Rapid and dynamic regulation of TGF-beta receptors on blood vessels and fibroblasts during ischemia-reperfusion injury

Roya Mortazavi-Haghighat1, Kayvan Taghipour-Khiabani1, Sam David2, Carolyn L. Kerrigan3, and Anie Philip1

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


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ABSTRACT
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The pathophysiological mechanisms involved in ischemia-reperfusion injury are poorly understood. Although transforming growth factor (TGF)-beta has been shown to provide protection against ischemia-reperfusion injury in different organ systems, little is known about the regulation of TGF-beta action during this process. Here we analyzed the effect of ischemia and reperfusion on the expression of TGF-beta 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-beta receptors and TGF-beta 1 in a cell type-specific manner. Furthermore, hypoxia upregulates type II TGF-beta receptor mRNA in skin fibroblasts in culture. Together, our results reveal that TGF-beta receptors and TGF-beta 1 are markedly increased under acute ischemic conditions in the blood vessels and fibroblasts of the skin. We conclude that TGF-beta action is enhanced under ischemic conditions and that it may represent an adaptive response to ischemic injury. The augmented TGF-beta responsiveness may be a critical determinant of the protective effect of TGF-beta during ischemia-reperfusion injury.

skin flap; pig; immunohistochemistry; Northern blot


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
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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-alpha (13, 14, 35). However, not all cytokines have detrimental effects, and some, such as transforming growth factor (TGF)-beta , have been shown to attenuate ischemic tissue damage. For example, external administration of TGF-beta has been demonstrated to provide protection against ischemia-reperfusion injury in organs such as heart and brain in animal models (18, 23, 24).

TGF-beta 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-beta (TGF-beta 1, -beta 2, and -beta 3) have been described, which are encoded by distinct genes (31). The most commonly expressed form is TGF-beta 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-beta receptors termed type I, type II, and type III are expressed on most cell types. The TGF-beta signal is transduced by the type I and II receptors, which are transmembrane serine/threonine kinases (12, 20). Type III TGF-beta receptor (betaglycan), which is a membrane proteoglycan, is believed to facilitate TGF-beta binding to the signaling receptors (21, 41).

Because TGF-beta is implicated in the protection of tissue from ischemic damage, it is important to understand the molecular basis of TGF-beta action under ischemic conditions. Hypoxia has been shown to upregulate the expression of TGF-beta (9) and to downregulate TGF-beta binding (10) in skin fibroblasts in culture. Upregulation of TGF-beta 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-beta receptors. Recently, upregulation of TGF-beta 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-beta has potent tissue protective effects during ischemia-reperfusion injury, our objective was to determine whether the expression of TGF-beta 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-beta receptors and TGF-beta 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.


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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.


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Fig. 1.   Experimental model. 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. 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 was created by clamp application on the artery. 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. 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 flaps (ischemic or reperfused) and at corresponding times from the control flaps (nonischemic) and unoperated buttock skin.

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-beta 1 from each group.

Antibodies used. Expression of type I, II, and III TGF-beta receptors and TGF-beta 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-beta 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-beta receptor. The anti-type III antibody and the anti-TGF-beta 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-beta 1 antibody involved coupling of TGF-beta 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-beta 1).

Immunohistochemistry. Immunohistochemical localization of type I, II, and III TGF-beta receptors and TGF-beta 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-beta 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.

Normal rabbit IgG (Lipshaw Immunon) at the same concentration as the primary antibody was used as a negative control for the immunostaining protocol. Additional procedural control involved incubation without the primary antibody, with the rest of the protocol unchanged. Antibody specificity was proven by absorbing each antibody with a 50× excess of immunizing peptide or protein (see Antibodies used) to the antibody. Briefly, the peptide or protein was incubated with the primary antibody overnight at 4°C, centrifuged, and applied to the tissue samples instead of the unabsorbed antibody. Positive staining on tissue sections was not observed under any of the above circumstances.

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-beta 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-beta receptors and TGF-beta 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.


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Immunohistochemical localization patterns of type I, II, and III TGF-beta receptors and TGF-beta 1. To analyze the regulation of TGF-beta receptors and TGF-beta 1 during ischemia-reperfusion injury, a pig skin flap model was used. Immunohistochemical localization of type I, II, and III TGF-beta receptors and TGF-beta 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-beta 1 in the ischemic flap, on the other hand, was not significantly different from that of the nonischemic control. Importantly, TGF-beta 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-beta 1 are shown in Table 1.


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Fig. 2.   Immunohistochemical localization of type I and II transforming growth factor (TGF)-beta receptors in pig skin. Immunostaining patterns of type I and II receptors in unoperated skin (aI and aII, respectively), nonischemic control flap (bI and bII, respectively), ischemic flap (cI and cII, respectively), reperfused flap (dI and dII, respectively), nonischemic control flap (eI and eII, respectively; higher magnification, 40× original), and ischemic flap (fI and fII, respectively; higher magnification, 40× original) are shown. The immunostaining patterns show dynamic regulation of type I and II receptors in a cell type-specific manner. Significant alterations are observed in blood vessels (large filled arrows), fibroblasts (small filled arrows), and stratum basale (open arrows). Control experiments in which the primary antibodies---anti-type I (g) and anti-type II (h) receptor antibodies---were preincubated with their respective immunizing peptides do not show any immunoreactivity. Sections g and h were counterstained with hematoxylin before mounting. The result shown is representative of samples collected at 1 h after ischemia and at 1 h after reflow from experimental flaps and at corresponding times from control flaps and unoperated skin.



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Fig. 3.   Immunohistochemical localization of type III TGF-beta receptors and TGF-beta 1 in pig skin. Immunostaining of type III receptors and TGF-beta 1 in unoperated skin (aIII and abeta 1, respectively), nonischemic control flap (bIII and bbeta 1, respectively), ischemic flap (cIII and cbeta 1, respectively), and reperfused flap (dIII and dbeta 1, respectively) is shown. The immunostaining patterns show dynamic regulation of type III receptors and TGF-beta 1 in a cell type-specific manner. Marked alterations are observed in blood vessels (large filled arrows), fibroblasts (small filled arrows), and stratum basale (open arrows). Control experiments in which the primary antibodies---anti-type III receptor (g) and anti-TGF-beta 1 (h) antibodies-were preincubated with their respective immunizing peptides do not show any immunoreactivity. Sections g and h were counterstained with hematoxylin before mounting. The result shown is representative of samples collected at 1 h after ischemia and at 1 h after reflow from experimental flaps and at corresponding times from control flaps and unoperated skin. SC, stratum corneum; SL, stratum lucidum; SG, stratum granulosum; SS, stratum spinosum; SB, stratum basale.


                              
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Table 1.   Semiquantitative analyses of immunoreactivity of type I, II, and III TGF-beta receptors and TGF-beta 1 in unoperated skin, nonischemic control flap, ischemic flap, and reperfused flap

The skin structures immunostained for type I , II, and III TGF-beta receptors and TGF-beta 1 ligand in the skin flaps were in general similar in the unoperated skin (Figs. 2 and 3). In the epidermis, no detectable immunostaining for type I, II, and III receptors and TGF-beta 1 was observed in the stratum corneum. The stratum lucidum showed the strongest immunostaining, whereas stratum granulosum and spinosum showed strong but less robust expression. However, these two layers displayed no dramatic alterations between groups. In the stratum basale, the immunostaining of the three receptors and TGF-beta 1 was moderate but showed marked differences in the expression of type I receptor and TGF-beta 1 under certain conditions. There was no significant difference in immunostaining patterns of type I, II, and III receptors and TGF-beta 1 between flaps exposed to 1 and 4 h of ischemia (data not shown). Data from flaps representing 1 h of ischemia and 1 h of reperfusion are shown in Table 1.

Type I and II TGF-beta 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-beta receptor and TGF-beta 1 expression. The immunoreactivity patterns of type III receptor and TGF-beta 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).

In the stratum basale, the type III immunostaining was not significantly different between the nonischemic control and ischemic flaps, as observed for type II but not type I in this cell layer. When the reperfused flap (Fig. 3dIII) was compared with the ischemic flap (Fig. 3cIII), the type III receptor immunoreactivity was unchanged in blood vessels, fibroblasts, and stratum basale. A higher magnification of the type III receptor immunostaining in the nonischemic control flap (eIII) and ischemic flap (fIII) is shown in Fig. 3 to highlight the alterations in immunoreactivity and cell types involved. When the type III receptor immunoreactivity in the nonischemic control flap (Fig. 3bIII) was compared with that of the unoperated skin (Fig. 3aIII), it was significantly increased in the control flap on blood vessels (P < 0.002) and fibroblasts (P < 0.0004) but not in the stratum basale.

In contrast to what was observed for type I, II, and III receptors, the TGF-beta 1 immunoreactivity in the ischemic flap was not significantly different from that of nonischemic control in blood vessels, fibroblasts, or stratum basale. More importantly, however, TGF-beta 1 immunostaining in the nonischemic control flap (Fig. 3bbeta 1) was markedly higher than that of the unoperated skin (Fig. 3abeta 1) on blood vessels (P < 0.0005) and fibroblasts (P < 0.005). Stratum basale showed no significant difference. Interestingly, TGF-beta 1 immunostaining in the reperfused flap (Fig. 3dbeta 1) compared with the ischemic flap (Fig. 3cbeta 1) was significantly higher in stratum basale (P < 0.05), with no significant difference in blood vessels and fibroblasts. In control experiments in which the anti-type III receptor (Fig. 3g) and anti-TGF-beta 1 (Fig. 3h) antibodies preincubated with their respective immunizing peptides were used, no detectable immunoreactivity was seen. The control slides were counterstained with Gill's hematoxylin to show the histology (Fig. 3, g and h).

Northern blot analysis of type II TGF-beta receptor expression. To determine whether hypoxia is able to regulate the expression of type II TGF-beta 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.


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Fig. 4.   Hypoxia upregulates type II TGF-beta receptor (R) mRNA expression in skin fibroblasts in vitro. Early-passage human skin fibroblasts were cultured and subjected to 2 h of hypoxia as described in MATERIALS AND METHODS. Total RNA was extracted, and Northern blot was done with a cDNA probe for the type II TGF-beta receptor. Type II receptor mRNA expression under normoxia (20% oxygen) and hypoxia (< 0.2% oxygen) conditions is shown (top). The 18S ribosomal RNA is shown to demonstrate equal loading of RNA (bottom).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta 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-beta receptors during ischemia and reperfusion. Our results demonstrate the occurrence of dynamic and cell type-specific regulation of TGF-beta receptors and TGF-beta 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-beta /TGF-beta 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-beta receptors on blood vessels (endothelial cells) and fibroblasts. Immunostaining of TGF-beta 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-beta receptors and TGF-beta 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-beta 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-beta 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-beta 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-beta receptors and TGF-beta 1, and thus enhanced TGF-beta signal transducing machinery, in endothelial cells and fibroblasts during ischemia-reperfusion provides an explanation at the molecular level for the potent effect of TGF-beta under these conditions, namely the tissue protective effect that TGF-beta 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-beta has been shown to be a key regulator of several endothelial responses important in attenuating ischemia reperfusion injury. For example, TGF-beta 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-beta 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-beta 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-beta 1 by global ischemia (nonischemic control vs. ischemia) and the enhanced expression of TGF-beta 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-beta 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-beta was effective under ischemic conditions would be predicted from our results, which show that the expression of the TGF-beta /TGF-beta receptor system is enhanced during ischemia. Thus the impaired wound healing observed in ischemic tissue is not likely due to decreased TGF-beta 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-beta receptors in skin cells under ischemic conditions. In in vitro culture studies with skin fibroblasts, hypoxia has been reported to induce TGF-beta 1 expression (9) and to decrease TGF-beta receptor mRNA and binding (10). The decreased expression of TGF-beta 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-beta 1 or all three TGF-beta 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-beta 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-beta /TGF-beta receptor system during ischemia remains to be determined. It is interesting to note in this regard that hypoxia and TGF-beta 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-1alpha and Smad3, a central mediator of TGF-beta action.

The cellular distribution of the three types of receptors was similar to that of TGF-beta 1. The concomitant expression profiles and the colocalization of the TGF-beta /TGF-beta 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-beta 1 ligand. The colocalization and the synchrony in the regulation of the type I and type II TGF-beta receptors on endothelial cells and fibroblasts are not consistent with the notion that activation of these receptors leads to distinct TGF-beta signaling pathways performing independent functions (4, 11) but suggest that they cooperate to initiate the TGF-beta signaling cascade.

In summary, our results demonstrate that the TGF-beta /TGF-beta 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-beta receptors and TGF-beta 1 as a rapid adaptive response to partial or global ischemia. Together, our results indicate that TGF-beta action is enhanced under ischemic conditions and provide an explanation at the molecular level for the potent effects of TGF-beta under these conditions. The augmented TGF-beta responsiveness may be an important determinant for the tissue protective effect of TGF-beta 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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Cell Physiol 282(5):C1161-C1169
0363-6143/02 $5.00 Copyright © 2002 the American Physiological Society




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