Oxygen in Acute and Chronic Wound Healing

S. Schreml; R.M. Szeimies; L. Prantl; S. Karrer; M. Landthaler; P. Babilas

Disclosures

The British Journal of Dermatology. 2010;163(2):257-268. 

In This Article

Chronic Wound Healing

Chronic wounds are defined as wounds that do not follow the well-defined stepwise process of physiological healing but are trapped in an uncoordinated and self-sustaining phase of inflammation (Fig. 1).[3] This impairs the constitution of anatomical and functional integrity in a physiologically appropriate length of time. The aetiology of chronic wounds is diverse, but more than 80% are associated with venous insufficiency, high blood pressure or diabetes mellitus.[3,57] Despite the different underlying aetiology, most chronic wounds show a similar behaviour and progress. This uniformity is due to consistent components of the multifactorial pathogenesis of most chronic wounds: local tissue hypoxia, bacterial colonization, repeated ischaemia-reperfusion injury and cellular as well as systemic changes of ageing (Fig. 2).[1,4,58,59]

Figure 2.

Chronic wound pathogenesis. Schematic representation of the elementary aetiological factors (blue), the resulting morphological correlates (green) and the consequent pathophysiology (red). The reciprocal interference of the pathophysiological factors is shown. These changes perpetuate inflammation in chronic wound pathogenesis.

Common causes of local wound tissue hypoxia are pathological alterations of the vascular bed (arteriosclerosis, micro- or macroangiopathy, venous hypertension), periwound fibrosis and a subsequent local reduction of tissue perfusion, or oedema, which increases the distance between capillaries (Fig. 2). Local tissue hypoxia has been widely accepted to impair wound healing profoundly. Mathematical models showing the importance of oxygen for physiological wound healing and ischaemic wounds have recently been published.[60,61] Sheffield measured a pO2 of 5–20 mmHg in chronic wound tissue as compared with 30–50 mmHg in control tissue.[62] Ahn and Mustoe[63] showed a wound healing deceleration of 80% in an ischaemic rabbit ear model evoked by a pO2 decrease from 40–45 mmHg to 28–30 mmHg. The initial implication of tissue hypoxia on the molecular level is the impairment of mitochondrial oxidative phosphorylation with a subsequently reduced ATP production. As a consequence, ATP-dependent membrane transport proteins such as Na+/K+-ATPase or Ca++-ATPase drop out, which leads to a loss of the transmembrane potential with subsequent cell swelling. Particularly intracellular accumulation of calcium ions activates a signal transduction pathway that ends up in cell membrane disruption,[64] which results in a promotion of inflammatory cascades via various signal pathways. Proinflammatory cytokines and chemokines such as TNF and IL-1 are released, which attracts and activates neutrophils and macrophages.[65] In addition, hypoxia induces a pronounced expression of endothelial adhesion molecules such as intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and the corresponding ligands leucocyte function-associated antigen-1 and very late antigen-4 that enhance the extravasation and invasion of neutrophils and macrophages into the wound site with a subsequent autocrine synthesis of proinflammatory cytokines such as IL-1α, IL-1β, IL-6 and TNF.[66] Growth factors and cytokines are released in a self-perpetuating manner, macrophages are attracted, and tissue degenerating enzymes, e.g. serine proteases (neutrophil-derived elastases, cathepsin) and MMPs, particularly MMP-8, are generated.[67] Wound fluid of chronic wounds has been demonstrated to show elevated levels of MMPs released from neutrophils (MMP-8) and fibroblasts (MMP-1, MMP-2, MMP-3, MMP-9, MMP-13).[68–70] In physiological wound healing, proteases are inhibited by α1-antiproteinase, SLPI, α2-macroglobulin and TIMPs, once all necrotic tissue and debris have been removed. In chronic wound healing, certain proteases (e.g. MMP-8 and MMP-9) exceed their inhibitors, leading to an excessive degradation of growth factors and ECM components such as collagen, fibronectin and vitronectin.[71] The breakdown products further promote inflammatory reaction,[72] changing the inflammation phase from a self-limiting to a self-sustaining process. Accordingly, the activity of neutrophils is long lasting in chronic wounds but is limited to the first 72 h in acute wound healing.[73,74]

Neutrophils and macrophages produce ROS like HO2, HO and O2−. As mentioned above, ROS in low concentrations provide signalling and defence against microorganisms and thus play an important role in acute wound healing. A prerequisite for this process is a delicate balance between the amount of oxidants and antioxidants, as high amounts of ROS impair wound healing due to oxidative damage. High amounts of ROS not only damage extracellular structure proteins, lipids and DNA, but also stimulate complex signal transduction pathways, leading to an enhanced expression of MMPs, serine proteases and inflammatory cytokines. The toxic effects of high amounts of ROS were shown by the severe endothelial damage in wounds of mice which lack the ROS-detoxifying enzyme peroxiredoxin-6.[75] The most effective antioxidant is nitric oxide (NO) that is produced by NO synthase in a strictly oxygen-dependent manner.[8] NO not only detoxifies ROS but also switches off nuclear factor-κB, an important transcriptional activator of inflammatory proteins.[76] Lymphocytes that invade wound tissue also activate the oxygen-dependent oxidoreductase thioredoxin as a protective mechanism against oxidative stress.[77] Under oxidative stress, macrophages express the oxygen-dependent haem oxygenase and cysteine transporter to protect themselves against ROS.[78] Thus, nearly all detoxification mechanisms are strictly oxygen dependent. Under hypoxic conditions, as prevalent in chronic wounds, the detoxification process is hindered, leading to a persistent and uncontrolled production of ROS and to a further potentiation of the inflammatory state. The resulting perpetual degradation of wound tissue impairs the maturation of wounds. However, hypoxia promotes wound healing not only by means of enhancing the inflammatory state but also through the impairment of countless other metabolic processes on the molecular, cellular and supracellular level. Siddiqui et al.[38] demonstrated in vitro that the collagen synthesis rate of human fibroblasts is decelerated under chronic hypoxia. α1-procollagen was significantly downregulated at the mRNA and at the protein level. Accordingly, Hunt et al.[79] showed in vivo that fibroblasts were actively proliferating only in wound tissue with a pO2 level of at least 15 mmHg. Jonsson et al.[80] demonstrated this causality in a clinical investigation, in which the amount of deposited collagen was proportional to the pO2 value present in the respective wound. In another ischaemic rabbit ear model, Wu et al.[81] demonstrated significantly impaired epithelial ingrowth and granulation tissue deposition under ischaemia.

Bacterial colonization, obligatory in all chronic wounds,[82] attracts leucocytes, which results in high levels of proinflammatory cytokines and proteases and therefore directly initiates and maintains the inflammatory cascade. Different authors investigated wound fluids from acute and chronic wounds and showed an increased level of proinflammatory cytokines and proteases as well as a decreased level of growth factors in chronic wounds.[65,83,84] Correspondingly, in healing wounds, a reduction in bacterial counts and markers of inflammation was reported.[85,86] A direct correlation between bacterial colonization and the hypoxic state of the wound was shown in numerous studies. Knighton et al.[87,88] compared the wound extent after subcutaneous inoculation of bacteria in hypoxic wounds with wounds in animals treated with oxygen and found an inversely proportional correlation between the wound extent and the wound oxygenation status. Grief et al.[89] performed a prospective study on 500 patients with colorectal resection. They compared the wound infection rates in two patient groups who had received 80% vs. 30% oxygen perioperatively and 2 h postoperatively. Wound infection rates were 5·2% (80% oxygen) and 11·2% (30% oxygen), respectively. Other studies showed that even a moderate decrease of the tissue oxygen level significantly increases the risk of infection.[90,91] Correspondingly, in an in vitro experiment, neutrophils were shown to lose their bacterial killing capacity at a pO2 level below 40 mmHg.[24,25]

In the past years, different authors postulated ischaemia-reperfusion injury as an important aetiological factor of chronic wounds.[58,92,93] Patients with impaired circulation due to venous insufficiency, arteriosclerosis, diabetes mellitus etc. suffer cyclic intervals of ischaemia and reperfusion in their lower legs when changing posture (leg elevation or leg dependency). As the leg position is changed repetitively, injury occurs in a self-potentiating cycle.[58,92] Ischaemia in combination with subsequent hypoxia induces a proinflammatory state (see above). With reperfusion, oedema is increased. Moreover, additional neutrophils flood into the wound tissue and transmigrate to the activated endothelium, which further contributes to the inflammatory vicious circle leading to cell death and tissue damage.[94] Besides, reperfusion accounts for partial reoxygenation with a subsequently enhanced production of ROS. In turn, ROS have a deleterious effect on vascular and cellular processes.[95] The impact of repetitive ischaemia-reperfusion injury on wound healing was demonstrated in animal models in which the tissue damage correlated with the number of ischaemia-reperfusion cycles.[93,96] Remarkably, repeated ischaemia-reperfusion cycles seem to be more deleterious for wound healing than prolonged phases of single ischaemia.[93,96]

The fact that ageing cells show reduced cell viability and proliferative capacity, altered patterns of gene expression and decreased response to growth factors is of great importance for our understanding of abnormal healing, as most chronic wounds occur in the elderly (average age over 60 years).[58,92,97,98] Senescent fibroblasts showed an increased generation of MMPs and a decreased release of MMP inhibitors,[99] which could explain the well-documented fact that MMP inhibitor (TIMP-1) and serine protease inhibitor (α1-antiproteinase, antileucoproteinase SLPI, α2-macroglobulin) activity is reduced in chronic wound fluids. The healing response in an aged organism is basically and essentially delayed[7] and additional pathogenetic factors such as local tissue hypoxia soon overpower response. Here, tissue hypoxia as a mainstay of chronic wound pathogenesis plays a crucial role because aged cells are significantly more susceptible to hypoxia than young adult cells. In an ischaemic rabbit ear model, aged human fibroblasts showed decelerated migration under stimulation with TGF-β, depression of PDGF receptor β, and decreased TGF-β1 mRNA expression compared with young controls.[81,100,101] Under hypoxic conditions, aged human keratinocytes showed a decelerated motility[101] and a decreased proliferation rate[102]in vitro compared with younger cells. Tandara et al.[103] demonstrated increased cell death of aged human fibroblasts compared with young adult cells if exposed to oxidant stress plus ischaemia, conditions that are analogous to chronic wounds.

Interestingly, cells of chronic wounds show signs of senescence even independently of a patient's age. Compared with fibroblasts taken from the healthy leg, fibroblasts harvested from the margin and bed of chronic wounds exhibited characteristics of premature senescence.[97,104] Agren et al.[105] showed in an in vitro setting a significantly decreased proliferative activity of fibroblasts of chronic wounds in comparison with fibroblasts isolated from acute wounds. These observations might be partially explained by the exposure of cells in chronic wounds to stress factors such as chronic inflammation and the respective proinflammatory cytokines (TNF, TGF-β), the presence of ROS,[106] and the aggressive proteolytic milieu caused by bacterial infection and toxins – factors that may accelerate cell senescence.[97] An additional explanation might be that fibroblasts are driven through countless cell divisions to induce wound healing. Due to ongoing stimulation, cells seem to lose their proliferative capacity. This causality could explain the success of wound debridement as this procedure removes senescent cells from the ulcer surface. These data demonstrate that stress factors apparent in chronic wounds, specifically hypoxia, and the resulting premature senescence create a vicious circle that is difficult to break, particularly in elderly patients.

From the clinical point of view, the main surface area of a common nonhealing wound shows extensive fibrin deposition and necrosis due to the prevalent inflammatory state. However, a typical hallmark of chronic wounds is the persistent occurrence of islands of granulation tissue or epithelialization within the inflammatory battlefield. Hunt et al.[79] showed that wound areas with actively proliferating fibroblasts were seen only at pO2 above 15 mmHg. Only rarely are these islands the origin of a structured healing process as, in most cases, they are overwhelmed by inflammation. The conditions in chronic wounds are changing repeatedly, not only temporally but also spatially. Thus, a chronic wound represents an extremely heterogeneous structure.

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