Faculty Bibliography

Diabetes is characterized by several poorly understood phenomena including dysfunctional wound healing and impaired vasculogenesis. p53, a master cell cycle regulator, is upregulated in diabetic wounds and has recently been shown to play a regulatory roles in vasculogenic pathways. We have previously described a novel method to topically silence target genes in a wound bed with small interfering (si)RNA. We hypothesized that silencing p53 results in improved diabetic wound healing and augmentation of vasculogenic mediators. Paired 4-mm stented wounds were created on diabetic db/db mice. Topically applied p53 siRNA, evenly distributed in an agarose matrix, was applied to wounds at postwound day 1 and 7 (matrix alone and nonsense siRNA served as controls). Animals were sacrificed at postwound days 10 and 24. Wound time to closure was photometrically assessed, and wounds were harvested for histology, immunohistochemistry, and immunofluorescence. Vasculogenic cytokine expression was evaluated via Western blot, reverse transcription-polymerase chain reaction, and enzyme-linked immunosorbent assay. The ANOVA/t-test was used to determine significance (p</= 0.05). Local p53 silencing resulted in faster wound healing with wound closure at 18+/-1.3 d in the treated group vs. 28+/-1.0 d in controls. The treated group demonstrated improved wound architecture at each time point while demonstrating near-complete local p53 knockdown. Moreover, treated wounds showed a 1.92-fold increase in CD31 endothelial cell staining over controls. Western blot analysis confirmed near-complete p53 knockdown in treated wounds. At day 10, VEGF secretion (enzyme-linked immunosorbent assay) was significantly increased in treated wounds (109.3+/-13.9 pg/mL) vs. controls (33.0+/-3.8 pg/mL) while reverse transcription-polymerase chain reaction demonstrated a 1.86-fold increase in SDF-1 expression in treated wounds vs. controls. This profile was reversed after the treated wounds healed and before closure of controls (day 24). Augmented vasculogenic cytokine profile and endothelial cell markers are associated with improved diabetic wound healing in topical gene therapy with p53 siRNA

OBJECTIVE: To attempt to mitigate the effects of irradiation on murine skin after high-dose radiation using a novel transcutaneous topical delivery system to locally inhibit gene expression with small interfering RNA (siRNA) against Smad3. DESIGN: Laboratory investigation. SETTING: University laboratory. SUBJECTS: Twenty-five wild-type C57 mice. INTERVENTION: In an isolated skin irradiation model, the dorsal skin of C57 wild-type mice was irradiated (45 Gy). Just before irradiation, Smad3 and nonsense siRNA were applied to 2 separate dorsal skin areas and then reapplied weekly. Skin was harvested after 1 and 4 weeks. Smad3 expression were assessed by immunohistochemistry, and collagen deposition and architecture was examined using picrosirius red collagen staining. MAIN OUTCOME MEASURES: Epidermal thickness was measured semiquantitatively at 4 weeks. Radiation-induced fibrosis was measured quantitatively via tensiometry. The Young modulus, a measure of cutaneous rigidity inversely related to elasticity, was determined, with normal irradiated skin serving as a control specimen. RESULTS: Murine skin treated with topical Smad3 siRNA demonstrated effective Smad3 inhibition at 1 week and persistent suppression at 4 weeks. Collagen deposition and epidermal thickness were significantly decreased in skin treated with Smad3 siRNA compared with control irradiated skin. Tensiometry demonstrated decreased tension in Smad3 siRNA-treated skin, with a Young modulus of 9.29 MPa (nonirradiated normal skin, 7.78 MPa) compared with nonsense (control) siRNA-treated skin (14.68 MPa). CONCLUSIONS: Smad3 expression can be effectively silenced in vivo using a novel topical delivery system. Moreover, cutaneous Smad3 inhibition mitigates radiation-induced changes in tissue elasticity, restoring a near-normal phenotype

BACKGROUND/AIMS: Neovascularization involves angiogenesis and vasculogenesis mediated by cytokines and soluble chemokines. The predominant stimulus is ischemia, however, recent data suggest that ionizing radiation (IR) has angiogenic potential. In this study we evaluated whether IR increases vascularity and perfusion in vivo. METHODS: In wild-type mice, a full-thickness, pedicled skin flap was created and isolated for localized irradiation at a dose of 5 Gy. Serial Doppler analysis of the flap was performed. The skin flaps were then harvested at various time points for vascularity and histologic analysis. Blood was concurrently harvested for serum and hematopoietic progenitor cell population analysis. RESULTS: IR to an ischemic flap augmented the angiogenic cytokines SDF-1 and VEGF. Serum MMP-9 and s-kit levels, which are critical for progenitor cell mobilization, were also increased. When hematopoietic progenitor cells were evaluated by Sca1+/Flk1+ cells, a correlate 2-fold increase was seen compared to controls. When the flaps were examined, both vascularity and perfusion were increased. CONCLUSION: In this study we demonstrate that local, low-dose IR upregulates angiogenic chemokines and results in progenitor cell mobilization to the systemic circulation. There is a resultant increase in the vascularity of the irradiated flap, suggesting that the pro-angiogenic effects of IR can be harnessed locally

Blood vessel growth is regulated by angiogenic and angiostatic CXC chemokines, and radiation is a vasculogenic stimulus. We investigated the effect of radiation on endothelial cell chemokine signaling, receptor expression, and migration and apoptosis. Human umbilical vein endothelial cells were exposed to a single fraction of 0, 5, or 20Gy of ionizing radiation (IR). All vasculogenic chemokines (CXCL1-3/5-8) increased 3-13-fold after 5 or 20Gy IR. 20Gy induced a marked increase (1.6-4-fold) in angiostatic CXC chemokines. CXCR4 expression increased 3.5 and 7-fold at 48h after 5 and 20Gy, respectively. Bone marrow progenitor cell chemotaxis was augmented by conditioned media from cells treated with 5Gy IR. Whereas 5Gy markedly decreased intrinsic cell apoptosis (0Gy=16%+/-3.6 vs. 5Gy=4.5%+/-0.3), 20Gy increased it (21.4%+/-1.2); a reflection of pro-survival angiogenic chemokine expression. Radiation induces a dose-dependent increase in pro-angiogenic CXC chemokines and CXCR4. In contrast, angiostatic chemokines and apoptosis were induced at higher (20Gy) radiation doses. Cell migration improved significantly following 5Gy, but not 20Gy IR. Collectively, these data suggest that lower doses of IR induce an angiogenic cascade while higher doses produce an angiostatic profile

BACKGROUND: The study of human autologous fat grafting has been primarily anecdotal. In this study, the authors aim to develop a murine model that recapitulates human fat grafting to study the fate of injected fat and the cell populations contained within. METHODS: The authors' method of fat harvesting and refinement has been described previously. The authors injected nude and tie2/lacZ mice with 2 ml of human lipoaspirate placed on the dorsal surface in a multipass, fan-like pattern. Fatty tissue was injected in small volumes of approximately 1/30 ml per withdrawal. The dorsal skin and associated fat was excised at various time points. Sections were stained with hematoxylin and eosin and cytochrome c oxidase IV. Transgenic tie2/lacZ samples were stained with X-galactosidase. At the 8-week time point, volumetric analysis was performed. RESULTS: Volumetric analysis at the 8-week time point showed 82 percent persistence of the original volume. Gross analysis showed it to be healthy, nonfibrotic, and vascularized. Hematoxylin and eosin analysis showed minimal inflammatory or capsular reaction, with viable adipocytes. Fat grafted areas were vascularized with multiple blood vessels. Cytochrome c oxidase IV human-specific stain and beta-galactosidase expression revealed these vessels to be of human origin. CONCLUSIONS: The authors have developed a murine model with which to study the fate of injected lipoaspirate. There is a high level of persistence of the grafted human fat, with minimal inflammatory reaction. The fat is viable and vascularized, demonstrating human-derived vessels in a mouse model. This model provides a platform for studying the populations of progenitor cells known to reside in lipoaspirate