Bone marrow-derived cell (BMDC) recruitment to diabetic wounds in GFP bone marrow chimeras is dramatically increased compared with nondiabetic wounds. (A): Representative bone marrow chimerism flow cytometry analysis of peripheral blood using non-GFP chimera siblings as negative controls. (B): Representative day 7 wound section stained with hematoxylin and eosin is shown in the upper half of the panel. A graphic of this section is shown in the lower half of the panel. Red cross-hatching represents the wound. Rectangles 1, 2, and 3 represent areas near the wound boundary imaged using confocal microscopy to obtain average recruited BMDC number for each animal. Scale bar = 1 mm. (C): Relative number of BMDCs (normalized using DAPI-stained nuclei to account for cell density differences) recruited to the wound site in unwounded nondiabetic controls (wt D0), unwounded diabetic (db D0), day 7 wounds from nondiabetic (wt D7) and diabetic (db D7) mice. Asterisk indicates p < .05. (D): Representative images of DAPI staining and GFP immunofluorescence of BMDCs in wound sections as shown in (B) from nondiabetic (wt) and diabetic (db) mice at day 0 and day 7 following wounding. Scale bar = 100 μm. Abbreviations: d, dermis, DAPI, 4′,6-diamidino-2-phenylindole; db, diabetic; D0, day 0; D7, day 7; ep, epithelium, es, eschar, Freq, frequency; FSC-H, forward scatter height; GFP, green fluorescent protein; gr, granulation tissue; m, muscle; neg cntl, negative control; SSC-H, side scatter height; wt, wild type.

Expression of HOXA3 during diabetic wound repair modulates the recruitment of bone marrow-derived cells (BMDCs). (A): Representative images of wound sections from diabetic mice with GFP immunofluorescent detection of BMDCs at days 0, 7, and 14 following wounding, treated with control (empty vector) or CMV-HOXA3 plasmid. Scale bar = 100 μm. (B): Relative number of BMDCs (normalized against 4′,6-diamidino-2-phenylindole [DAPI]-stained nuclei) in wounds from diabetic mice treated with control (DB+cntl) or CMV-HOXA3 plasmid (DB+A3) at day 0, day 7, and day 14 following wounding. Asterisk indicates p < .05. Abbreviations: DB+A3, diabetic wound treated with CMV-HOXA3; db+A3 D7, diabetic wound treated with CMV-HOXA3 at day 7; db+A3 D14, diabetic wound treated with CMV-HOXA3 at day 14; DB+cntl, diabetic wound treated with control; db+cntl D7, diabetic wound treated with control at day 7; db+cntl D14, diabetic wound treated with control at day 14; db D0, diabetic wound at day 0; GFP, green fluorescent protein.

Expression of HOXA3 in diabetic wounds suppresses inflammatory cell recruitment. (A): Representative images of sections from day 0, 7, and 14 wounds from nondiabetic and diabetic mice treated with control or CMV-HOXA3 plasmid. Upper panels of each group show GFP⁺ cells and lower panels show Cd45⁺ cells. Scale bar = 100 μm. (B): Bar graphs showing normalized relative numbers of GFP⁺ and GFP⁺Cd45⁺ (leukocyte) cells recruited to the wounds at days 0, 7, and 14 in nondiabetic and diabetic mice treated with control or CMV-HOXA3 plasmid. Abbreviations: D0, day 0; D7, day 7; D14, day 14; db+A3, diabetic wound treated with CMV-HOXA3; db+A3 D7, diabetic wound treated with CMV-HOXA3 at day 7; db+A3 D14, diabetic wound treated with CMV-HOXA3 at day 14; db+C and db+c, diabetic wound treated with control; db D0, diabetic wound at day 0; db D7, diabetic wound at day 7; db D14, diabetic wound at day 14; GFP, green fluorescent protein; hc+c, heterozygous control + control plasmid; non-db D0, nondiabetic wound at day 0; non-db D7, nondiabetic wound at day 7; non-db D14, nondiabetic wound at day 14.

Expression of HOXA3 in diabetic wounds stimulates mobilization of endothelial progenitor cells (EPCs). (A): Representative images of wound sections from nondiabetic mice (non-db) and diabetic mice (db) treated with control (empty vector) or CMV-HOXA3 plasmid. Upper panels show GFP⁺ bone marrow-derived cells at day 7 and lower panels show Cd34⁺ cells. Scale bar = 100 μm. (B): Percentage of total GFP⁺ cells that are double positive for Cd34 recruited to the wound at day 7 in nondiabetic mice treated with control plasmid, and diabetic mice treated with control (db+cntl) or CMV-HOXA3 (db+A3) plasmid. (C): Representative scatter plots of cleared peripheral blood from diabetic GFP chimeras at day 4 following wounding treated with control (db2+c) or CMV-HOXA3 (db7+A3) plasmid. Cells in the upper right quadrant are GFP⁺Cd133⁺Cd34⁺ (early EPC) cells. (D): Quantification of early EPCs in peripheral blood at day 4 following wounding of control-treated or CMV-HOXA3-treated diabetic wounds. (E): Representative scatter plots of cleared peripheral blood from diabetic GFP chimeras at day 7 following wounding treated with control (db2+c) or CMV-HOXA3 (db7+A3) plasmid. Cells in the upper right quadrant are GFP⁺Cd133⁺Cd34⁺ (early EPC) cells. (F): Quantification of early EPCs in peripheral blood at day 7 following wounding of control-treated or CMV-HOXA3-treated diabetic wounds. Abbreviations: APC, allophycocyanin; db+A3 D7, diabetic wound treated with CMV-HOXA3 at day 7; db+a3 and db+A3, diabetic wound treated with CMV-HOXA3 at day 3; db+C, diabetic wound treated with control; db+cntl, diabetic wound treated with control; db+cntl D7, diabetic wound treated with control at day 7; db1+C, diabetic wound 1 treated with control; db2+C, diabetic wound 2 treated with control; db7+A3, diabetic wound treated with CMV-HOXA3 at day 7; FMO, fluorescence minus one control; GFP, green fluorescent protein; non-db D7, nondiabetic wound at day 7; non db, nondiabetic wound; PB, peripheral blood; PE, phycoerythrin.

The HOXA3 transgene is not expressed in peripheral blood or bone marrow cells and has no effect on endogenous Hoxa3. (A): Quantitative reverse-transcription polymerase chain reaction (RT-PCR) of RNA isolated from wound tissue, peripheral blood, and bone marrow from diabetic mice treated with control or CMV-HOXA3 plasmid showing relative expression of the HOXA3 transgene at day 4 following wounding. (B): Quantitative RT-PCR of RNA isolated from wound tissue from diabetic mice treated with control or CMV-HOXA3 plasmid showing relative expression of endogenous Hoxa3. (C): Analysis of transgene expression 4 days after wounding and gene transfer. Transgene expression is shown in green in the upper left panel, white arrowhead indicates expression in hair follicle cells, yellow arrowhead indicates expression in basal keratinocytes, and red arrowhead indicates expression in leukocytes. Leukocytes are marked with red (Cd45) in upper middle panel, and Cd34⁺ cells are shown blue in upper right panel. Lower panels show merged channels as indicated. Scale bar = 100 μm. Abbreviations: +A3, plus HOXA3; BM, bone marrow; cntl, control; PB, peripheral blood; Tg, transgene; Wnd, wound tissue.

Expression of HOXA3 in diabetic wounds results in activation of proangiogenic genes and repression of proinflammatory genes at the wound site. (A): Schematic representation of microarray experiment. Mice were split into two groups; unwounded skin was harvested at day 0 and wounded skin, at day 4. Gene expression profiles of control-treated and HOXA3-treated wounds were compared. (B): NF-κB pathway member gene expression analysis in response to CMV-HOXA3 treatment. (C): Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) validation of selected genes that are central to NF-κB pathway signal transduction. (D): qRT-PCR of Tnfa expression in control-treated and CMV-HOXA3-treated wounds at days 0, 4, and 7. (E): Proangiogenic gene expression analysis in response to CMV-HOXA3 treatment of diabetic wounds. (F): qRT-PCR validation of selected genes known to promote endothelial progenitor cell (EPC) mobilization and recruitment to sites of neovascularization. (G): Model of HOXA3 function during wound repair. Expression of HOXA3 in diabetic wounds results in downregulation of members of the NF-κB pathway, leading to a decrease in Tnf-α expression. This results in reduced inflammation and a wound environment more conducive to repair and vascular regeneration. Abbreviations: +A3, plus HOXA3; cntl, control; downreg, downregulation; NF-κB, nuclear factor κB; qPCR, quantitative polymerase chain reaction; TNF-α, tumor necrosis factor α; upreg, upregulation.