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Neekhra A, Tran J, Pham K, Sharma A, Chwa M, Luthra S, a ALG, Mansoor S, Kuppermann BD, Kenney C. Memantine, Simvastatin, and Epicatechin Inhibit 7-Ketocholesterol-induced Apoptosis in Retinal Pigment Epithelial Cells But Not Neurosensory Retinal Cells In Vitro. JOVR [Internet]. 2020Oct.25 [cited 2024Jul.17];15(4):470–480. Available from: https://knepublishing.com/index.php/JOVR/article/view/7781
https://doi.org/10.18502/jovr.v15i4.7781
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authors
-
Aneesh Neekhra
Aneesh Neekhra
Gavin Herbert Eye Institute, University of California, Irvine, California
-
Julia Tran
Julia Tran
Gavin Herbert Eye Institute, University of California, Irvine, California
-
Khoa Pham
Khoa Pham
Gavin Herbert Eye Institute, University of California, Irvine, California
-
Ashish Sharma
Ashish Sharma
Gavin Herbert Eye Institute, University of California, Irvine, California
-
Marilyn Chwa
Marilyn Chwa
Gavin Herbert Eye Institute, University of California, Irvine, California
-
Saurabh Luthra
Saurabh Luthra
Gavin Herbert Eye Institute, University of California, Irvine, California
-
Ana L. Grmajo a
Ana L. Grmajo a
Gavin Herbert Eye Institute, University of California, Irvine, California
-
Saffar Mansoor
Saffar Mansoor
Gavin Herbert Eye Institute, University of California, Irvine, California
-
Baruch D. Kuppermann
Baruch D. Kuppermann
Gavin Herbert Eye Institute, University of California, Irvine, California
-
Cristina Kenney
Cristina Kenney
Department of Pathology and Laboratory Medicine, University of California Irvine, Irvine, CA, USA
Published Date
- Oct 25, 2020
Memantine, Simvastatin, and Epicatechin Inhibit 7-Ketocholesterol-induced Apoptosis in Retinal Pigment Epithelial Cells But Not Neurosensory Retinal Cells In Vitro
Abstract
Purpose: 7-ketocholesterol (7kCh), a natural byproduct of oxidation in lipoprotein deposits, is implicated in the pathogenesis of diabetic retinopathy and age-related macular degeneration (AMD). This study was performed to investigate whether several clinical drugs can inhibit 7kCh-induced caspase activation and mitigate its apoptotic effects on retinal cells in vitro.
Method: Two populations of retinal cells, human retinal pigment epithelial cells (ARPE-19) and rat neuroretinal cells (R28), were exposed to 7kCh in the presence of the following inhibitors: Z-VAD-FMK (pan-caspase inhibitor), simvastatin, memantine, epicatechin, and Z-IETD-FMK (caspase-8 inhibitor) or Z-ATAD-FMK (caspase-12 inhibitor). Caspase-3/7, -8, and -12 activity levels were measured by fluorochrome caspase assays to quantify cell death. IncuCyte live-cell microscopic images were obtained to quantify cell counts.
Results: Exposure to 7kCh for 24 hours significantly increased caspase activities for both ARPE-19 and R28 cells (P < 0.05). In ARPE cells, pretreatment with various drugs had significantly lower caspase-3/7, -8, and -12 activities, reported in % change in mean signal intensity (msi): Z-VAD-FMK (48% decrease, P < 0.01), memantine (decreased 47.8% at 1 μM, P = 0.0039 and 81.9% at 1 mM, P < 0.001), simvastatin (decreased 85.3% at 0.01 μM, P < 0.001 and 84.8% at 0.05 μM , P < 0.001) or epicatechin (83.6% decrease, P < 0.05), Z-IETD-FMK (68.1% decrease, P < 0.01), and Z-ATAD-FMK (47.7% decrease, P = 0.0017). In contrast, R28 cells exposed to 7kCh continued to have elevated caspase- 3/7, -8, and -12 activities (between 25.7% decrease and 17.5% increase in msi, P > 0.05) regardless of the pretreatment.
Conclusion: Several current drugs protect ARPE-19 cells but not R28 cells from 7kChinduced apoptosis, suggesting that a multiple-drug approach is needed to protect both cells types in various retinal diseases.
Keywords:
Epicatechin, 7-Ketocholesterol, Memantine
References
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2. Hinton DR, He S, Lopez PF. Apoptosis in surgically excised choroidal neovascular membranes in age-related macular degeneration. Arch Ophthalmol 1998;116:203–209.
3. Obulesu M, Lakshmi MJ. Apoptosis in Alzheimer’s disease: an understanding of the physiology, pathology and therapeutic avenues. Neurochem Res 2014;39:2301– 2312.
4. Samadi A, et al. Oxysterol species: reliable markers of oxidative stress in diabetes mellitus. J Endocrinol Invest 2019;42:7–17.
5. Thomas CN, et al. Caspases in retinal ganglion cell death and axon regeneration. Cell Death Discov 2017;3:17032.
6. Xu GZ, Li WW, Tso MO. Apoptosis in human retinal degenerations. Trans Am Ophthalmol Soc 1996;94:411– 430; discussion 430–431.
7. Wei Q, et al. Combination of bevacizumab and photodynamic therapy vs. bevacizumab monotherapy for the treatment of wet age-related macular degeneration: A meta-analysis of randomized controlled trials. Exp Ther Med 2018;16:1187–1194.
8. Pariente A, et al. Inflammatory and cell death mechanisms induced by 7-ketocholesterol in the retina. Implications for age-related macular degeneration. Exp Eye Res 2019;187:107746.
9. Rodriguez IR, Larrayoz IM. Cholesterol oxidation in the retina: implications of 7KCh formation in chronic inflammation and age-related macular degeneration. J Lipid Res 2010;51:2847–2862.
10. Yang C, et al. 7-Ketocholesterol disturbs RPE cells phagocytosis of the outer segment of photoreceptor and induces inflammation through ERK signaling pathway. Exp Eye Res 2019;189:107849.
11. Indaram M, et al. 7-Ketocholesterol increases retinal microglial migration, activation, and angiogenicity: a potential pathogenic mechanism underlying age-related macular degeneration. Sci Rep 2015;5:9144.
12. Larrayoz IM, et al. 7-ketocholesterol-induced inflammation: involvement of multiple kinase signaling pathways via NFkappaB but independently of reactive oxygen species formation. Invest Ophthalmol Vis Sci 2010;51:4942–4955.
13. Wang H, et al. Thy-1 Regulates VEGF-Mediated Choroidal Endothelial Cell Activation and Migration: Implications in Neovascular Age-Related Macular Degeneration. Invest Ophthalmol Vis Sci 2016;57:5525–5534.
14. Moreira EF, et al. 7-Ketocholesterol is present in lipid deposits in the primate retina: potential implication in the induction of VEGF and CNV formation. Invest Ophthalmol Vis Sci 2009;50:523–532.
15. Dulak J, et al. Vascular endothelial growth factor synthesis in vascular smooth muscle cells is enhanced by 7-ketocholesterol and lysophosphatidylcholine independently of their effect on nitric oxide generation. Atherosclerosis 2001;159:325–332.
16. Gramajo AL, et al. Mitochondrial DNA damage induced by 7-ketocholesterol in human retinal pigment epithelial cells in vitro. Invest Ophthalmol Vis Sci 2010;51:1164–1170.
17. Luthra S, et al. 7-Ketocholesterol activates caspases-3/7, - 8, and -12 in human microvascular endothelial cells in vitro. Microvasc Res 2008;75:343–350.
18. Luthra S, et al. Activation of caspase-8 and caspase-12 pathways by 7-ketocholesterol in human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 2006;47:5569– 5575.
19. Neekhra A, et al. Caspase-8, -12, and -3 activation by 7-ketocholesterol in retinal neurosensory cells. Invest Ophthalmol Vis Sci 2007;48:1362–1367.
20. Javitt NB, Javitt JC. The retinal oxysterol pathway: a unifying hypothesis for the cause of age-related macular degeneration. Curr Opin Ophthalmol 2009;20:151–157.
21. Brahmi F, et al. Prevention of 7-ketocholesterol-induced side effects by natural compounds. Crit Rev Food Sci Nutr 2018:1–20.
22. Dugas B, et al. Effects of oxysterols on cell viability, inflammatory cytokines, VEGF, and reactive oxygen species production on human retinal cells: cytoprotective effects and prevention of VEGF secretion by resveratrol. Eur J Nutr 2010;49:435–446.
23. Sun W, Seigel GM, Salvi RJ. Retinal precursor cells express functional ionotropic glutamate and GABA receptors. Neuroreport 2002;13:2421–2424.
24. Seigel GM. Review: R28 retinal precursor cells: the first 20 years. Mol Vis 2014;20:301–306.
25. Dunn KC, et al. ARPE-19, a human retinal pigment epithelial cell line with differentiated properties. Exp Eye Res 1996;62:155–169.
26. Moustafa MT, et al. Protective Effects of memantine on hydroquinone-treated human retinal pigment epithelium cells and human retinal muller cells. J Ocul Pharmacol Ther 2017;33:610–619.
27. Lee JW, Huang JD, Rodriguez IR. Extra-hepatic metabolism of 7-ketocholesterol occurs by esterification to fatty acids via cPLA2alpha and SOAT1 followed by selective efflux to HDL. Biochim Biophys Acta 2015;1851:605–619.
28. Rodriguez IR, et al. 7-ketocholesterol accumulates in ocular tissues as a consequence of aging and is present in high levels in drusen. Exp Eye Res 2014;128:151–155.
29. Jang ER, Lee CS. 7-ketocholesterol induces apoptosis in differentiated PC12 cells via reactive oxygen speciesdependent activation of NF-kappaB and Akt pathways. Neurochem Int 2011;58:52–59.
30. Pedruzzi E, et al. NAD(P)H oxidase Nox-4 mediates 7-ketocholesterol-induced endoplasmic reticulum stress and apoptosis in human aortic smooth muscle cells. Mol Cell Biol 2004;24:10703–10717.
31. Shimozawa M, et al. 7-Ketocholesterol enhances the expression of adhesion molecules on human aortic endothelial cells by increasing the production of reactive oxygen species. Redox Rep 2004;9:370–375.
32. Ong JM, et al. Oxysterol-induced toxicity in R28 and ARPE- 19 cells. Neurochem Res 2003;28:883–891.
33. Casson RJ. Possible role of excitotoxicity in the pathogenesis of glaucoma. Clin Exp Ophthalmol 2006;34:54–63.
34. Heinen-Kammerer T, et al. Added therapeutic value of memantine in the treatment of moderate to severe Alzheimer’s disease. Clin Drug Investig 2006;26:303–314.
35. Kim TW, et al. Neuroprotective effect of memantine in a rabbit model of optic nerve ischemia. Korean J Ophthalmol 2002;16:1–7.
36. Lipton SA, Rosenberg PA. Excitatory amino acids as a final common pathway for neurologic disorders. N Engl J Med 1994;330:613–622.
37. WoldeMussie E, et al. Neuroprotective effect of memantine in different retinal injury models in rats. J Glaucoma 2002;11:474–480.
38. Chen B, et al. Memantine attenuates cell apoptosis by suppressing the calpain-caspase-3 pathway in an experimental model of ischemic stroke. Exp Cell Res 2017;351:163–172.
39. Ota H, et al. Protective effects of NMDA receptor antagonist, memantine, against senescence of PC12 cells: a possible role of nNOS and combined effects with donepezil. Exp Gerontol 2015;72:109–116.
40. Jantas-Skotniczna D, Kajta M, Lason W. Memantine attenuates staurosporine-induced activation of caspase- 3 and LDH release in mouse primary neuronal cultures. Brain Res 2006;1069:145–153.
41. Seigel GM, et al. Neuronal gene expression and function in the growth-stimulated R28 retinal precursor cell line. Curr Eye Res 2004;28:257–269.
42. Jantas D, et al. An involvement of BDNF and PI3-K/Akt in the anti-apoptotic effect of memantine on staurosporineevoked cell death in primary cortical neurons. Apoptosis 2009;14:900–912.
43. Dias IHK, et al. Simvastatin reduces circulating oxysterol levels in men with hypercholesterolaemia. Redox Biol 2018;16:139–145.
44. Franke C, et al. Bcl-2 upregulation and neuroprotection in guinea pig brain following chronic simvastatin treatment. Neurobiol Dis 2007;25:438–445.
45. Johnson-Anuna LN, et al. Simvastatin protects neurons from cytotoxicity by up-regulating Bcl-2 mRNA and protein. J Neurochem 2007;101:77–86.
46. Kretz A, et al. Simvastatin promotes heat shock protein 27 expression and Akt activation in the rat retina and protects axotomized retinal ganglion cells in vivo. Neurobiol Dis 2006;21:421–430.
47. Miyahara S, et al. Simvastatin inhibits leukocyte accumulation and vascular permeability in the retinas of rats with streptozotocin-induced diabetes. Am J Pathol 2004;164:1697–1706.
48. Muck AO, Seeger H, Wallwiener D. Class-specific proapoptotic effect of statins on human vascular endothelial cells. Z Kardiol 2004;93:398–402.
49. Urbich C, et al. Double-edged role of statins in angiogenesis signaling. Circ Res 2002;90:737–744.
50. Weis M, et al. Statins have biphasic effects on angiogenesis. Circulation 2002;105:739–745.
51. Boucher K, et al. HMG-CoA reductase inhibitors induce apoptosis in pericytes. Microvasc Res 2006;71:91–102.
52. Fujino M, et al. Counteracting effects of high density lipoprotein-cholesterol subfractions on statin-induced growth arrest. Cardiovasc Drugs Ther 2005;19:113–118.
53. Krikorian R, et al. Concord grape juice supplementation improves memory function in older adults with mild cognitive impairment. Br J Nutr 2010;103:730–74.
54. Steffen Y, et al. Protein modification elicited by oxidized low-density lipoprotein (LDL) in endothelial cells: protection by (-)-epicatechin. Free Radic Biol Med 2007;42:955–970.
55. Steffen Y, Schewe T, Sies H. Epicatechin protects endothelial cells against oxidized LDL and maintains NO synthase. Biochem Biophys Res Commun 2005;331:1277– 1283.
56. Wang MH, et al. (-)-Epigallocatechin-3-gallate decreases the impairment in learning and memory in spontaneous hypertension rats. Behav Pharmacol 2012;23:771–780.
57. Zhang B, Osborne NN. Oxidative-induced retinal degeneration is attenuated by epigallocatechin gallate. Brain Res 2006;1124:176–187.
58. Li S, et al. Characterization of the responses of the caspase 2, 3, 6 and 8 genes to immune challenges and extracellular ATP stimulation in the Japanese flounder (Paralichthys olivaceus). BMC Vet Res 2019;15:20.
59. Li GY, Fan B, Su GF. Acute energy reduction induces caspase-dependent apoptosis and activates p53 in retinal ganglion cells (RGC-5). Exp Eye Res 2009;89:581–589.
60. Mansoor S, et al. Inhibition of apoptosis in human retinal pigment epithelial cells treated with benzo(e)pyrene, a toxic component of cigarette smoke. Invest Ophthalmol Vis Sci 2010;51:2601–2607.
61. Kozlowski MR. The ARPE-19 cell line: mortality status and utility in macular degeneration research. Curr Eye Res 2015;40:501–509.
2. Hinton DR, He S, Lopez PF. Apoptosis in surgically excised choroidal neovascular membranes in age-related macular degeneration. Arch Ophthalmol 1998;116:203–209.
3. Obulesu M, Lakshmi MJ. Apoptosis in Alzheimer’s disease: an understanding of the physiology, pathology and therapeutic avenues. Neurochem Res 2014;39:2301– 2312.
4. Samadi A, et al. Oxysterol species: reliable markers of oxidative stress in diabetes mellitus. J Endocrinol Invest 2019;42:7–17.
5. Thomas CN, et al. Caspases in retinal ganglion cell death and axon regeneration. Cell Death Discov 2017;3:17032.
6. Xu GZ, Li WW, Tso MO. Apoptosis in human retinal degenerations. Trans Am Ophthalmol Soc 1996;94:411– 430; discussion 430–431.
7. Wei Q, et al. Combination of bevacizumab and photodynamic therapy vs. bevacizumab monotherapy for the treatment of wet age-related macular degeneration: A meta-analysis of randomized controlled trials. Exp Ther Med 2018;16:1187–1194.
8. Pariente A, et al. Inflammatory and cell death mechanisms induced by 7-ketocholesterol in the retina. Implications for age-related macular degeneration. Exp Eye Res 2019;187:107746.
9. Rodriguez IR, Larrayoz IM. Cholesterol oxidation in the retina: implications of 7KCh formation in chronic inflammation and age-related macular degeneration. J Lipid Res 2010;51:2847–2862.
10. Yang C, et al. 7-Ketocholesterol disturbs RPE cells phagocytosis of the outer segment of photoreceptor and induces inflammation through ERK signaling pathway. Exp Eye Res 2019;189:107849.
11. Indaram M, et al. 7-Ketocholesterol increases retinal microglial migration, activation, and angiogenicity: a potential pathogenic mechanism underlying age-related macular degeneration. Sci Rep 2015;5:9144.
12. Larrayoz IM, et al. 7-ketocholesterol-induced inflammation: involvement of multiple kinase signaling pathways via NFkappaB but independently of reactive oxygen species formation. Invest Ophthalmol Vis Sci 2010;51:4942–4955.
13. Wang H, et al. Thy-1 Regulates VEGF-Mediated Choroidal Endothelial Cell Activation and Migration: Implications in Neovascular Age-Related Macular Degeneration. Invest Ophthalmol Vis Sci 2016;57:5525–5534.
14. Moreira EF, et al. 7-Ketocholesterol is present in lipid deposits in the primate retina: potential implication in the induction of VEGF and CNV formation. Invest Ophthalmol Vis Sci 2009;50:523–532.
15. Dulak J, et al. Vascular endothelial growth factor synthesis in vascular smooth muscle cells is enhanced by 7-ketocholesterol and lysophosphatidylcholine independently of their effect on nitric oxide generation. Atherosclerosis 2001;159:325–332.
16. Gramajo AL, et al. Mitochondrial DNA damage induced by 7-ketocholesterol in human retinal pigment epithelial cells in vitro. Invest Ophthalmol Vis Sci 2010;51:1164–1170.
17. Luthra S, et al. 7-Ketocholesterol activates caspases-3/7, - 8, and -12 in human microvascular endothelial cells in vitro. Microvasc Res 2008;75:343–350.
18. Luthra S, et al. Activation of caspase-8 and caspase-12 pathways by 7-ketocholesterol in human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 2006;47:5569– 5575.
19. Neekhra A, et al. Caspase-8, -12, and -3 activation by 7-ketocholesterol in retinal neurosensory cells. Invest Ophthalmol Vis Sci 2007;48:1362–1367.
20. Javitt NB, Javitt JC. The retinal oxysterol pathway: a unifying hypothesis for the cause of age-related macular degeneration. Curr Opin Ophthalmol 2009;20:151–157.
21. Brahmi F, et al. Prevention of 7-ketocholesterol-induced side effects by natural compounds. Crit Rev Food Sci Nutr 2018:1–20.
22. Dugas B, et al. Effects of oxysterols on cell viability, inflammatory cytokines, VEGF, and reactive oxygen species production on human retinal cells: cytoprotective effects and prevention of VEGF secretion by resveratrol. Eur J Nutr 2010;49:435–446.
23. Sun W, Seigel GM, Salvi RJ. Retinal precursor cells express functional ionotropic glutamate and GABA receptors. Neuroreport 2002;13:2421–2424.
24. Seigel GM. Review: R28 retinal precursor cells: the first 20 years. Mol Vis 2014;20:301–306.
25. Dunn KC, et al. ARPE-19, a human retinal pigment epithelial cell line with differentiated properties. Exp Eye Res 1996;62:155–169.
26. Moustafa MT, et al. Protective Effects of memantine on hydroquinone-treated human retinal pigment epithelium cells and human retinal muller cells. J Ocul Pharmacol Ther 2017;33:610–619.
27. Lee JW, Huang JD, Rodriguez IR. Extra-hepatic metabolism of 7-ketocholesterol occurs by esterification to fatty acids via cPLA2alpha and SOAT1 followed by selective efflux to HDL. Biochim Biophys Acta 2015;1851:605–619.
28. Rodriguez IR, et al. 7-ketocholesterol accumulates in ocular tissues as a consequence of aging and is present in high levels in drusen. Exp Eye Res 2014;128:151–155.
29. Jang ER, Lee CS. 7-ketocholesterol induces apoptosis in differentiated PC12 cells via reactive oxygen speciesdependent activation of NF-kappaB and Akt pathways. Neurochem Int 2011;58:52–59.
30. Pedruzzi E, et al. NAD(P)H oxidase Nox-4 mediates 7-ketocholesterol-induced endoplasmic reticulum stress and apoptosis in human aortic smooth muscle cells. Mol Cell Biol 2004;24:10703–10717.
31. Shimozawa M, et al. 7-Ketocholesterol enhances the expression of adhesion molecules on human aortic endothelial cells by increasing the production of reactive oxygen species. Redox Rep 2004;9:370–375.
32. Ong JM, et al. Oxysterol-induced toxicity in R28 and ARPE- 19 cells. Neurochem Res 2003;28:883–891.
33. Casson RJ. Possible role of excitotoxicity in the pathogenesis of glaucoma. Clin Exp Ophthalmol 2006;34:54–63.
34. Heinen-Kammerer T, et al. Added therapeutic value of memantine in the treatment of moderate to severe Alzheimer’s disease. Clin Drug Investig 2006;26:303–314.
35. Kim TW, et al. Neuroprotective effect of memantine in a rabbit model of optic nerve ischemia. Korean J Ophthalmol 2002;16:1–7.
36. Lipton SA, Rosenberg PA. Excitatory amino acids as a final common pathway for neurologic disorders. N Engl J Med 1994;330:613–622.
37. WoldeMussie E, et al. Neuroprotective effect of memantine in different retinal injury models in rats. J Glaucoma 2002;11:474–480.
38. Chen B, et al. Memantine attenuates cell apoptosis by suppressing the calpain-caspase-3 pathway in an experimental model of ischemic stroke. Exp Cell Res 2017;351:163–172.
39. Ota H, et al. Protective effects of NMDA receptor antagonist, memantine, against senescence of PC12 cells: a possible role of nNOS and combined effects with donepezil. Exp Gerontol 2015;72:109–116.
40. Jantas-Skotniczna D, Kajta M, Lason W. Memantine attenuates staurosporine-induced activation of caspase- 3 and LDH release in mouse primary neuronal cultures. Brain Res 2006;1069:145–153.
41. Seigel GM, et al. Neuronal gene expression and function in the growth-stimulated R28 retinal precursor cell line. Curr Eye Res 2004;28:257–269.
42. Jantas D, et al. An involvement of BDNF and PI3-K/Akt in the anti-apoptotic effect of memantine on staurosporineevoked cell death in primary cortical neurons. Apoptosis 2009;14:900–912.
43. Dias IHK, et al. Simvastatin reduces circulating oxysterol levels in men with hypercholesterolaemia. Redox Biol 2018;16:139–145.
44. Franke C, et al. Bcl-2 upregulation and neuroprotection in guinea pig brain following chronic simvastatin treatment. Neurobiol Dis 2007;25:438–445.
45. Johnson-Anuna LN, et al. Simvastatin protects neurons from cytotoxicity by up-regulating Bcl-2 mRNA and protein. J Neurochem 2007;101:77–86.
46. Kretz A, et al. Simvastatin promotes heat shock protein 27 expression and Akt activation in the rat retina and protects axotomized retinal ganglion cells in vivo. Neurobiol Dis 2006;21:421–430.
47. Miyahara S, et al. Simvastatin inhibits leukocyte accumulation and vascular permeability in the retinas of rats with streptozotocin-induced diabetes. Am J Pathol 2004;164:1697–1706.
48. Muck AO, Seeger H, Wallwiener D. Class-specific proapoptotic effect of statins on human vascular endothelial cells. Z Kardiol 2004;93:398–402.
49. Urbich C, et al. Double-edged role of statins in angiogenesis signaling. Circ Res 2002;90:737–744.
50. Weis M, et al. Statins have biphasic effects on angiogenesis. Circulation 2002;105:739–745.
51. Boucher K, et al. HMG-CoA reductase inhibitors induce apoptosis in pericytes. Microvasc Res 2006;71:91–102.
52. Fujino M, et al. Counteracting effects of high density lipoprotein-cholesterol subfractions on statin-induced growth arrest. Cardiovasc Drugs Ther 2005;19:113–118.
53. Krikorian R, et al. Concord grape juice supplementation improves memory function in older adults with mild cognitive impairment. Br J Nutr 2010;103:730–74.
54. Steffen Y, et al. Protein modification elicited by oxidized low-density lipoprotein (LDL) in endothelial cells: protection by (-)-epicatechin. Free Radic Biol Med 2007;42:955–970.
55. Steffen Y, Schewe T, Sies H. Epicatechin protects endothelial cells against oxidized LDL and maintains NO synthase. Biochem Biophys Res Commun 2005;331:1277– 1283.
56. Wang MH, et al. (-)-Epigallocatechin-3-gallate decreases the impairment in learning and memory in spontaneous hypertension rats. Behav Pharmacol 2012;23:771–780.
57. Zhang B, Osborne NN. Oxidative-induced retinal degeneration is attenuated by epigallocatechin gallate. Brain Res 2006;1124:176–187.
58. Li S, et al. Characterization of the responses of the caspase 2, 3, 6 and 8 genes to immune challenges and extracellular ATP stimulation in the Japanese flounder (Paralichthys olivaceus). BMC Vet Res 2019;15:20.
59. Li GY, Fan B, Su GF. Acute energy reduction induces caspase-dependent apoptosis and activates p53 in retinal ganglion cells (RGC-5). Exp Eye Res 2009;89:581–589.
60. Mansoor S, et al. Inhibition of apoptosis in human retinal pigment epithelial cells treated with benzo(e)pyrene, a toxic component of cigarette smoke. Invest Ophthalmol Vis Sci 2010;51:2601–2607.
61. Kozlowski MR. The ARPE-19 cell line: mortality status and utility in macular degeneration research. Curr Eye Res 2015;40:501–509.