Effects of follicle-stimulating hormone, insulin-like growth factor 1, fibroblast growth factor 2, and fibroblast growth factor 9 on sirtuins expression and histone deacetylase activity in bovine granulosa cells.

Granulosa cells (GC) are critical regulators of fertility. During the process of ovarian folliculogenesis, these cells undergo profound changes while producing steroid hormones that are important to control follicular growth, oocyte maturation, and ovulation. Sirtuins are enzymes that regulate several biological processes and have been associated with control of GC function. However, how sirtuins are regulated in GC during ovarian folliculogenesis remains to be unveiled. The present study was designed to investigate effects of hormones that control GC proliferation, differentiation, and steroidogenesis on expression of the seven members of the mammalian sirtuins family (SIRT1-7) and on histone deacetylase activity of nuclear sirtuins (SIRT1, 6, and 7) in GC. Bovine granulosa cells were isolated from small antral follicles (1-5 mm) and were treated with or without follicle-stimulating hormone (FSH), insulin-like growth factor 1 (IGF-1), and fibroblast growth factors 2 (FGF2) and 9 (FGF9). Following treatments, cell proliferation was determined via a cell analyzer, estradiol synthesis and histone deacetylase activity were determined via ELISA, and sirtuins mRNA expression was determined via qPCR. Treatments with FSH and IGF-1 stimulated cell proliferation while addition of FGF2 or FGF9 suppressed estradiol production stimulated by FSH plus IGF-1. In terms of treatments that regulated sirtuins expression in GC, fibroblast growth factors were the most impactful: FGF2 alone increased SIRT1 mRNA expression in comparison to several treatments and increased mRNA abundance of SIRT2 and SIRT7 when added to the combination of FSH and IGF-1; the addition of FGF9 to the combination of FSH and IGF-1 increased mRNA expression of SIRT2, SIRT3, SIRT4, SIRT6, and SIRT7 and increased mRNA expression of SIRT5 in comparison to the negative control group that received no treatment. Also, FGF2 alone increased histone deacetylase activity of sirtuins in comparison to all treatments that contained FSH and/or IGF-1. Furthermore, several correlations were observed between treatments and sirtuins expression and activity, between estradiol or GC numbers and sirtuins expression, and between expression of sirtuins. As FGF2 and FGF9 are considered anti-differentiation factors of GC that stimulate GC proliferation while suppressing estradiol production in combination with FSH and IGF-1, data of this study suggest that sirtuins are associated with control of differentiation of bovine GC.

Inhibition of miR-182-5p Targets FGF9 to Alleviate Osteoarthritis.

Background: The pathogenesis of osteoarthritis (OA) is complex and there is no specific drug for treatment. The aim of this study was to identify the molecular targets of OA therapy, focusing on the expression and biological functions of miR-182-5p and its target genes in OA. Methods: miR-182-5p and fibroblast growth factor 9 (FGF9) were overexpressed or knocked down in IL-1ß-induced chondrocytes. An OA knee model was performed by surgically destroying the medial meniscus. The gene expression of miR-182-5p and FGF9 was calculated. The protein FGF9 was tested by western blotting. Cell counting kit-8 (CCK8), plate cloning assay, and flow cytometry were conducted to evaluate cell proliferation and apoptosis. The expression of inflammatory factors, tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, and interleukin (IL)-8, was evaluated using enzyme-linked immunosorbent assay (ELISA). Dual-luciferase reporter assays validated the targeting relationship between miR-182-5p and FGF9. Hematoxylin-eosin (HE) and safranin O-fast Green (S-O) staining were utilized to access cartilage damage. Ki67 expression in cartilage was detected using immunohistochemistry (IHC). TdT-mediated dUTP nick-end labeling (TUNEL) assays were used to calculate the apoptosis rate of cartilage. Results: The expression of miR-182-5p was upregulated, and FGF9 was downregulated in the IL-1ß-induced chondrocytes. OA chondrocytes proliferation ability in the miR-182-5p mimics group was decreased, and the apoptosis rate and inflammatory factor were increased. Transfection with miR-182-5p inhibitor increased the proliferative ability and decreased the apoptosis rate in the IL-1ß-induced chondrocytes. Transfection with miR-182-5p inhibitor reversed IL-1ß-induced inflammatory factor release in chondrocytes. Targeted binding sites existed between miR-182-5p and FGF9. After overexpression of FGF9, the miR-182-5p effect on OA chondrocytes was reversed. The hyaline cartilage thickness and proteoglycan content decreased in OA rats, and this was reversed by miR-182-5p inhibitor treatment. Conclusions: miR-182-5p expression levels were increased in OA chondrocytes and regulated chondrocyte proliferation, apoptosis, and inflammation by targeting FGF9. miR-182-5p is a potential gene for OA treatment.

Fibroblast growth factor 9 reduces TBHP-induced oxidative stress in chondrocytes and diminishes mouse osteoarthritis by activating ERK/Nrf2 signaling pathway.

Osteoarthritis (OA) is a degenerative and progressive disease that affects joints. Pathologically, it is characterized by oxidative stress-mediated excessive chondrocyte apoptosis and mitochondrial dysfunction. Fibroblast growth factor 9 (FGF9) has been shown to exert antioxidant effects and prevent degenerative diseases by activating ERK-related signaling pathways. However, the mechanism of FGF9 in the pathogenesis of OA and its relationship with anti-oxidative stress and related pathways are unclear. In this study, mice with medial meniscus instability (DMM) were used as the in vivo model whereas TBHP-induced chondrocytes served as the in vitro model to explore the mechanism underlying the effects of FGF9 in OA and its association with anti-oxidative stress. Results showed that FGF9 reduced oxidative stress, apoptosis, and mitochondrial dysfunction in TBHP-treated chondrocytes and promoted the nuclear translocation of Nrf2 to activate the Nrf2/HO1 signaling pathway. Interestingly, silencing the Nrf2 gene or blocking the ERK signaling pathway abolished the antioxidant effects of FGF9. FGF9 treatment reduced joint space narrowing, cartilage ossification, and synovial thickening in the DMM model mice. In conclusion, the present findings demonstrate that FGF9 can inhibit TBHP-induced oxidative stress in chondrocytes through the ERK and Nrf2-HO1 signaling pathways and prevent the progression of OA in vivo.

Protective Effect of Ginsenosides Rg1 on Ischemic Injury of Cardiomyocytes After Acute Myocardial Infarction.

Acute myocardial infarction (AMI) leads to anoxia and ischemia of cardiomyocytes, followed by apoptosis. This study investigated the protective effect of ginsenoside Rg1 (Rg1) on myocardial ischemia injury in rats with AMI. Rats were randomly divided into five groups: group A (blank control group), group B (hypoxia/reoxygenation group), group C (hypoxia/reoxygenation + 10 mg/L Rg1), group D (hypoxia/reoxygenation + 20 mg/L Rg1) and group E (hypoxia/reoxygenation + 40 mg/L Rg1). The survival rate, apoptosis rate, expression of cyclin-dependent kinase 4 (CDK4), fibroblast growth factor 9 (FGF9), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), microvessel density and myocardial infarction area of rats in each group were compared. The expressions of CDK4 and FGF9, the contents of SOD and GSH-Px in groups C, D and E injected with Rg1 were significantly promoted compared to group B without Rg1 injection (P < 0.05). The survival rate of myocardial cells was significantly increased while the apoptosis rate was significantly decreased in group C, D, E compared to group B (P < 0.05). On the 3rd, 7th and 10th day following Rg1 treatment, the infarct area of E group was significantly decreased in three groups C, D, E, and the microvessel density of infarct area was significantly increased compared with group B (P < 0.05). So, Rg1 can improve the survival rate of myocardial cells, reduce the apoptosis rate and the area of myocardial infarction, and increase the microvessel density of infarct area, thus playing a protective role in ischemic myocardial cells of AMI rats.

Using a testis regeneration model, FGF9, LIF, and SCF improve testis cord formation while RA enhances gonocyte survival.

Implantation of testis cell aggregates from various donors under the back skin of recipient mice results in de novo formation of testis tissue. We used this implantation model to study the putative in vivo effects of six different growth factors on testis cord development. Recipient mice (n = 7/group) were implanted with eight neonatal porcine testis cell aggregates that were first exposed to a designated growth factor: FGF2 at 1 µg/mL, FGF9 at 5 µg/mL, VEGF at 3.5 µg/mL, LIF at 5 µg/mL, SCF at 3.5 µg/mL, retinoic acid (RA) at 3.5 × 10-5 M, or no growth factors (control). The newly developed seminiferous cords (SC) were classified based on their morphology into regular, irregular, enlarged, or aberrant. Certain treatments enhanced implant weight (LIF), implant cross-sectional area (SCF) or the relative cross-sectional area covered by SC within implants (FGF2). RA promoted the formation of enlarged SC and FGF2 led to the highest ratio of regular SC and the lowest ratio of aberrant SC. Rete testis-like structures appeared earlier in implants treated with FGF2, FGF9, or LIF. These results show that even brief pre-implantation exposure of testis cells to these growth factors can have profound effects on morphogenesis of testis cords using this implantation model.

Fibroblast growth factor 9 attenuates sepsis-induced fulminant hepatitis in mice.

Sepsis-induced fulminant hepatitis (FH) is a fatal syndrome that has a worse prognosis in clinical practice. Hence, seeking effective agents for sepsis-induced FH treatment is urgently needed. Fibroblast growth factors (FGFs) are vital for tissue homeostasis and damage repair in various organs including the liver. Our study aims to investigate the protective effects and potential mechanisms of FGF9 on lipopolysaccharide (LPS)/D-galactosamine (D-Gal)-induced FH in mice. We found that pre-treatment with FGF9 exhibited remarkable hepaprotective effects on liver damage caused by LPS/D-Gal, as manifested by the concomitant decrease in mortality and serum aminotransferase activities, and the attenuation of hepatocellular apoptosis and hepatic histopathological abnormalities in LPS/D-Gal-intoxicated mice. We further found that FGF9 alleviated the infiltration of neutrophils into the liver, and decreased the serum levels of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) in LPS/D-Gal-challenged mice. These effects can be explained at least in part by the inhibition of NF-κB signaling pathway. Meanwhile, FGF9 enhanced the antioxidative defense system in mice livers by upregulating the expression of NRF-2-related antioxidative enzymes, including glutamate-cysteine ligase catalytic subunit (GCLC), NAD(P)H: quinone oxidoreductase 1 (NQO-1), and heme oxygenase-1 (HO-1). These data indicate that FGF9 represents a promising therapeutic drug for ameliorating sepsis-induced FH via its anti-apoptotic and anti-inflammatory capacities.

Deletion of FGF9 in GABAergic neurons causes epilepsy.

Fibroblast growth factor 9 (FGF9) has long been assumed to modulate multiple biological processes, yet very little is known about the impact of FGF9 on neurodevelopment. Herein, we found that loss of Fgf9 in olig1 progenitor cells induced epilepsy in mice, with pathological changes in the cortex. Then depleting Fgf9 in different neural populations revealed that epilepsy was associated with GABAergic neurons. Fgf9 CKO in GABAergic neuron (CKOVGAT) mice exhibited not only the most severe seizures, but also the most severe growth retardation and highest mortality. Fgf9 deletion in CKOVGAT mice caused neuronal apoptosis and decreased GABA expression, leading to a GABA/Glu imbalance and epilepsy. The adenylate cyclase/cyclic AMP and ERK signaling pathways were activated in this process. Recombinant FGF9 proteoliposomes could significantly decrease the number of seizures. Furthermore, the decrease of FGF9 was commonly observed in serum of epileptic patients, especially those with focal seizures. Thus, FGF9 plays essential roles in GABAergic neuron survival and epilepsy pathology, which could serve as a new target for the treatment of epilepsy.

Fibroblast Growth Factor 9 Stimulates Neuronal Length Through NF-kB Signaling in Striatal Cell Huntington's Disease Models.

Proper development of neuronal cells is important for brain functions, and impairment of neuronal development may lead to neuronal disorders, implying that improvement in neuronal development may be a therapeutic direction for these diseases. Huntington's disease (HD) is a neurodegenerative disease characterized by impairment of neuronal structures, ultimately leading to neuronal death and dysfunctions of the central nervous system. Based on previous studies, fibroblast growth factor 9 (FGF9) may provide neuroprotective functions in HD, and FGFs may enhance neuronal development and neurite outgrowth. However, whether FGF9 can provide neuronal protective functions through improvement of neuronal morphology in HD is still unclear. Here, we study the effects of FGF9 on neuronal length in HD and attempt to understand the related working mechanisms. Taking advantage of striatal cell lines from HD knock-in mice, we found that FGF9 increases total neuronal length and upregulates several structural and synaptic proteins under HD conditions. In addition, activation of nuclear factor kappa B (NF-kB) signaling by FGF9 was observed to be significant in HD cells, and blockage of NF-kB leads to suppression of these structural and synaptic proteins induced by FGF9, suggesting the involvement of NF-kB signaling in these effects of FGF9. Taken these results together, FGF9 may enhance total neuronal length through upregulation of NF-kB signaling, and this mechanism could serve as an important mechanism for neuroprotective functions of FGF9 in HD.

FGF9 induces neurite outgrowth upon ERK signaling in knock-in striatal Huntington's disease cells.

AIMS: Huntington's disease (HD) is a neurodegenerative disease that causes deficits in neurite outgrowth, which suggests that enhancement of neurite outgrowth is a potential direction by which to improve HD. Our previous publications showed that fibroblast growth factor 9 (FGF9) provides anti-apoptosis and anti-oxidative functions in striatal cell models of HD through the extracellular signal-regulated kinases (ERK) pathway, and FGF9 also stimulates cytoskeletons to enhance neurite outgrowth via nuclear factor kappa B (NF-kB) signaling. In this study, we further demonstrate the importance of the ERK pathway for the neurite outgrowth induced by FGF9 in HD striatal models. MATERIALS AND METHODS: FGF9 was treated with ERK (U0126) or NF-kB (BAY11-7082) inhibitors in STHdhQ7/Q7 and STHdhQ111/Q111 striatal knock-in cell lines to examine neurite outgrowth, cytoskeletal markers, and synaptic proteins via immunofluorescence staining and Western blotting. NF-kB activity was analyzed by NF-kB promoter reporter assay. KEY FINDINGS: Here, we show that suppression of ERK signaling significantly inhibits FGF9-induced neurite outgrowth, cytoskeletal markers, and synaptic proteins in HD striatal cells. In addition, we also show suppression of ERK signaling significantly decreases FGF9-induced NF-kB activation, whereas suppression of NF-kB does not decrease FGF9-induced ERK signaling. These results suggest that FGF9 activates ERK signaling first, stimulates NF-kB upregulation, and then enhances neurite outgrowth in HD striatal cells. SIGNIFICANCE: We elucidate the more detailed mechanisms of neurite outgrowth enhanced by FGF9 in these HD striatal cells. This study may provide insights into targeting neurite outgrowth for HD therapy.

FGF9 induces functional differentiation to Schwann cells from human adipose derived stem cells.

Rationale: The formation of adipose-derived stem cells (ASCs) into spheres on a chitosan-coated microenvironment promoted ASCs differentiation into a mixed population of neural lineage-like cells (NLCs), but the underline mechanism is still unknown. Since the fibroblast growth factor 9 (FGF9) and fibroblast growth factor receptors (FGFRs) play as key regulators of neural cell fate during embryo development and stem cell differentiation, the current study aims to reveal the interplay of FGF9 and FGFRs for promoting peripheral nerve regeneration. Methods: Different concentration of FGF9 peptide (10, 25, 50, 100 ng/mL) were added during NLCs induction (FGF9-NLCs). The FGFR expressions and potential signaling were studied by gene and protein expressions as well as knocking down by specific FGFR siRNA or commercial inhibitors. FGF9-NLCs were fluorescent labeled and applied into a nerve conduit upon the injured sciatic nerves of experimental rats. Results: The FGFR2 and FGFR4 were significantly increased during NLCs induction. The FGF9 treated FGF9-NLCs spheres became smaller and changed into Schwann cells (SCs) which expressed S100ß and GFAP. The specific silencing of FGFR2 diminished FGF9-induced Akt phosphorylation and inhibited the differentiation of SCs. Transplanted FGF9-NLCs participated in myelin sheath formation, enhanced axonal regrowth and promoted innervated muscle regeneration. The knockdown of FGFR2 in FGF9-NLCs led to the abolishment of nerve regeneration. Conclusions: Our data therefore demonstrate the importance of FGF9 in the determination of SC fate via the FGF9-FGFR2-Akt pathway and reveal the therapeutic benefit of FGF9-NLCs.

Synergistic effects of laminin-1 peptides, VEGF and FGF9 on salivary gland regeneration.

Hyposalivation is associated with radiation therapy, Sjögren's syndrome and/or aging, and is a significant clinical problem that decreases oral health and overall health in many patients and currently lacks effective treatment. Hence, methods to regenerate salivary glands and restore saliva secretion are urgently needed. To this end, this study describes the modification of fibrin hydrogels with a combination of laminin-1 peptides (YIGSR and A99) and human growth factors (vascular endothelial growth factor and fibroblast growth factor 9) to enhance regeneration in a salivary gland injury mouse model. Our results indicate that these fortified hydrogels enhanced angiogenesis and neurogenesis while promoting formation of acinar structures, thereby leading to enhanced saliva secretion. Such functional recovery indicates salivary gland regeneration and suggests that our technology may be useful in promoting gland regeneration and reversing hyposalivation in a clinical setting. STATEMENT OF SIGNIFICANCE: We engineered Fibrin Hydrogels (FH) to contain multiple regenerative cues including laminin-1 peptides (L1p) and growth factors (GFs). L1p and GF modified FH were used to induce salivary gland regeneration in a wounded mouse model. Treatment with L1p and GF modified FH promoted salivary epithelial tissue regeneration, vascularization, neurogenesis and healing as compared to L1p-FH or FH alone. Results indicate that L1p and GF modified FH can be used for future therapeutic applications.

Fortifying Angiogenesis in Ischemic Muscle with FGF9-Loaded Electrospun Poly(Ester Amide) Fibers.

Delivery of angiogenic growth factors lessens ischemia in preclinical models but has demonstrated little benefit in patients with peripheral vascular disease. Augmenting the wrapping of nascent microvessels by mural cells constitutes an alternative strategy to regenerating a functional microvasculature, particularly if integrated with a sustained delivery platform. Herein, electrospun poly(ester amide) (PEA) nanofiber mats are fabricated for delivering a mural cell-targeting factor, fibroblast growth factor 9 (FGF9). Proof-of-principle is established by placing FGF9/FGF2-loaded PEA fiber mats on the chick chorioallantoic membrane and identifying enhanced angiogenesis by 3D power Doppler micro-ultrasound imaging. To assess the delivery system in ischemic muscle, FGF9-loaded PEA fiber mats are implanted onto the surface of the tibialis anterior muscle of mice with hindlimb ischemia. The system supplies FGF9 into the tibialis anterior muscle and yields a neo-microvascular network with enhanced mural cell coverage up to 28 days after injury. The regenerating muscle that receives FGF9 display near-normal sized myofibers and reduced interstitial fibrosis. Moreover, the mice demonstrate improved locomotion. These findings of locally released FGF9 from PEA nanofibers raise prospects for a microvascular remodeling approach to improve muscle health in peripheral vascular disease.

Hair follicle dermal condensation forms via Fgf20 primed cell cycle exit, cell motility, and aggregation.

Mesenchymal condensation is a critical step in organogenesis, yet the underlying molecular and cellular mechanisms remain poorly understood. The hair follicle dermal condensate is the precursor to the permanent mesenchymal unit of the hair follicle, the dermal papilla, which regulates hair cycling throughout life and bears hair inductive potential. Dermal condensate morphogenesis depends on epithelial Fibroblast Growth Factor 20 (Fgf20). Here, we combine mouse models with 3D and 4D microscopy to demonstrate that dermal condensates form de novo and via directional migration. We identify cell cycle exit and cell shape changes as early hallmarks of dermal condensate morphogenesis and find that Fgf20 primes these cellular behaviors and enhances cell motility and condensation. RNAseq profiling of immediate Fgf20 targets revealed induction of a subset of dermal condensate marker genes. Collectively, these data indicate that dermal condensation occurs via directed cell movement and that Fgf20 orchestrates the early cellular and molecular events.

Fibroblast Growth Factor 9 Suppresses Striatal Cell Death Dominantly Through ERK Signaling in Huntington's Disease.

BACKGROUND/AIMS: Huntington's disease (HD) is a heritable neurodegenerative disorder, and there is no cure for HD to date. A type of fibroblast growth factor (FGF), FGF9, has been reported to play prosurvival roles in other neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease. However, the effects of FGF9 on HD is still unknown. With many similarities in the cellular and pathological mechanisms that eventually cause cell death in neurodegenerative diseases, we hypothesize that FGF9 might provide neuroprotective functions in HD. METHODS: In this study, STHdhQ7/Q7 (WT) and STHdhQ111/Q111 (HD) striatal knock-in cell lines were used to evaluate the neuroprotective effects of FGF9. Cell proliferation, cell death and neuroprotective markers were determined via the MTT assay, propidium iodide staining and Western blotting, respectively. The signaling pathways regulated by FGF9 were demonstrated using Western blotting. Additionally, HD transgenic mouse models were used to further confirm the neuroprotective effects of FGF9 via ELISA, Western blotting and immunostaining. RESULTS: Results show that FGF9 not only enhances cell proliferation, but also alleviates cell death as cells under starvation stress. In addition, FGF9 significantly upregulates glial cell line-derived neurotrophic factor (GDNF) and an anti-apoptotic marker, Bcl-xL, and decreases the expression level of an apoptotic marker, cleaved caspase 3. Furthermore, FGF9 functions through ERK, AKT and JNK pathways. Especially, ERK pathway plays a critical role to influence the effects of FGF9 toward cell survival and GDNF production. CONCLUSIONS: These results not only show the neuroprotective effects of FGF9, but also clarify the critical mechanisms in HD cells, further providing an insight for the therapeutic potential of FGF9 in HD.

MicroRNA 221 expression in theca and granulosa cells: hormonal regulation and function.

Small noncoding RNA molecules (miRNA) regulate protein levels in a post-transcriptional manner by partial base pairing to the 3'-UTR of target genes thus mediating degradation or translational repression. Previous studies indicate that numerous miRNA regulate the biosynthesis of intraovarian hormones, and emerging evidence indicates that one of these, miRNA-221 (MIR221), may be a modulator of ovarian function. However, the hormonal control of ovarian MIR221 is not known. The objectives of this study were to investigate the developmental and hormonal regulation of MIR221 expression in granulosa (GC) and theca cell (TC) and its possible role in regulating follicular function. Bovine ovaries were collected from a local abattoir and GC and TC were obtained from small (<6 mm) and large (≥8 mm) follicles. In Exp. 1, GCs of small follicles had 9.7-fold greater (P < 0.001) levels of MIR221 than those of large follicles, and TCs of large follicles had 3.7-fold greater (P < 0.001) levels of MIR221 than those of small follicles. In large follicles, abundance of MIR221 was 66.6-fold greater (P < 0.001) in TCs than in GCs. In small follicles, MIR221 abundance did not differ (P = 0.14) between GC and TCs. In vitro Exp. 2, 3, and 4 revealed that treatment of bovine TCs with various steroids, phytoestrogens, IGF1, forskolin, and dibutyryl cyclic adenosine monophosphate had no effect (P > 0.35) on MIR221 expression, whereas treatment with fibroblast growth factor 9 (FGF9) and FGF2 increased (P < 0.001) TC MIR221 abundance 1.7- to 2.5-fold. In Exp. 5, FGF9 increased (P < 0.05) GC MIR221 abundance by 1.7- and 2.0-fold in small and large follicles, respectively. The role of MIR221 in GC steroidogenesis was investigated in Exp. 6 and it was found that transfection with a MIR221 mimic reduced (P < 0.01) GC estradiol and progesterone production induced by FSH and IGF1, whereas transfection with MIR221 inhibitor had little or no effect. We conclude that thecal MIR221 expression is increased by FGF9 and increased MIR221 may act to inhibit GC steroidogenesis in cattle.

Transcriptome profiling of bovine ovarian theca cells treated with fibroblast growth factor 9.

We reported previously that fibroblast growth factor 9 (FGF9) acts as an antidifferentiation factor, stimulating proliferation of granulosa cells (GCs) and theca cells (TCs) while suppressing hormone-induced steroidogenesis of these cells. How FGF9 acts to simultaneously suppress steroidogenesis and stimulate proliferation remains to be fully elucidated. Thus, this study was undertaken to clarify the effects of FGF9 on the TC transcriptome. Ovaries were obtained from beef heifers at a local abattoir, TCs were isolated from large antral follicles, and cultured with or without 30 ng/mL of FGF9 for 24 h in the presence of LH and IGF-1. After treatment, total RNA was extracted from TC and processed for microarray using Affymetrix GeneChip Bovine Genome Arrays (n = 4/group). Transcriptome analysis comparing FGF9-treated TC with control TC using 1.3-fold cutoff, and a P < 0.05 significance level identified 355 differentially expressed transcripts, with 164 elements upregulated and 191 elements downregulated by FGF9. The ingenuity pathway analysis (IPA) was used to investigate how FGF9 treatment affects molecular pathways, biological functions, and the connection between molecules in bovine TC. The IPA software identified 346 pathways in response to FGF9 in TC involved in several biological functions and unveiled interesting relationships among genes related to cell proliferation (eg, CCND1, FZD5, and MYB), antioxidation/cytoprotection (eg, HMOX1 and NQO1), and steroidogenesis (eg, CYP11A1 and STAR). Overall, genes, pathways, and networks identified in this study painted a picture of how FGF9 may regulate folliculogenesis, providing novel candidate genes for further investigation of FGF9 functions in ovarian follicular development.

FGF9 prevents pleural fibrosis induced by intrapleural adenovirus injection in mice.

Fibroblast growth factor 9 (FGF9) is necessary for fetal lung development and is expressed by epithelium and mesothelium. We evaluated the role of FGF9 overexpression on adenoviral-induced pleural injury in vivo and determined the biological effects of FGF9 on mesothelial cells in vitro. We assessed the expression of FGF9 and FGF receptors by mesothelial cells in both human and mouse lungs. Intrapleural injection of an adenovirus expressing human FGF9 (AdFGF9) or a control adenovirus (AdCont) was performed. Mice were euthanized at days 3, 5, and 14 Expression of FGF9 and markers of inflammation and myofibroblastic differentiation was studied by qPCR and immunohistochemistry. In vitro, rat mesothelial cells were stimulated with FGF9 (20 ng/ml), and we assessed its effect on proliferation, survival, migration, and differentiation. FGF9 was expressed by mesothelial cells in human idiopathic pulmonary fibrosis. FGF receptors, mainly FGFR3, were expressed by mesothelial cells in vivo in humans and mice. AdCont instillation induced diffuse pleural thickening appearing at day 5, maximal at day 14 The altered pleura cells strongly expressed α-smooth muscle actin and collagen. AdFGF9 injection induced maximal FGF9 expression at day 5 that lasted until day 14 FGF9 overexpression prevented pleural thickening, collagen and fibronectin accumulation, and myofibroblastic differentiation of mesothelial cells. In vitro, FGF9 decreased mesothelial cell migration and inhibited the differentiating effect of transforming growth factor-ß1. We conclude that FGF9 has a potential antifibrotic effect on mesothelial cells.

Ex vivo analysis of the contribution of FGF10+ cells to airway smooth muscle cell formation during early lung development.

BACKGROUND: Airway smooth muscle cells (ASMCs) have been widely studied during embryonic lung development. These cells have been shown to control epithelial bifurcation during branching morphogenesis. Fibroblast growth factor 10-positive (FGF10+ ) cells, originally residing in the submesothelial mesenchyme, contribute to ASMC formation in the distal lung. The reported work aims at monitoring the response of FGF10+ progenitors and differentiated ASMCs to growth factor treatment in real time using lineage tracing in the background of an air-liquid interface (ALI) culture system. RESULTS: FGF ligands impose divergent effects on iterative lung branching in vitro. Moreover, time-lapse imaging and endpoint analysis show that FGF9 treatment leads to amplification of the FGF10+ lineage and represses its differentiation to ASMCs. Sonic hedgehog (SHH) treatment reduces the amplification of this lineage and leads to decreased lung branching. Finally, differentiated ASMCs in proximal regions fail to expand upon FGF9 treatment. CONCLUSIONS: Our data demonstrate, in real time, that FGF9 is an important regulator of amplification, migration, and subsequent differentiation of ASMC progenitors during early lung development. The attained results agree with previous findings regarding ASMC formation and highlight the complexity of growth factor signaling networks in controlling mesenchymal cell-fate decisions in the developing mouse lung. Developmental Dynamics 246:531-538, 2017. © 2017 Wiley Periodicals, Inc.

Fibroblast growth factor 9 (FGF9) regulation of cyclin D1 and cyclin-dependent kinase-4 in ovarian granulosa and theca cells of cattle.

To determine the mechanism by which fibroblast growth factor 9 (FGF9) alters granulosa (GC) and theca (TC) cell proliferation, cell cycle proteins that regulate progression through G1 phase of the cell cycle, cyclin D1 (CCND1) and cyclin-dependent kinase-4 (CDK4; CCND1's catalytic partner), were evaluated. Ovaries were obtained from a local abattoir, GC were harvested from small (1-5 mm) and large (8-22 mm) follicles, and TC were harvested from large follicles. GC and TC were plated in medium containing 10% fetal calf serum followed by various treatments in serum-free medium. Treatment with 30 ng/mL of either FGF9 or IGF1 significantly increased GC numbers and when combined, synergized to further increase GC numbers by threefold. Abundance of CCND1 and CDK4 mRNA in TC and GC were quantified via real-time PCR. Alone and in combination with IGF1, FGF9 significantly increased CCND1 mRNA expression in both GC and TC. Western blotting revealed that CCND1 protein levels were increased by FGF9 in TC after 6 h and 12 h of treatment, but CDK4 protein was not affected. A mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway inhibitor, U0126, significantly reduced FGF9-induced CCND1 mRNA expression to basal levels. For the first time we show that CCND1 mRNA expression is increased by FGF9 in bovine TC and GC, and that FGF9 likely uses the MAPK pathway to induce CCND1 mRNA production in bovine TC.

Hybrid Biomaterial with Conjugated Growth Factors and Mesenchymal Stem Cells for Ectopic Bone Formation.

Bone is a highly vascularized tissue and efficient bone regeneration requires neovascularization, especially for critical-sized bone defects. We developed a novel hybrid biomaterial comprising nanocalcium sulfate (nCS) and fibrin hydrogel to deliver mesenchymal stem cells (MSCs) and angiogenic factors, vascular endothelial growth factor (VEGF) and fibroblast growth factor 9 (FGF9), to promote neovascularization and bone formation. MSC and growth factor(s)-loaded scaffolds were implanted subcutaneously into mice to examine their angiogenic and osteogenic potential. Micro CT, alkaline phosphatase activity assay, and histological analysis were used to evaluate bone formation, while immunohistochemistry was employed to assess neovessel formation. The presence of fibrin preserved the nCS scaffold structure and promoted de novo bone formation. In addition, the presence of bone morphogenic protein 2-expressing MSC in nCS and fibrin hydrogels improved bone regeneration significantly. While FGF9 alone had no significant effect, the combination FGF9 and VEGF conjugated in fibrin enhanced neovascularization and bone formation more than 4-fold compared to nCS with MSC. Overall, our results suggested that the combination of nCS (to support bone formation) with a fibrin-based VEGF/FGF9 release system (support vascular formation) is an innovative and effective strategy that significantly enhanced ectopic bone formation in vivo.