Perhaps the problem does not lie intrinsically in the HUVECs, but instead in either the source of the growth factor, or a design feature of your experiment (length of treatment, phenotypic assay, cell density and/or substratum selected). If you provide some more details about the type of GFs employed, and the types of read-outs you are using.

Dear Manindra even though David is right is his comment, I will simply discard the cell line and request another batch. Sometimes when you freeze the cells or the procedure to re culture them the cells are altered and the subsequent passages the cells no not recover. The results that you may obtein are unuseful.

good luck regards, 

Thank you very much for taking time for answering my question. 

Daniel, I intend to measure some activation markers and adhesion molecules upon the treatment of HUVECs with adipokines over a period of 24 to 36 hours. Cells were seeded in 24 well plates at a density of 80-100K cells/ well, starved in 0.5% EBM for 16h, and treatments added. Read outs were already documented molecules such as IL6, or VEGF, but I could not see any difference. Unfortunately, we did not see any significant change upon performing a cytokine array analysis. 

Juan, I appreciate your help. I shall try to get the new batch. Could it be maintained over time? Is there any suitable method to check the responsiveness of HUVECs in time/ dose dependent manner?

Dear Manindra yes CD31 or vWF are the key markers. However the issue is to get confluent cell culture in short time. Regards,

Manindra, perhaps the HUVEC would be responsive if slightly less crowded?  Sometimes cells are not responsive to GF when too confluent.  Maybe repeat, using 40-60K per well with 80-100K as "negative" control.  You could also try pre-coating well with gelatin, collagen I or fibronectin to see if it makes a difference.  Also, talk with Lonza tech services for suggestions.

So that's an extremely high cell density for HUVECs (around 50K/cm2). I'd anticipate that IL6 expression, and probably VEGFA mRNA, are elevated at high vs low cell density. So you may already be near maximal expression levels without further stimulation. Have you compared mRNA levels before and after serum starvation? You need to show that the serum starvation worked before you can ask whether addition of adiponectin or another adipocytokine changes gene expression.

Agree with Susan that collagen I or fibronectin could be important for the gene expression response.

Also note that many papers use mRNA levels as read-outs of gene expression. These changes don't necessary correlate with levels of protein, much less secreted protein. So unless the literature used the same cytokine array, it is hard to know what the expected result should be!

Dear Manindra

Before you change the conditions, you need to answer a fundamental question, how much like the in vivo condition do you want to simulate in culture. Confluent culture is one step towards the in vivo situation unless you are looking at post angioplasty repair and migration. If you have tried this experiment with multiple batches of HUVEC then that is probably the right answer. Previously published reports are sometimes hard to replicate as the media you are using and the culture conditions will e different, but if your culture conditions more accurately replicate a confluent monolayer then you may be closer to the 'truth'. I tend to plate at a moderate density and culture for 2-3 days before I use cells. I also use HCAEC as I am interested in cardiovascular disease. This allows the cells to form mature cell-cell junctions and lay down their own basement membrane proteins, so I hope they respond more authentically. I also do most of my experiments under physiological shear stress as well.

Try a few batches, test for immediate MAP kinase activation (~30 minutes), IL8 responds to most things in endos and VCAM1 to anything that regulates NFkB. If none of these change, then it is probably the reagents you are using...

Seeing VEGF is one of the GFs you are using, it might be worth trying to see if VEGFR2 is phosphorylated on a western blot in response to it? That way you know if the cells still have the receptors and whether they respond to it, at least in the short term. But yes, I find HUVECs are most responsive at around 50-60% confluent. 

I know I'll just be re-phrasing some of the comments here already, but... You should really consider you initial culture confluence: 50-60% area coverage is usually a good starting point. Also, as mentioned before, your readouts seem to be based on secreted protein levels. Keep in mind that you need (and this is an estimation) about 24h post induction to get soluble proteins differences in media, and another 24-48h (meaning 48-72h post induction) for physical readouts like tubulogenesis and sprouting (again, estimated times, might wanna keep an eye on your cultures every 12h or so).

As an extra-thought, even though 0.5% FBS should be low enough for your methods, FBS differs a bit from batch to batch. I'm assuming you have a nonstimulated control so you can "subtract" the background of your experiment vs your induced controls (data on ratio vs nonstimulated control)? If this is the case, it's possible you just need longer times to have better accumulation of your factors of interest in the media, but check your cells are somewhat proliferative during the experiment for optimal protein synthesis and secretion.