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772. Novel Functional Optical Imaging of Bone Repair in a Calvaria Defect Model
MUSCULO-SKELETAL GENE & CELL THERAPY II outcomes. Bohannon RW. Reference values for the Timed Up and Go Test: A Descriptive Meta-Analysis. Journal of Geriatric Physical Therapy, 2006;29(2):64-8.
769. AAV-Based shRNA Silencing of NF-κB Ameliorates Muscle Pathologies in mdx Mice
Qing Yang,1,2 Ying Tang,1 Kara Imbrogno,1 Aiping Lu,1 Anmin Chen,2 Fengjin Guo,2 Freddie H. Fu,1 Johnny Huard,1 Bing Wang.1 1 Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA; 2 Orthopaedic Surgery, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, Hubei, China. Duchenne muscular dystrophy (DMD) is an X-linked genetic muscle disease affecting 1 of every 3500 male births. Chronic inflammation, promoted by an up-regulated NF-kappaB (NF-κB) pathway, plays a key role in Duchenne muscular dystrophy (DMD) patients’ pathogenesis. Higher immune alertness, due to chronic inflammation and immune effector cell infiltration, challenges recombination adeno-associated viral (AAV) vector-mediated gene replacement treatment for DMD, especially in large animal models and clinical trials. We and others found that blocking the NF-κB pathway could be a viable solution to promote muscle regeneration and to decrease necrosis in mdx mice (a murine DMD model), by using transgenic and AAV-mediated transfer (dominant-negative forms of IKKα & β) approaches, without any side effect. Based on the small interference RNA (siRNA) technology used in this study, we examined whether an AAV vector carrying small hairpin RNA (shRNA) targeting the NF-κB/p65 subunit, a major subunit of NF-κB associated with inflammation in mdx mice, inhibits NF-κB activation as well as ameliorates pathological necrosis and regeneration in the locally-treated gastrocnemius (GAS) muscle of 1 and 4 month old mdx mice. At 1 month post-treatment, NF-κB/p65 levels in locally-treated muscles were significantly decreased by the efficient transfer of specific shRNA in both of the different treated age groups. This led to remarkable decreases in necrosis and myofiber damage compared to mdx mice treated by a phosphate-buffered saline (PBS) or a control AAV vector containing a scrambled shRNA. The shRNA silencing of the NF-κB pathway diminished the pathological myofiber regeneration in young mdx mice treated at 1 month of age, but it enhanced the capacity of myofiber regeneration in old mdx mice treated at 4 months of age. Moreover, quantitative analysis revealed that central nucleation of the myofibers of treated young mdx muscles was reduced from 67% to 34%, but centrally-located nuclei were not significantly reversed to the peripheral position in treated old mdx mice. Our findings indicate that AAV-mediated NF-κB/p65-shRNA has the capacity to ameliorate muscle pathologies in mdx mice by degrading mRNA of the NF-κB/p65 subunit.
770. Muscle Fiber Type-Predominant Promoter Activity in Lentiviral-Mediated Transgenic Mouse
Tomohiro Suga,1 En Kimura,1 Yuka Morioka,2 Masahito Ikawa,2 Sheng Li,3 Katsuhisa Uchino,1 Yuji Uchida,4 Satoshi Yamashita,1 Yasushi Maeda,1 Jeffrey S. Chamberlain,3 Makoto Uchino.1 1 Neurology, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan; 2Research Institute for Microbial Diseases, Osaka University, Suita, Japan; 3Department of Neurology, University of Washington School of Medicine, Seattle; 4Pharmacology, Division of Life Science, Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto, Japan.
Variations in gene promoter/enhancer activity in different muscle fiber types after gene transduction was noticed, but poorly analyzed. The murine stem cell virus (MSCV) promoter drives strong, stable gene expression in hematopoietic stem cells and several other cells, including cerebellar Purkinje cells, however it has not been studied in striate muscle. We injected a lentiviral vector carrying an MSCVS294
EGFP cassette (LvMSCV-EGFP) into tibialis anterior muscles and observed strong EGFP expression in muscle fibers, primary cultured myoblasts, and myotubes isolated from injected muscles. We also generated lentiviral-mediated transgenic mice carrying the MSCVEGFP cassette and detected transgene expression in striated muscles. LvMSCV-EGFP transgenic mice showed fiber type-dependent variations in expression: highest in type I and IIA, intermediate in type IID/X, and lowest in type IIB fibers. The soleus and diaphragm muscles, consisting mainly of type I and IIA, are most severely affected in the mdx mouse model of muscular dystrophy. Further analysis of this promoter may have the potential to achieve certain gene expression in severely affected muscles of mdx mice. The Lvmediated transgenic mouse may prove a useful tool for assessing the enhancer/promoter activities of a variety of different regulatory cassettes.
771. Noninvasive Monitoring of Whole Body Muscle Function in Dystrophin-Deficient Dogs
Jin-Hong Shin,1 Brian B. Greer,1 Chady H. Hakim,1 Zhihai He,2 Dongsheng Duan.1 1 Molecular Microbiology & Immunology, University of Missouri, Columbia, MO; 2Electrical and Computer Engineering, University of Missouri, Columbia, MO. Dystrophin-deficient dogs are highly valuable tools for evaluating gene/cell therapies to treat Duchenne muscular dystrophy (DMD). Unfortunately, robust muscle functional end points are poorly defined in the canine DMD models. Here, we described a set of non-invasive methods for whole body muscle function analysis in normal and affected dogs. Briefly, the gait and voluntary activity were recorded using high-resolution digital video recorders. Gait recoding was repeated three times on three different days for each dog. Voluntary activity monitoring was performed under low-lux red lighting between 7pm and 5am for each dog. The stride length, stride speed, range of the motion and overall activity were analyzed using a customer-developed program. Three normal and four affected dogs of a litter were examined. Affected dogs showed significantly reduced stride length (affected, 35.7 ± 8.5 cm; normal, 80.2 ± 6.1 cm; p <0.02) and stride speed (affected, 61.7 ± 16.4 cm/sec; normal, 156.5 ± 23.8 cm/sec; p <0.03). The range of the motion in forelimb (palm, forearm and wrist) and hind limb (sole, low leg and ankle) was also significantly decreased in affected dogs. The affected dogs also showed significantly less movement during overnight monitoring. In summary, our results suggest that whole body muscle function was significantly compromised in affected dogs. Non-invasive video recording may represent a convenient and reliable method to monitor disease progression and gene/cell therapy effect in canine models of DMD.
772. Novel Functional Optical Imaging of Bone Repair in a Calvaria Defect Model
Ilan Kallai,1 Nadav Kimelman-Bleich,1 Wafa Tawackoli,2 Xiaoyu Da,2 Anthony Oh,2 Gadi Pelled,1,2 Zulma Gazit,1,2 Dan Gazit.1,2 1 Hebrew University of Jerusalem, Jerusalem, Israel; 2SurgeryRegenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles. The establishment of new therapeutic approaches for tissue repair and regeneration could be accelerated if adequate technologies to monitor the repair process are developed. In cranio-maxillofacial surgery, 96,000 bone grafts are performed each year to regenerate bone loss due to trauma or disease. Autologous bone, usually used to treat these lesions, is not always available and necessitates additional surgery for its harvest. Alternatively, there is a large potential supply of craniofacial allografts, which are composed of non-vital bone. Yet, these grafts often fail to integrate due to the formation of scar tissue Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
MUSCULO-SKELETAL GENE & CELL THERAPY II around them. Little is known of the mechanism behind the induction of scar tissue by allografts. We hypothesized that the establishment of functional fluorescence imaging (FLI) of osteogenesis, angiogenesis and osteoclastogenesis in calvarial defects treated with allografts would provide new insights that would enable the development of novel therapies for allograft bio-integration. Methods: A criticalsize calvarial defect was created in FVB/n mice. Groups included transplantation of: allografts, autografts, and untreated defects (used as control). For blood flow and angiogenesis imaging the mice were injected with AngioSense 750 or Integrisense 750 on Days 5 and 12-post transplantation. FLI of angiogenesis was done using epiillumination 24 hours after probe injection. Signal quantification eliminated autofluorescence. For osteogenesis imaging the mice were injected with OsteoSense 680 or 800. Imaging with Osteosense was performed on Day 20, in vivo and then ex vivo following mice sacrifice. Finally, bone resorption activity was imaged using Cathepsin-K 680 probe. Bone formation and vasculogenesis were analyzed as well using Micro CT imaging and histological analysis. Results: Functional FLI of blood flow showed significantly higher signal on Day 6 than on Day 13 in both treated groups and was greater in both treated groups than in the control group.
Imaging of OsteoSense 680 on Day 20 in vivo and ex vivo showed no difference.[figure 2]Thus, although epi-illumination FLI is known for high tissue attenuation, in this model the signal is preserved. MicroCT and histological analysis performed on the calvaria samples corroborated the results of osteogenesis and angiogenesis detected by functional FLI. Discussion: Our findings indicate that functional FLI is a reliable tool for monitoring the repair of a calvarial bone defects. An evaluation and validation of new therapies involving allografts for the repair of calvarial injuries may be obtained using this system. Furthermore, a better understanding of the healing process in craniofacial defects treated with allografts may be reached.
Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
773. Comparison of Efficacy of a Novel DoubleStrand AAV2 Vector Harboring VEGF cDNA to AAVSS-VEGF: In Vitro Transgene Expression and In Vivo Application To Enhance Survival of Ischemic Flaps Xiao Tian Wang,1 Bella Avanessian,1 Qiangzhong Ma,1 Yuqing Tang,1 Heather Durfee,1 Paul Y. Liu.1 1 Surgical Research and Gene Therapy, Roger Williams Medical Center, Providence, RI.
Purpose: Conventional recombinant adeno-associated virus (rAAV) vectors are a single-stranded DNA vector, which needs to be converted to double-stranded DNA prior to transcription, thus delaying expression of transgene. Previously, we reported that rAAVVEGF gene transfer to rat ischemic flaps improved flap survival in PSRC meetings. Here we compare the transgene expression efficacy of single-stranded (SS) and double-stranded AAV (DS) mediated human VEGF gene transfer and the efficiency in enhancing ischemic flap survival. Materials and Methods: 1. 293 cells were transduced with AAV-DS-VEGF or AAV-SS-VEGF (MOI=1, 10, 50, 100, n=3). Non-treatment was as control. The supernatant was harvested at day 3 and 7 for human VEGF ELISA. 2. Primary DB/DB and DBm fibroblasts were transduced with AAV-DS-VEGF and AAV-SS-VEGF (MOI=100, n=4). Non-treatment was as control. Three days later, the supernatant was harvested for ELISA analysis. We performed in vitro wound healing assay by scratching transduced cells and recording the healing for 48 hours using an IncuCyte system. 3. AAV-SS-VEGF and AAV-DS-VEGF (n=10) were injected into a 7 cm x 3 cm area on the dorsum of the rat. The control rats received saline or AAV-SS-GFP injection. One week later, a flap of 10 cm x 3 cm was raised. One week post-surgery, we evaluated the flap viability and harvested the injected and surviving flap tissues for histology. Results: 1. At day 3 and 7, both AAV-SS-VEGF and AAV-DS-VEGF treated 293 cell supernatant contained a greater amount of VEGF compared to control at all MOIs. AAV-DS-VEGF treated cell supernatant had higher expression than AAV-SS-VEGF supernatant did at all MOIs tested. 2. AAV-SS-VEGF and AAV-DS-VEGF treated mouse fibroblasts (DB/DB and DBm) presented enhanced human VEGF expression compared to control. AAV-DS-VEGF treated cell supernatant showed higher VEGF expression than AAV-SS-VEGF group. Supernatant of DBm fibroblasts contained more VEGF than that of DB/DB cells. 3. In vitro wound healing was improved by use of either AAV-SSVEGF or AAV-DS-VEGF; no significant difference was found between two treatments. 4. AAV-DS-VEGF improved flap survival (57.2%±5.5%) compared to saline group (48%±5.1%) and AAV-SSGFP group (45.9±6.3%) significantly (p < 0.05, both comparisons). AAV-SS-VEGF treated flaps showed 50.7% ± 7.2% survival, which did not differ significantly compared to saline and AAV-SS-GFP groups. Discussion/Conclusions: AAV-DS-VEGF and AAV-GFPVEGF can successfully transduce primary diabetic and non-diabetic fibroblasts. AAV-DS-VEGF produces higher and earlier transgene expression. AAV-SS-VEGF and AAV-DS-VEGF enhanced in vitro wound healing. AAV-DS-VEGF improves flap survival compared to controls, but AAV-SS-VEGF did not significantly improve flap survival in the present study, which contrasts to our previous results of significant improvement in flap survival when it was injected 2 weeks prior to flap elevation.
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769. AAV-Based shRNA Silencing of NF-κB Ameliorates Muscle Pathologies in mdx Mice
Qing Yang,1,2 Ying Tang,1 Kara Imbrogno,1 Aiping Lu,1 Anmin Chen,2 Fengjin Guo,2 Freddie H. Fu,1 Johnny Huard,1 Bing Wang.1 1 Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA; 2 Orthopaedic Surgery, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, Hubei, China. Duchenne muscular dystrophy (DMD) is an X-linked genetic muscle disease affecting 1 of every 3500 male births. Chronic inflammation, promoted by an up-regulated NF-kappaB (NF-κB) pathway, plays a key role in Duchenne muscular dystrophy (DMD) patients’ pathogenesis. Higher immune alertness, due to chronic inflammation and immune effector cell infiltration, challenges recombination adeno-associated viral (AAV) vector-mediated gene replacement treatment for DMD, especially in large animal models and clinical trials. We and others found that blocking the NF-κB pathway could be a viable solution to promote muscle regeneration and to decrease necrosis in mdx mice (a murine DMD model), by using transgenic and AAV-mediated transfer (dominant-negative forms of IKKα & β) approaches, without any side effect. Based on the small interference RNA (siRNA) technology used in this study, we examined whether an AAV vector carrying small hairpin RNA (shRNA) targeting the NF-κB/p65 subunit, a major subunit of NF-κB associated with inflammation in mdx mice, inhibits NF-κB activation as well as ameliorates pathological necrosis and regeneration in the locally-treated gastrocnemius (GAS) muscle of 1 and 4 month old mdx mice. At 1 month post-treatment, NF-κB/p65 levels in locally-treated muscles were significantly decreased by the efficient transfer of specific shRNA in both of the different treated age groups. This led to remarkable decreases in necrosis and myofiber damage compared to mdx mice treated by a phosphate-buffered saline (PBS) or a control AAV vector containing a scrambled shRNA. The shRNA silencing of the NF-κB pathway diminished the pathological myofiber regeneration in young mdx mice treated at 1 month of age, but it enhanced the capacity of myofiber regeneration in old mdx mice treated at 4 months of age. Moreover, quantitative analysis revealed that central nucleation of the myofibers of treated young mdx muscles was reduced from 67% to 34%, but centrally-located nuclei were not significantly reversed to the peripheral position in treated old mdx mice. Our findings indicate that AAV-mediated NF-κB/p65-shRNA has the capacity to ameliorate muscle pathologies in mdx mice by degrading mRNA of the NF-κB/p65 subunit.
770. Muscle Fiber Type-Predominant Promoter Activity in Lentiviral-Mediated Transgenic Mouse
Tomohiro Suga,1 En Kimura,1 Yuka Morioka,2 Masahito Ikawa,2 Sheng Li,3 Katsuhisa Uchino,1 Yuji Uchida,4 Satoshi Yamashita,1 Yasushi Maeda,1 Jeffrey S. Chamberlain,3 Makoto Uchino.1 1 Neurology, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan; 2Research Institute for Microbial Diseases, Osaka University, Suita, Japan; 3Department of Neurology, University of Washington School of Medicine, Seattle; 4Pharmacology, Division of Life Science, Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto, Japan.
Variations in gene promoter/enhancer activity in different muscle fiber types after gene transduction was noticed, but poorly analyzed. The murine stem cell virus (MSCV) promoter drives strong, stable gene expression in hematopoietic stem cells and several other cells, including cerebellar Purkinje cells, however it has not been studied in striate muscle. We injected a lentiviral vector carrying an MSCVS294
EGFP cassette (LvMSCV-EGFP) into tibialis anterior muscles and observed strong EGFP expression in muscle fibers, primary cultured myoblasts, and myotubes isolated from injected muscles. We also generated lentiviral-mediated transgenic mice carrying the MSCVEGFP cassette and detected transgene expression in striated muscles. LvMSCV-EGFP transgenic mice showed fiber type-dependent variations in expression: highest in type I and IIA, intermediate in type IID/X, and lowest in type IIB fibers. The soleus and diaphragm muscles, consisting mainly of type I and IIA, are most severely affected in the mdx mouse model of muscular dystrophy. Further analysis of this promoter may have the potential to achieve certain gene expression in severely affected muscles of mdx mice. The Lvmediated transgenic mouse may prove a useful tool for assessing the enhancer/promoter activities of a variety of different regulatory cassettes.
771. Noninvasive Monitoring of Whole Body Muscle Function in Dystrophin-Deficient Dogs
Jin-Hong Shin,1 Brian B. Greer,1 Chady H. Hakim,1 Zhihai He,2 Dongsheng Duan.1 1 Molecular Microbiology & Immunology, University of Missouri, Columbia, MO; 2Electrical and Computer Engineering, University of Missouri, Columbia, MO. Dystrophin-deficient dogs are highly valuable tools for evaluating gene/cell therapies to treat Duchenne muscular dystrophy (DMD). Unfortunately, robust muscle functional end points are poorly defined in the canine DMD models. Here, we described a set of non-invasive methods for whole body muscle function analysis in normal and affected dogs. Briefly, the gait and voluntary activity were recorded using high-resolution digital video recorders. Gait recoding was repeated three times on three different days for each dog. Voluntary activity monitoring was performed under low-lux red lighting between 7pm and 5am for each dog. The stride length, stride speed, range of the motion and overall activity were analyzed using a customer-developed program. Three normal and four affected dogs of a litter were examined. Affected dogs showed significantly reduced stride length (affected, 35.7 ± 8.5 cm; normal, 80.2 ± 6.1 cm; p <0.02) and stride speed (affected, 61.7 ± 16.4 cm/sec; normal, 156.5 ± 23.8 cm/sec; p <0.03). The range of the motion in forelimb (palm, forearm and wrist) and hind limb (sole, low leg and ankle) was also significantly decreased in affected dogs. The affected dogs also showed significantly less movement during overnight monitoring. In summary, our results suggest that whole body muscle function was significantly compromised in affected dogs. Non-invasive video recording may represent a convenient and reliable method to monitor disease progression and gene/cell therapy effect in canine models of DMD.
772. Novel Functional Optical Imaging of Bone Repair in a Calvaria Defect Model
Ilan Kallai,1 Nadav Kimelman-Bleich,1 Wafa Tawackoli,2 Xiaoyu Da,2 Anthony Oh,2 Gadi Pelled,1,2 Zulma Gazit,1,2 Dan Gazit.1,2 1 Hebrew University of Jerusalem, Jerusalem, Israel; 2SurgeryRegenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles. The establishment of new therapeutic approaches for tissue repair and regeneration could be accelerated if adequate technologies to monitor the repair process are developed. In cranio-maxillofacial surgery, 96,000 bone grafts are performed each year to regenerate bone loss due to trauma or disease. Autologous bone, usually used to treat these lesions, is not always available and necessitates additional surgery for its harvest. Alternatively, there is a large potential supply of craniofacial allografts, which are composed of non-vital bone. Yet, these grafts often fail to integrate due to the formation of scar tissue Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
MUSCULO-SKELETAL GENE & CELL THERAPY II around them. Little is known of the mechanism behind the induction of scar tissue by allografts. We hypothesized that the establishment of functional fluorescence imaging (FLI) of osteogenesis, angiogenesis and osteoclastogenesis in calvarial defects treated with allografts would provide new insights that would enable the development of novel therapies for allograft bio-integration. Methods: A criticalsize calvarial defect was created in FVB/n mice. Groups included transplantation of: allografts, autografts, and untreated defects (used as control). For blood flow and angiogenesis imaging the mice were injected with AngioSense 750 or Integrisense 750 on Days 5 and 12-post transplantation. FLI of angiogenesis was done using epiillumination 24 hours after probe injection. Signal quantification eliminated autofluorescence. For osteogenesis imaging the mice were injected with OsteoSense 680 or 800. Imaging with Osteosense was performed on Day 20, in vivo and then ex vivo following mice sacrifice. Finally, bone resorption activity was imaged using Cathepsin-K 680 probe. Bone formation and vasculogenesis were analyzed as well using Micro CT imaging and histological analysis. Results: Functional FLI of blood flow showed significantly higher signal on Day 6 than on Day 13 in both treated groups and was greater in both treated groups than in the control group.
Imaging of OsteoSense 680 on Day 20 in vivo and ex vivo showed no difference.[figure 2]Thus, although epi-illumination FLI is known for high tissue attenuation, in this model the signal is preserved. MicroCT and histological analysis performed on the calvaria samples corroborated the results of osteogenesis and angiogenesis detected by functional FLI. Discussion: Our findings indicate that functional FLI is a reliable tool for monitoring the repair of a calvarial bone defects. An evaluation and validation of new therapies involving allografts for the repair of calvarial injuries may be obtained using this system. Furthermore, a better understanding of the healing process in craniofacial defects treated with allografts may be reached.
Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
773. Comparison of Efficacy of a Novel DoubleStrand AAV2 Vector Harboring VEGF cDNA to AAVSS-VEGF: In Vitro Transgene Expression and In Vivo Application To Enhance Survival of Ischemic Flaps Xiao Tian Wang,1 Bella Avanessian,1 Qiangzhong Ma,1 Yuqing Tang,1 Heather Durfee,1 Paul Y. Liu.1 1 Surgical Research and Gene Therapy, Roger Williams Medical Center, Providence, RI.
Purpose: Conventional recombinant adeno-associated virus (rAAV) vectors are a single-stranded DNA vector, which needs to be converted to double-stranded DNA prior to transcription, thus delaying expression of transgene. Previously, we reported that rAAVVEGF gene transfer to rat ischemic flaps improved flap survival in PSRC meetings. Here we compare the transgene expression efficacy of single-stranded (SS) and double-stranded AAV (DS) mediated human VEGF gene transfer and the efficiency in enhancing ischemic flap survival. Materials and Methods: 1. 293 cells were transduced with AAV-DS-VEGF or AAV-SS-VEGF (MOI=1, 10, 50, 100, n=3). Non-treatment was as control. The supernatant was harvested at day 3 and 7 for human VEGF ELISA. 2. Primary DB/DB and DBm fibroblasts were transduced with AAV-DS-VEGF and AAV-SS-VEGF (MOI=100, n=4). Non-treatment was as control. Three days later, the supernatant was harvested for ELISA analysis. We performed in vitro wound healing assay by scratching transduced cells and recording the healing for 48 hours using an IncuCyte system. 3. AAV-SS-VEGF and AAV-DS-VEGF (n=10) were injected into a 7 cm x 3 cm area on the dorsum of the rat. The control rats received saline or AAV-SS-GFP injection. One week later, a flap of 10 cm x 3 cm was raised. One week post-surgery, we evaluated the flap viability and harvested the injected and surviving flap tissues for histology. Results: 1. At day 3 and 7, both AAV-SS-VEGF and AAV-DS-VEGF treated 293 cell supernatant contained a greater amount of VEGF compared to control at all MOIs. AAV-DS-VEGF treated cell supernatant had higher expression than AAV-SS-VEGF supernatant did at all MOIs tested. 2. AAV-SS-VEGF and AAV-DS-VEGF treated mouse fibroblasts (DB/DB and DBm) presented enhanced human VEGF expression compared to control. AAV-DS-VEGF treated cell supernatant showed higher VEGF expression than AAV-SS-VEGF group. Supernatant of DBm fibroblasts contained more VEGF than that of DB/DB cells. 3. In vitro wound healing was improved by use of either AAV-SSVEGF or AAV-DS-VEGF; no significant difference was found between two treatments. 4. AAV-DS-VEGF improved flap survival (57.2%±5.5%) compared to saline group (48%±5.1%) and AAV-SSGFP group (45.9±6.3%) significantly (p < 0.05, both comparisons). AAV-SS-VEGF treated flaps showed 50.7% ± 7.2% survival, which did not differ significantly compared to saline and AAV-SS-GFP groups. Discussion/Conclusions: AAV-DS-VEGF and AAV-GFPVEGF can successfully transduce primary diabetic and non-diabetic fibroblasts. AAV-DS-VEGF produces higher and earlier transgene expression. AAV-SS-VEGF and AAV-DS-VEGF enhanced in vitro wound healing. AAV-DS-VEGF improves flap survival compared to controls, but AAV-SS-VEGF did not significantly improve flap survival in the present study, which contrasts to our previous results of significant improvement in flap survival when it was injected 2 weeks prior to flap elevation.
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