Amino Acids DOI 10.1007/s00726-017-2503-5
REVIEW ARTICLE
Hyperhomocysteinemia and cardiovascular disease in animal model Md. Abul Kalam Azad1,2 · Pan Huang1 · Gang Liu1,3 · Wenkai Ren1 · Tsegay Teklebrh1,2,4 · Wenxin Yan1,3 · Xihong Zhou1 · Yulong Yin1,3,5
Received: 20 July 2017 / Accepted: 4 October 2017 © Springer-Verlag GmbH Austria 2017
Abstract Hyperhomocysteinemia is an independent risk factor for cardiovascular disease and is associated with primary causes of mortality and morbidity throughout the world. Several studies have been carried out to evaluate the effects of a diet inducing cystathionine-β-synthase, methyltetrafolate, folic acid, and vitamin B supplemented with methionine on the homocysteine metabolism and in lowering the plasma total homocysteine levels. A large number of molecular and biomedical studies in numerous animals, such as mice, rabbits, and pigs, have sought to elevate the plasma total homocysteine levels and to identify a disease model for human hyperhomocysteinemia. However, a specific animal model is not suitable for hyperhomocysteinemia in terms of all aspects of cardiovascular disease. In this review article, the experimental progress of animal models with plasma total homocysteine levels is examined to identify a Handling Editor: J. D. Wade. * Gang Liu [email protected] 1
Key Laboratory of Agro‑ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, National Engineering Laboratory for Pollution Control and Waste Utilization in Livestock and Poultry Production, Changsha 410125, Hunan, China
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University of Chinese Academy of Sciences, Beijing 100049, China
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Taoyuan Agro‑ecosystem Research Station, Soil Molecular Ecology Section, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125, China
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School of Animal and Range Sciences, Haramaya University, 251 Haramaya, Dire Dawa, Ethiopia
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Animal Nutrition and Human Health Laboratory, School of Life Sciences, Hunan Normal University, Changsha 410081, Hunan, China
feasible animal model of hyperhomocysteinemia for different aspects. Keywords Hyperhomocysteinemia · Homocysteine · Animal model · Cardiovascular disease Abbreviations HC Homocysteine CVD Cardiovascular disease ATP Adenosine triphosphate MTHFR Methylenetetrahydrofolate reductase CBS Cystathionine-β-synthase THF Tetrahydrofolate NO Nitric oxide O2 Oxygen SAM Sulfur adenosylmethionine SAH S-Adenosylhomocysteine MAT Methionine adenosyltransferase MT Methyltransferase MS Methionine synthase
Introduction Homocysteine (HC) is a naturally occurring amino acid and a by-product of methionine metabolism, which is a risk factor for cardiovascular disease (CVD) when present in high levels in blood. HC levels are carefully regulated in pathways related to methionine ingestion and metabolism. Chronic renal failure is a morbid condition with high mortality rates and a prevalence of both hyperhomocysteinemia and CVD. CVD, predominantly coronary heart disease, is the major cause of morbidity and mortality worldwide. According to the World Health Organization, CVD was
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the most common underlying cause of death in 2012, accounting for an estimated 17.5 million (82% uncertainty interval) of 56 million total deaths (Global status report on noncommunicable diseases 2014). In recent years, the treatment of CVD has seen great advances through animal experiments. A huge amount of information has been generated with disease models in preclinical research and has outlined the pathogenesis, progression, and underlying mechanism of CVD at both molecular and cellular levels (Liu et al. 2017). Moreover, disease models have a vital role in the progress of various effective treatment strategies. HC treatment in CVD models has been developed in many animal species, including in murine (small animals) such as mice and rats (Ables et al. 2015; Glowacki et al. 2010; Zidan and Elnegris 2015), and in large animals, such as rabbits, swine, and dogs (Zhang et al. 2014; Sipahioglu et al. 2005; Leong et al. 2015). According to Cohen et al. (1994), small animal models are more acceptable for molecular research because they are inexpensive, and easy to handle and have a huge amount of available research literature. In this study, the progress made with various animal models with hyperhomocysteinemia is discussed to identify a feasible model for hyperhomocyseteinemia related to CVD.
Fig. 1 Homocysteine formation and metabolism (MAT methionine adenosyltransferase, MT methyltransferase, SAHH SAH hydrolase, MS methionine synthase, MTHFR 5, 10 methyltetrahydrofolate reductase, CBS cystathionine-β-synthase, THF tetrahydrofolate, SAM S-adenosylmethionine, SAH S-adenosylhomocysteine)
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HC production and metabolism A sulfur amino acid, HC, is synthesized from methionine via multi-step processes (Fig. 1). In the first step, methionine receives an adenosine group from ATP and then transforms to S-adenosylmethionine (SAM) by S-adenosylmethionine synthase (MAT). In the next step, S-adenosylhomocysteine (SAH) is produced from SAM by releasing methyl group acceptor molecules, which is catalyzed by methyltransferase (MT). Finally, SAM is hydrolyzed by S-adenosylhomocysteine hydrolase (SAHH) to yield the HC (Katko et al. 2012). HC is mainly metabolized via two key pathways (Fig. 1). When there is excess methionine, HC is metabolized via the sulfuration pathway by releasing cystathionine while vitamin B acts as a cofactor and is then converted to cysteine. Excess cysteine is oxidized to sulfates or taurine or is drained from the body via urine. Whenever the methionine concentrations are low, HC is metabolized via the methionine-conserving pathway. In most tissues, methyltetrahydrofolate and vitamin B12 play their roles as cofactors during the process of remethylation of HC to methionine. These two metabolism pathways are linked by SAM, which is the most vital source of the methyl group for all methylation reactions within the cell (Selhub 1999). Undoubtedly, it can be mentioned that high levels of HC are associated with reduced methylation potential, whereas folate and vitamin B 12 increase this methylation potential. The amount of dietary intake of methionine can
Hyperhomocysteinemia and cardiovascular disease in animal model
change the methionine concentration in the body and affect the rate of SAM synthesis and the metabolism of HC.
HC and CVD Hyperhomocysteinemia is defined as a medical condition that is characterized by an abnormal HC concentration in the blood. An HC concentration of 16 µmol/L in blood is termed mild, 16–30 µmol/L is considered intermediate, and greater than 100 µmol/L is known as hyperhomocysteinemia (Kang et al. 1992). In non-treated homocysteinuria, HC levels from 12 to 16 µmol/L are risk factors for CVD (David et al. 2002). Several meta-analyses have shown that the HC level in blood is the most important risk factor for CVD and stroke (David et al. 2002; Li et al. 2016; Clarke et al. 2012; Humphrey et al. 2008; Homocysteine Studies Collaboration 2002; Klerk et al. 2002). Observational studies have shown that the risk of ischemic heart disease and the risk of stroke increases by 32 and 59%, respectively, with each 5 µmol/L increase in serum HC levels (David et al. 2002). Several researchers have reported that methylenetetrahydrofolate reductase (MTHFR) enzyme is involved in HC metabolism and plays an important role in CVD. In contrast, the wildtype allele (CC) of MTHFR and the mutant allele (TT) had 25% higher HC concentrations and a 16% higher risk of CVD (Klerk et al. 2002). It was recently reported that the elevation of HC levels in blood serum reduces hyperhomocysteinemia and the risk of CVD (Clarke et al. 2010, 2012; Nandi and Mishra 2017; Wierzbicki 2016; Catena et al. 2014, 2015). Some studies have shown that folic acid, vitamin B, and vitamin B12 supplementation is inexpensive and effective at lowering the blood HC concentration (Li et al. 2016; Homocysteine Lowering Trialists’ Collaboration 1998; Marcus and Menon 2007; Clarke et al. 2010). In contrast, cystathionine β-synthase (CBS) and cystathionine gamma lyase enzymes play important roles in the pathway of decreasing HC levels to hydrogen sulfide (H2S) (Chang et al. 2008; Katko et al. 2012; Kamat et al. 2016; Nandi and Mishra 2017).
Animal model Epidemiological and experimental evidence indicates that hyperhomocysteinemia is highly related to an increased risk of vascular disease, which arises from unexpected HC metabolism. Severe hyperhomocysteinemia occurs due to rarer genetic defects and deficiencies in cystathionineβ-synthase, methylene THF reductase, or other enzymes involved in methyl-B12 synthesis and HC methylation. A lower level of hyperhomocysteinemia was found in fasting conditions because the methylation pathway suffered less
impairment (i.e., folate or B 12 deficiencies or methylene THF reductase thermolability). In addition, post-methionine loading may occur due to a heterozygous cystathionine-βsynthase defect or B6 defect (Selhub 1999).
Murine model of CVD Murine models are often used in CVD research because they have low maintenance costs and short gestation times, are easy to handle, allow genetic manipulation to generate transgenic strains, and are more suitable for “high-throughput” studies than large animal models (Recchia and Lionetti 2007). Considering these characteristics, small rodent models are mostly used for studies of cardiac physiology and disease, genetics, pharmacology, and long-term survival (Elnakish et al. 2012).
Murine model of total plasma HC levels In terms of pharmacological or pathological approaches, murine models can be used in various sectors, including dietary supplementation, genetic approaches, dietary modification, and genetic intervention with the purpose of lowering the total plasma HC levels of hyperhomocysteinemia. CBS is a genetic hyperhomocysteinemia model with gene deletion that has been used in studies of HC pathology in catalyzing HC to cystathionine. It has been found that the HC plasma levels in homozygous CBS-deficient mice (CBS−/−) are 50 times higher than those in wild-type mice and similar to those in human hyperhomocysteinemia. In contrast, heterozygous CBS-deficient mice (CBS−/+) have plasma HC levels approximately double of wild-type mice (Watanabe et al. 1995). Several studies with CBSdeficient mice showed that plasma HC levels elevated from 27.1 ± 5.2 µmol/L to 8.8 ± 1.1 µmol/L after 7 weeks and from 23.9 ± 3.0 µmol/L to 13.0 ± 2.3 µmol/L after 15 weeks in CBS+/− mice compared with CBS+/+ mice when both were fed a folate replacement, methionine diet (Dayal et al. 2001). In other murine models of severe hyperhomocysteinemia, total plasma HC concentrations were found to be 50-fold (205 ± 86 µmol/L vs. 3.9 ± 0.9 µmol/L in blood) and 20-fold (6.44 ± 3.86 nmol/mg vs. 0.34 ± 0.14 nmol/mg in cellular protein) higher in C BS−/− mice than in C BS+/+ mice (Watanabe et al. 1995; Robert et al. 2005). From this evidence of murine models associated with total plasma HC levels, homozygous CBS-deficient (CBS−/−) mice seem more suitable for the study of hyperhomocysteinemia. Methylenetetrahydrofolate reductase (MTHFR) is another genetic model related to mild hyperhomocysteinemia. It has been reported that MHTFR plays an integral role in the methionine metabolism cycle by supplying
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5-methylhydrofolate for the remethylation of HC to methionine (Fig. 1). Homozygous MTHFR mice showed moderate hyperhomocysteinemia (total HC of approximately 30 µmol/L), whereas MTHFR-deficient mice with a control diet were targeted for alteration of the MTHFR gene. The control diet resulted in lower plasma HC levels, but the survival rate decreased within the first 5 weeks of age. In another study, the early mortality rate of M THFR−/− mice was reduced from 83 to 23% with diet supplemented with betaine throughout pregnancy and the lactation period (Schwahn et al. 2004). The addition of betaine in the diet partially reverses the abnormal cerebellar development in MTHFR homozygous mice. Mice with MTHFR deficiency and heterozygous (MTHFR+/−) mice survive normally with higher plasma HC levels (5 µmol/L) than that (3 µmol/L) in wild-type homozygous (MTHFR−/−) mice (Chen et al. 2001). In several studies, MTHFR homozygous (MTHFR−/−) mice have been used to investigate the vascular effects of altered HC remethylation (Virdis et al. 2003; Devlin et al. 2004). Dietary supplementation is another most important approach to treat hyperhomocysteinemia. Most studies have indicated that dietary supplementation with methionine can regulate the HC metabolism in the HC cycle. Total methionine levels up to 12–20 g/kg with moderate hyperhomocysteinemia (i.e., plasma HC concentration of 18–60 µmol/L) can be achieved by the addition of 0.5% l-methionine to the mice’s water (Hofmann et al. 2001; Tan et al. 2006). Severe hyperhomocysteinemia (i.e., plasma HC concentration higher than 200 µmol/L) can be achieved by increasing methionine supplementation to 24.6 g/kg (Werstuck et al. 2001; Wang et al. 2004), but methionine intake of more than 20 g/kg may have some toxic effects and affect regular growth. A study of dietary methionine supplementation with a basal diet (22 g/kg) resulted in weight loss, and dietary methionine supplementation with a basal diet (44 g/kg) showed severe growth retardation and early death in apolipoprotein E-deficient (Apoe−/−) mice (Zhou et al. 2001). Considering these findings, excessive amounts of methionine may be fatal to humans (Cottington 2002). Interestingly, it has been found that dietary methionine restrictions in a rodent model increased life span despite higher heart-to-body weight ratios and insulin sensitivity, which are associated with CVD (Ables et al. 2015; Elshorbagy et al. 2010). Dietary methionine restriction with folic acid and vitamin B supplementation proved to be a remarkable approach to induce hyperhomocysteinemia in rodent models (Zidan and Elnegris 2015; Li et al. 2016; Homocysteine Lowering Trialists’ Collaboration 1998; Clarke et al. 2010). A high-methionine diet with different levels of vitamin B restriction may cause either mild hyperhomocysteinemia (plasma HC levels between 8 and 10 µmol/L) or very severe hyperhomocysteinemia (plasma
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HC levels more than 200 µmol/L) (Homocysteine Lowering Trialists’ Collaboration 1998; Gospe et al. 1995; Lentz et al. 2002; Dayal et al. 2006). Several studies found that a diet with varying methionine concentrations (8–14 g/kg) in combination with adequate restriction of folate, vitamin B12 or vitamin B6 can be used to lower plasma total HC levels to 10–90 µmol/L (Dayal et al. 2006; Homocysteine Lowering Trialists’ Collaboration 1998; Gospe et al. 1995; Lentz et al. 2002).
Large animal models Small animal models are tremendously useful for highthroughput screening. Most rodent models are not phylogenetically similar to humans, whereas some pathological features of certain diseases and their response to pharmacological treatments may not be reliable predictors. Testing of organs and organisms in a large animal model is a completely different challenge than testing in small animal models (Elnakish et al. 2012). The disease characteristics of a large animal that are comparable with those of humans can be obtained via a mechanistic insight into the biological and pathological process. Furthermore, large animals possess direct information about particular physiological events to observe the effects of particular variables, treatments, and modified factors when compared with smaller groups (Tsang et al. 2016).
Rabbit model of hyperhomocysteinemia The rabbit model has long been used in CVD research. Rabbits are comparatively better representative of human lipoprotein metabolism, endothelial dysfunction, and renal functions (Liao et al. 2017). In Japan and New Zealand, Watanabe heritable hyperlipidemic rabbits (WHHL rabbits) and white rabbits naturally have hypercholesterolemia and hyperhomocysteinemia, respectively (Sipahioglu et al. 2005; Buja et al. 1983; Jones et al. 2005). The endothelial dysfunction is associated with conditions of increases in superoxide anion (O2−) production. The reaction between this anion ( O2−) and endotheliumderived nitric oxide (NO) could lead to reduced bioavailability of NO. This kind of consequence can alter vascular function and lead to premature development of atherosclerosis. In contrast, hyperhomocysteinemia results in overproduction of O2-derived free radicals by the endothelium by a mechanism that is still unknown. A study with endothelium showed that 10 µmol/L of Tiron, vitamin C or vitamin E could control O2− level in HC-induced endothelial cells (Lang et al. 2000). A number of authors have suggested that the inhibitory effects of HC on endothelium-dependent relaxation is due
Hyperhomocysteinemia and cardiovascular disease in animal model
to an increase in O2− in the endothelial cell’s intracellular space and provide a possible mechanism for the endothelial dysfunction linked with hyperhomocysteinemia.
Porcine models of hyperhomocysteinemia A large animal generally closer to humans is a representative non-human primate, and in this sense the pig has several physiological and anatomical similarities to humans that make an appropriate animal model for biomedical research (Kawaguchi et al. 2011). Moreover, porcine models can be used as an alternative to monkeys and dogs, which helps in efforts to respond to animal welfare concerns and to minimize the use of these animals (Kakimoto et al. 2014). Pig models could be better than other animal models because the results of sulfur amino acid metabolism experiments showed similar results to those obtained in humans (Ambrosi et al. 1999). To evaluate the therapeutic effects of HC levels, porcine models are more convenient than other animal models. In a 4-month study of the combination of a folic acid (5 mg/ day) intake higher than the recommended daily allowance for humans (200 µg/L) and a high methionine intake lowered the HC levels to about 25–45%. Interestingly, folic acid therapy, the combination of folic acid and Vit-B and Vit-B12, lowered HC levels (Ambrosi et al. 1999). In the past few decades, the burden caused by the weight of pigs has fueled the rise of several modified miniature pigs that are only one-third their original weight and size. In general, these minipigs are smaller than domestic pigs and can be easily handled for any induced diet for experiments for which mice or rabbits are not feasible (Miyoshi et al. 2010). A novel microminipig that weighs only 7 kg was recently developed for biomedical research in Japan (Miyoshi et al. 2010). A study with microminipigs showed no significant change in plasma total HC levels as a result of sex or feeding regimen (Kakimoto et al. 2014). The same authors also showed that the intravenous injection of DL–HC reduced the plasma HC levels, followed by a quick return to pre-injection levels, which was similar to the findings in the minipig model (Ambrosi et al. 1999).
in which total plasma HC levels were elevated by dietary or genetic means showed alteration in other metabolites that could influence the vascular pathophysiology. All of the presently available genetic murine models of hyperhomocysteinemia produce significant alterations in folate and, SAM levels and in some other HC-related metabolites (Elmore and Matthews 2007). Comparatively large animal models, especially porcine models, could be better than other animal models as the results of sulfur amino acid metabolism experiments showed results similar to those obtained in humans (Ambrosi et al. 1999). The ideal animal model for CVD and hyperhomocysteinemia could represent human conditions metabolically and pathophysiologically. In this study, small animals such as mice and large animals such as rabbits and pigs were examined under the application of hyperhomocysteinemia. Murine models are mostly used in the basic research of CVD because of their genetic response, short life span, ease of handling, low space requirements, and lower cost. In contrast, large animals have several physiological and anatomical similarities to humans that could be useful for biomedical research. In some cases, large animal models have some difficulties in their body weight and size as well as experimental costs. In recent years, genetically modified microminipigs have made research more convenient in large animal models because they possess features similar to those of large pigs. Their smaller size and body weight would make them a better model for future research with a large animal model. In addition, all models in different aspects of a disease are not suitable for a specific animal species for all studies. Therefore, it is greatly important to choose an appropriate animal model to study any CVD; otherwise, it will mislead the research output. Acknowledgements This research was supported by National Natural Science Foundation of China (No. 31772642, 31672457, 31702125, 41771300), National Key Research and Development Program of China (2016YFD0500504), International Partnership Program of Chinese Academy of Sciences (161343KYSB20160008), and the Ministry of Science and Technology of the People’s Republic of China (2014BAD14B01). Compliance with ethical standards
Conclusions The development of convincing and useful animal models for hyperhomocysteinemia represents a major challenge. However, despite the hurdles, such models seem necessary to understand disease pathophysiology and to hasten the development of treatments based on new molecular targets. We have illustrated some of the complications of hyperhomocysteinemia and have suggested different approaches to elevate the total plasma HC levels in blood. Among the available models, murine models of hyperhomocysteinemia
Conflict of interest The author declares that there is no potential conflict of interest regarding the publication of this article. Ethical statements This review article does not contain any studies with human participants or animals performed by any of the authors.
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in a rabbit model. Transpl Proc 37(5):2371–2374. doi:10.1016/j. transproceed.2005.03.053 Tan H, Jiang X, Yang F, Li Z, Liao D, Trial J, Magera MJ, Durante W, Yang X, Wang H (2006) Hyperhomocysteinemia inhibits postinjury reendothelialization in mice. Cardiovasc Res 69(1):253– 262. doi:10.1016/j.cardiores.2005.08.016 Tsang HG, Rashdan NA, Whitelaw CBA, Corcoran BM, Summers KM, MacRae VE (2016) Large animal models of cardiovascular disease. Cell Biochem Funct 34:113–132. doi:10.1002/cbf.3173 Virdis A, Iglarz M, Neves MF, Amiri F, Touyz RM, Rozen R, Schiffrin EL (2003) Effect of hyperhomocysteinemia and hypertension on endothelial function in methylenetetrahydrofolate reductasedeficient mice. Arterioscler Thromb Vasc Biol 23(8):1352–1357. doi:10.1161/01.ATV.0000083297.47245.DA Wang L, Jhee KH, Hua X, DiBello PM, Jacobsen DW, Kruger WD (2004) Modulation of cystathionine beta-synthase level regulates total serum homocysteine in mice. Circ Res 94(10):1318–1324. doi:10.1161/01.RES.0000129182.46440.4a Watanabe M, Osada J, Aratani Y, Kluckman K, Reddick R, Malinow MR, Maeda N (1995) Mice deficient in cystathionine β-synthase: animal models for mild and severe homocyst(e)inemia. Proc Natl Acad Sci USA 92:1585–1589 Werstuck GH, Lentz SR, Dayal S, Hossain GS, Sood SK, Shi YY, Zhou J, Maeda N, Krisans SK, Malinow MR, Austin RC (2001) Homocysteine-induced endoplasmic reticulum stress causes dysregulation of the cholesterol and triglyceride biosynthetic pathways. J Clin Invest 107:1263–1273 Wierzbicki AS (2016) Homocysteine and cardiovascular disease: a review of the evidence. Diab Vasc Dis Res 4(2):143–149. doi:10.3132/dvdr.2007.033 Zhang S, Bai YY, Luo LM, Xiao WK, Wu HM, Ye P (2014) Association between serum homocysteine and arterial stiffness in elderly: a community-based study. J Geriatr Cardiol 11(1):32–38. doi:10.3969/j.issn.1671-5411.2014.01.007 Zhou Ji, Møller Jan, Danielsen Carl C, Bentzon Jacob, Ravn Hanne B, Austin Richard C, Falk E (2001) Dietary supplementation with methionine and homocysteine promotes early atherosclerosis but not plaque rupture in ApoE-deficient mice. Arterioscler Thromb Vasc Biol 21:1470–1476 Zidan RA, Elnegris HM (2015) Effect of homocysteine on the histological structure of femur in young male albino rats and the possible protective role of folic acid. J Histol Histopathol 2(1):16. doi:10.7243/2055-091x-2-16
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REVIEW ARTICLE
Hyperhomocysteinemia and cardiovascular disease in animal model Md. Abul Kalam Azad1,2 · Pan Huang1 · Gang Liu1,3 · Wenkai Ren1 · Tsegay Teklebrh1,2,4 · Wenxin Yan1,3 · Xihong Zhou1 · Yulong Yin1,3,5
Received: 20 July 2017 / Accepted: 4 October 2017 © Springer-Verlag GmbH Austria 2017
Abstract Hyperhomocysteinemia is an independent risk factor for cardiovascular disease and is associated with primary causes of mortality and morbidity throughout the world. Several studies have been carried out to evaluate the effects of a diet inducing cystathionine-β-synthase, methyltetrafolate, folic acid, and vitamin B supplemented with methionine on the homocysteine metabolism and in lowering the plasma total homocysteine levels. A large number of molecular and biomedical studies in numerous animals, such as mice, rabbits, and pigs, have sought to elevate the plasma total homocysteine levels and to identify a disease model for human hyperhomocysteinemia. However, a specific animal model is not suitable for hyperhomocysteinemia in terms of all aspects of cardiovascular disease. In this review article, the experimental progress of animal models with plasma total homocysteine levels is examined to identify a Handling Editor: J. D. Wade. * Gang Liu [email protected] 1
Key Laboratory of Agro‑ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, National Engineering Laboratory for Pollution Control and Waste Utilization in Livestock and Poultry Production, Changsha 410125, Hunan, China
2
University of Chinese Academy of Sciences, Beijing 100049, China
3
Taoyuan Agro‑ecosystem Research Station, Soil Molecular Ecology Section, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125, China
4
School of Animal and Range Sciences, Haramaya University, 251 Haramaya, Dire Dawa, Ethiopia
5
Animal Nutrition and Human Health Laboratory, School of Life Sciences, Hunan Normal University, Changsha 410081, Hunan, China
feasible animal model of hyperhomocysteinemia for different aspects. Keywords Hyperhomocysteinemia · Homocysteine · Animal model · Cardiovascular disease Abbreviations HC Homocysteine CVD Cardiovascular disease ATP Adenosine triphosphate MTHFR Methylenetetrahydrofolate reductase CBS Cystathionine-β-synthase THF Tetrahydrofolate NO Nitric oxide O2 Oxygen SAM Sulfur adenosylmethionine SAH S-Adenosylhomocysteine MAT Methionine adenosyltransferase MT Methyltransferase MS Methionine synthase
Introduction Homocysteine (HC) is a naturally occurring amino acid and a by-product of methionine metabolism, which is a risk factor for cardiovascular disease (CVD) when present in high levels in blood. HC levels are carefully regulated in pathways related to methionine ingestion and metabolism. Chronic renal failure is a morbid condition with high mortality rates and a prevalence of both hyperhomocysteinemia and CVD. CVD, predominantly coronary heart disease, is the major cause of morbidity and mortality worldwide. According to the World Health Organization, CVD was
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the most common underlying cause of death in 2012, accounting for an estimated 17.5 million (82% uncertainty interval) of 56 million total deaths (Global status report on noncommunicable diseases 2014). In recent years, the treatment of CVD has seen great advances through animal experiments. A huge amount of information has been generated with disease models in preclinical research and has outlined the pathogenesis, progression, and underlying mechanism of CVD at both molecular and cellular levels (Liu et al. 2017). Moreover, disease models have a vital role in the progress of various effective treatment strategies. HC treatment in CVD models has been developed in many animal species, including in murine (small animals) such as mice and rats (Ables et al. 2015; Glowacki et al. 2010; Zidan and Elnegris 2015), and in large animals, such as rabbits, swine, and dogs (Zhang et al. 2014; Sipahioglu et al. 2005; Leong et al. 2015). According to Cohen et al. (1994), small animal models are more acceptable for molecular research because they are inexpensive, and easy to handle and have a huge amount of available research literature. In this study, the progress made with various animal models with hyperhomocysteinemia is discussed to identify a feasible model for hyperhomocyseteinemia related to CVD.
Fig. 1 Homocysteine formation and metabolism (MAT methionine adenosyltransferase, MT methyltransferase, SAHH SAH hydrolase, MS methionine synthase, MTHFR 5, 10 methyltetrahydrofolate reductase, CBS cystathionine-β-synthase, THF tetrahydrofolate, SAM S-adenosylmethionine, SAH S-adenosylhomocysteine)
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HC production and metabolism A sulfur amino acid, HC, is synthesized from methionine via multi-step processes (Fig. 1). In the first step, methionine receives an adenosine group from ATP and then transforms to S-adenosylmethionine (SAM) by S-adenosylmethionine synthase (MAT). In the next step, S-adenosylhomocysteine (SAH) is produced from SAM by releasing methyl group acceptor molecules, which is catalyzed by methyltransferase (MT). Finally, SAM is hydrolyzed by S-adenosylhomocysteine hydrolase (SAHH) to yield the HC (Katko et al. 2012). HC is mainly metabolized via two key pathways (Fig. 1). When there is excess methionine, HC is metabolized via the sulfuration pathway by releasing cystathionine while vitamin B acts as a cofactor and is then converted to cysteine. Excess cysteine is oxidized to sulfates or taurine or is drained from the body via urine. Whenever the methionine concentrations are low, HC is metabolized via the methionine-conserving pathway. In most tissues, methyltetrahydrofolate and vitamin B12 play their roles as cofactors during the process of remethylation of HC to methionine. These two metabolism pathways are linked by SAM, which is the most vital source of the methyl group for all methylation reactions within the cell (Selhub 1999). Undoubtedly, it can be mentioned that high levels of HC are associated with reduced methylation potential, whereas folate and vitamin B 12 increase this methylation potential. The amount of dietary intake of methionine can
Hyperhomocysteinemia and cardiovascular disease in animal model
change the methionine concentration in the body and affect the rate of SAM synthesis and the metabolism of HC.
HC and CVD Hyperhomocysteinemia is defined as a medical condition that is characterized by an abnormal HC concentration in the blood. An HC concentration of 16 µmol/L in blood is termed mild, 16–30 µmol/L is considered intermediate, and greater than 100 µmol/L is known as hyperhomocysteinemia (Kang et al. 1992). In non-treated homocysteinuria, HC levels from 12 to 16 µmol/L are risk factors for CVD (David et al. 2002). Several meta-analyses have shown that the HC level in blood is the most important risk factor for CVD and stroke (David et al. 2002; Li et al. 2016; Clarke et al. 2012; Humphrey et al. 2008; Homocysteine Studies Collaboration 2002; Klerk et al. 2002). Observational studies have shown that the risk of ischemic heart disease and the risk of stroke increases by 32 and 59%, respectively, with each 5 µmol/L increase in serum HC levels (David et al. 2002). Several researchers have reported that methylenetetrahydrofolate reductase (MTHFR) enzyme is involved in HC metabolism and plays an important role in CVD. In contrast, the wildtype allele (CC) of MTHFR and the mutant allele (TT) had 25% higher HC concentrations and a 16% higher risk of CVD (Klerk et al. 2002). It was recently reported that the elevation of HC levels in blood serum reduces hyperhomocysteinemia and the risk of CVD (Clarke et al. 2010, 2012; Nandi and Mishra 2017; Wierzbicki 2016; Catena et al. 2014, 2015). Some studies have shown that folic acid, vitamin B, and vitamin B12 supplementation is inexpensive and effective at lowering the blood HC concentration (Li et al. 2016; Homocysteine Lowering Trialists’ Collaboration 1998; Marcus and Menon 2007; Clarke et al. 2010). In contrast, cystathionine β-synthase (CBS) and cystathionine gamma lyase enzymes play important roles in the pathway of decreasing HC levels to hydrogen sulfide (H2S) (Chang et al. 2008; Katko et al. 2012; Kamat et al. 2016; Nandi and Mishra 2017).
Animal model Epidemiological and experimental evidence indicates that hyperhomocysteinemia is highly related to an increased risk of vascular disease, which arises from unexpected HC metabolism. Severe hyperhomocysteinemia occurs due to rarer genetic defects and deficiencies in cystathionineβ-synthase, methylene THF reductase, or other enzymes involved in methyl-B12 synthesis and HC methylation. A lower level of hyperhomocysteinemia was found in fasting conditions because the methylation pathway suffered less
impairment (i.e., folate or B 12 deficiencies or methylene THF reductase thermolability). In addition, post-methionine loading may occur due to a heterozygous cystathionine-βsynthase defect or B6 defect (Selhub 1999).
Murine model of CVD Murine models are often used in CVD research because they have low maintenance costs and short gestation times, are easy to handle, allow genetic manipulation to generate transgenic strains, and are more suitable for “high-throughput” studies than large animal models (Recchia and Lionetti 2007). Considering these characteristics, small rodent models are mostly used for studies of cardiac physiology and disease, genetics, pharmacology, and long-term survival (Elnakish et al. 2012).
Murine model of total plasma HC levels In terms of pharmacological or pathological approaches, murine models can be used in various sectors, including dietary supplementation, genetic approaches, dietary modification, and genetic intervention with the purpose of lowering the total plasma HC levels of hyperhomocysteinemia. CBS is a genetic hyperhomocysteinemia model with gene deletion that has been used in studies of HC pathology in catalyzing HC to cystathionine. It has been found that the HC plasma levels in homozygous CBS-deficient mice (CBS−/−) are 50 times higher than those in wild-type mice and similar to those in human hyperhomocysteinemia. In contrast, heterozygous CBS-deficient mice (CBS−/+) have plasma HC levels approximately double of wild-type mice (Watanabe et al. 1995). Several studies with CBSdeficient mice showed that plasma HC levels elevated from 27.1 ± 5.2 µmol/L to 8.8 ± 1.1 µmol/L after 7 weeks and from 23.9 ± 3.0 µmol/L to 13.0 ± 2.3 µmol/L after 15 weeks in CBS+/− mice compared with CBS+/+ mice when both were fed a folate replacement, methionine diet (Dayal et al. 2001). In other murine models of severe hyperhomocysteinemia, total plasma HC concentrations were found to be 50-fold (205 ± 86 µmol/L vs. 3.9 ± 0.9 µmol/L in blood) and 20-fold (6.44 ± 3.86 nmol/mg vs. 0.34 ± 0.14 nmol/mg in cellular protein) higher in C BS−/− mice than in C BS+/+ mice (Watanabe et al. 1995; Robert et al. 2005). From this evidence of murine models associated with total plasma HC levels, homozygous CBS-deficient (CBS−/−) mice seem more suitable for the study of hyperhomocysteinemia. Methylenetetrahydrofolate reductase (MTHFR) is another genetic model related to mild hyperhomocysteinemia. It has been reported that MHTFR plays an integral role in the methionine metabolism cycle by supplying
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5-methylhydrofolate for the remethylation of HC to methionine (Fig. 1). Homozygous MTHFR mice showed moderate hyperhomocysteinemia (total HC of approximately 30 µmol/L), whereas MTHFR-deficient mice with a control diet were targeted for alteration of the MTHFR gene. The control diet resulted in lower plasma HC levels, but the survival rate decreased within the first 5 weeks of age. In another study, the early mortality rate of M THFR−/− mice was reduced from 83 to 23% with diet supplemented with betaine throughout pregnancy and the lactation period (Schwahn et al. 2004). The addition of betaine in the diet partially reverses the abnormal cerebellar development in MTHFR homozygous mice. Mice with MTHFR deficiency and heterozygous (MTHFR+/−) mice survive normally with higher plasma HC levels (5 µmol/L) than that (3 µmol/L) in wild-type homozygous (MTHFR−/−) mice (Chen et al. 2001). In several studies, MTHFR homozygous (MTHFR−/−) mice have been used to investigate the vascular effects of altered HC remethylation (Virdis et al. 2003; Devlin et al. 2004). Dietary supplementation is another most important approach to treat hyperhomocysteinemia. Most studies have indicated that dietary supplementation with methionine can regulate the HC metabolism in the HC cycle. Total methionine levels up to 12–20 g/kg with moderate hyperhomocysteinemia (i.e., plasma HC concentration of 18–60 µmol/L) can be achieved by the addition of 0.5% l-methionine to the mice’s water (Hofmann et al. 2001; Tan et al. 2006). Severe hyperhomocysteinemia (i.e., plasma HC concentration higher than 200 µmol/L) can be achieved by increasing methionine supplementation to 24.6 g/kg (Werstuck et al. 2001; Wang et al. 2004), but methionine intake of more than 20 g/kg may have some toxic effects and affect regular growth. A study of dietary methionine supplementation with a basal diet (22 g/kg) resulted in weight loss, and dietary methionine supplementation with a basal diet (44 g/kg) showed severe growth retardation and early death in apolipoprotein E-deficient (Apoe−/−) mice (Zhou et al. 2001). Considering these findings, excessive amounts of methionine may be fatal to humans (Cottington 2002). Interestingly, it has been found that dietary methionine restrictions in a rodent model increased life span despite higher heart-to-body weight ratios and insulin sensitivity, which are associated with CVD (Ables et al. 2015; Elshorbagy et al. 2010). Dietary methionine restriction with folic acid and vitamin B supplementation proved to be a remarkable approach to induce hyperhomocysteinemia in rodent models (Zidan and Elnegris 2015; Li et al. 2016; Homocysteine Lowering Trialists’ Collaboration 1998; Clarke et al. 2010). A high-methionine diet with different levels of vitamin B restriction may cause either mild hyperhomocysteinemia (plasma HC levels between 8 and 10 µmol/L) or very severe hyperhomocysteinemia (plasma
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HC levels more than 200 µmol/L) (Homocysteine Lowering Trialists’ Collaboration 1998; Gospe et al. 1995; Lentz et al. 2002; Dayal et al. 2006). Several studies found that a diet with varying methionine concentrations (8–14 g/kg) in combination with adequate restriction of folate, vitamin B12 or vitamin B6 can be used to lower plasma total HC levels to 10–90 µmol/L (Dayal et al. 2006; Homocysteine Lowering Trialists’ Collaboration 1998; Gospe et al. 1995; Lentz et al. 2002).
Large animal models Small animal models are tremendously useful for highthroughput screening. Most rodent models are not phylogenetically similar to humans, whereas some pathological features of certain diseases and their response to pharmacological treatments may not be reliable predictors. Testing of organs and organisms in a large animal model is a completely different challenge than testing in small animal models (Elnakish et al. 2012). The disease characteristics of a large animal that are comparable with those of humans can be obtained via a mechanistic insight into the biological and pathological process. Furthermore, large animals possess direct information about particular physiological events to observe the effects of particular variables, treatments, and modified factors when compared with smaller groups (Tsang et al. 2016).
Rabbit model of hyperhomocysteinemia The rabbit model has long been used in CVD research. Rabbits are comparatively better representative of human lipoprotein metabolism, endothelial dysfunction, and renal functions (Liao et al. 2017). In Japan and New Zealand, Watanabe heritable hyperlipidemic rabbits (WHHL rabbits) and white rabbits naturally have hypercholesterolemia and hyperhomocysteinemia, respectively (Sipahioglu et al. 2005; Buja et al. 1983; Jones et al. 2005). The endothelial dysfunction is associated with conditions of increases in superoxide anion (O2−) production. The reaction between this anion ( O2−) and endotheliumderived nitric oxide (NO) could lead to reduced bioavailability of NO. This kind of consequence can alter vascular function and lead to premature development of atherosclerosis. In contrast, hyperhomocysteinemia results in overproduction of O2-derived free radicals by the endothelium by a mechanism that is still unknown. A study with endothelium showed that 10 µmol/L of Tiron, vitamin C or vitamin E could control O2− level in HC-induced endothelial cells (Lang et al. 2000). A number of authors have suggested that the inhibitory effects of HC on endothelium-dependent relaxation is due
Hyperhomocysteinemia and cardiovascular disease in animal model
to an increase in O2− in the endothelial cell’s intracellular space and provide a possible mechanism for the endothelial dysfunction linked with hyperhomocysteinemia.
Porcine models of hyperhomocysteinemia A large animal generally closer to humans is a representative non-human primate, and in this sense the pig has several physiological and anatomical similarities to humans that make an appropriate animal model for biomedical research (Kawaguchi et al. 2011). Moreover, porcine models can be used as an alternative to monkeys and dogs, which helps in efforts to respond to animal welfare concerns and to minimize the use of these animals (Kakimoto et al. 2014). Pig models could be better than other animal models because the results of sulfur amino acid metabolism experiments showed similar results to those obtained in humans (Ambrosi et al. 1999). To evaluate the therapeutic effects of HC levels, porcine models are more convenient than other animal models. In a 4-month study of the combination of a folic acid (5 mg/ day) intake higher than the recommended daily allowance for humans (200 µg/L) and a high methionine intake lowered the HC levels to about 25–45%. Interestingly, folic acid therapy, the combination of folic acid and Vit-B and Vit-B12, lowered HC levels (Ambrosi et al. 1999). In the past few decades, the burden caused by the weight of pigs has fueled the rise of several modified miniature pigs that are only one-third their original weight and size. In general, these minipigs are smaller than domestic pigs and can be easily handled for any induced diet for experiments for which mice or rabbits are not feasible (Miyoshi et al. 2010). A novel microminipig that weighs only 7 kg was recently developed for biomedical research in Japan (Miyoshi et al. 2010). A study with microminipigs showed no significant change in plasma total HC levels as a result of sex or feeding regimen (Kakimoto et al. 2014). The same authors also showed that the intravenous injection of DL–HC reduced the plasma HC levels, followed by a quick return to pre-injection levels, which was similar to the findings in the minipig model (Ambrosi et al. 1999).
in which total plasma HC levels were elevated by dietary or genetic means showed alteration in other metabolites that could influence the vascular pathophysiology. All of the presently available genetic murine models of hyperhomocysteinemia produce significant alterations in folate and, SAM levels and in some other HC-related metabolites (Elmore and Matthews 2007). Comparatively large animal models, especially porcine models, could be better than other animal models as the results of sulfur amino acid metabolism experiments showed results similar to those obtained in humans (Ambrosi et al. 1999). The ideal animal model for CVD and hyperhomocysteinemia could represent human conditions metabolically and pathophysiologically. In this study, small animals such as mice and large animals such as rabbits and pigs were examined under the application of hyperhomocysteinemia. Murine models are mostly used in the basic research of CVD because of their genetic response, short life span, ease of handling, low space requirements, and lower cost. In contrast, large animals have several physiological and anatomical similarities to humans that could be useful for biomedical research. In some cases, large animal models have some difficulties in their body weight and size as well as experimental costs. In recent years, genetically modified microminipigs have made research more convenient in large animal models because they possess features similar to those of large pigs. Their smaller size and body weight would make them a better model for future research with a large animal model. In addition, all models in different aspects of a disease are not suitable for a specific animal species for all studies. Therefore, it is greatly important to choose an appropriate animal model to study any CVD; otherwise, it will mislead the research output. Acknowledgements This research was supported by National Natural Science Foundation of China (No. 31772642, 31672457, 31702125, 41771300), National Key Research and Development Program of China (2016YFD0500504), International Partnership Program of Chinese Academy of Sciences (161343KYSB20160008), and the Ministry of Science and Technology of the People’s Republic of China (2014BAD14B01). Compliance with ethical standards
Conclusions The development of convincing and useful animal models for hyperhomocysteinemia represents a major challenge. However, despite the hurdles, such models seem necessary to understand disease pathophysiology and to hasten the development of treatments based on new molecular targets. We have illustrated some of the complications of hyperhomocysteinemia and have suggested different approaches to elevate the total plasma HC levels in blood. Among the available models, murine models of hyperhomocysteinemia
Conflict of interest The author declares that there is no potential conflict of interest regarding the publication of this article. Ethical statements This review article does not contain any studies with human participants or animals performed by any of the authors.
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