Our interest is in endothelial cell injury and death. Endothelial cells
line the inside of all blood vessels. These vessels feed normal organs and
cancerous tumors, keeping them alive. We are focusing on DNA damage as a
cause of endothelial cell death because of the large number of agents that
damage DNA.
A diagram of what may happen when endothelial cells are damaged.
Anticancer drugs, radiation, and environmental substances are
among the common DNA damaging agents. A large group of agents that generate
reactive species from oxygen can also damage DNA. Many drugs are oxidants,
and oxidants are found in many places in the environment (air pollution
for example). Damage to normal endothelial cells impairs the operation of
the circulatory system.
In atherosclerosis debris ultimately accumulates where endothelium is damaged.
This can restrict blood flow and promote formation of dangerous clots. This
can cause heart attacks and stroke. Pulmonary fibrosis caused by the DNA-damaging
anticancer drug, bleomycin.
The accumulated collagen restricts gas exchange, leading to death of the afflicted individual.
On the other hand, it may be useful to kill endothelial cells in developing
tumors with standard DNA damaging anticancer drugs. Our goal is to manipulate
the sensitivity of endothelium to exogenous and endogenous DNA damaging
agents in order to inhibit injury to normal tissue, and to enhance tumor
killing by anticancer drugs.
The Cellular Environment Influences Cell Function
Potential sites of action of integrins on DNA damage and the endothelial cell response to it. How could one modulate the sensitivity of endothelium? Factors in the cellular environment also regulate the response to DNA damage. These factors activate receptors and transmit signals into the cell. These signals may affect the response to DNA damage. We have found that integrins, which are receptors for extracellular matrix proteins, inhibit DNA breakage and cell death caused by many agents in endothelium. Integrins are found on almost all cells. Thus, integrins are a handle by which we may manipulate DNA damage, cell death, tissue injury and anticancer therapy throughout the body. Integrins generally bind to RGD (arginine-glycine-aspartate) sequences matrix proteins and attach the cells to them.
One DNA strand breakage assay using in situ labeling of DNA breaks with DNA polymerases and a fluorescent nucleotide. A fluorescence microscope and image analysis gives us information on the extent of DNA breakage in the endothelial cells. A cardiovascular toxin from bacteria (LPS) was used here to cause DNA breakage. DNA damage is a lot less in cells grown on a collagen extracellular matrix that can activate integrin receptors.
The fact that integrin engagement can cause resistance to drug-induced DNA breakage suggests that the receptors can be used to manipulate the sensitivity of endothelial cells.
Cells Respond to DNA Damage
Once DNA is damaged, special signaling systems are activated. This could help cells resist injury, or it may actually contribute to the toxic impact of DNA damage. Poly (ADP-ribose) polymerase and the tumor suppressor, p53, are two molecules that are activated by DNA damage and that regulate apoptosis. Thus, DNA damage, DNA repair, and signals generated by the residual DNA breaks are likely to govern cell death through these systems. We are investigating the role of these two systems in DNA damage-induced cell death.
Fluorescent immunostaining for poly(ADP-ribose) in the nuclei of cultured endothelial cells treated with or without BLEOMYCIN (BLM). Poly(ADP-ribose) Polymerase (PARP) is an enzyme in cell nuclei that binds to the ends of DNA strand breaks. This turns the enzyme on, and it attaches polymers of ADP-ribose to proteins in the cell nucleus. You can see that the DNA damaging drug causes large amounts of poly(ADP-ribose) to be synthesized in cell nuclei. We can actually measure the relative amounts of polymer by computer image analysis of pictures like the ones below. This tells us about the activation of PARP in different situations. We are also using cells from mice that have had poly(ADP-ribose) polymerase deleted by knockout methods (PARP Knockout, mice donated by Dr. Csaba Szabo of the Inotek Corporation). They have very low staining. Wildtype and knockout cells are used to study the role of PARP in genotoxicity in endothelium. Cells lacking PARP may have entirely different reactions or sensitivity to DNA damage.
Nuclear localization of GFP fused to the PARP NLS.
PARP and other proteins can be manipulated by molecular biological methods to alter the cellular distribution of PARP, or to produce antagonists of the enzyme. These antagonists can be used to modulate or disrupt PARP in order to investigate its role in cell function. They also aid in determining viable strategies for new drug development. As an example, we cloned the DNA sequence encoding the Nuclear Localization Sequence (NLS) in PARP that is responsible for its concentration in cell nuclei. This domain was fused at the DNA level with Green Fluorescent Protein (GFP), originally derived by others from jellyfish mRNA. Plasmids containing these clones were transfected into endothelial cells, which then express the engineered proteins.
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