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Biology and Chemistry Research Atherosclerosis-Inducing Cytotoxin 7-Ketocholesterol Is Mitigated by Exposure to 70-Kilodalton Heat Shock Protein in THP-1 Human Monocyte Cells Anne Feng

ABSTRACT

The 70-kilodalton heat shock protein (HSP70) is a cytoprotective protein produced at an elevated concentration in individuals afflicted with atherosclerosis and has been linked to reduced risk of coronary artery disease. In contrast, 7-ketocholesterol (7KC) and other oxysterols have been implicated in accelerating the onset of atherosclerosis by inducing apoptosis in foam cells. As such, the goals of this study were to determine 1) whether HSP70 ameliorates the cytotoxic effects of 7KC on THP-1 human monocyte cells; 2) what dose of HSP70 maximizes cell viability; 3) whether incubation of cells in HSP70 before exposure to 7KC (“before” treatment) improves viability relative to simultaneous application of HSP70 with 7KC (“with” treatment). Cells were exposed to varying concentrations of HSP70 and 7KC and assayed for viability using a Trypan blue stain. Data indicate that the optimal concentration of HSP70 occurs at 0.8 μg/ml. The “before” treatment was not significantly better than the “with” treatment in the presence of 7KC; however, in the absence of 7KC, the “before” treatment improved cell viability. These results show that HSP70 opens a possible avenue of pursuit towards atherosclerosis therapy aimed specifically towards limiting the cytotoxic effect of 7KC. 7KC) within them to travel through the cytosol into the Introduction lysosomes, where they accumulate over time. Normally, the lysosome simply converts the cholesterol into Atherosclerosis is a cardiovascular disease characterized cholesteryl esters and transports them back out into the by the thickening of the arterial walls caused by cytosol, resulting in the formation of harmless lipid-laden cholesterol accumulation. Coronary heart disease, a form macrophages called foam cells (Figure 1). of atherosclerosis that occurs in the coronary arteries, is When high levels of 7KC are present in LDLs, currently the leading cause of death among both men the oxysterol stimulates atherosclerosis by acting as and women in the United States [1]. In 2008, nearly a a cytotoxin. Firstly, 7KC promotes an accelerated quarter (23%) of global deaths resulted from heart attacks accumulation of cholesterol [6]. In this case, the lysosome and strokes, both of which are strongly associated with becomes overwhelmed by the high cholesterol traffic, so atherosclerosis [2]. free cholesterol becomes trapped within the organelle. Oxysterols are oxidized derivatives of cholesterol that Furthermore, as more and more free cholesterol enters play a major role in a wide range of biological processes, the lysosome, the organelle begins to de-acidify [7], including the formation of bile acids and cholesterol thus reducing its ability to process the lipid. These metabolism [3]. Although oxysterols naturally occur in lipid-dense macrophages, or foam cells, now begin to extremely low levels compared to other sterols [4], they accumulate in a single location within the artery, forming are known to have a profound impact on the regulation a lipid-rich layer as they degrade; this contributes to the of cholesterol homeostasis, which they control by acting necrotic core (mass of dead cells) of the plaque that is as ligands to enzyme receptors [5]. As a result, certain characteristic of atherosclerosis [8]. Additionally, 7KC is oxysterols are crucial for proper functioning of many of also known to promote the differentiation of monocytes the body’s natural processes. Other oxysterols, however, into macrophages and cause lysosomal membrane can be detrimental to proper cellular function and have permeabilization, exacerbating the formation of the been implicated for their role in atherosclerosis. necrotic core. One of these damaging oxysterols is 7KC, a widely These cytotoxic effects of 7KC could be mitigated by studied oxysterol due to its relative abundance in the introduction of heat shock proteins (HSPs). HSPs atherosclerotic plaques that cause chronic inflammation are a class of highly conserved, ubiquitous proteins that and can lead to blockage of the arteries. In the bloodstream, function as chaperones to prevent protein misfolding. sterols and oxysterols often occur inside of low density At appropriate levels, HSPs serve a variety of important lipoproteins (LDLs), which cause inflammation signals functions within the arterial walls. Although HSP65 to be sent out from the artery walls. These signals attract has been proven capable of decreasing the sizes of monocytes that enter the vascular wall and differentiate atherosclerotic lesions in mice via mucosal administration into macrophages. The resulting macrophage cells then [9], to my knowledge, no other HSP has been tested as an engulf the LDLs through receptor-mediated endocytosis. atherosclerosis treatment. Among the remaining proteins, This process allows the LDLs and the oxysterols (including 34 | 2013-2014 | Volume 3


Biology and Chemistry Research HSP70 is the most likely to improve cell viability in the presence of oxysterols if delivered as a drug. HSP70 is a cytoprotective protein that is produced at an elevated concentration when an individual is afflicted with atherosclerosis [10]. Data also suggest that an elevated level of HSP70 prior to the presence of 7KC may also limit the risk of atherogenesis, as high levels of HSP70 were found in patients with low risk of coronary artery disease [11]. In the case of oxysterols, HSP70 expression is induced in the presence of intact oxidized LDLs (oxLDLs), into which oxysterols are typically packaged, but not the oxysterols themselves [12]. As a result, when the oxLDL surrounding the oxysterol is degraded, such as in parts of atherosclerotic lesions, the HSP70 response is no longer triggered, thus allowing a greater cytotoxic effect. The rescue mechanism could potentially be artificially induced by the introduction of HSP70 molecules; however, such a treatment may be a proverbial double edged sword, as HSP70 may also induce an inflammatory response, which contributes to lesion formation [13].

for the formation of the necrotic core of the lipid-rich plaque characteristic of atherosclerosis. As such, the goals of this study were to identify whether HSP70 is capable of improving the viability of THP1 human monocyte cells exposed to the oxysterol 7KC when added before and with the oxysterol, and what optimal dosage of HSP70 minimizes cell mortality.

Materials and Methods Cell culture THP-1 monocyte cells were obtained through a generous donation by the Mackman Lab at the University of North Carolina School of Medicine. Cells were cultured in a CO¬¬2 incubator at 37°C, 5% CO¬2 in RPMI-1640 media (Gibco) supplemented with 10% FBS (Thermo Scientific), 1 mM sodium pyruvate (Sigma), 1 mM HEPES (Sigma), 1% glucose (Sigma), and 1% penicillin/ streptomycin (Sigma), as recommended by the source lab. During maintenance of the cell line, cells were split every 3-4 days. HSP70 (MyBioSource) and 7KC (Sigma) were added to media as needed for each desired treatment level. Each independent treatment replicate was placed into an individual well on a standard 12-well tissue culture plate. The location of each plate within the incubator was randomized each day using a random number generator. Determination of THP-1 viability in response to treatment

Figure 1. The effect of 7KC on the onset of atherosclerosis. While normal cellular function persists with basal levels of 7KC, high levels of 7KC result in cholesterol accumulation, lysosomal deacidification, lysosomal membrane permeabilization, and increased monocyte differentiation. These factors ultimately lead to mass foam cell formation and apoptosis, allowing

To determine the optimal dosage of HSP70 to maximize cytoprotective effects against 7KC, THP-1 cells were exposed to either 0 μg/ml or 6 μg/ml of 7KC dissolved in 95% non-denatured ethanol. The 7KC dose concentration was selected through a combination of the results from Mathieu et al. [6], which suggested a concentration between 4 μg/ml and 10 μg/ml to produce detectable results, and from a preliminary experiment to determine the concentration within that range that reached LD50 at approximately 72 hours (73.06 ± 4.21 hours). Further preliminary experiments verified that the concentrations of ethanol added to the media had no detectable impact on cell viability (2.71% decrease at 96 hours, p > 0.05). Each treatment was also exposed to 0 μg/ml, 0.4 μg/ml, 0.8 μg/ml, 1.2 μg/ml, or 1.6 μg/ ml of HSP70. These treatment levels were based upon the concentration of HSP65 administered to decrease atherosclerotic lesion size in mice [9]. In order to distinguish the effects of adding HSP70 before or with the 7KC, the “before” treatments were incubated with their respective concentrations of HSP70 and media for 24 hours prior to the initial data collection point (at which Volume 3 | 2013-2014 | 35


Biology and Chemistry Research point 7KC was also added) while “with” treatments were incubated in media only. To minimize the effects of natural discrepancies between cells split into independent flasks during culture, all cells utilized in the experiment were pooled prior to distribution into individual treatments. Data was collected by sampling 20 μl of cells from each well at 24 hour intervals over a period of 96 hours. Cells were stained using 0.4% Trypan Blue dye, which allows for dead cells to appear dark blue while live cells remain clear, then counted on a hemocytometer to determine the cell viability. The entire experiment was repeated 3 times, with 6 replicates in the first trial and 4 in both of the following. The first two trials were performed on cells obtained from the same set of 3 tubes of frozen stock after 9 and 11 passages, respectively, whereas the third was performed on cells from a second set of 3 tubes after 6 passages. All frozen cells were obtained from the same lab.

control (Figure 3A); this confirms the results of Mathieu et al. [6]. Across all concentrations of HSP70, viability was significantly reduced by 24 hours except at the 0.8 μg/ ml dose, at which no significant difference was detected in viability between the presence and absence of 7KC until 48 hours (Figure 3B). By 48 hours, differences are detected with regards to 7KC treatment across all HSP70 treatments throughout the remainder of the experiment (Figure 3).

Statistical analyses Data were analyzed using JMP v.10.0.0 software (SAS Institute, Inc., 2012) and Microsoft Excel. Comparisons between treatments were performed on logarithmically transformed data using an ANOVA followed by the Tukey HSD test for preliminary data and all treatment variables investigated in the study (HSP70 timing, 7KC, and HSP70 concentration). The logarithmic transformation was performed to provide a normal error distribution, which is inherently assumed in statistical analyses utilized in this paper. Response to treatment was expressed in terms of the negative log of percent mortality, which serves as an indication of cell viability. Quantitative comparisons between treatments are expressed in terms of percent change relative to the control in transformed data (percent change calculated after transformation). In all cases, a p-value < 0.05 was set for statistical significance.

Results 70-kilodalton heat shock protein timing In the absence of 7KC, the “before” treatment resulted in higher viability than the “with” treatment at 0 hours (7.29%, p < 0.05), 24 hours (13.22%, p < 0.01), and 96 hours (32.47%, p < 0.001) (Figure 2). However, no difference was detected with the presence of 7KC between the “before” and “with” treatment. Thus, “before” and “with” treatments were grouped collectively for all further analyses. 7-ketocholesterol By 24 hours after incubation, 7KC acted as a potent cytotoxin at 6 μg/ml in the absence of HSP70, decreasing cell viability by 27.24% (p = 0.0023) compared to the 36 | 2013-2014 | Volume 3

Figure 2. Viability by HSP70 dose timing (before or with 7KC) over time. Data represents the collective analysis of all 7KC and HSP70 treatment levels. No significance was detected in the 6 μg/ml 7KC treatment. * indicates significant differences between treatments at each respective time point (p < 0.05); ** indicates p < 0.01; *** indicates p < 0.001. Error bars denote 1 SEM . 70-kilodalton heat shock protein concentration Prior to 72 hours, no differences were detected between any of the HSP70 treatments. At 96 hours, in all samples exposed to 7KC, cells exhibited significantly lower viability than the control regardless of HSP70 treatment level. The 0 μg/ml and the 0.8 μg/ml treatments appear to optimize cell viability. However, while 0 μg/ml treatment was not different from the next best concentration, the 0.8 μg/ml treatment had 93.85% higher viability (Figure 4). No differences were detected in the absence of 7KC.

Discussion Viability in the absence of 7KC was improved by the “before” treatment; viability in the presence of 7KC was not. As such, HSP70 may be intrinsically cytoprotective as the only difference between “before” and “with” treatments was 24 hours in HSP70-treated media. Since no significant difference was detected in HSP70 concentration at 0 μg/ ml 7KC, any inherent cytoprotective properties may be more affected by exposure duration than dosage strength. At all concentrations but 0.8 μg/ml HSP70, a significant difference was detected between presence and


Biology and Chemistry Research

Figure 4. Viability by HSP70 treatment level over time. THP-1 cells were exposed to varying levels of HSP70 over a span of 96 hours. Data represents both the “before” and the “with” treatment. No significance was detected in the negative control (0 μg/ml 7KC). Levels not connected by the same letter are significantly different (p < 0.05). The absence of letters indicates no significant differences. Error bars denote 1 SEM .

Figure 3. Viability by 7KC treatment over time. THP1 cells were exposed to either 0 μg/ml or 6 μg/ml 7KC over a span of 96 hours. Data represents both “before” treatment and “with” treatment. Figure summarizes data for cells exposed to 0 μg/ml (A) or 0.8 μg/ml (B) HSP70; cells exposed to all other HSP70 concentrations behaved similarly to the 0 μg/ml treatment. * indicates significant differences from the control (no 7KC) at each time point (p < 0.05); ** indicates p < 0.01; *** indicates p < 0.001. Error bars denote 1 SEM . absence of 7KC by 24 hours. As such, the data strongly suggest that the 0.8 μg/ml concentration optimizes cytoprotective effects, possibly totally negating the toxic effect of 7KC at 24 hours. In the presence of 7KC, both the 0 μg/ml and 0.8 μg/ ml HSP70 treatments optimized viability. This surprising since the viability at the intermediate treatment level, 0.4 μg/ml, was significantly lower than that of both the 0 μg/ ml and 0.8 μg/ml. Typically, it is expected that the dose response data will have a single peak at the optimal dosage if the optimal dose occurs within the range of treatments utilized. However, since this anomalous trend did not persist in the third trial, the behavior may have been an artifact of the cell stock used in the first two trials. The strange phenomena occurring at the 0 μg/ml and 0.4 μg/ml HSP70 are particularly because the population as a whole on average naturally fluctuates between these

two levels of HSP70 [14]. As such, if the anomalous behavior at the 0 μg/ml and 0.4 μg/ml concentrations are truly merely an anomaly, this study could provide an explanation as to why higher levels of HSP70 are linked to a less rapid onset of atherosclerosis [10]. However, regardless of the behavior of the data between 0 μg/ml and 0.4 μg/ml HSP70, 0.8 μg/ml HSP70 appears to optimal viability after analysis of both 7KC and HSP70 concentration.

Conclusion Oxysterols, specifically 7KC, have been implicated in accelerating the formation of atherosclerotic plaque. They do so by accelerating the rates of cholesterol endocytosis by macrophage cells (leading to lysosome deacidification), increasing lysosomal membrane permeabilization, and promoting monocyte differentiation [8]. While there currently are existing treatments for atherosclerosis, there are very few drugs presently in existence that specifically target 7KC and other oxysterols, despite their profound impact on atherogenesis. This study proposes the theory that the introduction of nonendogenous HSPs may mitigate the harmful effects of 7KC. Previous studies have linked high levels of HSP70 to reduced rates of coronary artery disease [11] and have shown that HSP70 is present in higher levels as a cytoprotectant in patients with atherosclerosis [10]. While another related heat shock protein, HSP65, has been shown to reduce the size of lesions in mice [9], no studies until now have been conducted to link HSP70 to Volume 3 | 2013-2014 | 37


Biology and Chemistry Research cytoprotective effects against 7KC or any other oxysterol. The first two goals of this study were to determine whether HSP70 may be used as a cytoprotective agent to mitigate the deleterious effects of 7KC on THP-1 cells and what dose of HSP70 optimizes these cytoprotective effects. At 24 hours, the data strongly suggests that the 0.8 μg/ml concentration is able to improve the viability of cells exposed to 7KC; its effects were profound enough to mask any cytotoxic effects of the 7KC treatment. However, beyond 24 hours, no dosage of HSP70 was capable of improving cell viability to the same extent. Furthermore, consistently throughout all 3 trials, the highest viability in cells exposed to 7KC occurred at 0.8 μg/ml HSP70. Both of these observations indicate that 0.8 μg/ml is most likely the optimal dosage of HSP70 to maximize viability against 7KC. The third goal of this study was to determine whether the timing of the HSP70 dosage results in any additional increased viability as compared to cells exposed to both substances simultaneously. The purpose of incorporating this element into the study was to determine whether HSP70 has any preventative capacities against the cytotoxic effects of 7KC. While it could not be concluded that HSP70 has any detectable effect on viability in the presence of 7KC, the data do indicate that the extra 24 hours of incubation in HSP70 improves viability in cells in the absence of 7KC. This could indicate inherent beneficial effects of prolonged exposure to HSP70 even in the absence of stressors. However, several key questions have yet to be answered. Firstly, it is unclear based on the results of this study whether the unexpected behavior of monocyte viability between 0 μg/ml and 0.4 μg/ml during trials involving the first cell stock was real or merely a result of unforeseen and unexplained experimental error. Furthermore, since, as stated previously, human HSP70 levels naturally fluctuate between these two concentrations, it may be interesting to characterize the viability of monocytes exposed to 7KC and treated with varying dosages of HSP70 within this range. Such an experiment, if performed on multiple cell stocks, would also confirm whether the surprising behavior observed in the first two trials truly was an anomaly. Most importantly, it remains to be determined why HSP70 has any impact on the viability of cells exposed to 7KC. As of now, there is little insight as to why heat shock proteins are linked to reduced symptoms of atherosclerosis and other vascular diseases; this study was among the first to link heat shock protein to any specific step in the atherogenesis process. Despite the data that link certain dosages of HSP70 to reduced cell mortality, this experiment has not been able to elucidate the specific mechanism by which HSP70 acts as a cytoprotectant. For instance, since no assay was performed in this study 38 | 2013-2014 | Volume 3

to determine the location of the heat shock protein relative to the cell, it is unknown whether the HSP70 physically entered the cell or activated a cytoprotective pathway through interactions with surface proteins. It is quite possible that the latter occurs, as it was shown by Johnson et al. [15] that in macaques, HSP72 and HSP73 (members of the HSP70 family) improve the viability of heat-stressed aortic cells without internalization (entering the cell), but further experimentation is necessary to substantiate this claim. This particular aspect at least could be determined in future experimentation through the use of fluorescently tagged HSP70. However, the eventual characterization of the entire pathway will be necessary before any pharmaceutical applications of HSP70 are possible to determine whether and how essential cellular functions are affected. The argument could be made that the increased viability response is not uniquely tailored towards damage induced by 7KC, but rather that HSP70 merely generally improves viability, which counterbalances the deleterious effects of the cytotoxin. However, this is unlikely to be the case. In 1990, it was verified that HSP70 improves the integrity of the lysosomal membrane [15]; since this directly counteracts one of the major cytotoxic effects of oxysterols (increased lysosomal permeabilization), the heat shock protein is most likely specifically cytoprotective against 7KC and other oxysterols. However, to validate this conjecture, as well as to identify the other positive tendencies of the HSP70, it will be necessary to characterize the specific pathways involved. In spite of certain questions that remain to be addressed, the conclusions of this study show that HSP70 opens a potential avenue of pursuit for future atherosclerosis treatment targeted specifically at mitigating the effects of oxysterols.

Acknowledgments I would like to thank Dr. Amy Sheck, Ph.D. for her mentorship and guidance throughout the research process; Dr. Floyd Bullard, Ph.D. for his expertise and guidance with statistical analysis; Ms. Korah Wiley for her advice and guidance during data collection; Research in Biology colleagues and Glaxo fellows ( Jackson Allen, Jovan Baslious, Madden Brewster, Joseph Kirollos, Hannah McShea, and Jennifer Wu) for their support, assistance with miscellaneous tasks during data collection, and peer review of all materials; Research in Biology seniors (William Ge, Jordan Harrison, Chelsey Lin, Ian Maynor, and Elizabeth Tsui) for their guidance and advice; William Ge for his aid with revision during literature search; Dr. A. Phillip Owens III, Ph.D. for his generous donation of THP-1 cells and advice on cell


Biology and Chemistry Research culture; Dr. Nigel Mackman for his generous donation of THP-1 cells; the Glaxo Endowment for financial support for the Research in Biology program.

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