1 Division of Plastic Surgery, Department of Surgery and 2 Department of Neuroscience, McGill University, Montreal, Quebec, Canada H3G 1A4; and 3 Section of Plastic and Reconstructive Surgery, Dartmouth Medical School, Dartmouth Hitchcock Medical Center, Lebanon, New Hampshire 03756-0001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The
pathophysiological mechanisms involved in ischemia-reperfusion
injury are poorly understood. Although transforming growth factor
(TGF)- has been shown to provide protection against
ischemia-reperfusion injury in different organ systems, little
is known about the regulation of TGF-
action during this process.
Here we analyzed the effect of ischemia and reperfusion on the
expression of TGF-
and its receptors in vivo with a pig skin flap
model. Analysis of unoperated skin, nonischemic control flap,
ischemic flap, and reperfused flap by immunohistochemistry
indicates that ischemia and reperfusion result in rapid and
dynamic regulation of type I, II, and III TGF-
receptors and
TGF-
1 in a cell type-specific manner. Furthermore, hypoxia
upregulates type II TGF-
receptor mRNA in skin fibroblasts in
culture. Together, our results reveal that TGF-
receptors and
TGF-
1 are markedly increased under acute ischemic conditions in the blood vessels and fibroblasts of the skin. We conclude that
TGF-
action is enhanced under ischemic conditions and that it may represent an adaptive response to ischemic injury. The augmented TGF-
responsiveness may be a critical determinant of the
protective effect of TGF-
during ischemia-reperfusion injury.
skin flap; pig; immunohistochemistry; Northern blot
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ISCHEMIC
INSULT, an inevitable consequence of vascular injury,
occlusion, or tissue transfer, has detrimental effects on tissue viability. Paradoxically, reestablishment of normal vascular flow can
incite continued, and often intensified, tissue injury
(15). Numerous studies with a variety of animal models
have shown that the deleterious effects of ischemic injury are
mediated by oxygen-derived free radicals such as superoxide anions
(6, 22, 37) and locally released cytokines such as
interleukin-1 and tumor necrosis factor- (13, 14, 35).
However, not all cytokines have detrimental effects, and some, such as
transforming growth factor (TGF)-
, have been shown to attenuate
ischemic tissue damage. For example, external administration of
TGF-
has been demonstrated to provide protection against
ischemia-reperfusion injury in organs such as heart and brain
in animal models (18, 23, 24).
TGF- is a member of a large family of multifunctional proteins
important in growth, differentiation, and development
(30-32). In mammals, three distinct isoforms of
TGF-
(TGF-
1, -
2, and -
3) have been described, which are
encoded by distinct genes (31). The most commonly
expressed form is TGF-
1, which has been shown to have potent effects
on immune modulation, endothelial adhesiveness, extracellular matrix
synthesis, and tissue repair (8, 27, 33). Three cloned
TGF-
receptors termed type I, type II, and type III are expressed on
most cell types. The TGF-
signal is transduced by the type I and II
receptors, which are transmembrane serine/threonine kinases (12,
20). Type III TGF-
receptor (betaglycan), which is a membrane
proteoglycan, is believed to facilitate TGF-
binding to the
signaling receptors (21, 41).
Because TGF- is implicated in the protection of tissue from
ischemic damage, it is important to understand the molecular basis of TGF-
action under ischemic conditions. Hypoxia has
been shown to upregulate the expression of TGF-
(9) and
to downregulate TGF-
binding (10) in skin fibroblasts
in culture. Upregulation of TGF-
after tissue ischemia has
been demonstrated in central nervous tissues (17) and
kidney (2). Less information is available on the effect of
tissue ischemia and reperfusion on the expression of TGF-
receptors. Recently, upregulation of TGF-
receptors after
ischemia was reported in the brain (1). However, in that study, the expression of receptors was analyzed
1 day after
the induction of ischemia. In addition, the effect of
reperfusion was not studied. Considering that TGF-
has potent tissue
protective effects during ischemia-reperfusion injury, our
objective was to determine whether the expression of TGF-
and its
receptors is regulated by acute ischemia and reperfusion in
different cell types of the skin. Our results with a pig skin flap
model show that ischemic conditions and reperfusion result in
rapid and dynamic regulation of type I, II, and III TGF-
receptors
and TGF-
1 in the skin. In addition, this regulation occurs in a cell
type-specific manner, with endothelial cells and fibroblasts exhibiting
the most marked alterations.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Surgical preparation and experimental design. Six female White Landrace pigs (10-14 wk old) were housed in a temperature-controlled (20-22°C) animal holding room. The protocol for use of pigs in this experiment was approved by the McGill University Animal Care Committee. All the pigs were offered the same commercial pig diet and tap water ad libitum. Food was withheld the evening before surgery. Animals were sedated with intramuscular injections of ketamine (20 mg/kg), xylazine (2 mg/kg), and atropine (1 mg). Animals were anesthetized with an intravenous injection of pentobarbital sodium (6 mg/kg). They were then intubated, and general anesthesia was maintained with spontaneous inhalation of oxygen (8 l/min) and halothane (0.5-1.0%).
In each pig, bilateral buttock skin flaps (10 × 18 cm) based on the vascular pedicle of the superficial circumflex artery and its accompanying vena comitans as well as the lateral femoral cutaneous nerve were elevated and later returned and sutured to their beds with 3-0 nylon skin sutures (Fig. 1). The procedure used was described previously by Kerrigan et al. (16). Briefly, the flap on one side was randomly assigned to 1 h of arterial occlusion with the flap on the contralateral side acting as a nonischemic control. Arterial ischemia in these island flaps was created by clamp application on the artery, which mimics the clinical scenario of an ischemic free flap. Complete occlusion of the vascular pedicle was achieved by application of an Acland V2 microvascular clamp to the branch of the circumflex iliac artery supplying the buttock flap and was verified by application of 10% sodium fluorescein dye (15 mg/kg). Absence of fluorescein in the skin 15 min after dye injection indicated complete occlusion of the vascular pedicle. After 1 h of ischemia, the microvascular clamps were removed to allow reflow. From each pig, skin biopsies (4 × 8-mm pieces from the central portion in the proximal third of the flap) were taken at 1 h after the induction of ischemia and at 1 h after reflow from the experimental flap (ischemic) and at corresponding times from the control flap (nonischemic) and unoperated buttock skin. This experiment was repeated in all six pigs. Thus each group (unoperated skin, nonischemic control flap, ischemic flap, and reperfusion flap) represents 6 animals or 12 flaps. In a similar manner, an additional pair of bilateral skin flaps (thoracic area) was created on each of the six pigs (i.e., 12 additional flaps). Global arterial ischemia in these flaps was achieved by clamping the thoracodorsal artery. Biopsies were collected after 4 h of ischemia and at 4 h after reflow. No necrosis was observed in the flaps during the ischemia-reperfusion time period that we tested.
|
Preparation of skin tissue sections.
Biopsies collected from the skin flaps and unoperated skin were fixed
in 4% paraformaldehyde for 8 h followed by immersion in 15%
sucrose for 30 h at 4°C. They were then embedded in Tissue Tek
compound and frozen in liquid nitrogen. Serial sections of the frozen
tissue were prepared with a cryostat. Each slide contained tissue
sections (in duplicates or triplicates) from all four groups: 1) unoperated skin, 2) nonischemic
control flap, 3) ischemic flap, and 4)
reperfusion flap. A minimum of 15 slides each were analyzed for type I,
II, and III receptors and TGF-1 from each group.
Antibodies used.
Expression of type I, II, and III TGF- receptors and TGF-
1 in
tissue sections was detected by immunohistochemistry with their
respective specific anti-peptide antibodies. The anti-type I and
anti-type II receptor antibodies (Santa Cruz Biotechnology, Santa Cruz,
CA) were both rabbit polyclonal IgGs. The anti-type I antibody
recognizes amino acids 158-179 of the precursor form of the type I
TGF-
receptor (ALK5) of human origin, whereas the anti-type II
antibody recognizes amino acids 550-565 of the precursor form of
the human type II TGF-
receptor. The anti-type III antibody and the
anti-TGF-
1 antibody were kind gifts from Dr. M. O'Connor-McCourt (Biotechnology Research Institute, Montreal, PQ, Canada). The peptide
sequences used and the procedure employed for the preparation of the
type III receptor antibody were exactly the same as described by
Moustakas et al. (26). Briefly, polyclonal rabbit antisera were raised against a COOH-terminal epitope of human type III receptor,
the immunoglobulins were prepared, and their ability to specifically
immunoprecipitate the receptor was tested. In comparison studies, this
antibody displayed the same specificity as the antibodies obtained from
Dr. A. Moustakas (Whitehead Institute for Medical Research, Cambridge,
MA). The procedure for the preparation of anti-TGF-
1
antibody involved coupling of TGF-
1 to keyhole limpet hemocyanin and
injection of rabbits and was described previously (25).
Normal rabbit IgG used as a negative control was obtained from Lipshaw
Immunon (Pittsburgh, PA). The immunizing peptides or protein (same as
used for the preparation of antibodies) that were used in control
experiments to show the specificity of antibodies during the
immunohistochemistry procedure were obtained from Santa Cruz
Biotechnology (type I and II peptide), synthesized locally at Sheldon
Biotechnology Institute (Montreal, PQ, Canada; type III peptide), or
bought from R&D Systems (Minneapolis, MN; TGF-
1).
Immunohistochemistry.
Immunohistochemical localization of type I, II, and III TGF-
receptors and TGF-
1 ligand was performed on 8-µm-thick cryostat sections of skin tissue placed onto gelatin-coated glass slides. The
sections were washed and permeabilized three times for 15 min each with
phosphate-buffered saline pH 7.5 (PBS) containing 0.1% Triton X-100.
Endogenous peroxidase activity was then quenched by treating the
sections with 1% H2O2 in 99% methanol for 45 min at room temperature. The sections were incubated in a humidified chamber for 3 h with blocking solution (PBS containing 1% normal goat serum, 0.3% Triton X-100, and 0.5% BSA) to block excess proteins and prevent nonspecific antibody binding. The primary antibodies diluted in the blocking solution were applied to the sections overnight
at 4°C in a humidified chamber. The anti-type I and anti-type II
antibodies were diluted 1:100 to a final concentration of 2 µg/ml,
whereas the anti-type III antibody and anti-TGF-
1 antibody were
diluted 1:400 and 1:250, respectively. The next day, slides were washed
two times with PBS containing 0.1% Triton X-100 and once with PBS
alone. The slides were incubated with biotinylated goat anti-rabbit
secondary antibody (diluted to 0.5% in PBS containing 0.5% BSA and
1.5% normal goat serum) for 1 h. The sections were washed two
times with PBS containing 0.1% Triton X-100 and once with PBS alone.
This was followed by incubation with avidin-biotin complex (ABC; Vector
Laboratories) diluted in PBS for 1 h. The slides were again washed
two times with PBS containing 0.1% Triton X-100 and once with PBS
alone. The brown color, indicating immunoreactivity, was developed with
0.05% of 3,3'-diaminobenzidine tetrahydrochloride (Sigma, St. Louis,
MO) in 0.1% H2O2 in PBS for 3 min. Sections
were rinsed in distilled water. Finally, the slides were dehydrated and
mounted with permount (Sigma). A corresponding set of slides was
counterstained with Gill's hematoxylin before mounting. All tissue
sections that were compared were treated at the same time and for the
same length of time.
Culture of skin fibroblasts. Early-passage skin fibroblasts were prepared from human skin tissue obtained at breast reduction surgery. The tissue was collected in Dulbecco's minimal essential medium (D-MEM), washed, and minced into pieces of <0.3 mm3. The explants were distributed into 25-cm2 tissue culture flasks (Costar, Cambridge, MA) and cultured in D-MEM supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin (GIBCO, Burlington, ON, Canada). The cultures were maintained at 37°C in an atmosphere of 5% CO2-95% air. Culture medium was changed every 3 days. Keratinocytes and other contaminating cells were removed by the first two or three subcultures. Cells from the fourth to tenth passages were used for experiments. The purity and homogeneity of the fibroblasts preparation were determined as described by Tam and Philip (39).
Culture of fibroblasts under hypoxic conditions. Skin fibroblasts growing as monolayers in 150-mm petri dishes (Starsted) and at ~70% confluence were exposed to hypoxia for 2 h with a Gas Pak system (BBL Gas Pak Plus system; Becton-Dickinson, Lincoln Park, NJ) at 37°C, as described by Detmar et al. (7). The Gas Pak system depletes oxygen by means of a palladium catalyst, and the oxygen concentration during hypoxia was <0.2%. Control cultures consisted of monolayers of fibroblasts at 70% confluence maintained under normoxic conditions (5% CO2-95% air) at 37°C.
Northern blot analysis.
Total RNA from skin fibroblasts cultured under normoxic and hypoxic
conditions was isolated by homogenization in 4 M guanidinium isothiocyanate, 25 mM sodium citrate, 0.5 M
N-lauroylsarcosine and 0.1 mercaptoethanol (Sigma) as
described previously by Chomczynski and Sacchi
(5). RNA (20 ug/well) was electrophoresed on a 1% agarose formaldehyde gel, transferred to nylon membrane (Boehringer Mannheim), and ultraviolet cross-linked. Membranes were prehybridized overnight at 42°C and transferred to fresh hybridization solution containing 32P-labeled probe. The type II TGF- receptor
probe was a 474-bp (521-995) fragment labeled with
[32P]dCTP with nick translation (GIBCO-BRL). After an
overnight hybridization, membrane was washed twice, and the blot was
exposed to Kodak X-ray film with an intensifying screen at
80°C for
94 h. Equivalent RNA loading and transfer were determined by
subsequent reprobing with 18S rRNA that was radiolabeled as above.
Scanning densitometry was performed to quantify relative mRNA abundance.
Evaluation of staining and statistical analysis.
The results of immunohistochemistry studies were assessed in a blinded
fashion by three separate investigators. The evaluation of positively
staining skin structures (cell types) was performed semiquantitatively
on an arbitrary scale ranging from 0 to 4 for each structure: 0, negative reaction; 1, positive reaction in a few cells; 2, reaction in
a moderate number of cells; 3, reaction in a large number of cells; 4, reaction in almost all cells. The Kruskal-Wallis test was used to
analyze the differences between groups (unoperated buttock skin,
nonischemic control flap, ischemic flap, and
reperfusion flap) in the expression of type I, II, and III TGF-
receptors and TGF-
1. Differences with a P value of <0.05
was considered significant (2-sided test). Analysis of the data using a
second nonparametric test, Monte Carlo estimates for the exact test,
gave results similar to those obtained by the Kruskal-Wallis test. SAS
version 8.0 statistical software (SAS Institute, 1999) was used for
computation and analysis.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immunohistochemical localization patterns of type I, II, and III
TGF- receptors and TGF-
1.
To analyze the regulation of TGF-
receptors and TGF-
1 during
ischemia-reperfusion injury, a pig skin flap model was used. Immunohistochemical localization of type I, II, and III TGF-
receptors and TGF-
1 ligand was performed in tissue sections prepared from nonischemic control flap, ischemic flap,
reperfused flap, and unoperated buttock skin. Comparative analysis of
immunostaining of type I, II, and III receptors revealed that
expression of these receptors was increased in the nonischemic
control flaps, ischemic flaps, and reperfused flaps compared
with the unoperated skin (Figs. 2 and
3). The skin structures that showed the
most dramatic increases were blood vessels (endothelial cells),
fibroblasts, and the basal layer of the epidermis. The immunoreactivity
of the three receptors were highest in the ischemic flap, with
the nonischemic control flap showing significantly lower
immunostaining. The expression of TGF-
1 in the ischemic
flap, on the other hand, was not significantly different from that of
the nonischemic control. Importantly, TGF-
1 expression in
the nonischemic control was markedly higher than in the
unoperated skin (Fig. 3). Semiquantitative evaluation of immunostaining
and statistical analysis of the differences between groups (unoperated,
nonischemic control, ischemic, and reperfused) for type
I, II, and III receptors and TGF-
1 are shown in Table
1.
|
|
|
Type I and II TGF- receptor expression.
The immunoreactivity patterns of type I and II receptors are shown in
Fig. 2. The expression of both type I and type II receptors was the
highest in the ischemic flap (Fig. 2, cI
and cII). The immunostaining of type I and II
receptors on blood vessels (P < 0.0004 and
P < 0.01, respectively) and fibroblasts
(P < 0.004 and P < 0.001, respectively) in the ischemic flap (Fig. 2,
cI and cII) was
significantly higher compared with the nonischemic control flap
(Fig. 2, bI and bII;
Table 1). A higher magnification of the nonischemic control
flap (eI and eII for
types I and II, respectively) and ischemic flap
(fI and fII for types I
and II, respectively) is also shown in Fig. 2 to emphasize the effect of ischemia on receptor expression and to better depict the
cell types involved. Although type I and II receptor staining was also increased in the stratum basale in the ischemic flap compared with the nonischemic control flap, it was significant
(P < 0.003) only for the type I receptor. The
immunoreactivity in the reperfused flap compared with ischemic
flap was significantly lower for the type I (Fig. 2,
dI vs. cI;
P < 0.01) and type II (Fig. 2,
dII vs. cII;
P < 0.01) receptors on fibroblasts with no significant difference in blood vessels and stratum basale. The expression of type
I and type II receptors in the nonischemic control flap (Fig.
2, bI and bII) compared
with the unoperated skin (Fig. 2, aI and
aII) was significantly increased in blood
vessels (P < 0.04 and P < 0.02 for
types I and II, respectively) and fibroblasts (P < 0.005 and P < 0.03 for types I and II, respectively)
but not in stratum basale (Table 1). No immunoreactivity was observed in control experiments when the type I (Fig. 2g) and type II
(Fig. 2h) antibodies preincubated with their respective
immunizing peptides were used. The control slides (Fig. 2, g
and h) were counterstained with Gill's hematoxylin before
mounting to show the histology.
Type III TGF- receptor and TGF-
1 expression.
The immunoreactivity patterns of type III receptor and TGF-
1 ligand
are shown in Fig. 3. As observed for type I and type II receptors, the
expression of the type III receptor was highest in the ischemic
flap (Fig. 3cIII), with the blood vessels
(P < 0.0009) and fibroblasts (P < 0.002) of this flap showing markedly higher immunoreactivity than those
of the nonischemic control flap (Fig.
3bIII; Table 1).
Northern blot analysis of type II TGF- receptor expression.
To determine whether hypoxia is able to regulate the expression of type
II TGF-
receptors in skin fibroblasts in vitro, early-passage human
skin fibroblasts were subjected to hypoxic conditions for 2 h
while the control cells remained under normoxic conditions. Expression
of the type II receptor was determined by Northern blot analysis. The
results shown in Fig. 4 demonstrate that
exposure to hypoxic condition for 2 h markedly increased the
abundance of type II receptor mRNA in early-passage skin fibroblasts.
This observation illustrates that acute exposure to hypoxia leads
to an increase in the expression of type II receptor in early-passage skin fibroblasts and supports the in vivo immunohistochemistry results
presented above.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The pathophysiological mechanisms involved in
ischemia-reperfusion injury, a common denominator in a variety
of clinical conditions from myocardial infarction and cerebral
ischemia to tissue transplantation and free tissue transfer,
are poorly understood. TGF- has been shown to provide protection
against ischemia-reperfusion injury in many organ systems and
is known to be a key regulator of the tissue repair process. However,
little is known about the regulation of TGF-
receptors during
ischemia and reperfusion. Our results demonstrate the
occurrence of dynamic and cell type-specific regulation of TGF-
receptors and TGF-
1 in vivo during ischemia-reperfusion in a
pig skin flap model. This model was designed to study the early dynamic
changes (1 and 4 h) during ischemia as well as
reperfusion. This time frame was chosen because it represents the
clinically significant period during which intervention with
therapeutic agents might have a beneficial effect on the deleterious
effects of injury. Thus the model we have used does not allow us to
evaluate the late effects of ischemia-reperfusion injury. The
bilateral skin flap design, with the contralateral flap acting as the
control, avoids animal-to-animal variation and allows us to tease out
the effect of ischemia from that of wounding (creation of the
flap), which is also associated with ischemia, on the
regulation of the TGF-
/TGF-
receptor system in vivo.
The most important finding in the present study is that global
ischemia resulted in rapid (within 1 h) upregulation of
type I, II, and III TGF- receptors on blood vessels (endothelial
cells) and fibroblasts. Immunostaining of TGF-
receptors in those
cell types was markedly higher in the ischemic skin flap
(subjected to global ischemia) than in the nonischemic
control flap. This increase was maintained at 4 h of
ischemia (data not shown). Importantly, creation of the flap
(which involves wounding and partial ischemia) resulted in an
increase in the expression of all three TGF-
receptors and TGF-
1
in endothelial cells and fibroblasts, as shown by significantly higher
immunostaining in the nonischemic control flap than in the
unoperated skin. The absence of a further increase in TGF-
1 expression in endothelial cells (blood vessels) and fibroblasts after
the induction of global ischemia is intriguing. It is possible that the expression of TGF-
l is already maximal because of the partial ischemia and wounding induced by the creation of the
flap, and subsequent global ischemia may have no further effect.
Although the expression of the TGF- receptors was increased on
endothelial cells and fibroblasts within 1 h of the induction of
ischemia, subsequent reperfusion led to a significant decrease in type I and type II receptors in fibroblasts, but this decrease was
not significant in blood vessels. The rapid upregulation of TGF-
receptors and TGF-
1, and thus enhanced TGF-
signal transducing machinery, in endothelial cells and fibroblasts during
ischemia-reperfusion provides an explanation at the molecular
level for the potent effect of TGF-
under these conditions, namely
the tissue protective effect that TGF-
exerts against
ischemia-reperfusion injury in several animal models (18,
23, 24). The endothelial cell is the principal cell type
involved in the development of ischemia reperfusion injury, and
TGF-
has been shown to be a key regulator of several endothelial
responses important in attenuating ischemia reperfusion injury.
For example, TGF-
has been demonstrated to potently inhibit
endothelial adhesiveness to polymorphonuclear leukocytes (18,
29) to inhibit free radical generation and preserve vasomotor
tone (19, 24).
Although the middle layers of the epidermis (granulosum, spinosum, and
lucidum) showed strong immunoreactivity for type I, II, and III
receptors and TGF-1, it was the stratum basale (basal keratinocytes)
that exhibited significant differences in immunoreactivity, specifically that for the type I receptor during ischemia and that for TGF-
1 after reflow. However, it is possible that the high
expression of the receptors and the ligand in the middle layers
precluded the detection of alterations in immunoreactivity. The
significance of the upregulation of type I receptors but not type II
and III receptors or TGF-
1 by global ischemia
(nonischemic control vs. ischemia) and the enhanced
expression of TGF-
1 but not type I, II, and III receptors after
reperfusion on basal keratinocytes is not known.
Although sustained ischemia results in the failure of wounds to
heal and hyperbaric oxygen has been documented to enhance wound
healing, ischemia in the initial phase of the wound healing process stimulates fibroplasia and angiogenesis. Therefore, occlusive dressings that create hypoxia have been shown to promote wound healing
(38). Interestingly, it was demonstrated recently that TGF- was capable of accelerating wound healing under both
nonischemic and ischemic conditions, whereas fibroblast
growth factor and platelet-derived growth factors were ineffective
under ischemic conditions (42). That TGF-
was
effective under ischemic conditions would be predicted from our
results, which show that the expression of the TGF-
/TGF-
receptor
system is enhanced during ischemia. Thus the impaired wound
healing observed in ischemic tissue is not likely due to
decreased TGF-
action but may be accounted for by the reduced supply
of nutrients and immune cells, increased production of oxygen free
radicals (37), decreased action of other growth factors
(42), or all of the above. Limited information is
available on the regulation of TGF-
receptors in skin cells under
ischemic conditions. In in vitro culture studies with skin fibroblasts, hypoxia has been reported to induce TGF-
1 expression (9) and to decrease TGF-
receptor mRNA and binding
(10). The decreased expression of TGF-
receptors in
vitro during hypoxia reported by Falanga et al. (10) is
not consistent with our results in vivo (Table 1) and in vitro (Fig. 4)
or with those of Ata et al. (1) in vivo in the brain.
Differences in experimental conditions may explain this discrepancy.
Although our study does not allow us to determine the precise temporal
relationship between the induction of ligand and receptors, the rapid
upregulation of TGF-1 or all three TGF-
receptors by partial or
global ischemia suggests that ischemia may have a
direct effect on the expression of these receptors and ligand. Our in vitro results demonstrating the upregulation of type II receptor
mRNA at 2 h of hypoxia in skin fibroblasts (Fig. 4) support this
conclusion. In the only other study that examined the regulation of
TGF-
receptors under ischemic conditions, receptor
expression was analyzed only at later time periods, namely, on days 1 and 3 after ischemia (1). Regulation of gene
expression by low oxygen concentration is now a well-documented
phenomenon (3, 28). Whether hypoxia-inducible factor
(HIF)-1, a master regulator of oxygen homeostasis (36,
40), is involved in the induction of the TGF-
/TGF-
receptor system during ischemia remains to be determined. It is
interesting to note in this regard that hypoxia and TGF-
were
recently reported to synergistically cooperate to induce vascular
endothelial growth factor expression (34) and that this
cooperation may involve a physical association between HIF-1
and
Smad3, a central mediator of TGF-
action.
The cellular distribution of the three types of receptors was similar
to that of TGF-1. The concomitant expression profiles and the
colocalization of the TGF-
/TGF-
receptor system in the same cell
types is consistent with numerous studies showing the heteromeric
complex formation of the three receptors and their high affinity for
the TGF-
1 ligand. The colocalization and the synchrony in the
regulation of the type I and type II TGF-
receptors on endothelial
cells and fibroblasts are not consistent with the notion that
activation of these receptors leads to distinct TGF-
signaling
pathways performing independent functions (4, 11) but
suggest that they cooperate to initiate the TGF-
signaling cascade.
In summary, our results demonstrate that the TGF-/TGF-
receptor
system is dynamically regulated during ischemia-reperfusion in
a cell type-specific manner in the skin. The data presented define an
increase in the expression of TGF-
receptors and TGF-
1 as a rapid
adaptive response to partial or global ischemia. Together, our
results indicate that TGF-
action is enhanced under ischemic conditions and provide an explanation at the molecular level for the
potent effects of TGF-
under these conditions. The augmented TGF-
responsiveness may be an important determinant for the tissue protective effect of TGF-
against ischemia-reperfusion injury.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Fatiha Boulemkahel for assistance in the immunocytochemistry procedure, Dr. M. O'Connor-McCourt, Biotechnology Research Institute, Montreal, for the gifts of anti-type II and anti-type III receptor antibodies, and Dr. E. Rahme, Department of Epidemiology, McGill University, for expert assistance in the statistical analysis of the data.
![]() |
FOOTNOTES |
---|
This research was supported by grants from the Canadian Institutes of Health Research (formerly Medical Research Council, Canada) (A. Philip and C. L. Kerrigan) and the Heart and Stroke Foundation, Quebec (A. Philip). A. Philip is a recipient of a Chercheur Boursier scholarship from the Fonds de la Recherche en santé du Québec (FRSQ).
Address for reprint requests and other correspondence: A. Philip, Montreal General Hospital, 1650 Cedar Ave., Montreal, PQ, Canada H3G 1A4 (E-mail: anie.philip{at}mcgill.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpcell.00413.2001
Received 27 August 2001; accepted in final form 19 December 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Ata, KA,
Lennmyr F,
Funa K,
Olsson Y,
and
Terent A.
Expression of transforming growth factor-beta1, 2, 3 isoforms and type I and II receptors in acute focal cerebral ischemia: an immunohistochemical study in rat after transient and permanent occlusion of middle cerebral artery.
Acta Neuropathol (Berl)
97:
447-455,
1999[ISI][Medline].
2.
Basile, DP,
Rovak JM,
Martin DR,
and
Hammerman MR.
Increased transforming growth factor-1 expression in regenerating rat renal tubules following ischemic injury.
Am J Physiol Renal Fluid Electrolyte Physiol
270:
F500-F509,
1996
3.
Bunn, HF,
and
Poyton RO.
Oxygen sensing and molecular adaptation to hypoxia.
Physiol Rev
76:
839-885,
1996
4.
Chen, RH,
Ebner R,
and
Derynck R.
Inactivation of the type II receptor reveals two receptor pathways for the diverse TGF-beta activities.
Science
260:
1335-1338,
1993[ISI][Medline].
5.
Chomczynski, P,
and
Sacchi N.
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal Biochem
162:
156-159,
1987[ISI][Medline].
6.
Cotran, RS,
and
Mayadas-Norton T.
Endothelial adhesion molecules in health and disease.
Pathol Biol (Paris)
46:
164-170,
1998[ISI][Medline].
7.
Detmar, M,
Brown LF,
Berse B,
Jackman RW,
Elicker BM,
Dvorak HF,
and
Claffey KP.
Hypoxia regulates the expression of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) and its receptors in human skin.
J Invest Dermatol
108:
263-268,
1997[Abstract].
8.
Diebold, RJ,
Eis MJ,
Yin M,
Ormsby I,
Boivin GP,
Darrow BJ,
Saffitz JE,
and
Doetschman T.
Early-onset multifocal inflammation in the transforming growth factor beta 1-null mouse is lymphocyte mediated.
Proc Natl Acad Sci USA
92:
12215-12219,
1995[Abstract].
9.
Falanga, V,
Qian SW,
Danielpour D,
Katz MH,
Roberts AB,
and
Sporn MB.
Hypoxia up-regulates the synthesis of TGF-1 by human dermal fibroblasts.
J Invest Dermatol
97:
634-637,
1991[Abstract].
10.
Falanga, V,
Takagi H,
Ceballos PI,
and
Pardes JB.
Low oxygen tension decreases receptor binding of peptide growth factors in dermal fibroblast cultures.
Exp Cell Res
213:
80-84,
1994[ISI][Medline].
11.
Feng, XH,
and
Derynck R.
Ligand-independent activation of transforming growth factor (TGF) beta signaling pathways by heteromeric cytoplasmic domains of TGF-beta receptors.
J Biol Chem
271:
13123-13129,
1996
12.
Franzen, P,
ten Dijke P,
Ichijo H,
Yamashita H,
Schulz P,
Heldin CH,
and
Miyazono K.
Cloning of a TGF beta type I receptor that forms a heteromeric complex with the TGF beta type II receptor.
Cell
75:
681-692,
1993[ISI][Medline].
13.
Gilmont, RR,
Dardano A,
Engle JS,
Adamson BS,
Welsh MJ,
Li T,
Remick DG,
Smith DJ, Jr,
and
Rees RS.
TNF-alpha potentiates oxidant and reperfusion-induced endothelial cell injury.
J Surg Res
61:
175-182,
1996[ISI][Medline].
14.
Herskowitz, A,
Choi S,
Ansari AA,
and
Wesselingh S.
Cytokine mRNA expression in postischemic/reperfused myocardium.
Am J Pathol
146:
419-428,
1995[Abstract].
15.
Kerrigan, CL,
and
Stotland MA.
Ischemia reperfusion injury: a review.
Microsurgery
14:
165-175,
1993[ISI][Medline].
16.
Kerrigan, CL,
Zelt RG,
Thomson JG,
and
Diano E.
The pig as an experimental animal in plastic surgery research for the study of skin flaps, myocutaneous flaps and fasciocutaneous flaps.
Lab Anim Sci
36:
408-412,
1986[Medline].
17.
Knuckey, NW,
Finch P,
Palm DE,
Primiano MJ,
Johanson CE,
Flanders KC,
and
Thompson NL.
Differential neuronal and astrocytic expression of transforming growth factor beta isoforms in rat hippocampus following transient forebrain ischemia.
Brain Res Mol Brain Res
40:
1-14,
1996[ISI][Medline].
18.
Lefer, AM,
Ma XL,
Weyrich AS,
and
Scalia R.
Mechanism of the cardioprotective effect of TGF-1 in feline myocardial ischemia and reperfusion.
Proc Natl Acad Sci USA
90:
1018-1022,
1993[Abstract].
19.
Lefer, AM,
Tsao P,
Aoki N,
and
Palladino MA.
Mediation of cardioprotection by transforming growth factor-.
Science
249:
61-64,
1990[ISI][Medline].
20.
Lin, HY,
Wang XF,
Ng-Eaton E,
Weinberg RA,
and
Lodish HF.
Expression cloning of the TGF-beta type II receptor, a functional transmembrane serine/threonine kinase.
Cell
68:
775-785,
1992[ISI][Medline].
21.
Lopez-Casillas, F,
Wrana JL,
and
Massague J.
Betaglycan presents ligand to the TGF beta signaling receptor.
Cell
73:
1435-1444,
1993[ISI][Medline].
22.
McCord, JM.
Oxygen-derived free radicals in post ischemic tissue injury.
N Engl J Med
312:
159-163,
1985[Abstract].
23.
McNeill, H,
Williams C,
Guan J,
Dragunow M,
Lawlor P,
Sirimanne E,
Nikolics K,
and
Gluckman P.
Neuronal rescue with transforming growth factor-beta 1 after hypoxic-ischaemic brain injury.
Neuroreport
5:
901-904,
1994[ISI][Medline].
24.
Mehta, JL,
Yang BC,
Strates BS,
and
Mehta P.
Role of TGF-beta1 in platelet-mediated cardioprotection during ischemia-reperfusion in isolated rat hearts.
Growth Factors
16:
179-190,
1999[ISI][Medline].
25.
Moulin, V,
Auger FA,
O'Connor-McCourt M,
and
Germain L.
Fetal and postnatal sera differentially modulate human dermal fibroblast phenotypic and functional features in vitro.
J Cell Physiol
171:
1-10,
1997[ISI][Medline].
26.
Moustakas, A,
Lin HY,
Henis YI,
Plamondon J,
O'Connor-McCourt MD,
and
Lodish HF.
The transforming growth factor beta receptors types I, II, and III form hetero-oligomeric complexes in the presence of ligand.
J Biol Chem
268:
22215-22218,
1993
27.
O'Kane, S,
and
Ferguson MW.
Transforming growth factor s and wound healing.
Int J Biochem Cell Biol
29:
63-78,
1997[ISI][Medline].
28.
Ratcliffe, PJ,
O'Rourke JF,
Maxwell PH,
and
Pugh CW.
Oxygen sensing, hypoxia-inducible factor-1 and the regulation of mammalian gene expression.
J Exp Biol
201:
1153-1162,
1998
29.
Rhodes, JM,
Engelmyer E,
Tilberg MS,
and
Gifford R.
Transforming growth factor 1 serves as an autocrine inhibitor of human endothelial cell/lymphocyte adhesion.
J Surg Res
59:
719-724,
1995[ISI][Medline].
30.
Roberts, AB.
Transforming growth factor-: activity and efficacy in animal models of wound healing.
Wound Repair Regen
3:
408-418,
1995.
31.
Roberts, AB.
Molecular, and cell biology of TGF-beta.
Miner Electrolyte Metab
24:
111-119,
1998[ISI][Medline].
32.
Roberts, AB,
Flanders KC,
Heine UI,
Jakowlew S,
Kondaiah P,
Kim SJ,
and
Sporn MB.
Transforming growth factor-beta: multifunctional regulator of differentiation and development.
Philos Trans R Soc Lond B Biol Sci
327:
145-154,
1990[ISI][Medline].
33.
Roberts, CJ,
Birkenmeier TM,
McQuillan JJ,
Akiyama SK,
Yamada SS,
Chen WT,
Yamada KM,
and
McDonald JA.
Transforming growth factor beta stimulates the expression of fibronectin and of both subunits of the human fibronectin receptor by cultured human lung fibroblasts.
J Biol Chem
263:
4586-4592,
1988
34.
Sanchez-Elsner T, Botella LM, Velasco B, Corbi A, Attisano L, and
Bernabeu C. Synergistic cooperation between hypoxia and
transforming growth factor- pathways on human vascular endothelial
growth factor gene expression. J Biol Chem:
38527-38535, 2001.
35.
Seekamp, A,
Warren JS,
Remick DG,
Till GO,
and
Ward PA.
Requirements for tumor necrosis factor-alpha and interleukin-1 in limb ischemic/reperfusion injury and associated lung injury.
Am J Pathol
143:
453-463,
1993[Abstract].
36.
Semenza, GL.
HIF-1: mediator of physiological and pathophysiological responses to hypoxia.
J Appl Physiol
88:
1474-1480,
2000
37.
Senel, O,
Cetinkale O,
Ozbay G,
Achioglu F,
and
Bulan R.
Oxygen free radicals impair wound healing in ischemic rat skin.
Ann Plast Surg
39:
516-523,
1997[ISI][Medline].
38.
Stadelmann, W,
Digenis AG,
and
Tobin GR.
Impediments to wound healing.
Am J Surg
176:
39S-47S,
1998[ISI][Medline].
39.
Tam, BYY,
and
Philip A.
Transforming growth factor-beta receptor expression on human skin fibroblasts: dimeric complex formation of type I and type II receptors and identification of glycosyl phosphatidylinositol-anchored transforming growth factor-beta binding proteins.
J Cell Physiol
176:
553-564,
1988.
40.
Wang, GL,
and
Semenza GL.
General involvement of hypoxia-inducible factor 1 in transcriptional response to hypoxia.
Proc Natl Acad Sci USA
90:
4304-4308,
1993[Abstract].
41.
Wang, XF,
Lin HY,
Ng-Eaton E,
Downward J,
Lodish HF,
and
Weinberg RA.
Expression cloning and characterization of the TGF-beta type III receptor.
Cell
67:
797-805,
1991[ISI][Medline].
42.
Wu, L,
and
Mustoe TA.
Effect of ischemia on growth factor enhancement of incisional wound healing.
Surgery
117:
570-576,
1995[ISI][Medline].
|
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |