I have performed gelatin zymography on HUVEC after treated with my compound of interest which suppose to be antiangiogenic.
Cells were cultured in serum-free media. The conditioned medium was collected for gelatin zymography. There is only one band representing pro-MMP-2 found at the end for all the groups (Untreated control and all treatment).
No MMP-9 and active MMP-2 were detected, even in the untreated control.
Is this normal? Anyone have any papers to support this too?
Many thanks in advance.
Hi Dylan,
Unstimulated HUVEC should only show proMMP-2, and no MMP-9 (active or pro) or active MMP-2. See for example Biochem J 296, 803-809 (1993). Active MMP-2 and MMP-9 are induced upon activation with eg phorbol ester (PMA).
In my hands unstimulated HUVECs do not express MMP-9 but readily do so when treated with pro-inflammatory cytokines.
Unstimulated and stimulated (PMA) HUVEC cells exhibit MMP-2 expression.
Ref: Roomi etal Oncology Report: 14(6), 1399-1404, 2005 (Open excess)
yes, retinoc acid (RA),PMA, or IL1 treatment shall trigger MMP-9 expression and detection in culture stn via casein, gelatin zymography or epitope detection
In connection with others answer, i have clarification, If active MMPs is not present in HUVEC cell then how HUVEC cells getting migrated and invaded to initiate angiogenesis or how tube formation takes place in HUVECs? Because MMPs are essential for migration and invasion of HUVECs.
You are right you need active MMPs, but they must only be active when migration is happening. And therefore you need a trigger which leads to MMP activation. In a normal state it is not unusual to only detect the latent forms.
Hi Dylan! In my experience HUVEC constitutively expressed only
MMP-2 an you need to activate MMP9 production. You can try to pre-stimulate HUVEC with 30 nM PMA under serum-free condition.
For more details, you can see this: http://www.ncbi.nlm.nih.gov/pubmed/26263024
Good luck!
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