Release Of Basic Fibroblast Growth Factor Research Articles

Controlled delivery of basic fibroblast growth factor (bFGF) using acoustic droplet vaporization stimulates endothelial network formation

The challenge of translating pro-angiogenic growth factors for therapeutic purposes has stimulated a myriad of biomaterials-based, delivery approaches. Many techniques rely on incorporating a growth factor into a hydrogel. The kinetics of release can be tuned based on the physiochemical properties of the growth factor and scaffold. We have developed an acoustically-responsive scaffold (ARS), whereby release of a growth factor is non-invasively and spatiotemporally controlled in an on-demand manner using focused ultrasound. An ARS consists of a fibrin matrix doped with a growth factor-loaded, sonosensitive emulsion. In this study, we used an ARS to investigate the impact of basic fibroblast growth factor (bFGF) release on endothelial tubule formation. The co-culture model of angiogenic sprouting consisted of endothelial cell-coated microbeads and dispersed fibroblasts. bFGF release correlated with the acoustic pressure applied while sprout length correlated with both the volume of bFGF-loaded emulsion in the ARS and acoustic pressure. Minimal bFGF release and sprouting were observed in the absence of ultrasound exposure. Staggering the release of bFGF via multiple ultrasound exposures did not affect sprouting. Additionally, sprouting did not display a dependence on the distance between each microbead and the ARS. Overall, these results highlight the potential of using ultrasound to control regenerative processes via the controlled delivery of a growth factor. Statement of SignificanceDue to the ineffectiveness of conventional routes of administration, implantable hydrogels are often used as matrices to deliver growth factors (GFs). Spatial control of release is typically realized using anisotropic constructs while temporal control is obtained by modifying matrix properties and GF-scaffold interactions. In this study, we demonstrate how focused ultrasound can be used to non-invasively and spatiotemporally control release of basic fibroblast growth factor (bFGF), in an on-demand manner, from a composite hydrogel. The acoustically-responsive scaffold (ARS) consists of a bFGF-loaded, monodispersed double emulsion embedded within a fibrin matrix. We demonstrate how controlled release of bFGF can stimulate endothelial network formation. These results may be of interest to groups working on controlled release strategies for GFs, especially in the context of stimulating angiogenesis.

Controlled basic fibroblast growth factor release device made of poly(ethyleneglycol) dimethacrylates for creating a subcutaneous neovascular bed for cell transplantation.

Subcutaneous space is a potential site for the transplantation of cells such as islets for treatment of type 1 diabetes. To enhance engraftment, an optimal space for the growth of the transplanted cells is needed along with neovascularization. In this study, we developed a device using a photocurable resin, poly(ethyleneglycol) dimethacrylates (PEGDM), for controlled release of basic fibroblast growth factor (bFGF) to create a subcutaneous neovascular bed in rats. The device consists of a disk-shaped capsule with micropores and is composed of tri(ethyleneglycol) dimethacrylate (TEGDM) and a drug formulation of PEGDM. The release rate was tuned by changing the number of pores and the composition of water and PEGDM in the drug formulation. bFGF released from devices incubated in phosphate-buffered saline (PBS) enhanced the growth of fibroblasts, indicating bioactivity of bFGF after release. Histological evaluation showed a significant increase in the extent of vasculature that was dependent on the amount of bFGF loaded into the device. A perfusion study using fluorescein isothiocyanate dextran 2000 kDa showed linear and capillary staining patterns, indicating potent functional vasculature. In conclusion, the controlled bFGF releasing device could provide a neovascular bed with the required vascularization in the subcutaneous space. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3017-3024, 2017.

BFGF- and CaPP-Loaded Fibrin Clots Enhance the Bioactivity of the Tendon-Bone Interface to Augment Healing

Background: Tendon-to-bone healing is a complex and slow process, and the rate of poor healing remains high. In recent years, several new strategies have been developed that enhance tendon-to-bone healing by increasing the bioactivity. Fibrin clots have been widely used to improve tissue healing and tissue engineering, Hypothesis: Modified fibrin clots can improve the bioactivity of the tendon-bone interface and histological appearance. Study Design: Controlled laboratory study. Methods: A total of 27 male New Zealand White rabbits were used. Of these, 3 were used for cell isolation, and the remaining 24 rabbits were divided into 2 groups (12 per group) for an in vivo partial patellectomy study. The setting time, degradation time, and basic fibroblast growth factor (bFGF) and ceramide-activated protein phosphatase (CaPP) release kinetics of bFGF- and CaPP-loaded fibrin clots were modified appropriately for early tendon-to-bone healing. In an in vitro experiment, the bFGF- and CaPP-loaded fibrin clots were assessed for cell migration and proliferation by microscopy, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, and DAPI (4′,6-diamidino-2-phenylindole) assay. Quantitative real-time reverse transcription polymerase chain reaction and a Western blot assay were performed to test for an induction effect of the bFGF- and CaPP-loaded fibrin clots. Finally, for the in vivo experiment, the rabbits were divided into 2 treatment groups: one with bFGF- and CaPP-loaded fibrin clots and one without bFGF- and CaPP-loaded fibrin clots after partial patellectomy in patella–patellar tendon sutured sites. A histological evaluation was performed at 2, 4, and 6 weeks after surgery. Results: The sitting time and degradation time of the bFGF- and CaPP-loaded fibrin clots were set at 15 seconds and more than 2 weeks, respectively, and the porosity was minimized to achieve the highest levels of cell migration and growth. In the bFGF-CaPP group of the in vitro experiment, cell proliferation increased to a greater extent relative to the control group (P < .05); the mRNA expression of osteopontin, alkaline phosphatase, runt-related transcription factor 2, vascular endothelial growth factor, and collagen type I was upregulated (P < .05); and the relative protein expression of these factors was enhanced (P < .05). In vivo, hematoxylin and eosin staining showed that the tendon-to-bone connections were more mature and more arranged when treated with bFGF- and CaPP-loaded fibrin clots than when untreated, and the histological scores were higher. Conclusion: bFGF- and CaPP-loaded fibrin clots enhanced cell migration and proliferation and the expression of related genes and proteins, which increased the bioactivity of the tendon-bone interface and resulted in the histological improvement of tendon-to-bone healing. Clinical Relevance: As fibrin clots have already been used in clinical practice, bFGF- and CaPP-loaded fibrin clots can be further used to augment healing in the early stages of tendon-to-bone healing.

Basic fibroblast growth factor eluting microspheres enhance distraction enterogenesis

PurposeThe purpose of this study was to determine if distraction enterogenesis using self-expanding polycaprolactone (PCL) springs is a potential therapy for short bowel syndrome. Sustained release basic fibroblast growth factor (bFGF) microspheres have been shown to induce angiogenesis and intestinal regeneration in tissue engineered scaffolds. We hypothesized that the provision of bFGF-loaded microspheres would increase angiogenesis and thereby enhance the process of enterogenesis. MethodsA 10-mm segment of rodent jejunum was isolated and an encapsulated PCL spring inserted. Blank or bFGF-loaded microspheres were delivered to the segment. After 4weeks, jejunal segments were assessed for lengthening, morphology, quantification of blood vessels, and ganglia. ResultsLengthened intestinal segments receiving bFGF microspheres demonstrated significantly increased microvascular density compared to those with blank microspheres. There were also significantly more submucosal and myenteric ganglia in the segments that received bFGF microspheres. Segments achieved similar lengthening and final muscular thickness in both blank and bFGF groups, but the bFGF microsphere caused a significant increase in luminal diameter of the jejunal segment. ConclusionSustained release bFGF microspheres enhanced distraction enterogenesis through improved vascularity. The synergy of growth factors such as bFGF with distraction enterogenesis may yield improved results for the future treatment of patients with short bowel syndrome.

In vitro stress effect on degradation and drug release behaviors of basic fibroblast growth factor--poly(lactic-co-glycolic-acid) microsphere.

ObjectiveTo study the degradation and basic fibroblast growth factor (bFGF) release activity of bFGF – poly(lactic-co-glycolic-acid) microsphere (bFGF-PLGA MS) under stress in vitro, including the static pressure and shearing force-simulating mechanical environment of the joint cavity.MethodFirst, bFGF-PLGA MSs were created. Meanwhile, two self-made experimental instruments (static pressure and shearing force loading instruments) were initially explored to provide stress-simulating mechanical environment of the joint cavity. Then, bFGF-PLGA MSs were loaded into the two instruments respectively, to study microsphere degradation and drug release experiments. In the static pressure loading experiment, normal atmospheric pressure loading (approximately 0.1 MPa), 0.35 MPa, and 4.0 MPa pressure loading and shaking flask oscillation groups were designed to study bFGF-PLGA MS degradation and bFGF release. In the shearing force loading experiment, a pulsating pump was used to give the experimental group an output of 1,000 mL/min and the control group an output of 10 mL/min to carry out bFGF-PLGA MS degradation and drug release experiments. Changes of bFGF-PLGA MSs, including microsphere morphology, quality, weight-average molecular weight of polymer, and microsphere degradation and bFGF release, were analyzed respectively.ResultsIn the static pressure loading experiment, bFGF-PLGA MSs at different pressure were stable initially. The trend of molecular weight change, quality loss, and bFGF release was consistent. Meanwhile, microsphere degradation and bFGF release rates in the 4.0 MPa pressure loading group were faster than those in the normal and 0.35 MPa pressure loading groups. It was the fastest in the shaking flask group, showing a statistically significant difference (P<0.0001). In the shearing force loading experiment, there were no distinctive differences in the rates of microsphere degradation and bFGF release between experimental and control group. Meanwhile, microsphere degradation and bFGF release rates by shaking flask oscillation were obviously faster than those by shearing force only (P<0.0001).ConclusionThere are significant effects on bFGF-PLGA MS degradation and bFGF release due to the interaction between extraction stress and time. Static pressure has a conspicuous influence on bFGF-PLGA MS degradation and release, especially at a pressure of 4.0 MPa. The shearing force has a slight effect on bFGF-PLGA MS degradation and drug release. On the contrary, shaking flask oscillation has a significantly distinctive effect.

Slow release of basic fibroblast growth factor (b-FGF) enhances mechanical properties of rat trachea

AimSevere tracheomalacia is a life-threatening disease, but symptoms usually improve with growth. The aims of this study were to investigate how slow release basic-Fibroblast Growth Factor (b-FGF) acts on tracheal cartilage, and whether growth-promoted trachea is more resistant against an increase in externally-applied pressure. MethodsBiodegradable gelatin hydrogel sheets soaked in 10μl of distilled water (sham) or 0.5 or 5μg/10μl of b-FGF solution were inserted behind the cervical trachea of three-week-old male Wistar rats. The cervical trachea was harvested 4weeks later. Extratracheal pressure was increased from 0 to 40 cmH2O in a chamber, while video-recording the internal lumen. The luminal area at each pressure was expressed as a proportion to that at 0 cmH2O. The amounts of collagen type II and glycosaminoglycan were measured by ELISA. ResultsThe luminal areas at 40 cmH2O in the control (no intervention), sham, and each of the b-FGF groups were 0.65, 0.62, 0.72, and 0.73, respectively. The amounts of collagen type II and glycosaminoglycan in each group were 127, 136, 193, 249μg/mg, respectively, and 15, 16, 19, 33μg/mg, respectively. There were significant differences between the control group and the FGF 5 group (P=0.02, 0.01, 0.01, for luminal area, collagen, and glycosaminoglycan, respectively). Conclusion5μg of slow-release b-FGF promotes matrix production (collagen type II and glycosaminoglycan). The growth-enhanced trachea was more resistant to collapse, suggesting that slowly released b-FGF might be useful in patients with severe tracheomalacia.

Bone Regeneration for Murine Femur Fracture by Gelatin Hydrogels Incorporating Basic Fibroblast Growth Factor with Different Release Profiles

Biodegradable hydrogels with three different water contents were prepared through the glutaraldehyde crosslinking of gelatin with an isoelectric point of 5.0 under varied reaction conditions. The objective of this study is to investigate the effect of time period of basic fibroblast growth factor (bFGF) release that is modified by the hydrogel water content, on the bone regeneration. A bone fracture was prepared at the femur bone of mice, while gelatin hydrogels incorporating bFGF or bFGF solution was applied at the fracture site. The gelatin hydrogels incorporating bFGF exhibited significantly stronger bone regeneration than the bFGF solution, although the extent depended on the water content of hydrogels. Bone regeneration induced by gelatin hydrogels incorporating bFGF increased with a decrease in their water content. Histological and biochemical examinations indicated that the area of bone tissue newly formed and the level of osteogenic genes were enhanced for the hydrogels incorporating bFGF of lower water content compared with those of higher one. It is possible that the hydrogels that slowly degraded released bFGF for a longer time period, resulting in an enhanced bone regeneration. It is concluded that the time profile of bFGF release is one of the factors contributing to the bFGF-induced bone regeneration.

The Effect of α‐Tocopherol and Selenium on Human Gingival Fibroblasts and Periodontal Ligament Fibroblasts In Vitro

The aim of the present study is to evaluate the effect of α-tocopherol and selenium on gingival fibroblasts (GFs) and periodontal ligament fibroblasts (PDLFs) in terms of proliferation, basic fibroblast growth factor (bFGF) release, collagen type I synthesis, and wound healing. Primary cultures of human GFs and PDLFs were isolated. Four test groups and a control group free of medication was formed. In group E, 60 μM α-tocopherol was used, and in groups ES1, ES2, and ES3, the combination of 60 μM α-tocopherol with 5 × 10(-9) M, 10 × 10(-9) M, and 50 × 10(-9) M selenium was used, respectively. Viability, proliferation, bFGF, and collagen type I synthesis from both cell types were evaluated at 24, 48, and 72 hours, and healing was compared on a new wound-healing model at 12, 24, 36, 48, and 72 hours. α-Tocopherol alone significantly increased the healing rate of PDLFs at 12 hours and increased bFGF and collagen type I release from GFs and PDLFs at 24, 48, and 72 hours. The α-tocopherol/selenium combination significantly enhanced the proliferation rate of both cells at 48 hours, decreased the proliferation of PDLFs at 72 hours, and increased the healing rate of GFs at 12 hours and PDLFs at 12 and 48 hours. bFGF and collagen type I synthesis was also increased in both cell types at 24, 48, and 72 hours by α-tocopherol/selenium combination. α-Tocopherol and α-tocopherol/selenium combination is able to accelerate the proliferation rate and wound-healing process and increase the synthesis of bFGF and collagen type I from both GFs and PDLFs.

Fabrication and characteristics of chitosan sponge as a tissue engineering scaffold.

Cells, growth factors, and scaffolds are the three main factors required to create a tissue-engineered construct. After the appearance of bovine spongiform encephalopathy (BSE), considerable attention has therefore been focused on nonbovine materials. In this study, we examined the properties of a chitosan porous scaffold. A porous chitosan sponge was prepared by the controlled freezing and lyophilization of different concentrations of chitosan solutions. The materials were examined by scanning electron microscopy, and the porosity, tensile strength, and basic fibroblast growth factor (bFGF) release profiles from chitosan sponge were examined in vitro. The morphology of the chitosan scaffolds presented a typical microporous structure, with the pore size ranging from 50 to 200 μm. The porosity of chitosan scaffolds with different concentrations was approximately 75–85%. A decreasing tendency for porosity was observed as the concentration of the chitosan increased. The relationship between the tensile properties and chitosan concentration indicated that the ultimate tensile strength for the sponge increased with a higher concentration. The in vitro bFGF release study showed that the higher the concentration of chitosan solution became, the longer the releasing time of the bFGF from the chitosan sponge was.

Increased basic fibroblast growth factor release and proliferation in xenotransplanted squamous cell carcinoma after combined irradiation/anti-vascular endothelial growth factor treatment

Novel strategies of cancer therapy combine irradiation and anti-angiogenic active compounds. However, little is known concerning the undesired cellular and molecular effects caused by this novel treatment concept. We used a mouse squamous cell carcinoma (SCC) xenotransplantation model to evaluate the potential undesired effects which compromise the success of this therapeutic combination. SCCs were subcutanously implanted in nude mice. Animals were treated with a fractionated irradiation scheme (5x4Gy) alone or in combination with daily injections of anti-vascular endothelial growth factor (VEGF) antibodies. Controls remained untreated. Before and after treatment, resonance imaging (MRI), ultrasound and near-infrared spectrometry were used to evaluate tumor vessel integrity. Finally, tumors were explanted and VEGF, basic fibroblast growth factor (bFGF), vessel density, proliferation and apoptotic activity were analyzed by immunohistochemistry. Irradiation caused VEGF release and we found evidence for VEGF-mediated vessel protection. In the tumors derived from the combined treatment, blood volume was decreased, and apoptotic indices were increased. Remarkably, bFGF levels and proliferative indices were also increased. Combined irradiation/anti-VEGF treatment resulted in the desired VEGF depletion and increased tumor cell apoptosis. Nonetheless, bFGF and proliferation also increased, possibly suggesting a compensatory response. The application of additional targeted drugs may help develop more effective SCC treatments.

Rhinovirus‐induced basic fibroblast growth factor release mediates airway remodeling features

BackgroundHuman rhinoviruses, major precipitants of asthma exacerbations, induce lower airway inflammation and mediate angiogenesis. The purpose of this study was to assess the possibility that rhinoviruses may also contribute to the fibrotic component of airway remodeling.MethodsLevels of basic fibroblast growth factor (bFGF) mRNA and protein were measured following rhinovirus infection of bronchial epithelial cells. The profibrotic effect of epithelial products was assessed by DNA synthesis and matrix metalloproteinase activity assays. Moreover, epithelial cells were exposed to supernatants from cultured peripheral blood mononuclear cells, obtained from healthy donors or atopic asthmatic subjects and subsequently infected by rhinovirus and bFGF release was estimated. bFGF was also measured in respiratory secretions from atopic asthmatic patients before and during rhinovirus-induced asthma exacerbations.ResultsRhinovirus epithelial infection stimulated mRNA expression and release of bFGF, the latter being positively correlated with cell death under conditions promoting rhinovirus-induced cytotoxicity. Supernatants from infected cultures induced lung fibroblast proliferation, which was inhibited by anti-bFGF antibody, and demonstrated increased matrix metalloproteinase activity. Rhinovirus-mediated bFGF release was significantly higher in an in vitro simulation of atopic asthmatic environment and, importantly, during rhinovirus-associated asthma exacerbations.ConclusionsRhinovirus infection induces bFGF release by airway epithelium, and stimulates stroma cell proliferation contributing to airway remodeling in asthma. Repeated rhinovirus infections may promote asthma persistence, particularly in the context of atopy; prevention of such infections may influence the natural history of asthma.

Heat Shock Protein 27 as a New Therapeutic Target for Radiation Sensitization of Head and Neck Squamous Cell Carcinoma

In a wide range of human cancers, increased levels of heat shock protein 27 (Hsp27) are closely associated with tumorigenesis, metastasis, resistance to anticancer therapeutics, and thus poor prognosis. In this study, we evaluate the radiosensitizing effects of Hsp27 gene silencing using OGX-427, a second-generation antisense oligonucleotide (ASO), on the radioresistant head and neck squamous cell carcinoma (HNSCC) SQ20B cells. In vitro, the downregulation of Hsp27 significantly enhanced radiation-induced apoptotic and clonogenic death, and promoted Akt inactivation. In vivo, combining OGX-427 with local tumor irradiation (5 x 2 Gy) led to a significant regression of SQ20B tumors related to a high rate of apoptosis and decreased levels of glutathione antioxidant defenses. Increasing the total radiation dose (15 x 2 Gy) significantly amplified the radiosensitizing effect of OGX-427. Treatment of tumors with OGX-427 plus radiation resulted in a decrease in angiogenesis associated with a reduced activation of the Akt pathway. Furthermore, the combined treatment enhanced the survival of SQ20B-bearing mice and showed no signs of acute and delayed toxicity. Our findings demonstrate for the first time that Hsp27 knockdown enhances the cytotoxic effects of radiotherapy in vivo and provide preclinical proof of principle for clinical trials using Hsp27 antisense technology in the treatment of patients with HNSCC radioresistant cancers.

Membrane Type-1 Matrix Metalloproteinase Potentiates Basic Fibroblast Growth Factor-Induced Corneal Neovascularization

Corneal neovascularization is one of the leading causes of blindness. The aim of this study was to evaluate the pro-angiogenic role of corneal fibroblast-derived membrane type-1 matrix metalloproteinase (MT1-MMP) on basic fibroblast growth factor (bFGF)-induced corneal neovascularization in vivo and in vitro. Immunohistochemical studies demonstrated that MT1-MMP was expressed in keratocytes and immortalized corneal fibroblast cell lines. Vascular endothelial growth factor protein levels were increased after bFGF-stimulation of wild-type fibroblast cells compared with MT1-MMP knockout fibroblast cells. Corneal vascularization was significantly increased after a combination of bFGF pellet implantation and naked MT1-MMP DNA injection in wild-type mouse corneas compared with either bFGF pellet implantation or naked MT1-MMP DNA-injected corneas. Western blotting analysis of the phosphorylation levels of the key signaling molecules (p38, JNK, and ERK) demonstrated that phosphorylation levels of both p38 and JNK were diminished after bFGF stimulation of MT1-MMP knockout cells compared with wild-type and MT1-MMP knockin cells. These results suggest that MT1-MMP potentiates bFGF-induced corneal neovascularization, likely by modulating the bFGF signal transduction pathway.

Vascularization Around Poly(tetrafluoroethylene) Mesh with Coating of Gelatin Hydrogel Incorporating Basic Fibroblast Growth Factor

For successful mesh hernia treatment with medical meshes, it is important to induce angiogenesis and fibroplasia around the site of the mesh implanted. The objective of this study is to combine a mesh with a gelatin hydrogel for basic fibroblast growth factor (bFGF) release and evaluate the angiogenic activity in vivo. The MotifMesh® (MM) of poly(tetrafluoroethylene) was treated with corona discharge to make the surface hydrophilic. This corona discharge treatment increased the bonding strength between the gelatin hydrogel coated and the mesh surface. When implanted into the back subcutis of mice, the MM coated with the gelatin hydrogel incorporating bFGF showed significant angiogenesis around the implanted site, in contrast to the MM alone and that without gelatin hydrogel or bFGF incorporation. It is concluded that the coating of hydrogel incorporating bFGF is a promising technology to give the mesh angiogenic properties.

Biodegradable elastomeric scaffolds with basic fibroblast growth factor release

Scaffolds that better approximate the mechanical properties of cardiovascular and other soft tissues might provide a more appropriate mechanical environment for tissue development or healing in vivo. An ability to induce local angiogenesis by controlled release of an angiogenic factor, such as basic fibroblast growth factor (bFGF), from a biodegradable scaffold with mechanical properties more closely approximating soft tissue could find application in a variety of settings. Toward this end biodegradable poly(ester urethane)urea (PEUU) scaffolds loaded with bFGF were fabricated by thermally induced phase separation. Scaffold morphology, mechanical properties, release kinetics, hydrolytic degradation and bioactivity of the released bFGF were assessed. The scaffolds had inter-connected pores with porosities of 90% or greater and pore sizes ranging from 34–173 μm. Scaffolds had tensile strengths of 0.25–2.8 MPa and elongations at break of 81–443%. Incorporation of heparin into the scaffold increased the initial burst release of bFGF, while the initial bFGF loading content did not change release kinetics significantly. The released bFGF remained bioactive over 21 days as assessed by smooth muscle mitogenicity. Scaffolds loaded with bFGF showed slightly higher degradation rates than unloaded control scaffolds. Smooth muscle cells seeded into the scaffolds with bFGF showed higher cell densities than for control scaffolds after 7 days of culture. The bFGF-releasing PEUU scaffolds thus exhibited a combination of mechanical properties and bioactivity that might be attractive for use in cardiovascular and other soft tissue applications.

Proangiogenic Role of ephrinB1/EphB1 in Basic Fibroblast Growth Factor-Induced Corneal Angiogenesis

Corneal neovascularization is a vision-threatening condition caused by various ocular pathological conditions. The aim of this study was to evaluate the function of the ephrin ligands and Eph receptors in vitro and in vivo in corneal angiogenesis in a mouse model. The Eph tyrosine kinase receptors and their ligands, ephrins, are expressed on the cell surface. The functions of Eph and ephrins have been shown to regulate axonal guidance, segmentation, cell migration, and angiogenesis. Understanding the roles of Eph and ephrin in corneal angiogenesis may provide a therapeutic intervention for the treatment of angiogenesis-related disorders. Immunohistochemical studies demonstrated that ephrinB1 and EphB1 were expressed in basic fibroblast growth factor (bFGF)-induced vascularized corneas. EphB1 was specifically colocalized with vascular endothelial marker CD31 surrounded by type IV collagen. EphrinB1 was expressed in corneal-resident keratocytes and neutrophils. Recombinant ephrinB1-Fc, which induces EphB receptor activation, enhanced bFGF-induced tube formation in an in vitro aortic ring assay and promoted bFGF-induced corneal angiogenesis in vivo in a corneal pocket assay. Synergistically enhanced and sustained activation of extracellular signal-regulated kinase was noted in vascular endothelial cell lines after stimulation with ephrin B1 and bFGF combinations. These results suggest that ephrinB1 plays a synergistic role in corneal neovascularization.

Cell-surface receptor for thyroid hormone and tumor cell proliferation

Integrin αVβ3 is a structural protein of the plasma membrane that transduces signals from extracellular matrix proteins and has recently been shown to contain a novel receptor for thyroid hormone. Thyroid hormone signals are converted by αVβ3 into mitogen-activated protein kinase (MAPK) (ERK1/2) activation and downstream intracellular events in the cell nucleus. The latter include post-translational modification of the nuclear thyroid hormone receptor (TRβ1) and complex cellular or tissue responses, such as hormone-induced angiogenesis via basic fibroblast growth factor release. The integrin receptor for thyroid hormone has been shown to mediate proliferative effects of the hormone on certain tumor cell lines, including murine glioma/glioblastoma cells and human breast cancer (MCF-7) cells. More than one mechanism may account for this hormonal action, but in vitro studies indicate a direct hormonal action on cellular proliferation. Other possible mechanisms involve indirect actions via the release of tumor growth factors and effects on cell migration. In the intact organism, support of tumor growth by thyroid hormone is postulated to include angiogenesis. Crosstalk between the integrin thyroid hormone receptor and the epidermal growth factor receptor on the plasma membrane may be another mechanism by which thyroid hormone may modify tumor cell growth. Tetraiodothyroacetic acid (tetrac) is an iodothyronine analog that has no agonist activity at the integrin receptor, but inhibits binding of l-thyroxine and 3,5,3´-triiodo-l-thyronine to the receptor, preventing MAPK activation and consequent actions downstream of MAPK. In vitro studies and a preliminary in vivo experiment indicate that tetrac blocks the action of thyroid hormone on tumor cell proliferation. Both unmodified tetrac and tetrac reformulated as a nanoparticle that does not gain access to the cell interior are under investigation in animal models as anticancer agents. Also under study is the susceptibility of other human cancer cell lines to induction of proliferation by physiological concentrations of thyroid hormone.

Lysophosphatidylcholine induces inflammatory activation of human coronary artery smooth muscle cells

Lysophosphatidylcholine (LPC) is the major bioactive lipid component of oxidized LDL, thought to be responsible for many of the inflammatory effects of oxidized LDL described in both inflammatory and endothelial cells. Inflammation-induced transformation of vascular smooth muscle cells from a contractile phenotype to a proliferative/secretory phenotype is a hallmark of the vascular remodeling that is characteristic of atherogenesis; however, the role of LPC in this process has not been fully described. The present study tested the hypothesis that LPC is an inflammatory stimulus in coronary artery smooth muscle cells (CASMCs). In cultured human CASMCs, LPC stimulated time- and concentration-dependent release of arachidonic acid that was sensitive to phospholipase A2 and C inhibition. LPC stimulated the release of arachidonic acid metabolites leukotriene-B4 and 6-keto-prostaglandin F1alpha, within the same time course. LPC was also found to stimulate basic fibroblast growth factor release as well as stimulating the release of the cytokines GM-CSF, IL-6, and IL-8. Optimal stimulation of these signals was obtained via palmitic acid-substituted LPC species. Stimulation of arachidonic acid, inflammatory cytokines and growth factor release, implies that LPC might play a multifactorial role in the progression of atherosclerosis, by affecting inflammatory processes.

Periurethral Injection of Sustained Release Basic Fibroblast Growth Factor Improves Sphincteric Contractility of the Rat Urethra Denervated by Botulinum-A Toxin

We evaluated the effects of sustained release basic fibroblast growth factor injection in rat urethra denervated by botulinum-A toxin (Wako Life Science, Osaka, Japan). A total of 30 female Sprague-Dawley rats underwent periurethral injection of 10 U botulinum-A toxin to induce chemical denervation of the urethral sphincter. Leak point pressure in the waking state was determined and a significant decrease in leak point pressure vs that in control rats was confirmed (mean +/- SD 58.7 +/- 6.2 vs 120.7 +/- 13.0 cm H(2)O, p <0.0001). Two weeks later 0, 50 and 200 microg basic fibroblast growth factor incorporating 200 microl gelatin hydrogels in 10 rats each were injected into the urethral sphincter, enabling sustained release of basic fibroblast growth factor for 2 weeks. Four weeks later injection leak point pressure measurement and histological evaluation of the urethra were performed. Leak point pressure in rats with 50 and 200 microg basic fibroblast growth factor injection was significantly higher than in rats with the 0 microg injection (82.7 +/- 9.0 vs 95.1 +/- 6.2 and 119.3 +/- 8.1 cm H(2)O, p = 0.0021 and <0.0001, respectively). Maximum cross-sectional area of the urethral smooth muscle layer in the 50 and 200 microg groups significantly increased compared with that in the urethra in the 0 micro group, which was considered 100% (114.1% +/- 15.8% and 132.5% +/- 13.4%, p = 0.029 and <0.0001, respectively). Similarly the cross-sectional area of the striated sphincter in the 50 and 200 microg groups was greater than the 100% in the 0 microg group (112.3% +/- 15.6% and 124.3% +/- 14.1%, p = 0.069 and 0.0007, respectively). Vascular density in the urethral peri-atrophic zone in the 50 and 200 microg groups was significantly higher than in the 0 microg group (p = 0.027 and <0.0001, respectively). Sustained release basic fibroblast growth factor injection in the chemically denervated urethral sphincter facilitates regeneration of the urethral muscles and improves sphincteric contractility. Endoscopic periurethral injection of basic fibroblast growth factor incorporating gelatin hydrogels may be an attractive therapy for stress urinary incontinence.

High density lipoproteins downregulate basic fibroblast growth factor production and release in minimally oxidated-LDL treated smooth muscle cells

Increase in plasma low density lipoprotein (LDL) levels and/or decrease in high density lipoprotein (HDL) levels are major risk factors for the development of atherosclerosis. An oxidative modification of LDL represents a key process in atherogenesis. It is well known that the LDL/HDL ratio is more important than the individual LDL and HDL levels to predict atherosclerosis. The purpose of our study was to investigate the effects of mildly oxidized LDL (minimally modified LDL: MM-LDL) and HDL, administrated alone or in combination, on the production and release of basic fibroblast growth factor (bFGF) by bovine aortic smooth muscle cells (SMCs) in culture. MM-LDL and HDL have opposite effects on aortic SMCs: MM-LDL increases both bFGF production and release and SMC proliferation, while HDL decreases both bFGF production and release and SMC proliferation. The effects of either MM-LDL or HDL on SMCs are mediated through a Gi-protein-coupled receptor. The simultaneous treatment of SMCs with MM-LDL and HDL (MM-LDL/HDL ratio = 4.0) produced the inhibition of MM-LDL effects. Our data suggest that the protective role of HDL could also be exerted through the inhibition of the pro-atherosclerotic effects of MM-LDL on SMCs.