Microcirculation in skeletal muscle.

The role of microcirculation in skeletal muscle is to provide the supply of oxygen and various nutrients and to remove waste products of muscle metabolism. As skeletal muscles are composed of different fibre types, this review tries to elucidate the question of capillary supply and flow with respect to these. It reviews the current knowledge of structure of microcirculation and its nervous, hormonal, and local (myogenic, metabolic and endothelial) control. It also discuss factors involved in the increase in blood flow and changes in microcirculation occurring during muscle contractions, exercise training, muscle hypertrophy and atrophy, hypoxia, ageing, hypertension, diabetes and limited blood supply.

Adaptation of skeletal muscle microvasculature to increased or decreased blood flow: role of shear stress, nitric oxide and vascular endothelial growth factor.

This review elucidates the roles of capillary haemodynamics, nitric oxide (NO) and vascular endothelial growth factor (VEGF) in the remodelling of skeletal muscle microcirculation in response to increased (electrical stimulation) or decreased (chronic ischaemia) blood flow. During early stages of stimulation-induced angiogenesis, up-regulation of VEGF and its receptor VEGF receptor 2 is dependent on shear stress and NO release, whereas later, involvement of NO in the expanding capillary bed appears to be VEGF/VEGF receptor 2 independent. Arteriolar growth most likely relies on mechanical wall stresses while growth factor involvement is less clear. By contrast, in muscles with restricted blood flow, increased VEGF/VEGF receptor 2 expression after ischaemia onset is not associated with changes in shear stress or hypoxia, or capillary growth. After several weeks, VEGF protein levels are lower than normal while modest angiogenesis takes place, a temporal mismatch that limits the utility of using growth factor levels during ischaemia to assess angiogenic potential. Chronic stimulation of ischaemic muscles restores their depressed endothelial-dependent arteriolar dilatation, increases capillary shear stress and VEGF receptor 2 and promotes capillary growth. In patients with peripheral vascular disease, electrical stimulation of ischaemic calf muscles increases blood flow, capillary surface area and muscle performance, offering an alternative 'endogenous' treatment to gene or cell therapy.

Vascular endothelial growth factor mRNA and protein do not change in parallel during non-inflammatory skeletal muscle ischaemia in rat.

Impaired blood flow is thought to induce a pro-angiogenic environment due to local hypoxia, yet prolonged mild ischaemia induces only modest capillary growth. We compared the expression of vascular endothelial growth factor (VEGF) mRNA and protein with capillary to fibre ratio (C: F) and muscle blood flow in extensor digitorum longus of rats that had undergone unilateral ligation of the common iliac artery. Resting blood flow during the first two weeks after ligation (3, 7 and 14 days) was decreased by approximately 60% but recovered partially after 5 weeks (36% reduction). Functional hyperaemia (9-fold increase in blood flow during contractions) was eliminated in the first week after ligation, with a moderate recovery seen after 14 and 35 days. Muscle histology confirmed the absence of tissue necrosis or inflammation. Both VEGF mRNA (60%, P<0.05) and protein levels (700%, P<0.01) increased during the initial phase of ischaemia (at 1 and 3 days), well before any overt angiogenesis, and both declined towards control levels by 7 days. A secondary increase in VEGF protein (by 60% at 14 days, P<0.05) preceded the 20% increase in C: F seen after 5 weeks, but occurred while VEGF transcript levels continued to decline (to 50% of control at 35 days, P<0.05). Thus, evaluation of neither VEGF mRNA nor protein is an adequate index of angiogenic potential in response to ischaemia. We conclude that VEGF alone is insufficient to induce angiogenesis in ischaemic conditions, and that effective angiotherapy requires intervention aimed at other cytokines.

ICAM-1 expression and leukocyte behavior in the microcirculation of chronically ischemic rat skeletal muscles.

In muscle microcirculation, short periods of ischemia followed by reperfusion are known to upregulate leukocyte and endothelial adhesion molecules, but little is known about leukocyte adherence and ICAM-1 expression during chronic ischemia or any likely effect of muscle activity which is recommended in chronic ischemia due to peripheral arterial disease. Leukocyte rolling and stationary adhesion were observed in post-capillary venules in ischemic and contralateral rat extensor digitorum longus (EDL) muscles 3 and 7 days after unilateral ligation of the common iliac artery and in 3-day ischemic EDLs that were electrically stimulated on days 1 and 2 post-ligation (7 x 15 min per day). ICAM-1 was localized immunohistochemically to venular vessels in all muscles. Following ligation, use of the ischemic leg was observed to be restricted for the first 3 days, returning to normal by 7 days. After 3 days, leukocyte rolling/adherence and ICAM-1 expression were no different in ischemic than control muscles, but all were increased in contralateral muscles. In ischemic muscles, electrical stimulation doubled the numbers of rolling leukocytes and upregulated ICAM-1 expression. After 7 days, increased muscle activity as a result of natural movement also resulted in greater ICAM-1 expression, a 4- to 5-fold increase in rolling leukocyte numbers and a 3-fold increase in stationary adherent leukocytes. Chronic ischemia thus increases ICAM-1 and leukocyte adherence in muscle microcirculation only when combined with contractile activity. Post-capillary venular endothelium may be modified by muscle acidosis when contractions are performed under low flow conditions or by changes in rheological (shear force) factors.

Changes in capillary shear stress in skeletal muscles exposed to long-term activity: role of nitric oxide.

OBJECTIVE: The purpose of this study was to establish whether suppression of angiogenesis by nitric oxide synthase (NOS) inhibition in skeletal muscles exposed to long-term activity can be explained by changes in capillary shear stress linked to the lack of nitric oxide production. METHODS: Capillary shear stress was calculated from diameters (d) and red blood cell velocities (V(rbc)) measured at rest and after acute contractions in epi-illuminated extensor digitorum longus muscles of control rats and those in which ankle flexors had been stimulated via implanted electrodes (10 Hz, 8 h x day(-1)) for 2 or 7 days without and with inhibition of nitric oxide synthase activity by N(omega)-nitro-L-arginine (L-NNA, 3-4 mg x day(-1) in drinking water). RESULTS: Neither chronic electrical stimulation nor L-NNA treatment altered capillary diameters. Capillary V(rbc) and shear stress (SS) were doubled in muscles after 2 days stimulation (298 +/- 22 microm x s(-1) and 11.4 +/- 1.0 dyne x cm(-2), respectively, p < .005) compared to controls (148 +/- 18 microm x s(-1) and 5.6 +/- 0.8 dyne x cm(-2)) but normalized after 7 days (153 +/- 27 microm x s(-1) and 6.2 +/- 1.0 dyne x cm(-2)), when the capillary bed is known to be enlarged. L-NNA, which increased blood pressure in all treated animals, abolished the increase in capillary SS after 2 days stimulation and decreased SS after 7 days. CONCLUSIONS: These data support a role for NO in the early elevation of capillary shear stress that initiates angiogenesis in stimulated muscles, likely via modulation of upstream vascular resistance, and could explain the lack of capillary growth in stimulated muscles when nitric oxide generation is suppressed.

Nitric oxide, VEGF, and VEGFR-2: interactions in activity-induced angiogenesis in rat skeletal muscle.

Vascular endothelial growth factor (VEGF) is considered to be important in promotion of capillary growth in skeletal muscles exposed to increased activity. We studied its interactions with nitric oxide (NO) by examining the expression of endothelial NO synthase (NOS), VEGF, and VEGF receptor-2 (VEGFR-2) proteins in relation to capillary growth in rat extensor digitorum longus muscles electrically stimulated for 2, 4, or 7 days with and without NOS inhibition by N(omega)-nitro-L-arginine (L-NNA, 3 mg/day). Stimulation increased all proteins from 2 days onward, concomitantly with capillary proliferation (labeling for proliferating cell nuclear antigen). Capillary-to-fiber ratio was elevated by 25% after 7 days. Concurrent oral administration of L-NNA did not affect the increase in endothelial NOS but depressed its activity, as shown by increased blood pressure and decreased arteriolar diameters in 2-day-stimulated muscles. NOS inhibition eliminated the increased expression of VEGFR-2 and VEGF proteins in muscles stimulated for 2 and 4 days but not for 7 days. However, it depressed capillary proliferation and the increase in C/F at all time points. We conclude that, in stimulated muscles, NO, generated by activation of neuronal NOS by muscle activity or endothelial NOS by increased blood flow and capillary shear stress, may increase capillary proliferation in the early stages of stimulation through upregulation of VEGFR-2 and VEGF. With longer stimulation, capillary growth appears to require NO, and high levels of VEGF and VEGFR-2 may be contributing to maintenance of the increased capillary bed.

Differential expression of Flk-1 and Flt-1 in rat skeletal muscle in response to chronic ischaemia: favourable effect of muscle activity.

To determine the involvement of vascular endothelial growth factor (VEGF) and its receptors Flk-1 and Flt-1 in capillary growth in ischaemic skeletal muscle, extensor digitorum longus muscles from hindlimbs of Sprague-Dawley rats were studied at 1, 2 and 5 week intervals after iliac artery ligation. Muscle VEGF protein levels (as determined by Western-blot analysis) increased only after 2 (60%) and 5 (80%) weeks, with more capillaries positively immunostained for VEGF than in control muscles. Ischaemia-induced angiogenesis was gradual, with capillary proliferation at 1 and 2 weeks and capillary:fibre ratio increased 20% after 5 weeks. This was associated with an initial doubling of Flk-1 protein after 1 week that declined below control levels by 5 weeks, whereas Flt-1 expression was elevated more than 40% at all time points. During more sustained ischaemia (femoral ligation 3 weeks after iliac ligation), VEGF protein level at 5 weeks was even higher, but Flt-1 and Flk-1 were unchanged from control levels and no capillary growth occurred. Intermittent electrical stimulation (10 Hz, 7x15 min/day) of these ischaemic muscles between weeks 3-5 did not elevate VEGF further, but increased Flk-1 by 32%, decreased Flt-1 by 71%, and led to significant capillary growth. These results demonstrate that during chronic muscle ischaemia Flk-1 and Flt-1 are regulated differentially and that electrical stimulation of ischaemic muscles can promote angiogenesis via Flk-1 up-regulation. Even when ischaemic muscle VEGF levels are high, capillary growth appears to be dependent on the presence of Flk-1.

The effect of chronic skeletal muscle stimulation on capillary growth in the rat: are sensory nerve fibres involved?

Indirect chronic electrical stimulation of skeletal muscle activates not only efferent but also afferent nerve fibres. To investigate effects specific to this on capillary growth, one of the earliest changes, cell proliferation and capillary ultrastructure were studied in ankle flexors of rats with and without deafferentation of the stimulated side. Two weeks after preganglionic section of dorsal roots L4-L6, the peroneal nerve was stimulated (10 Hz, 8 h day(-1)) for 2 or 7 days. Proliferating nuclei labelled by bromodeoxyuridine or proliferating cell nuclear antigen staining were colocalized to alkaline phosphatase-stained capillaries (Lc) or other interstitial nuclei (Li) in frozen sections of extensor digitorum longus. Capillary fine structure was examined in extensor hallucis proprius by transmission electron microscopy. The stimulation-induced increase in capillary and interstitial proliferation (Lc 9.9 +/- 1.9 %, Li 8.8 +/- 2.1 % vs. Lc 2.6 +/- 0.4 %, Li 1.9 +/- 0.3 % in controls, P < 0.05) was depressed at 2 days by dorsal root section (Lc 4.8 +/- 0.7 %, Li 3.2 +/- 0.9 %, P < 0.05), an effect likely to be mainly on fibroblasts; no depression was seen at 7 days. Dorsal root section reduced stimulation-induced capillary endothelial swelling at both time points. In contralateral muscles of intact rats, stimulation increased interstitial cell proliferation and capillary swelling, both effects being eliminated by dorsal root section. Capillary growth induced by stimulation (24 % increase in capillary : fibre ratio at 7 days) was unaffected by deafferentation. The reduction in capillary ultrastructural changes and interstitial proliferation in both stimulated and contralateral muscles implies that stimulation of afferent fibres leads directly to release of humoral factors and/or activation via dorsal roots of fibres that release humoral substances. Contralateral muscles are an inadequate control for the effects of chronic stimulation in the intact animal.

Differential involvement of MMP-2 and VEGF during muscle stretch- versus shear stress-induced angiogenesis.

Capillary growth in skeletal muscle occurs via the dissimilar processes of abluminal sprouting or longitudinal splitting, which can be initiated by muscle stretch and elevated shear stress, respectively. The distinct morphological hallmarks of these types of capillary growth suggest that discrete sets of angiogenic mediators play a role in each situation. Because proteolysis and proliferation are two key steps associated with capillary growth, we tested whether differences in the regulation of matrix metalloproteinases (MMPs) or VEGF may be associated with the two types of capillary growth. We found significant increases in MMP-2 total protein and percent activation, and membrane type-1 MMP mRNA levels, compared with controls after muscle stretch but not after shear stress stimulation. In contrast, VEGF protein and endothelial cell proliferation increased after either angiogenic stimulus. We observed that MMP-2 regulation occurs independent of VEGF signaling, because VEGF did not induce MMP-2 production or activation in isolated endothelial cells. Our data suggest that the involvement of MMPs in capillary growth is dependent on the nature of the angiogenic stimulus.