Supplemental Methods: Histopathology scoring of individual components of Valentino
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1 Supplementary Materials Online: Supplemental Methods: Histopathology scoring of individual components of Valentino synovitis grade and Mankin cartilage pathology scale Hemophilic synovitis was graded 0-10 points according to the system validated by Valentino et al. This scale awards 0-3 points for increasing synovial thickening, 0-3 points for increasing synovial vascularity and 0 or 1 point each for the presence of frank blood, discoloration by hemosiderin, synovial villous formation, and for gross cartilage erosion. In addition, the microscopic pathology in cartilage was scored using the modified Mankin scale This scale awards 0-14 points according to increasing degeneration of cartilage structure, cartilage cellularity, cartilage staining, and tidemark integrity. The Valentino and Mankin scores were combined to produce a Global Murine Hemarthrosis Score, as previously described 9. Gross structural changes in cartilage during the 8 week time course as graded by the Mankin score were minimal in this single hemarthrosis challenge, so that the trends for pathology as graded by the Global Score (included as Supplemental Figure 1) were similar to those graded by the Valentino score. The individual components of the Valentino synovitis grade, were also examined. (Supplemental Figure 2) As early as 24 hours after injury the clearance of blood from the synovial space in the WT and WT + Blood groups was nearly complete. This finding contrasts with the frank blood that dwelled in the joints of FIX -/- mice through at least day 3, by which time thickening of synovial lining cells was evident (Table 1, Figure 1B, Supplemental Figure 2). Synovitis, characterized by proliferative thickening of the fibroblast-like synoviocyte layer and dense subsynovial inflammatory infiltrate, developed in the joints of all FIX -/- mice, peaking on day 14 at a mean score of 4.9 out of a possible 10 points using the Valentino mouse synovitis scale. (Table 1, Figure 1A, 1B, Supplemental Figure 2) The corresponding peak synovitis score was 1.0 for the WT mice (day 5-7) and for the WT + Blood the peak synovitis
2 score was 1.4 (day 5). At the day 14 time point when FIX -/- mice demonstrated peak synovitis (day 14) and increasing neoangiogenesis, the WT + Blood group demonstrated strikingly better healing, with synovitis score only 0.33 and neovascular changes essentially resolved. (Table 1, Figure 1A, Figure 1B; Supplemental Figure 2) At no point during the 56 days of observation did the hemophilic mice completely resolve the synovitis, with persistent neovascularity contributing the most to the chronic histopathology. (Figure 1A, 1B, Supplemental Figure 2) In contrast with the FIX -/- findings, both groups of mice with normal thrombin-generating potential healed with minimal pathology. Supplemental Methods: Histopathologic evaluation of FIX -/- joint wound healing Sections were prepared with Prussian Blue stain for tissue iron. Iron staining was scored as not present (0); weak (1+); positive (2+); or strongly positive (3+) as previously described by Hoffman et al. 6 Sections were prepared for immunohistochemical staining and grading for macrophages and endothelial cells as previously described by Hoffman et al. 6 with some modifications. Macrophage infiltration was identified by immunostaining for CD68 using rat antimouse F4/80 (Serotec, Raleigh, NC, USA) at a 1:50 dilution, followed by biotinylated anti-rat IgG (Vector Laboratories, Burlingame, CA, USA) at a dilution of 1:100. The grading scheme used by Hoffman et al was used to evaluate the most heavily infiltrated portion of synovium: grade 0, no macrophages; 1+, scattered macrophages; 2+, lines of macrophages; 3+, clusters of macrophages; 4+, sheets of macrophages. Endothelial cells were identified by staining for VWF with a rabbit polyclonal primary antibody (abcam, Cambridge, MA, USA). Vessel profiles were defined as rounded or elongated spaces bounded by VWF-staining endothelial cells for serial quantification of vessel per high-powered field within synovium.
3 Supplemental Methods and Results: Dose finding study of recombinant nonacog beta pegol and unmodified rfix A dose-finding study of the effect of nonacog beta pegol (N9-GP) given early in response to induced joint hemorrhage was performed to determine an informative dose of N9-GP for subsequent study. A comparative study of the effect of N9-GP to preserve normal joint health employing the joint capsule puncture model was performed examining doses of 0.4 mg/kg (equivalent to 76 IU/kg), 0.75 mg/kg (143 IU/kg), 1.5 mg/kg (285 IU/kg) and 2.5 mg/kg (470 IU/kg) given intravenously at 20 minutes following injury to FIX -/- mice, in a fashion identical to induced hemarthrosis conditions used throughout the studies in this report. An additional group of FIX -/- mice received the same induced hemarthrosis followed by recombinant FIX (Benefix ) intravenously 20 minutes following injury. (Supplemental Table 1). In addition, one group of FIX -/- mice was infused with normal saline alone as an untreated hemophilic control group (NS). We have previously established that untreated hemophilia B mice never score less than 2/10 synovitis pathology score (Valentino scale) at two weeks following this bleeding challenge 8,23 ; in the same study we demonstrated that WT mice experiencing the same bleeding challenge do not score more than 2/10 synovitis when evaluated two weeks after induced hemarthrosis. The doses of 1.5 mg/kg (equivalent to ~280 Units/kg) 22 and 2.5 mg/kg (~470 Units/kg) each led to a mean synovitis score of <2/10 (untreated hemophilia B mice never score less than 2 at two weeks following this bleeding challenge) and similar partial protection from the development of iron deposition, inflammatory cell infiltrate and neoangiogenesis. (Supplemental Table 1) Because the dose of 1.5 mg/kg demonstrated clear efficacy but not complete protection, it was determined that comparing doses of 250 Units/kg of N9-GP and rfix in the subsequent studies likely would yield an informative range of therapeutic responses. We have previously demonstrated that plasma collected from FIX -/- mice that have received a dose of 250 units/kg of
4 unmodified recombinant FIX (Benefix) demonstrates the same clotting time in seconds in a onestage aptt assay as does the plasma of hemostatically normal wild type mice. Supplemental Methods and Results: Pharmacokinetic comparison of FIX measured in plasma and in synovial fluid We and others have previously shown that circulating FIX partitions into the synovial fluid and that intra-articular FIX can help maintain joint health of hemophilic mice following hemarthrosis 8,52,53. It was unknown whether N9-GP similarly could enter the joint space. To explore this we gave hemophilia B mice a dose of 250 IU/kg of either rfix or N9-GP by tail vein injection. Two hours later citrated plasma was collected followed immediately by euthanasia, harvest of the left knee joint, and lavage of the knee joint to collect synovial fluid lavage, as previously described 8. The lavage results in an estimated ten-fold dilution of the synovial fluid. Human FIX antigen concentration in plasma and in synovial fluid was measured using a sandwich ELISA as previously described 8. Consistent with previous pharmacokinetic evaluations in mice and humans 22,54, the mean incremental recovery in plasma of the N9-GP was higher than the unmodified rfix. The relative recovery in the synovial fluid was very similar for N9-GP when compared to unmodified rfix. The results suggest that the monopegylation of FIX does not alter the accessibility of the protein to the intraarticular space. (Table 2)
5 Supplemental Table 1: Pilot N9-GP dose-finding study Dose (mg/kg) N Synovitis Score N with Synovitis Score 2 (%)* N with Synovitis Score < 2 Iron CD68 Blood Vessels Normal Saline ±0.6 8/8 (100%) 0/8 2.6 ± ± ±5.3 N9-GP: 0.4mg/kg (76 IU/kg) N9-GP: 0.75mg/kg (143 IU/kg) N9-GP: 1.5mg/kg (285 IU/kg) N9-GP: 2.5mg/kg (470 IU/kg) ±1.3 5/5 (100%) 0/5 2.2 ± ± ± ±0.7 5/5 (100%) 0/5 1.6 ± ± ± ±1.0 3/5 (60%) 2/5 0.2 ± ± ± ±0.8 1/5 (20%) 4/5 0.4 ± ± ±4.7 rfix ±1.1 8/8 (100%) 0/8 1.8 ± ± ±6.3 (200 IU/kg) * Evaluated using the Valentino mouse synovitis pathology scale, untreated FIX-/- mice never demonstrate less than 2/10 synovitis at two weeks after a single joint capsule puncture, whereas hemostatically normal mice do not score more than 2/10.
6 Supplemental Table 2: Trabecular bone measurements vbmd [mgha] (% diff from WT) ConnDens [per mm 3 ] (% diff from WT) Tb.Sp [mm] (% diff from WT) Tb.N [mm] (% diff from WT) NS ±8.5 (-63.5%) 93.2 ±10.7 (-43.0%) 0.25 ±0.02 (-28.0%) 3.95 ±0.21 (-30.5%) N9-GP D ±13.2 (2.0%) ±20.1 (5.7%) 0.19 ±0.01 (3.3%) 5.07 ±0.29 (-1.6%) rfix D ±11.6 (-31.1%) ±32.7 (-21.0%) 0.25 ±0.03 (26.0%) 4.26 ±0.52 (-20.8%) rfix D0,1, ±18.5 (-40.8%) ±28.8 (7.3%) 0.21 ±0.01 (7.5%) 4.86 ±0.30 (-2.2%) rfix D ±9.4 (-4.5%) ±13.4 (24.1%) 0.18 ±0.01 (-4.5%) 5.42 ±0.12 (4.9%) WT ± ± ± ±0.20 Measurements are the mean ±SEM of each group, followed by the percent difference compared to WT control. Percent differences less than 5% are in bold.
7 Supplemental Table 3. Significant comparisons of Supplemental Figure 4. Table of Significance Synovitis Score Iron Score Macrophage Score Vessel Count / HPF NS vs N9-GP D0 #### #### #### # N9-GP D0, D7 #### #### ### # rfix D0 ## ## ## rfix D0, D7 ## ### ### rfix D 0, 1, 3 #### #### #### rfix D 0-13 #### #### #### # WT #### #### #### ### N9-GP D0 vs N9-GP D0, D7 rfix D0 * ** rfix D0, D7 ** ** rfix D 0, 1, 3 rfix D 0-13 WT N9-GP D0, D7 vs rfix D0 *** rfix D0, D7 ** *** rfix D 0, 1, 3 rfix D 0-13 WT rfix D0 vs rfix D0, D7 rfix D 0, 1, 3 ** rfix D 0-13 ** * WT ** *** ** rfix D0, D7 vs rfix D 0, 1, 3 rfix D 0-13 ** WT ** *** ** ** rfix D0, 1, 3 vs rfix D 0-13 WT rfix D0-13 vs WT *
8 Points that are significantly different from NS are indicated by #. Markers of significance are utilized as follows * P < 0.05, ** P < 0.01, *** P < 0.001; # P < 0.05, ## P < 0.01, ### P < 0.001, #### P <
9 Supplemental Figure 1: Joint wound healing: Global hemarthropathy score For the time-course study in hemostatically normal mice (WT + NS, ), normal mice challenged with intra-articular injection of autologous blood (WT + Blood, ) and hemophilia B mice (FIX -/-, ) joint cartilage pathology from Safranin-O stained sections was graded using the modified Mankin s score 49,50. The sum of the average Valentino synovitis score and modified Mankin s scores was calculated as the Global Score to represent the spectrum of bleeding-induced joint pathology, as previously described by Narkbunnam et al 9. The dynamic changes in the Global Score in WT and FIX -/- mice are shown to demonstrate that during the 8 week time course, the contribution to the Global Score of gross cartilage degenerative changes are minimal, as compared to the contribution of the synovitis component (Valentino synovitis grade, as shown in Figure 1A in the Text). Points that are significantly different from WT + Blood are indicated by *. Points that are significantly different from WT + NS are indicated by #. Markers of significance are utilized as follows ** P < 0.01, *** P < 0.001, **** P < ; # P < 0.05, ### P < 0.001, #### P < Supplemental Figure 2. Grading of individual components of the Valentino synovitis scale Figure 2A-C. The relative contribution of individual components of the Valentino scoring system to the overall pathology score are shown for the injured FIX -/- mice, for the injured WT mice (only NS instilled in the joint) and for the WT mice that received autologous citrated whole blood injected into the joint at the time of needle puncture (WT + Blood). Some WT mice having normal hemostatic potential did demonstrate modest neovascular and proliferative changes in the first week in response to the joint wound. The same changes were more marked and
10 persisted through the 2 nd to 8 th weeks in FIX -/- mice, accompanied with findings related to unresolved blood and heme iron contamination of the joint. Figures 2D-G. Synovial hyperplasia, neo-angiogenesis, blood present, and disocoloration were replotted for statistical comparison between hemostatically normal mice (WT + NS, ), normal mice challenged with intra-articular injection of autologous blood (WT + Blood, ) and hemophilia B mice (FIX -/-, ). Points that are significantly different from WT + Blood are indicated by *. Points that are significantly different from WT + NS are indicated by #. Markers of significance are utilized as follows * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < ; # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < Supplemental Figure 3: N9-GP dose-finding pilot study: Synovitis scale Hemostatically normal mice (WT) were treated with normal saline and FIX -/- mice were treated intravenously with nonacog beta pegol (N9-GP) or recombinant FIX (rfix) or NS at the doses shown 20 minutes following induced hemarthrosis. Two weeks later, histopathology within the injured joint was scored using the Valentino synovitis pathology score. The dashed line indicates the historical threshold value of 2, because hemostatically normal mice challenged with this injury do not develop synovitis > 2 using this grading system. Points that are significantly different from NS are indicated by #. Markers of significance are utilized as follows * P < 0.05, ** P < 0.01; # P < 0.05, ## P < 0.01, ### P < 0.001, #### P <
11 Supplemental Figure 4: Joint wound healing comparing once or twice weekly dosing with factor IX Unilateral hemarthrosis was induced in all treatment groups, followed twenty minutes later by tail vein infusion of first treatment or placebo (NS) at twenty minutes after wounding. Some treatment groups received repeated doses of rfix at later time points as indicated, including groups of mice treated with FIX at the end of the first week of wound healing to provide some hemostatic potential during the second week of joint wound healing. The conclusion that is evident from this data, when compared to the data included in the main text, is that doses given at day 7 do not improve the joint healing outcomes achieved by either FIX product. The critical period for support appears to include days 0-6. Treatment groups (6-8 mice/group) consisted of FIX -/- mice treated with a single dose of NS on day 0 (NS, ); FIX -/- mice treated with a single dose of N9-GP on day 0 (N9-GP d0, ); FIX -/- mice treated with a single dose of N9-GP on day 0 and day 7 (N9-GP d0,d7, ); FIX -/- mice treated with a single dose of rfix on day 0 (rfix d0, ); FIX -/- mice treated with rfix on day 0 and on day 7 (rfix d0,d7, Δ); FIX -/- mice treated with rfix on day 0, 1 and 3 (rfix d0,1,3, ); FIX -/- mice treated with rfix on day 0, 1,3,5,7,9,11,13 (rfix d0-13, ); and WT mice treated with NS on day 0 (WT, ). All parameters were graded from joint histology prepared from samples collected at two weeks following hemarthrosis. Due to the great number of statistically significant comparisons, all have been moved to Supplemental Table 3. Supplemental Figure 4A. Synovitis grade The Valentino mouse synovitis pathology grade was generated by examination of hematoxylin and eosin stained joint histology. Supplemental Figure 4B. Iron
12 The intensity of Prussian blue staining ferric iron was graded in the heaviest areas of synovial and subsynovial staining from joint samples. Supplemental Figure 4C. Macrophage Infiltration Macrophage infiltration was identified by immunostaining for CD68 graded in the most heavily infiltrated portion of joint synovial samples. Supplemental Figure 4D. Neoangiogenesis Endothelial cells were identified by staining for VWF and counted in the staining areas of the synovium and sub-synovium.
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