Advanced age and female sex protect cerebral arteries from mitochondrial depolarization and apoptosis during acute oxidative stress.

Aging increases reactive oxygen species (ROS) which can impair vascular function and contribute to brain injury. However, aging can also promote resilience to acute oxidative stress. Therefore, we tested the hypothesis that advanced age protects smooth muscle cells (SMCs) and endothelial cells (ECs) of posterior cerebral arteries (PCAs; diameter, ∼80 µm) during exposure to H2O2. PCAs from young (4-6 months) and old (20-26 months) male and female C57BL/6 mice were isolated and pressurized (~70 mm Hg) to evaluate cell death, mitochondrial membrane potential (ΔΨm), ROS production, and [Ca2+]i in response to H2O2 (200 µM, 50 min). SMC death and ΔΨm depolarization were greater in PCAs from males vs. females. Aging increased ROS in PCAs from both sexes but increased SMC resilience to death only in males. Inhibiting TRPV4 channels with HC-067047 (1 µM) or Src kinases with SU6656 (10 µM) reduced Ca2+ entry and SMC death to H2O2 most effectively in PCAs from young males. Activating TRPV4 channels with GSK1016790A (50 nM) evoked greater Ca2+ influx in SMCs and ECs of PCAs from young vs. old mice but did not induce cell death. However, when combined with H2O2, TRPV4 activation exacerbated EC death. Activating Src kinases with spermidine (100 µM) increased Ca2+ influx in PCAs from males vs. females with minimal cell death. We conclude that in males, chronic oxidative stress during aging increases the resilience of cerebral arteries, which contrasts with inherent protection in females. Findings implicate TRP channels and Src kinases as targets to limit vascular damage to acute oxidative injury.

Differential Effects of High Fat Diets on Resilience to H2O2-Induced Cell Death in Mouse Cerebral Arteries: Role for Processed Carbohydrates.

High fat, western-style diets increase vascular oxidative stress. We hypothesized that smooth muscle cells and endothelial cells adapt during the consumption of high fat diets to become more resilient to acute oxidative stress. Male C57Bl/6J mice were fed a western-style diet high in fat and processed carbohydrates (WD), a high fat diet that induces obesity (DIO), or their respective control (CD) and standard (SD) diets for 16 weeks. Posterior cerebral arteries (PCAs) were isolated and pressurized for study. During acute exposure to H2O2 (200 µM), smooth muscle cell and endothelial cell death were reduced in PCAs from WD, but not DIO mice. WD selectively attenuated mitochondrial membrane potential depolarization and vessel wall Ca2+ influx during H2O2 exposure. Selective inhibition of transient receptor potential (TRP) V4 or TRPC3 channels reduced smooth muscle cell and endothelial cell death in concert with the vessel wall [Ca2+]i response to H2O2 for PCAs from CD mice and eliminated differences between CD and WD. Inhibiting Src kinases reduced smooth muscle cell death along with [Ca2+]i response to H2O2 only in PCAs from CD mice and eliminated differences between diets. However, Src kinase inhibition did not alter endothelial cell death. These findings indicate that consuming a WD, but not high fat alone, leads to adaptations that limit Ca2+ influx and vascular cell death during exposure to acute oxidative stress.

Endothelial cells promote smooth muscle cell resilience to H2 O2 -induced cell death in mouse cerebral arteries.

AIM: Brain injury produces reactive oxygen species (ROS). However, little is known of how acute oxidative stress affects cell survival in the cerebral vascular supply. We hypothesized that endothelial cells (ECs) are more resilient to H2 O2 and protect vascular smooth muscle cells (SMCs) during acute oxidative stress. METHODS: Mouse posterior cerebral arteries (PCAs; diameter, ~80 µm) were exposed to H2 O2 (200 µM, 50 min, 37°C). Nuclear staining identified dead and live cells of intact and endothelium-disrupted vessels. SMC [Ca2+ ]i was assessed with Fura-2 fluorescence, and superoxide production was assessed by dihydroethidium and MitoSOX fluorescence. RESULTS: In response to H2 O2 : SMC death (21%) exceeded EC death (5%) and increased following endothelial disruption (to 48%) with a corresponding increase in SMC Ca2+ entry through transient receptor potential (TRP) channels. Whereas pharmacological inhibition of TRPV4 channels prevented SMC death and reduced Ca2+ entry for intact vessels, both remained elevated following endothelial disruption. In contrast, pharmacological inhibition or genetic deletion of TRPC3 prevented SMC death and attenuated Ca2+ entry for both intact and endothelium-disrupted vessels. Inhibiting gap junctions increased EC death (to 22%) while SMC death and [Ca2+ ]i responses were attenuated by inhibiting nitric oxide synthesis or scavenging superoxide/peroxynitrite. Inhibiting NADPH oxidases also prevented SMC Ca2+ entry and death. H2 O2 increased mitochondrial ROS production while scavenging mitochondria-derived superoxide prevented SMC death but not Ca2+ entry. CONCLUSIONS: During acute exposure of cerebral arteries to acute oxidative stress, ECs are more resilient than SMCs and the endothelium may protect SMCs by reducing Ca2+ entry through TRPC3 channels.

Myofibre injury induces capillary disruption and regeneration of disorganized microvascular networks.

Injury to skeletal muscle disrupts myofibres and their microvascular supply. While the regeneration of myofibres is well described, little is known of how the microcirculation is affected by skeletal muscle injury or its recovery during regeneration. Nevertheless, the microvasculature must also recover to restore skeletal muscle function. We aimed to define the nature of microvascular damage and time course of repair during muscle injury and regeneration induced by the myotoxin BaCl2 . To test the hypothesis that microvascular disruption occurred secondary to myofibre injury, isolated microvessels were exposed to BaCl2 or the myotoxin was injected into the gluteus maximus (GM) muscle of mice. In isolated microvessels, BaCl2 depolarized smooth muscle cells (SMCs) and endothelial cells while increasing intracellular calcium in SMCs but did not elicit death of either cell type. At 1 day post-injury (dpi) of the GM, capillary fragmentation coincided with myofibre degeneration while arteriolar and venular networks remained intact; neutrophil depletion before injury did not prevent capillary damage. Perfused capillary networks reformed by 5 dpi in association with more terminal arterioles and were dilated through 10 dpi. With no change in microvascular area or branch point number in regenerating capillary networks, fewer capillaries aligned with myofibres and were no longer organized into microvascular units. By 21 dpi, capillary orientation and microvascular unit organization were no longer different from uninjured GM. We conclude that following their disruption secondary to myofibre damage, capillaries regenerate as disorganized networks that remodel into microvascular units as regenerated myofibres mature. KEY POINTS: Skeletal muscle regenerates after injury; however, the nature of microvascular damage and repair is poorly understood. Here, the myotoxin BaCl2 , a standard experimental method of acute skeletal muscle injury, was used to investigate the response of the microcirculation to local injury of intact muscle. Intramuscular injection of BaCl2 induced capillary fragmentation with myofibre degeneration; arteriolar and venular networks remained intact. Direct exposure to BaCl2 did not kill microvascular endothelial cells or smooth muscle cells. Dilated capillary networks reformed by 5 days post-injury (dpi) in association with more terminal arterioles. Capillary orientation remained disorganized through 10 dpi. Capillaries realigned with myofibres and reorganized into microvascular units by 21 dpi, which coincides with the recovery of vasomotor control and maturation of nascent myofibres. Skeletal muscle injury disrupts its capillary supply secondary to myofibre degeneration. Reorganization of regenerating microvascular networks accompanies the recovery of blood flow regulation.

Apoptosis in resistance arteries induced by hydrogen peroxide: greater resilience of endothelium versus smooth muscle.

Reactive oxygen species (ROS) are implicated in cardiovascular and neurologic disorders including atherosclerosis, heart attack, stroke, and traumatic brain injury. Although oxidative stress can lead to apoptosis of vascular cells, such findings are largely based upon isolated vascular smooth muscle cells (SMCs) and endothelial cells (ECs) studied in culture. Studying intact resistance arteries, we have focused on understanding how SMCs and ECs in the blood vessel wall respond to acute oxidative stress induced by hydrogen peroxide, a ubiquitous, membrane-permeant ROS. We find that apoptosis induced by H2O2 is far greater in SMCs compared to ECs. For both cell types, apoptosis is associated with a rise in intracellular calcium concentration ([Ca2+]i) during H2O2 exposure. Consistent with their greater death, the rise in [Ca2+]i for SMCs exceeds that in ECs. Finding that disruption of the endothelium increases SMC death, we address how myoendothelial coupling and paracrine signaling attenuate apoptosis. Remarkably, conditions associated with chronic oxidative stress (advanced age, Western-style diet) protect SMCs during H2O2 exposure, as does female sex. In light of intracellular Ca2+ handling, we consider how glycolytic versus oxidative pathways for ATP production and changes in mitochondrial structure and function impact cellular resilience to H2O2-induced apoptosis. Gaining new insight into protective signaling within and between SMCs and ECs of the arterial wall can be applied to promote vascular cell survival (and recovery of blood flow) in tissues subjected to acute oxidative stress as occurs during reperfusion following myocardial infarction and thrombotic stroke.

Gene expression profiles of ion channels and receptors in mouse resistance arteries: Effects of cell type, vascular bed, and age.

OBJECTIVE: Receptors and ion channels of smooth muscle cells (SMCs) and endothelial cells (ECs) are integral to the regulation of vessel diameter and tissue blood flow. Physiological roles of ion channels and receptors in skeletal muscle and mesenteric arteries have been identified; however, their gene expression profiles are undefined. We tested the hypothesis that expression profiles for ion channels and receptors governing vascular reactivity vary with cell type, vascular bed, and age. METHODS: Mesenteric and superior epigastric arteries were dissected from Old (24-26 months) and Young (3-6 months) C57BL/6J mice. ECs and SMCs were collected for analysis with custom qRT-PCR arrays to determine expression profiles of 80 ion channel and receptor genes. Bioinformatics analyses were applied to gain insight into functional interactions. RESULTS: We identified 68 differences in gene expression with respect to cell type, vessel type, and age. Heat maps illustrate differential expression, and distance matrices predict patterns of coexpression. Gene networks based upon protein-protein interaction datasets and KEGG pathways illustrate biological processes affected by specific differences in gene expression. CONCLUSIONS: Differences in gene expression profiles are most pronounced between microvascular ECs and SMCs with subtle variations between vascular beds and age groups.

Targeted sequencing for high-resolution evolutionary analyses following genome duplication in salmonid fish: Proof of concept for key components of the insulin-like growth factor axis.

High-throughput sequencing has revolutionised comparative and evolutionary genome biology. It has now become relatively commonplace to generate multiple genomes and/or transcriptomes to characterize the evolution of large taxonomic groups of interest. Nevertheless, such efforts may be unsuited to some research questions or remain beyond the scope of some research groups. Here we show that targeted high-throughput sequencing offers a viable alternative to study genome evolution across a vertebrate family of great scientific interest. Specifically, we exploited sequence capture and Illumina sequencing to characterize the evolution of key components from the insulin-like growth (IGF) signalling axis of salmonid fish at unprecedented phylogenetic resolution. The IGF axis represents a central governor of vertebrate growth and its core components were expanded by whole genome duplication in the salmonid ancestor ~95Ma. Using RNA baits synthesised to genes encoding the complete family of IGF binding proteins (IGFBP) and an IGF hormone (IGF2), we captured, sequenced and assembled orthologous and paralogous exons from species representing all ten salmonid genera. This approach generated 299 novel sequences, most as complete or near-complete protein-coding sequences. Phylogenetic analyses confirmed congruent evolutionary histories for all nineteen recognized salmonid IGFBP family members and identified novel salmonid-specific IGF2 paralogues. Moreover, we reconstructed the evolution of duplicated IGF axis paralogues across a replete salmonid phylogeny, revealing complex historic selection regimes - both ancestral to salmonids and lineage-restricted - that frequently involved asymmetric paralogue divergence under positive and/or relaxed purifying selection. Our findings add to an emerging literature highlighting diverse applications for targeted sequencing in comparative-evolutionary genomics. We also set out a viable approach to obtain large sets of nuclear genes for any member of the salmonid family, which should enable insights into the evolutionary role of whole genome duplication before additional nuclear genome sequences become available.

Advanced age protects microvascular endothelium from aberrant Ca(2+) influx and cell death induced by hydrogen peroxide.

KEY POINTS: 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 (H2 O2 ) 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 H2 O2 ) 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 H2 O2 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. ABSTRACT: Endothelial cell Ca(2+) 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 Ca(2+) 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 [Ca(2+) ]i . At rest there was no difference in [Ca(2+) ]i between age groups. Compared to Young, the [Ca(2+) ]i response to maximal stimulation with acetylcholine (3 µm, 2 min) was ∼25% greater in Old, confirming signalling integrity with advanced age. Basal H2 O2 availability was ∼33% greater in Old while vascular catalase activity was reduced by half. Transient exposure to elevated H2 O2 (200 µm, 20 min) progressively increased [Ca(2+) ]i to ∼4-fold greater levels in endothelium of Young versus Old. With no difference between age groups at rest, Mn(2+) quench of Fura-2 fluorescence revealed 2-fold greater Ca(2+) influx in Young during elevated H2 O2 ; this effect was attenuated by ∼75% using ruthenium red (5 µm) as a broad-spectrum inhibitor of transient receptor potential channels. Prolonged exposure to H2 O2 (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 Ca(2+) influx during elevated oxidative stress. Protection from cell death during oxidative stress will sustain endothelial integrity during ageing.

Ovariectomy increases L-type Ca(2+) channel activity in porcine coronary smooth muscle.

OBJECTIVE: Sex hormone status has been demonstrated to play a role in the regulation of ion channel activity. We previously demonstrated increased L-type Ca channel current (ICa) in the coronary smooth muscle cells (SMCs) of male swine compared with female swine. In male swine, endogenous testosterone increases ICa in SMCs by enhanced expression of the pore-forming α1 subunit Cav1.2. Conversely, the role of sex hormones in female swine has not previously been investigated. Therefore, the purpose of the current study was to determine the effect of ovariectomy (OVX) on L-type Ca channel activity and expression in female Yucatan miniature swine. METHODS: Sexually mature female swine were obtained from a breeder and either left intact (intact female [IF]; n = 5) or ovariectomized (n = 6). RESULTS: Sensitivity to depolarization-induced contractions was increased by OVX. Accordingly, mean (SEM) ICa was enhanced in the OVX group (-9.5 [0.6] pA/pF) compared with the IF group (-4.5 [0.3] pA/pF), although L-type Ca channel α1 subunit (Cav1.2; α1c) messenger RNA (mRNA) and protein expressions were unchanged.Among the L-type Ca channel ß subunits, ß1 (188 [31]) and ß2a (561 [79]) had higher mRNA expression levels (target/18S) than ß3 (9 [1]) and ß4 (2 [0.1]). Although ß2a, ß3, and ß4 mRNA and protein expressions were not different between groups, protein expression of the ß1 subunit (Cavß1) was decreased in the OVX group compared with the IF group. CONCLUSIONS: Endogenous female hormones inhibit L-type Ca channel activity in coronary SMCs potentially via the up-regulation of Cavß1 subunit expression.

Aging impairs electrical conduction along endothelium of resistance arteries through enhanced Ca2+-activated K+ channel activation.

OBJECTIVE: Intercellular conduction of electrical signals underlies spreading vasodilation of resistance arteries. Small- and intermediate-conductance Ca(2+)-activated K(+) channels of endothelial cells serve a dual function by initiating hyperpolarization and modulating electrical conduction. We tested the hypothesis that regulation of electrical signaling by small- and intermediate-conductance Ca(2+)-activated K(+) channels is altered with advancing age. APPROACH AND RESULTS: Intact endothelial tubes (60 µm wide; 1-3 mm long) were freshly isolated from male C57BL/6 mouse (Young: 4-6 months; Intermediate: 12-14 months; Old: 24-26 months) superior epigastric arteries. Using dual intracellular microelectrodes, current was injected (± 0.1-3 nA) at site 1 while recording membrane potential (Vm) at site 2 (separation distance: 50-2000 µm). Across age groups, greatest differences were observed between Young and Old. Resting Vm in Old (-38 ± 1 mV) was more negative (P<0.05) than Young (-30 ± 1 mV). Maximal hyperpolarization to both direct (NS309) and indirect (acetylcholine) activation of small- and intermediate-conductance Ca(2+)-activated K(+) channels was sustained (ΔVm ≈-40 mV) with age. The length constant (λ) for electrical conduction was reduced (P<0.05) from 1630 ± 80 µm (Young) to 1320 ± 80 µm (Old). Inhibiting small- and intermediate-conductance Ca(2+)-activated K(+) channels with apamin+charybdotoxin or scavenging hydrogen peroxide (H2O2) with catalase improved electrical conduction (P<0.05) in Old. Exogenous H2O2 (200 µmol/L) in Young evoked hyperpolarization and impaired electrical conduction; these effects were blocked by apamin+charybdotoxin. CONCLUSIONS: Enhanced current loss through Ca2+-activated K+ channel activation impairs electrical conduction along the endothelium of resistance arteries with aging. Attenuating the spatial domain of electrical signaling will restrict the spread of vasodilation and thereby contribute to blood flow limitations associated with advanced age.