A. Leung

Fetal wound healing: implications for minimal scar formation.

Purpose of review The mid-gestation fetus is capable of regenerative healing with wound healing indistinguishable from surrounding skin. This review aims to evaluate the current knowledge of how the mid-gestation fetus heals without scar and the implications of these findings in efforts to recapitulate the fetal regenerative phenotype in the postnatal environment. Recent findings It has been over 30 years since the empirical observation that the fetus heals without scar; yet, the underlying mechanisms of this phenomenon have not been elucidated. Fetal wound healing is characterized by a distinct growth factor profile, an attenuated inflammatory response with an anti-inflammatory cytokine profile, an extracellular matrix rich in type III collagen and hyaluronan, attenuated biomechanical stress, and a potential role for stem cells. Current therapies to minimize scarring in postnatal wounds have attempted to recapitulate singular aspects of the fetal regenerative phenotype and have met with varying degrees of clinical success. We now have the molecular tools to more completely comprehend the fundamental mechanisms of fetal regenerative wound repair, which has the potential to provide insights into the identification of therapeutic targets to minimize the scar formation. Summary Successful therapies that help minimize postnatal scar formation can be realized through understanding the cellular and molecular mechanisms of fetal regenerative wound healing. These insights will have implications not only for cutaneous wound healing, but also potentially for any disease process characterized by excessive fibroplasia.

Role of salivary vascular endothelial growth factor (VEGF) in palatal mucosal wound healing.

The mucosa of alimentary tract heals more rapidly than cutaneous wounds. The underlying mechanisms of this enhanced healing have not been completely elucidated. Constant exposure to salivary growth factors has been shown to play a critical role in mucosal homeostasis and tissue repair. Angiogenesis also has an essential role in successful wound repair. One of the main angiogenic growth factors, vascular endothelial growth factor (VEGF), has a pleiotropic role in tissue repair via neovascularization, reepithelialization, and regulation of extracellular matrix. We have previously reported a critical role for salivary VEGF in bowel adaptation after small bowel resection. We hypothesize that salivary VEGF is an essential stimulus for oral mucosal tissue repair, and use the murine palatal wound model to test our hypothesis. In a loss‐of‐function experiment, we removed the primary source of VEGF production through selective submandibular gland (SMG) sialoadenectomy in a murine model and observed the effects on wound closure and neovascularization. We then performed a selective loss‐of‐function experiment using the protein VEGF‐Trap to inhibit salivary VEGF. In a gain‐of‐function experiment, we supplemented oral VEGF following SMG sialoadenectomy. After SMG sialoadenectomy, there was significant reduction in salivary VEGF level, wound closure, and vessel density. Lower levels of salivary VEGF were correlated with impaired neovascularization and reepithelialization. The selective blockade of VEGF using VEGF‐Trap resulted in a similar impairment in wound healing and neovascularization. The sole supplementation of oral VEGF after SMG sialoadenectomy rescued the impaired wound healing phenotype and restored neovascularization to normal levels. These data show a novel role for salivary‐VEGF in mucosal wound healing, and provide a basis for the development of novel therapeutics aimed at augmenting wound repair of the oral mucosa, as well as wounds at other sites in the alimentary tract.

Pseudotyped AAV Vector-Mediated Gene Transfer in a Human Fetal Trachea Xenograft Model: Implications for In Utero Gene Therapy for Cystic Fibrosis

Background Lung disease including airway infection and inflammation currently causes the majority of morbidities and mortalities associated with cystic fibrosis (CF), making the airway epithelium and the submucosal glands (SMG) novel target cells for gene therapy in CF. These target cells are relatively inaccessible to postnatal gene transfer limiting the success of gene therapy. Our previous work in a human-fetal trachea xenograft model suggests the potential benefit for treating CF in utero. In this study, we aim to validate adeno-associated virus serotype 2 (AAV2) gene transfer in a human fetal trachea xenograft model and to compare transduction efficiencies of pseudotyping AAV2 vectors in fetal xenografts and postnatal xenograft controls. Methodology/Principal Findings Human fetal trachea or postnatal bronchus controls were xenografted onto immunocompromised SCID mice for a four-week engraftment period. After injection of AAV2/2, 2/1, 2/5, 2/7 or 2/8 with a LacZ reporter into both types of xenografts, we analyzed for transgene expression in the respiratory epithelium and SMGs. At 1 month, transduction by AAV2/2 and AAV2/8 in respiratory epithelium and SMG cells was significantly greater than that of AAV2/1, 2/5, and 2/7 in xenograft tracheas. Efficiency in SMG transduction was significantly greater in AAV2/8 than AAV2/2. At 3 months, AAV2/2 and AAV2/8 transgene expression was >99% of respiratory epithelium and SMG. At 1 month, transduction efficiency of AAV2/2 and AAV2/8 was significantly less in adult postnatal bronchial xenografts than in fetal tracheal xenografts. Conclusions/Significance Based on the effectiveness of AAV vectors in SMG transduction, our findings suggest the potential utility of pseudotyped AAV vectors for treatment of cystic fibrosis. The human fetal trachea xenograft model may serve as an effective tool for further development of fetal gene therapy strategies for the in utero treatment of cystic fibrosis.

Pseudotyped adeno-associated viral vector tropism and transduction efficiencies in murine wound healing.

Cell specific gene transfer and sustained transgene expression are goals of cutaneous gene therapy for tissue repair and regeneration. Adeno‐associated virus serotype 2 (AAV2/2) mediated gene transfer to the skin results in stable transgene expression in the muscle fascicles of the panniculus carnosus in mice, with minimal gene transfer to the dermal or epidermal elements. We hypothesized that pseudotyped AAV vectors may have a unique and characteristic tropism and transduction efficiency profile for specific cells in the cutaneous wounds. We compared transduction efficiencies of cells in the epidermis, cells in the dermis, and the fascicles of the panniculus carnosus by AAV2/2 and three pseudotyped AAV vectors, AAV2/5, AAV2/7, and AAV2/8 in a murine excisional wound model. AAV2/5 and AAV2/8 result in significantly enhanced transduction of cells both in the epidermis and the dermis compared to AAV2/2. AAV2/5 transduces both the basilar and supra‐basilar keratinocytes. In contrast, AAV2/8 transduces mainly supra‐basilar keratinocytes. Both AAV2/7 and AAV2/8 result in more efficient gene transfer to the muscular panniculus carnosus compared to AAV2/2. The capsid of the different pseudotyped AAV vectors produces distinct tropism and efficiency profiles in the murine wound healing model. Both AAV2/5 and AAV2/8 administration result in significantly enhanced gene transfer. To further characterize cell specific transduction and tropism profiles of the AAV pseudotyped vectors, we performed in vitro experiments using human and mouse primary dermal fibroblasts. Our data demonstrate that pseudotyping strategy confers a differential transduction of dermal fibroblasts, with higher transduction of both human and murine cells by AAV2/5 and AAV2/8 at early and later time points. At later time points, AAV2/2 demonstrates increased transduction. Interestingly, AAV2/8 appears to be more efficacious in transducing human cells as compared to AAV2/5. The pseudotype‐specific pattern of transduction and tropism observed both in vivo and in vitro suggests that choice of AAV vectors should be based on the desired target cell and the timing of transgene expression in wound healing for gene transfer therapy in dermal wounds.