Enhanced KCl-mediated contractility and Ca2+ sensitization in porcine collateral-dependent coronary arteries persist after exercise training.

We have previously reported enhanced Ca2+ sensitivity of coronary arteries that is dependent upon collateral circulation for their blood supply. For the current study, we hypothesized that small collateral-dependent arteries would exhibit an enhanced KCl-mediated contractile response attributable to Ca2+ sensitization and increased Ca2+ channel current. Ameroid constrictors were surgically placed around the left circumflex (LCX) artery of female Yucatan miniature swine. Eight weeks postoperatively, pigs were randomized into sedentary or exercise-trained (treadmill run; 5 days/wk; 14 wk) groups. Small coronary arteries (150-300 µm luminal diameter) were isolated from myocardial regions distal to the collateral-dependent LCX and the nonoccluded left anterior descending arteries. Contractile tension and simultaneous measures of both tension and intracellular free Ca2+ levels (fura-2) were measured in response to increasing concentrations of KCl. In addition, whole cell Ca2+ currents were also obtained. Chronic occlusion enhanced contractile responses to KCl and increased Ca2+ sensitization in collateral-dependent compared with nonoccluded arteries of both sedentary and exercise-trained pigs. In contrast, smooth muscle cell Ca2+ channel current was not altered by occlusion or exercise training. Ca2+/calmodulin-dependent protein kinase II (CaMKII; inhibited by KN-93, 0.3-1 µM) contributed to the enhanced contractile response in collateral-dependent arteries of sedentary pigs, whereas both CaMKII and Rho-kinase (inhibited by hydroxyfasudil, 30 µM or Y27632, 10 µM) contributed to increased contraction in exercise-trained animals. Taken together, these data suggest that chronic occlusion leads to enhanced contractile responses to KCl in collateral-dependent coronary arteries via increased Ca2+ sensitization, a response that is further augmented with exercise training.NEW & NOTEWORTHY Small coronary arteries distal to chronic occlusion displayed enhanced contractile responses, which were further augmented after exercise training and attributable to enhanced calcium sensitization without alterations in calcium channel current. The calcium sensitization mediators Rho-kinase and CaMKII significantly contributed to enhanced contraction in collateral-dependent arteries of exercise-trained, but not sedentary, pigs. Exercise-enhanced contractile responses may increase resting arterial tone, creating an enhanced coronary flow reserve that is accessible during periods of increased metabolic demand.

Exercise training-induced adaptations in mediators of sustained endothelium-dependent coronary artery relaxation in a porcine model of ischemic heart disease.

OBJECTIVE: The aim of this study was to test the hypothesis that exercise training enhances sustained relaxation to persistent endothelium-dependent vasodilator exposure via increased nitric oxide contribution in small coronary arteries of control and ischemic hearts. METHODS: Yucatan swine were designated to a control group or a group in which an ameroid constrictor was placed around the proximal LCX. Subsequently, pigs from both groups were assigned to exercise (five days/week; 16 weeks) or SED regimens. Coronary arteries (~100-350 µm) were isolated from control pigs and from both nonoccluded and collateral-dependent regions of chronically-occluded hearts. RESULTS: In arteries from control pigs, training significantly enhanced relaxation responses to increasing concentrations of bradykinin (10(-10) -10(-7) M) and sustained relaxation to a single bradykinin concentration (30 nM), which were abolished by NOS inhibition. Training also significantly prolonged bradykinin-mediated relaxation in collateral-dependent arteries of occluded pigs, which was associated with more persistent increases in endothelial cellular Ca(2+) levels, and reversed with NOS inhibition. Protein levels for eNOS and p-eNOS-(Ser1179), but not caveolin-1, Hsp90, or Akt, were significantly increased with occlusion, independent of training state. CONCLUSIONS: Exercise training enhances sustained relaxation to endothelium-dependent agonist stimulation in small arteries of control and ischemic hearts by enhanced nitric oxide contribution and endothelial Ca(2+) responses.

Greater gains in strength and power with intraset rest intervals in hypertrophic training.

We sought to determine if hypertrophic training with intraset rest intervals (ISRs) produced greater gains in power compared with traditional rest (TRD) hypertrophic training. Twenty-two men (age 25 ± 5 years, height 179.71 ± 5.04 cm, weight 82.1 ± 10.6 kg, 6.5 ± 4.5 years of training) matched according to baseline characteristics were assigned to 12 weeks of training using TRD or ISR. Body composition, strength (1-repetition maximum [1RM] bench and squat), and power output (60% 1RM bench and squat, and vertical jump) were assessed at baseline, 4, 8, and 12 weeks. Determination of myosin heavy chain (MHC) percentage from the vastus lateralis was performed pretraining and posttraining. Body composition was analyzed by analysis of variance, whereas performance measures and MHC were analyzed by analysis of covariance with baseline values as the covariate. Data are presented as mean ± SD changes pre to post. The ISR produced greater power output in bench (TRD 32.8 ± 53.4 W; ISR 83.0 ± 49.9 W, p = 0.020) and vertical jump (TRD 91.6 ± 59.8 W; ISR 147.7 ± 52.0 W; p = 0.036) with squat power approaching significance (TRD 204.9 ± 70.2 W; ISR 282.1 ± 104.2 W; p = 0.053) after post hoc analysis (p < 0.10). The ISR produced greater gains in bench (TRD 9.1 ± 3.7 kg; ISR 15.1 ± 8.3 kg; p = 0.010) and squat (TRD 48.5 ± 17.4 kg; ISR 63.8 ± 12.0 kg; p = 0.002) strength. Both protocols produced significant gains in lean mass with no significant differences between groups (1.6 ± 2.1 kg; p = 0.869). The MHCIIx percentage decreased (-31.0 ± 24.5%; p = 0.001), whereas the MHCIIA percentage increased (28.9 ± 28.5%; p = 0.001) with no significant differences between groups. Results indicate that hypertrophy training with ISR produces greater gains in strength and power, with similar gains in lean mass and MHC alterations as TRD. The ISR may be best used in hypertrophic training for strength and power sports.

Exercise training-enhanced, endothelium-dependent dilation mediated by altered regulation of BK(Ca) channels in collateral-dependent porcine coronary arterioles.

OBJECTIVE: Test the hypothesis that exercise training increases the contribution of BK(Ca) channels to endothelium-mediated dilation in coronary arterioles from collateral-dependent myocardial regions of chronically occluded pig hearts and may function downstream of H2O2. METHODS: An ameroid constrictor was placed around the proximal left circumflex coronary artery to induce gradual occlusion in Yucatan miniature swine. Eight weeks postoperatively, pigs were randomly assigned to sedentary or exercise training (treadmill; 14 week) regimens. RESULTS: Exercise training significantly enhanced bradykinin-mediated dilation in collateral-dependent arterioles (~125 µm diameter) compared with sedentary pigs. The BK(Ca) -channel blocker, iberiotoxin alone or in combination with the H2O2 scavenger, polyethylene glycol catalase, reversed exercise training-enhanced dilation in collateral-dependent arterioles. Iberiotoxin-sensitive whole-cell K+ currents (i.e., BK(Ca)-channel currents) were not different between smooth muscle cells of nonoccluded and collateral-dependent arterioles of sedentary and exercise trained groups. CONCLUSIONS: These data provide evidence that BK(Ca)-channel activity contributes to exercise training-enhanced endothelium-dependent dilation in collateral-dependent coronary arterioles despite no change in smooth muscle BK(Ca)-channel current. Taken together, our findings suggest that a component of the bradykinin signaling pathway, which stimulates BK(Ca) channels, is enhanced by exercise training in collateral-dependent arterioles and suggest a potential role for H2O2 as the mediator.

Effect of exercise training on nitric oxide and superoxide/H2O2 signaling pathways in collateral-dependent porcine coronary arterioles.

Endothelial nitric oxide (NO) synthase (NOS) has been shown to contribute to enhanced vascular function after exercise training. Recent studies have revealed that relatively low concentrations of reactive oxygen species can contribute to endothelium-dependent vasodilation under physiological conditions. We tested the hypothesis that exercise training enhances endothelial function via endothelium-derived vasodilators, NO and superoxide/H(2)O(2), in the underlying setting of chronic coronary artery occlusion. An ameroid constrictor was placed around the proximal left circumflex coronary artery to induce gradual occlusion in Yucatan miniature swine. At 8 wk postoperatively, pigs were randomly assigned to sedentary (pen-confined) or exercise-training (treadmill-run: 5 days/wk for 14 wk) regimens. Exercise training significantly enhanced concentration-dependent, bradykinin-mediated dilation in cannulated collateral-dependent arterioles (∼130 µm diameter) compared with sedentary pigs. NOS inhibition reversed training-enhanced dilation at low bradykinin concentrations in collateral-dependent arterioles, although increased dilation persisted at higher bradykinin concentrations. Total and phosphorylated (Ser(1179)) endothelial NOS protein levels were significantly increased in arterioles from collateral-dependent compared with the nonoccluded region, independent of exercise. The H(2)O(2) scavenger polyethylene glycol-catalase abolished the training-enhanced bradykinin-mediated dilation in collateral-dependent arterioles; similar results were observed with the SOD inhibitor diethyldithiocarbamate. Fluorescence measures of bradykinin-stimulated H(2)O(2) levels were significantly increased by exercise training, independent of occlusion. The NADPH inhibitor apocynin significantly attenuated bradykinin-mediated dilation in arterioles of exercise-trained, but not sedentary, pigs and was associated with significantly increased protein levels of the NADPH subunit p67phox. These data provide evidence that, in addition to NO, the superoxide/H(2)O(2) signaling pathway significantly contributes to exercise training-enhanced endothelium-mediated dilation in collateral-dependent coronary arterioles.

Effects of exercise training on coronary collateralization and control of collateral resistance.

Coronary collateral vessels serve as a natural protective mechanism to provide coronary flow to ischemic myocardium secondary to critical coronary artery stenosis. The innate collateral circulation of the normal human heart is typically minimal and considerable variability occurs in extent of collateralization in coronary artery disease patients. A well-developed collateral circulation has been documented to exert protective effects upon myocardial perfusion, contractile function, infarct size, and electrocardiographic abnormalities. Thus therapeutic augmentation of collateral vessel development and/or functional adaptations in collateral and collateral-dependent arteries to reduce resistance into the ischemic myocardium represent a desirable goal in the management of coronary artery disease. Tremendous evidence has provided documentation for the therapeutic benefits of exercise training programs in patients with coronary artery disease (and collateralization); mechanisms that underlie these benefits are numerous and multifaceted, and currently under investigation in multiple laboratories worldwide. The role of enhanced collateralization as a major beneficial contributor has not been fully resolved. This topical review highlights literature that examines the effects of exercise training on collateralization in the diseased heart, as well as effects of exercise training on vascular endothelial and smooth muscle control of regional coronary tone in the collateralized heart. Future directions for research in this area involve further delineation of cellular/molecular mechanisms involved in effects of exercise training on collateralized myocardium, as well as development of novel therapies based on emerging concepts regarding exercise training and coronary artery disease.

Ca2+ sensitization and PKC contribute to exercise training-enhanced contractility in porcine collateral-dependent coronary arteries.

Exercise training enhances endothelium-dependent coronary vasodilatation, improving perfusion and contractile function of collateral-dependent myocardium. Paradoxically, studies from our laboratory have revealed increased Ca(2+)-dependent basal active tone in collateral-dependent arteries of exercise-trained pigs. In this study, we tested the hypothesis that exercise training enhances agonist-mediated contractile responses of collateral-dependent arteries by promoting Ca(2+) sensitization. Ameroid constrictors were surgically placed around the proximal left circumflex coronary (LCX) artery of female Yucatan miniature pigs. Eight weeks postoperatively, pigs were randomized into sedentary (pen confined) or exercise-training (treadmill run; 5 days/wk; 14 wk) groups. Arteries (∼150 µm luminal diameter) were isolated from the collateral-dependent and nonoccluded (left anterior descending artery supplied) myocardial regions, and measures of contractile tension or simultaneous tension and intracellular free Ca(2+) concentration levels (fura-2) were completed. Exercise training enhanced contractile responses to endothelin-1 in collateral-dependent compared with nonoccluded arteries, an effect that was more pronounced in the presence of nitric oxide synthase inhibition (N(ω)-nitro-l-arginine methyl ester; 100 µM). Contractile responses to endothelin-1 were not altered by coronary occlusion alone. Exercise training produced increased tension at comparable levels of intracellular free Ca(2+) concentration in collateral-dependent compared with nonoccluded arteries, indicative of exercise training-enhanced Ca(2+) sensitization. Inhibition of PKC (calphostin C; 1 µM), but not Rho-kinase (Y-27632, 10 µM; or hydroxyfasudil, 30 µM), abolished the training-enhanced endothelin-1-mediated contractile response. Exercise training also increased sensitivity to the PKC activator phorbol 12,13-dibutyrate in collateral-dependent compared with nonoccluded arteries. Taken together, these data reveal that exercise training enhances endothelin-1-mediated contractile responses in collateral-dependent coronary arteries likely via increased PKC-mediated Ca(2+) sensitization.

Effects of exercise training on cellular mechanisms of endothelial nitric oxide synthase regulation in coronary arteries after chronic occlusion.

Exercise training enhances agonist-mediated relaxation in both control and collateral-dependent coronary arteries of hearts subjected to chronic occlusion, an enhancement that is mediated in part by nitric oxide. The purpose of the present study was to elucidate exercise training-induced adaptations in specific cellular mechanisms involved in the regulation of endothelial nitric oxide synthase (eNOS) in coronary arteries of ischemic hearts. Ameroid constrictors were surgically placed around the proximal left circumflex coronary artery (LCX) of adult female Yucatan miniature swine. Eight weeks postoperatively, animals were randomized into sedentary (pen-confined) or exercise training (treadmill run; 5 days/wk; 14 wk) protocols. Coronary artery segments ( approximately 1.0 mm luminal diameter) were isolated from collateral-dependent (LCX) and control (nonoccluded left anterior descending) arteries 22 wk after ameroid placement. Endothelial cells were enzymatically dissociated, and intracellular Ca(2+) responses (fura 2) to bradykinin stimulation were studied. Immunofluorescence and laser scanning confocal microscopy were used to quantify endothelial cell eNOS and caveolin-1 cellular distribution under basal and bradykinin-stimulated conditions. Immunoblot analysis was used to determine eNOS, phosphorylated (p)-eNOS, protein kinase B (Akt), pAkt, and caveolin-1 protein levels. Bradykinin-stimulated nitrite plus nitrate (NOx; nitric oxide metabolites) levels were assessed via HPLC. Exercise training resulted in significantly enhanced bradykinin-mediated increases in endothelial Ca(2+) levels, NOx levels, and the distribution of eNOS-to-caveolin-1 ratio at the plasma membrane in endothelial cells of control and collateral-dependent arteries. Exercise training also significantly increased total eNOS and phosphorylated levels of eNOS (pSer(1179)) in collateral-dependent arteries. Total eNOS protein levels were also significantly increased in collateral-dependent arteries of sedentary animals. These data provide new insights into exercise training-induced adaptations in cellular mechanisms of nitric oxide regulation in collateral-dependent coronary arteries of chronically occluded hearts that contribute to enhanced nitric oxide production.

Neuropilin-1 is essential for enhanced VEGF(165)-mediated vasodilatation in collateral-dependent coronary arterioles of exercise-trained pigs.

BACKGROUND/AIMS: Exercise training enhances vasodilatation to vascular endothelial growth factor (VEGF(165)) in collateral-dependent coronary arterioles. Interaction of VEGF receptor 2 (VEGFR-2) and the non-tyrosine-kinase receptor, neuropilin-1 has been reported to potentiate VEGF(165)-mediated signaling. In the current study, we tested the hypotheses that neuropilin-1 mediates the exercise-enhanced VEGF(165)-mediated vasodilatation in collateral-dependent arterioles and that neuropilin-1 and/or VEGFR-2 protein levels are increased in these arterioles. METHODS: Ameroid occluders were surgically placed around the proximal left circumflex coronary artery of miniature swine. Eight weeks after surgery, the animals were randomized into sedentary or exercise training (treadmill run; 5 days/week; 14 weeks) protocols. Coronary arterioles (approximately 100 microm diameter) were isolated from both collateral-dependent and control (left anterior descending) myocardial regions and studied by in vitro videomicroscopy or frozen for immunoblot analysis. RESULTS: Exercise-enhanced VEGF(165)-mediated vasodilatation in collateral-dependent arterioles was reversed by inhibition of the VEGF(165)-neuropilin-1 interaction. VEGF(121), which does not interact with neuropilin-1, induced similar vasodilatation in arterioles from all treatment groups. Immunoblot revealed significantly elevated VEGFR-1, VEGFR-2 and neuropilin-1 protein levels in collateral-dependent arterioles of exercise-trained pigs. CONCLUSIONS: Neuropilin-1 plays a vital role in the exercise-enhanced VEGF(165)-mediated vasodilatation of collateral-dependent coronary arterioles and is associated with increased neuropilin-1 receptor protein levels.

Effects of gradual coronary artery occlusion and exercise training on gene expression in swine heart.

Gradual occlusion (O) of the swine left circumflex coronary artery (LCX) with an ameroid occluder results in complete O within 3 weeks, collateral vessel development, and compensatory hypertrophy. The purpose of this investigation was to determine the independent and combined effects of O and exercise training (E) on gene expression in the swine heart. Adult Yucatan miniature swine were assigned to one of the following groups (n=6-9/group): sedentary control (S), exercise-trained (E), sedentary swine subjected to LCX occlusion (SO), and exercise-trained swine with LCX occlusion (EO). Exercise consisted of progressive treadmill running conducted 5 d/wk for 16 weeks. Gene expression was studied in myocardium isolated from the collateral-dependent left ventricle free wall (LV) and the collateral-independent septum (SEP) by RNA blotting. E and O each stimulated cardiac hypertrophy independently (p<0.001) with no interaction. O but not E increased atrial natriuretic factor expression in the LV, but not in the SEP. E decreased the expression of beta-myosin heavy chain in the LV, but not in the SEP. E retarded the expression of collagen III mRNA in SEP; but not in the LV. Exercise training and coronary artery occlusion each stimulate cardiac hypertrophy independently and induce different patterns of gene expression.

Preclinical pharmacology of AL-12182, a new ocular hypotensive 11-oxa prostaglandin analog.

PURPOSE: The aim of this study was to determine selected in vivo ocular properties of AL-12182 (5,6-dihydro-4,5-didehydro-11-deoxy-11-oxa-16-(3-chlorophenoxy)-omega-tetranor-PGF(2alpha) isopropyl ester) and the in vitro profile of its free acid, AL-12180. METHODS: Previously documented radioligand binding and functional assays involving human ciliary muscle cells (h-CM), human trabecular meshwork (h-TM) and other cells, and porcine ocular arteries were utilized. For in vivo procedures, we utilized rabbits, cats, and nonhuman primates to measure hyperemia, pupil diameter, and intraocular pressure (IOP), respectively. RESULTS: AL-12180 exhibited the highest affinity for the FP-receptor (K(i) = 143 +/- 36 nM) and much lower affinity for DP-, EP(3)-, IP-, and TP-receptors, and for several nonprostanoid receptors, enzymes, neurotransmitter uptake sites, ion channels, and other regulatory sites. AL-12180 activated phospholipase C-mediated phosphoinositide hydrolysis (potency, EC(50) = 13.7-42.7 nM) through the FP-receptor in a variety of cells, such as h-CM, h-TM cells, human embryonic kidney cells expressing the cloned human ciliary body FP-receptor (HEK-FP), mouse 3T3 cells, and rat vascular smooth muscle cells. AL-8810, an FP-antagonist, blocked the effects of AL-12180 in h-CM cells (IC(50) = 8.7 microM). AL-12180 also stimulated the mobilization of intracellular Ca(2+) ([Ca(2+)](i)) in h-TM cells (EC(50) = 111 +/- 36 nM), h-CM cells (EC(50) = 11 nM), and in host cells expressing the cloned human ciliary body FP-receptor (EC(50) = 5.9 +/- 3.1 nM). AL-12180 lacked significant agonist activity at DP-, EP(2)-, EP(4)-, IP-, and TP-receptors in cell-based assays. However, AL-12180 contracted porcine central retinal and short posterior ciliary arteries in vitro with micromolar potencies that appeared to involve TP-receptor activation. in vivo, AL-12182 elicited dose-related hyperemia in the rabbit eye, miosis in the cat eye, and ocular hypotension in the nonhuman primate eye. CONCLUSIONS: AL-12180 is a relatively potent and selective FP-receptor agonist whose isopropyl ester prodrug (AL-12182) lowers IOP by as much as 40% following topical ocular dosing in a laser-induced nonhuman primate model of ocular hypertension.

Exercise training reverses downregulation of HSP70 and antioxidant enzymes in porcine skeletal muscle after chronic coronary artery occlusion.

Oxidative stress is associated with muscle fatigue and weakness in skeletal muscle of ischemic heart disease patients. Recently, it was found that endurance training elevates protective heat shock proteins (HSPs) and antioxidant enzymes in skeletal muscle in healthy subjects and antioxidant enzymes in heart failure patients. However, it is unknown whether coronary ischemia and mild infarct without heart failure contributes to impairment of stress proteins and whether exercise training reverses those effects. We tested the hypothesis that exercise training would reverse alterations in muscle TNF-alpha, oxidative stress, HSP70, SOD (Mn-SOD, Cu,Zn-SOD), glutathione peroxidase (GPX), and catalase (CAT) due to chronic coronary occlusion of the left circumflex (CCO). Yucatan swine were divided into three groups (n = 6 each): sedentary with CCO (SCO); 12 wk of treadmill exercise training following CCO (ECO); and sham surgery controls (sham). Forelimb muscle mass-to-body mass ratio decreased by 27% with SCO but recovered with ECO. Exercise training reduced muscle TNF-alpha and oxidative stress (4-hydroxynonenal adducts) caused by CCO. HSP70 levels decreased with CCO (-45%), but were higher with exercise training (+348%). Mn-SOD activity, Mn-SOD protein expression, and Cu,Zn-SOD activity levels were higher in ECO than SCO by 72, 82, and 112%, respectively. GPX activity was 177% greater in ECO than in SCO. CAT trended higher (P = 0.059) in ECO compared with SCO. These data indicate that exercise training following onset of coronary artery occlusion results in recovery of critical stress proteins and reduces oxidative stress.

Transcription factor Ets-1 regulates fibroblast growth factor-1-mediated angiogenesis in vivo: role of Ets-1 in the regulation of the PI3K/AKT/MMP-1 pathway.

We previously demonstrated that a modified secreted form of fibroblast growth factor 1 (FGF-1), a prototypic member of the FGF family, has the ability to stimulate angiogenesis in an in vivo model of angiogenesis, the so-called chick chorioallantoic membrane assay or CAM. We recently defined the importance of the phosphatidylinositol 3-kinase/AKT pathway in FGF-1-mediated angiogenesis in this model using specific pharmacological inhibitors. In our continuing efforts to define the molecular signaling pathway regulating FGF-1-induced angiogenesis in vivo, we utilized a transcription factor activity assay and identified transcription factor Ets-1 as a critical effector of FGF-1-induced angiogenesis. Both activity and mRNA expression levels of the Ets-1 molecule were increased in response to FGF-1 overexpression in CAMs, as documented by electrophoretic mobility shift assay (gel shift) and reverse transcription real-time PCR techniques, respectively. Furthermore, the delivery of Ets-1 antisense (AS) into CAM tissues effectively reduced angiogenesis in the CAM assay. In addition, both Ets-1 AS-treated chicken CAMs and cultured endothelial cells exhibited a reduction in matrix metalloproteinase 1 gene expression levels. The Ets-1 AS-treated endothelial cells also demonstrated a reduction in migration. These data suggest that Ets-1 activation is a requisite for FGF-1-mediated angiogenesis in vivo. Therefore, Ets-1 might be a potential target for the generation of inhibitor drugs for the treatment of FGF-dependent pathological angiogenesis such as metastatic tumors, rheumatoid arthritis and diabetic retinopathy.

Exercise training increases basal tone in arterioles distal to chronic coronary occlusion.

Endurance exercise training increases basal active tone in coronary arteries and enhances myogenic tone in coronary arterioles of control animals. Paradoxically, exercise training has also been shown to augment nitric oxide production and nitric oxide-mediated relaxation in coronary arterioles. The purpose of the present study was to examine the effect of exercise training on basal active tone of arterioles (approximately 150 microm ID) isolated from the collateral-dependent region of hearts exposed to chronic coronary occlusion. Ameroid occluders were surgically placed around the proximal left circumflex coronary artery of miniature swine. Arterioles were isolated from both the collateral-dependent and nonoccluded myocardial regions of sedentary (pen confined) and exercise-trained (treadmill run; 14 wk) pigs. Coronary tone was studied in isolated arterioles using microvessel myographs and standard isometric techniques. Exposure to nominally Ca2+-free external solution reduced resting tension in all arterioles; decreases were most profound (P < 0.05) in arterioles from the collateral-dependent region of exercise-trained animals. Furthermore, nitric oxide synthase (NOS) inhibition (N(omega)-nitro-L-arginine methyl ester; 100 microM) unmasked markedly increased nitric oxide-sensitive tone in arterioles from the collateral-dependent region of exercise-trained swine. Blockade of K+ channels revealed significantly enhanced K+ channel contribution to basal tone in collateral-dependent arterioles of exercise-trained pigs. Protein content of endothelial NOS (eNOS) and phosphorylated eNOS (pS1179), determined by immunoblot, was elevated in arterioles from exercise-trained animals with the greatest effect in collateral-dependent vasculature. Taken together, we demonstrate the interaction of opposing exercise training-enhanced arteriolar basal active tone, nitric oxide production, and K+ channel activity in chronic coronary occlusion, potentially enhancing the capacity to regulate blood flow to collateral-dependent myocardium.

GTP cyclohydrolase I gene transfer reverses tetrahydrobiopterin deficiency and increases nitric oxide synthesis in endothelial cells and isolated vessels from diabetic rats.

Nitric oxide (NO) synthesis in endothelial cells is impaired in diabetes. We previously showed that impaired NO synthesis in the spontaneously diabetic BB (BBd) rat is due to decreased levels of tetrahydrobiopterin (BH4), secondary to decreased expression of GTP cyclohydrolase I (GTPCH). The aim of this study was to utilize adenoviral GTPCH gene transfer to reverse BH4 deficiency and repair the ability of endothelial cells to produce NO. GTPCH gene transfer increased BH4 levels in BBd endothelial cells from 0.17 +/- 0.02 (mean +/-SE) to 73.37 +/- 14.42 pmol/million cells and NO production from 0.77 +/- 0.07 to 18.74 +/- 5.52 nmol/24 h/million cells. To demonstrate a functional effect of increasing BH4 concentrations in tissues, we transferred GTPCH into aortic rings from BBd and Zucker diabetic fatty (ZDF) rats, models of human type I and type II diabetes, respectively. GTPCH gene transfer led to a dose-dependent increase in acetylcholine-induced vasorelaxation, preventable by inhibiting NO synthase. Maximal relaxation of virus-treated rings (10(10) virus particles/ml) to acetylcholine was significantly higher than sham-treated rings (BBd 64% vs. 37%, P<0.005; ZDF 80% vs. 44%, P<0.05). This study demonstrates that GTPCH gene transfer can reverse BH4 deficiency in both type I and type II diabetes and provides an experimental basis for using gene therapy to treat cardiovascular complications in diabetic patients.

Altered calcium sensitivity contributes to enhanced contractility of collateral-dependent coronary arteries.

Coronary arteries distal to chronic occlusion exhibit enhanced vasoconstriction and impaired relaxation compared with nonoccluded arteries. In this study, we tested the hypotheses that an increase in peak Ca(2+) channel current density and/or increased Ca(2+) sensitivity contributes to altered contractility in collateral-dependent coronary arteries. Ameroid occluders were surgically placed around the proximal left circumflex coronary artery (LCX) of female miniature swine. Segments of epicardial arteries ( approximately 1 mm luminal diameter) were isolated from the LCX and nonoccluded left anterior descending (LAD) arteries 24 wk after Ameroid placement. Contractile responses to depolarization (10-100 mM KCl) were significantly enhanced in LCX compared with size-matched LAD arterial rings [concentration of KCl causing 50% of the maximal contractile response (EC(50)); LAD = 41.7 +/- 2.3, LCX = 34.3 +/- 2.7 mM]. However, peak Ca(2+) channel current was not altered in isolated smooth muscle cells from LCX compared with LAD (-5.29 +/- 0.42 vs. -5.68 +/- 0.55 pA/pF, respectively). Furthermore, whereas half-maximal activation of Ca(2+) channel current occurred at nearly the same membrane potential in LAD and LCX, half-maximal inactivation was shifted to a more positive membrane potential in LCX cells. Simultaneous measures of contractile tension and intracellular free Ca(2+) (fura 2) levels in arterial rings revealed that significantly more tension was produced per unit change in fura 2 ratio in LCX compared with LAD in response to KCl but not during receptor-agonist stimulation with endothelin-1. Taken together, our data indicate that coronary arteries distal to chronic occlusion display increased Ca(2+) sensitivity in response to high KCl-induced depolarization, independent of changes in whole cell peak Ca(2+) channel current. Unaltered Ca(2+) sensitivity in endothelin-stimulated arteries suggests more than one mechanism regulating Ca(2+) sensitization in coronary smooth muscle.

Exercise training enhances vasodilation responses to vascular endothelial growth factor in porcine coronary arterioles exposed to chronic coronary occlusion.

BACKGROUND: Chronic coronary occlusion (CCO) impairs endothelial function of distal collateral-dependent microvasculature; however, long-term exercise training (EX) seems to improve endothelial dysfunction. We hypothesized that EX enhances vasodilation responses to vascular endothelial growth factor (VEGF165), mediated via nitric oxide (NO), in arterioles exposed to CCO. METHODS AND RESULTS: The proximal left circumflex coronary artery (LCx) of female Yucatan miniswine was surgically instrumented with an ameroid occluder to induce CCO; 8 weeks after surgery, animals were randomized into 14-week sedentary (SED) or EX (treadmill; 5 d/wk) protocols. Coronary arterioles ( approximately 100 microm in diameter) were isolated from collateral-dependent (LCx) and nonoccluded (left anterior descending; LAD) perfused myocardium of SED and EX animals. Vasodilation was assessed by videomicroscopy and MacLab data acquisition. Responses to VEGF165 were unaffected by EX in nonoccluded LAD arterioles; in contrast, EX markedly enhanced VEGF165-induced vasodilation of collateral-dependent LCx arterioles (P<0.05; EX versus SED). Furthermore, VEGF165-induced vasodilation of EX LCx arterioles exceeded that of EX or SED LAD arterioles (P<0.05). Enhanced vasodilation of EX LCx arterioles was abolished by inhibition of NO synthase and tyrosine kinase activity. Combined inhibition of NO synthase and cyclooxygenase decreased VEGF165-induced vasodilation of all vessels. CONCLUSIONS: EX enhances VEGF165-induced vasodilation in arterioles distal to CCO; EX effects seem to be mediated through increases in NO.

Cell-based and direct gene transfer-induced angiogenesis via a secreted chimeric fibroblast growth factor-1 (sp-FGF-1) in the chick chorioallantoic membrane (CAM).

Fibroblast growth factor-1 (FGF-1) is a potent angiogenic factor; its structure lacks a signal peptide for secretion. We previously reported that the overexpression of a secreted version of FGF-1 (sp-FGF-1) in microvascular endothelial cells (ECs) enhances cell migration [Partridge et al. J Cell Biochem 2000; 78(3): 487]. In the current study, we have examined the angiogenic effects of sp-FGF-1 in chicken chorioallantoic membranes (CAMs). Two methods of examining the effects of sp-FGF-1 in CAMs were used: cell-mediated transfection via bovine ECs and direct gene transfection. In the cell-mediated gene transfection, those eggs that were implanted with a gelatin sponge seeded with ECs stably transfected to over-express sp-FGF-1 protein showed a significant increase in angiogenesis inside the sponge when compared to eggs treated with vector control-transfected ECs. In the direct gene transfer, eggs received sp-FGF-1 showed a significant increase in vascularization when compared to eggs received vector alone plasmids. These CAM models are useful both for studying molecular mechanisms of angiogenesis and for developing better gene therapy strategies.