Issekutz AC, Sapru K: Modulation of adjuvant arthritis in the rat by 587

2-methoxyestradiol: an effect independent of an anti-angiogenic action. Int 588

Immunopharmacol 2008, 8:708-716.

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47. Westra J, Brouwer E, van Roosmalen IA, Doornbos-van der Meer B, van Leeuwen MA, 590

Posthumus MD, Kallenberg CG: Expression and regulation of HIF-1alpha in 591

macrophages under inflammatory conditions; significant reduction of VEGF by 592

CaMKII inhibitor. BMC Musculoskelet Disord 2010, 11:61.

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48. Rice JW, Veal JM, Fadden RP, Barabasz AF, Partridge JM, Barta TE, Dubois LG, Huang 594

KH, Mabbett SR, Silinski MA, Steed PM, Hall SE: Small molecule inhibitors of 595

Hsp90 potently affect inflammatory disease pathways and exhibit activity in 596

models of rheumatoid arthritis. Arthritis Rheum 2008, 58:3765-3775.

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598

599

Figure legends 600

Figure 1. Experimental design of the sequence of events for the entire course of the 601

experiment. After the evaluations including measurements of paw edematous swelling and 602

pain threshold, the animals were sacrificed for histology and immunohistochemistry. 1D: one 603

dose; 3D: three doses; 3D6d: follow-up at the 6th day after 3 doses. CFA: complete Freund’s 604

adjuvant; HA: hyaluronan; No-tr: No treatment; SA: saline 605

606

Figure 2. Results of edema (A) and pain behavioral (B) assessments. Data were calculated 607

before treatment at the conditions of pre- and post-CFA-induced arthritis, after treatment at 608

conditions of one injection (1D), three injections (3D) and follow-up 6 d after 3D (3D6d) in 609

treatment (hyaluronan injection, HA), placebo (saline administration, SA) and control (sham 610

injection, No-tr) groups. Each bar represents the mean ± SD in body weight and mean ± SEM 611

in paw circumference and withdrawal threshold. #: p < 0.05, Student’s t-test for comparison 612

of pre- and post-arthritic condition before treatment. *p < 0.05, Bonferroni post hoc test for 613

comparison of difference between groups at dosages of 1D, 3D and 3D6d after treatment.

614

615

Figure 3. Histopathology of arthritis joints. Representative HE sections of the hind paws 616

obtained from adjuvant-induced arthritic animals treated with intra-articular three injections 617

for No-tr (A), SA (B), and HA (C). In rats without any treatment for No-tr group, wherein 618

cartilaginous tissue could not be clearly detected, bone damage was even greater and there 619

was massive inflammatory cells infiltrated in synovium (a). Similar changes were observed in 620

rats treated with SA. Cartilage erosion was more pronounced and the extensively expanded 621

synovial pannus was more densely infiltrated with mononuclear cells (b). In rats treated with 622

HA, the joints were much less inflamed, and lymphocyte accumulation (c) and cartilage 623

damage decreased. There was no sign of bone destruction (cart = cartilage; syn = synovial 624

tissue; see figure 2 for other definitions).

625

626

Figure 4. Representative immunohistochemical sections of HIF-1α immunoreactivity.

627

Sections obtained from the arthritic synovium treated with intra-articular three injections of 628

No-tr (A), SA (B), and HA (C) groups. At higher-power magnification, it is evident that these 629

positive (brown staining) immunoreactivities were clearly localized in both nucleus and 630

cytoplasm of arthritic synovium in the sections from No-tr (a) and SA (b) animals.

631

Administration of HA (c) to adjuvant-induced rat produced a marked reduction in the 632

immunostaining for HIF-1α quantitative analysis (D) of positive-labeled cells in synovium 633

for HIF-1α immunohistochemistry at the early phase of inflammation of each group was 634

presented in the average proportion of labeled neurons (mean ± SEM). ^* p < 0.05, showed 635

significant differences between groups when either SA or No-tr is compared with HA group 636

using Bonferroni post hoc test. Significant differences were found between HA vs SA groups 637

and HA vs No-tr groups. ^# p < 0.05, showed significant differences between dosages tested 638

by Bonferroni post hoc test (cart = cartilage; syn = synovial tissue; see figure 2 for other 639

definitions).

640

641

Figure 5. Representative immunohistochemical sections of iNOS immunoreactivity.

642

Sections obtained from the arthritic synovium treated with intra-articular three injections of 643

No-tr (A), SA (B), and HA (C) groups. At higher-power magnification, it is evident that these 644

positive (brown staining) immunoreactivities were clearly localized in both nucleus and 645

cytoplasm of arthritic synovium in the sections from No-tr (a) and SA (b) animals.

646

Administration of HA (c) to adjuvant-induced rat produced a marked reduction in the 647

immunostaining for iNOS. Quantitative analysis (D) of positive-labeled cells in synovium for 648

iNOS immunohistochemistry at the early phase of inflammation of each group was presented 649

in the average proportion of labeled neurons (mean ± SEM). ^* p < 0.05, showed significant 650

differences between groups when either SA or No-tr is compared with HA group using 651

Bonferroni post hoc test. Significant differences were found between HA vs SA groups and 652

HA vs No-tr groups. ^# p < 0.05, showed significant differences between dosages tested by 653

Bonferroni post hoc test (cart = cartilage; syn = synovial tissue; see figure 2 for other 654

definitions).

655

656

Figure 6. Representative immunohistochemical sections of MMP3 immunoreactivity.

657

Sections obtained from the arthritic synovium treated with intra-articular three injections of 658

No-tr (A), SA (B), and HA (C) groups. At higher-power magnification, it is evident that these 659

positive (brown staining) immunoreactivities were clearly localized in both nucleus and 660

cytoplasm of arthritic synovium in the sections from No-tr (a) and SA (b) animals.

661

Administration of HA (c) to adjuvant-induced rat produced a marked reduction in the 662

immunostaining for iNOS. Quantitative analysis (D) of positive-labeled cells in synovium for 663

MMP3 immunohistochemistry at the early phase of inflammation of each group was 664

presented in the average proportion of labeled neurons (mean ± SEM). ^* p < 0.05, showed 665

significant differences between groups when either SA or No-tr is compared with HA group 666

using Bonferroni post hoc test. Significant differences were found between HA vs SA groups 667

and HA vs No-tr groups. ^# p < 0.05, showed significant differences between dosages tested 668

by Bonferroni post hoc test (cart = cartilage; syn = synovial tissue; see figure 2 for other 669

definitions).

670 671

Table 1. Association of pain withdrawal threshold with the 672

immunoreactivity results given as γ-values 673

Pain withdrawal threshold

1D 3D 3D6d

HIF-1α -0.378** -0.848** -0.869**

iNOS -0.280* -0.782** -0.765**

MMP-3 -0.420** -0.823** -0.856**

Correlations were analyzed by Pearson correlation coefficients.

674

**: P<0.01; *:P<0.05.

675

Dose Group Intimal hyperplasia

Subintimal fibrosis

Lymphocytic

infiltration Vascularity Aggregate score

1D HA 2.45±0.11 2.60±0.11 1.50±0.11*^# 2.05±0.11 7.80±0.26*^# SA 2.60±0.11 2.60±0.11 2.50±0.11 2.10±0.12 9.10±0.31 No-tr 2.65±0.11 2.65±0.10 2.95±0.05 2.20±0.12 9.75±0.24

ap value among groups p>0.05 p>0.05 p<0.001 p>0.05 p<0.001

3D HA 2.50±0.11 2.70±0.11 1.40±0.13*^# 2.20±0.09 8.05±0.31*^# SA 2.80±0.09 2.70±0.10 2.55±0.11 2.15±0.11 9.55±0.28 No-tr 2.80±0.09 2.70±0.11 2.85±0.08 2.20±0.14 9.95±0.32

ap value among groups p>0.05 p>0.05 p<0.001 p>0.05 p<0.001

3D6d HA 2.50±0.11 2.50±0.11 1.40±0.11*^# 2.15±0.11 7.85±0.25*^# SA 2.70±0.11 2.60±0.10 2.77±0.10 2.20±0.14 9.6±0.36 No-tr 2.70±0.11 2.70±0.11 2.85±0.08 2.40±0.11 10.05±0.33

ap value among groups p>0.05 p>0.05 p<0.001 p>0.05 p<0.001

Values are mean± SEM. ^a: tested with Kruskal–Wallis test. *: p < 0.05, showed statistically significant differences between HA and SA groups; ^#

:

< 0.05, showed statistically significant differences between HA and No-tr groups; Mann-Whitney U-ranked tests were used for between-group