foundation Scholar halee patel oklahoma city, Oklahoma freshman, university of missouri-kansas city final research manuscripts

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1 w. T. Payne foundation Scholar halee patel oklahoma city, Oklahoma freshman, university of missouri-kansas city final research manuscripts

2 Effects of Oxidized LDL on Protein Expression in Macrophage Derived Foam Cells Halee Patel Oklahoma City, Oklahoma Freshman, University of Missouri-Kansas City, Kansas City, Missouri Mentor: Mike Kinter, Ph.D. Oklahoma Medical Research Foundation, Free Radical Biology and Aging Research Program Abstract Atherosclerosis is a vascular disease which involves a hardening of the arteries by the buildup of lipids, like cholesterol, in blood vessels. Initiation of atherosclerosis begins with the oxidative modification of low-density lipoprotein and the absorption of this oxidized low-density lipoprotein (oxldl) by macrophages to form foam cells. The foam cells accumulate into fatty streaks in the arteries and actively drive progression of the disease. The ability of macrophages to take up oxldl, which exhibits high toxicity, and maintain survival is not clearly understood. Our objective was to determine changes in the expression of the antioxidant proteins in response to the oxldl uptake. The effects on protein expression may illustrate how oxidative stress affects foam cells and how these cells subsequently contribute to the disease. The antioxidant proteins are a network of enzymes that metabolize and detoxify reactive species to prevent oxidation of other important biomolecules. Our study used the J774 cell line treated with oxldl, with unoxidized (native) LDL as a control. We treated the J774 cells in varying doses (0, 25, 50, and 75 μg/ml) of oxldl and native LDL (natldl) and for varying times. The cells were harvested after treatment and processed for analysis. Through gel electrophoresis, we obtained the protein samples which were digested with trypsin. Extracts of the digests were analyzed by mass spectrometry to quantify protein expression. Our results have found increase expression of peroxiredoxin-1, peroxiredoxin-4, superoxide dismutase-1, catalase, and thioredoxin reductase-1. Interestingly, other antioxidant enzymes such as the glutathione peroxidases and the other peroxiredoxins were not changed. Expression in the natldl treated cells did not change any protein that we studied. Future studies can now look at the functional effect of these changes. Understanding these changes in protein expression could be used to design strategies to affect and alter the progression of atherosclerosis. Introduction Cardiovascular disease has been the leading cause of death in the United States since There are different classifications of cardiovascular disease; however, atherosclerosis causes approximately 75% of all cardiovascular disease-related deaths. Atherosclerosis is the hardening of the arteries due to the buildup of lipids, such as cholesterol, in the arterial intima. Atherosclerosis has a chronic inflammatory component due to the accumulation of monocyte-derived macrophages. In Greek, the literal meaning of athero is gruel or paste and sclerosis is hardening. The gruel or pasty lesions that cause the hardening are composed of lipids, smooth muscle cells, T-cells, and macrophages. The development of atherosclerosis starts with the oxidation of the low-density lipoprotein (LDL) within the arterial wall. 1,2 LDL is a type of cholesterol that we normally call bad cholesterol. Oxidative modification of the LDL can be toxic to cells and is usually reversed by antioxidant proteins. Macrophages in the arterial intima take up the oxidized LDL (oxldl). This uptake produces a foamy appearance, and hence these cells are called foam cells. The foam cells coagulate to produce a fatty streak along the wall of the artery. 3,4 Figure 1 illustrates the formation of a fatty streak. This area of accumulation is called an atheroma which progresses in severity at later stages of the disease in which the vessel becomes narrow and less elastic. Ultimately, thrombosis blocks the flow of blood to produce a heart attack or stroke. The recognition of the role of the oxidized LDL in this process is a part of the oxidative hypothesis of atherosclerosis. This hypothesis began with the work of Brown and Goldstein, who recognized that LDL uptake is highly regulated unless the LDL has been modified. 5,6 Subsequent work by Figure 1. Initiation of Atherosclerosis: Foam Cell Formation. When LDL enters the arterial intima, it undergoes oxidative modification. Macrophages take up this oxldl and turn into foam cells which accumulate to form a fatty streak. Fatty streaks mature into advance lesions and drive the progression of atherosclerosis.

3 Figure 2: Multiplexed Assay used to Interrogate the Antioxidant Enzyme Pathway. Steinberg and Chisholm7 showed oxidation as a physiologically relevant way that LDL could be modified. A key part of these experiments was the high toxicity of oxidized LDL to cells such as fibroblasts, endothelial cells, and vascular smooth muscle cells. However, this observation of toxicity raises an important question. How does the macrophage take up large amounts of oxldl to generate foam cells and still maintain survival? Usually when cells such as endothelial cells and fibroblasts take up an oxidized molecule, they die. However, when macrophages take up oxldl and become foam cells, they continue their development of the disease. The hypothesis we are testing is that the oxldl uptake produces a compensatory change in antioxidant protein expression in foam cells that support this process. Materials and Methods Oxidation of LDL. Human LDL with a concentration of 5.0 mg protein/ml was obtained from Intracel (Frederick, Maryland). The LDL solution was dialyzed overnight in tris buffered saline (TBS) (20 mm tris and 150 mm NaCl with a ph of 7.8) to remove any preservatives. The LDL was then oxidized by dialysis against 5 nm CuSO4 overnight at room temperature. The oxidation reaction was stopped by transferring the oxldl dialysis tube to a new flask containing the stop buffer (TBS mm EDTA). Preparation of Protein Samples. Cell Culture. J774 cells were grown in DMEM +Selenium media with 10% FCS. A combination of dose and time experiments were carried out, in which the cells were treated with either oxldl or native LDL (as a control). Typical doses ranged from 0 to 150 μg/ml. Treatments were either 24 or 48 hours. After treatment, cells were scraped into TBS and centrifuged to obtain cell pellets. 0.5 ml of protease inhibitor was added to each of the cell pellets and before being centrifuged again. 0.5 ml of lysis buffer was added to each cell pellet making the solution viscous and nuclease reagent was added to clear the viscosity caused by the interaction of the SDS and DNA/RNA in the lysis buffer. A BioRad DC protein assay was performed with the homogenates to determine the volume needed of each cell solution in order to precipitate the protein. In small Eppendorf tubes, 10% SDS, the protein volume from the calculations, and internal standard were added for each protein sample. Cold acetone was added to each of the samples and the samples were placed in the freezer overnight to precipitate the protein.

4 Figure 3. The graphical representation of the mass spectrometry data which show an increase in protein of expression of peroxiredoxin-1 as the concentration of the oxldl is increased. Figure 4. Selected Increase in Peroxidases. a) Cat expression was increased as the concentration of oxldl increased. b) However, Gpx1 expression showed no change as the concentration of the oxldl increased. A similar lack of effect was seen for Gpx4. Figure 5. Visual Representation of Coordinated Response in Reductases. a) Txnrd1 b) Gsr c) G6pd increased their expression as the concentration of oxldl increased.

5 SDS Page. 100 µl of dilute sample buffer (50 mg DTT plus 1 ml Laemmli Sample Buffer diluted 1:1) was added to each dried sample. 20 µl of each sample was loaded onto a Criterion precast gel with 18 wells alternating samples with different concentrations of oxldl. Molecular markers were added in the first well as well as dilute sample buffer to wells on both sides of the loaded samples to keep the samples in place. The gel ran for approximately minutes or 1 ½ cm at 150 Volts. The gel was stained with Gel Code Blue Stain. Protein Digestion. Each lane of the gel was cut into approximately 1 mm3 pieces. 500 μl of wash reagent (50% ethanol/5% acetic acid) was added to each tube. The tubes were placed in a hot water bath at 50 C overnight to destain the gel pieces. Using the SRM digestion protocol, the gel pieces were completely destained, reduced and alkylated using DTT and iodoacetamide. The gel pieces were digested using trypsin at room temperature overnight. 200 μl of extraction reagent was added to the gel pieces. The protein extracts were transferred to another Eppendorf tube. 150 μl of acetic acid was added to the dried tubes. This solution was transferred to glass vials used in liquid chromatography-mass spectrometry. The mass spectrometer analyzed the proteins from Figure 2 in the samples. The above procedure was repeated for native LDL. Results A. Validation using Peroxiredoxin-1. Mass spectrometry is a relatively novel method for protein quantification, in comparison to the traditional Western blot analysis method. To validate this method, peroxiredoxin-1 (Prdx1) was studied first. Prdx1 is known for regulating levels of lipid hyrdroperoxides and peroxides. Previous data showed that increase in oxldl concentration did produce a significant increase in protein expression of Prdx1. 8 Reproducing these results using the LC-MS method was needed to demonstrate the validity of subsequent experiments. Our protein expression values were measured and expressed as ratios to the housekeeping protein voltage dependent anion channel-1 (Vdac1). These ratios were normalized to the 0 μg/ml values. Our results showed a significant increase in protein expression as the concentration of oxldl increased, with an overall 60% increase at the 75 μg/ ml concentration relative to the 0 μg/ml treatment. These data are summarized in Figure 3. The natldl treated cells showed no change in their protein expression of Prdx1. Data are not shown for clarity. B. Increased Expression of Catalase. Now that the LC-MS method was validated using Prdx1, the study was expanded to 24 other antioxidant proteins shown in Figure 2. The majority of the proteins were not affected by the oxldl treatment. Of the proteins that did change, two groups were particularly interesting to us, the peroxidases and the reductases. One of the peroxidases was catalase (Cat). Cat catalyzes the decomposition of hydrogen peroxide to water and oxygen. As shown in Figure 4, we saw an increase in protein expression as the concentration of the oxldl increased. Cat had a 50% increase from the 0 to the 75 μg/ml concentration with a statistical significance in the 50 and 75 μg/ml samples compared to the 0 μg/ml samples. In contrast, two other peroxidases, glutathione peroxidases-1 (Gpx1) and glutathione peroxidase-4 (Gpx4), showed no increase in protein expression. Gpx1 reduces hydrogen peroxide to water while Gpx4 reduces lipid hydroperoxides to their respective alcohols. C. Coordinated in Reductase Expression. In the reductases, there was a more coordinated response. Thioredoxin reductase-1 (Txnrd1) had an 80% increase in expression and glutathione reductase (Gsr) had a 60% increase in expression. Txnrd1 reduces thioredoxin, which gets oxidized in the process of repairing proteins. Gsr reduces glutathione, which similarly gets oxidized while repairing damaged molecules. Both proteins use NADPH in these processes, turning NADPH into NADP+. Glucose-6- phosphate dehydrogenase (G6pd) reduces NADP+ back to NADPH effectively reinitializing the system. G6pd had a 40% increase in protein expression as the concentration of the oxldl increased as seen in figure 5. Discussion The goal of this work was to examine changes in protein expression that occur when macrophages become lipid-loaded foam cells. We focused on the antioxidant proteins. The antioxidant proteins inhibit oxidation, making them a key part of any protective response against toxic molecules, such as the oxidized molecules in oxldl. Increase in antioxidant expression clearly indicates that the foam cell can produce a protective response against the oxldl. Under the protection of the antioxidant proteins, the cell cannot undergo apoptosis. Hence, the foam cell will maintain survival and continue the progression of atherosclerosis. One observation was the apparent selectivity of the effects. The LC-MS assay tested a set of 25 antioxidant and stress related proteins, yet changes were seen in only 8 of those proteins. Two aspects of the changes were the selective increase in the expression of the peroxidases and the more coordinated increase in the reductases. Of the peroxidases, Cat, Prdx1, and peroxredoxin-4 (Prdx4) were all increased. However, the other peroxiredoxins and the glutathione peroxidases were unaffected. The reason to why this happened is not known. We speculate that either there was a large upregulation in expression of other peroxidases which overshadowed the need for Gpx1 and Gpx4. It may also be due to the fact that the media we used is supplemented with selenium. Gpx1 and Gpx4 are selenium dependent proteins, containing amino acids that include selenium. In cell culture, media lacking selenium will not allow the cells to produce Gpx1. That being said, the selenium may have been used by other seleniumdependent proteins such as Txnrd1, causing Gpx1 to not be as well expressed. In contrast, both Txnrd1 and Gsr showed increased expression. In addition, G6pd, which provides the NADPH cofactor for these enzymes, also increased. This coordinated increase could indicate a favored role for the reductases in the protective response. This oxldl model was conducted in vitro in cell culture. We know that copper (II) sulfate is not the cause of oxidation in the human body as it was in our model. However, it produces a close replica of oxidative modification. The ongoing challenge in this area has been to find out the true source of this modification in the human body. Many speculate that free radicals may be involved, yet the source of the free radicals is not known. Even endothelial and smooth muscle cells might have a role with the oxidation. However, the source of oxidation is remains unknown. These results show a possible role for selected antioxidant proteins in this process. Future studies can now focus on the functional effects of the peroxidases and the reductases in the formation of foam cells. It would also be interesting to test the how the antioxidant proteins are regulated after oxldl treatment. Our long term goals would be to use manipulation of protein expression to prevent the disease by a reversal of oxldl uptake or the initiation of apoptosis of the foam cells. These further steps may aid us in understanding the complexity of the foam

6 cells and their ability to progress a dangerous disease. Acknowledgements A special thanks to Dr. Mike Kinter and all my colleagues, especially Caroline Kinter for all their support and making this an experience filled with both learning and fun. I would like to thank the W.T. Payne Foundation for supporting the Fleming Scholars Program and allowing me to participate in the program. Lastly, thank you OMRF for providing me with an amazing and unforgettable opportunity this summer! References 1. Quinn, M.T., Parthasarathy, S., Fong, L.G., and Steinberg, D. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, Steinbrecher, U.P., Witzum, J.L., Parthasarathy, S. and Steinberg, D. (1987) Arteriosclerosis 7, Schaffner, T., Taylor, K., Bartucci, E.J., Fischer-Dzoga, K., Beeson, J.H., Glagov, S., and Wissler, R.W. (1980) Am. J. Pathol. 100, Gerrity, R.G., and Naito, H.K. (1980) Artery 8, Brown, M.S., and Goldstein, J.L. (1976) Science 191, Basu, S.K., Goldstein, J.L., Anderson, R.G.W., Brown, M.S. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, Chisolm, G.M., and Steinberg, D. (2000) Free Rad. Biol. Med. 28, Conway, J.P., and Kinter, M. (2005) Mol. Cell. Proteomics 4,

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