Stuart Egginton

Unorthodox angiogenesis in skeletal muscle

OBJECTIVE The morphological pattern of angiogenesis occurring in mature, differentiated skeletal muscle in response to chronically increased muscle blood flow, muscle stretch or repetitious muscle contractions was examined to determine (a) whether capillary neoformation follows the generally accepted temporal paradigm, and (b) how the growth pattern is influenced by mechanical stimuli. METHODS Adult rats were treated for a maximum of 14 days either with the vasodilator prazosin, to elevate skeletal muscle blood flow, or underwent surgical removal of one ankle flexor, to induce compensatory overload in the remaining muscles, or had muscles chronically stimulated by implanted electrodes. Extensor digitorum longus and/or extensor hallucis proprius muscles were removed at intervals and processed for electron microscopy. A systematic examination of capillaries and their ultrastructure characterised the sequence of morphological changes indicative of angiogenesis, i.e., basement membrane disruption, endothelial cell (EC) sprouting and proliferation [immunogold labelling after bromodeoxyuridine (BrdU) incorporation]. RESULTS Capillary growth in response to increased blood flow occurred by luminal division without sprouting or basement membrane (BM) breakage. In stretched muscles, EC proliferation and abluminal sprouting gave rise to new capillaries, with BM loss only at sprout tips. These distinct mechanisms appear to be additive as in chronically stimulated muscles (increased blood flow with repetitive stretch and shortening during muscle contractions) both forms of capillary growth occurred. Endothelial cell numbers per capillary profile, mitotic EC nuclei, and BrdU labelling confirmed cell proliferation prior to overt angiogenesis. CONCLUSIONS Physiological angiogenesis within adult skeletal muscle progresses by mechanisms that do not readily conform to the consensus view of capillary growth, derived mainly from observations made during development, pathological vessel growth, or from in vitro systems. The temporal and spatial pattern of growth is determined by the polarity of the mechanical stimulus, i.e., by intra-luminal (increased shear stress) or abluminal (external stretch) stimuli.

Invited review: activity-induced angiogenesis.

The dynamic biochemical and mechanical environment around blood vessels during muscle activity generates powerful stimuli for vascular remodelling. Ultimately, this must lead to a coordinated expansion of various elements of the cardiovascular system in order to support enhanced aerobic exercise. Vascular endothelial growth factor plays a central role, and understanding how this is regulated in vivo by changes in transcription and stability of mRNA, production of protein and interaction with other growth factors, is a continuing challenge. Exercise hyperaemia leads to an increase in microvascular shear stress, which stimulates endothelial release of nitric oxide, whilst proteolytic modification of the extracellular matrix is induced by mechanical deformation during cyclical contractions or muscle overload. These components of the exercise response lead to different forms of capillary growth, and subsequent expansion of the microcirculation may not have the same functional outcome. In vitro and in vivo studies have shown a complex interplay between different cytokines, receptors and mural cells in directing the necessary tissue re-organisation. The mechanisms involved in arteriogenesis are less well-understood than those of angiogenesis, but application of these data to understanding vascular remodelling in response to exercise may help resolve a range of cardiovascular dysfunction.

Association between Shear Stress, Angiogenesis, and VEGF in Skeletal Muscles In Vivo

Objective: To investigate the hypothesis that capillary proliferation in skeletal muscles, induced by a long‐term increase in blood flow which elevates capillary shear stress, is associated with capillary expression of vascular endothelial growth factor (VEGF).

Inhibition of endogenous HIF inactivation induces angiogenesis in ischaemic skeletal muscles of mice.

Hypoxia‐inducible factor (HIF) modulates transcriptional control of several genes involved in vascular growth and cellular metabolism. HIF activity can be enhanced by suppression of prolyl and asparaginyl hydroxylase activity by dimethyloxalylglycine (DMOG). We have compared the effects of DMOG treatment and femoral artery ligation individually or in combination on HIF‐1α protein level, HIF‐dependent gene expression and capillary‐to‐fibre ratio (C: F) in extensor digitorum longus and tibialis anterior muscles of mice. Immunohistochemical examination revealed that HIF‐1α is present in non‐ischaemic mouse skeletal muscles, but its amount increased profoundly in response to the combination of DMOG treatment and ischaemia. Combined treatment resulted in 39% increase in C: F in ischaemic muscles (P < 0.0001 versus controls) whereas individual treatments produced little effect under our conditions. Combined treatment led to a significant increase in endogenous HIF‐1α protein (6.14 ± 1.1 versus 1.17 ± 0.2 in controls; P < 0.05) that was not apparent in mice treated with DMOG or femoral artery ligation alone. Ischaemia increased vascular endothelial growth factor (VEGF) protein production by 2.5‐fold (P < 0.05 versus controls), irrespective of DMOG treatment. However, production of the VEGF receptor Flk‐1 was more enhanced in ischaemic + DMOG‐treated muscles (P < 0.001 and P < 0.05 compared with controls and untreated ischaemic muscles, respectively), which may explain the intensive growth of capillaries in those muscles. The findings indicate that treatment with DMOG has a potential therapeutic use in promoting angiogenesis in ischaemic diseases, and perhaps for improving muscle recovery after injury, exercise or training.

Internal division of capillaries in rat skeletal muscle in response to chronic vasodilator treatment with alpha1-antagonist prazosin.

Abstract Chronic vasodilatation represents a stimulus for capillary growth associated with increased luminal shear stress. We have examined the ultrastructure of more than 2000 capillaries to establish whether the sequence of angiogenesis in response to this stimulus is similar to that described during development and under pathological circumstances. Administration of the α1-blocker prazosin to rats for 2 weeks led to a greater capillary length density in extensor hallucis proprius muscles without any change in capillary tortuosity: Jv(c,f)=262±54 compared with 350±17 mm–2, control compared with prazosin (P<0.002). There were obvious signs of endothelial cell (EC) activation after prazosin treatment, including an increased proportion of capillaries with rough endoplasmic reticulum, large cytoplasmic vacuoles, thickened endothelium and an irregular luminal surface. Capillaries from control muscles had a maximum of three ECs in cross section, whereas four ECs were noted in 0.8+0.5% of capillaries after 1 week (n.s.) and 2.5±0.9% after 2 weeks (P<0.01) of treatment. This could be due to elongation and/or migration of ECs, as cell proliferation has not been described at these time points. There was also an increase in the proportion of capillaries having a narrow, slit-like lumen (1.7±0.8% of controls; 7.1±1.9% at 1 week; 8.8±2.5% at 2 weeks; P<0.02), some of which were smaller in size (less than 2 μm diameter) than in controls (3–5 μm) and/or “seamless”, i.e. lacking EC junctions. These may represent newly formed vessels. Focal discontinuity of the basement membrane and abluminal EC processes were rarely seen, and capillary growth by abluminal sprouting appeared to be very infrequent (less than 0.001% of profiles). Of more importance was growth starting from the luminal side. Significantly more thin cytoplasmic processes were observed protruding into the lumen of capillaries after 1 week (47.5±6.2%, P<0.001) and 2 weeks of prazosin (34.2±5.5%, P<0.05) than in control vessels (16.7±3.9%). Some of these traversed the entire lumen and connected with endothelium of the opposite side, probably involving membrane fusion, resulting in the appearance of a double lumen. Individual capillaries with a complete double lumen were observed after 2 weeks’ prazosin but comparatively rarely, in only four out of six muscles. These findings indicate a pattern of luminal growth which is completely different from intussusceptive growth previously described during development, and from the abluminal capillary sprouting seen under pathological circumstances.

Molecular Evolution of Cytochrome c Oxidase Underlies High-Altitude Adaptation in the Bar-Headed Goose

Bar-headed geese (Anser indicus) fly at up to 9,000 m elevation during their migration over the Himalayas, sustaining high metabolic rates in the severe hypoxia at these altitudes. We investigated the evolution of cardiac energy metabolism and O(2) transport in this species to better understand the molecular and physiological mechanisms of high-altitude adaptation. Compared with low-altitude geese (pink-footed geese and barnacle geese), bar-headed geese had larger lungs and higher capillary densities in the left ventricle of the heart, both of which should improve O(2) diffusion during hypoxia. Although myoglobin abundance and the activities of many metabolic enzymes (carnitine palmitoyltransferase, citrate synthase, 3-hydroxyacyl-coA dehydrogenase, lactate dehydrogenase, and pyruvate kinase) showed only minor variation between species, bar-headed geese had a striking alteration in the kinetics of cytochrome c oxidase (COX), the heteromeric enzyme that catalyzes O(2) reduction in oxidative phosphorylation. This was reflected by a lower maximum catalytic activity and a higher affinity for reduced cytochrome c. There were small differences between species in messenger RNA and protein expression of COX subunits 3 and 4, but these were inconsistent with the divergence in enzyme kinetics. However, the COX3 gene of bar-headed geese contained a nonsynonymous substitution at a site that is otherwise conserved across vertebrates and resulted in a major functional change of amino acid class (Trp-116 → Arg). This mutation was predicted by structural modeling to alter the interaction between COX3 and COX1. Adaptations in mitochondrial enzyme kinetics and O(2) transport capacity may therefore contribute to the exceptional ability of bar-headed geese to fly high.

Capillary growth in overloaded, hypertrophic adult rat skeletal muscle: An ultrastructural study

We examined the early stages of angiogenesis in overloaded m. extensor digitorum longus following extirpation of the agonist m. tibialis anterior. Capillary‐to‐fibre ratio increased after 1 week (1.54 ± 0.02) vs. control (1.38 ± 0.06; P < 0.01) and resulted in a greater tortuosity of the capillary bed at 2 weeks, indicating the presence of lateral sprouts or anastomoses. Capillary endothelial cells (ECs) showed ultrastructural signs of activation, were thickened, and had irregular luminal and abluminal surfaces. The proportion of ECs with abluminal processes increased after overload (13.5 ± 0.6% vs. 2.0 ± 1.5%, 1 week vs. contralateral, P < 0.01; 12.5 ± 2.6% vs. 3.5 ± 0.6%, 2 weeks vs. contralateral, P < 0.01), whereas there was no significant change in proportion of luminal processes. Abluminal processes occurred in approximately 13% of capillaries in overloaded muscles (P < 0.01 v. control and contralateral), and most were associated with focal breakage of the basement membrane (BM). Small sprouts (<3 μm in diameter) comprised of one or two ECs sometimes lacked a lumen, and others had a slitlike or vacuolelike lumen between adjacent ECs or vacuolelike lumen formed by fusion of vesicles within a single EC. Endothelial mitosis was occasionally seen in nonsprouting capillaries with intact BM, increasing the average number of ECs per capillary from approximately 1.7 in control muscles to 2.1 after 1 week of overload (P < 0.05) when bromodeoxyuridine incorporation was also higher (P < 0.001). We conclude that muscle overload induces capillary growth by sprouting of existing capillaries, probably due to mechanical stretch acting from the abluminal side of the vessels. Anat. Rec. 252:49–63, 1998.

In vivo angiogenesis in adult rat skeletal muscle: early changes in capillary network architecture and ultrastructure.

Abstract.The individual structural stages in capillary growth have been identified during development and under pathological circumstances in adults (wound healing, tumors), but there are no data to indicate whether these steps are similar when angiogenesis is induced in a fully differentiated microvascular bed in normal, uninjured adult skeletal muscle. In this study changes in capillary ultrastructure were correlated with capillary density and network morphology to elucidate the sequelae of angiogenesis in adult rat extensor digitorum longus (EDL) muscle whose activity was increased by stimulation at 10 Hz (8 h/day). This resulted in an increased capillary/fiber (C/F) ratio (based on staining for alkaline phosphatase) after 4 days; by 7 days C/F ratio was increased further, by approximately 50%. The ultrastructure of capillary endothelium in both the EDL and extensor hallucis proprius (EHP) was similar to control muscles after 2 days of stimulation, whereas endothelial cells in some capillaries in muscle stimulated for 4 days revealed signs of metabolic activation such as proliferation of organelles (Golgi apparatus, endoplasmic reticulum, ribosomes and mitochondria) and fewer pinocytic vesicles. Luminal surfaces were often irregular with numerous pseudopodial processes. Basement membranes were always present but amorphous regions were observed, particularly near pericyte processes. Unusually small capillary profiles, with either a slit-like lumen or with cisternae but no lumen, probably represented capillary sprouts. The interstitium contained increased collagenous and granular extracellular matrix surrounding capillaries, and numerous activated fibroblasts which were closely apposed to many capillaries. Capillary growth in EHP was also evaluated by confocal microscopy using whole mounts. The complex pattern of vessels underwent remodelling between 2 and 7 days of stimulation, resulting in more tortuous capillaries with numerous sprouts and loops. These combined observations suggest that angiogenesis may occur by a combination of sprouting, intussusceptive growth and elongation; also, that activation of endothelial cells occurs at the same time as disturbance of basement membranes during the earliest phase of growth and remodelling of the capillary bed. These changes are postulated to occur in connection with increased shear stress and/or capillary wall tension, which have been demonstrated previously.

Lumen Formation In Vivo Versus In Vitro Observations

Lumen formation must accompany the de novo growth of blood vessels during embryological development, the production of new vessels (vasculogenesis), and the expansion or remodeling of the microcirculation in differentiated tissue (angiogenesis). The debate over lumen origin centers on whether this is an intracellular or intercellular phenomenon, entailing vesicle accretion or loss of endothelial cell (EC) contact, and whether this represents an intrinsic property of ECs or relies on extrinsic signals. In addition, recent in vivo data suggest that a third mechanism, that of longitudinal division, may be used to expand existing capillary networks. Importantly, more than one mechanism of lumen formation may be found in response to a given angiogenic signal. Tubule formation by ECs in a matrix is an increasingly popular form of in vitro angiogenesis assay, and it may offer insights into the mechanisms involved during growth in embryos or under pathological conditions in adults. Crucial to the validity of in vitro preparations is the extent to which tubule assembly and lumen formation mirrors that observed in vivo, although these data cannot elucidate the controls operative during adaptive remodeling of the vascular bed. Similar structures may be observed in vivo and in vitro, and may represent the situation found during angiogenesis and vasculogenesis, respectively. Lumen formation during angiogenesis, and tubule formation during EC culture, require the existence of cell polarity. As tubule formation is not a unique property of ECs, how this is developed is a key area where in vitro studies may extend our understanding of EC biology.

The use of power spectral analysis to determine cardiorespiratory control in the short-horned sculpin Myoxocephalus scorpius.

SUMMARY Anaesthesia and minor surgery to place electrocardiogram recording electrodes in the short-horned sculpin caused a decrease in mean normal beat (R–R) interval and heart rate variability (HRV), measured as the standard deviation in the R–R interval (SDRR). Mean R–R interval increased to a steady state value (1.9±2.9 s) 72 h post-surgery, but SDRR took 120 h to stabilise (0.56±0.09 s). Power spectral analysis applied to recordings of instantaneous heart rate showed no spectral peaks immediately after surgery, with the development of twin peaks (at 0.02 and 0.05 Hz) that also became stable 120 h post surgery. Bilateral cardiac vagotomy abolished the variability in beat-to-beat interval, and both the high and low frequency peaks, suggesting that much of the regulation of heart rate and HRV in sculpin was under parasympathetic, cholinergic control that was withdrawn as a result of surgical and handling stress. Rate of oxygen consumption \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(({\dot{M}}_{\mathrm{O}_{2}})\) \end{document} and heart rate (fH) were monitored simultaneously and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \({\dot{M}}_{\mathrm{O}_{2}}\) \end{document} showed a good correlation with both mean R–R interval (r2=–0.89) and SDRR (r2=0.93), although a more significant (ANCOVA, P=0.02) covariance existed between the post-surgical decrease in \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \({\dot{M}}_{\mathrm{O}_{2}}\) \end{document} and increase in SDRR. These data suggest that sculpin use fH as a way of moderating oxygen consumption, fine-tuned on a beat-to-beat basis by cholinergic control. We conclude that power spectral analysis is a useful method of determining HRV in fish, and that HRV is a more sensitive measure of recovery from disturbance than fH alone.