Guy P. Marti

Age-dependent impairment of HIF-1α expression in diabetic mice: Correction with electroporation-facilitated gene therapy increases wound healing, angiogenesis, and circulating angiogenic cells

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 (CACs) 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. CACs 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 CACs that contribute to the age‐dependent impairment of wound healing in db/db mice. J. Cell. Physiol. 217: 319–327, 2008.

Impaired angiogenesis and mobilization of circulating angiogenic cells in HIF‐1α heterozygous‐null mice after burn wounding

Hypoxia‐inducible factor 1 (HIF‐1) is a transcription factor that controls vascular responses to hypoxia and ischemia. In this study, mice that were heterozygous (HET) for a null allele at the locus encoding the HIF‐1α subunit (HET mice) and their wild‐type (WT) littermates were subjected to a thermal injury involving 10% of the body surface area. HIF‐1α protein levels were increased in burn wounds of WT but not of HET mice on day 2. The serum levels of stromal‐derived factor 1α, which binds to CXCR4, were increased on day 2 in WT but not in HET mice. Circulating angiogenic cells were also increased on day 2 in WT but not in HET mice and included CXCR4+Sca1+ cells. Laser Doppler perfusion imaging demonstrated increased blood flow in burn wounds of WT but not HET mice on day 7. Immunohistochemistry on day 7 revealed a reduced number of CD31+ vessels at the healing margin of burn wounds in HET as compared with WT mice. Vessel maturation was also impaired in wounds of HET mice as determined by the number of α‐smooth muscle actin‐positive vessels on day 21. The remaining wound area on day 14 was significantly increased in HET mice compared with WT littermates. The percentage of healed wounds on day 14 was significantly decreased in HET mice. These data delineate a signaling pathway by which HIF‐1 promotes angiogenesis during burn wound healing.

Current Insights into the role of HIF-1 in cutaneous wound healing.

Hypoxia Inducible Factor-1 (HIF-1) is considered the major coordinator of the cellular adaptive response to hypoxia. Over recent years, its activity in the context of wound healing has been the object of increasing investigation. On the molecular level, HIF-1 transcriptional target products have been shown to regulate the process of endothelial cell survival, migration and proliferation (VEGF, ANGPT-1, ANGPT-2, ANGPT-4, FGF-2, PlGF, PDGF-B, RGC-32), vascular smooth muscle cell migration and proliferation (FGF-2, EGF, PDGF, thrombospondin) and mobilization of Circulating Angiogenic Cells to the periphery (SFD-1/CXCR4). Studies on the effect of HIF-1 on the expression and activity of extracellular cell matrix modifying enzymes, such as MMPs and prolidase, have been conducted in the context of tumor angiogenesis and metastasis, and have resulted in controversial findings. A growing body of evidence suggests that HIF-1 also affects reepithelialization of the wound bed, through increasing keratinocyte migration, but decreasing their proliferation. Diminished HIF-1 levels and activity have been documented in conditions of impaired wound healing, such as wound healing in aged and in diabetic mice. The increasing number of studies on the role of HIF-1 in wound healing, apart from answering certain questions, has also raised an equal number, if not more. Clarifying the topics that still remain unclear could introduce a new era of HIF-1 targeted management of a wide range of problematic wounds.

Cellular GFP Toxicity and Immunogenicity: Potential Confounders in in Vivo Cell Tracking Experiments

Green Fluorescent protein (GFP), used as a cellular tag, provides researchers with a valuable method of measuring gene expression and cell tracking. However, there is evidence to suggest that the immunogenicity and cytotoxicity of GFP potentially confounds the interpretation of in vivo experimental data. Studies have shown that GFP expression can deteriorate over time as GFP tagged cells are prone to death. Therefore, the cells that were originally marked with GFP do not survive and cannot be accurately traced over time. This review will present current evidence for the immunogenicity and cytotoxicity of GFP in in vivo studies by characterizing these responses.

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.

Delivery of plasmid DNA expression vector for keratinocyte growth factor-1 using electroporation to improve cutaneous wound healing in a septic rat model

We have previously shown that wound healing was improved in a diabetic mouse model of impaired wound healing following transfection with keratinocyte growth factor‐1 (KGF‐1) cDNA. We now extend these findings to the characterization of the effects of DNA plasmid vectors delivered to rats using electroporation (EP) in vivo in a sepsis‐based model of impaired wound healing. To assess plasmid transfection and wound healing, gWIZ luciferase and PCDNA3.1/KGF‐1 expression vectors were used, respectively. Cutaneous wounds were produced using an 8 mm‐punch biopsy in Sprague–Dawley rats in which healing was impaired by cecal ligation‐induced sepsis. We used National Institutes of Health image analysis software and histologic assessment to analyze wound closure and found that EP increased expression of gWIZ luciferase vector up to 53‐fold compared with transfection without EP (p<0.001). EP‐assisted plasmid transfection was found to be localized to skin. Septic rats had a 4.7 times larger average wound area on day 9 compared with control (p<0.001). Rats that underwent PCDNA3.1/KGF‐1 transfection with EP had 60% smaller wounds on day 12 compared with vector without EP (p<0.009). Quality of healing with KGF‐1 vector plus EP scored 3.0±0.3 and was significantly better than that of 1.8±0.3 for treatment with vector alone (p<0.05). We conclude that both the rate and quality of healing were improved with DNA plasmid expression vector for growth factor delivered with EP to septic rats.

KGF-1 for Wound Healing in Animal Models

Keratinocyte growth factor-1 (KGF-1) is a member of the fibroblast growth factor (FGF) family FGF7 and is expressed in normal and wounded skin. KGF-1 is massively produced in the early stages of the wound healing process as well as during the later remodeling process (1, 2). We have studied the effects of the electroporation of a KGF-1 plasmid into excisional wounds of different rodent models mimicking diseases known to impair the normal wound healing process. We have used a genetically diabetic mouse model and a septic rat model in our experiments, and we have shown improvement of the healing rate (92% of the wounds are healed at day 12 vs. 40% of the control), the quality of epithelialization (histological score of 3.3 vs. 1.5), and the density of new blood vessels (85% more new blood vessels in the superficial layers than that of the control) (3, 4). Considering these results, we believe we can further explore the treatment modalities for using the electroporation-assisted transfection of DNA plasmid expression vectors of growth factors to enhance cutaneous wound healing.

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 HIFs 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 vascular endothelial growth factor 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 vascular endothelial growth factor 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.

Association of Increasing Burn Severity in Mice With Delayed Mobilization of Circulating Angiogenic Cells

OBJECTIVE To perform a systematic exploration of the phenomenon of mobilization of circulating angiogenic cells (CACs) in an animal model. This phenomenon has been observed in patients with cutaneous burn wounds and may be an important mechanism for vasculogenesis in burn wound healing. DESIGN We used a murine model, in which burn depth can be varied precisely, and a validated culture method for quantifying circulating CACs. SETTING Michael D. Hendrix Burn Research Center, Baltimore, Maryland. PARTICIPANTS Male 129S1/SvImJ mice, aged 8 weeks, and 31 patients aged 19-59 years with burn injury on 1% to 64% of the body surface area and evidence of hemodynamic stability. MAIN OUTCOME MEASURES Burn wound histological features, including immunohistochemistry for blood vessels with CD31 and alpha-smooth muscle actin antibodies, blood flow measured with laser Doppler perfusion imaging, and mobilization of CACs into circulating blood measured with a validated culture technique. RESULTS Increasing burn depth resulted in a progressive delay in the time to mobilization of circulating CACs and reduced mobilization of CACs. This delay and reduction in CAC mobilization was associated with reduced perfusion and vascularization of the burn wound tissue. Analysis of CACs in the peripheral blood of the human patients, using a similar culture assay, confirmed results previously obtained by flow cytometry, that CAC levels peak early after the burn wound. CONCLUSION If CAC mobilization and wound perfusion are important determinants of clinical outcome, then strategies designed to augment angiogenic responses may improve outcome in patients with severe burn wounds.

Tie2-dependent knockout of HIF-1 impairs burn wound vascularization and homing of bone marrow-derived angiogenic cells

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.