Evaluating PCSK9 as a New Point of Control for the Actions of Omega-3 LCPUFA on Blood Lipids
This article at a glance 
LDL levels provide an indication of the excess energy obtained from dietary intake that has been converted to fatty acids and cholesterol by the liver. Cholesterol and fatty acids (as triglycerides) are exported from the liver as VLDL, to distribute triglycerides and cholesterol-fatty acid esters to all peripheral tissues that require these lipids for membrane synthesis, or for storage in the adipose tissue, with the resulting LDL particles being taken up again mainly by the liver. However, in a context of concurrent chronic low-grade vascular inflammation, commonly resulting from a poor diet and a sedentary lifestyle, a sustained oxidative modification of the protein and lipid components that constitute LDL particles can cause the misdirection of LDL lipids into atheromatous sub-endothelial deposits via monocyte/macrophage-mediated retention. Over the course of a decades-long progression of vascular disease, this LDL-associated process is interlinked with pro-inflammatory vascular tissue remodeling and the formation of atherosclerotic lesions. 
Although LDL particle levels or LDL-cholesterol estimations in the circulation are increasingly considered a non-causal yet predictive biomarker of cardiovascular disease, interventions that achieve decreases in LDL appear to generally produce a favorable change in the risk of developing atherosclerotic cardiovascular disease. Current recommendations worldwide suggest both lifestyle changes and drug-based intervention to achieve lower LDL levels in people at risk of atherosclerotic cardiovascular disease. Both pharmacological interventions and lifestyle changes may, however, not achieve the desired targets for LDL reduction, as patient compliance is highly variable and a range of factors determines the individual response to LDL-lowering approaches. Although LDL particle levels or LDL-cholesterol estimations in the circulation are increasingly considered a non-causal yet predictive biomarker of cardiovascular disease, interventions that achieve decreases in LDL appear to generally produce a favorable change in the risk of developing atherosclerotic cardiovascular disease. Current recommendations worldwide suggest both lifestyle changes and drug-based intervention to achieve lower LDL levels in people at risk of atherosclerotic cardiovascular disease. Both pharmacological interventions and lifestyle changes may, however, not achieve the desired targets for LDL reduction, as patient compliance is highly variable and a range of factors determines the individual response to LDL-lowering approaches. Statin therapy achieves reductions in LDL-cholesterol levels through inhibition of the endogenous de novo biosynthesis of cholesterol. However, statin therapy also leads to increased PCSK9 levels, which reduce LDL uptake and hepatic clearance - an effect that counteracts its intended pharmacodynamic action. It has been suggested that further LDL lowering beyond that achieved by statins alone may be possible, and that reducing LDL levels below current target levels may offer further cardiovascular disease risk lowering. Newly developed PCSK9 inhibitors have been shown to achieve reduced LDL-cholesterol levels by increasing LDL clearance through removal of the inhibitory activity of PCSK9 on LDL receptor expression. Enhanced LDL clearance by PCSK9 inhibitors is heralded as a breakthrough approach in cardiovascular disease treatment that may eliminate statin resistance, and remove the residual cardiovascular disease risk that cannot be achieved by current LDL-cholesterol-lowering therapies. Modulation of LDL levels remains a major current topic in pharmacology, and understanding the contribution that PCSK9 makes to cardiovascular disease risk is worthy of evaluation. In line with this thinking, serum PCSK9 were recently shown to be associated with the future risk of cardiovascular disease in 60-year old people, independent of typical cardiovascular risk factors such as hypertension, diabetes, smoking, overweight, obesity, and physical inactivity. 
Although PCSK9 inhibition has been primarily considered as a promising drug target, its modulation by changes in dietary and lifestyle habits, and the physiological effects of these has been addressed to a very limited extent only. A recent study by Graversen and colleagues at the Department of Cardiology at Aalborg University, Denmark, has carried out a first careful evaluation of the effect of supplemental omega-3 LCPUFA intake on circulating PCSK9 levels in healthy Danish women (age 18 to 70 years). Half of the women were premenopausal (n=46), the other half postmenopausal (n=44). The study is a follow-up analysis of an earlier randomized, controlled, double-blinded study in which the influence of menopause on the distribution of omega-3 LCPUFA into platelets and adipose tissue was addressed. That study had demonstrated that in premenopausal women estrogen levels increased in response to omega-3 LCPUFA intake (2.2 g of omega-3 LCPUFA from fish oil, compared to control oil, daily for 12 weeks). The incorporation of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA) into platelet membranes and adipose tissue was nearly identical in premenopausal compared to postmenopausal women supplemented with fish oil. No marked changes in fatty acid levels were found in the control groups. 
Plasma PCSK9 levels were determined by ELISA in pre- and post-menopausal women supplemented with either fish oil or control oil (thistle oil) at baseline and after 12 weeks. Supplementation with fish oil induced a modest decrease in plasma PCSK9 levels that was similar in both pre- and postmenopausal women (a decrease of 29 ng/ml from baseline values of 255 and 286 ng/ml, respectively). In controls, the PCSK9 level did not change over the 12-week intervention period. The authors note that lowering of LDL level was not observed as a result of the interventions. This study shows that in healthy women a decrease in plasma PCSK9 levels can be induced by fish oil intake without any impact on LDL levels. Post-menopausal women did have significantly higher LDL levels at baseline compared to pre-menopausal women (on average 3.03 vs 2.26 mM, respectively). Current target serum levels for treatment of disorders of lipid metabolism are LDL-cholesterol levels <2.6 mM (100 mg/dl), pointing out that the LDL levels in these study participants are in a healthy range and can possibly not be lowered further. A recent study by Rodríguez-Pérez and coworkers reported a similar observation from a double-blind randomized controlled trial (COMIT trial) carried out in North-American middle-aged men and women with at least one component of metabolic syndrome (and average baseline LDL level of 3.41 mM). The trial assessed the effect on endothelial function, and plasma fatty acid and sterol profiles, of a 4-week daily intake of dietary vegetable oil types enriched with omega-3, omega-6 or omega-9 fatty acids. The group that received a DHA-supplemented high-oleic canola oil (DHA as 5% of fatty acids, 36 to 60 g oil per day as a shake-style beverage), had a doubling of plasma DHA level and a reduction in PCSK9 levels of approximately 10%, compared to people receiving canola oil or a high-oleic canola oil. Consumption of the DHA-enriched canola oil also increased HDL cholesterol level and markedly lowered triglycerides (1.30 mM from 1.61 mM). The intervention trial had previously reported an improved blood pressure. Although PCSK9 levels were decreased, no significant enhancement in the lowering of LDL-levels was observed in this study (all tested canola oil types induced a significantly lower LDL level). 
These new studies show that omega-3 LCPUFA supplementation, as fish oil or DHA, can reduce plasma PCSK9 levels. However, in both studies no differential LDL-cholesterol lowering effect was noted. Future studies will be needed to assess if the modulation of PCSK9 by supplemental omega-3 LCPUFA intake is of relevance to modulating LDL levels in individuals with elevated LDL levels, and in conjunction with other lipid-lowering approaches. PCSK9 not only regulates LDL clearance but is also important for VLDL secretion, a key determinant of triglyceride levels in blood. A recent Mendelian randomization study has provided support for a causal relationship between higher blood concentrations of triglycerides and increased cardiovascular disease risk. Since a well-documented benefit of omega-3 LCPUFA supplementation is the lowering of triglyceride levels, further studies on PCSK9 regulation by increased omega-3 LCPUFA intake will likely provide interesting new insight into the importance of omega-3 LCPUFA status for lipid metabolism and cardiovascular disease risk modification. Graversen CB, Lundbye-Christensen S, Thomsen B, Christensen JH, Schmidt EB. Marine n-3 polyunsaturated fatty acids lower plasma proprotein convertase subtilisin kexin type 9 levels in pre- and postmenopausal women: A randomised study. Vascul. Pharmacol. 2015;July 2. [PubMed] Rodríguez-Pérez C, Ramprasath VR, Pu S, Sabra A, Quirantes-Pine R, Segura-Carretero A, Jones PJ. Docosahexaenoic acid attenuates cardiovascular risk factors via a decline in proprotein convertase subtilisin/kexin type 9 (PCSK9) plasma levels. Lipids 2016;51(1):75-83. [PubMed] Worth Noting Abifadel M, Varret M, Rabes JP, Allard D, Ouguerram K, et al. Mutations in PCSK9 cause autosomal dominant hypercholesterolemia. Nat. Genet. 2003;34(2):154-156. [PubMed] Boekholdt SM, Hovingh GK, Mora S, Arsenault BJ, Amarenco P, et al. Very low levels of atherogenic lipoproteins and the risk for cardiovascular events: a meta-analysis of statin trials. J. Am. Coll. Cardiol. 2014;64(5):485-494. [PubMed] Bou Malham S, Goldberg AC. Cardiovascular risk reduction: the future of cholesterol lowering drugs. Curr. Opin. Pharmacol. 2016;27:62-69. [PubMed] Cohen JC. Emerging LDL therapies: Using human genetics to discover new therapeutic targets for plasma lipids. J. Clin. Lipidol. 2013;7(3 Suppl.):S1-5. [PubMed] Holmes MV, Asselbergs FW, Palmer TM, Drenos F, Lanktree MB, et al. Mendelian randomization of blood lipids for coronary heart disease. Eur. Heart J. 2015;36(9):539-550. [PubMed] Introduction to Lipids and Lipoproteins: http://www.ncbi.nlm.nih.gov/books/NBK305896/ Jones PJ, Senanayake VK, Pu S, Jenkins DJ, Connelly PW, Lamarche B, Couture P, Charest A, Baril-Gravel L, West SG, Liu X, Fleming JA, McCrea CE, Kris-Etherton PM. DHA-enriched high-oleic acid canola oil improves lipid profile and lowers predicted cardiovascular disease risk in the canola oil multicenter randomized controlled trial. Am. J. Clin. Nutr. 2014;100(1):88-97. [PubMed] Leander K, Malarstig A, Van't Hooft FM, Hyde C, Hellenius ML, Troutt JS, Konrad RJ, Ohrvik J, Hamsten A, de Faire U. Circulating PCSK9 predicts future risk of cardiovascular events independently of established risk factors. Circulation 2016;Feb 19. [PubMed] Lose JM, Dorsch MP, Bleske BE. Evaluation of proprotein convertase subtilisin/kexin type 9: focus on potential clinical and therapeutic implications for low-density lipoprotein cholesterol lowering. Pharmacotherapy 2013;33(4):447-460. [PubMed] Mayne J, Raymond A, Chaplin A, Cousins M, Kaefer N, Gyamera-Acheampong C, Seidah NG, Mbikay M, Chretien M, Ooi TC. Plasma PCSK9 levels correlate with cholesterol in men but not in women. Biochem. Biophys. Res. Commun. 2007;361(2):451-456. [PubMed] Morris PB, Ballantyne CM, Birtcher KK, Dunn SP, Urbina EM. Review of clinical practice guidelines for the management of LDL-related risk. J. Am. Coll. Cardiol. 2014;64(2):196-206. [PubMed] Poirier S, Mayer G. The biology of PCSK9 from the endoplasmic reticulum to lysosomes: new and emerging therapeutics to control low-density lipoprotein cholesterol. Drug Des. Devel. Ther. 2013;7:1135-1148. [PubMed] Senanayake VK, Pu S, Jenkins DA, Lamarche B, Kris-Etherton PM, West SG, Fleming JA, Liu X, McCrea CE, Jones PJ. Plasma fatty acid changes following consumption of dietary oils containing n-3, n-6, and n-9 fatty acids at different proportions: preliminary findings of the Canola Oil Multicenter Intervention Trial (COMIT). Trials 2014;15(136):1-12. [PubMed] Urban D, Pöss J, Böhm M, Laufs U. Targeting the proprotein convertase subtilisin/kexin type 9 for the treatment of dyslipidemia and atherosclerosis. J. Am. Coll. Cardiol. 2013;62(16):1401-1408. [PubMed] Witt PM, Christensen JH, Ewertz M, Aardestrup IV, Schmidt EB. The incorporation of marine n-3 PUFA into platelets and adipose tissue in pre- and postmenopausal women: a randomised, double-blind, placebo-controlled trial. Br. J. Nutr. 2010;104(3):318-325. [PubMed]
- Two recent studies have addressed if dietary supplementation with fish oil or DHA-enriched canola oil would modify the plasma levels of PCSK9, a pivotal regulator of LDL levels.
- Modest decreases in PCSK9 levels were achieved in both studies, demonstrating that this protein is susceptible to dietary intervention by omega-3 LCPUFA.
- New studies may assess if PCSK9 down-regulation by omega-3 LCPUFA supplementation is of benefit to people with elevated LDL levels.
LDL levels provide an indication of the excess energy obtained from dietary intake that has been converted to fatty acids and cholesterol by the liver. Cholesterol and fatty acids (as triglycerides) are exported from the liver as VLDL, to distribute triglycerides and cholesterol-fatty acid esters to all peripheral tissues that require these lipids for membrane synthesis, or for storage in the adipose tissue, with the resulting LDL particles being taken up again mainly by the liver. However, in a context of concurrent chronic low-grade vascular inflammation, commonly resulting from a poor diet and a sedentary lifestyle, a sustained oxidative modification of the protein and lipid components that constitute LDL particles can cause the misdirection of LDL lipids into atheromatous sub-endothelial deposits via monocyte/macrophage-mediated retention. Over the course of a decades-long progression of vascular disease, this LDL-associated process is interlinked with pro-inflammatory vascular tissue remodeling and the formation of atherosclerotic lesions. Although LDL particle levels or LDL-cholesterol estimations in the circulation are increasingly considered a non-causal yet predictive biomarker of cardiovascular disease, interventions that achieve decreases in LDL appear to generally produce a favorable change in the risk of developing atherosclerotic cardiovascular disease. Current recommendations worldwide suggest both lifestyle changes and drug-based intervention to achieve lower LDL levels in people at risk of atherosclerotic cardiovascular disease. Both pharmacological interventions and lifestyle changes may, however, not achieve the desired targets for LDL reduction, as patient compliance is highly variable and a range of factors determines the individual response to LDL-lowering approaches. Although LDL particle levels or LDL-cholesterol estimations in the circulation are increasingly considered a non-causal yet predictive biomarker of cardiovascular disease, interventions that achieve decreases in LDL appear to generally produce a favorable change in the risk of developing atherosclerotic cardiovascular disease. Current recommendations worldwide suggest both lifestyle changes and drug-based intervention to achieve lower LDL levels in people at risk of atherosclerotic cardiovascular disease. Both pharmacological interventions and lifestyle changes may, however, not achieve the desired targets for LDL reduction, as patient compliance is highly variable and a range of factors determines the individual response to LDL-lowering approaches. Statin therapy achieves reductions in LDL-cholesterol levels through inhibition of the endogenous de novo biosynthesis of cholesterol. However, statin therapy also leads to increased PCSK9 levels, which reduce LDL uptake and hepatic clearance - an effect that counteracts its intended pharmacodynamic action. It has been suggested that further LDL lowering beyond that achieved by statins alone may be possible, and that reducing LDL levels below current target levels may offer further cardiovascular disease risk lowering. Newly developed PCSK9 inhibitors have been shown to achieve reduced LDL-cholesterol levels by increasing LDL clearance through removal of the inhibitory activity of PCSK9 on LDL receptor expression. Enhanced LDL clearance by PCSK9 inhibitors is heralded as a breakthrough approach in cardiovascular disease treatment that may eliminate statin resistance, and remove the residual cardiovascular disease risk that cannot be achieved by current LDL-cholesterol-lowering therapies. Modulation of LDL levels remains a major current topic in pharmacology, and understanding the contribution that PCSK9 makes to cardiovascular disease risk is worthy of evaluation. In line with this thinking, serum PCSK9 were recently shown to be associated with the future risk of cardiovascular disease in 60-year old people, independent of typical cardiovascular risk factors such as hypertension, diabetes, smoking, overweight, obesity, and physical inactivity. Although PCSK9 inhibition has been primarily considered as a promising drug target, its modulation by changes in dietary and lifestyle habits, and the physiological effects of these has been addressed to a very limited extent only. A recent study by Graversen and colleagues at the Department of Cardiology at Aalborg University, Denmark, has carried out a first careful evaluation of the effect of supplemental omega-3 LCPUFA intake on circulating PCSK9 levels in healthy Danish women (age 18 to 70 years). Half of the women were premenopausal (n=46), the other half postmenopausal (n=44). The study is a follow-up analysis of an earlier randomized, controlled, double-blinded study in which the influence of menopause on the distribution of omega-3 LCPUFA into platelets and adipose tissue was addressed. That study had demonstrated that in premenopausal women estrogen levels increased in response to omega-3 LCPUFA intake (2.2 g of omega-3 LCPUFA from fish oil, compared to control oil, daily for 12 weeks). The incorporation of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA) into platelet membranes and adipose tissue was nearly identical in premenopausal compared to postmenopausal women supplemented with fish oil. No marked changes in fatty acid levels were found in the control groups. Plasma PCSK9 levels were determined by ELISA in pre- and post-menopausal women supplemented with either fish oil or control oil (thistle oil) at baseline and after 12 weeks. Supplementation with fish oil induced a modest decrease in plasma PCSK9 levels that was similar in both pre- and postmenopausal women (a decrease of 29 ng/ml from baseline values of 255 and 286 ng/ml, respectively). In controls, the PCSK9 level did not change over the 12-week intervention period. The authors note that lowering of LDL level was not observed as a result of the interventions. This study shows that in healthy women a decrease in plasma PCSK9 levels can be induced by fish oil intake without any impact on LDL levels. Post-menopausal women did have significantly higher LDL levels at baseline compared to pre-menopausal women (on average 3.03 vs 2.26 mM, respectively). Current target serum levels for treatment of disorders of lipid metabolism are LDL-cholesterol levels <2.6 mM (100 mg/dl), pointing out that the LDL levels in these study participants are in a healthy range and can possibly not be lowered further. A recent study by Rodríguez-Pérez and coworkers reported a similar observation from a double-blind randomized controlled trial (COMIT trial) carried out in North-American middle-aged men and women with at least one component of metabolic syndrome (and average baseline LDL level of 3.41 mM). The trial assessed the effect on endothelial function, and plasma fatty acid and sterol profiles, of a 4-week daily intake of dietary vegetable oil types enriched with omega-3, omega-6 or omega-9 fatty acids. The group that received a DHA-supplemented high-oleic canola oil (DHA as 5% of fatty acids, 36 to 60 g oil per day as a shake-style beverage), had a doubling of plasma DHA level and a reduction in PCSK9 levels of approximately 10%, compared to people receiving canola oil or a high-oleic canola oil. Consumption of the DHA-enriched canola oil also increased HDL cholesterol level and markedly lowered triglycerides (1.30 mM from 1.61 mM). The intervention trial had previously reported an improved blood pressure. Although PCSK9 levels were decreased, no significant enhancement in the lowering of LDL-levels was observed in this study (all tested canola oil types induced a significantly lower LDL level). These new studies show that omega-3 LCPUFA supplementation, as fish oil or DHA, can reduce plasma PCSK9 levels. However, in both studies no differential LDL-cholesterol lowering effect was noted. Future studies will be needed to assess if the modulation of PCSK9 by supplemental omega-3 LCPUFA intake is of relevance to modulating LDL levels in individuals with elevated LDL levels, and in conjunction with other lipid-lowering approaches. PCSK9 not only regulates LDL clearance but is also important for VLDL secretion, a key determinant of triglyceride levels in blood. A recent Mendelian randomization study has provided support for a causal relationship between higher blood concentrations of triglycerides and increased cardiovascular disease risk. Since a well-documented benefit of omega-3 LCPUFA supplementation is the lowering of triglyceride levels, further studies on PCSK9 regulation by increased omega-3 LCPUFA intake will likely provide interesting new insight into the importance of omega-3 LCPUFA status for lipid metabolism and cardiovascular disease risk modification. Graversen CB, Lundbye-Christensen S, Thomsen B, Christensen JH, Schmidt EB. Marine n-3 polyunsaturated fatty acids lower plasma proprotein convertase subtilisin kexin type 9 levels in pre- and postmenopausal women: A randomised study. Vascul. Pharmacol. 2015;July 2. [PubMed] Rodríguez-Pérez C, Ramprasath VR, Pu S, Sabra A, Quirantes-Pine R, Segura-Carretero A, Jones PJ. Docosahexaenoic acid attenuates cardiovascular risk factors via a decline in proprotein convertase subtilisin/kexin type 9 (PCSK9) plasma levels. Lipids 2016;51(1):75-83. [PubMed] Worth Noting Abifadel M, Varret M, Rabes JP, Allard D, Ouguerram K, et al. Mutations in PCSK9 cause autosomal dominant hypercholesterolemia. Nat. Genet. 2003;34(2):154-156. [PubMed] Boekholdt SM, Hovingh GK, Mora S, Arsenault BJ, Amarenco P, et al. Very low levels of atherogenic lipoproteins and the risk for cardiovascular events: a meta-analysis of statin trials. J. Am. Coll. Cardiol. 2014;64(5):485-494. [PubMed] Bou Malham S, Goldberg AC. Cardiovascular risk reduction: the future of cholesterol lowering drugs. Curr. Opin. Pharmacol. 2016;27:62-69. [PubMed] Cohen JC. Emerging LDL therapies: Using human genetics to discover new therapeutic targets for plasma lipids. J. Clin. Lipidol. 2013;7(3 Suppl.):S1-5. [PubMed] Holmes MV, Asselbergs FW, Palmer TM, Drenos F, Lanktree MB, et al. Mendelian randomization of blood lipids for coronary heart disease. Eur. Heart J. 2015;36(9):539-550. [PubMed] Introduction to Lipids and Lipoproteins: http://www.ncbi.nlm.nih.gov/books/NBK305896/ Jones PJ, Senanayake VK, Pu S, Jenkins DJ, Connelly PW, Lamarche B, Couture P, Charest A, Baril-Gravel L, West SG, Liu X, Fleming JA, McCrea CE, Kris-Etherton PM. DHA-enriched high-oleic acid canola oil improves lipid profile and lowers predicted cardiovascular disease risk in the canola oil multicenter randomized controlled trial. Am. J. Clin. Nutr. 2014;100(1):88-97. [PubMed] Leander K, Malarstig A, Van't Hooft FM, Hyde C, Hellenius ML, Troutt JS, Konrad RJ, Ohrvik J, Hamsten A, de Faire U. Circulating PCSK9 predicts future risk of cardiovascular events independently of established risk factors. Circulation 2016;Feb 19. [PubMed] Lose JM, Dorsch MP, Bleske BE. Evaluation of proprotein convertase subtilisin/kexin type 9: focus on potential clinical and therapeutic implications for low-density lipoprotein cholesterol lowering. Pharmacotherapy 2013;33(4):447-460. [PubMed] Mayne J, Raymond A, Chaplin A, Cousins M, Kaefer N, Gyamera-Acheampong C, Seidah NG, Mbikay M, Chretien M, Ooi TC. Plasma PCSK9 levels correlate with cholesterol in men but not in women. Biochem. Biophys. Res. Commun. 2007;361(2):451-456. [PubMed] Morris PB, Ballantyne CM, Birtcher KK, Dunn SP, Urbina EM. Review of clinical practice guidelines for the management of LDL-related risk. J. Am. Coll. Cardiol. 2014;64(2):196-206. [PubMed] Poirier S, Mayer G. The biology of PCSK9 from the endoplasmic reticulum to lysosomes: new and emerging therapeutics to control low-density lipoprotein cholesterol. Drug Des. Devel. Ther. 2013;7:1135-1148. [PubMed] Senanayake VK, Pu S, Jenkins DA, Lamarche B, Kris-Etherton PM, West SG, Fleming JA, Liu X, McCrea CE, Jones PJ. Plasma fatty acid changes following consumption of dietary oils containing n-3, n-6, and n-9 fatty acids at different proportions: preliminary findings of the Canola Oil Multicenter Intervention Trial (COMIT). Trials 2014;15(136):1-12. [PubMed] Urban D, Pöss J, Böhm M, Laufs U. Targeting the proprotein convertase subtilisin/kexin type 9 for the treatment of dyslipidemia and atherosclerosis. J. Am. Coll. Cardiol. 2013;62(16):1401-1408. [PubMed] Witt PM, Christensen JH, Ewertz M, Aardestrup IV, Schmidt EB. The incorporation of marine n-3 PUFA into platelets and adipose tissue in pre- and postmenopausal women: a randomised, double-blind, placebo-controlled trial. Br. J. Nutr. 2010;104(3):318-325. [PubMed]