*2.2. No Significant E*ff*ect of Ang II Administration on the Severity of Inflammatory Cell Infiltration in TNFtg Mice*

To assess the effect of Ang II on arthritis, exogenous Ang II (1.44 mg/kg/day) or water (H2O) was administered by osmotic pumps to the WT and TNFtg mice for 4 weeks. The treatment with Ang II did not significantly alter body weight (Figure 1B), but did induce hypertension (Figure A2). We monitored the severity of paw swelling in each limb during the experimental period. We found that the TNFtg mice exhibited severe swelling of the paws and that the severity of clinical arthritis was not affected by the Ang II infusion. The arthritis score and number of arthritic limbs at the age of 16 weeks are presented in Figure 1C,D.

To analyze the inflamed joints histologically, we performed hematoxylin and eosin (H&E) and Safranin O staining to determine the inflammatory cell infiltration and cartilage damage. In WT mice, Ang II administration did not cause any detectable histological changes (Figure 1E,F). TNFtg mice exhibited massive inflammatory cell infiltration, and Ang II administration did not affect the severity of inflammation in these mice, which is consistent with the arthritis score results (Figure 1C). Additionally, the severity of cartilage damage, represented by decreased staining of the cartilage matrix, was not affected by the administration of Ang II (Figure 1E,G).

#### *2.3. Exacerbation of Bone Erosion by Ang II Administration in TNFtg Mice*

We then examined the impact of Ang II on the erosive bone changes of the ankle. Bone erosion around the talus was quantified using micro-computed tomography (CT) and 3D image analysis software. The micro-CT analysis revealed that the destructive bone change was significantly more severe in the Ang II-infused TNFtg mice than in the H2O-infused TNFtg mice (Figure 2A). This aggravated bone erosion was revealed by the following quantitative analyses: the bone volume (BV) of the talus, the reduction rate of BV, and the eroded volume per repaired volume (Ev/Rpv) of the talus (Figure 2B–D). Tartrate-resistant acid phosphatase (TRAP)-stained images showed slightly increased osteoclast formation in the joints of the Ang II-infused TNFtg mice compared to those in the H2O-infused TNFtg mice (Figure 2E). Quantitative histological analyses also revealed increased bone erosion and slightly enhanced osteoclast formation around the talus (Figure 2F,G). These findings suggest that Ang II, imported to the joints from circulation, served as an osteoclast-activating factor in the arthritic joint, resulting in enhanced bone erosion without affecting the clinical severity of arthritis.

**Figure 2.** Exacerbation of bone erosion by angiotensin II (Ang II) administration in tumor necrosis factor-transgenic (TNFtg) mice. Ang II was administered by osmotic pumps to 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**) Representative 3D micro-computed tomography (CT) images of the talus. (**B**) Bone volume (BV) of the talus. (**C**) Rates of reduction in BV of Ang II-infused WT and TNFtg mice relative to H2O-infused control mice of each genotype. (**D**) Eroded volume per repaired volume (Ev/Rpv) of the talus. (**E**) Representative images of tartrate-resistant acid phosphatase (TRAP) staining around the talus bones of indicated mice; original magnification ×40. (**F**) Eroded surface per bone surface (ES/BS) around the taluses. (**G**) The number of osteoclasts per bone surface (N.Oc/BS) around the taluses. Values are the mean ± SEM. n.s., not significant. \*, *p* < 0.05.

#### *2.4. No Detectable Changes in the Trabecular and Cortical Bone Parameters with Ang II Administration*

Since both systemic inflammation and excess of Ang II have been reported to decrease the mass of systemic bones [4,11], we examined the bone properties of the tibia and vertebra and determined whether Ang II could synergistically enhance inflammation-mediated bone loss in the Ang II-administered arthritic mice. We assessed the tibia trabecular bone (secondary spongiosa, Figure 3A), the tibia cortical bone (midshaft of the tibia, Figure 3B), and the trabecular bone of the spine (fifth lumbar vertebra, Figure 3C) using micro-CT. The tibia trabecular bone tended to be decreased in the arthritic mice compared to that in the WT mice, although the difference was not statistically significant in the current set of experiments. The bone reduction rates with Ang II infusion were comparable between WT and TNFtg mice at approximately 30% (Figure 3D), indicating that the synergistic effect of inflammation and Ang II on osteopenia was not noticeable. In the tibia cortical bone, the presence of arthritis significantly increased bone loss, but the reduction rates with Ang II infusion were comparable between WT and TNFtg mice (Figure 3E). In the vertebral trabecular bone, the presence of arthritis did not significantly affect the bone volume (Figure 3F). Ang II administration tended to decrease bone volume by approximately 10%, but there was no significant difference in the reduction rates between WT and TNFtg mice (Figure 3F). The other analyzed parameters of the trabecular and cortical bones also indicated no significant effect of Ang II administration on bone properties (Figure A3). Collectively, these findings suggest that both inflammation and Ang II tended to decrease bone mass, but there was no apparent synergistic effect on the osteopenic phenotype.

**Figure 3.** No detectable changes in the trabecular and cortical bone parameters with Ang II administration. Angiotensin II (Ang II) was administered by osmotic pumps to WT (*n* = 6) and tumor necrosis factor-transgenic (TNFtg; *n* = 7) male mice from 12 to 16 weeks old. Water (H2O) was administered by osmotic pumps to WT (*n* = 6) and TNFtg (*n* = 4) male mice as controls. (**A**–**C**) Representative 2D micro-CT images of the tibia trabecular bone (**A**), the tibia cortical bone (**B**), and the trabecular bone of the spine (the fifth lumbar vertebra) (**C**). (**D**) Bone volume per total volume (BV/TV) and reduction rate of the tibia trabecular bone. (**E**) Cortical thickness (Ct.Th) and reduction rate of the tibia midshaft. (**F**) Bone volume per total volume (BV/TV) and reduction rate of the fifth lumbar vertebral trabecular bone. Values are the mean ± SEM. n.s., not significant. \*, *p* < 0.05.
