Expression of RAS components was upregulated in the joints of SKGc mice and the PBMCs and BdCs of r-axSpA patients
To determine the expression of RAS components in the arthritic joints of an animal model of SpA, SKG mice were injected with curdlan to generate SKGc mice (Fig. 1a). The expression of RAS components in the joints of SKGc mice was assessed using RT-qPCR and western blotting. The expression levels of AGT, ACE, and AT1R were significantly higher in the joints of SKGc mice than in those of wild type (WT) or SKG mice (p = 0.025, 0.034, and0.014, respectively; Fig. 1b). Western blotting revealed that the protein levels of AGT, ACE, and AT1R in the ankle joints of SKGc mice were higher than those in WT or SKG mice (Fig. S1).
We also compared the expression of RAS molecules between patients with r-axSpA and control participants. First, we identified the RAS molecules in the PBMCs of patients with r-axSpA and control participants. The expression levels of AGT, ACE, AT1R, AT2R, and NEP were significantly higher (p = 0.003, 0.0037, 0.005, 0.025, and 0.0306, respectively) in the PBMCs of patients with r-axSpA than in those of the control participants (Fig. 1c). Next, we compared the expression of RAS molecules in BdCs between patients with r-axSpA and control participants. The expression levels of all RAS molecules, except for AT2R, were significantly higher (p = 0.027, 0.003, 0.05, and 0.032, respectively) in r-axSpA patients than in control participants (Fig. 1d).
ARBs, but not ACEis, inhibited bone erosion and systemic bone loss in SKGc mice
RAS affects the development of inflammation [32]. Hence, we investigated whether RAS modulators, such as ARBs and ACEis, influence the development of arthritis in SKGc mice. First, we monitored the severity of paw swelling in each limb and compared the clinical arthritis scores among SKGc groups treated with the vehicle, ARB, or ACEi (SKGc-saline, SKGc-ARB, or SKGc-ACEi, respectively). No significant differences were observed between the groups (Fig. 2a). Next, we measured MPO activity using IVIS; however, we did not find any differences between the groups (Fig. 2b).
Additionally, joint tissues were stained with hematoxylin and eosin to determine the degree of inflammatory cell infiltration in the ankle joints of SKGc mice. Compared to that in WT mice, SKGc-saline mice showed increased inflammatory cell infiltration in the joints. There was no difference in the degree of inflammatory cell infiltration among SKGc-saline, SKGc-ARB, and SKGc-ACEi mice, which is consistent with the results on the clinical arthritis score and MPO activity (Fig. S2). Thus, RAS modulators did not significantly affect the severity of arthritis in SKGc mice.
Next, we performed CT of arthritic joints to assess the effect of RAS modulation on the development of erosion and abnormal bone formation. The CT erosion score was significantly lower in SKGc-ARB mice (p = 0.033) and higher in SKGc-ACEi mice than in SKGc-saline mice (p = 0.898) (Fig 2c and d, left). CT bone formation scores decreased in SKGc-ARB mice (p = 0.515) and increased in SKGc-ACEi mice (p = 0.516) compared to that in SKGc-saline mice (Fig. 2c and d, right).
Furthermore, bone CT was used to investigate the effect of RAS modulation on bone remodeling. Bone mineral density (BMD) was significantly higher in the SKGc-ARB group (p = 0.011) and lower in the SKGc-ACEi group (p = 0.462) than in the SKGc-saline group (Fig. 2e and f). Differences were not observed in the BMD measurement of mice in the WT-saline, WT-ARB, and WT-ACEi groups treated with each RAS modulator (to determine whether these drugs affected BMD in a non-inflammatory environment) (Fig. S3a and b).
Next, we measured the expression levels of bone cell-related molecules in the joints of SKGc mice. The expression levels of OC differentiation markers, such as TRAP, NFATc, and cathepsin K, were significantly lower in SKGc-ARB mice than in SKGc-saline mice (p = 0.006, 0.006, and 0.006, respectively); however, differences were not observed between SKGc-ACEi and SKGc-saline mice (p = 0.522, 0.670, and 0.394, respectively) (Fig. 2g). Similarly, the expression of OB differentiation markers such as BMP2, RUNX2, and RANKL was significantly lower in SKGc-ARB mice (p = 0.006, 0.006, and 0.006, respectively) but higher in SKGc-ACEi mice than in SKGc-saline mice (p = 0.286, 0.088, and 0.394, respectively) (Fig. 2h).
ARB and ACEi showed opposite effects on osteoclast differentiation from mouse primary bone marrow macrophages
To verify the direct role of RAS in bone cells, we applied RAS modulators in bone cell culture systems. First, we administered ARB or ACEi to the BMMs of mice. TRAP staining revealed that OC differentiation was significantly inhibited by ARB (p = 0.006), but significantly promoted by ACEi (p = 0.004), compared to that in the controls (Fig. 3a). ACEi increased the numbers of TRAP-positive multinucleated OCs and OCs with more than 30 nuclei per cell (p = 0.002; Fig. S4). Differences in cell viability were not observed among the groups in the CCK8 assay (Fig. 3b). RT-qPCR analysis revealed that ARB significantly lowered (p = 0.006, 0.028, 0.009, 0.009, and 0.009, respectively) whereas ACEi increased (p = 0.006, 0.028, 0.009, 0.021, and 0.009, respectively) the expression of OC differentiation markers, such as TRAP, NFATc, cathepsin K, DC-STAMP, and OC-STAMP compared to that in the controls (Fig. 3c).
To investigate the signals acting upstream of the above-mentioned factors, we measured the expression level of TRAF6 in RAW 264.7 cells treated with saline, ARB, or ACEi; ARB inhibited the expression of TRAF6, but ACEi did not (Fig. 3d). To further investigate the mechanism underlying the inhibition, cells were treated with MG132, a proteasome inhibitor, which restored TRAF6 expression (Fig. 3e), suggesting that the effect of RAS modulators on OCs was mediated via TRAF6 ubiquitination, resulting in degradation by the proteasome.
Ang 1-7 facilitated osteoclast differentiation from mouse primary bone marrow macrophages
The conflicting effects of ARB and ACEi led us to hypothesize that contrary to previous reports [22, 33, 34], Ang 1-7, rather than Ang II, might play a dominant role in OC differentiation. To verify this, we first confirmed that Ang 1-7 levels increased significantly (p = 0.021) after the treatment of OC progenitors with ACEi (Fig. 4a).
To examine the effect of Ang 1-7 on OC differentiation, we treated the cells with saline, Ang 1-7, or Ang II. TRAP staining revealed that Ang 1-7 enhanced OC differentiation (p = 0.004; Fig. 4b) and the expression of target molecules, including TRAP, NFATc, cathepsin K, DC-STAMP, OC-STAMP, OSCAR, and Blimp1 (p = 0.020, 0.018, 0.017, 0.019, 0.017, 0.019, and 0.019, respectively), to an extent similar to that observed with Ang II (Fig. 4c). Both Ang II and Ang 1-7 increased the expression of TRAF6, contrary to that observed with ARB (Fig. 4D).
We hypothesized that the increase in OC differentiation after treatment with Ang II was caused by Ang 1-7 derived from Ang II, and not by Ang II itself. Therefore, we simultaneously administered Ang II and ACE2i to OC progenitor cells to inhibit the conversion. This diminished the promoting effect of Ang II on OC differentiation to a level similar to that of the control group (Fig. 4e). Furthermore, Ang 1-7 levels decreased when ACE2i was used in combination with Ang II compared to Ang II alone (Fig. S5). Additionally, the NEP inhibitor (NEPi), administered with Ang I, completely blocked OC differentiation (p = 0.002; Fig. 4f), implying that NEP might be a critical enzyme involved in Ang 1-7 production, at least in the bone milieu. OC differentiation was inhibited by NEPi (p = 0.004) without changes in cell viability (Fig. S6a and b), and the mRNA expression of target molecules, including TRAP, NFATc, cathepsin K, DC-STAMP, and OC-STAMP,decreased after NEPi treatment (p = 0.008, 0.008, 0.008, 0.008, and 0.009, respectively; Fig. S6c). These results, combined with the finding that ACEi promoted OC differentiation, suggested that Ang 1-7, rather than Ang II, plays a major role in OC differentiation.
Furthermore, a Mas receptor (MasR) inhibitor (MasRi) was used to assess OC differentiation, as MasR is the major receptor of Ang 1-7. MasRi treatment significantly increased OC differentiation (p = 0.009; Fig. 4g). Additionally, to determine whether Ang 1-7 acted on the AT1 receptor, we treated OC progenitor cells simultaneously with Ang 1-7 and ARB, which suppressed the Ang 1-7-induced promotion of OC differentiation (Fig. 4b and c). To verify this further, ACEi and ARB were simultaneously administered to the cells. We observed that OC differentiation was inhibited without changes in cell viability (Fig. S7a and b), and expression of the OC differentiation marker decreased (Fig. S7c). Thus, the Ang 1-7/AT1 axis is the major pathway involved in OC differentiation.
ARB and ACEi exerted opposite effects on osteoblast differentiation from mouse primary osteoblast progenitor cells
To examine the effect of ARB and ACEi on OB differentiation, mouse OB progenitor cells were treated with RAS modulators in an in vitro culture system. Intracellular ALP activity assay and alizarin red staining (ARS) were performed to assess OB differentiation and mineralization, respectively. Intracellular ALP activity in OBs was suppressed by ARB (p = 0.021) but promoted by ACEi (p = 0.11), suggesting that ARB and ACEi exerted opposite effects on OB differentiation despite the lack of statistical significance (Fig. 5a). ARS analysis revealed that ARB significantly inhibited (p = 0.021), whereas ACEi promoted (p = 0.043) mineralization (Fig. 5b) without changes in cell viability (Fig. 5c). Consistent with these findings, we observed that compared to the vehicle, ARB significantly decreased the expression of bone formation markers, including BMP2, RUNX2, RANKL, and osteocalcin, in OBs (p = 0.021, 0.020, 0.021, and 0.021, respectively), whereas ACEi significantly increased their expression levels (p = 0.043, 0.248, 0.149, and 0.021, respectively) (Fig. 5d). Experiments using the human SaOS2 cell line produced similar results (Fig. S8a and b).
Additionally, Ang 1-7 increased OB differentiation and mineralization to a degree similar to that of Ang II (Fig. 5e and f). When cells were co-stimulated by ARB and ACEi, OB differentiation and mineralization were inhibited without changes in cell viability (Fig. S9a–c).
ARB inhibited osteoblast differentiation from BdCs of r-axSpA patients
We investigated whether RAS modulators affect the differentiation of OBs obtained from patients with r-axSpA. ARB and ACEi were administered to the BdCs of biologic-naïve r-axSpA patients and control participants. ALP, ARS, and Von Kossa staining revealed that ARB significantly inhibited OB differentiation and mineralization in biologic-naïve r-axSpA patients (p = 0.0058, 0.042, 0.003, and 0.0144, respectively), whereas ACEi did not (Fig 6a and b, right panels). Significant changes were not observed in the OBs of the control participants treated with ARB or ACEi (Fig. 6a and b, left panels). Additionally, HA staining and bright field imaging showed that ARB inhibited the mineralization of OBs in the r-axSpA patients, but not in the control participants (Fig. 6c).