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Reduced angiogenesis and delay in wound healing in angiotensin II type 1a receptor-deficient mice
Available online at
www.sciencedirect.com Biomedicine & Pharmacotherapy 63 (2009) 627e634
Original article
Reduced angiogenesis and delay in wound healing in angiotensin II type 1a receptor-deficient mice Maya Kurosaka a,b, Tatsunori Suzuki a, Kanako Hosono a, Yuji Kamata a,b, Akiyoshi Fukamizu c, Hidero Kitasato d, Yoshikuni Fujita b, Masataka Majima a,* a Department of Pharmacology, Kitasato University School of Medicine, Kanagawa, Japan Department of Internal Medicine, Kitasato University School of Medicine, Kanagawa, Japan c Department of Biology, Faculty of Science Tsukuba University, Ibaraki, Japan d Department of Microbiology, Kitasato University School of Allied Health Science, Kanagawa, Japan b
Received 17 November 2008; accepted 14 January 2009 Available online 14 February 2009
Abstract Angiotensin II (Ang II) is a bioactive peptide that plays important roles in blood pressure regulation and saltewater homeostasis. Recently, Ang II was reported to function in the promotion of angiogenesis. Since the wound healing process is highly dependent upon angiogenesis, we employed Ang II receptor knockout mice (AT1a/) to investigate whether or not Ang II facilitates angiogenesis and wound healing via AT1a receptor signaling. In comparison to wild-type (WT) mice, wound healing and wound-induced angiogenesis were significantly suppressed in AT1a/ mice, and these mice exhibited reduced expression of CD31 in wound granulation tissues. In comparison to vehicle-treated mice, wound healing was delayed significantly in mice treated with an AT1-R antagonist and this delay was accompanied by the reduced expression of vascular endothelial growth factor in wound granulation tissues. These findings suggest that Ang IIeAT1a signaling plays a crucial role in wound healing and wound-induced angiogenesis. Ó 2009 Elsevier Masson SAS. All rights reserved. Keywords: Angiogenesis; Angiotensin II; Wound healing
1. Introduction Angiotensin II (Ang II) is an octapeptide derived from the renineangiotensinealdosterone system. It is involved in blood pressure control and saltewater homeostasis, being generally regarded as a systemic hormone that acts to release aldosterone and to enhance reabsorption of sodium. Recently, it has been proposed that Ang II is also formed and is active in peripheral tissues such as the heart, kidney and brain, where it can function as a growth factor [1,2]. Although this peptide has been implicated in cellular proliferation, migration, inflammation and growth factor biosynthesis, it is now also * Corresponding author. Department of Molecular Pharmacology, Kitasato University Graduate School of Medical Sciences, 1-15-1 Kitasato, Sagamihara 228-8555, Kanagawa, Japan. Tel.: þ81 42 778 8822; fax: þ81 42 778 7604. E-mail address: [email protected] (M. Majima). 0753-3322/$ - see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biopha.2009.01.001
clear that Ang II is an inducer of angiogenesis. Angiogenesis is observed in many physiological processes, including embryonic development and endometrial proliferation during the menstrual cycle. It is also seen in various pathological states such as chronic inflammation, cancer and wound healing [3e5]. Recent in vitro reports have shown that Ang II promotes angiogenesis via the proliferation and migration of vascular cells such as smooth muscle cells [6,7] and pericytes [8,9]. In addition, several in vivo studies have revealed that Ang II accelerates angiogenesis [10e14]. Ang II exerts its biological activity through G proteincoupled receptors. There are two subtypes of Ang II receptors, the Ang II type1 receptor (AT1-R) and the Ang II type2 receptor (AT2-R). AT1-R is distributed widely throughout human tissues and is found in cardiac, vascular, endocrine, kidney and brain cells, where it mediates the most well-known physiological activities of Ang II. AT1-R has two splicing
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variants in mice, namely AT1a and AT1b; the former is known to mediate many of the physiological activities regulated by AT1 signaling. In contrast, AT2-R is considered to have the opposite effect to AT1-R; it is found in fetal tissue and its levels decrease after birth. Previously, we reported that endogenous Ang II stimulates angiogenesis in sponge implantation models [15,16] and demonstrated that administration of an AT1-R antagonist suppressed bFGF-induced angiogenesis in sponge granulomas. In a tumor implantation model, Fujita et al. found that Ang II promotes tumorassociated angiogenesis via signaling of an AT1a receptor (AT1a-R) [17]. In order to identify the pathophysiological functions of AT1a-R in vivo, we developed mice genetically lacking AT1a-R [18]. The wound healing process requires reformation of epithelium to cover exposed tissues and angiogenesis plays a key role in granulation formation and re-epithelialization. Since Ang II is believed to be a regulator of angiogenesis, we have attempted to clarify the role of AT1a-R signaling in wound-induced angiogenesis and wound healing. In this study, we have used a drug that blocks AT1, as well as AT1aR-deficient (AT1a/) mice. 2. Materials and methods 2.1. Evaluation of the wound healing process The AT1-R antagonist TCV-116 was a kind gift from Takeda Chemical Industries (Osaka, Japan). Male 8-week-old C57BL/6 wild-type mice (WT) were purchased from Clea Japan (Tokyo, Japan) and AT1a/ mice were developed, as reported previously [16]. All mice had free access to tap water and rodent chow. Animals were housed individually with a 12 h light/dark cycle and at a constant temperature (25 1 C) and humidity (60 5%). All experiments were performed in accordance with the guidelines for animal experiments of Kitasato University School of Medicine. On day 0 and following anesthetization with diethylether, 8 mm diameter full-thickness excisional skin wounds were formed on either side of the dorsal midline using an 8-mm biopsy punch (Kai Industries, Tokyo, Japan). TCV-116 was administered orally (100 mg/kg/day) from day 0 until the end of the experiment and the wound healing process was evaluated in comparison to vehicle-treated mice. At each time point, the wounds were photographed digitally and wound areas were calculated using the image analysis software Scion Image (Scion Corporation). Changes in wound area were expressed as a percentage of the wound area at day 0. The wound closure process in AT1a/ mice was compared with WT mice in the same manner. 2.2. Immunohistochemistry For some immunohistochemical studies, the wounds were excised completely at day 3 and then fixed with 4% paraformaldehyde in 0.1 mol/L sodium phosphate buffer (pH 7.4). Samples were embedded in paraffin, after which sections were prepared and mounted on glass slides. After removal of paraffin
with xylene, the slides were placed in cold (4 C) acetone and then sections were used for hematoxylin and eosin (H&E) or immunohistochemical staining. For immunostaining, the sections were exposed to dilute normal horse serum and then incubated with rabbit antiserum against mouse CD31 (Santa Cruz Biotechnology, Santa Cruz, CA), mouse VEGF (Santa Cruz Biotechnology) or mouse AT1-R (Santa Cruz Biotechnology). Immune complexes were detected using an LSAB þ System-HRP kit (DakoCytomation, Carpinteria, CA). Wound-induced angiogenesis in granulation tissues was estimated by microvessel density (MVD), according to established methods described previously [19]. Areas with the greatest neovascularization were selected under a 40 field and then individual microvessels were counted 10 times under a 200 field, to obtain average numbers. Vascular endothelial cells were distinguished from other connective tissue elements by CD31 immunostaining. MVD was expressed as the number of vessels/mm2. 2.3. Total RNA isolation and reverse transcription Transcripts encoding VEGF, CD31 and glyceraldehyde3-phosphate dehydrogenase (GAPDH) were quantified by RTPCR analysis. Total RNA was isolated from wound tissue using TRIzol reagent (Invitrogen, Carlsbad, CA) and RNA abundance was determined from the 260/280 ratio measured using a BioPhotometer (Eppendorf, Hamburg, Germany). cDNA synthesis reactions (40 ml) contained 1 mg of total RNA, 200 U of ReverTra Ace (reverse transcriptase, Toyobo, Osaka, Japan), 40 nmol of dNTP mixture (Toyobo), 20 U of RNase inhibitor (Toyobo) and 20 pmol of Oligo(dT)20. The reactions were incubated initially at 30 C for 10 min and then at 42 C for 40 min, followed by the inactivation of the reaction at 99 C for 5 min. 2.4. Real-time quantitative PCR analysis Real-time PCR primers were designed using Primer3 software (http://primer3.sourceforge.net/). Primers used were as follows: for VEGF, 50 -GAGAGAGGCCGAAGTCCTTT-30 (forward) and 50 -TTGGAACCGGCATCTTTATC-30 (reverse); for CD31, 50 -ACTTCTGAACTCCAACAGCGA-30 (forward) and 50 -CCATGTTCTGGGGGTCTTTAT-30 (reverse); and for GAPDH, 50 -ACATCAAGAAGGTGGTGAAGC-30 (forward) and 50 -AAGGTGGAAGAGTGGGAGTTG-30 (reverse). Genespecific primers and cDNA solutions were added to SYBR Premix Ex Taq (Takara, Otsu, Japan) and subjected to PCR amplification in the Opticon 2 system (Bio-Rad, Hercules, CA). PCR reactions were incubated initially at 95 C for 10 s and then cycled 40 times using the following conditions: 95 C for 3 s and 60 C for 20 s. PCR reactions (20 ml) contained 2 ml of cDNA solution, 0.2 ml of 20 mM forward and reverse primers and 10 ml of SYBR Premix Ex Taq. The threshold cycle (Ct) values were processed for further calculations according to the comparative Ct method. The DCt value was determined by the normalization of the target gene expression
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levels against the housekeeping gene GAPDH and calculating 2DCt.
3.2. Effects of an AT1-R antagonist on wound-induced angiogenesis
2.5. Statistical analyses
On day 3, wound granulation tissues were isolated and examined histologically. Granulation tissues in TCV116-treated mice were atrophic, exhibiting a lower cell density than vehicle-treated mice (Fig. 1b). The granulation tissues were examined immunohistochemically with CD31 antibody and the wounds of TCV-116-treated mice contained fewer CD31-positive microvessels than vehicle-treated mice (Fig. 2a). Furthermore, MVD in granulation tissues of TCV116-treated mice was reduced significantly in comparison to vehicle-treated mice (Fig. 2b). Immunohistochemical localization detected the presence of AT1-R and VEGF in fibroblast-like cells of wound granulation tissues (Fig. 3a and b, respectively) and there were markedly fewer VEGF-expressing cells in TCV-116-treated mice than in vehicle-treated mice (Fig. 3b). Real-time RT-PCR analysis also showed significantly reduced VEGF mRNA expression in mice treated with TCV-116, compared to mice treated with vehicle alone (Fig. 3c).
Data were expressed as means SEM. Multiple group comparisons were performed via factorial analysis of variance (ANOVA), followed by Scheffe’s test. Comparisons between two groups were performed using the ManneWhitney test. p Values < 0.05 were considered statistically significant. 3. Results 3.1. Effects of an AT1-R antagonist on wound healing To examine the role of AT1-R signaling with respect to wound-induced angiogenesis, we evaluated the wound healing process in WT mice during the treatment with the AT1a-R antagonist TCV-116. Wound area was measured from day 0 to day 13 and then expressed as a percentage of wound area at day 0 (Fig. 1a). On days 2, 4 and 10, the wound areas of vehicle-treated mice had reduced to 58, 44 and 0% (i.e., completely closure), respectively. In contrast, on days 2, 4 and 10, the wound areas in mice treated with TCV-116 had decreased to 68, 54 and 4%, respectively. Clearly, wound closure was delayed significantly between day 1 and day 6 in TCV-116-treated mice compared to vehicle-treated mice.
3.3. Delay in wound healing in AT1a receptor-deficient mice In order to assess the significance of angiotensin II and AT1a receptor signaling in wound repair, we evaluated the wound healing process in AT1a/ and WT mice for 14 days.
Fig. 1. Effects of the AT1-R antagonist TCV-116 on wound healing. (a) Time course of wound closure in TCV-116- and vehicle-treated mice. Data are shown as means SEM. **p < 0.01 and *p < 0.05 versus vehicle-treated mice (n ¼ 20 for TCV-116-treated mice; n ¼ 16 for vehicle-treated mice); (b) histological appearance of skin wounds on day 3 after wounding in TCV-116- (right) and vehicle-treated mice (left) (H&E staining). W, wound; Gr, granulation tissue. Scale bar ¼ 300 mm.
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Fig. 2. Effects of the AT1-R antagonist TCV-116 on wound-induced angiogenesis. (a) Immunohistochemical staining of CD31 in wound granulation tissues from TCV-116- and vehicle-treated mice on day 3. Reduced numbers of CD31-positive microvessels (red arrows) in the granulation tissues of TCV-116-treated mice compared with vehicle-treated mice. Scale bar ¼ 100 mm; (b) MVD in granulation tissues on day 3. Data are shown as means SEM; significantly different from vehicle-treated mice ( p < 0.01); (c) CD31 mRNA levels determined by real-time RT-PCR in the wound granulation tissues of TCV-116- and vehicle-treated mice on day 3. Data are shown as means SEM; significantly different from vehicle-treated mice ( p < 0.05).
In comparison to WT mice, AT1a/ mice demonstrated a significant delay in wound healing, especially during the early phase of the healing process (Fig. 4a). 3.4. Suppression of wound-induced angiogenesis in AT1a receptor-deficient mice On day 3, we compared the histological appearance of wounds in WT and AT1a/ mice. WT mice exhibited thick layers of granulation tissue that contained large numbers of fibroblasts, neutrophils and macrophages (Fig. 4b). In contrast, these tissues were poorly formed in AT1a/ mice (Fig. 4b). Although immunostaining of CD31-positive microvessels indicated the presence of abundant microvessel assembles in wound granulation tissues of WT mice, few were observed in AT1a/ mice (Fig. 5a). Moreover, AT1a/ mice demonstrated a significantly lower MVD at day 3 than WT mice (Fig. 5b). Real-time RT-PCR analysis also showed significantly lower CD31 mRNA levels in AT1a/ mice than in WT mice (Fig. 5c). Although immunostaining detected the presence of VEGF in the fibroblast-like cells of wound granulation tissues, the levels in AT1a/ mice were markedly lower than in WT mice (Fig. 6). 4. Discussion Cutaneous wound healing is traditionally classified into three overlapping phases: (1) the inflammatory phase, which is
characterized as platelet aggregation and recruitment of inflammatory cells to the wound site; (2) the tissue formation phase, which is when granulation tissue formation and epithelialization occur; and (3) the tissue remodeling phase, when collagen deposition is remodeled [20]. Angiogenesis is fundamental to wound repair, since it is responsible for the provision of oxygen and nutrients to healing tissue, as well as for the successful formation of granulation tissues. Recent studies have demonstrated that Ang II accelerates proliferation, migration and growth factor synthesis in vascular cells. Although these findings suggest that Ang II has some proangiogenic effect, the involvement of Ang II and AT1-R signaling in wound-induced angiogenesis remains to be elucidated. In the present study, we examined the role of Ang II and AT1-R signaling in wound-induced angiogenesis. We have shown that wound healing was delayed significantly in AT1-R antagonist-treated mice (Fig. 1a) and this result suggests that Ang II and AT1-R signaling promotes wound healing. Immunohistochemical analysis indicated that AT1R-expressing cells were abundant in wound granulation tissues (Fig. 3a), but not in normal skin regions (data not shown), suggesting that recruitment of AT1-R-expressing cells to the wound site represents a crucial step in the healing process. In comparison to vehicle-treated mice, TCV-116-treated mice demonstrated significantly reduced MVD and lower CD31 mRNA levels (Fig. 2c). These results indicate that Ang II and AT1-R signaling induces angiogenesis in wound granulation tissues, a process required for wound repair.
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Fig. 3. Immunohistochemical detection of AT1-R in wound tissues and reduced expression of VEGF in TCV-116-treated mice. (a) A representative immunohistochemical staining of AT1-R in wound granulation tissues of vehicle-treated mice on day 3. Scale bar ¼ 300 mm (left) and 100 mm (right); (b) Immunohistochemical staining of VEGF in wound granulation tissues of TCV-116- and vehicle-treated mice on day 3. Poor expression of VEGF in TCV-116-treated mice compared with vehicle-treated mice. Scale bar ¼ 100 mm; (c) real-time RT-PCR of VEGF mRNA levels in wound granulation tissues of TCV-116- and vehicletreated mice on day 3. VEGF mRNA levels were significantly lower in mice treated with TCV-116 in comparison to vehicle solution ( p < 0.05).
Previously, we used an in vivo sponge angiogenesis model to demonstrate that Ang II-induced angiogenesis correlated with increased expression of the potent proangiogenic factor VEGF [15,16]. We also used an antisense oligodeoxynucleotide complementary to VEGF mRNA to determine that VEGF plays a significant role in Ang II-induced angiogenesis [16]. In addition, we demonstrated that the wound healing process is dependent upon VEGF, since topical application of VEGFneutralizing antibody to wound granulation tissues suppressed the wound closure process [5,21]. VEGF is known to be an endothelial cell-specific mitogen that acts as inducer of angiogenesis [22e24]. In the present experiment, we observed reduced levels of VEGF mRNA in the wounds of TCV-116treated mice compared to vehicle-treated mice (Fig. 3c). In addition, immunohistochemical analysis detected many VEGF-expressing fibroblast-like cells in the granulation tissues of mice treated with vehicle alone, whereas only a few VEGF-expressing cells were found in TCV-116-treated mice (Fig. 3b). Taken together with our previous finding that topical
application of VEGF-neutralizing antibody to wound granulation tissues significantly delays healing [5], our present results suggest that suppression of angiogenesis, followed by decreased levels of VEGF, contribute to delay of wound closure in AT1-R antagonist-treated mice. In the present study, we attempted to investigate Ang II receptor signaling further and this is the first report to examine wound healing in AT1a/ mice (Fig. 4a). The delay in wound healing of AT1a/ mice was essentially the same as seen with TCV-116 treatment, which suggests that AT1a is the primary target of the AT1-R antagonist. Immunostaining with CD31 antibody demonstrated that the MVD was significantly lower in AT1a/ mice than in WT mice (Fig. 5b) and RT-PCR analysis revealed reduced CD31 mRNA levels in AT1a/ mice relative to WT mice (Fig. 5c). These results show that wound-induced angiogenesis is promoted by AT1a-R signaling. Immunohistological examination detected a large number of VEGF-expressing cells in the wound granulation tissues of WT mice, whereas much lower levels of expression
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Fig. 4. Delayed wound healing in AT1a receptor knockout mice. (a) Time course of wound healing in AT1a/ and WT mice. Data are shown as means SEM; *p < 0.05 versus WT mice (n ¼ 12 for AT1a/ mice; n ¼ 12 for WT mice); (b) histological appearance of wound tissues in AT1a/ (right) and WT (left) mice on day 3 (H&E staining). W, wound; Gr, granulation tissue. Scale bar ¼ 300 mm.
Fig. 5. Inhibition of wound-induced angiogenesis in AT1a receptor knockout mice. (a) Immunohistochemical staining of CD31 in wound granulation tissues of AT1a/ (right) and WT (left) mice on day 3. Reduced numbers of CD31-positive microvessels (indicated by red arrows) were observed in the granulation tissues of AT1a/ mice compared to WT mice. Scale bar ¼ 100 mm; (b) MVD in granulation tissues on day 3. Data are shown as means SEM; significantly different from WT mice ( p < 0.01); (c) real-time RT-PCR of CD31 mRNA levels in wound granulation tissues of AT1a/ and WT mice on day 3. Significantly different from WT mice ( p < 0.01).
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Fig. 6. Suppression of VEGF expression in AT1a receptor knockout mice. Immunohistological detection of VEGF in wound granulation tissues of AT1a/ and WT mice on day 3. AT1a/ mice exhibit lower numbers of VEGF-expressing cells in comparison to WT mice. Scale bar ¼ 100 mm.
were observed in AT1a/ mice (Fig. 6). These results also suggest that inhibition of angiogenesis, followed by reduced induction of VEGF, contribute to the delay of wound healing in AT1a/ mice. Previously, we demonstrated that primary cultured fibroblasts prepared from granulation tissues could express VEGF mRNA in response to Ang II [16]. Research in rat heart endothelial cells has indicated that protein kinase C may also be involved in Ang II-induced VEGF up-regulation [25]. We have shown that the protein kinase C inhibitors, H7 and GFX, attenuate expression of VEGF in primary fibroblasts. Further, we determined that NF-B and AP-1 are the transcription factors responsible for Ang IImediated induction of VEGF mRNA in fibroblasts. The human VEGF gene promoter has been shown to contain potential binding sites for signal protein-1 (SP-1), AP-1 and AP-2 [26]. The mouse VEGF promoter contains not only binding sites for SP-1, AP-1 and AP-2, but also for NF-B [27]. A link between the activation of AT1a-R and the transcription factors, NF-B and AP-1, may be crucial for active wound healing, since this process is highly dependent upon angiogenesis. Taken together, our findings lead us to conclude that AT1aR signaling enhances VEGF expression and the formation of wound granulation tissues. In addition, AT1-R antagonists are commonly used as therapeutic agents and thus, we consider it important to recognize the possibility that patients undergoing treatment with these products may experience delays in wound healing. Acknowledgments We thank Michiko Ogino, Kyoko Yoshikawa, and Osamu Katsumata for their technical assistance. This work was supported by research grants (#12470529 and #12670094), by a High-tech Research Center grant from Ministry of Education, Culture, Sports, Science and Technology, Japan. This study was also supported by an Integrative Research Program of the Graduate School of Medical Science, Kitasato University. We are also grateful to Dr. Patrick Hughes for linguistic assistance in the preparation of the manuscript.
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Original article
Reduced angiogenesis and delay in wound healing in angiotensin II type 1a receptor-deficient mice Maya Kurosaka a,b, Tatsunori Suzuki a, Kanako Hosono a, Yuji Kamata a,b, Akiyoshi Fukamizu c, Hidero Kitasato d, Yoshikuni Fujita b, Masataka Majima a,* a Department of Pharmacology, Kitasato University School of Medicine, Kanagawa, Japan Department of Internal Medicine, Kitasato University School of Medicine, Kanagawa, Japan c Department of Biology, Faculty of Science Tsukuba University, Ibaraki, Japan d Department of Microbiology, Kitasato University School of Allied Health Science, Kanagawa, Japan b
Received 17 November 2008; accepted 14 January 2009 Available online 14 February 2009
Abstract Angiotensin II (Ang II) is a bioactive peptide that plays important roles in blood pressure regulation and saltewater homeostasis. Recently, Ang II was reported to function in the promotion of angiogenesis. Since the wound healing process is highly dependent upon angiogenesis, we employed Ang II receptor knockout mice (AT1a/) to investigate whether or not Ang II facilitates angiogenesis and wound healing via AT1a receptor signaling. In comparison to wild-type (WT) mice, wound healing and wound-induced angiogenesis were significantly suppressed in AT1a/ mice, and these mice exhibited reduced expression of CD31 in wound granulation tissues. In comparison to vehicle-treated mice, wound healing was delayed significantly in mice treated with an AT1-R antagonist and this delay was accompanied by the reduced expression of vascular endothelial growth factor in wound granulation tissues. These findings suggest that Ang IIeAT1a signaling plays a crucial role in wound healing and wound-induced angiogenesis. Ó 2009 Elsevier Masson SAS. All rights reserved. Keywords: Angiogenesis; Angiotensin II; Wound healing
1. Introduction Angiotensin II (Ang II) is an octapeptide derived from the renineangiotensinealdosterone system. It is involved in blood pressure control and saltewater homeostasis, being generally regarded as a systemic hormone that acts to release aldosterone and to enhance reabsorption of sodium. Recently, it has been proposed that Ang II is also formed and is active in peripheral tissues such as the heart, kidney and brain, where it can function as a growth factor [1,2]. Although this peptide has been implicated in cellular proliferation, migration, inflammation and growth factor biosynthesis, it is now also * Corresponding author. Department of Molecular Pharmacology, Kitasato University Graduate School of Medical Sciences, 1-15-1 Kitasato, Sagamihara 228-8555, Kanagawa, Japan. Tel.: þ81 42 778 8822; fax: þ81 42 778 7604. E-mail address: [email protected] (M. Majima). 0753-3322/$ - see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biopha.2009.01.001
clear that Ang II is an inducer of angiogenesis. Angiogenesis is observed in many physiological processes, including embryonic development and endometrial proliferation during the menstrual cycle. It is also seen in various pathological states such as chronic inflammation, cancer and wound healing [3e5]. Recent in vitro reports have shown that Ang II promotes angiogenesis via the proliferation and migration of vascular cells such as smooth muscle cells [6,7] and pericytes [8,9]. In addition, several in vivo studies have revealed that Ang II accelerates angiogenesis [10e14]. Ang II exerts its biological activity through G proteincoupled receptors. There are two subtypes of Ang II receptors, the Ang II type1 receptor (AT1-R) and the Ang II type2 receptor (AT2-R). AT1-R is distributed widely throughout human tissues and is found in cardiac, vascular, endocrine, kidney and brain cells, where it mediates the most well-known physiological activities of Ang II. AT1-R has two splicing
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variants in mice, namely AT1a and AT1b; the former is known to mediate many of the physiological activities regulated by AT1 signaling. In contrast, AT2-R is considered to have the opposite effect to AT1-R; it is found in fetal tissue and its levels decrease after birth. Previously, we reported that endogenous Ang II stimulates angiogenesis in sponge implantation models [15,16] and demonstrated that administration of an AT1-R antagonist suppressed bFGF-induced angiogenesis in sponge granulomas. In a tumor implantation model, Fujita et al. found that Ang II promotes tumorassociated angiogenesis via signaling of an AT1a receptor (AT1a-R) [17]. In order to identify the pathophysiological functions of AT1a-R in vivo, we developed mice genetically lacking AT1a-R [18]. The wound healing process requires reformation of epithelium to cover exposed tissues and angiogenesis plays a key role in granulation formation and re-epithelialization. Since Ang II is believed to be a regulator of angiogenesis, we have attempted to clarify the role of AT1a-R signaling in wound-induced angiogenesis and wound healing. In this study, we have used a drug that blocks AT1, as well as AT1aR-deficient (AT1a/) mice. 2. Materials and methods 2.1. Evaluation of the wound healing process The AT1-R antagonist TCV-116 was a kind gift from Takeda Chemical Industries (Osaka, Japan). Male 8-week-old C57BL/6 wild-type mice (WT) were purchased from Clea Japan (Tokyo, Japan) and AT1a/ mice were developed, as reported previously [16]. All mice had free access to tap water and rodent chow. Animals were housed individually with a 12 h light/dark cycle and at a constant temperature (25 1 C) and humidity (60 5%). All experiments were performed in accordance with the guidelines for animal experiments of Kitasato University School of Medicine. On day 0 and following anesthetization with diethylether, 8 mm diameter full-thickness excisional skin wounds were formed on either side of the dorsal midline using an 8-mm biopsy punch (Kai Industries, Tokyo, Japan). TCV-116 was administered orally (100 mg/kg/day) from day 0 until the end of the experiment and the wound healing process was evaluated in comparison to vehicle-treated mice. At each time point, the wounds were photographed digitally and wound areas were calculated using the image analysis software Scion Image (Scion Corporation). Changes in wound area were expressed as a percentage of the wound area at day 0. The wound closure process in AT1a/ mice was compared with WT mice in the same manner. 2.2. Immunohistochemistry For some immunohistochemical studies, the wounds were excised completely at day 3 and then fixed with 4% paraformaldehyde in 0.1 mol/L sodium phosphate buffer (pH 7.4). Samples were embedded in paraffin, after which sections were prepared and mounted on glass slides. After removal of paraffin
with xylene, the slides were placed in cold (4 C) acetone and then sections were used for hematoxylin and eosin (H&E) or immunohistochemical staining. For immunostaining, the sections were exposed to dilute normal horse serum and then incubated with rabbit antiserum against mouse CD31 (Santa Cruz Biotechnology, Santa Cruz, CA), mouse VEGF (Santa Cruz Biotechnology) or mouse AT1-R (Santa Cruz Biotechnology). Immune complexes were detected using an LSAB þ System-HRP kit (DakoCytomation, Carpinteria, CA). Wound-induced angiogenesis in granulation tissues was estimated by microvessel density (MVD), according to established methods described previously [19]. Areas with the greatest neovascularization were selected under a 40 field and then individual microvessels were counted 10 times under a 200 field, to obtain average numbers. Vascular endothelial cells were distinguished from other connective tissue elements by CD31 immunostaining. MVD was expressed as the number of vessels/mm2. 2.3. Total RNA isolation and reverse transcription Transcripts encoding VEGF, CD31 and glyceraldehyde3-phosphate dehydrogenase (GAPDH) were quantified by RTPCR analysis. Total RNA was isolated from wound tissue using TRIzol reagent (Invitrogen, Carlsbad, CA) and RNA abundance was determined from the 260/280 ratio measured using a BioPhotometer (Eppendorf, Hamburg, Germany). cDNA synthesis reactions (40 ml) contained 1 mg of total RNA, 200 U of ReverTra Ace (reverse transcriptase, Toyobo, Osaka, Japan), 40 nmol of dNTP mixture (Toyobo), 20 U of RNase inhibitor (Toyobo) and 20 pmol of Oligo(dT)20. The reactions were incubated initially at 30 C for 10 min and then at 42 C for 40 min, followed by the inactivation of the reaction at 99 C for 5 min. 2.4. Real-time quantitative PCR analysis Real-time PCR primers were designed using Primer3 software (http://primer3.sourceforge.net/). Primers used were as follows: for VEGF, 50 -GAGAGAGGCCGAAGTCCTTT-30 (forward) and 50 -TTGGAACCGGCATCTTTATC-30 (reverse); for CD31, 50 -ACTTCTGAACTCCAACAGCGA-30 (forward) and 50 -CCATGTTCTGGGGGTCTTTAT-30 (reverse); and for GAPDH, 50 -ACATCAAGAAGGTGGTGAAGC-30 (forward) and 50 -AAGGTGGAAGAGTGGGAGTTG-30 (reverse). Genespecific primers and cDNA solutions were added to SYBR Premix Ex Taq (Takara, Otsu, Japan) and subjected to PCR amplification in the Opticon 2 system (Bio-Rad, Hercules, CA). PCR reactions were incubated initially at 95 C for 10 s and then cycled 40 times using the following conditions: 95 C for 3 s and 60 C for 20 s. PCR reactions (20 ml) contained 2 ml of cDNA solution, 0.2 ml of 20 mM forward and reverse primers and 10 ml of SYBR Premix Ex Taq. The threshold cycle (Ct) values were processed for further calculations according to the comparative Ct method. The DCt value was determined by the normalization of the target gene expression
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levels against the housekeeping gene GAPDH and calculating 2DCt.
3.2. Effects of an AT1-R antagonist on wound-induced angiogenesis
2.5. Statistical analyses
On day 3, wound granulation tissues were isolated and examined histologically. Granulation tissues in TCV116-treated mice were atrophic, exhibiting a lower cell density than vehicle-treated mice (Fig. 1b). The granulation tissues were examined immunohistochemically with CD31 antibody and the wounds of TCV-116-treated mice contained fewer CD31-positive microvessels than vehicle-treated mice (Fig. 2a). Furthermore, MVD in granulation tissues of TCV116-treated mice was reduced significantly in comparison to vehicle-treated mice (Fig. 2b). Immunohistochemical localization detected the presence of AT1-R and VEGF in fibroblast-like cells of wound granulation tissues (Fig. 3a and b, respectively) and there were markedly fewer VEGF-expressing cells in TCV-116-treated mice than in vehicle-treated mice (Fig. 3b). Real-time RT-PCR analysis also showed significantly reduced VEGF mRNA expression in mice treated with TCV-116, compared to mice treated with vehicle alone (Fig. 3c).
Data were expressed as means SEM. Multiple group comparisons were performed via factorial analysis of variance (ANOVA), followed by Scheffe’s test. Comparisons between two groups were performed using the ManneWhitney test. p Values < 0.05 were considered statistically significant. 3. Results 3.1. Effects of an AT1-R antagonist on wound healing To examine the role of AT1-R signaling with respect to wound-induced angiogenesis, we evaluated the wound healing process in WT mice during the treatment with the AT1a-R antagonist TCV-116. Wound area was measured from day 0 to day 13 and then expressed as a percentage of wound area at day 0 (Fig. 1a). On days 2, 4 and 10, the wound areas of vehicle-treated mice had reduced to 58, 44 and 0% (i.e., completely closure), respectively. In contrast, on days 2, 4 and 10, the wound areas in mice treated with TCV-116 had decreased to 68, 54 and 4%, respectively. Clearly, wound closure was delayed significantly between day 1 and day 6 in TCV-116-treated mice compared to vehicle-treated mice.
3.3. Delay in wound healing in AT1a receptor-deficient mice In order to assess the significance of angiotensin II and AT1a receptor signaling in wound repair, we evaluated the wound healing process in AT1a/ and WT mice for 14 days.
Fig. 1. Effects of the AT1-R antagonist TCV-116 on wound healing. (a) Time course of wound closure in TCV-116- and vehicle-treated mice. Data are shown as means SEM. **p < 0.01 and *p < 0.05 versus vehicle-treated mice (n ¼ 20 for TCV-116-treated mice; n ¼ 16 for vehicle-treated mice); (b) histological appearance of skin wounds on day 3 after wounding in TCV-116- (right) and vehicle-treated mice (left) (H&E staining). W, wound; Gr, granulation tissue. Scale bar ¼ 300 mm.
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Fig. 2. Effects of the AT1-R antagonist TCV-116 on wound-induced angiogenesis. (a) Immunohistochemical staining of CD31 in wound granulation tissues from TCV-116- and vehicle-treated mice on day 3. Reduced numbers of CD31-positive microvessels (red arrows) in the granulation tissues of TCV-116-treated mice compared with vehicle-treated mice. Scale bar ¼ 100 mm; (b) MVD in granulation tissues on day 3. Data are shown as means SEM; significantly different from vehicle-treated mice ( p < 0.01); (c) CD31 mRNA levels determined by real-time RT-PCR in the wound granulation tissues of TCV-116- and vehicle-treated mice on day 3. Data are shown as means SEM; significantly different from vehicle-treated mice ( p < 0.05).
In comparison to WT mice, AT1a/ mice demonstrated a significant delay in wound healing, especially during the early phase of the healing process (Fig. 4a). 3.4. Suppression of wound-induced angiogenesis in AT1a receptor-deficient mice On day 3, we compared the histological appearance of wounds in WT and AT1a/ mice. WT mice exhibited thick layers of granulation tissue that contained large numbers of fibroblasts, neutrophils and macrophages (Fig. 4b). In contrast, these tissues were poorly formed in AT1a/ mice (Fig. 4b). Although immunostaining of CD31-positive microvessels indicated the presence of abundant microvessel assembles in wound granulation tissues of WT mice, few were observed in AT1a/ mice (Fig. 5a). Moreover, AT1a/ mice demonstrated a significantly lower MVD at day 3 than WT mice (Fig. 5b). Real-time RT-PCR analysis also showed significantly lower CD31 mRNA levels in AT1a/ mice than in WT mice (Fig. 5c). Although immunostaining detected the presence of VEGF in the fibroblast-like cells of wound granulation tissues, the levels in AT1a/ mice were markedly lower than in WT mice (Fig. 6). 4. Discussion Cutaneous wound healing is traditionally classified into three overlapping phases: (1) the inflammatory phase, which is
characterized as platelet aggregation and recruitment of inflammatory cells to the wound site; (2) the tissue formation phase, which is when granulation tissue formation and epithelialization occur; and (3) the tissue remodeling phase, when collagen deposition is remodeled [20]. Angiogenesis is fundamental to wound repair, since it is responsible for the provision of oxygen and nutrients to healing tissue, as well as for the successful formation of granulation tissues. Recent studies have demonstrated that Ang II accelerates proliferation, migration and growth factor synthesis in vascular cells. Although these findings suggest that Ang II has some proangiogenic effect, the involvement of Ang II and AT1-R signaling in wound-induced angiogenesis remains to be elucidated. In the present study, we examined the role of Ang II and AT1-R signaling in wound-induced angiogenesis. We have shown that wound healing was delayed significantly in AT1-R antagonist-treated mice (Fig. 1a) and this result suggests that Ang II and AT1-R signaling promotes wound healing. Immunohistochemical analysis indicated that AT1R-expressing cells were abundant in wound granulation tissues (Fig. 3a), but not in normal skin regions (data not shown), suggesting that recruitment of AT1-R-expressing cells to the wound site represents a crucial step in the healing process. In comparison to vehicle-treated mice, TCV-116-treated mice demonstrated significantly reduced MVD and lower CD31 mRNA levels (Fig. 2c). These results indicate that Ang II and AT1-R signaling induces angiogenesis in wound granulation tissues, a process required for wound repair.
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Fig. 3. Immunohistochemical detection of AT1-R in wound tissues and reduced expression of VEGF in TCV-116-treated mice. (a) A representative immunohistochemical staining of AT1-R in wound granulation tissues of vehicle-treated mice on day 3. Scale bar ¼ 300 mm (left) and 100 mm (right); (b) Immunohistochemical staining of VEGF in wound granulation tissues of TCV-116- and vehicle-treated mice on day 3. Poor expression of VEGF in TCV-116-treated mice compared with vehicle-treated mice. Scale bar ¼ 100 mm; (c) real-time RT-PCR of VEGF mRNA levels in wound granulation tissues of TCV-116- and vehicletreated mice on day 3. VEGF mRNA levels were significantly lower in mice treated with TCV-116 in comparison to vehicle solution ( p < 0.05).
Previously, we used an in vivo sponge angiogenesis model to demonstrate that Ang II-induced angiogenesis correlated with increased expression of the potent proangiogenic factor VEGF [15,16]. We also used an antisense oligodeoxynucleotide complementary to VEGF mRNA to determine that VEGF plays a significant role in Ang II-induced angiogenesis [16]. In addition, we demonstrated that the wound healing process is dependent upon VEGF, since topical application of VEGFneutralizing antibody to wound granulation tissues suppressed the wound closure process [5,21]. VEGF is known to be an endothelial cell-specific mitogen that acts as inducer of angiogenesis [22e24]. In the present experiment, we observed reduced levels of VEGF mRNA in the wounds of TCV-116treated mice compared to vehicle-treated mice (Fig. 3c). In addition, immunohistochemical analysis detected many VEGF-expressing fibroblast-like cells in the granulation tissues of mice treated with vehicle alone, whereas only a few VEGF-expressing cells were found in TCV-116-treated mice (Fig. 3b). Taken together with our previous finding that topical
application of VEGF-neutralizing antibody to wound granulation tissues significantly delays healing [5], our present results suggest that suppression of angiogenesis, followed by decreased levels of VEGF, contribute to delay of wound closure in AT1-R antagonist-treated mice. In the present study, we attempted to investigate Ang II receptor signaling further and this is the first report to examine wound healing in AT1a/ mice (Fig. 4a). The delay in wound healing of AT1a/ mice was essentially the same as seen with TCV-116 treatment, which suggests that AT1a is the primary target of the AT1-R antagonist. Immunostaining with CD31 antibody demonstrated that the MVD was significantly lower in AT1a/ mice than in WT mice (Fig. 5b) and RT-PCR analysis revealed reduced CD31 mRNA levels in AT1a/ mice relative to WT mice (Fig. 5c). These results show that wound-induced angiogenesis is promoted by AT1a-R signaling. Immunohistological examination detected a large number of VEGF-expressing cells in the wound granulation tissues of WT mice, whereas much lower levels of expression
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Fig. 4. Delayed wound healing in AT1a receptor knockout mice. (a) Time course of wound healing in AT1a/ and WT mice. Data are shown as means SEM; *p < 0.05 versus WT mice (n ¼ 12 for AT1a/ mice; n ¼ 12 for WT mice); (b) histological appearance of wound tissues in AT1a/ (right) and WT (left) mice on day 3 (H&E staining). W, wound; Gr, granulation tissue. Scale bar ¼ 300 mm.
Fig. 5. Inhibition of wound-induced angiogenesis in AT1a receptor knockout mice. (a) Immunohistochemical staining of CD31 in wound granulation tissues of AT1a/ (right) and WT (left) mice on day 3. Reduced numbers of CD31-positive microvessels (indicated by red arrows) were observed in the granulation tissues of AT1a/ mice compared to WT mice. Scale bar ¼ 100 mm; (b) MVD in granulation tissues on day 3. Data are shown as means SEM; significantly different from WT mice ( p < 0.01); (c) real-time RT-PCR of CD31 mRNA levels in wound granulation tissues of AT1a/ and WT mice on day 3. Significantly different from WT mice ( p < 0.01).
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Fig. 6. Suppression of VEGF expression in AT1a receptor knockout mice. Immunohistological detection of VEGF in wound granulation tissues of AT1a/ and WT mice on day 3. AT1a/ mice exhibit lower numbers of VEGF-expressing cells in comparison to WT mice. Scale bar ¼ 100 mm.
were observed in AT1a/ mice (Fig. 6). These results also suggest that inhibition of angiogenesis, followed by reduced induction of VEGF, contribute to the delay of wound healing in AT1a/ mice. Previously, we demonstrated that primary cultured fibroblasts prepared from granulation tissues could express VEGF mRNA in response to Ang II [16]. Research in rat heart endothelial cells has indicated that protein kinase C may also be involved in Ang II-induced VEGF up-regulation [25]. We have shown that the protein kinase C inhibitors, H7 and GFX, attenuate expression of VEGF in primary fibroblasts. Further, we determined that NF-B and AP-1 are the transcription factors responsible for Ang IImediated induction of VEGF mRNA in fibroblasts. The human VEGF gene promoter has been shown to contain potential binding sites for signal protein-1 (SP-1), AP-1 and AP-2 [26]. The mouse VEGF promoter contains not only binding sites for SP-1, AP-1 and AP-2, but also for NF-B [27]. A link between the activation of AT1a-R and the transcription factors, NF-B and AP-1, may be crucial for active wound healing, since this process is highly dependent upon angiogenesis. Taken together, our findings lead us to conclude that AT1aR signaling enhances VEGF expression and the formation of wound granulation tissues. In addition, AT1-R antagonists are commonly used as therapeutic agents and thus, we consider it important to recognize the possibility that patients undergoing treatment with these products may experience delays in wound healing. Acknowledgments We thank Michiko Ogino, Kyoko Yoshikawa, and Osamu Katsumata for their technical assistance. This work was supported by research grants (#12470529 and #12670094), by a High-tech Research Center grant from Ministry of Education, Culture, Sports, Science and Technology, Japan. This study was also supported by an Integrative Research Program of the Graduate School of Medical Science, Kitasato University. We are also grateful to Dr. Patrick Hughes for linguistic assistance in the preparation of the manuscript.
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