Anti-PCSK9 monoclonal antibody attenuates high-fat diet and zymosan-induced vascular inflammation in C57BL/6 mice by modulating TLR2/NF-ƙB signaling pathway

Document Type : Original Article


1 Department of Pharmacology, School of Pharmaceutical Education & Research (SPER), Jamia Hamdard, New Delhi - 110062, India

2 Department of Pharmaceutical Chemistry, SPS, DPSRU, New Delhi-110017


Objective(s): Excess intake of a high-fatty diet (HFD) together with zymosan administration mediates vasculitis response which leads to impaired serum lipid levels and causes arterial stiffness. In the development of new cholesterol-lowering medications, PCSK9 inhibitor (proprotein convertase subtilisin/kexin type 9) is an emerging therapeutic. The goal of the present study was to see whether anti-PCSK9 mAb1 might prevent vasculitis in C57BL/6 mice by blocking TLR2/NF-B activation in HFD and Zymosan-induced vasculitis. 
Materials and Methods: Protein-protein molecular docking was performed to validate the binding affinity of anti-PCSK9 mAb1 against TLR2. Under the experimental study, mice were randomly allocated to the following groups: Group I: standard mice diet (30 days) + Zymosan vehicle (sterile PBS solution of 5mg/ml on 8th day); Group II: HFD (30 days) + Zymosan ( single IP dose 80 mg/kg on day 8th); Group III: HFD+Zymosan + anti-PCSK9 mAb1 (6 mg/kg, s.c. on 10th and 20th days); Group IV: HFD+Zymosan+anti-PCSK9 mAb1 (10 mg/kg, s.c. on 10th and 20th days).
Results: In comparison with the low dose of anti-PCSK9 mAb1 (6 mg/kg), the high dose of anti-PCSK9 mAb1 (10 mg/kg) together with HFD and Zymosan inhibited vasculitis more effectively by decreasing aortic TLR2 and NF-B levels, reducing serum TNF- and IL-6, and up-regulating liver LDLR levels, which down-regulated serum LDL-C and improved serum lipids levels. Histopathological studies showed that anti-PCSK9 mAb1 treatment reduced plaque accumulation in the aorta of mice.
Conclusion: These findings indicate that anti-PCSK9 mAb1 has therapeutic potential in reducing HFD and Zymosan-induced vascular inflammation.  


1. Steven S, Frenis K, Oelze M, Kalinovic S, Kuntic M, Bayo Jimenez MT, et al. Vascular inflammation and oxidative stress: major triggers for cardiovascular disease. Oxid Med Cell Longev 2019; 2019: 1-26. 
2. Wolf D, Ley K. Immunity and inflammation in atherosclerosis. Circu Res 2019; 124: 315-327.
3. Libby P, Ridker PM, Maseri A. Inflammation and atherosclerosis. Circulation 2002; 105: 1135-1143.
4. Madan M, Amar S. Toll-like receptor-2 mediates diet and/or pathogen associated atherosclerosis: proteomic findings. PloS One 2008; 3: e3204.
5. Zhu YJ, Wang C, Song G, Zang SS, Liu YX, Li L. Toll-like receptor-2 and-4 are associated with hyperlipidemia. Mol Med Rep 2015; 12: 8241-8246.
6. Goulopoulou S, McCarthy CG, Webb RC. Toll-like receptors in the vascular system: sensing the dangers within. Pharmacol Rev 2016; 68: 142-167.
7. Biswas S, Zimman A, Gao D, Byzova TV, Podrez EA. TLR2 plays a key role in platelet hyperreactivity and accelerated thrombosis associated with hyperlipidemia. Circ Res 2017; 121: 951-962.
8. Hovland A, Jonasson L, Garred P, Yndestad A, Aukrust P, Lappegård KT, et al. The complement system and toll-like receptors as integrated players in the pathophysiology of atherosclerosis. Atherosclerosis 2015; 241: 480-494.
9. Duan Y, Zeng L, Zheng C, Song B, Li F, Kong X, et al. Inflammatory links between high fat diets and diseases. Front Immunol 2018; 9: 1-10.
10. Han Q, Yeung SC, Ip MS, Mak JC. Dysregulation of cardiac lipid parameters in high-fat high-cholesterol diet-induced rat model. Lipids Health Dis 2018; 17: 1-10.
11. Walker AE, Breevoort SR, Durrant, JR, Liu Y, Machin DR, Dobson PS,  et al. The pro-atherogenic response to disturbed blood flow is increased by a western diet, but not by old age. Sci Rep 2019; 9: 1-11 
12. Liu Y, Zhang HG, Jia Y, Li XH. Panax notoginseng saponins attenuate atherogenesis accelerated by zymosan in rabbits. Biol Pharm Bull 2010; 33: 1324-1330
13. Malik P, Berisha SZ, Santore J, Agatisa-Boyle C, Brubaker G, Smith JD. Zymosan-mediated inflammation impairs in vivo reverse cholesterol transport. J Lipid Res 2011; 52: 951-957.
14. Feingold KR, Moser AH, Shigenaga JK, Patzek SM, Grunfeld C. Inflammation stimulates the expression of PCSK9. Biochem Biophys Res Commun 2008; 374: 341-344.
15. Xiao HB, Sun ZL, Zhang HB, Zhang DS. Berberine inhibits dyslipidemia in C57BL/6 mice with lipopolysaccharide induced inflammation. Pharmacol Rep 2012; 64: 889-895.
16. Underhill, DM, Macrophage recognition of zymosan particles. J Endotoxin Res 2003; 9: 176-180.
17. Sato M, Sano H, Iwaki D, Kudo K, Konishi M, Takahashi H, et al. Direct binding of Toll-like receptor 2 to zymosan, and zymosan-induced NF-κB activation and TNF-α secretion are down-regulated by lung collectin surfactant protein A. J Immunol 2003; 171: 417-425.
18. Yuan Z, Liao Y, Tian G, Li H, Jia Y, Zhang H, et al. Panax notoginseng saponins inhibit Zymosan A induced atherosclerosis by suppressing integrin expression, FAK activation and NF-kappaB translocation. J Ethnopharmacol 2011; 138: 150-155.
19. Araújo FA, Rocha MA, Mendes JB, Andrade SP. Atorvastatin inhibits inflammatory angiogenesis in mice through down regulation of VEGF, TNF-α and TGF-β1. Biomed Pharmacother 2010; 64: 29-34.
20. Preiss D, Seshasai SR, Welsh P, Murphy SA, Ho JE, Waters DD, et al. Risk of incident diabetes with intensive-dose compared with moderate-dose statin therapy: a meta-analysis. Jama 2011; 305: 2556-64. 21. Dadu RT and Ballantyne CM.  Lipid lowering with PCSK9 inhibitors. Nat Rev Cardiol 2014; 11: 563-573
22. Chan JC, PiperDE, Cao Q, Liu D, King C, Wang W, et al. A proprotein convertase subtilisin/kexin type 9 neutralizing antibody reduces serum cholesterol in mice and nonhuman primates. Proc Natl Acad Sci 2009; 106: 9820-9825.
23. Bittner V.  Pleiotropic effects of PCSK9 (proprotein convertase subtilisin/kexin type 9) inhibitors? Circulation 2016; 134:1695-1696.
24. Navarese EP, Kołodziejczak M, Kereiakes DJ, Tantry U.S, O’connor C, Gurbel PA. Proprotein convertase subtilisin/kexin type 9 monoclonal antibodies for acute coronary syndrome: a narrative review. Ann Intern Med 2016; 164: 600-607
25. Filippatos TD, Christopoulou EC, Elisaf MS. Pleiotropic effects of proprotein convertase subtilisin/kexin type 9 inhibitors? Curr Opin Lipido 2018; 29:333-339
26. Lee JS, Mukhopadhyay P, Matyas C, Trojnar E, Paloczi J, Yang YR, et al. PCSK9 inhibition as a novel therapeutic target for alcoholic liver disease. Sci Rep 2019; 9: 1-6.
27. Frostegård J, Ahmed S, Hafström I, Ajeganova S, Rahman M. Low levels of PCSK9 are associated with remission in patients with rheumatoid arthritis treated with anti-TNF-α: potential underlying mechanisms. Arthritis Res Ther 2021; 23:1-9.
28. Sasidharan SR, Joseph JA, Anandakumar S, Venkatesan V, Ariyattu Madhavan CN, Agarwal A. An experimental approach for selecting appropriate rodent diets for research studies on metabolic disorders. Biomed Res 2013; 2013: 1-9. 
29. Arya P, Bhandari U. Involvement of the toll-like receptors-2/nuclear factor-kappa B signaling pathway in atherosclerosis induced by high-fat diet and zymosan A in C57BL/6 mice. Indian J Pharmacol 2020; 52: 203-209.
30. Liang S, Hosur KB, Lu S, Nawar HF, Weber BR, Tapping RI, Connell TD, Hajishengallis G. Mapping of a microbial protein domain involved in binding and activation of the TLR2/TLR1 heterodimer. J Immunol 2009; 182: 2978-2985.
31. Vakser IA. Protein-protein docking: From interaction to interactome. Biophys J 2014; 107: 1785-1793.
32. Kang JY, Nan X, Jin MS, Youn SJ, Ryu YH, Mah S, et al. Recognition of lipopeptide patterns by Toll-like receptor 2-Toll-like receptor 6 heterodimer. Immunity 2009; 31: 873-884.
33. Kühnast S, van der Hoorn JW, Pieterman EJ, van den Hoek AM, Sasiela WJ, Gusarova V, et al. Alirocumab inhibits atherosclerosis, improves the plaque morphology, and enhances the effects of a statin. J Lipid Res 2014; 55: 2103-2112.
34. Bhandari U, Kumar V, Khanna N, Panda BP. The effect of high-fat diet-induced obesity on cardiovascular toxicity in Wistar albino rats. Hum Exp Toxicol 2011; 30: 1313-1321.
35. Demacker PN, Hijmans AG, Vos-Janssen HE, Van’t Laar A, Jansen AP. A study of the use of polyethylene glycol in estimating cholesterol in high-density lipoprotein. Clin Chem 1980; 26: 1775-1779.
36. Foster LB and Dunn RT. Stable reagents for determination of serum triglycerides by a colorimetric Hantzsch condensation method. Clin Chem 1973; 19: 338-340.
37. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem 1972; 18: 499-502.
38. Kazemi T, Hajihosseini M, Moossavi M, Hemmati M, Ziaee M. Cardiovascular risk factors and Atherogenic indices in an Iranian population: Birjand east of Iran. Clin Med Insights Cardiol 2018; 12:1-6.
39. Boehmer ED, Meehan MJ, Cutro BT, Kovacs EJ. Aging negatively skews macrophage   TLR2-and TLR4-mediated pro-inflammatory responses without affecting the IL-2-stimulated pathway. Mech Ageing Dev 2005; 12: 1305-1313.
40. Pushpan CK, Shalini V, Sindhu G, Rathnam P, Jayalekshmy A, Helen A. Attenuation of atherosclerotic complications by modulating inflammatory responses in hypercholesterolemic rats with dietary Njavara rice bran oil. Biomed Pharmacother 2016; 83: 1387-1397.
41. May K, Kraemer F, Chen J, Cooper A. ELISA measurement of LDL receptors. J lipid Res 1990; 31: 1683-1691. 
42. Naiki Y, Sorrentino R, Wong MH, Michelsen KS, Shimada K, Chen S, et al. TLR/MyD88 and liver X receptor α signaling pathways reciprocally control Chlamydia pneumoniae-induced acceleration of atherosclerosis. J Immunol 2008; 181: 7176-7185.
43. Bhat OM, Kumar PU, Giridharan NV, Kaul D, Kumar MM, Dhawan V. Interleukin-18- induced atherosclerosis involves CD36 and NF-κB crosstalk in Apo E−/− mice. J Cardiol 2015; 66: 28-35.
44. Venegas-Pino DE, Banko N, Khan MI, Shi Y, Werstuck GH. Quantitative analysis and characterization of atherosclerotic lesions in the murine aortic sinus. J Vis Exp 2013; 82: 1-11.
45. Kajava AV, Vasselon T. A network of hydrogen bonds on the surface of TLR2 controls ligand positioning and cell signaling. J Biol Chem 2010; 285: 6227-6234.
46. Balasubramanian PK, Kim J, Son K, Durai P, Kim Y. 3, 6‐Dihydroxyflavone: A potent inhibitor with anti‐inflammatory activity targeting toll‐like receptor 2. Bull Korean Chem Soc 2019; 40: 51-55.
47. Koymans KJ, Feitsma LJ, Bisschop A, Huizinga EG, van Strijp JA, de Haas CJ, et al. Molecular basis determining species specificity for TLR2 inhibition by staphylococcal superantigen-like protein 3 (SSL3). Vet Res 2018; 49: 1-5.
48. Zhang YG, Zhang HG, Zhang GY, Fan JS, Li XH, Liu YH, et al. Panax notoginseng saponins attenuate atherosclerosis in rats by regulating the blood lipid profile and an anti‐inflammatory action. Clin Exp Pharmacol Physiol 2008; 35: 1238-1244.
49. Li Y, Shen S, Ding S, Wang L. Toll-like receptor 2 downregulates the cholesterol efflux by activating the nuclear factor‑κB pathway in macrophages and may be a potential therapeutic target for the prevention of atherosclerosis. Exp Ther Med 2018; 15: 198-204
50. Li B, Xia Y, Hu B. Infection and atherosclerosis: TLR-dependent pathways. Cell Mol Life Sci 2020; 77: 2751-2769.
51. Roshan MH, Tambo A, Pace NP. The role of TLR2, TLR4, and TLR9 in the pathogenesis of atherosclerosis. Inter J Inflam 2016; 2016: 1-11.
52. Edfeldt K, Swedenborg J, Hansson GK, Yan ZQ. Expression of toll-like receptors in human atherosclerotic lesions: a possible pathway for plaque activation. Circulation 2002; 105:1158-1161.
53. Bhaskar S, Sudhakaran PR and Helen A. Quercetin attenuates atherosclerotic inflammation and adhesion molecule expression by modulating TLR-NF-κB signaling pathway. Cell Immunol 2016; 310: 131-140.
54. Ascer E, Bertolami MC, Venturinelli ML, Buccheri V, Souza J, Nicolau JC, et al. Atorvastatin reduces proinflammatory markers in hypercholesterolemic patients. Atherosclerosis 2004; 177: 161-166.
55. Zhang Y, Ma KL, Ruan XZ, Liu BC. Dysregulation of the low-density lipoprotein receptor pathway is involved in lipid disorder-mediated organ injury. Int J Biol Sci 2016; 12: 569-579.
56. Catapano AL, Pirillo A, Norata GD. Vascular inflammation and low‐density lipoproteins: is  cholesterol the link? A lesson from the clinical trials. Br J Pharmacol 2017; 174: 3973-3985.
57. Ruan XZ, Moorhead JF, Tao JL, Ma KL, Wheeler DC, Powis SH, et al. Mechanisms of dysregulation of low-density lipoprotein receptor expression in vascular smooth muscle cells by inflammatory cytokines. Arterioscler Thromb Vasc Biol 2006; 26: 1150-1155.