGF-β2 and likely involved in TGF-β2-mediated EndMT, we performed CRISPR/Cas9 gene editing to genetically deplete Snail expression in MS-1 cells. We hypothesized that the depletion of Snail would be sufficient to inhibit TGF-β2-induced EndMT. As shown in Figure 2A, we generated Snail knockout cells in two steps. Firstly, Cas9 was ectopically expressed by infecting MS-1 cells with a Cas9 expressing lentivirus. Since there is a blasticidin resistance cassette in the pLV-Cas9 construct, we checked the expression of Cas9 by Western blot analysis in blasticidin resistant cells (Figure 2D). Subsequently, we introduced sgRNAs that specifically targeted Snail to disrupt its protein expression. This procedure was also performed by infection with lentiviral particles carrying the AA19 pLKO.1-Snail-sgRNA construct, which includes a puromycin expression cassette. Cas9-expressing cells were again infected with gRNA containing lentivirus and further selected with puromycin. Two complementary sgRNA oligos targeting murine Snail were designed with a predicted low off-target activity (Figure 2B,C). After introducing two independent Snail sgRNAs in Cas9 expressing MS-1 cells, Snail protein expression was abrogated (Figure 2D)15.
Deficiency of Snail inhibits TGF-β2-induced EndMT in MS-1 cells
To demonstrate the function of Snail in TGF-β2-mediated EndMT, we performed an EndMT assay in Snail-depleted cells and compared it with parental MS-1 cells. As shown in Figure 3A, the knockout of Snail was sufficient to inhibit the fibroblast-like cell morphology driven by TGF-β2 in MS-1 cells15. In addition, the TGF-β2-mediated decline in Pecam-1 and enhancement of Sm22α were completely blocked in Snail-depleted MS-1 cells. In summary, we demonstrated that Snail is critical for TGF-β2-mediated EndMT in MS-1 cells (Figure 3B)15.
Figure 1. TGF-β2 induces EndMT and Snail expression in MS-1 cells. A. Effects of TGF-β2 and/or TGF-β type I receptor kinase inhibitor SB-431542 on cell morphology. Brightfield images of MS-1 cells upon treatment with TGF-β2 (1 ng/mL) and/or SB-431542 (SB, 5 µM, administered 30 min prior to TGF-β2) for 2 days. Scale bar represents 200 µm. B. Immunofluorescence staining of Pecam-1 (green) and Sm22α (red) in MS-1 cells cultured in medium containing TGF-β2 (1 ng/mL) for 3 days. Nuclei are visualized in blue (DAPI). Scale bar: 50 µm. C. Western blot with whole cell lysate of TGF-β2 stimulated MS-1 cells. The expression of Snail, but not Slug, was enhanced by TGF-β2 stimulation, as previously reported in Ma et al15. D. Quantification of Snail expression by integrating the results from three independent western blot experiments. Please click here to view a larger version of this figure.
Figure 2. Depletion of Snail by CRISPR-Cas9 gene editing. A. Scheme depicting how to generate Snail knockout cells. Bsd: Blasticidin. Puro: Puromycin. B. Oligonucleotides of two independent sgRNAs targeting Snail using CHOPCHOP (http://chopchop.cbu.uib.no/) and Cas-OFFinder (http://www.rgenome.net/cas-offinder/). C. The predicted off-target activity of the two gRNAs for Snail using Cas-OFFinder (http://www.rgenome.net/cas-offinder/). D. Cas9 and Snail expression in wild type (WT) and Cas9-overexpressed MS-1 measured by Western blot analysis. E. Knockout of Snail with two independent gRNAs in MS-1 cells as measured by Western blot analysis. We reported similar results in Ma et al15. Please click here to view a larger version of this figure.
Figure 3. Genetic depletion of Snail inhibits TGF-β2-induced EndMT in MS-1 cells. A. Brightfield images of MS-1 cells upon treatment with TGF-β2 (0.1 ng/mL) for 3 days in wildtype (WT, upper panel) and Snail knocked out (lower panel) cells. Scale bar represents 200 µm. B. Immunofluorescent staining for Pecam-1 (green), Sm22α (red) and nuclei (blue) of MS-1 cells cultured in medium containing TGF-β2 (1 ng/mL) for 3 days. Depletion of Snail abrogated TGF-β2-induced decrease of Pecam-1 and increase of Sm22α expression. Scale bar represents 50 µm. C. Schematic representation of the effect of Snail knockout on TGF-β-induced EndMT in MS-1 cells. TGF-β stimulates the expression of Snail through Smad pathway by phosphorylating Smad2/3 and further drives EndMT. Knocking out Snail using CRISPR/Cas9-based gene editing abrogated TGF-β-mediated EndMT. Please click here to view a larger version of this figure.
Understanding the mechanism of EndMT is critical for modulating this process and targeting EndMT-related diseases. Here, we described methods to perform a TGF-β-induced EndMT assay and interrogate the role of the EndMT-TF Snail in TGF-β-triggered EndMT, by performing CRISPR/Cas9-mediated stable gene depletion of Snail from cells. The depletion of Snail using CRISPR/Cas9 approach successfully abrogated TGF-β2 driven EndMT in MS-1 cells (Figure 3C). To study the effects of any cytokines, like TGF-β, on EndMT, ECs were exposed to cytokines and then the occurrence of EndMT was assessed according to morphological changes and endothelial and mesenchymal marker expression changes in cells. TGF-β2 strongly induced EndMT in MS-1 cells accompanied by a strong increase in the expression of the transcription factor Snail. The EndMT-TFs induced by TGF-β can differ according to the species or tissue-specific endothelial cell type. For example, we observed that Snail but not Slug was significantly upregulated by TGF-β in MS-1 cells, while in human umbilical vein endothelial cells (HUVECs), both Snail and Slug are increased after exposure to TGF-β20.
We assessed the extent of the EndMT process in two ways by examining cell morphology changes and then by investigating changes in EndMT-related markers expression. After TGF-β exposure for 3 days, cells underwent EndMT with consistent morphological variations and changes in the expression of EndMT-related markers. In addition to the immunofluorescence staining we performed here, marker variations can also be monitored by western blotting at the protein expression level or by qRT-PCR (Real-Time Quantitative Reverse Transcription PCR) at the gene levels21. In addition to these two time- and cost-saving methods that we showed in this protocol, there are other methods to examine EndMT. For example, performing transcriptome analysis (by RNA sequencing or qPCR) to compare the expression levels of endothelial- and mesenchymal-related genes between treated and control cells can precisely assess EndMT22,23. In addition, EndMT often involves the stable loss of barrier function, which can be assessed by impedance spectroscopy24. Furthermore, additional proof of the acquisition of stem cell-like properties by EndMT-derived cells may be examined. For instance, under specific culture conditions, EndMT mesenchymal-like cells can be further differentiated into osteoblasts, chondrocytes, adipocytes or (myo)fibroblasts. Therefore, additional analysis to confirm the differentiation into different cell types belonging to the mesoderm lineage (i.e., gene expression and matrix staining) is useful for demonstrating the multipotent nature of EndMT-derived cells. Finally, the EndMT assessment methods are not limited to in vitro studies, but can be extrapolated to investigate the relation between EndMT and some diseases in vivo or in ex vivo organs. In this sense, the use of endothelial-specific lineage tracing strategies is broadly extended to EndMT-related research25.
To investigate the role of Snail during EndMT, in this study CRISPR/Cas9 gene editing was used to knock out this gene. The data showed that TGF-β2 failed to mediate EndMT in Snail deficient MS-1 cells. This observation demonstrated that Snail is essential for TGF-β2 induced EndMT in MS-1 cells. We used an independent U6-driven sgRNA expression cassette to introduce specific sgRNAs for Cas9 to target Snail. In addition to this method, Ran et al.26 described another strategy for cloning the sgRNA oligos sequence into the Cas9 scaffold to generate a construct containing both Cas9 and gRNAs. Emerging novel approaches allow for CRISPR/Cas to incorporate additional functions. For example, double or triple knockouts can be achieved by delivering more sgRNAs into cells expressing Cas927. The engineered Cas13 protein targets and digests RNA molecules without disrupting endogenous DNA28. Besides knocking out genes with CRISPR/Cas, short hairpin RNAs (shRNAs) can be used as alternatives to stably knock down targeted gene expression29. For all CRISPR/Cas gene editing methods, off-target cleavage should always be taken into consideration. In addition, small interfering RNAs (siRNAs) transiently silence gene expression and the siRNA concentration is diluted with cell division30. Both these methods partially suppress targeted gene expression. In contrast, ectopic gene expression is also used to verify gene function during EndMT/EMT31. This approach can determine if the upregulation of a gene is sufficient to elicit an EndMT response. Therefore, currently there are a multitude of technical strategies that can be used to identify and verify potential regulators of EndMT. Besides, transcriptomic analysis can be a good option in the identification and comprehensive analysis of EndMT related regulators. We recommend using different complementary approaches to investigate the modulation of EndMT.
In summary, we introduced a workflow to identify factors that may play functional roles during TGF-β-induced EndMT. This method can also be used to study whether other stimuli (i.e., cytokines, growth factors, mechanical stimuli, cell-cell interactions) can modulate EndMT, and the interplay of TGF-β with other stimuli. In addition, we highlighted an approach using CRIPSR/Cas gene editing to elucidate whether a certain gene is required for TGF-β-induced EndMT. To illustrate this methodology, we used the strong EndMT inducer TGF-β2 in MS-1 cells, but the protocols can be adapted to other cytokines and other cell types. We expect that this detailed protocol described will serve as a stepping stone for future EndMT-related studies.
The authors have nothing to disclose.
The research was supported by CGC.NL and the Netherlands Cardio Vascular Research Initiative: the Dutch Heart Foundation, the Dutch Federation of University Medical Centers, the Netherlands Organization for Health Research and Development, and the Royal Netherlands Academy of Sciences Grant awarded to the Phaedra-Impact (http://www.phaedraresearch.nl). JM is supported by the Chinese Scholarship Council. GSD is supported by a Trampoline grant from AFM-Telethon , FOP Italia and a grant from La Fundació La Marató de TV3 (#202038).
|0.45 μm filter
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|10× T4 DNA ligase buffer
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|12 mm round glass slice
|20 mL syringe
|BD Eclipse, USA
|Vector Laboratories, USA
|VECTASHIELD Antifade Mounting Media
|Dr. M Gonçalves, Leiden University Medical Center, Netherlands
|Serva Electrophoresis, USA
|GE Healthcare, USA
|Cell signaling, USA
|Buffer O (10X)
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|Thermofisher Scientific, USA
|Leica Microsystems, Germany
|Thermo Fisher Scientific, USA
|Donkey anti-rat Alexa 488
|Thermo Fisher Scientific, USA
|Thermo Fisher Scientific, USA
|Leica Microsystems, Germany
|Goat anti-rabbit Alexa 594
|LabNed Plasmid kit
|MS-1 cell line
|American Type Culture Collection (ATCC)
|QIAquick gel extraction kit
|Cell signaling, USA
|T4 DNA ligase
|Thermofisher Scientific, USA
|TC20 automated Cell Counter
|Joachim Nickel, University of Wurzburg
|Other commercial recommendation: 302-B2, R&D systems
|Tocris Bioscience, UK
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