Active Immunization Using TRPM2 Peptide Vaccine Attenuates Atherosclerotic Progression in a Mouse Model of Atherosclerosis

Abstract

Background/Objective: Atherosclerosis is one of the leading causes of cardiovascular diseases and mortality around the world. One exciting strategy for atherosclerosis treatment is immunotherapy, especially active immunization. Active immunization relies on the delivery of antigens in a vaccine platform to introduce humoral and cellular immunity, alleviating atherosclerotic progression. Transient receptor potential channel isoform M2 (TRPM2) is an ROS-activated Ca²⁺-permeable ion channel that can promote atherosclerosis via stimulating vascular inflammation. In the present study, we developed a strategy of active immunization with the TRPM2 E3 domain peptide in a vaccine platform, aiming to induce the endogenous production of anti-TRPM2 blocking antibody in mice in vivo, consequently inhibiting TRPM2 channel activity to alleviate atherosclerotic progression. Methods: ApoE knockout mice were fed with a high cholesterol diet to develop atherosclerosis. The mice were injected with or without the E3 peptide vaccines, followed by analysis of atherosclerotic lesion by en face Oil Red O staining of the whole aorta and histologic analysis of thin tissue sections from aortic roots. Results: The results show that immunization with a pig TRPM2 E3 region-based peptide (P1) could effectively alleviate high cholesterol diet-induced atherosclerosis in ApoE knockout mice. We worked out the best vaccine formulation for the most effective atheroprotection, namely P1 at the dose of 67.5 µg per mouse (2.5 mg/kg body weight) with aluminum salts as adjuvant. Conclusions: The present study provides a novel target TRPM2 for peptide vaccine-based anti-atherosclerotic strategy and lays the foundation for future preclinical/clinical trials using TRPM2 E3 P1 vaccine for a potential therapeutic option against atherosclerosis.

Keywords: TRPM2; atherosclerosis; immunization; peptide vaccine.

Figure 1

Experimental schedule (a) and the amino acid sequences of peptide antigens (b).

Figure 2

A pig TRPM2 E3 region-based peptide vaccine P1 ameliorates the development of atherosclerotic plaques in mouse whole aortas. (a) Quantification of atherosclerotic plaques (left) and representative images (right) of Oil Red O staining of whole aortas. (b–d) The effect of high cholesterol diet feeding and E3 region-based peptide vaccines on serum LDL cholesterol level (b), serum cholesterol level (c) and triglyceride level (d). Data are shown as mean ± SD (n = 5–11), * p < 0.05; *** p < 0.001.

Figure 3

Titration of TRPM2 E3-based polyclonal antibody in mice and effectiveness of TM2E3-P1 in suppressing H₂O₂-stimulated Ca²⁺ entry in endothelial cells. (a) Titration of polyclonal antibodies produced in mice in response to P1 peptide vaccine. The mice were immunized with P1 peptide vaccine or KLH control. The polyclonal antiserum was taken from the mice, followed by Elisa-based titration vs. synthetic P1 peptide. Shown are titration curves of the antiserum at different dilutions. (b–e) Polyclonal antibody TM2E3-P1 (compared with pre-immune IgG) could effectively inhibit the H₂O₂-stimulated Ca²⁺ entry in mouse H5V endothelial cells (b,c) and human HUVEC endothelial cells (d,e). The cells were bathed in 0Ca²⁺-PSS and challenged by 500 µM H₂O₂, which elicited the first cytosolic Ca²⁺ rise due to intracellular Ca²⁺ release. Then, 2 mM Ca²⁺ was added back to initiate the second Ca²⁺ rise, which was due to Ca²⁺ entry. Shown were representative time course (b,d) and data summary for Ca²⁺ entry (c,e). Controls had no H₂O₂ treatment. Mean ± SEM (n = 3–7); ns, not significant; **, p < 0.01; ***, p < 0.001.

Figure 4

Immunization with P1 peptide vaccine reduced the development of atherosclerotic plaques in thin tissue sections of aortic roots. (a) Representative tissue section images (left) and data summary (right) of aortic roots stained with Oil Red O. The atherosclerotic lesion area is visualized as red. (b) Representative tissue section images (left) and data summary for necrotic sore size (right) of aortic roots stained with Masson’s trichrome. The collagen is stained in blue. The size of the necrotic core is quantified using Image J. Quantification was performed in five random visual fields of multiple slides prepared from five to eight mice. Scale bars are as indicated. Data are shown as Mean ± SEM (n = 5–8 in (a), n = 25–29 in (b)), ** p < 0.01.

Figure 5

Comparison of different P1 vaccine doses and adjuvants in suppressing atherosclerotic progression based on en face Oil Red O staining of whole aortas. Two different dosages of P1 peptide vaccines were tested, 67.5 µg per mouse (2.5 mg/kg body weight) (a,c) and 135 µg per mouse (5 mg/kg body weight) (b,d). Two different adjuvants, aluminum hydroxide adjuvant (a,b) and Freund’s adjuvant (c,d), were also evaluated. Shown is the quantification of atherosclerotic plaques based on en face Oil Red O staining of whole aortas. The plaque area is quantified by using Image J. Data are shown as mean ± SD (n = 6–10), * p < 0.05; ** p < 0.01; ***, p < 0.001.

Figure 6

Comparison of different P1 vaccine doses and adjuvants in suppressing atherosclerotic progression based on the staining on thin tissue sections of aortic roots. (a) Representative images (left) and data summary of Oil Red O staining of thin tissue sections of mouse aortic roots. (b) Representative images (left) and data summary of Masson’s trichrome staining of thin tissue sections of mouse aortic roots. (c) Representative images (left) and data summary of immunohistochemical stains of CD68, MPO and PCNA on thin tissue sections of mouse aortic roots. The mice were treated with two different P1 vaccine doses (67.5 µg or 135 µg per mouse) and two different adjuvants (aluminum hydroxide adjuvant or Freund’s adjuvant). Quantification was performed in five random visual fields of multiple slides prepared from three to ten mice. Scale bar, 100 µm as indicated. Mean ± SD (n = 3–10 in (a), n = 40–120 in (b)), * p < 0.05; ** p < 0.01; *** p < 0.001.