We did a few days ago. We thought it might be due to smooth muscle cells contamination so did not use them. But, we did not verify further.

Ah ha - I suspect you did not have contamination - when/if you repeat your procedure, please let us know (Thanks for sharing)

I think that little positivity for alpha actin is not uncommon in endothelial lines. The endothelial phenotype of the cells is not 'set in stone' and your cells might shift a little bit in the endothelial to mesenchymal spectrum. I think you'll have a little EndMT just because the cells don't see laminar flow anymore. Other factors might also contribute like TGF-b in your serum, perhaps a lack of VEGF, oxygen radical stress in the hyperoxic culture environment, the high stiffness of the tissue culture plastic, collagen coatings and so on. What culture conditions are you using? The susceptibility of the cells to this differs per isolate, maybe due to epigenetic factors (although in your inbred rats variability might be less).

To truly push the cells to a mesenchymal phenotype is difficult however, so I wouldn't worry too much. If the staining is homogenous and dimly positive, I wouldn't worry about contamination. Mostly you can see easily it by eye too, although for rat it is less clear than in human or pig.

I agree that nothing is 100% in biology. Maybe our endothelial cells express some a-actin but we did not look into it more thoroughly. We were trying to do knockdown and knock-in experiments and did not want to take any risk then.

I agree to Hendrik and Ru-Jeng. It is very likely you find some alpha actin in your endothelial cells.

Endothelial cells cultivated under artifical tissue culture change a little biz phenotype according to used media, supplements and coating. So its easily possible that they express alpha actin.

We have clearly seen low level expression of alpha- vascular smooth muscle actin in cultured lymphatic endothelial cells by multiple modalities (message and protein) that are >95% pure by all other classic lEC markers. Indeed in a number of lymphatic development studies done in situ "LECs" begin to express significantly more aVSM actin when various growth factor pathways are altered. This is of course all work in Lymphatic not blood vessel endothelium.

Thanks Dave - I take the distinction between lymphatic and microvascular EC - Since these are low passage cells I am not convinced this is phenotypic drift. We are presently growing up EC from macrovascular and microvascular sources to make direct comparisons. Need to take some lymphatic EC as well :)

Just an update. One of my colleague just claimed that he saw some primary cultures change phenotype and demonstrate smooth muscle actin. He believes primary culture, especially from premature animals or fetus, may have such potential to do so and it will be interested to do further studies to prove it.

Interesting - we have seen the smooth muscle actin in microvascular EC from adult rats (indeed, the juveniles have phenotypic differences). I wonder if there is something in the media that is potentiating the expression.

Pericytes (that express alpha smooth muscle actin) are a common contaminant when I isolate primary rat brain endothelial cells. One way we cope with it is by adding puromycine during the early days of our cultures. Also our ECs are very poor proliferators such as pericytes may overgrow our cultures.

Be sure that you indeed have endos and not the pericytes outgrowing from your cultures. You can try lectin staining to further confirm that you may have ECs.

Thanks Abraham - we are seeing it in microvascular EC isolated now from mesentery, skeletal muscle and aorta - none of these are vessels thought to be endowed with pericytes to the extent you are likely to see from brain or retina. '"Curiouser and curiouser" cried Alice'

Dear Virginia, thanks for your reply. Have you investigated several markers for your endothelial cells at the time (PECAM-1, VE-cadherin, vWF)? What about DiI-AcLDL uptake? You can also try PDGFRbeta to confirm the pericytes origin.

YEs - they express PECAM-1, VE-cadherin, vWK, show Dil-ACLDL uptake and form tubes on matrigel. We have not yet probed for PDGFRbeta - in the process of isolating fresh cells. The microvascular phenotype is not cobblestone at confluence while those from the aorta of those animals is - if the microvascular EC are stem-cell-like, is it not possible we are getting pericytes with differentiation?