Fibroblast Growth Factor 23 Neutralizing Antibody Ameliorates Abnormal Renal Phosphate Handling in Sickle Cell Disease Mice.

We assessed the involvement of fibroblast growth factor 23 (FGF23) in phosphaturia in sickle cell disease (SCD) mice. Control and SCD mice were treated with FGF23 neutralizing antibody (FGF23Ab) for 24 hours. Serum ferritin was significantly increased in SCD mice and was significantly reduced in female but not male SCD mice by FGF23Ab. FGF23Ab significantly reduced increased erythropoietin in SCD kidneys. Serum intact FGF23 was significantly increased in SCD female mice and was markedly increased in SCD male mice; however, FGF23Ab significantly reduced serum intact FGF23 in both genotypes and sexes. Serum carboxy-terminal-fragment FGF23 (cFGF23) was significantly reduced in SCD IgG male mice and was markedly but not significantly reduced in SCD IgG female mice. FGF23Ab significantly increased cFGF23 in both sexes and genotypes. Serum 1,25-dihydroxyvitamin D3 was significantly increased in SCD IgG and was further significantly increased by FGF23Ab in both sexes and genotypes. Significantly increased blood urea nitrogen in SCD was not reduced by FGF23Ab. The urine phosphate (Pi)/creatinine ratio was significantly increased in SCD in both sexes and was significantly reduced by FGF23Ab. Increased SCD kidney damage marker kidney injury molecule 1 was rescued, but sclerotic glomeruli, increased macrophages, and lymphocytes were not rescued by short-term FGF23Ab. FGF23Ab significantly reduced increased phospho-fibroblast growth factor receptor 1, αKlotho, phosphorylated extracellular signal-regulated kinase, phosphorylated serum/glucocorticoid-regulated kinase 1, phosphorylated sodium-hydrogen exchanger regulatory factor-1, phosphorylated janus kinase 3, and phosphorylated transducer and activator of transcription-3 in SCD kidneys. The type II sodium Pi cotransporter (NPT2a) and sodium-dependent Pi transporter PiT-2 proteins were significantly reduced in SCD kidneys and were increased by FGF23Ab. We conclude that increased FGF23/FGF receptor 1/αKlotho signaling promotes Pi wasting in SCD by downregulating NPT2a and PIT2 via modulation of multiple signaling pathways that could be rescued by FGF23Ab.

FGF receptor inhibitor BGJ398 partially rescues osteoarthritis-like phenotype in older high molecular weight FGF2 transgenic mice via multiple mechanisms.

We have used Basic Fibroblast Growth Factor (FGF2) transgenic mice as experimental models for human X-linked hypophosphatemia (XLH)-related degenerative osteoarthritis (OA) to investigate the pathogenesis of the disease and to test potential pharmacotherapies for treatment. This study tested the efficacy of BJG398, a small molecule fibroblast growth factor receptor tyrosine kinase (FGFRTK) inhibitor, to rescue the knee joint osteoarthritis phenotype in High Molecular Weight fibroblast growth factor 2 transgenic (HMWTgFGF2) mice. BJG398 was administered in vivo to 8-month-old female HMWTgFGF2 mice for six weeks. Histomorphometry, immunohistochemistry and micro-CT were used to examine the knee joints in BGJ398-treated and control mice. We assessed: Fibroblast Growth Factor 23 (FGF23) expression and FGFR1 activity; Matrix metalloproteinase 13 (MMP13) and Aggrecanase2 (ADAMTS5) expression; then signaling by SMAD1/5/8-pSMAD6, pERK1/2 and Runt-related transcription factor 2 (RUNX2). Using PrimePCR arrays, we identified a contributing role for major target genes in the TGFB/BMP2 signaling pathway that were regulated by BGJ398. BGJ398 inhibited HMWFGF2/FGF23-induced increase in bone morphogenic protein receptor-1, bone morphogenic protein-2 and 4 and Serine peptidase inhibitor, clade E, member 1. The results from Micro-CT and histology show BGJ398 treatment rescued the OA changes in subchondral bone and knee articular cartilage of HMWTgFGF2 mice. The gene expression and signal transduction results provide convincing evidence that HMWFGF2 generates OA through FGFRTK with characteristic downstream signaling that defines OA, namely: increased FGF23-FGFR1 activity with BMP-BMPR, activation of pSMAD1/5/8-RUNX2 and pERK signaling pathways, then upregulation of MMP13 and ADAMTS5 to degrade matrix. BGJ398 treatment effectively reversed these OA molecular phenotypes, providing further evidence that the OA generated by HMWFGF2 in the transgenic mice is FGFR-mediated and phenocopies the OA found in the Hyp mouse homolog of XLH with a spontaneous mutation in the Phex (phosphate regulating endopeptidase on the X chromosome) gene and human XLH-OA. Overall, the results obtained here explain how the pleotropic effects of FGF2 emanate from the different functions of HMW protein isoforms for cartilage and bone homeostasis, and the pathogenesis of XLH-degenerative osteoarthropathy. BGJ398 inhibits HMWFGF2-induced osteoarthritis via multiple mechanisms. These results provided important scientific evidence for the potential application of BGJ398 as a therapeutic agent for osteoarthritis in XLH.

Fibroblast Growth Factor 2 High Molecular Weight Isoforms in Dentoalveolar Mineralization.

Transgenic mice overexpressing human high molecular weight fibroblast growth factor 2 (HMWFGF2) isoforms in osteoblast and odontoblast lineages (HMWTg) exhibit decreased dentin and alveolar bone mineralization, enlarged pulp chamber, and increased fibroblast growth factor 23 (FGF23). We examined if the alveolar bone and dentin mineralization defects in HMWTg mice resulted from increased FGF23 expression and whether an FGF23 neutralizing antibody could rescue the hypomineralization phenotype. HMWTg and VectorTg control mice were given subcutaneous injections of FGF23 neutralizing antibody twice/week starting at postnatal day 21 for 6 weeks. Since Calcitriol (1,25D) have direct effects in promoting bone mineralization, we also determined if 1,25D protects against the defective dentin and alveolar bone mineralization. Therefore, HMWTg mice were given subcutaneous injections of 1,25D daily or concomitantly with FGF23 neutralizing antibody for 6 weeks. Our results showed that HMWTg mice displayed thickened predentin, alveolar bone hypomineralization, and enlarged pulp chambers. FGF23 neutralizing antibody and 1,25D monotherapy partially rescued the dentin mineralization defects and the enlarged pulp chamber phenotype in HMWTg mice. 1,25D alone was not sufficient to rescue the alveolar bone hypomineralization. Interestingly, HMWTg mice treated with both FGF23 neutralizing antibody and 1.25D further rescued the enlarged pulp chamber size, and dentin and alveolar bone mineralization defects. We conclude that the dentin and alveolar bone mineralization defects in HMWTg mice might result from increased FGF23 expression. Our results show a novel role of HMWFGF2 on dentoalveolar mineralization.

Endogenous FGF-2 levels impact FGF-2/BMP-2 growth factor delivery dosing in aged murine calvarial bone defects.

Bone repair in elderly mice has been shown to be improved or negatively impacted by supplementing the highly osteogenic bone morphogenetic protein-2 (BMP-2) with fibroblast growth factor-2 (FGF-2). To better predict the outcome of FGF-2 supplementation, we investigated whether endogenous levels of FGF-2 play a role in optimal dosing of FGF-2 for augmenting BMP-2 activity in elderly mice. In vivo calvarial bone defect studies in Fgf2 knockout mice with wildtype controls were conducted with the growth factors delivered in a highly localized manner from a biomimetic calcium phosphate/polyelectrolyte multilayer coating applied to a bone graft substitute. Endogenous FGF-2 levels were measured in old mice versus young and found to decrease with age. Optimal dosing for improving bone defect repair correlated with levels of endogenous FGF-2, with a larger dose of FGF-2 required to have a positive effect on bone healing in the Fgf2 knockout mice. The same dose in wildtype old mice, with higher levels of FGF-2, promoted chondrogenesis and increased osteoclast activity. The results suggest a personalized medicine approach, based on a knowledge of endogenous levels of FGF-2, should guide FGF-2 supplementation in order to avoid provoking excessive bone resorption and cartilage formation, both of which inhibited calvarial bone repair.

Gait disturbances and muscle dysfunction in fibroblast growth factor 2 knockout mice.

Fibroblast growth factor 2 (FGF2) is important in musculoskeletal homeostasis, therefore the impact of reduction or Fgf2 knockout on skeletal muscle function and phenotype was determined. Gait analysis as well as muscle strength testing in young and old WT and Fgf2KO demonstrated age-related gait disturbances and reduction in muscle strength that were exacerbated in the KO condition. Fgf2 mRNA and protein were significantly decreased in skeletal muscle of old WT compared with young WT. Muscle fiber cross-sectional area was significantly reduced with increased fibrosis and inflammatory infiltrates in old WT and Fgf2KO vs. young WT. Inflammatory cells were further significantly increased in old Fgf2KO compared with old WT. Lipid-related genes and intramuscular fat was increased in old WT and old Fgf2KO with a further increase in fibro-adipocytes in old Fgf2KO compared with old WT. Impaired FGF signaling including Increased ß-Klotho, Fgf21 mRNA, FGF21 protein, phosphorylated FGF receptors 1 and 3, was observed in old WT and old Fgf2KO. MAPK/ ERK1/2 was significantly increased in young and old Fgf2KO. We conclude that Fgf2KO, age-related decreased FGF2 in WT mice, and increased FGF21 in the setting of impaired Fgf2 expression likely contribute to impaired skeletal muscle function and sarcopenia in mice.

Creating a Comprehensive Approach to Exposing Underrepresented Pre-health Professions Students to Clinical Medicine and Health Research.

BACKGROUND/PURPOSE: With the urgency to create more equitable health care, increased research and early exposure to health interventions and clinical medicine are imperative. Health disparities continue to persist nationwide, particularly in underserved areas and among traditionally disadvantaged populations. In addition to the need to eliminate health disparities, increasing the diversity among health professionals to more accurately reflect the US population is essential. METHODS: The health professions partnership at the School of Medicine and the School of Dental Medicine is a comprehensive pipeline designed to increase the preparation of underrepresented students for health careers. Through this health professions pipeline's Health Disparities Clinical Summer Research Fellowship Program (HDCSRFP), undergraduate students are exposed to health disparities research and clinical skills over seven weeks. Over the course of the program, participants conducted a research project, gained clinical exposure by shadowing community physicians and other health professionals, and received mentoring by health professional faculty and students. At the conclusion of the program, participants presented their research projects during a poster symposium. RESULTS: A total of 121 program participants between 2008 and 2018 each conducted a research project focused on reducing health inequities within specific populations, particularly in urban settings. The health professions pipeline has been instrumental in increasing the aptitude and competitiveness of these students pursuing health careers through participation in research, clinical medicine, and enrichment activities. Specifically, 92% of the 79 program participants identified who completed undergraduate studies before the end of the 2018 fall semester pursued a career or further studies within a health profession. Forty-six percent of these college graduates were accepted or matriculated in medical school by the end of 2018. CONCLUSION: The HDCSRFP, like the other health professions partnership pipeline programs, serves as a model for other educational programs to expose students to the field of medicine and health research, and to increase diversity within health professions.

Inhibition of FGFR Signaling Partially Rescues Osteoarthritis in Mice Overexpressing High Molecular Weight FGF2 Isoforms.

Fibroblast growth factor 2 (FGF2) and fibroblast growth factor receptors (FGFRs) are key regulatory factors in osteoarthritis (OA). HMWTg mice overexpress the high molecular weight FGF2 isoforms (HMWFGF2) in osteoblast lineage and phenocopy both Hyp mice (which overexpress the HMWFGF2 isoforms in osteoblasts and osteocytes) and humans with X-linked hypophosphatemia (XLH). We previously reported that, similar to Hyp mice and XLH subjects who develop OA, HMWTg mice also develop an OA phenotype associated with increased degradative enzymes and increased FGFR1 compared with VectorTg mice. Therefore, in this study, we examined whether in vivo treatment with the FGFR tyrosine kinase inhibitor NVP-BGJ398 (BGJ) would modulate development of the OA phenotype in knee joints of HMWTg mice. VectorTg and HMWTg mice (21 days of age) were treated with vehicle or BGJ for 13 weeks. Micro-computed tomography images revealed irregular shape and thinning of the subchondral bone with decreased trabecular number and thickness within the epiphyses of vehicle-treated HMWTg knees, which was partially rescued following BGJ treatment. Articular cartilage thickness was decreased in vehicle-treated HMWTg mice, and was restored to the cartilage thickness of VectorTg mice in the BGJ-treated HMWTg group. Increased OA degradative enzymes present in HMWTg vehicle-treated joints decreased after BGJ treatment. OA in HMWTg mice was associated with increased Wnt signaling that was rescued by BGJ treatment. This study demonstrates that overexpression of the HMWFGF2 isoforms in preosteoblasts results in osteoarthropathy that can be partially rescued by FGFR inhibitor via reduction in activated Wnt signaling.

A Health Professions Pipeline for Underrepresented Students: Middle and High School Initiatives.

The Department of Health Career Opportunity Programs at UConn Health has developed the Aetna Health Professions Partnership Initiative (Aetna HPPI), a formal education consortium offering a comprehensive program of educational enrichment and support activities for underrepresented and first-generation students. The purpose is to identify and develop a diverse applicant pool of students who will eventually enter a health professions career with a focus on medicine and dental medicine. Activities are conducted for students in middle school through college. The achievements of the middle and high school pipeline programs and their impact on producing a more diversified health professions workforce were examined. The students are recruited from the greater Hartford, CT area and come from backgrounds traditionally underrepresented in healthcare, first-generation college families and modest family means. Program elements include a 30-week academic year Saturday Academy and a 6-week summer academic enrichment program aimed at preparing students for successful entrance into college, and a Parental Seminar Series for parents. Some of the activities include science, math, language arts, PSAT, SAT and ACT preparation, college tours, career counseling, mentoring by health professionals, and cultural experiences. Data analysis and tracking of the students in the academy have revealed some significant achievements. All seniors in the academy have graduated from high school. The SAT scores of the academy students have consistently stood above the average for the rest of the Hartford School District. In addition, the graduating seniors have a high rate of college matriculation.

Ablation of low-molecular-weight FGF2 isoform accelerates murine osteoarthritis while loss of high-molecular-weight FGF2 isoforms offers protection.

FGF2 is an essential growth factor implicated in osteoarthritis (OA), and deletion of full-length FGF2 (Fgf2ALLKO ) leads to murine OA. However, the FGF2 gene encodes both high-molecular-weight (HMW) and low-molecular-weight (LMW) isoforms, and the effects of selectively ablating individual isoforms, as opposed to total FGF2, has not been investigated in the context of OA. We undertook this study to examine whether mice lacking HMW FGF2 (Fgf2HMWKO ) or LMW FGF2 (Fgf2LMWKO ) develop OA and to further characterize the observed OA phenotype in Fgf2ALLKO mice. Fgf2HMWKO mice never developed OA, but 6- and 9-month-old Fgf2LMWKO and Fgf2ALLKO mice displayed signs of OA, including eroded articular cartilage, altered subchondral bone and trabecular architecture, and increased OA marker enzyme levels. Even with mechanical induction of OA, Fgf2HMWKO mice were protected against OA, whereas Fgf2LMWKO and Fgf2ALLKO displayed OA-like changes of the subchondral bone. Before exhibiting OA symptoms, Fgf2LMWKO or Fgf2ALLKO joints displayed differential expression of genes encoding key regulatory proteins, including interleukin-1ß, insulin-like growth factor 1, bone morphogenetic protein 4, hypoxia-inducible factor 1, B-cell lymphoma 2, Bcl2-associated X protein, a disintegrin and metalloproteinase with thrombospondin motifs 5, ETS domain-containing protein, and sex-determining region Y box 9. Moreover, Fgf2LMWKO OA cartilage exhibited increased FGF2, FGF23, and FGFR1 expression, whereas Fgf2HMWKO cartilage had increased levels of FGFR3, which promotes anabolism in cartilage. These results demonstrate that loss of LMW FGF2 results in catabolic activity in joint cartilage, whereas absence of HMW FGF2 with only the presence of LMW FGF2 offers protection from OA.

FGF2 crosstalk with Wnt signaling in mediating the anabolic action of PTH on bone formation.

The mechanisms of the anabolic effect of parathyroid hormone (PTH) in bone are not fully defined. The bone anabolic effects of PTH require fibroblast growth factor 2 (FGF2) as well as Wnt signaling and FGF2 modulates Wnt signaling in osteoblasts. In vivo PTH administration differentially modulated Wnt signaling in bones of wild type (WT) and in mice that Fgf2 was knocked out (Fgf2KO). PTH increased Wnt10b mRNA and protein in WT but not in KO mice. Wnt antagonist SOST mRNA and protein was significantly higher in KO group. However, PTH decreased Sost mRNA significantly in WT as well as in Fgf2KO mice, but to a lesser extent in Fgf2KO. Dickhopf 2 (DKK2) is critical for osteoblast mineralization. PTH increased Dkk2 mRNA in WT mice but the response was impaired in Fgf2KO mice. PTH significantly increased Lrp5 mRNA and phosphorylation of Lrp6 in WT but the increase was markedly attenuated in Fgf2KO mice. PTH increased ß-catenin expression and Wnt/ß-catenin transcriptional activity significantly in WT but not in Fgf2KO mice. These data suggest that the impaired bone anabolic response to PTH in Fgf2KO mice is partially mediated by attenuated Wnt signaling.

FGF23 Regulates Wnt/ß-Catenin Signaling-Mediated Osteoarthritis in Mice Overexpressing High-Molecular-Weight FGF2.

Although humans with X-linked hypophosphatemia (XLH) and the Hyp mouse, a murine homolog of XLH, are known to develop degenerative joint disease, the exact mechanism that drives the osteoarthritis (OA) phenotype remains unclear. Mice that overexpress high-molecular-weight fibroblast growth factor (FGF) 2 isoforms (HMWTg mice) phenocopy both XLH and Hyp, including OA with increased FGF23 production in bone and serum. Because HMWTg cartilage also has increased FGF23 and there is cross-talk between FGF23-Wnt/ß-catenin signaling, the purpose of this study was to determine if OA observed in HMWTg mice is due to FGF23-mediated canonical Wnt signaling in chondrocytes, given that both pathways are implicated in OA pathogenesis. HMWTg OA joints had decreased Dkk1, Sost, and Lrp6 expression with increased Wnt5a, Wnt7b, Lrp5, Axin2, phospho-GSK3ß, Lef1, and nuclear ß-catenin, as indicated by immunohistochemistry or quantitative PCR analysis. Chondrocytes from HMWTg mice had enhanced alcian blue and alkaline phosphatase staining as well as increased FGF23, Adamts5, Il-1ß, Wnt7b, Wnt16, and Wisp1 gene expression and phospho-GSK3ß protein expression as indicated by Western blot, compared with chondrocytes of vector control and chondrocytes from mice overexpressing the low-molecular-weight isoform, which were protected from OA. Canonical Wnt inhibitor treatment rescued some of those parameters in HMWTg chondrocytes, seemingly delaying the initially accelerated chondrogenic differentiation. FGF23 neutralizing antibody treatment was able to partly ameliorate OA abnormalities in subchondral bone and reduce degradative/hypertrophic chondrogenic marker expression in HMWTg joints in vivo. These results demonstrate that osteoarthropathy of HMWTg is at least partially due to FGF23-modulated Wnt/ß-catenin signaling in chondrocytes.

FGF23 Neutralizing Antibody Partially Improves Bone Mineralization Defect of HMWFGF2 Isoforms in Transgenic Female Mice.

Mice overexpressing high molecular weight FGF2 isoforms (HMWTg) in osteoblast lineage phenocopy human X-linked hypophosphatemic rickets (XLH) and a Hyp murine model of XLH demonstrating increased FGF23/FGF receptor signaling and hypophosphatemic rickets/osteomalacia. Because HMWFGF2 was upregulated in bones of Hyp mice and abnormal FGF23 signaling is important in XLH, HMWTg mice were used to examine the effect of the FGF23 neutralizing antibody (FGF23Ab). Eight-week-old female Vector control mice and HMWTg mice were treated with FGF23Ab or control IgG. A single injection of FGF23Ab rescued abnormal hypophosphatemia in HMWTg. The decreased type II sodium-dependent phosphate co-transporter (Npt2a) was rescued by FGF23Ab treatment. Inappropriately low serum 1,25(OH)2 D in HMWTg mice was normalized by FGF23Ab treatment, which is accompanied by increased anabolic vitamin D hydroxylase Cyp27b1 and decreased catabolic vitamin D hydroxylase Cyp24 mRNA in kidney. Long-term treatment with FGF23Ab normalized femur length and significantly increased vertebrae BMD and BMC, and femur BMC in HMWTg mice compared to IgG-treated HMWTg mice. Micro-computed tomography (µCT) revealed increased cortical porosity and decreased cortical apparent density in the HMWTg-IgG group compared with the Vector-IgG group; however, FGF23Ab treatment rescued defective cortical mineralization, decreased porosity, and increased apparent density in HMWTg mice. Bone histomorphometry analysis showed FGF23Ab treatment decreased osteoid volume, increased intra-label thickness, mineralization apposition rate, and bone formation rate in HMWTg mice. FGF23Ab improved disorganized double labeling in femurs from HMWTg mice. Quantitative real-time PCR analysis of tibia shafts showed FGF23Ab treatment normalized the osteocalcin (Ocn) mRNA expression in HMWTg mice, but further increased expression of SIBLING protein-related and pyrophosphate-related genes that are important in matrix mineralization, suggesting that HMWFGF2 modulates these genes independent of FGF23. We conclude that FGF23Ab partially rescued hypophosphatemic osteomalacia in HMWTg. However, long-term treatment with FGF23Ab further increased SIBLING protein-related genes and pyrophosphate-related genes in bone that could contribute to incomplete rescue of the mineralization defect. © 2018 American Society for Bone and Mineral Research.

Inhibition of FGFR Signaling Partially Rescues Hypophosphatemic Rickets in HMWFGF2 Tg Male Mice.

Transgenic mice harboring high molecular weight fibroblast growth factor (FGF)2 isoforms (HMWTg) in osteoblast lineage cells phenocopy human X-linked hypophosphatemic rickets (XLH) and Hyp murine model of XLH demonstrating increased FGF23/FGF receptor signaling and hypophosphatemic rickets. Because HMWFGF2 was upregulated in bones of Hyp mice and abnormal FGF receptor (FGFR) signaling is important in XLH, HMWTg mice were used to examine the effect of the FGFR inhibitor NVP-BGJ398, now in clinical trials for cancer therapy, on hypophosphatemic rickets. Short-term treatment with NVP-BGJ398 rescued abnormal FGFR signaling and hypophosphatemia in HMWTg. Long-term treatment with NVP-BGJ398 normalized tail, tibia, and femur length. Four weeks NVP-BGJ398 treatment significantly increased total body bone mineral density (BMD) and bone mineral content (BMC) in HMWTg mice; however, at 8 weeks, total body BMD and BMC was indistinguishable among groups. Micro-computed tomography revealed decreased vertebral bone volume, trabecular number, and increased trabecular spacing, whereas femur trabecular tissue density was increased; however, NVP-BGJ398 rescued defective cortical bone mineralization, increased thickness, reduced porosity, and increased endosteal perimeter and cortical tissue density in HMWTg. NVP-BGJ398 improved femur cancellous bone, cortical bone structure, growth plate, and double labeling in cortical bone and also increased femur trabeculae double labeled surface, mineral apposition rate, bone formation rate, and osteoclast number and surface in HMWTg. The decreased NPT2a protein that is important for renal phosphate excretion was rescued by NVP-BGJ398 treatment. We conclude that NVP-BGJ398 partially rescued hypophosphatemic rickets in HMWTg. However, long-term treatment with NVP-BGJ398 further increased serum FGF23 that could exacerbate the mineralization defect.

FGF2 High Molecular Weight Isoforms Contribute to Osteoarthropathy in Male Mice.

Humans with X-linked hypophosphatemia (XLH) and Hyp mice, the murine homolog of the disease, develop severe osteoarthropathy and the precise factors that contribute to this joint degeneration remain largely unknown. Fibroblast growth factor 2 (FGF2) is a key regulatory growth factor in osteoarthritis. Although there are multiple FGF2 isoforms the potential involvement of specific FGF2 isoforms in joint degradation has not been investigated. Mice that overexpress the high molecular weight FGF2 isoforms in bone (HMWTg mice) phenocopy Hyp mice and XLH subjects and Hyp mice overexpress the HMWFGF2 isoforms in osteoblasts and osteocytes. Given that Hyp mice and XLH subjects develop osteoarthropathies we examined whether HMWTg mice also develop knee joint degeneration at 2, 8, and 18 mo compared with VectorTg (control) mice. HMWTg mice developed spontaneous osteoarthropathy as early as age 2 mo with thinning of subchondral bone, osteophyte formation, decreased articular cartilage thickness, abnormal mineralization within the joint, increased cartilage degradative enzymes, hypertrophic markers, and angiogenesis. FGF receptors 1 and 3 and fibroblast growth factor 23 were significantly altered compared with VectorTg mice. In addition, gene expression of growth factors and cytokines including bone morphogenetic proteins, Insulin like growth factor 1, Interleukin 1 beta, as well as transcription factors Sex determining region Y box 9, hypoxia inducible factor 1, and nuclear factor kappa B subunit 1 were differentially modulated in HMWTg compared with VectorTg. This study demonstrates that overexpression of the HMW isoforms of FGF2 in bone results in catabolic activity in joint cartilage and bone that leads to osteoarthropathy.

Loss of Bone in Sickle Cell Trait and Sickle Cell Disease Female Mice Is Associated With Reduced IGF-1 in Bone and Serum.

Characterization of the bone phenotype of 24-week-old female transgenic sickle cell disease (SCD), sickle cell trait (SCT) revealed significant reductions in bone mineral density and bone mineral content relative to control with a further significant decreased in SCD compared with SCT. By microcomputed tomography, femur middiaphyseal cortical area was significantly reduced in SCT and SCD. Cortical thickness was significantly decreased in SCD vs control. Diaphysis structural stiffness and strength were significantly reduced in SCT and SCD. Histomorphometry showed a significant increase in osteoclast perimeter in SCD and significantly decreased bone formation in SCD and SCT compared with control with a further significant decrease in SCD compared with SCT. Collagen-I mRNA was significantly decreased in tibiae from SCT and SCD and osterix, Runx2, osteoclacin, and Dmp-1 mRNA were significantly decreased in tibiae of SCD compared with control. Serum osteocalcin was significantly decreased and ferritin was significantly increased in SCD compared with control. Igf1 mRNA and serum IGF1 were significantly decreased in SCD and SCT. IGF1 protein was decreased in bone marrow stromal cells from SCT and SCD cultured in osteogenic media. Crystal violet staining revealed fewer cells and significantly reduced alkaline phosphatase positive mineralized nodules in SCT and SCD that was rescued by IGF1 treatment. We conclude that reduced bone mass in SCD and SCT mice carries architectural consequences that are detrimental to the mechanical integrity of femoral diaphysis. Furthermore reduced IGF1 and osteoblast terminal differentiation contributed to reduced bone formation in SCT and SCD mice.

FGFR Inhibitor Ameliorates Hypophosphatemia and Impaired Engrailed-1/Wnt Signaling in FGF2 High Molecular Weight Isoform Transgenic Mice.

High molecular weight FGF2 transgenic (HMWTg) mouse phenocopies the Hyp mouse, homolog of human X-linked hypophosphatemic rickets with hypophosphatemis, and abnormal FGF23, FGFR, Klotho signaling in kidney. Since abnormal Wnt signaling was reported in Hyp mice we assessed whether Wnt signaling was impaired in HMWTg kidneys and the effect of blocking FGF receptor (FGFR) signaling. Bone mineral density and bone mineral content in female HMWTg mice were significantly reduced. HMWTg mice were gavaged with FGFR inhibitor NVP-BGJ398, or vehicle and were euthanized 24 h post treatment. Serum phosphate was significantly reduced and urine phosphate was significantly increased in HMWTg and was rescued by NVP-BGJ398. Analysis of kidneys revealed a significant reduction in Npt2a mRNA in HMWTg that was significantly increased by NVP-BGJ398. Increased FGFR1, KLOTHO, P-ERK1/2, and decreased NPT2a protein in HMWTg were rescued by NVP-BGJ398. Wnt inhibitor Engrailed-1 mRNA and protein was increased in HMWTg and was decreased by BGJ398. Akt mRNA and protein was decreased in HMWTg and was increased by NVP-BGJ398. The active form of glycogen synthase 3 beta (pGSK3-ß) and phosphor-ß-catenin were increased in HMWTg and were both decreased by NVP-BGJ398 while decreased active-ß-catenin in HMWTg was increased by NVP-BGJ398. We conclude that FGFR blockade rescued hypophosphatemia by regulating FGF and WNT signaling in HMWTg kidneys. J. Cell. Biochem. 117: 1991-2000, 2016. © 2016 Wiley Periodicals, Inc.

Accelerated fracture healing in transgenic mice overexpressing an anabolic isoform of fibroblast growth factor 2.

The effect of targeted expression of an anabolic isoform of basic fibroblast growth factor (FGF2) in osteoblastic lineage on tibial fracture healing was assessed in mice. Closed fracture of the tibiae was performed in Col3.6-18 kDaFgf2-IRES-GFPsaph mice in which a 3.6 kb fragment of type I collagen promoter (Col3.6) drives the expression of only the 18 kD isoform of FGF2 (18 kDaFgf2/LMW) with green fluorescent protein-sapphire (GFPsaph) as well as Vector mice (Col3.6-IRES-GFPsaph, Vector) that did not harbor the FGF2 transgene. Radiographic, micro-CT, DEXA, and histologic analysis of fracture healing of tibiae harvested at 3, 10 and 20 days showed a smaller fracture callus but accelerated fracture healing in LMWTg compared with Vector mice. At post fracture day 3, FGF receptor 3 and Sox 9 mRNA were significantly increased in LMWTg compared with Vector. Accelerated fracture healing was associated with higher FGF receptor 1, platelet derived growth factors B, C, and D, type X collagen, vascular endothelial cell growth factor, matrix metalloproteinase 9, tartrate resistant acid phosphatase, cathepsin K, runt-related transcription factor-2, Osterix and Osteocalcin and lower Sox9, and type II collagen expression at 10 days post fracture. We postulate that overexpression of LMW FGF2 accelerated the fracture healing process due to its effects on factors that are important in chondrocyte and osteoblast differentiation and vascular invasion.

Age-Related Changes in FGF-2, Fibroblast Growth Factor Receptors and ß-Catenin Expression in Human Mesenchyme-Derived Progenitor Cells.

FGF-2 stimulates preosteoblast replication, and knockout of the FGF-2 gene in mice resulted in osteopenia with age, associated with decreased Wnt-ß-Catenin signaling. In addition, targeted expression of FGF-2 in osteoblast progenitors increased bone mass in mice via Wnt-ß-Catenin signaling. We posited that diminution of the intrinsic proliferative capacity of human mesenchyme-derived progenitor cells (HMDPCs) with age is due in part to reduction in FGF-2. To test this hypothesis HMDPCs from young (27-38), middle aged (47-56), and old (65-76) female human subjects were isolated from bone discarded after orthopedic procedures. HMDPCs cultures were mostly homogeneous with greater than 90% mesenchymal progenitor cells, determined by fluorescence-activated cell sorting. There was a progressive decrease in FGF-2 and FGFR1 mRNA and protein in HMDPCs with age. Since FGF-2 activates ß-catenin, which can enhance bone formation, we also assessed its age-related expression in HMDPCs. An age-related decrease in total-ß-Catenin mRNA and protein expression was observed. However there were increased levels of p-ß-Catenin and decreased levels of activated-ß-Catenin in old HMDSCs. FGF-2 treatment increased FGFR1 and ß-Catenin protein, reduced the level of p-ß-Catenin and increased activated-ß-Catenin in aged HMDPCs. In conclusion, reduction in FGF-2 expression could contribute to age-related impaired function of HMDPCs via modulation of Wnt-ß-catenin signaling.

Effects of low dose FGF-2 and BMP-2 on healing of calvarial defects in old mice.

There is an age-associated reduction in the bone healing activity of bone morphogenetic protein-2 (BMP-2) that is currently addressed by administering higher doses of BMP-2 in elderly patients. The unwanted medical complications from high dose BMP-2 motivated this investigation to determine whether the addition of a low dose of fibroblast growth factor 2 (FGF-2) could enhance the ability of a lower dose of BMP-2 to heal calvarial bone defects in old mice (18-20 months old). FGF-2 (5 ng) and BMP-2 (2 µg) were administered by a controlled release two-phase biomaterial scaffold placed into the bone defect. FGF-2 released more rapidly and completely in vitro than BMP-2 (40% vs 2%). In vivo, both BMP-2 and FGF-2+BMP-2 groups formed more new bone in calvarial defects than scaffold alone (p < 0.001) or FGF-2 only groups (p < 0.01). The overall total volume of new bone was not statistically increased by the addition of FGF-2 to BMP-2 as measured by microCT, but the pattern of bone deposition was different. In old mice, but not young, there was enhanced bony fill in the central bone defect area when the BMP-2 was supplemented with FGF-2. Histological analysis of the center of the defect revealed an increased bone volume (%BV/TV (p = 0.004)) from the addition of FGF-2. These studies suggest that combining a low dose of FGF-2 with a low dose of BMP-2 has the potential to increase bone healing in old mice relative to BMP-2 alone.

Knockout of nuclear high molecular weight FGF2 isoforms in mice modulates bone and phosphate homeostasis.

We previously reported that targeted overexpression of the fibroblast growth factor 2 (FGF2) high molecular weight (HMW) isoforms in osteoblastic lineage cells in mice resulted in phenotypic changes, including dwarfism, rickets, osteomalacia, hypophosphatemia, increased serum parathyroid hormone, and increased levels of the phosphatonin FGF23 in serum and bone. This study examined the effects of genetically knocking out the FGF2HMW isoforms (HMWKO) on bone and phosphate homeostasis. HMWKO mice were not dwarfed and had significantly increased bone mineral density and bone mineral content in femurs and lumbar vertebrae when compared with the wild-type (WT) littermates. Micro-computed tomography analysis of femurs revealed increased trabecular bone volume, thickness, number, and connective tissue density with decreased trabecular spacing compared with WT. In addition, there was significantly decreased cortical porosity and increased cortical thickness and sub-periosteal area in femurs of HMWKO. Histomorphometric analysis demonstrated increased osteoblast activity and diminished osteoclast activity in the HMWKO. In vitro bone marrow stromal cell cultures showed there was a significant increase in alkaline phosphatase-positive colony number at 1 week in HMWKO. At 3 weeks of culture, the mineralized area was also significantly increased. There was increased expression of osteoblast differentiation marker genes and reduced expression of genes associated with impaired mineralization, including a significant reduction in Fgf23 and Sost mRNA. Normal serum phosphate and parathyroid hormone were observed in HMWKO mice. This study demonstrates a significant negative impact of HMWFGF2 on biological functions in bone and phosphate homeostasis in mice.