Biomedical subjects

O Hudlicka

Publications and source records attributed to O Hudlicka.

At least 19 recordsLinked to original sources

PubMed2006

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.

Adenosine↗

PubMed2005

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.

Animals↗

PubMed2005

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.

Animals↗

PubMed2004

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.

Actins↗

PubMed2004

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.

Aged↗

PubMed2003

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.

Animals↗

PubMed2003

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.

Actins↗

PubMed2002

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.

Animals↗

PubMed2001

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.

Animals↗

PubMed2001

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.

Alkaline Phosphatase↗

PubMed2000

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.

Animals↗

PubMed1998

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 microm 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.

Animals↗

PubMed1998

Alterations in small arterioles precede changes in limb skeletal muscle after myocardial infarction.

We tested the hypothesis that alterations in arterioles in locomotor skeletal muscles in rats with myocardial infarction (MI), but before development of congestive heart failure (CHF), precede structural and functional changes commonly observed in limb muscle in association with CHF. Resting diameters of third- (A3) and fourth-order arterioles (A4) in extensor digitorum longus (EDL) muscle were significantly smaller in rats with nonfailing small and medium-sized MI compared with control animals. Dilation of A4 in response to 10(-4) M adenosine was significantly attenuated in both groups (P < 0.05), whereas dilation of A3 was unaltered. Microvessels from both groups of infarcted rats constricted to all doses of acetylcholine (10(-9), 10(-8), and 10(-7) M) and showed a significantly exaggerated vasoconstrictor response to norepinephrine (10(-9), 10(-8), and 10(-7) M) compared with microvessels in control rats (P < 0.05). Peak isometric tension of combined tibialis anterior and EDL muscles and muscle fatigue (final/peak tension x 100), measured during 5-min isometric supramaximal twitch contractions at 4 Hz, were similar in control and MI rats (218 +/- 7 vs. 213 +/- 15 g/g muscle and 52 +/- 1 vs. 51 +/- 9%, respectively; n = 5 for both). There was also no difference with respect to the proportion of oxidative fibers or capillary-to-fiber ratios. Our results indicate that, in rats with left ventricular dysfunction but without failure, decreased diameter and perturbations in reactivity of small arterioles precede alterations in skeletal muscle performance often seen at a later date in association with CHF. These findings are consistent with the notion of aberrant endothelial and smooth muscle function and may contribute to the maintenance of blood pressure after MI but before CHF.

Acetylcholine↗

PubMed1998

Is physiological angiogenesis in skeletal muscle regulated by changes in microcirculation?

Physiological angiogenesis occurs in female reproductive organs, in growing antlers as a result of long-term exposure to cold and possibly hypoxia, and due to increased activity (training) in skeletal and cardiac muscle. The common denominator is increased blood flow, which may result in increased velocity of flow and/or diameters in arterioles and capillaries, increased capillary pressure and increased capillary hematocrit. Increased velocity would lead to increased shear stress, while increased pressure and/or diameters would increase wall tension. Either of these factors may cause a disturbance of the endothelium on the luminal side of vessels. In addition, increased contractile activity during training could cause changes on the abluminal side (for example, modification of the capillary basement membrane or the extracellular matrix induced by stretch/relaxation). In order to elucidate the role of these individual factors in angiogenesis, microcirculation was studied in skeletal muscles which were exposed to: (a) increased activity by chronic electrical stimulations; (b) long-term increase in blood flow by various vasodilators; (c) long-term administration of CoCl2 to increase hematocrit; and (d) long-term stretch, achieved by removal of agonist muscles. Capillary growth, demonstrated as an increased capillary/fiber ratio, as determined by histochemical staining and by electron microscopy, occurred in (a), (b), and (d), but not (c). Capillary proliferation, estimated by labeling index for bromodeoxyuridine of capillary-linked nuclei, occurred in (a), but not in (b). Chronic electrical stimulation resulted in an increase in the diameter of capillaries, a transient widening of arterioles, and no change in venules. Capillary hematocrit and the velocity of red blood cells (Vrbc) were also increased. Calculated shear stress and capillary wall tension were higher in stimulated muscles than in control muscles. Long-term increase in blood flow, induced by administration of the alpha 1-blocker prazosin, caused increased Vrbc with no change in diameters and increased only capillary shear stress. Stretched muscles had decreased blood flow, but longer sarcomeres initially caused concomitant stretch of capillaries. Increased shear stress/wall tension/stretch may initiate angiogenesis by damaging the luminal side of endothelial cells and/or their basement membrane, or by releasing growth factors or other humoral agents (prostaglandins and/or nitric oxide). Immunohistochemistry in stimulated or stretched muscles showed no evidence for expression of mRNA for basic fibroblast growth factor (bFGF), or the growth factor itself, but a low molecular mass endothelial cell-stimulating angiogenic factor (ESAF) (77) was increased in (a), (b), and (d). The involvement of prostaglandins and nitric oxide was demonstrated by the finding of attenuated incorporation of BrdU into capillary-linked nuclei in stimulated muscles after administration of indomethacin or L-NNA. Thus, changes in the microcirculation leading to increased shear stress and/or capillary wall tension may stimulate proliferation of endothelial cells either directly, or by release of various humoral factors. However, extravascular mechanical factors have also to be taken into account.

Animals↗

PubMed1998

Growth of arterioles in chronically stimulated adult rat skeletal muscle.

OBJECTIVE: The purpose of this study was to test the hypothesis that capillary growth induced by chronic electrical stimulation of skeletal muscle is accompanied by the growth of small arterioles. METHODS: Lower limb flexor muscles of Sprague-Dawley rats were stimulated by electrodes implanted in the vicinity of the peroneal nerve at 10 Hz for 8 h/d for 2 and 7 days. Cryostat sections from the proximal, middle, and distal regions of the extensor digitorum longus muscle (EDL) were fluorescently immunolabeled with alpha-smooth muscle actin (alpha SMA) and myosin heavy chain (MHC) to identify mature (alpha SMA and MHC-positive) and immature (alpha SMA-positive, MHC-negative) arterioles. The fluorescent derivative of the lectin Griffonia simplicifolia I (GSI) was used to identify all microvessels, including arterioles, capillaries, and venules. RESULTS: The number of vessels positive for GSI or alpha SMA surrounding muscle fibers was similar in all three muscle regions (proximal, middle, distal). The mean values +/- SEM for GSI-positive vessels from all regions were similar in control (4.3 +/- 0.07) and 2-day stimulated (4.7 +/- 0.08) but higher in 7-day stimulated muscles (6.7 +/- 0.1, p < 0.05), thus confirming the previous findings on capillary growth. A similar increase was found in the number of alpha SMA positive vessels < or = 10 microns outer diameter (1.3 +/- 0.09 versus 0.4 +/- 0.03 around muscle fibers in controls). The density of terminal arterioles (< or = 10 microns) was slightly but not significantly higher after 2 days of stimulation (19.5 +/- 4 versus 15.6 +/- 2 profiles/mm2 in control muscles) and significantly higher after 7 days (33 +/- 7). While a similar increase was observed in the density of preterminal arterioles > 10 microns (17 +/- 3 control, 22 +/- 3 at 2 days and 40 +/- 5 at 7 days), the density of MHC-positive vessels muscles stimulated for 7 days was unchanged. Seven-day stimulated muscle also had a fivefold higher density of microvessel profiles < or = 10 microns that were only partially surrounded by alpha SMA. This considerably exceeds the relative increase in the number of capillaries and thus supports the concept of arteriolar growth by transformation from capillaries. CONCLUSIONS: Chronic electrical stimulation results in an early increase in the number of immature (MHG-negative), but not mature (MHC-positive) arterioles, a process that accompanies the increase in capillarization. The great increase in the number of microvessels only partially covered by alpha SMA suggests arteriolization of capillaries as a contributing mechanism in this growth.

Actins↗

PubMed1997

Capillary red blood cell flow and activation of white blood cells in chronic muscle ischemia in the rat.

Increased activity of ischemic skeletal muscles in which functional hyperemia is impaired has been linked with capillary endothelial swelling postcapillary white blood cell (WBC) adherence. The perfusion pattern of capillaries under these conditions and time course of WBC activation is not known. Capillary microcirculation was studied by videomicroscopy at rest and after muscle contractions (1 Hz, 10 min) in extensor digi-torum longus muscles of pentobarbital sodium-anesthetized rat during the early stages of chronic ischemia (unilateral ligation of the common iliac artery for 3 days) and in ischemic muscles subjected to increased activity (7 days of ischemia or 3 days of ischemia plus indirect electrical stimulation via planted electrodes, 10 Hz, 7 x 10 min on-90 min off/day) to investigate how perfusion was affected. All ischemic muscles had more intermittently flowing capillaries than did unoperated control) muscles. Temporal heterogeneity of perfusion at rest, assessed by velocity, time spent stationary, and stop/start frequency of red blood cells, was similar to control values in ischemic muscles but greater in ischemic muscles subjected to additional activity. Hyperemic responses to contractions were severely blunted in all ischemic groups. The portion of morphologically nonspherical WBCs, taken to indicate activation, was 24 +/- 3% in venous blood after 3 days of ischemia vs. 14 +/- 1% in control muscles and increased further by 7 days (42 +/- 2%) when activated cells were also found in arterial blood. Thus increased muscular activity may exacerbate the adverse effects of ischemia on capillary perfusion, and WBC activation, evident before endothelial swelling is apparent, provides the potential as a circulating signal for capillary swelling in the ischemic and other muscles.

Animals↗

PubMed1996

In vivo pericyte-endothelial cell interaction during angiogenesis in adult cardiac and skeletal muscle.

Pericytes (PC) exert an inhibitory effect on endothelial cell (EC) proliferation in vitro and withdrawal of PC occurs prior to EC proliferation during pathological capillary growth in vivo. Using stereological analyses of PC and capillary EC fine structure, we have studied their relationship during the early stages of physiological angiogenesis using two in vivo models. In rat skeletal muscle where capillary growth was induced by indirect electrical stimulation, there was a reduction in the relative area of contact between pericytes and the capillary abluminal surface (22% vs 30% for normal controls; P < 0.05) and pericyte surface: volume ratio (12.5 vs 14.9 microns-1 for normal controls; P < 0.05) after 3 days of stimulation, at a time prior to the appearance of new capillaries. Further withdrawal of pericyte processes was evident at the point where an actual increase in capillary numbers was observed (7 days stimulation; PC surface: volume ratio = 10.4 microns-1), although the degree of PC-EC interdigitation increased. Similar changes, but to a lesser extent, were observed in both left ventricular myocardium and papillary muscles of pigs following 4-5 weeks chronic heart rate reduction. Although PC coverage of capillaries in the heart was found to be less than that in skeletal muscle, the relative contact area between PC and capillaries also showed a reduction in paced vs sham-operated control heart (papillary: 15% vs 20%; ventricle: 12% vs 16%; P < 0.05). Interdigitation of PC and EC was absent in cardiac muscle. These data suggest that retraction of PC may play a permissive role in controlling angiogenesis in vivo in normal adult tissue.

Animals↗

PubMed1996

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

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.

Age Factors↗