Delivery Of Fibroblast Growth Factor Research Articles

Distribution of fibroblast growth factor-2 (FGF-2) within model excisional wounds following topical application

To characterise the magnitude and distribution of fibroblast growth factor-2 (FGF-2) following topical application in hypromellose gel and film formulations or a solution in an animal wound model, in order to assess the potential of this route for treatment of chronic wounds. Topical formulations of FGF-2 were applied to punch biopsy wounds, and FGF-2 levels within the wound measured. Each 12 mm diameter wound received 0.3 microg FGF-2 in solution, a 7% (w/w) hypromellose gel, a dried hypromellose film on Melolin-backing or a saline control. After 2, 5 or 8 h the wounds were horizontally dissected into four sections (surface granulation, subcutaneous fat, superficial muscle and deep muscle) which were then analysed for FGF-2 concentration using ELISA. Confocal microscopy was used to evaluate the distribution of FGF-2 within the wound. There were significant differences in the mean FGF-2 levels with respect to formulation and time following application (P < 0.05). FGF-2 penetrated faster into tissue when formulated as a solution than as a gel or a film. There did not appear to be a significant difference between the gel and the film with respect to total concentrations achieved in the tissue, although confocal microscopy showed differences in FGF-2 distribution within the wound. Delivery of FGF-2 to wounds in a solution gave the greatest increase in tissue FGF-2 concentration when measured by ELISA and visualised using confocal microscopy. Gel and film formulations prolonged the release of FGF-2 into the wound, although FGF-2 levels were not significantly different from controls when measured by ELISA. Confocal microscopy highlighted the differences in the penetration and distribution of the FGF-2 within the wound when released from different formulations.

Comparison of vascular endothelial growth factor and fibroblast growth factor-2 in a swine model of endothelial dysfunction☆☆☆

Growth factor based angiogenesis, with or without cell therapy, is a promising therapeutic modality for patients with coronary artery disease. We compared the relative efficacies of surgically delivered vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) in a swine model of hypercholesterolemia-induced endothelial dysfunction which captures many of the pathophysiologic abnormalities of human coronary disease. Yucatan mini-swine (20-30 kg), fed a high cholesterol diet (total 20 weeks), underwent circumflex ameroid placement to create chronic myocardial ischemia, followed three weeks later by perivascular administration of VEGF (2 microg; n=6), FGF-2 (100 microg; n=6), or placebo (n=7) in the ischemic territory. Normocholesterolemic animals (n=7) served as controls. Four weeks later, endothelial function, collateral-dependent perfusion, as well as myocardial protein and mRNA levels of angiogenic mediators were assessed. Endothelial dysfunction was observed in all hypercholesterolemic animals as impaired microvessel relaxation in response to adenosine diphosphate and VEGF. VEGF administration improved baseline-adjusted collateral-dependent perfusion at rest (-0.03+/-0.05 vs -0.12+/-0.04, VEGF vs placebo, p=0.09), but FGF-2 delivery caused a significantly greater improvement in perfusion compared to either group (+0.15+/-0.03, p<0.05 vs HC-placebo and HC-VEGF) at rest. Molecular analysis revealed increased eNOS expression (135%+/-8%, p=0.03 vs placebo) in all growth factor treated animals and increased expression of FGF-2 receptor, FGFR1 (65+/-26%, p=0.04 vs placebo), in FGF-2 treated animals. No significant changes were demonstrated in other angiogenic mediators including Akt, Syndecan-4. In the setting of hypercholesterolemic endothelial dysfunction, FGF-2 is more effective than VEGF at enhancing collateral-dependent perfusion and thus, may be a better candidate than VEGF for angiogenic therapy in patients with end-stage CAD.

Combined therapy with human cord blood cell transplantation and basic fibroblast growth factor delivery for treatment of myocardial infarction

Transplanting cord blood-derived cells has been shown to augment neovascularization in ischaemic tissue. To test whether sustained delivery of basic fibroblast growth factor (bFGF) enhances the efficacy of angiogenic cord blood mononuclear cell (CBMNC) transplantation therapy in treating myocardial infarction. Three weeks after myocardial infarction, Sprague-Dawley rats were randomised to either injection of medium only (control), CBMNC transplantation, sustained bFGF delivery, or combined CBMNC transplantation and sustained bFGF delivery. Six weeks after treatment, tissue formation, neovascularization, and apoptotic activity in the infarct regions were evaluated by histology and immunohistochemistry. Left ventricular (LV) dimensions and function were evaluated by magnetic resonance imaging. Combined bFGF delivery and CBMNC transplantation significantly enhanced neovascularization in the ischaemic myocardium, as compared with either therapy alone. The enhanced neovascularization was likely due to increased VEGF and bFGF expression. The combined therapy also exhibited a reduced infarct area and apoptosis in the ischaemic myocardium, as compared with either individual therapy. The combined therapy did not attenuate LV dilation or increase ejection fraction significantly over either individual therapy. This study demonstrates that sustained bFGF delivery enhances the angiogenic efficacy of CBMNC transplantation in rat myocardial infarction models.

Healing of full-thickness defects of the articular cartilage in rabbits using fibroblast growth factor-2 and a fibrin sealant

We have investigated in vitro the release kinetics and bioactivity of fibroblast growth factor-2 (FGF-2) released from a carrier of fibrin sealant. In order to evaluate the effects of the FGF-2 delivery mechanism on the repair of articular cartilage, full-thickness cylindrical defects, 5 mm in diameter and 4 mm in depth, which were too large to undergo spontaneous repair, were created in the femoral trochlea of rabbit knees. These defects were then filled with the sealant. Approximately 50% of the FGF-2 was released from the sealant within 24 hours while its original bioactivity was maintained. The implantation of the fibrin sealant incorporating FGF-2 successfully induced healing of the surface with hyaline cartilage and concomitant repair of the subchondral bone at eight weeks after the creation of the defect. Our findings suggest that this delivery method for FGF-2 may be useful for promoting regenerative repair of full-thickness defects of articular cartilage in humans.

Vascular Endothelial Growth Factor Localization in the Adult

Although vascular endothelial growth factor (VEGF) has been well studied in both developmental and pathological angiogenesis, its role in mature blood vessels is poorly understood. A growing body of observations, including the side effects of anti-VEGF therapies as well as the role of soluble VEGFR1 in preeclampsia, points to an important role for VEGF in maintenance of stable blood vessels. To better understand the potential function of VEGF in mature vessels, a survey of VEGF localization in adult mice was conducted. In adult VEGF-lacZ mice, VEGF was expressed in a cell-specific manner by cells overlying fenestrated and sinusoidal blood vessels, including podocytes, choroid plexus epithelium, and hepatocytes, as well as in tissues with high metabolic demands or with secretory functions, such as cardiac and skeletal myocytes, Leydig cells, prostatic epithelium, and salivary serous epithelium. VEGF was not detected in most endothelium but was specifically expressed by aortic endothelial cells where VEGFR2 was found to be phosphorylated, indicating an autocrine loop. Additionally, VEGFR2 was constitutively phosphorylated in the liver, lung, adipose, and kidney in vivo, providing evidence consistent with a role for VEGF in adult tissues. These observations support the concept that VEGF acts in the adult to stabilize mature vessels.

Autologous cartilage grafts enhanced by a novel transplant medium using fibrin sealant and fibroblast growth factor.

To introduce and assess a system for the delivery of fibroblast growth factor to autologous cartilage grafts using fibrin sealant and analyze whether this "enhancement" results in reduced rates of cartilage resorption and greater preservation of normal architectural features compared with "unenhanced" cartilage grafts. Auricular cartilage segments measuring 1 cm(2) were harvested from 12 New Zealand white rabbits, morselized, and implanted into the subcutaneous dorsum of the upper back for 3 months. The conditions included (1) cartilage alone; (2) cartilage + fibrin sealant; (3/4) cartilage + acidic or basic fibroblast growth factor (aFGF or bFGF); and (5/6) cartilage + fibrin sealant + aFGF or bFGF. Subsequent to graft harvest, gross and microscopic assessments were performed to assess size, structural integrity, and architectural features, with comparisons performed between each of the conditions. The mean areas of the harvested cartilage grafts treated with fibrin sealant + aFGF or bFGF were 1.23 cm(2) and 1.19 cm(2), respectively, while the corresponding value for the untreated (ie, cartilage alone) specimens was 1.03 cm(2). The percentage of decrease in size was 45% for the untreated specimens and 0% for the specimens treated with fibrin sealant + aFGF or fibrin sealant + bFGF. Cartilage treated with fibrin sealant + bFGF had the greatest quantity of elastin fibers of the 6 conditions. Cartilage grafts treated with fibrin sealant alone demonstrated the most intense ground substance staining on a computerized measure of pixel intensity. Our findings demonstrated significant improvements in graft quality using fibroblast growth factor and fibrin sealant or even fibrin sealant alone. These findings may justify changes in how cartilage grafts are prepared and delivered for facial augmentation procedures to reduce graft resorption and maintain the structural integrity of the cartilage. Further trials will be performed to elucidate the optimal growth factor concentrations for maximum structural and architectural benefits.

Evaluation of high‐pressure retrograde coronary venous delivery of FGF‐2 protein

Delivery of angiogenic factors to ischemic myocardium remains a practical challenge. We evaluated the efficiency and efficacy of delivery of fibroblast growth factor-2 (FGF-2) protein via high-pressure retrograde injection into the anterior interventricular vein (AIV) in a porcine model of chronic myocardial ischemia. Labeled FGF-2 protein was delivered to the myocardium of three pigs via the AIV and the left anterior descending (LAD) coronary artery in three others. At 1 hr, the amount of protein in the left ventricle and the LAD region was quantified. Copper stents were implanted in the LAD of 25 pigs, resulting in chronic myocardial ischemia. At 4 weeks, microsphere-derived myocardial blood flow was assessed at rest and during pacing. In eight pigs (AIV FGF), FGF-2 protein (6 microg/kg) was delivered via high-pressure retrograde injection into the AIV. Six pigs (intracoronary FGF) received the same amount of FGF-2 by intracoronary delivery. Five pigs (AIV saline) received a placebo injection into the AIV and six pigs (control) served as controls. Four weeks later, myocardial blood flow was reassessed. At 1 hr, significantly more FGF remained in the left ventricle (1.3 vs. 0.82 microg; P < 0.04) and in the LAD region (1.2 vs. 0.64 microg; P = 0.03) after AIV compared to intracoronary delivery. Four weeks after treatment, resting LAD blood flow (normalized to right ventricular flow) improved slightly in the AIV FGF and intracoronary FGF arms (1.32-1.37 for both; P = 0.11), while it decreased significantly in the AIV saline (1.32-1.23; P = 0.02) and the control arms (1.32-1.19; P = 0.0004). Pacing LAD blood flow decreased significantly in the control arm (1.30-1.23; P < 0.05), but did not change significantly in the other three arms. High-pressure retrograde injection into the AIV may represent an efficient and effective means for delivering angiogenic factors to ischemic myocardium.

Cochlear delivery of fibroblast growth factor 1 and its effects on apoptosis and cell cycling in noise-exposed guinea pig ears.

Acidic fibroblast growth factor 1 (FGF-1) is a mitogen and antiapoptotic factor synthesized by cochlear neurons and transported to the organ of Corti. The objectives of this investigation were threefold: (1) to develop an animal model to study the cochlear effects of intratympanic delivery of FGF-1; (2) to determine the distribution, in the mature mammalian cochlea, of FGF-1 and the receptor, FGFR3, to which it binds with high affinity; and (3) to examine the effect of exogenous FGF-1 on cochlear apoptotic and cell-cycling markers in noise and non-noise-exposed guinea pigs ears. Fifteen adult Hartley guinea pigs were divided into three groups. Group 1 animals (n = 5) underwent direct placement of FGF-1 in phosphate buffered saline (PBS) (20 pg/mL) soaked Gelfoam pledgets to the right round window membrane. Phosphate buffered saline-soaked Gelfoam pledgets were placed on the left round window membrane as a control. In group 2 animals (n = 5), surgical placement of either FGF-1 or PBS was followed by exposure to 120 dB of white noise for 2 hours. Group 3 animals (n = 5) were subjected to identical noise conditions prior to undergoing round window application of either FGF-1 or PBS. All groups were allowed to recover in a noise-controlled environment for 12 hours following surgery. Anti-FGF-1-stained Western blots and optical densitometry analyses were used to quantitate passage of FGF-1 into cochlear perilymph. Standard in situ immunohistochemical techniques were used to stain each cochlea for FGF-1 and FGFR3, apoptotic markers p53 and p21, Bcl-2, and the cell-cycling marker proliferating cell nuclear antigen (PCNA). Tissue sections were subjected to the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling technique (TUNEL) for apoptosis. Western blot and optical densitometry analyses of cochlear perilymph showed increased concentrations of FGF-1 in 10 of 14 experimental cochleas. Cochlear perilymph FGF-1 was consistently bound to heparan sulphate proteoglycan (HSPG). Immunoreactivity of both FGF-1 and FGFR3 was observed in spiral ganglion neurons, inner and outer hair cells, pillar cells, and Dieter and Hensen's cells. Specific FGF-1 immunostaining to the distal portion of the pars pectinata of the basilar membrane was noted in noise-exposed animals only. Bcl-2 and PCNA immunostaining was not detected in any group. There was no significant nuclear immunoreactivity to proapoptotic markers, p53 and p21, in any group. Semiquantitative analysis of TUNEL staining in block sections of all cochleas demonstrated a 340% increase in nuclear immunoreactivity of noise-exposed outer hair cells and organ of Corti cells. There was no difference between FGF-1 treated and control ears subjected to TUNEL staining. Exogenous FGF-1 crosses the round window membrane and is bound to HSPG in cochlear perilymph. The specific immunoreactivity of the pars pectinata to FGF-1 may represent a unique reservoir for cochlear FGF-1 in noise-exposed ears of the guinea pig. Noise induces apoptosis of organ of Corti cells as demonstrated with the TUNEL technique. PCNA, Bcl-2, p53, and p21 in noise-exposed and non-noise-exposed guinea pig cochleas are not affected by exogenous FGF-1. Noise-induced hair cell apoptosis appears to be independent of the p53 pathway. Lack of immunoreactivity to Bcl-2 supports the concept that the apoptotic mechanism is likely to involve C-Jun-N-terminal kinase- or caspase-dependent pathways. Exogenous FGF-1 does not alter apoptosis or cell cycling in the mature guinea pig cochlea within 12 hours of acute acoustic trauma.

Fibroblast Growth Factor-2 Inhibits Myelin Production by Oligodendrocytes in Vivo

Fibroblast growth factor-2 (FGF-2) controls in part the timely differentiation of oligodendrocytes into the myelin-producing cells of the CNS. However, although differentiated oligodendrocytes express FGF receptors (R), the effect of FGF-2 on myelin-producing oligodendrocytes in vivo was unknown. In the present study, we show that delivery of FGF-2 into the cerebrospinal fluid of anaesthetized rat pups, aged postnatal day (P) 6 to 9, induced a severe loss of myelin in the caudal anterior medullary velum (AMV). Furthermore, we show that the caudal AMV was myelinated at the time of treatment, so the effects of FGF-2 represent a loss of myelin and not delayed differentiation. This was confirmed by injection of platelet-derived growth factor-AA (PDGF-AA), a factor known to affect the differentiation of PDGF-αR expressing oligodendrocyte progenitors, but which did not induce myelin loss in the caudal AMV and did not affect differentiated oligodendrocytes, which do not express PDGF-αR. Compared to controls treated with saline or PDGF-AA, FGF-2 induced an accumulation of PLP protein and MBP mRNA within the somata of myelin-producing oligodendrocytes. The results indicate that FGF receptor signalling disrupts myelin production in differentiated oligodendrocytes in vivo and interrupted the transport of myelin-related gene products from the oligodendrocyte cell body to their myelin sheaths.

The role of FGF and VEGF in angioblast induction and migration during vascular development.

The embryonic vasculature forms by the processes of vasculogenesis and angiogenesis. Angioblasts (endothelial cell precursors) appear to be induced by fibroblast growth factor 2 (FGF-2). The angioblasts contributing to the dorsal aortae arise by an epithelial to mesenchymal transformation of cells originating from the splanchnic mesoderm. QH-l and vascular endothelial growth factor receptor 2 (VEGFR-2) both appear to label these cells as they adopt a mesenchymal morphology. Since VEGFR-2 is the earliest known VEGF receptor this suggests that VEGF is not involved in angioblast induction. VEGF does appear to be critical, however, for growth and morphogenesis of angioblasts into the initial vascular pattern. Controlled delivery of FGF-2 from beads and aggregates of cells transfected with quail VEGF have been used in our laboratory to study the role of these growth factors in angioblast induction and migration. We have induced cells from the epithelial quail somite to differentiate into angioblasts with FGF-2 both in the embryo and in culture. This is a useful model system to study the origins of endothelial cells that are normally more diffusely induced during gastrulation by an obscure process probably involving signals from the embryonic endoderm. The origins of arterial versus venous endothelial cells is also poorly understood but recent findings on the distribution of ephrins and Eph receptors suggest that molecular differences exist prior to the onset of circulation. Finally, studies on the role of growth factors in such diverse phenomena as stem cell biology, angiogenesis, and molecular medicine in addition to vascular development suggest multiple roles for FGF-2 and VEGF in vascular development.

Enhanced angiogenesis and unfavorable remodeling in injured porcine coronary artery lesions: effects of local basic fibroblast growth factor delivery.

There is interest in the role of growth factors in the genesis of arterial remodeling. We studied local administration of basic fibroblast growth factor (bFGF) to coronary lesions to determine whether there is a difference in remodeling and whether neovascularization could be induced in such stenoses and distal myocardium. Pigs were randomized to balloon infusion of either saline or bFGF at each thermally injured arterial site. After the animals were killed, their internal elastic lamina, neointima, and lumen areas were measured. Capillaries were counted in the arteries and myocardium. There was a greater loss of lumen and internal elastic in the bFGF group. The neointima, media, and myocardium in the bFGF treated arteries had statistically more capillaries. This study showed that local intracoronary bFGF, at a dose that results in arterial luminal revascularization in injured segments, adversely affects arterial remodeling. Thus, the angiogenic response to exogenous bFGF may be offset by concomitant shrinkage of injured arterial segments.