Inducible deletion of endothelial cell Efnb2 delays capillary regeneration and attenuates myofibre reinnervation following myotoxin injury in mice.

Acute injury of skeletal muscle disrupts myofibres, microvessels and motor innervation. Myofibre regeneration is well characterized, however its relationship with the regeneration of microvessels and motor nerves is undefined. Endothelial cell (EC) ephrin-B2 (Efnb2) is required for angiogenesis during embryonic development and promotes neurovascular regeneration in the adult. We hypothesized that, following acute injury to skeletal muscle, loss of EC Efnb2 would impair microvascular regeneration and the recovery of neuromuscular junction (NMJ) integrity. Mice (aged 3-6 months) were bred for EC-specific conditional knockout (CKO) of Efnb2 following tamoxifen injection with non-injected CKO mice as controls (CON). The gluteus maximus, tibialis anterior or extensor digitorum longus muscle was then injured with local injection of BaCl2. Intravascular staining with wheat germ agglutinin revealed diminished capillary area in the gluteus maximus of CKO vs. CON at 5 days post-injury (dpi); both recovered to uninjured (0 dpi) level by 10 dpi. At 0 dpi, tibialis anterior isometric force of CKO was less than CON. At 10 dpi, isometric force was reduced by half in both groups. During intermittent contractions (75 Hz, 330 ms s-1, 120 s), isometric force fell during indirect (sciatic nerve) stimulation whereas force was maintained during direct (electrical field) stimulation of myofibres. Neuromuscular transmission failure correlated with perturbed presynaptic (terminal Schwann cells) and postsynaptic (nicotinic acetylcholine receptors) NMJ morphology in CKO. Resident satellite cell number on extensor digitorum longus myofibres did not differ between groups. Following acute injury of skeletal muscle, loss of Efnb2 in ECs delays capillary regeneration and attenuates recovery of NMJ structure and function. KEY POINTS: The relationship between microvascular regeneration and motor nerve regeneration following skeletal muscle injury is undefined. Expression of Efnb2 in endothelial cells (ECs) is essential to vascular development and promotes neurovascular regeneration in the adult. To test the hypothesis that EfnB2 in ECs is required for microvascular regeneration and myofibre reinnervation, we induced conditional knockout of Efnb2 in ECs of mice. Acute injury was then induced by BaCl2 injection into gluteus maximus, tibialis anterior or extensor digitorum longus (EDL) muscle. Capillary regeneration was reduced at 5 days post-injury (dpi) in gluteus maximus of conditional knockout vs. controls; at 10 dpi, neither differed from uninjured. Nerve stimulation revealed neuromuscular transmission failure in tibialis anterior with perturbed neuromuscular junction structure. Resident satellite cell number on EDL myofibres did not differ between groups. Conditional knockout of EC Efnb2 delays capillary regeneration and attenuates recovery of neuromuscular junction structure and function.

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.

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.

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.

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.

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.

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.

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.