FGF-2 Gene Transfer Research Articles

Dual FGF-2 and SOX9 gene transfer via RAAV Vectors: effects on the chondrogenic and metabolic processes in human bone marrow aspirates

Dual FGF-2 and SOX9 gene transfer via RAAV Vectors: effects on the chondrogenic and metabolic processes in human bone marrow aspirates

Effects of rAAV-mediated FGF-2 gene transfer and overexpression upon the chondrogenic differentiation processes in human bone marrow aspirates.

BackgroundApplication of genetically modified bone marrow concentrates in articular cartilage lesions is a promising approach to enhance cartilage repair by stimulating the chondrogenic differentiation processes in sites of injury.MethodIn the present study, we examined the potential benefits of transferring the proliferative and pro-chondrogenic basic fibroblast growth factor (FGF-2) to human bone marrow aspirates in vitro using the clinically adapted recombinant adeno-associated virus (rAAV) vectors to monitor the biological and chondrogenic responses over time to the treatment compared with control (lacZ) gene application.ResultsEffective, significant FGF-2 gene transfer and expression via rAAV was established in the aspirates relative to the lacZ condition (from ~ 97 to 36 pg rhFGF-2/mg total proteins over an extended period of 21 days). Administration of the candidate FGF-2 vector led to prolonged increases in cell proliferation, matrix synthesis, and chondrogenesis but also to hypertrophic and terminal differentiation in the aspirates.ConclusionThe present evaluation shows the advantages of rAAV-mediated FGF-2 gene transfer to conveniently modify bone marrow concentrates as a future approach to directly treat articular cartilage lesions, provided that expression of the growth factor is tightly regulated to prevent premature hypertrophy in vivo.

Opposing effects of Sca-1(+) cell-based systemic FGF2 gene transfer strategy on lumbar versus caudal vertebrae in the mouse.

Our previous work showed that a Sca-1+ cell-based FGF2 therapy was capable of promoting robust increases in trabecular bone formation and connectivity on the endosteum of long bones. Past work reported that administration of FGF2 protein promoted bone formation in red marrow but not in yellow marrow. The issue as to whether the Sca-1+ cell-based FGF2 therapy is effective in yellow marrow is highly relevant to its clinical potential for osteoporosis, as most red marrows in a person of an advanced age, are converted to yellow marrows. Accordingly, this study sought to compare the osteogenic effects of this stem cell-based FGF2 therapy on red marrow-filled lumbar vertebrae with those on yellow marrow-filled caudal vertebrae of young adult W41/W41 mice. The Sca-1+ cell-based FGF2 therapy drastically increased trabecular bone formation in lumbar vertebrae, but the therapy not only did not promote bone formation but instead caused substantial loss of trabecular bone in caudal vertebrae. The lack of an osteogenic response was not due to insufficient engraftment of FGF2-expressing Sca-1+ cells or inadequate FGF2 expression in caudal vertebrae. Previous studies have demonstrated that recipient mice of this stem cell-based FGF2 therapy developed secondary hyperparathyroidism and increased bone resorption. Thus, the loss of bone mass in caudal vertebrae might in part be due to an increase in resorption without a corresponding increase in bone formation. In conclusion, the Sca-1+ cell-based FGF2 therapy is osteogenic in red marrow but not in yellow marrow.

Direct FGF-2 Gene Transfer via Recombinant Adeno-Associated Virus Vectors Stimulates Cell Proliferation, Collagen Production, and the Repair of Experimental Lesions in the Human ACL

Background: Basic fibroblast growth factor (FGF-2) is a powerful stimulator of fibroblast proliferation and type I/III collagen production. Hypothesis: Overexpression of FGF-2 via direct recombinant adeno-associated virus (rAAV) vector–mediated gene transfer enhances the healing of experimental lesions to the human anterior cruciate ligament (ACL). Study Design: Controlled laboratory study. Methods: rAAV vectors carrying a human FGF-2 sequence or the lacZ marker gene were applied to primary human ACL fibroblasts in vitro and to intact or experimentally injured human ACL explants in situ to evaluate the efficacy and duration of transgene expression and the potential effects of FGF-2 treatment upon the proliferative, metabolic, and regenerative activities in these systems. Results: Sustained, effective dose-dependent lacZ expression was achieved in all systems tested (up to 96% ± 2% in vitro and 80%-85% in situ for at least 30 days). rAAV allowed for continuous FGF-2 production both in vitro and in the intact ACL in situ (32.7 ± 1.4 and 33.1 ± 0.8 pg/mL/24 h, respectively, ie, up to 41-fold more than in the controls at day 30; always P ≤ .001), leading to significantly and durably enhanced levels of proliferation and type I/III collagen production vis-à-vis lacZ (at least 3- and 4-fold increases at day 30, respectively; always P ≤ .001). Most notably, rAAV FGF-2 promoted a significant, long-term production of the factor in experimental ACL lesions (92.7 ± 3.9 pg/mL/24 h, ie, about 5-fold more than in the controls; P ≤ .001) associated with enhanced levels of proliferation and type I/III collagen synthesis (at least 2- and 4-fold increases at day 30, respectively; always P ≤ .001). Remarkably, the FGF-2 treatment allowed for a decrease in the amplitude of such lesions possibly because of the increased expression in contractile α–smooth muscle actin, ligament-specific transcription factor scleraxis, and nuclear factor–κB for proliferation and collagen deposition, which are all markers commonly induced in response to injury. Conclusion: Efficient, stable FGF-2 expression via rAAV enhances the healing of experimental human ACL lesions by activating key cellular and metabolic processes. Clinical Relevance: This approach has potential value for the development of novel, effective treatments for ligament reconstruction.

Effect of adenovirus-mediated basic fibroblast growth factor gene transfer in vivo on oligodendrocyte cell numbers throughout ventrolateral white matter following spinal cord injury in rats.

To study the effect of adenovirus-mediated basic fibroblast growth factor(FGF-2) gene transfer in vivo on oligodendrocyte cell numbers throughout ventrolateral white matter following spinal cord injury in rats. Thirty-two adult female Sprague Dawley rats were injured with the Infinite Horizon Impactor, and then were randomly assigned to four groups: FGF-2-Adts high-titer group (1.27x10(7) pfu/rat), FGF-2-Adts intermediate-titre group (6.37x10(6) pfu/rat), FGF-2-Adts low-titer group (3.18 x 10(6) pfu/rat), and green fluorescent protein (GFP)-Adts group (5.9x10(7) pfu/rat). The transgenic expression in vivo was detected with fluorescence microscopy. The locomotor function of the hindlimbs of rats was evaluated using Rivlin plate. Slides mounted with tissue sections were processed for immunohistochemical detection and quantification of oligodendrocytes (CC1(+)) in the ventral lateral funiculi (VLF) of injured spinal cords. One week after spinal cord injury, GFP showed that many cells had expressed objective gene in vivo and the angles of the occlusal plane of rats in FGF-2 groups were significantly higher than in GFP-Adts group. Also, there was a significant difference among the FGF-2-Adts treatment groups for the volume of gray matter sparing. However, there were no significant differences for total white matter sparing. Stereological quantification of total CC1(+) cell numbers in the spared VLF showed a significant reduction in numbers with GFP controls compared to all other groups 4 weeks after injury. In contrast, the FGF-2 Adts intermediate-titer group had significantly more CC1(+) cells when compared to both the FGF-2-Adts high- and low-titer groups. Adenovirus-mediated FGF-2 gene transfer can promote the functional recovery of the injured spinal cord by enhancing the proliferation and/or differentiation of oligodendrocytes.

Correction for Kiyota et al., FGF2 gene transfer restores hippocampal functions in mouse models of Alzheimer's disease and has therapeutic implications for neurocognitive disorders

Correction for Kiyota et al., FGF2 gene transfer restores hippocampal functions in mouse models of Alzheimer's disease and has therapeutic implications for neurocognitive disorders

FGF2 gene transfer restores hippocampal functions in mouse models of Alzheimer's disease and has therapeutic implications for neurocognitive disorders

The adult hippocampus plays a central role in memory formation, synaptic plasticity, and neurogenesis. The subgranular zone of the dentate gyrus contains neural progenitor cells with self-renewal and multilineage potency. Transgene expression of familial Alzheimer's disease-linked mutants of β-amyloid precursor protein (APP) and presenilin-1 leads to a significant inhibition of neurogenesis, which is potentially linked to age-dependent memory loss. To investigate the effect of neurogenesis on cognitive function in a relevant disease model, FGF2 gene is delivered bilaterally to the hippocampi of APP+presenilin-1 bigenic mice via an adenoassociated virus serotype 2/1 hybrid (AAV2/1-FGF2). Animals injected with AAV2/1-FGF2 at a pre- or postsymptomatic stage show significantly improved spatial learning in the radial arm water maze test. A neuropathological investigation demonstrates that AAV2/1-FGF2 injection enhances the number of doublecortin, BrdU/NeuN, and c-fos-positive cells in the dentate gyrus, and the clearance of fibrillar amyloid-β peptide (Aβ) in the hippocampus. AAV2/1-FGF2 injection also enhances long-term potentiation in another APP mouse model (J20) compared with control AAV2/1-GFP-injected littermates. An in vitro study confirmed the enhanced neurogenesis of mouse neural stem cells by direct AAV2/1-FGF2 infection in an Aβ oligomer-sensitive manner. Further, FGF2 enhances Aβ phagocytosis in primary cultured microglia, and reduces Aβ production from primary cultured neurons after AAV2/1-FGF2 infection. Thus, our data indicate that virus-mediated FGF2 gene delivery has potential as an alternative therapy of Alzheimer's disease and possibly other neurocognitive disorders.

Metabolic Activities and Chondrogenic Differentiation of Human Mesenchymal Stem Cells Following Recombinant Adeno-Associated Virus–Mediated Gene Transfer and Overexpression of Fibroblast Growth Factor 2

The genetic manipulation of bone marrow-derived mesenchymal stem cells (MSCs) is an attractive approach to produce therapeutic platforms for settings that aim at restoring articular cartilage defects. Here, we examined the effects of recombinant adeno-associated virus (rAAV)-mediated overexpression of human fibroblast growth factor 2 (hFGF-2), a mitogenic factor also known to influence MSC differentiation, upon the proliferative and chondrogenic activities of human MSCs (hMSCs) in a three-dimensional environment that supports chondrogenesis in vitro. Prolonged, significant FGF-2 synthesis was noted in rAAV-hFGF-2-transduced monolayer and aggregate cultures of hMSCs, leading to enhanced, dose-dependent cell proliferation compared with control treatments (rAAV-lacZ transduction and absence of vector administration). Chondrogenic differentiation (proteoglycans, type-II collagen, and SOX9 expression) was successfully achieved in all types of aggregates, without significant difference between conditions. Most remarkably, application of rAAV-hFGF-2 reduced the expression of type-I and type-X collagen, possibly due to increased levels of matrix metalloproteinase-13, a key matrix-degrading enzyme. FGF-2 overexpression also decreased mineralization and the expression of osteogenic markers such as alkaline phosphatase, with diminished levels of RUNX-2, a transcription factor for osteoblast-related genes. Altogether, the present findings show the ability of rAAV-mediated FGF-2 gene transfer to expand hMSCs with an advantageous differentiation potential for future, indirect therapeutic approaches that aim at treating articular cartilage defects in vivo.

522. Enhanced Repair of Articular Cartilage Defects in Rabbits by Overexpression of Human FGF-2 Via rAAV-Mediated Gene Transfer

Therapeutic gene transfer may be a means to enhance the healing of articular cartilage damage. We tested the hypotheses that FGF-2 gene transfer via rAAV modulates the chondrocytic phenotype and stimulates the healing of osteochondral defects in rabbit knee joints. A human basic fibroblast growth factor cDNA was cloned in rAAV. Rabbit chondrocytes were transduced by rAAV and encapsulated in alginate (constructs) to evaluate the synthesis of FGF-2, viable cell numbers, proteoglycans, and DNA contents. In vivo, osteochondral defects (3.2 mm, femoro-patellar groove) received 10 |[igrave]|l rAAV-hFGF-2 or rAAV-lacZ. After 4 months, the distal femora were removed, fixed, and decalcified for histological analyses (safranin O, hematoxylin eosin, type-II collagen) and to monitor transgene expression. The sections were graded using a repair scoring system. In the FGF-2 constructs, FGF-2 synthesis was elevated for 14 days and detected until day 26, leading to a significant increase in cell densities (P < 0.001). The proteoglycan content was not significantly different between the FGF-2 and lacZ constructs (P = 0.233), while the DNA content in the FGF-2 constructs was significantly higher (P < 0.001). In vivo, there were no adverse reactions in the knees and transgene expression was seen in all defects. At 4 months, tissue healing was enhanced in the FGF-2-treated defects, with an improved total score (P < 0.01), filling (P < 0.05), architecture (P < 0.01), cell morphology (P < 0.001), and a more intense type-II collagen staining. The data show that rAAV-mediated FGF-2 production improves the healing of osteochondral defects, suggesting that rAAV have the potential to enhance cartilage repair by direct application to sites of damage. (See Table 1)

Postischemic Intraventricular Administration of FGF-2 Expressing Adenoviral Vectors Improves Neurologic Outcome and Reduces Infarct Volume after Transient Focal Cerebral Ischemia in Rats

Fibroblast growth factor (FGF)-2 is a potent neurotrophic and angiogenic peptide. To examine possible protective effects of FGF-2 gene expression against transient focal cerebral ischemia in rats, a replication defective, recombinant adenovirus vector expressing FGF-2, was injected intraventricularly 2 hours after middle cerebral artery occlusion (MCAO). The treatment group showed significant recovery compared with the vehicle-treated groups in terms of serial neurologic severity scores over the 35 days after MCAO. Further, 2,3,5-triphenyltetrazolium chloride staining showed that FGF-2 gene transfer decreased infarct volume by 44% as compared with that in the vehicle-treated groups at 2 days after MCAO. The same tendency of gene transfer effects on infarct volume was confirmed at 35 days after MCAO with hematoxylin/eosin staining. Enzyme-linked immunosorbent assay revealed that FGF-2 concentration was increased significantly at 2 days after MCAO, not only in cerebrospinal fluid but also in cerebral substance in the lesioned and treated animals. These results suggested that FGF-2 gene transfer using these adenoviral vectors might be a useful modality for the treatment of occlusive cerebrovascular disease even after the onset of stroke.

Fibroblast growth factor-2 gene delivery stimulates axon growth by adult retinal ganglion cells after acute optic nerve injury

Basic fibroblast growth factor (or FGF-2) has been shown to be a potent stimulator of retinal ganglion cell (RGC) axonal growth during development. Here we investigated if FGF-2 upregulation in adult RGCs promoted axon regrowth in vivo after acute optic nerve injury. Recombinant adeno-associated virus (AAV) was used to deliver the FGF-2 gene to adult RGCs providing a sustained source of this neurotrophic factor. FGF-2 gene transfer led to a 10-fold increase in the number of axons that extended past 0.5 mm from the lesion site compared to control nerves. Detection of AAV-mediated FGF-2 protein in injured RGC axons correlated with growth into the distal optic nerve. The response to FGF-2 upregulation was supported by our finding that FGF receptor-1 (FGFR-1) and heparan sulfate (HS), known to be essential for FGF-2 signaling, were expressed by adult rat RGCs. FGF-2 transgene expression led to only transient protection of injured RGCs. Thus the effect of this neurotrophic factor on axon extension could not be solely attributed to an increase in neuronal survival. Our data indicate that selective upregulation of FGF-2 in adult RGCs stimulates axon regrowth within the optic nerve, an environment that is highly inhibitory for regeneration. These results support the hypothesis that key factors involved in axon outgrowth during neural development may promote regeneration of adult injured neurons.

Involvement of VEGF-C/flt-4 system in therapeutic angiogenesis of FGF-2 gene transfer in murine ischemic hind limb

Involvement of VEGF-C/flt-4 system in therapeutic angiogenesis of FGF-2 gene transfer in murine ischemic hind limb

1082. Recombinant Sendai Virus-Mediated Gene Therapy for Experimental Critical Limb Ischemia: FGF-2 Gene Transfer Can Stimulate Endogenous HGF Expression, Regardless to Hypoxia-Mediated down Regulation in Ischemic Limbs

1082. Recombinant Sendai Virus-Mediated Gene Therapy for Experimental Critical Limb Ischemia: FGF-2 Gene Transfer Can Stimulate Endogenous HGF Expression, Regardless to Hypoxia-Mediated down Regulation in Ischemic Limbs

A Novel Recombinant Basic Fibroblast Growth Factor and Its Secretion

Basic fibroblast growth factor (FGF-2) is a pleiotropic mitogen which plays an important role in cell growth, differentiation, migration, and survival in different cells and organ systems. Recently, several clinical applications for FGF-2 gene transfer are being evaluated in wound healing and collateral artery development to relieve myocardial and peripheral ischemia due to the ability of FGF-2 to regulate the growth and function of vascular cells. However, FGF-2 lacks a classical hydrophobic secretion signal peptide, the FGF-2 chimeras containing various signal sequences have been explored. In this study, a novel recombinant 4sFGF-2 was constructed by replacing nine residues from the amino-terminus of native FGF-2 (Met1 to Leu9) with eight amino acid residues of signal peptide of FGF-4 (Met1 to Ala8) to better increase the secretion level of FGF-2. When the recombinant FGF-2 gene, cloned into the expression vector with CMV promoter, was expressed in COS-7 cells, the recombinant 4sFGF-2 was highly secreted into the media. The secreted 4sFGF-2 showed the same biological activity as the native FGF-2 in the dose-response effects on DNA synthesis and cell growth of rat aortic smooth muscle cells (RASMCs) and NIH3T3 cells. The 4sFGF-2 also was able to activate MAPK as wild FGF-2 in RASMCs. These results indicate that a novel recombinant 4sFGF-2 may be useful as clinical applicability of angiogenic growth factor gene transfer.