Exogenous FGF-2 Research Articles

Macrophages modulate fibrosis during newt lens regeneration

BackgroundPrevious studies have suggested that macrophages are present during lens regeneration in newts, but their role in the process is yet to be elucidated.MethodsHere we generated a transgenic reporter line using the newt, Pleurodeles waltl, that traces macrophages during lens regeneration. Furthermore, we assessed early changes in gene expression during lens regeneration using two newt species, Notophthalmus viridescens and Pleurodeles waltl. Finally, we used clodronate liposomes to deplete macrophages during lens regeneration in both species and tested the effect of a subsequent secondary injury after macrophage recovery.ResultsMacrophage depletion abrogated lens regeneration, induced the formation of scar-like tissue, led to inflammation, decreased iris pigment epithelial cell (iPEC) proliferation, and increased rates of apoptosis in the eye. Some of these phenotypes persisted throughout the last observation period of 100 days and could be attenuated by exogenous FGF2 administration. A distinct transcript profile encoding acute inflammatory effectors was established for the dorsal iris. Reinjury of the newt eye alleviated the effects of macrophage depletion, including the resolution of scar-like tissue, and re-initiated the regeneration process.ConclusionsTogether, our findings highlight the importance of macrophages for facilitating a pro-regenerative environment in the newt eye by regulating fibrotic responses, modulating the overall inflammatory landscape, and maintaining the proper balance of early proliferation and late apoptosis of the iPECs.

Cancer-associated fibroblasts promote gastric cancer cell proliferation by paracrine FGF2-driven ribosome biogenesis

The cancer-associated fibroblast (CAF)-derived secretome plays critical roles in tumor progression by remodelling tumor microenvironment. Tumorigenesis is accompanied by the transformation of normal fibroblasts (NF) into CAF, leading to significant changes in their secretome. This work aims to identify the differential components of secretome between NFs and CAFs and reveal their functions in gastric cancer (GC). Firstly, our molecular typing studies and immune infiltration analysis showed that CAF infiltration level was increased and showed a significant association with clinical characteristics and poor prognosis of GC patients. Secondly, RNA-seq analysis revealed that a total of 1531 genes showed significant expression changes between NF and CAF. According to the annotation of the Human Protein Atlas (HPA) database, 147 genes encode secreted proteins, including FGF2. Particularly, the cell co-culture and RNA sequencing studies confirmed that exogenous recombinant FGF2 protein treatment promoted GC cell proliferation by enhancing ribosome biogenesis. The rescue assay showed that CAF-secreted FGF2 protein promotes GC cell growth and proliferation in a FGFR1-dependent manner. Our finding provides evidence that targeting blockade of CAF-derived FGF2 protein might be a promising treatment for GC.

FGF2 Rescued Cisplatin-Injured Granulosa Cells through the NRF2-Autophagy Pathway.

Premature ovarian failure (POF) is a complicated disorder related to the apoptosis of granulosa cells. The incidence of chemotherapy-associated POF is rising dramatically owing to the increasing proportion of cancer in adolescents. According to previous studies, oxidative stress caused by chemotherapeutic agents plays an important role in the development of POF. However, the exact effects of nuclear factor-erythroid 2-related factor2 (NRF2), a pivotal anti-oxidative factor, are still unknown in chemotherapy-associated POF. Firstly, we manipulated NRF2 expressions on a genetic or pharmaceutical level in cisplatin-injured granulosa cell models. The results indicate that the increasing NRF2 in cisplatin-injured cells was just compensatory and not enough to resist the accumulated stress. Upregulation of NRF2 could protect granulosa cells against cisplatin via elevating autophagic level by using an autophagic activator (rapamycin) and inhibitor (chloroquine). Additionally, exogenous FGF2 exerted a protective role by increasing NRF2 expression and promoting its nuclear translocation. Meanwhile, the results in cisplatin-POF mice models were consistent with what was found in injured cells. In conclusion, our research proved that FGF2 rescued cisplatin-injured granulosa cells through the NRF2-autophagy pathway and might provide a possible alternative treatment choice by targeting NRF2 for POF patients who are intolerant or unsuitable to FGF2.

Exogenous FGF-1 Differently Regulates Oligodendrocyte Replenishment in an SCI Repair Model and Cultured Cells.

We studied the phenotypes in an oligodendrocyte genesis site at the acute stage of spinal cord injury, when we observed regenerated ascending neurites. Pan-oligodendrocyte marker OLIG2+ cells were more in fibroblast growth factor (FGF)-1-treated rats (F group) than in non-treated (T group) in this site, while the number of NG2+OX42- oligodendrocyte progenitor cell (OPC), CNPase+ OPC, Nkx2.2+ OPC, and APC+ remyelinating oligodendrocytes was less in the F group. Paradoxically, when we label the rats with pulsed bromodeoxyuridine (BrdU), we found that the mitotic NKX2.2+ OPC cells are more in the F group than in the T group. We tested the embryonic spinal cord mixed culture. FGF treatment resulted in more NG2(+) CNPase (+) than non-FGF-1-treated culture, while the more mature NG2(-) CNPase(+) cell numbers were reduced. When we block the FGF receptor in the injured rat model, the NG2+OX42- cell numbers were increased to be comparable to non-FGF-1 rats, while this failed to bring back the APC+ mature oligodendrocyte cell numbers. As migration of OPC toward injury is a major factor that was absent from the cell culture, we tested 8 mm away from the injury center, and found there were more NG2+ cells with FGF-1 treatment. We proposed that it was possibly a combination of migration and proliferation that resulted in a reduction in the NG2+ OPC population at the oligodendrocyte genesis site when FGF-1 was added to the spinal cord injury in vivo.

Patterns of NFkB activation resulting from damage, reactive microglia, cytokines, and growth factors in the mouse retina

Müller glia are a cellular source for neuronal regeneration in vertebrate retinas. However, the capacity for retinal regeneration varies widely across species. Understanding the mechanisms that regulate the reprogramming of Müller glia into progenitor cells is key to reversing the loss of vision that occurs with retinal diseases. In the mammalian retina, NFkB signaling promotes glial reactivity and represses the reprogramming of Müller glia into progenitor cells. Here we investigate different cytokines, growth factors, cell signaling pathways, and damage paradigms that influence NFkB-signaling in the mouse retina. We find that exogenous TNF and IL1β potently activate NFkB-signaling in Müller glia in undamaged retinas, and this activation is independent of microglia. By comparison, TLR1/2 agonist indirectly activates NFkB-signaling in Müller glia, and this activation depends on the presence of microglia as Tlr2 is predominantly expressed by microglia, but not other types of retinal cells. Exogenous FGF2 did not activate NFkB-signaling, whereas CNTF, Osteopontin, WNT4, or inhibition of GSK3β activated NFkB in Müller glia in the absence of neuronal damage. By comparison, dexamethasone, a glucocorticoid agonist, suppressed NFkB-signaling in Müller glia in damaged retinas, in addition to reducing numbers of dying cells and the accumulation of reactive microglia. Although NMDA-induced retinal damage activated NFkB in Müller glia, optic nerve crush had no effect on NFkB activation within the retina, whereas glial cells within the optic nerve were responsive. We conclude that the NFkB pathway is activated in retinal Müller glia in response to many different cell signaling pathways, and activation often depends on signals produced by reactive microglia.

Regulation of Transplanted Cell Homing by FGF1 and PDGFB after Doxorubicin Myocardial Injury.

There is no effective treatment for the total recovery of myocardial injury caused by an anticancer drug, doxorubicin (Dox). In this study, using a Dox-induced cardiac injury model, we compared the cardioprotective effects of ventricular cells harvested from 11.5-day old embryonic mice (E11.5) with those from E14.5 embryos. Our results indicate that tail-vein-infused E11.5 ventricular cells are more efficient at homing into the injured adult myocardium, and are more angiogenic, than E14.5 ventricular cells. In addition, E11.5 cells were shown to mitigate the cardiomyopathic effects of Dox. In vitro, E11.5 ventricular cells were more migratory than E14.5 cells, and RT-qPCR analysis revealed that they express significantly higher levels of cytokine receptors Fgfr1, Fgfr2, Pdgfra, Pdgfrb and Kit. Remarkably, mRNA levels for Fgf1, Fgf2, Pdgfa and Pdgfb were also found to be elevated in the Dox-injured adult heart, as were the FGF1 and PDGFB protein levels. Addition of exogenous FGF1 or PDGFB was able to enhance E11.5 ventricular cell migration in vitro, and, whereas their neutralizing antibodies decreased cell migration. These results indicate that therapies raising the levels of FGF1 and PDGFB receptors in donor cells and or corresponding ligands in an injured heart could improve the efficacy of cell-based interventions for myocardial repair.

FGF2 disruption enhances thermogenesis in brown and beige fat to protect against adiposity and hepatic steatosis

ObjectiveFibroblast growth factor 2 (FGF2) has been reported to play divergent roles in white adipogenic differentiation, however, whether it regulates thermogenesis of fat tissues remains largely unknown. We therefore aimed to investigate the effect of FGF2 on fat thermogenesis and elucidate the underlying mechanisms. MethodsFGF2-KO and wild-type (WT) mice were fed with chow diet and high-fat diet (HFD) for 14 weeks. The brown and white fat mass, thermogenic capability, respiratory exchange ratio, and hepatic fat deposition were determined. In vitro experiments were conducted to compare the thermogenic ability of FGF2-KO- with WT-derived brown and white adipocytes. Exogenous FGF2 was supplemented to in vitro-cultured WT brown and ISO-induced beige adipocytes. The FGFR inhibitor, PPARγ agonist, and PGC-1α expression lentivirus were used with the aid of technologies including Co-IP, ChIP, and luciferase reporter assay to elucidate the mechanisms underlying the FGF2 regulation of thermogenesis. ResultsFGF2 gene disruption results in increased thermogenic capability in both brown and beige fat, supporting by increased UCP1 expression, enhanced respiratory exchange ratio, and elevated thermogenic potential in response to cold exposure. Thus, the deletion of FGF2 protects mice from high fat-induced adiposity and hepatic steatosis. Mechanistically, in vitro investigations indicated FGF2 acts in autocrine/paracrine fashions. Exogenous FGF2 supplementation inhibits both PGC-1α and PPARγ expression, leading to suppression of UCP1 expression in brown and beige adipocytes. ConclusionsThese findings demonstrate that FGF2 is a novel thermogenic regulator, suggesting a viable potential strategy for using FGF2-selective inhibitors in combat adiposity and associated hepatic steatosis.

Role of TGF\u03b21 and WNT6 in FGF2 and BMP4-driven endothelial differentiation of murine embryonic stem cells

Embryonic stem cells (ES) are a valuable source of endothelial cells. By co-culturing ES cells with the stromal PA6 cells, the endothelial commitment can be achieved by adding exogenous FGF2 or BMP4. In this work, the molecular pathways that direct the differentiation of ES cells toward endothelium in response to FGF2 are evaluated and compared to those activated by BMP4. To this purpose the genes expression profiles of both ES/PA6 co-cultures and of pure cultures of PA6 cells were obtained by microarray technique at different time points. The bioinformatics processing of the data indicated TGFβ1 as the most represented upstream regulator in FGF2-induced endothelial commitment while WNT pathway as the most represented in BMP4-activated endothelial differentiation. Loss of function experiments were performed to validate the importance of TGFβ1 and WNT6 respectively in FGF2 and BMP4-induced endothelial differentiation. The loss of TGFβ1 expression significantly impaired the accomplishment of the endothelial commitment unless exogenous recombinant TGFβ1 was added to the culture medium. Similarly, silencing WNT6 expression partially affected the endothelial differentiation of the ES cells upon BMP4 stimulation. Such dysfunction was recovered by the addition of recombinant WNT6 to the culture medium. The ES/PA6 co-culture system recreates an in vitro complete microenvironment in which endothelial commitment is accomplished in response to alternative signals through different mechanisms. Given the importance of WNT and TGFβ1 in mediating the crosstalk between tumor and stromal cells this work adds new insights in the mechanism of tumor angiogenesis and of its possible inhibition.

Extracellular Matrix Composition Is Altered Alongside Dysmorphology in the Developing Palate

Normal morphogenesis of the primary palate involves directed outgrowth of the facial primordia until they meet and fuse. Gradients of cellular proliferation induced by growth factors are the current paradigm for directed outgrowth, but active cellular migration and physical force propagated through the extracellular matrix (ECM) likely also direct growth. Previously we showed that two of the main components of the ECM, collagen IV and laminin, are thinner in regions of the frontonasal process (FNP) undergoing growth, suggesting tissue stiffness may passively constrain growth in certain directions. We have also shown that increasing Fibroblast growth factor (FGF) signaling in the FNP of chick embryos prior to palate fusion caused overgrowth and disrupted cellular organization. Since cells form and modify the ECM, and since the ECM is required for cellular organization and movement, the effects of FGF signaling on morphogenesis may be due in part to active feedback between ECM and cells. Our goal was to examine the changes to the ECM in the FNP following alteration of FGF signaling. Blocking FGF signaling with SU5402 caused hypoplasia of the FNP and increased the concentration of collagen IV and laminin in regions of expected outgrowth. Conversely, increasing FGF signaling with exogenous FGF2 caused overgrowth of the FNP and decreased concentration of the ECM components. Analysis using transmission electron microscopy of the FGF2 treated samples suggests increasing FGF signaling also decreases the thickness of the epithelium and basement membrane. These results provide preliminary evidence that ECM may play an important role in facial morphogenesis, and that FGF signaling may affect an active biomechanical feedback system between cells and ECM. In the future I will further characterize ECM composition and compliance across the FNP using Fourier transform infrared spectroscopy and atomic force microscopy to generate 3-dimensional maps of biomechanical properties in the face. I will also use confocal microscopy to map cellular organization across the face. I will overlay these maps onto representative reconstructions of the face to correlate regional variation in biomechanical properties and cellular organization with dysmorphologies that occur following exogenous modulation of FGF signaling.

FGF2: a key regulator augmenting tendon-to-bone healing and cartilage repair.

Ligament/tendon and cartilage injuries are clinically common diseases that perplex most clinicians.Because of the lack of blood vessels and nerves, their self-repairing abilities are rather poor. Therefore, surgeries are necessary and also widely used to treat ligament/tendon or cartilage injuries. However, after surgery, there are still many problems that affect healing. In recent years, it has been found that exogenous FGF2 plays an important role in the repair of ligament/tendon and cartilage injuries and exerts a synergistic effect with endogenous FGF2. Therefore, FGF2 can be used as a new type of biomolecule to accelerate tendon-to-bone healing and cartilage repair after injury.

Neural regulation in tooth regeneration of Ambystoma mexicanum

The presence of nerves is an important factor in successful organ regeneration in amphibians. The Mexican salamander, Ambystoma mexicanum, is able to regenerate limbs, tail, and gills when nerves are present. However, the nerve-dependency of tooth regeneration has not been evaluated. Here, we reevaluated tooth regeneration processes in axolotls using a three-dimensional reconstitution method called CoMBI and found that tooth regeneration is nerve-dependent although the dentary bone is independent of nerve presence. The induction and invagination of the dental lamina were delayed by denervation. Exogenous Fgf2, Fgf8, and Bmp7 expression could induce tooth placodes even in the denervated mandible. Our results suggest that the role of nerves is conserved and that Fgf+Bmp signals play key roles in axolotl organ-level regeneration. The presence of nerves is an important factor in successful organ regeneration in amphibians. The Mexican salamander, Ambystoma mexicanum, is able to regenerate limbs, tail, and gills when nerves are present. However, the nervedependency of tooth regeneration has not been evaluated. Here, we reevaluated tooth regeneration processes in axolotls using a three-dimensional reconstitution method called CoMBI and found that tooth regeneration is nerve-dependent although the dentary bone is independent of nerve presence. The induction and invagination of the dental lamina were delayed by denervation. Exogenous Fgf2, Fgf8, and Bmp7 expression could induce tooth placodes even in the denervated mandible. Our results suggest that the role of nerves is conserved and that Fgf+Bmp signals play key roles in axolotl organ-level regeneration.

DNA demethylation is a driver for chick retina regeneration

ABSTRACT Cellular reprogramming resets the epigenetic landscape to drive shifts in transcriptional programmes and cell identity. The embryonic chick can regenerate a complete neural retina, after retinectomy, via retinal pigment epithelium (RPE) reprogramming in the presence of FGF2. In this study, we systematically analysed the reprogramming competent chick RPE prior to injury, and during different stages of reprogramming. In addition to changes in the expression of genes associated with epigenetic modifications during RPE reprogramming, we observed dynamic changes in histone marks associated with bivalent chromatin (H3K27me3/H3K4me3) and intermediates of the process of DNA demethylation including 5hmC and 5caC. Comprehensive analysis of the methylome by whole-genome bisulphite sequencing (WGBS) confirmed extensive rearrangements of DNA methylation patterns including differentially methylated regions (DMRs) found at promoters of genes associated with chromatin organization and fibroblast growth factor production. We also identified Tet methylcytosine dioxygenase 3 (TET3) as an important factor for DNA demethylation and retina regeneration, capable of reprogramming RPE in the absence of exogenous FGF2. In conclusion, we demonstrate that injury early in RPE reprogramming triggers genome-wide dynamic changes in chromatin, including bivalent chromatin and DNA methylation. In the presence of FGF2, these dynamic modifications are further sustained in the commitment to form a new retina. Our findings reveal active DNA demethylation as an important process that may be applied to remove the epigenetic barriers in order to regenerate retina in mammals. Abbreviations bp: Base pair; DMR: Differentially methylated region; DMC: Differentially methylated cytosines; GFP: Green fluorescent protein; PCR: Polymerase chain reaction. TET: Ten-eleven translocation; RPE: retinal pigment epithelium.

Tanycyte-Like Cells Derived From Mouse Embryonic Stem Culture Show Hypothalamic Neural Stem/Progenitor Cell Functions.

Tanycytes have recently been accepted as neural stem/progenitor cells in the postnatal hypothalamus. Persistent retina and anterior neural fold homeobox (Rax) expression is characteristic of tanycytes in contrast to its transient expression of whole hypothalamic precursors. In this study, we found that Rax+ residual cells in the maturation phase of hypothalamic differentiation in mouse embryonic stem cell (mESC) cultures had similar characteristics to ventral tanycytes. They expressed typical neural stem/progenitor cell markers, including Sox2, vimentin, and nestin, and differentiated into mature neurons and glial cells. Quantitative RT-PCR analysis showed that Rax+ residual cells expressed Fgf-10, Fgf-18, and Lhx2, which are expressed by ventral tanycytes. They highly expressed tanycyte-specific genes Dio2 and Gpr50 compared with Rax+ early hypothalamic progenitor cells. Therefore, Rax+ residual cells in the maturation phase of hypothalamic differentiation were considered to be more differentiated and similar to late progenitor cells and tanycytes. They self-renewed and formed neurospheres when cultured with exogenous FGF-2. Additionally, these Rax+ neurospheres differentiated into three neuronal lineages (neurons, astrocytes, and oligodendrocytes), including neuropeptide Y+ neuron, that are reported to be differentiated from ventral tanycytes toward the arcuate nuclei. Thus, Rax+ residual cells were multipotent neural stem/progenitor cells. Rax+ neurospheres were stably passaged and retained high Sox2 expression even after multiple passages. These results suggest the successful induction of Rax+ tanycyte-like cells from mESCs [induced tanycyte-like (iTan) cells]. These hypothalamic neural stem/progenitor cells may have potential in regenerative medicine and as a research tool.

Fgf- and Bmp-signaling regulate gill regeneration in Ambystoma mexicanum

Gill regeneration has not been well studied compared to regeneration of other appendages, such as limb and tail regeneration. Here, we focused on axolotl gill regeneration and found that Fgf- and Bmp-signaling are involved in their gill regeneration mechanism. Axolotls have three pairs of gill rami, and each gill ramus has multiple gill filaments. The gills consist of mesenchyme rich in extracellular matrix and epidermis. The gill nerves are supplied from the trigeminal ganglia located in the head. Denervation resulted in no gill regeneration responses. Nerves and gills express Bmp and Fgf genes, and treating animals with Fgf- and Bmp-signaling inhibitors results in phenotypes similar to those seen in denervated gills. Inducing an accessory appendage is a standard assay in amphibian regeneration research. In our study, an accessory gill could be induced by lateral wounding, suggesting that thin axon fibers and mesenchymal Fgfs and Bmps contributed to the induction of the accessory structure. Such accessory gill induction was inhibited by the denervation. Exogenous Fgf2+Fgf8+Bmp7, which have been determined to function as a regeneration inducer in urodele amphibians, could compensate for the effects denervation has on accessory blastema formation. Our findings suggest that regeneration of appendages in axolotls is regulated by common Fgf- and Bmp-signaling cascades.

Translocation of Exogenous FGF1 and FGF2 Protects the Cell against Apoptosis Independently of Receptor Activation

FGF1 and FGF2 bind to specific cell-surface tyrosine kinase receptors (FGFRs) and activate intracellular signaling that leads to proliferation, migration or differentiation of many cell types. Besides this classical mode of action, under stress conditions, FGF1 and FGF2 are translocated in a receptor-dependent manner via the endosomal membrane into the cytosol and nucleus of the cell. However, despite many years of research, the role of translocated FGF1 and FGF2 inside the cell remains unclear. Here, we reveal an anti-apoptotic activity of intracellular FGF1 and FGF2, which is independent of FGFR activation and downstream signaling. We observed an inhibition of cell apoptosis induced by serum starvation or staurosporine upon treatment with exogenous FGF1 or FGF2, despite the presence of highly potent FGFR inhibitors. Similar results were found when the tyrosine kinase of FGFR1 was completely blocked by a specific mutation. Moreover, the anti-apoptotic effect of the growth factors was abolished by known inhibitors of the translocation of FGF1 and FGF2 from the endosomes to the interior of the cell. Interestingly, FGF2 showed higher anti-apoptotic activity than FGF1. Since FGF2 is not phosphorylated by PKCδ and is present inside the nucleus longer than is FGF1, we speculated that the different activities could reflect their diverse nuclear export kinetics. Indeed, we observed that FGF1 mutations preventing binding to nucleolin and therefore phosphorylation in the nucleus affect the anti-apoptotic activity of FGF1. Taken together, our data indicate that the translocation of FGF1 and FGF2 protects cells against apoptosis and promotes cell survival.

FGF2-dependent mesenchyme and laminin-111 are niche factors in salivary gland organoids.

Epithelial progenitor cells are dependent upon a complex 3D niche to promote their proliferation and differentiation during development, which can be recapitulated in organoids. The specific requirements of the niche remain unclear for many cell types, including the proacinar cells that give rise to secretory acinar epithelial cells that produce saliva. Here, using ex vivo cultures of E16 primary mouse submandibular salivary gland epithelial cell clusters, we investigated the requirement for mesenchymal cells and other factors in producing salivary organoids in culture. Native E16 salivary mesenchyme, but not NIH3T3 cells or mesenchymal cell conditioned medium, supported robust protein expression of the progenitor marker Kit and the acinar/proacinar marker AQP5, with a requirement for FGF2 expression by the mesenchyme. Enriched salivary epithelial clusters that were grown in laminin-enriched basement membrane extract or laminin-111 together with exogenous FGF2, but not with EGF, underwent morphogenesis to form organoids that displayed robust expression of AQP5 in terminal buds. Knockdown of FGF2 in the mesenchyme or depletion of mesenchyme cells from the organoids significantly reduced AQP5 levels even in the presence of FGF2, suggesting a requirement for autocrine FGF2 signaling in the mesenchyme cells for AQP5 expression. We conclude that basement membrane proteins and mesenchyme cells function as niche factors in salivary organoids.

FGF2-mediated attenuation of myofibroblast activation is modulated by distinct MAPK signaling pathways in human dermal fibroblasts

BackgroundPrevious human and animal studies have demonstrated the ability of exogenously administered basic fibroblast growth factor (FGF2) to act as an antifibrotic agent in the skin. Though the activity of FGF2 as an anti-scarring agent is well-established for fibrotic skin wounds, the mechanisms by which FGF2 exerts these actions are not entirely understood. Canonical FGF2 signaling proceeds in part via FGFR/MAPK pathways in human dermal fibroblasts, and FGF2 has been described to prevent or reverse the fibroblast-to-myofibroblast transition, which is driven by TGFβ signaling and understood to be an important step in the formation of a fibrotic scar in vivo. Thus, we set out to investigate the antagonistic effects of FGF2 on TGFβ signaling as well as the broader effects of MAPK inhibition on the TGFβ-mediated induction of myofibroblast gene expression. ObjectiveTo better understand the effects of FGF2 signaling pathways on myofibroblastic gene expression and cell phenotypes. MethodsHuman dermal fibroblasts were cultured in vitro in the presence of FGF2, TGFβ, and/or MAPK inhibitors, and the effects of these agents were investigated by molecular biology techniques including qRT-PCR, immunofluorescence, Western blot, and flow cytometry. ResultsFGF2 inhibited TGFβ-mediated fibroblast activation, resulting in more rapidly proliferating, spindle-shaped cells, compared to the more slowly proliferating, flatter TGFβ-treated cells. Treatment with FGF2 also attenuated TGFβ-mediated increase in expression of myofibroblast markers smooth muscle α-actin, calponin, transgelin, connective tissue growth factor, ED-A fibronectin, and collagen I. FGF2-mediated antagonism of the TGFβ-mediated fibroblast-to-myofibroblast transition was reversed by small molecule inhibition of ERK or JNK, and it was potentiated by inhibition of p38. MAPK inhibition was demonstrated to have qualitatively similar effects even in the absence of exogenous FGF2, and small molecule inhibition of p38 MAPK was sufficient to attenuate TGFβ-mediated fibroblast activation. ConclusionsInhibition of select MAPK signaling pathways can reverse or potentiate anti-fibrotic FGF2 effects on human dermal fibroblasts, as well as exert their effects independently of exogenous FGF2 supplementation.

210 Syndecan-4/fgf-2/pkca signalling regulates vascular smooth muscle cell calcification via cross-talk with tgfb

Vascular smooth muscle cells (VSMCs) were induced to mineralise with β-glycerophosphate (β-GP). Controls were cultured without β-GP. FGF-2 mRNA (~40-fold increase, P<0.001) and protein (~2-fold increase, P<0.05) expression are significantly...

Abstract 241: An Increased Response to Injury by Smooth Muscle Cells of Human Veins at Valve Sites

Objective: Venous valves are prone to injury, thrombosis and fibrosis. We compared the behavior and gene expression of smooth muscle cells (SMCs) in the valve sinus vs non-valve sites to elucidate biological differences associated with vein valves. Methods: SMC migration was measured using 2.5 mm 2 explants of the intima/media of valve sinus segments (without valve leaflets) vs. non-valve segments of human saphenous veins. Proliferation and death of SMCs was determined by staining for Ki67 and TUNEL, respectively. Proliferation and migration of passaged valve vs non-valve SMCs was determined by cell counts and using microchemotaxis chambers. Global gene expression in valve vs non-valve intima/media was determined by RNA-Seq. Results: Valve SMCs demonstrated greater proliferation within tissue explants compared to non-valve SMCs (19.3±5.4% vs. 6.8±2.0% Ki67 positive nuclei at 4 days, respectively; mean ± SEM, 5 veins; P&lt;.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&lt;.0001). Cell death was not different (39.6±16.1% vs. 41.5±16.0% TUNEL positive cells, respectively, at 4 days, 5 veins). Cultured valve SMCs also proliferated faster than non-valve SMCs in response to PDGF-BB (2.9±0.2 vs. 2.1±0.2 fold of control, respectively; P&lt;.001; N=5 vein’s paired cells). This was also true for migration (6.5±1.2 vs. 4.4±0.8 fold of control, respectively; P&lt;.001; N=7 vein’s paired cells). Blockade of FGF2 inhibited the increased responses of valve SMCs, but had no effect on non-valve SMCs. Exogenous FGF2 increased migration of valve, but not non-valve SMCs. Unexpectedly, blockade of FGF2 did not block migration of valve or non-valve SMCs from tissue explants. 37 genes were differentially expressed by valve compared to non-valve intimal/medial tissue (11 veins). Conclusions: Valve, compared to non-valve, SMCs have greater rates of migration and proliferation, which may in part explain the propensity for pathological lesion formation in valves. While FGF2 mediates these effects in cultured SMCs, the mediators of these stimulatory effects in valve wall tissue remain unidentified. Here, the newly identified differentially expressed genes may play a role.

Efficient regulation of branching morphogenesis via fibroblast growth factor receptor 2c in early-stage embryonic mouse salivary glands

Salivary gland (SG) defects have a wide range of health implications, including xerostomia, bacterial infections, and oral health issues. Branching morphogenesis is critical for SG development. A clear understanding of the mechanisms underlying this process will accelerate SG regeneration studies. Fibroblast growth factor receptor 2 (FGFR2) interacts with multiple fibroblast growth factors (FGFs), which promote development. FGFR2 consists of two isoforms, FGFR2b and FGFR2c. FGFR2b is critical for SG development, but little is known about the expression and function of FGFR2c. We investigated the expression of all FGFR family members in fetal SGs between embryonic day 12.5 (E12.5) and E18.5. Based on RT-PCR, we observed an increase in the expression of not only Fgfr2b, but also Fgfr2c in early-stage embryonic mouse SGs, suggesting that FGFR2c is related to SG development. The branch number decreased in response to exogenous FGF2 stimulation, and this effect was suppressed by a mouse anti-FGFR2c neutralizing antibody (NA) and siRNA targeting FGFR2c, whereas FGFR2b signaling was not inhibited. Moreover, the expression of marker genes related to EMT was induced by FGF2, and this expression was suppressed by the NA. These results suggested that branching morphogenesis in SGs is regulated by FGFR2c, in addition to FGFR2b. Interestingly, FGFR2c signaling also led to increased fgf10 expression, and this increase was suppressed by the NA. FGFR2c signaling regulates branching morphogenesis through the activation of FGFR2b signaling via increased FGF10 autocrine. These results provide new insight into the mechanisms by which crosstalk between FGFR2b and FGFR2c results in efficient branching morphogenesis.