International Journal of
Molecular Sciences Article
Broad-Spectrum Inhibition of the CC-Chemokine Class Improves Wound Healing and Wound Angiogenesis Anisyah Ridiandries 1,2 , Christina Bursill 1,2, *,† and Joanne Tan 1,2, *,† 1 2
* †
Heart Research Institute, 7 Eliza Street, Newtown, Sydney 2042, NSW, Australia; [email protected] Sydney Medical School, University of Sydney, Camperdown, Sydney 2050, NSW, Australia Correspondence: [email protected] (C.B.); [email protected] (J.T.); Tel.: +61-2-8208-8905 (C.B.); +61-2-8208-8900 (J.T.); Fax: +61-2-9565-5584 (C.B. & J.T.) These authors contributed equally to this work.
Academic Editors: Allison Cowin and Terrence Piva Received: 17 August 2016; Accepted: 10 January 2017; Published: 13 January 2017
Abstract: Angiogenesis is involved in the inflammation and proliferation stages of wound healing, to bring inflammatory cells to the wound and provide a microvascular network to maintain new tissue formation. An excess of inflammation, however, leads to prolonged wound healing and scar formation, often resulting in unfavourable outcomes such as amputation. CC-chemokines play key roles in the promotion of inflammation and inflammatory-driven angiogenesis. Therefore, inhibition of the CC-chemokine class may improve wound healing. We aimed to determine if the broad-spectrum CC-chemokine inhibitor “35K” could accelerate wound healing in vivo in mice. In a murine wound healing model, 35K protein or phosphate buffered saline (PBS, control) were added topically daily to wounds. Cohorts of mice were assessed in the early stages (four days post-wounding) and in the later stages of wound repair (10 and 21 days post-wounding). Topical application of the 35K protein inhibited CC-chemokine expression (CCL5, CCL2) in wounds and caused enhanced blood flow recovery and wound closure in early-mid stage wounds. In addition, 35K promoted neovascularisation in the early stages of wound repair. Furthermore, 35K treated wounds had significantly lower expression of the p65 subunit of NF-κB, a key inflammatory transcription factor, and augmented wound expression of the pro-angiogenic and pro-repair cytokine TGF-β. These findings show that broad-spectrum CC-chemokine inhibition may be beneficial for the promotion of wound healing. Keywords: chemokine; angiogenesis; wound; healing; inflammation
1. Introduction Wound healing is a complex multistep process comprised of three overlapping but distinct phases: inflammation, proliferation and remodelling. It begins with the inflammation phase, where neutrophils and macrophages are recruited to the wound site to remove cell debris and phagocytose infectious pathogens. This is followed by the proliferation stage, where re-epithelialization and the formation of granulation tissue begin to close the wound. Angiogenesis, the formation of new blood vessels, is important during both the inflammation and proliferation phases, forming angiogenic capillary sprouts that allow for recruitment of inflammatory cells to the wound for debris removal, invasion of the fibrin/fibronectin-rich wound clot, and reorganization of a microvascular network to maintain the new granulation tissue being formed [1]. Finally, during the remodelling phase, fibroblasts reorganize the collagen matrix, forming a closed wound [2]. All three phases must occur in the proper sequence Int. J. Mol. Sci. 2017, 18, 155; doi:10.3390/ijms18010155
www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2017, 18, 155
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and time frame in order for a wound to heal successfully. Excessive macrophage accumulation during the inflammation and proliferation stages may prolong the inflammatory response leading to delayed wound healing and scar formation at the injury site. Macrophage phenotype has also been reported to change throughout the wound healing process, alternating between inflammatory M1 and repair M2 phenotypes [3]. The regulation of these phenotypes has been shown to affect wound functionality and the extent of scar tissue [4]. Wounds that exhibit impaired healing frequently enter a state of pathological inflammation, with most chronic wounds developing into ulcers. The incidence of chronic wounds is associated with ischemia, diabetes mellitus, venous stasis disease or pressure [5]. Chronic, non-healing wounds greatly impact a person’s quality of life and, when left untreated, sometimes lead to amputation, resulting in an enormous health care burden [6,7]. While current therapies provide some relief, there is a need for the continued development of novel therapies that address the debilitating effects of impaired wound healing. CC-chemokines are small inflammatory cytokines that have been shown to play a key role in the induction of inflammation and inflammation-mediated angiogenesis. In the wound healing process, macrophage infiltration is highly regulated by CC-chemokine gradients released by hyper-proliferating keratinocytes, fibroblasts and other macrophages [8,9]. In human wounds, a host of CC-chemokines including CCL1, CCL2, CCL3, CCL4, CCL5 and CCL7 are expressed during the first week after injury and high levels of CCL2 have been found in human burns [10,11]. Additionally, studies in excisional wounds have found localized expression of CCL2 and CCL3 in the epidermis, while CCL3 is expressed in follicular epithelium and sebaceous glands [12]. CC-chemokines regulate key angiogenic processes such as the recruitment of inflammatory cells to the wound, to provide support for proliferating and migrating cells [13], and the formation of granulation tissue [9]. Furthermore, incubation with the recombinant CC-chemokines CCL2 and CCL5 stimulate angiogenic functions such as endothelial cell tubulogenesis and migration [14–18]. Therefore, manipulation of the CC-chemokine family may modulate key wound healing processes to confer benefits. To date, studies have inhibited a single CC-chemokine and shown modest or no effect on wound healing. CCL2 knockout mice have delayed re-epithelialization and reduced angiogenesis in the early stages of wound repair, whilst the CCL3 knockout mice have normal wound healing [19]. Similarly, CCR1 knockout models have no alteration in wound healing [20]. Inhibition of a single CC-chemokine, however, may not be as effective in modulating wound healing due to redundancies in CC-chemokine signalling [21,22]. A broad-spectrum CC-chemokine inhibition approach may therefore have increased efficacy. The broad-spectrum CC-chemokine inhibitor “35K” is a 35 kDa soluble protein produced by the Vaccinia virus that uniquely inhibits only the CC-chemokine class [23]. It recognizes and binds to common structural features shared by most CC-chemokines, preventing binding to their cognate receptors [24]. Broad-spectrum CC-chemokine inhibition using 35K inhibits a host of inflammatory diseases including atherosclerosis, acute peritonitis, hepatitis and liver fibrosis [25–28]. Given the inhibitory effects of 35K on inflammatory-mediated diseases and the role of CC-chemokines in angiogenesis, we sought to elucidate the effect of broad-spectrum CC-chemokine inhibition on wound healing. We found that topical application of 35K enhanced wound closure, blood flow perfusion and neovascularisation at the early-mid stages of wound healing. Augmentation of wound angiogenesis by 35K was mediated via an increase in the pro-angiogenic and pro-repair cytokine TGF-β. In addition, 35K treatment inhibited wound levels of inflammatory transcription factor NF-κB and reduced collagen deposition at the late stages of wound healing in the 35K-treated wounds, suggesting a reduction in scar formation. Taken together, these findings show that broad-spectrum inhibition of CC-chemokines may be beneficial for the promotion of wound healing by suppressing inflammation and promoting wound closure and neovascularisation and reducing scar formation.
Int. J. Mol. Sci. 2017, 18, 155 Int. J. Mol. Sci. 2017, 18, 155
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2. Results 2. Results 2.1.Topical Topical35K 35KIncreases Increases Wound Blood Perfusion and Rate Wound Closure Early-Mid Stage 2.1. Wound Blood Perfusion and thethe Rate of of Wound Closure in in Early-Mid Stage Wound Repair Wound Repair Topicaltreatment treatmentofofwounds woundswith with35K 35Kprotein proteincaused causedsignificantly significantlyfaster fasterwound woundclosure closure Topical throughout early to mid-stages of wound healing at Day 7 (42%, p < 0.05) and8Day p< throughout thethe early to mid-stages of wound healing at Day 7 (42%, p < 0.05) and Day (33%,8 p(33%, < 0.05) 0.05) compared to PBS wounds treated wounds (Figure 1a). Using Laser imaging, Doppler it imaging, it wasthat revealed compared to PBS treated (Figure 1a). Using Laser Doppler was revealed 35K that 35K protein augmented wound blood(Figure perfusion 1b). Doppler The Laser Doppler protein augmented wound blood perfusion 1b).(Figure The Laser Index (LDI),Index used(LDI), as a used as markerangiogenesis of wound angiogenesis and as determined the ratio of 35K:PBS wounds, was marker of awound and determined the ratio ofas35K:PBS wounds, was significantly significantly in the 35K treated in the early stages following wounding (Day 3,to4), elevated in theelevated 35K treated wounds in the wounds early stages following wounding (Day 3, 4), compared compared to PBS control (values aboveincreased 100% indicate increased wound blood perfusion with PBS control wounds (valueswounds above 100% indicate wound blood perfusion with 35K treatment 35K treatment relativeIn tothe PBSlater control). stages of theprocess, woundat healing process, at seven days relative to PBS control). stagesInofthe thelater wound healing seven days post-wounding, post-wounding, blood to perfusion to decline wounds withto35K, compared the PBS blood perfusion started decline started in wounds treatedinwith 35K,treated compared the PBS treatedtocontrol treated control wounds. After Day 7,fluctuated blood perfusion fluctuated the baseline andperfusion very little wounds. After Day 7, blood perfusion along the baselinealong and very little blood blood perfusion was detected. There were no significant increases decreases was detected. There were no significant increases or decreases to theor end point. to the end point.
Figure1.1.Topical Topical35K 35K increasesthe the rate wound closure and wound bloodperfusion perfusionininearly-mid early-mid Figure increases rate ofof wound closure and wound blood stage wound repair. Two full thickness wounds were created on C57Bl/6J mice (n = 7–12). Mice stage wound repair. Two full thickness wounds were created on C57Bl/6J mice (n = 7–12). Mice received received daily topical application of 35K protein nM) or PBS (vehicle). (a) representative images daily topical application of 35K protein (200 nM) (200 or PBS (vehicle). (a) representative images of the of the wounds andblood wound blood using perfusion Laser Doppler imaging (red) blood to lowflow); (blue) wounds and wound perfusion Laserusing Doppler imaging (high (red) to(high low (blue) blood flow); wound area was from the average of threemeasurements daily diameteralong measurements (b) wound area(b) was calculated fromcalculated the average of three daily diameter the x-, yalong the x-, yand z-axes. Wound closure is expressed as a percentage of initial wound areacircles at Day and z-axes. Wound closure is expressed as a percentage of initial wound area at Day 0. Black 0. PBS Blacktreated circleswounds; are PBS treated wounds; are 35K treated and wound (c) the 35K:PBS are grey triangles aregrey 35Ktriangles treated wounds; and (c)wounds; the 35K:PBS blood wound blood ratio flow was perfusion ratio was determined usingimaging Laser Doppler imaging (LDI). Dataasis flow perfusion determined using Laser Doppler (LDI). Data is represented represented as mean ± SEM. Pointsline above the dotted line represent improvement with 35K mean ± SEM. Points above the dotted represent an improvement withan 35K treatment. Statistical treatment. Statistical analysis was performed by an unpaired two-tailed t-test. * p < 0.05 compared analysis was performed by an unpaired two-tailed t-test. * p < 0.05 compared to PBS treated wounds.to PBS treated wounds.
Int. J. Mol. Sci. 2017, 18, 155 Int. J. Mol. Sci. 2017, 18, 155
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2.2. Formation in in Early Early Stage Stage Wounds Wounds but butDecreases 2.2. Inhibition Inhibition of of CC-Chemokines CC-Chemokines by by 35K 35K Increases Increases Neovessel Neovessel Formation NeovesselsNeovessels in Late Stage Wounds Decreases in Late Stage Wounds To investigate investigate wound wound angiogenesis, angiogenesis, neovessels neovessels and and arterioles arterioles were were assessed. assessed. In In the the early early stages stages To of wound wound healing healing (Day (Day 4), 4), there there was was an an increase increase in in the the presence presence of of wound wound neovessels neovessels in in the the 35K 35K of treated treated wounds wounds as as determined determined by by CD31+ CD31+ staining staining (182%, (182%, pp
Molecular Sciences Article
Broad-Spectrum Inhibition of the CC-Chemokine Class Improves Wound Healing and Wound Angiogenesis Anisyah Ridiandries 1,2 , Christina Bursill 1,2, *,† and Joanne Tan 1,2, *,† 1 2
* †
Heart Research Institute, 7 Eliza Street, Newtown, Sydney 2042, NSW, Australia; [email protected] Sydney Medical School, University of Sydney, Camperdown, Sydney 2050, NSW, Australia Correspondence: [email protected] (C.B.); [email protected] (J.T.); Tel.: +61-2-8208-8905 (C.B.); +61-2-8208-8900 (J.T.); Fax: +61-2-9565-5584 (C.B. & J.T.) These authors contributed equally to this work.
Academic Editors: Allison Cowin and Terrence Piva Received: 17 August 2016; Accepted: 10 January 2017; Published: 13 January 2017
Abstract: Angiogenesis is involved in the inflammation and proliferation stages of wound healing, to bring inflammatory cells to the wound and provide a microvascular network to maintain new tissue formation. An excess of inflammation, however, leads to prolonged wound healing and scar formation, often resulting in unfavourable outcomes such as amputation. CC-chemokines play key roles in the promotion of inflammation and inflammatory-driven angiogenesis. Therefore, inhibition of the CC-chemokine class may improve wound healing. We aimed to determine if the broad-spectrum CC-chemokine inhibitor “35K” could accelerate wound healing in vivo in mice. In a murine wound healing model, 35K protein or phosphate buffered saline (PBS, control) were added topically daily to wounds. Cohorts of mice were assessed in the early stages (four days post-wounding) and in the later stages of wound repair (10 and 21 days post-wounding). Topical application of the 35K protein inhibited CC-chemokine expression (CCL5, CCL2) in wounds and caused enhanced blood flow recovery and wound closure in early-mid stage wounds. In addition, 35K promoted neovascularisation in the early stages of wound repair. Furthermore, 35K treated wounds had significantly lower expression of the p65 subunit of NF-κB, a key inflammatory transcription factor, and augmented wound expression of the pro-angiogenic and pro-repair cytokine TGF-β. These findings show that broad-spectrum CC-chemokine inhibition may be beneficial for the promotion of wound healing. Keywords: chemokine; angiogenesis; wound; healing; inflammation
1. Introduction Wound healing is a complex multistep process comprised of three overlapping but distinct phases: inflammation, proliferation and remodelling. It begins with the inflammation phase, where neutrophils and macrophages are recruited to the wound site to remove cell debris and phagocytose infectious pathogens. This is followed by the proliferation stage, where re-epithelialization and the formation of granulation tissue begin to close the wound. Angiogenesis, the formation of new blood vessels, is important during both the inflammation and proliferation phases, forming angiogenic capillary sprouts that allow for recruitment of inflammatory cells to the wound for debris removal, invasion of the fibrin/fibronectin-rich wound clot, and reorganization of a microvascular network to maintain the new granulation tissue being formed [1]. Finally, during the remodelling phase, fibroblasts reorganize the collagen matrix, forming a closed wound [2]. All three phases must occur in the proper sequence Int. J. Mol. Sci. 2017, 18, 155; doi:10.3390/ijms18010155
www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2017, 18, 155
2 of 15
and time frame in order for a wound to heal successfully. Excessive macrophage accumulation during the inflammation and proliferation stages may prolong the inflammatory response leading to delayed wound healing and scar formation at the injury site. Macrophage phenotype has also been reported to change throughout the wound healing process, alternating between inflammatory M1 and repair M2 phenotypes [3]. The regulation of these phenotypes has been shown to affect wound functionality and the extent of scar tissue [4]. Wounds that exhibit impaired healing frequently enter a state of pathological inflammation, with most chronic wounds developing into ulcers. The incidence of chronic wounds is associated with ischemia, diabetes mellitus, venous stasis disease or pressure [5]. Chronic, non-healing wounds greatly impact a person’s quality of life and, when left untreated, sometimes lead to amputation, resulting in an enormous health care burden [6,7]. While current therapies provide some relief, there is a need for the continued development of novel therapies that address the debilitating effects of impaired wound healing. CC-chemokines are small inflammatory cytokines that have been shown to play a key role in the induction of inflammation and inflammation-mediated angiogenesis. In the wound healing process, macrophage infiltration is highly regulated by CC-chemokine gradients released by hyper-proliferating keratinocytes, fibroblasts and other macrophages [8,9]. In human wounds, a host of CC-chemokines including CCL1, CCL2, CCL3, CCL4, CCL5 and CCL7 are expressed during the first week after injury and high levels of CCL2 have been found in human burns [10,11]. Additionally, studies in excisional wounds have found localized expression of CCL2 and CCL3 in the epidermis, while CCL3 is expressed in follicular epithelium and sebaceous glands [12]. CC-chemokines regulate key angiogenic processes such as the recruitment of inflammatory cells to the wound, to provide support for proliferating and migrating cells [13], and the formation of granulation tissue [9]. Furthermore, incubation with the recombinant CC-chemokines CCL2 and CCL5 stimulate angiogenic functions such as endothelial cell tubulogenesis and migration [14–18]. Therefore, manipulation of the CC-chemokine family may modulate key wound healing processes to confer benefits. To date, studies have inhibited a single CC-chemokine and shown modest or no effect on wound healing. CCL2 knockout mice have delayed re-epithelialization and reduced angiogenesis in the early stages of wound repair, whilst the CCL3 knockout mice have normal wound healing [19]. Similarly, CCR1 knockout models have no alteration in wound healing [20]. Inhibition of a single CC-chemokine, however, may not be as effective in modulating wound healing due to redundancies in CC-chemokine signalling [21,22]. A broad-spectrum CC-chemokine inhibition approach may therefore have increased efficacy. The broad-spectrum CC-chemokine inhibitor “35K” is a 35 kDa soluble protein produced by the Vaccinia virus that uniquely inhibits only the CC-chemokine class [23]. It recognizes and binds to common structural features shared by most CC-chemokines, preventing binding to their cognate receptors [24]. Broad-spectrum CC-chemokine inhibition using 35K inhibits a host of inflammatory diseases including atherosclerosis, acute peritonitis, hepatitis and liver fibrosis [25–28]. Given the inhibitory effects of 35K on inflammatory-mediated diseases and the role of CC-chemokines in angiogenesis, we sought to elucidate the effect of broad-spectrum CC-chemokine inhibition on wound healing. We found that topical application of 35K enhanced wound closure, blood flow perfusion and neovascularisation at the early-mid stages of wound healing. Augmentation of wound angiogenesis by 35K was mediated via an increase in the pro-angiogenic and pro-repair cytokine TGF-β. In addition, 35K treatment inhibited wound levels of inflammatory transcription factor NF-κB and reduced collagen deposition at the late stages of wound healing in the 35K-treated wounds, suggesting a reduction in scar formation. Taken together, these findings show that broad-spectrum inhibition of CC-chemokines may be beneficial for the promotion of wound healing by suppressing inflammation and promoting wound closure and neovascularisation and reducing scar formation.
Int. J. Mol. Sci. 2017, 18, 155 Int. J. Mol. Sci. 2017, 18, 155
3 of 15 3 of 14
2. Results 2. Results 2.1.Topical Topical35K 35KIncreases Increases Wound Blood Perfusion and Rate Wound Closure Early-Mid Stage 2.1. Wound Blood Perfusion and thethe Rate of of Wound Closure in in Early-Mid Stage Wound Repair Wound Repair Topicaltreatment treatmentofofwounds woundswith with35K 35Kprotein proteincaused causedsignificantly significantlyfaster fasterwound woundclosure closure Topical throughout early to mid-stages of wound healing at Day 7 (42%, p < 0.05) and8Day p< throughout thethe early to mid-stages of wound healing at Day 7 (42%, p < 0.05) and Day (33%,8 p(33%, < 0.05) 0.05) compared to PBS wounds treated wounds (Figure 1a). Using Laser imaging, Doppler it imaging, it wasthat revealed compared to PBS treated (Figure 1a). Using Laser Doppler was revealed 35K that 35K protein augmented wound blood(Figure perfusion 1b). Doppler The Laser Doppler protein augmented wound blood perfusion 1b).(Figure The Laser Index (LDI),Index used(LDI), as a used as markerangiogenesis of wound angiogenesis and as determined the ratio of 35K:PBS wounds, was marker of awound and determined the ratio ofas35K:PBS wounds, was significantly significantly in the 35K treated in the early stages following wounding (Day 3,to4), elevated in theelevated 35K treated wounds in the wounds early stages following wounding (Day 3, 4), compared compared to PBS control (values aboveincreased 100% indicate increased wound blood perfusion with PBS control wounds (valueswounds above 100% indicate wound blood perfusion with 35K treatment 35K treatment relativeIn tothe PBSlater control). stages of theprocess, woundat healing process, at seven days relative to PBS control). stagesInofthe thelater wound healing seven days post-wounding, post-wounding, blood to perfusion to decline wounds withto35K, compared the PBS blood perfusion started decline started in wounds treatedinwith 35K,treated compared the PBS treatedtocontrol treated control wounds. After Day 7,fluctuated blood perfusion fluctuated the baseline andperfusion very little wounds. After Day 7, blood perfusion along the baselinealong and very little blood blood perfusion was detected. There were no significant increases decreases was detected. There were no significant increases or decreases to theor end point. to the end point.
Figure1.1.Topical Topical35K 35K increasesthe the rate wound closure and wound bloodperfusion perfusionininearly-mid early-mid Figure increases rate ofof wound closure and wound blood stage wound repair. Two full thickness wounds were created on C57Bl/6J mice (n = 7–12). Mice stage wound repair. Two full thickness wounds were created on C57Bl/6J mice (n = 7–12). Mice received received daily topical application of 35K protein nM) or PBS (vehicle). (a) representative images daily topical application of 35K protein (200 nM) (200 or PBS (vehicle). (a) representative images of the of the wounds andblood wound blood using perfusion Laser Doppler imaging (red) blood to lowflow); (blue) wounds and wound perfusion Laserusing Doppler imaging (high (red) to(high low (blue) blood flow); wound area was from the average of threemeasurements daily diameteralong measurements (b) wound area(b) was calculated fromcalculated the average of three daily diameter the x-, yalong the x-, yand z-axes. Wound closure is expressed as a percentage of initial wound areacircles at Day and z-axes. Wound closure is expressed as a percentage of initial wound area at Day 0. Black 0. PBS Blacktreated circleswounds; are PBS treated wounds; are 35K treated and wound (c) the 35K:PBS are grey triangles aregrey 35Ktriangles treated wounds; and (c)wounds; the 35K:PBS blood wound blood ratio flow was perfusion ratio was determined usingimaging Laser Doppler imaging (LDI). Dataasis flow perfusion determined using Laser Doppler (LDI). Data is represented represented as mean ± SEM. Pointsline above the dotted line represent improvement with 35K mean ± SEM. Points above the dotted represent an improvement withan 35K treatment. Statistical treatment. Statistical analysis was performed by an unpaired two-tailed t-test. * p < 0.05 compared analysis was performed by an unpaired two-tailed t-test. * p < 0.05 compared to PBS treated wounds.to PBS treated wounds.
Int. J. Mol. Sci. 2017, 18, 155 Int. J. Mol. Sci. 2017, 18, 155
4 of 15 4 of 14
2.2. Formation in in Early Early Stage Stage Wounds Wounds but butDecreases 2.2. Inhibition Inhibition of of CC-Chemokines CC-Chemokines by by 35K 35K Increases Increases Neovessel Neovessel Formation NeovesselsNeovessels in Late Stage Wounds Decreases in Late Stage Wounds To investigate investigate wound wound angiogenesis, angiogenesis, neovessels neovessels and and arterioles arterioles were were assessed. assessed. In In the the early early stages stages To of wound wound healing healing (Day (Day 4), 4), there there was was an an increase increase in in the the presence presence of of wound wound neovessels neovessels in in the the 35K 35K of treated treated wounds wounds as as determined determined by by CD31+ CD31+ staining staining (182%, (182%, pp
