Rational selection of an ideal oncolytic virus to address current limitations in clinical translation.

Oncolytic virus therapy (OVT) is a promising modality that leverages the propensity of natural or engineered viruses to selectively replicate in and kill cancer cells. Over the past decade, (pre)clinical studies have focused on the development and testing of adenovirus, herpes simplex virus, and vaccinia virus-based vectors. These studies have identified barriers to success confronting the field. Here, we propose a set of selection criteria or ideal properties of a successful oncolytic virus, which include lack of pathogenicity, low seroprevalence, selectivity (infection and replication), transgene carrying capacity, and genome stability. We use these requirements to analyze the oncolytic virus landscape, and then identify a potentially optimal species for platform development - vesicular stomatitis virus.

IL-10 promotes endothelial progenitor cell infiltration and wound healing via STAT3.

Endothelial progenitor cells (EPCs) contribute to de novo angiogenesis, tissue regeneration, and remodeling. Interleukin 10 (IL-10), an anti-inflammatory cytokine that primarily signals via STAT3, has been shown to drive EPC recruitment to injured tissues. Our previous work demonstrated that overexpression of IL-10 in dermal wounds promotes regenerative tissue repair via STAT3-dependent regulation of fibroblast-specific hyaluronan synthesis. However, IL-10's role and specific mode of action on EPC recruitment, particularly in dermal wound healing and neovascularization in both normal and diabetic wounds, remain to be defined. Therefore, inducible skin-specific STAT3 knockdown mice were studied to determine IL-10's impact on EPCs, dermal wound neovascularization and healing, and whether it is STAT3-dependent. We show that IL-10 overexpression significantly elevated EPC counts in the granulating wound bed, which was associated with robust capillary lumen density and enhanced re-epithelialization of both control and diabetic (db/db) wounds at day 7. We noted increased VEGF and high C-X-C motif chemokine 12 (CXCL12) levels in wounds and a favorable CXCL12 gradient at day 3 that may support EPC mobilization and infiltration from bone marrow to wounds, an effect that was abrogated in STAT3 knockdown wounds. These findings were supported in vitro. IL-10 promoted VEGF and CXCL12 synthesis in primary murine dermal fibroblasts, with blunted VEGF expression upon blocking CXCL12 in the media by antibody binding. IL-10-conditioned fibroblast media also significantly promoted endothelial sprouting and network formation. In conclusion, these studies demonstrate that overexpression of IL-10 in dermal wounds recruits EPCs and leads to increased vascular structures and faster re-epithelialization.

The Pediatric Surgeon-Scientist: Succeeding in Today's Academic Environment.

BACKGROUND: Pediatric surgeons have long been advocates of basic science research. However, new challenges facing the scientific community have threatened the success of academic surgeons pursuing basic science careers. The purpose of this study was to compare academic pediatric surgeons' perceptions of their ability to effectively conduct basic science research to those of other surgical subspecialties. METHODS: An online survey was distributed to all members of the Association for Academic Surgery and Society of University Surgeons. A total of 1033 members (41%) responded, and 137 (13.3%) were pediatric surgeons. Comparisons were made between the five most-represented surgical subspecialties. Data are presented as reporting percentage and P values by Student's t-test. RESULTS: Among the specialists studied, pediatric surgeons are those most likely to believe that surgeons can succeed as basic scientists in today's research environment. Pediatric surgery reported the highest rates of National Institutes of Health funding of all surgical specialties and the lowest rates of perceived external pressures related to clinical demands, hospital administrative duties, and work-life balance concerns than their surgical peers. CONCLUSIONS: Pediatric surgeons have a more optimistic perspective on the state of basic science research in surgery while exhibiting an enhanced ability to overcome the challenges that surgeon-scientists currently face. Our findings suggest that pediatric surgery may provide a model for succeeding in basic science in today's challenging surgical research environment.

The Future of Basic Science in Academic Surgery: Identifying Barriers to Success for Surgeon-scientists.

OBJECTIVE: The aim of this study was to examine the challenges confronting surgeons performing basic science research in today's academic surgery environment. SUMMARY OF BACKGROUND DATA: Multiple studies have identified challenges confronting surgeon-scientists and impacting their ability to be successful. Although these threats have been known for decades, the downward trend in the number of successful surgeon-scientists continues. Clinical demands, funding challenges, and other factors play important roles, but a rigorous analysis of academic surgeons and their experiences regarding these issues has not previously been performed. METHODS: An online survey was distributed to 2504 members of the Association for Academic Surgery and Society of University Surgeons to determine factors impacting success. Survey results were subjected to statistical analyses. We also reviewed publicly available data regarding funding from the National Institutes of Health (NIH). RESULTS: NIH data revealed a 27% decline in the proportion of NIH funding to surgical departments relative to total NIH funding from 2007 to 2014. A total of 1033 (41%) members responded to our survey, making this the largest survey of academic surgeons to date. Surgeons most often cited the following factors as major impediments to pursuing basic investigation: pressure to be clinically productive, excessive administrative responsibilities, difficulty obtaining extramural funding, and desire for work-life balance. Surprisingly, a majority (68%) did not believe surgeons can be successful basic scientists in today's environment, including departmental leadership. CONCLUSIONS: We have identified important barriers that confront academic surgeons pursuing basic research and a perception that success in basic science may no longer be achievable. These barriers need to be addressed to ensure the continued development of future surgeon-scientists.

Angiopoietin-1 improves endothelial progenitor cell-dependent neovascularization in diabetic wounds.

BACKGROUND: The diabetic phenotype of wound healing is in part characterized by impaired neovascularization and deficient endothelial progenitor cell (EPC) recruitment. Angiopoietin-1 (Ang-1) is a potent mobilizer of EPCs from the bone marrow (BM). A suggested mechanism for EPC mobilization from the BM is mediated by matrix metalloproteinase 9 (MMP-9) and stem cell factor (SCF). Taken together, we hypothesized that overexpression of Ang-1 in diabetic wounds will recruit EPCs and improve neovascularization and wound healing. METHODS: An endothelial lineage BM-labeled murine model of diabetes was developed to track BM-derived EPCs. FVBN mice were lethally irradiated and then reconstituted with BM from syngeneic Tie2/LacZ donor mice. Diabetes was induced with streptozotocin. Dorsal wounds in BM-transplanted mice were treated with Ad-Ang-1, Ad-GFP, or phosphate-buffered saline. At day 7 after injury, wounds were harvested and analyzed. A similar experiment was conducted in EPC mobilization deficient MMP-9 -/- mice to determine whether the effects of Ang-1 were EPC-dependent. RESULTS: Overexpression of Ang-1 resulted in greatly improved re-epithelialization, neovascularization, and EPC recruitment in diabetic BM-transplanted wounds at day 7. Ang-1 treatment resulted in increased serum levels of proMMP-9 and SCF but had no effect on vascular endothelial growth factor levels. According to our FACS results, peripheral blood EPC (CD34(+)/Cd133(+)/Flk1(+)) counts at day 3 after wounding showed impaired EPC mobilization in MMP-9 -/- mice compared with those of wild-type controls. EPC mobilization was rescued by SCF administration, validating this model for EPC-mobilization-deficient mechanistic studies. In MMP-9 -/- mice, Ad-Ang-1 accelerated re-epithelialization in a similar manner, but had no effect on neovascularization. CONCLUSION: Our results show that Ang-1 administration results in improved neovascularization which is dependent on EPC recruitment and has direct effects on wound re-epithelialization. These data may represent a novel strategy to correct the phenotype of impaired diabetic neovascularization and may improve diabetic wound healing.

Comparison of interleukin 10 homologs on dermal wound healing using a novel human skin ex vivo organ culture model.

BACKGROUND: Anti-inflammatory cytokine interleukin (IL)-10 has been shown to induce regenerative healing in postnatal wounds. A viral homolog of IL-10 produced by human cytomegalovirus (CMV IL-10) similarly generates potent immunoregulatory effects, but its effects on wound healing have not been investigated. Currently, there are limited cost-effective methods of screening vulnerary therapeutics. Taken together, we aim to develop and validate a novel human ex vivo dermal wound model and hypothesize that CMV IL-10 will enhance dermal wound healing. METHODS: Full-thickness circular (6-mm) explants were taken from surgical skin samples and 3-mm full-thickness wounds were created. Explants were embedded in collagen I matrix and maintained in specially formulated media with the epidermis at air-liquid interface, and treated with human IL-10 or CMV IL-10 (200 ng/mL). The viability of cultured explants was validated by histology and lactate dehydrogenase (LDH) activity. Epithelial gap, epithelial height, basal keratinocyte migration, vascular endothelial growth factor levels, and neovascularization were measured at days 3 and 7 to determine IL-10 effects on wound healing. RESULTS: Culture explants at day 7 appeared similar to fresh skin in morphology, cell, and vessel density. By day 14, the epidermis separated from the dermis and the cell density diminished. Day 7 wounds appeared viable with advancing epithelial and basal keratinocyte migration with no evidence of necrosis. Cytotoxicity analysis via the quantification of LDH revealed no differences between controls and treated groups. There was a slight increase in the quantity of LDH in media at day 3; however, this decreased at day 5 and continued to decline up to day 21. CMV IL-10 treatment resulted in a significant decrease in the epithelial gap and an increase in epithelial height. There were no differences in the rates of basal keratinocyte migration at day 7 between treated and control groups. Interestingly, human IL-10 increased vascular endothelial growth factor expression and neovascularization compared with controls. CONCLUSIONS: The human ex vivo wound model provides a simple and viable design to study dermal wound healing. Both IL-10 homologs demonstrate vulnerary effects. The viral homolog demonstrates enhanced effects on wound closure compared with human IL-10. These data represent a novel tool that can be used to screen therapeutics, such as CMV IL-10, before preclinical studies.

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.