Fibroblast Growth Factor Activity Research Articles

1527-P: Anorexia Induced by the Central Action of Fibroblast Growth Factor 1 (FGF1) Is Dependent on Reduced GABA Receptor Activation

Previously, we reported that in multiple rodent models of type 2 diabetes, remission of hyperglycemia lasting for weeks can be induced by a single intracerebroventricular (icv) injection of FGF1. This effect is accompanied by a potent but transient anorexic response that is sufficiently robust as to hinder efforts to translate these findings towards the clinic. To understand the mechanism underlying FGF1-induced anorexia, we considered 1) our recent finding that icv FGF1 induces acute and sustained inhibition of hypothalamic AgRP neurons, and 2) prior work showing that parabrachial nucleus that express calcitonin gene-related peptide (PBNCGRP neurons) are both highly anorexigenic and tonically inhibited by GABAergic projections from AgRP neurons. We therefore hypothesized that FGF1-induced anorexia involves activation PBNCGRPneurons arising from reduced GABAergic input. Consistent with this hypothesis, we report that PBNCGRPneurons are robustly activated following icv FGF1 injection (based on c-Fos staining) via a mechanism that appears to be indirect, since the c-Fos induction is not associated with FGF receptor (FGFR) activation (detected by pERK staining in the PBN). We next asked whether the anorexic response to icv FGF1 administration can be blunted by systemic administration of a GABAA receptor agonist. Consistent with this hypothesis, we found that continuous subcutaneous infusion of the GABAA receptor agonist Bretazenil blunts the anorexic response to icv FGF1 injection by ~50% at a dose that has no effect intake in control mice. Similarly, the effect of FGF1 to induce c-Fos in the PBN was largely blocked by Bretazenil, and our preliminary findings indicate that CGRP neurons are among those in which this effect occurred. Together, these data suggest that FGF1-induced anorexia results from reduced GABAergic inhibition of PBNCGRP neurons. Disclosure J.M.Scarlett: None. K.M.Alonge: None. B.N.Phan: None. G.J.Morton: Research Support; Novo Nordisk A/S. M.W.Schwartz: Consultant; NodThera, Research Support; Novo Nordisk A/S. Funding U.S. Department of Defense (PRMRP W81XWH-21-1-0635); National Institutes of Health (DK083042)

THE MAIN CLINICAL AND PATHOGENETIC FEATURES OF THE COMORBID COURSE OF NON-ALCOHOLIC STEATOHEPATITIS, OBESITY AND HYPERTENSION

SUMMARY. The incidence of nonalcoholic steatohepatitis (NASH) in the population is 20–40 %, in obese patients – 50–90 %. The presence of NASH significantly reduces the quality of life of patients. The presence of concomitant arterial hypertension in patients can cause a cascade of reactions of mutual burdens, which will lead to the progression of all comorbid diseases. The aim – to investigate the differences between clinical and laboratory parameters in the comorbid course of NASH on the background of obesity. In addition, we studied the state of hepatic circulation and the functional state of the endothelium depending on the presence of comorbid hypertension. Material and Methods. Systematic review with further analysis, comparison, systematization and generalization of scientific literature in MEDLINE, Cochrane and PubMed databases of relevant articles on the study of psychological characteristics of women of childbearing age diagnosed with psychogenic infertility. Results. In recent years, NASH has increasingly been seen as an additional independent risk factor for cardiovascular disease and a predictor of its complications. The course of NASH under conditions of comorbidity with obesity in comparison with the isolated course is characterized by a predominance of hypertension of II degree (60.0 %), increased variability of systolic blood pressure (SBP) during the day with increasing duration of “pressure load” during the day. The comorbid course of NASH with obesity and hypertension (HT) there is a significant activation of the processes of liver tissue fibrosis, the content of protein-bound oxyproline in the blood, fibronectin, hexosamines, sialic acids, fucose, not bound to protein due to the activation of fibroblast growth factor, in response to which there is usually inadequate compensatory activation of collagenolysis (increase in the content of free oxyproline in the blood, which was inhibited by excessively activated proteinase-inhibitory system). Conclusions. The comorbidity of NASH with HT and obesity is a powerful pathogenetic factor that worsens the quality of life of patients to a much greater extent than the severity of a single disease. It's crucial to have multidisciplinary approach in the treatment of people with comorbidity of HT with NASH on the background of obesity.

FGF2 activity regulates operant alcohol self-administration and mesolimbic dopamine transmission

Fibroblast growth factor 2 (FGF2) is involved in the development and maintenance of the brain dopamine system. We previously showed that alcohol exposure alters the expression of FGF2 and its receptor, FGF receptor 1 (FGFR1) in mesolimbic and nigrostriatal brain regions, and that FGF2 is a positive regulator of alcohol drinking. Here, we determined the effects of FGF2 and of FGFR1 inhibition on alcohol consumption, seeking and relapse, using a rat operant self-administration paradigm. In addition, we characterized the effects of FGF2-FGFR1 activation and inhibition on mesolimbic and nigrostriatal dopamine neuron activation using in vivo electrophysiology. We found that recombinant FGF2 (rFGF2) increased the firing rate and burst firing activity of dopaminergic neurons in the mesolimbic and nigrostriatal systems and led to increased operant alcohol self-administration. In contrast, the FGFR1 inhibitor PD173074 suppressed the firing rate of these dopaminergic neurons, and reduced operant alcohol self-administration. Alcohol seeking behavior was not affected by PD173074, but this FGFR1 inhibitor reduced post-abstinence relapse to alcohol consumption, albeit only in male rats. The latter was paralleled by the increased potency and efficacy of PD173074 in inhibiting dopamine neuron firing. Together, our findings suggest that targeting the FGF2-FGFR1 pathway can reduce alcohol consumption, possibly via altering mesolimbic and nigrostriatal neuronal activity.

FIRST INTERIM ANALYSIS OF THE INTERNATIONAL X-LINKED HYPOPHOSPHATAEMIA (XLH) REGISTRY: ORTHOPAEDIC BASELINE CHARACTERISTICS

X-Linked Hypophosphataemia (XLH) is a rare, progressive, hereditary phosphate-wasting disorder characterised by excessive activity of fibroblast growth factor 23. The International XLH Registry was established to provide information on the natural history of XLH and impact of treatment on patient outcomes. The cross-sectional orthopaedic data presented are from the first interim analysis.The XLH Registry (NCT03193476) was initiated in August 2017, aims to recruit 1,200 children and adults with XLH, and will run for 10 years. At the time of analysis (Last Patient In: 30/11/2020; Database Lock: 29/03/2021) 579 subjects diagnosed with XLH were enrolled from 81 hospital sites in 16 countries (360 (62.2%) children, 217 (37.5%) adults, and 2 subjects of unknown age).Of subjects with retrospective clinical data available, skeletal deficits were the most frequently self-reported clinical problems for children (223/239, 93.3%) and adults (79/110, 71.8%). Retrospective fracture data were available for 183 subjects (72 children, 111 adults); 50 had a fracture (9 children, 41 adults). In children, fractures tended to occur in tibia/fibula and/or wrist; only adults reported large bone fractures. Joint conditions were noted for 46 subjects (6 children, 40 adults). For adults reporting osteoarthritis, knees (60%), hips (42.5%), and shoulders (22.5%) were the most frequently affected joints. Retrospective orthopaedic surgery data were collected for 151 subjects (52 children, 99 adults). Osteotomy was the most frequent surgery reported (n=108); joint replacements were recorded for adults only.This is the largest set of orthopaedic data from XLH subjects collected to date. Longitudinal information collected during the 10-year Registry duration will generate real-world evidence which will help to inform clinical practice.Authors acknowledge the contribution of all International XLH Registry Steering Committee members.

Effects of gallic acid on gingival wounds.

Our aim in this study was to investigate the effect of Gallic acid (GA) on gingival tissue injury. Twenty rats were categorized into two groups. In the burn group, an excisional wound area was created by removing a 4 mm diameter flap from the left molar region in the mucoperiosteal region of the gingiva. In the Burn+gallic acid group, 1.2 mg/ml GA was administered as irrigation for 1 week. Animals were sacrificed under anesthesia at the end of experiment. Malondialdehyde (MDA), myeloperoxidase (MPO) and glutathione (GSH) levels were measured. Hematoxylin Eosin, fibroblast growth factor (FGF) and epidermal growth factor (EGF) immunostaining was applied to tissues. MDA and MPO levels increased, and GSH, epithelization, FGF and EGF expression levels were decreased. Gallic acid treatment improved these scores. Degenerated gingival epithelium, disintegrity in epithelial and connective tissue fibers, edema and inflammatory cells were observed in the burn group. Gallic acid treatment after burn improved the pathologies. After burn injury, FGF and EGF activity was increased in Gallic acid-treated groups. We suggest that GA has the potential for better healing outcomes in oral wounds. GA seems to have promising therapeutic efficacy in enhancing oral wound healing.

Activated FGFR3 suppresses bone regeneration and bone mineralization in an ovariectomized mouse model

BackgroundPostmenopausal osteoporosis is a widespread health concern due to its prevalence among older adults and an associated high risk of fracture. The downregulation of bone regeneration delays fracture healing. Activated fibroblast growth factor receptor 3 (FGFR3) accelerates bone regeneration at juvenile age and downregulates bone mineralization at all ages. However, the impact of FGFR3 signaling on bone regeneration and bone mineralization post-menopause is still unknown. This study aimed to evaluate the impact of FGFR3 signaling on bone regeneration and bone mineralization during menopause by developing a distraction osteogenesis (DO) mouse model after ovariectomy (OVX) using transgenic mice with activated FGFR3 driven by Col2a1 promoter (Fgfr3 mice).MethodsThe OVX or sham operations were performed in 8-week-old female Fgfr3 and wild-type mice. After 8 weeks of OVX surgery, DO surgery in the lower limb was performed. The 5-day-latency period followed by performing distraction for 9 days. Bone mineral density (BMD) and bone regeneration was assessed by micro-computed tomography (micro-CT) scan and soft X-ray. Bone volume in the distraction area was also evaluated by histological analysis after 7 days at the end of distraction. Osteogenic differentiation and mineralization of bone marrow-derived mesenchymal stem cells (BMSCs) derived from each mouse after 8 weeks of the OVX or sham operations were also evaluated with and without an inhibitor for FGFR3 signaling (meclozine).ResultsBMD decreased after OVX in both groups, and it further deteriorated in Fgfr3 mice. Poor callus formation after DO was also observed in both groups with OVX, and the amount of regenerated bone was further decreased in Fgfr3 mice. Similarly, histological analysis revealed that Fgfr3 OVX mice showed lower bone volume. Osteogenic differentiation and mineralization of BMSCs were also deteriorated in Fgfr3 OVX mice. An inhibitor for FGFR3 signaling dramatically reversed the inhibitory effect of OVX and FGFR3 signaling on BMSC mineralization.ConclusionUpregulated FGFR3 decreased newly regenerated bone after DO and BMD in OVX mice. FGFR3 signaling can be a potential therapeutic target in patients with postmenopausal osteoporosis.

Direct and indirect effects of fibroblast growth factor 23 on the heart.

Fibroblast growth factor (FGF)23 is a bone-derived phosphotropic hormone that regulates phosphate and mineral homeostasis. Recent studies have provided evidence that a high plasma concentration of FGF23 is associated with cardiac disease, including left ventricular hypertrophy (LVH), heart failure, atrial fibrillation, and cardiac death. Experimental studies have shown that FGF23 activates fibroblast growth factor receptor 4 (FGFR4)/phospholipase Cγ/calcineurin/nuclear factor of activated T-cells signaling in cardiomyocytes and induces cardiac hypertrophy in rodents. Activation of FGFR4 by FGF23 normally requires the co-receptor α-klotho, and klotho-independent signaling occurs only under conditions characterized by extremely high FGF23 concentrations. Recent studies have demonstrated that FGF23 activates the renin-angiotensin-aldosterone system (RAAS) and induces LVH, at least in part as a result of lower vitamin D activation. Moreover, crosstalk between FGF23 and RAAS results in the induction of cardiac hypertrophy and fibrosis. In this review, we summarize the results of studies regarding the relationships between FGF23 and cardiac events, and describe the potential direct and indirect mechanisms whereby FGF23 induces LVH.

Piezo1 (Piezo-Type Mechanosensitive Ion Channel Component 1)-Mediated Mechanosensation in Macrophages Impairs Perfusion Recovery After Hindlimb Ischemia in Mice.

Angiogenesis is a promising strategy for those with peripheral artery disease. Macrophage-centered inflammation is intended to govern the deficiency of the angiogenic response after hindlimb ischemia. However, little is known about the mechanism of macrophage activation beyond signals from cytokines and chemokines. We sought to identify a novel mechanical signal from the ischemic microenvironment that provokes macrophages and the subsequent inflammatory cascade and to investigate the potential role of Piezo-type mechanosensitive ion channels (Piezo) on macrophages during this process. Myeloid cell-specific Piezo1 (Piezo-type mechanosensitive ion channel component 1) knockout (Piezo1ΔMΦ) mice were generated by crossing Piezo1fl/fl (LysM-Cre-/-; Piezo1 flox/flox) mice with LysM-Cre transgenic mice to assess the roles of Piezo1 in macrophages after hindlimb ischemia. Furthermore, in vitro studies were carried out in bone marrow-derived macrophages to decipher the underlying mechanism. We found that tissue stiffness gradually increased after hindlimb ischemia, as indicated by Young's modulus. Compared to Piezo2, Piezo1 expression and activation were markedly upregulated in macrophages from ischemic tissues in concurrence with increased tissue stiffness. Piezo1ΔMΦ mice exhibited improved perfusion recovery by enhancing angiogenesis. Matrigel tube formation assays revealed that Piezo1 deletion promoted angiogenesis by enhancing FGF2 (fibroblast growth factor-2) paracrine signaling in macrophages. Conversely, activation of Piezo1 by increased stiffness or the agonist Yoda1 led to reduced FGF2 production in bone marrow-derived macrophages, which could be blocked by Piezo1 silencing. Mechanistically, Piezo1 mediated extracellular Ca2+ influx and activated Ca2+-dependent CaMKII (calcium/calmodulin-dependent protein kinase II)/ETS1 (ETS proto-oncogene 1) signaling, leading to transcriptional inactivation of FGF2. This study uncovers a crucial role of microenvironmental stiffness in exacerbating the macrophage-dependent deficient angiogenic response. Deletion of macrophage Piezo1 promotes perfusion recovery after hindlimb ischemia through CaMKII/ETS1-mediated transcriptional activation of FGF2. This provides a promising therapeutic strategy to enhance angiogenesis in ischemic diseases.

FGF23 Actions in CKD-MBD and other Organs During CKD.

Fibroblast growth factor 23 (FGF23) is a new endocrine product discovered in the past decade. In addition to being related to bone diseases, it has also been found to be related to kidney metabolism and parathyroid metabolism, especially as a biomarker and a key factor to be used in kidney diseases. FGF23 is upregulated as early as the second and third stages of chronic kidney disease (CKD) in response to relative phosphorus overload. The early rise of FGF23 has a protective effect on the body and is essential for maintaining phosphate balance. However, with the decline in renal function, eGFR (estimated glomerular filtration rate) declines, and the phosphorus excretion effect caused by FGF23 is weakened. It eventually leads to a variety of complications, such as bone disease (Chronic Kidney Disease-Mineral and Bone Metabolism Disorder), vascular calcification (VC), and more. Monoclonal antibodies against FGF23 are currently used to treat genetic diseases with increased FGF23. CKD is also a state of increased FGF23. This article reviews the current role of FGF23 in CKD and discusses the crosstalk between various organs under CKD conditions and FGF23. Studying the effect of hyperphosphatemia on different organs of CKD is important. The prospect of FGF23 for therapy is also discussed.

Molecular and Immunohistochemical assessment of Fibroblast Growth Factor Receptor 3 (FGFR3) gene as a survival time predictor in bladder cancer patients

BACKGROUND: Mutation signatures contributing to the tumorigenesis of bladder cancer (BC) are complex and heterogeneous, resulting in unpredictable progression, recurrence, and time survival. Clinically, useful prognostic and predictive biomarkers for both disease recurrence and surveillance are therefore needed. Activating fibroblast growth factor receptor 3 (FGFR3) mutations are regarded as early drivers in the molecular pathogenesis of BC. AIM: The aim of the present study is to evaluate the frequency and distribution pattern of FGFR3 mutation in urine sediments of BC patients in relation to its immunohistochemical (IHC) and molecular expression and to determine the prognostic and predictive value of FGFR3 relative to BC. PATIENTS AND METHODS: One hundred and sixty patients with diagnosed BC and 80 healthy controls were recruited. Urine samples were collected from all participants. DNA was extracted and FGFR3 mutations were examined in exons 7, 10, and 15 by polymerase chain reaction. IHC for FGFR3 expression and fluorescence in situ hybridization technique for assessment of gene amplification was also applied in tissue sections. RESULTS: Ninety-eight (61.3%) patients were mutant in exon 7, 82 (51.3%) were mutant in exon 10, while only 14 (8.8%) were mutant in exon 15. Univariate logistic regression analysis revealed that mutations in the three exons of FGFR3 were statistically associated with BC and could be used as predictor and/or prognostic parameters for BC. Receiver operating characteristic analysis showed that the mutation of exons 7 and 10 could be used as diagnostic biomarkers for BC. Our findings confirm that FGFR3 mutations are associated with tumors of low grade and stage. The prevalence of mutations was significantly associated with recurrence and survival time of patients for all exons. Kaplan–Meier analysis revealed a significant association between mutant patients in exon 10 and survival time. Our findings suggest that estimation of FGFR3 expression and gene amplification could serve as a prognostic indicator in the follow-up of BC patients. It could also be utilized for molecular targeted therapy in BC. CONCLUSION: Our data confirmed the feasibility of FGFR3 mutation detection in urine sediment. FGFR3 genetic mutations are independent prognostic factors for tumor recurrence and the genetic alternation of FGFR3 could be used for prediction of survival time of BC patients.

Paricalcitol Improved Cardiac Hypertrophy and Fibrosis through Upregulation of Fibroblast Growth Factor-23 and Downregulation of Transforming Growth Factor-beta in a Rat Model of Isoproterenol-Induced Cardiomyopathy

Acute cardiomyopathy is a significant global health concern and one of the leading causes of death in developed countries. Prior studies have shown an association between acute cardiomyopathy and low vitamin D levels. Although paricalcitol, a vitamin D receptor (VDR) activator, has demonstrated clinical benefits in patients with advanced kidney disease, its effect on cardiac remodeling in cardiomyopathy is unknown. This study aimed to investigate the relative effects of paricalcitol on cardiomyopathy in rats. Wistar–Kyoto rats were administered vehicle (sham control group) or isoproterenol to induce cardiomyopathy. Rats administered isoproterenol were subsequently treated with paricalcitol (experimental group) or vehicle (isoproterenol group). Picrosirius red and immunofluorescence staining were used to analyze cardiac fibrosis and hypertrophy. Immunohistochemistry staining was used to confirm the molecular mechanisms involved in isoproterenol-induced cardiomyopathy in rats. Injection of paricalcitol could reduce collagen and transforming growth factor-beta 1 (TGF-β1) levels while activating fibroblast growth factor receptor 1 (FGFR1) and fibroblast growth factor-23 (FGF23) without the help of Klotho, thereby reducing myocardial hypertrophy and fibrosis. As a VDR activator, paricalcitol reduces isoproterenol-induced cardiac fibrosis and hypertrophy by reducing the expression of TGF-β1 and enhancing the expression of VDR, FGFR1, and FGF23.

Meclozine ameliorates bone mineralization and growth plate structure in a mouse model of X‑linked hypophosphatemia.

X-linked hypophosphatemic rickets (XLH) is characterized by hypo-mineralization of the bone due to hypophosphatemia. XLH is caused by abnormally high levels of fibroblast growth factor 23, which trigger renal phosphate wasting. Activated fibroblast growth factor receptor 3 (FGFR3) signaling is considered to be involved in XLH pathology. Our previous study revealed that meclozine attenuated FGFR3 signaling and promoted longitudinal bone growth in an achondroplasia mouse model. The present study aimed to examine whether meclozine affected the bone phenotype in a mouse model of XLH [X-linked hypophosphatemic (Hyp) mice]. Meclozine was administered orally to 7-day-old Hyp mice for 10 days, after which the mice were subjected to blood sampling and histological analyses of the first coccygeal vertebra, femur and tibia. Villanueva Goldner staining was used to assess bone mineralization, hematoxylin and eosin staining was used to determine the growth plate structure and tartrate-resistant acid phosphatase staining was used to measure osteoclast activity. The osteoid volume/bone volume of cortical bone was lower in meclozine-treated Hyp mice compared with untreated Hyp mice. Meclozine treatment improved the abnormally thick hypertrophic zone of the growth plate and ameliorated the downregulation of osteoclast surface/bone surface in Hyp mice. However, meclozine had only a marginal effect on mineralization in the trabecular bone and on calcium and phosphate plasma levels. A 10-day-tratment with meclozine partially ameliorated bone mineralization in Hyp mice; hence, meclozine could alleviate XLH symptoms.

High-Dose Intravenous Iron with Either Ferric Carboxymaltose or Ferric Derisomaltose: A Benefit-Risk Assessment.

The intravenous iron formulations ferric carboxymaltose (FCM) and ferric derisomaltose (FDI) offer the possibility of administering a large amount of iron in one infusion. This results in faster correction of anemia and the formulations being better tolerated than oral iron formulations. This triad of logistic advantages, improved patient convenience, and fast correction of anemia explains the fact that intravenous iron formulations nowadays are frequently prescribed worldwide in the treatment of iron deficiency anemia. However, these formulations may result in hypophosphatemia by inducing a strong increase in active fibroblast growth factor-23 (FGF-23), a hormone that stimulates renal phosphate excretion. This effect is much more pronounced with FCM than with FDI, and therefore the risk of developing hypophosphatemia is remarkably higher with FCM than with FDI. Repeated use of FCM may result in severe osteomalacia, which is characterized by bone pain, Looser zones (pseudofractures), and low-trauma fractures. Intravenous iron preparations are also associated with other adverse effects, of which hypersensitivity reactions are the most important and are usually the result of a non-allergic complement activation on nanoparticles of free labile iron—Complement Activation-Related Pseudo-Allergy (CARPA). The risk on these hypersensitivity reactions can be reduced by choosing a slow infusion rate. Severe hypersensitivity reactions were reported in < 1% of prospective trials and the incidence seems comparable between the two formulations. A practical guideline has been developed based on baseline serum phosphate concentrations and predisposing risk factors, derived from published cases and risk factor analyses from trials, in order to establish the safe use of these formulations.

Activating transcription factor 4 aggravates angiotensin II-induced cell dysfunction in human vascular aortic smooth muscle cells via transcriptionally activating fibroblast growth factor 21.

Abdominal aortic aneurysm (AAA) is a life-threatening disorder worldwide. Fibroblast growth factor 21 (FGF21) was shown to display a high level in the plasma of patients with AAA; however, its detailed functions underlying AAA pathogenesis are unclear. An in vitro AAA model was established in human aortic vascular smooth muscle cells (HASMCs) by angiotensin II (Ang-II) stimulation. Cell counting kit-8, wound healing, and Transwell assays were utilized for measuring cell proliferation and migration. RT-qPCR was used for detecting mRNA expression of FGF21 and activating transcription factor 4 (ATF4). Western blotting was utilized for assessing protein levels of FGF21, ATF4, and markers for the contractile phenotype of HASMCs. ChIP and luciferase reporter assays were implemented for identifying the binding relation between AFT4 and FGF21 promoters. FGF21 and ATF4 were both upregulated in Ang-II-treated HASMCs. Knocking down FGF21 attenuated Ang-II-induced proliferation, migration, and phenotype switch of HASMCs. ATF4 activated FGF21 transcription by binding to its promoter. FGF21 overexpression reversed AFT4 silencing-mediated inhibition of cell proliferation, migration, and phenotype switch. ATF4 transcriptionally upregulates FGF21 to promote the proliferation, migration, and phenotype switch of Ang-II-treated HASMCs.

Safety of terminally gamma-ray-sterilized screws coated with fibroblast growth factor 2-calcium phosphate composite layers in non-human primates.

Screws coated with fibroblast growth factor 2 (FGF-2)-calcium phosphate (CP) composite layers exhibit enhanced soft tissue and bone formation and angiogenesis because of the biological activity of FGF-2. Furthermore, the mitogenic activity of the FGF-2 within the composite layers remains unchanged after gamma-ray sterilization, which may improve the storage stability prior to clinical use. However, the in vivo safeties of these screws as spinal implants remain unknown. Here, a randomized controlled trial, involving non-human primates, investigated the safety of using FGF-2-CP composite layer-coated screws after either gamma-ray sterilization or aseptic processing. Titanium alloy screws coated with FGF-2-CP composite layers and subjected to either gamma-ray sterilization at 25kGy (GS group) or aseptic storage (AS group) were implanted into the vertebral bodies of two cynomolgus monkeys exceeding 12weeks (day 99). Physiological, histological, and radiographic investigations were performed to evaluate the safeties of the screws. There were no serious adverse events, such as surgical site infection, significant loss of body weight, or abnormal blood test results. No radiolucent areas were observed around the screws from the GS or AS group throughout the study. In the intraosseous region, no significant differences were observed in bone and fibrous tissue apposition rates and rate of bone formation between the two groups (p = 0.49, 0.77, and 0.11, respectively). Neither tumor lesions nor accumulation of lymphocytes and neutrophils were observed in either group. Our data suggest that FGF-2-CP composite layer-coated screws subjected to terminal gamma-ray sterilization are as safe as those fabricated in aseptic processing.

FGF 10 Inhibited Spinal Microglial Activation in Neuropathic Pain via PPAR-γ/NF-κB Signaling

Overactivated microglia in the spinal cord leads to neuropathic pain sensitivity. The FGF 10, a Fibroblast Growth Factor (FGFs) that is prevalent in neurons, has been demonstrated to suppress microglial polarization. The objective of this study was to investigate the role of FGF 10 in neuropathic pain and the underlying regulatory mechanisms. Immunofluorescence staining and western blot detection revealed that FGF 10 expression was upregulated in the ipsilateral spinal dorsal horn of Spared Nerve Injury (SNI) rat models and was mainly detected in neurons and microglia. To test the anti-microgliosis actions of FGF 10, SNI rats were intrathecally administered with different concentrations of recombinant FGF 10. Behavioral tests and immunostaining results showed that FGF 10 relieved hyperalgesia in SNI rats and inhibited microglial activity in the ipsilateral spinal dorsal horn in a dose-dependent manner. Besides, BV2 cells were cultured and treated with LPS to activate microglia to explore the underlying mechanisms of FGF 10-induced analgesic effects in vitro. As a result, FGF 10 administration suppressed the LPS-induced microglial augmentation in a dose-dependent manner, followed by increased PPAR-γ and decreased NFκB phosphorylation (p-NFκB) levels. Moreover, PPAR-γ agonist (pioglitazone) and antagonist (GW9662) were administrated into spinal cords of SNI rats, revealing that pioglitazone had similar anti-nociceptive and anti-microglial effects to FGF 10. Conversely, GW9662 reversed all beneficial effects of FGF 10 on SNI rats. In addition, phosphorylated levels of NFκB were reduced by pioglitazone or FGF 10 treatment but raised by GW9662 administration in FGF 10-treated SNI rats. Our findings show that FGF 10 has analgesic effects in rats after peripheral nerve injury and justify the role of PPAR-γ/NFκB signaling in FGF 10-regulated anti-microgliosis.

Genetic and functional analyses of TBX4 reveal novel mechanisms underlying pulmonary arterial hypertension

BackgroundPulmonary arterial hypertension (PAH) is a fatal disease, with approximately 10% of cases associated with genetic variants. Recent genetic studies have reported pathogenic variants in the TBX4 gene in patients with PAH, especially in patients with childhood-onset of the disease, but the pathogenesis of PAH caused by TBX4 variant has not been fully uncovered. MethodsWe analysed the TBX4 gene in 75 Japanese patients with sporadic or familial PAH using a PCR-based bidirectional sequencing method. Detected variants were evaluated using in silico analyses as well as in vitro analyses including luciferase assay, immunocytochemistry and chromatin immunoprecipitation (ChIP) whether they have altered function. We also analysed the function of TBX4 using mouse embryonic lung explants with inhibition of Tbx4 expression. ResultsPutative pathogenic variants were detected in three cases (4.0%). Our in vitro functional analyses revealed that TBX4 directly regulates the transcriptional activity of fibroblast growth factor 10 (FGF10), whereas the identified TBX4 variant proteins failed to activate the FGF10 gene because of disruption of nuclear localisation signal or poor DNA-binding affinity. Furthermore, ex vivo inhibition of Tbx4 resulted in insufficiency of lung morphogenesis along with specific downregulation of Tie2 and Kruppel-like factor 4 expression. ConclusionOur results implicate variants in TBX4 as a genetic cause of PAH in a subset of the Japanese population. Variants in TBX4 may lead to PAH through insufficient lung morphogenesis by disrupting the TBX4-mediated direct regulation of FGF10 signalling and pulmonary vascular endothelial dysfunction involving PAH-related molecules.

Cyclic and dimeric fibroblast growth factor 2 variants with high biomedical potential

Fibroblast growth factor 2 (FGF2) is a pleiotropic protein engaged in the regulation of key cellular processes in a wide spectrum of cells. FGF2 is an important object of basic research as well as a molecule used in regenerative medicine, in vitro cell culture maintenance, and as an anticancer drug carrier. However, the unsatisfactory stability and pleiotropic activities of the wild-type FGF2 largely limit its use as a medical product. To overcome these limitations, we have designed a set of FGF2-based macromolecules via sortase A-mediated cyclization and oligomerization. We obtained heparin-switchable FGF2 variants with enhanced stability and improved ability to stimulate cell proliferation and migration. We have shown that stimulation of glucose uptake by adipocytes is modulated by the architecture of FGF2 oligomers. Moreover, we used hyper-stable FGF2 variants for the construction of highly effective drug carriers for selective killing of FGFR1-overproducing cancer cells. The strategy for FGF2 engineering presented in this work provides novel insights into the design of growth factor variants for regenerative and anti-cancer precise medicine.

A case report to assess the safety and efficacy of Burosumab, an investigational antibody to FGF23, in a single pediatric patient with Epidermal Nevus Syndrome and associated hypophosphatemic rickets

Epidermal Nevus Syndrome (ENS), also known as Cutaneous Skeletal Hypophosphatemia Syndrome or Linear Sebaceous Nevus Syndrome, is caused by a mosaic somatic mutation of RAS (Rat Sarcoma genes) which leads to abnormally elevated levels of fibroblast growth factor 23 (FGF23). FGF23 is a major regulator in phosphate homeostasis. There are multiple disorders, along with Epidermal Nevus Syndrome (ENS), that result in unusually high circulating levels of FGF23. This increase ultimately leads to renal phosphate wasting and reduced levels of 1,25-dihydroxy vitamin D. Across these disorders, the clinical symptoms are similar and often include osteomalacia (hypophosphatemic rickets in children), muscle weakness, fatigue, joint deformities, bone pain, and fractures. Burosumab (KRN23), is an IgG1 monoclonal antibody that binds to the FGF23 receptor and inhibits the activity of FGF23. This leads to an increase in serum phosphate levels. Burosumab emerged as a potential therapy in FGF23 overactivity disorders. Burosumab was successful in the treatment of X-linked hypophosphatemia (XLH) and is now FDA-approved for its treatment. Studies have indicated that Burosumab therapy in subjects with XLH consistently increases and sustains serum phosphorus levels and tubular reabsorption of phosphate without a major impact on urine calcium levels or vitamin D metabolism. We studied the effect of Burosumab treatment in a single pediatric patient with Epidermal Nevus Syndrome. Serum phosphorus rose gradually as we titrated the dose of Burosumab upwards. During treatment, a persistent elevation of parathyroid hormone levels was noted along with a persistent elevation of serum calcium. We presumed the patient had tertiary hyperparathyroidism. However, after the removal of three parathyroid glands, the pathology came back with a single enlarged parathyroid adenoma. Subsequently, his calcium and PTH, and phosphorus levels stabilized while taking only Burosumab.ClinicalTrials.gov NCT04320316.

Abstract 1792: FGFR3 mediated PARP1 tyrosine 158 phosphorylation promotes PARP inhibitor resistance

Abstract Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi), which induce DNA damage by inhibiting PARP1 enzymatic activity and trapping PARP on the damaged DNA, are used to eliminate BRCA1/2-mutated (BRCAm) cancer. However, clinical observations suggest that BRCAm tumors develop PARPi resistance. Current strategies to overcome PARPi resistance include impeding multiple DNA repair pathways to induce excessive DNA damage. Here, we propose a novel strategy targeting oncogenic receptor tyrosine kinases to enhance PARP trapping. By developing triple-negative breast cancer (TNBC) cells with acquired talazoparib resistance, we observed a high prevalence of activated fibroblast growth factor receptor 3 (FGFR3) kinase in these cells through kinase antibody array analysis. Mass spectrometry analysis and in vitro kinase assay suggested that FGFR3 phosphorylated PARP1 at tyrosine residues 158 and 176. Biochemistry studies suggested that only PARP1 tyrosine 158 phosphorylation contributes to PARPi resistance in the cells we developed. We then developed a monoclonal antibody against tyrosine 158 phosphorylated PARP1, and found that high-level PARP1 tyrosine 158 phosphorylation positively correlated with PARPi resistance in breast cancer patient-derived xenograft models. We further demonstrated that the combination of FGFR inhibitor and PARPi delayed DNA repair with prolonged PARP trapping. Moreover, synergy between PARPi and FGFR inhibition was observed in multiple TNBC cell lines with PARPi resistance in vitro. The combination of PARPi and FGFR inhibitor also showed synergism in vivo, and treatment with the combination of PARPi and FGFR inhibitor was tolerated in mouse models. These findings reveal that PARP1 tyrosine 158 phosphorylation facilitates resolving of the PARPi-induced PARP-trapping, and that the tyrosine 158 phosphorylated PARP1 may be an effective biomarker to indicate FGFR3 mediated PARPi resistance. Citation Format: MeiKuang Chen, Yuan Gao, Weiya Xia, Yu-Han Wang, Jennifer K. Litton, Yu-Yi Chu, Funda Meric-Bernstam, Helen Piwnica-Worms, Banu Arun, Jordi Rodon Ahnert, Yongkun Wei, Wei-Chao Chang, Hung-Ling Wang, Coya Tapia, Constance T. Albarracin, Shao-Chun Wang, Ying-Nai Wang, Gabriel N. Hortobagyi, Chunru Lin, Liuqing Yang, Dihua Yu, Mien-Chie Hung. FGFR3 mediated PARP1 tyrosine 158 phosphorylation promotes PARP inhibitor resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1792.