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Endoplasmic reticulum stress and glycogen synthase kinase-3β activation in apolipoprotein E-deficient mouse models of accelerated atherosclerosis

Interview with Dr. Werstuck
Basic Sciences, Pharmacology, Genomics and Cardiovascular Pathology

Q: Dr. Werstuck, congratulations on this most intriguing and stimulating study on endoplasmic reticulum (ER) stress, glycogen synthase kinase GSK3β, and atherosclerosis, published in print in the first 2012 edition of ATVB. Would you mind summarizing the key findings of the study for us?

A: Our study published in the January 2012 edition of ATVB examined the role of ER stress signaling and Glycogen Synthase Kinase (GSK)-3β in the development of atherosclerosis in an ApoE deficient mouse model. We found that several different conditions, known to induce atherosclerosis – including hyperglycemia, hyperhomocysteinemia and high fat diet - were also associated with ER stress and increased GSK3β activity. Further, we found that inhibition of GSK3β with a small molecule, valproate, significantly attenuated hepatic lipid accumulation and reduced atherosclerotic plaque size. GSK3β activity level appeared to be correlated to its phosphorylation state. This study suggests that ER stress signaling through GSK3β may represent a common pathway in the development of atherosclerosis for several different cardiovascular risk factors.

Q: First of all, we might have to define ER stress and the unfolded protein response (UPR) a little bit more. What exactly is meant by these terms?

A:One third of all proteins synthesized, in a typical cell, are folded and processed in the endoplasmic reticulum (ER). ER stress is a condition in which the protein processing capacity of the ER is overwhelmed and unfolded proteins begin to accumulate in the ER lumen. The Unfolded Protein Response (UPR) is a cellular self defense mechanism that is initiated in conditions of ER stress. The role of the UPR is to: i) decrease the load of proteins that require folding, by inhibiting protein translation, ii) increase the protein folding capacity of the cell by increasing the expression of protein chaperones, and iii) to initiate programmed cell death (apoptosis) pathways if ER homeostasis cannot be achieved.

Q: You are certainly one of the experts on this particular topic and aspect of atherosclerosis. Since when have you devoted your research efforts on this and which changes have you noted in the field over this time period?   

A: Our understanding of ER stress and the UPR has increased substantially over the past decade. This has predominately been a result of studies published by the laboratories of Dr. David Ron (University of Cambridge), Dr. Randal Kaufmann (University of Michigan) and others who have significantly advanced the field. In addition, it has become increasingly evident that ER stress and UPR pathways play a central role in the development of a number of human pathologies including, neurodegenerative disorders, diabetes, obesity, cancer, and cardiovascular disease. Therefore, in addition to being an interesting cellular self defense mechanism, the UPR has become a target for new drug therapies.

Q: As much as I recall, in the past we had some hints of a link between ER stress, the UPR and atherosclerosis. For instance, ER stress induces the UPR in endothelial cells in regions susceptible to atherosclerosis and several cardiovascular risk factors have been associated with the UPR. Also, you and your group found that ER stress conditions promote lipid accumulation, which is very relevant for atherosclerosis. However, how ER stress, the UPR, and atherosclerosis fit together and particularly the nature of the molecular networks mediating this link has remained unknown – is this correct?

A: This is correct. While our understanding of proximal UPR signaling is relatively clear. The downstream networks and signaling factors that link ER stress and the UPR to atherosclerotic processes have only recently begun to be investigated. Increasing evidence suggests that many established molecular pathways (such as the Wnt pathway, the PI3K/AKT pathway, and multiple MAPK pathways) are activated by ER stress. Accumulating evidence suggests that ER stress signaling leads to many hallmark features of atherosclerosis including lipid accumulation, inflammation and apoptosis However, the mechanisms by which ER stress induces these processes is not understood. A better understanding of these mechanistic links may lead to the identification of new anti-atherosclerotic drug targets.

Q: In this study then, in order to shed further light on the molecular interplay between ER stress, the URP and atherosclerosis, you set out with three murine models of pro-atherosclerotic conditions. Please tell us more about these and why one model would not suffice for the goals of your study.

A: We established three mouse models of accelerated atherosclerosis in ApoE-/- mice. A group of mice were injected with streptozocin to induce hyperglycemia, another group was given drinking water supplemented with 0.5% w/v methionine to induce hyperhomocysteinemia and the final group of mice was fed a high fat diet to induce relative dyslipidemia.
To date, many studies delineating molecular mechanisms involved in atherosclerosis have focused on one risk cardiovascular factor such as hyperglycemia or dyslipidemia. However it is evident from clinical trials that there are multiple risk factors which promote atherosclerosis development. We chose to investigate multiple models of atherosclerosis because we hypothesize that ER stress signaling may be a unifying molecular mechanism by which multiple risk factors drive the disease.

Q: Did you observe ER stress in these models in the current study? Where and how did you measure ER stress?

A: We observed indications of ER stress in all three mouse models in our study. Actually, to identify conditions of ER stress we look for increased levels of UPR proteins in atherosclerotic lesions as well as in liver tissue. Specific UPR markers include the Glucose Regulated Protein (GRP) 78, GRP94, Protein Disulphide Isomerase (PDI) and C/EBP-homologous protein (CHOP).

Q: Which “tools” and/or parameters are most suitable to measure ER stress and the UPR, especially for in vivo and for in vitro studies? Are there any gold standards?

A: To date, researchers have quantified UPR markers as a measure of ER stress using combinations of Western blotting and immunohistochemical analysis. There are a number of UPR proteins for which commercial antibodies are available; GRP78/94, PDI, CHOP, Calreticulin, phospho-PERK and ATF4 for example. Quantitative RT-PCR can also be used to measure these and other UPR markers such as XBP1. Because each cell type or tissue expresses a slightly different UPR program, there really is no gold standard that will work in all situations.

Q: You also had an intervention arm in each pro-atherosclerotic model and control group: valproate. How did you get to this?  

A: In previous studies we, and others, had identified valproate as a small orally available molecule with GSK3 inhibitory properties. This study was designed to take advantage of these properties in order to study the role of GSK3 in ER stress induced atherosclerosis. 

Q: Apparently, there were differences in valproate “acceptance” among the different models as reflected in the plasma concentrations. Please tell us more about this.

A: It appeared as though plasma concentrations of valproate in the high fat model were lower than the other groups, the reason for this is not clear. We speculate this could be due to a number of factors. First, it is possible that the severe dyslipidemia alters the ability to uptake valproate from the digestive system. Second, only circulating, plasma valproate levels were measured. It is possible that condition of severe dyslipidemia actually promote uptake and retention of valproate into cells. Finally,  the high fat concentration in the plasma samples may have affected the ability of the AxSYM system to detect and quantify valproate.

Q: What about the effects of valproate on atherosclerosis in the different models?  

A: As expected, hyperhomocysteinemia, hyperglycemia and high fat diet each accelerated the development of atherosclerotic lesions relative to controls. In each of these models, valproate supplementation reduced lesion area, volume and necrotic area.

Q: Given the molecular target of valproate, how did it influence GSK3 activity in the aorta? Did these effects correlate with the effects on atherosclerosis?

A: GSK3 activity within the aorta wall was not directly measured. However, through immunohistochemical staining, it appeared as though valproate supplementation induced phosphorylation of GSK3β at serine 9, which is indicative of decreased activity of the enzyme. Phosphorylation of GSK3β at serine 9 correlated to decreased lesion size and percent necrotic area. The mechanism by which valproate inhibits GSK3 is not fully understood, especially in in vivo models. It does appear that valproate is both a direct and indirect GSK3 inhibitor.

Q: You described GSK3 as a link between ER stress and lipid accumulation. What is the molecular basis for this?

A: To date our understanding of ER stress and GSK3’s role in lipid accumulation is limited. However, evidence from our lab suggests a number of lipid biosynthesis markers are regulated by ER stress induced GSK3. In both in vitro and in vivo models, GSK3 inhibition or deletion results in decreased triglyceride and cholesterol accumulation, as well as decreased SREBP, HMG-CoA Reductase and Fatty Acid Synthase (FAS) expression. Valproate can attenuate these effects without altering ER stress levels. Together, these results suggest that ER stress signals through GSK3 to induce lipid accumulation.

Q: Likely for practical reasons but likely also for other reasons, you also examined liver samples and studied ER stress, lipid accumulation as well as the role of GSK3β. Please tell us more about this.

A: Due to the abundance and relative cellular consistency of hepatic tissue we examined GSK3 activity, phosphorylation, and lipid accumulation specifically in this tissue. ER stress was also examined in hepatic tissue as well as in the aorta. We found that pro-atherosclerotic stimuli induced hepatic GSK3β activity, altered its phosphorylation state and caused increased hepatic steatosis. Valproate supplementation and subsequent GSK3β inhibition resulted in changes to GSK3β phosphorylation and attenuation of hepatic steatosis.

Q: Valproate prevented lipid accumulation in the liver in all models but not in the high-fat one. Did this correlate with the low plasma levels observed in this model?

A: It is possible hepatic lipid accumulation in the high fat diet model was not attenuated by valproate due to its low plasma levels. Alternatively, the dyslipidemia and subsequent lipid uptake by the liver may have been too severe or have progressed too far to observe the effects of valproate on its total lipid content.

Q: Interestingly, valproate attenuated hepatic GSK3β activation even in high fat-fed mice but even so, it did not change hepatic lipid levels in this model. Is this a misunderstanding or is there a “disconnect”?

A:This is an interesting observation that we cannot fully explain at this time. Although valproate had an effect on GSK3β activity in the high fat model, it apparently was not sufficient to attenuate the development of hepatic steatosis. It is most likely that the severe dyslipidemia in this model promotes excessive lipid uptake by the liver that overwhelms any effect of GSK3 inhibition.

Q: You examined the phosphorylation status of GSK3β, which have activating effects if involving Tyr216 or inhibiting effects of involving Ser9. Consistent with the effects on its enzymatic activities, all three pro-atherosclerotic risk factor models increased activating phosphor-Tyr216- GSK3β levels and valproate increased inhibitory phosphor-Ser9- GSK3β levels in the liver. Did this translate into the aortic wall?

A:Using immunohistochemical staining techniques, we assessed the phosphorylation state of GSK3β in the aortic well. Consistent with out observations in the liver, staining was more intense using an antibody towards phosphor-Tyr216 GSK3β with proatherogenic stimuli while phosphor-Ser9 GSK3β staining was more intense in the groups supplemented with valproate.

Q: What are your primary conclusions from this study?

A: Our study suggests that multiple cardiovascular risk factors induce ER stress, which promote atherosclerotic lesion development by signaling through GSK3.

Q: How do you see the translation of the current findings into the clinic?

A: ER stress signaling and/or GSK3 may prove to be key therapeutic targets in managing atherosclerosis development clinically. A number of chemical chaperones, including 4-phenylbutyric acid (4PBA), have been shown to attenuate ER stress. These are currently being investigated for their therapeutic potential. Interestingly, recent clinical studies have found that epileptic patients that are treated with valproate are at significantly lower risk of myocardial infarction and stroke compared to individuals taking other medications. As our knowledge of these pathways increases, we will identify additional targets and potential therapies.

Q: What is the next step? What is on the horizon? Into which direction do you see the field of ER stress and UPR in atherosclerosis moving? 

A: What is significantly lacking in the field of ER stress and UPR signaling in atherosclerosis is a better understanding of mechanisms acting downstream of UPR induction. Next steps include identifying specific UPR pathways that control hallmark features of atherosclerosis –lipid accumulation and inflammation and identifying the specific role of GSK3. Once there is a better mechanistic understanding of ER stress induced atherosclerosis therapeutics can be developed and applied clinically.

Q: Dr. Werstuck, thank you so, so much for this great interview! We deeply appreciate that you brought us your wonderful study much closer and gave us so much additional information and expertise.

A: Thank you to the European Cardiology Society Atherosclerosis Working Group’s and your interest and for selecting our publication as the article of the month!          

The content of this article reflects the personal opinion of the author/s and is not necessarily the official position of the European Society of Cardiology.

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