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(American Journal of Pathology. 2004;165:1653-1662.)
© 2004 American Society for Investigative Pathology

Effects of Angiotensin II Receptor Signaling during Skin Wound Healing

Hikaru Takeda, Yohtaro Katagata, Yutaka Hozumi and Shigeo Kondo

From the Department of Dermatology, Yamagata University School of Medicine, Yamagata, Japan


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
The tissue angiotensin (Ang) system, which acts independently of the circulating renin Ang system, is supposed to play an important role in tissue repair in the heart and kidney. In the skin, the role of the system for wound healing has remained to be ascertained. Our study demonstrated that oral administration of selective AngII type-1 receptor (AT1) blocker suppressed keratinocyte re-epithelization and angiogenesis during skin wound healing in rats. Immunoprecipitation and Western blot analysis indicated the existence of AT1 and AngII type-2 receptor (AT2) in cultured keratinocytes and myofibroblasts. In a bromodeoxyuridine incorporation study, induction of AT1 signaling enhanced the incorporation into keratinocytes and myofibroblasts. Wound healing migration assays revealed that induction of AT1 signaling accelerated keratinocyte re-epithelization and myofibroblasts recovering. In these experiments, induction of AT2 signaling acted vice versa. Taken together, our study suggests that skin wound healing is regulated by balance of opposing signals between AT1 and AT2.

Angiotensin (Ang) II is the active biological octapeptide of the systemic renin Ang system, which is a circulating humoral system responsible for blood pressure regulation and salt-water homeostasis. AngII exerts its effects through binding the specific receptors. There are two major subtypes of AngII receptors: type 1 (AT1) and type 2 (AT2).1 Most of the physiological actions of AngII are regulated through AT1. Recently, in addition to the classical role, AngII has attracted attention for its novel role as a growth factor. Growth-modulating effects of these receptors were reported in cardiac vascular endothelial cells, smooth muscle cells, and fibroblasts, where AT1 caused cell proliferation and synthesis of extracellular matrix proteins, whereas AT2 acted in an anti-proliferative manner.2-5 Despite the fact that the role of AngII and its receptors has been well discussed in the cardiovascular field, a few reports can be found in the dermatological field. Literature has shown that the topical administration of AngII accelerated skin wound healing,6-8 an increased level of AngII or Ang-converting enzyme in the rat skin1,9 during wound healing, and the existence of AngII receptors by harvesting whole wounded skin.10-13 However, the mechanism of AngII on skin wound healing has remained unknown. In this report, we studied the mechanism of AngII receptor signaling using selective AT1 or AT2 blockers to assess the effects of AngII receptor signaling during skin wound healing.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Agents, Animals, and Wound Healing Experiments in Vivo

Candesartan (CV-11974) and Candesartan cilexetil (TCV-116) were kind gifts from Takeda Chemical Industries (Osaka, Japan). Candesartan is a potent and selective AT1 blocker that binds tightly to and dissociates slowly from AT1. Candesartan cilexetil is a prodrug, and is administered orally and converted rapidly and completely to its active form, candesartan, by the enzyme esterase at the time of intestinal absorption.14 PD-123319 ditrifluoroacetate (Sigma Japan, Tokyo, Japan) is a selective AT2 blocker and used in experiments in vitro.2,3 Sprague-Dawley male rats (250 to 300 g, ~10 weeks of age) were purchased from SLC Inc., Japan and housed on a 12:12-hour light:dark cycle. Food and water were available ad libitum. The experiments were performed according to the institutional guidelines for care and use of laboratory animals.

TCV-116, 1 mg/kg/day (n = 5) or 10 mg/kg/day (n = 5), suspended with gum arabic in a volume of 2 ml/kg water or vehicle (n = 5) were orally administered to the rats once a day starting day 0 until the end of the experiment. On day 0, a full-thickness skin wound was made on the dorsal site of each rat using 8-mm biopsy trepan (Kai Industries, Seki, Japan). The wounds had 50 µl of 70% ethanol once a day starting day 0 until day 5 after injury without bandaging. No wounds had infections. The wounds were totally excised at each selected time point (day 3, 6, 9, 12, and 15 after wounding), and were fixed in 10% formalin, embedded in paraffin, and offered for hematoxylin and eosin (H&E) or immunohistochemical staining. Rats were anesthetized with pentobarbital sodium (50 mg/kg i.p.) during wounding and tissue collection.

Analysis of Re-Epithelization in Vivo

Using the H&E sections, the extent of re-epithelization of wounds was evaluated by measuring the epidermal migration from the normal wound margin to the point where the migrating epithelium stopped processing.6 The re-epithelization index was determined by the percentage of the new epithelium present in the total wound.

Analysis of Vascular Growth in Vivo

To detect the blood vessels, immunohistochemical staining using the rabbit anti-von Willebrand factor polyclonal antibody (DAKO Japan, Kyoto, Japan) was performed on the sections by the labeled streptavidin-biotin method.15 The reaction was visualized using 3-amino-9-ethylcarbazole (DAKO Japan) as chromogen and the slides were counterstained with hematoxylin and mounted in aqueous mounting medium for examination. The average number of blood vessels with a diameter of more than 4 µm per square millimeter in the wound site was calculated as vascular density. Two of the authors independently counted the number of blood vessels without any information on each section.

Cell Culture

Keratinocytes were cultured as previously described.16 In brief, epidermal strips were isolated from the surgically removed rat dorsal skin using dispase (1.2 U/ml, Sigma Japan, Tokyo, Japan) for 24 hours at 4°C. After incubation of epidermal strips in 0.05% trypsin/0.02% ethylenediaminetetraacetic acid for 25 minutes at room temperature, keratinocytes were plated on culture dishes (78.5 cm2; Falcon Plastics, Becton-Dickinson Labware, Franklin Lakes, NJ) at ~4.0 x 104 cells/cm2 in Defined Keratinocyte Serum-Free Medium (Life Technologies, Inc., Grand Island, NY) with the addition of the growth supplement included with the medium. Calcium level, as stated by the manufacturer, was lower than 0.1 mmol/L. The cells were grown as monolayers at 37°C in a humidified incubator with 5% CO2/95% air.

Myofibroblasts from rat cutaneous granulation tissue were obtained as previously described.17 The cells within 17 passages were used because myofibroblasts were maintained in culture for more than 17 passages. Myofibroblasts were grown to confluence and subcultured on culture dishes (78.5 cm2, Falcon Plastics) at 37°C in a humidified incubator with 5% CO2/95% air in Dulbecco’s modified medium with 10% fetal bovine serum (Life Technologies, Inc.), penicillin (100 U/ml), streptomycin (50 µg/ml), kanamycin (50 µg/ml), and hydrocortisone (0.4 µg/ml).

Immunoprecipitation and Western Blot Analyses

The epidermal strips as a source of keratinocytes, cultured keratinocytes, and myofibroblasts were washed twice with ice-cold phosphate-buffered saline, and harvested and lysed in lysis buffer containing 10 mmol/L Tris-HCl, 10 mmol/L ethylenediaminetetraacetic acid, 0.4 mmol/L phenylmethyl sulfonyl fluoride, 1 µg/ml leupeptin, and 1% Triton X-100. After they were incubated for 1 hour at 4°C, the cell lysates were centrifuged at 12,000 x g for 10 minutes, and the supernatant was collected.18 Proteins were precleared with protein G agarose for 30 minutes at 4°C. Then anti-AT1 (affinity-purified rabbit polyclonal anti-AT1; Santa Cruz Biotechnology, Santa Cruz, CA) or AT2 antibodies (affinity-purified goat polyclonal anti-AT2, Santa Cruz) were added to the precleared samples and incubated for 1 hour at 4°C. Immune complexes were boiled in sodium dodecyl sulfate-sample buffer, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis,19 transferred to polyvinylidene difluoride membrane,20 and immunoblotted with anti-AT1 or AT2 antibodies, respectively. After incubation with secondary antibodies, immunoreactive proteins were detected by the enhanced chemiluminescence reaction (Amersham Bioscience Japan, Tokyo, Japan). Staining of standard proteins on the polyvinylidene difluoride membranes was performed with 0.04% Coomassie brilliant blue R-250.

Bromodeoxyuridine (BrdU) Labeling and Immunohistochemical Staining

Keratinocytes and myofibroblasts were plated on culture disks (0.79 cm2; Sumitomo Chemical, Tokyo, Japan) at ~1.5 x 102 cells/cm2. Subsequently, keratinocytes and myofibroblasts were exposed to AngII (100 pg/ml; Wako, Tokyo, Japan), CV-11974 (10–7 mol/L), CV-11974 (10–7 mol/L with AngII 100 pg/ml), PD-123319 (10–5 mol/L, dissolved in 0.9% saline), PD-123319 (10–5 mol/L with AngII 100 pg/ml), or vehicle (0.9% saline with 0.1 N Na2CO3), respectively, with BrdU (15 µg/ml) in the medium for 24 hours and fixed in 100% ethanol. The BrdU immunohistochemistry was performed using a cell proliferation kit (Amersham Bioscience Japan). After quenching endogenous peroxidase with 3% H2O2, the DNA in cells was subsequently denatured with denaturing solution for 30 minutes, followed by a 1-hour incubation with biotinylated mouse monoclonal anti-BrdU antibody at 37°C. Disks were incubated with streptavidin-peroxidase for 10 minutes at room temperature. The incorporation of BrdU into the genomic DNA of proliferating cells was detected by staining with 3-amino-9-ethylcarbazole. Finally, the disks were counterstained with hematoxylin and mounted in aqueous mounting medium. Negative disks were not incubated with anti-BrdU antibody. Cells with red-stained nuclei by 3-amino-9-ethylcarbazole were identified as cells with BrdU incorporation. The BrdU labeling index (percentage) was determined by the number of BrdU-positive nuclei in cells and analyzed and scored by two different observers under light microscopy. The experiments were done in triplicate.

In Vitro Wound Healing Migration Assays

Wound healing migration assays were performed as previously described.21 In brief, keratinocytes or myofibroblasts were seeded on 24-well multiplates and grown to confluence. After 60 minutes of incubation with AngII (100 pg/ml), CV-11974 (10–7 mol/L), CV-11974 (10–7 mol/L with AngII 100 pg/ml), PD-123319 (10–5 mol/L), PD-123319 (10–5 mol/L with AngII 100 pg/ml), or vehicle in the medium, respectively, the monolayers of keratinocytes or myofibroblasts were wounded with a cell scraper and photographed at different times by a phase contrast microscope equipped with Olympus Camedia C-3030ZOOM digital camera (Olympus, Tokyo, Japan). The re-epithelization index (percentage) was determined by the percentage of new epithelium present in the initial wound using image analysis software program Analytical Imaging Station for Windows (Imaging Research Inc., Willowdale, Canada). The experiments were done in triplicate.

Statistical Analysis

Statistical significance of the quantitative measurement was assessed by Turkey’s multiple range test. Each time point was analyzed separately, and two-tailed {alpha}-levels of P ≤ 0.05 are significant.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Effects of AT1-Signaling Blockade on Re-Epithelization in Vivo

The effect of AT1 blocker on re-epithelization in vivo was evaluated kinetically (Figure 1, A and B) . On day 12, the control group and the group with TCV-116, 1 mg/kg/day, showed 100% re-epithelization of the wound (Figure 1A) . However, the group with TCV-116, 10 mg/kg/day, showed delayed re-epithelization and delayed dermal repair. At all time points, administration of TCV-116 suppressed the keratinocyte re-epithelization dose dependently (Figure 1B) .



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  		Figure 1. Effects of AT1 blocker on re-epithelization and angiogenesis during skin wound healing. A: Histological findings of the skin wounds on day 12 after wounding (H&E staining). The control group and the group with TCV-116, 1 mg/kg, showed 100% re-epithelization. The group with TCV-116, 10 mg/kg, showed delayed re-epithelization and delayed dermal repair. The crust still attached on the surface. Arrows indicate the re-epithelized edges. B: Time course of re-epithelization. The re-epithelization index (percentage of the wound site with new epithelization) was evaluated microscopically using tissue sections stained with H&E. Rats were orally administered with vehicle or TCV-116, 1 or 10 mg/kg/day. Data are represented as mean ± SD (n = 5). *, Significantly different from controls (P < 0.05). Administration of TCV-116 suppressed the re-epithelization dose dependently and postponed the day of 100% re-epithelization. C: Immunohistochemical staining for von Willebrand factor of the wound bed on day 9. The control wound showed many blood vessels. Administration of TCV-116 suppressed vascular growth dose dependently. D: Time course of angiogenesis. Number of blood vessels was evaluated microscopically using tissue sections immunohistochemically stained for von Willebrand factor. Each time point was analyzed separately. Data are represented as mean ± SD (n = 5). *, Significantly different from controls (P ≤ 0.05). Administration of TCV-116, 1 mg/kg/day, slightly (on days 3, 6, 12, and 15) or significantly (day 9) suppressed vascular growth. At all time points, administration of TCV-116, 10 mg/kg/day, significantly inhibited vascular growth. Original magnifications: x10 [A (top layer)]; x20 [A (bottom layer), C].

 
Effects of AT1-Signaling Blockade on Vascular Growth in Vivo

The effect of AT1 blocker on vascular growth at the wound bed was evaluated kinetically (Figure 1, C and D) . On day 9 after wounding, the control group showed many blood vessels in the dermis, however, the administration of TCV-116 suppressed angiogenesis dose dependently (Figure 1C) . Administration of TCV-116, 1 mg/kg/day, slightly suppressed angiogenesis at all time points except for the significant suppression on day 9 (Figure 1D) . Administration of TCV-116, 10 mg/kg/day, significantly suppressed angiogenesis at all time points.

Immunoprecipitation and Western Blot Analysis

To analyze the effects of AngII receptor signaling on keratinocytes and myofibroblasts, we examined the expression of AngII receptors using immunoprecipitation and Western blot analysis (Figure 2) . The results showed the signals of both AT1 and AT2 in the epidermal strips, cultured keratinocytes, and myofibroblasts. The signal of AT1 is prominent compared to that of AT2 in the epidermal strips, keratinocytes, and myofibroblasts.



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  		Figure 2. Immunoprecipitation and Western blot analysis of AngII receptors. In the epidermal strips, cultured keratinocytes, and myofibroblasts, the signals of both AT1 and AT2 were detected. The signal of AT1 is prominent compared to that of AT2. SM, size marker; ES, epidermal strips; CK, cultured keratinocytes; MF, myofibroblasts.

 
Effects of Stimulation or Blockade of AngII Receptor Signaling on BrdU Incorporation into Cultured Keratinocytes

We investigated the level of cell proliferation by using BrdU labeling index. BrdU, a thymidine analog, can be incorporated into DNA in the S phase of cell cycle.16 Hence, the cells that traversed S phase can be detected with an anti-BrdU antibody. Based on the analysis of AngII receptor expression, keratinocytes were exposed to AngII, CV-11974, CV-11974 with AngII (selective AT2 signaling), PD-123319, PD-123319 with AngII (selective AT1 signaling), or vehicle, respectively, for 24 hours and offered for analyzing BrdU labeling index (percentage) (Figure 3, A and B) . The presence of AngII significantly enhanced (P < 0.05) the BrdU incorporation into keratinocytes compared to the control. The similar enhancing effect was recognized in the group of PD-123319 with AngII. In contrast, the presence of CV-11974 with AngII significantly suppressed (P < 0.05) the BrdU incorporation compared to the control or the group with AngII. These results suggest that AT1 signaling accelerates and AT2 signaling inhibits BrdU incorporation into keratinocytes, respectively.



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  		Figure 3. BrdU incorporation and wound healing migration assays of cultured keratinocytes. A: Detection of BrdU incorporation by immunohistochemical staining. The BrdU-labeled nuclei stained red. CV, CV-11974; PD, PD-123319. B: Effects of stimulation or blockade of AngII receptor signaling on BrdU incorporation. Data are represented as mean ± SD (n = 3). *, Significantly different from controls (P ≤ 0.05). {diamondsuit}, Significantly different from AngII-treated group (P ≤ 0.05). AngII or PD-123319 with AngII enhanced BrdU incorporation. CV-11974 with AngII suppressed BrdU incorporation. C: Wound healing migration assays. The phase contrast microscopic findings at 40 hours after scraping. D: Results of wound healing migration assays. Data are represented as mean ± SD (n = 3). *, Significantly different from controls (P ≤ 0.05). {diamondsuit}, Significantly different from AngII-treated group (P ≤ 0.05). AngII or PD-123319 with AngII accelerated re-epithelization. CV-11974 with AngII inhibited keratinocyte re-epithelization. Original magnifications: x20 (A); x10 (C).

 
Wound Healing Migration Assays of Cultured Keratinocytes

The re-epithelizing movements of keratinocytes were recorded starting from wounding until one of the wounds was re-epithelized (40 hours later) (Figure 3, C and D) . At all time points except for 16 hours after scraping, the presence of AngII accelerated re-epithelization compared to the control. The similar accelerating effect was recognized in the group of PD-123319 with AngII. In contrast, the presence of CV-11974 with AngII significantly suppressed re-epithelization compared to the control or the group with AngII. The results suggest that AT1 signaling accelerates and AT2 signaling inhibits the re-epithelization of keratinocytes, respectively.

Effects of Stimulation or Blockade of AngII Receptor Signaling on BrdU Incorporation into Cultured Myofibroblasts

Based on the analysis of AngII receptor expression, myofibroblasts were exposed to AngII, CV-11974, CV-11974 with AngII (selective AT2 signaling), PD-123319, PD-123319 with AngII (selective AT1 signaling), or vehicle, respectively, for 24 hours and offered for analyzing BrdU labeling index (percentage) (Figure 4, A and B) . The presence of AngII significantly enhanced (P < 0.05) the BrdU incorporation into myofibroblasts compared to the control. The similar enhancing effect was recognized in the group of PD-123319 with AngII. In contrast, the presence of CV-11974 with AngII significantly suppressed (P < 0.05) the BrdU incorporation compared to the control group or the group with AngII. The results suggest that AT1 signaling enhances and AT2 signaling suppresses the BrdU incorporation into myofibroblasts, respectively.



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  		Figure 4. BrdU incorporation and wound healing migration assays of cultured myofibroblasts. A: Detection of BrdU incorporation by immunohistochemical staining. The BrdU-labeled nuclei stained red. CV, CV-11974; PD, PD-123319. B: Effects of stimulation or blockade of AngII receptor signaling on BrdU incorporation. Data are represented as mean ± SD (n = 3). *, Significantly different from controls (P ≤ 0.05). {diamondsuit}, Significantly different from AngII-treated group (P ≤ 0.05). AngII or PD-123319 with AngII enhanced BrdU incorporation. CV-11974 with AngII suppressed BrdU incorporation. C: Wound healing migration assays. The phase contrast microscopic findings at 36 hours after scraping. D: Results of wound healing migration assays. Data are represented as mean ± SD (n = 3). *, Significantly different from controls (P ≤ 0.05). {diamondsuit}, Significantly different from AngII-treated group (P ≤ 0.05). AngII or PD-123319 with AngII accelerated myofibroblast recovering. CV-11974 with AngII inhibited recovering. Original magnifications: x20 (A); x10 (C).

 
Wound Healing Migration Assays of Cultured Myofibroblasts

The recovering movements of myofibroblasts were recorded starting from wounding until one of the wounds was recovered (38 hours later) (Figure 4, C and D) . At all of the time points except for 12 hours after scraping, the presence of AngII significantly accelerated (P < 0.05) recovering compared to the control. The similar accelerating effect was recognized in the group of PD-123319 with AngII. On the contrary, the presence of CV-11974 with AngII significantly inhibited (P < 0.05) recovering compared to the control or the group with AngII. The results suggest that AT1 signaling accelerates and AT2 signaling inhibits the recovering of myofibroblasts, respectively.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
It has been proposed that exogenous administration of AngII is a potent accelerator of cutaneous wound repair.7,8 These reports showed that topical administration of AngII could accelerate skin wound healing by stimulating dermal repair including angiogenesis and epidermal repair in vivo. However, the mechanism of AngII-induced acceleration of skin wound healing is poorly understood. In the present report, we studied the effects of AngII on skin wound healing focusing on AngII receptor-mediated signaling.

AngII, the effector peptide of the renin-Ang system, is now known to have growth regulating properties in various cell types.22 AngII exerts its actions by binding the specific receptors. Two major AngII receptors with the seven transmembrane domains, namely, AT1 and AT2, have been identified. AT1 is expressed in the brain, kidney, liver, heart, adrenal gland, ovary, testis, and vascular smooth muscle cells.23 AT2 is abundantly and widely expressed in fetal tissues. Most of the AngII-induced actions are mediated by AT1. AT1 activates many signaling pathways and can stimulate tyrosine phosphorylation [through c-Src, Pyk2, and transactivation of epidermal growth factor (EGF) receptor (EGFR)] and activation of phospholipases and protein kinase C.24 A recent study showed that AngII-induced extracellular signal-regulated kinase (ERK) activation was mediated by EGFR transactivated via AT1.18 In contrast, AT2 stimulates none of these (except p38 MAPK) and appears to involve protein tyrosine phosphatases (SHP-1), MAP kinases (p38 and ERK1/2), and G{alpha}s (independent of ß{gamma} G proteins).

EGFR is an 1186-amino acid glycoprotein containing a single transmembrane domain, an extracellular portion involved in ligand binding, and an intracellular portion harboring the tyrosine kinase domain.25 The C-terminal sequences harbor autophosphorylation sites that are important for the initiation of downstream signaling.26 Stimulation of EGFR results in the simultaneous activation of multiple signaling pathways including the RAS-RAF-MEK-MAPK, the PLC-{gamma}/PKC, the PI-3K/AKT cascades, and the JAK and STATS pathways, and these pathways are often functionally interlinked. The known mammalian ligands that act as direct agonists for EGFR include transforming growth factor (TGF)-{alpha}, heparin-binding EGF-like growth factor (HB-EGF), amphiregulin, betacellulin, epiregulin, and epigen.27 These ligands are synthesized as transmembrane precursors and must therefore be proteolytically cleaved by metalloproteases to release the mature growth factor. The agonist-occupied EGFR undergoes gene transcription and mitogenesis. In addition to its cognate ligands, EGFR is activated by stimuli that do not directly interact with EGFR ectodomain including G protein-coupled receptor (GPCR) ligands, other receptor tyrosine kinase agonists, cytokines, chemokines, and cell adhesion elements.26 This process is termed EGFR transactivation and the GPCR-induced EGFR transactivation is stimulated by bradykinin, endothelin (ET)-1, lysophosphatidic acid, interleukin-8, carbachol stimulation, or AngII. Literature has shown that AT1 stimulates processing of pro-HB-EGF by metalloproteinases including a disintegrin and metalloprotease 12 (ADAM 12), and the released HB-EGF transactivates EGFR.28 AngII-induced transactivation of EGFR is essential for the activation of downstream Sr/Thr kinases such as ERK and subsequent protein synthesis.29

Our in vivo study showed that administration of AT1 blocker suppressed rat skin wound healing by inhibiting re-epithelization, dermal repair, and angiogenesis. The process of re-epithelization consists of keratinocyte migration and proliferation. Keratinocytes undergo profound changes in morphology as they migrate.30 The cytoskeleton is critical to these changes and cell migration. EGF-induced activation of PLC-{gamma} stimulates cell motility by releasing PIP2-bound gelsolin from the membrane, thereby restoring its ability to bind, sever, and cap polymerized actin filaments, a process required for filopodia/lamellipodia extension and retraction in motile cells.31,32 Thus, EGFR-mediated activation of PLC-{gamma} is believed to be critical for the reorganization of the actin cytoskeleton and contribute to the initiation of the asymmetric motile phenotype.33

For keratinocytes to migrate, they need to detach themselves from the basal lamina to which they are attached through the hemidesmosomes, anchoring contacts linking laminin via integrin {alpha}6ß4 to the keratinocyte’s keratin filament network.34 A fraction of EGFR combines with the hemidesmosomal integrin {alpha}6ß4 in normal keratinocytes.35 Activation of EGFR causes tyrosine phosphorylation of the ß4 cytoplasmic domain by activating Fyn and disruption of hemidesmosomes and releasing keratinocytes for migration.

The process of re-epithelization is also impaired without the appropriate expression and action of enzymes needed to dissolve substrates and matrix materials for keratinocytes to migrate. Collagenase-1 (matrix metalloproteinase-1) is needed for keratinocyte migration on type I collagen and is up-regulated in keratinocytes at the very edge of the wound.30 Keratinocyte contact with type I collagen is sufficient to induce collagenase-1 expression, whereas sustained enzyme production requires EGFR activation by HB-EGF as an obligatory intermediate step, thereby maintaining collagenase-1-dependent migration during the re-epithelization.36

Autocrine keratinocyte-derived EGFR ligands including TGF-{alpha}, amphiregulin, and HB-EGF contribute to keratinocyte proliferation.37 Literature demonstrated that EGFR activation is indispensable for DNA synthesis38 and cell cycle progression from the G1 to S phase39 in cultured keratinocytes and suggests an essential role of EGFR activation in keratinocyte proliferation.

These data suggest that EGFR activation is important for keratinocyte re-epithelization including migration and proliferation. Accelerated epidermal repair by topical administration of AngII in vivo7,8 may be because of induction of AT1 signaling and at least in part because of transactivation of EGFR. Consistent with this notion, our experiments in vivo showed that administration of AT1 blocker suppressed keratinocyte re-epithelization during skin wound healing. In vitro experiments showed that induction of AT1 signaling enhanced keratinocyte BrdU incorporation and keratinocyte re-epithelization in wound healing migration assays. In contrast, induction of AT2 signaling acted vice versa. Therefore, we conclude that AT1 signaling accelerates keratinocyte proliferation and migration and AT2 signaling suppresses keratinocyte proliferation and migration.

The process by which fibroblasts leave their collagen-rich environment to enter the wound site requires them to modify their behavior and characteristics.30 Early during injury, contraction of dermal myofibroblasts rapidly reduces dermal defects and decreases the area requiring re-epithelization. Later in the injury response, dermal myofibroblasts are stimulated to migrate into the wound and proliferate. AngII increases the TGF-ß1 gene and protein output in cardiac fibroblasts and, especially, in myofibroblasts.40 In cardiac fibroblasts, AngII via AT1 and TGF-ß1 increased fibronectin secretion, smooth muscle {alpha}-actin synthesis, and formation of actin stress fibers and enhanced attachment of fibroblasts to a fibronectin matrix and thereby stimulated and modified fibroblasts into a myofibroblast-like phenotype.41 The blockade of AT1 signaling impaired fibroblast proliferation, consequent differentiation into myofibroblasts, and the synthesis of TGF-ß1.42 These data suggests AngII via AT1 stimulates TGF-ß1 expression, thereby resulting in modification of fibroblast phenotype into myofibroblasts and contributing tissue re-modeling in wound healing. A recent study showed that the endothelial cell-derived vasoconstrictor ET-1 stimulates colonic subepithelial myofibroblast contraction and migration via ET receptor mediated myosin phosphorylation that may be regulated by both calcium and rhoA signaling pathways.43 AngII via AT1 stimulates ET-1 gene expression44 and RAS/RAF/ERK pathways are at least in part involved in AngII-induced proliferation and ET-1 gene expression in rat cardiac fibroblasts45 and aortic smooth muscle cells.46 These data suggest that induction of AT1 signaling is important for dermal repair including transformation of fibroblasts into myofibroblasts. Supporting the notion, our experiments showed that induction of AT1 signaling enhanced myofibroblast BrdU incorporation and myofibroblast recovering in wound healing migration assays. In contrast, induction of AT2 signaling acted vice versa. Therefore, we conclude that AT1 signaling accelerates myofibroblast proliferation and migration and AT2 signaling suppresses myofibroblast proliferation and migration. Recent studies have shown that AngII via AT1 contributes fibrotic response after tissue injury.47 Inhibiting AT1 signaling through the use of AT1 blocker or AngII-converting enzyme inhibitor has proven very useful in the treatment of cardiac hypertrophy47 or renal failure48 because they prevent pathological fibrosis. In the skin, overinduction of AT1 signaling may contribute to cause the pathological fibrotic response such as scarring.

In the experiments using NIH3T3 fibroblasts that were transiently co-transfected with rat AT1 or AT2 expression vectors, the presence of AngII enhanced or reduced the EGFR-induced mitogen activated proliferation kinase (MAPK) activation in the cells expressing AT1 or AT2, respectively.49 In the cells expressing both AngII subtype receptors, AngII stimulated an enhancement of EGFR-induced MAPK activation. Consistent with the results, our experiments indicated that the presence of AngII resulted in induction of AT1 signaling in keratinocytes and myofibroblasts expressing both AngII receptors.

Another aspect for wound healing is neovascularization. Endothelial cell migration and tube formation in response to vascular endothelial growth factor (VEGF) play an important role in the process of angiogenesis. AngII via AT1 stimulates processing of pro-HB-EGF by metalloproteinases, and the released HB-EGF transactivates EGFR to induce angiogenesis via the combined effect of angiopoietin-2 and VEGF, whereas AT2 attenuates them by blocking EGFR phosphorylation in cardiac microvascular endothelial cells expressing both AT1 and AT2.2 Stimulation of AT2 signaling suppresses the phosphorylation of Akt and its downstream effector endothelial NO synthase that is pivotal to VEGF-induced angiogenesis and inhibits VEGF-induced endothelial cell migration and tube formation of endothelial cells.50 Recent studies demonstrated a powerful capacity of VEGF to increase AngII-converting enzyme in human umbilical vein endothelial cells, suggesting a synergistic relationship between renin Ang system and VEGF.51 ET-1 is secreted not only from endothelial cells but also from all rat vascular smooth muscle cells52 and induces angiogenic responses through ET(B) receptor and that stimulates neovascularization in concert with VEGF in cultured endothelial cells.53 TGF-ß1 stimulates ET-1 secretion from vascular smooth muscle cells and endothelial cells.52 These data suggest that AngII stimulates vascular growth via AT1 in skin wound healing. Consistent with this notion, the results of our study showed that blockade of AT1 signaling suppressed angiogenesis during skin wound healing in vivo. Taken together, AT1 signaling accelerates proliferation and migration of both keratinocytes and myofibroblasts and AT2 signaling suppresses these effects. In other words, skin wound healing is regulated by the balance of opposing signals between AT1 and AT2.


   Footnotes
 
Address reprint requests to Hikaru Takeda, M.D., Department of Dermatology, Yamagata University, School of Medicine, 2-2-2, Iida-Nishi, Yamagata 990-9585, Japan. E-mail: hitakeda{at}med.id.yamagata-u.ac.jp

Accepted for publication July 19, 2004.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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