(M1230-11-65) Anti-inflammatory Effects of Bone-Targeted Angiotensin1-7 Delivery in Adjuvant Arthritic Rat Model

Purpose: The renin-angiotensin (Ang) system (RAS) has two major arms, the “Classical Arm” and the “Protective Arm”. The balanced RAS is responsible for regulating inflammation and maintaining blood pressure. The classical arm composed of Ang II/Ang-converting enzyme 1 (ACE1)/Ang II type 1 receptor (AT1R) is mainly involved in vasoconstriction, vascular smooth muscle, and hypertrophy of the heart vessel wall (1). In contrast, the protective arm plays a significant role in vasodilation, anti-inflammation, and anti-oxidation through the action of Ang1-7 on the Mas receptor (MasR) (2, 3).Ang1-7 is very effective in managing inflammatory diseases, such as arthritis (4). However, its activity is hampered by a short half-life due to rapid degradation. We developed a bone-seeking Ang1-7 conjugate (Ang Conj), and we hypothesized that it can outperform the native peptide actions due to its longer half-life and circulation time in the body in a rat model of arthritis.

Methods: We used male Sprague-Dawley rats to induce adjuvant arthritis (AA). They were injected with Mycobacterium butyricum at the tail base. Arthritic animals were divided into 3 groups and treated with subcutaneous injection of normal saline (inflamed control), Ang1-7 (0.6 mg/kg) or Ang Conj. (equivalent dose of Ang1-7). A group of healthy rats received the vehicle as the control group. The drug administration was done thrice weekly for 14 days upon the emergence of arthritis. Animals were monitored every other day for their body weight and arthritis index and were euthanized after 14 days; their tissues were harvested and analyzed for mRNA and protein level of the RAS components by qPCR and western blot, respectively.

Results: Adjuvant arthritis emerged 8-12 days after induction affecting weight gain and arthritis index. (Fig.1). The mean±SEM results of the body weight show that the body weight change percentage compared to the first day was significantly (50.1 ± 2.5) higher in the control group than in the inflamed group (12.6 ± 5.5). Treatment with Ang1-7 (31.6 ± 5.3) or Ang Conj. (34.2 ± 4.5) could significantly revert this trend compared to the inflamed group (Fig.1A). The AI, a measure of signs and symptoms of arthritis, gradually increased in inflamed rats and decreased in Ang Conj.-treated rats (Fig.1B). The results of mRNA expression analysis of ACE1, ACE2, MasR, and AT1R, using qPCR in the heart and kidney show in the rats’ hearts of inflamed rats, the expression level of ACE1 and AT1R significantly increased despite ACE2. Treatment with Ang Conj could reverse the increased expression of ACE1 and AT1R significantly. MasR and ACE2 expressions were significantly increased in Ang Conj. and Ang1-7-treated rats compared to the inflamed group (Fig.2B). The same trend was seen in kidney tissue (Fig.2A). Plotting the ratios of ACE2/ACE1 and MasR/AT1R depicts that they both increased in the heart via Ang Conj. treatment, significantly in the heart and kidney (Fig.2C&D). Fig.3 represents the protein analysis in heart and kidney tissues and their ratio (Fig.3C&D). In both tissues, ACE1 and AT1R reverted back to normal after treatment with Ang Conj. and a significant increase is seen in ACE2 and MasR compared to inflamed rats after Ang Conj. treatment in the kidney (Fig.3B) and heart (Fig.3A) tissues.

Conclusion: The results of this study suggest that inflammation alters the balance of the RAS components at the cellular level. The examined Ang1-7 and Ang Conj. restore the imbalances in the RAS caused by inflammation. This suggests that the bone-targeted delivery of Ang1-7 can be used as a modality for inflammatory diseases.

References: 1. Cassis P, Conti S, Remuzzi G, Benigni A. Angiotensin receptors as determinants of life span. Pflügers Archiv-European Journal of Physiology. 2010;459(2):325-32.
2. Farag E, Maheshwari K, Morgan J, Esa WAS, Doyle DJ. An update of the role of renin angiotensin in cardiovascular homeostasis. Anesthesia & Analgesia. 2015;120(2):275-92.
3. Farag E, Sessler DI, Ebrahim Z, Kurz A, Morgan J, Ahuja S, et al. The renin angiotensin system and the brain: new developments. Journal of Clinical Neuroscience. 2017;46:1-8.
4. da Silveira KD, Coelho FM, Vieira AT, Sachs D, Barroso LC, Costa VV, et al. Anti-inflammatory effects of the activation of the angiotensin-(1–7) receptor, MAS, in experimental models of arthritis. The Journal of Immunology. 2010;185(9):5569-76.

Figure 1. Effect of the arthritis induction and treatment with Ang-(1-7) or Ang. Conj. on weight change (A). The effect of Ang1-7 and Ang. Conj. administration on the emergence of arthritis. Different letters denote the significance level between groups (p < 0.05). Statistical analysis was done using one-way ANOVA with Tukey’s post hoc test.



Figure 2. qPCR analysis of the RAS components for ACE1, ACE2, MasR, and AT1R in the kidney (A), heart (B), and their ratio (C & D). Different letters denote the significance level between groups (p < 0.05). Statistical analysis was done using one-way ANOVA with Tukey’s post hoc test.



Figure 3. Western blot analysis of the RAS components for ACE1, ACE2, MasR, and AT1R in the kidney (A), heart (B), and their ratio (C & D). Different letters denote the significance level between groups (p < 0.05). Statistical analysis was done using one-way ANOVA with Tukey’s post hoc test.