Attenuation of changes in capillary fine structure and leukocyte adhesion improves muscle performance following chronic ischaemia in rats.

Acute ischaemia-reperfusion disrupts capillary fine structure and increases leukocyte adhesion in postcapillary venules. We determined whether chronic muscle ischaemia has similar consequences, and whether it is possible to ameliorate its effect on muscle performance. Following ischaemia (unilateral ligation, common iliac artery) rat hindlimb muscles were examined without other intervention or following treatment with an xanthine oxidase inhibitor (allopurinol), a Na(+)/H(+) exchange blocker (amiloride), or an oxygen free radical scavenger (vitamin E). No significant leukocyte adhesion or rolling, nor changes in capillary fine structure were observed 3 days postsurgery, when limb use was limited. However, leukocyte rolling and adhesion almost trebled by 7 days (P < 0.001), when normal gait was largely restored. Capillary fine structure was disturbed over a similar time course, e.g. relative endothelial volume (control 46%, 7 days 61%; P < 0.05), that resolved by 5 weeks. Where activity was increased by mild electrical stimulation 3 days after ligation muscles showed enhanced capillary swelling (endothelial volume 66% versus 50%, P < 0.005), but improved fatigue index (52% versus 16%, P < 0.001) as a result of greater blood flow. Muscle fatigue after ligation was related to the extent of contraction-induced hyperaemia (R(2) = 0.725), but not capillary swelling. Amiloride, and to a lesser extent allopurinol but not vitamin E, significantly decreased leukocyte rolling and adhesion, as well as capillary endothelial swelling. We conclude that increased activity of ischaemic muscles on recovery is likely to accentuate acidosis accompanying changes in microcirculation and contribute to enhanced muscle fatigue, whereas formation of oxygen free radicals may be attenuated by endogenous protective mechanisms.

Microcirculation in rat soleus muscle after eccentric exercise: the effect of nifedipine.

This paper explores the role of the calcium entry blocker nifedipine in the explanation of eccentric exercise-induced fibre damage by changes in skeletal muscle microcirculation. Eccentric exercise (EE) was induced by indirect stimulation of rat soleus muscle in its lengthening phase during cycling. Muscle damage was assessed by histology, electron microscopy and muscle tension 48 h later. Diameters of arterioles and venules, their response to dilator and constrictor stimuli and pattern of capillary flow were measured in epiiluminated muscles using intravital microscopy. Tetanic tension developed by EE muscles was lower (8.60 +/- 1.02, means +/- SEM, n = 8 N g(-1) wet weight compared to 12.25 +/- 0.56 in controls, P < 0.01). Electron microscopy showed changes similar to those in muscles exposed to EE by downhill running (Z line streaming, disruption of sarcolemma, swollen tubules). A total of 16% of muscle fibres were damaged, and fibre areas and interstitial space were enlarged. Capillary red blood cell flow showed tendency to a greater intermittency. Large venules were narrower, but arterioles and smaller venules had diameters similar to control muscles. Vessel dilatation to topically applied 10(-4) M adenosine was attenuated. Daily administration of calcium entry blocker nifedipine by gavage (2 mg/kg/day in two equal doses) removed the narrowing of venules, restored the dilator response of all vessels to adenosine and increased capillary:fibre ratio. The percentage of damaged fibres decreased to 4.7 and the size of the interstitial space and fibre areas was normalized. Thus muscle damage caused by eccentric exercise was attenuated by nifedipine due to its beneficial effect on muscle microcirculation, which was impaired by eccentric exercise.

Angiogenesis in ischaemic and hypertrophic hearts induced by long-term bradycardia.

Angiogenesis and improved left ventricular function as a consequence of long-term bradycardia were first demonstrated in normal hearts, either electrically paced (rabbits, pigs) or treated with a selective sinus blocking drug alinidine (rats). Here we review the evidence that chronic heart rate reduction can have similar effects in the heart with compromised vascular supply, due to either hypertensive or haemodynamic overload hypertrophy (rats, rabbits) or ischaemic damage (rats, rabbits, pigs). Bradycardia induced over several weeks increased capillarity in all hypertrophied hearts, and in border and remote left ventricular myocardium of infarcted hearts. In some, but not all cases, coronary blood flow was improved by heart rate reduction, suggesting enlargement of the resistance vasculature in some circumstances. Cardiac or left ventricular function indices, which were depressed by hypertrophy or ischaemic damage, were preserved or even enhanced by chronic heart rate reduction. The expansion of the capillary bed in the vascularly compromised heart induced by bradycardia may be stimulated by mechanical stretch of the endothelium and/or VEGF activated by chamber dilation and myocyte stretch. The increased number of capillaries and more homogeneous distribution of capillary perfusion would support the better pump function, even in the absence of higher coronary flow. The beneficial impact of chronic heart rate reduction on myocardial angiogenesis and function in cardiac hypertrophy and infarction may be major factor in the success of beta-blockers in treatment of human heart failure.

A new model of peripheral arterial disease: sustained impairment of nutritive microcirculation and its recovery by chronic electrical stimulation.

OBJECTIVES: To develop a model of peripheral arterial disease (PAD) in rat skeletal muscle with sustained impairment of microcirculatory perfusion, and to ascertain whether increased muscle activity can reverse the impairment. METHODS: Three weeks after iliac ligation in rats, the ipsilateral femoral artery was ligated (double ligation, DL), and in some animals, muscle activity was increased by electrical stimulation for 2 weeks (10 Hz, 15 min on, 85 mins off, 7 times per day). Diameter changes of precapillary arterioles to vasoactive agonists and capillary perfusion (flow intermittency, capillary red cell velocity [V(rbc)], and diameters) were measured in extensor digitorum longus muscle and compared with 5 weeks iliac only ligation (single ligation, SL) and controls. Total muscle endothelial nitric oxide synthase (eNOS) was estimated by Western blotting. RESULTS: Whereas single ligation increased intermittency of capillary flow with little effect on V(rbc) and shear stress, DL completely eliminated increases in V(rbc) and shear stress after muscle contractions. Arterial dilation to sodium nitroprusside was attenuated similarly in SL and DL; in SL, acetylcholine induced constriction and bradykinin an attenuated dilation, but in DL vessels were unresponsive to either. Chronic stimulation returned all microcirculatory parameters in DL to normal and increased levels of eNOS protein by 75%. CONCLUSIONS: Femoral artery ligation following iliac ligation impairs arteriolar vasodilator capacity, capillary perfusion, and shear-dependent function of microcirculatory endothelium more than iliac ligation alone and is more representative of long-standing ischemia in PAD. Chronic intermittent electrical stimulation can normalize these derangements.

Arteriolar endothelial dysfunction is restored in ischaemic muscles by chronic electrical stimulation.

Chronic intermittent electrical stimulation (15 min on, 85 min off, seven times per day) eliminated endothelial dysfunction of pre-capillary arterioles in ischaemic rat ankle flexor muscles. Responses to acetylcholine were restored from constriction to dilation, and the reduced dilation to bradykinin was corrected by 1 week of stimulation. Administration of the NOS inhibitor N(omega)-nitro-L-arginine for 1 week impaired arteriolar reactivity in a similar way to ischaemia, and dilator function was likewise restored by chronic stimulation. This suggests that nitric oxide production in the microcirculation is depressed by chronic ischaemia and that chronic electrical stimulation can specifically reverse this deficit. Stimulation applied to ischaemic muscles for 2 weeks also increased the numbers of microvessels immunostained for alpha-smooth muscle actin and the numbers of eNOS-positive microvessels and capillaries. These findings help to elucidate the mechanism of the beneficial effect of exercise in the treatment of peripheral vascular diseases by showing that muscle activity can improve both function and structural capacity of the microvasculature.

Chronic transcutaneous electrical stimulation of calf muscles improves functional capacity without inducing systemic inflammation in claudicants.

OBJECTIVES: To assess whether electrical stimulation of ischaemic calf muscles in claudicants causes a systemic inflammatory response and to evaluate effects of its chronic application on muscle function and walking ability. DESIGN: Prospective randomised controlled trial of calf muscle stimulation. MATERIALS AND METHODS: Stable claudicants were randomised to receive either active chronic low frequency (6 Hz) motor stimulation (n=15) or, as a control treatment, submotor transcutaneous electrical nerve (TENS) stimulation (n=15) of calf muscles in one leg, 3 x 20 min per day for four weeks. Leucocyte activation was quantified by changes in cell morphology, vascular permeability by urinary albumin:creatinine ratio (ACR), calf muscle function by isometric twitch contractions and walking ability by treadmill performance pre- and post-intervention. RESULTS: Acute active muscle stimulation activated leucocytes less (28% increase) than a standard treadmill test (81% increase) and did not increase ACR. Chronic calf muscle stimulation significantly increased pain-free walking distance by 35 m (95% CI 17, 52, P<0.001) and maximum walking distance by 39 m (95% CI 7, 70, P<0.05) while control treatment had no effect. Active stimulation prevented fatigue of calf muscles during isometric electrically evoked contractions by abolishing the slowing of relaxation that was responsible for loss of force. CONCLUSIONS: Chronic electrical muscle stimulation is an effective treatment for alleviating intermittent claudication which, by targeted activation of a small muscle mass, does not engender a significant systemic inflammatory response.

Modulation of physiological angiogenesis in skeletal muscle by mechanical forces: involvement of VEGF and metalloproteinases.

Growth factors are involved in physiological angiogenesis in female reproductive organs but their role in capillary growth in skeletal muscles during activity or exercise training is not proven. Evidence suggests that increases in muscle blood flow and accompanying capillary shear stress and/or wall tension, or mechanical stress due to sarcomere length changes during contraction/relaxation cycles are closely linked with angiogenesis. Time-dependent studies of rat muscles in models with increased shear stress (chronic vasodilator treatment with alpha(1) antagonist prazosin), altered sarcomere length (stretch-induced overload with no increase in blood flow), or both (chronic electrical muscle stimulation) showed a similar increase in capillary supply in all models but by different modes of growth. With prazosin, it occurred by intra-luminal splitting of vessels, with stretch by abluminal sprouting, and in stimulated muscles by both methods. Whole muscle matrix metalloproteinase-2 (MMP-2) was elevated during sprouting growth induced by extravascular tensile forces but not during splitting growth induced by shear. Vascular endothelial growth factor (VEGF) protein was elevated at capillary sites in all three models but with different time courses. With shear as the stimulus, the increase occurred early although there was little capillary proliferation; it matched the rise in proliferation in stretched muscles but lagged behind proliferation in stimulated muscles. Mechanical forces therefore influence MMP and VEGF expression and capillary growth patterns in skeletal muscle differentially depending upon whether they act intra- or ab-luminally. In exercise-trained muscles, the type of capillary growth remains to be determined but the most likely stimuli for angiogenesis are increased blood flow and shear forces to vessel supplying the active fibres, probably linked with metabolic factors.

Differences in local environment determine the site of physiological angiogenesis in rat skeletal muscle.

The specificity in location of angiogenesis to either glycolytic or oxidative fibre types, or muscle regions, was examined in the tibialis anterior (TA) and extensor digitorum longus (EDL) muscles of rat. Angiogenesis was induced by mechanical means either with (chronic muscle stimulation) or without (muscle stretch by overload) changes in blood flow, treatments which invoked only minor changes in fibre type and fibre size. Proliferation estimated by PCNA labelling of cells co-localised with capillaries was very rare in control muscles, where it occurred mainly in the glycolytic regions, but was increased in both models of angiogenesis. However, when labelled capillaries were scored according to the type of surrounding fibres, only muscle stimulation significantly accentuated proliferation of capillaries surrounded by glycolytic fibres. We conclude that while mechanical stimuli are important for proliferation in glycolytic regions in both models, capillary growth occurs specifically around glycolytic fibres in that region when the angiogenic stimulus includes increased blood flow and/or increased metabolic demand.

Remodeling in the microcirculation of rat skeletal muscle during chronic ischemia.

OBJECTIVE: To establish the time course and extent of remodeling of terminal microcirculation in ischemic rat skeletal muscle during prolonged low flow that does not lead to inflammation. METHODS: One common iliac artery was ligated via laparotomy in adult Sprague-Dawley rats and extensor digitorum longus (EDL) muscles removed at intervals (1, 2, and 5 weeks) postsurgery. Serial frozen EDL sections were stained to show capillaries (alkaline phosphatase), cell proliferation (antibody to proliferating cell nuclear antigen [PCNA]), terminal microvessels (antibodies to alpha-smooth muscle actin (alpha-SMA) or endothelial nitric oxide synthase [eNOS]), and macrophages (antibodies to infiltrating and resident macrophages). Total muscle eNOS protein was quantified by standard Western blotting techniques. RESULTS: Capillary proliferation was very limited in ischemic EDLs, with a modest 12% increase in the capillary/fiber ratio after 5 weeks, preceded at 2 weeks by increased numbers of PCNA-positive nuclei at capillary sites. There was no muscle necrosis or evidence of inflammation, based on macrophage staining. The number of terminal microvessels that were positive for alpha-SMA and <10 microm in diameter was fewer in ischemic EDLs at all time points, whereas the number of larger positive vessels was unchanged. eNOS-positive vessels <10 microm in diameter were stained similarly throughout ischemic muscles as the controls, and showed a similar increase in vessel/fiber ratio as the capillaries. The total eNOS protein level was similar to that in controls in ischemic EDLs after 1 and 2 weeks, but was 28% lower after 5 weeks. CONCLUSIONS: Prolonged, moderate flow reduction to skeletal muscles does not necessarily lead to inflammation or extensive capillary growth. Based on eNOS staining, the terminal microcirculation remains intact, but the loss of alpha-SMA immunoreactivity may indicate remodeling involving the "deinvestment" of microvessels by smooth muscle.

Hypoxia and expression of VEGF-A protein in relation to capillary growth in electrically stimulated rat and rabbit skeletal muscles.

To investigate the role of hypoxia as a stimulus to the early upregulation of vascular endothelial growth factor (VEGF) in fast skeletal muscles during chronic low frequency stimulation, blood flow, oxygen consumption, VEGF expression and capillary : fibre ratio were measured in chronically stimulated tibialis anterior and extensor digitorum longus (EDL) muscles in rabbits and rats. No differences were found in blood flow, oxygen consumption and extraction between rabbit muscles stimulated for 2 or 4 days (8 h on-16 h off) and controls. Muscle P(O(2)) polarographically measured immediately at the end of stimulation on day 2 was also no different from control under resting conditions (10.7 +/- 1.6 vs. 9.5 +/- 1.2 Torr, n.s.). Unlike control muscles, however, P(O(2)) in 2 day stimulated muscles did not increase immediately after a further acute bout of contractions. This difference was not apparent after similar acute contractions in 4 day stimulated muscles. The involvement of VEGF in early angiogenesis in stimulated muscles was studied in serial cryosections of rat EDL. The proportion of capillaries positively immunostained for VEGF increased from 25 +/- 1 % to 40 +/- 1 % (P < 0.05) in muscles removed on day 2 immediately at the end of chronic stimulation; it decreased slightly after 16 h rest, and increased again after 4 days of stimulation. Capillary : fibre ratio was unchanged throughout the experiment. Capillary cell proliferation increased only after the rest period on day 2 (20-fold increase) and day 4 (12-fold increase), indicating angiogenesis in progress. Thus the timing of transient hypoxia and increase in capillary-linked VEGF in stimulated muscles, albeit in different species, was similar, and increased VEGF staining and capillary cell proliferation occurred even after the hypoxia had resolved. This suggests (1) a connection between hypoxia and VEGF during the early stages of stimulation, although ensuing capillary proliferation may thereafter rapidly correct for local hypoxia, and (2) that the subsequent angiogenesis and VEGF expression are dependent on factors other than hypoxia.

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). METHODS: Adult rats received prazosin in drinking water ( approximately 2 mg per day) or had extensor digitorum longus (EDL) muscles stimulated by implanted electrodes for up to 14 days. At intervals, serial frozen sections of EDL were stained for alkaline phosphatase to identify capillaries, proliferating cell nuclear antigen (PCNA), and VEGF-A protein. Shear stress was estimated from capillary red blood cell velocities and diameters, measured by direct observation of epi-illuminated EDL. RESULTS: Chronic stimulation and prazosin treatment both increased capillary: fiber ratio by approximately 40% after 14 days. In stimulated muscles, the percentage of capillaries positively stained for VEGF increased within 3 to 4 days, while the density of PCNA-positive capillaries had increased 20-fold after 2 days. With prazosin, VEGF-positive capillaries increased after 2 and 4 days, accompanied by a threefold increase in PCNA. By 14 days, PCNA labeling and VEGF were still high in stimulated muscles, but no longer different from controls with prazosin. After 3 to 4 days of treatment, capillary shear stress in resting muscle was 57% higher than in controls as a result of stimulation, but 4 times higher with prazosin. CONCLUSIONS: Higher capillary shear stress with prazosin than with stimulation may upregulate VEGF expression in the early stages of treatment. Greater proliferation of capillaries preceding a higher proportion of VEGF-positive capillaries in stimulated muscles, in the presence of a modest increase in shear stress, suggests that angiogenesis was initiated by other factors in addition to shear stress.

Growth of arterioles precedes that of capillaries in stretch-induced angiogenesis in skeletal muscle.

Arteriolar growth accompanying capillary angiogenesis has been linked with hemodynamic factors resulting from increased blood flow. Here we describe the growth of arterioles occurring in rat skeletal muscles stretched by an overload due to the removal of agonist muscles, where blood flow was not increased, and we provide morphological evidence for the type of cells involved in this growth. Rat extensor digitorum longus (EDL) and extensor hallucis proprius (EHP) were overloaded by unilateral extirpation of their agonist, tibialis anterior. EDL muscles were taken for immunohistochemistry in cryostat sections to mark endothelial cells (Griffonia simplicifolia I, GSI lectin), smooth muscle cells and pericytes (alpha smooth muscle actin, alphaSMA), and "mature" arterioles (smooth muscle myosin heavy chains). EHP muscles were used for corresponding evaluation by confocal and electron microscopy. The number of capillaries surrounding muscle fibers was not significantly different after 1 week of stretch but was higher after 2 weeks (5.15 +/- 0.2 vs 4.3 +/- 0.2 in controls, P < 0.05). Similarly, capillary density (CD) and capillary/fiber ratio (C/F) gradually increased (CD 778 +/- 86 at 2 weeks vs 593 +/- 35 mm(-2) in controls, C/F 2.07 +/- 0.13 vs 1.38 +/- 0.06, respectively). In contrast, the number of alphaSMA-positive vessels around fibers increased after 1 week (2.16 +/- 0.09 vs 0.25 +/- 0.02 in controls) and was lower after 2 weeks (1.42 +/- 0.24, P < 0.05, vs 1 week). Arteriolar density was higher at 1 (110.9 +/- 7.5 mm(-2)) and 2 weeks (70.7 +/- 12.1) with respect to controls (31.0 +/- 1.6 mm(-2)). The increased density was greater in alphaSMA-positive vessels <10 microm in diameter (controls 18.0 +/- 1.04, 1 week 77.2 +/- 4.5, 2 wk 42.2 +/- 9.0 mm(-2)) than in vessels >10 microm (13.0 +/- 0.8, 33.7 +/- 4.0, 29.5 +/- 4.7 mm(-2)). Electron microscopy showed "activated" (TEM fine structure) and proliferating (immunogold labeling for BrdU) fibroblasts in the vicinity of capillaries, some of which were embedded in the capillary basement membrane, consistent with a transformation into pericytes and possibly later smooth muscle cells. Confocal microscopy indicated that some mesenchymal cells became GSI positive and formed extended processes which contacted capillaries via tapered endings. Growth of arterioles in stretched muscles appears to involve proliferation of fibroblasts, which may migrate toward capillaries and precedes any apparent increase in capillarization.

Alterations in reactivity of small arterioles in rat skeletal muscle as a result of chronic ischaemia.

In a model of chronic hind limb ischaemia, we examined whether impaired muscle blood flow, particularly during exercise, is partly due to modification of the reactivity of skeletal muscle resistance vessels by prolonged low blood flow. Two or 5 weeks after unilateral iliac artery ligation, terminal (A4) and preterminal (A3) arterioles of extensor digitorum longus muscle were viewed by intravital microscopy using epi-illumination, and diameter changes to topical application of endothelium-dependent (bradykinin, acetylcholine) and endothelium-independent (adenosine, sodium nitroprusside and noradrenaline) agonists measured. Chronic ischaemia had no effect on resting diameters of A3 or A4 vessels. Two weeks after ligation, dilation to bradykinin was attenuated by 75% for A3 and 50% for A4 arterioles (p < 0.01 vs. control) and responses to acetylcholine were reversed from dilation to constriction (A3: control diameter change +29%, 2-week-ligated -17%; A4: control 18%, 2-week-ligated -13%). Five weeks after ligation, these effects were still apparent and, additionally, dilation to adenosine and sodium nitroprusside and constriction to noradrenaline were reduced. Thus, impaired dilation, most likely due to endothelial dysfunction, is an early manifestation of altered reactivity in the microcirculation of chronically ischaemic muscles, with functional impairment of vascular smooth muscle as a later consequence. These changes occurred despite modest improvements in muscle blood flow and perfusion pressure over the same time. These changes will act to the detriment of blood flow in contracting muscles and could limit the outcome of interventions to restore flow such as angioplasty or surgical bypass.

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.

Selective long-term electrical stimulation of fast glycolytic fibres increases capillary supply but not oxidative enzyme activity in rat skeletal muscles.

Glycolytic fibres in rat extensor digitorum longus (EDL) and tibialis anterior (TA) were selectively activated, as demonstrated by glycogen depletion, by indirect electrical stimulation via electrodes implanted in the vicinity of the peroneal nerve using high frequency (40 Hz) trains (250 ms at 1 Hz) and low voltage (threshold of palpable contractions). This regime was applied 10 times per day, each bout being of 15 min duration with 60 min recovery, for 2 weeks. Cryostat sections of muscles were stained for alkaline phosphatase to depict capillaries, succinate dehydrogenase (SDH) to demonstrate oxidative fibres, and periodic acid-Schiff reagent (PAS) to verify glycogen depletion. Specific activity of hexokinase (HK), 6-phosphofructokinase, pyruvate kinase, glycogen phosphorylase and cytochrome c oxidase (COX) were estimated separately in homogenates of the EDL and the predominantly glycolytic cortex and oxidative core of the TA. Stimulation increased the activity of HK but not that of oxidative enzymes in fast muscles. Comparison of changes in oxidative capacity and capillary supply showed a dissociation in the predominantly glycolytic TA cortex. Here, COX was 3.9+/-0.68 microM min(-1) (g wet wt)-1 in stimulated muscles compared with 3.7+/-0.52 microM min(-1) (g wet wt)-1 in contralateral muscles (difference not significant), while the percentage of oxidative fibres (those positively stained for SDH) was also similar in stimulated (14.0+/-2.8 %) and contralateral (12.2 +/-1.9 %) muscles. In contrast, the capillary to fibre ratio was significantly increased (2.01+/-0.12 vs. 1.55+/-0.04, P<0.01). We conclude that capillary supply can be increased independently of oxidative capacity, possibly due to haemodynamic factors, and serves metabolite removal to a greater extent than substrate delivery.

Matrix metalloproteinase activity is required for activity-induced angiogenesis in rat skeletal muscle.

Proteolysis of the capillary basement membrane is a hallmark of inflammation-mediated angiogenesis, but it is undetermined whether proteolysis plays a critical role in the process of activity-induced angiogenesis. Matrix metalloproteinases (MMPs) constitute the major class of proteases responsible for degradation of basement membrane proteins. We observed significant elevations of mRNA and protein levels of both MMP-2 and membrane type 1 (MT1)-MMP (2.9 +/- 0.7- and 1.5 +/- 0.1-fold above control, respectively) after 3 days of chronic electrical stimulation of rat skeletal muscle. Inhibition of MMP activity via the inhibitor GM-6001 prevented the growth of new capillaries as assessed by the capillary-to-fiber ratio (1.34 +/- 0.08 in GM-6001-treated muscles compared with 1.69 +/- 0.03 in control 7-day-stimulated muscles). This inhibition correlated with a significant reduction in the number of capillaries with observable breaks in the basement membrane, as assessed by electron microscopy (0.27 +/- 0.27% in GM-6001-treated muscles compared with 3.72 +/- 0.65% in control stimulated muscles). Proliferation of capillary-associated cells was significantly elevated by 2 days and remained elevated throughout 14 days of stimulation. Capillary-associated cell proliferation during muscle stimulation was not affected by MMP inhibition (80.3 +/- 9.3 nuclei in control and 63.5 +/- 8.5 nuclei in GM-6001-treated animals). We conclude that MMP proteolysis of capillary basement membrane proteins is a critical component of physiological angiogenesis, and we postulate that capillary-associated proliferation precedes and occurs independently of endothelial cell sprout formation.

The role of pericytes in controlling angiogenesis in vivo.

In order to evaluate the interaction between endothelial cells and the perivascular pericytes during physiological angiogenesis, stereological analysis of fine structure was performed on samples of rat skeletal muscle where capillary growth was induced to a similar extent by three different interventions (indirect electrical stimulation, vasodilatation by alpha 1-blockade, stretch due to synergist extirpation). There was a significant reduction in the relative area of contact between pericytes and the capillary abluminal surface with stimulation, and withdrawal of pericyte processes coincided with an increase in anatomical capillary supply. These data indicate that pericytes may play an anti-angiogenic role in vivo in normal adult tissue similar to that proposed for in vitro models of angiogenesis, with their retraction during increased muscle activity possibly releasing endothelial cells from their contact inhibition. However, following long-term peripheral vasodilatation expansion of the capillary bed was accompanied by a co-ordinated increase in pericytes, such that coverage of capillaries was similar to that in control muscles. In addition, growth of capillaries following prolonged stretch resulted in a slightly greater increase in the pericyte population, suggesting they may be permissive for endothelial cell migration. Thus, the role of pericytes in controlling physiological angiogenesis is dependent on the nature of the initial stimulus, suggesting that in vitro data have to be interpreted with caution when discussing the mechanism of capillary growth in vivo.

Effect of indomethacin on capillary growth and microvasculature in chronically stimulated rat skeletal muscles.

1. Capillary proliferation and microvessel diameters were studied in rat ankle flexors subjected to chronic electrical stimulation by implanted electrodes (10 Hz, 0.3 ms pulse width, up to 6 V, 8 h day-1) for 2 or 7 days with or without concurrent indomethacin treatment ( approximately 2 mg day-1 in drinking water) to study the role of prostaglandins in the microcirculation in relation to capillary growth. 2. Diameters of terminal arterioles, capillaries and confluent venules were measured in epi-illuminated muscles, together with capillary red cell velocity, to evaluate whether changes in capillary pressure and/or shear stress participate in capillary growth via release of prostaglandins. 3. Cell proliferation was detected following bromodeoxyuridine (BrdU) incorporation and immuno-staining of frozen sections. Labelling was assessed as the percentage of all interstitial nuclei (Haematoxylin-stained) that were BrdU positive. By comparison with serial sections stained for alkaline phosphatase, from which the capillary-to-fibre ratio (C:F) was obtained, labelling was derived for nuclei colocalised either to capillaries or to other non-capillary interstitial cells. 4. C:F increased to 1.89 +/- 0.06 from 1.47 +/- 0.04 in controls only after 7 days stimulation; indomethacin reduced this to 1.55 +/- 0.07. Capillary labelling increased from 2.9 +/- 0.5 % in controls to 11.3 +/- 2.2 % after 2 days stimulation and 10.6 +/- 0.8 % after 7 days. The increase was attenuated by indomethacin at both time points (to 5.8 +/- 1.6 % and 4.2 +/- 0.5 %, respectively). 5. Non-capillary interstitial labelling (2.0 +/- 0.4 % in controls) increased to 9.5 +/- 2.7 % after 2 days stimulation and was back to normal after 7 days (3.2 +/- 0.7 %). Indomethacin depressed the increase at 2 days to 4.0 +/- 1.3 % and had no effect at 7 days (2.9 +/- 0.13 %). Labelling in sham-operated rats with or without indomethacin or in vehicle-treated animals was no different from controls. 6. Arteriolar and venular diameters were increased by 2 days of stimulation but unchanged after 7 days. Indomethacin increased diameters of arterioles after 2 days and venules after 7 days in sham-operated animals, but had no effect on diameters of either vessel type in stimulated muscles. 7. Capillary diameters did not change during acute muscle contractions whereas red cell velocity did. Calculated shear stress in capillaries was thereby increased by 75 %. 8. Thus during chronic electrical stimulation both capillary growth and the cell proliferation that precedes it were attenuated by indomethacin. Transient stimulation-induced increases in arteriolar and venular diameters, which were unaffected by indomethacin, do not implicate increased capillary pressure as a factor in prostaglandin release and capillary growth. Estimations of increases in capillary shear stress during muscle contractions and of a 45 % higher value even at rest after chronic stimulation for 7 days suggest that shear stress is a more likely stimulus for prostaglandin release in chronically stimulated muscles.

Inhibition of capillary growth in chronically stimulated rat muscles by N(G)-nitro-l-arginine, nitric oxide synthase inhibitor.

Shear stress causes release of nitric oxide (NO) from microvascular endothelial cells in vivo and stimulates their growth in vitro. After chronic electrical stimulation of lower hind limb skeletal muscles in the rat, measurements of capillary diameters and red blood cell velocity indicated that shear stress is increased in these vessels as a potential source of NO. This study therefore investigated whether NO is involved in capillary growth in stimulated muscles. Control rats or those stimulated for 2 or 7 days were treated with the NO synthase inhibitor, N(G)-nitro-l-arginine (l-NNA, 10 mg.day(-1) in drinking water), or water alone. After bromodeoxyuridine (BrdU) administration, extensor digitorum longus muscles were removed and frozen. Capillary supply was assessed in cryostat sections as capillary:fiber (C:F) ratio after staining for alkaline phosphatase; proliferation of capillary-linked and interstitial nuclei was evaluated by immunostaining for BrdU incorporation. C:F was not increased after 2 days of stimulation but the increase after 7 days (1.88 +/- 0.50 vs control 1.45 +/- 0.04, P < 0.001) was abolished by l-NNA (1.55 +/- 0.04, NS). The labeling index for BrdU-positive nuclei colocalized with capillaries as a percentage of total interstitial nuclei increased in muscles stimulated for 2 days (11.3 +/- 2.2%) and 7 days (10.6 +/- 0.8%) compared with controls (2.9 +/- 0.5%, P < 0.01) and was eliminated by l-NNA at both time points (3.1 +/- 0.6 and 1.0 +/- 0.6%, respectively; both P < 0.05 vs stimulated). A transient increase in BrdU labeling of interstitial nuclei not associated with capillaries (possibly fibroblasts) after 2 but not 7 days stimulation was eliminated by l-NNA treatment. These results suggest that NO is involved in capillary growth in chronically stimulated muscles possibly via its shear-stress-induced release from capillaries or from interstitial fibroblasts.

Arteriolar reactivity and capillarization in chronically stimulated rat limb skeletal muscle post-MI.

The purpose of this study was to assess whether electrical stimulation-induced increases in muscular activity could improve capillary supply and correct previously documented abnormal vasodilator and vasoconstrictor responses of arterioles in limb skeletal muscle post-myocardial infarction (MI). Extensor digitorum longus (EDL) muscle from rats with surgically induced MI ( approximately 30% of the left ventricle) was chronically stimulated (Stim) 8 h/day for 6 +/- 1 days, at 11 wk post-MI. Third- (3A) and fourth-order (4A) arterioles in EDL from nine MI rats and four MI+Stim rats were compared with those of 11 controls (Con). Compared with Con rats, MI alone caused a reduction in the resting diameter of 3A and 4A arterioles, which was completely reversed by MI+Stim. However, Stim did not correct the attenuated vasodilator response to 10(-4) M adenosine seen in 4A arterioles from MI rats compared with Con. The constrictor response of both 3A and 4A vessels in MI rats to low doses of acetylcholine (10(-9) M, 10(-8) M) and norepinephrine (10(-9) M) was accentuated in MI+Stim. The proportion of oxidative fibers in EDL was unaffected by MI or MI+Stim combination. However, Stim significantly increased (P < 0.05) the capillary-to-fiber ratio in this muscle compared with Con. Thus, although the increase in muscle activity induced by chronic electrical stimulation normalized the reduction in resting vessel diameter seen after MI, it failed to correct the abnormalities in vasoreactivity of these same vessels.