Effects of Homocysteine on Proliferation, Necrosis, and Apoptosis of Vascular Smooth Muscle Cells in Culture and Influence of Folic Acid

Thrombosis Research 104 (2001) 207 – 213

REGULAR ARTICLE

Effects of Homocysteine on Proliferation, Necrosis, and Apoptosis of Vascular Smooth Muscle Cells in Culture and Influence of Folic Acid

Michele Buemi1, Demetrio Marino2, Giuseppe Di Pasquale3, Fulvio Floccari1, Antonella Ruello1, Carmela Aloisi1, Francesco Corica1, Massimino Senatore1, Adolfo Romeo1 and Nicola Frisina1 1 Department of Internal Medicine, Faculty of Medicine, University of Messina, Messina, Italy; 2Institute of General Physiology, Faculty of Science, University of Messina, Messina, Italy and 3Institute of Pediatrics, Faculty of Medicine, University of Messina, Messina, Italy (Received 25 January 2001 by Editor R. Lorenzet; revised/accepted 8 August 2001)

Abstract Background: It is known that hyperhomocysteinemia is associated with an increased risk of vascular disease, yet little is known about the pathogenic mechanisms underlying the action of homocysteine (Hcy) itself. Methods: We evaluated the effects of Hcy on cell proliferation, apoptosis, and necrosis in smooth muscle cells (SMCs) cultured for 24 h with different amounts of Hcy. The percentage of apoptotic and necrotic cells from the culture was evaluated using two different techniques: annexin V–FITC and propidium iodide (PI) fluorescence and apoptosis TUNEL assay. Results: The addition of 10 mM/l of Hcy to the medium was followed by a significant increase in cell proliferation and death, through apoptosis and necrosis, respectively. Notwithstanding this apparent balance, a significant increase was found in the total number of cells present in Hcy-treated culture, thus demonstrating a positive dose-dependent correlation with Hcy concentrations in the culture medium. The addition of folic acid to the culture medium significantly reduced both Hcy concentrations in media and the effects of Corresponding author: Prof. Michele Buemi, MD, Via Salita Villa Contino, 30, 98100 Messina, Italy. Tel: +39 (90) 2930 586; Fax: +39 (90) 2935 162; E-mail: .

Hcy on the proliferation/apoptosis/necrosis balance of cells in culture. The percentages for apoptotic cells and for cells with a necrotic morphology continued to increase as Hcy concentrations increased, although the absolute values were lower in the culture treated than in that not treated with folic acid. Conclusions: In the presence of folic acid, at increasing concentrations of Hcy, the total number of cells in culture showed increases far less relevant with respect to the control. Also the percentage of apoptotic cells to that of cells with a necrotic morphology, although conserving the tendency to increase to growth of the concentrations of Hcy, have shown absolute values that were lower in the folic acid-treated cultures. D 2001 Elsevier Science Ltd. All rights reserved. Key Words: Homocysteine; Atherosclerosis; Vascular myocytes; Apoptosis

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omocysteine (Hcy), an amino acid containing sulfhydryl, is an intermediate product in the methionine conservation cycle and in the one-carbon metabolic and transsulfuration pathways [1]. The recent discovery of a close correlation between the plasma Hcy levels and cardiovascular disease has prompted studies on its possible use in the prevention of cardiovascular morbidity and mortality [2]. Moreover,

0049-3848/01/$ – see front matter D 2001 Elsevier Science Ltd. All rights reserved. PII S0049-3848(01)00363-2

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in a large recent prospective study, hyperhomocysteinemia was defined as an independent risk factor for myocardial infarction, stroke, and atherosclerosis [3,4]. Moreover, data reported in literature demonstrate that therapy with folates, vitamin B6, and vitamin B12 can reduce hyperhomocysteinemia, and therefore have a direct protective effect on the vessel wall [5–7]. However, the mechanism by which hyperhomocysteinemia induces vascular remodeling is unclear. Hcy appears to promote arterial wall growth through a number of mechanisms. In their study on the effect of Hcy on the growth of vascular muscle cells and endothelial cells, Tsai et al. [8] found a 25% increase in DNA synthesis in rat aortic smooth muscle cells (SMCs). On the other hand, in human umbilical vein endothelial cells, Hcy has been found to reduce DNA synthesis in a dose-dependent manner. These findings suggest that Hcy has a growth-promoting effect on vascular SMCs and an inhibitory effect on endothelial cell growth. This combination could lead to atherosclerosis. It was recently demonstrated that cardiovascular remodeling during atherosclerosis expresses an imbalance between proliferative and cell death pathways [9]. A cell can die from necrosis, death being due to external forces, and occurring too rapidly to allow the activation of a protective mechanism. In nature, there is another type of death: apoptosis, a condition in which cells shrink rather than swell, with a 30% loss in their volume in less than an hour. Chromatin condenses under the nuclear membrane and the cell fragments. As the cell does not expel its contents, inflammatory phenomena are obviated. It is not yet known whether necrosis and apoptosis are a continuum to each other, or whether they are completely independent processes [10,11]. The accumulation of cells is the main cause of intimal thickening in vascular disease. This process depends on the proliferation of SMCs and macrophages, even if the rate of proliferation is slow. Likewise, cell death is also involved in atherogenesis. Recently reported findings in humans demonstrate that apoptosis plays a role in cell death in lesions from atherosclerosis, as do findings in an animal model of lesions from atherosclerosis and in the arteries of newborn lambs [12]. The aim

of the present study was therefore to investigate apoptosis and necrosis in human vascular myocyte cultures exposed to increasingly high Hcy concentrations. A further end-point was to establish whether folic acid influences the proliferation/apoptosis/necrosis balance.

1. Materials and Method Human vascular myocytes (A617 from the human femoral artery) were cultured following the Ross’ method [13] in a single layer at 37 C in a humidified atmosphere of 5% CO2 in modified Eagle’s medium (MEM), supplemented with 10% (v/v) fetal calf serum (FCS), 100 U/ml penicillin, 0.1 mg/ml streptomycin, 20 mM tricine buffer, and 1% (v/v) nonessential amino acid solution. The total cysteine concentration was within the physiological range, and a concentration of CN-Cbl 500 nmol/l was used. The culture medium was changed every third day. Cells between the 4th and 10th passages were used. SMCs were identified for growth behavior and morphology using a monoclonal antibody specific for smooth muscle actin. The actin isoform typical of SMC was also identified. Cells, growing from explants after 12 – 16 days and piling up after confluency, contained numerous myofilaments and dense bodies, observed by transmission electron microscopy. The cultures were incubated for 48 h. The study was made on glass plates from 24 wells with inoculations of 3  105 cells/well in 1 ml of culture medium. Apoptosis, necrosis, and cell proliferation were evaluated 24 h after the addition of Hcy (Sigma, St. Louis, MO, USA) at the following concentrations: 10, 15, and 20 mM. In a second batch of cultures, we studied the effects of increasing concentrations of Hcy (10, 15, and 20 mM) and folic acid at a concentration of 15 mM. To the third batch of culture media, we added folic acid alone, at concentrations of 5, 10, and 15 mM. These concentrations were chosen because in previous experiments we had found them to be highly effective. Hcy concentrations in culture medium were evaluated at Time 0 and 24 h after the addition of Hcy in all cultures. This assay was performed with high-pressure liquid chromatography.

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1.1. Evaluation of Cell Proliferation

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An index of cell proliferation was determined by measuring the proliferating cell nuclear antigens that are expressed only in proliferating cells. The monoclonal antibodies (anti-Ki-67, clone K1-S5, Roche, Germany) to cell cycleassociated antigens, which are mouse monoclonal antibodies, react with nuclear antigens expressed only in proliferating cells. They do not react with cytoplasmic antigens or with resting cells.

cals). Positive cells were visualized using a peroxidase substrate enhancer and a metal-enhanced DAB substrate (Boehringer Mannheim Biochemicals). SMCs were split into the medium and grown on glass coverslips. A terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay was performed as suggested by the manufacturer. Cells were counterstained with 2% methyl green pyronin. Control cultures were prepared without Hcy or folic acid in the medium.

1.2. Apoptotic and Necrotic Cell Count

1.3. Statistical Analysis

The percentages of apoptotic cells in culture were evaluated using two different techniques: annexin V–FITC and propidium iodide (PI) fluorescence and apoptosis TUNEL assay (Roche).

The statistical analysis of data was made using an SPSS statistical software package, release 7.5 (SPSS, Chicago, IL, USA). Student’s t test for paired and unpaired data was used to evaluate the difference between variables studied during the treatment periods with respect to baseline values (washout). Pearson’s correlation coefficient was used to investigate the relationships between the variables studied. Data are expressed as a mean ± S.D. P values of < .05 were considered statistically significant.

1.2.1. Annexin V–FITC and PI Annexin V–FITC conjugates allow the identification and qualification of cell surface changes that occur early during the apoptotic process when flow cytometry is used. These conjugates facilitate the rapid fluorometric quantification of apoptotic cells. By simultaneously staining cells with annexin V–FITC and the nonvital dye, PI, it is possible to distinguish between intact cells (FITC , PI ), early apoptotic (FITC+, PI ), and late apoptotic or necrotic cells (FITC+, PI+). After being washed twice in ice-cold PBS and pelletted by centrifugation at 1000  g for 10 min, cells were resuspended at 106 cells/ 100 ml in a binding buffer (HEPES buffer 10 mM, pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2), stained with 10 ml annexin V (1 mg/ml in binding buffer) and 10 ml PI (50 ml in binding buffer), and incubated in the dark for 15 min at room temperature. Then, after adding 500 ml of binding buffer, the cells were processed by flow cytometry, 10,000 cells per sample were collected, and data were acquired with LYSYS II phenotyping analysis software [14]. 1.2.2. TUNEL Assay This assay was performed with an in situ cell detection kit (Boehringer Mannheim Biochemi-

2. Results 2.1. Effects of Hcy on Proliferation in Vascular SMCs The effects of Hcy on SMC were determined by growing human SMC from the femoral arteries for 24 h with or without Hcy in the culture medium. The standard culture medium used in all experiments contained physiological concentrations of cysteine and high concentrations of CN-B1. Cell proliferation increased significantly in the presence of increasing concentrations of Hcy in a dose-dependent manner: on adding only 10 mM of Hcy to SMC cultures, the number of cells in culture after 24-h exposure increased by 60% with respect to the control ( P < .001). At Hcy concentrations of 16 and 20 mM, the mean overall number of cells in culture was, respectively, 62% ( P < .001) and 63% ( P < .001) higher than the that for the control (Fig. 1).

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2.3. Effects of Hcy on Necrosis Hcy increased the percentage of cells with a necrotic morphology present in culture, values being statistically significant for Hcy concentrations of 15 (20%, P < .003) and 20 mM (22%, P < .003) (Fig. 3). 2.4. Effects of Folic Acid on Proliferation, Apoptosis, and Necrosis in Vascular SMC

Fig. 1. Effect of Hcy concentration on proliferation in SMC cultures. Data are expressed as D% vs. control. White columns: Hcy-treated cultures. Gray columns: Hcy + folic acid (10 mM)-treated cultures. * P < .05, vs. control; # P < .05, vs. only Hcy.

2.2. Effects of Hcy on Apoptosis The mean percentage of apoptotic cells present in our control culture was 4%, but it was 16 ( P < .003) in cultures treated with Hcy 10 mM, with progressive, but minor, increases for Hcy concentrations of 15 (18%, P < .002) and 20 mM (19% P < .002) (Fig. 2).

Fig. 2. Effect of Hcy addition on apoptotic cell percentage in SMC cultures. Data are expressed as D% vs. control. White columns: Hcy-treated cultures. Gray columns: Hcy + folic acid (10 mM)-treated cultures. * P < .05, vs. control; # P < .05, vs. only Hcy.

The addition of folic acid to the medium had no statistically significant effect on the proliferation of cells in culture. Percentages for apoptotic and necrotic cells increased to a statistically significant extent. 2.5. Effects of Folic Acid on Proliferation, Apoptosis, and Necrosis in Vascular SMCs Treated With Hcy The addition of folic acid to the culture medium containing Hcy reduced the effects of Hcy on the proliferation/apoptosis/necrosis balance of cells in culture: in the presence of folic acid, a statistically significant increase was observed in the mean total number of cells in culture. The percentages of apoptotic cells and cells with a necrotic morphology continued to increase as Hcy concentrations increased, although the

Fig. 3. Effect of Hcy addition on necrotic cell percentage in SMC cultures. Data are expressed as D% vs. control. White columns: Hcy-treated cultures. Gray columns: Hcy + folic acid (10 mM)-treated cultures. * P < .05, vs. control; # P < .05, vs. only Hcy.

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Fig. 4. Effect of Folic acid on Hcy concentrations in SMC cultures. White columns: Hcy-treated cultures at Time 0. Pale gray columns: Hcy + folic acid at 24 h. Dark gray columns: Hcy-treated cultures at 24 h. * P < .05, vs. control; # P < .05, vs. only Hcy.

absolute values were lower in folic acid-treated cell cultures. 2.6. Effects of Folic Acid on Hcy Concentrations in Culture Medium The addition of folic acid to the culture medium containing Hcy led to a statistically significant reduction in the Hcy concentration (Fig. 4).

3. Discussion Numerous studies indicate that increase in plasma levels of Hcy, even if only slight, is an independent risk factor for vascular disease [1,2]. The atherogenous properties of Hcy may be responsible for the increased vascular risk in hyperhomocysteinemic patients. Several factors may be responsible for the increased risk of atherosclerosis in the presence of high Hcy concentrations: the direct cytotoxic effect of Hcy on the endothelial cells, stimulation of proliferation of vascular muscular cells, stimulation of oxidative stress, direct prothrombotic effect, and a direct effect on genes GRP 78/BIP [15–20]. In order to clarify the pathogenesis of vascular lesions, we evaluated the effect of exposure to increasing Hcy concentrations on the proliferation/apoptosis/necrosis balance in SMCs in cul-

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ture. In physiological conditions, at a vascular level, there is an exact balance between proliferation and cell death [9,21]. A selective increase in proliferation with respect to cell death can lead to hyperplasia, while an increase in apoptosis leads to atrophy. Death, a somewhat unpopular topic in our society, can play a positive role, at least as far as cells are concerned. It is crucial to normal tissue turnover: in adult humans, the number of cells making up tissue must be kept constant while guaranteeing cell renewal and regulating the delicate balance between cellular proliferation, differentiation, and death. Although there is a close correlation between the survival of a cell and its host, we do not yet understand the relation between these two processes. As has recently emerged in studies conducted on vascular SMC cultures, it is not yet known whether necrosis and apoptosis may be a continuum to each other or completely independent processes [10]. The present study therefore evaluated the effects of low concentrations of Hcy (10–20 mM) on cell proliferation in cultures, but also the entire proliferation/death balance. Previous studies in vitro on this aspect have often involved the use of extremely high Hcy concentrations, which are often difficult to achieve in vivo. The most important finding in our study was the constant increase in the total number of cells present in the Hcy-treated cultures, at concentrations of only 10 mM. We believe that the further increases in the total populations of cells in cultures treated with higher concentrations indicate a dose-related response pattern in the tendency of cells to proliferate in the presence of Hcy. Moreover, the percentage of apoptotic cells and that of cells with a necrotic morphology in culture increased significantly in the presence of increasingly high Hcy concentrations. Notwithstanding this increase, the total number of cells present in culture treated with Hcy increased significantly, thus revealing dose-dependent positive correlation with the concentrations of Hcy in the culture medium. Our data are in line with the observation made by Tsai et al. [8] concerning one of the possible atherogenic mechanisms. They demonstrate, however, that stimulation of proliferation of

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SMCs by Hcy occurs at concentrations as low as 10–20 mM. Furthermore, our data clearly demonstrate that the effects of the exposure of cells in culture to Hcy are correlated not only with the promotor effect that this toxin has on proliferation, but also, and above all, with the breakdown of the complex balance between cell proliferation and death, due to apoptosis and necrosis, respectively. Many studies in vivo have shown the utility of folic acid supplementation in subjects with hyperhomocysteinemia [2]. Woo et al. [7] and Bellamy et al. [22] have also demonstrated that folic acid supplementation can lower Hcy levels in these subjects. Our in vitro data are in agreement with those obtained in vivo by Vermeulen et al. [6]: the addition of folic acid to the culture medium caused a significant reduction in the effects of Hcy on the proliferation/apoptosis/necrosis balance of cells in culture. In the presence of folic acid, at increasing concentrations of Hcy the total number of cells in culture increased far less markedly than in the control. Moreover, the ratio of apoptotic cells to cells with a necrotic morphology conserved the tendency to increase as Hcy concentrations increased, but absolute values were lower in the folic acid-treated cultures. The presence of folic acid in the culture medium may therefore favor the catabolism of Hcy, via the remethylation of Hcy in methionine, and the effect of the 5-methylthetrahydro-homocysteinemethyl-transferase (methionine synthesis) and of the 5,10-methylene-tetraidrofolate reductase. The protective effect of folic acid against the toxic action of Hcy may therefore be linked to an enzymatic reduction in the concentrations of the toxin. We found that the addition of folic acid to the medium caused a statistically significant reduction in the concentration of Hcy in the medium (Fig. 4). In conclusion, our findings demonstrate that the promotor effect of Hcy on SMC proliferation (even at concentrations of 10–20 mM) depends on the breakdown in the overall balance between cell proliferation and death. They, moreover, provide in vitro confirmation of the utility of folic acid administration in the prevention of vascular risk linked to hyperhomocysteinemia.

References 1. Majors A, Ehrhart A, Pezacka EH. Homocysteine as a risk factor for vascular disease. Arterioscler, Thromb, Vasc Biol 1997;17:2074–81. 2. Chambers JC, Obeid OA, Refsum H, Ueland P, Hackett D, Hooper J, Turner RM, Thompson SG, Kooner JS. Plasma homocysteine concentrations and risk of coronary heart disease in UK Indian Asian and European men. Lancet 2000;355(9203):523–7 (February 12). 3. Welch G, Loscalzo J. Homocysteine and atherothrombosis. N Engl J Med 1998;338:1042–50. 4. Stampfer MJ, Malinow R, Willet WC, Newcomer LM, Upson B, Ullman D, Tishler PV, Hennekens CH. A prospective study of plasma homocysteine and risk of myocardial infarction in US physicians. JAMA, J Am Med Assoc 1992;268:877–81. 5. Chambers JC, Obeid OA, Kooner JS. Physiological increments in plasma homocysteine induce vascular endothelial dysfunction in normal human subjects. Arterioscler, Thromb, Vasc Biol 1999;19(12):2922–7. 6. Vermeulen EG, Stehouwer CD, Twisk JW, van der Berg M, de Jong SC, Mackaay AJ, van Campen CM, Visser FC, Jakobs CA, Bulterjis EJ, Rauwerda JA. Effect of homocysteine-lowering treatment with folic acid plus vitamin B6 on progression of subclinical atherosclerosis: a randomised, placebocontrolled trial. Lancet 2000;355(9203):517–22 (February 12). 7. Woo KS, Chook P, Lolin YI, Sanderson JE, Metreweli C, Celermajer DS. Folic acid improves arterial endothelial function in adults with hyperhomocysteinemia. J Am Coll Cardiol 1999;34(7):2002–6. 8. Tsai JC, Perrella MA, Yoshizumi M, Hsieh CM, Haber E, Schlegel R, Lee ME. Promotion of vascular smooth muscle cells growth by homocysteine: a link to atherosclerosis. Proc Natl Acad Sci USA 1994;97:146–53. 9. Buemi M, Corica F, Marino D, Medici MA, Aloisi C, Di Pasquale G, Ruello A, Sturiale A, Senatore M, Frisina N. Cardiovascular remodelling apoptosis and drugs. Am J Hypertens 2000;13:450–4.

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10. Raffray M, Cohen GM. Apoptosis and necrosis in toxicology: a continuum or distinct modes of cell death? Pharmacol Ther 1997; 75:153–77. 11. Champagne MJ, Dumas P, Orlov SN, Bennet MR, Hamet P, Tremblay J. Protection against necrosis but not apoptosis by HSPs in vascular smooth muscle cells: evidence for distinct modes of cell death. Hypertension 1999; 33:906–13. 12. Cai WJ, Devaux B, Schaper W, Schaper J. The role of Fas/APO 1 and apoptosis in the development of human atherosclerotic lesions. Atherosclerosis 1997;131:177–86. 13. Ross R. Growth of smooth muscle in culture and formation of elastic fibers. J Cell Biol 1991; 50:172–7. 14. Sun XM, Snowden RT, Skilleter DN, Dinsdate D, Ormerod MG, Cohen GM. A flow cytometric method for the separation and quantitation of normal and apoptotic thymocytes. Ann Biochem 1992;204:351–6. 15. Tsai JC, Wang H, Perrella MA, Yoshizumi M, Sibinga NE, Tan LC, Haber E, Chang TH, Schlegel R, Lee ME. Induction of cyclin A gene expression by homocysteine in vascular smooth muscle cells. J Clin Invest 1996;97: 146–53. 16. McCully KS. Vascular pathology of homocysteinemia: implications for the pathogenesis of arteriosclerosis. Am J Pathol 1969;56: 111–28.

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17. Rolland PH, Friggi A, Barlatier A, Piquet P, Latrille V, Faye MM, Guillou J, Charpiot P, Bodard H, Ghiringhelli O. Hyperhomocysteinemia-induced vascular damage in the minipig. Captopril-hydrochlorothiazide combination prevents elastic alterations. Circulation 1995;91:1161–74. 18. Stamler JS, Osborne JA, Jaraki O, Rabboni LE, Mullins M, Singel D, Loscalyo S. Adverse vascular effects of homocysteine are modulated by endothelium-derived relaxing factor and related oxides of nitrogen. J Clin Invest 1993;91:308–18. 19. Welch GN, Upchurch GR, Lo Scalzo J. Hyperhomocysteinemia and atherothrombosis. Ann NY Acad Sci 1997;811:48–58. 20. Heinecke JW. Superoxide-mediated oxidation of low density lipoprotein by thiols. In: Cerrutti PA, Fridovich I, McCord JM, editors. Oxy-radicals in molecular biology and pathology. New York: Alan R. Liss, 1988. pp. 443–57. 21. Mulvany MJ, Baumbach GL, Aalkjaer C, Heagerty M, Korsgaard N, Schiffrin EL, Heistad DD. Vascular remodeling. Hypertension 1996;28:505–6. 22. Bellamy MF, McDowell LF, Ramsey MW, Brownlee M, Newcombe RG, Lewis MJ. Oral folate enhances endothelial function in hyperhomocysteinaemic subjects. Eur J Clin Invest 1999;29(8):659–62 (Aug).