Factor VIII restores bone parameters and modulates muscle proteo-metabolome in Factor VIII knockout male mice

TL;DR

Factor VIII deficiency in mice causes bone issues and muscle changes, but FVIII treatment can restore bone health, though not all muscle effects are reversed.

Contribution

First comprehensive characterization of musculoskeletal effects in FVIII knockout mice and the impact of FVIII supplementation.

Findings

FVIII deficiency leads to osteoporotic bone changes and reduced vascularization in mice.

FVIII treatment reverses bone abnormalities but not all muscle fiber type changes.

Muscle proteomic and metabolomic differences were observed and partially corrected by FVIII.

Abstract

In addition to its role in hemostasis, Factor VIII (FVIII) has recently been shown to potentially impact angiogenesis, inflammation, osteopenia, and sarcopenia. This was explored here by studying the musculoskeletal development of FVIII knockout (FVIII-/-) male mice. These animals developed an osteoporotic phenotype with significant bone microarchitectural alteration, reduced vascularization, and a lower osteoblastic population. Proteomic analyses revealed differentiating bone metabolism-related proteins between FVIII-/- and wildtype mice. Weekly infusions of recombinant FVIII protein reversed this phenotype. Surprisingly, younger FVIII-/- mice had heavier muscles with larger fibers, shifted from type IIx to type IIb, not reversed by FVIII treatment. Significant proteomic and metabolomic differences between wildtype and FVIII-/- muscles were observed, some of which were reduced by FVIII treatment. This study provides the first comprehensive full-phenotypic characterization of bones and muscles in FVIII-/- mice and demonstrates the benefits of FVIII supplementation to normalize their musculoskeletal phenotype.

Figures

Recombinant FVIII restores bone and vascular phenotypes of *FVIII*^*-/-*^ mice. a Quantitative bone parameters, X-ray tomodensitometry analysis. Bone density images were obtained using the DataViewer software (Bruker) and represent sagittal, coronal, and transversal views of femurs. 3D images represent a section corresponding to the distal growth plate of the analyzed femurs. Light grey: WT mice, dark grey: *FVIII*^*-/-*^ mice, dark brown: *FVIII*^*-/-*^ mice treated with pegylated recombinant FVIII (recFVIII); BV/TV bone volume/total volume; Tb.Th trabecular thickness; Tb. N trabecular number

Proteomics and metabolomics profiling of bones collected from *FVIII*^*-/-*^ and WT mice and results of protein regulation. a Experimental procedure: after protein extraction from fresh frozen bone, collected proteins were enzymatically digested to obtain peptide suspensions analyzed by LC-MS, combined with data processing. Bones extracted from mouse legs were cut in three fragments (femur, knee joint, tibia/fibula) and analyzed independently. b Clustering of joints, tibias, and femurs and differentiation between *FVIII*^*-/-*^ and WT based on 84 proteins (significant difference *P* < 10^−^^5^

Skeletal muscle phenotype of *FVIII*^*-/-*^ mice and effects of recombinant FVIII. a Experimental protocol: mice were weighed before sacrifice, quadriceps and tibilias anterior muscles were harvested, weighed, and snap frozen. Mid and distal sections of muscles were analyzed by immunohistochemistry for fiber’s cross-sectional area (laminin staining in red) and myosin composition (red: type I fibers; green: type IIa fibers; black: type IIx fibers; orange: type IIb fibers). b Quadriceps and c tibialis anterior phenotype: weight of muscles (mg of muscle per g of mice), mean fiber size (cross sect

Proteomic and metabolomic profiling of skeletal muscles collected from *FVIII*^*-/-*^ and WT mice. *FVIII*^*-/-*^ mice were treated or not with 200 IU/kg of recombinant pegylated FVIII once per week for 6 weeks. a PCA plot of proteomics and metabolomics data obtained from fresh frozen skeletal muscles of WT and *FVIII*^*-/-*^ mice treated or not with recFVIII. b Volcano plot analysis of differentially expressed proteins in *FVIII*^*-/-*^ skeletal muscles compared to the control group and compared to treatment with recFVIII (|log_2_FC| > 1, *P* value adjusted < 0.05). c Protein enrichment in th

Effect of recombinant FVIII on injured skeletal muscle of *FVIII*^*-/-*^ mice. a Experimental procedure: 100 μL of cardiotoxin (100 μmol/L) was injected in the tibialis anterior muscle, followed by and iv injection of 200 IU/kg of recombinant pegylated FVIII (recFVIII), 30 min after the injury and then every week (total of 4 injections) at the same dose until sacrifice at day 28. Injection of cardiotoxin induced a complete destruction of myofibers at day 4, followed by complete muscle regeneration at day 28 (black and white bar = 500 μmol/L). b Tibialis anterior phenotype 28 days after injury,

Full text

Introduction

After a bleeding injury, the hemostatic process is rapidly activated to avoid major blood loss. This process implies local vasoconstriction and an accumulation of pro-coagulation factors, followed by platelet activation and aggregation, ultimately triggering the coagulation cascade to generate a fibrin clot.^1^ These mechanisms of hemostasis are highly conserved in vertebrates, and dysfunction at any of these steps can heavily compromise the life of the affected individual.^2^

Hemophilia A (HA) is a rare, genetically inherited blood-clotting disorder associated with the X chromosome, with an incidence of 1 in 5 000 males. HA implies the dysfunction or quantitative expression reduction of the coagulation Factor VIII (FVIII). The hemostatic role of FVIII has been described as necessary for factor X activation and, finally, thrombin generation.^3^ Depending on plasma FVIII activity, HA can be classified as severe (less than 1% of activity), moderate (between 1% and 5%), or mild (6% to 40%) disorder.^4^ Individuals affected by severe or moderate HA suffer from spontaneous joint and muscle bleedings, and recurrent joint bleedings can result in the development of hemophilic arthropathy.^5^ As a consequence of these side effects, patients with HA tend to have a sedentary lifestyle that may lead to a loss of muscle and bone mass.^6^ Between 2010 and 2020, more than 30 clinical studies on four continents, involving more than 1 000 patients with HA or HB, have described this bone disorder (reviewed in ref.^7^), characterized^7^ by reduced bone mineral density and abnormal blood levels of bone markers such as osteoprotegerin, vitamin D, parathyroid hormone, and calcium.^8,9^ Recently, muscular abnormalities have also been identified in patients with HA, mostly early-onset sarcopenia.^6,10,11^

During the last decades, prophylactic therapies have been developed that imply intravenous (IV) administration of recombinant FVIII (recFVIII) protein. More recently, drugs with different mechanisms of action have been developed, including assets that mimic FVIII activity or balance thrombin generation.^5,12^ Moreover, gene therapy is increasingly being considered for the treatment of HA.^5,12^ Such new non-factor replacement therapies have improved the quality of life of individuals with HA, notably by reducing bleeding episodes. However, several studies have questioned the ability of these treatments to overcome inflammation or address reduced muscle and bone mass associated with HA, suggesting that FVIII or thrombin might show activities beyond hemostasis, although issues remain controversial.^7,13^

The development of murine models for HA, such as FVIII knockout mice (FVIII^-/-^), has allowed us to demonstrate the association between undetectable levels of FVIII and the presence of an osteoporotic phenotype with decreased bone strength.^14,15^ In addition, the induction of joint bleedings in FVIII^-/-^ animals was shown to result in a more severe osteoporosis phenotype, indicating a potential role of FVIII in bone remodeling.^14,16^ Physiologically, bone remodeling is the result of a balance between osteoblastic anabolic activities (bone deposition) and osteoclast-associated catabolism (bone resorption). It has been suggested that FVIII could play a direct^16^ or indirect role in the development of osteoblasts through the production of thrombin and activation of PAR1 receptors on the surface of osteoblastic precursors,^17^^,18^ the latter observation being controversial.^19^ In contrast, HA has also been associated with high bone turnover due to increased osteoclast activity, resulting in upregulation of osteoblast response and increased levels of bone-alkaline phosphatase in patients with low bone mineral density (BMD).^20^ Nevertheless, the precise mechanisms of action of FVIII in bone remodeling remain unclear. Consequently, a better understanding of the extra-hemostatic role of FVIII in bone metabolism is needed.

The present work aimed at: (i) phenotyping musculoskeletal tissues in the context of HA; (ii) deciphering the molecular mechanisms associated with FVIII extra-hemostatic activities in muscle and bone remodeling; iii) analyzing the biological effect of recombinant FVIII administration on the musculoskeletal system, using a FVIII^-/-^ mouse model.

Results

The osteoporotic bone phenotype of FVIII-/- mice is associated with a dysregulation of the vascular tree of the growth plate and a reduction of osteoblastic cells

The bone phenotype of FVIII^-/-^ mice and wild-type (WT) mice was compared by using X-ray micro tomodensitometry. Five-week-old (“juvenile”) FVIII^-/-^ mice (Fig. 1a) exhibited significantly lower bone parameters compared to age-matched WT mice, including trabecular bone volume (BV/TV) and trabecular numbers (Tb.N). This effect was associated with larger trabecular spaces (Tb.Sp), a modification even more pronounced in 14-week-old adult mice. However, there was no significant difference in trabecular thickness at both ages. BMD was lower in juvenile and young adult FVIII^-/-^ mice, without reaching significance.Fig. 1FVIII^-/-^ mice suffer from osteoporosis and show altered bone vascularization with reduced osteoblastic cells. a Bone parameters, X-ray tomodensitometry analysis. Bone density images were obtained using the DataViewer software (Bruker) and represent sagittal, coronal, and transversal views of femurs. Parameters were analyzed at the growth plate level of the distal epiphysis of the femurs (metaphyseal). The same point of reference (chrondocyte seam) at the growth plate was established for each sample, from an offset of 0.215 mm from the growth plate and a trabecular volume of 1.5 mm^3^ was selected for the analysis of bone morphometric parameters. b Bone vascularization parameters analyzed from trabecular bone sections (Endomucin in green, NG2 in purple, α- SMA in red, Dapi in blue). Light grey: WT mice, dark grey: FVIII^-/-^ mice, BMD bone mineral density, BV/TV bone volume/total volume, Tb.Th trabecular thickness, Tb. N trabecular number, Tb.Sp trabecular space, Cor coronal, Sag sagittal, Trans transversal, up to 11 mice per group, one to four sections per bone analyzed. Mice were 5 (juvenile) or 14 (young adult) weeks old at sacrifice. *P < 0.05, **P < 0.01, ***P < 0.001, ns not significant. Statistical test used: non-parametric Mann-Whitney test. Mean ± SD

Recent data have coupled bone remodeling with angiogenesis.^21–23^ Based on these data, a comparison of the bone vascular tree in FVIII^-/-^ and WT mice was performed by immunohistochemistry. Veins and capillaries were stained with endomucin, and perivascular cells and arteries were stained with antibodies against nerve/glial-antigen 2 (NG2) and α-smooth muscle actin (SMA), respectively (Fig. S1). Interestingly, low bone microarchitectural parameters were associated with a reduction of trabecular bone vascularization. In FVIII^-/-^ juvenile mice compared to WT mice, there was a significant reduction of veins and capillaries. Perivascular cells were also lower in FVIII^-/-^ mice without reaching significance. Arteries were not affected. In adult mice, veins, capillaries and perivascular cells were lower in FVIII^-/-^ animals compared to WT mice, but without reaching significance. Interestingly, the number of arteries was significantly reduced in FVIII^-/-^ animals compared to WT mice (Fig. 1b).

To investigate further the trabecular bone loss in FVIII^-/-^ mice, osterix^+^ or Runx2^+^ osteoblasts, and TRAP^+^ osteoclasts were quantified on bone trabecular tissue sections using ratios corresponding to cell length or cell numbers by bone millimeter^24,25^ (Fig. 2a). Osteoblasts and osteoclasts were located close to bone vessels (Fig. S2). In juvenile animals, a significant reduction of the osteoclastic population was observed in FVIII^-/-^ mice compared to WT mice (Fig. 2b). Reduction in the osteoblastic population was even more pronounced in juvenile FVIII^-/-^ animals compared to WT mice. Interestingly, this phenotype was no longer observed in adult mice, and even reversed, suggesting an early contribution of FVIII during bone growth, followed by compensatory mechanisms (Fig. 2).Fig. 2FVIII^-/-^ mice show reduced osteoblastic cells. a Histological identification of bone cells. Decalcified long bones from FWIII^-/-^ and WT mice were used to analyze histomorphometric parameters. Osteoclasts (red) were identified by histoenzymatic staining. Osteoblast precursors and differentiated osteoblasts were detected by immunohistochemistry. Osteoblast precursors were positive for osterix (yellow, arrows), and differentiated osteoblasts were identified by Runx2-positive staining (brown, arrowheads). Veins and capillaries expressed endomucin were stained in blue. b Bone cells were quantified by ImageJ, B bone, Pm perimeter, Ob osteoblast, Oc osteoclast; up to 11 mice per group, one to two sections per bone analyzed. Mice were 5 (juvenile) or 14 (young adult) weeks old at sacrifice. Results are expressed as mean ± SD. Statistical test used: non-parametric Mann-Whitney test. *P < 0.05, **P <0.01, ***P < 0.001, ns non significant

Intravenous administration of recombinant FVIII protein restores bone microarchitectural and vascularization parameters of FVIII-/- mice

To assess the therapeutic effect of recombinant FVIII on the FVIII^-/-^ phenotype, juvenile animals (6 weeks at treatment initiation) were treated with 200 IU/kg of recFVIII (pegylated FVIII, JIVI^®^, Bayer, Leverkusen, Germany), weekly for 6 weeks (Fig. S3). Plasma FVIII activity was detectable 72 h after treatment with recFVIII (Fig. S4). Bone parameters were analyzed by X-ray tomodensitometry, and bone vascularization, osteoclast and osteoblast infiltration by immunohistochemistry (Fig. 3). As shown in Fig. 3a, the BV/TV ratio of FVIII^-/-^ mice was higher after 6 weeks of treatment by 1.87-fold compared to untreated animals. Similarly, Tb. N was higher by 1.47-fold in treated FVIII^-/-^ mice in comparison to the untreated group. Consequently, Tb. Sp was significantly lower post-treatment. Of note, trabecular thickness was not affected by recFVIII treatment. BMD in FVIII^-/-^ treated mice was higher than untreated FVIII^-/-^ mice, without reaching significance.Fig. 3. Recombinant FVIII restores bone and vascular phenotypes of FVIII^-/-^ mice. a Quantitative bone parameters, X-ray tomodensitometry analysis. Bone density images were obtained using the DataViewer software (Bruker) and represent sagittal, coronal, and transversal views of femurs. 3D images represent a section corresponding to the distal growth plate of the analyzed femurs. Light grey: WT mice, dark grey: FVIII^-/-^ mice, dark brown: FVIII^-/-^ mice treated with pegylated recombinant FVIII (recFVIII); BV/TV bone volume/total volume; Tb.Th trabecular thickness; Tb. N trabecular number; Tb.Sp trabecular space; Cor coronal; Sag sagittal; Trans transversal. b Bone vascularization parameters analyzed from trabecular bone sections (Endomucin in green, NG2 in red, α-SMA in orange). c Histological identification of bone cells. Decalcified long bones from FWIII^-/-^ and WT mice were used to analyze histomorphometric parameters. Osteoclasts (red) were identified by histoenzymatic staining. Osteoblast precursors and differentiated osteoblasts were detected by immunohistochemistry. Osteoblast precursors were positive for osterix (yellow, arrows), and differentiated osteoblasts were identified by Runx2 positive staining (brown, arrow heads). Veins and capillaries expressed endomucin, stained in blue. Bone cells were quantified by ImageJ; B bone; Pm perimeter; Ob osteoblast; Oc osteoclast; up to 15 mice per group, one to four sections per bone analyzed. Mice were 12 to 17 (young adult) weeks old at sacrifice. Statistical test used: non-parametric Kruskal-Wallis with Dunn’s multiple comparison test. Results are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ns non significant

The recombinant FVIII used contains a pegylated fragment that might have impacted leukocyte biology,^26^ which in turn could affect bone remodeling. The biological effect of a recombinant non-pegylated FVIII (Elocta®, SOBI, Stockholm, Sweden) on bone parameters in FVIII^-/-^ mice was therefore investigated. Bone microarchitectural parameters, including main bone parameters, were significantly upregulated following the administration of recombinant non-pegylated FVIII (Fig. S5a, b), demonstrating that the therapeutic benefit of recFVIII on bone remodeling was associated with the FVIII fragment and not related to the pegylated chain.

As expected, FVIII^-/-^ mice treated with recFVIII showed a higher number of veins, capillaries, and perivascular cells, without reaching significance, but attaining the levels of WT mice (Fig. 3b). Surprisingly, the number of arteries was not statistically different in treated FVIII^-/-^ mice compared to untreated FVIII^-/-^ mice, but significantly lower compared to WT mice (Fig. 3b). Treatment with recFVIII dramatically reduced the osteoclastic population compared to untreated FVIII^-/-^ and WT mice. In parallel, osteoblasts, either with osterix or Runx2 staining, were higher in FVIII^-/-^ treated mice compared to untreated mice and reached significance compared to WT mice (Fig. 3c).

FVIII-/- mice show an altered bone proteomic profile compared to WT mice

The proteomic profiles of bones collected from adult (12 to 17 weeks old at sacrifice) WT mice and FVIII^-/-^ mice were evaluated by liquid chromatography and mass spectrometry (LC-MS) (Fig. 4). Femur, knee joint and tibia/fibula were analyzed separately (Fig. 4a). In general, more than 7 000 protein groups were identified and used for the pathway enrichment analysis. Principal component analysis (PCA) demonstrated a strong clustering of bone fragments collected from WT and FVIII^-/-^ mice with low inter-animal variability (Fig. 4b, MANOVA, P < 10^−^^5^). Pathway enrichment analysis identified numerous proteins associated with abnormal appendicular skeleton (167 out of 523), osteoblast (57 out of 154), and trabecular bone (91 out of 215) morphology, as well as cartilage development (70 out of 197) and long bone morphology (126 out of 376) (Table 1, Table S1). For 84 proteins differentiating sample groups, the trend towards lower expression was observed in FVIII^-/-^ compared to WT mice (P < 0.001 for Joint and Tibia) (Fig. 4b, Fig. S6).Fig. 4. Proteomics and metabolomics profiling of bones collected from FVIII^-/-^ and WT mice and results of protein regulation. a Experimental procedure: after protein extraction from fresh frozen bone, collected proteins were enzymatically digested to obtain peptide suspensions analyzed by LC-MS, combined with data processing. Bones extracted from mouse legs were cut in three fragments (femur, knee joint, tibia/fibula) and analyzed independently. b Clustering of joints, tibias, and femurs and differentiation between FVIII^-/-^ and WT based on 84 proteins (significant difference P < 10^−^^5^). c Modulated proteins of femurs, joints, and tibias in FVIII^-/-^ mice compared to the WT group (significant difference, P < 0.001). Analyses were performed on young adult mice (12 to 17 weeks old at sacrifice). n = 4–5 mice per group. Statistical test used: multiple ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001Table 1Main enrichment functions identified and related to bone remodeling associated with the modulated proteins in bone samplesThe Mammalian Phenotype Ontology (Monarch)Description# in set/pathwayFDR^1^MP:0009250Abnormal appendicular skeleton morphology175 of 5231.00e−04MP:0004986Abnormal osteoblast morphology58 of 1542.60e−04MP:0000130Abnormal trabecular bone morphology94 of 2152.60e−04MP:0000164Abnormal cartilage development74 of 1971.10e−03MP:0003723Abnormal long bone morphology133 of 3721.10e−031: False Discovery RateAnalyses were performed on young adult mice (12 to 17 weeks old at sacrifice). n = 4 to 5 mice per group

In bone samples from FVIII^-/-^ vs. WT mice, a statistically significant difference in the expression of numerous proteins was observed: 37 proteins in femur, 20 proteins in joint, and 22 proteins in tibia samples (Fig. 4c, Fig. S7). While most of these proteins did not overlap between bone fragments, similar trends of up- or down-regulation were observed, independently of the bone fragment. Among 37 proteins, 33 were upregulated and 4 were downregulated in femur samples of FVIII^-/-^ mice. Interestingly, 31 of 33 proteins were also upregulated in tibias and joints of FVIII^-/-^ mice. Similarly, 3 of 4 proteins were downregulated in all three bone fragments.

Several proteins related to osteoblast differentiation, osteoclast differentiation, bone formation, and mineralization were dysregulated in the femurs of FVIII^-/-^ mice (Table 2). These proteins are involved in two major canonical signaling pathways in osteoblast differentiation, respectively, Wnt and Bmp. TNFRS11b (osteoprotegerin) and Receptor-type tyrosine-protein phosphatase epsilon (Ptpre/PTPɛ), which are involved in the regulation of osteoclasts differentiation, were down- and upregulated, respectively (Table 2).Table 2. Major dysregulated proteins in FVIII^-/-^ femurs compared to WT miceMajor dysregulated proteins in FVIII^-/-^ bonesFemur FVIII^-/-^ vs WTOsteoblasts differentiationBone morphogenetic protein receptor type-2 (Bmpr2)−0.512 9Catenin beta-1 (Ctnnb1)−0.024 8Core-binding factor subunit beta (cbfb)−0.030 9Signal transducer and activator of transcription 1 (Stat1)−0.196 2Sclerostin (Sost)−0.065 0Mothers against decapentaplegic homolog 1 (Smad1)1.029 0Transmembrane protein 119 (Tmem119)−0.076 9Osteoclasts differentiationTumor necrosis factor receptor superfamily member 11B (Tnfrsf11b = Osteoprotegerin/OPG)−0.345 7Receptor-type tyrosine-protein phosphatase epsilon (Ptpre/PTPɛ)0.546 3Mineralization, bone formationAlcaline phosphatase (Alpl)−0.093 4Osteocalcin (Bglap)−0.101 4Collagen alpha-1(X) chain (Col10a1)−0.100 3Collagen alpha-1(XXVII) chain (Col27a1)−0.120 0Bone sialoprotein 2 (lbsp)−0.066 6Phosphoethanolamine/phosphocholine phosphatase (Phospho1)−0.263 3Sulfate transporter (Slc26a2)−0.209 9Analyses were performed on young adult mice (12 to 17 weeks old at sacrifice). n = 4 to 5 mice per group

Pathway enrichment analysis indicated a strong link between upregulated proteins associated to cellular nitrogen compound metabolic process, linked to embryonic lethality (Fig. S8).

There were no strong links between regulated proteins in tibias and joints of FVIII^-/-^ mice (Fig. S9). The combined pathway enrichment analysis for all significantly regulated proteins did not reveal additional pathways nor links between proteins (Fig. S9).

Adult FVIII-/- mice exhibit an altered skeletal muscle phenotype compared to WT mice, restored at the proteome and metabolome levels by recFVIII

The skeletal muscle phenotype of FVIII^-/-^ mice was examined using quadriceps and tibialis anterior muscles of juvenile (4-week-old) and adult mice (11-week-old). Muscle mid and distal cross-sectional areas and fiber myosin composition were analyzed (Fig. 5a, Fig. S10a, b). Surprisingly, the quadriceps muscles of juvenile FVIII^-/-^ mice were significantly heavier compared to WT mice, with higher fibers mean cross-sectional area (Fig. 5b). There was no significant difference in myosin subtype composition between the two groups (Fig. S11a). Adult mice had similar muscle weight as well as fiber mean cross-sectional area. Nevertheless, there were significantly fewer type IIx fibers in adult FVIII^-/-^ mice compared to WT mice in contrast to type IIb fibers (Fig. 5b and Fig. S11a). Similar results were observed in tibialis anterior muscles, which were heavier in juvenile FVIII^-/-^ mice compared to WT mice, and showed higher fiber mean cross-sectional area, but without a significant difference in myosin subtype composition. In adult FVIII^-/-^ mice, there was no significant difference in tibialis anterior muscle weight, but significantly higher mean cross-sectional area. As for quadriceps, adult FVIII^-/-^ tibialis anteriors showed a significant reduction in type IIx fibers, in favor of significantly more type IIb fibers (Fig. 5c and Fig. S11b).Fig. 5. Skeletal muscle phenotype of FVIII^-/-^ mice and effects of recombinant FVIII. a Experimental protocol: mice were weighed before sacrifice, quadriceps and tibilias anterior muscles were harvested, weighed, and snap frozen. Mid and distal sections of muscles were analyzed by immunohistochemistry for fiber’s cross-sectional area (laminin staining in red) and myosin composition (red: type I fibers; green: type IIa fibers; black: type IIx fibers; orange: type IIb fibers). b Quadriceps and c tibialis anterior phenotype: weight of muscles (mg of muscle per g of mice), mean fiber size (cross sectional area of mid and distal sections of the muscle) and quantitation of fiber types (n = 8 to 11 mice per group, mice were 4 (juvenile) or 11 (young adult) weeks old at sacrifice non parametric Mann-Withney test). d Mice were treated by IV 200 IU/kg of recombinant pegylated FVIII (recfVIII) once per week for 6 weeks, and the phenotype of quadriceps and tibialis anterior was studied by histological assessment (n = 6 to 11 mice per group, young adult mice aged 12 to 17 weeks old at sacrifice, non-parametric Kruskal-Wallis with Dunn’s multiple comparison test). *P < 0.05, **P < 0.01, ***P < 0.001, ns non significant. Mean ± SD

Histological characterization of skeletal muscles was compared between treated and untreated animals to assess the therapeutic effect of recFVIII on the skeletal muscle phenotype of FVIII^-/-^ mice (Fig. 5d and Fig. S11c). No modification of fiber subtypes was observed after treatment with recFVIII. However, histological assessment revealed significantly lower mean muscle fiber cross-sectional areas in recFVIII-treated vs. untreated FVIII^-/-^ mice for both quadriceps and tibialis anterior muscles. Myosin subtype composition was similar in all groups (Fig. 5d and Fig. S11c).

To better understand the functional impact of FVIII deficiency on skeletal muscles, proteomic and metabolomic profiles were assessed, using the same procedures as for bones (Fig. 4a). PCA demonstrated a strong differentiation between FVIII^-/-^, WT and recFVIII-treated FVIII^-/-^ mice (Fig. 6a and Fig. S12). Muscles from untreated and treated FVIII^-/-^ mice showed sub-clustering with 2 untreated and 2 treated samples mixed with other groups. Volcano plot analysis revealed a significant modulation of protein expression in the different groups (Fig. 6b). FVIII^-/-^ mice showed downregulation of multiple pathways, including those related to translation, cellular respiration, ATP synthesis, and rRNA binding (Table 3 and Fig. 6c). Proteomic analysis of myosin isoforms confirmed the histological findings. There was significantly less myosin-1 isoform (MYH1), the major component of type IIx fibers, in FVIII^-/-^ mice compared to WT mice. Conversely, there was more myosin-4 isoform (MYH4), the major component of type IIb fibers, in FVIII^-/-^ mice compared with WT mice (Fig. S13). Significant regulations were also observed for the metabolomic profile (Fig. 6d). A dysregulation of muscular metabolism, including higher levels of succinic acid, anserine, and phenylalanine was observed in FVIII^-/-^ mice and has been altered by administration of recFVIII down to WT mice levels.Fig. 6. Proteomic and metabolomic profiling of skeletal muscles collected from FVIII^-/-^ and WT mice. FVIII^-/-^ mice were treated or not with 200 IU/kg of recombinant pegylated FVIII once per week for 6 weeks. a PCA plot of proteomics and metabolomics data obtained from fresh frozen skeletal muscles of WT and FVIII^-/-^ mice treated or not with recFVIII. b Volcano plot analysis of differentially expressed proteins in FVIII^-/-^ skeletal muscles compared to the control group and compared to treatment with recFVIII (|log_2_FC| > 1, P value adjusted < 0.05). c Protein enrichment in the four selected groups was differentially expressed in FVIII^-/-^ mice compared to the WT group, visualized as the relative changes to the mean value for all mouse groups, with significance tested against a zero mean using unpaired t-test. d Main metabolites affected by the FVIII deficiency compared to WT mice muscles and the recFVIII-treated group. Analyses were performed on young adult mice (12 to 17 weeks old at sacrifice). n = 4 to 5 mice per group. Statistical test used: one-sample t-test, Kruskal-Wallis test for multiple comparisons. Ns non significant, P < 0.05, P < 0.01, P < 0.001, ****P < 0.000 1Table 3Main enrichment functions identified and related to muscle metabolism associated with the proteins modulated in muscle samplesGO^1^ Biological processDescription# in set/pathwayFDR^2^GO:0006412Translation162 of 3909.87e−13GO:0043043Peptide biosynthetic process168 of 4132.96e−12GO:0002181Cytoplasmic translation94 of 1275.59e−12GO:0045333Cellular respiration116 of 2044.00e−11GO:0009060Aerobic respiration106 of 1685.35e−11GO:0006119Oxidative phosphorylation84 of 1208.24e−11GO:0043604Amide biosynthetic process187 of 5229.04e−11GO:0042776Proton motive force-driven mitochondrial ATP synthesis53 of 623.43e−09GO:0034645Cellular macromolecule biosynthetic process200 of 7814.92e−09GO:0015986Proton motive force-driven ATP synthesis55 of 695.42e−09GO:0015980Energy derivation by oxidation of organic compounds140 of 2755.42e−09GO:0022900Electron transport chain68 of 1252.63e−08GO:0006754ATP biosynthetic process61 of 853.07e−08GO Molecular functionDescription****# in set/pathway****FDR**GO:0003735Structural constituent of ribosome85 of 1754.04e−13GO:0019843rRNA binding30 of 752.16e−05GO:0009055Electron transfer activity35 of 563.90e−041: Gene Ontology2: False Discovery RateAnalyses were performed on young adult mice (12 to 17 weeks old at sacrifice). n = 4 to 5 mice per group

Recombinant FVIII modulates the infiltration of macrophages and the composition of myosin of injured skeletal muscles in adult FVIII-/- mice

The potential contribution of FVIII in muscle inflammation and regeneration was studied by treating adult FVIII^-/-^ mice with injured-muscles with recFVIII. WT and FVIII^-/-^ mice were anesthetized before injection of 100 µL of cardiotoxin (10 μmol/L) in the tibialis anterior muscle, followed or not by IV administration of 200 IU/kg rec FVIII 30 min after injury and then weekly. Animals were sacrificed at day (D) 7 for macrophagic infiltration analysis and D28 for muscle regeneration analysis (Fig. 7a). As previously described,^27^ this protocol allows for complete muscle destruction (by D4), followed by early macrophagic recruitment and myofiber regeneration, with complete restoration of muscle integrity at D30. Recently regenerated myofibers are easily identified by the presence of centered nuclei (Fig. 7a and 7b, Fig. S10c).Fig. 7. Effect of recombinant FVIII on injured skeletal muscle of FVIII^-/-^ mice. a Experimental procedure: 100 μL of cardiotoxin (100 μmol/L) was injected in the tibialis anterior muscle, followed by and iv injection of 200 IU/kg of recombinant pegylated FVIII (recFVIII), 30 min after the injury and then every week (total of 4 injections) at the same dose until sacrifice at day 28. Injection of cardiotoxin induced a complete destruction of myofibers at day 4, followed by complete muscle regeneration at day 28 (black and white bar = 500 μmol/L). b Tibialis anterior phenotype 28 days after injury, weight of muscles (mg of muscle per g of mice), mean fiber size (cross sectional area of mid and distal sections of the muscle) and quantitation of fiber types (green: fibers type IIa; black: fibers type IIx; orange: fibers type IIb); regenerated myofibers were identified by laminin and DAPI staining. c Macrophagic infiltration 7 days after injury: percentage of macrophages type 1 (F4/80^+^; CD163^-^; pink, arrow) and percentage of macrophages type 2 (F4/80^+^; CD163^+^; orange or brown, arrow head) over total cells of the muscle. Dark grey: FVIII^-/-^ mice, light grey: WT mice, dark brown: FVIII^-/-^ mice treated with recFVIII. Analyses were performed on young adult mice (13 to 16 weeks old at sacrifice for muscle phenotype and 9 weeks old for macrophagic infiltration). For muscle phenotype: n = 4 to 5 mice per group, 4 sections per muscle (2 mid sections and 2 distal sections). For macrophagic infiltration: n = 4 to 9 mice per group 3 sections per muscle (mid sections). Statistical test used: non-parametric Kruskal-Wallis with Dunn’s multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001, ns non significant. Mean ± SD

Histomorphometric analysis of tibialis anterior muscles at D28 did not show any significant difference in muscle weight between FVIII^-/-^ untreated and treated mice (Fig. 7b), but the weight of muscles from treated mice was significantly lower than for WT mice. The mean fiber cross-sectional area after injury was not significantly different in FVIII^-/-^ untreated or treated mice, but both were significantly lower than in WT mice. The percentage of type IIb fibers was significantly lower in treated vs. untreated FVIII^-/-^ mice. However, there was no difference in type IIa fibers and type IIx fibers in treated FVIII^-/-^ mice compared to the untreated group. Interestingly, treated FVIII^-/-^ mice had a significantly higher proportion of type IIa fibers than WT mice, with not significantly fewer type IIx and type IIb fibers (Fig. 7b and Fig. S14).

F4/80^+^/CD163^-^ type-1 and F4/80^+^/ CD163^+^ type-2 macrophages were analyzed at D7, which showed a lower infiltration of type-1 macrophages in FVIII^-/-^ mice compared to WT mice. This lower infiltration was partly reversed in treated FVIII^-/-^ mice, with no significant difference compared to WT mice. Type-2 macrophage infiltration was similar in all groups (Fig. 7c).

Discussion

Osteopenia,^28^ sarcopenia,^6,10,11^ abnormalities of angiogenesis^29^ and inflammation processes^30^ are frequent complications in individuals with HA. Although this was considered consecutive to the sedentary lifestyle of these patients, recent insights on the biology of FVIII suggest that extra-hemostatic activities of this protein may partly contribute to these clinical manifestations.^7^ The presented data show an in-depth characterization of the bone, muscle, and vascular phenotype of FVIII-deficient mice compared to WT mice, and the effect of recFVIII treatment on the specific tissues.

The acquisition of bone mass is crucial in the first years of human life, with peak bone mass generally reached shortly after adulthood, before the age of 30. Abnormalities in bone formation during this period lead to premature osteoporosis and an increased risk of fracture.^31^ It therefore seemed particularly important to study bone metabolism in juvenile and young adult mice to assess the potential role of FVIII and FVIII deficiency.

In FVIII^-/-^ mice, the osteoporotic phenotype, which has been previously described,^14^ was associated with a significant reduction of the bone-associated vascular network, with a significant reduction of veins, capillarie,s and perivascular cells, in contrast to a normal amount of arteries. These in vivo impacts of FVIII deficiency are in agreement with previously published in vitro observations.^32,33^ NG2 staining identifies pericytes, located at the abluminal surface of blood vessels, enwrapping endothelial cells through dedicated intercellular junctions.^34^ The key role of pericytes in the homeostasis and physiology of blood vessels has been identified from pathological situations.^35^ Ischemic stroke, infarction, and degenerative contexts can be associated with pericyte atresia and decrease, leading to blood vessels disorganization^36^ via the loss of pericyte interactions with endothelial cells.^36–38^ Bone growth and remodeling have been functionally associated with specific subtypes of endothelial cells.^23^ The negative effect of FVIII deficiency on non-arterial vessels located at the growth plate level, observed in male FVIII^-/-^ mice, could explain their osteoporotic phenotype, with a reduction of bone formation in the early stages of development, resulting from a reduction of osteoblastic cells, as previously described in FVIII^-/-^ male mice.^39^ In 2009, our team demonstrated the inhibition of osteoclastogenesis in vitro when the VWF-FVIII complex was added to a culture medium containing RANKL from mesenchymal cells.^40^ Conversely, this study shows a reduction in osteoclast length in juvenile FVIII^-/-^ mice. The works done by Baud’huin et al. in 2009 used FVIII-VWF complex purified from human plasma and not recombinant molecules, and consequently, a potential contamination by an unknown co-factor cannot be excluded. In addition, in this work, we demonstrated that recombinant FVIII (Octocog α, kindly provided by CSL Behring) alone at the doses used had no effect on RANK-mediated osteoclastogenesis. In 2024, Battafarano et al. observed in ex vivo experiments increased osteoclastogenesis from osteoclast precursors obtained from patients with HA. Surprisingly, they also demonstrated decreased mineralization capacity of control osteoblasts in the presence of FVIII.^41^ Bone remodeling is the result of a functional balance between bone cells with catabolic and anabolic activities functionally interconnected and involving a compensation mechanism in vivo. Therefore, the inhibition of osteoclastogenesis observed in juvenile FVIII^-/-^ mice can be associated to a decreased number of osteoblast precursors and mature osteoblasts, as shown in Fig. 2, osteoblast progenitors being the most abundant source of RANKL in mouse.^42^

Thus, these data demonstrate the functional impact of FVIII deficiency in bone growth and the association of altered vascular trees and osteoblast differentiation at the growth plate, resulting in an altered bone remodeling in FVIII^-/-^ mice.

Interestingly, treatment of adult FVIII^-/-^ mice by recFVIII fully restored bone microarchitectural parameters similar to those of WT animals. Bone vasculature was also partially restored, with an impact on veins and capillaries, yet a negative effect on arteries. As osteoblasts and osteoclasts are close to blood vessels, improvement of bone vasculature may therefore enable the establishment of new zones of cell proliferation, demonstrated by higher numbers of osteoblast precursors and fewer osteoclasts after treatment with recFVIII. This strengthens a crucial functional contribution of FVIII in bone remodeling and vascular tree homeostasis. Especially in young patients with HA, FVIII might be crucial to support bone growth and remodeling.

Bone proteomic analyses from FVIII^-/-^ and WT mice confirmed the phenotypic differences observed, highlighting the lower expression of proteins involved in appendicular skeleton, osteoblast, and trabecular morphology as well as cartilage development and long bone morphology.

Two canonical signaling pathways in osteoblast differentiation were downregulated in the femurs of FVIII^-/-^ mice. Lower levels of Stat1, β-catenin, and Cbfb, belonging to the Wnt pathway, as well as lower levels of Tmem119 and Bmpr2, belonging to the Bmp pathway, were observed in FVIII^-/-^ mice. Interestingly, a higher amount of Smad1 was observed in FVIII^-/-^ mice, probably corresponding to an accumulation of the non-phosphorylated form due to lack of activation by Bmpr2^43,44^ (Fig. S15). On the other hand, lower levels of Tnfrsf11b, which acts as a decoy receptor for TNFSF11/RANKL, thereby inhibiting osteoclast activation, and higher levels of PTPɛ were found, suggesting an increase in osteoclast maturation.^45^ This results in a decrease in many proteins (osteocalcin, collagen α1, lbsp, phospho1, slc26a2, Alpl) involved in mineralization and bone formation (Table S1). Sclerostin is produced by osteocytes and inhibits the Wnt pathway associated with osteoblast differentiation during bone remodeling.^46^ Reduced sclerostin levels in the femurs of FVIII^-/-^ mice may be a consequence of reduced osteocyte formation, as evidenced by reduced bone mineralization proteins. Surprisingly, two studies found increased plasma levels of sclerostin in children with HA, suggesting high bone turnover.^47,48^ It should be noted that these studies only included children and that one of the two studies included patients with mild, moderate, and severe HA.^47^ It would be interesting to kinetically measure plasma sclerostin levels in juvenile and adult FVIII^-/-^ mice to confirm these results. Furthermore, an upregulation of proteins belonging to the cellular nitrogen metabolic process was observed. Dysregulation of this metabolic process has been associated with cancer^49^ and embryonic lethality.^50^ Overall, these data demonstrate the functional impact of FVIII deficiency on bone proteomic profiles, related to the osteoporotic phenotype of FVIII^-/-^ mice.

Bone tissue is strongly associated with the physiology of skeletal muscles, as they enable muscle contractions by acting as an anchor for muscular bundles. In addition, bones are the main reservoir of mineral ions mandatory for nerve influx and rhabdomyocyte contraction. There is limited data on the biological effect of FVIII on skeletal muscles, yet repeated bleeding within skeletal muscles may affect tissue integrity and muscle regeneration. Recently, quadriceps contractile properties were studied in a small series of patients with moderate or severe HA, showing a reduced contraction strength compared with the control group.^10,11^ The skeletal muscle phenotype of FVIII^-/-^ mice was therefore investigated here. Surprisingly, young FVIII^-/-^ mice exhibit an altered skeletal muscle phenotype with heavier muscles, larger fibers, and a fiber type IIx to type IIb switch. In addition, skeletal muscle proteomic and metabolomic profiling confirmed differences between FVIII^-/-^ and WT muscle fibers. The alterations detected in FVIII^-/-^ mice skeletal muscles were partly reversed by recFVIII. More precisely, FVIII^-/-^ mice showed a significant downregulation of mitochondrial activities such as translation, cellular respiration, ATP synthesis, and rRNA binding, which could lead to mitochondrial dysfunction and decreased muscle strength.^51–53^

Rhabdomyocytes are characterized by metabolic, electrical, and contractile diversity.^54,55^ Most mammalian skeletal muscles have 4 categories of muscle fibers. Type I fibers have a low ATPase activity, slow twitch, high oxidative and low glycolytic activities, leading to a relatively fatigue-resistant profile. Fast-twitch type II fibers are composed of several entities: (i) type IIa, relatively fatigue-resistant through high oxidative and glycolytic capacities; (ii) type IIb, major in rodents,^56^ fatigue-sensitive with high ATPase activity, low oxidative and high glycolytic capacities; (iii) type IIx (or IId), intermediate between IIa and IIb.^57^ A significant reduction of IIx fibers, compensated by more IIb fibers, was observed in FVIII^-/-^ compared to WT muscles. This modified type IIx/type IIb balance can be associated with the proteomic and metabolomic changes observed, particularly with changes in myosin isoform.^58^ More metabolites involved in ATP synthesis were observed in FVIII^-/-^ muscles. Carnitine derivatives and amino acids can be used to produce AcetylCoa, while succinic acid is an intermediate metabolite in the citric acid cycle pathway, contributing to ATP formation (Fig. S16). Besides, low inosine and proline levels were observed in FVIII^-/-^ mice, which might be overused to produce ATP in abnormal conditions.^59–61^ These data point toward a greater need for ATP production, possibly associated with poorer metabolic economy.^53^ Anserin and spermidine are two muscle metabolites characterized by antioxidant properties.^62,63^ Higher levels in FVIII^-/-^ mice may be necessary to balance the high reactive oxygen species secondary to high ATP production. Treatment by recFVIII partially restores metabolite levels, in turn reducing ATP synthesis. Thus, FVIII^-/-^ mice appear to need greater energy, hence having a poorer metabolic economy phenotype. This is consistent with the clinical observation of a quadriceps strength deficit in individuals with HA^10,11^, although this should be cautiously interpreted. Indeed, we studied the quadriceps and tibialis anterior muscles, while the muscle fiber composition of the body’s various muscles is tailored to muscle functions. For example, postural muscles are enriched with highly vascularized slow fibers dependent on oxidative metabolism,^64^, which differs from the muscles we analyzed. The effects of FVIII deficiency we observed in the quadriceps and tibialis anterior muscles may therefore not be applicable to all muscles in the body.

Type IIb fibers are the muscle fibers with the largest size and the most intense ATPase activity.^56,65^ They also present the lowest capillary density.^66,67^ Thus, the phenotypic and metabolic abnormalities found in this study could be secondary to early abnormalities in vascularization through reduced capillary density, as found in bone analysis. These abnormalities translate into early sarcopenia,^68^ as found in patients with HA.^10,11^ The abnormalities found in FVIII^-/-^ mice could thus be attributed to early abnormalities of vascularization in bone and muscle, compensated for in adulthood. Minor effects of recFVIII on muscle phenotype suggest that metabolic abnormalities derive from early vascularization defects, whereas major enhancement of bone phenotype derives from FVIII-dependent mechanisms in the regulation of bone metabolism.

The link between FVIII and vascularization is the subject of increasing research. Several teams have now demonstrated that FVIII, synthesized by liver endothelial cells in particular,^69^ appears to be necessary for the formation of intact vascular tubes and regulating vascular permeability^29,32,33,70,71^, and could be responsible for multiple organic anomalies.

The recent evidence of chronic low-grade inflammation in plasma from patients with HA^30,72^ has been attributed to subclinical microbleeds. Nieuwenhuizen et al.^73^ demonstrated a defect in macrophage polarization in a hemarthrosis model. Here, 7 days after induction of muscle injury, a lower recruitment of pro-inflammatory type-1, but not type-2, macrophages was demonstrated in FVIII^-/-^ mice, partially restored by recFVIII. It could be interesting to further investigate the effects of FVIII treatment on macrophage infiltration and differentiation at an earlier stage. FVIII biological activity could be impacted by the inflammation induced by cardiotoxin inoculation and might contribute to promoting the regeneration of muscle fibers characterized by low twitch properties. This study again demonstrates a functional impact of recFVIII in a context of injured muscles and new evidence of the biological effects of FVIII on inflammation.

The presented data is, to our knowledge, the first report describing bone, vascular, and muscular alterations, as well as molecular musculoskeletal dysregulation using a mouse model of HA. These bone and vascular modifications could be partly reversed by administration of recFVIII, yet skeletal muscle abnormalities were only partially corrected by FVIII supplementation in injured conditions. This observation is in favor of FVIII supplementation in individuals with HA to preserve bone tissue and prevent bone loss, particularly in the early stages of development. Further investigations are necessary to further explore the biological effect of FVIII on the biology of skeletal muscles in health and HA.

Materials and methods

Sex as a biological variable

Severe hemophilia A, corresponding to a total deficiency of coagulation FVIII, is an X-linked condition that almost exclusively affects men. Consequently, in order to reproduce the disease as faithfully as possible, and also to avoid possible interference from female hormones, only male mice were used in this study.

No excess mortality nor any external bleeding was observed in FVIII^-/-^ animals compared to WT mice. However, after sacrifice, during tissue collection, intramuscular bleeding, or joint degradations were observed, confirming the hemorrhagic phenotype of FVIII^-/-^ mice.

Ethics and animal experimentation

All studies were approved by the institutional committee of Ethics and Animal Experimentation of Nantes University and were conducted in accordance with the French and EU guidelines for animal care (Ref.: AFAPIS#21869-2019080716454315v7). FVIII^-/-^ mice were produced by Jackson Laboratory (B6;129S-F8tm1Kaz/J, Bar Harbor, ME) in a C57BL/6J and 129S genetic mixed background. In order to establish a homogeneous C57BL6/J background, FVIII^-/-^ mice were backcrossed with C57BL/6J wild-type mice (Jackson Laboratory), and the resulting FVIII^+/-^ heterozygous mice were then crossed at least 10 times to reduce the theoretical proportion of 129S background to less than 0.1%.^74^ Finally, crosses from new FVIII^+/-^ heterozygous mice were performed to obtain FVIII^+/+^ and FVIII^-/-^ in a similar and comparable background (Charles Rivers Laboratories). Animals were used between 5 and 25 weeks after birth. In order to analyze the period of musculoskeletal capital formation during growth, bone and muscle analyses were carried out in juvenile mice (less than 8 weeks old). To determine the medium-term impact of FVIII deficiency, analyses were also carried out on young adult mice (11 to 14 weeks).

The age distribution of comparable mice between groups was similar, with no difference in interpretation between groups.

Mice treated with recombinant FVIII for 6 weeks were also studied. For technical reasons, treatment was initiated at 6 weeks at the earliest. Treated mice were sacrificed as adults at the end of the sixth week of treatment, i.e., mice between 12 and 17 weeks of age. The age distribution of mice was comparable between groups.

Mice under anesthesia were sacrificed by cervical dislocation at different time points.

Recombinant Factor VIII treatment

The effect of recombinant FVIII (recFVIII) on bone and muscle phenotypes of FVIII^+/+^ and FVIII^-/-^ mice was analyzed by weekly IV injection of 4 IU of rFVIII (FVIIIr peg, Jivi®, BAYER, Leverkusen, Germany, or FVIIIr fc, Elocta®, SOBI, Stockholm, Sweden) corresponding to approximately 200 IU/kg in 20 µL, for 4 to 6 weeks.

Factor VIII activity assay

One stage FVIII clotting activity was based on the activated partial thromboplastin time (aPTT) using FVIII immunodepleted plasma and an aPTT reagent (Stago, Asnières, France).

X-ray tomodensitometry (Micro-CT) analysis

Bone morphometric parameters were obtained using a Skyscan 1276 CMOS in vivo micro-CT scanner (Bruker, Kontich, Belgium). The femurs of WT and FVIII^-/-^ mice were used to compare bone morphometric, structural, and mineral parameters. All samples were scanned using the same parameters (pixel size 9 μm, 50 kV, 0.5 mm Aluminum filter). Scanner reconstruction was carried out using the NRecon software, and the analyses were performed using CTAn, CTVox, and DataViewer software (Bruker XRM solutions software). Bone mineral density (BMD), trabecular bone volume (BV/TV), trabecular thickness (Tb.Th), trabecular spaces (Tb.Sp), and trabecular numbers (Tb.N) were analyzed at the growth plate level of the distal epiphysis of femurs (metaphyseal). The same point of reference (chondrocyte seam) at the growth plate was established for each sample, and, from an offset of 0.215 mm from the growth plate to the metaphysis, a trabecular volume of 1.5 mm was selected for the analysis of bone morphometric parameters.

Skeletal tissue preparation for immunostaining

Sample preparation for bone vascular architecture analysis was performed as reported by Kusumbe et al.^22^ Briefly, tibias from WT and FVIII^-/-^ mice were fixed immediately after dissection in cold paraformaldehyde 4% (wt/vol) (Sigma-Aldrich, St Louis, MO) for 4 h at 4 °C under agitation. Then, samples were washed 3 times in cold PBS (CS1PBS00-01, Eurobio Scientific, Les Ulis, France) and decalcified by using a DCAL solution (0.5 mol/L EDTA, pH 7.4–7.6, Sigma-Aldrich) at 4°C under agitation. After 24 h of decalcification, samples were again washed 3 times with cold PBS and incubated in a cryoprotectant solution of sucrose 20% (S7903, Sigma-Aldrich) and polyvinylpyrrolidone (PVP) 2% (P5288, Sigma-Aldrich) in PBS at 4 °C for 24 h under agitation. Finally, bone samples were washed with PBS and embedded in a warm EMB solution of gelatin 8% (G1890, Sigma-Aldrich), sucrose 20% and PVP % in PBS. Samples were then preserved at −80°C.

Bone cryosection and immunostaining

Blocks containing tibia samples were cut in a precooled cryostat (Leica CM1950, Leica Biosystem, Washington DC) in serial sections of 30 µm, placed in a slide (Polysine Adhesion Microscope Slide, Epredia, PHC group, Tokyo, Japan) and stored at −20°C. For immunostaining, samples were dried at room temperature (RT) and surrounded with a PAP pen to limit the staining area. Samples were then rehydrated with PBS for 5 minutes, permeabilized with Triton X-100 0.3% (T8787, Sigma-Aldrich) for 20 minutes, and blocked with fresh goat serum 10% and BSA 5% in PBS for 30 minutes. Primary antibodies were added at the corresponding concentration and incubated at 4 °C for 12 h. Endomucin (V.7C7, sc-65495, Santa Cruz Biotechnology, Dallas, TX) dilution 1/50. NG2 (546930, MA5-24247, Invitrogen, Waltham, MA) dilution 1/100. α-SMA (clone1A4, C6198, Sigma-Aldrich) coupled to Cy3, dilution 1/400). Secondary antibodies for endomucin and NG2 (AlexaFluor 647, A-21247, Invitrogen) were incubated for 1 h at RT in the dark. Finally, sections were counterstained with a nuclear marker (ProLong Gold Antifade Mountant with DNA Stain DAPI, ThermoFisher, Waltham, MA). Immunofluorescent stains were analyzed at high resolution with a Nikon A/TiE1 confocal microscope (Nikon Instruments, Tokyo, Japan), with a Galvano scanner. Z-stacks of images were processed and reconstructed with NIS-Element AR software. All quantifications were done with ImageJ software (NIH, Washington, DC) (2 sections per bone). Endomucin, NG2, and α-SMA analyses were carried out on the bone marrow at the metaphyseal level. Analyses were carried out on a 0.53*1.26 mm surface and expressed as the percentage of positive cells per marrow area (percentage/mm²) for endomucin and NG2 analyses and the number of positive cells per marrow area (number/mm²) for α-SMA.

Osteoblasts, osteoclasts, and endomucin staining

Immediately after dissection, femurs were fixed in formalin for at least 24 h. Samples were then decalcified as previously described and embedded in paraffin wax. Immunohistochemical staining was performed on serial 3 µm (Polysine^TM^ Adhesion Microscope Slide, Epredia, PHC group, Japan) sections using an indirect immunoperoxydase technique. Tissue sections were dewaxed and rehydrated by successive immersions in xylene, decreasing ethanol concentration bath, and water.

Tartrate Resistance Acid Phosphatase (TRAP) staining

TRAP Staining Solution Mix (Sodium acetate trihydrate [S7670], L-(+) Tartaric Acid [Sigma T-1807], distilled water, Glacial Acetic Acid [Merck, Darmstadt, Germany]), adjusted at pH 4.7–5.0, was pre-warmed in staining dishes. Slides with rehydrated samples were immersed in TRAP staining solution mix and incubated with Naphthol AS-MX Phosphate Substrate mix (Napthol AS-MX Phosphate N-4875, Sigma; Ethoxyethanol 128082, Merck) and Fast Red Violet LB Salt (HY-D1491A, MedChemTronica Sollentuna, Sweden) at 37 °C in a water bath for 1 h. Slides were then rinsed with distilled water as described.^75^

Endomucin and Osterix immune detection

Antigen retrieval was performed by immersing samples in Epitope Retrieval Solution 2 (pH 9) (AR9640, Leica) and heating at 60 °C for 20 hours. Slides were then cooled 20 min at room temperature in antigen retrieval buffer and rinsed with tap water. Endomucin (V.7C7, sc-65495, Santa Cruz), Osterix (polyclonal, ab22552, Abcam, Cambridge, UK), and anti-rat (ab7099, Abcam) were diluted (respectively 1/500, 1/2 000, and 1/2 000) in Primary Antibody Diluent (Leica AR9352). Immunostaining was performed with the bond detection kit (DS9800, Leica) on Leica Biosystems BOND RX. Briefly, endogenous peroxidase was blocked 5 min by Peroxide Block (Leica), prior to incubation with monoclonal antibodies (15 min for Endomucin and 30 min for Osterix). Antigen expression was detected by incubation with labeled polymer horseradish peroxidase and diaminobenzidine to yield a brown color and PolyDetector Yellow (BSB-0365, BioSB) for 20 min at room temperature for the yellow signal. Sections were then counterstained with hematoxylin and treated with ChromoProtector (BSB-0153, BioSB, Santa Barbara, CA) before mounting to preserve the yellow color (1 to 2 sections per bone). Sections were mounted with xylene-based mounting medium (HistoCore Spectra CV X1, 3801733, Leica).

Runx2 immune detection

Antigen retrieval was performed by immersing samples in Epitope Retrieval Solution 2 (pH 9) (AR9640, Leica) and heating at 60 °C for 20 hours. Slides were then cooled 20 min at room temperature in antigen retrieval buffer and rinsed with tap water. Runx2 (EPR14334, ab192256, Abcam, Cambridge, UK) was diluted 1/2 000 in Primary Antibody Diluent (Leica AR9352). Immunostaining was performed with the bond detection kit (DS9800, Leica) on Leica Biosystems BOND RX. Briefly, endogenous peroxidase was blocked 5 min by Peroxide Block (Leica), prior to incubation for 30 minutes at RT. Antigen expression was detected by incubation with labeled polymer horseradish peroxidase and diaminobenzidine to yield a brown color. Sections were then counterstained with hematoxylin. Sections were mounted with xylene-based mounting medium (HistoCore Spectra CV X1, 3801733, Leica) in HistoCore SPECTRA ST automated.

Muscle tissue preparation and immunostaining

Tibialis anterior and quadriceps muscles from WT and FVIII^-/-^ mice were dissected and weighed systematically to be related to total animal weight. Then, muscle samples were cryopreserved with OCT (Superfrost^TM^ Plus, Epredia, PHC group) in small cryomolds, quickly frozen in a solution of Isopentane (PHR1661, Sigma-Aldrich), cooled in liquid nitrogen, and stored at −80 °C. Blocks containing muscle samples were cut in a precooled cryostat (Leica CM1950, Leica Biosystem) in serial sections (10 µm for myosin subtypes, 5 µm for macrophages), placed in a slide (Superfrost^TM^ Plus) and stored at −20 °C (mid and distal sections with two sections for each cutting level for myosin subtype analysis; mid-section with 3 sections spaced by 100 µm for macrophage analysis).

For myosin subtypes immunostaining, samples were dried at room temperature (RT), surrounded with a PAP pen to limit the staining area, and blocked with fresh goat serum 10% and BSA 5% in PBS for 45 minutes. Primary antibodies were added to the sample at the corresponding concentration and incubated at 4 °C overnight. Laminin monoclonal antibody (A5, MA1-06100, Thermofisher) at a dilution of 1/200 was used to determine muscle fiber number and areas. For muscle myosin composition, myosin type I (16F2, BA-D5 monoclonal antibody, Developmental Studies Hybridoma Bank®, Iowa City, IA), myosin type IIa (15l1, SC-71 monoclonal antibody, Developmental Studies Hybridoma Bank, Iowa City, IA) and myosin type IIb (1514, BF-F3 monoclonal antibody, Developmental Studies Hybridoma Bank) were used at 2 μg/mL. Secondary antibodies for laminin (AlexaFluor^TM^647, A-21247, Invitrogen), myosin Type I (AlexaFluor^TM^647, A-21242, Invitrogen), myosin Type IIa (AlexaFluor^TM^488, A-21121, Invitrogen), and myosin Type IIb (AlexaFluor^TM^594, A-11032, Invitrogen) at a dilution of 1/1 000 were incubated for 45 minutes at RT in darkness. Images were captured on an Operetta CLS High Content Analysis System (Revvity) and analyzed using Harmony® software and ImageJ (NIH).

For immunostaining of macrophagic populations, samples were dried at RT and fixed 10 min with paraformaldehyde 4% (wt/vol). Antigen retrieval was then performed by immersing samples in Epitope Retrieval Solution 1 (pH 6) (AR9961, Leica) and heating at 95 °C for 20 min. Slides were then cooled 20 min at room temperature in antigen retrieval buffer and then rinsed with tap water. F4/80 (D2S9R, CST 70076, Cell Signaling Technology, Danver, MA) and CD 163 (EPR19518, ab182422, Abcam) antibodies were used to identify M1 and M2 macrophages. Antibodies were diluted in Primary Antibody Diluent solution (AR9352, Leica) at 1/500 and incubated on samples for 1 hour at RT. Red immunostaining (F4/80) was performed with the red bond detection kit (DS9390, Leica). Briefly, after Ab incubation, antigen expression was detected by incubation with labeled polymer alkaline phosphatase. CD163 staining was performed with the bond detection kit (DS9800, Leica). Antigen expression was detected by incubation with labeled polymer horseradish peroxidase and either with or without PolyDetector Yellow (BSB-0365, BioSB) for 20 min at room temperature.

Sections were then counterstained with hematoxylin and treated with ChromoProtector (BSB-0153, BioSB) before mounting. Thus, CD 163-positive cells were stained either in yellow (with PolyDetector Yellow) or in brown (without PolyDetector Yellow). Sections were mounted with xylene-based mounting medium (HistoCore Spectra CV X1, 3801733, Leica) in an HistoCore SPECTRA ST automated. Images were captured on a Hamamatsu digital slide scanner (Hamamatsu Photonics, Shizuoka Prefecture, Japan) and analyzed using ImageJ software (NIH).

Muscle injury by Cardiotoxin

Muscle injury was induced using the snake venom cardiotoxin (Cardiotoxin L8102-1, Latoxan Laboratory, Porte les Valence, France) as described by Guardiola et al.^27^ Briefly, after animal anesthesia with isoflurane/air (1.3–1.8, 1 L/min) and buprenorphine (0.05 mg/kg), a solution of 100 µL cardiotoxin at 10 μmol/L in PBS was injected intramuscularly with a 30 Gauge Hamilton microsyringe in the left tibialis anterior muscle.

Proteomic and metabolomic profiling

Proteomic and metabolomic profiling was carried out according to the procedures described by Bian et al.^76,77^ Proteins extracted from fresh frozen bones and skeletal muscles were enzymatically digested to obtain peptide suspensions that were analyzed by liquid chromatography-mass spectrometry (LC-MS) combined with data processing. Leg bones were cut in three fragments (femur, knee joint, tibia/fibula), and analyzed separately.

LC-MS metabolomics

Frozen quadriceps samples were lysed with Precellys Evolution tissue homogenizer (Bertin Technologies, Montigny le Bretonneux, France) using Tissue homogenizing CKMix tubes, 2 mL (KT03961-1-009.2). The three fragments of bones were homogenized, and 50 µL of the homogenized solutions were mixed with 50 µL of an internal reference standard solution composed of heavy labeled metabolite standards representing lipids, steroids, amino acids, organic acids, Vitamin B6, and peptides diluted in 95:5 acetonitrile (ACN): water (%, v/v). The proteins were precipitated by adding 500 µL of ice-cold 95:5 ACN: water (%, v/v) to the mixture. Mixtures were shaken in low-bind 96 deep well plates (Eppendorf, Montesson, France) at 1 500 r/min for 5 min using a deep well plate mixer (Eppendorf, MixMate). After that, samples were centrifuged at 3 500 r/min for 10 min at 5°C with Allegra X-15R Beckman Coulter (Miami, FL). A volume of 500 µL of supernatant was transferred to 96 deep well plates with the CyBio SELMA 96 pipetting robot (Analytik, Jena, Germany). Precipitated protein pellets were stored and used for proteomics sample preparation and LC-MS analysis. Collected supernatants with metabolites were evaporated to dryness under vacuum with the Vacuum Evaporation System (Thermo Scientific SpeedVac SRF110, Refrigerated Centrifugal Vacuum Concentrator). Metabolites were reconstituted in 50 µL of ACN with 0.1% formic acid and mixed for 10 min at 1 500 r/min. 100 µL of 0.1% formic acid in water was added to the final solution that was used for LC-MS analysis.

Untargeted micro-flow LC-MS metabolomic measurements were completed on a 1290 Infinity II LC system (Agilent, Waldbronn, Germany) coupled to an Orbitrap Exploris 240 high-resolution accurate-mass mass spectrometer (HRAM MS) (Thermo Scientific, Bremen, Germany). Metabolites were separated on ACQUITY UPLC HSS T3 (1.8 µm, 1.0 × 150 mm, Waters, Guyancourt, France) at 40 °C using the following gradient elutions: 0 min – 1% B, 50 µL/min; 15 min – 99% B, 50 µL/min; 15.1–99%, 120 µL/min; 8 min – 99% B, 120 µL/min; 18.1–1% B, 120 µL/min; 20 min – 1% B, 50 µL/min. Mobile phase A was 0.1% FA in water, mobile phase B was 0.1% FA in ACN.

Data were acquired in MS1 Full scan mode in the range of 100–1 000 m/z at 60 000 resolution with alternating positive and negative polarity. The ion source was H-ESI, ion voltage 3 500 V for acquisition in positive polarity and 2 500 V for acquisition in negative polarity. Sheath gas was 25, auxiliary gas, 5, sweep gas, 0, ion transfer tube temperature, 325 °C, vaporizer temperature, 75 °C, internal mass calibration, Easy-IC, scan-to-scan mode.

For metabolite identification, the Acquire X workflow (Thermo Scientific) was run separately in positive and negative modes on pooled QC samples that were made by mixing equal volumes from all studied muscle or bone samples, respectively. After 3 replicate blank runs and a pooled QC Full Scan run, 4 injections of the pooled QC sample in data-dependent acquisition (DDA) mode with subsequent exclusion of already fragmented ions before each next injection were completed. Mass tolerance (low and high) was set to 10 ppm. Data were acquired with Orbitrap MS1 resolution, 30 000; scan range, 100–1 000 m/z; minimum intensity, 5 000. The number of dependent MS2 scans was 5; MS2 resolution, 15 000; isolation window, 1.5 m/z; HCD collision energy type was normalized; HCD collision energy (%) was 15, 50, 150; RF Lens (%), 70; AGC target, standard. During DDA, dynamic exclusion was made after 1 scan, duration, 2.5 s; exclusion mass width (low and high), 5 ppm; isotopes were excluded. Acquire X data were acquired with an exclusion override factor of 3, exclusion list peak window extension of 0, exclusion duration of 10 s, enabled automatic adding of isotopes, and exclusion list minimum intensity of 5e4.

Compound Discoverer 3.3 SP3 (Thermo Scientific) was used to process .raw files, identify and quantify metabolites after QC-based batch normalization. The same software was used for statistical analysis. Compounds were annotated in the order of matches in (i) mzVault, including an internally generated database and mzCloud library, (ii) Metabolika search, (iii) ChemSpider search, (iv) MassList search, and (v) predicted compositions. Advanced data processing was performed with Qlucore Omics Explorer software (Qlucore, Lund, Sweden).

LC-MS proteomics

Precipitated protein pellets, after removal of metabolite extracts, were reconstituted in 60 µL 1 mol/L Guanidinium chloride (GHD)/40 mmol/L Dithiothreitol (DTT)/300 mmol/L tris(hydroxymethyl)aminomethane (Tris) at pH 8 for cysteine reduction. Samples were incubated for 30 min at 37 °C with continuous shaking. After that, 60 µL of 200 mmol/L 2-chloracetamide (CAA) was added to induce cysteine carbamidomethylation. Samples were incubated for 30 min at RT in the dark. To stop the reaction, 100 µL of 1 mol/L GHD/40 mmol/L DTT/300 mmol/L Tris was added to the mixture, and samples were mixed for 10 min. Proteins were digested with 175 µL of 1 µg/µL trypsin solution. Digestion was completed overnight for 18 h at 37 ℃ with continuous mixing. To stop the reaction, 100 µL of 2% FA in water was added to the solution. Digested samples were loaded onto solid phase extraction (SPE) Oasis HLB 96-well plates (Waters), washed, and eluted with 200 µL of 50/50 (v/v) ACN at 0.1% FA in water. After drying in the Vacuum Evaporation System (Thermo SpeedVac SRF110, Refrigerated Centrifugal Vacuum Concentrator), peptides were reconstituted in 200 µL of 0.1% FA in water.

Untargeted micro-flow LC-MS proteomics measurements were run on a Vanquish Neo UHPLC system (Thermo Scientific, Germering, Germany) coupled to an Orbitrap Exploris 480 (Thermo Scientific, Bremen, Germany). The separation was done on ACQUITY UPLC CSH columns (1.7 µm, 0.3 mm × 150 mm, Waters) at 60 °C using a flow rate of 20 µL/min.

Muscle samples were analyzed on the system configured for direct column injection in a forward flush direction. Gradient elutions were 0 min – 1% B, 0.9 min – 5% B, 1 min – 5%, 52 min – 20% B, 82 min – 36% B, 82.1 min – 99% B and 90 min – 99%. The separation column was equilibrated with 3 column volumes by pressure-controlled mode at 1 200 bar. Mobile phase A was 0.1% FA in water, mobile phase B was 0.1% FA in ACN. Sample loading was set to pressure control at 1 200 bars, autosampler temperature was set to 7 °C.

Bone samples were analyzed with the Vanquish Neo UHPLC system configured in trap-and-elute injection workflow. Samples were loaded onto the Acclaim PepMap (2 µm, 1 mm × 2.1 mm) trap column with combined control of maximum pressure 800 bars or maximum flow 200 µL/min. Gradient elution was of 0 min – 2% B, 0.2 min – 5% B, 5.1 min – 7%, 5.2 min – 7% B, 35.2 min – 22% B, 55.2 min – 40% B, 56 min – 99% B, 60 min – 99%. After the end of gradient, the trap column was washed with 1 column volume of strong wash (0.1% FA in ACN and 3% DMSO) and re-equilibrated with 3 column volumes of weak wash (0.1% FA in water). The separation column was equilibrated with 3 column volumes at a constant flow of 20 µL/min. Mobile phase A was 0.1% FA in water, and mobile phase B, 0.1% FA in ACN.

The ion source was H-ESI operated at ion voltage: 3 500 V, sheath gas: 25, auxiliary gas: 5, sweep gas: 5, ion transfer tube temperature: 325 °C, vaporizer temperature: 75 °C. FAIMS was set to On at CV -50 V. Data were acquired in DDA mode with an MS1 range of 360–1 300 m/z at 120 000 resolution, time 50 ms, normalized AGC target, 300%, RF Lens, 40%, internal mass calibration, Easy-IC. Minimum intensity for MS2 events was 1^4^, charge states 2-6, polarity, positive. The number of dependent scans was 20. Dynamic exclusion was set to exclude after 1 scan, with an exclusion duration of 120 s, mass tolerance low and high of 25 × 10^-6^. The isolation window was 1.6 m/z, normalized HCD collision energy of 28%, Orbitrap MS2 resolution at 15 000, first mass at 120 m/z, accumulation time at 50 ms, RF lens at 50% and normalized AGC target at 300%.

Data were processed with Proteome Discoverer software (version 3.0 SP1) using multistep Sequest HT^78^ with INFERYS^79^ rescoring algorithms, followed by Percolator,^80^ MS Amanda 2.0,^81^ followed by Target Decoy PSM validator. The CHIMERYS search engine, followed by Percolato, was added to the workflow to process bone sample results. The false discovery rate (FDR) target was set to 1% for all search engines. The search was completed against the mouse Fasta file with protein isoforms that contain 25 477 protein entries. For quantification purposes, muscle samples were compared between WT and FVIII^-/-^ groups, and bone samples were compared for each bone part separately between WT and FVIII^-/-^. Data were median-normalized for all samples. The Search Tool for the Retrieval of Interacting Genes/Proteins (String-db)^82,83^ was used for pathways analysis. The Qlucore software was used for statistical data processing.

Statistical analyses

Quantitative data were expressed as mean (standard deviation [SD]). For quantitative variables, the difference in medians between two groups was assessed using the Wilcoxon-Mann-Whitney test. Comparison of the means of two independent or unrelated groups was assessed with an unpaired t-test. The difference between more than two groups was assessed using Kruskal–Wallis tests with Dunn’s multiple comparisons test or the one-way analysis of variance (ANOVA). Error bars show mean ± SD to value of the mean. A P-value ≤ 0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism 10 software (GraphPad Software, La Jolla, CA).

Supplementary information

FVIII^-/-^ mice show altered bone vascularization, restored by recFVIII treatment FVIII^-/-^ mice show altered osteoclastic and osteoblastic infiltration, which is not restored by recFVIII treatment Experimental procedure Kinetics of plasma FVIII activity in WT mice, FVIII-/- mice with or without pegylated recombinant FVIII injection Similar bone beneficial effect of recombinant non-pegylated FVIII compared to recombinant pegylated FVIII Combined list of the 84 proteins downregulated in bones of FVIII^-/-^ mice compared to WT mice (p < 1e−5) Protein profiling of joints and tibias collected from FVIII^-/-^ and WT mice Pathway enrichment analysis indicates a strong link between up-regulated proteins that belong to the cellular nitrogen compound metabolic process and are linked to embryonic lethality Combined list of proteins regulated in femur, tibia, and joint of FVIII^-/-^ vs. WT mice Muscle phenotype characterization Quantitation of fiber types in quadriceps and tibialis anterior muscles Proteomic profiling of skeletal muscles collected from adult WT, FVIII^-/-^, and FVIII^-/-^ mice treated with recFVIII Proteomic profiling of skeletal muscles myosin isoforms from adult WT, FVIII^-/-^, and FVIII^-/-^ mice treated with recFVIII Quantitation of fiber types in tibialis anterior muscles after cardiotoxin injection Reduced osteoblastic maturation and increased osteoclastic maturation result in mineralization defects in FVIII^-/-^ mice Cellular energy production pathways from different sources to the citric acid cycle in the mitochondria Proteomic data of bone collected from adult and FVIII knockout mice