Study finds that arteries adapt to oxidative stress caused by aging.
Although the causes of many age-related diseases remain unknown, oxidative stress is thought to be the main culprit. Oxidative stress has been linked to cardiovascular and neurodegenerative diseases including diabetes, hypertension and age-related cancers. However, researchers at the University of Missouri recently found that aging actually offered significant protection against oxidative stress. These findings suggest that aging may trigger an adaptive response to counteract the effects of oxidative stress on blood vessels.
“Molecules known as reactive oxygen species, or ROS, play an important role in regulating cellular function,” said Steven Segal, a professor of medical pharmacology and physiology at the MU School of Medicine and senior author of the study. “However, the overproduction of ROS can help create a condition referred to as oxidative stress, which can alter the function of cells and interfere with their growth and reproduction.”
To understand the effects of aging on the function of blood vessels when they are exposed to oxidative stress, Segal’s team studied the inner lining, or endothelium, of small resistance arteries. Resistance arteries are important to cardiovascular function because they regulate both the amount of blood flow into tissues and systemic blood pressure.
“We studied the endothelium from resistance arteries of male mice at 4 months and 24 months of age, which correspond to humans in their early 20s and mid-60s,” Segal said. “We first studied the endothelium under resting conditions and in the absence of oxidative stress. We then simulated oxidative stress by adding hydrogen peroxide. When oxidative stress was induced for 20 minutes, the endothelial cells of the younger mice had abnormal increases in calcium when compared to the endothelial cells of the older mice. This finding is important because when calcium gets too high, cells can be severely damaged.”
When oxidative stress was extended to 60 minutes, Segal’s team found that the death of endothelial cells in the younger mice was seven times greater than those from the older mice. These findings indicated that with advancing age, the endothelium had adapted to preserve cellular integrity when confronted with oxidative stress.
“The most surprising thing we found is that the endothelium was much less perturbed by oxidative stress during advanced age when compared to younger age,” Segal said. “This finding contrasts with the generally held belief that the functional integrity of the endothelium is compromised as we age. Our study suggests that blood vessels adapt during the aging process to regulate ROS and minimize cell death when subjected to an abrupt increase in oxidative stress. This adaptation helps to ensure that the arteries of older individuals can still do their jobs.”
“Although more studies are needed to identify the mechanism by which the endothelium adapts to advanced age, our study provides evidence that the natural tendency of the body is to adapt to oxidative stress during healthy aging,” Segal said.
Funding: Funding for the study was provided by the National Institutes of Health (R37-HL041026, R01-HL086483, F32-HL107050, F32-HL118836, K99-AG047198 and K01-AG041208). The content of the article is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.
Source: Jeffrey Hoelscher – University of Missouri
Image Credit: The image is credited to Kelvinsong and is licensed CC BY-SA 3.0
Original Research: Full open access research for “Advanced age protects microvascular endothelium from aberrant Ca2+ influx and cell death induced by hydrogen peroxide” by Boerman, Erik J. Behringer, Rebecca L. Shaw, Timothy L. Domeier and Steven S. Segal in Journal of Physiology. Published online May 1 2015 doi:10.1113/JP270169
Advanced age protects microvascular endothelium from aberrant Ca2+ influx and cell death induced by hydrogen peroxide
- Calcium signalling in endothelial cells of resistance arteries is integral to blood flow regulation. Oxidative stress and endothelial dysfunction can prevail during advanced age and we questioned how calcium signalling may be affected.
- Intact endothelium was freshly isolated from superior epigastric arteries of Young (∼4 months) and Old (∼24 months) male C57BL/6 mice. Under resting conditions, with no difference in intracellular calcium levels, hydrogen peroxide (H2O2) availability was ∼1/3 greater in endothelium of Old mice while vascular catalase activity was reduced by nearly half.
- Compared to Old, imposing oxidative stress (200 μm H2O2) for 20 min increased intracellular calcium to 4-fold greater levels in endothelium of Young in conjunction with twice the calcium influx. Prolonged (60 min) exposure to H2O2 induced 7-fold greater cell death in endothelium of Young.
- Microvascular adaptation to advanced age may protect endothelial cells during elevated oxidative stress to preserve functional viability of the intima.
Endothelial cell Ca2+ signalling is integral to blood flow control in the resistance vasculature yet little is known of how its regulation may be affected by advancing age. We tested the hypothesis that advanced age protects microvascular endothelium by attenuating aberrant Ca2+ signalling during oxidative stress. Intact endothelial tubes (width, ∼60 μm; length, ∼1000 μm) were isolated from superior epigastric arteries of Young (3–4 months) and Old (24–26 months) male C57BL/6 mice and loaded with Fura-2 dye to monitor [Ca2+]i. At rest there was no difference in [Ca2+]i between age groups. Compared to Young, the [Ca2+]i response to maximal stimulation with acetylcholine (3 μm, 2 min) was ∼25% greater in Old, confirming signalling integrity with advanced age. Basal H2O2 availability was ∼33% greater in Old while vascular catalase activity was reduced by half. Transient exposure to elevated H2O2 (200 μm, 20 min) progressively increased [Ca2+]i to ∼4-fold greater levels in endothelium of Young versus Old. With no difference between age groups at rest, Mn2+ quench of Fura-2 fluorescence revealed 2-fold greater Ca2+ influx in Young during elevated H2O2; this effect was attenuated by ∼75% using ruthenium red (5 μm) as a broad-spectrum inhibitor of transient receptor potential channels. Prolonged exposure to H2O2 (200 μm, 60 min) induced ∼7-fold greater cell death in endothelium of Young versus Old. Thus, microvascular endothelium can adapt to advanced age by reducing Ca2+ influx during elevated oxidative stress. Protection from cell death during oxidative stress will sustain endothelial integrity during ageing.
“Advanced age protects microvascular endothelium from aberrant Ca2+ influx and cell death induced by hydrogen peroxide” by Boerman, Erik J. Behringer, Rebecca L. Shaw, Timothy L. Domeier and Steven S. Segal in Journal of Physiology. Published online May 1 2015 doi:10.1113/JP270169
About Hydrogen Peroxide
H2O2 signals growth. H2O2 functions as a signalling molecule in the intracellular propagation of both physiological and oncogenic growth signals (Fig. 1). The induction of cell proliferation by several growth factors (such as EGF, PDGF, nerve growth factor (NGF) and insulin) correlates with a transient increase of intracellular H2O2, whereas antioxidant treatments prevent DNA synthesis29. Similarly, cellular transformation following the expression of activated oncogenes (such as mutated Ras30 or overexpressed myc31) is associated with increased intracellular H2O2 and is prevented by treatment with antioxidants32, 33. Furthermore, H2O2 mediates angiogenic signalling and has been implicated in the so-called angiogenic switch, which allows non-invasive and poorly vascularized tumours to become highly invasive and angiogenic tumours (through direct activation of the transcription factor hypoxia-inducible factor (HIF))34.The function of H2O2as a mitogenic or angiogenic signalling molecule is supported by its documented activity on various well established signal transduction proteins that are involved in mitogenesis or angiogenesis.
Mitochondrial H2O2 controls lifespan. In regard to H2O2 scavenging, overexpression of catalase or GPX together with SOD has been demonstrated to increase oxidative-stress resistance and lifespan in some transgenic models of C. elegans and D. melanogaster56. Remarkably, transgenic mice that overexpress catalase in mitochondria show a specifically increased scavenging activity in mitochondria and a prolonged lifespan57. Likewise, deletion of p66Shc in mice results in the decreased formation of mitochondrial H2O2 (Ref. 48), which correlates with delayed ageing58, 59, a reduced incidence of ageing-associated degenerative diseases58, 59, 60, 61 and increased lifespan47. Thus, genetic mammalian models of increased scavenging or decreased production of mitochondrial H2O2 directly implicate mitochondrial H2O2 in ageing and lifespan determination.
Alterations in metabolism and mitochondrial ROS production have also been proposed as mechanisms that control lifespan by other genetic pathways (such as certain endocrine signalling pathways62; see the Review by Kahn in this issue) and other ‘ageing’ genes (such as grisea in fungi; Indy, stunted and methuselah in flies; isp–1, mev-1 and clk-1 in worms; and MclK1 (also known as Coq7) in mammals, which is mainly involved in mitochondrial energetic metabolism)63. Notably, some progeric (that is, accelerated ageing) syndromes in humans, such as those due to chromosome 21 trisomy or DNA-repair gene mutations, are also characterized by increased intracellular levels of H2O2 (Ref. 64). However, transgenic mice bearing error-prone polymerase- exhibited a progeric phenotype and a shortened lifespan without increased mitochondrial ROS release65, 66. This finding might indicate that mitochondrial DNA mutation is downstream of ROS accumulation and suggests that specific mechanisms other than random mitochondrial genome mutations are responsible for ROS accumulation in mitochondria.
H2O2 regulation during caloric restriction. Caloric restriction (CR) is the only non-genetic way to increase lifespan in various species, including mammalian species. Many studies demonstrate an increased lifespan in calorie-restricted mice, although this effect is strain specific67. Considering the two mouse strains that are associated with reduced body size, CR extends the lifespan of the Ames dwarf mouse but not of the growth-hormone-receptor mutant, which is associated with reduced mitochondrial metabolism68. Indeed, the role of mitochondria in the mechanism underlying the effect of CR on lifespan has been actively investigated in different species and conflicting results have been reported, precluding a firm conclusion.
In flies, CR does not affect the number of mitochondria, although it alters mitochondrial morphology and, in vitro, alters enzyme activities69. In cells that are grown in serum from calorie-restricted rats, mitochondrial biogenesis is increased and bioenergetics efficiency is improved while ROS generation is reduced70. Similarly, mitochondrial biogenesis is increased in tissues from calorie-restricted mice, which is probably due to the increased transcription of nuclear-encoded mitochondrial genes71. Initial studies reported a reduction in the metabolic rate of calorie-restricted mice72. However, when normalized to their lean body mass, other reports show the opposite73, 74. In all of these studies, however, metabolism is assessed using whole-body determinations, which may underestimate important variations in the metabolic rates of individual tissues. Furthermore, other studies in rodents show decreased mitochondrial H2O2 production and mitochondrial and/or nuclear oxidative DNA damage after a long period of CR (more than 1 year)75. Notably, no differences are seen after a short period (4 months), which highlights the importance of time for the manifestation of the effects of CR on ROS metabolism75.
Studies on mitochondria that were isolated from the skeletal muscle of calorie-restricted rats reveal a reduction in H2O2 production and in the age-dependent decline of respiratory capacity76. In calorie-restricted yeast, however, oxygen consumption and ROS production are both increased. Sod1, Sod2, catalase and Gpx are overexpressed in calorie-restricted yeast; however, expression profile studies did not reveal gross modifications in the expression of most genes that are involved in the antioxidant defence77. In conclusion, at least in mammals, caloric intake regulates lifespan through a complex combination of processes that also includes reduced oxygen metabolism and, consequently, mitochondrial H2O2 production78
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