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| Author(s): |
Gustavo Luis Tripodi
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ) |
| Defense date: | 2021-12-15 |
| Examining board members: |
Dulcineia Saes Parra Abdalla;
Denise Morais da Fonseca;
Ricardo José Giordano;
Patricia Moriel
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| Advisor: | Dulcineia Saes Parra Abdalla; Eduardo Lani Volpe da Silveira |
| Abstract | |
Introduction: Atherosclerosis is a chronic inflammatory disease resulting from changes in the wall of medium and large-caliber arteries and associated with several risk factors, among which hyperlipidemias stand out, ie, the increase in plasma lipoproteins, but also other comorbidities, such as Metabolic Syndrome. Among the lipoproteins, low-density lipoprotein (LDL) is of great relevance in atherosclerosis. Different isoforms of modified LDL (LDLm) are originated through lipolysis, glycation and proteolysis, in addition to oxidation, varying in density and electronegativity, being better called electronegative LDL [LDL (-)]. Considering the conformational differences between the ApoB-100 structure of native LDL and LDL (-), in an initial study, our group developed a monoclonal antibody (2C7) from the immunization of Balb/c mice with human LDL (-). In a next step, the epitope recognized by the anti-LDL monoclonal antibody (-) was mapped using phage display. The binding peptide of anti-LDL monoclonal antibodies (-) was named p2C7. This peptide does not represent linear sequence regions of human ApoB-100, but conformational microdomains of LDL (-) ApoB-100 epitopes, making them candidates for the immunomodulation of atherogenesis. Therefore, investigating the immunomodulation induced by p2C7 peptide mimetics of LDL (-) as it represents an immunodominant epitope of LDL (-) could open new future therapeutic perspectives for the immunomodulation of atherosclerosis. Objective: To evaluate the immunomodulation promoted by p2C7 in vivo, using C57BL/6 LDLr -/- mice, and human plasma samples. In addition, in the internship (BEPE), held at the Karolinska Institute (December 2019 to March 2021), immunometabolism as a mediator of Cardiovascular Diseases was studied. In part II-A, the results of the initially proposed study are described. In part II-B, the results that were developed later are presented, expanding the scope of the project, approaching the vascular inflammation involved in the abdominal aortic aneurysm through bioinformatics tools. In part II-C, the results of the study of the involvement of the enzyme indoleamine 2,3 dioxygenase (IDO) in non-alcoholic steatohepatitis (NASH) and atherosclerosis in ApoE-/- and ApoE/IDO/double mice are presented -knockout. Methodology: The presence of anti-p2C7 antibodies in human plasma samples with or without Metabolic Syndrome was evaluated. We measured circulating TNF in the same samples and proceeded with linear regressions associating inflammatory parameters with levels of anti-p2C7 antibodies. C57BL/6 LDLr -/- mice were immunized with p2C7 and the adjuvants Alum or Montanide ISA 720, analyzing the antibody titers against p2C7 and LDL (-), the production of cytokines (IL-10, IL-4, IL -2, IL-6, IFNγ, IL-17, TNFα) and antibody-secreting cells. C57BL/6 LDLr -/- mice were tolerized against mimotope peptides with intravenous injections (caudal vein) and challenged with immunization containing LDL (-) + Alum. Antibody titers against p2C7 and LDL (-) and cytokine production (TNF-α, IFNγ, IL-12, IL-6, IL-10 and MCP-1) were evaluated. The mice were kept on a hypercholesterolemic diet for 3 months for atherosclerotic plaque formation. After this period, the mice were euthanized, evaluating the formation of atherosclerotic plaque in the abdominal artery and aortic arch, as well as the production of cytokines (TNF-α, IFNγ, IL-12, IL-6, IL-10 and MCP -1). C57BL/6 LDLr -/- mice were immunized with OVA-p2C7 and, after a 3-month hypercholesterolemic diet for atherosclerotic plaque formation, inflammatory parameters were evaluated and 18F-FDG uptake was evaluated by PET/CT. Results: Immunization with p2C7 (free) was not able to induce a humoral response, with no detectable titers of antibodies reactive to p2C7 or LDL (-) being observed in any immunized mouse, as well as no detectable antibody-secreting cells for the LDL (-). The group immunized with Alum or Montanide + p2C7 had a significant increase in TNF-α production when compared to the other groups. The tolerance protocol was successfully performed, as the tolerized mice had lower antibody titers than controls for the epitope used. Only mice tolerated with p2C7 showed a significant increase in the production of IL-6, IL-12, IL-10, TNF-α, IFNγ and MCP 1 after a hypercholesterolemic diet. Active immunization with OVA-p2C7 was able to reduce TNF production induced by the hypercholesterolemic diet, as well as to reduce 18F-FDG uptake. Conclusion: the p2C7 epitope is highly expressed in LDL (-) of patients with higher cardiovascular risk. Furthermore, active immunization with p2C7 is also a promising tool to prevent and regulate inflammation caused by LDL (-) in the course of atherosclerosis. (AU) | |
| FAPESP's process: | 17/06568-0 - Immunization of LDLr - / - mice with electronegative LDL mimetic peptides: a potential therapeutic strategy for atherosclerosis |
| Grantee: | Gustavo Luis Tripodi |
| Support Opportunities: | Scholarships in Brazil - Doctorate |