Effect of L-homocysteine on endothelial cell-cell junctions following F-actin stabilization through tropomyosin-1 overexpression

Gagat,Maciej; Grzanka,Dariusz; Izdebska,Magdalena; Grzanka,Alina

doi:10.3892/ijmm.2013.1357

Effect of L-homocysteine on endothelial cell-cell junctions following F-actin stabilization through tropomyosin-1 overexpression

Authors:
- Maciej Gagat
- Dariusz Grzanka
- Magdalena Izdebska
- Alina Grzanka
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Affiliations: Department of Histology and Embryology, Nicolaus Copernicus University in Toruń, Collegium Medicum in Bydgoszcz, 85-092 Bydgoszcz, Poland, Department of Clinical Pathomorphology, Nicolaus Copernicus University in Toruń, Collegium Medicum in Bydgoszcz, 85-092 Bydgoszcz, Poland
Published online on: April 22, 2013 https://doi.org/10.3892/ijmm.2013.1357
Pages: 115-129

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Abstract

Since the identification of actin in non‑muscle cells, it has been suggested that the regulation of the mechanical behaviors of the actin cytoskeleton regulates cellular shape changes and the generation of forces during cell migration and division. The maintenance of cell shape and polarity are important in the formation of cell-cell junctions. The aim of the present study was to determine the effect of L‑homocysteine thiolactone hydrochloride on EA.hy926 endothelial cells in the context of the maintenance cell-cell junctions through the stabilization of filamentous actin cytoskeleton (F‑actin). The actin filaments were stabilized by the overexpression of tropomyosin-1, which has the ability to stabilize actin filaments in muscle and non-muscle cells. The stabilization of F-actin induced a significant decrease in the percentage of late apoptotic and necrotic cells following treatment with L-homocysteine. Moreover, the migratory potential of the endothelial cells was greater in the cells overexpressing tropomyosin-1 treated with L-homocysteine. Additionally, our results indicated that the stabilization of F-actin in the EA.hy926 cells significantly increased the expression of junctional β‑catenin, as compared to the cells not overexpressing tropomyosin‑1. Similarly, the fluorescence intensity of junctional α-catenin was also increased in the cells with stabilized F‑actin cytoskeleton. However, this increase was only slightly higher than that observed in the EA.hy926 cells not overexpressing tropomyosin-1. Furthermore, the analysis of Zonula occludens (ZO)‑1 relative fluorescence demonstrated a statistically significant decrease in the cell-cell junction areas among the cells with stabilized F-actin cytoskeleton in comparison to the cells not overexpressing tropomyosin-1. Our results indicate that the stabilization of F-actin does not affect the migratory potential of cells, and consequently protects the EA.hy926 cells against the L-homocysteine-induced decrease in cell mobility. Moreover, it is suggested that α‑catenin may participate in the suppression of actin polymerization in the area of cell-cell junctions. It can be hypothesized that the stabilization of F-actin strengthens endothelial adherens and tight junctions by increasing the number of cell-cell junctions due to the amplification of β-catenin and the ZO‑1 fluorescence signal. However, ZO-1 stabilizes the endothelial barrier function through the stabilization of F-actin and F-actin itself stabilizes the localization of ZO-1.

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1.	Pollard TD: Cytoskeletal functions of cytoplasmic contractile proteins. J Supramol Struct. 5:317–334. 1976. View Article : Google Scholar : PubMed/NCBI
2.	Clarke M and Spudich JA: Nonmuscle contractile proteins: the role of actin and myosin in cell motility and shape determination. Annu Rev Biochem. 46:797–822. 1977. View Article : Google Scholar : PubMed/NCBI
3.	Stossel TP: Contractile proteins in cell structure and function. Annu Rev Med. 29:427–457. 1978. View Article : Google Scholar : PubMed/NCBI
4.	Stricker J, Falzone T and Gardel ML: Mechanics of the F-actin cytoskeleton. J Biomech. 43:9–14. 2010. View Article : Google Scholar : PubMed/NCBI
5.	Zhang J, Betson M, Erasmus J, Zeikos K, Bailly M, Cramer LP and Braga VM: Actin at cell-cell junctions is composed of two dynamic and functional populations. J Cell Sci. 118:5549–5562. 2005. View Article : Google Scholar : PubMed/NCBI
6.	Homeister JW and Willis MS: Atherosclerosis: Pathogenesis, Genetics and Experimental Models. Encyclopedia of Life Sciences 2010. John Wiley & Sons Ltd; Chichester: 2010, View Article : Google Scholar
7.	Boushey CJ, Beresford SA, Omenn GS and Motulsky AG: A quantitative assessment of plasma homocysteine as a risk factor for vascular disease. Probable benefits of increasing folic acid intakes. JAMA. 274:1049–1057. 1995. View Article : Google Scholar : PubMed/NCBI
8.	Nygård O, Vollset SE, Refsum H, et al: Total plasma homocysteine and cardiovascular risk profile. The Hordaland Homocysteine Study. JAMA. 274:1526–1533. 1995.PubMed/NCBI
9.	Ueland PM, Refsum H, Beresford SA and Vollset SE: The controversy over homocysteine and cardiovascular risk. Am J Clin Nutr. 72:324–332. 2000.PubMed/NCBI
10.	Tehlivets O: Homocysteine as a risk factor for atherosclerosis: is its conversion to s-adenosyl-L-homocysteine the key to deregulated lipid metabolism? J Lipids. 2011:7028532011. View Article : Google Scholar : PubMed/NCBI
11.	McDowell IF and Lang D: Homocysteine and endothelial dysfunction: a link with cardiovascular disease. J Nutr. 130:369S–372S. 2000.PubMed/NCBI
12.	Heydrick SJ, Weiss N, Thomas SR, Cap AP, Pimentel DR, Loscalzo J and Keaney JF Jr: L-Homocysteine and L-homocystine stereospecifically induce endothelial nitric oxide synthase-dependent lipid peroxidation in endothelial cells. Free Radic Biol Med. 36:632–640. 2004. View Article : Google Scholar
13.	Dejana E: Endothelial cell-cell junctions: happy together. Nat Rev Mol Cell Biol. 5:261–270. 2004. View Article : Google Scholar : PubMed/NCBI
14.	Hoelzle MK and Svitkina T: The cytoskeletal mechanisms of cell-cell junction formation in endothelial cells. Mol Biol Cell. 23:310–323. 2012. View Article : Google Scholar : PubMed/NCBI
15.	Bazzoni G, Martínez Estrada O and Dejana E: Molecular structure and functional role of vascular tight junctions. Trends Cardiovasc Med. 9:147–152. 1999. View Article : Google Scholar : PubMed/NCBI
16.	Braga VM: Cell-cell adhesion and signalling. Curr Opin Cell Biol. 14:546–556. 2002. View Article : Google Scholar : PubMed/NCBI
17.	Matter K and Balda MS: Signalling to and from tight junctions. Nat Rev Mol Cell Biol. 4:225–236. 2003. View Article : Google Scholar : PubMed/NCBI
18.	Wheelock MJ and Johnson KR: Cadherin-mediated cellular signaling. Curr Opin Cell Biol. 15:509–514. 2003. View Article : Google Scholar : PubMed/NCBI
19.	Harris ES and Nelson WJ: VE-cadherin: at the front, center, and sides of endothelial cell organization and function. Curr Opin Cell Biol. 22:651–658. 2010. View Article : Google Scholar : PubMed/NCBI
20.	Harris TJ and Tepass U: Adherens junctions: from molecules to morphogenesis. Nat Rev Mol Cell Biol. 11:502–514. 2010. View Article : Google Scholar : PubMed/NCBI
21.	Yonemura S: Cadherin-actin interactions at adherens junctions. Curr Opin Cell Biol. 23:515–522. 2011. View Article : Google Scholar : PubMed/NCBI
22.	Nwariaku FE, Liu Z, Zhu X, et al: NADPH oxidase mediates vascular endothelial cadherin phosphorylation and endothelial dysfunction. Blood. 104:3214–3220. 2004. View Article : Google Scholar : PubMed/NCBI
23.	Kinumi T, Ogawa Y, Kimata J, Saito Y, Yoshida Y and Niki E: Proteomic characterization of oxidative dysfunction in human umbilical vein endothelial cells (HUVEC) induced by exposure to oxidized LDL. Free Radic Res. 39:1335–1344. 2005. View Article : Google Scholar : PubMed/NCBI
24.	Xu Y, Arai H, Murayama T, Kita T and Yokode M: Hypercholesterolemia contributes to the development of atherosclerosis and vascular remodeling by recruiting bone marrow-derived cells in cuff-induced vascular injury. Biochem Biophys Res Commun. 363:782–787. 2007. View Article : Google Scholar : PubMed/NCBI
25.	Javanmard SH and Dana N: The effect of interferon γ on endothelial cell nitric oxide production and apoptosis. Adv Biomed Res. 1:692012.
26.	Bouïs D, Hospers GA, Meijer C, Molema G and Mulder NH: Endothelium in vitro: a review of human vascular endothelial cell lines for blood vessel-related research. Angiogenesis. 4:91–102. 2001.PubMed/NCBI
27.	Edgell CJ, Reisner HM and Graham JB: Endothelial cell hybrids and the suspension of factor VIII related antigen expression. Br J Haematol. 46:613–620. 1980. View Article : Google Scholar : PubMed/NCBI
28.	Edgell CJ, Haizlip JE, Bagnell CR, Packenham JP, Harrison P, Wilbourn B and Madden VJ: Endothelium specific Weibel-Palade bodies in a continuous human cell line, EA.hy926. In Vitro Cell Dev Biol. 26:1167–1172. 1990. View Article : Google Scholar : PubMed/NCBI
29.	Claise C, Chalas J, Edeas M, Abella A, Khalfoun Y, Laurent D and Lindenbaum A: Comparison of oxidized low-density lipoprotein toxicity on EA.hy 926 cells and human vein endothelial cells: influence of antioxidant systems. Cell Mol Life Sci. 53:156–161. 1997. View Article : Google Scholar : PubMed/NCBI
30.	Vrekoussis T, Stathopoulos EN, De Giorgi U, et al: Modulation of vascular endothelium by imatinib: a study on the EA.hy 926 endothelial cell line. J Chemother. 18:56–65. 2006. View Article : Google Scholar : PubMed/NCBI
31.	Rosenkranz AC, Lob H, Breitenbach T, Berkels R and Roesen R: Endothelial antioxidant actions of dihydropyridines and angiotensin converting enzyme inhibitors. Eur J Pharmacol. 529:55–62. 2006. View Article : Google Scholar : PubMed/NCBI
32.	Pittenger MF, Kazzaz JA and Helfman DM: Functional properties of non-muscle tropomyosin isoforms. Curr Opin Cell Biol. 6:96–104. 1994. View Article : Google Scholar : PubMed/NCBI
33.	Perry SV: Vertebrate tropomyosin: distribution, properties and function. J Muscle Res Cell Motil. 22:5–49. 2001. View Article : Google Scholar : PubMed/NCBI
34.	Cooper JA: Actin dynamics: tropomyosin provides stability. Curr Biol. 12:R523–R525. 2002. View Article : Google Scholar : PubMed/NCBI
35.	Gunning PW, Schevzov G, Kee AJ and Hardeman EC: Tropomyosin isoforms: divining rods for actin cytoskeleton function. Trends Cell Biol. 15:333–341. 2005. View Article : Google Scholar : PubMed/NCBI
36.	Lin JJ, Eppinga RD, Warren KS and McCrae KR: Human tropomyosin isoforms in the regulation of cytoskeleton functions. Adv Exp Med Biol. 644:201–222. 2008. View Article : Google Scholar : PubMed/NCBI
37.	Plazar N and Jurdana M: Hyperhomocysteinemia: relation to cardiovascular disease and venous thromboembolism. Pathophysiology and Clinical Aspects of Venous Thromboembolism in Neonates, Renal Disease and Cancer Patients. Abdelaal MA: InTech; Rijeka: pp. 17–34. 2012
38.	Ridker PM, Manson JE, Buring JE, Shih J, Matias M and Hennekens CH: Homocysteine and risk of cardiovascular disease among postmenopausal women. JAMA. 281:1817–1821. 1999. View Article : Google Scholar : PubMed/NCBI
39.	Upchurch GR Jr, Welch GN, Fabian AJ, Freedman JE, Johnson JL, Keaney JF Jr and Loscalzo J: Homocyst(e)ine decreases bioavailable nitric oxide by a mechanism involving glutathione peroxidase. J Biol Chem. 272:17012–17017. 1997. View Article : Google Scholar : PubMed/NCBI
40.	Nagai Y, Tasaki H, Takatsu H, Nihei S, Yamashita K, Toyokawa T and Nakashima Y: Homocysteine inhibits angiogenesis in vitro and in vivo. Biochem Biophys Res Commun. 281:726–731. 2001. View Article : Google Scholar : PubMed/NCBI
41.	Zhang C, Cai Y, Adachi MT, Oshiro S, Aso T, Kaufman RJ and Kitajima S: Homocysteine induces programmed cell death in human vascular endothelial cells through activation of the unfolded protein response. J Biol Chem. 276:35867–35874. 2001. View Article : Google Scholar : PubMed/NCBI
42.	Lee SJ, Kim KM, Namkoong S, et al: Nitric oxide inhibition of homocysteine-induced human endothelial cell apoptosis by down-regulation of p53-dependent Noxa expression through the formation of S-nitrosohomocysteine. J Biol Chem. 280:5781–5788. 2005. View Article : Google Scholar : PubMed/NCBI
43.	Suhara T, Fukuo K, Yasuda O, et al: Homocysteine enhances endothelial apoptosis via upregulation of Fas-mediated pathways. Hypertension. 43:1208–1213. 2004. View Article : Google Scholar : PubMed/NCBI
44.	Mercié P, Garnier O, Lascoste L, et al: Homocysteine-thiolactone induces caspase-independent vascular endothelial cell death with apoptotic features. Apoptosis. 5:403–411. 2000.PubMed/NCBI
45.	Rodríguez-Nieto S, Chavarría T, Martínez-Poveda B, Sánchez-Jiménez F, Rodríguez Quesada A and Medina MA: Anti-angiogenic effects of homocysteine on cultured endothelial cells. Biochem Biophys Res Commun. 293:497–500. 2002.PubMed/NCBI
46.	Wejksza K, Rzeski W and Turski WA: Kynurenic acid protects against the homocysteine-induced impairment of endothelial cells. Pharmacol Rep. 61:751–756. 2009. View Article : Google Scholar : PubMed/NCBI
47.	Vincent PA, Xiao K, Buckley KM and Kowalczyk AP: VE-cadherin: adhesion at arm’s length. Am J Physiol Cell Physiol. 286:C987–C997. 2004.
48.	Drees F, Pokutta S, Yamada S, Nelson WJ and Weis WI: α-catenin is a molecular switch that binds E-cadherin-β-catenin and regulates actin-filament assembly. Cell. 123:903–915. 2005.
49.	Yamada S, Pokutta S, Drees F, Weis WI and Nelson WJ: Deconstructing the cadherin-catenin-actin complex. Cell. 123:889–901. 2005. View Article : Google Scholar : PubMed/NCBI
50.	Millán J, Cain RJ, Reglero-Real N, et al: Adherens junctions connect stress fibres between adjacent endothelial cells. BMC Biol. 8:112010.PubMed/NCBI
51.	Fanning AS, Ma TY and Anderson JM: Isolation and functional characterization of the actin binding region in the tight junction protein ZO-1. FASEB. 16:1835–1837. 2002.PubMed/NCBI
52.	Itoh M, Nagafuchi A, Moroi S and Tsukita S: Involvement of ZO-1 in cadherin-based cell adhesion through its direct binding to alpha catenin and actin filaments. J Cell Biol. 138:181–192. 1997. View Article : Google Scholar : PubMed/NCBI
53.	Fanning AS, Jameson BJ, Jesaitis LA and Anderson JM: The tight junction protein ZO-1 establishes a link between the trans-membrane protein occludin and the actin cytoskeleton. J Biol Chem. 273:29745–29753. 1998. View Article : Google Scholar : PubMed/NCBI
54.	Wittchen ES, Haskins J and Stevenson BR: Protein interactions at the tight junction. Actin has multiple binding partners, and ZO-1 forms independent complexes with ZO-2 and ZO-3. J Biol Chem. 274:35179–35185. 1999. View Article : Google Scholar : PubMed/NCBI
55.	Van Itallie CM, Fanning AS, Bridges A and Anderson JM: ZO-1 stabilizes the tight junction solute barrier through coupling to the perijunctional cytoskeleton. Mol Biol Cell. 20:3930–3940. 2009.PubMed/NCBI
56.	Chen VC, Li X, Perreault H and Nagy JI: Interaction of zonula occludens-1 (ZO-1) with alpha-actinin-4: application of functional proteomics for identification of PDZ domain-associated proteins. J Proteome Res. 5:2123–2134. 2006. View Article : Google Scholar : PubMed/NCBI
57.	Etournay R, Zwaenepoel I, Perfettini I, Legrain P, Petit C and El-Amraoui A: Shroom2, a myosin-VIIa- and actin-binding protein, directly interacts with ZO-1 at tight junctions. J Cell Sci. 120:2838–2850. 2007. View Article : Google Scholar : PubMed/NCBI