Louis D. Le

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.

Adenoviral-mediated gene transfer of insulin-like growth factor 1 enhances wound healing and induces angiogenesis

BACKGROUND Chronic wounds are characterized by a wound healing and neovascularization deficit. Strategies to increase neovascularization can significantly improve chronic wound healing. Insulin-like growth factor (IGF)-1 is reported to be a keratinocyte mitogen and is believed to induce angiogenesis via a vascular endothelial growth factor (VEGF)-dependent pathway. Using a novel ex vivo human dermal wound model and a diabetic-impaired wound healing murine model, we hypothesized that adenoviral overexpression of IGF-1 (Ad-IGF-1) will enhance wound healing and induce angiogenesis through a VEGF-dependent pathway. METHODS Ex vivo: 6-mm full-thickness punch biopsies were obtained from normal human skin, and 3-mm full-thickness wounds were created at the center. Skin explants were maintained at air liquid interface. Db/db murine model: 8-mm full-thickness dorsal wounds in diabetic (db/db) mice were created. Treatment groups in both human ex vivo and in vivo db/db wound models include 1×10(8) particle forming units of Ad-IGF-1 or Ad-LacZ, and phosphate buffered saline (n=4-5/group). Cytotoxicity (lactate dehydrogenase) was quantified at days 3, 5, and 7 for the human ex vivo wound model. Epithelial gap closure (hematoxylin and eosin; Trichrome), VEGF expression (enzyme-linked immunosorbent assay), and capillary density (CD 31+CAPS/HPF) were analyzed at day 7. RESULTS In the human ex vivo organ culture, the adenoviral vectors did not demonstrate any significant difference in cytotoxicity compared with phosphate buffered saline. Ad-IGF-1 overexpression significantly increases basal keratinocyte migration, with no significant effect on epithelial gap closure. There was a significant increase in capillary density in the Ad-IGF-1 wounds. However, there was no effect on VEGF levels in Ad-IGF-1 samples compared with controls. In db/db wounds, Ad-IGF-1 overexpression significantly improves epithelial gap closure and granulation tissue with a dense cellular infiltrate compared with controls. Ad-IGF-1 also increases capillary density, again with no significant difference in VEGF levels in the wounds compared with control treatments. CONCLUSIONS In two different models, our data demonstrate that adenoviral-mediated gene transfer of IGF-1 results in enhanced wound healing and induces angiogenesis via a VEGF-independent pathway. Understanding the underlying mechanisms of IGF-1 effects on angiogenesis may help produce novel therapeutics for chronic wounds or diseases characterized by a deficit in neovascularization.

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.

The congenital diaphragmatic hernia composite prognostic index correlates with survival in left-sided congenital diaphragmatic hernia.

PURPOSE We developed the congenital diaphragmatic hernia congenital prognostic index (CDH-CPI) to incorporate all known prognostic variables into a single composite index to improve prognostic accuracy. The purpose of this study is to examine the ability of the CDH-CPI to predict survival in patients with left-sided congenital diaphragmatic hernia and to determine if the index has a stronger correlation with survival than each of the individual components. METHODS A retrospective review of patients with left-sided congenital diaphragmatic hernia between 2004 and 2010 was conducted. Ten prenatal parameters of the CDH-CPI were collected, total score was tabulated, and patients stratified according to total score and survival. RESULTS Sixty-four patients with a prenatal diagnosis of left-sided congenital diaphragmatic hernia were identified. Patients with a CDH-CPI score of 8 or higher had a significantly higher survival than patients with a CDH-CPI score of lower than 8. The CDH-CPI has the strongest correlation with survival compared with the individual parameters measured. The CDH-CPI correlates with extracorporeal membrane oxygenation use, and 75% of patients with a score of 5 or lower were placed on extracorporeal membrane oxygenation. CONCLUSIONS The CDH-CPI accurately stratifies survival in left-sided congenital diaphragmatic hernia. The amalgamation of 10 prenatal parameters of the CDH-CPI may be a better prenatal predictor than any single prognostic variable currently used.

Submucosal gland development in the human fetal trachea xenograft model: implications for fetal gene therapy☆

BACKGROUND/PURPOSE Our previous work in a human-fetal trachea xenograft model suggests potential benefits of treating cystic fibrosis in utero. The target for postnatal gene therapy in cystic fibrosis is tracheal submucosal glands (SMGs). The aim of this study was to determine if SMG development in our model recapitulates normal trachea development and its validity for studying fetal gene transfer. METHODS Fetal tracheas were divided into developmental phases: early, mid, and late. Fetal tracheas were xenografted onto immunocompromised mice and analyzed for SMG developmental staging and mucopolysaccharide production. RESULTS There were no significant differences in gland number, size, or density from early through late phase between groups. Xenografted tracheas demonstrated a similar progression through the stages of SMG development as controls after an initial phase shift. Control and xenografted tracheas demonstrated characteristic patterns of acidic mucin production at the base of the SMGs. CONCLUSIONS Fetal trachea xenograft SMG recapitulates normal development and is a valid model for studying human fetal gene transfer. The accessibility of SMG stem cells in early tracheal development may afford a unique window of opportunity for gene transfer. This model has the benefit of providing access to human fetal tracheas in vivo and permits the study of novel fetal gene therapy strategies.

Interleukin-10 regulates fetal extracellular matrix hyaluronan production.

BACKGROUND/PURPOSE Mid-gestational (E14.5) fetal wounds heal regeneratively with attenuated inflammation and high levels of hyaluronan (HA) in their extracellular matrix (ECM), whereas late-gestational (E18.5) fetal wounds heal with scarring. IL-10 plays an essential role in the fetal regenerative phenotype and is shown to recapitulate scarless wound healing postnatally. We hypothesize a novel role of IL-10 as a regulator of HA in the ECM. METHODS Murine fetal fibroblasts (FFb) from C57Bl/6 and IL-10-/- mice were evaluated in vitro. Pericellular matrix (PCM) and HA synthesis were quantified using a particle exclusion assay and ELISA. The effects of hyaluronidase and hyaluronan synthase (HAS) inhibitor (4-methylumbelliferone[4-MU]) were evaluated. An ex vivo fetal forearm culture incisional wound model comparing mid-gestation and late-gestation fetuses was used to evaluate IL-10s effect on HA-rich ECM production with pentachrome and immunohistochemistry. RESULTS FFb produce a robust HA-rich PCM which is IL-10 dependent and attenuated with hyaluronidase and HAS inhibition. Mid-gestation fetal wounds produce more ground substance and HA than late-gestation fetal wounds. IL-10 in late-gestation fetal wounds results in elevated ground substance levels and HA staining. CONCLUSIONS Our data demonstrate that IL-10 regulates an HA-rich ECM deposition, suggesting a novel non-immunoregulatory mechanism of IL-10 in mediating regenerative wound healing.

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.

Interleukin-10-mediated regenerative postnatal tissue repair is dependent on regulation of hyaluronan metabolism via fibroblast-specific STAT3 signaling.

The cytokine IL‐10 has potent antifibrotic effects in models of adult fibrosis, but the mechanisms of action are unclear. Here, we report a novel finding that IL‐10 triggers a signal transducer and activator of transcription 3(STAT3)–dependent signaling pathway that regulates hyaluronan (HA) metabolism and drives adult fibroblasts to synthesize an HA‐rich pericellular matrix, which mimics the fetal regenerative wound healing phenotype with reduced fibrosis. By using cre‐lox‐mediated novel, inducible, fibroblast‐, keratinocyte‐, and wound‐specific STAT3‐knockdown postnatal mice—plus syngeneic fibroblast cell‐transplant models—we demonstrate that the regenerative effects of IL‐10 in postnatal wounds are dependent on HA synthesis and fibroblast‐specific STAT3‐dependent signaling. The importance of IL‐10‐induced HA synthesis for regenerative wound healing is demonstrated by inhibition of HA synthesis in a murine wound model by administering 4‐methylumbelliferone. Although IL‐10 and STAT3 signaling were intact, the antifibrotic repair phenotype that is induced by IL‐10 overexpression was abrogated in this model. Our data show a novel role for IL‐10 beyond its accepted immune‐regulatory mechanism. The opportunity for IL‐10 to regulate a fibroblast‐specific formation of a regenerative, HA‐rich wound extracellular matrix may lead to the development of innovative therapies to attenuate postnatal fibrosis in organ systems or diseases in which dysregulated inflammation and HA intersect.—Balaji, S., Wang, X., King, A., Le, L. D., Bhattacharya, S. S., Moles, C. M., Butte, M. J., de Jesus Perez, V.A., Liechty, K. W., Wight, T. N., Crombleholme, T. M., Bollyky, P. L., Keswani, S. G. Interleukin‐10‐mediated regenerative postnatal tissue repair is dependent on regulation of hyaluronan metabolism via fibroblast‐specific STAT3 signaling. FASEB J. 31, 868–881 (2017). www.fasebj.org

Prenatal and postnatal urologic complications of sacrococcygeal teratomas

PURPOSE Urologic complications are an underappreciated sequelae of the mass effect of sacrococcygeal teratoma (SCT) and its resection. The goal of this study was to evaluate the incidence and severity of urologic complications in patients with SCT. METHODS A retrospective review of patients with a prenatal diagnosis of SCT and postnatal referral for SCT at a single institution during a 5-year period (2004-2009) was performed. The presence of prenatal and postnatal urologic abnormalities were collected and analyzed. RESULTS Thirty patients were identified for inclusion in the study. Twenty-two patients were diagnosed prenatally, 3 patients were referred for resection of the tumor, and 5 patients were initially treated elsewhere and referred after resection for urologic complications. Of the 20 patients with subsequent follow-up included in this study, 9 had neurogenic bladder, 5 of whom also had associated renal injury. All patients with renal injury had a higher grade Altman type II/III lesion. CONCLUSION Urologic problems appear to be common in patients with SCT. Higher grade SCTs should alert the surgeon to possible urologic sequelae, particularly neurogenic bladder and renal injury. Careful urologic evaluation and management of the genitourinary tract should be included in prenatal and preoperative counseling.

Urologic co-morbidities associated with sacrococcygeal teratoma and a rational plan for urologic surveillance

Sacrococcygeal teratoma (SCT) is one of the most common neonatal and fetal tumors. SCT pelvic mass effect and the need for aggressive surgical resection, create potential for urologic co‐morbidity. We reviewed our experience with SCTs and propose a rational plan for urologic surveillance.