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First published online 24 May 2005
doi: 10.1242/jcs.02399

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HOXA3 induces cell migration in endothelial and epithelial cells promoting angiogenesis and wound repair

Surgical Research Laboratory, Department of Surgery, University of California San Francisco, San Francisco General Hospital, 1001 Potrero Avenue, San Francisco, CA 94110, USA

View larger version (43K): [in a new window]   Fig. 1. HOXA3 induces endothelial cell migration and angiogenesis in vivo. (a) Semi-quantitative RT-PCR of a 900 bp product corresponding to HOXA3 in HMEC transfected with control or HOXA3 expression plasmid (upper panel). 250 bp ladder in the first lane represents from top to bottom: 1000 bp, 750 bp and 500 bp. Lower panel, western blot for the 46 kDa HOXA3/myc/His fusion protein expressed by HMEC-1 transfected with HOXA3 myc/His cDNA. (b) Migration of control or HOXA3-transfected HMEC in modified Boyden chamber coated with collagen I. As a positive control, 50 ng VEGF165 was added to the lower chambers. Migration was assessed after 4 hours and results are expressed as the mean±s.d. **P<0.05 relative to migration of control cells. (c) CAM tissue (original magnification x15) 72 hours after application of control (ß-gal, left-hand panel) or HOXA3 expression plasmids (middle panel), or recombinant VEGF165 (right-hand panel). (d) Quantification of angiogenesis induced by control or HOXA3 expression plasmids, or in the presence of 50 ng of VEGF165. **P<0.05 compared with levels in control cells. Results are the mean±s.d. of 12 experiments.

View larger version (30K): [in a new window]   Fig. 2. HOXA3 upregulates MMP-14 and uPAR in endothelial cells. (a) Northern blot analysis of uPAR and MMP-14 expression in control and HOXA3-transfected HMEC-1. Bottom panel shows relative RNA loading visualized by ethidium bromide staining of rRNA. (b) Western blot analysis of uPAR protein in control and HOXA3-transfected EC (upper panel). Lower panel, western blot of MMP-14 in cell lysates from control or HOXA3-transfected HMEC-1.

View larger version (97K): [in a new window]   Fig. 3. Requirement of uPAR for HOXA3-dependent invasion of endothelial cells. (a) Control-transfected HMEC after 48 hours in fibrin gels (original magnification x100). Extensive invasion of fibrin gels is observed after 48 hours in HMEC-1 stably expressing HOXA3 (b). (c) Invasion of HOXA3-expressing HMEC-1, 48 hours after addition of function-blocking antibodies against uPAR. (d) Migration was quantified by measuring the distance from the edge of the cytodex bead to the most distal point of migration in the gel, control (open bar), +HOXA3 (black bar), or +HOXA3 plus 25 µg of a function blocking antibody against uPAR (hatched bar). Results are the mean±s.d. of four experiments. **P<0.05 when compared with migration of control cells.

View larger version (82K): [in a new window]   Fig. 4. HOXA3 is upregulated in response to injury in angiogenic vessels. (a) In situ hybridization for HOXA3 expression was performed on sections of control (unwounded) mouse skin (original magnification x400). Little or no signal (purple/black) was detected in resting endothelial cells (arrows). Note the strong positive signal in the adjacent hair follicle (h). (b) In situ hybridization in tissue harvested 1 day following administration of a full-thickness incisional wound shows an increase in mRNA expression in vessels immediately adjacent to the site of the wound. (c) By 4 days following wounding, strong signals were visible in endothelial cells of neovessels within the granulation tissue, as well as other (unidentified) cells in the region. (d) Sense strand control at day 4. (e) Semi-quantitative RT-PCR of HOXA3 mRNA levels 7 days following wounding in heterozygous (wt) and diabetic (db/db) mice. 1 kb ladder in lane 1 represents 2 kb, 1.6 kb, 1 kb, 0.5 kb, 0.4 kb, 0.34 kb, 0.3 kb and 0.22 kb (top to bottom). The 900 bp band corresponding to HOXA3 mRNA is abundantly expressed in wild type but not diabetic wound tissue.

View larger version (30K): [in a new window]   Fig. 5. HOXA3 upregulates MMP-14 and uPAR gene expression in vivo during wound repair. (a) Western blot of HOXA3/myc/His fusion protein in diabetic wound tissue harvested at 4 days after treatment with control or HOXA3 expression plasmids. (b) Analysis of murine MMP-14 expression (relative to 18S RNA expression) in heterozygous (wt), untreated Leprdb/Leprdb homozygotes (db) or homozygotes treated with HOXA3 expression plasmids (db +HOXA3). **P<0.05 when compared with expression in untreated homozygotes. Results are the mean±s.d. of two experiments. (c) Quantitative real time PCR analysis of murine uPAR mRNA expression (relative to GUS expression) in Leprdb/Leprdb homozygotes treated with control or HOXA3 expression plasmids. **P<0.01 compared with expression in control cells. Results are the mean±s.d. of four experiments. (d) Western blot analysis of uPAR expression in control or HOXA3-treated wounds.

View larger version (38K): [in a new window]   Fig. 6. HOXA3 promotes angiogenesis and wound repair in vivo. (a-c) Effect of HOXA3 treatment on wound closure. Gross appearance of 2.5 cm wounds in Leprdb/Leprdb mice, 14 days after treatment with (a) control plasmid, or (b) HOXA3 expression plasmid. (c) Wound closure in control- (pink) and HOXA3-treated (blue) diabetic wounds. Wound area was measured every 7 days following excision of a 2.5 cm diameter wound in Leprdb/Leprdb mice. Mice treated with HOXA3 showed a significantly (**P<0.05) greater degree of closure at days 7, 14, 21, 28, 35 and 42 days when compared with closure of control plasmid-treated Leprdb/Leprdb wounds (HOXA3, n=10; control, n=10). (d-f) Analysis of vascular density in Leprdb/Leprdb wounds 4 days following treatment with control (d) or HOXA3 (e) expression plasmids. 10 µm cryosections were stained with antibodies against CD31 (original magnification x200). (f) Histological scores (±s.d.) of tissue sections from wounds 7 days after administration of control or HOXA3 expression plasmids. Scores include quantification of vascular density, epithelial and fibroblast cell density, collagen deposition, and granulation tissue formation. **P<0.05 compared with score in control cells.

View larger version (61K): [in a new window]   Fig. 7. HOXA3 promotes keratinocyte migration in a uPAR-dependent manner. Photomicrographs of BALB/c MK keratinocytes stably transfected with control cDNA (original magnification x40) (a) or HOXA3 construct (b), 48 hours after administration of a scratch wound (denuded area indicated with black box outline). (c) Quantitative analysis of migration following scratch wounds in control or HOXA3-expressing keratinocytes in the presence or absence of function-blocking antibodies against murine uPAR. Results are the means±s.d. of 12 experiments.