for Murine Annexin II
Alina Predescu, Biology, University of Maryland Baltimore County,
1000 Hilltop Circle, Baltimore, MD 21250
Dr. Sandra McLeskey, Department of Adult Health Nursing, School of Nursing,
University of Maryland Baltimore, Baltimore MD 21201
Angiogenesis is the
process of blood vessel formation. In adults it is found at the level
of wound healing and the female reproductive cycle. However, pathological
angiogenesis occurs in the formation of the microvessels that supply tumors.
Our study focuses on the molecular differences between the blood vessels
in the tumor compared with the ones in the rest of the body. We want to
know the importance of genes that are expressed in the tumor endothelial
cells, having to do with digestion of fibrin. More fibrin is found in
tumor extracellular matrix than in extracellular matrix of the normal
tissues, because the tumor-associated blood vessels are abnormally leaky.
They allow the extravasation of fibrinogen. The tumor-associated endothelial
cells have to digest fibrin during the formation of new blood vessels.
One of the genes upregulated in tumor endothelial cells is tissue plasminogen
activator (tPA), which activates plasminogen into plasmin, which degrades
fibrin. Normally inactive, tPA is activated by annexin II. Therefore,
in order to shut down the activation of tPA, annexin II needs to be inactivated.
A way to achieve this is to produce antisense mRNA for annexin II that
would bind to the sense annexin II mRNA so it won’t be translated
into the protein annexin II. For this, a vector that will produce antisense
annexin II mRNA needs to be constructed. The vector will be used to infect
tumor-associated endothelial cells, shutting down their synthesis of annexin
II protein.
Mouse cDNA for the coding region of annexin II was amplified by RT-PCR
from total RNA extracted from a murine hemangioma cell line (EOMA) using
primers with appended Sal I restriction sites. The cDNA was gel purified
and digested with Sal I to obtain Sal I sticky ends. The retroviral vector
pLNCX2 was digested in the multiple cloning site with Sal I and the 5’
phosphates were removed with alkaline phosphatase to prevent self-ligation.
The insert and the plasmid were ligated and HB 101 bacteria were transformed.
Plasmids will be isolated from transformed bacteria and directionality
of inserts determined. Plasmids with inserts in the antisense direction
will be sequenced to assure there were no mutations introduced by the
RT-PCR. Ecotropic pachaging cells will be transfected with the vector.
The infectious virus they will produce will be used in vitro and in vivo
angiogenesis experiments to stop expression of annexin II. These experiments
will demonstrate the importance of tPA activation in angiogenesis.
