**4.1.5 Measurement of cytokine mRNA**

Total RNA was extracted from the infrapatellar fat pad with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol and was then reversetranscribed into cDNA with the SuperScript First-Strand Synthesis System for RT-PCR kit (Invitrogen). To measure rabbit TNFα, IL-6, vascular endothelial growth factor (VEGF), transforming growth factor beta (TGFβ), and β-actin mRNA levels, we used the quantitative RT-PCR method of real-time quantitative PCR.

### **4.2 Result after joint injection of ADM in AIA rabbit 4.2.1 Adrenomedullin concentration in plasma**

We measured the plasma ADM concentration before and 15, 30, 60 and 120 minutes after intra-articular injection of 3 μg ADM (*n* = 6). No significant change, however, was observed in the plasma concentration of ADM (Figure (Figure1).1). The intra-articular injection of ADM did not therefore increase the level of ADM in plasma.

Fig. 8. Sequential concentrations of plasma ADM following intra-articular ADM injection in rabbits with antigen-induced arthritis.

Whole-blood samples (total 1 ml) were taken from a peripheral artery in the rabbit ear using a 22-gauge needle before and 15, 30, 60 and 120 minutes after intra-articular injection of 3 μg

Role of Adrenomedullin in Patients with Rheumatoid Arthritis 183

Fig. 10. Macroscopic pathology of joint swelling in rabbits with antigen-induced arthritis

injections of saline for 7 days. Photograph taken 8 days after arthritis onset.

**4.2.3 Histological findings** 

**(a)** Photograph taken before arthritis onset. **(b)** Photograph taken 24 hours after arthritis onset. **(c)** The left knee of the rabbit with antigen-induced arthritis (AIA) was treated with daily intra-articular injections of 1 ng ADM for 7 days and the right knee was treated with daily intra-articular injections of saline for 7 days. Photograph taken 8 days after arthritis onset. **(d)** The left knee of the rabbit with AIA was treated with daily intra-articular injections of 3 μg ADM for 7 days and the right knee was treated with daily intra-articular

The infrapatellar fat pads harvested from control knees on day 8 showed a dense inflammatory reaction, including edematous changes in the synovial interstitium, intracellular edema in the infrapatellar fat pads, hyperplasia of synovial surface cells and widespread infiltration of inflammatory cells in the infrapatellar fat pads (Fig. 11d,e,f). In contrast, these inflammatory reactions were suppressed in the knees treated with ADM for 7 days. In particular, edematous changes in the synovial interstitium, intracellular edema in the infrapatellar fat pads and infiltration of inflammatory cells in the deep

adrenomedullin (AM). The plasma AM concentration was measured using an immunoenzymometric assay kit (*n* = 6). White circles, plasma AM levels in rabbits; black circles, average plasma AM levels at each time point after intra-articular injection of 3 μg AM. Data expressed as the mean ± SEM. Each of plasma ADM level was not significantly.

#### **4.2.2 Joint swelling**

To evaluate the anti-inflammatory effect of ADM on arthritis, we used calipers to measure joint swelling in ADM-treated knees and compared the swelling with that at the same level on the contralateral knees, treated with saline. In rabbits with AIA treated with daily injections of ADM or saline into the knee joint spaces for 7 days, 3 μg ADM significantly reduced joint swelling compared with contralateral knees after day 5. No significant decrease in joint swelling was observed, however, in knees treated with <0.1 μg ADM (Fig. 9a and 10).

Fig. 9. Adrenomedullin reduced joint swelling in rabbits with antigen-induced arthritis.

**(a)** Joint swelling progress in rabbits with AIA treated with daily intra-articular injections of ADM or saline for 7 days (*n* = 5 in each group). Daily intra-articular injections of 3 μg ADM significantly decreased joint swelling compared with contralateral knees after day 5. No significant decrease in joint swelling was observed in knees treated with <0.1 μg ADM.

**(b)** Joint swelling progress in rabbits with AIA treated with daily intra-articular injections of ADM or saline for 20 days (*n* = 3 in each group). Daily intra-articular injections of 0.1 μg and 3 μg ADM showed a tendency to reduce joint swelling throughout the experiment, and significantly decreased joint swelling on days 12 and 16 and on days 8, 12 and 16, respectively, compared with contralateral control knees. Daily intra-articular injections of 1 ng and 0.01 μg ADM did not ameliorate joint swelling throughout the experiments (data not shown). Data expressed as the mean ± standard error of the mean. \**P* < 0.05 and \*\**P* < 0.01, compared with contralateral knees.

In rabbits with AIA treated for 20 days with daily injections of ADM or saline into the knee joint spaces, 0.1 μg and 3 μg ADM showed a tendency to reduce joint swelling throughout the experiment – and significantly decreased joint swelling on days 12 and 16 and on days 8, 12 and 16, respectively, compared with contralateral knees (Fig. 9b). Daily intra-articular injections of 1 ng and 0.01 μg ADM, however, did not ameliorate joint swelling (data not shown).

adrenomedullin (AM). The plasma AM concentration was measured using an immunoenzymometric assay kit (*n* = 6). White circles, plasma AM levels in rabbits; black circles, average plasma AM levels at each time point after intra-articular injection of 3 μg AM. Data expressed as the mean ± SEM. Each of plasma ADM level was not significantly.

To evaluate the anti-inflammatory effect of ADM on arthritis, we used calipers to measure joint swelling in ADM-treated knees and compared the swelling with that at the same level on the contralateral knees, treated with saline. In rabbits with AIA treated with daily injections of ADM or saline into the knee joint spaces for 7 days, 3 μg ADM significantly reduced joint swelling compared with contralateral knees after day 5. No significant decrease in joint swelling was observed, however, in knees treated with <0.1 μg ADM (Fig.

Fig. 9. Adrenomedullin reduced joint swelling in rabbits with antigen-induced arthritis.

**(a)** Joint swelling progress in rabbits with AIA treated with daily intra-articular injections of ADM or saline for 7 days (*n* = 5 in each group). Daily intra-articular injections of 3 μg ADM significantly decreased joint swelling compared with contralateral knees after day 5. No significant decrease in joint swelling was observed in knees treated with <0.1 μg ADM. **(b)** Joint swelling progress in rabbits with AIA treated with daily intra-articular injections of ADM or saline for 20 days (*n* = 3 in each group). Daily intra-articular injections of 0.1 μg and 3 μg ADM showed a tendency to reduce joint swelling throughout the experiment, and significantly decreased joint swelling on days 12 and 16 and on days 8, 12 and 16, respectively, compared with contralateral control knees. Daily intra-articular injections of 1 ng and 0.01 μg ADM did not ameliorate joint swelling throughout the experiments (data not shown). Data expressed as the mean ± standard error of the mean. \**P* < 0.05 and \*\**P* < 0.01,

In rabbits with AIA treated for 20 days with daily injections of ADM or saline into the knee joint spaces, 0.1 μg and 3 μg ADM showed a tendency to reduce joint swelling throughout the experiment – and significantly decreased joint swelling on days 12 and 16 and on days 8, 12 and 16, respectively, compared with contralateral knees (Fig. 9b). Daily intra-articular injections of 1 ng and 0.01 μg ADM, however, did not ameliorate joint

**4.2.2 Joint swelling** 

compared with contralateral knees.

swelling (data not shown).

9a and 10).

Fig. 10. Macroscopic pathology of joint swelling in rabbits with antigen-induced arthritis

**(a)** Photograph taken before arthritis onset. **(b)** Photograph taken 24 hours after arthritis onset. **(c)** The left knee of the rabbit with antigen-induced arthritis (AIA) was treated with daily intra-articular injections of 1 ng ADM for 7 days and the right knee was treated with daily intra-articular injections of saline for 7 days. Photograph taken 8 days after arthritis onset. **(d)** The left knee of the rabbit with AIA was treated with daily intra-articular injections of 3 μg ADM for 7 days and the right knee was treated with daily intra-articular injections of saline for 7 days. Photograph taken 8 days after arthritis onset.

### **4.2.3 Histological findings**

The infrapatellar fat pads harvested from control knees on day 8 showed a dense inflammatory reaction, including edematous changes in the synovial interstitium, intracellular edema in the infrapatellar fat pads, hyperplasia of synovial surface cells and widespread infiltration of inflammatory cells in the infrapatellar fat pads (Fig. 11d,e,f). In contrast, these inflammatory reactions were suppressed in the knees treated with ADM for 7 days. In particular, edematous changes in the synovial interstitium, intracellular edema in the infrapatellar fat pads and infiltration of inflammatory cells in the deep

Role of Adrenomedullin in Patients with Rheumatoid Arthritis 185

Fig. 12. Histological analysis of infrapatellar fat pad harvested from rabbit knees 21 days

**4.2.4 Collagen volume in infrapatella fat pads treated with Adrenomedullin** 

were not observed in knees treated with low-dose ADM.

Rabbits with antigen-induced arthritis (AIA) were treated with daily injections of ADM or saline (control) into the knee joint spaces for 20 days. **(a)**, **(b)**, **(c)** AIA rabbit knee was treated with daily intra-articular injections of 3 μg AM for 20 days. (a) Low-magnification image (×100). (b), (c) High-magnification images (×400) of the superficial portion and the deep portion of (a), respectively. **(d)**, **(e)**, **and (f)** the contralateral knee of (a), (b) and (c) was treated with daily intra-articular injections of saline for 20 days. (d) Lowmagnification image (×100). (e), (f) High-magnification images (×400) of the superficial portion and the deep portion of (d), respectively. Arrows indicate inflammatory cells. Bar

To observe the effect of ADM on fibrosis of the infrapatellar fat pads harvested on day 21, we examined the collagen volume ratio of the infrapatellar fat pad histologically using Mallory–Azan staining. The collagen volume ratio was significantly increased in ADMtreated knees by 39% and 31% at 0.1 μg and 3 μg ADM, respectively, compared with control knees (Fig. 13 and 14). The effects of ADM on these pathological tissue changes, however,

after arthritis onset

= 50 μm.

portion of the infrapatellar fat pads were significantly reduced (Fig. 11a,b,c). The infrapatellar fat pads harvested from control knees on day 21 also showed a severe inflammatory reaction. Edematous changes in the synovial interstitium, hyperplasia of synovial surface cells and widespread infiltration of inflammatory cells throughout the infrapatellar fat pads were observed (Fig. 12d,e,f). In the knees treated with ADM for 20 days, these inflammatory reactions were ameliorated. ADM treatment significantly suppressed infiltration of inflammatory cells in the deep portion of the infrapatellar fat pads (Fig. 12a,b,c).

Fig. 11. Histological analysis of infrapatellar fat pad harvested from rabbit knees 8 days after arthritis onset

Rabbits with antigen-induced arthritis (AIA) were treated with daily injections of ADM or saline (control) into the knee joint spaces for 7 days. Tissues were sectioned longitudinally perpendicular to the patella ligament in the middle of the tissue, and were stained with H & E. **(a)**, **(b)**, **(c)** AIA rabbit knee was treated with daily intra-articular injections of 3 μg ADM for 7 days. (a) Low-magnification image (×100). (b), (c) High-magnification images (×400) of the superficial portion and the deep portion of (a), respectively. **(d)**, **(e)**, **and (f)** the contralateral knee of (a), (b) and (c) was treated with daily intra-articular injections of saline for 7 days. (d) Low-magnification image (×100). (e), (f) High-magnification images (×400) of the superficial portion and the deep portion of (d), respectively. Arrows indicate inflammatory cells. Bar = 50 μm.

portion of the infrapatellar fat pads were significantly reduced (Fig. 11a,b,c). The infrapatellar fat pads harvested from control knees on day 21 also showed a severe inflammatory reaction. Edematous changes in the synovial interstitium, hyperplasia of synovial surface cells and widespread infiltration of inflammatory cells throughout the infrapatellar fat pads were observed (Fig. 12d,e,f). In the knees treated with ADM for 20 days, these inflammatory reactions were ameliorated. ADM treatment significantly suppressed infiltration of inflammatory cells in the deep portion of the infrapatellar fat

Fig. 11. Histological analysis of infrapatellar fat pad harvested from rabbit knees 8 days after

Rabbits with antigen-induced arthritis (AIA) were treated with daily injections of ADM or saline (control) into the knee joint spaces for 7 days. Tissues were sectioned longitudinally perpendicular to the patella ligament in the middle of the tissue, and were stained with H & E. **(a)**, **(b)**, **(c)** AIA rabbit knee was treated with daily intra-articular injections of 3 μg ADM for 7 days. (a) Low-magnification image (×100). (b), (c) High-magnification images (×400) of the superficial portion and the deep portion of (a), respectively. **(d)**, **(e)**, **and (f)** the contralateral knee of (a), (b) and (c) was treated with daily intra-articular injections of saline for 7 days. (d) Low-magnification image (×100). (e), (f) High-magnification images (×400) of the superficial portion and the deep portion of (d), respectively. Arrows indicate

pads (Fig. 12a,b,c).

arthritis onset

inflammatory cells. Bar = 50 μm.

Fig. 12. Histological analysis of infrapatellar fat pad harvested from rabbit knees 21 days after arthritis onset

Rabbits with antigen-induced arthritis (AIA) were treated with daily injections of ADM or saline (control) into the knee joint spaces for 20 days. **(a)**, **(b)**, **(c)** AIA rabbit knee was treated with daily intra-articular injections of 3 μg AM for 20 days. (a) Low-magnification image (×100). (b), (c) High-magnification images (×400) of the superficial portion and the deep portion of (a), respectively. **(d)**, **(e)**, **and (f)** the contralateral knee of (a), (b) and (c) was treated with daily intra-articular injections of saline for 20 days. (d) Lowmagnification image (×100). (e), (f) High-magnification images (×400) of the superficial portion and the deep portion of (d), respectively. Arrows indicate inflammatory cells. Bar = 50 μm.
