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Fibroblast Growth Factor-2
Fibroblast growth factor-2 (FGF-2) is a protein of the FGF family. The Fibroblast growth factor (FGF) family, which includes 23 proteins, is involved in diverse functions in the embryo and the adult. FGF-2 plays a role in a multitude of developmental and biological processes, including limb development, tissue repair, mesoderm induction, lung development and maintenance of neuron survival. The wide range of roles this protein plays in development and differentiation has brought increasing attention to the clinical potential uses.

Alternative initiation sites for translation on the FGF-2 mRNA produces distinct isoforms, which are differentially sorted in cells. High molecular weight FGF-2 isoforms are not secreted from the cell, but are transported to the nucleus where they inhibit cell migration and induce cell transformation or growth arrest in a cell type- and dose-dependent fashion. Whereas the low molecular weight (18 kDa) FGF-2 can be secreted to the extracellular medium, by an unknown, non-canonical secretory pathway, where it acts by binding to and activating cell-surface tyrosine kinase receptors and ultimately causes upregulation of cell proliferation and migration. Receptor-mediated endocytosis allows exogenous FGF-2 to be transported to the nuclei of target cells, this and other downstream signals from FGF-2-FGFR binding are important for the transmission of the mitogenic signal.

Functions
FGF-2 plays a role in multiple biological systems and is important in the development of the embryo through adulthood. During embryonic development, FGF-2 regulates cell proliferation, migration, and differentiation. In the adult, the protein mainly functions in tissue repair. FGF-2 is found in many tissues including skeletal muscle, cardiac and gastrointestinal smooth muscle, the dermis of the skin, the kidney, the lung, neurons, glial cells, and all vascular endothelial cells of blood vessels and capillaries. FGF-2 is expressed at low levels in most tissues, but expressed at high levels in the brain.

Angiogenesis
FGF-2 is involved in the formation of new blood vessels, called angiogenesis. Angiogenesis is important for biological processes, such as embryo development and wound healing. In tumor growth, penetration of branching blood vessels from angiogenesis supplies the tumor with nutrients and oxygen and aids in its spread. FGF-2 modulates angiogenesis by regulating the production of essential molecules involved in the steps leading to blood vessel formation, namely: interstitial collagenase, urokinase type plasmninogen activator (uPA), plasminogen activator inhibitor (PAI-1), uPA receptor, and TGFbeta-1. In vitro studies have shown that FGF-2 induces endothelial wall proliferation. The induced endothelial cells have been shown to form vascular structures and may be fundamental in embryonic development .The activity of FGF-2 on angiogenesis can be regulated by transforming growth factor beta (TGF-beta). In-vitro studies have shown that the concentration of TGF-beta has an effect on FGF-2 activity. Low concentrations of TGF-beta stimulates FGF-2, and low concentrations have an inhibitory effect.

Reproduction System
FGF-2 may regulate Sertoli cells. These cells reside in the seminiferous tubules of the testes and function to nourish sperm cells during spermatogenesis. However, studies have shown that FGF-2 is not necessary for fertility as male FGF-2 knockout mice showed normal fertility.

Muscle and Skeleton
Interaction of FGF-2 and FGFR1 has shown to be involved in the control of the growth and differentiation of skeletal muscle. FGF-2 plays an inhibitory role in skeletal muscle differentiation. Defective high FGF-2 release may cause muscular disorders. FGF-2 has also been shown to cause osteoblast proliferation and differentiation and increase TGF-beta production. FGF-2 enhances healing in bone exposed to high levels of irradiation.

Nervous System
Expression of FGF-2 has been shown in the nervous systems of many species. The time and place at which expression begins is dependent on the species of interest. In human brains, FGF-2 expression is strong in the central nervous system. In fact, among the FGF family, FGF-2 is the most abundant in the central nervous system. FGF-2 has been shown to stimulate growth of hippocampal neurons in vitro. FGF-2 also plays a great role in neuroprotection and neurogenesis. FGF-2 prevents cell death and maintains survival by interfering with the expression of NMDA receptors. The activation of NMDA receptors causes opening of Ca2+ ion channels and an influx of Ca2+. A transduction cascade is activated and produces nitric oxide (NO), free radicals, and excitotoxicity, leading to apoptosis. FGF-2 has been shown to be upregulated following brain injury and stroke

Mechanism
FGF-2 has been identified as having a role in a number of biological systems, such as neurogenesis, angiogenesis, myogenesis, chondrogenesis, hematopoiesis, and apoptosis. However, its exact molecular mechanisms are only known in a few of these, namely angiogenesis, myogenesis, hematopoiesis, and apoptosis.

FGF molecules bind to FGF receptors (FGFRs) which are receptor tyrosine kinases with a single-spanning transmembrane domain, and an extracellular domain for binding with FGFs, heparan sulfates, and cell adhesion molecules. The most common signalling pathway downstream of FGFRs is the MAPK/ERK cascade that stimulates expression of or activation of transcription factors, such as c-Myc and CREB ; the other pathway is the PLCγ cascade, which activates PKC which feeds into the MAPK cascade. Heparan sulfate proteoglycans are required for high affinity binding of FGFs to FGFRs with lower affinities for ligand binding and for formation of dimers between FGF-FGFR complexes for autophosphorylation. FGF-2 in particular has been found to bind with higher specificity to FGFR1b, FGFR1c, FGFR2c, FGFR3c, and FGFR4.

Heparin and heparan sulfates have been shown to be required in FGF signaling through a number of experiments. Heparin modulates activity of FGFs, protects them from thermal denaturation and degradation, and provides an extracellular reservoir from which FGFs can be released in response to certain triggers. Heparan sulfates are able to differentiate between FGF molecules and direct them to the appropriate FGF receptor. However, the role of heparin and heparan sulfates on the regulation of FGF-2 activity in cells has not been fully established. It has been suggested that heparin increases the survival of mesencephalic progenitor cells through FGF-2 signaling. However, upon looking at the relationships between different subtypes of neural cells, the FGFR types they express, and the varying degrees to which heparin binds to these receptors, it was concluded that FGF regulation through heparin most likely differs from target cell to target cell and from receptor to receptor.

Prostaglandin F2α (PGF2α) has been shown to regulate FGF-2 and FGFR expression in osteoblasts. In a knockout of FGF-2 in osteoblasts, it was determined that PGF2α increases Ras and MAPK cascade activity, and Bcl2 and c-Myc levels in wildtype osteoblasts, but not in FGF-2 -/- cells. Levels of p53 in wildtype cells were increased due to PGF2α, and increased levels of phosphorylated p53 were found in FGF-2 -/- cells. Additionally, the FGF-2-FGFR1 complex was found to be responsible for the p53 effects from PGF2α. Therefore, it is likely that this same complex is responsible for the increased Ras and MAPK cascade activities and the increase in Bcl2 and c-Myc levels, without which the cell could undergo apoptosis due to low levels of Bcl2.

The role of FGF-2 in angiogenesis has been heavily studied. FGF-2 stimulates endothelial cell migration and proliferation, and has been implicated in embryonic vascular development. It has been shown to regulate levels of transcriptional growth factor β (TGFβ), namely by activating latent TGFβ after the activation of urokinase-type plasminogen activator (uPA). The newly activated TGFβ activates PAI1 production, which in turn deactivates uPA and therefore subsequent production of TGFβ. TGFβ is a biphasic regulator of FGF-2 itself - meaning that high levels of TGFβ inhibit FGF-2-induced angiogenesis and low levels increase angiogenesis. TGFβ also increases levels of vascular endothelial growth factor (VEGF), the protein most important in angiogenesis. The addition of physiologically relevant levels of FGF-2 to osteoblasts increases VEGF transcription and production through a pathway independent of that of TGFβ. Also, neurons have been shown to secrete vesicles containing FGF-2 and VEGF, potentially in order to promote vascular growth towards them.

In satellite cells - muscle-resident stem cells - FGF-2 has been demonstrated to induce Grb2- and PKC-mediated phosphorylation of MAPK/ERK. The MAPK/ERK phosphorylation cascade is important in many cellular functions, like differentiation, proliferation, and survival, and can lead to cancer if not properly regulated. The Grb2-mediated pathway was shown to be the faster of the two, whereas PKC-mediated phosphorylation results in a slower but more sustained cascade. The combination of Grb2-specific siRNA and PKC inhibitors almost completely suppressed FGF-2-induced MAPK/ERK phosphorylation, confirming that MAPK/ERK phosphorylation in this case is dependent upon both the Grb2- and PKC-mediated pathways.

FGF-2 has been shown to reduce secretion and production of stromal cell-derived factor-1 (SDF1) in bone marrow stromal cells. SDF1 is a critical regulator of hematopoiesis during development and after birth, and stromal cells are the main source of SDF1 in bone marrow. FGF-2 was found to affect the levels of SDF1 through one specific FGFR isoform, FGFR1 IIIc, and that the mechanism of action for decreasing production of SDF1 was an acceleration of SDF1 mRNA decay and not a direct effect on transcription. This accelerated mRNA decay may be due to the effects of FGF-2 on the MAPK/ERK phosphorylation cascade, which has been implicated in regulation of mRNA stability. The higher levels of FGF-2 may result in overstimulation of the cascade, and thereby hyperdegradation of mRNA.

Developmental Roles
During development, the biological roles that FGF-2 has on embryogenesis are different than the FGF-2 found in adults. In general, FGF-2 takes part during the early developmental stage is axis patterning, induction or maintenance of cell lineages and caudalization.

Mesoderm induction
FGF-2 takes a role in mesoderm induction because it is expressed before and throughout the initiation of the mesoderm formation. FGF-2 affects mesoderm induction in two ways: it can be expressed intracellularly, or be exported via an alternate pathway that is activated at a specific timeframe during the early stages of embryogenesis  FGF-2 relies on the protein, MAPK, which stimulates the mesodermal germ layer formation. On the other hand, FGF-2 can interact with PKC, which will diminish the FGF-2 signalling and terminates the effects of FGF-2 on mesoderm induction.

Nervous System
FGF-2 can be found expressed in the ventricular epithelium during embryonic neurogenesis. It regulates the proliferation, lineage commitment, and differentiation of the neural stem cell. When neural stem cells are cultured with the presence of FGF-2, they will proliferate and maintain its multipotency characteristics; however, the subsequent lineage commitments are also influenced by the exposure of FGF-2. Even though studies have shown that FGF-2 can induce neural crest, but it must do so in concert with other signalling proteins such as Wnts, eFGF, noggin, and chordin. FGF-2 is capable to induce neuron formation, such as 5-HT neurons specifically in the hindbrain area.

FGF-2 is one of the many signalling factors as listed above that could prompt the development of neural plate and sectioning into anterior and posterior compartments. FGF-2 will then also change the cell fate along the anterior-posterior axis of the neural tube. Evidence shows that the specific patterning of the neural tube is a step wise process and FGF-2 is one of the many contributing signals, where later on it will also regulate the fate of other mature neurons.

In chick and frog embryos, the neural tube caudalizes in the presence of FGF-2. Caudalization will then induce the posterior cascades of neural development in the dorsal ectoderm. Studies have shown that FGF-2 influences the neural tube indirectly by either altering the paraxial mesoderm, or changing the primary localized markers. Also, FGF-2 caudalizes the hindbrain without mesoderm induction, and induces spinal cord formation. In a forebrain explant experiment, FGF-2 induced the expression of hindbrain markers in the prospective forebrain and the amount of posterior markers increased as higher concentrations of FGF-2 were administered. Because FGF-2 expresses markers of the more posterior fate rather than the anterior fate of the prospective hindbrain, it demonstrated true neural patterning activity, particularly caudalization.

Future Uses
FGF-2 expression levels have been implicated in multiple neurological disorders, angiogenesis, wound healing, cancer, and in peripheral nervous system injury. FGF-2 is known to be an effective growth factor that protects neurons and cardiac muscle cells against excitotoxic and ischemic injury and promotes neuron survival. It is released in large amounts by bone marrow-derived cells, as well as by growing axons and endoneurial fibroblasts during development and regeneration of the peripheral nervous system (PNS). It strongly increases early glial proliferation, which can potentially improve PNS regeneration for use in treatment of peripheral nerve damage.

Increases of immunoreactive detection of FGF-2 were found in reactive astrocytes in both white and grey matter and at the border of adjacent preserved tissue in contused rat spinal cord at 72 hours, 2 and 3 weeks post-surgery. It is possible that the FGF-2 triggers wound repair events in the area of trauma, and leads to neurotrophism and neuronal plasticity in the neighboring regions, another function that could lend to an FGF-2 related treatment of nerve tissue damage. In animal models of Parkinson’s, where neurotoxin-induced dopaminergic (DA) neuron death, co-transplantation of fetal DA cells with FGF-2 expressing cells or exogenous FGF-2 resulted in increased survival and functional integration of grafted DA neurons resulting in improved behavioral performance. Grafting of FGF-2 (and other neurotrophic factors) somatic gene transferred DA progenitor cells could be one possible strategy for Parkinson’s treatment in the future.

Therapeutic potential and value for FGF-2 lie in the fact that it is a neurotrophic factor and a neural progenitor mitogen. It functions in stem cell culture to help repress BMP signaling and sustain undifferentiated proliferation of hESCs as well as increases the number of neural progenitors and neurons in neural stem cell culture.

Neural progenitors native to human hippocampus and neocortex that were previously only shown to generate glia are also able to generate neurons but only post FGF-2 exposure. . It is conceivable that FGF-2 may illustrate an invaluable co-transplantation factor for future use in neural stem cell transplantation for treatment of PNS injury, Alzheimer’s, Parkinson’s and many other neurological disorders. Separately, FGF-2 has also been shown to have a proliferative and motile effect on keratinocytes, stimulate wound healing and augment skin-derived mesenchymal stem cells. FGF-2 treatment was shown to be effective in radiation-exposed skin using CLAWN miniature pigs as a model organism for human skin and soft tissue damage post-radiation. (D9) It’s use as a topical treatment for second degree burn wounds has also been evaluated, where significantly faster wound healing as well as larger maximal scar extension, scar retraction to maximal scar extension ratio and elasticity were found in FGF-2 treated group versus untreated. Recent studies have attempted to elucidate the mechanism by which FGF-2 treatment supports scarless wound healing by induction of myofibroblast apoptosis but sparing the same effect on fibroblasts.

Big Idea


As FGF-2 has been implicated in neurogenesis, regulation of nerve cell survival, and angiogenesis, it has naturally been studied in reference to ischemic stroke, caused by a loss of blood flow to the brain. FGF-2 mRNA and protein levels have been shown to be upregulated in human brains and sera post-ischemic stroke, particularly in astrocytes and endothelial cells in the penumbra and the point of infarction, suggesting involvement in astrocytic regulation of neuronal survival and higher angiogenic activity from brain endothelial cells. A recent clinical trial with a recombinant form of FGF-2 (Trafermin) showed an advantageous trend toward treatment with a dosage of 5 mg intravenously infused into patients showing onset of stroke symptoms within 6 hours, over a 24 hour period. However, the trial was not taken to completion as the data did not support a statistically significant benefit.

Drugs targeting the brain face the challenge of crossing the blood brain barrier (BBB), a highly restrictive diffusion barrier composed of endothelial cells with extensive tight junctions. These tight junctions prevent influx of many molecules in the blood through the BBB.

Treatment for ischemic stroke may be made more efficient by direct delivery across the BBB. For example, the partial agonist hexafin-2 (IV) has been shown to exert an FGF-2 mimetic effect on FGFRs, and if a motif for BBB penetration is added to this peptide, like the TAT viral-derived peptide, this agonist could cross the BBB and exert effects directly on the cells that need it post-stroke, like astrocytes, neural progenitors, and endothelial cells. Assuming hexafin-2 has a similar receptor affinity as FGF-2, a dosage of <5-10 mg over 8 hours to stroke-induced rats or monkeys showing symptoms within 6 hours of the stroke may promote angiogenesis, neuroprotection and proliferation to a greater extent than by just natural upregulation of endogenous FGF-2. Stroke would be induced through photochemical induction of thrombi in the left parietal lobe by injection of rose-bengal dye and shining of 560-nm-wavelength light. Motor functions of the model animal would be expected to be impaired on the right side of the body within hours of stroke induction. Additional changes in the animal can include sensory impairment and behavioural changes. The effects of this treatment would be tested through behavioral analysis, examining the magnitude of the radius of dead neural cells around the infarction through brain slices, and examining VEGF expression through VEGF mRNA bound to superparamagnetic iron oxide nanoparticles through MRI.