Brewing complications: the effect of acute ethanol exposure on wound healing

Abstract

Ethanol consumption is linked to a higher incidence of traumatic wounds and increases the risk for morbidity and mortality following surgical or traumatic injury. One of the most profound effects of acute ethanol exposure on wound healing occurs during the inflammatory response, and altered cytokine production is a primary component. Acute ethanol exposure also impairs the proliferative response during healing, causing delays in epithelial coverage, collagen synthesis, and blood vessel regrowth. The accumulated data support the paradigm that acute ethanol intoxication prior to injury significantly diminishes a patient’s ability to heal efficiently.

Introduction

Traumatic injury remains the primary cause for emergency room visits each year in the United States. Most traumatic injuries are sustained following motor vehicle crashes, weapon-related injuries, burns, and falls. Nearly all of these injuries result in a severe wound, and many will require an additional surgical procedure. Successful wound repair is therefore critical to patient outcomes for this population. Out of the millions of trauma patients treated in the emergency room each year, over half are intoxicated at the time of injury [1,2,3]. Although most studies to date have focused on the effect of chronic alcohol abuse, a significant fraction of trauma patients exhibits binge or acute ethanol use prior to injury [4,5,6]. In such patients, ethanol clearance is generally achieved within a short time-frame, yet ethanol exposure has been shown to impact morbidity and mortality significantly in trauma patients. Recent studies suggest that even a single isolated incidence of acute ethanol exposure, if it occurs at the time of injury, can perturb the response to trauma significantly. Although ethanol exposure certainly influences many systemic parameters, including hemodynamic and metabolic processes, this review will focus on those changes that acute ethanol induces at the wound site itself. The consequences of acute ethanol exposure at the time of injury on downstream wound-healing events are the topic of this review.

THE PROCESS OF WOUND REPAIR

Wound repair involves a dynamic interaction among cytokines, growth factors, cells, and the ECM through three overlapping phases of inflammation, proliferation, and remodeling [7, 8] (Fig. 1). The cascade of events begins immediately after injury with the aggregation of platelets, initiation of the clotting cascade, and aggregation of fibrin, an insoluble blood-derived protein resulting from fibrinogen cleavage [7]. The fibrin clot prevents further blood loss and provides a transient matrix, upon which cells can attach and migrate (Fig. 1). During the inflammatory phase, neutrophils and monocytes infiltrate into the wound bed. Neutrophils are the first immune cells to arrive and serve as the initial line of defense against invading pathogens through phagocytosis and the production of ROS [9]. Macrophages (derived from monocytes) believed to play a vital role in repair, enter the wound bed shortly after neutrophils. Macrophages participate in host defense through antigen presentation and phagocytosis and secrete growth factors that stimulate angiogenesis and the formation of fibrous tissue [10,11,12]. As inflammation gives way to proliferation, the fibrin clot must be remodeled and replaced to allow for cellular migration and proliferation [13]. The fibrinolytic pathway involves a tightly coordinated protease network that begins with the conversion of plasminogen to plasmin by a PA. Plasmin then attacks fibrin at multiple sites, causing its degradation into inactive remnants. Dysregulation of these proteolytic proteins is part of various pathobiologic states, such as cancer, fibrosis, arthritis, and delayed wound healing [14,15,16].

Figure 1.

Schematic of the process of wound repair. Uninjured skin is composed of an outer layer of epidermal cells (keratinocytes) and a dermal layer composed of fibroblasts, ECM components, and vasculature. Immediately following wounding, degranulation of platelets occurs following damage to the vasculature (1). Hemostasis is then achieved through the release of clotting factors and fibrin, which acts as a provisional matrix for the migration of immune cells (2). Neutrophils are the first immune cells to arrive at the wound site, followed by macrophages, both of which participate in host defense (3). The initial fibrin matrix is degraded slowly and replaced by newly synthesized ECM components by the fibroblasts (4). The robust fibroblast and endothelial cell proliferation result in the synthesis of fibrillar collagen and revascularization (5). Vessel regression occurs along with matrix remodeling, and 80–90% of the skin’s original integrity is restored (6).

The proliferative phase of healing involves wound closure, angiogenesis, and matrix deposition and is initiated in response to stimulatory factors produced initially during the inflammatory phase (Fig. 1) [9, 17,18,19]. In skin, regeneration of the epithelium occurs through cellular proliferation, migration, and differentiation [7, 20]. As alluded to above, the effective migration of keratinocytes along the provisional matrix requires degradation of the fibrin clot and modification of the ECM via the production of proteolytic molecules [21].

The development of a new capillary bed, or angiogenesis, provides oxygen and nutrient support to the rapidly proliferating cells within healing wounds. During the proliferative phase of wound healing, capillary growth continues until the vascular density reaches nearly three times that of uninjured normal tissue [22]. During the resolution phase of repair, most of the new capillaries regress, leaving behind a residual vascularity that is similar or slightly higher than uninjured tissue [23].

Similar to other angiogenic situations, the magnitude of neovascularization in wounds is dependent on many factors, including levels of growth factors, cell responsiveness, cell-to-cell interaction, cell-to-ECM interaction, and the activity of proteases [24,25,26]. VEGF is a potent, directly acting angiogenic factor that is capable of stimulating endothelial cell migration and activation in vitro and angiogenesis in vivo [27, 28]. Several pieces of evidence suggest that VEGF is the single-most important mediator of angiogenesis in wounds. First, the time course of production of VEGF parallels the time course of angiogenesis [29]. Second, neutralization of VEGF decreases wound angiogenic activity significantly [29,30,31]. Finally, the blockade of VEGF receptors inhibits wound angiogenesis significantly [32, 33]. Taken together, these findings support the concept that the proliferative phase of wound angiogenesis is sustained primarily by VEGF, which is produced by multiple cell types within the wound, including macrophages, endothelial cells, and keratinocytes [30, 34, 35].

Soluble VEGF induces angiogenesis through the binding of two tyrosine kinase cell-surface receptors, VEGFR1 and VEGFR2 [36]. Although VEGF interacts with both receptors, functional differences between VEGFR1 and VEGFR2 exist. VEGFR1 binds VEGF with an affinity tenfold higher than VEGFR2, yet the tyrosine kinase activity of VEGFR2, upon binding VEGF, is tenfold higher than VEGFR1 [37]. Most studies suggest that VEGFR2 is the key receptor responsible for endothelial proliferation and migration during wound angiogenesis [33]. Interestingly, VEGF receptors have also been shown to be present on keratinocytes, implying that VEGF may play a role in the reepithelialization of wounds as well as angiogenesis [38].

The robust angiogenesis in wounds supports the restitution of a durable dermis, which is an essential component of adequate repair. Collagen is the primary structural component of the skin and composes most of the dermal ECM [20]. The dermal matrix also contains elastic fibers, fibronectin, glycosaminoglycans, and proteoglycans, all of which are restored to some degree by the healing process [7]. During wound repair, fibroblasts within the dermis exhibit a significant proliferative response. Fibroblasts produce the majority of ECM in wounds and convert the fibrin-rich provisional matrix into a secondary provisional matrix [7, 39]. As the fibroblasts migrate from the periphery into the center of the wound, they lyse the fibrin clot and replace it with fibronectin and hyaluronic acid, which develops into early granulation tissue. Fibronectin is a critical matrix component in healing wounds, as it serves to bind migrating cells and acts as a scaffold for newly synthesized collagen fibers. The secondary matrix is replaced first by collagen type III, followed by collagen type I.

The ECM in wound beds provides the scaffold upon which cells migrate and also acts as a reservoir for growth factors. Cell adhesion and migration are critical processes for wound closure, angiogenesis, and scar formation and are dependent on an intact ECM [15, 40,41,42]. The ECM composition depends on an equilibrium between protein synthesis and degradation. In healing wounds, the degradation of ECM components is mediated predominantly by MMPs, a family of proteases that are involved in the hydrolysis of ECM components [43,44,45]. Three groups of MMPs are named according to their respective substrates: collagenases, gelatinases, and stromelysins [20]. The collagenases are involved in the degradation of native fibrillar collagen, such as types I, II, and III [46, 47]. Stromelysins, on the other hand, vary in their substrate specificity and are involved in the release of matrix-bound proteins [48]. Gelatinases participate in the degradation of basement membrane proteins and denatured collagen fragments [49].

During the resolution phase of healing, most of the newly formed capillaries regress via apoptosis. The dermis is remodeled slowly through a continued balance of degradative and synthetic activity, along with the cross-linking of collagen. This remodeling process yields an organized matrix that re-establishes the strength and flexibility of the wounded tissue [17, 20, 22, 50]. In normally healing wounds, ∼90% of the original strength of the skin is restored upon completion of tissue repair [20].

Models of ethanol exposure

Established models of ethanol exposure include oral chronic administration, oral gavage, and i.p. injection [51]. For chronic ethanol exposure, a liquid diet containing ethanol is commonly used in rats and mice to produce a sustained level of intoxication. Alternatively, daily oral gavage or intragastric administration may also be used daily to achieve chronic intoxication, although the process is much more tedious and can cause physiologic alterations in the gut compared with the liquid ethanol diet [52].

For acute ethanol exposure, oral gavage can be used, especially in larger animals, such as rats or rabbits. When used in smaller animals, such as the mouse, oral gavage frequently leads to esophageal refluxes and thus, loss of ethanol. To provide accurate dosing as well as for the ease of administration, i.p. injection is often used for acute ethanol administration in smaller animals.

Metabolism of ethanol

Several mechanisms may play a part in the molecular and cellular changes that are caused by ethanol during complex processes such as tissue repair [53]. The effects of ethanol on cellular activity may be directly a result of the ethanol itself via changes in membrane fluidity or of the products of its metabolism via its oxidation. Three enzyme systems are involved in ethanol oxidation: ADH, the MEOS, and catalase. Ethanol is oxidized predominantly by the ADH pathway, with the MEOS and catalase pathways accounting for only a small fraction of ethanol metabolism. NADH, acetaldehyde, and acetate metabolites generated from the breakdown of ethanol induce tissue damage through the production of ROS, lipid peroxidation, and alterations in signal transduction [53,54,55].

Acetaldehyde is the primary oxidation product of ethanol and is generated by all three pathways. The toxicity of acetaldehyde is mediated by its capacity to form protein adducts, thus contributing to the decrease in oxygen use, enzyme activity, DNA repair mechanisms, free-radical formation, and depleted glutathione levels [54, 56, 57]. ROS is a term that describes radicals and other nonradical reactive oxygen derivatives, including hydrogen peroxide, superoxide, and NO, which may participate in reactions giving rise to free radicals that are damaging to organic substrates. At low levels, ROS are believed to be beneficial and serve as cellular messengers to promote such actions as cell motility, angiogenesis, and establishment of the ECM [58]. On the contrary, ROS, in excess, are believed to be detrimental for wound healing and have been implicated in the pathology of chronic wounds [59,60,61]. Likewise, production of protein adducts via metabolism of ethanol into acetaldehyde can potentially lead to aberrant cell function. However, the production of protein adducts may be much less robust in a model of acute ethanol exposure and hence, contribute less to perturbations in cellular function.

EFFECTS OF ETHANOL EXPOSURE ON THE INFLAMMATORY RESPONSE IN WOUNDS

Acute ethanol exposure has been shown to perturb many of the processes that are critical to appropriate wound healing, including inflammation, angiogenesis, and restitution of the ECM. Interestingly, only a few studies describe local effects of acute ethanol exposure on the inflammatory processes in the healing wound. The available information comes primarily from studies of burn or excisional injury models in rodents. Chemokines that attract neutrophils (KC, MIP-2) and macrophages (MCP-1) are known to be produced at sites of burn injury [62, 63]. In this system, ethanol exposure affects at least one neutrophil chemoattractant, KC, which is increased in burn wounds of mice exposed to ethanol yet is without a corresponding change in the number of neutrophils [62]. In excisional wounds, prior acute ethanol exposure seems to result in a distinct functional impairment in inflammation [64]. In a murine excisional wound model, acute ethanol exposure caused a significant reduction in the wound levels of two potent neutrophil chemoattractants, MIP-2 and KC, at 12 h after injury. This reduction in the level of neutrophil chemokines corresponded with a 40% decrease in neutrophil infiltration at the wound site. In support of the idea that acute ethanol exposure might seriously impair local immune function in wounds, many studies demonstrate the ability of ethanol to perturb functions that are known to be critical at the wound site. For example, in splenic macrophages, cytokine production is dysregulated after exposure to ethanol and burn to a greater degree than burn alone [65]. Acute ethanol exposure inhibits the production of the proinflammatory mediators IL-8 and TNF-α from human mononuclear cells following LPS challenge, thus potentially suppressing the migration and activation of neutrophils [66]. Furthermore, monocyte function is impaired significantly by acute ethanol exposure, as stimulated monocytes exhibited a down-regulation of proinflammatory molecules TNF-α and IL-1β and up-regulation of immunoinhibitory molecules TGF-β and IL-10 [67]. Acute ethanol exposure suppresses TLR-mediated signaling, thus contributing to the dysregulation of macrophage function by diminishing their response to microbial pathogens [68,69,70,71].

Beyond effects on immune cells themselves, another mechanism by which ethanol might influence inflammation is by altering the adhesion of leukocytes to endothelium. Leukocyte-endothelial interactions are critical to the movement of white blood cells into tissues, including wounds [72]. Studies in LPS and carrageenan-induced models of inflammation suggest that ethanol exposure blocks endothelial activation, thus modulating leukocyte-endothelial interactions [73]. Ethanol exposure has also been shown to inhibit the ability of activated monocytes to adhere to endothelium via a direct effect on monocytes [74].

Regulated inflammation is important for successful healing, and excessive or restrained leukocyte infiltration can influence repair negatively. Given myriad potential effects of acute ethanol exposure on immune function and inflammation, it seems plausible that acute ethanol exposure might predispose patients to increased wound infection. Indeed, individuals with acute ethanol exposure have been shown to be more susceptible to infection, particularly in the lung [75,76,77]. Ethanol exposure was also shown to be a contributing factor toward the enhanced rates of mortality following Pseudomonas aeruginosa infection in mice exposed to acute ethanol prior to burn injury [62]. Ethanol-related alterations in immune cell function within the wound bed also probably set the stage for subsequent changes in the proliferative response [78].

ACUTE ETHANOL AND THE PROLIFERATIVE PHASE OF REPAIR

Few investigations have attempted to assess the effects of ethanol on the proliferative phase of wound healing in vivo. This is quite astounding considering that ethanol consumption increases the frequency and severity of injuries, thus complicating patient care administration. Acute, nonchronic ethanol exposure is seen frequently in trauma victims. Even short-term ethanol exposure can yield detrimental effects on cellular functions through a variety of mechanisms, including changes in membrane fluidity, mRNA stability, transcription factors, post-translational modifications, and cell-surface receptor expression. Below, we review the influence of acute ethanol exposure on wound angiogenesis and ECM repair.

ACUTE ETHANOL AND WOUND ANGIOGENESIS

Despite the clinical evidence suggesting that acute ethanol exposure impairs wound healing, studies pertaining to ethanol exposure and angiogenesis, particularly wound angiogenesis, are scarce. Although some research has focused on ethanol exposure and gastric mucosal angiogenesis, no studies have assessed the effects of ethanol treatment on dermal wound angiogenesis. The effect of ethanol exposure on endothelial cell signaling and angiogenesis has also received minimal experimental attention. Studies in other cell types suggest that receptors and signals important to endothelial cell function can be affected by ethanol exposure. For example, many studies demonstrate that ethanol exposure can enhance or suppress protein kinase C, a signaling pathway that is important in endothelial cell responses, depending on the dose of ethanol [79, 80]. Ethanol has also been implicated in membrane-lipid remodeling, lipid-derived signals, and production of lipid mediators [81]. One study determined that 5% of membrane phospholipids was phosphatidylethanol in ethanol-treated endothelial cells, compared with only 0.5–1% in those cells not treated with ethanol. This change in phosphotidylethanol composition is believed to alter membrane properties and influence intracellular signaling [82].

Studies in our own lab suggest that angiogenesis and the vascularity of wounds are markedly reduced by a single exposure to ethanol and that this effect involves multiple components (Fig. 2) [83]. The direct effect of ethanol on endothelial cell responsiveness has been examined further in a well-characterized in vitro cord-formation assay, in which endothelial cells differentiate into cord-like structures in the presence of VEGF. Exposure to 100 mg/dl ethanol caused a significant inhibition of the formation of cord-like structures in response to VEGF. The level of cord formation by cells exposed to VEGF and ethanol averaged just 8.33% of that of control cells exposed to VEGF alone [83, 84]. We then investigated whether acute ethanol exposure caused a transient or stable inhibition of endothelial cell cord formation in vitro. Endothelial cells were incubated in flasks with ethanol (100 mg/dl) or media alone for 8 h. After 8 h, the media were removed and replaced with fresh ethanol-free media for another 8 h, and the cells were subjected to the cord-forming assay. Even after a period of 8 h in ethanol-free media, the ability of endothelial cells to form cord-like structures was impaired significantly. Endothelial cells incubated with ethanol and VEGF exhibited a 60% reduction in percent cord formation compared with cells incubated with VEGF alone [84].

Figure 2.

Probable effects of ethanol on reparative angiogenesis. Ethanol inhibits the hypoxia-associated translocation of HIF-1α and subsequent production of VEGF. Ethanol also decreases expression and phosphorylation (p) of VEGFR2 in endothelial cells. Through a combination of these effects, endothelial function is impaired, including the ability to form cord-like structures, ultimately resulting in a reduction in capillary density, wound vascularity, and angiogenesis.

To determine if changes in receptor levels might be involved in the ability of ethanol to inhibit cord formation, we examined the effect of ethanol on endothelial VEGFR1 and VEGFR2 levels on in vitro functioning. VEGFR1 is thought to play a minor role as a regulator in angiogenesis, as blocking the receptor does not prevent the differentiation of endothelial cells, and activating the receptor in the absence of VEGFR2 activation fails to stimulate proliferation or differentiation and only minimally stimulates migration [37]. VEGFR2, on the other hand, is thought to be critically important in angiogenesis, as homozygous knockout mice are nonviable in utero. Although blockade of VEGFR2 prevents differentiation and proliferation of endothelial cells, activation of VEGFR2 in the absence of VEGFR1 activation yields normal endothelial cell proliferation, migration, and differentiation [37]. Studies of the effects of ethanol on VEGF receptors demonstrate that in vitro ethanol exposure causes decreased expression of endothelial VEGFR2 but does not impact the expression of VEGFR1 significantly [84]. This decrease in receptor expression may partially explain the decreased angiogenesis observed with ethanol during wound healing, as VEGFR2 has a much greater impact on proliferation, migration, and differentiation of endothelial cells during angiogenesis. A decrease in receptor expression is not the only mechanism explaining the role of ethanol on endothelial cells however. In the same studies, endothelial cells exposed to ethanol also displayed a reduction in VEGFR2 phosphorylation. Thus, not only were less VEGFR2 present on endothelial cells exposed to ethanol, but the receptors that were present displayed defects in phosphorylation as well, implying a second level at which ethanol might be impairing endothelial cell functioning. These findings suggest that acute ethanol exposure might cause a decrease in angiogenesis by limiting the production and signaling of key receptors, predominantly by impairing the VEGFR2 receptor and its downstream cascade.

Perhaps not surprisingly, the observed decrease in wound capillary density that follows ethanol exposure is accompanied by an increased level of hypoxia, and hypoxia-sensing mechanisms are affected by ethanol, as endothelial cells exposed to ethanol showed a significant decrease in nuclear protein expression of HIF-1α, which is a hypoxia-induced transcription factor composed of an oxygen-sensing α subunit and a constitutively expressed β subunit. The α subunit is stabilized in the presence of hypoxia, iron chelators, and divalent cations [85]. When oxygen levels begin to decline, the hydroxylation of HIF-1α decreases and allows for HIF-1 stabilization [86]. HIF-1α and -β subsequently migrate into the nucleus to transactivate genes, for instance, VEGF, which contain hypoxia response elements. Following ethanol exposure, HIF-1α nuclear translocation in endothelial cells was reduced significantly, suggesting a defect in the translocation or increased degradation of HIF-1α [84].

ETHANOL METABOLISM, MEMBRANE FLUIDITY, AND WOUND ANGIOGENESIS

To better understand the mechanisms responsible for the effect of ethanol on wound angiogenesis, the importance of membrane fluidity and ethanol metabolism in these impairments has been studied. To determine the contribution of membrane fluidity versus ethanol metabolism to the decrease in wound vascularity in vivo, we used two distinct pharmacologic agents [84]. First, we injected mice i.p. with 1.4 g/kg t-butanol, a tertiary alcohol that is known to induce alterations in membrane fluidity but is not metabolized into toxic, metabolic by-products, such as acetaldehyde. In contrast to the response to ethanol, acute exposure of mice to t-butanol did not result in a decrease in wound vascularity (unpublished results). However, in vitro experiments using t-butanol in culture with endothelial cells resulted in a significant inhibition of endothelial cell cord formation, presumably through changes in membrane fluidity, even in the presence of VEGF. This discrepancy suggests that the actions of alcohols differ with respect to their applied models. In the in vitro experiments, the t-butanol was applied directly to the culture media, which may allow for direct changes in membrane fluidity. In our mouse model, the t-butanol is distributed throughout all tissues via the bloodstream and may not exert such intense changes in membrane fluidity as it does in vitro. To assess further whether ethanol metabolism may be involved in the inhibition of endothelial cell differentiation into capillary tubes, we incubated endothelial cells with ethanol and 4-MP, an inhibitor of ADH [87]. The addition of 4-MP resulted in a partial reversal of the negative effect of ethanol on VEGF-induced cord formation by endothelial cells. This result suggests that ethanol metabolism plays a role but is not exclusively responsible for the decrease in endothelial cell differentiation in vitro. Together, the data suggest that ethanol induces additional changes in vitro, such as alterations in membrane fluidity, in combination with the production of acetaldehyde. Even minor changes in membrane fluidity may exacerbate the effects of alcohol metabolism by impairing specific protein–protein interactions required for cellular processes, such as angiogenesis, during the wound-healing process. Changes in membrane fluidity may perturb receptor-mediated signaling by inhibiting receptor dimerization and impairing the recruitment of the proper intracellular molecules required for angiogenesis following VEGF binding.

Endothelial cells on the periphery of the wound are exposed to ethanol via the bloodstream. Although the effect of ethanol on membrane fluidity may be minimal, receptor-mediated cell signaling may be impaired. Furthermore, ethanol may also increase membrane concentration of phosphotidyl ethanol [82]. This represents a subfraction of membrane phospholipids that at high levels, have been shown to induce changes in intracellular signaling and vascular permeability. Endothelial cells also express functional ADH, which is believed to have evolved from retinoid metabolism and removal of lipid peroxidation aldehydes [88]. This supports the idea that ethanol metabolism by endothelial cells at the wound site may contribute to the ethanol-mediated toxicity, resulting in aberrant endothelial cell function and vascular leakage associated with acute ethanol exposure.

ETHANOL AND ANGIOGENESIS IN OTHER SYSTEMS

Beyond the described observations in healing wounds, the effect of acute ethanol on angiogenesis has received a modest amount of experimental attention in other systems relevant to healing. Early studies demonstrated that ethanol-induced gastric injury resulted in an angiogenic response, thus suggesting that ethanol might promote angiogenesis [89]. In these studies, gastric injury resulted in angiogenesis adjacent to the damaged gastric mucosa along with significant increases in VEGF, which was also shown to be the mediator responsible for the injury-induced angiogenesis. In many ways, this model system is similar to most other injury models, as tissue injury and resulting hypoxia evoke VEGF-mediated angiogenesis in nearly all tissues [90, 91]. Interestingly, ethanol has also been shown to enhance angiogenesis in pathologic situations. In a chick CAM assay of tumor growth, the addition of ethanol was shown to increase angiogenesis and tumor growth [92]. Ethanol exposure between 10 and 20 mM (equivalent to 45 and 90 mg/dl, respectively) was also shown to enhance angiogenesis in the CAM assay alone, although this effect was not proven to be direct [93]. Similarly, in a rat model pertinent to macular degeneration, choroidal neovascularization of the eye was shown to increase with long-term ethanol exposure [94]. Several in vitro studies suggest that relatively high doses of ethanol [as much as 1% (800 mg/dL)] can enhance the angiogenic response of isolated endothelial cells [95]. Several caveats to these studies should be considered. First, these concentrations are well in excess of what the cells would experience normally during intoxication. Second, many of these experiments used endothelial cell lines, including transformed or fused cells, and thus, may not translate to the response of primary cells. Moreover, similar to other cell types, the effect of ethanol on endothelial cells appears to be highly dose-dependent, as 20 mM induces an angiogenic response in vitro, and 10 mM does not [96]. Another study of ethanol and endothelial cells demonstrated that 0.4% ethanol (315 mg/dl) increased cell migration and induced in vitro angiogenesis in endothelial cells; however, this dose is quite high [97]. Pertinent to issues with dosage is the finding that ethanol metabolism itself, at high concentrations, produces metabolic byproducts that are angiogenic. A study conducted by Murray and Wilson [98] demonstrated that metabolites of glycolytic and oxidative metabolic pathways have angiogenic properties. In these studies, some of these metabolites induced a robust angiogenic response in the chick CAM in vivo and in chick embryonic capillary endothelial cells in vitro, whereas solely oxidative metabolites did not. Our own studies suggest that acute ethanol exposure at 100 mg/dl, a level found frequently in intoxicated persons, is inhibitory to reparative angiogenesis in vivo [83]. Taken together, the accumulated data strongly suggest that ethanol can modulate angiogenesis at sites of injury, but that length and level of exposure, as well as the type of angiogenic response (i.e., physiologic or pathologic), may dictate the effect of ethanol on the process of capillary growth.

ACUTE ETHANOL AND RESTITUTION OF THE EXTRACELLULAR MATRIX

Similar to the effects of ethanol on angiogenesis, the influence of ethanol on the restoration of the ECM involves many components of the process. Analysis by RT-PCR, performed at a limited number of time-points (Days 5 and 10), suggested that ethanol exposure causes a decrease in the production of collagen type I but does not affect levels of collagen type III [99]. Thus, one way that ethanol decreases collagen content in wounds seems to be by direct inhibition of collagen mRNA synthesis. Another factor that may be involved in the ethanol-mediated effects on ECM repair is the levels of protease activity and the rate of dissolution of the fibrin clot. Wounds from mice exposed to ethanol had significantly increased levels of active u-PA, suggesting that increased plasmin levels could accelerate fibrin degradation in these wounds. In addition, the level of MMP-8, a collagen type I proteinase, was 2.2-fold higher in wounds from mice exposed to ethanol than control (P<0.05). Overall then, the influence of acute ethanol on dermal ECM repair is complex and multifactorial (Fig. 3).

Figure 3.

Probable effects of ethanol on ECM reconstitution in wounds. Ethanol impairs synthesis of collagen at the mRNA and post-translational level. Additionally, ethanol increases production and activity of collagen degradation enzymes, including MMP-8, u-PA, and t-PA. Glutathione, a substrate for ROS that modulates MMP activity, is depleted by ethanol, removing inhibition of the degradative enzymes. The cumulative effect is a decrease in collagen content as a result of a reduction in production and an increase in breakdown.

Several investigations support the idea that ethanol alters the production of ECM components and relevant proteases [100]. Stephens et al. [101] demonstrated that a 1% v/v (∼800 mg/dL) dose of ethanol inhibited collagen synthesis significantly by fibroblasts in vitro. Although this concentration appears to be well out of normal physiologic range, there are substantial clinical implications for this study. Unlike the epidermis, mucosal epithelial cells may be in direct contact with ethanol and thus, can be exposed to high concentrations immediately upon ingestion. An 80-proof drink, for instance, is 40% ethanol, which is the equivalent of 31,500 mg/dL. Tissues that might have direct exposure include intraoral and intestinal epithelium, suggesting a potential mechanism for increased intestinal anastomotic leakage as a result of ethanol exposure.

Beyond direct effects on ECM synthesis, other studies have established that ethanol exposure results in higher levels of MMP and serine protease activity, leading to the degradation of the epithelial ECM. Glutathione acts as a substrate for multiple antioxidant systems that reduce the ROS, which are generated during the metabolism of ethanol. Velasquez et al. [102] demonstrated that ethanol exposure in rats resulted in the depletion of glutathione levels, which presumably led to increased sepsis-mediated lung dysfunction via excess MMP activity. Lois et al. [103] examined this mechanism further in rat lungs and established that chronic ethanol ingestion yields an increase in the activation of MMP-9 and MMP-2, members of the gelatinase class of MMPs involved in the degradation of basement membrane components. These studies demonstrate that ethanol exposure increases MMP activation via glutathione depletion, consequently increasing the degradation of alveolar epithelium. Similar to our findings in wounds, ethanol has been shown to increase the production of the t-PA and u-PA in endothelial cells [104] and increase the levels of endogenous PA-mediated fibrinolytic activity in this cell type [105]. One can speculate that the ethanol may up-regulate the PAs directly themselves or may up-regulate growth factors such as VEGF and fibroblast growth factor-2 that modulate PA expression.

Several studies have identified functional abnormalities in wound structure as a potential complication if the patient consumes alcohol prior to injury, which may be explained by these established alterations in ECM production and matrix proteolytic activity [106, 107]. Specifically, several studies have shown increased anastomotic leakage rates in patients undergoing intestinal surgeries following alcohol use [107,108,109]. Although there are a fair number of studies showing decreased strength in intestinal or mucosal healing, there are relatively few detailing similar effects in other tissues [110]. Preliminary data from our own studies suggest that a single dose of ethanol prior to wounding can reduce the tensile strength of murine skin at 14 and 28 days after wounding. Further studies are required to assess whether alcohol exposure also compromises barrier integrity directly, possibly through defects in cell-matrix interactions. Overall, studies of ethanol and ECM production support the concept that ethanol can modify ECM production and proteases, an outcome that may have implications for higher rates of morbidity and mortality in intoxicated patients.

ACUTE ETHANOL AND EPITHELIAL CLOSURE OF WOUNDS

Epithelial closure is assessed frequently in healing wounds, and this component of healing is appreciated easily by external observations. Despite extreme perturbations of wound angiogenesis and ECM development, the effect of ethanol exposure on wound closure per se is surprisingly rather modest. In animal models, acute ethanol exposure appears to cause only a slight yet significant delay in wound re-epithelialization [83]. As wounds heal, the most important functions of epithelial coverage are to provide a barrier to infectious agents and to reduce water loss from exposed tissue [111]. The completion of epithelial closure provides little strength to the healing wound, as most strength is derived from dermal connective tissue repair. Therefore, the described ethanol-induced changes in angiogenesis and ECM synthesis, rather than changes in epithelial repair, probably play key roles in the observed increases in anastomotic leakage and wound breakdown that occur in patients exposed to ethanol.

PERSISTENCE OF EFFECTS

One intriguing observation is that the effects of acute ethanol exposure on healing can be detected days after the ethanol has been cleared from the circulation. The long-lasting effect of a single ethanol exposure suggests that early perturbations may have prolonged effects. One possibility is that early effects have enduring consequences for healing. The known interconnections among the three phases of wound healing support this possibility. For example, alterations in the early inflammatory phase have been shown to profoundly affect the proliferative response in healing wounds [10]. As described above, acute ethanol exposure can alter the local inflammatory response in various ways [52, 96]. Thus, an influence of ethanol on key inflammatory patterns may have indirect downstream effects on epithelial cell migration and proliferation. In addition to inflammatory changes, acute ethanol exposure may mediate its effects directly on those cells involved in the proliferative phase of wound healing. In support of the idea that ethanol inhibits endothelial cell function directly, acute ethanol exposure has been shown recently to inhibit endothelial cell activation in vivo in a model of acute inflammation [63], and we have shown a decrease in VEGFR2 receptor number and function as described above. If several days are required for endothelial cell recovery, the direct consequences of acute ethanol exposure might reasonably include delayed capillary outgrowth in wounds, increased hypoxia, and prolonged impairment of wound healing. Lastly, research in liver and neuronal systems has identified epigenetic changes associated with alcohol. Transcriptional regulation of genes by DNA methylation and histone acetylation, methylation, and phosphorylation has been described [97]. If epigenetic changes to any myriad genes involved in the wound-healing process occur, this would clearly be a further explanation of how alcohol seems to exert its influence on wounds weeks after it has been cleared from the environment.

CLINICAL RELEVANCE

Clinicians are cognizant of the fact that ethanol exposure, whether chronic or acute, can induce multiple changes in various systems, including neurologic, cardiovascular, hepatic, respiratory, immune, and gastrointestinal. As ethanol is such a promiscuous drug, the direct and indirect effects on wound healing are difficult to decipher (Fig. 4). Our understanding of the effect of ethanol exposure on tissue repair has improved dramatically over the last decade, yet further investigations must be performed to determine the specific effects on various cells involved in wound repair, particularly, neutrophils, macrophages, keratinocytes, endothelial cells, and fibroblasts. Although an underlying disparity exists between human injury and animal models intended to replicate the human situation, the rodent wound models used in many of the studies of ethanol and healing replicate the human wound-healing process almost identically. Nevertheless, human studies using tissue from intoxicated patients will be needed to obtain accurate information about how ethanol induces cellular alterations during wound healing in patients. Moreover, the impact of the systemic effects of ethanol exposure on those cells involved in tissue repair requires additional consideration. The observations of delayed wound closure, impaired angiogenesis, and reduced collagen content, all occurring several days after ethanol clearance, suggest that ethanol exposure induces a long-term effect on the cells located on the periphery of the wound and that this effect renders these cells incapable of normal, physiologic function [83]. Ultimately, a complete understanding of the specific ethanol-mediated changes that occur during the wound healing will be fundamental to ameliorating the high incidence of morbidity and mortality among intoxicated patients.

Figure 4.

Schematic of the metabolic, hemodynamic, and pathologic consequences of ethanol consumption prior to injury. Following injury, the metabolic, hemodynamic, and immunologic alterations are exacerbated by ethanol exposure. These effects interact with one another and negatively influence the capacity of the body to heal. The net result of these effects is an increase in wound-healing complications.

ACKNOWLEDGMENTS

This publication was supported by grants RO1-GM50875 (L. A. D.), P20-GM078426 (L. A. D.), and 5T32AA013527 (K. A. R., M. J. R.).

DISCLOSURES

The contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institute of General Medical Sciences, National Institute on Alcohol Abuse and Alcoholism, or National Institutes of Health.