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Accumulation of Advanced Glycation End Products in Canine Atherosclerosis
J. Comp. Path. 2010, Vol. 143, 65e69
Available online at www.sciencedirect.com
www.elsevier.com/locate/jcpa
SHORT PAPER
Accumulation of Advanced Glycation End Products in Canine Atherosclerosis K. Chiers, V. Vandenberge and R. Ducatelle Laboratory of Veterinary Pathology, Department of Pathology, Bacteriology and Poultry Diseases, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium
Summary Atherosclerosis is an uncommon lesion in animals and particularly in dogs. Prominent atherosclerotic lesions of the coronary arteries are described in three dogs. These comprised an expansion of the tunica media by the accumulation of foam cells and/or cholesterol crystals, with subsequent narrowing of the vascular lumen. Immunohistochemical analysis revealed the accumulation of advanced glycation end products (AGEs) in foam cells, macrophages and lymphocytes. As in man, these findings suggest a possible role of AGEs in the development of canine atherosclerosis. Ó 2009 Elsevier Ltd. All rights reserved. Keywords: advanced glycation end products (AGEs); atherosclerosis; coronary artery; dog; immunohistochemistry
Advanced glycation end products (AGEs) are a heterogeneous and complex group of compounds resulting from glycation and oxidation of proteins, lipids and nucleic acids (Bierhaus et al., 1998). In man, AGEs accumulate during life, but this can be enhanced by hyperglycaemic conditions (McCance et al., 1993), smoking or intake of heated food (Smit and Lutgers, 2004). The accumulation of AGEs may induce several biological effects: (1) AGEs may covalently cross-link with proteins, changing their functional characteristics, (2) AGEs may suppress cellular antioxidant defence mechanisms and allow the release of oxygen radicals, and (3) AGEs can bind to a range of cellular receptors resulting in cellular dysfunction. It is now evident that AGEs play an important role in several pathologies of human organs and tissues including the cardiovascular, renal, orthopaedic, periodontal, ocular and nervous tissue (Smit and Lutgers, 2004); however, little is known about AGE accumulation in the tissues of domestic animals. The present report describes the presence of AGEs in three cases of spontaneously arising canine atherosclerosis. The first case was an 11-year-old neutered male standard poodle presented with paralysis of the left Correspondence to: K. Chiers (e-mail: [email protected]). 0021-9975/$ - see front matter doi:10.1016/j.jcpa.2009.12.006
hindlimb with gradual involvement of the right hindlimb. Neurological examination revealed asymmetrical paraparesis. Both hindlimbs were cold with absent arterial pulse. Ultrasonographic examination of the heart and abdomen revealed a mottled appearance to the myocardium and the presence of a linear accumulation of hyperechoic material along the luminal surface of the aortic wall at the level of the aortic bifurcation. A diagnosis of ischaemic neuromyopathy caused by a saddle (aorticeiliac) thrombus was suggested. The dog was treated with aspirin, morphine, broad-spectrum antibiotics and fluid therapy (Hartmann’s solution), but died suddenly within 2 days of initiation of treatment. Case 2 was a 10-year-old intact male schnauzer referred to a private clinic for surgical correction of a ruptured right cruciate ligament. The animal died unexpectedly during anaesthesia. The third case was a 12-year-old intact male toy poodle presented with acute onset blindness, anorexia, dullness and right-sided hemiparesis. The dog was treated with prednisolone and non-steroidal anti-inflammatory drugs. Magnetic resonance imaging of the brain was performed and a neoplasm in the left cerebral hemisphere was suspected. The dog was humanely destroyed. Ó 2009 Elsevier Ltd. All rights reserved.
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Fig. 1. Heart from case 1 showing severe atherosclerosis of coronary arteries.
The three dogs were subject to necropsy examination and samples from gross lesions were fixed in 10% neutral buffered formalin, embedded in paraffin wax, sectioned (4 mm) and stained by haematoxylin and eosin (HE), Von Kossa’s method and Van Gieson’s stain. Immunohistochemistry (IHC) was undertaken to assess expression of smooth muscle actin (mouse anti-human smooth muscle actin; Dako, Glostrup, Denmark), CD3 (rabbit anti-human CD3; Dako), CD20 (rabbit anti-human CD20; Dako), MAC387 (mouse anti-human MAC387;
Abcam, Cambridge, UK) and elastin (IgG1 mouse anti-bovine elastin; Novocastra Laboratories, Newcastle upon Tyne, UK). AGEs were demonstrated using monoclonal mouse antibody 6D12 (IgG1; Cosmo Bio, Tokyo, Japan) with subsequent incubation of the linking reagent (Envision Link + rabbit anti-mouse IgG, Dako) and 3,30 -diaminobenzidine. The elastin immunolabelling served as an isotype-matched control for the immunohistochemical procedure. Coronary samples from three age-matched dogs without arteriosclerosis were included. In all three affected dogs, coronary arteries and their branches were prominent, cord-like and had a thickened, firm wall (Fig. 1), often with severe narrowing of the lumen. Microscopically, the lesions consisted of thickening of the tunica media due to accumulation of foam cells, cholesterol crystals, mineralization and intra- and extracellular lipids as well as mild mononuclear infiltration (Fig. 2a). In some lesions there was fibrinoid degeneration (Fig. 3). Van Gieson’s staining revealed the presence of collagen in the subintimal region of some large arteries (Fig. 4a). The inflammatory infiltrate consisted of moderate numbers of CD20+ B lymphocytes and CD3+ T lymphocytes, as well as many reactive macrophages (Figs. 5e7). IHC for smooth muscle actin and elastin expression demonstrated the disruption of the tunica media by the infiltration of the foam cells and cholesterol crystals and the loss of integrity of the lamina elastica interna and/or externa in some arteries (Figs. 8 and 9). AGEs were demonstrated immunohistochemically in all atherosclerotic lesions. The presence of AGEs was mainly restricted to foam cells, surrounding cholesterol crystals and extracellular lipids as well as to macrophages and T and B lymphocytes (Fig. 10). Athererosclerotic lesions were not present and AGEs were not detected in the coronary arteries of age-matched control dogs (Figs. 2b and 4b). In cases 1 and 3, atherosclerotic lesions, with similar morphology, were also observed in the abdominal
Fig. 2. (a) Coronary artery from case 2 showing severe atherosclerotic lesion with dominance of foam cells, cholesterol clefts and narrowing of lumen. HE. Bar, 250 mm. (b) Coronary artery of an age-matched normal dog. HE. Bar, 500 mm.
Glycation End Products in Canine Atherosclerosis
Fig. 3. Coronary artery from case 3 showing severe atherosclerosis of the coronary artery with foam cells ([), cholesterol clefts (=) and fibrinoid necrosis (*). HE. Bar, 100 mm.
aorta. A saddle thrombus was present in the distal abdominal aorta extending into the right iliac artery in dog 1. The thrombus was predominantly composed of fibrin, red blood cells and neutrophils. Focal left ventricular myocardial fibrosis was present in case 2. The third dog had focal haemorrhage (2.5 cm diameter) in the left cranial cerebrum. Cerebrovascular atherosclerosis was not observed. In the dogs of the present study, the deposition of AGEs in atherosclerotic lesions was similar to that described in man (Nakamura et al., 1993). This finding may suggest that AGE formation may also contribute to the development of atherosclerosis in dogs. In man, it is commonly accepted that accumulation of AGEs accelerates the development of atherosclerosis. This may be explained by considering the complex effects of AGEs, which include: (1) effects on the extracellular matrix such as collagen crosslinking, enhanced matrix production, quenching of nitric oxide and endothelial trapping of low density lipoproteins (LDLs) and immunoglobulin (Ig) G,
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(2) effects on lipoproteins including reduced LDL receptor recognition and increased susceptibility of LDLs to oxidation, (3) enhanced release of inflammatory cytokines and growth factors by mononuclear phagocytes, (4) proliferation and production of fibronectin by smooth muscle cells, and (5) modulation of vascular tone and expression of adhesion molecules and chemokines by vascular endothelial cells (Basta et al., 2004). AGEs induce expression of two important oxidized LDL (OxLDL) receptors: macrophage scavenger receptors class A and CD36. The increased expression of these receptors leads to enhanced uptake of OxLDLs, resulting in the transformation of foam cells (Goldin et al., 2006). Foam cells were abundant in all advanced canine atherosclerotic lesions and AGEs were most prominent in the lipid core of these lesions, which may suggest an important role for AGEs in the transformation of macrophages into foam cells. Atherosclerosis in dogs is rare and almost always associated with hypothyroidism or diabetes mellitus (Hess et al., 2003). The pathogenesis of atherosclerosis in hypothyroidism is not characterized, but the association is recognized in hypothyroid rats, in which levels of glycation and oxidation products were decreased (Pamplona et al., 1999). In contrast, Nanda et al. (2008) found that lipid peroxidation was pronounced in hyperlipidaemic hypothyroid patients. Those authors proposed that increased lipid peroxidation enhances the process of protein glycation in these patients. However, in euthyroid dogs, deprivation of essential fatty acids also induces atherosclerotic lesions (McCullagh et al., 1976), which is believed to be due to changes in cholesterol and lipoprotein metabolism. It has recently been shown that small-dense LDLs are more susceptible to glycation and therefore may contribute to atherosclerosis (Younis et al., 2009). Accelerated atherosclerosis is one of the major complications in diabetic people. It is now evident that hyperglycaemia and oxidant stress contribute to increased
Fig. 4. (a) Coronary artery from case 2 showing mild deposition of collagen in the atherosclerotic lesion. Van Gieson’s stain. Bar, 250 mm. (b) Coronary artery of an age-matched normal dog. Van Gieson’s stain. Bar, 500 mm.
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Fig. 5. Coronary artery from case 3 showing numerous MAC387+ reactive macrophages in an atherosclerotic lesion. IHC. Bar, 100 mm.
Fig. 8. Coronary artery from case 3 labelled for elastin expression and showing loss of integrity of the lamina elastic interna and externa in an atherosclerotic lesion. IHC. Bar, 100 mm.
Fig. 6. Coronary artery from case 3 showing low numbers of CD3+ T lymphocytes infiltrating an atherosclerotic lesion. IHC. Bar, 100 mm.
Fig. 9. Coronary artery from case 3 labelled for smooth muscle actin expression and showing disruption of the tunica media by infiltrating foam cells in an atherosclerotic lesion. IHC. Bar, 100 mm.
Fig. 7. Coronary artery from case 3 showing low numbers of CD20+ B lymphocytes infiltrating an atherosclerotic lesion. IHC. Bar, 100 mm.
Fig. 10. Coronary artery from case 3 showing foam cells (*), macrophages (=) and lymphocytes ([) with strong cytoplasmic expression of AGEs. IHC. Bar, 50 mm.
Glycation End Products in Canine Atherosclerosis
formation of AGEs (Basta et al., 2004). In the present study, the dogs were examined for cause of death and/ or presenting clinical signs, so histological examination or ancillary assays for demonstration of hypothyroidism or diabetes were not performed. However, none of the animals had clinical signs or gross lesions indicative of these diseases. It is therefore not possible to define any underlying causes of atherosclerosis in these cases. Nevertheless, atherosclerosis can occur without the background of an endocrinopathy (Kagawa et al., 1998; Sako et al., 2003). Other risk factors for the development of atherosclerosis in dogs include obesity, male gender and advanced age (Kagawa et al., 1998). All dogs in the present study were male and aged greater than 10 years, but only one dog was considered overweight. This is the first study demonstrating AGE deposition in natural cases of canine atherosclerosis and the findings suggest a possible role for AGEs in the development of these lesions.
Acknowledgments We are grateful to C. Puttevils, D. Ameye and S. Loomans for assistance with the samples and performance of the histochemical stains and IHC.
References Basta G, Schmidt AM, De Caterina R (2004) Advanced glycation end products and vascular inflammation: implications for accelerated atherosclerosis in diabetes. Cardiovascular Research, 63, 582e592. Bierhaus A, Hofmann MA, Ziegler R, Nawroth PP (1998) AGEs and their interaction with AGE-receptors in vascular disease and diabetes mellitus. I. The age concept. Cardiovascular Research, 37, 586e600. Goldin A, Beckman JA, Schmidt AM, Creager MA (2006) Advanced glycation end products: sparking the development of diabetic vascular injury. Circulation, 114, 597e605. Hess RS, Kass PH, Van Winkle TJ (2003) Association between diabetes mellitus, hypothyroidism or hyperadrenocorticism, and atherosclerosis in dogs. Journal of Veterinary Internal Medicine, 17, 489e494.
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Kagawa Y, Hirayama K, Uchida E, Izumisawa Y, Yamaguchi M et al. (1998) Systemic atherosclerosis in dogs: histopathological and immunohistochemical studies of atherosclerotic lesions. Journal of Comparative Pathology, 118, 195e206. McCance DR, Dyer DG, Dunn JA, Bailie KE, Thorpe SR et al. (1993) Maillard reaction products and their relation to complications in insulin-dependent diabetes mellitus. Journal of Clinical Investigation, 91, 2470e2478. McCullagh KG, Ehrhart LA, Butkus A (1976) Experimental canine atherosclerosis and its prevention e dietary induction of severe coronary, cerebral, aortic, and iliac atherosclerosis and its prevention by safflower oil. Laboratory Investigation, 34, 394e405. Nakamura Y, Horii Y, Nishino T, Shiiki H, Sakaguchi Y et al. (1993) Immunohistochemical localization of advanced glycosylation end products in coronary atheroma and cardiac tissue in diabetes mellitus. American Journal of Pathology, 143, 1649e1656. Nanda N, Bobby Z, Hamide A (2008) Oxidative stress and protein glycation in primary hypothyroidism. Male/ female difference. Clinical and Experimental Medicine, 8, 101e108. Pamplona R, Portero-Otin M, Ruiz C, Bellmunt MJ, Requena JR et al. (1999) Thyroid status modulates glycoxidative and lipoxidative modification of tissue proteins. Free Radical Biology and Medicine, 27, 901e910. Sako T, Uchida E, Kagawa Y, Hirayama K, Nakade T et al. (2003) Immunohistochemical detection of apolipoprotein A-I and B-100 in canine atherosclerotic lesions. Veterinary Pathology, 40, 328e331. Smit AW, Lutgers HL (2004) The clinical relevance of advanced glycation end products (AGE) and recent developments in pharmaceutics to reduce AGE accumulation. Current Medicinal Chemistry, 11, 2767e2784. Younis N, Charlton-Menys V, Sharma R, Soran H, Durrington PN (2009) Glycation of LDL in nondiabetic people: small dense LDL is preferentially glycated both in vivo and in vitro. Atherosclerosis, 202, 162e168.
June 26th, 2009 ½ Received, Accepted, December 16th, 2009
Available online at www.sciencedirect.com
www.elsevier.com/locate/jcpa
SHORT PAPER
Accumulation of Advanced Glycation End Products in Canine Atherosclerosis K. Chiers, V. Vandenberge and R. Ducatelle Laboratory of Veterinary Pathology, Department of Pathology, Bacteriology and Poultry Diseases, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium
Summary Atherosclerosis is an uncommon lesion in animals and particularly in dogs. Prominent atherosclerotic lesions of the coronary arteries are described in three dogs. These comprised an expansion of the tunica media by the accumulation of foam cells and/or cholesterol crystals, with subsequent narrowing of the vascular lumen. Immunohistochemical analysis revealed the accumulation of advanced glycation end products (AGEs) in foam cells, macrophages and lymphocytes. As in man, these findings suggest a possible role of AGEs in the development of canine atherosclerosis. Ó 2009 Elsevier Ltd. All rights reserved. Keywords: advanced glycation end products (AGEs); atherosclerosis; coronary artery; dog; immunohistochemistry
Advanced glycation end products (AGEs) are a heterogeneous and complex group of compounds resulting from glycation and oxidation of proteins, lipids and nucleic acids (Bierhaus et al., 1998). In man, AGEs accumulate during life, but this can be enhanced by hyperglycaemic conditions (McCance et al., 1993), smoking or intake of heated food (Smit and Lutgers, 2004). The accumulation of AGEs may induce several biological effects: (1) AGEs may covalently cross-link with proteins, changing their functional characteristics, (2) AGEs may suppress cellular antioxidant defence mechanisms and allow the release of oxygen radicals, and (3) AGEs can bind to a range of cellular receptors resulting in cellular dysfunction. It is now evident that AGEs play an important role in several pathologies of human organs and tissues including the cardiovascular, renal, orthopaedic, periodontal, ocular and nervous tissue (Smit and Lutgers, 2004); however, little is known about AGE accumulation in the tissues of domestic animals. The present report describes the presence of AGEs in three cases of spontaneously arising canine atherosclerosis. The first case was an 11-year-old neutered male standard poodle presented with paralysis of the left Correspondence to: K. Chiers (e-mail: [email protected]). 0021-9975/$ - see front matter doi:10.1016/j.jcpa.2009.12.006
hindlimb with gradual involvement of the right hindlimb. Neurological examination revealed asymmetrical paraparesis. Both hindlimbs were cold with absent arterial pulse. Ultrasonographic examination of the heart and abdomen revealed a mottled appearance to the myocardium and the presence of a linear accumulation of hyperechoic material along the luminal surface of the aortic wall at the level of the aortic bifurcation. A diagnosis of ischaemic neuromyopathy caused by a saddle (aorticeiliac) thrombus was suggested. The dog was treated with aspirin, morphine, broad-spectrum antibiotics and fluid therapy (Hartmann’s solution), but died suddenly within 2 days of initiation of treatment. Case 2 was a 10-year-old intact male schnauzer referred to a private clinic for surgical correction of a ruptured right cruciate ligament. The animal died unexpectedly during anaesthesia. The third case was a 12-year-old intact male toy poodle presented with acute onset blindness, anorexia, dullness and right-sided hemiparesis. The dog was treated with prednisolone and non-steroidal anti-inflammatory drugs. Magnetic resonance imaging of the brain was performed and a neoplasm in the left cerebral hemisphere was suspected. The dog was humanely destroyed. Ó 2009 Elsevier Ltd. All rights reserved.
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Fig. 1. Heart from case 1 showing severe atherosclerosis of coronary arteries.
The three dogs were subject to necropsy examination and samples from gross lesions were fixed in 10% neutral buffered formalin, embedded in paraffin wax, sectioned (4 mm) and stained by haematoxylin and eosin (HE), Von Kossa’s method and Van Gieson’s stain. Immunohistochemistry (IHC) was undertaken to assess expression of smooth muscle actin (mouse anti-human smooth muscle actin; Dako, Glostrup, Denmark), CD3 (rabbit anti-human CD3; Dako), CD20 (rabbit anti-human CD20; Dako), MAC387 (mouse anti-human MAC387;
Abcam, Cambridge, UK) and elastin (IgG1 mouse anti-bovine elastin; Novocastra Laboratories, Newcastle upon Tyne, UK). AGEs were demonstrated using monoclonal mouse antibody 6D12 (IgG1; Cosmo Bio, Tokyo, Japan) with subsequent incubation of the linking reagent (Envision Link + rabbit anti-mouse IgG, Dako) and 3,30 -diaminobenzidine. The elastin immunolabelling served as an isotype-matched control for the immunohistochemical procedure. Coronary samples from three age-matched dogs without arteriosclerosis were included. In all three affected dogs, coronary arteries and their branches were prominent, cord-like and had a thickened, firm wall (Fig. 1), often with severe narrowing of the lumen. Microscopically, the lesions consisted of thickening of the tunica media due to accumulation of foam cells, cholesterol crystals, mineralization and intra- and extracellular lipids as well as mild mononuclear infiltration (Fig. 2a). In some lesions there was fibrinoid degeneration (Fig. 3). Van Gieson’s staining revealed the presence of collagen in the subintimal region of some large arteries (Fig. 4a). The inflammatory infiltrate consisted of moderate numbers of CD20+ B lymphocytes and CD3+ T lymphocytes, as well as many reactive macrophages (Figs. 5e7). IHC for smooth muscle actin and elastin expression demonstrated the disruption of the tunica media by the infiltration of the foam cells and cholesterol crystals and the loss of integrity of the lamina elastica interna and/or externa in some arteries (Figs. 8 and 9). AGEs were demonstrated immunohistochemically in all atherosclerotic lesions. The presence of AGEs was mainly restricted to foam cells, surrounding cholesterol crystals and extracellular lipids as well as to macrophages and T and B lymphocytes (Fig. 10). Athererosclerotic lesions were not present and AGEs were not detected in the coronary arteries of age-matched control dogs (Figs. 2b and 4b). In cases 1 and 3, atherosclerotic lesions, with similar morphology, were also observed in the abdominal
Fig. 2. (a) Coronary artery from case 2 showing severe atherosclerotic lesion with dominance of foam cells, cholesterol clefts and narrowing of lumen. HE. Bar, 250 mm. (b) Coronary artery of an age-matched normal dog. HE. Bar, 500 mm.
Glycation End Products in Canine Atherosclerosis
Fig. 3. Coronary artery from case 3 showing severe atherosclerosis of the coronary artery with foam cells ([), cholesterol clefts (=) and fibrinoid necrosis (*). HE. Bar, 100 mm.
aorta. A saddle thrombus was present in the distal abdominal aorta extending into the right iliac artery in dog 1. The thrombus was predominantly composed of fibrin, red blood cells and neutrophils. Focal left ventricular myocardial fibrosis was present in case 2. The third dog had focal haemorrhage (2.5 cm diameter) in the left cranial cerebrum. Cerebrovascular atherosclerosis was not observed. In the dogs of the present study, the deposition of AGEs in atherosclerotic lesions was similar to that described in man (Nakamura et al., 1993). This finding may suggest that AGE formation may also contribute to the development of atherosclerosis in dogs. In man, it is commonly accepted that accumulation of AGEs accelerates the development of atherosclerosis. This may be explained by considering the complex effects of AGEs, which include: (1) effects on the extracellular matrix such as collagen crosslinking, enhanced matrix production, quenching of nitric oxide and endothelial trapping of low density lipoproteins (LDLs) and immunoglobulin (Ig) G,
67
(2) effects on lipoproteins including reduced LDL receptor recognition and increased susceptibility of LDLs to oxidation, (3) enhanced release of inflammatory cytokines and growth factors by mononuclear phagocytes, (4) proliferation and production of fibronectin by smooth muscle cells, and (5) modulation of vascular tone and expression of adhesion molecules and chemokines by vascular endothelial cells (Basta et al., 2004). AGEs induce expression of two important oxidized LDL (OxLDL) receptors: macrophage scavenger receptors class A and CD36. The increased expression of these receptors leads to enhanced uptake of OxLDLs, resulting in the transformation of foam cells (Goldin et al., 2006). Foam cells were abundant in all advanced canine atherosclerotic lesions and AGEs were most prominent in the lipid core of these lesions, which may suggest an important role for AGEs in the transformation of macrophages into foam cells. Atherosclerosis in dogs is rare and almost always associated with hypothyroidism or diabetes mellitus (Hess et al., 2003). The pathogenesis of atherosclerosis in hypothyroidism is not characterized, but the association is recognized in hypothyroid rats, in which levels of glycation and oxidation products were decreased (Pamplona et al., 1999). In contrast, Nanda et al. (2008) found that lipid peroxidation was pronounced in hyperlipidaemic hypothyroid patients. Those authors proposed that increased lipid peroxidation enhances the process of protein glycation in these patients. However, in euthyroid dogs, deprivation of essential fatty acids also induces atherosclerotic lesions (McCullagh et al., 1976), which is believed to be due to changes in cholesterol and lipoprotein metabolism. It has recently been shown that small-dense LDLs are more susceptible to glycation and therefore may contribute to atherosclerosis (Younis et al., 2009). Accelerated atherosclerosis is one of the major complications in diabetic people. It is now evident that hyperglycaemia and oxidant stress contribute to increased
Fig. 4. (a) Coronary artery from case 2 showing mild deposition of collagen in the atherosclerotic lesion. Van Gieson’s stain. Bar, 250 mm. (b) Coronary artery of an age-matched normal dog. Van Gieson’s stain. Bar, 500 mm.
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Fig. 5. Coronary artery from case 3 showing numerous MAC387+ reactive macrophages in an atherosclerotic lesion. IHC. Bar, 100 mm.
Fig. 8. Coronary artery from case 3 labelled for elastin expression and showing loss of integrity of the lamina elastic interna and externa in an atherosclerotic lesion. IHC. Bar, 100 mm.
Fig. 6. Coronary artery from case 3 showing low numbers of CD3+ T lymphocytes infiltrating an atherosclerotic lesion. IHC. Bar, 100 mm.
Fig. 9. Coronary artery from case 3 labelled for smooth muscle actin expression and showing disruption of the tunica media by infiltrating foam cells in an atherosclerotic lesion. IHC. Bar, 100 mm.
Fig. 7. Coronary artery from case 3 showing low numbers of CD20+ B lymphocytes infiltrating an atherosclerotic lesion. IHC. Bar, 100 mm.
Fig. 10. Coronary artery from case 3 showing foam cells (*), macrophages (=) and lymphocytes ([) with strong cytoplasmic expression of AGEs. IHC. Bar, 50 mm.
Glycation End Products in Canine Atherosclerosis
formation of AGEs (Basta et al., 2004). In the present study, the dogs were examined for cause of death and/ or presenting clinical signs, so histological examination or ancillary assays for demonstration of hypothyroidism or diabetes were not performed. However, none of the animals had clinical signs or gross lesions indicative of these diseases. It is therefore not possible to define any underlying causes of atherosclerosis in these cases. Nevertheless, atherosclerosis can occur without the background of an endocrinopathy (Kagawa et al., 1998; Sako et al., 2003). Other risk factors for the development of atherosclerosis in dogs include obesity, male gender and advanced age (Kagawa et al., 1998). All dogs in the present study were male and aged greater than 10 years, but only one dog was considered overweight. This is the first study demonstrating AGE deposition in natural cases of canine atherosclerosis and the findings suggest a possible role for AGEs in the development of these lesions.
Acknowledgments We are grateful to C. Puttevils, D. Ameye and S. Loomans for assistance with the samples and performance of the histochemical stains and IHC.
References Basta G, Schmidt AM, De Caterina R (2004) Advanced glycation end products and vascular inflammation: implications for accelerated atherosclerosis in diabetes. Cardiovascular Research, 63, 582e592. Bierhaus A, Hofmann MA, Ziegler R, Nawroth PP (1998) AGEs and their interaction with AGE-receptors in vascular disease and diabetes mellitus. I. The age concept. Cardiovascular Research, 37, 586e600. Goldin A, Beckman JA, Schmidt AM, Creager MA (2006) Advanced glycation end products: sparking the development of diabetic vascular injury. Circulation, 114, 597e605. Hess RS, Kass PH, Van Winkle TJ (2003) Association between diabetes mellitus, hypothyroidism or hyperadrenocorticism, and atherosclerosis in dogs. Journal of Veterinary Internal Medicine, 17, 489e494.
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Kagawa Y, Hirayama K, Uchida E, Izumisawa Y, Yamaguchi M et al. (1998) Systemic atherosclerosis in dogs: histopathological and immunohistochemical studies of atherosclerotic lesions. Journal of Comparative Pathology, 118, 195e206. McCance DR, Dyer DG, Dunn JA, Bailie KE, Thorpe SR et al. (1993) Maillard reaction products and their relation to complications in insulin-dependent diabetes mellitus. Journal of Clinical Investigation, 91, 2470e2478. McCullagh KG, Ehrhart LA, Butkus A (1976) Experimental canine atherosclerosis and its prevention e dietary induction of severe coronary, cerebral, aortic, and iliac atherosclerosis and its prevention by safflower oil. Laboratory Investigation, 34, 394e405. Nakamura Y, Horii Y, Nishino T, Shiiki H, Sakaguchi Y et al. (1993) Immunohistochemical localization of advanced glycosylation end products in coronary atheroma and cardiac tissue in diabetes mellitus. American Journal of Pathology, 143, 1649e1656. Nanda N, Bobby Z, Hamide A (2008) Oxidative stress and protein glycation in primary hypothyroidism. Male/ female difference. Clinical and Experimental Medicine, 8, 101e108. Pamplona R, Portero-Otin M, Ruiz C, Bellmunt MJ, Requena JR et al. (1999) Thyroid status modulates glycoxidative and lipoxidative modification of tissue proteins. Free Radical Biology and Medicine, 27, 901e910. Sako T, Uchida E, Kagawa Y, Hirayama K, Nakade T et al. (2003) Immunohistochemical detection of apolipoprotein A-I and B-100 in canine atherosclerotic lesions. Veterinary Pathology, 40, 328e331. Smit AW, Lutgers HL (2004) The clinical relevance of advanced glycation end products (AGE) and recent developments in pharmaceutics to reduce AGE accumulation. Current Medicinal Chemistry, 11, 2767e2784. Younis N, Charlton-Menys V, Sharma R, Soran H, Durrington PN (2009) Glycation of LDL in nondiabetic people: small dense LDL is preferentially glycated both in vivo and in vitro. Atherosclerosis, 202, 162e168.
June 26th, 2009 ½ Received, Accepted, December 16th, 2009