Exogenous Fibroblast Growth Factor-2 Research Articles

FGF2 promotes the proliferation of injured granulosa cells in premature ovarian failure via Hippo-YAP signaling pathway

Young women undergoing anticancer treatment are at risk of premature ovarian failure (POF). Endometrial-derived stem cells (EnSCs) have demonstrated significant therapeutic potential for treating ovarian insufficiency, although the underlying mechanisms remain to be fully understood. This study aims to further investigate the therapeutic effects of EnSCs, particularly through the paracrine action of fibroblast growth factor 2 (FGF2), on POF. The findings show that exogenous FGF2 enhances the survival of ovarian granulosa cells damaged by cisplatin. FGF2 stimulates the proliferation of these damaged cells by suppressing the Hippo signaling pathway and activating YAP expression. In vivo experiments also revealed that FGF2 treatment significantly improves ovarian reserve and endocrine function in mice with POF. These results suggest that FGF2 can boost the proliferative capacity of damaged ovarian granulosa cells through the Hippo-YAP signaling pathway, providing a theoretical foundation for using EnSCs and FGF2 in clinical treatments for POF.

Scleral remodeling in early adulthood: the role of FGF-2

Emmetropization, a natural process of ocular elongation, is closely associated with scleral remodeling. The Fibroblast growth factor-2 (FGF-2) was reported involved in scleral remodeling in myopia models. Herein, we aimed to investigate the role of scleral fibroblast-to-myofibroblast differentiation and FGF-2 in scleral remodeling during maturation. Our findings revealed that the posterior scleral fibroblasts (SFs) from mature guinea pigs exhibit increased stiffness compared to those from young guinea pigs. Moreover, mature SFs displayed decreased cell proliferation but increased levels of α-SMA, matrix metalloproteinase 2 (MMP2), and collagen 1, when compared to young SFs. Additionally, the mRNA expression of scleral Fgf-2, Fgf receptor 1 (Fgfr1), Fgfr2, Fgfr3, and Fgfr4 was increased in mature SFs. Notably, exogenous FGF-2 showed increased cell proliferation and led to decreased expression of α-SMA, MMP2, and collagen 1 in mature SFs. Overall, our findings highlight the influence of maturation on SFs from posterior scleral shells, resulting in increased stiffness and the manifestation of fibroblast-to-myofibroblast differentiation during development. Exogenous FGF-2 increased cell proliferation and reversed the age-related fibroblast-to-myofibroblast differentiation, suggesting a potential role of FGF-2 in regulating scleral remodeling.

A single administration of FGF2 after renal ischemia-reperfusion injury alleviates post-injury interstitial fibrosis.

Despite lack of clinical therapy in acute kidney injury (AKI) or its progression to chronic kidney disease (CKD), administration of growth factors shows great potential in the treatment of renal repair and further fibrosis. At an early phase of AKI, administration of exogenous fibroblast growth factor 2 (FGF2) protects against renal injury by inhibition of mitochondrial damage and inflammatory response. Here, we investigated whether this treatment attenuates the long-term renal interstitial fibrosis induced by ischemia-reperfusion (I/R) injury. Unilateral renal I/R with contralateral nephrectomy was utilized as an in vivo model for AKI and subsequent CKD. Rats were randomly divided into four groups: Sham-operation group, I/R group, I/R-FGF2 group and FGF2-3D group. These groups were monitored for up to 2 months. Serum creatinine, inflammatory response and renal histopathology changes were detected to evaluate the role of FGF2 in AKI and followed renal interstitial fibrosis. Moreover, the expression of vimentin, α-SMA, CD31 and CD34 were examined. Two months after I/R injury, the severity of renal interstitial fibrosis was significantly attenuated in both of I/R-FGF2 group and FGF2-3D group, compared with the I/R group. The protective effects of FGF2 administration were associated with the reduction of high-mobility group box 1 (HMGB1)-mediated inflammatory response, the inhibition of transforming growth factor beta (TGF-β1)/Smads signaling-induced epithelial-mesenchymal transition and the maintenance of peritubular capillary structure. A single dose of exogenous FGF2 administration 1 h or 3 days after reperfusion inhibited renal fibrogenesis and thus blocked the transition of AKI to CKD. Our findings provided novel insight into the role of FGF signaling in AKI-to-CKD progression and underscored the potential of FGF-based therapy for this devastating disease.

FGF2 positively regulates osteoclastogenesis via activating the ERK-CREB pathway

Fibroblast growth factor 2 (FGF2) plays crucial roles in the growth and development of several tissues. However, its function in bone homeostasis remains controversial. Here, we found that exogenous FGF2 supplementation inhibited the mineralization of bone marrow stromal cells (BMSCs), at least partially, via up-regulating the gene expression of osteoclastogenesis. The FGF receptor (FGFR) allosteric antagonist SSR128129E modestly, whereas the FGFR tyrosine kinase inhibitor AZD4547 significantly antagonized the effects of FGF2. Mechanistically, FGF2 stimulated ERK phosphorylation, and the ERK signaling inhibitor PD325901 strongly blocked FGF2 enhancement of osteoclastogenesis. Moreover, the phosphorylation of CREB was also activated in response to FGF2, thereby potentiating the interaction of p-CREB with the promoter region of Rankl gene. Notably, FGF2-deficient BMSCs exhibited higher mineralization capability and lower osteoclastogenic gene expression. Correspondingly, FGF2-knockout mice showed increased bone mass and attenuated expression of osteoclast-related markers, which were associated with moderate inhibition of the ERK signaling. In conclusion, FGF2 positively regulates osteoclastogenesis via stimulating the ERK-CREB pathway. These findings establish the importance of FGF2 in bone homeostasis, hinting the potential use of FGF2/ERK/CREB specific inhibitors to fight against bone-related disorders, such as osteoporosis.

High Expression of FGF2/SDC1 Signaling Contributes to Poor Clinical Outcome in Hodgkin Lymphoma Patients and SDC1 siRNA Suppresses Proliferation, Migration, and Invasion in Hodgkin Lymphoma Cells

Our recent studies have shown that Fibroblast Growth Factor-2 (FGF2) and Syndecan-1 (SDC1) expressions are upregulated, by 45- and 24-fold respectively, in CD30+ cells from tissues of relapsed versus relapsed-free Hodgkin lymphoma (HL) patients (p<0.005; Fig. 1A). Normal control and relapsed-free groups showed similar FGF2 and SDC1 expression profiles in tissue samples. The circulating HL cells in peripheral blood lymphocytes (PBL) have demonstrated that FGF2 and SDC1 mRNA were upregulated, by nearly 7000- and 9000-fold respectively, in relapsed versus relapsed-free HL patient samples (p<0.0001; Fig. 1B). Preliminary Kaplan-Meier analyses indicate that the HL patient group (n=4) with elevated co-expression of FGF2 and SDC1 had shortened survival by six-fold when compared to the majority of patients (n=11) with significantly lowered expression of FGF2/SDC1 (p < 0.05). This suggests that high levels of FGF2/SDC1 contribute to poor prognosis in HL patients. Western blot analyses showed that shed SDC1 regulate the HL cell division rate by a negative feedback mechanism. However, secretion of FGF2 and an abundance of four FGF receptor (FGFR) proteins were not associated with the cell division rate.Among eleven HL cell lines examined, HD-MyZ represented the best model for relapsed/refractory HL due to its highest cell proliferation rate (35 hour doubling time) and overexpression of FGF2 and SDC1, by 60- and 150-fold respectively, when compared with the B lymphoblast cell line, C1RB7. Immunofluorescence and co-immunoprecipitation studies in normal fibroblasts and HL cells demonstrate that FGF2 and SDC1 are co-localized on the cell membrane and bind together as ligand and receptor. Additionally, FGF2 binds to FGFRs in HL cells with lower affinity than in normal fibroblasts with predominant binding to FGFR1, suggesting that FGF2 signaling largely involves both SDC1 and FGFR1. Fluorescence microscopy studies showed that FGFR1 is constitutively phosphorylated in HL cells, and it stimulates cell proliferation by an autocrine pathway. Treatment with exogenous FGF2 in the absence of serum resulted in phosphorylation of both AKT and ERK, thereby activating FGF2-mediated cell proliferation. FGF2 induction also upregulated mRNA expressions of SDC1 (12-fold), proliferation marker- CCND1 (242-fold) and metastatic marker- MMP9 (67-fold) compared to unstimulated cells.To examine the molecular mechanism, whereby SDC1 is involved in the proliferation and metastasis of HL cells, SDC1 knockdown (KD) was achieved near 100% with 1nM siRNA after 48 hours of transfection. The SDC1 KD condition did not affect cell morphology but significantly attenuated cell proliferation by two-fold (p < 0.05; Fig. 2A) by downregulating BCL2 (87%), CCND1 (74%), and CDK4 (64%), indicating that SDC1 has pivotal functional roles in proliferation of HL cells. Transwell chamber assays showed that SDC1 KD reduced migration of HL cells by two-fold (p < 0.05; Fig. 2B) and invasion by three-fold (p < 0.005; Fig. 2C) by downregulating metastatic markers MMP9, ADAM9, ITGB3, and VEGF . The flow cytometry data demonstrated that 50% of HL cells expressed residual SDC1 on the cell membrane after SDC1 KD for 48 hours, which potentially maintains the normal HL cells morphology and cellular functions. However, four days of prolonged SDC1 KD caused an induction of apoptosis by upregulating CASP3, and continuous KD for 17 days caused near eradication of surviving HL cells.In conclusion, our results provide evidence that increased FGF2/SDC1 expression and signaling may drive and maintain the hyperproliferation of HL cells, thereby contributing to disease progression and poor prognosis in HL patients. Therefore, a novel method to inhibit altered FGF2/SDC1 signaling offers an excellent potential for treating high-risk and refractory HL patient populations. [Display omitted] DisclosuresGoy:Genentech: Research Funding; Acerta: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics / J&J: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Feldman:Janssen: Speakers Bureau; Celgene: Speakers Bureau; AbbVie: Speakers Bureau; Bristol-Myers Squibb: Consultancy; Kite Pharma: Speakers Bureau; Seattle Genetics: Honoraria, Research Funding, Speakers Bureau; Pharmacyclics: Speakers Bureau. Leslie:KITE pharma: Speakers Bureau; celgene: Speakers Bureau; seattle genetics: Speakers Bureau. Pecora:COTA: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Caladrius Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Caffeine Inhibits Direct and Indirect Angiogenesis in Zebrafish Embryos.

In this study, we report the effects of caffeine on angiogenesis in zebrafish embryos both during normal development and after exposure to Fibroblast Growth Factor 2 (FGF2). As markers of angiogenesis, we measured the length and width of intersegmental vessels (ISVs), performed whole-mount in situ hybridization with fli1 and cadh5 vascular markers, and counted the number of interconnecting vessels (ICVs) in sub-intestinal venous plexus (SIVP). In addition, we measured angiogenesis after performing zebrafish yolk membrane (ZFYM) assay with microinjection of fibroblast growth factor 2 (FGF2) and perivitelline tumor xenograft assay with microinjection of tumorigenic FGF2-overexpressing endothelial (FGF2-T-MAE) cells. The results showed that caffeine treatment causes a shortening and thinning of ISVs along with a decreased expression of the vascular marker genes and a decrease in the number of ICVs in the SIVP. Caffeine was also able to block angiogenesis induced by exogenous FGF2 or FGF2-producing cells. Overall, our results are suggestive of the inhibitory effect of caffeine in both direct and indirect angiogenesis.

Exogenous FGF-2 prolongs endothelial connection in multilayered human skeletal muscle cell sheet

Angiogenesis is a pressing issue in tissue engineering associated with restoration of blood supply to ischemic tissues and promotion of rapid vascularization of tissue-engineered grafts. Fibroblast growth factor-2 (FGF-2) plays a vital role in processes such as angiogenesis and is an attractive candidate for tissue engineering. While skeletal muscle tissue engineering is established, the role of FGF-2 in endothelial function to promote angiogenesis after transplantation is unclear. Here, a culture system comprising a five-layered sheet of human skeletal muscle cells co-incubated on green fluorescent protein-expressing human umbilical vein endothelial cells (GFP-HUVECs) mimicking invivo angiogenesis was used to investigate the role of FGF-2 in vascularization of engineered tissues. The basal level of FGF-2 in cultured media of skeletal muscle cell sheets was undetectable. Therefore, cell sheets co-incubated with GFP-HUVECs were exogenously treated with 10ng/mL FGF-2, and endothelial network formation was evaluated. After prolonged culture, the endothelial network length and connectivity increased following treatment with FGF-2 as compared with control treatment. The numbers of medium and long endothelial networks significantly increased inside the sheet longer than 0.2 and 0.4cm, respectively, after FGF-2 treatment. Time-lapse microscopy monitoring dynamic endothelial behavior revealed that FGF-2-mediated maintenance of endothelial connection and retardation of endothelial network disconnection after 72h. The present study suggests the precise role of FGF-2 in maintaining endothelial connection and the extent of the endothelial network in skeletal muscle cell sheets. This understanding can be applied to design invitro pre-vascularized tissue and graft integration prospects.

Effect of fibroblast growth factor 2 on degenerative endplate chondrocyte: From anabolism to catabolism

BackgroundEndplate degeneration is characterized by an unbalance between the anabolism and catabolism of endplate chondrocyte (CH). Fibroblast growth factor 2 (FGF2) has been shown to promote cartilage repair by increasing articular CH anabolic activity. We aimed to explore the effect of FGF2 on the metabolism of endplate CH to elucidate whether FGF2 could be used as a therapy to delay the endplate degeneration. MethodsWe collected the endplate tissue from the patients and tested the collagen II mRNA level as the anabolic marker and the MMP-13 and TIMP-4 expression as the catabolic markers. The FGF2, FGF receptor 1 (FGFR1), and FGFR3 mRNA expression of the endplate tissue were also analyzed. Besides, we treated the CHs with exogenic FGF2 protein, measured the markers mentioned above, the proliferation and the apoptosis of the CHs. To compare the effect of FGF2 on the CHs with or without degeneration, we also induced CHs degeneration by interleukin-1β (IL-1β) stimulation and used the FGF2 protein to treat the degenerative CHs. ResultsSeverely degenerative endplate had a lower collagen II and TIMP-4 mRNA level, but it expressed a more massive amount of MMP-13, FGF2, and FGFR1. FGF2 supplement upregulated the FGFR1/FGFR3, TIMP-4, collagen II expression, and promoted the CHs proliferation. In the first 24 h of IL-1β treatment, the FGF2 mRNA expression was suppressed, but it significantly increased 48 h later. Meanwhile, the FGFR1 was upregulated, and FGFR3 was inhibited by IL-1β treatment. Interestingly, the FGF2 protein supplement accelerated the degenerative CHs catabolism by decreasing collagen II and TIPM-4 expression but increasing MMP-13. However, the FGF2 could promote the anabolism process in case of the blocking of FGFR1. The FGF2 supplement could also promote the proliferation and inhibited the apoptosis of degenerative CHs, which could be magnified by FGFR1 blocking. ConclusionsThe results demonstrate that FGF2 is upregulated in the highly degenerative endplate. The supplement of FGF2 contributes to the anabolism in the early phase of endplate degeneration. In the later stage of endplate degeneration, FGF2 turns to accelerate the catabolism, which can be rejected by the reasonable use of FGFR1 inhibitors.

Pentraxin 3 inhibits fibroblast growth factor 2 induced osteoclastogenesis in rheumatoid arthritis

BackgroundSynovial fibroblasts (SFs) act as key effector cells mediating synovial inflammation and joint destruction in rheumatoid arthritis (RA). Fibroblast growth factor 2 (FGF2) and its receptors (FGFRs) play important roles in RASF-mediated osteoclastogenesis. Pentraxin 3 (PTX3) is a soluble pattern recognition receptor with nonredundant roles in inflammation and innate immunity. PTX3 is produced by various cell types, including SFs and is highly expressed in RA. However, the role of PTX3 in FGF2-induced osteoclastogenesis in RA and the underlying mechanism have been poorly elucidated. MethodsWe first determined the expression of FGF2 and RANKL in synovial tissue and synovial fluid of RA patients. We then examined the effect of PTX3 on RASF osteoclastogenesis induced by endogenous and exogenous FGF2 in isolated RASF cells treated with FGF2 and/or recombinant PTX3 (rPTX3). Thirdly, we analyzed the effect of PTX3 on FGF2 binding to FGFR-1 and HSPG receptors on RASFs. Lastly, we evaluated joint morphology after injection of rPTX3 into collagen-induced arthritis (CIA) mice. ResultsFGF2 was confirmed to be highly expressed in both synovial tissue and synovial fluid of RA patients. FGF2 promoted cell proliferation and increased the expressions of RANKL and ICAM-1 and RANKL/OPG to induce osteoclastogenesis in RASF, while anti-FGF2 neutralized this effect. PTX3 significantly inhibited FGF2-induced RASF cell growth and osteoclastogenesis by preventing the interaction of 125I-FGF2 and FGFRs on the same cells. In addition, administration of rPTX3 significantly ameliorated cartilage and bone destruction in mice with CIA. ConclusionsPTX3 exhibited an inhibitory effect on the autocrine and paracrine stimulation of FGF2 on SFs, and ameliorated bone destruction in CIA mice. PTX3 may be implicated in bone destruction in RA, which may provide theoretical evidence and potential therapeutic targets for RA treatment.

Down-Regulation of Fibroblast Growth Factor 2 (FGF2) Contributes to the Premature Senescence of Mouse Embryonic Fibroblast.

BackgroundFreshly isolated mouse embryonic fibroblasts (MEFs) have great proliferation capacity but quickly enter senescent state after several rounds of cell cycle, a process called premature senescence. Cellular senescence can be induced by various stresses such as telomere erosion, DNA damage, and oncogenic signaling. But the contribution of other molecules, such as growth factors, to cellular senescence is incompletely understood. This study aimed to compare the gene expression difference between non-senescent and senescent MEFs to identify the key molecule(s) involved in the spontaneous senescence of MEFs.Material/MethodsPrimary MEFs were isolated from E12.5 pregnant C57/BL6 mice. The cells were continuously cultured in Dulbecco’s Modified Eagle Medium for 9 passages. SA-β-Gal staining was used as an indicator of cell senescence. The supernatant from primary MEFs (P1 medium) or Passage 6 MEFs (P6 medium) were used to culture freshly isolated MEFs to observe the effects on cell senescence state. Gene expression profiles of primary and senescent MEFs were investigated by RNA-Seq to find the key genes involved in cell senescence. Adipocyte differentiation assay was used to evaluate the stemness of MEFs cultured in FGF2-stimulated medium.ResultsThe senescence of MEFs cultured in the P1 medium was alleviated when compared to the P6 medium. Downregulation of FGF2 expression was revealed by RNA-Seq and further confirmed by real-time quantitative polymerase chain reaction and western blot. FGF2-stimulated medium also had anti-senescence function and could maintain the differentiation ability of MEFs.ConclusionsThe premature senescence of MEFs was at least partially caused by FGF2 deficiency. Exogenous FGF2 could alleviate the senescent phenotype.

Human Caesarean scar-derived feeder cells: a novel feeder cell type for culturing human pluripotent stem cells without exogenous basic fibroblast growth factor supplementation.

In a feeder-dependent culture system of human pluripotent stem cells (hPSCs), coculture with mouse embryonic fibroblasts may limit the clinical use of hPSCs. The aim of this study was to determine the feasibility of using human Caesarean scar fibroblasts (HSFs) as feeder cells for the culture of hPSCs. HSFs were isolated and characterised and cocultured with hPSCs, and the pluripotency, differentiation ability and karyotypic stability of hPSCs were determined. Inactivated HSFs expressed genes (including inhibin subunit beta A (INHBA), bone morphogenetic protein 4 (BMP4), fibroblast growth factor 2 (FGF2), transforming growth factor-β1 (TGFB1), collagen alpha-1(I) (COL1A1) and fibronectin-1 (FN1) that have been implicated in the maintenance of hPSC pluripotency. When HSFs were used as feeder cells, the pluripotency and karyotypic stability of hPSC lines did not change after prolonged coculture. Interestingly, exogenous FGF2 could be omitted from the culture medium when HSFs were used as feeder cells for hESCs but not hiPSCs. hESCs cocultured with HSF feeder cells in medium without FGF2 supplementation maintained their pluripotency (as confirmed by the expression of pluripotency markers and genes), differentiated invitro into embryonic germ layers and maintained their normal karyotype. The present study demonstrates that HSFs are a novel feeder cell type for culturing hPSCs and that supplementation of exogenous FGF2 is not necessary for the Chula2.hES line.

Enhancing the Efficacy of Stem Cell Therapy with Glycosaminoglycans.

SummaryHuman mesenchymal stem cell (hMSC) therapy offers significant potential for osteochondral regeneration. Such applications require their ex vivo expansion in media frequently supplemented with fibroblast growth factor 2 (FGF2). Particular heparan sulfate (HS) fractions stabilize FGF2-FGF receptor complexes. We show that an FGF2-binding HS variant (HS8) accelerates the expansion of freshly isolated bone marrow hMSCs without compromising their naivety. Importantly, the repair of osteochondral defects in both rats and pigs is improved after treatment with HS8-supplemented hMSCs (MSCHS8), when assessed histologically, biomechanically, or by MRI. Thus, supplementing hMSC culture media with an HS variant that targets endogenously produced FGF2 allows the elimination of exogenous growth factors that may adversely affect their therapeutic potency.

Exogenous FGF-2 improves biological activity of endothelial progenitor cells exposed to high glucose conditions

Purpose: To investigate the effects of exogenous basic fibroblast growth factor -2 (FGF-2) on the biological activity of endothelial progenitor cells (EPCs) exposed to high glucose conditions.Materials and Methods: 1) Bone marrow EPCs from C57BL/6 mice were isolated and cultured in vitro. EPC purity was identified by flow cytometry and immunofluorescence staining. 2) Apoptosis was detected by TUNEL assay. Migration and tube formation ability was detected by Transwell chamber and Matrigel assays, respectively. The expression and activation of β-catenin was detected by Western blot. 3) Doppler flowmetry was used to detect the effect of FGF2 on blood flow recovery in ischemic hind limbs of mice.Results: 1) FGF-2 treatment reversed high glucose induced growth inhibition of EPCs. FGF-2 treatment also increased migration and tube formation ability of EPCs even in high glucose conditions. 2) Western blot analysis demonstrated that the percentage of activated β-catenin/total β-catenin in the high glucose group were significantly lower than that in the control group, while FGF-2 treatment reversed high glucose induced β-catenin inhibition. 3) In vivo experiments demonstrated that the blood flow recovery in ischemic hind limbs of mice was significantly improved after FGF-2 treatment.Conclusion: Exogenous FGF-2 could play a role in the functional repair of damaged EPC exposed to high glucose conditions, via the activation of the Wnt/β-catenin signaling pathway.

FGF-2 targets sclerostin in bone and myostatin in skeletal muscle to mitigate the deleterious effects of glucocorticoid on musculoskeletal degradation

AimMyokines are associated with regulation of bone and muscle mass. However, limited information is available regarding the impact of myokines on glucocorticoid (GC) mediated adverse effects on the musculoskeletal system. This study investigates the role of myokine fibroblast growth factor-2 (FGF-2) in regulating GC-induced deleterious effects on bone and skeletal muscle. MethodsPrimary osteoblast cells and C2C12 myoblast cell line were treated with FGF-2 and then exposed to dexamethasone (GC). FGF-2 mediated attenuation of the inhibitory effect of GC on osteoblast and myoblast differentiation and muscle atrophy was assessed through quantitative PCR and western blot analysis. Further, FGF-2 was administered subcutaneously to dexamethasone treated mice to collect bone and skeletal muscle tissue for in vivo analysis of bone microarchitecture, mechanical strength, histomorphometry and for histological alterations in treated tissue samples. Key findingsFGF-2 abrogated the dexamethasone induced inhibitory effect on osteoblast differentiation by modulating BMP-2 pathway and inhibiting Wnt antagonist sclerostin. Further, dexamethasone induced atrophy in C2C12 cells was mitigated by FGF-2 as evident from down regulation of atrogenes expression. FGF-2 prevented GC-induced impairment of mineral density, biomechanical strength, trabecular bone volume, cortical thickness and bone formation rate in mice. Additionally, skeletal muscle tissue from GC treated mice displayed weak myostatin immunostaining and reduced expression of atrogenes following FGF-2 treatment. SignificanceFGF-2 mitigated GC induced effects through inhibition of sclerostin and myostatin expression in bone and muscle respectively. Taken together, this study exhibited the role of exogenous FGF-2 in sustaining osteoblastogenesis and inhibiting muscle atrophy in presence of glucocorticoid.

Fibroblast Growth Factor 2 Modulates Hippocampal Microglia Activation in a Neuroinflammation Induced Model of Depression.

Recent studies indicate that disturbed structure and function of microglia can cause depression and associated neurogenesis impairments. Our previous work has demonstrated that exogenous fibroblast growth factor 2 (FGF2) reverses the depressive-like behaviors and the impaired hippocampal neurogenesis in a neuroinflammatory model of depression. However, whether and how the antidepressant effects of FGF2 involve the modulation of microglia activation has not been elucidated. In this study, to examine the effects of FGF2 on microglia activation, exogenous FGF2 was supplemented to the lateral ventricle of rats during the neuroinflammatory state induced by central lipopolysaccharides (LPS) administrations. It was found that FGF2 infusions reversed the LPS-induced depressive-like behaviors and inhibited the hippocampal microglia activation. In LPS-treated rats, FGF2 decreased the level of pro-inflammatory cytokines including interlukin-1β (IL-1β), IL-6 and tumor necrosis factor (TNF)-α, increased the level of IL-10, the anti-inflammatory cytokine and reversed the decreased expression of CX3CL1, a chemokine mainly expressed by neurons and keeping microglia in surveillance. Further, we examined the effects of inhibited FGF2 signaling by administration of SU5402, an FGFR inhibitor. It was found that SU5402 itself evoked depressive-like behaviors, induced microglia activation, increased production of pro-inflammatory cytokines including IL-1β, IL-6 and TNF-α, and decreased the expression of CX3CL1. Two lines of results that FGF2 signaling and FGFR inhibitor can effectively but oppositely modulate the regulation of microglia and the generation of depressive-like behavior, suggesting that microglia-regulated mechanisms may underlie the antidepressant role of FGF2. The present data provide novel insights into the understanding of mechanism of neuroinflammation-associated depression and may serve as a novel mechanism-based target for the treatment of inflammation-related depression.

FGF-2 is a driving force for chromosomal instability and a stromal factor associated with adverse clinico-pathological features in prostate cancer

BackgroundThere is mounting evidence to suggest that stromal cells play an integral role in the progression of prostate cancer (PCa). One of the most frequently altered growth factors in PCa is fibroblast growth factor-2 (FGF-2). It has previously been proposed that early stages of PCa are characterized by a primarily exogenous, that is, stromal cell-derived FGF-2 production, whereas advanced tumors rely more on an autocrine FGF-2 production. Prostate cancer progression is characterized by an increase of genomic instability including aneuploidy and structural chromosomal alterations. Herein, we address 2 problems that have not been comprehensively answered. First, we ask whether exogenous FGF-2 can directly drive genomic instability to promote PCa progression. Second, we investigate whether and to what extent stromal FGF-2 expression is maintained in advanced PCa and whether this influences tumor progression and patient prognosis. MethodsIn vitro experiments to investigate the role of FGF-2 in numerical and structural chromosomal instability were performed using immunofluorescence microscopy, fluorescence in situ hybridization and single cell electrophoresis. A human patient-derived xenograft mouse model recapitulating osteoblastic PCa bone metastasis was used for in vivo validation experiments. The prognostic role of stromal FGF-2 expression was analyzed using immunohistochemical staining of a tissue microarray with primary tumor specimens from 162 predominantly high-risk patients with PCa. ResultsOur results show that FGF-2 not only rapidly induces mitotic defects and numerical chromosomal imbalances but also an enhanced DNA breakage to promote chromosomal instability. Using the patient-derived xenograft model, we show that a deregulation of the FGF axis results in an increase of mitotic aberrations as well as DNA damage checkpoint activation in vivo. The FGFR inhibitor dovitinib was found to reduce numerical chromosomal instability as well as DNA breakage, thus underscoring the relevance of the FGF axis in promoting genomic instability. An overexpression of tumor cell-associated FGF-2 was detected in 52 of 162 patients (32.1%), whereas a stromal overexpression was found in 27 of 165 patients (16%). Remarkably, a strong stromal FGF-2 expression was associated with a significantly higher clinical stage and higher biochemical recurrence rate. Patients with strong stromal FGF-2 expression also had a significantly worse biochemical recurrence-free survival. ConclusionsOur results underscore that exogenous FGF-2 can shape PCa cell genomes and that stromal FGF-2 expression is detectable in a sizeable proportion of advanced PCa where it is associated with adverse clinico-pathological features. Our results highlight the impact of the tumor stroma on malignant progression and provide a rationale for a further exploration of components of the tumor stroma as therapeutic targets in PCa.

FGF2 Enhances Odontoblast Differentiation by αSMA+ Progenitors In Vivo

The goal of this study was to examine the effects of early and limited exposure of perivascular cells expressing α (αSMA) to fibroblast growth factor 2 (FGF2) in vivo. We performed in vivo fate mapping by inducible Cre-loxP and experimental pulp injury in molars to induce reparative dentinogenesis. Our results demonstrate that early delivery of exogenous FGF2 to exposed pulp led to proliferative expansion of αSMA-tdTomato+ cells and their accelerated differentiation into odontoblasts. In vivo lineage-tracing experiments showed that the calcified bridge/reparative dentin in FGF2-treated pulps were lined with an increased number of Dspp+ odontoblasts and devoid of BSP+ osteoblasts. The increased number of odontoblasts derived from αSMA-tdTomato+ cells and the formation of reparative dentin devoid of osteoblasts provide in vivo evidence for the stimulatory effects of FGF signaling on odontoblast differentiation from early progenitors in dental pulp.

Regulation of vascular smooth muscle cell calcification by syndecan-4/FGF-2/PKCα signalling and cross-talk with TGFβ.

AimsVascular calcification is a major cause of morbidity and mortality. Fibroblast growth factor-2 (FGF-2) plays an instructive role in osteogenesis and bone development, but its role in vascular calcification was unknown. Therefore, we investigated the involvement of FGF-2 in vascular calcification and determined the mechanism by which it regulates this process.Methods and resultsWe demonstrate that FGF-2 expression is increased in vascular smooth muscle cells (VSMCs) induced to deposit a mineralized matrix by incubation with β-glycerophosphate. FGF-2 is also localized to sites of calcification within human atherosclerotic plaques. The expression of syndecan-4, a heparan sulfate proteoglycan which regulates FGF-2 signalling, is also increased in mineralizing VSMCs and co-localizes with FGF-2 in human calcified atherosclerotic plaques. Exogenous FGF-2 inhibits VSMC mineralization, and this inhibition is reduced when syndecan-4 expression is knocked-down using siRNA. Biochemical inhibition of FGFR signalling using a pan FGFR inhibitor (BGJ398) or knocking-down syndecan-4 expression in VSMCs using siRNA increases VSMC mineralization. These increases are prevented by inhibiting transforming growth factor-β (TGFβ) signalling with SB431542, suggesting cross-talk between FGF-2 and TGFβ signalling is crucial for the regulation of VSMC mineralization. Syndecan-4 can also regulate FGF-2 signalling directly via protein kinase Cα (PKCα) activation. Biochemical inhibition of PKCα activity using Gö6976, or siRNA-mediated suppression of PKCα expression increases VSMC mineralization; this increase is also prevented with SB431542. Finally, the ability of FGF-2 to inhibit VSMC mineralization is reduced when PKCα expression is knocked-down.ConclusionThis is the first demonstration that syndecan-4 promotes FGF-2 signalling, and in turn, suppresses VSMC mineralization by down-regulating TGFβ signalling. Our discoveries that FGF-2 and syndecan-4 expression is increased in mineralizing VSMCs and that PKCα regulates FGF-2 and TGFβ signalling in VSMCs suggests that the syndecan-4/FGF-2/TGFβ signalling axis could represent a new therapeutic target for vascular calcification.

Smooth muscle cells of human veins show an increased response to injury at valve sites

ObjectiveVenous valves are essential but are prone to injury, thrombosis, and fibrosis. We compared the behavior and gene expression of smooth muscle cells (SMCs) in the valve sinus vs nonvalve sites to elucidate biologic differences associated with vein valves. MethodsTissue explants of fresh human saphenous veins were prepared, and the migration of SMCs from explants of valve sinus vs nonvalve sinus areas was measured. Proliferation and death of SMCs were determined by staining for Ki67 and terminal deoxynucleotidyl transferase dUTP nick end labeling. Proliferation and migration of passaged valve vs nonvalve SMCs were determined by cell counts and using microchemotaxis chambers. Global gene expression in valve vs nonvalve intima-media was determined by RNA sequencing. ResultsValve SMCs demonstrated greater proliferation in tissue explants compared with nonvalve SMCs (19.3% ± 5.4% vs 6.8% ± 2.0% Ki67-positive nuclei at 4 days, respectively; mean ± standard error of the mean, five veins; P < .05). This was also true for migration (18.2 ± 2.7 vs 7.5 ± 3.0 migrated SMCs/explant at 6 days, respectively; 24 veins, 15 explants/vein; P < .0001). Cell death was not different (39.6% ± 16.1% vs 41.5% ± 16.0% terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells, respectively, at 4 days, five veins). Cultured valve SMCs also proliferated faster than nonvalve SMCs in response to platelet-derived growth factor subunit BB (2.9 ± 0.2-fold vs 2.1 ± 0.2-fold of control, respectively; P < .001; n = 5 pairs of cells). This was also true for migration (6.5 ± 1.2-fold vs 4.4 ± 0.8-fold of control, respectively; P < .001; n = 7 pairs of cells). Blockade of fibroblast growth factor 2 (FGF2) inhibited the increased responses of valve SMCs but had no effect on nonvalve SMCs. Exogenous FGF2 increased migration of valve but not of nonvalve SMCs. Unlike in the isolated, cultured cells, blockade of FGF2 in the tissue explants did not block migration of valve or nonvalve SMCs from the explants. Thirty-seven genes were differentially expressed by valve compared with nonvalve intimal-medial tissue (11 veins). Peptide-mediated inhibition of SEMA3A, one of the differentially expressed genes, increased the number of migrated SMCs of valve but not of nonvalve explants. ConclusionsValve compared with nonvalve SMCs have greater rates of migration and proliferation, which may in part explain the propensity for pathologic lesion formation in valves. Whereas FGF2 mediates these effects in cultured SMCs, the mediators of these stimulatory effects in the valve wall tissue remain unclear but may be among the differentially expressed genes discovered in this study. One of these genes, SEMA3A, mediates a valve-specific inhibitory effect on the injury response of valve SMCs.

Exogenous FGF2 reverses depressive-like behaviors and restores the suppressed FGF2-ERK1/2 signaling and the impaired hippocampal neurogenesis induced by neuroinflammation

Our previous work demonstrated that neuroinflammation evoked by triple repeated central LPS challenges inhibited adult hippocampal neurogenesis that were correlated with the depressive-like behavioral symptoms induced by neuroinflammation. These findings suggest that hippocampal neurogenesis might be one of biological mechanisms underlying depression induced by neuroinflammation and targeting neurogenesis might lead to new therapeutic strategies for the treatment of depression. In this study, we manipulated adult hippocampal neurogenesis using fibroblast growth factor 2 (FGF2), one crucial molecule modulating cell proliferation and survival in central nervous system, and investigate the involvement and the potential therapeutic effects of FGF2 on neuroinflammation-induced depression. Central lipopolysaccharides (LPS) challenges were used as previously to evoke the neuroinflammatory state in the brain of rat. Exogenous FGF2 was infused into lateral ventricle during the neuroinflammatory state. It was found that the protein expression of FGF2 in hippocampus was inhibited by neuroinflammation. The activation of extracellular signal-regulated kinase (ERK), the downstream molecule of FGF2, was also inhibited by neuroinflammation. Exogenous FGF2 infusions prevented the decrease in phosphorylation of ERK1/2 under neuroinflammation state. Exogenous FGF2 reversed depressive-like behaviors and the impaired hippocampal neurogenesis induced by neuroinflammation. These findings provide evidence that the FGF2-ERK1/2 pathway is involved in the pathophysiology of depressive-like behaviors, and manipulating the neurogenesis pathway is a viable therapeutic approach to inflammation-associated depression.