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.

Angiogenesis precedes myogenesis during regeneration following biopsy injury of skeletal muscle.

BACKGROUND: Acute injury to skeletal muscle damages myofibers and fragment capillaries, impairing contractile function and local perfusion. Myofibers and microvessels regenerate from satellite cells and from surviving microvessel fragments, respectively, to restore intact muscle. Established models of injury have used myotoxins and physical trauma to demonstrate the concurrence of myogenesis and angiogenesis during regeneration. In these models, efferocytosis removes cellular debris while basal laminae persist to provide guidance during myofiber and microvessel regeneration. It is unknown whether the spatiotemporal coupling between myofiber and microvascular regeneration persists when muscle tissue is completely removed and local guidance cues are lost. METHODS: To test whether complete removal of skeletal muscle tissue affects the spatiotemporal relationship between myogenesis and angiogenesis during regeneration, subthreshold volumetric muscle loss was created with a biopsy punch (diameter, 2 mm) through the center of the gluteus maximus (GM) in adult mice. Regeneration into the void was evaluated through 21 days post-injury (dpi). Microvascular perfusion was evaluated in vivo by injecting fluorescent dextran into the circulation during intravital imaging. Confocal imaging and histological analyses of whole-mount GM preparations and tissue cross-sections assessed the growth of microvessels and myofibers into the wound. RESULTS: A provisional matrix filled with PDGFRα+ and CD45+ cells spanned the wound within 1 dpi. Regenerating microvessels advanced from the edges of the wound into the matrix by 7 dpi. Nascent microvascular networks formed by 10 dpi with blood-perfused networks spanning the wound by 14 dpi. In striking contrast, the wound remained devoid of myofibers at 7 and 10 dpi. Myogenesis into the wound was apparent by 14 dpi and traversed the wound by 21 dpi. Regenerated myofibers and microvessels were disorganized compared to the uninjured muscle. CONCLUSIONS: Following punch biopsy of adult skeletal muscle, regenerating microvessels span the wound and become perfused with blood prior to myofiber regeneration. The loss of residual guidance cues with complete tissue removal disrupts the spatiotemporal correspondence between microvascular and myofiber regeneration. We conclude that angiogenesis precedes myogenesis during regeneration following subthreshold volumetric muscle loss.

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.

Differential hyperpolarization to substance P and calcitonin gene-related peptide in smooth muscle versus endothelium of mouse mesenteric artery.

OBJECTIVE: We sought to define how sensory neurotransmitters substance P and calcitonin gene-related peptide (CGRP) affect membrane potential of vascular smooth muscle and endothelium. METHODS: Microelectrodes recorded membrane potential of smooth muscle from pressurized mouse mesenteric arteries (diameter, ~150 µm) and in endothelial tubes. RESULTS: Resting potential was similar (~ -45 mV) for each cell layer. Substance P hyperpolarized smooth muscle and endothelium ~ -15 mV; smooth muscle hyperpolarization was abolished by endothelial disruption or NO synthase inhibition. Blocking KCa channels (apamin + charybdotoxin) attenuated hyperpolarization in both cell types. CGRP hyperpolarized endothelium and smooth muscle ~ -30 mV; smooth muscle hyperpolarization was independent of endothelium. Blocking KCa channels prevented hyperpolarization to CGRP in endothelium but not smooth muscle. Inhibiting KATP channels with glibenclamide or genetic deletion of KIR 6.1 attenuated hyperpolarization in smooth muscle but not endothelium. Pinacidil (KATP channel agonist) hyperpolarized smooth muscle more than endothelium (~ -35 vs. ~ -20 mV). CONCLUSIONS: Calcitonin gene-related peptide elicits greater hyperpolarization than substance P. Substance P hyperpolarizes both cell layers through KCa channels and involves endothelium-derived NO in smooth muscle. Endothelial hyperpolarization to CGRP requires KCa channels, while KATP channels mediate hyperpolarization in smooth muscle. Differential K+ channel activation in smooth muscle and endothelium through sensory neurotransmission may selectively tune mesenteric blood flow.

Functionalizing biomaterials to promote neurovascular regeneration following skeletal muscle injury.

During embryogenesis, blood vessels and nerves develop with similar branching structure in response to shared signaling pathways guiding network growth. With both systems integral to physiological homeostasis, dual targeting of blood vessels and nerves to promote neurovascular regeneration following injury is an emerging therapeutic approach in biomedical engineering. A limitation to this strategy is that the nature of cross talk between emergent vessels and nerves during regeneration in an adult is poorly understood. Following peripheral nerve transection, intraneural vascular cells infiltrate the site of injury to provide a migratory pathway for mobilized Schwann cells of regenerating axons. As Schwann cells demyelinate, they secrete vascular endothelial growth factor, which promotes angiogenesis. Recent advances point to concomitant restoration of neurovascular architecture and function through simultaneous targeting of growth factors and guidance cues shared by both systems during regeneration. In the context of traumatic injury associated with volumetric muscle loss, we consider the nature of biomaterials used to engineer three-dimensional scaffolds, functionalization of scaffolds with molecular signals that guide and promote neurovascular growth, and seeding scaffolds with progenitor cells. Physiological success is defined by each tissue component of the bioconstruct (nerve, vessel, muscle) becoming integrated with that of the host. Advances in microfabrication, cell culture techniques, and progenitor cell biology hold great promise for engineering bioconstructs able to restore organ function after volumetric muscle loss.

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.

Aging alters spontaneous and neurotransmitter-mediated Ca2+ signaling in smooth muscle cells of mouse mesenteric arteries.

OBJECTIVE: Aging impairs MA dilation by reducing the ability of sensory nerves to counteract sympathetic vasoconstriction. This study tested whether altered SMC Ca2+ signals to sympathetic (NE) and sensory (CGRP) neurotransmitters underlie aging-related deficits in vasodilation. METHODS: MAs from young and old mice were pressurized and loaded with Fluo-4 dye for confocal measurement of SMC Ca2+ sparks and waves. Endothelial denudation resolved the influence of ECs. SMCs were immunolabeled for RyR isoforms and compared with transcript levels for RyRs and CGRP receptor components. RESULTS: SMCs from young vs old mice exhibited more spontaneous Ca2+ spark sites with no difference in Ca2+ waves. NE reduced spark sites and increased waves for both groups; addition of CGRP restored sparks and reduced waves only for young mice. Endothelial denudation attenuated Ca2+ responses to CGRP for young but not old mice, which were already attenuated, suggesting a diminished role for ECs with aging. CGRP receptor expression was similar between ages with increased serum CGRP in old mice, where RyR1 expression was replaced by RyR3. CONCLUSION: With aging, we suggest that altered RyR expression in SMCs contributes to impaired ability of sensory neurotransmission to restore Ca2+ signaling underlying vasomotor control during sympathetic activation.

Female sex and Western-style diet protect mouse resistance arteries during acute oxidative stress.

A Western-style diet (WD; high in fat and carbohydrates) increases vascular oxidative stress. We hypothesized that vascular cells adapt to a WD by developing resilience to oxidative stress. Male and female C57BL/6J mice (4 wk of age) were fed a control diet (CD) or a WD for 16-20 wk. Superior epigastric arteries (SEAs; diameter, ~125 µm) were isolated and pressurized for study. Basal reactive oxygen species production was greatest in SEAs from males fed the WD. During exposure to H2O2 (200 µM, 50 min), propidium iodide staining identified nuclei of disrupted endothelial cells (ECs) and smooth muscle cells (SMCs). For mice fed the CD, death of SMCs (21%) and ECs (6%) was greater (P < 0.05) in SEAs from males than females (9% and 2%, respectively). WD consumption attenuated cell death most effectively in SEAs from males. With no difference at rest, H2O2 increased intracellular Ca2+ concentration ([Ca2+]i) to the greatest extent in SEAs from males, as shown by fura 2 fluorescence. Selective disruption of the endothelium (luminal air bubble) increased [Ca2+]i and SMC death during H2O2 exposure irrespective of sex; the WD reduced both responses most effectively in males. Nonselective transient receptor potential (TRP) channel inhibition (ruthenium red, 5 µM) attenuated the rise of [Ca2+]i, as did selective inhibition of TRP vanilloid type 4 (TRPV4) channels (HC-067047, 1 µM), which also attenuated cell death. In contrast, inhibition of voltage-gated Ca2+ channels (diltiazem, 50 µM) was without effect. Thus, for resistance arteries during acute oxidative stress: 1) ECs are more resilient than (and can protect) SMCs, 2) vessels from females are inherently more resilient than those from males, and 3) a WD increases vascular resilience by diminishing TRPV4 channel-dependent Ca2+ entry.

Barium chloride injures myofibers through calcium-induced proteolysis with fragmentation of motor nerves and microvessels.

BACKGROUND: Local injection of BaCl2 is an established model of acute injury to study the regeneration of skeletal muscle. However, the mechanism by which BaCl2 causes muscle injury is unresolved. Because Ba2+ inhibits K+ channels, we hypothesized that BaCl2 induces myofiber depolarization leading to Ca2+ overload, proteolysis, and membrane disruption. While BaCl2 spares resident satellite cells, its effect on other tissue components integral to contractile function has not been defined. We therefore asked whether motor nerves and microvessels, which control and supply myofibers, are injured by BaCl2 treatment. METHODS: The intact extensor digitorum longus (EDL) muscle was isolated from male mice (aged 3-4 months) and irrigated with physiological salt solution (PSS) at 37 °C. Myofiber membrane potential (Vm) was recorded using sharp microelectrodes while intracellular calcium concentration ([Ca2+]i) was evaluated with Fura 2 dye. Isometric force production of EDL was measured in situ, proteolytic activity was quantified by calpain degradation of αII-spectrin, and membrane disruption was marked by nuclear staining with propidium iodide (PI). To test for effects on motor nerves and microvessels, tibialis anterior or gluteus maximus muscles were injected with 1.2% BaCl2 (50-75 µL) in vivo followed by immunostaining to evaluate the integrity of respective tissue elements post injury. Data were analyzed using Students t test and analysis of variance with P ≤ 0.05 considered statistically significant. RESULTS: Addition of 1.2% BaCl2 to PSS depolarized myofibers from - 79 ± 3 mV to - 17 ± 7 mV with a corresponding rise in [Ca2+]i; isometric force transiently increased from 7.4 ± 0.1 g to 11.1 ± 0.4 g. Following 1 h of BaCl2 exposure, 92 ± 3% of myonuclei stained with PI (vs. 8 ± 3% in controls) with enhanced cleavage of αII-spectrin. Eliminating Ca2+ from PSS prevented the rise in [Ca2+]i and ameliorated myonuclear staining with PI during BaCl2 exposure. Motor axons and capillary networks appeared fragmented within 24 h following injection of 1.2% BaCl2 and morphological integrity deteriorated through 72 h. CONCLUSIONS: BaCl2 injures myofibers through depolarization of the sarcolemma, causing Ca2+ overload with transient contraction, leading to proteolysis and membrane rupture. Motor innervation and capillarity appear disrupted concomitant with myofiber damage, further compromising muscle integrity.

Advanced age protects resistance arteries of mouse skeletal muscle from oxidative stress through attenuating apoptosis induced by hydrogen peroxide.

KEY POINTS: Vascular oxidative stress increases with advancing age. We hypothesized that resistance vessels develop resilience to oxidative stress to protect functional integrity and tested this hypothesis by exposing isolated pressurized superior epigastric arteries (SEAs) of old and young mice to H2 O2 . H2 O2 -induced death was greater in smooth muscle cells (SMCs) than endothelial cells (ECs) and lower in SEAs from old vs. young mice; the rise in vessel wall [Ca2+ ]i induced by H2 O2 was attenuated with ageing, as was the decline in noradrenergic vasoconstriction; genetic deletion of IL-10 mimicked the effects of advanced age on cell survival. Inhibiting NO synthase or scavenging peroxynitrite reduced SMC death; endothelial denudation or inhibiting gap junctions increased SMC death; delocalization of cytochrome C activated caspases 9 and 3 to induce apoptosis. Vascular cells develop resilience to H2 O2 during ageing by preventing Ca2+ overload and endothelial integrity promotes SMC survival. ABSTRACT: Advanced age is associated with elevated oxidative stress and can protect the endothelium from cell death induced by H2 O2 . Whether such protection occurs for intact vessels or differs between smooth muscle cell (SMC) and endothelial cell (EC) layers is unknown. We tested the hypothesis that ageing protects SMCs and ECs during acute exposure to H2 O2 (200 µm, 50 min). Mouse superior epigastric arteries (SEAs; diameter, ∼150 µm) were isolated and pressurized to 100 cmH2 O at 37˚C. For SEAs from young (4 months) mice, H2 O2 killed 57% of SMCs and 11% of ECs in males vs. 8% and 2%, respectively, in females. Therefore, SEAs from males were studied to resolve the effect of ageing and experimental interventions. For old (24 months) mice, SMC death was reduced to 10% with diminished accumulation of [Ca2+ ]i in the vessel wall during H2 O2 exposure. In young mice, genetic deletion of IL-10 mimicked the protective effect of ageing on cell death and [Ca2+ ]i accumulation. Whereas endothelial denudation or gap junction inhibition (carbenoxolone; 100 µm) increased SMC death, inhibiting NO synthase (l-NAME, 100 µm) or scavenging peroxynitrite (FeTPPS, 5 µm) reduced SMC death along with [Ca2+ ]i . Despite NO toxicity via peroxynitrite formation, endothelial integrity protects SMCs. Caspase inhibition (Z-VAD-FMK, 50 µm) attenuated cell death with immunostaining for annexin V, cytochrome C, and caspases 3 and 9 pointing to induction of intrinsic apoptosis during H2 O2 exposure. We conclude that advanced age reduces Ca2+ influx that triggers apoptosis, thereby promoting resilience of the vascular wall during oxidative stress.

Recovery of blood flow regulation in microvascular resistance networks during regeneration of mouse gluteus maximus muscle.

KEY POINTS: Skeletal muscle regenerates after injury, however the recovery of its microvascular supply is poorly understood. We injured the gluteus maximus muscle in mice aiming to investigate the recovery of blood flow regulation in microvascular resistance networks. We hypothesized that blood flow regulation recovers in concert with myofibre regeneration. Microvascular perfusion ceased within 1 day post injury and was restored at 5 days coincident with the appearance of new myofibres; however, the resistance network was dilated and unresponsive to vasoactive agents. Spontaneous vasomotor tone, endothelium-dependent dilatation and adrenergic vasoconstriction increased at 10 days in concert with myofibre regeneration. Vasomotor control recovered at 21 days, when regenerated myofibres matured and active force production stabilized. Functional vasodilatation in response to muscle contraction recovered at 35 days. Physiological integrity of microvascular smooth muscle and endothelium recovers in parallel with myofibre regeneration. Additional time is required to restore the efficacy of signalling between myofibres and microvascular networks controlling their oxygen supply. ABSTRACT: Myofibre regeneration after skeletal muscle injury is well-studied, although little is known about how microvascular perfusion is restored. The present study aimed to evaluate the recovery of blood flow regulation during skeletal muscle regeneration. In anaesthetized male C57BL/6J mice (aged 4 months), the gluteus maximus muscle (GM) was injured by local injection of barium chloride solution (1.2%, 75 µL). Functional integrity of the resistance network was evaluated at 5, 10, 21 and 35 days post-injury vs. Control by measuring internal diameter of feed arteries, first-, second- and third-order arterioles supplying the GM using intravital microscopy. The resting diameters of all branch orders were significantly greater (P < 0.05) than Control at 5 and 10 days and recovered to Control by 21 days, as did spontaneous vasomotor tone. Vasodilatation to ACh and vasoconstriction to phenylephrine (10-9 to 10-5  m) were absent at 5 days, increased at 10 days and recovered to Control by 21 days; reactivity improved in a distal-to-proximal gradient. Across branch orders, functional vasodilatation to single tetanic contraction (100 Hz, 500 ms) and to rhythmic twitch contractions (4 Hz, 30 s) was impaired at 5 days, improved through 21 days and was not different from Control at 35 days. Peak force development (g) was 60% of Control at 10 days and recovered by 21 days. Diminished vasomotor tone during the initial stages of regeneration promotes tissue perfusion as myofibre recovery begins. Recovery of tone and vasomotor responses to agonists occur in concert with myofibre regeneration. Delayed recovery of functional vasodilatation indicates that additional time is required to restore signalling between contracting myofibres and their vascular supply.

Microvascular mechanisms limiting skeletal muscle blood flow with advancing age.

Effective oxygen delivery to active muscle fibers requires that vasodilation initiated in distal arterioles, which control flow distribution and capillary perfusion, ascends the resistance network into proximal arterioles and feed arteries, which govern total blood flow into the muscle. With exercise onset, ascending vasodilation reflects initiation and conduction of hyperpolarization along endothelium from arterioles into feed arteries. Electrical coupling of endothelial cells to smooth muscle cells evokes the rapid component of ascending vasodilation, which is sustained by ensuing release of nitric oxide during elevated luminal shear stress. Concomitant sympathetic neural activation inhibits ascending vasodilation by stimulating α-adrenoreceptors on smooth muscle cells to constrict the resistance vasculature. We hypothesized that compromised muscle blood flow in advanced age reflects impaired ascending vasodilation through actions on both cell layers of the resistance network. In the gluteus maximus muscle of old (24 mo) vs. young (4 mo) male mice (corresponding to mid-60s vs. early 20s in humans) inhibition of α-adrenoreceptors in old mice restored ascending vasodilation, whereas even minimal activation of α-adrenoreceptors in young mice attenuated ascending vasodilation in the manner seen with aging. Conduction of hyperpolarization along the endothelium is impaired in old vs. young mice because of "leaky" membranes resulting from the activation of potassium channels by hydrogen peroxide released from endothelial cells. Exposing the endothelium of young mice to hydrogen peroxide recapitulates this effect of aging. Thus enhanced α-adrenoreceptor activation of smooth muscle in concert with electrically leaky endothelium restricts muscle blood flow by impairing ascending vasodilation in advanced age.

Calcitonin gene-related peptide hyperpolarizes mouse pulmonary artery endothelial tubes through KATP channel activation.

The sensory neurotransmitter calcitonin gene-related peptide (CGRP) is associated with vasodilation of systemic arteries through activation of ATP-sensitive K+ (KATP) channels in smooth muscle cells (SMCs); however, its effects on endothelial cell (EC) membrane potential ( Vm) are unresolved. In pulmonary arteries (PAs) of C57BL/6J mice, we questioned whether CGRP would hyperpolarize ECs as well as SMCs. Intact PAs were isolated and immunostained for CGRP to confirm sensory innervation; vessel segments (1-2 mm long, ∼150 µm diameter) with intact or denuded endothelium were cannulated and pressurized to 16 cmH2O at 37°C. Increasing concentrations (10-10-10-6 M) of CGRP progressively dilated PAs preconstricted with UTP (10-5 M); SMCs hyperpolarized similarly (Δ Vm ∼20 mV) before and after endothelial denudation. To study native intact PA ECs, SMCs were dissociated to isolate endothelial tubes, and their integrity was confirmed by vital dye uptake, nuclear staining, and reproducible electrical and intracellular Ca2+ responses to acetylcholine (10-5 M) over 2 h. Increasing [CGRP] hyperpolarized ECs in a manner similar to SMCs, with each cell layer demonstrating robust immunostaining for CGRP receptor proteins. Increasing concentrations (10-10-10-6 M) of pinacidil, a KATP channel agonist, resulted in progressive hyperpolarization of SMCs of intact PAs (Δ Vm ∼30 mV), which was blocked by glibenclamide (10-6 M), as was hyperpolarization of ECs and SMCs to CGRP. Inhibition of protein kinase A with protein kinase inhibitor (10-5 M) also inhibited hyperpolarization to CGRP. We demonstrate [CGRP]-dependent hyperpolarization of ECs for the first time while validating freshly isolated PA endothelial tubes as an experimental model. Redundant electrical signaling to CGRP in ECs and SMCs implies an integral role for KATP channels in PA dilation.

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.

Biophysical properties of microvascular endothelium: Requirements for initiating and conducting electrical signals.

OBJECTIVE: Electrical signaling along the endothelium underlies spreading vasodilation and blood flow control. We use mathematical modeling to determine the electrical properties of the endothelium and gain insight into the biophysical determinants of electrical conduction. METHODS: Electrical conduction data along endothelial tubes (40 µm wide, 2.5 mm long) isolated from mouse skeletal muscle resistance arteries were analyzed using cable equations and a multicellular computational model. RESULTS: Responses to intracellular current injection attenuate with an axial length constant (λ) of 1.2-1.4 mm. Data were fitted to estimate the axial (ra ; 10.7 MΩ/mm) and membrane (rm ; 14.5 MΩ∙mm) resistivities, EC membrane resistance (Rm ; 12 GΩ), and EC-EC coupling resistance (Rgj ; 4.5 MΩ) and predict that stimulation of ≥30 neighboring ECs is required to elicit 1 mV of hyperpolarization at distance = 2.5 mm. Opening Ca2+ -activated K+ channels (KCa ) along the endothelium reduced λ by up to 55%. CONCLUSIONS: High Rm makes the endothelium sensitive to electrical stimuli and able to conduct these signals effectively. Whereas the activation of a group of ECs is required to initiate physiologically relevant hyperpolarization, this requirement is increased by myoendothelial coupling and KCa activation along the endothelium inhibits conduction by dissipating electrical signals.

Calcium and electrical dynamics in lymphatic endothelium.

KEY POINTS: Endothelial cell function in resistance arteries integrates Ca2+ signalling with hyperpolarization to promote relaxation of smooth muscle cells and increase tissue blood flow. Whether complementary signalling occurs in lymphatic endothelium is unknown. Intracellular calcium and membrane potential were evaluated in endothelial cell tubes freshly isolated from mouse collecting lymphatic vessels of the popliteal fossa. Resting membrane potential measured using intracellular microelectrodes averaged ∼-70 mV. Stimulation of lymphatic endothelium by acetylcholine or a TRPV4 channel agonist increased intracellular Ca2+ with robust depolarization. Findings from Trpv4-/- mice and with computational modelling suggest that the initial mobilization of intracellular Ca2+ leads to influx of Ca2+ and Na+ through TRPV4 channels to evoke depolarization. Lymphatic endothelial cells lack the Ca2+ -activated K+ channels present in arterial endothelium to generate endothelium-derived hyperpolarization. Absence of this signalling pathway with effective depolarization may promote rapid conduction of contraction along lymphatic muscle during lymph propulsion. ABSTRACT: Subsequent to a rise in intracellular Ca2+ ([Ca2+ ]i ), hyperpolarization of the endothelium coordinates vascular smooth muscle relaxation along resistance arteries during blood flow control. In the lymphatic vasculature, collecting vessels generate rapid contractions coordinated along lymphangions to propel lymph, but the underlying signalling pathways are unknown. We tested the hypothesis that lymphatic endothelial cells (LECs) exhibit Ca2+ and electrical signalling properties that facilitate lymph propulsion. To study electrical and intracellular Ca2+ signalling dynamics in lymphatic endothelium, we excised collecting lymphatic vessels from the popliteal fossa of mice and removed their muscle cells to isolate intact LEC tubes (LECTs). Intracellular recording revealed a resting membrane potential of ∼-70 mV. Acetylcholine (ACh) increased [Ca2+ ]i with a time course similar to that observed in endothelium of resistance arteries (i.e. rapid initial peak with a sustained 'plateau'). In striking contrast to the endothelium-derived hyperpolarization (EDH) characteristic of arteries, LECs depolarized (>15 mV) to either ACh or TRPV4 channel activation. This depolarization was facilitated by the absence of Ca2+ -activated K+ (KCa ) channels as confirmed with PCR, persisted in the absence of extracellular Ca2+ , was abolished by LaCl3 and was attenuated ∼70% in LECTs from Trpv4-/- mice. Computational modelling of ion fluxes in LECs indicated that omitting K+ channels supports our experimental results. These findings reveal novel signalling events in LECs, which are devoid of the KCa activity abundant in arterial endothelium. Absence of EDH with effective depolarization of LECs may promote the rapid conduction of contraction waves along lymphatic muscle during lymph propulsion.

Rapid versus slow ascending vasodilatation: intercellular conduction versus flow-mediated signalling with tetanic versus rhythmic muscle contractions.

KEY POINTS: In response to exercise, vasodilatation ascends from downstream arterioles into upstream feed arteries (FAs). We hypothesized that the signalling events underlying ascending vasodilatation variy with the intensity and duration of skeletal muscle contraction. In the gluteus maximus muscle of C57BL/6 mice, brief tetanic contraction evoked rapid onset vasodilatation (ROV) (<1 s) throughout the resistance network. Selective damage to endothelium midway between FAs and primary arterioles eliminated ROV only in FAs. Blocking SKCa and IKCa channels attenuated ROV, implicating hyperpolarization as the underlying signal. During rhythmic twitch contractions, slow onset vasodilatation (10-15 s) in FAs remained intact following loss of ROV and was eliminated following nitric oxide synthase inhibition. Tetanic contraction initiates hyperpolarization that conducts along endothelium into FAs. Rhythmic twitch contractions stimulate FA endothelium to release nitric oxide in response to elevated shear stress secondary to metabolic dilatation of arterioles. Complementary endothelial signalling pathways for ascending vasodilatation ensure increased oxygen delivery to active skeletal muscle. ABSTRACT: In response to exercise, vasodilatation initiated within the microcirculation of skeletal muscle ascends the resistance network into upstream feed arteries (FAs) located external to the tissue. Ascending vasodilatation (AVD) is essential for reducing FA resistance that otherwise restricts blood flow into the microcirculation. In the present study, we tested the hypothesis that signalling events underlying AVD vary with the intensity and duration of muscle contraction. In the gluteus maximus muscle of anaesthetized male C57BL/6 mice (aged 3-4 months), brief tetanic contraction (100 Hz for 500 ms) evoked rapid onset vasodilatation (ROV) in FAs that peaked within 4 s. By contrast, during rhythmic twitch contractions (4 Hz), slow onset vasodilatation (SOV) of FAs began after ∼10 s and plateaued within 30 s. Selectively damaging the endothelium with light-dye treatment midway between a FA and its primary arteriole eliminated ROV in the FA along with conducted vasodilatation of the FA initiated on the arteriole using ACh microiontophoresis. Superfusion of SKCa and IKCa channel blockers UCL 1684 + TRAM 34 attenuated ROV, implicating endothelial hyperpolarization as the underlying signal. Nevertheless, the SOV of FAs during rhythmic contractions persisted until inhibition of nitric oxide synthase with Nω -nitro-l-arginine methyl ester. Thus, ROV of FAs reflects hyperpolarization of downstream arterioles that conducts along the endothelium into proximal FAs. By contrast, SOV of FAs reflects the local production of nitric oxide by the endothelium in response to luminal shear stress, which increases secondary to arteriolar dilatation downstream. Thus, AVD ensures increased oxygen delivery to active muscle fibres by reducing upstream resistance via complementary signalling pathways that reflect the intensity and duration of muscle contraction.

Impact of Aging on Calcium Signaling and Membrane Potential in Endothelium of Resistance Arteries: A Role for Mitochondria.

Impaired blood flow to peripheral tissues during advanced age is associated with endothelial dysfunction and diminished bioavailability of nitric oxide (NO). However, it is unknown whether aging impacts coupling between intracellular calcium ([Ca2+]i) signaling and small- and intermediate K+ channel (SKCa/IKCa) activity during endothelium-derived hyperpolarization (EDH), a signaling pathway integral to dilation of the resistance vasculature. To address the potential impact of aging on EDH, Fura-2 photometry and intracellular recording were applied to evaluate [Ca2+]i and membrane potential of intact endothelial tubes (width, 60 µm; length, 1-3 mm) freshly isolated from superior epigastric arteries of young (4-6 mo) and old (24-26 mo) male C57BL/6 mice. In response to acetylcholine, intracellular release of Ca2+ from the endoplasmic reticulum (ER) was enhanced with aging. Further, treatment with the mitochondrial uncoupler FCCP evoked a significant increase of [Ca2+]i with membrane hyperpolarization in an SKCa/IKCa-dependent manner in the endothelium of old but not young mice. We conclude that the ability of resistance artery endothelium to release Ca2+ from intracellular stores (ie, ER and mitochondria) and hyperpolarize Vm via SKCa/IKCa activation is augmented as compensation for reduced NO bioavailability during advanced age.