BRIEF COMMUNICATION
Pivotal role of ATP in macrophages fast tracking wound repair and regeneration Girish J. Kotwal, PhD1,2; Harshini Sarojini, PhD1,2; Sufan Chien, MD1,2 1. Department of Surgery, University of Louisville School of Medicine, MDR Bldg. 3rd floor, Room 316/328E, 511 South Floyd Street, Louisville, KY, 40202, USA, 2. Research and Development Department, Noveratech, Louisville, Kentucky
Reprint requests: Girish J. Kotwal, Ph.D., Department of Surgery, University of Louisville, School of Medicine, MDR Bldg. 3rd floor, Room 316/328E, 511 South Floyd Street, Louisville, KY 40202, USA. Tel: 1502 852 0722; Fax: 1502 327 7466; Email: [email protected] Manuscript received: April 23, 2015 Accepted in final form: June 2, 2015 DOI:10.1111/wrr.12323
Abstract Chronic wounds occurring during aging or diabetes pose a significant burden to patients. The classical four-phase wound healing process has a 3–6 day lag before granulation starts to appear and it requires an intermediate step of activation of resident fibroblasts during the remodeling phase for production of collagen. This brief communication discusses published articles that demonstrate how the entire wound healing process can be fast tracked by intracellular ATP delivery, which triggers a novel pathway where alternatively activated macrophages play absolutely critical and central roles. This novel pathway involves an increase in proinflammatory cytokines (TNF, IL-1b, IL-6) and a chemokine (MCP-1) release. This is followed by activation of purinergic receptor (a family of plasma membrane receptors found in almost all mammalian cells), production of platelets and platelet microparticles, and activation of ATP-dependent chromatin remodeling enzymes. The end result is a massive influx and in situ proliferation of macrophages, increases in vascular endothelial growth factors that promote neovascularization, and most prominently, the direct production of collagen.
INTRODUCTION The classical wound healing process
Wound healing is a complex process in which, although still somewhat debated, macrophages play essential direct and indirect roles.1 The classical wound healing process involves several phases—hemostasis, inflammation, proliferation, and remodeling—which results in a 3–6 day lag before the wound starts to closed.2 This progression of phases seems to be “carved in stone” and no one has ever been able to shorten it. During the inflammatory phase, proinflammatory cytokines (IL-1b, IL-6, and TNF-a) are secreted to recruit peripheral circulating monocytes and white blood cells. Monocytes differentiate into macrophages, which are classically activated macrophage after stimulation with interferon gamma and lipopolysaccharide to a cytotoxic phenotype. These macrophages secrete vascular endothelial growth factors and promote proliferation of endothelial cells, skeletal myoblasts, and fibroblasts in a process referred to as the reepithelialization and phagocytosis of surrounding dead cells and debris, while secreting IL-10 to suppress further influx of macrophages. This phase is followed by angiogenesis, myotubule formation, collagen production, and finally a remodeling phase that involves collagen remodeling.3 Alternately activated wound healing macrophages
Extensive murine studies support the proposal that a temporal variability occurs during the wound healing process, 724
where selected phenotypic traits occur as the healing wound progresses from the classically activated early phase to a later alternatively activated phase. This aspect of wound healing has been reviewed by Brancato and Albina4 who suggest that the lack of pure phenotype of macrophages in vivo means that macrophage activation could be appropriately described as a continuous spectrum of distinct continuous phenotypic features. Wound macrophages do express the alternatively activated phenotype, so they could be termed wound-healing macrophages. Macrophage phenotype switching
The macrophages in the wound change in phenotype as wound healing progresses. Successful completion of the full wound healing process requires that macrophages undergo phenotypic as well as functional switching or transitions from M1 (proinflammatory) to M2 (anti-inflammatory or wound healing) phenotypes; this transition is influenced by the local microenvironment.5 The M2 phenotype is characterized by distinct surface receptors (CD68, CD163, CD206) and several sub phenotypes (M2a, M2b, M2c, and M2d). Switching from the M1 to the M2 phenotype depends on down-regulation of IL-10 and the up-regulation of IL-4 and IL-13 and occurs in stages in response to specific mediators. The M1 to M2a transition is induced by IL-4R-a dependent activation. Macrophage polarization, or the acquisition of distinct phenotypes, must occur in an orderly manner for effective wound healing. Macrophage polarization is regulated by a C 2015 by the Wound Healing Society Wound Rep Reg (2015) 23 724–727 V
Kotwal et al.
Pivotal role of ATP in wound
Table 1. Multiple activities of intracellular ATP delivery Activities Rapid granulation tissue generation, increased macrophages MAC387, CD68, and CD163 Increases cytokines (TNF, IL-1beta, Il-6, IL-4, IL-13), chemokines (MCP-1) Increased VEGFs, VSGFs Increased neovascularization (CD31) Increased healing Increased collagen synthesis Increased PCNA Increased BRG/BRM
complex process involving adenosine-mediated “switching” that aids in regulation of angiogenesis during the final stages of wound healing—viz. the repair process.5 Another layer of complexity arises from the presence of specific macrophage phenotypes associated with each tissue type.6 Our focus is on wound healing events occurring in the skin, so this article focuses on the resident and infiltrating macrophages of the skin. Fast-track wound healing following intracellular ATP delivery
In sharp contrast to the classical wound healing process that takes place with all current interventions, the fasttrack process involves the introduction of adenosine triphosphate (Mg-ATP), encapsulated within minute unilamellar lipid vesicles, directly to the wound. This simple innovation of intracellular ATP delivery via fusogenic vesicles (ATP-Vesicles) significantly enhances wound healing in the skin of rodent models.7 The same enhancement was observed in a rabbit model, where treatment of full-thickness skin wounds with ATP-vesicles resulted not only in improved healing but also in an extremely rapid tissue regeneration.8,9 In contrast to the traditional 3–6 day lag, granulation tissue started to appear within 24 hours—a phenomenon never seen or reported previously with any other treatment modality. The early growth is mainly composed of macrophages, which show active proliferation in situ. Collagen is produced and neovascularization is enhanced. Reepithelializing tissue tunnels then through the granulation tissue.8 The top of the granulation tissue finally falls off, revealing a perfectly healed wound. This healing enhancement appears to be more prominent in ischemic wounds. A similar growth pattern was not seen following treatment with free ATP or with empty vehicles, or with the only FDA-approved prescription growth factor for wound dressing—Regranex.10 Most importantly, the growth has a self-limiting feature, so that no hypertrophic scar or any other unusual growth remains, even after two years.11 This type of healing is totally different from the conventional process known to the wound care community, where fibrin, platelets, and red blood cells are the main compoC 2015 by the Wound Healing Society Wound Rep Reg (2015) 23 724–727 V
References Howard et al., 2014,11 Novak and Koh, 20133
Howard et al., 201411
Chiang et al., 2007,7 Wang et al., 20098 Wang et al., 20098 Wang et al., 2009,8 2010,9 Howard et al., 201411 Chiang et al., 2007,7 Howard et al., 201411 Howard et al., 201411 Wang et al., 200914
nents of the early provisional matrix. This matrix is then gradually replaced by granulation tissue during the proliferation phase after the 3–6 day lag.12,13 We dissected the individual steps involved in this novel wound healing process by testing several individual activities of the ATP delivery system.11 In vitro studies revealed an increase in chemokine MCP-1 accompanied by a reduction in the anti-inflammatory cytokine IL-10 levels following ATP-vesicle treatment of human macrophage cells.11 As suggested earlier,11 this explained the large influx of macrophages observed following the ATP-vesicle introduction. The predominant macrophage phenotype was M2, consistent with the observed increase in IL-13R. The vascular specific growth factors also increased, consistent with the observed neovascularization.7 Increases were also observed in platelets at the wound site at a very early time.9 We originally inferred that the stem cell and macrophage influxes and accumulations and the phenotypic differentiation of the incoming macrophages were a result of the increased energy supply.14 Our recent studies comparing ATP-vesicles with Regranex-treated wounds have further refined the likely mechanism underlying the fast-track healing. The rapid granulation and the accompanying tissue generation, accompanied by the extraordinary in situ macrophage proliferation (as determined by increased PCNA synthesis and BrdU stain), appear to contribute to the collagen synthesis without the need for intermediate fibroblast generation at the wound site.11 These multiple responses to the ATP vesicles are summarized in Table 1. Based on earlier studies by Wang et al.,8 the phenomenon of macrophage accumulation in response to ATP vesicles has been attributed to the ATP-driven rearrangement of the chromatin structure within the 12 component SWI/SNF complex, which includes the BRG1 or Brm ATPase subunits that contribute to cell proliferation.15 Increased levels of BRG1 and BRM in wounds treated with ATP-vesicles indicate that the fast-track mechanism could be associated with ATPase activity that contributes to the in situ proliferation of the macrophages. A summary of our current understanding of the mechanism of wound healing following ATP delivery is detailed in Figure 1. 725
Pivotal role of ATP in wound
Kotwal et al.
Figure 1. Schematic illustration of the proposed complex cascades by which intracellular ATP delivery causes extremely rapid tissue regeneration while overgranulation is prevented. (1) Stem/progenitor cell attraction mostly via purinergic receptor activation. (2) Leukocyte chemotaxis caused by ATP. (3) Enhanced platelet accumulation by ATP. (4) Monocyte accumulation and activation from the above processes and the platelet-derived growth factors further enhance monocyte activation. (5) Monocyte transformation to macrophages via platelet and platelet microparticles and the MCP-1 pathways. (6) Massive cell accumulation caused by in situ macrophage proliferation driven by ATP-dependent chromatin remodeling complex SW1/SNF. (7) Energy provided by ATP-vesicles nourishes massive cell accumulation and enhances their function. (8) Activated M1 macrophages exert their phagocytic function but also secret MCP-1 resulting in further cell accumulation. (9) Activated M2 macrophages produce collagen directly via alternative pathway and enhance neovascularization via various cytokines and growth factors. (10) Up-regulated apoptosis keeps the growth in check and maintains the balance between proliferation and regression.
The full delineation of the steps involved in this proliferation will provide a clearer picture of the actual mechanism. Our current hypothesis that it is the energy increase introduced by the added ATP encapsulated in the vesicles contributes to the more rapid rate of wound healing. Although our results were obtained with acute wounds, the accumulation, survival, and function of massive amounts of cells in a wound cavity totally devoid of blood supply may represent a new hope for treatment of chronic wounds, which would be expected to benefit from any enhancement of neovascularization.
CONCLUSION The rabbit wound model studies described here confirm that intracellular delivery of ATP to a wound activates macrophages and results in the initiation of gross healing 726
within 24 h. The macrophages, secrete collagen directly into extracellular matrix that rapidly fills the wound space. Conventional wound healing, by contrast, starts only after a 3–6 day lag, and is a less direct process, where the wound space is first filled with a provisional matrix consisting of red cells trapped in a fibrin mesh. The two unique features of the ATP delivery healing process—elimination of the lag time and the ability to support cell survival and proliferation in a wound cavity without any blood supply in the early days following injury—have never been achieved with any other treatment. The rapid tissue generation following intracellular ATP delivery is a major advance that awaits full elucidation of its underlying biological mechanism. Whether it is the provision of energy to the microenvironment or the adenosine-mediated macrophage phenotype switching, or both, that hastens the wound healing process remains to be established. C 2015 by the Wound Healing Society Wound Rep Reg (2015) 23 724–727 V
Kotwal et al.
DISCLOSURES The authors received support in part from the following funding awarded to Sufan Chien; R01DK74566, R44A R52984, R43HL114235, R43GM106639, R43DK104625b and R43DK105692 from the National Institutes of Health and in part from the Kentucky Cabinet for Economic Development, Office of Entrepreneurship, under the Grant agreements KSTC-184-512-12-138, KSTC-184-512-14-174 with the Kentucky Science and Technology Corporation. Noveratech, LLC was founded by SC and is a co-inventor of a US issued patent on the intracellular ATP delivery technique.
REFERENCES 1. Martin P, Leibovich SJ. Inflammatory cells during wound repair: the good, the bad and the ugly. Trends Cell Biol 2005; 15: 599–607. 2. Delavary BM, van der Veer WM, van Egmond M, Niessen FB, Beelen RH. Macrophages in skin injury and repair. Immunobiology 2011; 216: 753–62. 3. Novak ML, Koh TJ. Phenotypic transitions of macrophages orchestrate tissue repair. Am J Pathol 2013; 183: 1352–63. 4. Brancato SK, Albina JE. Wound macrophages as key regulators of repair: origin, phenotype, and function. Am J Pathol 2011; 178: 19–25. 5. Ferrante CJ, Leibovich SJ. Regulation of macrophage pollarization and wound healing. Adv Wound Care 2012; 1: 10–16. 6. Davies LC, Jenkins SJ, Allen JE, Taylor PR. Tissue-resident macrophages. Nat Immunol 2013; 14: 986–95.
C 2015 by the Wound Healing Society Wound Rep Reg (2015) 23 724–727 V
Pivotal role of ATP in wound
7. Chiang B, Essick E, Ehringer W, Murphree S, Hauck MA, Li M, et al. Enhancing skin wound healing by direct intracellular ATP delivery. Am J Surg 2007; 193: 213–18. 8. Wang J, Zhang Q, Wan R, Mo Y, Li M, Tseng M, et al. Intracellular ATP-delivery enhanced skin wound healing in rabbits. Ann Plastic Surg 2009; 62: 180–6. 9. Wang J, Wan R, Mo Y, Li M, Zhang Q, Chien S. Intracellular delivery of ATP enhanced healing process in full-thickness skin wounds in diabetic animals. Am J Surg 2010; 199: 823–32. 10. Cohen MA, Eaglstein WH. Recombinant human plateletderived growth factor gel speeds healing of acute fullthickness punch biopsy wounds. J Am Acad Dermatol 2001; 45: 857–62. 11. Howard JD, Sarojini H, Wan R, Chien S. Rapid granulation tissue regeneration by intracellular ATP delivery-a comparison with regranex. PLoS One 2014; 9: 1–14. 12. Levenson SM, Demetriou AA. Metabolic factors. In: Cohen IK, Diegelmann RF, Lindblad WJ, eds. Wound healing: Biochemical & clinical aspects. Philadelphia: Saunders, 1992: 248–73. 13. Schaffer M, Witte M, Becker HD. Models to study ischemia in chronic wounds. Int J Low Extrem Wounds 2002; 1: 104–11. 14. Chien S. Intracellular ATP delivery using highly fusogenic liposomes. Methods Mol Biol 2010; 605: 377–92. 15. Wang X, Sansam CG, Thom CS, Metzger D, Evans JA, Nguyen PT, et al. Oncogenesis caused by loss of the SNF5 tumor suppressor is dependent on activity of BRG1, the ATPase of the SWI/SNF chromatin remodeling complex. Cancer Res 2009; 69: 8094–101.
727
Pivotal role of ATP in macrophages fast tracking wound repair and regeneration Girish J. Kotwal, PhD1,2; Harshini Sarojini, PhD1,2; Sufan Chien, MD1,2 1. Department of Surgery, University of Louisville School of Medicine, MDR Bldg. 3rd floor, Room 316/328E, 511 South Floyd Street, Louisville, KY, 40202, USA, 2. Research and Development Department, Noveratech, Louisville, Kentucky
Reprint requests: Girish J. Kotwal, Ph.D., Department of Surgery, University of Louisville, School of Medicine, MDR Bldg. 3rd floor, Room 316/328E, 511 South Floyd Street, Louisville, KY 40202, USA. Tel: 1502 852 0722; Fax: 1502 327 7466; Email: [email protected] Manuscript received: April 23, 2015 Accepted in final form: June 2, 2015 DOI:10.1111/wrr.12323
Abstract Chronic wounds occurring during aging or diabetes pose a significant burden to patients. The classical four-phase wound healing process has a 3–6 day lag before granulation starts to appear and it requires an intermediate step of activation of resident fibroblasts during the remodeling phase for production of collagen. This brief communication discusses published articles that demonstrate how the entire wound healing process can be fast tracked by intracellular ATP delivery, which triggers a novel pathway where alternatively activated macrophages play absolutely critical and central roles. This novel pathway involves an increase in proinflammatory cytokines (TNF, IL-1b, IL-6) and a chemokine (MCP-1) release. This is followed by activation of purinergic receptor (a family of plasma membrane receptors found in almost all mammalian cells), production of platelets and platelet microparticles, and activation of ATP-dependent chromatin remodeling enzymes. The end result is a massive influx and in situ proliferation of macrophages, increases in vascular endothelial growth factors that promote neovascularization, and most prominently, the direct production of collagen.
INTRODUCTION The classical wound healing process
Wound healing is a complex process in which, although still somewhat debated, macrophages play essential direct and indirect roles.1 The classical wound healing process involves several phases—hemostasis, inflammation, proliferation, and remodeling—which results in a 3–6 day lag before the wound starts to closed.2 This progression of phases seems to be “carved in stone” and no one has ever been able to shorten it. During the inflammatory phase, proinflammatory cytokines (IL-1b, IL-6, and TNF-a) are secreted to recruit peripheral circulating monocytes and white blood cells. Monocytes differentiate into macrophages, which are classically activated macrophage after stimulation with interferon gamma and lipopolysaccharide to a cytotoxic phenotype. These macrophages secrete vascular endothelial growth factors and promote proliferation of endothelial cells, skeletal myoblasts, and fibroblasts in a process referred to as the reepithelialization and phagocytosis of surrounding dead cells and debris, while secreting IL-10 to suppress further influx of macrophages. This phase is followed by angiogenesis, myotubule formation, collagen production, and finally a remodeling phase that involves collagen remodeling.3 Alternately activated wound healing macrophages
Extensive murine studies support the proposal that a temporal variability occurs during the wound healing process, 724
where selected phenotypic traits occur as the healing wound progresses from the classically activated early phase to a later alternatively activated phase. This aspect of wound healing has been reviewed by Brancato and Albina4 who suggest that the lack of pure phenotype of macrophages in vivo means that macrophage activation could be appropriately described as a continuous spectrum of distinct continuous phenotypic features. Wound macrophages do express the alternatively activated phenotype, so they could be termed wound-healing macrophages. Macrophage phenotype switching
The macrophages in the wound change in phenotype as wound healing progresses. Successful completion of the full wound healing process requires that macrophages undergo phenotypic as well as functional switching or transitions from M1 (proinflammatory) to M2 (anti-inflammatory or wound healing) phenotypes; this transition is influenced by the local microenvironment.5 The M2 phenotype is characterized by distinct surface receptors (CD68, CD163, CD206) and several sub phenotypes (M2a, M2b, M2c, and M2d). Switching from the M1 to the M2 phenotype depends on down-regulation of IL-10 and the up-regulation of IL-4 and IL-13 and occurs in stages in response to specific mediators. The M1 to M2a transition is induced by IL-4R-a dependent activation. Macrophage polarization, or the acquisition of distinct phenotypes, must occur in an orderly manner for effective wound healing. Macrophage polarization is regulated by a C 2015 by the Wound Healing Society Wound Rep Reg (2015) 23 724–727 V
Kotwal et al.
Pivotal role of ATP in wound
Table 1. Multiple activities of intracellular ATP delivery Activities Rapid granulation tissue generation, increased macrophages MAC387, CD68, and CD163 Increases cytokines (TNF, IL-1beta, Il-6, IL-4, IL-13), chemokines (MCP-1) Increased VEGFs, VSGFs Increased neovascularization (CD31) Increased healing Increased collagen synthesis Increased PCNA Increased BRG/BRM
complex process involving adenosine-mediated “switching” that aids in regulation of angiogenesis during the final stages of wound healing—viz. the repair process.5 Another layer of complexity arises from the presence of specific macrophage phenotypes associated with each tissue type.6 Our focus is on wound healing events occurring in the skin, so this article focuses on the resident and infiltrating macrophages of the skin. Fast-track wound healing following intracellular ATP delivery
In sharp contrast to the classical wound healing process that takes place with all current interventions, the fasttrack process involves the introduction of adenosine triphosphate (Mg-ATP), encapsulated within minute unilamellar lipid vesicles, directly to the wound. This simple innovation of intracellular ATP delivery via fusogenic vesicles (ATP-Vesicles) significantly enhances wound healing in the skin of rodent models.7 The same enhancement was observed in a rabbit model, where treatment of full-thickness skin wounds with ATP-vesicles resulted not only in improved healing but also in an extremely rapid tissue regeneration.8,9 In contrast to the traditional 3–6 day lag, granulation tissue started to appear within 24 hours—a phenomenon never seen or reported previously with any other treatment modality. The early growth is mainly composed of macrophages, which show active proliferation in situ. Collagen is produced and neovascularization is enhanced. Reepithelializing tissue tunnels then through the granulation tissue.8 The top of the granulation tissue finally falls off, revealing a perfectly healed wound. This healing enhancement appears to be more prominent in ischemic wounds. A similar growth pattern was not seen following treatment with free ATP or with empty vehicles, or with the only FDA-approved prescription growth factor for wound dressing—Regranex.10 Most importantly, the growth has a self-limiting feature, so that no hypertrophic scar or any other unusual growth remains, even after two years.11 This type of healing is totally different from the conventional process known to the wound care community, where fibrin, platelets, and red blood cells are the main compoC 2015 by the Wound Healing Society Wound Rep Reg (2015) 23 724–727 V
References Howard et al., 2014,11 Novak and Koh, 20133
Howard et al., 201411
Chiang et al., 2007,7 Wang et al., 20098 Wang et al., 20098 Wang et al., 2009,8 2010,9 Howard et al., 201411 Chiang et al., 2007,7 Howard et al., 201411 Howard et al., 201411 Wang et al., 200914
nents of the early provisional matrix. This matrix is then gradually replaced by granulation tissue during the proliferation phase after the 3–6 day lag.12,13 We dissected the individual steps involved in this novel wound healing process by testing several individual activities of the ATP delivery system.11 In vitro studies revealed an increase in chemokine MCP-1 accompanied by a reduction in the anti-inflammatory cytokine IL-10 levels following ATP-vesicle treatment of human macrophage cells.11 As suggested earlier,11 this explained the large influx of macrophages observed following the ATP-vesicle introduction. The predominant macrophage phenotype was M2, consistent with the observed increase in IL-13R. The vascular specific growth factors also increased, consistent with the observed neovascularization.7 Increases were also observed in platelets at the wound site at a very early time.9 We originally inferred that the stem cell and macrophage influxes and accumulations and the phenotypic differentiation of the incoming macrophages were a result of the increased energy supply.14 Our recent studies comparing ATP-vesicles with Regranex-treated wounds have further refined the likely mechanism underlying the fast-track healing. The rapid granulation and the accompanying tissue generation, accompanied by the extraordinary in situ macrophage proliferation (as determined by increased PCNA synthesis and BrdU stain), appear to contribute to the collagen synthesis without the need for intermediate fibroblast generation at the wound site.11 These multiple responses to the ATP vesicles are summarized in Table 1. Based on earlier studies by Wang et al.,8 the phenomenon of macrophage accumulation in response to ATP vesicles has been attributed to the ATP-driven rearrangement of the chromatin structure within the 12 component SWI/SNF complex, which includes the BRG1 or Brm ATPase subunits that contribute to cell proliferation.15 Increased levels of BRG1 and BRM in wounds treated with ATP-vesicles indicate that the fast-track mechanism could be associated with ATPase activity that contributes to the in situ proliferation of the macrophages. A summary of our current understanding of the mechanism of wound healing following ATP delivery is detailed in Figure 1. 725
Pivotal role of ATP in wound
Kotwal et al.
Figure 1. Schematic illustration of the proposed complex cascades by which intracellular ATP delivery causes extremely rapid tissue regeneration while overgranulation is prevented. (1) Stem/progenitor cell attraction mostly via purinergic receptor activation. (2) Leukocyte chemotaxis caused by ATP. (3) Enhanced platelet accumulation by ATP. (4) Monocyte accumulation and activation from the above processes and the platelet-derived growth factors further enhance monocyte activation. (5) Monocyte transformation to macrophages via platelet and platelet microparticles and the MCP-1 pathways. (6) Massive cell accumulation caused by in situ macrophage proliferation driven by ATP-dependent chromatin remodeling complex SW1/SNF. (7) Energy provided by ATP-vesicles nourishes massive cell accumulation and enhances their function. (8) Activated M1 macrophages exert their phagocytic function but also secret MCP-1 resulting in further cell accumulation. (9) Activated M2 macrophages produce collagen directly via alternative pathway and enhance neovascularization via various cytokines and growth factors. (10) Up-regulated apoptosis keeps the growth in check and maintains the balance between proliferation and regression.
The full delineation of the steps involved in this proliferation will provide a clearer picture of the actual mechanism. Our current hypothesis that it is the energy increase introduced by the added ATP encapsulated in the vesicles contributes to the more rapid rate of wound healing. Although our results were obtained with acute wounds, the accumulation, survival, and function of massive amounts of cells in a wound cavity totally devoid of blood supply may represent a new hope for treatment of chronic wounds, which would be expected to benefit from any enhancement of neovascularization.
CONCLUSION The rabbit wound model studies described here confirm that intracellular delivery of ATP to a wound activates macrophages and results in the initiation of gross healing 726
within 24 h. The macrophages, secrete collagen directly into extracellular matrix that rapidly fills the wound space. Conventional wound healing, by contrast, starts only after a 3–6 day lag, and is a less direct process, where the wound space is first filled with a provisional matrix consisting of red cells trapped in a fibrin mesh. The two unique features of the ATP delivery healing process—elimination of the lag time and the ability to support cell survival and proliferation in a wound cavity without any blood supply in the early days following injury—have never been achieved with any other treatment. The rapid tissue generation following intracellular ATP delivery is a major advance that awaits full elucidation of its underlying biological mechanism. Whether it is the provision of energy to the microenvironment or the adenosine-mediated macrophage phenotype switching, or both, that hastens the wound healing process remains to be established. C 2015 by the Wound Healing Society Wound Rep Reg (2015) 23 724–727 V
Kotwal et al.
DISCLOSURES The authors received support in part from the following funding awarded to Sufan Chien; R01DK74566, R44A R52984, R43HL114235, R43GM106639, R43DK104625b and R43DK105692 from the National Institutes of Health and in part from the Kentucky Cabinet for Economic Development, Office of Entrepreneurship, under the Grant agreements KSTC-184-512-12-138, KSTC-184-512-14-174 with the Kentucky Science and Technology Corporation. Noveratech, LLC was founded by SC and is a co-inventor of a US issued patent on the intracellular ATP delivery technique.
REFERENCES 1. Martin P, Leibovich SJ. Inflammatory cells during wound repair: the good, the bad and the ugly. Trends Cell Biol 2005; 15: 599–607. 2. Delavary BM, van der Veer WM, van Egmond M, Niessen FB, Beelen RH. Macrophages in skin injury and repair. Immunobiology 2011; 216: 753–62. 3. Novak ML, Koh TJ. Phenotypic transitions of macrophages orchestrate tissue repair. Am J Pathol 2013; 183: 1352–63. 4. Brancato SK, Albina JE. Wound macrophages as key regulators of repair: origin, phenotype, and function. Am J Pathol 2011; 178: 19–25. 5. Ferrante CJ, Leibovich SJ. Regulation of macrophage pollarization and wound healing. Adv Wound Care 2012; 1: 10–16. 6. Davies LC, Jenkins SJ, Allen JE, Taylor PR. Tissue-resident macrophages. Nat Immunol 2013; 14: 986–95.
C 2015 by the Wound Healing Society Wound Rep Reg (2015) 23 724–727 V
Pivotal role of ATP in wound
7. Chiang B, Essick E, Ehringer W, Murphree S, Hauck MA, Li M, et al. Enhancing skin wound healing by direct intracellular ATP delivery. Am J Surg 2007; 193: 213–18. 8. Wang J, Zhang Q, Wan R, Mo Y, Li M, Tseng M, et al. Intracellular ATP-delivery enhanced skin wound healing in rabbits. Ann Plastic Surg 2009; 62: 180–6. 9. Wang J, Wan R, Mo Y, Li M, Zhang Q, Chien S. Intracellular delivery of ATP enhanced healing process in full-thickness skin wounds in diabetic animals. Am J Surg 2010; 199: 823–32. 10. Cohen MA, Eaglstein WH. Recombinant human plateletderived growth factor gel speeds healing of acute fullthickness punch biopsy wounds. J Am Acad Dermatol 2001; 45: 857–62. 11. Howard JD, Sarojini H, Wan R, Chien S. Rapid granulation tissue regeneration by intracellular ATP delivery-a comparison with regranex. PLoS One 2014; 9: 1–14. 12. Levenson SM, Demetriou AA. Metabolic factors. In: Cohen IK, Diegelmann RF, Lindblad WJ, eds. Wound healing: Biochemical & clinical aspects. Philadelphia: Saunders, 1992: 248–73. 13. Schaffer M, Witte M, Becker HD. Models to study ischemia in chronic wounds. Int J Low Extrem Wounds 2002; 1: 104–11. 14. Chien S. Intracellular ATP delivery using highly fusogenic liposomes. Methods Mol Biol 2010; 605: 377–92. 15. Wang X, Sansam CG, Thom CS, Metzger D, Evans JA, Nguyen PT, et al. Oncogenesis caused by loss of the SNF5 tumor suppressor is dependent on activity of BRG1, the ATPase of the SWI/SNF chromatin remodeling complex. Cancer Res 2009; 69: 8094–101.
727
