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author:("mart1, Guy P.")
1.  Strengthening the Skin with Topical Delivery of Keratinocyte Growth Factor-1 Using a Novel DNA Plasmid 
Molecular Therapy  2014;22(4):752-761.
Fragile skin, susceptible to decubitus ulcers and incidental trauma, is a problem particularly for the elderly and for those with spinal cord injury. Here, we present a simple approach to strengthen the skin by the topical delivery of keratinocyte growth factor-1 (KGF-1) DNA. In initial feasibility studies with the novel minimalized, antibiotic-free DNA expression vector, NTC8385-VA1, the reporter genes luciferase and enhanced green fluorescent protein were delivered. Transfection was documented when luciferase expression significantly increased after transfection. Microscopic imaging of enhanced green fluorescent protein–transfected skin showed green fluorescence in hair follicles, hair shafts, and dermal and superficial epithelial cells. With KGF-1 transfection, KGF-1 mRNA level and protein production were documented with quantitative reverse transcriptase–polymerase chain reaction and immunohistochemistry, respectively. Epithelial thickness of the transfected skin in the KGF group was significantly increased compared with the control vector group (26 ± 2 versus 16 ± 4 µm) at 48 hours (P = 0.045). Dermal thickness tended to be increased in the KGF group (255 ± 36 versus 162 ± 16 µm) at 120 hours (P = 0.057). Biomechanical assessment showed that the KGF-1–treated skin was significantly stronger than control vector–transfected skin. These findings indicate that topically delivered KGF-1 DNA plasmid can increase epithelial thickness and strength, demonstrating the potential of this approach to restore compromised skin.
doi:10.1038/mt.2014.2
PMCID: PMC3982499  PMID: 24434934
2.  Tie2-dependent knockout of HIF-1 impairs burn wound vascularization and homing of bone marrow-derived angiogenic cells 
Cardiovascular Research  2011;93(1):162-169.
Aims
Hypoxia-inducible factor 1 (HIF-1) is a heterodimer composed of HIF-1α and HIF-1β subunits. HIF-1 is known to promote tissue vascularization by activating the transcription of genes encoding angiogenic factors, which bind to receptors on endothelial cells (ECs) and bone marrow-derived angiogenic cells (BMDACs). In this study, we analysed whether HIF-1 activity in the responding ECs and BMDACs is also required for cutaneous vascularization during burn wound healing.
Methods and results
We generated mice with floxed alleles at the Hif1a or Arnt locus encoding HIF-1α and HIF-1β, respectively. Expression of Cre recombinase was driven by the Tie2 gene promoter, which is expressed in ECs and bone marrow cells. Tie2Cre+ and Tie2Cre− mice were subjected to burn wounds of reproducible diameter and depth. Deficiency of HIF-1α or HIF-1β in Tie2-lineage cells resulted in delayed wound closure, reduced vascularization, decreased cutaneous blood flow, impaired BMDAC mobilization, and decreased BMDAC homing to burn wounds.
Conclusion
HIF-1 activity in Tie2-lineage cells is required for the mobilization and homing of BMDACs to cutaneous burn wounds and for the vascularization of burn wound tissue.
doi:10.1093/cvr/cvr282
PMCID: PMC3243042  PMID: 22028336
Hypoxia; Wound healing; Conditional knockout; Angiogenesis
3.  Hypoxia and Hypoxia-Inducible Factor in the Burn Wound 
The importance of hypoxia-inducible factor (HIF) in promoting angiogenesis and vasculogenesis during wound healing has been demonstrated. It is widely accepted that HIF activity can be promoted by many factors, including hypoxia in the wound or cytokines from inflammatory cells infiltrating the wound. However, there has not been a systematic exploration of the relationship between HIF activity and hypoxia in the burn wound. The location of the hypoxic tissue has not been clearly delineated. The time course of the appearance of hypoxia and the increased activity of HIF and appearance of HIF’s downstream transcription products has not been described. The aim of this study was to utilize pimonidazole, a specific tissue hypoxia marker, to characterize the spatial and temporal course of hypoxia in a murine burn model and correlate this with the appearance of HIF-1α and its important angiogenic and vasculogenic transcription products VEGF and SDF-1. Hypoxia was found in the healing margin of burn wounds beginning at 48 hours after burn and peaking at day 3 after burn. On sequential sections of the same tissue block, positive staining of HIF-1α, SDF-1, and VEGF all occurred at the leading margin of the healing area and peaked at day 3, as did hypoxia. Immunohistochemical analysis was used to explore the characteristics of the hypoxic region of the wound. The localization of hypoxia was found to be related to cell growth and migration, but not to proliferation or inflammatory infiltration.
doi:10.1111/j.1524-475X.2010.00656.x
PMCID: PMC3075089  PMID: 21362088
ypoxia; Hypoxia-inducible factor -10α; Burn; Wound Healing; Ki67 Cell Proliferation; Keratin17
4.  Age-dependent Impairment of HIF-1α̣Expression in Diabetic Mice: Correction with Electroporation-facilitated Gene Therapy Increases Wound Healing, Angiogenesis, and Circulating Angiogenic Cells 
Journal of cellular physiology  2008;217(2):319-327.
Wound healing is impaired in elderly patients with diabetes mellitus. We hypothesized that age-dependent impairment of cutaneous wound healing in db/db diabetic mice: (a) would correlate with reduced expression of the transcription factor hypoxia-inducible factor 1α (HIF-1α) as well as its downstream target genes; and (b) could be overcome by HIF-1α replacement therapy. Wound closure, angiogenesis, and mRNA expression in excisional skin wounds were analyzed and circulating angiogenic cells were quantified in db/db mice that were untreated or received electroporation-facilitated HIF-1α gene therapy. HIF-1α mRNA levels in wound tissue were significantly reduced in older (4–6 months) as compared to younger (1.5–2 months) db/db mice. Expression of mRNAs encoding the angiogenic cytokines vascular endothelial growth factor (VEGF), angiopoietin 1 (ANGPT1), ANGPT2, platelet derived growth factor B (PDGF-B), and placental growth factor (PLGF) was also impaired in wounds of older db/db mice. Intradermal injection of plasmid gWIZ-CA5, which encodes a constitutively active form of HIF-1α, followed by electroporation, induced increased levels of HIF-1α mRNA at the injection site on day 3 and increased levels of VEGF, PLGF, PDGF-B, and ANGPT2 mRNA on day 7. Circulating angiogenic cells in peripheral blood increased 10-fold in mice treated with gWIZ-CA5. Wound closure was significantly accelerated in db/db mice treated with gWIZ-CA5 as compared to mice treated with empty vector. Thus, HIF-1α gene therapy corrects the age-dependent impairment of HIF-1α expression, angiogenic cytokine expression, and circulating angiogenic cells that contribute to the age-dependent impairment of wound healing in db/db mice.
doi:10.1002/jcp.21503
PMCID: PMC2716010  PMID: 18506785
Aging; Angiogenesis; Diabetes; Wound Healing
5.  Aging impairs the mobilization and homing of bone marrow-derived angiogenic cells to burn wounds 
Impaired wound healing in the elderly represents a major clinical problem. Delineating the cellular and molecular mechanisms by which aging impairs wound healing may lead to the development of improved treatment strategies for elderly patients with non-healing wounds. Neovascularization is an essential step in wound healing, and bone marrow-derived angiogenic cells (BMDACs) play an important role in vascularization. Using a mouse full-thickness burn wound model, we demonstrate that perfusion and vascularization of burn wounds were impaired by aging and were associated with dramatically reduced mobilization of BMDACs bearing the cell surface molecules CXCR4 and Sca1. Expression of stromal-derived factor 1 (SDF-1), the cytokine ligand for CXCR4, was significantly decreased in peripheral blood and burn wounds of old mice. Expression of hypoxia-inducible factor (HIF)-1α was detected in burn wounds from young (2-month-old), but not old (2-year-old), mice. When BMDACs from young donor mice were injected intravenously, homing to burn wound tissue was impaired in old recipient mice, whereas the age of the BMDAC donor mice had no effect on homing. Our results indicate that aging impairs burn wound vascularization by impairing the mobilization of BMDACs and their homing to burn wound tissue as a result of impaired HIF-1 induction and SDF-1 signaling.
doi:10.1007/s00109-011-0754-2
PMCID: PMC3587336  PMID: 21499736
Burn wound healing; Aging; Neovascularization; Bone marrow-derived angiogenic cells; Hypoxia-inducible factor 1; Stromal-derived factor 1

Results 1-5 (5)