Attenuation of flightless I improves wound healing and enhances angiogenesis in a murine model of type 1 diabetes
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Skin lesions and ulcerations are severe complications of diabetes that often result in leg amputations. In this study we investigated the function of the cytoskeletal protein flightless I (FLII) in diabetic wound healing. We hypothesised that overexpression of FLII would have a negative effect on diabetic wound closure and modulation of this protein using specific FLII-neutralising antibodies (FnAb) would enhance cellular proliferation, migration and angiogenesis within the diabetic wound.
Using a streptozotocin-induced model of diabetes we investigated the effect of altered FLII levels through Flii genetic knockdown, overexpression or treatment with FnAb on wound healing. Diabetic wounds were assessed using histology, immunohistochemistry and biochemical analysis. In vitro and in vivo assays of angiogenesis were used to assess the angiogenic response.
FLII levels were elevated in the wounds of both diabetic mice and humans. Reduction in the level of FLII improved healing of murine diabetic wounds and promoted a robust pro-angiogenic response with significantly elevated von Willebrand factor (vWF) and vascular endothelial growth factor (VEGF)-positive endothelial cell infiltration. Diabetic mouse wounds treated intradermally with FnAb showed improved healing and a significantly increased rate of re-epithelialisation. FnAb improved the angiogenic response through enhanced formation of capillary tubes and functional neovasculature. Reducing the level of FLII led to increased numbers of mature blood vessels, increased recruitment of smooth muscle actin-α-positive cells and improved tight junction formation.
Reducing the level of FLII in a wound may be a potential therapeutic approach for the treatment of diabetic foot ulcers.
KeywordsAngiogenesis Diabetes Flightless I FLII Flightless neutralising antibodies Type 1 diabetes VEGF Wound healing
Haematoxylin and eosin
Smooth muscle actin-α
Vascular endothelial growth factor
von Willebrand factor
Up to 15% of people with diabetes can expect to develop a non-healing ulcer , and 3% will have a lower-limb amputation . Despite numerous approaches the therapeutic results are still very limited. Impaired healing of diabetic wounds is caused by complex factors such as abnormal keratinocyte and fibroblast migration, proliferation, differentiation and apoptosis, and decreased vascularisation . Chronic hyperglycaemia also leads to impaired angiogenesis, which further contributes to the delayed wound healing seen in patients with diabetes [4, 5].
The cytoskeletal protein flightless I (FLII) [6, 7] regulates the dynamic remodelling of the actin cytoskeleton  by severing and bundling actin filaments ; it is involved in cellular processes, including migration and proliferation, which are important in wound healing . The defining feature of FLII is its homology with two families of proteins, the gelsolin protein family and the leucine-rich repeat (LRR) protein family , providing the capacity to be involved in a variety of signalling pathways [7, 8, 11, 12]. FLII downregulates IL-1/Toll-like receptor (TLR)4 signalling of the TLR pathway, suggesting that FLII is involved in the regulation of the innate immune response . FLII interacts directly with MyD88, an intracellular adaptor protein immediately downstream of most TLRs, and modulates the activity of nuclear factor of κ light polypeptide gene enhancer in B cells 1 (NF-κB) [13, 14, 15], pro-inflammatory caspases  and TNF-α . FLII has been identified as an important regulator of wound repair, affecting cell proliferation, motility and matrix deposition . Flii gene heterozygous knockout leads to improved wound healing, whereas its overexpression is associated with impaired healing of wounds . FLII-neutralising antibodies (FnAb) have the capacity to increase cellular proliferation and migration in vitro [17, 18] and promote the rate of healing in cutaneous wounds in vivo .
Using human samples and genetic mouse models with low (Flii+/−), normal (Flii+/+), or high (FliiTg/Tg) levels of FLII, or FnAb, which binds to extracellular FLII and reduces its local expression, the effect of FLII on diabetic wound healing was investigated. These studies suggest that FLII adversely affects healing in diabetic wounds and that reducing its activity leads to improved wound outcomes and enhanced angiogenesis.
Mouse monoclonal anti-FLII antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rat anti-mouse CD31 antibody (BD Pharmingen, North Ryde, NSW, Australia), anti-smooth muscle actin-α (α-SMA), rabbit polyclonal anti-vascular endothelial growth factor (VEGF) and rabbit polyclonal anti-von Willebrand factor (vWF) antibodies were obtained from Abcam (Cambridge, UK). Alexa-488- and Alexa-594-tagged secondary antibodies were obtained from Invitrogen (Mulgrave, VIC, Australia) and mouse monoclonal anti-FLII-neutralising antibodies (FnAb)  were obtained from MAbSA (Adelaide, SA, Australia).
Human wound and skin collection
Prior to recruitment to the study, participants were screened using relevant investigations including blood tests, quantitative microbiology, duplex and advanced wound diagnostics in the form of toe pressure, ankle brachial index and transcutaneous oxygen pressure. Participants with non-diabetic acute wounds (acute trauma wounds such as those seen in the emergency department [≤6 weeks old]), diabetic wounds (type 1 or type 2 diabetes, diabetic ulcer [≥6 weeks old]) and normal unwounded skin (healthy age-matched volunteers) were included in the study. Exclusion criteria for the diabetic wounds included active infection or cellulitis in the area biopsied, drugs that impair wound healing such as steroids and immunosuppressive drugs, ulcer of malignant nature and renal failure/dialysis with GFR <30 ml/min. As part of the inclusion criteria all patients with diabetic wounds displayed presence of pulses and/or ankle brachial index >0.7, and had a diabetic ulcer and toe pressure >40 mmHg. Informed consent was obtained from each patient and the study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in approval by the Health Service Human Research Ethics Committee and Central Northern Adelaide Health Service Ethics of Human Research Committee. The three study groups were: diabetic ulcer (n = 6, mean age 56); acute wound (n = 6, mean age 49); and normal skin (n = 6, mean age 45).
Murine model of type 1 diabetes
Flii heterozygous null (FliiTm1Hdc; Flii+/−)  and transgenic overexpressing (Tg[Flii]2Hdc; FliiTg/Tg) [17, 21] mice on a BALB/c background were provided by Ruth M. Arkell, Australian National University, Canberra, ACT, Australia . Wild-type (WT) animals (Flii+/+) were obtained from the BALB/c colony used for maintenance of the null strain (The University of Adelaide, Adelaide, SA, Australia). All animal experiments were approved by the Women’s and Children’s Health Research Institute’s Animal Ethics Committee and principles of laboratory animal care were followed. Female Flii+/−, WT and FliiTg/Tg mice of 12–16 weeks of age, weighing 20–35 g were given one intraperitoneal  injection of streptozotocin (STZ) (Sigma-Aldrich, St Louis, MO, USA) at 50 mg/kg for five consecutive days as described previously . Diabetes was confirmed by assessing the fasting blood glucose levels on 0, 15, 30 and 60 days post STZ injection. After 6 weeks of confirmed diabetes, excisional wounds were created on the dorsal surface of the mice as described below. Age-matched non-diabetic control animals were treated with an equivalent dose of vehicle.
Murine model of diabetic wound healing
Two 6 mm wounds, one on each side of the midline, were created on the shaved dorsa of mice. Digital photographs were taken of the wounds at 0, 7, 14 and 21 days post wounding. Wounds were harvested at 0, 7, 14 and 21 days and fixed in 10% wt/vol. buffered formalin. In a separate cohort, wounded diabetic WT mice were (n = 10; 20 wounds in total) were injected intradermally around the wound margins with 200 μl of FnAb 50 μg/ml at day 1, 2 and 3. Control diabetic mice were treated with an equivalent dosage of IgG antibodies (n = 10, 20 wounds in total).
Skin explant outgrowth assay
Punch biopsies, 2 mm, were taken from the excised skin of diabetic and non-diabetic mice. Biopsies were placed in ice-cold Nutrient Mixture F12 (Sigma-Aldrich) and 1% (vol./vol.) penicillin-streptomycin solution (Sigma-Aldrich) and cultured as described previously  in either normal (5.5 mmol/l) or high (25 mmol/l) glucose media.
Sections were deparaffinised in two changes of xylene and re-hydrated in two changes of absolute alcohol, 1 min each, after which they were transferred into 70% (vol./vol.) and 30% (vol./vol.) alcohol for 1 min each. Sections were brought to water, rinsed in ×1 PBS and placed into 250 ml target retrieval solution as described previously . Primary antibodies (1:100) were applied and incubated overnight at 4°C. Detection was by species-specific secondary antibodies (1:200). AnalySIS software package (Munster, Germany) was used to measure fluorescence intensity per unit area as described previously . Control immunostaining was carried out by omitting primary or secondary antibodies for verification of staining and non-specific binding.
Aortic ring assay
Dissection of the aorta, serum starvation, embedding and feeding of the aortic rings, imaging and quantifying microvessel sprouts was as described previously . A total of 15 rings per mouse were obtained. The microvessel growth from the aorta rings was quantified on days 5, 6, 7 and 8 post embedding by live phase-contrast microscopy. Emerging microvessels were counted and the data was plotted as mean microvessel numbers per ring as described previously .
Isolation and culture of HUVEC
The collection of primary HUVEC for use in this study was given ethical clearance from relevant committees and consent was obtained from all participants in accordance with the Declaration of Helsinki. HUVEC were isolated from umbilical veins by collagenase digestion as previously described  and used at passage two for experiments.
Matrigel tube formation assay
Passage 1-2 HUVEC were cultured on gelatine-plated T25 flasks and maintained in culture media containing 10% (vol./vol.) FCS in EGM-2 BulletKit (Lonza, Basel, Switzerland). HUVEC were seeded on angiogenesis plate (1 μ-Slide Angiogenesis ibiTreat; Ibidi, Munich, Germany) coated with 12 μl of Matrigel (354234, BD Biosciences, Bedford, MA, USA). The number of tubes was counted as previously described [22, 27] and data were derived from an average of four donors.
Matrigel plug assay
Two plugs containing different ligands (FnAb or IgG antibodies were mixed by pipetting; 500 μl at 50 μg/ml) were injected into each mouse to avoid differences between the individual mice . The plug on the left side of the abdomen contained Matrigel compound (354248, BD Biosciences) mixed with 50 μg/ml of FnAb and the plug on the right (control) contained an equivalent dose of rabbit IgG. The Matrigel plugs were removed after 7 days; half of the plug was snap frozen in liquid nitrogen and used for blood vessel visualisation by immunofluorescence and the other half fixed in 10% (vol./vol.) formalin and processed for histology. Quantification in Matrigel plug assay was as described previously  using Image ProPlus 5.1 (Media Cybernetics, Rockville, MD, USA). Matrigel plugs retrieved from Flii+/−, WT and FliiTg/Tg mice were fixed in 4% (wt/vol.) paraformaldehyde/1.25% (wt/vol.) glutaraldehyde and processed for electron microscopy.
After washing, specimens were dehydrated in a graded series of ethanol (70–100%) followed by further dehydration in propylene oxide for 30 min and treated further as previously described . Electron micrographs of six samples of Matrigel plugs harvested from Flii+/−, WT and FliiTg/Tg mice were captured at two different magnifications (×3,800 and ×88,000). Blood capillaries were examined focusing on the tight junction region, which seals the intercellular space between adjacent endothelial cells. The overall width of the outer leaflets of the cell membrane of adjacent endothelial cells forming tight junctions was measured using Analysis5 iTEM software (SIS, Tokyo, Japan).
Protein was extracted from day 7 WT diabetic mouse wounds treated with either IgG control antibodies or FnAb using standard protein extraction protocols . Protein, 50 μg, was subjected to SDS-PAGE separation and immunoblotting. Densitometry was performed on bands within the linear range and fold changes in levels calculated from this data as previously described .
Statistical differences were determined using the Student’s t test to compare between two groups or an ordinary two-way ANOVA with Tukey’s multiple comparisons test when comparisons between more than two groups were required. A p value <0.05 was considered significant.
Cytoskeletal protein FLII is increased in human diabetic wounds
Inhibitory effect of FLII on cellular growth and migration
Skin explants (2 mm) were collected from diabetic and non-diabetic mice and incubated in normal (5.5 mmol/l) or high (25 mmol/l) glucose-supplemented media for 7 days (Fig. 1d–h). On day 2 post culture a halo appeared around the explants. The cellular growth and migration potential was determined by measuring the distance between the explant and the end of the growing halo. Diabetic explants displayed a significant reduction in their capacity to grow and migrate compared with non-diabetic skin tissue (Fig. 1e, f).
FLII deficiency improves diabetic wound healing
Diabetes increased FLII level in response to wounding
FLII deficiency leads to upregulation of pro-angiogenic VEGF expression in diabetic wounds
Increased levels of FLII leads to increased numbers of endothelial cells in diabetic wounds
To determine the effect of FLII on endothelial cell recruitment, healing wounds from non-diabetic and diabetic Flii+/−, WT and FliiTg/Tg mouse wounds were stained for vWF (ESM Fig. 2c and Fig. 4b) and CD31 (ESM Fig. 2f, g and Fig. 4c, d). Day 7 non-diabetic mice Flii+/− wounds showed a 1.7-fold increase in the expression of vWF (Fig. 4b) and CD31 (Fig. 4c) compared with WT as determined by immunohistochemistry. Low FLII led to markedly increased endothelial cell numbers in diabetic wounds compared with diabetic WT controls (Fig. 4b, d).
Attenuation of Flii gene expression improves new blood vessel formation in vivo
Pro-angiogenic effects of FnAb in vitro
Reducing FLII using FnAb enhanced formation of mature blood vessels in vivo
Matrigel plugs were mixed with either IgG control (50 μg/ml) or FnAb (50 μg/ml) and injected under the skin of the abdominal area of WT mice (ESM Fig. 3c, d and Fig. 6b). The vessels were categorised into three groups: (1) vessels containing erythrocytes; (2) vessels without erythrocytes; and (3) vessel-like structure but without a cavity. FnAb resulted in a fourfold increase in the length of functional vessels that contained erythrocytes compared with IgG control (Fig. 6b). Adding FnAb (50 μg/ml) into Matrigel plugs enhanced the formation of new blood vessels (Fig. 6b).
FnAb contributes to enhanced α-SMA+/CD31+ cell recruitment in vivo
Matrigel plugs supplemented with either IgG control (50 μg/ml) or FnAb (50 μg/ml) were injected under the skin of WT mice and harvested at day 7 post injection and subsequently stained with antibodies to establish the level of α-SMA and endothelial cell (CD31+) recruitment (Fig. 6c). The number of cells positive for α-SMA was lower in IgG-treated Matrigel plugs compared with FnAb-treated Matrigel plug sections (Fig. 6d).
FLII antibodies increase VEGF expression in diabetic wounds
The effect of reduced FLII on VEGF levels in diabetic wounds was assessed by immunoblotting and immunostaining with anti-VEGF antibody the day 7 WT diabetic wounds treated with either IgG control antibodies or FnAb (ESM Fig. 3e and Fig. 6e, f). FnAb resulted in significant upregulation of VEGF (Fig. 6e–g).
Accelerated diabetic wound closure is induced by FnAb in vivo
Human diabetic wounds have elevated levels of FLII, a cytoskeletal protein previously shown to inhibit cellular migration, adhesion and proliferation and to negatively impact on the wound repair process . To determine the extent of the involvement of FLII in diabetic wound healing, mice with low Flii+/−, normal (WT) and high (FliiTg/Tg) expression of Flii gene were rendered diabetic and assessed for their healing characteristics. Diabetes was associated with a delayed wound healing phenotype with larger wound area and, as observed in human patients, elevated levels of FLII in the wounds. Reduced FLII significantly improved the healing of diabetic and non-diabetic wounds, whereas FLII overexpression decreased the rate of re-epithelialisation under both diabetic and non-diabetic conditions. Given that rapid epithelialisation and closure of disrupted dermal matrix are key determinants of successful wound healing these results suggest that decreasing the level of FLII in diabetic wounds may exert a positive effect on wound healing.
Diabetic wounds fail to heal because of several contributing factors, including abnormal keratinocyte and fibroblast migration, proliferation, differentiation, and apoptosis and decreased vascularisation . Given that FLII inhibits cellular migration  and vascular cell migration is an important component of new vessel formation and vascular maturation  we investigated whether altering Flii gene expression could affect angiogenesis in diabetic wounds. Diabetes itself caused a downregulation in both VEGF and vWF during the early stages of wound healing, which in turn coincided with high levels of FLII. New blood vessel formation was further impaired in FLII-overexpressing diabetic wounds, with reduced numbers of endothelial cells and decreased VEGF and vWF being observed. In FLII-deficient diabetic wounds, the levels of vWF were elevated twofold at days 7 and 14, suggesting that lowering the level of FLII in diabetic wounds could have a positive effect on angiogenesis and therefore healing. In support of this, robust sprouting of endothelial cells was observed from aortic rings obtained from Flii+/− mice, whereas in contrast aortic rings from FliiTg/Tg mice showed minimal microvessel outgrowth suggesting that FLII inhibits angiogenic processes. FLII deficiency was also associated with enhanced cell-to-cell interactions between endothelial cells with physiologically normal tight junctions being observed when FLII levels were reduced, and disrupted cell-to-cell interactions with significantly wider distances observed in FLII-overexpressing mice wounds. These findings support previous observations of impaired hemidesmosome formation in FLII-overexpressing mice  and suggest the importance of FLII in endothelial cell tight junction formation, which is important for blood vessel stability and leakage prevention.
FLII is secreted by both fibroblasts and macrophages [10, 17] and is present in acute and chronic human wound fluid . FLII is secreted through a non-classic late endosome/lysosome-mediated pathway by at least two of the major cell types found in wounds, fibroblasts and macrophages , and is able to bind to lipopolysaccharide and alter macrophage activation . Treatment of wound fluid with FnAb decreases the level of available FLII present in the wound fluid, which implies that FnAb can ‘mop up’ extracellular FLII and neutralise its activity in vivo .
As wounds in FLII-deficient mice showed improved healing, we assessed the effect of using FnAb as a means of decreasing local levels of FLII within the wound environment. Diabetic wounds treated with FnAb showed accelerated wound closure with a 1.9-fold decrease in average wound size observed in day 3 and day 7 WT FnAb-treated diabetic wounds. Similar effects have been reported in previous studies where FnAb improved the rate of incisional , excisional  and burn wound repair . Dose-dependent effects of FnAb on cellular proliferation have also previously been demonstrated  and the addition of FnAb to human chronic wound fluids can reduce their inhibitory effect on cell proliferation in vitro . Both of these effects could contribute positively to improved wound outcomes.
Angiogenic responses to the FnAb were also assessed, with FnAb leading to significantly increased capillary tube formation and longer and more mature blood vessel formation compared with IgG controls. FnAb-treated Matrigel plugs showed longer vessels containing erythrocytes, which indicated that these vessels were sufficiently mature to receive blood flow, with flow being a critical determinant of vessel maintenance and durability . In the Matrigels treated with FnAb, the expression of α-SMA-positive cells was also significantly higher, suggesting that FnAb may be able to promote the migration of these cells towards endothelial cells, hence stimulating the formation of functionally mature blood vessels.
In summary, the actin cytoskeletal protein FLII is upregulated in both human and mouse diabetic wounds. Reducing its activity either by genetic knockdown or by the application of neutralising antibodies leads to improved wound healing. One mechanism via which FLII may be actively promoting improved wound healing is via its pro-angiogenic effects. Identification of novel wound-healing targets and understanding how these targets may affect wound healing is the first step in the development of new therapies that will improve the quality of life of patients with diabetes and reduce the costs of diabetic wound management.
The authors thank L. Waterhouse for excellent electron microscopy expertise, the Animal Care Facility at the Women’s and Children’s Hospital, Adelaide, the mothers and staff at the Women’s & Children’s Hospital, and Burnside War Memorial Hospital for collection of umbilical cords.
AJC is supported by the National Health and Medical Research Council Senior Research Fellowship (number 1002009). ZK is supported by the National Health and Medical Research Council Early Career Fellowship (number 1036509). CSB is supported by a Heart Foundation Fellowship.
Duality of interest
AJC is a shareholder in a company developing intellectual property associated with FnAb treatment of wounds. All other authors declare that there is no duality of interest associated with their contribution to this manuscript.
NR and ZK made substantial contribution to the design, acquisition and analysis of data and have drafted the manuscript. EM, SLA, CSB, RMA and RF made substantial contribution to the interpretation of data and revision of the manuscript. AJC made substantial contribution to the design and drafting of the manuscript. All authors have reviewed and approved the final version.
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