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Angiotensin II type 1 receptor antagonist attenuates lacrimal gland, lung, and liver fibrosis in a murine model of chronic graft-versus-host disease.

Yaguchi S, Ogawa Y, Shimmura S, Kawakita T, Hatou S, Satofuka S, Nakamura S, Imada T, Miyashita H, Yoshida S, Yaguchi T, Ozawa Y, Mori T, Okamoto S, Kawakami Y, Ishida S, Tsubota K - PLoS ONE (2013)

Bottom Line: Tissue RAS is present in the lacrimal gland, lung, and liver, and is known to be involved in the fibrotic pathogenesis of the lung and liver.First, we examined the localization and expression of RAS components in the lacrimal glands using immunohistochemistry and quantitative real-time polymerase chain reaction (PCR).We conclude that RAS is involved in fibrotic pathogenesis in the lacrimal gland and that AT1R antagonist has a therapeutic effect on lacrimal gland, lung, and liver fibrosis in cGVHD model mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan.

ABSTRACT
Chronic graft-versus-host disease (cGVHD), a serious complication following allogeneic HSCT (hematopoietic stem cell transplantation), is characterized by systemic fibrosis. The tissue renin-angiotensin system (RAS) is involved in the fibrotic pathogenesis, and an angiotensin II type 1 receptor (AT1R) antagonist can attenuate fibrosis. Tissue RAS is present in the lacrimal gland, lung, and liver, and is known to be involved in the fibrotic pathogenesis of the lung and liver. This study aimed to determine whether RAS is involved in fibrotic pathogenesis in the lacrimal gland and to assess the effect of an AT1R antagonist on preventing lacrimal gland, lung, and liver fibrosis in cGVHD model mice. We used the B10.D2→BALB/c (H-2(d)) MHC-compatible, multiple minor histocompatibility antigen-mismatched model, which reflects clinical and pathological symptoms of human cGVHD. First, we examined the localization and expression of RAS components in the lacrimal glands using immunohistochemistry and quantitative real-time polymerase chain reaction (PCR). Next, we administered an AT1R antagonist (valsartan; 10 mg/kg) or angiotensin II type 2 receptor (AT2R) antagonist (PD123319; 10 mg/kg) intraperitoneally into cGVHD model mice and assessed the fibrotic change in the lacrimal gland, lung, and liver. We demonstrated that fibroblasts expressed angiotensin II, AT1R, and AT2R, and that the mRNA expression of angiotensinogen was greater in the lacrimal glands of cGVHD model mice than in controls generated by syngeneic-HSCT. The inhibition experiment revealed that fibrosis of the lacrimal gland, lung, and liver was suppressed in mice treated with the AT1R antagonist, but not the AT2R antagonist. We conclude that RAS is involved in fibrotic pathogenesis in the lacrimal gland and that AT1R antagonist has a therapeutic effect on lacrimal gland, lung, and liver fibrosis in cGVHD model mice. Our findings point to AT1R antagonist as a possible target for therapeutic intervention in cGVHD.

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Fibrotic changes in control and cGVHD murine lacrimal glands.A: Light microscopy of cGVHD lacrimal glands showing the infiltration of inflammatory cells around medium-sized interlobular ducts, and a severely fibrotic interstitium that is more intense in the periductal areas. Inset: Infiltrated inflammatory cells (white arrowhead) and interstitial fibroblasts (red arrowhead). B: Collagen deposition was quantified as the ratio of the blue-stained area to the total stained area in Mallory staining and expressed as % fibrotic area. Collagen deposition was more severe in the cGVHD lacrimal glands compared with the controls (p = 0.000005). C: Immunostaining for CD45 and HSP47 in lacrimal glands shows intense brown staining of many cells in the interstitium and periductal areas in the cGVHD lacrimal glands. CD45-positive cells indicate infiltrating inflammatory cells. HSP47-positive cells are spindle-shaped cells with oval nuclei, which indicate fibroblasts (inset). D: The mean densities of CD45+ inflammatory cells and HSP47+ fibroblasts were significantly higher in the cGVHD compared with control lacrimal glands (CD45+ inflammatory cells; p = 0.001, HSP47+ fibroblasts; p = 0.0001). E: Quantitative real-time PCR revealed that the HSP47, collagen type I alpha 1, collagen type I alpha 2, and collagen type III alpha 1 expressions in the cGVHD group were higher than in the control group (HSP47; p = 0.004, collage type I alpha 1; p = 0.0002, collagen type I alpha 2; p = 0.03, collagen type III alpha 1; p = 0.002). Magnification, A: X200, C: X200. Scale bar, A: 50 µm, C: 50 µm. The results represent the mean ± SD. B: n = 9; D: n = 8; E: n = 9 in each group. E: The vertical axis shows the expression ratio of mRNAs. Fold change shows the expression ratio of mRNAs in the control and cGVHD group. The expression level in the control group was defined as 1. *p<0.05, **p<0.01 by Student's t-test.
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pone-0064724-g001: Fibrotic changes in control and cGVHD murine lacrimal glands.A: Light microscopy of cGVHD lacrimal glands showing the infiltration of inflammatory cells around medium-sized interlobular ducts, and a severely fibrotic interstitium that is more intense in the periductal areas. Inset: Infiltrated inflammatory cells (white arrowhead) and interstitial fibroblasts (red arrowhead). B: Collagen deposition was quantified as the ratio of the blue-stained area to the total stained area in Mallory staining and expressed as % fibrotic area. Collagen deposition was more severe in the cGVHD lacrimal glands compared with the controls (p = 0.000005). C: Immunostaining for CD45 and HSP47 in lacrimal glands shows intense brown staining of many cells in the interstitium and periductal areas in the cGVHD lacrimal glands. CD45-positive cells indicate infiltrating inflammatory cells. HSP47-positive cells are spindle-shaped cells with oval nuclei, which indicate fibroblasts (inset). D: The mean densities of CD45+ inflammatory cells and HSP47+ fibroblasts were significantly higher in the cGVHD compared with control lacrimal glands (CD45+ inflammatory cells; p = 0.001, HSP47+ fibroblasts; p = 0.0001). E: Quantitative real-time PCR revealed that the HSP47, collagen type I alpha 1, collagen type I alpha 2, and collagen type III alpha 1 expressions in the cGVHD group were higher than in the control group (HSP47; p = 0.004, collage type I alpha 1; p = 0.0002, collagen type I alpha 2; p = 0.03, collagen type III alpha 1; p = 0.002). Magnification, A: X200, C: X200. Scale bar, A: 50 µm, C: 50 µm. The results represent the mean ± SD. B: n = 9; D: n = 8; E: n = 9 in each group. E: The vertical axis shows the expression ratio of mRNAs. Fold change shows the expression ratio of mRNAs in the control and cGVHD group. The expression level in the control group was defined as 1. *p<0.05, **p<0.01 by Student's t-test.

Mentions: In the lacrimal glands of the cGVHD model mice, hematoxylin-eosin staining showed inflammatory cell infiltration around the medium-sized interlobular ducts. Mallory staining showed a severely fibrotic interstitium in these periductal areas. These pathogenic fibrotic changes were more severe in the cGVHD compared with the control group (Figure 1A). We confirmed that the phenotype of the cGVHD mouse lacrimal gland closely resembles that of clinical samples of patients suffering from cGVHD [4]. The collagen deposition was also more severe in the cGVHD compared with the control lacrimal glands (p = 0.000005) (Figure 1B).


Angiotensin II type 1 receptor antagonist attenuates lacrimal gland, lung, and liver fibrosis in a murine model of chronic graft-versus-host disease.

Yaguchi S, Ogawa Y, Shimmura S, Kawakita T, Hatou S, Satofuka S, Nakamura S, Imada T, Miyashita H, Yoshida S, Yaguchi T, Ozawa Y, Mori T, Okamoto S, Kawakami Y, Ishida S, Tsubota K - PLoS ONE (2013)

Fibrotic changes in control and cGVHD murine lacrimal glands.A: Light microscopy of cGVHD lacrimal glands showing the infiltration of inflammatory cells around medium-sized interlobular ducts, and a severely fibrotic interstitium that is more intense in the periductal areas. Inset: Infiltrated inflammatory cells (white arrowhead) and interstitial fibroblasts (red arrowhead). B: Collagen deposition was quantified as the ratio of the blue-stained area to the total stained area in Mallory staining and expressed as % fibrotic area. Collagen deposition was more severe in the cGVHD lacrimal glands compared with the controls (p = 0.000005). C: Immunostaining for CD45 and HSP47 in lacrimal glands shows intense brown staining of many cells in the interstitium and periductal areas in the cGVHD lacrimal glands. CD45-positive cells indicate infiltrating inflammatory cells. HSP47-positive cells are spindle-shaped cells with oval nuclei, which indicate fibroblasts (inset). D: The mean densities of CD45+ inflammatory cells and HSP47+ fibroblasts were significantly higher in the cGVHD compared with control lacrimal glands (CD45+ inflammatory cells; p = 0.001, HSP47+ fibroblasts; p = 0.0001). E: Quantitative real-time PCR revealed that the HSP47, collagen type I alpha 1, collagen type I alpha 2, and collagen type III alpha 1 expressions in the cGVHD group were higher than in the control group (HSP47; p = 0.004, collage type I alpha 1; p = 0.0002, collagen type I alpha 2; p = 0.03, collagen type III alpha 1; p = 0.002). Magnification, A: X200, C: X200. Scale bar, A: 50 µm, C: 50 µm. The results represent the mean ± SD. B: n = 9; D: n = 8; E: n = 9 in each group. E: The vertical axis shows the expression ratio of mRNAs. Fold change shows the expression ratio of mRNAs in the control and cGVHD group. The expression level in the control group was defined as 1. *p<0.05, **p<0.01 by Student's t-test.
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Related In: Results  -  Collection

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pone-0064724-g001: Fibrotic changes in control and cGVHD murine lacrimal glands.A: Light microscopy of cGVHD lacrimal glands showing the infiltration of inflammatory cells around medium-sized interlobular ducts, and a severely fibrotic interstitium that is more intense in the periductal areas. Inset: Infiltrated inflammatory cells (white arrowhead) and interstitial fibroblasts (red arrowhead). B: Collagen deposition was quantified as the ratio of the blue-stained area to the total stained area in Mallory staining and expressed as % fibrotic area. Collagen deposition was more severe in the cGVHD lacrimal glands compared with the controls (p = 0.000005). C: Immunostaining for CD45 and HSP47 in lacrimal glands shows intense brown staining of many cells in the interstitium and periductal areas in the cGVHD lacrimal glands. CD45-positive cells indicate infiltrating inflammatory cells. HSP47-positive cells are spindle-shaped cells with oval nuclei, which indicate fibroblasts (inset). D: The mean densities of CD45+ inflammatory cells and HSP47+ fibroblasts were significantly higher in the cGVHD compared with control lacrimal glands (CD45+ inflammatory cells; p = 0.001, HSP47+ fibroblasts; p = 0.0001). E: Quantitative real-time PCR revealed that the HSP47, collagen type I alpha 1, collagen type I alpha 2, and collagen type III alpha 1 expressions in the cGVHD group were higher than in the control group (HSP47; p = 0.004, collage type I alpha 1; p = 0.0002, collagen type I alpha 2; p = 0.03, collagen type III alpha 1; p = 0.002). Magnification, A: X200, C: X200. Scale bar, A: 50 µm, C: 50 µm. The results represent the mean ± SD. B: n = 9; D: n = 8; E: n = 9 in each group. E: The vertical axis shows the expression ratio of mRNAs. Fold change shows the expression ratio of mRNAs in the control and cGVHD group. The expression level in the control group was defined as 1. *p<0.05, **p<0.01 by Student's t-test.
Mentions: In the lacrimal glands of the cGVHD model mice, hematoxylin-eosin staining showed inflammatory cell infiltration around the medium-sized interlobular ducts. Mallory staining showed a severely fibrotic interstitium in these periductal areas. These pathogenic fibrotic changes were more severe in the cGVHD compared with the control group (Figure 1A). We confirmed that the phenotype of the cGVHD mouse lacrimal gland closely resembles that of clinical samples of patients suffering from cGVHD [4]. The collagen deposition was also more severe in the cGVHD compared with the control lacrimal glands (p = 0.000005) (Figure 1B).

Bottom Line: Tissue RAS is present in the lacrimal gland, lung, and liver, and is known to be involved in the fibrotic pathogenesis of the lung and liver.First, we examined the localization and expression of RAS components in the lacrimal glands using immunohistochemistry and quantitative real-time polymerase chain reaction (PCR).We conclude that RAS is involved in fibrotic pathogenesis in the lacrimal gland and that AT1R antagonist has a therapeutic effect on lacrimal gland, lung, and liver fibrosis in cGVHD model mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan.

ABSTRACT
Chronic graft-versus-host disease (cGVHD), a serious complication following allogeneic HSCT (hematopoietic stem cell transplantation), is characterized by systemic fibrosis. The tissue renin-angiotensin system (RAS) is involved in the fibrotic pathogenesis, and an angiotensin II type 1 receptor (AT1R) antagonist can attenuate fibrosis. Tissue RAS is present in the lacrimal gland, lung, and liver, and is known to be involved in the fibrotic pathogenesis of the lung and liver. This study aimed to determine whether RAS is involved in fibrotic pathogenesis in the lacrimal gland and to assess the effect of an AT1R antagonist on preventing lacrimal gland, lung, and liver fibrosis in cGVHD model mice. We used the B10.D2→BALB/c (H-2(d)) MHC-compatible, multiple minor histocompatibility antigen-mismatched model, which reflects clinical and pathological symptoms of human cGVHD. First, we examined the localization and expression of RAS components in the lacrimal glands using immunohistochemistry and quantitative real-time polymerase chain reaction (PCR). Next, we administered an AT1R antagonist (valsartan; 10 mg/kg) or angiotensin II type 2 receptor (AT2R) antagonist (PD123319; 10 mg/kg) intraperitoneally into cGVHD model mice and assessed the fibrotic change in the lacrimal gland, lung, and liver. We demonstrated that fibroblasts expressed angiotensin II, AT1R, and AT2R, and that the mRNA expression of angiotensinogen was greater in the lacrimal glands of cGVHD model mice than in controls generated by syngeneic-HSCT. The inhibition experiment revealed that fibrosis of the lacrimal gland, lung, and liver was suppressed in mice treated with the AT1R antagonist, but not the AT2R antagonist. We conclude that RAS is involved in fibrotic pathogenesis in the lacrimal gland and that AT1R antagonist has a therapeutic effect on lacrimal gland, lung, and liver fibrosis in cGVHD model mice. Our findings point to AT1R antagonist as a possible target for therapeutic intervention in cGVHD.

Show MeSH
Related in: MedlinePlus