Lifespan, body size and oxygen : aerobic metabolism and oxidative stress in cultured fibroblasts /

The allometric scaling relationship observed between metabolic rate (MR) and species body mass can be partially explained by differences in cellular MR (Porter & Brand, 1995). Here, I studied cultured cell lines derived from ten mammalian species to determine whether cells propagated in an identical environment exhibited MR scaling. Oxidative and anaerobic metabolic parameters did not scale significantly with donor body mass in cultured cells, indicating the absence of an intrinsic MR setpoint. The rate of oxygen delivery has been proposed to limit cellular metabolic rates in larger organisms (West et al., 2002). As such cells were cultured under a variety of physiologically relevant oxygen tensions to investigate the effect of oxygen on cellular metabolic rates. Exposure to higher medium oxygen tensions resulted in increased metabolic rates in all cells. Higher MRs have the potential to produce more reactive oxygen species (ROS) which could cause genomic instability and thus reduced lifespan. Longer-lived species are more resistant to oxidative stress (Kapahi et al, 1999), which may be due to greater antioxidant and/or DNA repair capacities. This hypothesis was addressed by culturing primary dermal fibroblasts from eight mammalian species ranging in maximum lifespan from 5 to 120 years. Only the antioxidant manganese superoxide dismutases (MnSOD) positively scaled with species lifespan (p<0.01). Oxidative damage to DNA is primarily repaired by the base excision repair (BER) pathway. BER enzyme activities showed either no correlation or as in the case of polymerase p correlated, negatively with donor species (p<0.01 ). Typically, mammalian cells are cultured in a 20% O2 (atmospheric) environment, which is several-fold higher than cells experience in vivo. Therefore, the secondary aim of this study was to determine the effect of culturing mammalian cells at a more physiological oxygen tension (3%) on BER, and antioxidant, enzyme activities. Consistently, standard culture conditions induce higher antioxidant and DNA ba.se excision repair activities than are present under a more physiological oxygen concentration. Therefore, standard culture conditions are inappropriate for studies of oxidative stress-induced activities and species differences in fibroblast DNA BER repair capacities may represent differences in ability to respond to oxidative stress. An interesting outcome firom this study was that some inherent cellular properties are maintained in culture (i.e. stress responses) while others are not (i.e. MR).

A Survey of hMLH1 mRNA Splicing Profiles in Human Cell Lines: Comparing Primary Cultured Fibroblasts Before and After Oxidative Stress and Transiently versus Stably Demethylated Colon Cancer Derived Cell Lines

Most human genes undergo alternative splicing and loss of splicing fidelity is associated with disease. Epigenetic silencing of hMLH 1 via promoter cytosine methylation is causally linked to a subset of sporadic non-polyposis colon cancer and is reversible by 5-aza-2' -deoxycytidine treatment. Here I investigated changes in hMLHI mRNA splicing profiles in normal fibroblasts and colon cancer-derived human cell lines. I established the types and frequencies of hMLHI mRNA transcripts generated under baseline conditions, after hydrogen peroxide induced oxidative stress, and in acutely 5-aza-2' -deoxycytidine-treated and stably derepressed cancer cell lines. I found that hMLHI is extensively spliced under all conditions including baseline (50% splice variants), the splice variant distribution changes in response to oxidative stress, and certain splice variants are sensitive to 5- aza-2' -deoxycytidine treatment: Splice variant diversity and frequency of exon 17 skipping correlates with the level of hMLHI promoter methylation suggesting a link between promoter methylation and mRNA splicing.

Expression and function of endothelin and its receptors in vascular adventitial fibroblasts

Objective: The adventitia has been recognized to play important roles in vascular oxidative stress, remodelling and contraction. We recently demonstrated that adventitial fibroblasts are able to express endothelin-1 (ET-1) in response to angiotensin II (ANG II). However, the mechanisms by which ANG II induces ET-1 expression are unknown. It is also unclear whether the ET-1 receptors are expressed in the adventitia. We therefore examined the role of oxidative stress in the regulation of ET-1. We also investigated the expression of both the ETA and ETB receptors and the roles of these two types of receptors in collagen synthesis and ET-1 clearance in adventitial fibroblasts. Methods and Results: Adventitial fibroblasts were isolated and cultured from the thoracic mouse aorta. Cells were treated with ANG II (lOOnM), ET-1 (lOpM), NADPH oxidase inhibitor apocynin (lOOfiM), the superoxide anion scavenger tempol (lOOfiM), the ANG II receptor antagonists (100[aM), losartan (AT| receptor) and PD 1233 19 (AT2 receptor), the ET-1 receptor antagonists (lOOuM), BQ123 (ETA receptor) and BQ788 (ETB receptor), and the ETB receptor agonist (lOOnM) Sarafotoxin 6C. ET-1 peptide levels were determined by ELISA, while ETA ,ETB and collagen levels were determined by Western blot. ANG II increased ET-1 peptide levels in a time-dependent manner reaching significance when incubated for 24 hours. NAD(P)H oxidase inhibitor, apocynin, as well as the superoxide scanverger, tempol, significantly reduced ANG Il-induced ET-1 peptide levels while over-expression of SOD1 (endogenous antioxidant enzyme) significantly decreased ANG Il-induced collagen I expression, therefore implicating reactive oxygen species in the mediation of ET-1. ANG II increased ETA receptor protein as well as collagen in a similar fashion, reaching significance after 4, 6, and 24 hours treatment. ANG II induced collagen was reduced while in the presence of the ETA receptor antagonist suggesting the role of the ETa receptor in the regulation of the extracellular matrix. ANG II treatment also increased ETB receptor protein levels in a time-dependent manner. ANG II treatment in the presence of the ETB receptor antagonist significantly increased ET-1 peptide levels. On another hand, the ETB receptor agonist, Sarafotoxin 6C, significantly decreased ET-1 peptide levels. These data implicate the role of the ETb receptor in the clearance of the ET-1 peptide. Conclusion: ANG II-induced increases of ET-1 peptide appears to be mediated by reactive oxygen species derived from NAD(P)H oxidase. Both the ETA and ETB receptors are expressed in adventitial fibroblasts. The ETA receptor subtype mediates collagen I expression, while the ETB receptor may play a protective role through increasing the clearance of the ET- 1 peptide.

The roles of ERK1/2 and PI3K in abnormal vascular functions in angiotensin II-infused hypertensive rats

Background: Ang II plays a major role in cardiovascular regulation. Recently, it has become apparent that vascular superoxide anion may play an important role in hypertension development. Treatment with antisense NAD(P)H oxidase or SOD decreased BP in Ang II-infused rats. Wang et al recently reported mice which lack one of the subunits of NAD(P)H oxidase developed hypertension at a much lower extent when compared to the wild type animals infused with Ang II, indicating that superoxide anion contributes to elevation in BP in the Ang II-infused hypertensive model. In the Ang II-infused hypertensive model, altered reactivity of blood vessels is often associated with the elevation of systolic blood pressure. We have observed abnormal tension development and impaired endothelium-dependent relaxation in the isolated aorta of Ang II-infused and DOCA-salt hypertensive rats. Recently, several other cellular signal molecules, including ERK1I2 and PI3K, have been determined to play important roles in the regulation of smooth muscle contraction and relaxation. ERKl/2 and PI3K pathways are also reported to contribute to Ang II induced cell growth, hypertrophy, remodeling and contraction. Moreover, these signaling pathways have shown ROS-sensitive properties. Therefore, the aim of the present study is to investigate the roles of ERKl12 and PI3K in vascular oxidative stress, spontaneous tone and impaired endothelium relaxation in Ang II-infused hypertensive model. Hypothesis: We hypothesize that the activation of ERKl12 and PI3K are elevated in response to an Ang II infusion for 6 days. The elevated activation of phospho-ERKl/2 and PI3K mediated the increased level of vascular superoxide anion, the abnormal vascular contraction and impaired endothelium-dependent vascular relaxation in Ang II-infused hypertensive rats. Methods: Vascular superoxide anion level is measured by lucigenin chemiluminescence. Spontaneous tone and ACh-induced endothelium-dependent relaxation was measured by isometric tension recording in organ chamber. The activity of ERK pathway will be measured by its Western blot of phosphorylation of ERK. PI3K activity was evaluated indirectly by Western blot of the phosphorylation of PDKl, a downstream protein of PI3K signaling pathway. The role of each pathway was also addressed via comparing the responses to the specific inhibitors. Results: Superoxide anion was markedly increased in the isolated thoracic aorta from Ang II-infused rats. There was spontaneous tone developed in rings from Ang II-induced hypertensive but not sham-operated normotensive rats. ACh-induced endothelium-dependent relaxation function is impaired in Ang II-infused hypertensive rats. Superoxide dismutase and NAD(P)H oxidase inhibitor, apocynin, inhibited the abnormal spontaneous tone and ameliorated impaired endothelium-dependent relaxation. The expression of phopho-ERKII2 was enhanced in Ang II-infused rats, indicating the activity of ERK1I2 could be increased. MEK1I2 inhibitors, PD98059 and U126, but not their inactive analogues, SB203580 and U124, significantly reduced the vascular superoxide anion in aortas from Ang II-infused rats. The MEK1I2 inhibitors reduced the spontaneous tone and improved the impaired endothelium-dependent relaxation in aorta of hypertension. These findings supported the role of ERKII2 signaling pathway in vascular oxidative stress, spontaneous tone and impaired endothelium-dependent relaxation in Ang II-infused hypertensive rats. The amount of phospho-PDK, a downstream protein of PI3K was increased in Ang II rats indicating the activity of PI3K activity was elevated. Strikingly, PI3K significantly inhibited the increase of superoxide anion level, abnormal spontaneous tone and restored endothelium-dependent relaxation in Ang II-infused hypertensive rats. These findings indicated the important role of PI3K in Ang II-infused hypertensive rats. Conclusion: ERKII2 and PI3K signaling pathways are sustained activated in Ang II-infused hypertensive rats. The activated ERKII2 and PI3K mediate the increase of vascular superoxide anion level, vascular abnormal spontaneous tone and impaired endothelium-dependent relaxation.

Intracellular antioxidant and DNA repair enzymes as correlates of stress resistance and longevity in vertebrates

In animals, both stress resistance and longevity appear to be influenced by the insulin/insulin-like growth factor-l signaling (lIS) pathway, the basic organization of which is highly conserved from invertebrates to vertebrates. Reduced lIS or genetic disruption of the lIS pathway leads to the activation of forkhead box transcription factors, which is thought to upregulate the expression of genes involved in enhancing stress resistance, including perhaps key antioxidant enzymes as well as DNA repair enzymes. Enhanced antioxidant and DNA repair capacities may underlie the enhanced cellular stress resistance observed in long-lived animals, however little data is available that directly supports this idea. I used three. experimental approaches to test the association of intracellular antioxidant and DNA base excision repair (BER) capacities with stress resistance and longevity: (1) a comparison of multiple vertebrate endotherm species of varying body masses and longevities; (2) a comparison of long-lived Snell dwarf mice and their normallittermates; and (3) a comparison of hypometabolic animals undergoing hibernation or estivation with their active counterparts. The activities of the five major intracellular antioxidant enzymes as well as the two rate-limiting enzymes in the BER pathway, apurininc/apyrimidinic (AP) endonuclease and polymerase ~, were measured. These measurements were performed in one or more of the following: (1) cultured dermal fibroblasts; (2) brain tissue; (3) heart tissue; (4) liver tissue. My results indicate that antioxidant enzymes are not universally upregulated in association with enhanced stress resistance and longevity. I also did not find that BER enzyme activity was positively correlated with longevity, in an inter-species context, though there was evidence for enhanced BER in long-lived Snell dwarf mice. Thus, while there were instances in which enhanced antioxidant and BER enzyme activities were associated with increased stress resistance and/or longevity, this was not universally the case, indicating that other mechanisms must be involved. These results suggest the need to re-examine existing 'oxidative stress' hypotheses of longevity and probe further into the molecular physiology of longevity to discover its mechanistic basis.