Journals of Gerontology: BIOLOGICAL SCIENCES, 2015, 1–5 doi:10.1093/gerona/glu244 Brief report
Brief report
Aging Exacerbates Pressure-Induced Mitochondrial Oxidative Stress in Mouse Cerebral Arteries Zsolt Springo,1,2,* Stefano Tarantini,1,* Peter Toth,1,* Zsuzsanna Tucsek,1 Akos Koller,2 William E. Sonntag,1,3 Anna Csiszar,1,2,3 and Zoltan Ungvari1,2,3 1 Reynolds Oklahoma Center on Aging, Donald W. Reynolds Department of Geriatric Medicine, University of Oklahoma Health Sciences Center. 2Department of Pathophysiology and Gerontology, Medical School and Szentágothai Research Center, University of Pecs, Hungary. 3The Peggy and Charles Stephenson Cancer Center, University of Oklahoma Health Sciences Center.
*These authors contributed equally to this work. Address correspondence to Zoltan Ungvari, MD, PhD, Reynolds Oklahoma Center on Aging, Department of Geriatric Medicine, University of Oklahoma Health Sciences Center, 975 N.E. 10th Street, BRC 1303, Oklahoma City, OK 73104. Email: [email protected]
Abstract Epidemiological studies demonstrate that in addition to the increased prevalence of hypertension in old patients, the deleterious cerebrovascular effects of hypertension (including atherosclerosis, stroke, and vascular cognitive impairment) are also exacerbated in elderly individuals. The cellular mechanisms by which aging and hypertension interact to promote cerebrovascular pathologies are not well understood. To test the hypothesis that aging exacerbates high pressure–induced mitochondrial oxidative stress, we exposed isolated segments of the middle cerebral arteries of young (3 months) and aged (24 months) C57BL/6 mice to 60 or 140 mmHg intraluminal pressure and assessed changes in mitochondrial reactive oxygen species production using a mitochondriatargeted redox-sensitive fluorescent indicator dye (MitoSox) by confocal microscopy. Perinuclear MitoSox fluorescence was significantly stronger in high pressure–exposed middle cerebral arteries compared with middle cerebral arteries of the same animals exposed to 60 mmHg, indicating that high pressure increases mitochondrial reactive oxygen species production in the smooth muscle cells of cerebral arteries. Comparison of young and aged middle cerebral arteries showed that aging exacerbates high pressure–induced mitochondrial reactive oxygen species production in cerebral arteries. We propose that increased mechanosensitive mitochondrial oxidative stress may potentially exacerbate cerebrovascular injury and vascular inflammation in aging.
Key Words: Hypertension—Middle cerebral artery—Oxidative stress—Mitochondrion—Free radicals. Decision Editor: Rafael de Cabo, PhD
Hypertension is known to exert deleterious effects on the cerebral circulation (1–3). The elderly individuals (≥65 years of age) have the highest prevalence of hypertension and these individuals are more likely to develop cerebrovascular pathologies
(4). Epidemiological studies demonstrate that in addition to the increased prevalence of hypertension in old patients, the deleterious cerebrovascular effects of hypertension are also exacerbated in the elderly patients (5–7).
© The Author 2015. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: [email protected].
1
2
Journals of Gerontology: BIOLOGICAL SCIENCES, 2015, Vol. 00, No. 00
There is strong evidence that oxidative stress, by promoting inflammation and pathological vascular remodeling and inducing activation of matrix metalloproteinases, plays a central role in hypertension-induced atherosclerosis and vascular injury. Among the potential sources of reactive oxygen species (ROS), the role of NADPH oxidases in hypertension-induced vascular oxidative stress has been well documented (reviewed in ref. 8). Recent evidence also implicates mitochondrial oxidative stress in hypertension-induced vascular pathologies (9). Despite the paramount importance of mitochondrial oxidative stress in the aging process in general and vascular aging in particular (10), it is not well understood how hypertension and aging interact to regulate mitochondrial ROS (mtROS) production in the cerebrovasculature. The mechanisms by which hypertension promotes vascular oxidative stress are likely multifaceted. Multiple lines of evidence suggest that in addition to circulating factors (eg, angiotensin II), changes in the hemodynamic environment associated with hypertension activate vascular ROS production in the peripheral circulation (11). A primary role for high intraluminal pressure in upregulation of ROS generation is supported by the findings that in aortic-banded rats (in which only blood vessels proximal to the coarctation are exposed to high pressure), regional increases in blood pressure result in selective increases in O2•− production in peripheral arteries (12). Furthermore, exposure of isolated peripheral arteries to high pressure in vitro was shown to increase cellular ROS production (11,13). Despite these advances, the role of high intraluminal pressure per se in regulation of mtROS production in cerebral arteries and its alterations in aging remain elusive. The present study was designed to test the hypothesis that aging exacerbates high pressure–induced mitochondrial oxidative stress in cerebral arteries. To test our hypothesis, we exposed isolated segments of the middle cerebral arteries (MCAs) of young and aged C57BL/6 mice to normal or high intraluminal pressure ex vivo and assessed changes in mtROS production using a mitochondriatargeted redox-sensitive fluorescent indicator dye by confocal microscopy.
(Living Systems Instrumentation, Burlington, VE). The absence of leaks was verified by observing no changes in intraluminal pressure for more than 3 minutes upon turning off the pressure servo-control system. To characterize mechanosensitive mitochondrial oxidative stress, pressure-induced changes in mitochondrial O2•− production in MCAs were measured using MitoSOX Red (Invitrogen, Carlsbad CA), a mitochondrion-specific hydroethidine-derivative fluorescent dye (10,14–19), as previously reported (20). In brief, cannulated MCAs were loaded with MitoSox (3 µmol/L for 30 minutes). Hoechst 33258 was used for nuclear staining. After loading, the chamber was washed out five times with warm Krebs buffer, and the vessels were allowed to equilibrate at 60 mmHg for another 20 minutes. Then, MCAs from the same animals were pressurized to 60 or 140 mmHg (normal pressure and high pressure groups, respectively) for 4 hours. After the experimental period, confocal images of the wall of the pressurized MCAs were captured using a Leica SP2 MP confocal laser scanning microscope. Average perinuclear MitoSox fluorescence intensities were assessed using the Metamorph software. In separate experiments, to assess pressure-induced cellular peroxide production, we used the cell-permeant oxidative fluorescent indicator dye CM-H2DCFDA (5 (and 6)-chloromethyl-2′,7′dichlorodihydrofluorescein diacetate-acetyl ester, Invitrogen) as we previously reported (17). In brief, pressurized MCAs were incubated with CM-H2DCFDA (5 µM, at 37°C, for 4 hours). At the end of the incubation period, CM-H2DCFDA fluorescence was assessed by confocal microscopy.
Methods
Despite recent advances (21–28), the mechanisms underlying the increased vulnerability of aged vessels to injury remain elusive. We used the mitochondria-targeted redox-sensitive dye MitoSox to examine pressure-induced production of O2•− by mitochondria in mouse cerebral arteries. We found that perinuclear MitoSox fluorescence (Figure 1B and C) was significantly stronger in high pressure– exposed MCAs compared with MCAs of the same animals exposed to 60 mmHg, indicating that high pressure increases mtROS production in the smooth muscle cells of cerebral arteries. Comparison of MCAs isolated from aged mice as isolated from young mice showed that aging significantly increases high pressure– induced mtROS production in cerebral arteries (Figure 1D). As illustrated in Figure 1E, a significant cellular heterogeneity with respect to pressure-induced mitochondrial oxidative stress was observed. Representative confocal images shown in Figure 1F suggest that high pressure–induced mtROS production is associated with increased cellular peroxide levels (represented by the intense 2′,7′-dichlorofluorescein staining).
Animals Young (3 months, n = 10) and aged (24 months, n = 10) male C57BL/6 mice were purchased from the National Institute on Aging. All mice were maintained under specific pathogen-free barrier conditions. Water and normal laboratory diet (D12450B, Research Diets Inc.; New Brunswick, NJ) were available ad libitum. All procedures were approved by the Institutional Animal Use and Care Committees of the participating institutions.
Measurement of Pressure-Induced mtROS Production in Isolated MCAs Mice were decapitated, the brains were removed, and segments of the MCAs were isolated using microsurgery instruments for imaging studies, as reported (2,3). Two segments of MCAs from the same animal were mounted onto two glass micropipettes in an organ chamber (Figure 1A) in oxygenated (21% O2, 5% CO2, 75% N2) Krebs’ buffer (composed of [in mmol/L]: 110.0 NaCl, 5.0 KCl, 2.5 CaCl2, 1.0 MgSO4, 1.0 KH2PO4, 5.5 glucose, and 24.0 NaHCO3, pH ~7.4; at 37°C) and pressurized to 10 mmHg. The hydrodynamic resistance of the micropipettes was matched. Inflow and outflow pressures were controlled and measured by a pressure servo-control system
Statistical Analysis Data were analyzed by one-way analysis of variance. A p value less than .05 was considered statistically significant. Data are expressed as mean ± SEM.
Results Aging Exacerbates Pressure-Induced Mitochondrial Oxidative Stress in Mouse Cerebral Arteries
Discussion The results of this study demonstrate for the first time that high pressure elicits increased mitochondrial production of ROS in smooth
Journals of Gerontology: BIOLOGICAL SCIENCES, 2015, Vol. 00, No. 00
3
Figure 1. Aging exacerbates hypertension-induced mitochondrial ROS (mtROS) production in cerebral arteries. (A) Schematic representation of the vessel culture setup used to image isolated MCAs exposed to high pressure in vitro. (B and C) Representative confocal images showing stronger perinuclear MitoSox staining (representing mtROS production; red fluorescence) in high pressure–exposed MCAs isolated from aged mice compared with MCAs isolated from young mice. Hoechst 33258 (blue fluorescence) was used for nuclear staining (original magnification: ×20). Bar graphs (D) are summary data. Data are means ± SEM (n = 5 in each group). *p < .05. (E) Representative image showing heterogeneity in MitoSox staining of smooth muscle cells (identified by the single centrally located spindle-shaped nuclei that are perpendicular to the long axis of the artery) in the wall of a pressurized MCA. Arrows and arrowheads point to cells with high and low mtROS production, respectively. (F) Representative confocal images showing that high pressure–induced mtROS production is associated with increased cellular peroxide levels (represented by the intense 2′,7′-dichlorofluorescein staining). (G) Proposed scheme depicting mechanisms of exacerbation of hypertension-induced vascular oxidative stress in aging and its pathophysiological consequences.
muscle cells located in the cerebral arteries and that this effect is significantly exacerbated in aging (Figure 1). Several lines of evidence suggest that changes in the hemodynamic environment associated with hypertension, either directly or indirectly, can activate ROS production in arteries from other vascular beds as well, including vessels from the coronary and
mesenteric circulation and the skeletal muscle (11,13,29–33). It is likely that increased wall tension–dependent cellular stretch is the primary mechanical stimulus for increased pressure-induced vascular ROS production. In support of this concept, previous studies reported that exposure of isolated arterial rings to in vitro stretching also increases ROS generation (34), mimicking the effects of
4
Journals of Gerontology: BIOLOGICAL SCIENCES, 2015, Vol. 00, No. 00
high pressure. Moreover, earlier studies also show that in cultured endothelial and smooth muscle cells, subjected to in vitro stretching, ROS production also significantly increases (reviewed in ref. 9). It is likely that mitochondria may release ROS in response to strain by virtue of their attachment to cytoskeletal proteins. The molecular mechanisms underlying mechanosensitive mtROS production in cerebral arteries are presently unknown. Mechanotransduction of pressure in vascular smooth muscle cells is known to involve diverse signaling pathways (eg, integrin-mediated signal transduction, Ca2+ signaling, activation of protein kinase C, Rho kinases, and mitogen-activated protein kinases) (35). The role of these pathways in regulation of mechanosensitive mtROS production should be elucidated in future studies. Our working hypothesis is that mechanical forces can be transmitted from the cell membrane to the mitochondria via cytoskeletal tension. Further studies are needed to characterize the molecular mechanotransducer in the mitochondrial membrane (eg, the possible involvement of mitochondrial KATP channels). The sites of mechanosensitive O2•− production in the mitochondria are also subject of future investigation. Potential sites include complex I, the entry point for electrons from NADH reduced nicotinamide adenine dinucleotide into the respiratory chain, and complex III, which funnels electrons from the CoQ pool to cytochrome c. In addition, the ROS-generating enzyme NADPH oxidase 4 (NOX4) is localized to the mitochondrial membrane, it can be activated by mechanical forces (36), and its activation increases mitochondrial oxidative stress (37). The expression of NOX4 is reported to be increased in hypertension (3,38), and an important study demonstrated that mitochondrial-located NOX4 is a major source of pressure overload-induced oxidative stress in the heart (39). Importantly, high levels of angiotensin II can further exacerbate NOX4-dependent ROS production (37,38). In addition to the direct generation of ROS by NOX4, it is also possible that activation of NOX4 also upregulates ROS production by the mitochondrial electron transports chain (40,41). These possibilities warrant further investigation. The mechanisms by which aging exacerbates pressure-induced mtROS production in cerebral arteries also remain elusive. We propose that aging may affect both mechanotransduction of pressure and alter downstream ROS producing mechanisms (eg, by altering the mitochondrial phenotype). Indeed, vascular aging is known to be associated with increased basal mtROS production (10); thus, it is likely that mtROS production stimulated by high pressure will also be exacerbated. In addition, abnormal ROS detoxification (including impaired activation of Nrf2-dependent antioxidant response in aging) (42) is likely to contribute to age-related exacerbation of hypertension-induced mitochondrial oxidative stress. It is significant that aging impairs myogenic adaptation of proximal cerebral arteries to hypertension, which likely enables the penetration of high pressure into the thinned-walled smaller vessels, exacerbating hypertension-induced oxidative damage (2,3). Importantly, a significant cellular heterogeneity with respect to pressure-induced mitochondrial oxidative stress was observed (Figure 1E). One may speculate that age-related changes in cellular phenotype (eg, activation of the senescence program) differentially alter the behavior of individual cells in response to the mechanical stimulation. It is also possible that heterogeneous changes in the extracellular matrix of aged cerebral arteries also contribute to this phenomenon. To understand fully the cellular specificity and complexity of the tissue microenvironment under hypertensive conditions in aged arteries (eg, the link between increased mtROS production and development of an inflammatory phenotype), it will be necessary to measure molecular signatures with single cell resolution in subsequent studies.
The consequences of increased propensity for pressure-induced mtROS generation in aged arteries are likely multiple (Figure 1G). There is increasing evidence supporting a role for mitochondriaderived ROS in chronic low-grade inflammation, including NF-κB activation and development of atherosclerosis, which is associated with aging in the cardiovascular system (10,43). The current view is that O2•−, overproduced in the aged mitochondria, is dismutated to H2O2 by Mn-SOD. It is likely the increased release of H2O2 that activates NF-κB, and likely many other redox-sensitive factors, in the cytoplasm (O2•− is membrane impermeable, whereas H2O2 easily penetrates the mitochondrial membranes) (10). Future studies should also investigate the interaction among aging, chronic hypertension, and mtROS production and its role in activation of matrix metalloproteinases and increased vulnerability of aged arteries to hypertension-induced injury (including stroke, aneurysm formation as well as vascular rarefaction).
Funding This work was supported by grants from the American Heart Association (to P.T., S.T., A.C., and Z.U.), the Oklahoma Center for the Advancement of Science and Technology (to A.C., Z.U., and W.E.S.), the Hungarian National Science Research Fund (OTKA) K 108444 and grants: Developing Competitiveness of Universities in the South Transdanubian Region, “Identification of new biomarkers…,” SROP-4.2.2.A-11/1/KONV-2012-0017 and “Complex examination of neuropeptides…,” SROP-4.2.2.A-11/1/KONV-2012-0024 to A.K., the National Center for Complementary and Alternative Medicine (R01-AT006526 to Z.U.), the National Institute on Aging (R01-AG031085 to A.C.; R01-AG038747 to W.E.S.; R01-NS056218 to A.C. and W.E.S.), the Ellison Medical Foundation (to W.E.S.), and the Arkansas Claude Pepper Older Americans Independence Center at University of Arkansas Medical Center (P30 AG028718 to A.C.). Z.S. is a Copastoeck Fellow supported by a fellowship from the European Union and the State of Hungary, cofinanced by the European Social Fund in the framework of TÁMOP 4.2.4. A/2-11-12012-0001 “National Excellence Program.”
References 1. Csiszar A, Tucsek Z, Toth P, et al. Synergistic effects of hypertension and aging on cognitive function and hippocampal expression of genes involved in β-amyloid generation and Alzheimer’s disease. Am J Physiol Heart Circ Physiol. 2013;305:H1120–H130. doi:10.1152/ajpheart.00288.2013. 2. Toth P, Csiszar A, Tucsek Z, et al. Role of 20-HETE, TRPC channels, and BKCa in dysregulation of pressure-induced Ca2+ signaling and myogenic constriction of cerebral arteries in aged hypertensive mice. Am J Physiol Heart Circ Physiol. 2013;305:H1698–H1708. doi:10.1152/ajpheart.00377.2013. 3. Toth P, Tucsek Z, Sosnowska D, et al. Age-related autoregulatory dysfunction and cerebromicrovascular injury in mice with angiotensin IIinduced hypertension. J Cereb Blood Flow Metab. 2013;33:1732–1742. doi:10.1038/jcbfm.2013.143. 4. Aronow WS, Fleg JL, Pepine CJ, et al. ACCF/AHA 2011 expert consensus document on hypertension in the elderly: a report of the American College of Cardiology Foundation Task Force on Clinical Expert Consensus Documents. Circulation. 2011;123:2434–2506. doi:10.1161/ CIR.0b013e31821daaf6. 5. Brickman AM, Reitz C, Luchsinger JA, et al. Long-term blood pressure fluctuation and cerebrovascular disease in an elderly cohort. Arch Neurol. 2010;67:564–569. doi:10.1001/archneurol.2010.70. 6. Reitz C, Tang MX, Manly J, Mayeux R, Luchsinger JA. Hypertension and the risk of mild cognitive impairment. Arch Neurol. 2007;64:1734–1740. doi:10.1001/archneur.64.12.1734.
Journals of Gerontology: BIOLOGICAL SCIENCES, 2015, Vol. 00, No. 00
7. Kuller LH, Lopez OL, Jagust WJ, et al. Determinants of vascular dementia in the Cardiovascular Health Cognition Study. Neurology. 2005;64:1548– 1552. doi:10.1212/01.WNL.0000160115.55756.DE. 8. Montezano AC, Touyz RM. Oxidative stress, Noxs, and hypertension: experimental evidence and clinical controversies. Ann Med. 2012;44(suppl 1):S2–S16. doi:10.3109/07853890.2011.653393. 9. Dikalov SI, Ungvari Z. Role of mitochondrial oxidative stress in hypertension. Am J Physiol Heart Circ Physiol. 2013;305:H1417–H1427. doi:10.1152/ajpheart.00089.2013. 10. Ungvari Z, Orosz Z, Labinskyy N, et al. Increased mitochondrial H2O2 production promotes endothelial NF-kappaB activation in aged rat arteries. Am J Physiol Heart Circ Physiol. 2007;293:H37–H47. 11. Ungvari Z, Csiszar A, Huang A, Kaminski PM, Wolin MS, Koller A. High pressure induces superoxide production in isolated arteries via protein kinase C-dependent activation of NAD(P)H oxidase. Circulation. 2003;108:1253–1258. 12. Ungvari Z, Csiszar A, Kaminski PM, Wolin MS, Koller A. Chronic high pressure-induced arterial oxidative stress: involvement of protein kinase C-dependent NAD(P)H oxidase and local renin-angiotensin system. Am J Pathol. 2004;165:219–226. 13. Huang A, Sun D, Kaley G, Koller A. Superoxide released to high intraarteriolar pressure reduces nitric oxide-mediated shear stress- and agonistinduced dilations. Circ Res. 1998;83:960–965. 14. Robinson KM, Janes MS, Beckman JS. The selective detection of mitochondrial superoxide by live cell imaging. Nat Protocol. 2008;3:941–947. doi:10.1038/nprot.2008.56. 15. Ungvari Z, Podlutsky A, Sosnowska D, et al. Ionizing radiation promotes the acquisition of a senescence-associated secretory phenotype and impairs angiogenic capacity in cerebromicrovascular endothelial cells: role of increased DNA damage and decreased DNA repair capacity in microvascular radiosensitivity. J Gerontol A Biol Sci Med Sci. 2013;68:1443–1457. doi:10.1093/gerona/glt057. 16. Ungvari Z, Labinskyy N, Mukhopadhyay P, et al. Resveratrol attenuates mitochondrial oxidative stress in coronary arterial endothelial cells. Am J Physiol Heart Circ Physiol. 2009;297:H1876–H1881. doi:10.1152/ajpheart.00375.2009. 17. Csiszar A, Podlutsky A, Podlutskaya N, et al. Testing the oxidative stress hypothesis of aging in primate fibroblasts: is there a correlation between species longevity and cellular ROS production? J Gerontol A Biol Sci Med Sci. 2012;67:841–852. 18. Csiszar A, Sosnowska D, Wang M, Lakatta EG, Sonntag WE, Ungvari Z. Age-associated proinflammatory secretory phenotype in vascular smooth muscle cells from the non-human primate Macaca mulatta: reversal by resveratrol treatment. J Gerontol A Biol Sci Med Sci. 2012;67:811–820. doi:10.1093/gerona/glr228. 19. Mukhopadhyay P, Rajesh M, Haskó G, Hawkins BJ, Madesh M, Pacher P. Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy. Nat Protocol. 2007;2:2295–2301. doi:10.1038/nprot.2007.327. 20. Csiszar A, Labinskyy N, Perez V, et al. Endothelial function and vascular oxidative stress in long-lived GH/IGF-deficient Ames dwarf mice. Am J Physiol Heart Circ Physiol. 2008;295:H1882–H1894. doi:10.1152/ajpheart.412.2008. 21. Tucsek Z, Gautam T, Sonntag WE, et al. Aging exacerbates microvascular endothelial damage induced by circulating factors present in the serum of septic patients. J Gerontol A Biol Sci Med Sci. 2013;68:652–660. doi:10.1093/gerona/gls232. 22. Tucsek Z, Toth P, Sosnowska D, et al. Obesity in aging exacerbates bloodbrain barrier disruption, neuroinflammation, and oxidative stress in the mouse hippocampus: effects on expression of genes involved in beta-amyloid generation and Alzheimer’s disease. J Gerontol A Biol Sci Med Sci. 2014;69:1212–1226. doi:10.1093/gerona/glt177. 23. Tucsek Z, Toth P, Tarantini S, et al. Aging exacerbates obesity-induced cerebromicrovascular rarefaction, neurovascular uncoupling, and cognitive decline in mice. J Gerontol A Biol Sci Med Sci. 2014;69:1212–1226. doi:10.1093/gerona/glt177. 24. Ungvari Z, Tucsek Z, Sosnowska D, et al. Aging-induced dysregulation of dicer1-dependent microRNA expression impairs angiogenic capacity of
5
rat cerebromicrovascular endothelial cells. J Gerontol A Biol Sci Med Sci. 2013;68:877–891. doi:10.1093/gerona/gls242. 25. Valcarcel-Ares MN, Gautam T, Warrington JP, et al. Disruption of Nrf2 signaling impairs angiogenic capacity of endothelial cells: implications for microvascular aging. J Gerontol A Biol Sci Med Sci. 2012;67:821–829. 26. Banki E, Sosnowska D, Tucsek Z, et al. Age-related decline of autocrine PACAP impairs angiogenic capacity of rat cerebromicrovascular endothelial cells. J Gerontol A Biol Sci Med Sci. 2014. doi:10.1093/gerona/ glu1116. 27. Csiszar A, Pinto JT, Gautam T, et al. Resveratrol encapsulated in novel fusogenic liposomes activates Nrf2 and attenuates oxidative stress in cerebromicrovascular endothelial cells from aged rats. J Gerontol A Biol Sci Med Sci. 2014. doi:10.1093/gerona/glu029. 28. Csiszar A, Sosnowska D, Tucsek Z, et al. Circulating factors induced by caloric restriction in the nonhuman primate Macaca mulatta activate angiogenic processes in endothelial cells. J Gerontol A Biol Sci Med Sci. 2013;68:235–249. doi:10.1093/gerona/gls158. 29. Wei EP, Kontos HA, Christman CW, DeWitt DS, Povlishock JT. Superoxide generation and reversal of acetylcholine-induced cerebral arteriolar dilation after acute hypertension. Circ Res. 1985;57:781–787. 30. De Bruyn VH, Nuno DW, Cappelli-Bigazzi M, Dole WP, Lamping KG. Effect of acute hypertension in the coronary circulation: role of mechanical factors and oxygen radicals. J Hypertens. 1994;12:163–172. 31. Ghaleh B, Hittinger L, Kim SJ, et al. Selective large coronary endothelial dysfunction in conscious dogs with chronic coronary pressure overload. Am J Physiol. 1998;274(2 Pt 2):H539–H551. 32. Kinugawa S, Post H, Kaminski PM, et al. Coronary microvascular endothelial stunning after acute pressure overload in the conscious dog is caused by oxidant processes: the role of angiotensin II type 1 receptor and NAD(P)H oxidase. Circulation. 2003;108:2934–2940. 33. Jacobson A, Yan C, Gao Q, et al. Aging enhances pressure-induced arterial superoxide formation. Am J Physiol Heart Circ Physiol. 2007;293:H1344– H1350. doi:10.1152/ajpheart.00413.2007. 34. Oeckler RA, Kaminski PM, Wolin MS. Stretch enhances contraction of bovine coronary arteries via an NAD(P)H oxidase-mediated activation of the extracellular signal-regulated kinase mitogen-activated protein kinase cascade. Circ Res. 2003;92:23–31. 35. Ungvari Z, Wolin MS, Csiszar A. Mechanosensitive production of reactive oxygen species in endothelial and smooth muscle cells: role in microvascular remodeling? Antioxid Redox Signal. 2006;8:1121–1129. 36. Touyz RM, Montezano AC. Vascular Nox4: a multifarious NADPH oxidase. Circ Res. 2012;110:1159–1161. doi:10.1161/CIRCRESAHA.112.269068. 37. Dai DF, Chen T, Szeto H, et al. Mitochondrial targeted antioxidant peptide ameliorates hypertensive cardiomyopathy. J Am Coll Cardiol. 2011;58:73–82. doi:10.1016/j.jacc.2010.12.044. 38. Briones AM, Tabet F, Callera GE, et al. Differential regulation of Nox1, Nox2 and Nox4 in vascular smooth muscle cells from WKY and SHR. J Am Soc Hypertens. 2011;5:137–153. doi:10.1016/j.jash.2011.02.001. 39. Kuroda J, Ago T, Matsushima S, Zhai P, Schneider MD, Sadoshima J. NADPH oxidase 4 (Nox4) is a major source of oxidative stress in the failing heart. Proc Natl Acad Sci USA. 2010;107:15565–15570. doi:10.1073/ pnas.1002178107. 40. Dikalov SI, Nazarewicz RR. Angiotensin II-induced production of mitochondrial reactive oxygen species: potential mechanisms and relevance for cardiovascular disease. Antioxid Redox Signal. 2013;19:1085–1094. doi:10.1089/ars.2012.4604. 41. Dikalova AE, Bikineyeva AT, Budzyn K, et al. Therapeutic targeting of mitochondrial superoxide in hypertension. Circ Res. 2010;107:106–116. doi:10.1161/CIRCRESAHA.109.214601. 42. Ungvari Z, Bailey-Downs L, Sosnowska D, et al. Vascular oxidative stress in aging: a homeostatic failure due to dysregulation of NRF2-mediated antioxidant response. Am J Physiol Heart Circ Physiol. 2011;301:H363– H372. 43. Ungvari Z, Kaley G, de Cabo R, Sonntag WE, Csiszar A. Mechanisms of vascular aging: new perspectives. J Gerontol A Biol Sci Med Sci. 2010;65:1028–1041. doi:10.1093/gerona/glq113.
Brief report
Aging Exacerbates Pressure-Induced Mitochondrial Oxidative Stress in Mouse Cerebral Arteries Zsolt Springo,1,2,* Stefano Tarantini,1,* Peter Toth,1,* Zsuzsanna Tucsek,1 Akos Koller,2 William E. Sonntag,1,3 Anna Csiszar,1,2,3 and Zoltan Ungvari1,2,3 1 Reynolds Oklahoma Center on Aging, Donald W. Reynolds Department of Geriatric Medicine, University of Oklahoma Health Sciences Center. 2Department of Pathophysiology and Gerontology, Medical School and Szentágothai Research Center, University of Pecs, Hungary. 3The Peggy and Charles Stephenson Cancer Center, University of Oklahoma Health Sciences Center.
*These authors contributed equally to this work. Address correspondence to Zoltan Ungvari, MD, PhD, Reynolds Oklahoma Center on Aging, Department of Geriatric Medicine, University of Oklahoma Health Sciences Center, 975 N.E. 10th Street, BRC 1303, Oklahoma City, OK 73104. Email: [email protected]
Abstract Epidemiological studies demonstrate that in addition to the increased prevalence of hypertension in old patients, the deleterious cerebrovascular effects of hypertension (including atherosclerosis, stroke, and vascular cognitive impairment) are also exacerbated in elderly individuals. The cellular mechanisms by which aging and hypertension interact to promote cerebrovascular pathologies are not well understood. To test the hypothesis that aging exacerbates high pressure–induced mitochondrial oxidative stress, we exposed isolated segments of the middle cerebral arteries of young (3 months) and aged (24 months) C57BL/6 mice to 60 or 140 mmHg intraluminal pressure and assessed changes in mitochondrial reactive oxygen species production using a mitochondriatargeted redox-sensitive fluorescent indicator dye (MitoSox) by confocal microscopy. Perinuclear MitoSox fluorescence was significantly stronger in high pressure–exposed middle cerebral arteries compared with middle cerebral arteries of the same animals exposed to 60 mmHg, indicating that high pressure increases mitochondrial reactive oxygen species production in the smooth muscle cells of cerebral arteries. Comparison of young and aged middle cerebral arteries showed that aging exacerbates high pressure–induced mitochondrial reactive oxygen species production in cerebral arteries. We propose that increased mechanosensitive mitochondrial oxidative stress may potentially exacerbate cerebrovascular injury and vascular inflammation in aging.
Key Words: Hypertension—Middle cerebral artery—Oxidative stress—Mitochondrion—Free radicals. Decision Editor: Rafael de Cabo, PhD
Hypertension is known to exert deleterious effects on the cerebral circulation (1–3). The elderly individuals (≥65 years of age) have the highest prevalence of hypertension and these individuals are more likely to develop cerebrovascular pathologies
(4). Epidemiological studies demonstrate that in addition to the increased prevalence of hypertension in old patients, the deleterious cerebrovascular effects of hypertension are also exacerbated in the elderly patients (5–7).
© The Author 2015. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: [email protected].
1
2
Journals of Gerontology: BIOLOGICAL SCIENCES, 2015, Vol. 00, No. 00
There is strong evidence that oxidative stress, by promoting inflammation and pathological vascular remodeling and inducing activation of matrix metalloproteinases, plays a central role in hypertension-induced atherosclerosis and vascular injury. Among the potential sources of reactive oxygen species (ROS), the role of NADPH oxidases in hypertension-induced vascular oxidative stress has been well documented (reviewed in ref. 8). Recent evidence also implicates mitochondrial oxidative stress in hypertension-induced vascular pathologies (9). Despite the paramount importance of mitochondrial oxidative stress in the aging process in general and vascular aging in particular (10), it is not well understood how hypertension and aging interact to regulate mitochondrial ROS (mtROS) production in the cerebrovasculature. The mechanisms by which hypertension promotes vascular oxidative stress are likely multifaceted. Multiple lines of evidence suggest that in addition to circulating factors (eg, angiotensin II), changes in the hemodynamic environment associated with hypertension activate vascular ROS production in the peripheral circulation (11). A primary role for high intraluminal pressure in upregulation of ROS generation is supported by the findings that in aortic-banded rats (in which only blood vessels proximal to the coarctation are exposed to high pressure), regional increases in blood pressure result in selective increases in O2•− production in peripheral arteries (12). Furthermore, exposure of isolated peripheral arteries to high pressure in vitro was shown to increase cellular ROS production (11,13). Despite these advances, the role of high intraluminal pressure per se in regulation of mtROS production in cerebral arteries and its alterations in aging remain elusive. The present study was designed to test the hypothesis that aging exacerbates high pressure–induced mitochondrial oxidative stress in cerebral arteries. To test our hypothesis, we exposed isolated segments of the middle cerebral arteries (MCAs) of young and aged C57BL/6 mice to normal or high intraluminal pressure ex vivo and assessed changes in mtROS production using a mitochondriatargeted redox-sensitive fluorescent indicator dye by confocal microscopy.
(Living Systems Instrumentation, Burlington, VE). The absence of leaks was verified by observing no changes in intraluminal pressure for more than 3 minutes upon turning off the pressure servo-control system. To characterize mechanosensitive mitochondrial oxidative stress, pressure-induced changes in mitochondrial O2•− production in MCAs were measured using MitoSOX Red (Invitrogen, Carlsbad CA), a mitochondrion-specific hydroethidine-derivative fluorescent dye (10,14–19), as previously reported (20). In brief, cannulated MCAs were loaded with MitoSox (3 µmol/L for 30 minutes). Hoechst 33258 was used for nuclear staining. After loading, the chamber was washed out five times with warm Krebs buffer, and the vessels were allowed to equilibrate at 60 mmHg for another 20 minutes. Then, MCAs from the same animals were pressurized to 60 or 140 mmHg (normal pressure and high pressure groups, respectively) for 4 hours. After the experimental period, confocal images of the wall of the pressurized MCAs were captured using a Leica SP2 MP confocal laser scanning microscope. Average perinuclear MitoSox fluorescence intensities were assessed using the Metamorph software. In separate experiments, to assess pressure-induced cellular peroxide production, we used the cell-permeant oxidative fluorescent indicator dye CM-H2DCFDA (5 (and 6)-chloromethyl-2′,7′dichlorodihydrofluorescein diacetate-acetyl ester, Invitrogen) as we previously reported (17). In brief, pressurized MCAs were incubated with CM-H2DCFDA (5 µM, at 37°C, for 4 hours). At the end of the incubation period, CM-H2DCFDA fluorescence was assessed by confocal microscopy.
Methods
Despite recent advances (21–28), the mechanisms underlying the increased vulnerability of aged vessels to injury remain elusive. We used the mitochondria-targeted redox-sensitive dye MitoSox to examine pressure-induced production of O2•− by mitochondria in mouse cerebral arteries. We found that perinuclear MitoSox fluorescence (Figure 1B and C) was significantly stronger in high pressure– exposed MCAs compared with MCAs of the same animals exposed to 60 mmHg, indicating that high pressure increases mtROS production in the smooth muscle cells of cerebral arteries. Comparison of MCAs isolated from aged mice as isolated from young mice showed that aging significantly increases high pressure– induced mtROS production in cerebral arteries (Figure 1D). As illustrated in Figure 1E, a significant cellular heterogeneity with respect to pressure-induced mitochondrial oxidative stress was observed. Representative confocal images shown in Figure 1F suggest that high pressure–induced mtROS production is associated with increased cellular peroxide levels (represented by the intense 2′,7′-dichlorofluorescein staining).
Animals Young (3 months, n = 10) and aged (24 months, n = 10) male C57BL/6 mice were purchased from the National Institute on Aging. All mice were maintained under specific pathogen-free barrier conditions. Water and normal laboratory diet (D12450B, Research Diets Inc.; New Brunswick, NJ) were available ad libitum. All procedures were approved by the Institutional Animal Use and Care Committees of the participating institutions.
Measurement of Pressure-Induced mtROS Production in Isolated MCAs Mice were decapitated, the brains were removed, and segments of the MCAs were isolated using microsurgery instruments for imaging studies, as reported (2,3). Two segments of MCAs from the same animal were mounted onto two glass micropipettes in an organ chamber (Figure 1A) in oxygenated (21% O2, 5% CO2, 75% N2) Krebs’ buffer (composed of [in mmol/L]: 110.0 NaCl, 5.0 KCl, 2.5 CaCl2, 1.0 MgSO4, 1.0 KH2PO4, 5.5 glucose, and 24.0 NaHCO3, pH ~7.4; at 37°C) and pressurized to 10 mmHg. The hydrodynamic resistance of the micropipettes was matched. Inflow and outflow pressures were controlled and measured by a pressure servo-control system
Statistical Analysis Data were analyzed by one-way analysis of variance. A p value less than .05 was considered statistically significant. Data are expressed as mean ± SEM.
Results Aging Exacerbates Pressure-Induced Mitochondrial Oxidative Stress in Mouse Cerebral Arteries
Discussion The results of this study demonstrate for the first time that high pressure elicits increased mitochondrial production of ROS in smooth
Journals of Gerontology: BIOLOGICAL SCIENCES, 2015, Vol. 00, No. 00
3
Figure 1. Aging exacerbates hypertension-induced mitochondrial ROS (mtROS) production in cerebral arteries. (A) Schematic representation of the vessel culture setup used to image isolated MCAs exposed to high pressure in vitro. (B and C) Representative confocal images showing stronger perinuclear MitoSox staining (representing mtROS production; red fluorescence) in high pressure–exposed MCAs isolated from aged mice compared with MCAs isolated from young mice. Hoechst 33258 (blue fluorescence) was used for nuclear staining (original magnification: ×20). Bar graphs (D) are summary data. Data are means ± SEM (n = 5 in each group). *p < .05. (E) Representative image showing heterogeneity in MitoSox staining of smooth muscle cells (identified by the single centrally located spindle-shaped nuclei that are perpendicular to the long axis of the artery) in the wall of a pressurized MCA. Arrows and arrowheads point to cells with high and low mtROS production, respectively. (F) Representative confocal images showing that high pressure–induced mtROS production is associated with increased cellular peroxide levels (represented by the intense 2′,7′-dichlorofluorescein staining). (G) Proposed scheme depicting mechanisms of exacerbation of hypertension-induced vascular oxidative stress in aging and its pathophysiological consequences.
muscle cells located in the cerebral arteries and that this effect is significantly exacerbated in aging (Figure 1). Several lines of evidence suggest that changes in the hemodynamic environment associated with hypertension, either directly or indirectly, can activate ROS production in arteries from other vascular beds as well, including vessels from the coronary and
mesenteric circulation and the skeletal muscle (11,13,29–33). It is likely that increased wall tension–dependent cellular stretch is the primary mechanical stimulus for increased pressure-induced vascular ROS production. In support of this concept, previous studies reported that exposure of isolated arterial rings to in vitro stretching also increases ROS generation (34), mimicking the effects of
4
Journals of Gerontology: BIOLOGICAL SCIENCES, 2015, Vol. 00, No. 00
high pressure. Moreover, earlier studies also show that in cultured endothelial and smooth muscle cells, subjected to in vitro stretching, ROS production also significantly increases (reviewed in ref. 9). It is likely that mitochondria may release ROS in response to strain by virtue of their attachment to cytoskeletal proteins. The molecular mechanisms underlying mechanosensitive mtROS production in cerebral arteries are presently unknown. Mechanotransduction of pressure in vascular smooth muscle cells is known to involve diverse signaling pathways (eg, integrin-mediated signal transduction, Ca2+ signaling, activation of protein kinase C, Rho kinases, and mitogen-activated protein kinases) (35). The role of these pathways in regulation of mechanosensitive mtROS production should be elucidated in future studies. Our working hypothesis is that mechanical forces can be transmitted from the cell membrane to the mitochondria via cytoskeletal tension. Further studies are needed to characterize the molecular mechanotransducer in the mitochondrial membrane (eg, the possible involvement of mitochondrial KATP channels). The sites of mechanosensitive O2•− production in the mitochondria are also subject of future investigation. Potential sites include complex I, the entry point for electrons from NADH reduced nicotinamide adenine dinucleotide into the respiratory chain, and complex III, which funnels electrons from the CoQ pool to cytochrome c. In addition, the ROS-generating enzyme NADPH oxidase 4 (NOX4) is localized to the mitochondrial membrane, it can be activated by mechanical forces (36), and its activation increases mitochondrial oxidative stress (37). The expression of NOX4 is reported to be increased in hypertension (3,38), and an important study demonstrated that mitochondrial-located NOX4 is a major source of pressure overload-induced oxidative stress in the heart (39). Importantly, high levels of angiotensin II can further exacerbate NOX4-dependent ROS production (37,38). In addition to the direct generation of ROS by NOX4, it is also possible that activation of NOX4 also upregulates ROS production by the mitochondrial electron transports chain (40,41). These possibilities warrant further investigation. The mechanisms by which aging exacerbates pressure-induced mtROS production in cerebral arteries also remain elusive. We propose that aging may affect both mechanotransduction of pressure and alter downstream ROS producing mechanisms (eg, by altering the mitochondrial phenotype). Indeed, vascular aging is known to be associated with increased basal mtROS production (10); thus, it is likely that mtROS production stimulated by high pressure will also be exacerbated. In addition, abnormal ROS detoxification (including impaired activation of Nrf2-dependent antioxidant response in aging) (42) is likely to contribute to age-related exacerbation of hypertension-induced mitochondrial oxidative stress. It is significant that aging impairs myogenic adaptation of proximal cerebral arteries to hypertension, which likely enables the penetration of high pressure into the thinned-walled smaller vessels, exacerbating hypertension-induced oxidative damage (2,3). Importantly, a significant cellular heterogeneity with respect to pressure-induced mitochondrial oxidative stress was observed (Figure 1E). One may speculate that age-related changes in cellular phenotype (eg, activation of the senescence program) differentially alter the behavior of individual cells in response to the mechanical stimulation. It is also possible that heterogeneous changes in the extracellular matrix of aged cerebral arteries also contribute to this phenomenon. To understand fully the cellular specificity and complexity of the tissue microenvironment under hypertensive conditions in aged arteries (eg, the link between increased mtROS production and development of an inflammatory phenotype), it will be necessary to measure molecular signatures with single cell resolution in subsequent studies.
The consequences of increased propensity for pressure-induced mtROS generation in aged arteries are likely multiple (Figure 1G). There is increasing evidence supporting a role for mitochondriaderived ROS in chronic low-grade inflammation, including NF-κB activation and development of atherosclerosis, which is associated with aging in the cardiovascular system (10,43). The current view is that O2•−, overproduced in the aged mitochondria, is dismutated to H2O2 by Mn-SOD. It is likely the increased release of H2O2 that activates NF-κB, and likely many other redox-sensitive factors, in the cytoplasm (O2•− is membrane impermeable, whereas H2O2 easily penetrates the mitochondrial membranes) (10). Future studies should also investigate the interaction among aging, chronic hypertension, and mtROS production and its role in activation of matrix metalloproteinases and increased vulnerability of aged arteries to hypertension-induced injury (including stroke, aneurysm formation as well as vascular rarefaction).
Funding This work was supported by grants from the American Heart Association (to P.T., S.T., A.C., and Z.U.), the Oklahoma Center for the Advancement of Science and Technology (to A.C., Z.U., and W.E.S.), the Hungarian National Science Research Fund (OTKA) K 108444 and grants: Developing Competitiveness of Universities in the South Transdanubian Region, “Identification of new biomarkers…,” SROP-4.2.2.A-11/1/KONV-2012-0017 and “Complex examination of neuropeptides…,” SROP-4.2.2.A-11/1/KONV-2012-0024 to A.K., the National Center for Complementary and Alternative Medicine (R01-AT006526 to Z.U.), the National Institute on Aging (R01-AG031085 to A.C.; R01-AG038747 to W.E.S.; R01-NS056218 to A.C. and W.E.S.), the Ellison Medical Foundation (to W.E.S.), and the Arkansas Claude Pepper Older Americans Independence Center at University of Arkansas Medical Center (P30 AG028718 to A.C.). Z.S. is a Copastoeck Fellow supported by a fellowship from the European Union and the State of Hungary, cofinanced by the European Social Fund in the framework of TÁMOP 4.2.4. A/2-11-12012-0001 “National Excellence Program.”
References 1. Csiszar A, Tucsek Z, Toth P, et al. Synergistic effects of hypertension and aging on cognitive function and hippocampal expression of genes involved in β-amyloid generation and Alzheimer’s disease. Am J Physiol Heart Circ Physiol. 2013;305:H1120–H130. doi:10.1152/ajpheart.00288.2013. 2. Toth P, Csiszar A, Tucsek Z, et al. Role of 20-HETE, TRPC channels, and BKCa in dysregulation of pressure-induced Ca2+ signaling and myogenic constriction of cerebral arteries in aged hypertensive mice. Am J Physiol Heart Circ Physiol. 2013;305:H1698–H1708. doi:10.1152/ajpheart.00377.2013. 3. Toth P, Tucsek Z, Sosnowska D, et al. Age-related autoregulatory dysfunction and cerebromicrovascular injury in mice with angiotensin IIinduced hypertension. J Cereb Blood Flow Metab. 2013;33:1732–1742. doi:10.1038/jcbfm.2013.143. 4. Aronow WS, Fleg JL, Pepine CJ, et al. ACCF/AHA 2011 expert consensus document on hypertension in the elderly: a report of the American College of Cardiology Foundation Task Force on Clinical Expert Consensus Documents. Circulation. 2011;123:2434–2506. doi:10.1161/ CIR.0b013e31821daaf6. 5. Brickman AM, Reitz C, Luchsinger JA, et al. Long-term blood pressure fluctuation and cerebrovascular disease in an elderly cohort. Arch Neurol. 2010;67:564–569. doi:10.1001/archneurol.2010.70. 6. Reitz C, Tang MX, Manly J, Mayeux R, Luchsinger JA. Hypertension and the risk of mild cognitive impairment. Arch Neurol. 2007;64:1734–1740. doi:10.1001/archneur.64.12.1734.
Journals of Gerontology: BIOLOGICAL SCIENCES, 2015, Vol. 00, No. 00
7. Kuller LH, Lopez OL, Jagust WJ, et al. Determinants of vascular dementia in the Cardiovascular Health Cognition Study. Neurology. 2005;64:1548– 1552. doi:10.1212/01.WNL.0000160115.55756.DE. 8. Montezano AC, Touyz RM. Oxidative stress, Noxs, and hypertension: experimental evidence and clinical controversies. Ann Med. 2012;44(suppl 1):S2–S16. doi:10.3109/07853890.2011.653393. 9. Dikalov SI, Ungvari Z. Role of mitochondrial oxidative stress in hypertension. Am J Physiol Heart Circ Physiol. 2013;305:H1417–H1427. doi:10.1152/ajpheart.00089.2013. 10. Ungvari Z, Orosz Z, Labinskyy N, et al. Increased mitochondrial H2O2 production promotes endothelial NF-kappaB activation in aged rat arteries. Am J Physiol Heart Circ Physiol. 2007;293:H37–H47. 11. Ungvari Z, Csiszar A, Huang A, Kaminski PM, Wolin MS, Koller A. High pressure induces superoxide production in isolated arteries via protein kinase C-dependent activation of NAD(P)H oxidase. Circulation. 2003;108:1253–1258. 12. Ungvari Z, Csiszar A, Kaminski PM, Wolin MS, Koller A. Chronic high pressure-induced arterial oxidative stress: involvement of protein kinase C-dependent NAD(P)H oxidase and local renin-angiotensin system. Am J Pathol. 2004;165:219–226. 13. Huang A, Sun D, Kaley G, Koller A. Superoxide released to high intraarteriolar pressure reduces nitric oxide-mediated shear stress- and agonistinduced dilations. Circ Res. 1998;83:960–965. 14. Robinson KM, Janes MS, Beckman JS. The selective detection of mitochondrial superoxide by live cell imaging. Nat Protocol. 2008;3:941–947. doi:10.1038/nprot.2008.56. 15. Ungvari Z, Podlutsky A, Sosnowska D, et al. Ionizing radiation promotes the acquisition of a senescence-associated secretory phenotype and impairs angiogenic capacity in cerebromicrovascular endothelial cells: role of increased DNA damage and decreased DNA repair capacity in microvascular radiosensitivity. J Gerontol A Biol Sci Med Sci. 2013;68:1443–1457. doi:10.1093/gerona/glt057. 16. Ungvari Z, Labinskyy N, Mukhopadhyay P, et al. Resveratrol attenuates mitochondrial oxidative stress in coronary arterial endothelial cells. Am J Physiol Heart Circ Physiol. 2009;297:H1876–H1881. doi:10.1152/ajpheart.00375.2009. 17. Csiszar A, Podlutsky A, Podlutskaya N, et al. Testing the oxidative stress hypothesis of aging in primate fibroblasts: is there a correlation between species longevity and cellular ROS production? J Gerontol A Biol Sci Med Sci. 2012;67:841–852. 18. Csiszar A, Sosnowska D, Wang M, Lakatta EG, Sonntag WE, Ungvari Z. Age-associated proinflammatory secretory phenotype in vascular smooth muscle cells from the non-human primate Macaca mulatta: reversal by resveratrol treatment. J Gerontol A Biol Sci Med Sci. 2012;67:811–820. doi:10.1093/gerona/glr228. 19. Mukhopadhyay P, Rajesh M, Haskó G, Hawkins BJ, Madesh M, Pacher P. Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy. Nat Protocol. 2007;2:2295–2301. doi:10.1038/nprot.2007.327. 20. Csiszar A, Labinskyy N, Perez V, et al. Endothelial function and vascular oxidative stress in long-lived GH/IGF-deficient Ames dwarf mice. Am J Physiol Heart Circ Physiol. 2008;295:H1882–H1894. doi:10.1152/ajpheart.412.2008. 21. Tucsek Z, Gautam T, Sonntag WE, et al. Aging exacerbates microvascular endothelial damage induced by circulating factors present in the serum of septic patients. J Gerontol A Biol Sci Med Sci. 2013;68:652–660. doi:10.1093/gerona/gls232. 22. Tucsek Z, Toth P, Sosnowska D, et al. Obesity in aging exacerbates bloodbrain barrier disruption, neuroinflammation, and oxidative stress in the mouse hippocampus: effects on expression of genes involved in beta-amyloid generation and Alzheimer’s disease. J Gerontol A Biol Sci Med Sci. 2014;69:1212–1226. doi:10.1093/gerona/glt177. 23. Tucsek Z, Toth P, Tarantini S, et al. Aging exacerbates obesity-induced cerebromicrovascular rarefaction, neurovascular uncoupling, and cognitive decline in mice. J Gerontol A Biol Sci Med Sci. 2014;69:1212–1226. doi:10.1093/gerona/glt177. 24. Ungvari Z, Tucsek Z, Sosnowska D, et al. Aging-induced dysregulation of dicer1-dependent microRNA expression impairs angiogenic capacity of
5
rat cerebromicrovascular endothelial cells. J Gerontol A Biol Sci Med Sci. 2013;68:877–891. doi:10.1093/gerona/gls242. 25. Valcarcel-Ares MN, Gautam T, Warrington JP, et al. Disruption of Nrf2 signaling impairs angiogenic capacity of endothelial cells: implications for microvascular aging. J Gerontol A Biol Sci Med Sci. 2012;67:821–829. 26. Banki E, Sosnowska D, Tucsek Z, et al. Age-related decline of autocrine PACAP impairs angiogenic capacity of rat cerebromicrovascular endothelial cells. J Gerontol A Biol Sci Med Sci. 2014. doi:10.1093/gerona/ glu1116. 27. Csiszar A, Pinto JT, Gautam T, et al. Resveratrol encapsulated in novel fusogenic liposomes activates Nrf2 and attenuates oxidative stress in cerebromicrovascular endothelial cells from aged rats. J Gerontol A Biol Sci Med Sci. 2014. doi:10.1093/gerona/glu029. 28. Csiszar A, Sosnowska D, Tucsek Z, et al. Circulating factors induced by caloric restriction in the nonhuman primate Macaca mulatta activate angiogenic processes in endothelial cells. J Gerontol A Biol Sci Med Sci. 2013;68:235–249. doi:10.1093/gerona/gls158. 29. Wei EP, Kontos HA, Christman CW, DeWitt DS, Povlishock JT. Superoxide generation and reversal of acetylcholine-induced cerebral arteriolar dilation after acute hypertension. Circ Res. 1985;57:781–787. 30. De Bruyn VH, Nuno DW, Cappelli-Bigazzi M, Dole WP, Lamping KG. Effect of acute hypertension in the coronary circulation: role of mechanical factors and oxygen radicals. J Hypertens. 1994;12:163–172. 31. Ghaleh B, Hittinger L, Kim SJ, et al. Selective large coronary endothelial dysfunction in conscious dogs with chronic coronary pressure overload. Am J Physiol. 1998;274(2 Pt 2):H539–H551. 32. Kinugawa S, Post H, Kaminski PM, et al. Coronary microvascular endothelial stunning after acute pressure overload in the conscious dog is caused by oxidant processes: the role of angiotensin II type 1 receptor and NAD(P)H oxidase. Circulation. 2003;108:2934–2940. 33. Jacobson A, Yan C, Gao Q, et al. Aging enhances pressure-induced arterial superoxide formation. Am J Physiol Heart Circ Physiol. 2007;293:H1344– H1350. doi:10.1152/ajpheart.00413.2007. 34. Oeckler RA, Kaminski PM, Wolin MS. Stretch enhances contraction of bovine coronary arteries via an NAD(P)H oxidase-mediated activation of the extracellular signal-regulated kinase mitogen-activated protein kinase cascade. Circ Res. 2003;92:23–31. 35. Ungvari Z, Wolin MS, Csiszar A. Mechanosensitive production of reactive oxygen species in endothelial and smooth muscle cells: role in microvascular remodeling? Antioxid Redox Signal. 2006;8:1121–1129. 36. Touyz RM, Montezano AC. Vascular Nox4: a multifarious NADPH oxidase. Circ Res. 2012;110:1159–1161. doi:10.1161/CIRCRESAHA.112.269068. 37. Dai DF, Chen T, Szeto H, et al. Mitochondrial targeted antioxidant peptide ameliorates hypertensive cardiomyopathy. J Am Coll Cardiol. 2011;58:73–82. doi:10.1016/j.jacc.2010.12.044. 38. Briones AM, Tabet F, Callera GE, et al. Differential regulation of Nox1, Nox2 and Nox4 in vascular smooth muscle cells from WKY and SHR. J Am Soc Hypertens. 2011;5:137–153. doi:10.1016/j.jash.2011.02.001. 39. Kuroda J, Ago T, Matsushima S, Zhai P, Schneider MD, Sadoshima J. NADPH oxidase 4 (Nox4) is a major source of oxidative stress in the failing heart. Proc Natl Acad Sci USA. 2010;107:15565–15570. doi:10.1073/ pnas.1002178107. 40. Dikalov SI, Nazarewicz RR. Angiotensin II-induced production of mitochondrial reactive oxygen species: potential mechanisms and relevance for cardiovascular disease. Antioxid Redox Signal. 2013;19:1085–1094. doi:10.1089/ars.2012.4604. 41. Dikalova AE, Bikineyeva AT, Budzyn K, et al. Therapeutic targeting of mitochondrial superoxide in hypertension. Circ Res. 2010;107:106–116. doi:10.1161/CIRCRESAHA.109.214601. 42. Ungvari Z, Bailey-Downs L, Sosnowska D, et al. Vascular oxidative stress in aging: a homeostatic failure due to dysregulation of NRF2-mediated antioxidant response. Am J Physiol Heart Circ Physiol. 2011;301:H363– H372. 43. Ungvari Z, Kaley G, de Cabo R, Sonntag WE, Csiszar A. Mechanisms of vascular aging: new perspectives. J Gerontol A Biol Sci Med Sci. 2010;65:1028–1041. doi:10.1093/gerona/glq113.
