β2AR Antagonists and β2AR Gene Deletion Both Promote Skin Wound Repair Processes

Skin wound healing is a complex process requiring the coordinated, temporal orchestration of numerous cell types and biological processes to regenerate damaged tissue. Previous work has demonstrated that a functional β-adrenergic receptor autocrine/paracrine network exists in skin, but the role of β2-adrenergic receptor (β2AR) in wound healing is unknown. A range of in vitro (single-cell migration, immunoblotting, ELISA, enzyme immunoassay), ex vivo (rat aortic ring assay), and in vivo (chick chorioallantoic membrane assay, zebrafish, murine wild-type, and β2AR knockout excisional skin wound models) models were used to demonstrate that blockade or loss of β2AR gene deletion promoted wound repair, a finding that is, to our knowledge, previously unreported. Compared with vehicle-only controls, β2AR antagonism increased angiogenesis, dermal fibroblast function, and re-epithelialization, but had no effect on wound inflammation in vivo. Skin wounds in β2AR knockout mice contracted and re-epithelialized faster in the first few days of wound repair in vivo. β2AR antagonism enhanced cell motility through distinct intracellular signalling mechanisms and increased vascular endothelial growth factor secretion from keratinocytes. β2AR antagonism promoted wound repair processes in the early stages of wound repair, revealing a possible new avenue for therapeutic intervention.


INTRODUCTION
Wound healing is a complex process requiring the coordinated, temporal orchestration of numerous cell types and processes to initiate repair (Shaw and Martin, 2009). The epidermis can synthesize and secrete a number of proteins (Boyce, 1994) including epinephrine (Schallreuter et al., 1992;Pullar et al., 2006b), a ligand for the b-adrenergic receptors (bARs): b1-adrenergic receptor (b1AR), b2AR, and b3AR (Wallukat, 2002). They are G protein-coupled receptors highly expressed on all cell lineages in the skin (McSwigan et al., 1981;Steinkraus et al., 1992;Izeboud et al., 1999;Iaccarino et al., 2002;de Coupade et al., 2004); therefore, an autocrine and paracrine bAR network exists in the epidermis and dermis, respectively. Previously, nonselective activation or blockade of the bARs has revealed a regulatory role for bARs in keratinocyte migration and re-epithelialization. bAR activation decreased the rate of human keratinocyte (HK) migration in vitro (Pullar et al., 2003(Pullar et al., , 2006aPullar and Isseroff, 2005), whereas a bAR antagonist promoted HK migration, by blocking the autocrine epinephrine-mediated decrease in migration (Pullar et al., 2006b). In excised human skin, bAR activation delayed wound re-epithelialization (Pullar et al., 2006a), whereas bAR antagonism promoted skin re-epithelialization (Pullar et al., 2006b) in an ex vivo model of chronic wound reepithelialization (Kratz, 1998).
Here the effect of b2AR antagonism and loss of the b2AR on the main processes in wound repair were investigated. The in vitro, ex vivo, and in vivo models allowed a thorough mechanistic investigation of skin wound repair at both the cellular and the tissue level, using a combination of pharmacological and genetic approaches.

b2AR antagonism promotes angiogenesis
Angiogenesis is an essential process in acute wound healing (Tonnesen et al., 2000), and endothelial cell (EC) migration is a critical process in angiogenesis (Eming et al., 2007). To determine whether a b2AR antagonist altered EC migration, single-cell migration (SCM) assays were performed with primary human dermal microvascular ECs (HDMECs) in the presence and absence of b2AR antagonist; however, there was no effect on migration rate ( Figure 1a).
To determine whether b2AR antagonism could alter cell outgrowth from the cut edge of rat aorta (Smith and Staton, 2006), aortic pieces were embedded into the basement membrane extract and incubated in the presence or absence of a b2AR antagonist for 5 days. b2AR antagonism increased aortic outgrowth by 66% after 5 days ( Figure 1b).
To investigate angiogenesis in a more complex, multicellular environment in vivo, the chick chorioallantoic membrane (CAM) assay, a model of embryonic angiogenesis (Smith and Staton, 2006), was selected. b2AR antagonist treatment increased angiogenesis by 48% in the CAM assay, between days 5 and 9 post fertilization ( Figure 1c).
Finally, to determine whether b2AR antagonism could modulate wound angiogenesis in vivo, two full-thickness 6mm wounds were created in the dorsal skin of wild-type (b2AR þ / þ ) and b2AR knockout (b2AR À/À ) mice (Chruscinski et al., 1999). The wounds were treated immediately after wounding and then daily, topically, with hydrogel alone or hydrogel containing 0.1% b2AR antagonist, and photographed daily. To evaluate the amount of angiogenesis in the dermis, 7-mm-thick sections from the wound center, excised after 5 days, were immunostained with an antibody to an EC marker (CD31), revealing the degree of angiogenesis in the mouse wounds (Yoon et al., 2009). b2AR antagonist treatment increased angiogenesis by 26% ( Figure 1d). The amount of angiogenesis in b2AR À/À wounds was equal to that observed in control b2AR þ / þ wounds (Figure 1d), whereas b2AR antagonist treatment of b2AR À/À wounds had no effect (results not shown).
Vascular endothelial growth factor (VEGF) is a potent proangiogenic factor in acute wounds (Barrientos et al., 2008), and bAR activation can increase VEGF expression in human choroid ECs (Steinle et al., 2008). To determine whether b2AR antagonism increased VEGF secretion from HDMECs, HKs, human dermal fibroblasts (HDFs), neutrophils, and macrophages, ELISAs were performed on control and b2AR antagonist-treated supernatants. VEGF secretion was not detected in HDMECs or HDF supernatants. Although VEGF secretion was detected in the supernatants of neutrophils and macrophages, b2AR antagonist treatment had no effect (results not shown). However, bAR antagonist treatment increased the amount of VEGF secreted from HKs after 24 hours by 23% (Figure 1e). b2AR antagonism promotes human dermal fibroblast migration in vitro and function in vivo Within a few days of wounding, fibroblasts proliferate and migrate into the wound bed (Shaw and Martin, 2009). A nonselective bAR agonist increased HDF migration via the src-dependent transactivation of EGFR (Pullar and Isseroff, 2006). To determine the effect of b2AR antagonism on HDF migration, SCM was performed in the presence and absence of a highly selective b2AR antagonist. b2AR antagonism increased the speed of HDF migration by 27% (Figure 2a). In addition, DFs were isolated from the skin of b2AR þ / þ and b2AR À/À mice. Although a b2AR antagonist increased the migration speed of b2AR þ / þ murine DFs (MDFs) by 49%, the speed of b2AR À/À MDFs was increased by 46% compared with b2AR þ / þ MDFs (Figure 2b). The b2AR antagonist had no additional effect on b2AR À/À MDF migration speed, as expected ( Figure 2b). Extracellular signal-regulated kinase (ERK) has an important role in cell migration (Klemke et al., 1997). b2AR antagonism increased b2AR þ / þ MDF ERK phosphorylation by 90% (Figure 2c).
Expression of a-smooth muscle actin (SMA) is a reliable marker of myofibroblast differentiation, and the rate of wound contraction can directly correlate with SMA expression (Hinz et al., 2001). Sections (7 mm thick) from the wound center, excised after 5 days, were immunostained with an antibody to SMA. Populations of SMA-stained fibroblasts were observed below the wound epithelial margins, 5 days post wounding. The area of SMA staining was increased by 60% in b2AR antagonist-treated wounds in b2AR þ / þ mice and was similar in control b2AR À/À and b2AR þ / þ wounds ( Figure 3b).
Myofibroblasts synthesize collagen III early in the wound repair process, which is later replaced by collagen I in more mature scars (Hinz, 2007). Sections (7 mm thick) from the wound center, excised after 5 days, were immunostained with an antibody to collagen III. b2AR antagonist treatment increased the area of collagen III-stained neo-dermis by 20% in b2AR þ / þ wounds (Figure 3c), whereas control b2AR À/À and b2AR þ / þ wounds had similar areas of www.jidonline.org 2077 collagen III staining (Figure 3c). To examine this further, sections were stained with picrosirius red to assess total collagen deposition in the neo-dermis (Junqueira et al., 1979). b2AR antagonist treatment increased the area of collagen-stained neo-dermis, within 1 mm 2 of the wound edge, by 29% in b2AR þ / þ wounds (Figure 3d), whereas control b2AR À/À and b2AR þ / þ wounds had similar areas of collagen staining (Figure 3d). b2AR antagonism has no effect on wound inflammation in vivo Neutrophils and macrophages are recruited to the wound site to quell bacterial infections, where they secrete a cocktail of growth factors and cytokines to activate resident keratinocytes and fibroblasts (Martin, 1997).
To determine whether a b2AR antagonist could alter the guidance of inflammatory cells to a wound, a zebrafish wound model was used. Indeed, zebrafish are known to have two b2AR genes (Steele et al., 2011), but their role in tissue regeneration is unknown. Zebrafish larvae are transparent and zebrafish neutrophils are identifiable 48 hours post fertilization. Upon transection of the caudal fin, neutrophils are guided to the wound within minutes, but peak neutrophil recruitment occurs after approximately 6 hours (Renshaw et al., 2006). When the wounded zebrafish larvae were immersed in pond water containing a b2AR antagonist, there was no effect on neutrophil guidance to the caudal fin wound ( Figure 4a).
To determine whether b2AR antagonist treatment altered wound inflammation in the mouse excisional skin wound model, 7-mm-thick sections from the wound center, excised after 3 days, were immunostained with either an antibody to a polymorphonuclear cell marker (Ly6G-6C) (Fleming et al., 1993) or a macrophage marker (F4-80) (Austyn and Gordon, 1981). Topical b2AR antagonist treatment or the absence of the receptor (b2AR À/À ) had no effect on the number of polymorphonuclear cells or macrophages recruited to the wound site either 3 or 5 days post wounding (Figure 4b and c; day 5 results not shown).
b2AR antagonism promotes keratinocyte migration in vitro and re-epithelialization in vivo Keratinocyte migration from the wound edges is a critical process to restore the barrier function of the epidermis, and they are a valuable source for growth factors in the wound (Shaw and Martin, 2009).
A b2AR antagonist increased keratinocyte migration significantly by 12% (Figure 5a). To confirm that murine keratinocytes (MKs) respond similarly to a b2AR antagonist, MKs were isolated from newborn b2AR þ / þ and b2AR À/À mice and the effect of a b2AR antagonist on motility was determined. A b2AR antagonist enhanced MK SCM by 28% (Figure 5b). b2AR À/À MKs migrated 16% faster than b2AR þ / þ MKs, whereas a b2AR antagonist had no additional effect on their migration, confirming the absence of the b2AR (Figure 5b). A b2AR antagonist-mediated 2.5-fold increase in ERK phosphorylation, known to have a pivotal role in pro-migratory signaling pathways (Klemke et al., 1997), underpinned its effect on MK motility (Figure 5c).
Tyrosine hydroxylase, the enzyme controlling the ratelimiting step for catecholamine biosynthesis (Nagatsu et al., 1964), was expressed in MKs (Figure 5d), and 100 ± 10 and 75 ± 8 pg mg À1 protein of epinephrine was measured by enzyme immunoassay in extracts from MKs. MKs similar to HKs (Pullar et al., 2006b), therefore, can also synthesize epinephrine, generating an autocrine and paracrine adrenergic network in the murine epidermis and dermis, respectively.
Finally, to determine the effect of a b2AR antagonist on wound re-epithelialization, 7-mm-thick sections from the wound center, excised after 3 and 5 days, were stained with hematoxylin and eosin, and the linear distance covered by new epithelium was determined. b2AR antagonist treatment increased b2AR þ / þ wound re-epithelialization by 60 and 56% within 3 and 5 days post wounding, respectively ( Figure 5e). In addition, re-epithelialization of b2AR À/À wounds was increased by 64% 5 days post wounding ( Figure 5e).

DISCUSSION
b2AR antagonism enhanced angiogenesis ex vivo and in vivo and increased VEGF secretion from HKs. b2AR antagonism increased the migration rate of HDFs in vitro, while increasing SMA expression, collagen III expression, and contractile ability in murine wounds in vivo. In addition, b2AR À/À wounds contracted faster in the first few days post wounding. In contrast, b2AR antagonism had no effect on wound inflammation in vivo. Finally, b2AR antagonism or loss of b2AR increased keratinocyte migration in vitro and promoted re-epithelialization in vivo within 5 days of wounding. In summary, b2AR antagonism promoted wound repair, as summarized in the Supplementary Appendix S1 online.
The mechanism underpinning the b2AR antagonistmediated increase in angiogenesis could be partly attributed to the b2AR antagonist-mediated increase in VEGF secretion from HKs, known to be an important source of VEGF (Rossiter et al., 2004) and other growth factors in wounds (Werner and Grose, 2003;Barrientos et al., 2008). Indeed, the addition of autologous cultured keratinocytes can aid healing of chronic venous leg ulcers (Liu et al., 2004) and the addition of VEGF promotes healing in chronic diabetic wounds (Galiano et al., 2004b;Brem et al., 2009); therefore, the b2AR antagonist-mediated increase in HK VEGF secretion could be highly beneficial for wound repair. In the complex wound environment, b2AR antagonist treatment could also increase the secretion of other pro-angiogenic factors such as fibroblast growth factor 2 or basic fibroblast growth factor or plateletderived growth factor (Battegay et al., 1994), from wound cells, which could contribute to the enhanced angiogenesis observed in vivo. In contrast, post-ischemic angiogenesis was impaired in b2AR À/À mice (Ciccarelli et al., 2011).
The increase in wound contraction observed in b2AR À/À wounds (Figure 3a) occurred in the absence of an increase in SMA-expressing cells in the wound bed (Figure 3b), in contrast to the observed increase in both wound contraction and SMA expression in the b2AR antagonist-treated b2AR þ / þ wounds. Although the rate of wound contraction is thought to correlate directly with SMA expression (Hinz et al., 2001), there are several reports describing that wound contraction can occur in the absence of SMA-expressing myofibroblasts. Wounds in vanadate-treated rats (Ehrlich et al., 1999) and rat wounds treated with a Smad blocker, SB-505124 (Au and Ehrlich, 2010), contracted normally, but SMAexpressing cells were not detected in the granulation tissue. Finally, a personal communication from JJ Tomasek reports that SMA knockout mice heal normally (Hinz, 2007). It appears that wound contraction is not correlated with SMA expression in b2AR À/À mouse wounds and only b2AR antagonist treatment can increase SMA expression (Figure 3b).
In addition, blockade of autocrine, endogenously synthesized epinephrine (Figure 5d), inhibitory to keratinocyte migration (Pullar et al., 2003(Pullar et al., , 2006a, likely contributed to the ability of the b2AR antagonist to promote MK migration in vitro and re-epithelialization in vivo. Although b2AR antagonist treatment did not alter acute wound inflammation or the secretion of VEGF from inflammatory cells, b2AR blockade has been demonstrated to attenuate the hyper-inflammatory response to traumatic injury in mice (Rough et al., 2009). It is possible, therefore, that b2AR antagonist treatment could reduce persistent or hyper-inflammation, which will be tested in a chronic wound model.
In conclusion, here we report that b2AR antagonism promotes wound angiogenesis, DF function, and re-epithelialization, enhancing wound repair, thus revealing a possible new avenue of therapeutic intervention. To our knowledge, this is previously unreported.

Animals
The b2AR-deficient FVB/N (b2AR À/À ) mice were a kind gift from Dr Brian Kobilka, Stanford University (Stanford, CA) (Chruscinski et al., 1999). The congenic controls, FVB/N (b2AR þ / þ ) mice, were purchased from The Jackson Laboratory (Bar Harbor, ME). The genotypes of the b2AR þ / þ and b2AR -/mice were confirmed by PCR, using published primers (Chruscinski et al., 1999). All animals used in the study were females between 8 and 12 weeks of age. The University of California, Davis Institutional Animal Care and Use Committee (IACUC) approved all animal protocols. Adult zebrafish were maintained, as described (Westerfield, 1994), in compliance with the Animals (Scientific Procedures; www.legislation.gov.uk/ukpga/1986/14/contents) Act, 1986. Embryos were harvested and raised in egg water (Westerfield, 1994), at 28.5 1C, until the required developmental stage. Staging was performed in accordance with (Kimmel et al., 1995).
www.jidonline.org 2081 Dermal pieces were allowed to attach for 5 minutes, before the addition of 4 ml of fibroblast growth medium, which consists of DMEM (basal medium), 0.5% antibiotic solution (25 U ml À1 penicillin and 25 mg ml À1 streptomycin; Sigma Aldrich), and 10% fetal calf serum (Invitrogen). The plates were incubated at 37 1C in a humidified atmosphere of 5% CO 2 for 48 hours before adding an additional 6 ml of fibroblast growth medium per dish. Primary human neonatal DFs were purchased from Invitrogen and cultured as described previously (Pullar and Isseroff, 2006).
Primary HDMECs were purchased from Invitrogen. Cells were grown in EC growth media supplemented with microvascular growth supplement (PromoCell, Heidelburg, Germany) and antibiotics (25 U ml À1 penicillin, 25 mg ml À1 streptomycin; Invitrogen) at 37 1C with 5% CO 2 /95% air in a humidified atmosphere. Cells were grown on attachment factor containing 0.1% gelatin (Invitrogen), and experiments were conducted using cell passages between 3 and 7.
Primary neonatal HKs were purchased from Invitrogen and cultured as described previously (Pullar and Isseroff, 2005). At least three keratinocytes strains, between passages 3 and 6, were used in all experiments.

SCM assay
Plastic cell culture dishes (35 mm) were coated with collagen I (60 mg ml À1 ) (Invitrogen) in phosphate-buffered saline for 1 hour at 37 1C. MDFs, isolated from both b2AR À/À and b2AR þ / þ mice, and HDFs were plated at a density of 25 cells per mm 2 in fibroblast growth medium, whereas MKs, HKs, and HDMECs were plated at a density of 50 cells per mm 2 , in their appropriate complete media (see culture methods), for 2 hours at 37 1C. Cells were incubated with complete media alone (control) or with media containing 10 nM b2AR antagonist (ICI 118,551; Tocris Biosciences, Bristol, UK) at time 0. The 35-mm dishes were placed in a heating chamber, designed to maintain the medium at 37 1C, and images were taken every 10 minutes for 1 hour with the Improvision software (Perkin Elmer, Cambridge, UK), as described previously (Pullar et al., 2006b).

Cell treatments for immunoblotting
MKs or MDFs plated at a density of 1 Â 10 6 , isolated from both b2AR À/À and b2AR þ / þ mice, were incubated with either murine keratinocyte complete growth medium (MK) or fibroblast growth medium (MDF) alone (control and lysates for catecholamine synthesis enzyme detection), or with media containing 10 nM B2AR antagonist (ICI, 118551), for 10 minutes. Lysates were prepared and electrophoresed, as previously described for phosphorylated and total ERK (Pullar et al., 2003) and tyrosine hydroxylase (Pullar et al., 2006b).

Enzyme immunoassay for the quantitative determination of epinephrine in small sample volumes
MKs (1 Â 10 7 ), isolated from both b2AR À/À and b2AR þ / þ mice, were extracted in 100 ml 0.1 N HCl and sonicated on ice for 10 minutes. Extracts were tested in triplicate in an epinephrine enzyme immunoassay (Invitrogen) according to the manufacturer's instructions.

ELISA
Cells were plated on collagen I (30 mg ml À1 )-coated 6-well plates and incubated for 24 hours in the appropriate complete media to reach 70/80% confluence. Cells were washed with Hank's balanced salt solution and serum starved for 24 hours in the appropriate basal media. Cells were then incubated with either basal media alone or media containing 10 mM b2AR antagonist for 6, 24, or 48 hours. After each time point, media were collected and stored at À80 1C for analysis with a Duoset VEGF-A ELISA kit (R&D Systems, Abingdon, UK).

Rat aortic ring assay
Animals were killed using an approved schedule 1 method. The abdominal and aortic arcs were extracted from adult Wistar rats and placed into sterile phosphate-buffered saline. The fibro-adipose tissue was then removed, and the aortas were washed in warm, sterile phosphate-buffered saline three times. Aortas were cut into uniform, 1-mm sections, placed into 80 ml of basement membrane extract (R&D Systems), and incubated at 37 1C for 30 minutes. Aortas were incubated in EC basal media supplemented with 2% microvascular growth supplement for 3 days, and then with media alone or media containing 10 mM b2AR antagonist (ICI 118,551) for a further 5 days. Aortic endothelial outgrowth was imaged every 24 hours, using a Nikon eclipse microscope and the Improvision Openlab software. The area of outgrowth was analyzed using the Improvision Volocity software.

Zebrafish caudal fin wound model
Zebrafish larvae, 72 hours post-fertilization, were immersed in pond water containing 2% tricaine, and complete transection of the tail was performed with a sterile scalpel blade. After wounding, the larvae were incubated in the presence or absence of 500 mM b2AR antagonist (ICI 118,551) for 6 hours, and washed and stained using a fluorescein-tyramide signal amplification method (Perkin Elmer) before fixation in 4% paraformaldehyde. Tails were mounted and photographed on a Nikon TE-2000E inverted microscope at Â 20 magnification using a fluor objective. The number of neutrophils recruited to each wounded tail was recorded.

CAM assay
Fertilized eggs were obtained, 1 day post fertilization, from local hatcheries and incubated at 37 1C in a humidified environment for 48 hours. On day 3, approximately 5 ml of albumin was removed from the obtuse poles of the eggs using a 21 G cannula. A square window (2 Â 2 cm 2 ) was opened into the shell and shell membrane using a Dremel tool (Dremel, Uxbridge, UK). The window was then sealed with Parafilm and incubated horizontally for 48 hours at 37 1C. On day 5, water or 100 mM b2AR antagonist (ICI 118,551) in water was evaporated onto the center of a sterile 13-mm coverslip and then placed face down onto the CAM. CAMs were photographed through the coverslip center, every 24 hours, until day 9 using a stereomicroscope (Prior Scientific, Cambridge, UK). Angiogenesis was analyzed by counting the total number of branch points per field of view through the center of the coverslip.
Murine wound model b2AR À/À and b2AR þ / þ mice were anesthetized by intraperitoneal injection of ketamine (100 mg kg À1 )/xylazine (10 mg kg À1 ) (Pfizer, Sandwich, UK). Back skin was shaved and two circular, full-thickness 6-mm excisional wounds were created 2 cm apart, in the center of the back, using a sterile 6-mm biopsy punch (SMS, Camberley, UK) to mark the skin for surgical excision. Wounds were treated topically with 100 ml of hydrogel alone (Duoderm, ConvaTec, Uxbridge, UK) or containing 0.1% (1 mM) selective b2AR antagonist (ICI 118,551) (a concentration previously demonstrated to accelerate mouse epidermal barrier recovery; Denda et al., 2003) immediately after wounding and daily thereafter until harvesting (n ¼ 5-7 mice per group, two wounds per mouse). Each mouse was housed separately after wounding until wound harvest. Wounds were left uncovered and digitally photographed, daily, to determine wound contraction over time. Wounds were harvested 3 and 5 days post wounding by carefully applying an 8-mm punch (SMS) around the original wound and lightly pressing to form an outline on the skin. Scissors were used to excise the wound without damaging the delicate wound bed.
For histological analysis, the wounds were fixed in an IHC zinc fixative (BD Biosciences). The zinc-fixed biopsies were bisected, to ensure that sections were taken from the center of the wound, dehydrated through an ethanol-xylene series, and embedded in paraffin. Cross-sections, 7 mm thick, were stained with the hematoxylin-eosin technique or with picrosirius red, to visualize collagen in the neo-dermis (Junqueira et al., 1979), as described by Dapson et al. (2011). Sections were immunostained with an antibody against SMA (Dako, Ely, UK), collagen III (Rockland, Lorne Laboratories, Reading, UK), Ly6G-6C (a neutrophil marker; BD Pharmingen, Oxford, UK), F4-80 (a macrophage marker; Serotec, Kidlington, UK), or CD31 (an EC marker; BD Pharmingen), followed by DAB detection (BD Pharmingen) according to the manufacturer's protocols.
Specimens that were damaged in the histological process or otherwise non-interpretable were excluded from the study. Reepithelialization was determined by examining all hematoxylin-and eosin-stained sections by light microscopy, using a Q-imaging Retiga-EX camera attached to a Nikon T-100 inverted microscope, controlled by the Improvision software. Image J was used to measure the linear distance covered by new epithelium and the linear distance between the original wound edges, to determine the percentage of reepithelialization. Wound closure was quantified by measuring the wound area of each wound from the digital pictures taken every day with Image J. Each picture was calibrated individually. Images of picrosirius red-stained sections were captured on an Olympus BX51 upright microscope equipped for dark field. The area of the collagenstained neo-dermis was analyzed by measuring the red-stained area within 1 mm 2 of each wound edge on the dark-field pictures with the Image J software. Images of the SMA-stained wounds were captured on a Nikon SMZ-U upright microscope with the Nikon ACT-1 software. Images of the collagen III-stained wounds were captured on a Nikon Eclipse inverted microscope with the NIS-Elements software. NIH Image J was used to determine the area of SMA and collagen III staining in each wound to calculate the mean area of SMA and collagen III staining in each group. Images of the CD31-( Â 40 magnification), Ly-6G-6C-, and F4-80-stained ( Â 60 magnification) wounds were also captured on the Nikon Eclipse microscope. The number of stained cells/vessels in each image was counted in a double-blind manner, and the average cell/vessel number was calculated for each group. For all image analysis, 10 fields were selected from the dermis below the wound margins and across the wound bed using a template to ensure that images were captured from similar areas in each wound.

Statistical analysis
Unless mentioned in a specific method, categorical variables were compared with a two-tailed Fisher's exact test or a one-way analysis of variance, followed by the Dunnett's test, whereas each continuous variable test group was compared with the control using the twotailed Student's t-test for unpaired data with unequal variance. Po0.05 (*) was significant and Po0.01 (**) highly significant.

CONFLICT OF INTEREST
CEP/RRI are coinventors on a patent held at University of California, Davis, entitled ''b2AR modulation of wound healing''. The authors declare that there are no other competing interests.