Preparation of Gelatin Microspheres Encapsulated with bFGF for Therapeutic Angiogenesis in a Canine Ischemic Hind Limb

There is urgent need for the treatment of limb ischemia. In order to avoid the risk of genetic materials or injury in collection of implanted cells, a basic fibroblast growth factor (bFGF) sustained release system using cross-linked gelatin microspheres was developed for therapeutic angiogenesis. In this study, gelatin microspheres (MSs) and the complex of MSs and bFGF (MSs–bFGF) were prepared. MSs and MSs–bFGF were analyzed for morphology, particle size, in vitro bFGF release and the bioactivity of the released medium. MSs–bFGF was intramuscularly implanted into the ischemic hind limb of a dog and free bFGF, empty MSs and untreated animals were used as controls. Histological examination was performed for angiogenesis evaluation. After immersion in an aqueous solution, the un-cross-linked MSs became deformed and adhered together. The cross-linked MSs showed a more stable character both in vivo and in vitro. The bFGF released from MSs remained bioactive. The histological examination indicated that the densities of micro-vessels in the MSs–bFGF-treated hind limb muscle were significantly greater than that in the untreated control, free bFGF and empty MSs groups. The MSs–bFGF sustained release system was a simple, safe and effective way to achieve therapeutic angiogenesis in an ischemic limb.     

Development of bFGF-Chitosan Matrices and Their Interactions with Human Dermal Fibroblast Cells

Chitosan (CH) is a naturally derived, biodegradable polymer of glucosamine with a variable frequency of N-acetyl-D-glucosamine units, and has been demonstrated to have numerous pharmacological and wound-healing properties. Biodegradable chitosan films were fabricated using a solvent casting technique and investigated for skin tissue-engineering applications. Basic fibroblast growth factor (bFGF) was incorporated into the CH matrices (1 μg/film) by 3 methods: adsorption, entrapment and covalent binding. Release rates and biological activity of the incorporated bFGF were monitored. Human dermal fibroblasts (HDF cells) were used as an in vitro model for cell response to CH and bFGF-CH films. Cell attachment, growth and acid-soluble collagen quantification were employed as an assessment of cell function. The fibroblasts were found to remain viable on the chitosan films and scaffolds. CH films without bFGF were compatible with HDF cells; however, the fibroblasts did not proliferate. The release profile of adsorbed and bound bFGF from CH films were similar (indicating that binding was not efficient) while entrapped bFGF was not released in the time frame studied. The concentration of bFGF released to the cell culture medium was not high enough to stimulate HDF proliferation. However, cell attachment was significantly increased in chitosan films with bFGF adsorbed onto the surface as compared to control surfaces. HDF cells grown on CH films produced significantly more collagen than those on control surfaces.     

A Calcium-Cross-Linked Hydrogel Based on Alginate-Modified Atelocollagen Functions as a Scaffold Material

We have developed a scaffold material consisting of a covalently-bonded structure of alginate and atelocollagen (AtCol). Addition of calcium ions caused the material to form a hydrogel (alginate-modified AtCol gel). The condition of the alginate-modified AtCol gel could be controlled by the feed ratio of alginate and the activating reagent. Measurement of temporal stability in culture medium suggested that covalent bonding between alginate and AtCol might contribute to the structural stability of the alginate-modified AtCol gel. Culture with endothelial cells indicated that cell adhesiveness on the alginate-modified AtCol gel was similar to that on native collagen. Collagenase digestion revealed that the alginate-modified AtCol gel had considerable ability to retain basic fibroblast growth factor. Additionally, active cell migration into alginate-modified AtCol was detected by in vitro assay using endothelial cells. These findings indicate that this gel material can be expected to function as a scaffold for inducing vascular in-growth.     

Covalently-Immobilized Vascular Endothelial Growth Factor Promotes Endothelial Cell Tubulogenesis in Poly(ethylene glycol) Diacrylate Hydrogels

The development and use of functional tissue-engineered products is currently limited by the challenge of incorporating microvasculature. To this end, we have investigated strategies to facilitate vascularization in scaffold materials, in this case poly(ethylene glycol) (PEG) hydrogels. These hydrogels are hydrophilic and resist protein adsorption and subsequent non-specific cell adhesion, but can be modified to contain cell-adhesive ligands and growth factors to support cell and tissue function. Additionally, the hydrogel matrix can include proteolytically degradable peptide sequences in the backbone of the structure to allow cells to control scaffold biodegradation, allowing three-dimensional migration. Vascular endothelial growth factor (VEGF), a potent angiogenic signal, and the cell-adhesive peptide RGDS were each covalently attached to PEG monoacrylate linkers. PEGylated RGDS and VEGF were then covalently immobilized in PEG-diacrylate (PEGDA) hydrogels in 2D and 3D. Immobilized VEGF increased endothelial cell tubulogenesis on the surface of non-degradable PEGDA hydrogels 4-fold compared to controls without the growth factor. Endothelial cell behavior in 3D collagenase-degradable hydrogels modified with RGDS and VEGF was observed using time-lapse confocal microscopy. Bulk immobilization of VEGF in 3D collagenase-degradable RGDS-modified hydrogels increased endothelial cell motility 14-fold and cell–cell connections 3-fold. Covalent incorporation of PEGylated VEGF in PEG hydrogels can be a useful tool to promote endothelial cell migration, cell–cell contact formation and tubulogenesis in an effort to produce vascularized tissue-engineered constructs.     

Influence of gelatin complexation on cell proliferation activity and proteolytic resistance of basic fibroblast growth factor

The objective of this study is to investigate the influence of gelatin complexation on the biological activity of basic fibroblast growth factor (bFGF) and its resistance to trypsin digestion. When bFGF was mixed at 37°C with acidic gelatin with an isoelectric point(IEP)of 5.0, the activity to promote in vitro proliferation of BHK cells became lower compared with that of free bFGF,in contrast to mixing with the basic gelatin with an IEP of 9.0. A maximum reduction in the bFGF activity was observed for the bFGF-gelatin complex prepared at a mixing molar ratio of 1/1. The bFGF activity of cell proliferation reduced at the initial period after mixing with the acidic gelatin at 37°C, followed by no substantial change. Complexation with the acidic gelatin at 4°C had no influence on the bFGF activity, irrespective of the bFGF/gelatin ratio and complexation time. The biological activity of bFGF was reduced by the trypsin treatment, but the reduced extent was suppressed through gelatin complexation at 37°C. In an electrophoresis study, the protective effect of gelatin complexation on the trypsin digestion was also confirmed in terms of the molecular weight loss. It is possible that the complexing gelatin covers bFGF molecules, resulting in suppression of their interaction with the cell surface receptor as well as protection from their enzymatic attack.     

Heparin-modified dendrimer cross-linked collagen matrices for the delivery of basic fibroblast growth factor (FGF-2)

Tissue integration between a tissue-engineered corneal equivalent and the host eye is of critical importance in ensuring long-term implant success. A novel dendrimer cross-linked collagen scaffold has previously shown good corneal epithelial cell compatibility in vitro particularly when the highly functional dendrimer cross-linkers were functionalized to introduce additional biological groups. Herein we investigated heparinization of these materials and their potential to facilitate the delivery of basic fibroblast growth factor (FGF-2) in an active form, ultimately for use as a corneal tissue scaffold. Collagen gels cross-linked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) chemistry, and varying amounts of polypropyleneimine octaamine generation 2 (G2) dendimer and heparin were synthesized. Swelling studies and differential scanning calorimetry characterization indicated higher gel stability with the introduction of dendrimer cross-linking, which was not compromised by heparin integration. Dendrimer cross-linked gels with or without heparin gave multiple denaturation peaks, as did the heparinized EDC gels. This is thought to be the result of the heterogeneous cross-linking possible between collagen, the dendrimer and heparin. Release of FGF-2 from collagen gels showed typical first-order kinetics, with an initial burst followed by a prolonged gradual release. Heparinized dendrimer cross-linked gels released approx. 40% of the growth factor over a 2-week period, with significance maintenance of growth factor activity. Incorporation of heparin resulted in somewhat prolonged release from these systems.     

Drug Release from Interpenetrating Polymer Networks Based on Poly(ethylene glycol) Methyl Ether Acrylate and Gelatin

In order to develop new materials for biomedical and pharmaceutical applications, interpenetrating polymer networks (IPNs) based on poly(ethylene glycol) methyl ether acrylate (PEGMEA) and gelatin were synthesized. These two materials were cross-linked sequentially using N,N′-methylene bisacrylamide (NMBA) and glutaraldehyde (Glu). Two series of IPNs gels were synthesized by applying different amounts of PEGMEA and gelatin in the initial feed. Sequential IPNs were prepared by polymerizing and cross-linking PEGMEA in the presence of gelatin using redox initiators (e.g., ammonium peroxydisulfate (APS) and N,N,N′,N′-tetramethyl ethylenediamine (TEMED)), as well as NMBA as the cross-linking agent. Gelatin in firm gel was then cross-linked with 1% glutaraldehyde. The swelling kinetics, mechanical properties and drug-release behavior of these IPNs were analyzed. The surface properties were examined by scanning electron microscopy. The results indicated that the swelling ratio decreased with an increase in the content of both PEGMEA and gelatin in the IPNs. PEGMEA/gelatin-based full-IPNs had a significantly higher shear modulus (G) and cross-linking density (ρ) when the content of PEGMEA was increased. The drug loading was very high due to the full-IPN structure. The drug-release velocity was mainly affected by the content of PEGMEA.     

Cellular Behavior on Epidermal Growth Factor (EGF)-Immobilized PCL/Gelatin Nanofibrous Scaffolds

Nano-scaled poly(ε-caprolactone) (PCL) and PCL/gelatin fibrous scaffolds with immobilized epidermal growth factor (EGF) were prepared for the purpose of wound-healing treatments. The tissue scaffolds were fabricated by electrospinning and the parameters that affect the electrospinning process were optimized. While the fiber diameters were 488 ± 114 nm and 663 ± 107 nm for PCL and PCL/gelatin scaffolds, respectively, the porosities were calculated as 79% for PCL and 68% for PCL/gelatin scaffolds. Electrospun PCL and PCL/gelatin scaffolds were first modified with 1,6-diaminohexane to introduce amino groups on their surfaces, then EGF was chemically conjugated to the surface of nanofibers. The results obtained from Attenuated Total Reflectance Fourier Transform Infrared (ATR–FT-IR) spectroscopy and quantitative measurements showed that EGF was successfully immobilized on nanofibrous scaffolds. L929 mouse fibroblastic cells were cultivated on both neat and EGF-immobilized PCL and PCL/gelatin scaffolds in order to investigate the effect of EGF on cell spreading and proliferation. According to the results, especially EGF-immobilized PCL/gelatin scaffolds exerted early cell spreading and superior and rapid proliferation compared to EGF-immobilized PCL scaffolds and neat PCL, PCL/gelatin scaffolds. Consequently, EGF-immobilized PCL/gelatin scaffolds could potentially be employed as novel scaffolds for skin tissueengineering applications.     

In Vivo Validation of Biological Responses of bFGF Released from Microspheres Formulated by Blending Poly-Lactide-co-Glycolide and Poly(ethylene glycol)-Grafted-Chitosan in Hamster Cheek Pouch Microcirculatory Models

Microspheres formulated from blending poly(lactide-co-glycolide) (PLGA) and poly(ethylene glycol)-grafted-chitosan (PEG-g-CHN), using a modified in-emulsion-solvent-evaporation method, were investigated for the delivery of protein. A model protein, bovine serum albumin (BSA), was incorporated into the PLGA/PEG-g-CHN microspheres and both initial burst and release kinetics could be modulated by varying the PEG-g-CHN content. Basic fibroblast growth factor (bFGF) was formulated into the microspheres containing 5% PEG-g-CHN and the bFGF contents in the releasates were determined by a receptor-based ELISA with their in vitro bioactivities validated by fibroblast cell culture. The in vivo effect of the bFGF microspheres formulation was evaluated in a hamster cheek pouch model using a 7 day exposure (e.g., before significant vascular remodeling was expected). Using intravital microscopy, the tissue showed no evidence of inflammation with any formulation; deliberate activation of a preconditioning response linked to inflammation was attenuated by BSA microspheres alone. Vasoactive responses (receptor-dependant and independent constriction and dilation) linked to nitric oxide were attenuated, and constriction to endothelin was enhanced in bFGF and not BSA containing microspheres. PLGA/PEG-g-CHN blended microspheres were also demonstrated to be non-inflammatory and non-thrombogenic in vivo by observing the vascular changes in the cheek pouch. In conclusion, the addition of PEG-g-CHN to PLGA microspheres can serve as a sustained delivery vehicle for bFGF and the released protein provides vasoactive changes consistent with chronic bFGF exposure.     

Development of a Wound Dressing Composed of Hyaluronic Acid and Collagen Sponge with Epidermal Growth Factor

This study was designed to investigate the effect of a wound dressing composed of hyaluronic acid (HA) and collagen (Col) sponge containing epidermal growth factor (EGF) on various parameters of wound healing in vitro and in vivo. High-molecular-weight (HMW) HA solution, hydrolyzed low-molecular-weight (LMW) HA solution and heat-denatured Col solution were mixed, followed by freeze-drying to obtain a spongy sheet. Cross-linkage between Col molecules was induced by UV irradiation to the spongy sheet (Type-I dressing). In a similar manner, a spongy sheet containing EGF was prepared (Type-II dressing). The efficacy of these products was firstly evaluated in vitro. Fibroblast proliferation was assessed in culture medium in the presence or absence of a piece of each wound dressing. EGF stimulated cell proliferation after UV irradiation and dry sterilization at 110°C for 1 h. In the second experiment, fibroblasts-embedded Col gels were elevated to the air–liquid interface to create a wound surface model, on which wound dressings were placed and cultured for 1 week. Cell proliferation and the production of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) were investigated. With Type-II dressings, the amounts of VEGF and HGF released from fibroblasts in the Col gel were significantly increased compared with Type-I dressing. Next, the efficacy of these products was evaluated in vivo using Sprague–Dawley (SD) rats. Wound conditions after 1 and 2 weeks of treatment with the wound dressings were evaluated based on the gross and histological appearances. Type-II dressings promoted a decrease in wound size, re-epithelialization and granulation tissue formation associated with angiogenesis. These findings indicate that the combination of HA, Col and EGF promotes wound healing by stimulating fibroblast function.     

Synthesis,Swelling and Drug-Release Behaviour of a Poly(N,N-diethylacrylamide-co-(2-dimethylamino) ethyl methacrylate) Hydrogel

In this study, poly(N,N-diethylacrylamide-co-(2-dimethylamino) ethyl methacrylate) (poly(DEA-co-DMAEMA)) hydrogels were synthesized by changing the initial DEA/DMAEMA mol ratio. The hydrogels were characterized by Fourier transform infrared (FT-IR) spectroscopy and scanning electron microscopy (SEM). In comparison with the PDEA hydrogel, the equilibrium swelling ratio (ESR) and lower critical solution temperature (LCST) of the hydrogels increase with the increase of DMAEMA content in the feed. The deswelling and reswelling kinetics and cytotoxicity of the different composition ratios of DEA to DMAEMA in the co-polymerized hydrogels were also investigated in detail. The absorption and release behaviour of the model drug, bovine serum albumin, were found to be dependent on hydrogel composition and environment temperature, which suggests that these materials have potential application as intelligent drug carriers.     

Improved Healing of Tympanic Membrane Perforations with Basic Fibroblast Growth Factor

Abstract

We have investigated the effects of basic fibroblast growth factor (FGF) on the healing of tympanic membrane (TM) perforations. In the first series of experiments, a simple, round I-mm perforation was made in the membrane and the effects of basic FGF examined. In a second series of experiments, basic FGF was tested on 2-mm perforations in which the borders were folded inward in order to delay normal healing. Topical applications of saline or basic FGF were administered onto gelfoam overlays of the TM perforations in 51 guinea pigs by delivering 5μl aliquots of PBS or 5μl of PBS containing 1 μg of basic FGF on the day of surgery and daily thereafter. Repair of the lesions was evaluated 3, 5 or 8 days after surgery. The results show that basic FGF mediates faster healing of TM perforations by inducing rapid proliferation of the subepithelial connective tissue layer.     

Biological Feature of Collagen-Chitosan Membrane with Basic Fibroblast Growth Factor for the Culture of Human Fibroblast

INTRODUCTION　　Collagen present in the extracellular matrix is the most promising natural poly-mer for tissue engineering.It has many expected biological features,includinggrowth promotion,biological stability,low antigenicity and cytotoxic properties.Collagen is frequently used material for cell culture carriers in various fields.Al-though collagen possess excellent biocompatibility,the chemical treatment makesthe reconstituted collagen very low tensile strength and easily biodegraded b…     

碱性成纤维细胞生长因子（bFGF）mRNA的表达在部分恶性肿瘤中的检测

本文用地高辛为标记物的Ｎｏｒｔｈｅｒｎｂｌｏｔ技术检测了各种恶性肿瘤５１例，良性肿瘤１７例，肿瘤组织中ｂＦＧＦｍＲＮＡ的表达。结果以地高辛抗体－碱性磷酸酶显色体系显色，以硝纤薄膜条带上７．０处出现蓝紫色条带为阳性，无条带者为阴性。结果肺癌阳性率为４２．８６％（３／７）；胃癌阳性率为７２．７３％（８／１１）：肝癌阳性率为７３．３３％（１１／１５）；前列腺癌阳性率为７７．７８％（７／９），乳腺癌阳性率为５５．５６％（５／９），全部恶性肿瘤阳性率为３４例，阳性率为６６．６７％，而良性肿瘤仅有１例阳性，阳性率为９．３％。用　检验恶性肿瘤组与良性肿瘤组有明显差别，Ｐ＜０，０１。由此可以说明ｂＦＧＦｍＲＮＡ在恶性肿瘤中强烈表达，而在良性肿瘤中表达较弱或不表达，从而ｂＦＧＦｍＲＮＡ可以用来诊断恶性肿瘤。     

A Reaction-Diffusion Model of Basic Fibroblast Growth Factor Interactions with Cell Surface Receptors

Basic fibroblast growth factor (FGF-2) is a potent angiogenic growth factor involved in the development of diseases such as cancer, atherosclerosis, and heart and limb ischemia, as well as normal wound healing and tissue development. Despite being one of the most heavily studied angiogenic growth factors, the binding kinetics and signaling pathways of FGF-2 are still incompletely understood. In this study, we address the role of the low-affinity heparan sulfate proteoglycans (HSPGs), the identity of the minimal signaling complex leading to FGF-2 activity, and the importance of FGF-2 dimerization using a mathematical model of FGF-2 diffusion and ligand-receptor binding. Unique model features include the degradation of internalized cell surface species, the binding of a second FGF-2 ligand to a high-affinity FGF receptor (FGFR), and the dimerization of FGF-2 ligands. All experimentally determined reaction rates and diffusivity values are scaled to 37 degrees C. Our model results suggest that FGF-2-induced cellular response is the result of a temporal combination of triads (FGF-2/HSPG/FGFR complexes), double triads (2 FGF-2/HSPG/FGFR complexes), and FGF-2-bound HSPGs (FGF-2/HSPG complexes). Moreover, ligand dimerization is shown to potentially regulate FGF-2 activity by shifting the distribution of signaling complexes from the less stable triads to the more stable double triads.     

Secretion of Basic Fibroblast Growth Factor (FGF-2) by WEHI-3B Myelomonocytic Leukemia Cells

In order to investigate the role of Fibroblast Growth Factors in hematopoietic cells, we studied the expression of FGF-1, FGF-2, FGF-3, FGF-4, FGF-5 and FGF-6 mRNAs both in murine myelomonocytic leukemia WEHI-3B and in a murine stromal cell line SR-4987. Secretion of FGF-2 in the cell culture supernatant was also studied. Expression of mRNA encoding for the above-mentioned FGFs was analyzed by RT-PCR. The production of FGF-2 in the conditioned media of WEHI-3B and SR-4987 cell cultures was evaluated by techniques of affinity chromatography, chromatofocusing and immunoblotting. The biological activity of FGF-2 was checked on SR-4987 cells by a agar clonogenic assay. In both cell lines mRNA was found encoding for FGF-1, FGF-2 and FGF-6 and WEHI-3B cells express also mRNA for FGF-3 (int-2) and FGF-4 (K-FGF/hst). Furthermore, supernatant from WEHI-3B cells was found to stimulate dramatically the agar clonogenicity of SR-4987 cells which have a very poor basal capacity for growth in agar. The clonogenic activity of WEHI-3B conditioned medium is due to FGF-2 secreted into cell culture supernatant whereas SR-4987 cells, although express FGF-2 mRNA, do not seem able to secrete this factor. The expression in myeloid leukemia cells of oncogene-related factors such as FGF-3, FGF-4 and FGF-6 together with the secretion of FGF-2 able to support a positive regulation of bone marrow stromal cells function suggest that FGFs may have an important role in sustaining the leukemogenic process and related disorders.     

白血病抑制因子(LIF)与bFGF协同促进小鼠外胚间充质干细胞(EMSCs)体外自我维持

白血病抑制因子（leukemia inhibitor factor，LIF）可以抑制小鼠胚胎干细胞分化，维持其自我更新，因而用于该干细胞的体外扩增。由于不同的胚胎或成体干细胞分化特性相差很大，因此，LIF可能对不同的干细胞有不同的作用。对新鲜分离的小鼠外胚间充质细胞（ectomesenchymal cells，EMCs）在有或无LIF的培养条件下，观察了细胞生长的特性，表型变化等，并通过检测其中外胚间充质干细胞（ectomesenchymal stem cells，EMSCs）的水平，探讨UF与EMSCs的关系。结果表明：（1）LIF的受体gp130表达于未分化与早期分化的EMSCs表面，提示LIF对于EMSCs具有潜在的调节作用；（2）LIF促进小鼠EMSCs的自我维持，bFGF则对此具有协同作用。因此，UF与bFGF联合对体外培养和扩增小鼠外胚间充质干细胞，以及对其深入研究和应用具有重要意义。     

Increased Basic Fibroblast Growth Factor (bFGF) Accumulation and Distinct Patterns of Localization in Isoproterenol-Induced Cardiomyocyte Injury

Basic FGF is a multifunctional protein which promotes regeneration in several tissues. To investigate involvement in cardiac injury-repair, bFGF accumulation and localization was examined in hearts of rats injected with a single high dose of isoproterenol. The bFGF content of cardiac extracts was analyzed at 6 and 24 hours as well as 1, 4, and 6 weeks by western blotting of heparin-sepharose-bound fractions. The 18 kilodalton bFGF species showed an approximately 2-fold increase in extracts from treated animals compared to non-treated controls. A transient rise in a 21–23 kilodalton bFGF species was seen at 24 hours after treatment. An induction of bFGF mRNA was also observed in treated animals. To localize bFGF in vivo, immunofluorescent labelling with specific antibodies was used at 4–24 hours and 1–4 weeks after treatment. Simultaneous labelling for the cytoskeletal proteins vinculin or vimentin was employed to identify viable myocytes or non-muscle interstitial cells, respectively. Necrotic myocytes, identified by loss of vinculin, displayed a pronounced increase in cytoplasmic anti-bFGF staining compared to adjacent normal myocytes. This increase occurred prior to and may play a role in promoting mobile cell migration and proliferation in areas of necrosis. Viable cardiomyocytes adjacent to fibrotic regions displayed strong pericellular anti-bFGF staining and, occasionally, were also stained by anti-vimentin antibodies, suggesting reexpression of an embryonic phenotype and thus an attempt for regeneration. These data showing increased accumulation and distinct patterns of localization of bFGF in the hearts of isoproterenol-treated animals suggest that this growth factor plays a role in short-term as well as long term response of the myocardium to injury.     

Spontaneous Formation of a Hydrogel Composed of Water-Soluble Phospholipid Polymers Grafted with Enantiomeric Oligo(lactic acid) Chains

We designed and synthesized water-soluble biocompatible and biodegradable polymers composed of 2-methacryroyloxyethyl phosphorylcholine and oligo(L- or D-lactic acid) macromonomers to develop an injectable hydrogel matrix. Aqueous solutions containing the polymers with oligo(L-lactic acid) (OLLA) and oligo(D-lactic acid) (ODLA) chains underwent spontaneous gelation when mixed together. This was due to the formation of a stereocomplex between the OLLA and ODLA side-chains, which act as cross-linking components in the hydrogel. Therefore, the hydrogel could be re-dissolved in a buffer solution by hydrolysis of the oligo(lactic acid) chains. We obtained an injectable, biocompatible and degradable hydrogel, and we anticipate that it will be used in applications involving the controlled release of bioactive molecules and cell-based tissue engineering.     

A Possible Role of Multidrug Resistance-Associated Protein (MRP) in Basic Fibroblast Growth Factor Secretion by AIDS-Associated Kaposi's Sarcoma Cells: A Survival Molecule?

Kaposi's sarcoma (KS) is considered a disorder of cytokines. Basic fibroblast growth factor (bFGF) is produced by AIDS-associated KS (AIDS-KS) cells and supports their growth in an autocrine and paracrine manner. bFGF lacks a signal sequence; therefore, its mechanism of secretion is unclear. In this study, we investigate the role of two important members of ATP-binding cassette transport proteins, the P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP), in the secretion of bFGF from AIDS-KS cells. Expression of P-gp and MRP was examined at both the protein and the mRNA levels by flow cytometry and RT-PCR respectively. Intracellular and secreted bFGF was measured by ELISA. AIDS-KS cells expressed MRP at both the mRNA and the protein levels; however, no P-gp expression was detected at either the mRNA or the protein level, Probenecid, a putative inhibitor of MRP efflux function, in a concentration-dependent manner, inhibited bFGF secretion, with a concomitant increase in intracellular bFGF, demonstrating that probenecid blocks bFGF secretion without inhibiting its synthesis. In addition, probenecid induced apoptosis in AIDS-KS cells. AIDS-KS cells expressed fas, bcl-2, and bcl-xL genes but lacked fasL and bax gene expression. These data suggest that bFGF is secreted from AIDS-KS cells via a probenecid-sensitive transporter, most likely by MRP. Furthermore, probenecid appears to induce apoptosis in AIDS-KS cells by depriving them of the growth promoting activity of bFGF. These data suggest that MRP may play a role as a survival molecule in AIDS-KS cells.