Topical Gene Delivery to Murine Skin

Yu, Wei; Kashani‐Sabet, Mohammed; Liggitt, Denny; Moore, Dan H.; Heath, Timothy D.; Debs, Robert J.

doi:10.1046/j.1523-1747.1999.00513.x

Cited by 79 publications

(36 citation statements)

References 31 publications

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“…[31][32][33][34] The longer duration of expression of the HoxD3 transgene in our current study is likely attributed to the methylcellulose carrier that retains DNA at the wound site and slows diffusion of the plasmid into the wound and tissue fluid. A recent study also suggested that a major limitation of plasmid DNA gene transfer was the inability of plasmid DNA to effectively enter the nucleus.…”

Section: Discussionmentioning

confidence: 79%

HoxD3 Accelerates Wound Healing in Diabetic Mice

Hansen¹,

Myers²,

Charboneau³

et al. 2003

The American Journal of Pathology

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Poorly healing diabetic wounds are characterized by diminished collagen production and impaired angiogenesis. HoxD3, a homeobox transcription factor that promotes angiogenesis and collagen synthesis, is up-regulated during normal wound repair whereas its expression is diminished in poorly healing wounds of the genetically diabetic (db/db) mouse. To determine whether restoring expression of HoxD3 would accelerate diabetic wound healing, we devised a novel method of gene transfer, which incorporates HoxD3 plasmid DNA into a methylcellulose film that is placed on wounds created on db/db mice. The HoxD3 transgene was expressed in endothelial cells, fibroblasts, and keratinocytes of the wounds for up to 10 days. More importantly, a single application of HoxD3 to db/db mice resulted in a statistically significant acceleration of wound closure compared to control-treated wounds. Furthermore, we also observed that the HoxD3-mediated improvement in diabetic wound repair was accompanied by increases in mRNA expression of the HoxD3 target genes, Col1A1 and ␤3-integrin leading to enhanced angiogenesis and collagen deposition in the wounds. Although HoxD3-treated wounds also show improved re-epithelialization as compared to control db/db wounds, this effect was not due to direct stimulation of keratinocyte migration by HoxD3. Finally, we show that despite the dramatic increase in collagen synthesis and deposition in HoxD3-treated wounds, these wounds showed normal remodeling and we found no evidence of abnormal wound healing. These results indicate that HoxD3 may provide a means to directly improve collagen deposition, angiogenesis and closure in poorly healing diabetic wounds. Wound healing is an intricate process involving the communication and interaction between fibroblasts, endothelial cells, keratinocytes, inflammatory cells, and the extracellular matrix. Disruption of these interactions can impair angiogenesis and/or collagen synthesis resulting in wounds that heal slowly and incompletely.

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Section: Discussionmentioning

confidence: 79%

HoxD3 Accelerates Wound Healing in Diabetic Mice

Hansen¹,

Myers²,

Charboneau³

et al. 2003

The American Journal of Pathology

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“…Each of the two wounds was topically treated once daily (days 0 -5) with either empty plasmid (pEXV) or expression plasmid pEXVracV12 (constitutively active Rac1) (21). Twenty-five microliters of naked plasmid DNA (10 g) in sterile Tris-EDTA, pH 8.0, (TE) were administered onto the wound area and spread evenly using the pipette tip.…”

Section: Rac1 Gene Transfermentioning

confidence: 99%

Oxidant-induced Vascular Endothelial Growth Factor Expression in Human Keratinocytes and Cutaneous Wound Healing

Sen

Khanna

Babior

et al. 2002

Journal of Biological Chemistry

291

218

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Neutrophils and macrophages, recruited to the wound site, release reactive oxygen species by respiratory burst. It is commonly understood that oxidants serve mainly to kill bacteria and prevent wound infection. We tested the hypothesis that oxidants generated at the wound site promote dermal wound repair. We observed that H 2 O 2 potently induces vascular endothelial growth factor (VEGF) expression in human keratinocytes. Deletion mutant studies with a VEGF promoter construct revealed that a GC-rich sequence from bp ؊194 to ؊50 of the VEGF promoter is responsible for the H 2 O 2 response. It was established that at M concentrations oxidant induces VEGF expression and that oxidant-induced VEGF expression is independent of hypoxia-inducible factor (HIF)-1 and dependent on Sp1 activation. To test the effect of NADPH oxidase-generated reactive oxygen species on wound healing in vivo, Rac1 gene transfer was performed to dermal excisional wounds left to heal by secondary intention. Rac1 gene transfer accelerated wound contraction and closure. Rac1 overexpression was associated with higher VEGF expression both in vivo as well in human keratinocytes. Interestingly, Rac1 gene therapy was associated with a more well defined hyperproliferative epithelial region, higher cell density, enhanced deposition of connective tissue, and improved histological architecture. Overall, the histological data indicated that Rac1 might be an important stimulator of various aspects of the repair process, eventually enhancing the wound-healing process as a whole. Taken together, the results of this study indicate that wound healing is subject to redox control.

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“…Epidermal keratinocytes and dendritic cells have the ability to take up and express naked plasmid DNA upon intradermal in vivo injection and topical application, [1][2][3][4][5][6][7] making them attractive targets for gene therapy and DNA vaccination.…”

Section: Introductionmentioning

confidence: 99%

Uptake and trafficking of DNA in keratinocytes: evidence for DNA-binding proteins

et al. 2004

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The skin is an interesting organ for human gene therapy due to accessibility, immunologic potential and synthesis capabilities. In this study, we attempted to visualize and measure the uptake of naked FITC-labeled plasmid by FACS analysis detecting up to 15% internalization in a doseand time-dependent manner. Cycloheximide treatment inhibited the uptake by 490%, suggesting a protein-mediated uptake. The inhibition of different internalization pathways demonstrated that blocking macropinocytosis (by amiloride and N,N-dimethylamylorid) reduced DNA uptake by 485%, while the inhibition of clathrin-coated pits (by chlorpromazine) and caveolae (by nystatin/filipin III) did not limit the uptake. Colocalization studies using confocal laser microscopy revealed a time-dependent accumulation of plasmid DNA in endosomes and lysosomes. When a green fluorescent protein (GFP) expression vector was used, specific GFP-RNA became detectable by reverse transcriptase-PCR, whereas measurable amounts of protein could not be identified in FACS experiments. To detect the potential DNA receptors on the keratinocyte surface, membrane proteins were extracted and subjected to South-Western blotting using digoxigenin-labeled calf thymus and l-phage DNA. Two DNA-binding proteins, ezrin and moesin, known as plasma membrane-actin linkers, were identified by one-and two-dimensional-South-Western blots and matrix-assisted laser desorption and ionizationmass spectrometry. Ezrin and moesin are functionally associated with a number of transmembrane receptors such as the EGF, CD44 or ICAM-1 receptor. Taken together, naked plasmid DNA seems to enter human keratinocytes through different pathways, mainly by macropinocytosis. Two DNA-binding proteins were identified that seemed to be involved in binding/trafficking of internalized DNA.

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Topical Gene Delivery to Murine Skin

Cited by 79 publications

References 31 publications

HoxD3 Accelerates Wound Healing in Diabetic Mice

HoxD3 Accelerates Wound Healing in Diabetic Mice

Oxidant-induced Vascular Endothelial Growth Factor Expression in Human Keratinocytes and Cutaneous Wound Healing

Uptake and trafficking of DNA in keratinocytes: evidence for DNA-binding proteins

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