Mitochondrial biogenesis is involved in the control of cell metabolism, signal transduction, and regulation of mitochondrial reactive oxygen species (ROS) production. Despite the central role of mitochondria in cellular aging and endothelial physiology, there are no studies extant investigating age-related alterations in mitochondrial biogenesis in blood vessels. Electronmicroscopy and confocal microscopy (en face Mitotracker staining) revealed that in aortas of F344 rats, a decline in mitochondrial biogenesis occurs with aging. In aged vessels, the expression of the mitochondrial biogenesis factors (including mitochondrial transcription factor A and peroxisome proliferator-activated receptor-γ coactivator-1) was decreased. The vascular expression of complex I, III, and IV significantly declined with age, whereas aging did not alter the expression of complex II and V. Cytochrome c oxidase (COX) expression/activity exhibited the greatest age-related decline, which was associated with increased mitochondrial ROS production in the aged vessels. In cultured coronary arterial endothelial cells, a partial knockdown of COX significantly increased mitochondrial ROS production. In conclusion, vascular aging is characterized by a decline in mitochondrial mass in the endothelial cells and an altered expression of components of the mitochondrial electron transport chain likely due to a dysregulation of mitochondrial biogenesis factors. We posit that impaired mitochondrial biogenesis and downregulation of COX may contribute to the increased mitochondrial oxidative stress in aged endothelial cells.
- vascular senescence
mitochondria are highly dynamic organelles, and their biogenesis is likely to be involved in the regulation of cell metabolism and signal transduction (19). In addition, there is a growing body of evidence that mitochondrial biogenesis is a key regulator of mitochondrial reactive oxygen species (ROS) production (28). Recent studies suggest that mitochondrial oxidative stress plays an important role in cardiovascular dysfunction in various pathophysiological states, including diabetes (6, 17, 25, 32, 38, 42).
Since Harman (22) proposed the original mitochondrial theory of aging, it has been widely accepted that mitochondrial oxidative stress is a major factor in the pathophysiology of aging in many organs, including skeletal muscle, heart, and the brain (reviewed recently elsewhere in Refs. 1, 8, and 46). There is evidence that in these organs, mitochondrial biogenesis is dysregulated, and it is thought that the resulting decline in cellular mitochondrial mass may contribute to the increased mitochondrial generation of ROS. Recent studies by this and other laboratories (43, 45) showed that aged endothelial and smooth muscle cells exhibit increased mitochondrial ROS production. There is growing evidence that aging promotes vascular inflammation (9, 10, 12, 14–16, 43) and development of atherosclerosis and that mitochondrial oxidative stress may contribute to proinflammatory phenotypic alterations in aged vessels. In this context we have demonstrated that increased mitochondrial ROS production promotes endothelial NF-κB activation, which contributes to the upregulation of adhesion molecules and increased monocyte adhesiveness in the aged arteries (43). Despite the emerging role of mitochondria in endothelial pathophysiological alteration in aging and the relationship between mitochondrial biogenesis and mitochondrial ROS production, there are no studies extant investigating aging-induced alterations in vascular mitochondrial biogenesis.
Thus the present study was designed to test the hypothesis that aging leads to dysregulation of vascular mitochondrial biogenesis. To achieve that goal, we determined whether aging is associated with alterations in mitochondrial mass in the endothelial cells (confocal microscopy, electron microscopy), altered vascular expression of the nuclear-encoded components of the electron transport chain, and mitochondrial biogenesis factors. We also sought to determine whether selective downregulation of cytochrome c oxidase (COX) may contribute to the development of mitochondrial oxidative stress in endothelial cells.
All animal use protocols were approved by the Institutional Animal Care and Use Committee of the New York Medical College (Valhalla, NY). Male Fisher 344 rats [ages, 3 (young) and 24 (aged) mo old; n = 30; purchased from the National Institute of Aging; kept under pathogen-free conditions] were used, as previously described (15, 16, 43). All animals were disease free with no signs of systemic inflammation and/or neoplastic alterations. The animals were euthanized by an overdose of pentobarbital sodium as previously described (15, 16), and the carotid arteries and aortas were isolated for subsequent studies.
Mitochondrial content in the endothelial cells was assessed by selectively loading the mitochondria with the red fluorescent dye Mitotracker (Invitrogen, Carlsbad, CA). Sytox green (Invitrogen) was used as nuclear counterstaining. Optical sections of the endothelial cells of en face vessel preparations were captured at ×60 magnification using a Bio-Rad confocal microscope. The ratio of mitochondrial area densities to cytoplasmic volume was calculated using the Zeiss Axionvision imaging software (Carl Zeiss, Gottingen, Germany).
Carotid arteries (n = 3 animals in each group) were fixed using Karnowsky's method (23). Thin sections were obtained with an ultramicrotome, stained with osmium tetroxide, and examined with a transmission electron microscope (Zeiss Leo 906) as previously described (5). Cytoplasmic volume densities were obtained in a blinded fashion using the principles of Weibel (47). Data were expressed as volume density (volume of mitochondria per cytoplasmic volume).
Measurement of cytochrome c oxidase enzyme activity.
COX enzyme activity was measured spectrophotometrically from vascular homogenates as previously reported (20). The results were normalized to sample protein concentration.
Western blot analysis.
Western blot analysis was performed as previously described (14, 15). Primary antibodies directed against porin, complex I, complex II, complex III, complex V (Molecular Probes/Invitrogen), and cytochrome c oxidase subunit IV (COX-IV; No. 4844, Cell Signaling) were used. Anti-β-actin (No. 6276, Abcam) was used for normalization purposes.
Measurement of mitochondrial O2•− and H2O2 production.
MitoSox (Invitrogen), a mitochondrion-specific hydroethidine-derivative fluorescent dye, was used to assess mitochondrial O2•− production in the vascular endothelial cells in situ as previously described (11, 43) using the modified protocols from the laboratory of Pacher and colleagues (30, 31). In brief, aortic segments (en face) were incubated with MitoSox (5 × 10−6 mol/l) in Krebs buffer solution containing (in mM) 118 NaCl, 4.7 KCl, 1.5 CaCl2, 25 NaHCO3, 1.1 MgSO4, 1.2 KH2PO4, and 5.6 glucose at 37°C; gassed with 95% room air-5% CO2. MitoSox was protected from light and exposure to air. All the experiments were run on the same day using a fresh MitoSox stock solution. Interestingly, we have noticed that in a HEPES buffer, MitoSox was loaded in part to the nucleus; therefore, we used Krebs buffer solution in the present experiments. Confocal microscopy showed that under the aforementioned experimental conditions, MitoSox was selectively loaded into the mitochondria (30, 31). The time course (for 30 min) of buildup of perinuclear MitoSox fluorescence was obtained, and the slope factor was calculated as previously described (11). In some experiments vessels coincubated with 1 μmol/l carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP, a potent uncoupler of oxidative phosphorylation, which effectively attenuates mitochondria-derived ROS production; Invitrogen) (11, 43).
In separate experiments, H2O2 production was measured fluorometrically in aortic segments using the Amplex red/horseradish peroxidase assay as we reported (11). H2O2 generation rate was compared by measuring the time course of the buildup of resorufin fluorescence for 60 min in the presence and absence of FCCP. Appropriate corrections for background signals were applied, and standard curves generated using known amounts of H2O2 were used to calculate the rate of H2O2 production (in nmol/min). Data were normalized to tissue mass and reported as relative differences in H2O2 production (normalized to the mean value of H2O2 generation by young vessels). We have recently reported that resorufin fluorescence in this assay shows an excellent linear correlation with H2O2 concentrations in the range of 10−9 to 3 × 10−6 mol/l H2O2 (11).
Studies on cultured endothelial cells.
Primary human coronary arterial endothelial cells (CAECs; Cell Applications) were cultured as previously described (43). To mimic the aged endothelial phenotype, a partial downregulation of COX-IV in CAECs was achieved by RNA interference using proprietary small-interfering (si)RNA sequences (Superarray) and the Amaxa Nucleofector technology (Amaxa, Gaithersburg, MD), as we have previously reported (7, 13). A siRNA dose, which resulted in ∼30% knockdown while preserving cell viability, was selected based on pilot experiments. Cell density at transfection was 30%. Specific gene silencing was verified with Western blot analysis as described (7, 16). Free radical measurements were performed using CAECs transfected with anti-COX-IV siRNA on day 2 after the transfection, when gene silencing was optimal (Fig. 3, C and D). As a positive control, COX was also inhibited by KCN (1 mmol/l). Mitochondria-derived ROS production was assessed in CEACs using the MitoSox and Amplex red assays, as previously described (11, 43).
Quantitative real-time PCR.
We have used a quantitative real-time (qRT)-PCR technique to analyze mRNA expression of mitochondrial biogenesis factors [mitochondrial transcription factor A (Tfam), nuclear respiratory factor-1 (NRF-1), peroxisome proliferator-activated receptor-γ coactivator-1 (PGC-1α), mitofusin-1/2 (Mfn-1/2)] in carotid arteries of 3- and 24-mo-old rats, using the Strategen MX3000 as previously reported (43). Total RNA was isolated with Mini RNA Isolation Kit (Zymo Research) and was reverse transcribed using Superscript III RT (Invitrogen). Efficiency of the PCR reaction was determined using dilution series of a standard vascular sample. Quantification was performed using the ΔΔCT method. The housekeeping gene β-actin was used for internal normalization. Fidelity of the PCR reaction was determined by melting temperature analysis and visualization of product on a 2% agarose gel.
Data were normalized to the respective control mean values and are expressed as means ± SE. Statistical analyses of data were performed by Student's t-test or by two-way ANOVA followed by the Tukey post hoc test, as appropriate. P < 0.05 was considered statistically significant.
Decreased mitochondrial content in the endothelial cells in aged arteries.
Mitotracker staining showed that mitochondria are localized in the perinuclear region in vascular endothelial cells (Fig. 1, A and B). The ratio of mitochondrial area densities to cytoplasmic volume in Mitotracker-labeled endothelial cells was significantly lower in aged arteries than in young vessels (Fig. 1, A–C). The distribution of mitochondria in endothelial cells was unaltered by aging (Fig. 1C, inset). An analysis of electron microscopic images of endothelial cells confirmed that the volume of mitochondria per cytoplasmic volume is lower (by ∼20%) in aged vessels (Fig. 1, E and H) than in young arteries (Fig. 1, D and H). Smooth muscle cell mitochondrial content was also decreased in aged vessels (Fig. 1G) as compared with young controls (Fig. 1F). Western blot analysis revealed that compared with young vessels in aged vessels, the expression of porin was significantly decreased (Fig. 1, I and J). The levels of porin, which controls the diffusion of small metabolites through the outer membrane, are thought to correlate with the cellular mitochondrial volume.
Altered mitochondrial composition in aged arteries.
Western blot analysis showed that the expression of complex I (Fig. 2A), complex III (Fig. 2C), and complex IV (Fig. 2E) is decreased in aged arteries compared with young vessels. Expression of complex II (Fig. 2B) and complex V (Fig. 2D) did not change with age.
Decreased COX expression and activity in aged arteries.
Importantly, among the components of electron transport chain investigated, the relative expression of complex IV exhibited the greatest age-related decline (Fig. 2E). Downregulation of COX protein expression was associated with a decreased COX activity in homogenates of aged vessels (Fig. 2F).
Increased mitochondrial O2•− and H2O2 production in aged arteries.
Endothelial MitoSox fluorescence was measured in en face aorta preparations. In endothelial cells of aged aortas, there was an increased MitoSox fluorescence (Fig. 3A), which was attenuated by FCCP. Similar results were obtained when FCCP-sensitive H2O2 production was compared in young and aged aortic segments (Fig. 3B).
Downregulation and inhibition of COX increase mitochondrial oxidative stress in endothelial cells.
In cultured CAECs, partial downregulation of COX (by siRNA, Fig. 3, C and D) or pharmacological inhibition of COX (by KCN) cells resulted in a significant rise in mitochondrial O2•−/H2O2 production, as assessed by the MitoSox and Amplex red/horseradish peroxidase assays, respectively (Fig. 3, E and F).
Age-related changes in the expression of mitochondrial biogenesis factors.
qRT-PCR measurements revealed that the expression of the mitochondrial biogenesis factor Tfam (Fig. 4A) and PGC-1α (Fig. 4B) is significantly decreased in aged arteries compared with young vessels. The expression of NRF-1 (Fig. 4B), Mfn-1 (Fig. 4D), and Mfn-2 (Fig. 4E) also tended to decrease in aged vessels; however, the difference did not reach statistical significance.
The results of this study, in which we examined the effect of aging on mitochondrial biogenesis and mitochondrial ROS generation in endothelial cells, permit several new and potentially important insights to be drawn with respect to the role of mitochondria in vascular aging.
Here we report that aged endothelial cells contain significantly less mitochondria than young ones (Fig. 1). Electron micrographic analysis (Fig. 1, D–H) and assessment of porin content (Fig. 1, I and J) in whole vessel homogenates suggest that mitochondrial mass also declines aged smooth muscle cells. Similar age-related decline in mitochondrial content has been previously reported in the skeletal muscle (24). The majority of mitochondrial proteins are encoded in the nucleus, synthesized in the cytosol, and imported into mitochondria (the mtDNA codes for only 13 proteins essential for oxidative phosphorylation as well as the structural RNAs for mitochondrial protein synthesis). In the present study we assessed age-related alterations in representative nuclear-encoded subunits of components of the electron transport chain. It seems that not only the gross mitochondrial mass is decreased in aging, but there is also a dysregulation of the relative expression level of the different electron transport chain components in aged vessels (Fig. 2). The vascular expression of complex I, complex III, and complex IV significantly declined with age, whereas aging did not alter the expression of complex II and complex V (Fig. 2). Interestingly, the expression of the nuclear-encoded COX subunit IV exhibited the greatest age-related decline in relation to the other examined electron transport chain complexes (Fig. 2E). Downregulation of COX expression was also accompanied by a significant age-dependent decline in vascular COX activity (Fig. 2F), which may reflect impaired mitochondrial oxidative capacities in aged vessels. Of note, in skeletal muscle of aged rats, mitochondrial dysregulation is accompanied by a 30% reduction in ATP production (29). Whether this is the case in aged blood vessels is yet to be determined. Mitochondrial mass in most tissues including the skeletal muscle is normally regulated in accordance with metabolic activity of the cells. One may hypothesize that if bioenergetic demand for ATP were to decrease with age, the mitochondrial mass would also be downregulated. In that case, the decrease in mitochondrial mass would be a marker of an aging endothelial cell, rather than a mediator of its aging phenotype. Although age-related alterations in the bioenergetic demand of vascular cells has not been characterized, one can assume that maintenance of vascular tone, transport, and secretory function of vascular cells would require comparable amounts of ATP in young and old animals. Moreover, caloric restriction, which slows down metabolic activity, was shown to increase mitochondrial biogenesis (37). Thus we believe that the observed decline in mitochondrial mass plays a pathophysiological role in aged vessels.
There is increasing evidence that alterations in mitochondrial biogenesis are associated with mitochondrial dysfunction in various organs in aging (reviewed elsewhere in Refs. 1 and 8). In the present study we have evidence that in the endothelial cells of aged arteries, there is a substantially increased mitochondrial ROS production (Fig. 3, A and B), extending our previous findings (43). Previous studies also documented that the release of ROS from isolated skeletal-muscle mitochondria significantly increases with age (29). It is thought that mitochondrial proliferation reduces the flow of electrons per unit mitochondria, if the cellular energy demand is unchanged, which per se attenuates mitochondrial ROS production. This view is supported by our recent findings that mitochondrial biogenesis induced by activation of SIRT1 significantly decreases both basal and stimulated mitochondrial O2•− generation in endothelial cells (Csiszar and Ungvari, unpublished). A similar reduction in mitochondrial ROS production was observed in HeLa cells and primary hepatocytes when mitochondrial biogenesis was induced using sera from caloric-restricted animals (28). On the basis of the aforementioned findings, we posit that an impaired mitochondrial biogenesis in aging may contribute to an increased mitochondrial oxidative stress. The mitochondrial hypothesis of aging predicts that if impaired mitochondrial biogenesis is important in eliciting mitochondrial oxidative stress and thereby determining the rate of cardiovascular aging, then the mitochondrial content in the cells of long-lived animals should be maintained for an extended period of time. Our recent data showing no decline in cardiac mitochondrial mass in the longest-living rodent, the naked mole rat (Heterocephalus glaber, maximum life-span potential, >28 yr) (9) in a 24-yr time frame, seems to support this premise. Recently, we have shown that increased mitochondria-derived H2O2 generation in aging promotes endothelial NF-κB activation and that proinflammatory gene expression in aged arteries can be inhibited by attenuation of mitochondrial H2O2 production (43). In this context, it is important to note that the same stimuli that elicit mitochondrial biogenesis (i.e., SIRT1 activators and caloric restriction) (26, 28) also inhibit NF-κB activation in aged vessels.
In aged endothelial cells, the precise sites of ROS production in the electron transport chain are still a matter of debate, but it seems that both complex I and complex III are capable of producing O2•−, particularly when the electron transport is inhibited (1, 4, 21, 43). An impairment of complex IV activity is expected to increase the fraction of upstream electron carriers in a reduced state, thereby increasing the rate of univalent reduction of oxygen and subsequently the generation of O2•− (and subsequently H2O2) from the aforementioned sites. Thus we hypothesized that a relative decrease in complex IV expression/activity could, at least in part, contribute to the increased ROS production in aged endothelial cells. The findings that specific, partial downregulation of COX (by siRNA) or that pharmacological inhibition of COX in endothelial cells resulted in a significant rise in mitochondrial O2•− production and increased mitochondrion-derived cellular H2O2 levels (Fig. 3) support this premise.
Mitochondrial biogenesis requires the choreographed expression of diverse transcription activators. To determine whether the decreased numbers of mitochondria in aged endothelial cells could be a consequence of altered mitochondrial biogenesis factor expression, we used qRT-PCR to examine the relative mRNA expression levels of some of these factors. NRF-1 activates the transcription of a number of the nuclear-encoded components of the electron transport chain and also regulates Tfam, which is responsible for the transcription of mtDNA-encoded genes (18). The regulatory function of NRF-1 and other mitochondrial biogenesis factors can be modulated by coactivators such as PGC-1α. Mfns are mitochondrial membrane proteins that participate in mitochondrial fusion in mammalian cells and regulate the expression nuclear-encoded subunits of many oxidative phosphorylation complexes (41). We found that Tfam and PGC-1α are significantly downregulated in aged vessels (Fig. 4). The expression of NRF-1, Mfn-1, and Mfn-2 also tended to decrease in aged vessels; however, the differences did not reach statistical significance (Fig. 4). There is solid evidence that alterations in the expression of these factors modulate mitochondrial biogenesis activity (33–37), and we have previously confirmed that in cultured endothelial cells, the expression of Tfam correlates with COX expression (Csiszar and Ungvari, unpublished data). Previous studies raised the possibility that the expression of many mitochondrial biogenesis factors can be regulated by NO bioavailability (2, 37). Because in aged rat arteries bioavailability of NO is significantly decreased (12, 14), one can hypothesize that impaired NO bioavailability in aging may contribute to age-related alterations in mitochondrial mass in blood vessels. Accordingly, previous studies showed that in endothelial NO synthase knockout mice, mitochondrial biogenesis is impaired (27). It should be noted that NO can play a multifaceted role in the regulation of mitochondrial ROS generation. For example, recent studies indicate that a partial inhibition of COX by NO leads to an accumulation of reduced cytochrome c and, consequently, to an increase in electron flux through the enzyme population not inhibited by NO (39). In agreement with the results of the laboratory of Moncada and colleagues (40), we have previously reported that an inhibition of NO synthase does not result in an increased O2•− generation, when the cells are incubated at 21% O2-79% room air (43). However, under hypoxic conditions, endogenous NO seems to directly regulate mitochondrial O2•− production (40).
In conclusion, vascular aging is characterized by a decline in mitochondrial mass in the endothelial cells and an altered expression of components of the mitochondrial electron transport chain likely due to a dysregulation of mitochondrial biogenesis factors (Fig. 4F). Aging-induced alterations in mitochondrial phenotype, including the downregulation of COX, are likely to contribute to increased mitochondrial oxidative stress in aged endothelial cells. Because mitochondrial oxidative stress promotes vascular inflammation in aging, interventional treatments that normalize mitochondrial biogenesis [e.g., SIRT1 activators (3) and caloric restriction] may attenuate mitochondrial oxidative stress in aged endothelial cells exerting vasoprotective effects in the elderly.
This work was supported by American Heart Association Grants 0430108N and 0435140N; National Heart, Lung, and Blood Institute Grants HL-077256 and HL-43023; and Philip Morris USA and Philip Morris International.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2008 by the American Physiological Society