**1. Introduction**

Rheumatoid arthritis is a chronic inflammatory disorder that can cause painful swelling and bone erosion in the inflamed joints [1]. The accumulation of joint damage results in long-lasting pain and deformity of the affected joints [2]. Persistent systemic inflammation in rheumatoid arthritis can also cause tissue damage in organs such as the lungs, heart, eyes, and bone [3]. Increased inflammatory cytokines affect bone metabolism throughout the body and decrease bone mass and strength, leading to increased risks of osteoporosis and fracture [4]. Joint deformities impair activities of daily life and thus exacerbate osteoporosis in patients with rheumatoid arthritis. This highlights the importance of resolving joint damage and systemic bone loss issues in these patients.

Angiotensin II (Ang II) is the main effector peptide of the renin-angiotensin system (RAS), which causes vasoconstriction and an increase in blood pressure [5,6]. The RAS is one of the most important systems in cardiovascular control [5]. Ang II is generated from its precursor angiotensinogen through serial enzymatic processes mediated by renin and angiotensin-converting enzyme (ACE). Ang II is converted mainly in the kidneys and lungs. Recent studies have shown that Ang II is generated locally in various tissues such as the brain, vascular wall, and reproductive tract [5,7]. Locally produced Ang II plays a role in many physiological and pathological processes such as hypertension, inflammation, tissue fibrosis, and oxidative stress [8], and it fulfills a key function in bone metabolism [6,9]. Animal studies have revealed that Ang II and Ang II type1 receptor (AT1R) are expressed locally within bone tissue and regulate bone mass [6,9,10]. Additionally, studies using mouse models have indicated that the local RAS is involved in the pathogenesis of several bone diseases, such as postmenopausal [11], age-related [12], and glucocorticoid-induced osteoporosis [13].

Components of the RAS are expressed at elevated levels in the synovium of patients with rheumatoid arthritis compared to those in the non-inflamed synovium [9,14,15]. However, the role of the RAS in the mechanisms underlying bone erosion and inflammation-mediated bone loss in arthritic conditions remains unclear. In the present study, we investigated the in vivo effects of Ang II on bone erosion and systemic bone loss in a tumor necrosis factor (TNF)-induced arthritis model. We also aimed to assess whether the deletion of AT1R ameliorates the bone erosion and systemic bone loss caused by arthritis.

#### **2. Results**

#### *2.1. Increased AT1R Expression in the Joint Tissue of Tumor Necrosis Factor-Transgenic (TNFtg) Mice*

As a first step to explore the effect of Ang II on arthritis, the expression of *Agtr1a*, encoding AT1R, in the joints was assessed by quantitative polymerase chain reaction (qPCR). *Agtr1a* mRNA was detected in the joint tissues from both wild-type (WT) and TNFtg mice, and the expression levels were 2.5-fold elevated in the tissues of the TNFtg mice compared with those in the WT mice (Figure 1A). Increased AT1R protein expression was also shown in arthritic joint tissues by Western blotting (Figure A1A). Immunohistochemical stain of the arthritic joints revealed AT1R expression in the tissues, including proliferated synovial cells (Figure A2B).

**Figure 1.** No significant effect of angiotensin II (Ang II) administration on the severity of inflammatory cell infiltration and cartilage damage in tumor necrosis factor-transgenic (TNFtg) mice. Ang II was administered by osmotic pumps to wild-type (WT; *n* = 6) and TNFtg (*n* = 7) male mice from 12 to 16 weeks of age. Water (H2O) was administered by osmotic pumps to WT (*n* = 6) and TNFtg (*n* = 4) male mice as controls. (**A**) Quantitative real-time PCR analysis. *Agtr1a* mRNA expression levels in the right ankle joint tissue were determined. (**B**) Body weight at the age of 16 weeks. (**C**) Arthritis scores at the age of 16 weeks. (**D**) The numbers of arthritic paws with an arthritis score of 2 or higher. (**E**) Representative images of stained sections around the talus bones of indicated mice; sections were stained with hematoxylin and eosin (H&E) and Safranin O; original magnification ×40. (**F**,**G**) Histological scores of inflammation (**F**) and cartilage damage (**G**). Values are the mean ± SEM. n.s., not significant. \*, *p* < 0.05.
