Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-β Signaling

This method can help answer key questions in vascular and cancer biology fields, such as mechanisms of endothelial plasticity in various pathological conditions, including cancer. The main advantage of this technique is that robust induction of in vitro EndMT is accomplished by treatment with TGF-in MS-1 endothelial cells. First grow the MS-1 cells in standard culture conditions and avoid the culture from being confluent.

Next, on a 10 cm dish, wash the MS-1 cells with 1x phosphate-buffered saline. Then add 1 mL of trypsin to the plate and incubate the plate at 37 degrees Celsius for five minutes. Then add 9 mL of culture media to the trypsinized cells for detachment.

Then collect the trypsinized cell suspension in a 15 mL tube. Centrifuge the cell suspension at 300 to 400 x G for five minutes at room temperature. Next, use an automatic cell counter to count the number of viable cells.

Then plate the MS-1 cells on uncoated standard culture plates for long-term culture. After 24 hours, stimulate the endothelial cells with a final concentration of 1 ng per milliliter of TGF-2. Then incubate the culture plate at 37 degrees Celsius with 5%carbon dioxide.

After 48 hours of treatment, replace the medium with fresh, pre-warmed culture medium containing TGF-2 For the downstream analysis, plate the MS-1 cells on an 8-well culture chamber slide pre-coated with gelatin. After 24 hours, add 10 M of ROCK inhibitor to the cell culture. After an hour, stimulate the endothelial cells with a final concentration of 1 ng per milliliter of TGF-2.

Then incubate the cells at 37 degrees Celsius. For a 72 hours long culture, replace the old medium with fresh TGF-2-containing medium after 48 hours. After one whole day of TGF-treatment, remove the medium.

Then fix the cells with 4%paraformaldehyde and phosphate-buffered saline for 20 minutes at room temperature. After the fixing period is over, rinse the cells with phosphate-buffered saline. Then incubate the cells with 0.2%Triton X-100 for five minutes at room temperature.

After five minutes, again rinse the cells with 1x phosphate-buffered saline three times. After three washes, incubate the cells with diluted fluorescent phallotoxin containing blocking buffer for an hour at room temperature. Post incubation, wash the cells with 1x phosphate-buffered saline, thrice.

Then stain the cell nuclei using cyanine nucleic acid binding dye for five minutes at room temperature. After five minutes, rinse the slide, and mount a cover slip on the slide using slide mounting media. Observe the cells under a confocal microscope.

The cells show reorganization of the thick stress fibers upon TGF-2 treatment. This indicates the potency of TGF-2 in inducing EndMT in endothelial cells. After 72 hours of TGF-2 treatment, discard the medium and fix the cells with 0.5 to 1 ml of 50%cold methanol and 50%acetone for five minutes.

Then rinse the cells with 1x phosphate-buffered saline, thrice. After the three washes are complete, incubate the cells with primary antibodies in blocking buffer overnight at 4 degrees Celsius, in the dark. The following day, again wash the cells with 1x phosphate-buffered saline, thrice.

Then incubate the cells with green dye conjugated secondary antibody to VE-cadherin in blocking buffer, for one hour in the dark. Again wash the cells with 1x phosphate-buffered saline, thrice. Then stain the cell nuclei using cyanine nucleic acid binding dye for five minutes at room temperature.

After five minutes, rinse the slide, and mount a cover slip on the slide using slide mounting medium. Then observe the cells under a confocal microscope. The cells show decreased expression of VE-cadherin, and increased smooth muscle actin expression upon TGF-2 treatment for 48 to 72 hours.

Before the collagen gel contraction assay, culture the MS-1 cells in the presence and absence of TGF-2 for 72 hours. Then prepare the type 1 collagen gel on ice. Then mix 800 l of the collagen gel solution with 200 mL of the control, or TGF-2-treated endothelial cells.

Next, add 1 mL of this mixture to each well of a 12-well culture plate. Let the gel solidify at 37 degrees Celsius for 30 to 60 minutes. After the solidification of the gel, add 1 mL of MEM Alpha, containing 10%fetal calf serum, 50 units per milliliter of penicillin, and 50 g per milliliter of streptomycin, to float the gel.

Then detach the gel from the wall of the well, and incubate the floating gel at 37 degrees Celsius for 48 hours. After 48 hours of incubation, scan the images of the gel from the bottom of the plate using a scanner. Distinct morphological changes are visible, transforming the cells from the cobblestone to the mesenchymal spindle shaped architecture due to exposure to TGF-2 treatment for 72 hours.

Interestingly, TGF beta 2 treatment for 48 to 72 hours shows an increase in both the the mRNA and the protein expression levels of smooth muscle actin in the MS-1 cells. The gel contraction assay also shows that upon TGF-2 treatment, the collagen type 1 gel contracts significantly. Interestingly, deprivation of TGF-2 decreases the smooth muscle actin to basal level and the cells resume the cobblestone morphology, indicating that EndMT is a dynamic process.

In fact, inhibiting microRNA 31 by LNA microRNA inhibitor using the MS-1 cells, attenuates the induction of mesenchymal markers. However, the inhibitor does not affect the induction of TGF-target genes such as Fibronectin 1, PAI-1, and Smad 7. Following this procedure as a method like gene expression, knockdown, or microarray modulation, can be performed in order to answer additional questions like specific gene and microRNA function in EndMT.