Xiao Tian Wang

Liposome‐mediated transfer of vascular endothelial growth factor cDNA augments survival of random‐pattern skin flaps in the rat

Tissue engineering is an application for gene therapy that is in its infancy. We show that simple liposomal‐mediated gene transfer could result in a potentially useful biological effect in the field of wound healing. cDNA encoding the 165 amino acid form of vascular endothelial growth factor complexed to commercially available liposomes was injected into rat skin 1 week before raising a random pattern 3 × 10 cm flap. The flap survival was enhanced by 14 percent, and was accomplished without accessing the arterial inflow of the territory. These results were statistically significant (p < 0.002) and reproducible. No adverse effects were seen. Histological analysis of the angiogenesis localized much of the new vessel formation to the area around the hair follicles. Polymerase chain reaction amplification of extracted flap tissue confirmed the presence of the transgene.

PDGF gene therapy enhances expression of VEGF and bFGF genes and activates the NF-kappaB gene in signal pathways in ischemic flaps.

Background: Gene therapy is a novel approach for enhancing the viability of ischemic flaps. Expression of growth factor genes pertinent to angiogenesis and activation of genes of relevant signal pathways are imperative for improving flap viability. The authors investigated the gene expression profiles of growth factors and signal transduction pathways in ischemic flaps after PDGF gene therapy. Methods: Twenty Sprague-Dawley rats were divided into two groups. The experimental group (n = 10) received the plasmid vector containing the PDGF cDNA injected into the dermis of the flap area, whereas the control group (n = 10) received the physiologic saline. Seven days later, a dorsal random flap was raised. Seven days after surgery, flap viability was assessed, and expression of VEGF, bFGF, TGF-&bgr;1, NF-&kgr;B, Erk2, Stat1, and Smad2 genes of the NF-&kgr;B, MAPK, JAK-STAT, and Smad pathways was assessed by quantitative analysis of the products of reverse-transcriptase polymerase chain reaction. Results: Transfer of exogenous PDGF gene significantly improved flap viability (p = 0.011). Levels of expression of VEGF and bFGF genes in the flap were significantly elevated after PDGF gene transfer (p = 0.0001 and p = 0.001, respectively). Expression of the NF-&kgr;B gene was significantly elevated (p = 0.041). In contrast, expression of TGF-&bgr;1, and Erk2, Stat1, and Smad2 genes was not changed. Conclusions: Transfer of exogenous PDGF gene to ischemic flaps promotes expression of VEGF and bFGF genes and activation of NF-&kgr;B gene in addition to its effects on the PDGF gene. The finding implies that transfer of the gene of one growth factor ultimately improves the expression of the genes of multiple growth factors. Activation of the NF-&kgr;B gene suggests that the NF-&kgr;B pathway may be important in enhancement of flap viability and will likely be a target of future efforts of regulation of signaling process in treatment of ischemic flaps.

Tendon Healing In Vitro: Adeno-associated Virus-2 Effectively Transduces Intrasynovial Tenocytes with Persistent Expression of the Transgene, but Other Serotypes Do Not

Background: Transfer of exogenous growth factor genes to injured tendons offers a promising method for strengthening tendon repairs. Adeno-associated virus vectors have advantages of being both nonpathogenic and nontoxic. The authors explored the efficiency of transduction of intrasynovial tenocytes with different serotypes of adeno-associated virus (AAV) and the persistency of its expression of a growth factor transgene. Methods: Tenocytes were obtained from cultures of rat intrasynovial tendons and distributed to 82 wells in eight culture plates and to 30 culture dishes. The tenocytes in the wells were treated with AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, and AAV8 vectors containing the lacZ gene, and plasmid vectors (pCMVβ-lacZ). The tenocytes were stained with in situ β-galactosidase 5 days later. The basic fibroblast growth factor (bFGF) gene was cloned to the AAV2 vector to construct the AAV2-bFGF vector, which transduced tenocytes in culture dishes. Expression of the transgene was measured over 3 weeks and analyzed statistically. Results: AAV2 effectively delivered exogenous genes to proliferating intrasynovial tenocytes. In contrast, other tested adeno-associated viruses transduced tenocytes minimally or not at all. The efficiency of gene transfer by AAV2, indicated by the percentage of cells with positive β-galactosidase staining, was significantly greater than that by a plasmid vector (p = 0.001). Expression of the bFGF gene in tenocytes transduced with the AAV2-bFGF was significantly higher than that in the control over the 3-week period (p < 0.01). Conclusions: Gene transfer to tenocytes by AAV2 is more efficient than that by a plasmid vector. However, other adeno-associated virus serotypes cannot effectively transduce tenocytes. The bFGF gene can be delivered to intrasynovial tenocytes by the AAV2 vector effectively, and the gene transfer significantly increases expression of bFGF gene over 3 weeks.

Basic FGF or VEGF gene therapy corrects insufficiency in the intrinsic healing capacity of tendons

Tendon injury during limb motion is common. Damaged tendons heal poorly and frequently undergo unpredictable ruptures or impaired motion due to insufficient innate healing capacity. By basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF) gene therapy via adeno-associated viral type-2 (AAV2) vector to produce supernormal amount of bFGF or VEGF intrinsically in the tendon, we effectively corrected the insufficiency of the tendon healing capacity. This therapeutic approach (1) resulted in substantial amelioration of the low growth factor activity with significant increases in bFGF or VEGF from weeks 4 to 6 in the treated tendons (p < 0.05 or p < 0.01), (2) significantly promoted production of type I collagen and other extracellular molecules (p < 0.01) and accelerated cellular proliferation, and (3) significantly increased tendon strength by 68–91% from week 2 after AAV2-bFGF treatment and by 82–210% from week 3 after AAV2-VEGF compared with that of the controls (p < 0.05 or p < 0.01). Moreover, the transgene expression dissipated after healing was complete. These findings show that the gene transfers provide an optimistic solution to the insufficiencies of the intrinsic healing capacity of the tendon and offers an effective therapeutic possibility for patients with tendon disunion.

Enhancement of flap survival and changes in angiogenic gene expression after AAV2‐mediated VEGF gene transfer to rat ischemic flaps

Necrosis of surgically transferred flaps due to ischemia is a serious wound problem. We evaluated the improvement of flap survival and changes in angiogenic gene expression profiles after transfer of the VEGF gene by means of adeno‐associated virus type 2 (AAV2) vector to rat ischemic flaps. Thirty rats were divided into one experimental group, one AAV2‐GFP group, and one saline group. AAV2‐VEGF or AAV2‐GFP were injected intradermally into the rat dorsum in the AAV2‐VEGF or AAV2‐GFP group. The saline group received saline injection. A 3 × 10 cm flap was raised in each rat two weeks post‐injection. One week after surgery, flap viability was evaluated. Angiogenesis real‐time PCR array was performed to analyze the expression of angiogenesis‐associated genes. The AAV2‐VEGF treatment significantly improved flap survival (p<0.05). Immunohistochemical staining showed increased VEGF expression in AAV2‐VEGF treated flaps. The PCR array identified remarkable changes in 6 out of the 84 angiogenesis‐associated genes in AAV2‐VEGF treated flaps. Particularly, EGF, PDGF‐A and VEGF‐B genes were up‐regulated in these flaps. In contrast, FGF2 gene expression was down‐regulated. In conclusion, AAV2‐VEGF improves flap survival and affects the expression of a series of endogenous growth factor genes, which likely play critical roles in the enhancement of ischemic flap survival.

Application of AAV2-Mediated bFGF Gene Therapy on Survival of Ischemic Flaps : Effects of Timing of Gene Transfer

Necrosis of surgically transferred flaps is a major problem in reconstructive surgery. We investigated efficacy of a new vector system—adeno-associated viral 2 (AAV2)-mediated bFGF gene transfer to enhance survival of the ischemic flap. Thirty-eight Sprague-Dawley rats were divided into 3 gene therapy groups and 1 nontreated control of 9 or 10 each. 7.5 × 1010 AAV2-bFGF viral particles were injected to the dorsum of each of the 29 rats; these rats were divided into 3 groups according to the timing of flap elevation. At the time of surgery, 1 week, and 2 weeks after surgery, flaps of 3 × 7 cm were raised. One week after surgery, flap viability was measured. Vascularization and immunohistochemical staining of the bFGF were evaluated of histologic sections. Flap viability was significantly improved by the AAV2-bFGF gene therapy at the time of surgery, and the flaps with the greatest survival area were found in the rats injected with AAV2-bFGF, 2 weeks before surgery. However, flap viability was significantly decreased by the gene therapy 1 week before surgery. Histologically, vascularity was increased in the groups with AAV2-bFGF injection and immunohistochemical staining showed greatly enhanced bFGF expression by gene transfer. The novel approach of AAV2-bFGF gene therapy shows encouraging manifestations in improving survival of flaps when the flaps are prefabricated during or 2 weeks before surgery.