Alcohol exposure and mechanisms of tissue injury and repair.

Abstract

Tissue injury owing to acute and chronic alcohol consumption has extensive medical consequences, with the level and duration of alcohol exposure affecting both the magnitude of injury and the time frame to recovery. While the understanding of many of the molecular processes disrupted by alcohol has advanced, mechanisms of alcohol-induced tissue injury remain a subject of intensive research. Alcohol has multiple targets, as it affects diverse cellular and molecular processes. Some mechanisms of tissue damage as a result of alcohol may be common to many tissue types, while others are likely to be tissue specific. Here, we present a discussion of the alcohol-induced molecular and cellular disruptions associated with injury or recovery from injury in bone, muscle, skin, and gastric mucosa. In every case, the goal of characterizing the sites of alcohol action is to devise potential measures for protection, prevention, or therapeutic intervention.

Full text

Page 1

Alcohol Exposure and Mechanisms of Tissue Injury and
Repair
M. Katherine Jung, John J. Callaci, Kristen L. Lauing, Jeffrey S. Otis, Katherine A. Radek,
Michael K. Jones, and Elizabeth J. Kovacs
Tissue injury owing to acute and chronic alcohol consumption has extensive medical conse-
quences, with the level and duration of alcohol exposure affecting both the magnitude of injury
and the time frame to recovery. While the understanding of many of the molecular processes dis-
rupted by alcohol has advanced, mechanisms of alcohol-induced tissue injury remain a subject of
intensive research. Alcohol has multiple targets, as it affects diverse cellular and molecular pro-
cesses. Some mechanisms of tissue damage as a result of alcohol may be common to many tissue
types, while others are likely to be tissue specific. Here, we present a discussion of the alcohol-
induced molecular and cellular disruptions associated with injury or recovery from injury in bone,
muscle, skin, and gastric mucosa. In every case, the goal of characterizing the sites of alcohol
action is to devise potential measures for protection, prevention, or therapeutic intervention.
Key Words: Acute Alcohol, Alcoholic Myopathy, Angiogenesis, Antioxidant, Binge Alcohol,
Bone Fracture Repair, Canonical Wnt Signaling, Chronic Alcohol, Cytoprotection, Extracellular
Matrix, Fracture Non Union, Gastric Mucosa, Glutathione, Inflammation, Myopathy, Orthopedic
Trauma, p34cdc2Kinase, Oxidative Stress, Survivin, Tissue Injury, Wound Healing.
A
lar and molecular functions. Alcohol itself alters biological
function by direct interaction with cellular components, and
also because of the direct effect of alcohol metabolism on the
systemic oxidative and inflammatory state. Characterization
of the cellular and molecular processes that are disrupted after
exposure to alcohol is necessary to understanding and treating
LCOHOL AFFECTS VIRTUALLY every organ and
tissue in the body, with multifactorial actions on cellu-
or preventing its pathophysiological effects. Throughout this
report, the term alcohol refers to ethanol.
The metabolism of alcohol results in the generation of acet-
aldehyde and reactive oxygen (and other) species, biochemical
moieties that damage healthy tissue. The oxidative stress
resulting from these reactive oxygen and nitrogen species orig-
inates in many organs and tissues and varies in severity
depending on the systemic inflammatory and oxidative state,
and on systemic and local immune function. In addition, in
different tissues, the magnitude and duration of oxidative
stress depends on the metabolic state of the cells and on the
ability of those tissues to metabolize and clear alcohol and its
by-products. Secondary sources of oxidative stress result from
increased endotoxin leakage and increased release of pro-
inflammatory cytokines from both immune and nonimmune
cells responding to alcohol. Tissue-specific variation in local
inflammatory⁄immune function and in the response to sys-
temic factors may contribute to organ-specific differences in
the pathological effects of alcohol. Overall, the presence of
oxidative stress is balanced by cellular stress response systems,
both those that prevent accumulation of oxidative stress and
those that repair damage caused by oxidative species.
In addition to, and separate from, the consequences of
alcohol metabolism, direct interactions of alcohol with molec-
ular components affect physiological function. Alcohol has
been shown to modify signal transduction at multiple sites
through its interaction with cell membranes (Dolganiuc et al.,
2006; Szabo et al., 2007) as well as with signaling proteins
(Goral and Kovacs, 2005; Higashi et al., 1996; Resnicoff
et al., 1994; Saso et al., 1997) and ion channels (Dopico,
From the Division of Metabolism and Health Effects (MKJ),
National Institute on Alcohol Abuse and Alcoholism, Bethesda, Mary-
land; Alcohol Research Program, Burn & Shock Trauma Institute,
Department of Orthopaedic Surgery (JJC, KLL), Loyola University
Stritch School of Medicine, Maywood, Illinois; Alcohol Research Pro-
gram, Burn & Shock Trauma Institute, Department of Surgery
(EJK), Loyola University Stritch School of Medicine, Maywood, Illi-
nois; Division of Pulmonary, Allergy, and Critical Care Medicine
(JSO), Emory University School of Medicine, Atlanta, Georgia; VA
San Diego Healthcare System and Division of Dermatology, Depart-
ment of Medicine (KAR), University of California, San Diego, Cali-
fornia; and Research Healthcare Group, VA Long Beach Healthcare
System, Long Beach, California and Department of Medicine
(MKJ), University of California, Irvine, California.
Received for publication June 1, 2010; accepted August 22, 2010.
Reprint requests: M. Katherine Jung, PhD, Division of Metabo-
lism and Health Effects, National Institute on Alcohol Abuse and
Alcoholism, 5635 Fishers Lane, Bethesda, MD 20892-9304; Tel.: 301-
443-8744; Fax: 301-594-0673; E-mail: jungma@mail.nih.gov
Present address: Alcohol Research Program, Burn & Shock
Trauma Institute, Department of Surgery (KAR), Loyola University
Stritch School of Medicine, Maywood, Illinois.
Copyright ? 2010 by the Research Society on Alcoholism.
DOI: 10.1111/j.1530-0277.2010.01356.x
Alcoholism: Clinical and Experimental Research
Vol. 35, No. 3
March 2011
392Alcohol Clin Exp Res, Vol 35, No 3, 2011: pp 392–399

Page 2

2003). Modification of the function of receptors and other sig-
naling molecules leads to altered function of multiple signal-
ing pathways that mediate many essential processes. This
review presents examples of the tissue-specific harm resulting
from the alteration of basic signaling pathways by alcohol
and by enhanced oxidative stress.
Both chronic and acute alcohol consumption have the
potential to impair health and well-being. In addition to being
a risk factor for the events that lead up to injuries and acci-
dents, alcohol also leads to increased morbidity and mortality
after those injuries. Alcohol-related diagnoses are associated
with longer hospital stays, more complications, and greater
medical expense. Nearly 50% of adult emergency room visits
are associated with alcohol consumption, and, surprisingly,
most of these patients are not chronic alcohol abusers, but
rather those who consume alcohol on an acute or binge basis.
As shown herein, acute alcohol has a negative impact on
recovery after injury or illness. The cumulative tissue injury
resulting from chronic alcohol exposure also results in signifi-
cant detriment to overall health and to an impaired capacity
to fend off illness.
With the understanding that some mechanisms of tissue
damage because of alcohol may be common to multiple tissue
types, while others are likely to be tissue specific, we present a
discussion of some of the organ systems and pathways
affected by alcohol. This review focuses on mechanisms by
which different levels of alcohol exposure yield tissue-specific
injury and how the altered pathways affect repair processes in
muscle, bone, gastric mucosa, and skin.
Among the clinical implications of tissue-specific alcohol
effects is the fact that injury is often not limited to a single tis-
sue; for example, a fractured bone may also be associated
with skin and muscle or tendon damage. Moreover, devising
therapeutic strategies to help accelerate the repair process in
one tissue or organ system may result in a deleterious effect
on others. Hence, gaining knowledge about how the response
to alcohol-induced injury differs among these tissues and
organs may aid in the design of treatments that will benefit
the patient as a whole.
BINGE ALCOHOL EXPOSURE IMPAIRS BONE
FRACTURE HEALING BY INHIBITION OF
CANONICAL WNT SIGNALING
Social binge drinking of high alcohol content beverages by
nonalcoholics is a commonly observed phenomenon in col-
lege-aged and older adults in the U.S.A. Approximately 40%
of all orthopedic trauma patients are intoxicated at the time
of hospital admission (Levy et al., 1996), and delayed or
incomplete development of the fracture callus is observed in
these intoxicated patients (Frost, 1989a,b). Thus, alcohol con-
sumption increases the risk for incurring a traumatic injury
(Savola et al., 2005), is associated with higher incidences of
clinical complications following orthopedic trauma (Levy
et al., 1996), and also has an inhibitory effect on the fracture
repair process (Janicke-Lorenz and Lorenz, 1984). Despite
these statistics, little is known about the mechanisms underly-
ing alcohol-induced effects on bone fracture repair. Early
studies in both alcoholic patients and in rodent models of
chronic alcohol consumption demonstrated an inhibition
of bone fracture healing (Janicke-Lorenz and Lorenz, 1984;
Kristensson et al., 1980). The lack of normal fracture callus
formation in both intoxicated patients and rodents suggests
that alcohol inhibits bone fracture repair at an early stage in
the healing process. More recent studies demonstrate that the
production of inflammatory cytokines interleukin-1 (IL-1)
and tumor necrosis factor alpha (TNFa) at the site of fracture
injury may be associated with normal bone repair, that
an alcohol-related modulation of this local inflammatory
response may inhibit repair, and that fracture healing in
alcohol-exposed animals can be improved by the administra-
tion of IL-1 and TNF antagonists (Perrien et al., 2004). Recent
reports demonstrate that impairments in osteoinduction, a
process by which an externally fixed fracture is stretched
along the long axis of the bone to achieve greater limb
length, may contribute to deficient bone repair in alcohol-
exposed rodents. This hypothesis supports the supposition
that bone formation is the primary target of alcohol during
bone healing (Trevisiol et al., 2007).
Global transcriptome analysis demonstrated that the
canonical Wnt pathway, which regulates bone formation, is
targeted by alcohol exposure in rat vertebral bone (Himes
et al., 2008). Alcohol exposure decreased the expression of
several genes in bone associated with canonical Wnt signaling
including those coding for Lrp5 and b-catenin (Himes et al.,
2008). As canonical Wnt signaling plays an important role in
bone fracture repair (Chen et al., 2007), the effects of binge
alcohol exposure on canonical Wnt signaling activity during
the fracture repair process were evaluated. Using a binge alco-
hol exposure model (Callaci et al., 2004), male C57BL⁄6 mice
were given alcohol for 3 days and then subjected to a mid-
shaft, stabilized tibia fracture, shown previously to heal by
both intramembranous and endochondral ossification, similar
to stabilized human fractures (Le et al., 2001). The fracture
callus was recovered from animals at 3, 6, 9, and 14 days
postinjury and analyzed using histological, biomechanical,
and molecular analysis. Binge alcohol exposure prior to frac-
ture injury decreased callus size at all time points examined.
Callus composition was markedly different between control
and alcohol-treated animals, with decreases in both cartilage
and boney components of the callus at 6, 9, and 14 days
postfracture. Material properties of the callus also differed
between control and alcohol-treated animals. Biomechanical
4-point bending analysis revealed a significant decrease in cal-
lus strength in alcohol-treated mice at 14 days postinjury.
Molecular analysis revealed that b-catenin protein expression
in callus from alcohol-treated mice was significantly decreased
at postfracture days 9 and 14. The alcohol-induced changes in
Wnt⁄b-catenin signaling resulted in spatial and quantitative
changes in downstream transcriptional activation of Wnt tar-
get gene expression in transgenic T-cell factor reporter mice
(TCF transgenic mice).
ALCOHOL, TISSUE INJURY, AND REPAIR
393

Page 3

The canonical Wnt signaling pathway plays a crucial role
in bone formation (Gong et al., 2001), mesenchymal stem cell
differentiation (Hill et al., 2005), and bone fracture repair
(Chen et al., 2007). Recent data implicate the canonical Wnt
signaling pathway as a cellular target in alcohol-induced bone
loss (Himes et al., 2008) and the current data suggest deregu-
lation of this pathway is also responsible for alcohol-induced
deficient bone fracture repair (Fig. 1). Alcohol exposure prior
to fracture resulted in decreased callus size and altered cellular
composition, indicating that alcohol may inhibit both carti-
lage and bone formation in the fracture callus. Osteoblast and
chondrocyte cells share a common mesenchymal stem cell
precursor, which requires tight regulation of b-catenin expres-
sion and activity for proper differentiation. These observa-
tions suggest that binge alcohol exposure prior to fracture
injury may target the canonical Wnt pathway during the
differentiation of mesenchymal precursors into bone and
cartilage-forming cells, ultimately causing a decrease in the
available pool of functional osteoblasts and chondrocytes
available for normal fracture healing. Additionally, b-catenin
signaling appears to be essential for normal osteoblast func-
tion and proliferation, which may also indicate that alcohol is
capable of affecting the function of mature osteoblasts to pro-
duce osteoid in the callus. Evidence from TCF transgenic
reporter mouse studies suggests that, in addition to alcohol’s
detrimental effects on callus tissue constitution, the spatial
organization of activated Wnt⁄b-catenin signaling in the cal-
lus is disrupted. At postfracture day 6, active Wnt signaling
was predominantly located in the marrow cavity of alcohol-
treated animals rather than in the soft tissue comprising the
callus as found in saline-treated controls, suggesting that alco-
hol may cause a delay in the differentiation of mesenchymal
precursors into chondrocytes and osteoblasts following frac-
ture injury. Quantification of b-catenin protein levels in the
callus showed that an acute, 3-day binge exposure prior to
injury is sufficient to cause long-term effects on Wnt signaling,
as noted by the sharp decline in b-catenin protein levels in cal-
lus tissue at days 9 and 14 postfracture. This suggests that
alcohol may not only have immediate consequences on the
repair process by deregulating stem cell differentiation at
the early stages of healing, but may also trigger significant
downstream changes in osteoblast cells that persist long after
alcohol exposure has ceased. Future studies directed toward
the association between canonical Wnt signaling and alcohol
exposure may reveal valuable therapeutic targets to alcohol-
abusing patients sustaining orthopedic injuries.
GLUTATHIONE RESTORATION AS AN
INTERVENTION FOR ALCOHOLIC MYOPATHY
The severity of skeletal muscle derangements because of
alcohol abuse is directly proportional to the quantity and
duration of alcohol consumption. Acute alcoholic myopathy,
which may occur after single or multiple episodes of binge
drinking, is an extremely rare condition that affects approxi-
mately 1% of alcoholics and may manifest in myalgia, muscle
weakness and pain, renal impairment with myoglobinuria,
and rhabdomyolysis. In contrast, chronic alcoholic myopathy
has been estimated to occur in up to 70% of alcoholics and is
reportedly more common than other alcohol-induced dis-
eases, such as liver cirrhosis and cardiomyopathy. Chronic
alcoholic myopathy is characterized by severe atrophy and
muscle dysfunction, but appears to be a phenomenon specific
to skeletal muscles with a predominantly glycolytic, fast-
twitch phenotype such as the plantaris, while characteristically
oxidative, slow-twitch muscles such as the soleus are largely
spared. The fundamental cause(s) of the disease is unknown;
however, the diverse toxicology of chronic alcohol consump-
tion would suggest that the etiology and pathology are likely
multifactorial. Indeed, the development of alcoholic myopa-
thy has been attributed to acetaldehyde protein adduct forma-
tion, gene dysregulation, altered growth hormone production,
decreased muscle protein synthesis, increased muscle-derived
and circulating catabolic factors, and oxidant stress (for
comprehensive reviews, see Ferna ´ ndez-Sola ` et al., 2007; Lang
et al., 2005).
Despite an alcoholic’s generally poor diet and high caloric
content of alcohol in excess, nutritional status does not
Injury
Injury + Alcohol
Deficient Fracture
Repair
Normal Fracture
Repair
Mesenchymal
Stem Cell
Alcohol
Wnt target gene
expression: MSC
differentiation
β β-Catenin
Wnt
Frizzled
Wnt
Frizzled
β β-Catenin
β β-Catenin
β β-Catenin
β β-Catenin
β β-Catenin
Fig. 1. Alcohol targets Wnt signaling during bone fracture repair. Bone
fracture triggers the mobilization of mesenchymal stem cells from local and
distant compartments to the site of injury, where they undergo differentiation
into osteoblasts and chondrocytes under the control of canonical Wnt signal-
ing. Wnt proteins bind to surface frizzled receptors, leading to stabilization
and translocation of cytosolic b-catenin to the nucleus. Nuclear b-catenin
activates transcription of Wnt-related genes necessary for bone and carti-
lage formation, and its expression is tightly regulated throughout the repair
process. Binge alcohol exposure prior to injury decreases protein levels of
b-catenin and disrupts its precise pattern of expression, decreases down-
stream Wnt target gene expression, and ultimately causes decreased for-
mation of osteoblasts and chondrocytes within the fracture callus. These
effects lead to alcohol-induced deficient bone repair and fracture nonunion.
394
JUNG ET AL.

Page 4

appear to play a leading role in the development of alcoholic
myopathy. However, reduced plasma and muscle levels of
several essential antioxidants have been reported in alcoholics
with myopathy and likely contribute to the development
of the disease (Ferna ´ ndez-Sola `
Peters, 1992). Accordingly, research has used antioxidant or
antioxidant cofactor supplementation to abate the surge in
alcohol-induced reactive oxygen species, often with limited
effectiveness. For example, providing alcoholics dietary
supplements of zinc, an essential cofactor for superoxide
dismutase and glutathione peroxidase, did not alleviate
symptoms of alcoholic myopathy (Ferna ´ ndez-Sola ` et al.,
1998). Further, a-tocopherol supplementation had little effect
on the rates of protein synthesis or the total protein content in
skeletal muscle (Koll et al., 2003). Moreover, precursors of the
nonvitamin antioxidant glutathione have been used to rectify
alcohol-induced derangements to lung and skeletal muscle tis-
sue (Otis and Guidot, 2009; Otis et al., 2007; Velasquez et al.,
2002).
Glutathione is a thiol-based, trimeric amino acid compound
comprised of glycine, glutamate, and cysteine and is the
principle nucleophilic scavenger of free radicals in cells.
Additionally, glutathione stabilizes other antioxidants, main-
tains proteins in a reduced and, therefore, functional state,
attenuates redox-sensitive catabolic factors, and preserves
healthy biomembranes by limiting alcohol-induced lipid per-
oxidation (Wu et al., 2004). Glutathione levels can be depleted
in certain disease states, but restoration is achievable with pre-
cursor compounds such as S-adenosyl methionine (SAMe),
N-acetylcysteine (NAC), or L-2-oxothiazolidine-4-carboxylate
(OTC or procysteine). Specifically, chronic alcohol ingestion
decreasesskeletalmusclelevelsofglutathione andcysteineand
decreases the enzyme activities of glutathione peroxidase and
glutathione reductase(seeFig. 2)(Ferna ´ ndez-Sola ` et al.,2002;
OtisandGuidot,2009;Otisetal.,2007;Wuetal.,2004).
Muscle glutathione levels in rats fed alcohol for 6 weeks
were replenished by procysteine administration, while oxidant
stress and the expression of 2 catabolic factors, atrogin-1 and
transforming growth factor b1 (TGFb1), were decreased (Otis
et al., 2007). Interestingly, oxidant stress and the induction of
atrogin-1 and TGFb1occurred in the absence of clinically sig-
nificant myopathy, suggesting that ‘‘pro-atrophy’’ programs
may be remedied with glutathione restoration before the
development of overt atrophy. Because the severity of myopa-
thy progresses along a continuum as alcohol abuse persists,
muscle alterations were also seen in rats that consumed alco-
hol for up to 35 weeks, a duration of abuse that produced
overt muscle atrophy (Otis and Guidot, 2009). Long-term
alcohol ingestion created an overall catabolic state in atro-
phied rat plantaris muscles, as evidenced by oxidant stress
and by the production of catabolic members of the interleu-
kin-6 (IL-6) family (i.e., IL-6 and oncostatin M) and compo-
nents of the ubiquitin proteasome system [i.e., atrogin-1 and
muscle RING-finger protein-1 (MuRF1)]. However, glutathi-
one restoration was insufficient to attenuate these catabolic
factors, but rather stimulated the production of several
et al., 2002; Ward and
anabolic factors [i.e., insulin-like growth factor 1 (IGF-1), cili-
ary neurotrophic factor, and cardiotrophin-1] and ultimately
led to increased plantaris fiber area in alcohol-fed rats. Taken
together, these data reveal important temporal associations
between early alcohol-induced oxidant stress and the induc-
tion of catabolic factors and resultant muscle atrophy.
Further, if these salutary physiological responses to glutathi-
one supplementation in animal models translate to the clinical
setting, then glutathione replacement provided before the clin-
ical onset of myopathy, and perhaps even after atrophy is
established, could significantly improve muscle mass and
function in chronic alcoholics.
IMPACT OF ACUTE ALCOHOL EXPOSURE ON
DERMAL WOUND HEALING
The detrimental impact of alcohol exposure on tissue repair
has been evident for decades, influencing multiple pathways
across a wide range of tissues. These changes have been attrib-
uted to the metabolism of alcohol and production of toxic
metabolites that directly impact normal cellular function
required for efficient wound repair. Intoxicated patients have
a higher incidence of associated injuries, many of which sus-
tain traumatic cutaneous wounds that demonstrate a correla-
tion between alcohol intoxication and wound healing
complications. For example, a blood alcohol level (BAL) of
>200 mg⁄dl has been associated with a 2.6-fold increase in
the incidence of wound-related infections (Gentilello et al.,
1993). Although most studies have focused on chronic alco-
hol, the awareness of the frequency of acute alcohol abuse
has led to the development of clinically relevant acute alcohol
Fig. 2. Glutathione metabolism in alcoholic skeletal muscle. Alcoholic
myopathy is characterized by increased formation of superoxide (O??
and hydrogen peroxide (H2O2), and reactive oxygen species. Glutathione
cycling is also altered suggesting a reduced capacity to sequester these
alcohol-induced oxidants. Supplementing the diets of alcoholics with L-2-
oxothiazolidine-4-carboxylate, a proform of L-cysteine (procysteine), normal-
izes thiol levels of cysteine and glutathione. Enzymes that catalyze these
reactions are the following: (1) glutathione-dependent thiodisulfide, thiol-
transferase, and nonenzymatic reactions; (2) protein disulfide isomerase; (3)
c-glutamylcysteine synthetase; (4) glutathione synthetase; (5) glutathione
peroxidase; and (6) glutathione reductase. Arrows (›, fl) denote direction of
change as a result of chronic alcohol abuse. Other abbreviations used:
ADP, adenosine diphosphate; ATP, adenosine triphosphate; H+, hydrogen
ion; H2O, water; NADP+, nicotinamide adenine dinucleotide phosphate;
NADPH, reduced form of NADP+; Pi, inorganic phosphate.
2), lipid
ALCOHOL, TISSUE INJURY, AND REPAIR
395

Page 5

exposure models. Until recently, a comprehensive analysis
identifying the effects of acute alcohol exposure on tissue
repair was not established in vivo.
Wound repair is comprised of 3 overlapping phases that
orchestrate a variety of cellular functions, with the end point
being healed tissue up to 90% of its original integrity (Bern-
stein et al., 1996). The inflammatory phase initiates the heal-
ing process and involves the recruitment of proinflammatory
cells into the wound site where they participate in host defense
through secretion of chemokines and cytokines. This cascade
subsequently activates the next phase, the proliferative phase,
to increase cellular migration and proliferation to repopulate
the wound bed that is devoid of epithelial, vascular, and
matrix-producing cells. The final phase allows for remodeling
and reorganization of newly synthesized matrix molecules to
regain near-normal functional capacity of the injured tissue.
Thus, any disruption between the delicate balance of cellular
recruitment and⁄or activation can lead to devastating defects
in tissue regeneration.
The development of an acute alcohol model in mice in con-
junction with an established wound healing model identified
multipledefectsinthewoundrepairprocessfollowingonedose
of acute alcohol 30 minutes prior to excisional injury. This
model parallels a binge alcohol model yielding a blood alcohol
concentrationof100 mg⁄dlthatrepresentsmanyoftheclinical
patients thatfrequentthe emergencyroomwithtraumainjury.
Wounds from alcohol-treated mice exhibited a significant
reduction in the myeloperoxidase activity of neutrophils
compared to saline controls, indicating a defect in neutrophil
function. In addition, the levels of 2 major proinflammatory
chemokines involved in macrophage recruitment, macrophage
inflammatoryprotein-2(MIP-2)andKC,themurinehomolog
of human interleukin-8, were significantly reduced compared
to their saline-treated counterparts (Fitzgerald et al., 2007).
Subsequent studies revealed that early defects in the inflam-
matory phase following acute alcohol exposure may have
contributed to a delay in wound closure (Radek et al., 2005).
Wounds from alcohol-treated mice were approximately
50% less re-epithelialized after 2 days following wounding
compared to saline-treated mice. However, wounds from both
groups were completely re-epithelialized after 5 days, indicat-
ingatransienteffectofalcoholonkeratinocytemigration.
As angiogenesis is a critical element of the wound repair
process, as it restores the underlying vasculature to restore
oxygenation of the wound bed, an assessment of vascular den-
sity was made in wounds from alcohol-treated mice. Previous
studies demonstrated that direct intragastric alcohol exposure
(50% v⁄v) can actually promote vascular endothelial growth
factor(VEGF)-inducedangiogenesis ingastricepithelia(Jones
et al., 1999). However, these concentrations are far beyond
what would be available to cutaneous tissue following inges-
tion. In the presence of acute alcohol exposure, wound vascu-
larity was significantly reduced up to 10 days following
wounding compared to saline controls, despite near-normal
levels of the pro-angiogenic cytokine, VEGF, and the wounds
of alcohol-treatedmice werenotably morehypoxic.
Subsequent in vitro studies revealed that the defect in vascu-
larity seen with acute alcohol in vivo is attributed, in part, to
reduced phosphorylation of the VEGF receptor, critical to a
signaling pathway involved in endothelial cell proliferation
and differentiation into capillaries (Radek et al., 2008). Mark-
ers of matrix integrity were also assessed and revealed that
wounds from alcohol-treated animals had significantly less
collagen, while exhibiting an increase in matrix proteolytic
activity. Stimulation of fibroblasts, the matrix-producing cells
of the skin, with alcohol (100 mg⁄dl) in vitro also reduced the
gene expression of collagen type I (Radek et al., 2007). Previ-
ously, alcohol exposure (>5% v⁄v) diminished the ability of
fibroblaststoproliferate andsufficientlyproduce collagentype
I in the presence of TGF-b1 in vitro (Stephens et al., 1996).
Together, these data demonstrate that acute alcohol exposure
renders fibroblasts unable to properly synthesize the required
matrix molecules to re-establish dermal stability. This defect
can reduce the capacity of endothelial cells to properly migrate
to form capillary networks within the newly synthesized
matrix, culminating in a functionally defective wound bed that
is more vulnerable to wound dehiscence and infection.
One of the major difficulties involved in deciphering the
direct effects of alcohol from indirect effects on the various cell
populations involved in wound repair is because of the pro-
miscuity of alcohol and its metabolites. Collectively, the
prolonged consequences of alcohol exposure on dermal tissue
repair (Fig. 3) evoke concern for intoxicated trauma patients
who may succumb to opportunistic infections after injury
and⁄or surgical intervention. Ultimately, more detailed insight
into the specific alcohol-mediated changes on cell signaling
pathways that occur during tissue repair will be essential to
develop more effective treatment strategies to limit physio-
logical perturbations in intoxicated patients.
ROLE OF SURVIVIN IN ADAPTIVE
CYTOPROTECTION AGAINST ALCOHOL-INDUCED
GASTRIC INJURY
In 1983, Andre Robert and colleagues first described the
phenomenon they termed ‘‘adaptive cytoprotection’’ whereby
preingestion (or experimental preadministration in animal
models) of ‘‘mild-irritant’’ alcohol concentrations (approxi-
mately 10–20% v⁄v) resulted in the preservation of gastric
mucosal (stomach) integrity against the damaging effects of
subsequent ingestion (administration) of strong (‡50% v⁄v)
alcohol (Robert et al., 1983). They and numerous other
groups have since verified this phenomenon and attributed it
to an increase in the production of prostaglandins. Although
there is no doubt that prostaglandins (particularly prostaglan-
din E2and prostacyclin) play important contributory roles in
gastric adaptive cytoprotection, several studies suggest that
other, prostaglandin-independent, factors also participate in
this phenomenon. These include nitric oxide, vagal innerva-
tion, sensory nerves, blood flow, calcium (Ca2+) influx, heat
shock proteins, and a physical barrier resulting from mucosal
surface exfoliation (Jones et al., 2008).
396
JUNG ET AL.

Page 6

Survivin is a recently discovered protein that possesses a
dual function as both a regulator of cell division and cell sur-
vival. This protein is highly expressed in most organ tissues
during embryonic development where it plays an essential role
in organ tissue remodeling through the regulation of cell pro-
liferation and apoptosis. Following the completion of devel-
opment, survivin expression disappears in most adult
differentiated organ tissues where its function is presumably
no longer needed. Interestingly, however, survivin is highly
expressed in all known forms of human cancer thus classify-
ing it as a ‘‘universal tumor antigen’’ (Andersen et al., 2007).
This has opened a floodgate of investigation into the most
efficacious means by which to universally target⁄inhibit survi-
vin expression as a component of cancer therapy. Neverthe-
less, survivin has also been shown to be a factor in liver
regeneration (Deguchi et al., 2002), angiogenesis (O’Connor
et al., 2000), and vascular injury and repair (Conte and
Altieri, 2006; Simosa et al., 2005). In at least some normal
adult organ tissues, therefore, survivin may be a crucial player
in maintaining the physiologically important balance between
cellular proliferation and apoptosis.
Survivin is expressed in normal, noncancerous, adult-
differentiated gastric mucosa (Chiou et al., 2003). In addition,
in response to exposure of gastric epithelial cells to cytoprotec-
tive alcohol concentrations, survivin accumulation is required
for the full cytoprotective effect against subsequent exposure
to cytotoxic alcohol concentrations (Jones et al., 2008). The
latter was demonstrated by showing that: (i) the cytoprotec-
tion produced by preexposure to ‘‘mild irritant’’ alcohol
against subsequent exposure to cytotoxic alcohol was blocked
by inhibiting survivin accumulation⁄expression using small
interfering RNA (an experimental strategy used to attenuate
targeted protein expression); and (ii) forced overexpression of
survivin by transfection, or gene transfer, produced substan-
tial cytoprotection against exposure to a cytotoxic alcohol
concentration even in the absence of preexposure to ‘‘mild irri-
tant’’ alcohol.
How do survivin expression levels accumulate in the gastric
mucosa in response to cytoprotective alcohol ingestion or
exposure? The complete answer to this question, at present,
remains moot as the mechanism(s) by which survivin
expression levels accumulate in gastric epithelial cells grown
in the laboratory and the mechanism(s) by which survivin
expression levels accumulate in the mammalian gastric
mucosa, including that of humans, are likely to differ some-
what. For instance, exposure of gastric epithelial cells to cyto-
protective alcohol results in an increase in survivin protein
levels by virtue of being stabilized against normal degradation
via phosphorylation of amino acid residue, threonine-34. Evi-
dence suggests that this modification occurs or is enhanced,
following cytoprotective alcohol exposure, by the induction⁄
activation of one or more regulatory kinases, including
the cell cycle-dependent kinase, p34cdc2(Jones et al., 2008).
The same modification has been observed in the gastric
mucosa from experimental animals administered mild irritant
alcohol; further, preliminary data also suggest that de novo
transcription of the survivin gene may be involved in gastric
cytoprotection to whole animals.
A key question remaining is how cytoprotective alcohol
exposure enhances the activation of the kinase(s) that lead(s)
to survivin stabilization⁄accumulation in gastric epithelial
cells. One possibility, supported by as yet unpublished data, is
that this may occur via Ca2+mobilization and the resulting
activation of calmodulin-dependent kinase, CaM kinase II.
Nevertheless, an alternative possibility that the activities
of upstream membrane-associated kinases (e.g., receptor
Fig. 3. Detrimental effects of acute alcohol exposure on cutaneous wound repair. Acute alcohol exposure disrupts the balance of cellular processes to
favor diminished cellular function leading to matrix degradation. Impaired immune cell function, vascularity, and matrix regeneration contribute to the increase
in susceptibility to wound infection. EC, endothelial cell, VEGF, vascular endothelial growth factor.
ALCOHOL, TISSUE INJURY, AND REPAIR
397

Page 7

kinases) are influenced by physical perturbations in the sur-
face membranes of cells exposed to cytoprotective alcohol
cannot be ruled out.
SUMMARY
The examples presented here attest to the multifactorial
and multisystemic mechanisms by which, even after a single
acute or binge exposure, alcohol leads to tissue damage. In
gastric mucosa and in bone, alcohol exposure alters basic sig-
naling processes. In the repair of skin damage following injury
or surgery, alcohol disrupts signaling in a broader context, in
multiple tissue types. The derangement of the inflammatory
response by alcohol leads to altered cytokine and chemokine
production by multiple cell types and this, in turn, influences
the responses of other cell types. Disruption of multiple
molecular processes by alcohol contributes to myopathy and
muscle atrophy, with the accumulation of oxidative stress
playing a major role.
In every case, the goal of characterizing the sites of alcohol
action is to identify potential targets for intervention, either
preventative or therapeutic. Recovery from bone injury after
alcohol exposure is adversely affected by alterations in the
Wnt signaling pathway, which is essential to the repair pro-
cess. Undisturbed Wnt signaling is essential to bone forma-
tion, as disruption of mesenchymal stem cell differentiation
compromises both bone and callus formation. Restoration or
protection of Wnt signaling may improve the prognosis
for recovery from bone fractures sustained with alcohol
exposure.
Preclinical testing of methods for correcting alcoholic
myopathy has generated promisingresults. The causes of alco-
hol-induced myopathy are multifaceted, with the disruption of
oxidative balance playing a significant role. Alcohol affects
levels of antioxidant compounds as well as the activity of
enzymes involved in oxidative balance. Significantly, restora-
tion of the antioxidant compound glutathione by precursor
supplementation restores oxidative balance. Supplementation
of the glutathione precursor after binge alcohol consumption,
prior to the appearance of muscleatrophy, has the potential to
prevent or reduce muscle damage. Further, replenishing gluta-
thione by precursor supplementation even in the presence of
full-blown atrophy after chronic alcohol consumption restores
some measures of metabolic balance in an animal model, sug-
gestingthe potential for correction of cumulative tissue injury.
The derangements of the inflammatory response in the
presence of alcohol consumption adversely affect the pro-
cess of wound healing. Many aspects of the inflammatory
response are essential to proper healing of dermal wounds,
potentially providing multiple therapeutic targets. The many
cytokines and chemokines released in response to tissue dam-
age and the many cell types that are mobilized during inflam-
mation all contribute to the process of wound healing.
Normalization of the inflammatory response in the presence
of alcohol would improve the outlook for recovery after der-
mal injury, particularly in the context of wound infections.
The fact that mechanisms of injury and of repair processes
differ among tissues brings to the fore the potential for com-
plications in patients with multiple injuries. For example, tar-
geting survivin in tumor tissue has the potential to lead to
unanticipated or undesired elimination of necessary survivin
functions, including protection of gastric mucosa. Survivin
protein function is essential to the development of cytoprotec-
tion. Maintenance of survivin levels result from modification
of the kinase activity of p34cdc2after modest alcohol exposure.
Other evidence suggests modification at the expression level
may also be occurring.
The effects of acute alcohol exposure on the healing
process persist many days out in wound healing, in recovery
from bone fracture (both reported here), as well as after
burn injury (Messingham et al., 2002), and other forms of
traumatic injury. While the sustained disruption of healing
is discouraging, it presents multiple potential approaches for
intervention. The response to a short-term alcohol state sug-
gests that a seminal process is altered, leading to branching
downstream effects. Identifying and targeting the primary
disruption would be ideal, but if missed, the potential to
intervene to restore downstream functions may also be con-
sidered.
ACKNOWLEDGMENTS
This research was supported by NIH grants R01AA016138
(JJC), T32AA013527 (KLL), K01AA017190 (JSO), R01AA
014946 (MKJ), P30AA019373 (EJK, KAR), R01AA012034
(EJK), the Ralph and Marian C Falk Medical Research Trust
(EJK), the Margaret A. Baima Endowment Fund for Alcohol
Research (EJK), and the VA Biomedical Laboratory
Research & Development Service (MKJ).
REFERENCES
Andersen MH, Svane IM, Becker JC, Straten PT (2007) The universal charac-
ter of the tumor-associated antigen survivin. Clin Cancer Res 13:5991–5994.
Bernstein E, Bolten LL, Mauviel A, Frank T, McGrath JA, Uitto J (1996)
Principles of Cutaneous Surgery. McGraw Hill, New York.
Callaci JJ, Juknelis D, Patwardhan A, Sartori M, Frost N, Wezeman FH
(2004) The effects of binge alcohol exposure on bone resorption and biome-
chanical and structural properties are offset by concurrent bisphosphonate
treatment. Alcohol Clin Exp Res 28:182–191.
Chen Y, Whetstone HC, Lin AC, Nadesan P, Wei Q, Poon R, Alman BA
(2007) Beta-catenin signaling plays a disparate role in different phases of
fracture repair: implications for therapy to improve bone healing. PLoS
Med 4:e249.
Chiou SK, Moon WS, Jones MK, Tarnawski AS (2003) Survivin expression
in the stomach: implications for mucosal integrity and protection. Biochem
Biophys Res Commun 305:374–379.
Conte MS, Altieri DC (2006) Survivin regulation of vascular injury. Trends
Cardiovasc Med 16:114–117.
Deguchi M, Shiraki K, Inoue H, Okano H, Ito T, Yamanaka T, Sugimoto K,
Sakai T, Ohmori S, Murata K, Furusaka A, Hisatomi H, Nakano T (2002)
Expression of survivin during liver regeneration. Biochem Biophys Res
Commun 297:59–64.
Dolganiuc A, Bakis G, Kodys K, Mandrekar P, Szabo G (2006) Acute etha-
nol treatment modulates toll-like receptor-4 association with lipid rafts.
Alcohol Clin Exp Res 30:76–85.
398
JUNG ET AL.

Page 8

Dopico AM (2003) Ethanol sensitivity of BK(Ca) channels from arterial
smooth muscle does not require the presence of the beta 1-subunit. Am J
Physiol Cell Physiol 284:C1468–C1480.
Ferna ´ ndez-Sola ` J, Garcia G, Elena M, Tobias E, Sacanella E, Estruch R,
Nicolas JM (2002) Muscle anti-oxidant status in chronic alcoholism.
Alcohol Clin Exp Res 26:1858–1862.
Ferna ´ ndez-Sola ` J, Preedy VR, Lang CH, Gonzalez-Reimers E, Arno M, Lin
JC, Wiseman H, Zhou S, Emery PW, Nakahara T, Hashimoto K, Hirano
M, Santolaria-Ferna ´ ndez F, Gonza ´ lez-Herna ´ ndez T, Fatjo ´ F, Sacanella E,
Estruch R, Nicola ´ s JM, Urbano-Ma ´ rquez A (2007) Molecular and cellular
events in alcohol-induced muscle disease. Alcohol Clin Exp Res 31:1953–
1962.
Ferna ´ ndez-Sola ` J, Villegas E, Nicolas JM, Deulofeu R, Antunez E, Sacanella
E, Estruch R, Urbano-Ma ´ rquez A (1998) Serum and muscle levels of alpha-
tocopherol, ascorbic acid, and retinol are normal in chronic alcoholic myop-
athy. Alcohol Clin Exp Res 22:422–427.
Fitzgerald DJ, Radek KA, Chaar M, Faunce D, DiPietro LA, Kovacs EJ
(2007) Effects of acute ethanol exposure on the early inflammatory response
after excisional injury. Alcohol Clin Exp Res 31:317–323.
Frost HM (1989a) The biology of fracture healing. An overview for clinicians.
Part I. Clin Orthop Relat Res 248:283–293.
Frost HM (1989b) The biology of fracture healing. An overview for clinicians.
Part II. Clin Orthop Relat Res 248:294–309.
Gentilello LM, Cobean RA, Walker AP, Moore EE, Wertz MJ, Dellinger EP
(1993) Acute ethanol intoxication increases the risk of infection following
penetrating abdominal trauma. J Trauma 34:669–674.
Gong Y, Slee RB, Fukai N, Rawadi G, Roman-Roman S, Reginato AM,
Wang H, Cundy T, Glorieux FH, Lev D, Zacharin M, Oexle K, Marcelino
J, Suwairi W, Heeger S, Sabatakos G, Apte S, Adkins WN, Allgrove J,
Arslan-Kirchner M, Batch JA, Beighton P, Black GC, Boles RG, Boon
LM, Borrone C, Brunner HG, Carle GF, Dallapiccola B, De Paepe A,
Floege B, Halfhide ML, Hall B, Hennekam RC, Hirose T, Jans A, Ju ¨ ppner
H, Kim CA, Keppler-Noreuil K, Kohlschuetter A, LaCombe D, Lambert
M, Lemyre E, Letteboer T, Peltonen L, Ramesar RS, Romanengo M,
Somer H, Steichen-Gersdorf E, Steinmann B, Sullivan B, Superti-Furga A,
Swoboda W, van den Boogaard MJ, Van Hul W, Vikkula M, Votruba M,
Zabel B, Garcia T, Baron R, Olsen BR, Warman ML; Osteoporosis-
Pseudoglioma Syndrome Collaborative Group. (2001) LDL receptor-related
protein 5(LRP5) affects bone accrual and eye development. Cell 107:513–
523.
Goral J, Kovacs EJ (2005) In vivo ethanol exposure down-regulates TLR2-,
TLR4-, and TLR9-mediated macrophage inflammatory response by limit-
ing p38 and ERK1⁄2 activation. J Immunol 174:456–463.
Higashi K, Hoshino M, Nomura T, Saso K, Ito M, Hoek JB (1996) Interac-
tion of protein phosphatases and ethanol on phospholipase C-mediated
intracellular signal transduction processes in rat hepatocytes: role of protein
kinase A. Alcohol Clin Exp Res 20:320A–324A.
Hill TP, Taketo MN, Birchmeier W, Hartmann C (2005) Canonical Wnt⁄
b-catenin signaling prevents osteoblasts from differentiating into chondro-
cytes. Dev Cell 8:727–738.
Himes R, Wezeman FH, Callaci JJ (2008) Identification of novel bone-specific
molecular targets of binge alcohol and ibandronate by transcriptome analy-
sis. Alcohol Clin Exp Res 32:1167–1180.
Janicke-Lorenz J, Lorenz R (1984) Alcoholism and fracture healing. A radio-
logical study in the rat. Arch Orthop Trauma Surg 103:286–289.
Jones MK, Itani RM, Wang H, Tomikawa M, Sarfeh IJ, Szabo S, Tarnawski
AS (1999) Activation of VEGF and Ras genes in gastric mucosa during
angiogenic response to ethanol injury. Am J Physiol 276:G1345–G1355.
Jones MK, Padilla OR, Webb NA, Norng M (2008) The anti-apoptosis pro-
tein, survivin, mediates gastric epithelial cell cytoprotection against ethanol-
induced injury via activation of the p34cdc2cyclin dependent kinase. J Cell
Physiol 215:750–764.
Koll M, Beeso JA, Kelly FJ, Simanowski UA, Seitz HK, Peters TJ, Preedy
VR (2003) Chronic alpha-tocopherol supplementation in rats does not ame-
liorate either chronic or acute alcohol-induced changes in muscle protein
metabolism. Clin Sci (Lond) 104:287–294.
Kristensson H, Lunden A, Nilsson BE (1980) Fracture incidence and diagnos-
tic roentgen in alcoholics. Acta Orthop Scand 51:205–207.
Lang CH, Frost RA, Summer AD, Vary TC (2005) Molecular mechanisms
responsible for alcohol-induced myopathy in skeletal muscle and heart. Int J
Biochem Cell Biol 37:2180–2195.
Le AX, Miclau T, Hu D, Helms JA (2001) Molecular aspects of healing in sta-
bilized and non-stabilized fractures. J Orthop Res 19:78–84.
Levy RS, Hebert CK, Munn BG, Barrack RL (1996) Drug and alcohol use in
orthopedic trauma patients: a prospective study. J Orthop Trauma 10:21–
27.
Messingham KAN, Faunce DE, Kovacs EJ (2002) Alcohol, injury and cellu-
lar immunity. Alcohol 28:137–149.
O’Connor DS, Schechner JS, Adida C, Mesri M, Rothermel AL, Li F, Nath
AK, Pober JS, Altieri DC (2000) Control of apoptosis during angiogenesis
by survivin expression in endothelial cells. Am J Pathol 156:393–398.
Otis JS, Brown LAS, Guidot DM (2007) Oxidant-induced atrogin-1 and
TGFb1precede alcoholic myopathy in rats. Muscle Nerve 36:842–848.
Otis JS, Guidot DM (2009) Procysteine stimulates expression of key anabolic
factors and reduces plantaris atrophy in alcohol-fed rats. Alcohol Clin Exp
Res 33:1450–1459.
Perrien DS, Wahl EC, Hogue WR, Feige U, Aronson J, Ronis MJJ, Badger
TM, Lumpkin CK (2004) IL-1 and TNF antagonists prevent inhibition of
fracture healing by ethanol in rats. Toxicol Sci 82:656–660.
Radek KA, Kovacs EJ, DiPietro LA (2007) Matrix proteolytic activity during
wound healing: modulation by acute ethanol exposure. Alcohol Clin Exp
Res 31:1045–1052.
Radek KA, Kovacs EJ, Gallo RL, DiPietro LA (2008) Acute ethanol expo-
sure disrupts VEGF receptor cell signaling in endothelial cells. Am J Physiol
Heart Circ Physiol 295:H174–H184.
Radek KA, Matthies AM, Burns AL, Heinrich SA, Kovacs EJ, Dipietro LA
(2005) Acute ethanol exposure impairs angiogenesis and the proliferative
phase of wound healing. Am J Physiol Heart Circ Physiol 289:H1084–
H1090.
Resnicoff M, Rubini M, Baserga R, Rubin R (1994) Ethanol inhibits insulin-
like growth factor-1-mediated signalling and proliferation of C6 rat glioblas-
toma cells. Lab Invest 71:657–662.
Robert A, Nezamis JE, Lancaster C, Davis JP, Field SO, Hanchar AJ (1983)
Mild irritants prevent gastric necrosis through ‘‘adaptive cytoprotection’’
mediated by prostaglandins. Am J Physiol 245:G113–G121.
Saso K, Moehren G, Higashi K, Hoek JB (1997) Differential inhibition of epi-
dermal growth factor signaling pathways in rat hepatocytes by long-term
ethanol treatment. Gastroenterology 112:2073–2088.
Savola O, Niemela O, Hillbom M (2005) Alcohol intake and the pattern of
trauma in young adults and working aged people admitted after trauma.
Alcohol Alcohol 40:269–273.
Simosa HF, Wang G, Sui X, Peterson T, Narra V, Altieri DC, Conte MS
(2005) Survivin expression is up-regulated in vascular injury and identifies a
distinct cellular phenotype. J Vasc Surg 41:682–690.
Stephens P, al-Khateeb T, Davies KJ, Shepherd JP, Thomas DW (1996) An
investigation of the interaction between alcohol and fibroblasts in wound
healing. Int J Oral Maxillofac Surg 25:161–164.
Szabo G, Dolganiuc A, Dai Q, Pruett SB (2007) TLR4, ethanol, and lipid
rafts: a new mechanism of ethanol action with implications for other
receptor-mediated effects. J Immunol 178:1243–1249.
Trevisiol CH, Turner RT, Pfaff JE, Hunter JC, Nemagh PG, Hardin K, Ho
E, Iwaniec UT (2007) Impaired osteoinduction in a rat model for chronic
alcohol abuse. Bone 41:175–180.
Velasquez A, Bechara RI, Lewis JF, Malloy J, McCaig L, Brown LA, Guidot
DM (2002) Glutathione replacement preserves the functional surfactant
phospholipid pool size and decreases sepsis-mediated lung dysfunction in
ethanol-fed rats. Alcohol Clin Exp Res 26:1245–1251.
Ward RJ, Peters TJ (1992) The anti-oxidant status of patients with
either alcohol-induced liver damage or myopathy. Alcohol Alcohol 27:
359–365.
Wu G, Fang Y-Z, Yang S, Lupton JR, Turner ND (2004) Glutathione metab-
olism and its implications for health. J Nutr 134:489–492.
ALCOHOL, TISSUE INJURY, AND REPAIR
399