Dynamic reciprocity in the wound microenvironment

Here, we define dynamic reciprocity (DR) as an ongoing, bidirectional interaction among cells and their surrounding microenvironment. In this review, we posit that DR is especially meaningful during wound healing as the DR‐driven biochemical, biophysical, and cellular responses to injury play pivotal roles in regulating tissue regenerative responses. Such cell–extracellular matrix interactions not only guide and regulate cellular morphology, but also cellular differentiation, migration, proliferation, and survival during tissue development, including, e.g., embryogenesis, angiogenesis, as well as during pathologic processes including cancer, diabetes, hypertension, and chronic wound healing. Herein, we examine DR within the wound microenvironment while considering specific examples across acute and chronic wound healing. This review also considers how a number of hypotheses that attempt to explain chronic wound pathophysiology may be understood within the DR framework. The implications of applying the principles of DR to optimize wound care practice and future development of innovative wound healing therapeutics are also briefly considered.     

Fibroblast response to hypoxia: the relationship between angiogenesis and matrix regulation.

A number of studies have demonstrated the critical role of angiogenesis for successful wound repair in the surgical patient. Vascular disruption from tissue injury due to trauma or surgery leads to a hypoxic zone in the healing wound. In this dynamic process, angiogenesis is vital for the delivery of oxygen, nutrients, and growth factors necessary to initiate the synthetic processes of wound healing. Fibroblasts, invading the wound early in the healing process, are involved in extracellular matrix (ECM) deposition as well as wound contraction. However, the exact mechanisms by which important genes are regulated remain unknown. In order to examine these processes, we studied the effects of hypoxia on fibroblasts for the expression of VEGF, type IalphaI collagen, and matrix-metalloproteinase-3, three genes essential for the regulation of angiogenesis, ECM deposition, and ECM degradation in wound healing. Primary cell cultures of normal human dermal fibroblasts (NHDFs) were placed in hypoxia for varying periods of time. Northern blot hybridization was performed with [alpha32P]dCTP-labeled cDNA probes for VEGF, type IalphaI collagen, and MMP-3. The results demonstrated a time-dependent VEGF mRNA upregulation (470% of baseline) under hypoxia. Type IalphaI collagen increased (170% of baseline) at 24 h, but was then abruptly downregulated to 3.8% of baseline at 48 h. MMP-3 was incrementally downregulated to 2.2% of baseline at 48 h. These experiments focused on the effect of hypoxia on genes thought to play a role in wound repair. VEGF upregulation in the hypoxic microenvironment of the early wound may serve to stimulate angiogenesis. Type IalphaI collagen, though upregulated early on, was abruptly downregulated at 48 h. This downregulation may reflect the in vivo requirement for angiogenesis to deliver oxygen for successful hydroxylation and collagen synthesis in the wound. MMP-3, also downregulated at 48 h, may also implicate the need for angiogenesis. These data support the theory that hypoxia-driven angiogenesis is critical for ECM formation and remodeling in successful soft tissue repair. Furthermore, they may represent the role of hypoxia as an important regulator to efficiently balance these complex processes in the healing wound.     

Early wound healing exhibits cytokine surge without evidence of hypoxia

OBJECTIVE: To ascertain the spatial and temporal relation of wound hypoxia to the cell types involved, expression of selected angiogenic cytokines, the proliferative status of cells in the wound site, and angiogenesis. SUMMARY BACKGROUND DATA: Hypoxia is considered to drive the angiogenic response by upregulating angiogenic cytokines observed during wound healing. But this correlation has not been shown on a cell-to-cell basis in vivo because of limitations in measuring tissue PO2 at the cellular level. METHODS: Using punch biopsy wounds in rats as a wound healing model, the distributions of vascular endothelial growth factor, transforming growth factor-beta, tumor necrosis factor-alpha, and pimonidazole adducts (as a hypoxia marker) were followed immunohistochemically during the healing process. RESULTS: Hypoxia was absent on day 1 after wounding, even though angiogenesis and maximal expression of cytokines were observed in the wounds. Hypoxia peaked in the granulation tissue stage at day 4 and correlated with increased cellularity and cellular proliferation. Hypoxia started to decrease after day 4 and was limited to the remnant blood vessels and epithelial layer in the scar tissue. CONCLUSIONS: Induction of angiogenic cytokines early during wound healing may be due to triggering mechanisms other than hypoxia. Alternatively, the unique pattern of development and decline of cellular hypoxia as wound cellularity and proliferation regress suggest its involvement in initiating vascular regression during the later stages of healing.     

Role of alpha(v) integrins and angiogenesis during wound repair.

Angiogenesis, the formation of new blood vessels from pre-existing blood vessels, is thought to be critical for wound repair. Yet few studies have critically examined dermal wound repair in a system in which angiogenesis was impaired. Since alpha(v)-containing integrins are critical for angiogenesis, we administered either an alpha(v) integrin blocking antibody or cyclic Arg-Gly-Asp peptide into a murine excisional wound model to restrict wound angiogenesis. Although both methods markedly decreased wound angiogenesis, decreased angiogenesis had no significant effect on wound epithelization, contraction, or ultimate wound closure. These results suggest that if other cellular components of wound healing are intact, moderate impairment of angiogenesis alone does not necessarily retard normal wound healing.     

The relationship between protease/anti-protease profile, angiogenesis and re-epithelialisation in acute burn wounds

In the management of partial thickness burns, it is difficult to balance between conservative management and surgical intervention. Our hypothesis was that a triangular relationship exists between protease/anti-protease profile at the burn wound surface, angiogenesis and re-epithelialisation. By manipulation of the biochemical profile at the wound level, we determined to affect the nature and extent of angiogenesis and resulting re-epithelialisation. We performed a randomised longitudinal observational study on partial thickness burns in adult patients presenting to two regional burns units.

Our results demonstrated that a high-protease wound environment is associated with lower levels of the angiogenic factor VEGF, a lower more uniform change in wound bloodflow and a uniform well healed wound with an architecturally normal epidermis. In addition, we found that a low protease wound environment is associated with higher levels of the angiogenic factor VEGF, a higher wound bloodflow throughout the wound healing period and a more chaotic, hypercellular, overkeratinised, and chaotic thickened epidermis.     

Mechanisms influencing cellular physiology as a concept of treatment for wound healing disturbances

AIM: Recent knowledge about repair mechanisms in different types of tissue is the basic of actual therapeutic efforts. Center of several experimental and clinical approaches is the influencing of angiogenesis with an also distinct meaning concerning wound healing. Therefore, application of growth factors, gene transfer, and employment of genetically manipulated cells often aim at angiogenesis. Nevertheless, manipulation of angiogenesis also leads to secondary problems such as hyperpermeability followed by impairment of local wound milieu. Our study was done to identify mechanisms to protect from disturbances of endothelial barrier function. METHOD: In a first experimental investigation on cultured endothelial cells, the influence of plasma-transglutaminase (Factor XIII) to endothelial barrier function was studied. In a second step, the influence of Factor XIII on wound healing properties was investigated in patients with a chronic venous ulceration. RESULTS: Activated Factor XIII (FXIIIA*) led to a dose-dependent reduction of endothelial cell permeability of 30 % compared to control with a maximum effect using 1 to 5 U/mL. Clinical investigation revealed a nearly complete reduction of wound secretion. CONCLUSION: Experimental studies revealed that activated Factor XIII stabilizes endothelial barrier under basic conditions as well as under conditions of induced hyperpermeability. Clinical study revealed that Factor XIII also distinctly reduces wound secretion. Therefore, plasma-transglutaminase may offer a new therapeutic option to treat the local or generalized leakage-syndrome.     

Relationship between lymphangiogenesis and exudates during the wound-healing process of mouse skin full-thickness wound

We considered the relationship among exudate, wound area, angiogenesis, lymphangiogenesis, and reepithelialization during wound healing. Full-thickness wounds were made on the dorsum of mice. The weight of exudate absorbed into the dressing as well as the wound area was determined daily. Sections of the wounds were stained with anti-LYVE-1 and CD31 antibodies. Indian ink was injected into the wound for observing the movement of the exudate on days 3, 5, and 7 after wounding. New epithelium completely covered the wound on day 11. The quantity of exudate peaked on day 1, and then rapidly decreased until it was undetectable on day 11. Most of the Indian ink injected into the wound was retained within the wound and did not flow into the surrounding tissue. New blood vessels showed a uniform distribution in the granulation tissue on day 5. New lymphatics appeared in the granulation tissue approximately 2 days later than the blood vessels and they were distributed toward the center of the granulation tissue on day 11. Thus, reduction of exudate from the wound appears to be related to blood vessels, not lymphatics. However, increasing lymphatics may play a role in the late phase of the wound-healing process.     

Hox D3 expression in normal and impaired wound healing

BACKGROUND: We have previously shown that Hox D3 and Hox B3 can promote angiogenesis. As angiogenesis is essential for wound healing, we examined expression of these genes in the vasculature following wounding in normal and genetically diabetic adult mice with impaired healing. METHODS: In situ hybridization was performed on tissues taken 0, 1, 4, 7, and 14 days following administration of linear wounds in wild-type and genetically diabetic mice. Expression of Hox D3 and Hox B3, angiogenesis, and synthesis of type I collagen were assessed in the wound. RESULTS: Hox B3 was expressed in endothelial cells (ECs) of both medium and small vessels in unwounded tissue, whereas little Hox D3 was detected in resting ECs. Hox D3 expression was significantly upregulated by 1 day after wounding in ECs of vessels immediately adjacent to the wound site, and expression was maintained for at least 7 days. In the diabetic mice, expression of Hox B3 was similar to that of wild-type mice. In contrast, expression of Hox D3 in ECs was significantly lower and delayed during wound repair in diabetic mice. In cultured microvascular ECs, Hox D3 selectively induced high levels of collagen I mRNA expression. Hox D3-deficient wounds of diabetic animals also displayed a reduction in expression and deposition of type I collagen. CONCLUSIONS: These results suggest that reduced angiogenesis and type I collagen in diabetic mice with impaired wound healing may be related to deficient Hox D3 expression, and restoring Hox D3 expression may enhance angiogenesis and wound repair.     

Cultured autologous keratinocytes on a cell-free dermis in the treatment of full-thickness wounds.

The purpose of this study was to establish a method for transplantation of autologous keratinocytes on an allogenic cell-free dermis. From four healthy volunteers two full thickness skin grafts, 1 x 1 cm, were taken. The epidermis was separated from the dermis enzymatically and the cells of the dermal part were removed by incubation in Triton X-100. Keratinocytes were seeded on a cell-free dermis and the combined graft transplanted back to one of the wounds of the donor of the keratinocytes. The other wound was covered with cell-free dermis without keratinocytes. After 2, 3, 4 and 6 weeks, respectively, the grafted wounds were removed from the subjects and investigated histologically and immunohistochemically regarding re-epithelialisation, fibroblast ingrowth and angiogenesis. The wounds covered with cell-free dermis and keratinocytes showed a complete epidermal coverage 2 weeks after transplantation, in contrast to the wounds covered with un-seeded dermis which only showed epidermal coverage at the wound edges. There was also a marked difference concerning fibroblast ingrowth and angiogenesis. In this study we have shown that autologous keratinocytes can be seeded on a cell-free dermis and transplanted as a kerato-dermal graft which stimulate re-epithelialisation as well as fibroblast ingrowth and angiogenesis in the wound.     

Nitric Oxide and Wound Healing

Nitric oxide is a short-lived free radical that acts at the molecular, cellular, and physiologic level. Since its discovery almost 20 years ago it has proven itself as an important element in wound healing. This review highlights many of the important aspects of nitric oxide in wound healing, including a review of the basic biology of nitric oxide, its role as part of the cytokine cascade and as a promoter of angiogenesis, as well as its more recently elucidated role in apoptosis.     

Fucoidan promotes angiogenesis and accelerates wound healing through AKT/Nrf2/HIF-1α signalling pathway

After skin injury, wound repair involves a complex process in which angiogenesis plays a crucial role. Previous research has indicated that fucoidan may aid in wound healing; we therefore hypothesised that fucoidan may speed up the process by promoting angiogenesis. In this study, we investigated the potential molecular mechanism underlying fucoidan's ability to accelerate wound healing by promoting angiogenesis. Using a full-cut wound model, we observed that fucoidan significantly intensified wound closure and promoted granulation formation and collagen deposition. Immunofluorescence staining revealed that fucoidan also promoted wound angiogenesis, specifically by accelerating the migration of new blood vessels to the middle area of the wound. Furthermore, fucoidan demonstrated the ability to enhance the proliferation of human umbilical vein endothelial cells (HUVECs) damaged by hydrogen peroxide (H₂O₂) and to improve the formation of endothelial tubes. Mechanistic studies revealed that fucoidan upregulated the protein levels of the AKT/Nrf2/HIF-1α signalling pathway, which plays a crucial role in angiogenesis. This was further confirmed using the inhibitor LY294002, which reversed the promotion of endothelial tube formation by fucoidan. Overall, our findings suggest that fucoidan can promote angiogenesis via the AKT/Nrf2/HIF-1α signalling pathway and accelerate wound healing.     

Activated protein C prevents inflammation yet stimulates angiogenesis to promote cutaneous wound healing

Activated protein C (APC) is a serine protease that plays a central role in physiological anticoagulation, and has more recently been shown to be a potent anti-inflammatory mediator. Using cultured human cells, we show here that APC up-regulates the angiogenic promoters matrix metalloproteinase-2 in skin fibroblasts and umbilical vein endothelial cells, vascular endothelial growth factor in keratinocytes and fibroblasts, and monocyte chemoattractant protein-1 in fibroblasts. In the chick embryo chorioallantoic membrane assay, APC promoted the granulation/remodeling phases of wound healing by markedly stimulating angiogenesis as well as promoting reepithelialization. In a full-thickness rat skin-healing model, a single topical application of APC enhanced wound healing compared to saline control. APC-treated wounds had markedly more blood vessels on day 7 and a significantly lower infiltration of neutrophils at days 4 and 7. The broad spectrum matrix metallo-proteinase, GM6001, prevented the ability of APC to promote wound healing. In summary, our results show that APC promotes cutaneous wound healing via a complex mechanism involving stimulation of angiogenesis and inhibition of inflammation. These unique properties of APC make it an attractive therapeutic agent to promote the healing of chronic wounds.     

紫黄生肌膏对慢性感染创面血管生成及血管内皮生长因子和血管生成素-1表达的影响

目的:观察紫黄生肌膏对大鼠慢性感染性创面血管生成及血管内皮生长因子(VEGF)和血管生成素(Ang)-1表达的影响.方法:将84只SD大鼠随机分为4组,分别是空白组、模型组、贝复济组、紫黄生肌膏组,造模成功后,除空白组外,其余组每天换药前在创面上涂粪液1 mL,保留30 min后予以清洁换药,于用药后3、7、14 d观...     

Topical vascular endothelial growth factor reverses delayed wound healing secondary to angiogenesis inhibitor administration

The prevention of new blood vessel growth is an increasingly attractive strategy to limit tumor growth. However, it remains unclear whether anti-angiogenesis approaches will impair wound healing, a process thought to be angiogenesis dependent. Results of previous studies differ as to whether angiogenesis inhibitors delay wound healing. We evaluated whether endostatin at tumor-inhibiting doses delayed excisional wound closure. C57/BL6J mice were treated with endostatin or phosphate-buffered solution 3 days prior to the creation of two full-thickness wounds on the dorsum. Endostatin was administered daily until wound closure was complete. A third group received endostatin, but also had daily topical vascular endothelial growth factor applied locally to the wound. Wound area was measured daily and the wounds were analyzed for granulation tissue formation, epithelial gap, and wound vascularity. Endostatin-treated mice showed a significant delay in wound healing. Granulation tissue formation and wound vascularity were significantly decreased, but reepithelialization was not effected. Topical vascular endothelial growth factor application to wounds in endostatin-treated mice resulted in increased granulation tissue formation, increased wound vascularity, and wound closure approaching that of control mice. This study shows that the angiogenesis inhibitor endostatin delays wound healing and that topical vascular endothelial growth factor is effective in counteracting this effect.     

Angiogenic effects of suture biomaterials. An experimental study in rats

Angiogenesis is a limiting step of inflammation and wound healing. Although laboratory tests for the evaluation of the angiogenetic effects of biomaterials are available, no investigations have been performed. In this study we examine the angiogenetic effect of suture biomaterials in the rat mesenteric window model. Absolute controls had laparotomy only, controls had mesenteric window wounding, test groups had the insertion of either a thread of collagen, polyglactin 910, polyglycolic acid salt, silk or of a titanium clip. We considered the percentage area of the mesenteric window covered by neovessels. After 7 days, negligible angiogenesis was found in absolute controls, significant angiogenesis in controls, collagen, polyglactin 910 and polyglycolic acid salt treated rats, without differences between treatments. A significant increase of angiogenesis in comparison to the control group was found in rats treated with silk and titanium. Ultramicroscopy of the neovessels showed specific changes of their architecture in titanium-treated rats. In conclusion, angiogenesis during wound healing is differently influenced by the suture material used. Silk and titanium stimulated angiogenesis in a different way.     

A single local application of recombinant human basic fibroblast growth factor accelerates initial angiogenesis during wound healing in rabbit ear chamber

Local angiogenic therapy with recombinant human basic fibroblast growth factor (rhbFGF) has been used to promote wound healing. To obtain useful information for the development of optimal angiogenic therapy, we chronologically evaluated the effects of a single local application of rhbFGF on angiogenesis in a rabbit ear chamber model of wound healing by observing the subcutaneous vessel bed intravitally. New vessel formation during wound healing was macroscopically and microscopically evaluated for 5 wk. Each rabbit ear chamber received a single dose of 6 microg rhbFGF (treatment B1: n = 13), 18 microg rhbFGF (treatment B2: n = 16), or physiological saline as control (n = 13). At 1 wk the newly vascularized area was significantly larger in groups B1 and B2 than in control. At 2 wk, the vascularized areas in groups B1, B2, and control were similar. At 5 wk, the percentage of rabbits with complete vascularization was significantly larger in group B1 than in control. Capillary density at 5 wk was similar among the three groups. These results suggest that locally applied rhbFGF accelerated angiogenesis during early wound healing in rabbits; however, this effect was transient and no increase in capillary density occurred at the completion of vascularization.     

Cutaneous window for in vivo observations of organs and angiogenesis

BACKGROUND AND OBJECTIVE: The continuous observation in experimental animals of internal organs and processes, such as wound healing and angiogenesis, has been achieved using a variety of transparent windows and chambers. Our objective was to develop procedures for these observations using disposable material for the window and simple surgical techniques. METHODS: For observation of wound healing in the mouse kidney, the kidney was externalized and a wedge was excised. An oval window of polyvinyl chloride film was sutured in place in the skin over the wound. The progress of healing of the wound was observed through the window over 10 days. For observation of angiogenesis, adrenocortical cells were transplanted beneath fascia and muscle and a window was sewn into the skin above the site of transplantation. RESULTS: Clear observations could be made using these cutaneous windows over the period of the experiments. Healing of a wound in the kidney was photographed and measured. The growth of new blood vessels over the site of adrenocortical cell transplantation was observed. CONCLUSIONS: Continuous in vivo observations of organs such as the kidney and processes such as angiogenesis can be made in experimental animals using this simple technique.     

Wound repair and regeneration

The skin is the biggest organ of the human being and has many functions. Therefore, the healing of a skin wound displays an extraordinary mechanism of cascading cellular functions which is unique in nature. As healing and regeneration processes take place in all parts of the human body, this review focuses on the healing processes of the skin and highlights the classical wound healing phases. While regeneration describes the specific substitution of the tissue, i.e. the superficial epidermis, mucosa or fetal skin, skin repair displays an unspecific form of healing in which the wound heals by fibrosis and scar formation. The first stage of acute wound healing is dedicated to hemostasis and the formation of a provisional wound matrix, which occurs immediately after injury and is completed after some hours. Furthermore, this phase initiates the inflammatory process. The inflammatory phase of the wound healing cascade gets activated during the coagulation phase and can roughly be divided into an early phase with neutrophil recruitment and a late phase with the appearance and transformation of monocytes. In the phase of proliferation the main focus of the healing process lies in the recovering of the wound surface, the formation of granulation tissue and the restoration of the vascular network. Therefore, next to the immigration of local fibroblasts along the fibrin network and the beginning of reepithelialization from the wound edges, neovascularization and angiogenesis get activated by capillary sprouting. The formation of granulation tissue stops through apoptosis of the cells, characterizing a mature wound as avascular as well as acellular. During the maturation of the wound the components of the extracellular matrix undergo certain changes. The physiological endpoint of mammalian wound repair displays the formation of a scar, which is directly linked to the extent of the inflammatory process throughout wound healing.     

Nitric oxide and wound repair

NO produced by both iNOS and eNOS plays many important roles in wound healing, from the inflammatory phase through to scar remodeling. NO has cytostatic, chemotactic, and vasodilatory effects during early wound repair, regulates proliferation and differentiation of several cell types, modulates collagen deposition and angiogenesis, and affects wound contraction. The data accumulated thus far indicates that the timing, level, and site of NO production are highly coordinated in normal wound repair. Defining states resulting from either inadequate substrate or depressed enzyme expression appear to contribute to impaired wound repair; however, NO represents only one factor in the complex process of wound healing. Approaches to improve NO availability may be of therapeutic value.