The role of NADPH oxidase in age-related collateral growth impairment Nandita Chittajallu1, Mary J. Wenning2, Bruce Henry2, Matthew DiStasi2, Randall G. Bills2, Joseph L. Unthank2, Steven J. Miller, PhD 1Pike High School, Pike, IN, and 2Department of Surgery, Indiana University School of Medicine, Indianapolis, IN ABSTRACT Due to cardiovascular disease or injury, arteries may become blocked, decreasing blood flow and thus oxygen delivery to tissues and organs. However, pre-existing arteries that bypass the blockage site may enlarge to compensate for the decreased blood flow. This process, known as collateral growth, is dependent on the production of nitric oxide which is stimulated by decreased blood flow in the bypass arteries. Collateral growth is impaired during aging, and this occurs in part because of an increase in reactive oxygen species (ROS) which interfere with nitric oxide production. The major source of ROS produced in arteries is thought to be an enzyme called NADPH oxidase, which exists in several forms and locations in the artery wall. The goal of this project was to determine how the major types of NADPH oxidase regulate collateral growth by measuring temporal changes in their expression/activity and localization during flow-mediated vascular remodeling. By using quantitative PCR, it was determined that the basal arterial expression of Nox4 and p47phox increases with age. There was also a significant increase p47phox in aged rat collaterals at 7 days in comparison to those of young rat collaterals. This suggested that they are the sources of chronic oxidative stress and therefore inhibitors to collateral growth. This knowledge will allow for the development of targeted approaches to promote collateral growth in aged patients. METHODS Figure 6. Using quantitative PCR, Nox2, Nox4, and p47phox mRNA expression was determined in mesenteric arteries from young and aged WKY rats. (* p<0.05 vs. young). Nox4 and p47phox mRNA expression was increased with age. Rat Mesenteric Collateral Growth Model Figure 3. Collateral Growth Models - A model of arterial obstruction with a pre-existing collateral pathway was created in the ileal arteries of young (2-3 month) and retired breeder (aged; 10-12 month) male Wistar Kyoto WKY rats. At 1, 3, and 7 days, respectively, digital images of collateral and same animal control arteries (maximally dilated) were obtained and compared to diameters at day 0. SUMMARY A specific inhibitor of Nox stimulated collateral development in aged rats (Fig.5). Basal arterial expression of Nox4 and p47phox, but not Nox2, was increased with age (Fig.6). Collateral expression of p47phox was normalized to control levels in the young, but not the aged rats, at 7 days (Figs. 7 & 8). CONCLUSIONS Excessive Nox activity in aged rats inhibits collateral growth. Nox4 and p47phox appear to be the main sources of chronic oxidative stress in aged arteries. Increased expression of p47phox in collateral arteries may contribute to the age-related impairment in collateral development. FURTHER STUDIES Performing additional experiments in other vascular beds is warranted to ensure that observations are not simply coincidental. The effects of long-term (>7 days) elevated flow on Nox expression also will be investigated. Therapy with siRNA to inhibit expression of specific Nox isoforms/subunits responsible for oxidant stress in aged arteries may be a method to promote collateral development in patients with PAD. ACKNOWLEDGEMENTS Thank you to Project SEED and its funders for providing the opportunity to gain experience in research. Also thank you to Dr. Miller’s lab for all of the knowledge they have passed on and fond memories that have been made over the course of the summer. INTRODUCTION Peripheral Arterial Disease (PAD) is an arterial occlusive disease that affects 8 to 12 million people in the United States and its incidence increases with age. Collateral growth, the enlargement of pre-existing bypass arteries, provides an alternative pathway for blood flow in response to arterial occlusions and can partially or completely compensate for the blockage. Flow and shear-mediated production of nitric oxide (NO) and reactive oxygen species (ROS) play a role in vascular events such as dilation and remodeling (Fig. 1). The balance between basal NO and ROS is altered by disease and aging, which inhibits collateral growth. NADPH oxidase (Nox) is the primary source of vascular ROS. It exists as multiple isoforms: Nox1, Nox2, Nox 4, Nox 5 (Fig. 2). Previous work suggests that Nox regulates collateral growth in young and aged animals, but the role of specific Nox isoforms is still unclear. Shear Stress ROS NO Dilation and Remodeling Nox eNOS Figure 1. Illustration of the important role of shear stress dependent NO and ROS production on the regulation of vascular tone and structure. Example of Successful Collateral Growth Day 0 Day 7 Artery Vein Artery Vein Figure 4. Representative micrographs showing the increase in diameter between the same collateral artery in a young rat on the day of ligation (left) and 7 days post-ligation (right). The diameter has increased 26% from 223 µm to 303 µm. No significant diameter change occurred in collaterals from aged rats or in any control arteries at either age. Figure 7. Changes in collateral (high flow) artery Nox mRNA expression at 1, 3, and 7 days were determined as compared to control (normal flow) arteries (*, +, #: p<0.05 vs. control, 1 day, 3 day; n=4-6). Mesenteric Collateral Artery Nox Expression is Time Dependent in Young Rats Vascular Nox isoforms/subunits Figure 2. Diagram depicting NADPH oxidase (Nox) isoforms and subunits present in the vasculature. Nox5 is present in humans, but not rodents . Quantitative PCR – RNA was isolated from harvested arteries and reverse transcribed to produce cDNA. The cDNA is combined with fluorescent dual-labeled primers/probes to specify the section that must be amplified. Based on the levels of cDNA produced, it is possible to determine how much message was expressed. The changes in collateral Nox mRNA were normalized to hexokinase-1 and expressed as a collateral/control ratio. RESULTS Nox inhibition promotes collateral growth in aged rat arteries Figure 5. A specific peptide inhibitor of Nox (gp91ds-tat) promoted collateral artery growth in WKY aged rats. The aged control not receiving ds-tat was administered a control peptide via osmotic minipump. Aged + ds-tat was administered gp91ds-tat at 1 mg/kg/day (*p<0.05 vs.. control, n=3-6) Figure 8. Changes in collateral (high flow) artery Nox mRNA expression at 1, 3, and 7 days were determined as compared to control (normal flow) arteries. (*, +; p<0.05 vs. control, 1 day; n=3-6). Mesenteric Collateral Artery Nox Expression is Time Dependent in Aged Rats OBJECTIVES The goal of this project was to assess the differential temporal changes in expression between the various NADPH oxidase isoforms during unsuccessful collateral growth in aged rats to determine the role of the specific isoforms/subunits.