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Hyperhomocysteinemia and myocardial remodeling in the sand rat, Psammomys obesus
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Hyperhomocysteinemia and myocardial remodeling in the sand rat, Psammomys obesus Billel Chaouada,b, Elara N. Moudilouc, Adel Ghoula, Fouzia Zerrouka, Anissa Moulahouma, ⁎ Khira Othmani-Mecifa, Mohamed El Hadi Cherifid, Jean-Marie Exbrayatc, Yasmina Benazzouga, a Biochemistry and Remodeling of the Extracellular Matrix, Laboratory of Cellular and Molecular Biology, Faculty of Biological Sciences, Houari Boumediene University of Science and Technology (USTHB), Bab Ezzouar, El Alia, 16111, Algiers, Algeria b University Djilali Bounaama of Khemis Miliana, Faculty of Natural and Life Sciences and Earth Sciences, Theniet El Had Road, 44225, Khemis Miliana, Algeria c UMRS 449, General Biology – Reproduction and Comparative Development, Lyon Catholic University, UDL, EPHE, PSL, 10, Place des Archives, 69288, Lyon Cedex 02, France d Central Laboratory of Biology, EPH Bologhine Ibn Ziri, Algiers, Algeria
A R T I C LE I N FO
A B S T R A C T
Keywords: Apoptosis Fibrosis Hyperhomocysteinemia Myocardium Psammomys obesus
Objective: Numerous studies have shown that a methionine-rich diet induces hyperhomocysteinemia (Hhcy), a risk factor for cardiovascular diseases. The objective of the present study was to determine the involvement of Hhcy in cardiac remodeling in the sand rat Psammomys obesus. Materials and methods: An experimental Hhcy was induced, in the sand rat Psammomys obesus, by intraperitoneal injection of 300 mg/kg of body weight/day of methionine for 1 month. The impact of Hhcy on the cellular and matricial structures of the myocardium was analyzed with histological techniques (Masson trichrome and Sirius red staining). Immunohistochemistry allowed us to analyze several factors involved in myocardial remodeling, such as fibrillar collagen I and III, metalloproteases (MMP-2 and -9) and their inhibitors (TIMP-1 and -2), TGF-β1 and activated caspase 3. Results: Our results show that Hhcy induced by an excess of methionine causes, in the myocardium of Psammomys obesus, a significant accumulation of fibrillar collagens I and III at the interstitial and perivascular scales, indicating the appearance of fibrosis, which is associated with an immuno-expression increase of TGF-β1, MMP-9 and TIMP-2 and an immuno-expression decrease of MMP-2 and TIMP-1. Also, Hhcy induces apoptosis of some cardiomyocytes and cardiac fibroblasts by increasing of activated caspase 3 expression. These results highlight a remodeling of cardiac tissue in hyperhomocysteinemic Psammomys obesus.
1. Introduction In our days, cardiovascular diseases such as myocardial infarction and atherosclerosis are the leading causes of death and morbidity in the world (Pagidipati and Gaziano, 2013). These diseases are caused by several risk factors, including smoking (Burns, 2003), hyperlipidemia (Nelson, 2013), high blood pressure (Roberts, 1987) and hyperhomocysteinemia (Hhcy) (Maurer et al., 2010; Refsum et al., 1998). It has been shown that Hhcy alone, without the intervention of other known risk factors, is responsible for 10% of heart failure in humans (Baszczuk et al., 2014). The history of homocysteine as a potential risk factor for cardiovascular diseases began in the 1960s when McCully observed vascular disorders in children with a hereditary deficiency of cystathionine-β-Synthetase, a key enzyme in homocysteine metabolism
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(McCully, 1969). In myocardium, Hhcy is strongly associated with tissue remodeling characterized by the accumulation of extracellular matrix elements (ECM), in particular fibrillary collagens (Raaf et al., 2011; Zhi et al., 2013), but also with hypertrophy and apoptosis of the cardiomyocytes (Levrand et al., 2007; Wei et al., 2010). This accumulation of collagens results from an imbalance between the activity of some matrix metalloproteinases (MMPs), particularly MMP-2 and -9 and their inhibitors, TIMP-1 and -2 (Kumar et al., 2013; Takawale et al., 2017). According to several studies, transforming growth factor β (TGF-β) is the leading mediator involved in cardiac fibrosis in diabetes (Yue et al., 2017), myocardial infarction (Matsumoto-ida et al., 2006) and Hhcy (Raaf et al., 2011). Psammomys obesus or sand rat is recognized as an excellent
Corresponding author. E-mail address: [email protected] (Y. Benazzoug).
https://doi.org/10.1016/j.acthis.2019.07.008 Received 19 March 2019; Received in revised form 22 July 2019; Accepted 25 July 2019 0065-1281/ © 2019 Elsevier GmbH. All rights reserved.
Please cite this article as: Billel Chaouad, et al., Acta Histochemica, https://doi.org/10.1016/j.acthis.2019.07.008
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immunoturbidimetric method was used for C-reactive protein (CRP) (Eda et al., 1998).
polygenic animal model of metabolic syndrome closely resembling that observed in humans (Walder et al., 2002). This desert rodent is unique in the study of both obesity, insulin resistance, type 2 diabetes (Ziv et al., 1999), dyslipidemia (Walder et al., 2002) and atherosclerosis (Aouichat-Bouguerra et al., 2004). Several cardiac alterations have been reported in Psammomys obesus subjected to a high-fat diet (Sahraoui et al., 2016). However, there is no study on cardiac remodeling during Hhcy has been performed in this species. The present work consists of studying and analyzing some histopathological and immuno-histochemical factors involved in myocardial remodeling in hyperhomocysteinemic sand rats by daily administration of methionine. We analyzed the fibrillar collagens type I and III, the metalloproteinases MMP-2 and -9 and their tissue inhibitors TIMP-1 and -2, the TGF-β1 and the activated caspase 3.
2.4. Histological and histochemical analysis The histological and histochemical study was performed with 7 control rats and 7 Hhcy rats. At the end of the experiment, the animals were euthanized by intraperitoneal injection of 70 mg/kg of body weight of Ketamine and then dissected. The organs removed were quickly fixed in Bouin's solution for 2 days and then dehydrated in alcohol baths of increasing degree (50°, 70°, 90°, and 100°). The organs were then cleared in 2 butanol baths and embedded in paraffin. The organs were cut off using a Lab-Kite microtome. The 5μm-thick cuts obtained were spread on Superfrost-plus glass slides. After drying for 24 h at 37 °C, the slides were subjected to topographic staining (Masson trichrome) and selective histochemical staining of fibrillar collagens (Sirius red).
2. Materials and methods 2.1. Biological material and experimental protocol
2.5. Immunohistochemistry This study was carried out in Psammomys obesus, an athero-sensible animal model of the gerbillidea class. This desert rodent was caught in the M'Sila region (Algeria) in July - August 2016 (sexual rest). After an adaptation period of 15 days in the laboratory (temperature 25 °C, 50% humidity, 12-h light/dark cycle), the adult rats were divided into two groups of 15 animals each, a control group and a treated group of mean body weight of 144 g and 145 g respectively. The 2 lots were fed with halophilic plants and subjected to an injection of physiological water (0.9% NaCl) for the control lot and methionine (Sigma-Aldrich, 64340, St. Louis, MO 63103 USA), previously dissolved in physiological water, at a dose of 300 mg/kg of body weight/day for the treated lot. During the 30-day experiment, the animals were weekly weighted. All experiments were carried out in accordance with the Algerian legislation (Law number 12-235/2012) relating to animal protection, the recommendations of the Algerian Association of Experimental Animal Sciences (AASEA 45/DGLPAG/DVA/SDA/14) and the EU Directive 2010/63/EU for animal experiments. Blood samples were collected at the beginning (T0) and at the end (T30) of the experiment from the retro-orbital sinus of the eyes using a previously heparinized Pasteur pipette. Blood collected on heparinized tubes was centrifuged at 3000 rpm for 10 min. The collected plasma was stored at −80 °C for the determination of some biochemical plasma parameters.
The immunohistochemistry technique was performed using the indirect avidin-biotin-peroxidase amplification method (Vectastain Elite Universal Kit, PK-6200, Vector Laboratories, CA, USA) to detect and localize fibrillar collagens type I and III, MMP-2 and -9, TIMPs 1 and 2, TGF-β1 and activated caspase 3 in the cardiac sections. 5μm-thick histological sections of the heart were deparaffinized in cyclohexane and then rehydrated in ethanol baths of decreasing degree. In order to block the activity of endogenous peroxidases, the sections were incubated for 40 min in a 3% H2O2 solution. After washing with PBSx1 for 3 min, the sections were incubated in a 2% BSA solution for 60 min in order to block non-specific binding sites. The primary antibody was applied on the sections overnight at 4 °C in the following dilutions: 1/ 100 for anti-collagen I (rabbit polyclonal - Abcam ab34710, Cambridge, UK), anti-collagen III (rabbit polyclonal - Abcam ab7778, Cambridge, UK), anti-TGF-β1 (rabbit polyclonal - Abcam ab92486) and activated anti-caspase 3 (rabbit monoclonal - Abcam, ab32042), 1/25 for antiMMP-2 (rabbit polyclonal - Abcam ab37150, Cambridge, UK), antiMMP-9 (rabbit polyclonal - Abcam ab38898, Cambridge, UK), antiTIMP 1 (mouse monoclonal - Santa Cruz sc-21734) and anti-TIMP-2 (mouse monoclonal - Santa Cruz sc-271932). Antigen retrieval in a basic citrate solution (H-3300, Vector Laboratories, CA, USA) was required for anti-TGF-β1 and anti-TIMP-1 and -2 antibodies. After application of the antibodies, the sections were rinsed twice with PBSx1 for 5 min and incubated 60 min with the secondary biotinylated antibody (Anti-Mouse/Anti-Rabbit IgG, Vector Laboratories, CA, USA). Another PBSx1 wash was required before and after application for 60 min of the avidin-biotin-peroxidase complex. After the revelation with NovaRed (peroxidase substrate, SK-4805, Vector Laboratories, CA, USA) and the hematoxylin QS counter-staining (H-3404, Vector Laboratories, CA, USA), the sections were dehydrated and then mounted in Permount (Fisher Chemical, USA). Negative control was performed for each protein analyzed by the omission of the primary antibody.
2.2. Determination of total plasma homocysteine The quantitative determination of total plasma homocysteine was performed by the Architect Homocysteine assay kit (Axis-Shield Diagnostics Ltd, Dundee, UK) using CMIA (chemiluminescent microparticle immunoassay) technology. The oxidized form of homocysteine present in the sample was reduced to free homocysteine by the action of dithiothreitol. Total free homocysteine was transformed into S-adenosyl-L-homocysteine (SAH) by recombinant SAH-hydrolase in the presence of adenosine in excess. SAH and S-adenosyl-L-cysteine labelled with acridinium competed to fill the binding sites on the anti-SAH monoclonal antibody. After washing, magnetic separation and triggering reactions, the light emitted by the acridinium was measured in relative units of light by the optical system of Architect analyzer. An indirect relationship exists between the homocysteine present in the sample and the amount of light emitted.
2.6. Morphometric study and quantification of fibrillar collagens The myocardium histological and histochemical analysis of control and treated animals is completed by a morphometric study. This study was performed with AxioVision 4.8 software from CARL ZEISS, after calibration and at x1000 magnification. The measurements were made on transversely cut cardiomyocytes with clearly visible nuclei for cellular parameters (large and small axes, surface) and on longitudinally cut cardiomyocytes for nuclear diameter and surface evaluation. For each parameter analyzed, we performed 100 measurements (on average 15–17 randomly selected per animal). In addition, Sirius red stained sections were used to quantify fibrillar collagens at interstitial and
2.3. Determination of some biochemical plasma parameters Different plasma parameters were quantified by the enzymatic colorimetric method: glucose (Trinder, 1969), cholesterol (Richmond, 1973), triglycerides (Fossati and Prencipe, 1982) and HDL (Warnick and Wood, 1995). Colorimetric methods were also performed for total proteins (Doumas et al., 1981) and albumin (Rodkey, 1965). The 2
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perivascular level using Threshold function in imageJ software (1.47uNational institutes of health. USA). For each control and treated animal (N = 7), the quantification of interstitial collagens was performed by measuring the percentage of the stained surface related to the total surface area over 6 to 8 sections of 19000μm2 tissue randomly selected at x1000 magnification. The quantification of perivascular collagens was performed by the ratio stained surface area to total surface area of the blood vessel. We selected 3 to 4 blood vessels distributed in the myocardial tissue for each control and treated animal (N = 7). Immunohistochemistry and imageJ software allowed us to quantify type I (14–15 measurements/animal) and type III (10–12 measurements/animal) collagens on 19000μm2 tissue sections randomly selected at x1000 magnification.
Table 1 Body weight and some biochemical plasma parameters in control and Hhcy sand rats. Parameters
2.7. Statistical analysis Quantitative results were analyzed by STATISTICA 6.1 (StatSoft, Inc.). The values were expressed as a mean affected from standard error to mean (SEM). The Mann-Whitney test was used to evaluate the difference between the parameters of control and treated animals. When the values of P were lower than 0.05, the difference was considered statistically significant.
T0 values
After 30 days of experimentation Control Group
Hhcy Groupe 9/6 129.77 ± 7.68 (p < 0.05) 0.71 ± 0.06 (p < 0.0001) 47.57 ± 2.20 (p < 0.05)
Sex (male/female) Body weight (g)
16/14 144.62 ± 6.57
7/8 157.33 ± 7.28
Glycemia (g/l)
0.96 ± 0.03
1.07 ± 0.04
Proteinemia (g/l)
53.76 ± 1.08
55.18 ± 1.73
Lipids Cholesterolemia (g/l) Triglyceridemia (g/l) HDL (g/l) Albumin (g/l)
0.41 ± 0.02 0.59 ± 0.07 0.22 ± 0.01 27.34 ± 0.60
0.46 ± 0.03 0.59 ± 0.10 0.25 ± 0.02 28.04 ± 0.52
CRP (mg/l)
0.04 ± 0.01
0.03 ± 0.01
0.44 ± 0.06 (ns) 0.44 ± 0.09 (ns) 0.27 ± 0.03 (ns) 24.22 ± 1.23 (p < 0.05) 0.06 ± 0.01 (p < 0.05)
The values represent the mean ± SEM. The statistical analysis is performed by the Mann-Whitney test. ns: non-significant difference between the Control and Treated group. p < 0.05 and p < 0.0001: significant difference between the Control (n = 15) and Treated group (n = 15).
3. Results
in blood glucose (p < 0.0001) and a 13.79% decrease in proteinemia (p < 0.05) were recorded. Lipid parameters (cholesterol, triglyceride, and HDL) did not appear to be altered consequently to methionine administration. A decrease (p < 0.05) was also observed in plasma albumin of 13.62%, while the CRP dosage, a marker of inflammation, showed a 50% increased (p < 0.05) in Hhcy rats.
3.1. Quantification of total plasma homocysteine The baseline value of homocysteinemia determined in all sand rats (n = 30) at the beginning of the experiment was 1.52 ± 0.15 μmol/l. The homocysteinemia recorded at the end of the experiment (30 days) in control and subjected to methionine (Met) is shown in Fig. 1. Our results show an increase (p < 0.0001) of 875.64% in plasma homocysteine in rats subjected to methionine, inducing hyperhomocysteinemia. This parameter increases from 2.34 ± 0.23 μmol/l in the control group to 22.83 ± 5.20 μmol/l in the treated group which then became hyperhomocysteinemic (Hhcy).
3.3. Histo-morphometry of the myocardium The increase in plasma homocysteine concentration in sand rats caused significant cardiac alterations at both the cellular and matrix level. Matrix alterations were characterized by fibrosis (Fig. 2b and c) which resulted in a focused accumulation of fibrillar collagens at the interstitial (Fig. 3A) and perivascular (Fig. 3B) levels with a decrease in cell density in the fibrotic regions. The semi-quantification of fibrillar collagens at the interstitial level (Fig. 3A) showed an increase of 632.90% (P < 0.0001) in Hhcy rats compared to control rats (17.15 ± 1.3% vs 2.34 ± 0.09%). At the perivascular level (Fig. 3B), the semi-quantification of fibrillar collagens showed an increase of 83.18% (P < 0.0001) in the Hhcy rats compared to the controls (38.67 ± 1.13% vs 21.11 ± 0.36%). Vacuolation of the cytoplasm was observed in some cardiomyocytes (Fig. 2c). Many of these cells were hypertrophied, but others were atrophied in fibrotic areas particularly (Fig. 2c). These results were confirmed by the morphometric study which shows an increase (P < 0.0001) of all the cellular and nuclear parameters of cardiomyocytes in Hhcy rats compared to controls (Table 2).
3.2. Impact of hyperhomocysteinemia on body weight and some biochemical plasma parameters Table 1 shows the evolution of body weight and some biochemical plasma parameters in control and Hhcy sand rats. At the end of the experiment, the body weight of Hhcy rats was decreased by 17.52% compared to that of controls. A 33.64% decrease
3.4. Immunohistochemical analysis of collagens I and III In order to determine the type of collagens implicated in this cardiac fibrosis, an immunohistochemical study of fibrillar collagen I and III was performed (Fig. 4). In the control group (N = 7), a weak immunostaining of collagens I and III was observed in the interstitial tissue between cardiomyocytes. The semi-quantification by imageJ showed that collagens I and III occupied 9.78 ± 0.33% and 7.74 ± 0.26% of the myocardium surface respectively. In the interstitial tissue, collagen I was more present than collagen III. In the treated group (N = 7), a significant accumulation of these two types of collagens was observed between the cardiac cells. In Hhcy
Fig. 1. Homocysteinemia in control (n = 15) and treated (n = 15) animals. The statistical analysis is performed by the Mann-Whitney test. p < 0.0001: significant difference. 3
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Fig. 2. Myocardial histology of control animals (a) and Hhcy (b, c). Masson trichrome staining. The myocardium of control rats consists of several cardiomyocytes (CM) surrounded by weak vascularized connective tissue (CT). This tissue is composed of a few collagen fibres and many cardiac fibroblasts (CF). The myocardium of Hhcy rats is characterized by significant diffuse interstitial fibrosis (black arrows), hypertrophy of some cardiomyocytes (brown arrow) and vacuolation in their cytoplasm (yellow arrows). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
Fig. 3. Heart histological sections of control and Hhcy rats stained by Sirius red and quantification of the fibrillar collagens at the interstitial (A) and perivascular (B) level by imageJ software. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). In control rats, fibrillar collagens cover a small percentage of the myocardium (a) and blood vessels (c). In Hhcy rats, there is a significant accumulation of fibrillar collagens in the myocardium (b) and vascular adventitia (d). The statistical analysis is performed by the Mann-Whitney test. p < 0.0001: significant difference between the control (n = 7) and Hhcy (n = 7) group. 4
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Table 2 Cardiomyocytes morphometric study of some cellular and nuclear parameters in control and Hhcy rats. Control
Hhcy
Cellular parameters
Major axis (μm) Small axis (μm) Surface area (μm2)
20.67 ± 0.56 13.32 ± 0.32 223.98 ± 10.15
28.39 ± 0.85 (p < 0.0001) 16.69 ± 0.53 (p < 0.0001) 394.58 ± 20.29 (p < 0.0001)
Nuclear parameters
Major axis (μm) Small axis (μm) Surface area (μm2)
9.52 ± 0.14 4.08 ± 0.07 30.90 ± 0.70
12.71 ± 0.14 (p < 0.0001) 4.48 ± 0.06 (p < 0.0001) 45.04 ± 0.49 (p < 0.0001)
The values represent the mean ± SEM. The statistical analysis is performed by the Mann-Whitney test. p < 0.0001: All cellular and nuclear parameters (n = 100) of cardiomyocytes are statistically different between the control (n = 7) and Hhcy groups (n = 7).
fibrosis in Psammomys obesus, via a simultaneous increase in the expression of collagen I and III, indicating a modulation of the biochemical composition of myocardial ECM.
rats, collagens I and III represented respectively 28.86 ± 0.76% and 27.66 ± 0.83% of the myocardial surface area. Therefore, our results show a significant increase (p < 0.0001) of 195.09% and 257.36% in the surface area occupied by collagen I and III respectively in Hhcy rats compared to controls. This result highlighted a change in the proportion of the two collagen types in the interstitial tissue. We noted that collagen I was more represented than collagen III in interstitial tissue in both control and Hhcy animals. However, the analysis of our results indicates that Psammomys obesus Hhcy showed a variation in the increase of the two types of collagen. Indeed, collagen III was 3.6 times more important than in the control group, while collagen I was 2.7 times more important. Our results showed that hyperhomocysteinemia induced myocardial
3.5. Study of the biochemical composition modulation of ECM via some MMPs and their inhibitors In order to study the biochemical composition modulation of the myocardial extracellular matrix during Hhcy, we analyzed, by immunohistochemistry, the involvement of matrix metalloproteinases 2 and 9 and their tissue inhibitors 1 and 2 (Fig. 5). Our results show a variability in the immunohistochemical staining intensity of MMPs and TIMPs analyzed between the control and Hhcy groups. In the control
Fig. 4. Immunohistochemistry of collagens I (A) and III (B) in the heart of control rats and subjected to methionine and semi-quantification by imageJ software. In control rats, a low accumulation of collagen I (a) and III (c) between cardiomyocytes is observed and quantified. A significant accumulation of collagens I (b) and III (d) is reported and semi-quantified in the myocardium of Hhcy rats. The statistical analysis is performed by the Mann-Whitney test. p < 0.0001: significant difference between the control (n = 7) and Hhcy (n = 7) group. 5
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Fig. 5. Immunohistochemistry of MMP-2 and -9 and TIMP-1 and -2 in the heart of the control and Hhcy rats. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). Black arrows (strong cytoplasmic immunostaining of cardiomyocytes). Blue arrows (low cytoplasmic immunostaining of cardiomyocytes). Yellow arrows (low interstitial immunostaining). Green arrows (strong interstitial immunostaining). Red arrows (strong cytoplasmic immunostaining of inflammatory cells).
activated caspase 3 was reported in the cytoplasm of cardiomyocytes and cardiac fibroblasts (Fig. 6d).
group, a strong homogeneous immunostaining of MMP-2 in cardiomyocytes (Fig. 5a) was observed. In Hhcy rats, the immunostaining of MMP-2 was more or less important in cardiomyocytes with a heterogeneous intensity (Fig. 5b). Indeed, we noticed on the same section, strongly and weakly immunostained cardiomyocytes. No MMP-2 labeling was observed in cardiac fibroblasts and in the ECM in both groups of animals. The immunostaining of MMP-9 showed essentially a matrix distribution in both groups of animals with a higher rate and intensity in Hhcy rats versus control rats (Fig. 5c and d). At the cardiomyocytes level (Fig. 5c and d), a weak immunostaining of MMP-9 was observed in the two groups of Psammomys. Immunohistochemical analysis of the tissue inhibitors of these MMPs showed a very strong granular immunostaining of TIMP-1 in cardiomyocytes in the control group (Fig. 5e) and a weak one in the Hhcy group (Fig. 5f). No immunostaining of TIMP-1 was reported in cardiac fibroblasts or in the ECM in either group of animals. The results did not show any immunostaining of TIMP-2 in cardiomyocytes and in cardiac fibroblasts in the control group, contrarily to the Hhcy group, in which a strong immunostaining was observed in the cardiomyocytes (Fig. 5h). In addition, a strong granular immunostaining of TIMP-2 was reported in inflammatory cells (Fig. 5g) in both control and Hhcy animals. Results showed, therefore, that Hhcy caused in cardiac tissue an increase in the immuno-expression of MMP-9 and TIMP-2, associated with a decrease in the immuno-expression of MMP-2 and TIMP-1. The MMP-9/TIMP-1 ratio was increased, while the MMP-2/TIMP-2 ratio was decreased.
4. Discussion The present work showed that the administration of methionine at a dose of 300 mg/kg body weight/day for only 30 days generates Hhcy in Psammomys obesus. Sharma et al. (2007) reported in Wistar rats, an Hhcy after 4 weeks of administration of a high methionine dose (1 g/ kg/day). According to the same authors, lower doses of methionine (100, 250 and 500 mg/kg/day) cause Hhcy after 8 weeks. Moderate Hhcy was recorded by Kirac et al. (2013) in rats exposed to a high dose of methionine (1 g/kg/day) for 4 weeks. Raaf et al. (2011) and Ghoul et al. (2017) reported Hhcy in Wistar rats exposed to excess methionine (200 mg/kg/day) only from the 5th and 3rd month respectively. A diet containing 1.7% methionine causes Hhcy in male Sprague-Dawley rats within 4 weeks (Woo et al., 2006). These results show that a low dose administration of methionine for a short time produces Hhcy in Psammomys obesus compared to the Wistar rat. We noted a decrease of body weight in Hhcy rats during the experiment, but this body weight increases in control animals. Our results are in agreement with those of Zhou et al. (2001) and Velez-Carrasco et al. (2008) who reported a significant decrease in body weight in apoE−/− and wild mice exposed to excess methionine for 8 months and 8 weeks respectively. Some studies indicate an increase in body weight due to methionine (Raaf et al., 2011; Chiba et al., 2016) while others (Sharma et al., 2007; Singh et al., 2008; Xu et al., 2011) suggest that methionine excess does not appear to have any impact on body weight. In plasma, we recorded a significant decrease in glycemia and proteinemia in Hhcy rats. Lipid parameters (cholesterol, triglycerides and HDL) are not altered by methionine administration. Our result is similar to that of Zhang et al. (2016) who report no difference in glycemia between the general population and individuals with moderate Hhcy. In a large cohort of hypertensive patients, a small non-significant decrease in glycemia is reported between HHcy and normoHcy (Qin et al., 2017). The effects of Hhcy on plasma lipids reported in the bibliography are highly variable (Ozkan et al., 2002; Girelli et al., 2006; Naono et al., 2009; Qin et al., 2017; Strauss et al., 2017). Some studies involve oxidative stress (Au-Yeung et al., 2004) and
3.6. The TGF-β1 and activated caspase-3 The histological and immunohistochemical results obtained oriented this work to analyzing fibrosis via TGF-β1 and apoptosis via activated caspase 3. In control animals, the immunostaining of TGF-β1 was very weak at the cytoplasmic and perinuclear level in cardiomyocytes but also in some cardiac fibroblasts (Fig. 6a). High-intensity cytoplasmic immunostaining was observed in cardiomyocytes of Hhcy rats (Fig. 6b). Immunohistochemical analysis of activated caspase 3 showed very low levels of apoptotic cells in the heart of control animals (Fig. 6c). In contrast, in Hhcy animals, a strong immunostaining of 6
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Fig. 6. Immunohistochemistry of TGF-β1 and activated caspase 3 in the heart of the control and Hhcy rats. In the control group, a very low immunostaining of TGF-β1 is reported in cardiomyocytes, in cytoplasmic (black star) and perinuclear (black arrow) positions and in some cardiac fibroblasts (red arrow). Highly intense cytoplasmic immuno-staining is observed in the cardiomyocytes of Hhcy rats (red star). Except the few immune cells that immuno-express activated caspase 3 in controls (yellow arrow), no cardiomyocytes (green star) or cardiac fibroblasts (green arrow) express activated caspase 3. In Hhcy rats, strong immunolabelling of activated caspase 3 is observed in cardiomyocytes (blue star) and fibroblasts (blue arrow). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
development of perivascular and interstitial fibrosis associated with an increase in the diameter of cardiac myocytes. This same diet administered for 20 weeks also leads to the development of perivascular and interstitial fibrosis in Sprague-Dawley rats (Devi et al., 2006). Obese rabbits with abnormally high plasma Hcy levels have an accumulation of collagen I and III in myocardium associated with an increase in TGFβ1 expression (Carroll and Tyagi, 2005). Our result highlights the sensitivity of Psammomys obesus to methionine administration. Indeed, the myocardium of HHcy sand rats is the seat of both interstitial and perivascular fibrosis after use of a lower dose and shorter duration than those cited in the literature (Joseph et al., 2003; Devi et al., 2006; Zulli et al., 2006; Raaf et al., 2011). Myocardial fibrosis results from an imbalance between the synthesis and degradation of ECM elements, particularly fibrillar collagens types I and III (Ricard-blum et al., 2018). In Hhcy rats, we recorded an increase of TGF-β1 expression in cardiomyocytes, one of the main profibrotic mediators. The mechanisms underlying the installation of fibrosis via TGF-1 would involve the activation of the Smad 2/3 signalling pathway. Indeed, a study conducted in rats showed that TGF-β1 is responsible for myocardial fibrosis by activating the Smad 2/3 signalling pathway after experimental induction of myocardial infarction (Wang et al., 2017). According to these authors, the Smad-binding element (SBE) or CAGA box has been identified in the proximity of promoters of the COL1A2 and COL3A1 genes in humans. Hhcy can directly induce TGF-β1 expression via angiotensin II AT-1 receptor activation (Yao and Sun, 2014). Indeed, a recent study showed that Hcy can bind and directly activate the AT-1 receptor of angiotensin II causing agonist effects (Li et al., 2018). In cardiomyocytes and fibroblasts, Ang II binds to the AT-1 receptor and induces the expression of TGF-β1 by activation of
inflammation (Lazzerini et al., 2007) in the deleterious effects of Hhcy. In Hhcy rats, we recorded a decrease in plasma albumin, which has antioxidant properties (Taverna et al., 2013) and an increase in CRP, a marker of inflammation (Dhingra et al., 2007). An elevated plasma Hcy level associated with a decrease in plasma albumin has been previously reported (Ozkan et al., 2002; Valli et al., 2008). According to Głowacki and Jakubowski (2004), in the Hhcy case, one of the homocysteinylated forms of albumin, strongly increased, is more likely to be hydrolyzed by proteolytic enzymes than the native forms. This may explain the decrease in total plasma albumin that we recorded in Hhcy rats. Our results for CRP are in agreement with those reported by Pang et al. (2017) in rats rendered Hhcy by a diet containing 2% methionine for 4 weeks. Sharma et al. (2007) also reported a dose- and time-dependent increase in CRP in rats subjected to methionine doses ranging from 0.1 to 1 g/kg for 4 and 8 weeks. In the myocardium, Hhcy induces several cellular and matrix alterations. We observed in the Hhcy group a vacuolization of the cytoplasm of some cardiomyocytes with myofibril degradation suggesting autophagic cell death. Several studies highlight the involvement of Hhcy in the autophagy of different cell types, particularly neurons (Zhang et al., 2017), astrocytes (Tripathi et al., 2016) and hepatocytes (Yang et al., 2018). Our study showed the development of interstitial and perivascular fibrosis in the myocardium of Hhcy animals, due to the accumulation of fibrillar collagens I and III. These results are in agreement with those reported by several authors. Indeed, fibrosis is reported in the myocardium in Wistar rats subjected to methionine at a rate of 200 mg/kg body weight/day for 6 months (Raaf et al., 2011). Joseph et al. (2003) recorded in Wistar-Kyoto rats (9 g/kg homocysteine, 10 weeks), the
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Yasmina Benazzoug: Directorate of Labour in Algiers, Editing correction. Jean Marie Exbrayat: Directorate of Labour in Lyon. Editing correction.
the intracellular signalling pathway NAD(P)H oxidase/PKC/P38MAPK/AP-1 (Lim and Zhu, 2006). These data suggest that Hcy induces the synthesis of TGF-β1 in cardiac cells by the same mechanism as Ang II. By autocrine/paracrine pathway, TGF-β1 stimulates fibroblast proliferation, synthesis of ECM elements and cardiomyocyte hypertrophy (Rosenkranz, 2004). Epigenetic regulation of hcy by DNA methylation is one of the main processes that affect gene expression without altering the DNA sequence (Zhang, 2018). DNMTs, or DNA methyltransferases, are the key enzymes that enable DNA methylation in animals and convert S-adenosylmethionine (SAM) to S-adenosyl-homocysteine (SAH) during homocysteine metabolism (Miranda and Jones, 2007). The SAM/tissue SAM/SAH ratio, disrupted during Hhcy, affects the overall methylation of DNA or that of specific genes (Zhang, 2018). Similarly, Pan et al. showed that TGF-β1 induces COL1A1 expression in cardiac fibroblasts with a decrease in overall DNMT activity, particularly DNMT1 and DNMT3a, and hypomethylation of the COL1A1 promoter (Pan et al., 2013). According to several authors, the MMP/TIMP balance is important in maintaining ECM homeostasis (Givvimani et al., 2010; Kandasamy et al., 2010; Kumar et al., 2013). We found in rats HHcy, an increase in the MMP-9 and TIMP-2 immuno-expression and a decrease in the MMP2 and TIMP-1 immuno-expression leading to an increase in the MMP-9/ TIMP-1 ratio and a decrease in the MMP-2/TIMP-2 ratio. An increase in MMP-2 levels in the heart associated with a decrease in its active fraction has been reported in Wistar rats subjected to methionine excess for 6 months (Raaf et al., 2011). These authors also reported an increase in TIMP-2 expression and a decrease in TIMP-1 expression. Other studies have shown that Hcy at different doses (30 and 100μM) increases the MMP-2 and -9, and TIMP-1expression in the HL-1 cardiomyocyte line (Mishra et al., 2009). Similarly, Rosenberger et al. (2011) showed an increase in the MMP-2 and -9 expression in the myocardium of Hhcy mice. According to Münch et al. (2016), cardiac fibrosis is positively correlated with plasma MMP-9 concentration and negatively with MMP-2 concentration. In the heart, Hhcy activates MMP-9 which degrades connexin-43 and causes arrhythmia (Moshal et al., 2007). We have also shown that Hhcy induces apoptosis in cardiomyocytes by increasing the expression of activated caspase 3. In vitro studies on cardiomyocytes from CBS+/− mice incubated in the presence of Hcy (100 μM) for 24 h show that Hhcy initiates apoptosis by increasing the expression of activated caspase 3 (Wang et al., 2012). These effects are associated with the activation of the p38 MAPK signalling pathway, decreased thioredoxin expression and increased ROS production, particularly intracellular generation of peroxynitrite (Levrand et al., 2007).
Funding This work was supported by the Cnepru project F00220130015 (DGRSDT-Ministry of Higher Education and Scientific Research. Algeria). Declaration of Competing Interest None of the authors have any conflicts of interests. Acknowledgements We thank Leila Khedis and Caroline Bouchot for their technical support. We also thank Dr Zibouch. References Aouichat-Bouguerra, S., Benazzoug, Y., Bekkhoucha, F., Bourdillon, M.C., 2004. Effect of high glucose concentration on collagen synthesis and cholesterol level in the phenotypic modulation of aortic cultured smooth muscle cells of sand rat (Psammomys obesus). Exp. Diab. Res. 5, 227–235. https://doi.org/10.1080/15438600490489793. Au-Yeung, K.K.W., Woo, C.W.H., Sung, F.L., Yip, J.C.W., Siow, Y.L., Karmin, O., 2004. Hyperhomocysteinemia activates nuclear factor-κB in endothelial cells via oxidative stress. Circ. Res. 94, 28–36. https://doi.org/10.1161/01.RES.0000108264.67601.2C. Baszczuk, A., Musialik, K., Kopczynski, J., Thielemann, A., Kopczynski, Z., Kesy, L., Dopierała, G., 2014. Hyperhomocysteinemia, lipid and lipoprotein disturbances in patients with primary hypertension. Adv. Med. Sci. 59, 68–73. https://doi.org/10. 1016/j.advms.2013.08.001. Burns, D.M., 2003. Epidemiology of smoking-induced cardiovascular disease. Prog. Cardiovasc. Dis. 46, 11–29. https://doi.org/10.1016/S0033-0620(03)00079-3. Carroll, J.F., Tyagi, S.C., 2005. Extracellular matrix remodeling in the heart of the homocysteinemic obese rabbit. Am. J. Hypertens. 18, 692–698. https://doi.org/10. 1016/j.amjhyper.2004.11.035. Chiba, T., Suzuki, S., Sato, Y., Itoh, T., Umegaki, K., 2016. Evaluation of methionine content in a high-fat and choline-deficient diet on body weight gain and the development of non-alcoholic steatohepatitis in mice. PLoS One 11, 1–17. https://doi.org/ 10.1371/journal.pone.0164191. Devi, S., Kennedy, R.H., Joseph, L., Shekhawat, N.S., Melchert, R.B., Joseph, J., 2006. Effect of long-term hyperhomocysteinemia on myocardial structure and function in hypertensive rats. Cardiovasc. Pathol. 15, 75–82. https://doi.org/10.1016/j.carpath. 2005.11.001. Dhingra, R., Gona, P., Nam, B., D’Agostino, R.B., Wilson, P.W.F., Benjamin, E.J., O’Donnell, C.J., 2007. C-Reactive protein, inflammatory conditions, and cardiovascular disease risk. Am. J. Med. 120, 1054–1062. https://doi.org/10.1016/j.amjmed. 2007.08.037. Doumas, B.T., Bayse, D.D., Carter, R.J., Peters, T., Schaffer, R., 1981. A candidate reference method for determination of total protein in serum I. Development and validation. Clin. Chem. 27, 1642–1650. Eda, S., Kaufmann, J., Roos, W., Pohl, S., 1998. Development of a new microparticleenhanced turbidimetric assay for C-reactive protein with superior features in analytical sensitivity and dynamic range. J. Clin. Lab. Anal. 12, 137–144. https://doi.org/ 10.1002/(SICI)1098-2825(1998)12:3<137::AID-JCLA2>3.0.CO;2-6. Fossati, P., Prencipe, L., 1982. Serum triglycerides determined colorimetrically with an enzyme that produces hydrogen peroxide. Clin. Chem. 28, 2077–2080. Ghoul, A., Moudilou, E., Cherifi, M.E.H., Zerrouk, F., Chaouad, B., Moulahoum, A., Aouichat-bouguerra, S., Othmani, K., Exbrayat, J., Benazzoug, Y., 2017. The role of homocysteine in seminal vesicles remodeling in rat. Folia Histochem. Cytobiol. 55, 62–73. https://doi.org/10.5603/FHC.a2017.0010. Girelli, D., Martinelli, N., Olivieri, O., Pizzolo, F., Friso, S., Faccini, G., Bozzini, C., 2006. Hyperhomocysteinemia and mortality after coronary artery bypass grafting. PLoS One 1, e83. https://doi.org/10.1371/journal.pone.0000083. Givvimani, S., Tyagi, N., Sen, U., Mishra, P.K., Qipshidze, N., Munjal, C., Vacek, J.C., Abe, O.A., Tyagi, S.C., 2010. MMP-2/TIMP-2/TIMP-4 versus MMP-9/TIMP-3 in transition from compensatory hypertrophy and angiogenesis to decompensatory heart failure*. Arch. Physiol. Biochem. 116, 63–72. https://doi.org/10.3109/13813451003652997. Głowacki, R., Jakubowski, H., 2004. Cross-talk between Cys34 and lysine residues in human serum albumin revealed by N-homocysteinylation. J. Biol. Chem. 279, 10864–10871. https://doi.org/10.1074/jbc.M313268200. Joseph, J., Joseph, L., Shekhawat, N.S., Devi, S., Wang, J., Melchert, R.B., Hauer-jensen, M., Kennedy, R.H., Physiol, A.J., Circ, H., Hyper-, R.H.K., 2003. Hyperhomocysteinemia leads to pathological ventricular hypertrophy in normotensive rats. Am. J. Physiol. Heart Circ. Physiol. 285, H679–86. https://doi.org/10. 1152/ajpheart.00145.2003. Kandasamy, A.D., Chow, A.K., Ali, M.A.M., Schulz, R., 2010. Matrix metalloproteinase-2
5. Conclusions In summary, this work shows that in the short term, Hhcy induced by an excess of methionine induces in sand rats a remodeling of cardiac tissue characterized by the development of interstitial and perivascular fibrosis and by cardiomyocyte hypertrophy. This fibrosis, resulting from the accumulation of fibrillar collagens I and III and the modulation of their expression, is associated with an increase in the MMP-9/TIMP-1 ratio and a decrease in the MMP-2/TIMP-2 ratio, an increase in the expression of TGF-β1 and the induction of cardiomyocytes apoptosis. Author statement The Co-authors researchers who contributed to this work are as follow: Elara Moudilou: Development of immunohistochemistry protocol. Adel GHOUL and Fouzia Zerrouk: Assistance in animal experiments. Anissa Moulahoum and Khira Othmani-Mecif: Assistance in histological technique. Mohamed El Hadi Cherifi: dosage of Homocysteine and some biochemical plasma parameters. 8
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Hyperhomocysteinemia and myocardial remodeling in the sand rat, Psammomys obesus Billel Chaouada,b, Elara N. Moudilouc, Adel Ghoula, Fouzia Zerrouka, Anissa Moulahouma, ⁎ Khira Othmani-Mecifa, Mohamed El Hadi Cherifid, Jean-Marie Exbrayatc, Yasmina Benazzouga, a Biochemistry and Remodeling of the Extracellular Matrix, Laboratory of Cellular and Molecular Biology, Faculty of Biological Sciences, Houari Boumediene University of Science and Technology (USTHB), Bab Ezzouar, El Alia, 16111, Algiers, Algeria b University Djilali Bounaama of Khemis Miliana, Faculty of Natural and Life Sciences and Earth Sciences, Theniet El Had Road, 44225, Khemis Miliana, Algeria c UMRS 449, General Biology – Reproduction and Comparative Development, Lyon Catholic University, UDL, EPHE, PSL, 10, Place des Archives, 69288, Lyon Cedex 02, France d Central Laboratory of Biology, EPH Bologhine Ibn Ziri, Algiers, Algeria
A R T I C LE I N FO
A B S T R A C T
Keywords: Apoptosis Fibrosis Hyperhomocysteinemia Myocardium Psammomys obesus
Objective: Numerous studies have shown that a methionine-rich diet induces hyperhomocysteinemia (Hhcy), a risk factor for cardiovascular diseases. The objective of the present study was to determine the involvement of Hhcy in cardiac remodeling in the sand rat Psammomys obesus. Materials and methods: An experimental Hhcy was induced, in the sand rat Psammomys obesus, by intraperitoneal injection of 300 mg/kg of body weight/day of methionine for 1 month. The impact of Hhcy on the cellular and matricial structures of the myocardium was analyzed with histological techniques (Masson trichrome and Sirius red staining). Immunohistochemistry allowed us to analyze several factors involved in myocardial remodeling, such as fibrillar collagen I and III, metalloproteases (MMP-2 and -9) and their inhibitors (TIMP-1 and -2), TGF-β1 and activated caspase 3. Results: Our results show that Hhcy induced by an excess of methionine causes, in the myocardium of Psammomys obesus, a significant accumulation of fibrillar collagens I and III at the interstitial and perivascular scales, indicating the appearance of fibrosis, which is associated with an immuno-expression increase of TGF-β1, MMP-9 and TIMP-2 and an immuno-expression decrease of MMP-2 and TIMP-1. Also, Hhcy induces apoptosis of some cardiomyocytes and cardiac fibroblasts by increasing of activated caspase 3 expression. These results highlight a remodeling of cardiac tissue in hyperhomocysteinemic Psammomys obesus.
1. Introduction In our days, cardiovascular diseases such as myocardial infarction and atherosclerosis are the leading causes of death and morbidity in the world (Pagidipati and Gaziano, 2013). These diseases are caused by several risk factors, including smoking (Burns, 2003), hyperlipidemia (Nelson, 2013), high blood pressure (Roberts, 1987) and hyperhomocysteinemia (Hhcy) (Maurer et al., 2010; Refsum et al., 1998). It has been shown that Hhcy alone, without the intervention of other known risk factors, is responsible for 10% of heart failure in humans (Baszczuk et al., 2014). The history of homocysteine as a potential risk factor for cardiovascular diseases began in the 1960s when McCully observed vascular disorders in children with a hereditary deficiency of cystathionine-β-Synthetase, a key enzyme in homocysteine metabolism
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(McCully, 1969). In myocardium, Hhcy is strongly associated with tissue remodeling characterized by the accumulation of extracellular matrix elements (ECM), in particular fibrillary collagens (Raaf et al., 2011; Zhi et al., 2013), but also with hypertrophy and apoptosis of the cardiomyocytes (Levrand et al., 2007; Wei et al., 2010). This accumulation of collagens results from an imbalance between the activity of some matrix metalloproteinases (MMPs), particularly MMP-2 and -9 and their inhibitors, TIMP-1 and -2 (Kumar et al., 2013; Takawale et al., 2017). According to several studies, transforming growth factor β (TGF-β) is the leading mediator involved in cardiac fibrosis in diabetes (Yue et al., 2017), myocardial infarction (Matsumoto-ida et al., 2006) and Hhcy (Raaf et al., 2011). Psammomys obesus or sand rat is recognized as an excellent
Corresponding author. E-mail address: [email protected] (Y. Benazzoug).
https://doi.org/10.1016/j.acthis.2019.07.008 Received 19 March 2019; Received in revised form 22 July 2019; Accepted 25 July 2019 0065-1281/ © 2019 Elsevier GmbH. All rights reserved.
Please cite this article as: Billel Chaouad, et al., Acta Histochemica, https://doi.org/10.1016/j.acthis.2019.07.008
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immunoturbidimetric method was used for C-reactive protein (CRP) (Eda et al., 1998).
polygenic animal model of metabolic syndrome closely resembling that observed in humans (Walder et al., 2002). This desert rodent is unique in the study of both obesity, insulin resistance, type 2 diabetes (Ziv et al., 1999), dyslipidemia (Walder et al., 2002) and atherosclerosis (Aouichat-Bouguerra et al., 2004). Several cardiac alterations have been reported in Psammomys obesus subjected to a high-fat diet (Sahraoui et al., 2016). However, there is no study on cardiac remodeling during Hhcy has been performed in this species. The present work consists of studying and analyzing some histopathological and immuno-histochemical factors involved in myocardial remodeling in hyperhomocysteinemic sand rats by daily administration of methionine. We analyzed the fibrillar collagens type I and III, the metalloproteinases MMP-2 and -9 and their tissue inhibitors TIMP-1 and -2, the TGF-β1 and the activated caspase 3.
2.4. Histological and histochemical analysis The histological and histochemical study was performed with 7 control rats and 7 Hhcy rats. At the end of the experiment, the animals were euthanized by intraperitoneal injection of 70 mg/kg of body weight of Ketamine and then dissected. The organs removed were quickly fixed in Bouin's solution for 2 days and then dehydrated in alcohol baths of increasing degree (50°, 70°, 90°, and 100°). The organs were then cleared in 2 butanol baths and embedded in paraffin. The organs were cut off using a Lab-Kite microtome. The 5μm-thick cuts obtained were spread on Superfrost-plus glass slides. After drying for 24 h at 37 °C, the slides were subjected to topographic staining (Masson trichrome) and selective histochemical staining of fibrillar collagens (Sirius red).
2. Materials and methods 2.1. Biological material and experimental protocol
2.5. Immunohistochemistry This study was carried out in Psammomys obesus, an athero-sensible animal model of the gerbillidea class. This desert rodent was caught in the M'Sila region (Algeria) in July - August 2016 (sexual rest). After an adaptation period of 15 days in the laboratory (temperature 25 °C, 50% humidity, 12-h light/dark cycle), the adult rats were divided into two groups of 15 animals each, a control group and a treated group of mean body weight of 144 g and 145 g respectively. The 2 lots were fed with halophilic plants and subjected to an injection of physiological water (0.9% NaCl) for the control lot and methionine (Sigma-Aldrich, 64340, St. Louis, MO 63103 USA), previously dissolved in physiological water, at a dose of 300 mg/kg of body weight/day for the treated lot. During the 30-day experiment, the animals were weekly weighted. All experiments were carried out in accordance with the Algerian legislation (Law number 12-235/2012) relating to animal protection, the recommendations of the Algerian Association of Experimental Animal Sciences (AASEA 45/DGLPAG/DVA/SDA/14) and the EU Directive 2010/63/EU for animal experiments. Blood samples were collected at the beginning (T0) and at the end (T30) of the experiment from the retro-orbital sinus of the eyes using a previously heparinized Pasteur pipette. Blood collected on heparinized tubes was centrifuged at 3000 rpm for 10 min. The collected plasma was stored at −80 °C for the determination of some biochemical plasma parameters.
The immunohistochemistry technique was performed using the indirect avidin-biotin-peroxidase amplification method (Vectastain Elite Universal Kit, PK-6200, Vector Laboratories, CA, USA) to detect and localize fibrillar collagens type I and III, MMP-2 and -9, TIMPs 1 and 2, TGF-β1 and activated caspase 3 in the cardiac sections. 5μm-thick histological sections of the heart were deparaffinized in cyclohexane and then rehydrated in ethanol baths of decreasing degree. In order to block the activity of endogenous peroxidases, the sections were incubated for 40 min in a 3% H2O2 solution. After washing with PBSx1 for 3 min, the sections were incubated in a 2% BSA solution for 60 min in order to block non-specific binding sites. The primary antibody was applied on the sections overnight at 4 °C in the following dilutions: 1/ 100 for anti-collagen I (rabbit polyclonal - Abcam ab34710, Cambridge, UK), anti-collagen III (rabbit polyclonal - Abcam ab7778, Cambridge, UK), anti-TGF-β1 (rabbit polyclonal - Abcam ab92486) and activated anti-caspase 3 (rabbit monoclonal - Abcam, ab32042), 1/25 for antiMMP-2 (rabbit polyclonal - Abcam ab37150, Cambridge, UK), antiMMP-9 (rabbit polyclonal - Abcam ab38898, Cambridge, UK), antiTIMP 1 (mouse monoclonal - Santa Cruz sc-21734) and anti-TIMP-2 (mouse monoclonal - Santa Cruz sc-271932). Antigen retrieval in a basic citrate solution (H-3300, Vector Laboratories, CA, USA) was required for anti-TGF-β1 and anti-TIMP-1 and -2 antibodies. After application of the antibodies, the sections were rinsed twice with PBSx1 for 5 min and incubated 60 min with the secondary biotinylated antibody (Anti-Mouse/Anti-Rabbit IgG, Vector Laboratories, CA, USA). Another PBSx1 wash was required before and after application for 60 min of the avidin-biotin-peroxidase complex. After the revelation with NovaRed (peroxidase substrate, SK-4805, Vector Laboratories, CA, USA) and the hematoxylin QS counter-staining (H-3404, Vector Laboratories, CA, USA), the sections were dehydrated and then mounted in Permount (Fisher Chemical, USA). Negative control was performed for each protein analyzed by the omission of the primary antibody.
2.2. Determination of total plasma homocysteine The quantitative determination of total plasma homocysteine was performed by the Architect Homocysteine assay kit (Axis-Shield Diagnostics Ltd, Dundee, UK) using CMIA (chemiluminescent microparticle immunoassay) technology. The oxidized form of homocysteine present in the sample was reduced to free homocysteine by the action of dithiothreitol. Total free homocysteine was transformed into S-adenosyl-L-homocysteine (SAH) by recombinant SAH-hydrolase in the presence of adenosine in excess. SAH and S-adenosyl-L-cysteine labelled with acridinium competed to fill the binding sites on the anti-SAH monoclonal antibody. After washing, magnetic separation and triggering reactions, the light emitted by the acridinium was measured in relative units of light by the optical system of Architect analyzer. An indirect relationship exists between the homocysteine present in the sample and the amount of light emitted.
2.6. Morphometric study and quantification of fibrillar collagens The myocardium histological and histochemical analysis of control and treated animals is completed by a morphometric study. This study was performed with AxioVision 4.8 software from CARL ZEISS, after calibration and at x1000 magnification. The measurements were made on transversely cut cardiomyocytes with clearly visible nuclei for cellular parameters (large and small axes, surface) and on longitudinally cut cardiomyocytes for nuclear diameter and surface evaluation. For each parameter analyzed, we performed 100 measurements (on average 15–17 randomly selected per animal). In addition, Sirius red stained sections were used to quantify fibrillar collagens at interstitial and
2.3. Determination of some biochemical plasma parameters Different plasma parameters were quantified by the enzymatic colorimetric method: glucose (Trinder, 1969), cholesterol (Richmond, 1973), triglycerides (Fossati and Prencipe, 1982) and HDL (Warnick and Wood, 1995). Colorimetric methods were also performed for total proteins (Doumas et al., 1981) and albumin (Rodkey, 1965). The 2
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perivascular level using Threshold function in imageJ software (1.47uNational institutes of health. USA). For each control and treated animal (N = 7), the quantification of interstitial collagens was performed by measuring the percentage of the stained surface related to the total surface area over 6 to 8 sections of 19000μm2 tissue randomly selected at x1000 magnification. The quantification of perivascular collagens was performed by the ratio stained surface area to total surface area of the blood vessel. We selected 3 to 4 blood vessels distributed in the myocardial tissue for each control and treated animal (N = 7). Immunohistochemistry and imageJ software allowed us to quantify type I (14–15 measurements/animal) and type III (10–12 measurements/animal) collagens on 19000μm2 tissue sections randomly selected at x1000 magnification.
Table 1 Body weight and some biochemical plasma parameters in control and Hhcy sand rats. Parameters
2.7. Statistical analysis Quantitative results were analyzed by STATISTICA 6.1 (StatSoft, Inc.). The values were expressed as a mean affected from standard error to mean (SEM). The Mann-Whitney test was used to evaluate the difference between the parameters of control and treated animals. When the values of P were lower than 0.05, the difference was considered statistically significant.
T0 values
After 30 days of experimentation Control Group
Hhcy Groupe 9/6 129.77 ± 7.68 (p < 0.05) 0.71 ± 0.06 (p < 0.0001) 47.57 ± 2.20 (p < 0.05)
Sex (male/female) Body weight (g)
16/14 144.62 ± 6.57
7/8 157.33 ± 7.28
Glycemia (g/l)
0.96 ± 0.03
1.07 ± 0.04
Proteinemia (g/l)
53.76 ± 1.08
55.18 ± 1.73
Lipids Cholesterolemia (g/l) Triglyceridemia (g/l) HDL (g/l) Albumin (g/l)
0.41 ± 0.02 0.59 ± 0.07 0.22 ± 0.01 27.34 ± 0.60
0.46 ± 0.03 0.59 ± 0.10 0.25 ± 0.02 28.04 ± 0.52
CRP (mg/l)
0.04 ± 0.01
0.03 ± 0.01
0.44 ± 0.06 (ns) 0.44 ± 0.09 (ns) 0.27 ± 0.03 (ns) 24.22 ± 1.23 (p < 0.05) 0.06 ± 0.01 (p < 0.05)
The values represent the mean ± SEM. The statistical analysis is performed by the Mann-Whitney test. ns: non-significant difference between the Control and Treated group. p < 0.05 and p < 0.0001: significant difference between the Control (n = 15) and Treated group (n = 15).
3. Results
in blood glucose (p < 0.0001) and a 13.79% decrease in proteinemia (p < 0.05) were recorded. Lipid parameters (cholesterol, triglyceride, and HDL) did not appear to be altered consequently to methionine administration. A decrease (p < 0.05) was also observed in plasma albumin of 13.62%, while the CRP dosage, a marker of inflammation, showed a 50% increased (p < 0.05) in Hhcy rats.
3.1. Quantification of total plasma homocysteine The baseline value of homocysteinemia determined in all sand rats (n = 30) at the beginning of the experiment was 1.52 ± 0.15 μmol/l. The homocysteinemia recorded at the end of the experiment (30 days) in control and subjected to methionine (Met) is shown in Fig. 1. Our results show an increase (p < 0.0001) of 875.64% in plasma homocysteine in rats subjected to methionine, inducing hyperhomocysteinemia. This parameter increases from 2.34 ± 0.23 μmol/l in the control group to 22.83 ± 5.20 μmol/l in the treated group which then became hyperhomocysteinemic (Hhcy).
3.3. Histo-morphometry of the myocardium The increase in plasma homocysteine concentration in sand rats caused significant cardiac alterations at both the cellular and matrix level. Matrix alterations were characterized by fibrosis (Fig. 2b and c) which resulted in a focused accumulation of fibrillar collagens at the interstitial (Fig. 3A) and perivascular (Fig. 3B) levels with a decrease in cell density in the fibrotic regions. The semi-quantification of fibrillar collagens at the interstitial level (Fig. 3A) showed an increase of 632.90% (P < 0.0001) in Hhcy rats compared to control rats (17.15 ± 1.3% vs 2.34 ± 0.09%). At the perivascular level (Fig. 3B), the semi-quantification of fibrillar collagens showed an increase of 83.18% (P < 0.0001) in the Hhcy rats compared to the controls (38.67 ± 1.13% vs 21.11 ± 0.36%). Vacuolation of the cytoplasm was observed in some cardiomyocytes (Fig. 2c). Many of these cells were hypertrophied, but others were atrophied in fibrotic areas particularly (Fig. 2c). These results were confirmed by the morphometric study which shows an increase (P < 0.0001) of all the cellular and nuclear parameters of cardiomyocytes in Hhcy rats compared to controls (Table 2).
3.2. Impact of hyperhomocysteinemia on body weight and some biochemical plasma parameters Table 1 shows the evolution of body weight and some biochemical plasma parameters in control and Hhcy sand rats. At the end of the experiment, the body weight of Hhcy rats was decreased by 17.52% compared to that of controls. A 33.64% decrease
3.4. Immunohistochemical analysis of collagens I and III In order to determine the type of collagens implicated in this cardiac fibrosis, an immunohistochemical study of fibrillar collagen I and III was performed (Fig. 4). In the control group (N = 7), a weak immunostaining of collagens I and III was observed in the interstitial tissue between cardiomyocytes. The semi-quantification by imageJ showed that collagens I and III occupied 9.78 ± 0.33% and 7.74 ± 0.26% of the myocardium surface respectively. In the interstitial tissue, collagen I was more present than collagen III. In the treated group (N = 7), a significant accumulation of these two types of collagens was observed between the cardiac cells. In Hhcy
Fig. 1. Homocysteinemia in control (n = 15) and treated (n = 15) animals. The statistical analysis is performed by the Mann-Whitney test. p < 0.0001: significant difference. 3
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Fig. 2. Myocardial histology of control animals (a) and Hhcy (b, c). Masson trichrome staining. The myocardium of control rats consists of several cardiomyocytes (CM) surrounded by weak vascularized connective tissue (CT). This tissue is composed of a few collagen fibres and many cardiac fibroblasts (CF). The myocardium of Hhcy rats is characterized by significant diffuse interstitial fibrosis (black arrows), hypertrophy of some cardiomyocytes (brown arrow) and vacuolation in their cytoplasm (yellow arrows). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
Fig. 3. Heart histological sections of control and Hhcy rats stained by Sirius red and quantification of the fibrillar collagens at the interstitial (A) and perivascular (B) level by imageJ software. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). In control rats, fibrillar collagens cover a small percentage of the myocardium (a) and blood vessels (c). In Hhcy rats, there is a significant accumulation of fibrillar collagens in the myocardium (b) and vascular adventitia (d). The statistical analysis is performed by the Mann-Whitney test. p < 0.0001: significant difference between the control (n = 7) and Hhcy (n = 7) group. 4
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Table 2 Cardiomyocytes morphometric study of some cellular and nuclear parameters in control and Hhcy rats. Control
Hhcy
Cellular parameters
Major axis (μm) Small axis (μm) Surface area (μm2)
20.67 ± 0.56 13.32 ± 0.32 223.98 ± 10.15
28.39 ± 0.85 (p < 0.0001) 16.69 ± 0.53 (p < 0.0001) 394.58 ± 20.29 (p < 0.0001)
Nuclear parameters
Major axis (μm) Small axis (μm) Surface area (μm2)
9.52 ± 0.14 4.08 ± 0.07 30.90 ± 0.70
12.71 ± 0.14 (p < 0.0001) 4.48 ± 0.06 (p < 0.0001) 45.04 ± 0.49 (p < 0.0001)
The values represent the mean ± SEM. The statistical analysis is performed by the Mann-Whitney test. p < 0.0001: All cellular and nuclear parameters (n = 100) of cardiomyocytes are statistically different between the control (n = 7) and Hhcy groups (n = 7).
fibrosis in Psammomys obesus, via a simultaneous increase in the expression of collagen I and III, indicating a modulation of the biochemical composition of myocardial ECM.
rats, collagens I and III represented respectively 28.86 ± 0.76% and 27.66 ± 0.83% of the myocardial surface area. Therefore, our results show a significant increase (p < 0.0001) of 195.09% and 257.36% in the surface area occupied by collagen I and III respectively in Hhcy rats compared to controls. This result highlighted a change in the proportion of the two collagen types in the interstitial tissue. We noted that collagen I was more represented than collagen III in interstitial tissue in both control and Hhcy animals. However, the analysis of our results indicates that Psammomys obesus Hhcy showed a variation in the increase of the two types of collagen. Indeed, collagen III was 3.6 times more important than in the control group, while collagen I was 2.7 times more important. Our results showed that hyperhomocysteinemia induced myocardial
3.5. Study of the biochemical composition modulation of ECM via some MMPs and their inhibitors In order to study the biochemical composition modulation of the myocardial extracellular matrix during Hhcy, we analyzed, by immunohistochemistry, the involvement of matrix metalloproteinases 2 and 9 and their tissue inhibitors 1 and 2 (Fig. 5). Our results show a variability in the immunohistochemical staining intensity of MMPs and TIMPs analyzed between the control and Hhcy groups. In the control
Fig. 4. Immunohistochemistry of collagens I (A) and III (B) in the heart of control rats and subjected to methionine and semi-quantification by imageJ software. In control rats, a low accumulation of collagen I (a) and III (c) between cardiomyocytes is observed and quantified. A significant accumulation of collagens I (b) and III (d) is reported and semi-quantified in the myocardium of Hhcy rats. The statistical analysis is performed by the Mann-Whitney test. p < 0.0001: significant difference between the control (n = 7) and Hhcy (n = 7) group. 5
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Fig. 5. Immunohistochemistry of MMP-2 and -9 and TIMP-1 and -2 in the heart of the control and Hhcy rats. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). Black arrows (strong cytoplasmic immunostaining of cardiomyocytes). Blue arrows (low cytoplasmic immunostaining of cardiomyocytes). Yellow arrows (low interstitial immunostaining). Green arrows (strong interstitial immunostaining). Red arrows (strong cytoplasmic immunostaining of inflammatory cells).
activated caspase 3 was reported in the cytoplasm of cardiomyocytes and cardiac fibroblasts (Fig. 6d).
group, a strong homogeneous immunostaining of MMP-2 in cardiomyocytes (Fig. 5a) was observed. In Hhcy rats, the immunostaining of MMP-2 was more or less important in cardiomyocytes with a heterogeneous intensity (Fig. 5b). Indeed, we noticed on the same section, strongly and weakly immunostained cardiomyocytes. No MMP-2 labeling was observed in cardiac fibroblasts and in the ECM in both groups of animals. The immunostaining of MMP-9 showed essentially a matrix distribution in both groups of animals with a higher rate and intensity in Hhcy rats versus control rats (Fig. 5c and d). At the cardiomyocytes level (Fig. 5c and d), a weak immunostaining of MMP-9 was observed in the two groups of Psammomys. Immunohistochemical analysis of the tissue inhibitors of these MMPs showed a very strong granular immunostaining of TIMP-1 in cardiomyocytes in the control group (Fig. 5e) and a weak one in the Hhcy group (Fig. 5f). No immunostaining of TIMP-1 was reported in cardiac fibroblasts or in the ECM in either group of animals. The results did not show any immunostaining of TIMP-2 in cardiomyocytes and in cardiac fibroblasts in the control group, contrarily to the Hhcy group, in which a strong immunostaining was observed in the cardiomyocytes (Fig. 5h). In addition, a strong granular immunostaining of TIMP-2 was reported in inflammatory cells (Fig. 5g) in both control and Hhcy animals. Results showed, therefore, that Hhcy caused in cardiac tissue an increase in the immuno-expression of MMP-9 and TIMP-2, associated with a decrease in the immuno-expression of MMP-2 and TIMP-1. The MMP-9/TIMP-1 ratio was increased, while the MMP-2/TIMP-2 ratio was decreased.
4. Discussion The present work showed that the administration of methionine at a dose of 300 mg/kg body weight/day for only 30 days generates Hhcy in Psammomys obesus. Sharma et al. (2007) reported in Wistar rats, an Hhcy after 4 weeks of administration of a high methionine dose (1 g/ kg/day). According to the same authors, lower doses of methionine (100, 250 and 500 mg/kg/day) cause Hhcy after 8 weeks. Moderate Hhcy was recorded by Kirac et al. (2013) in rats exposed to a high dose of methionine (1 g/kg/day) for 4 weeks. Raaf et al. (2011) and Ghoul et al. (2017) reported Hhcy in Wistar rats exposed to excess methionine (200 mg/kg/day) only from the 5th and 3rd month respectively. A diet containing 1.7% methionine causes Hhcy in male Sprague-Dawley rats within 4 weeks (Woo et al., 2006). These results show that a low dose administration of methionine for a short time produces Hhcy in Psammomys obesus compared to the Wistar rat. We noted a decrease of body weight in Hhcy rats during the experiment, but this body weight increases in control animals. Our results are in agreement with those of Zhou et al. (2001) and Velez-Carrasco et al. (2008) who reported a significant decrease in body weight in apoE−/− and wild mice exposed to excess methionine for 8 months and 8 weeks respectively. Some studies indicate an increase in body weight due to methionine (Raaf et al., 2011; Chiba et al., 2016) while others (Sharma et al., 2007; Singh et al., 2008; Xu et al., 2011) suggest that methionine excess does not appear to have any impact on body weight. In plasma, we recorded a significant decrease in glycemia and proteinemia in Hhcy rats. Lipid parameters (cholesterol, triglycerides and HDL) are not altered by methionine administration. Our result is similar to that of Zhang et al. (2016) who report no difference in glycemia between the general population and individuals with moderate Hhcy. In a large cohort of hypertensive patients, a small non-significant decrease in glycemia is reported between HHcy and normoHcy (Qin et al., 2017). The effects of Hhcy on plasma lipids reported in the bibliography are highly variable (Ozkan et al., 2002; Girelli et al., 2006; Naono et al., 2009; Qin et al., 2017; Strauss et al., 2017). Some studies involve oxidative stress (Au-Yeung et al., 2004) and
3.6. The TGF-β1 and activated caspase-3 The histological and immunohistochemical results obtained oriented this work to analyzing fibrosis via TGF-β1 and apoptosis via activated caspase 3. In control animals, the immunostaining of TGF-β1 was very weak at the cytoplasmic and perinuclear level in cardiomyocytes but also in some cardiac fibroblasts (Fig. 6a). High-intensity cytoplasmic immunostaining was observed in cardiomyocytes of Hhcy rats (Fig. 6b). Immunohistochemical analysis of activated caspase 3 showed very low levels of apoptotic cells in the heart of control animals (Fig. 6c). In contrast, in Hhcy animals, a strong immunostaining of 6
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Fig. 6. Immunohistochemistry of TGF-β1 and activated caspase 3 in the heart of the control and Hhcy rats. In the control group, a very low immunostaining of TGF-β1 is reported in cardiomyocytes, in cytoplasmic (black star) and perinuclear (black arrow) positions and in some cardiac fibroblasts (red arrow). Highly intense cytoplasmic immuno-staining is observed in the cardiomyocytes of Hhcy rats (red star). Except the few immune cells that immuno-express activated caspase 3 in controls (yellow arrow), no cardiomyocytes (green star) or cardiac fibroblasts (green arrow) express activated caspase 3. In Hhcy rats, strong immunolabelling of activated caspase 3 is observed in cardiomyocytes (blue star) and fibroblasts (blue arrow). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
development of perivascular and interstitial fibrosis associated with an increase in the diameter of cardiac myocytes. This same diet administered for 20 weeks also leads to the development of perivascular and interstitial fibrosis in Sprague-Dawley rats (Devi et al., 2006). Obese rabbits with abnormally high plasma Hcy levels have an accumulation of collagen I and III in myocardium associated with an increase in TGFβ1 expression (Carroll and Tyagi, 2005). Our result highlights the sensitivity of Psammomys obesus to methionine administration. Indeed, the myocardium of HHcy sand rats is the seat of both interstitial and perivascular fibrosis after use of a lower dose and shorter duration than those cited in the literature (Joseph et al., 2003; Devi et al., 2006; Zulli et al., 2006; Raaf et al., 2011). Myocardial fibrosis results from an imbalance between the synthesis and degradation of ECM elements, particularly fibrillar collagens types I and III (Ricard-blum et al., 2018). In Hhcy rats, we recorded an increase of TGF-β1 expression in cardiomyocytes, one of the main profibrotic mediators. The mechanisms underlying the installation of fibrosis via TGF-1 would involve the activation of the Smad 2/3 signalling pathway. Indeed, a study conducted in rats showed that TGF-β1 is responsible for myocardial fibrosis by activating the Smad 2/3 signalling pathway after experimental induction of myocardial infarction (Wang et al., 2017). According to these authors, the Smad-binding element (SBE) or CAGA box has been identified in the proximity of promoters of the COL1A2 and COL3A1 genes in humans. Hhcy can directly induce TGF-β1 expression via angiotensin II AT-1 receptor activation (Yao and Sun, 2014). Indeed, a recent study showed that Hcy can bind and directly activate the AT-1 receptor of angiotensin II causing agonist effects (Li et al., 2018). In cardiomyocytes and fibroblasts, Ang II binds to the AT-1 receptor and induces the expression of TGF-β1 by activation of
inflammation (Lazzerini et al., 2007) in the deleterious effects of Hhcy. In Hhcy rats, we recorded a decrease in plasma albumin, which has antioxidant properties (Taverna et al., 2013) and an increase in CRP, a marker of inflammation (Dhingra et al., 2007). An elevated plasma Hcy level associated with a decrease in plasma albumin has been previously reported (Ozkan et al., 2002; Valli et al., 2008). According to Głowacki and Jakubowski (2004), in the Hhcy case, one of the homocysteinylated forms of albumin, strongly increased, is more likely to be hydrolyzed by proteolytic enzymes than the native forms. This may explain the decrease in total plasma albumin that we recorded in Hhcy rats. Our results for CRP are in agreement with those reported by Pang et al. (2017) in rats rendered Hhcy by a diet containing 2% methionine for 4 weeks. Sharma et al. (2007) also reported a dose- and time-dependent increase in CRP in rats subjected to methionine doses ranging from 0.1 to 1 g/kg for 4 and 8 weeks. In the myocardium, Hhcy induces several cellular and matrix alterations. We observed in the Hhcy group a vacuolization of the cytoplasm of some cardiomyocytes with myofibril degradation suggesting autophagic cell death. Several studies highlight the involvement of Hhcy in the autophagy of different cell types, particularly neurons (Zhang et al., 2017), astrocytes (Tripathi et al., 2016) and hepatocytes (Yang et al., 2018). Our study showed the development of interstitial and perivascular fibrosis in the myocardium of Hhcy animals, due to the accumulation of fibrillar collagens I and III. These results are in agreement with those reported by several authors. Indeed, fibrosis is reported in the myocardium in Wistar rats subjected to methionine at a rate of 200 mg/kg body weight/day for 6 months (Raaf et al., 2011). Joseph et al. (2003) recorded in Wistar-Kyoto rats (9 g/kg homocysteine, 10 weeks), the
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Yasmina Benazzoug: Directorate of Labour in Algiers, Editing correction. Jean Marie Exbrayat: Directorate of Labour in Lyon. Editing correction.
the intracellular signalling pathway NAD(P)H oxidase/PKC/P38MAPK/AP-1 (Lim and Zhu, 2006). These data suggest that Hcy induces the synthesis of TGF-β1 in cardiac cells by the same mechanism as Ang II. By autocrine/paracrine pathway, TGF-β1 stimulates fibroblast proliferation, synthesis of ECM elements and cardiomyocyte hypertrophy (Rosenkranz, 2004). Epigenetic regulation of hcy by DNA methylation is one of the main processes that affect gene expression without altering the DNA sequence (Zhang, 2018). DNMTs, or DNA methyltransferases, are the key enzymes that enable DNA methylation in animals and convert S-adenosylmethionine (SAM) to S-adenosyl-homocysteine (SAH) during homocysteine metabolism (Miranda and Jones, 2007). The SAM/tissue SAM/SAH ratio, disrupted during Hhcy, affects the overall methylation of DNA or that of specific genes (Zhang, 2018). Similarly, Pan et al. showed that TGF-β1 induces COL1A1 expression in cardiac fibroblasts with a decrease in overall DNMT activity, particularly DNMT1 and DNMT3a, and hypomethylation of the COL1A1 promoter (Pan et al., 2013). According to several authors, the MMP/TIMP balance is important in maintaining ECM homeostasis (Givvimani et al., 2010; Kandasamy et al., 2010; Kumar et al., 2013). We found in rats HHcy, an increase in the MMP-9 and TIMP-2 immuno-expression and a decrease in the MMP2 and TIMP-1 immuno-expression leading to an increase in the MMP-9/ TIMP-1 ratio and a decrease in the MMP-2/TIMP-2 ratio. An increase in MMP-2 levels in the heart associated with a decrease in its active fraction has been reported in Wistar rats subjected to methionine excess for 6 months (Raaf et al., 2011). These authors also reported an increase in TIMP-2 expression and a decrease in TIMP-1 expression. Other studies have shown that Hcy at different doses (30 and 100μM) increases the MMP-2 and -9, and TIMP-1expression in the HL-1 cardiomyocyte line (Mishra et al., 2009). Similarly, Rosenberger et al. (2011) showed an increase in the MMP-2 and -9 expression in the myocardium of Hhcy mice. According to Münch et al. (2016), cardiac fibrosis is positively correlated with plasma MMP-9 concentration and negatively with MMP-2 concentration. In the heart, Hhcy activates MMP-9 which degrades connexin-43 and causes arrhythmia (Moshal et al., 2007). We have also shown that Hhcy induces apoptosis in cardiomyocytes by increasing the expression of activated caspase 3. In vitro studies on cardiomyocytes from CBS+/− mice incubated in the presence of Hcy (100 μM) for 24 h show that Hhcy initiates apoptosis by increasing the expression of activated caspase 3 (Wang et al., 2012). These effects are associated with the activation of the p38 MAPK signalling pathway, decreased thioredoxin expression and increased ROS production, particularly intracellular generation of peroxynitrite (Levrand et al., 2007).
Funding This work was supported by the Cnepru project F00220130015 (DGRSDT-Ministry of Higher Education and Scientific Research. Algeria). Declaration of Competing Interest None of the authors have any conflicts of interests. Acknowledgements We thank Leila Khedis and Caroline Bouchot for their technical support. We also thank Dr Zibouch. References Aouichat-Bouguerra, S., Benazzoug, Y., Bekkhoucha, F., Bourdillon, M.C., 2004. Effect of high glucose concentration on collagen synthesis and cholesterol level in the phenotypic modulation of aortic cultured smooth muscle cells of sand rat (Psammomys obesus). Exp. Diab. Res. 5, 227–235. https://doi.org/10.1080/15438600490489793. Au-Yeung, K.K.W., Woo, C.W.H., Sung, F.L., Yip, J.C.W., Siow, Y.L., Karmin, O., 2004. Hyperhomocysteinemia activates nuclear factor-κB in endothelial cells via oxidative stress. Circ. Res. 94, 28–36. https://doi.org/10.1161/01.RES.0000108264.67601.2C. Baszczuk, A., Musialik, K., Kopczynski, J., Thielemann, A., Kopczynski, Z., Kesy, L., Dopierała, G., 2014. Hyperhomocysteinemia, lipid and lipoprotein disturbances in patients with primary hypertension. Adv. Med. Sci. 59, 68–73. https://doi.org/10. 1016/j.advms.2013.08.001. Burns, D.M., 2003. Epidemiology of smoking-induced cardiovascular disease. Prog. Cardiovasc. Dis. 46, 11–29. https://doi.org/10.1016/S0033-0620(03)00079-3. Carroll, J.F., Tyagi, S.C., 2005. Extracellular matrix remodeling in the heart of the homocysteinemic obese rabbit. Am. J. Hypertens. 18, 692–698. https://doi.org/10. 1016/j.amjhyper.2004.11.035. Chiba, T., Suzuki, S., Sato, Y., Itoh, T., Umegaki, K., 2016. Evaluation of methionine content in a high-fat and choline-deficient diet on body weight gain and the development of non-alcoholic steatohepatitis in mice. PLoS One 11, 1–17. https://doi.org/ 10.1371/journal.pone.0164191. Devi, S., Kennedy, R.H., Joseph, L., Shekhawat, N.S., Melchert, R.B., Joseph, J., 2006. Effect of long-term hyperhomocysteinemia on myocardial structure and function in hypertensive rats. Cardiovasc. Pathol. 15, 75–82. https://doi.org/10.1016/j.carpath. 2005.11.001. Dhingra, R., Gona, P., Nam, B., D’Agostino, R.B., Wilson, P.W.F., Benjamin, E.J., O’Donnell, C.J., 2007. C-Reactive protein, inflammatory conditions, and cardiovascular disease risk. Am. J. Med. 120, 1054–1062. https://doi.org/10.1016/j.amjmed. 2007.08.037. Doumas, B.T., Bayse, D.D., Carter, R.J., Peters, T., Schaffer, R., 1981. A candidate reference method for determination of total protein in serum I. Development and validation. Clin. Chem. 27, 1642–1650. Eda, S., Kaufmann, J., Roos, W., Pohl, S., 1998. Development of a new microparticleenhanced turbidimetric assay for C-reactive protein with superior features in analytical sensitivity and dynamic range. J. Clin. Lab. Anal. 12, 137–144. https://doi.org/ 10.1002/(SICI)1098-2825(1998)12:3<137::AID-JCLA2>3.0.CO;2-6. Fossati, P., Prencipe, L., 1982. Serum triglycerides determined colorimetrically with an enzyme that produces hydrogen peroxide. Clin. Chem. 28, 2077–2080. Ghoul, A., Moudilou, E., Cherifi, M.E.H., Zerrouk, F., Chaouad, B., Moulahoum, A., Aouichat-bouguerra, S., Othmani, K., Exbrayat, J., Benazzoug, Y., 2017. The role of homocysteine in seminal vesicles remodeling in rat. Folia Histochem. Cytobiol. 55, 62–73. https://doi.org/10.5603/FHC.a2017.0010. Girelli, D., Martinelli, N., Olivieri, O., Pizzolo, F., Friso, S., Faccini, G., Bozzini, C., 2006. Hyperhomocysteinemia and mortality after coronary artery bypass grafting. PLoS One 1, e83. https://doi.org/10.1371/journal.pone.0000083. Givvimani, S., Tyagi, N., Sen, U., Mishra, P.K., Qipshidze, N., Munjal, C., Vacek, J.C., Abe, O.A., Tyagi, S.C., 2010. MMP-2/TIMP-2/TIMP-4 versus MMP-9/TIMP-3 in transition from compensatory hypertrophy and angiogenesis to decompensatory heart failure*. Arch. Physiol. Biochem. 116, 63–72. https://doi.org/10.3109/13813451003652997. Głowacki, R., Jakubowski, H., 2004. Cross-talk between Cys34 and lysine residues in human serum albumin revealed by N-homocysteinylation. J. Biol. Chem. 279, 10864–10871. https://doi.org/10.1074/jbc.M313268200. Joseph, J., Joseph, L., Shekhawat, N.S., Devi, S., Wang, J., Melchert, R.B., Hauer-jensen, M., Kennedy, R.H., Physiol, A.J., Circ, H., Hyper-, R.H.K., 2003. Hyperhomocysteinemia leads to pathological ventricular hypertrophy in normotensive rats. Am. J. Physiol. Heart Circ. Physiol. 285, H679–86. https://doi.org/10. 1152/ajpheart.00145.2003. Kandasamy, A.D., Chow, A.K., Ali, M.A.M., Schulz, R., 2010. Matrix metalloproteinase-2
5. Conclusions In summary, this work shows that in the short term, Hhcy induced by an excess of methionine induces in sand rats a remodeling of cardiac tissue characterized by the development of interstitial and perivascular fibrosis and by cardiomyocyte hypertrophy. This fibrosis, resulting from the accumulation of fibrillar collagens I and III and the modulation of their expression, is associated with an increase in the MMP-9/TIMP-1 ratio and a decrease in the MMP-2/TIMP-2 ratio, an increase in the expression of TGF-β1 and the induction of cardiomyocytes apoptosis. Author statement The Co-authors researchers who contributed to this work are as follow: Elara Moudilou: Development of immunohistochemistry protocol. Adel GHOUL and Fouzia Zerrouk: Assistance in animal experiments. Anissa Moulahoum and Khira Othmani-Mecif: Assistance in histological technique. Mohamed El Hadi Cherifi: dosage of Homocysteine and some biochemical plasma parameters. 8
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