Canine Coronary Arteries Research Articles

Leptin receptors are expressed in coronary arteries, and hyperleptinemia causes significant coronary endothelial dysfunction

Obesity is associated with marked increases in plasma leptin concentration, and hyperleptinemia is an independent risk factor for coronary artery disease. As a result, the purpose of this investigation was to test the following hypotheses: 1) leptin receptors are expressed in coronary endothelial cells; and 2) hyperleptinemia induces coronary endothelial dysfunction. RT-PCR analysis revealed that the leptin receptor gene is expressed in canine coronary arteries and human coronary endothelium. Furthermore, immunocytochemistry demonstrated that the long-form leptin receptor protein (ObRb) is present in human coronary endothelium. The functional effects of leptin were determined using pressurized coronary arterioles (<130 microm) isolated from Wistar rats, Zucker rats, and mongrel dogs. Leptin induced pharmacological vasodilation that was abolished by denudation and the nitric oxide synthase inhibitor N(omega)-nitro-l-arginine methyl ester and was absent in obese Zucker rats. Intracoronary leptin dose-response experiments were conducted in anesthetized dogs. Normal and obese concentrations of leptin (0.1-3.0 microg/min ic) did not significantly change coronary blood flow or myocardial oxygen consumption; however, obese concentrations of leptin significantly attenuated the dilation to graded intracoronary doses of acetylcholine (0.3-30.0 microg/min). Additional experiments were performed in canine coronary rings, and relaxation to acetylcholine (6.25 nmol/l-6.25 micromol/l) was significantly attenuated by obese concentrations of leptin (625 pmol/l) but not by physiological concentrations of leptin (250 pmol/l). The major findings of this investigation were as follows: 1) the ObRb is present in coronary arteries and coupled to pharmacological, nitric oxide-dependent vasodilation; and 2) hyperleptinemia produces significant coronary endothelial dysfunction.

Amiodarona causa vasodilatação dependente do endotélio em artérias coronárias caninas

To assess the vasodilating effects of amiodarone on canine coronary arteries by using solutions of amiodarone dissolved in polysorbate 80 or water. Rings of coronary arteries, with or without intact endothelium, were immersed in Krebs solution and connected to a transducer for measuring the isometric force promoted by a vascular contraction. The arteries were exposed to increasing concentrations of polysorbate 80, amiodarone dissolved in water, amiodarone dissolved in polysorbate 80, and a commercial presentation of amiodarone (Cordarone). The experiments were conducted in the presence of the following enzymatic blockers: only indomethacin, Nomega-nitro-L-arginine associated with indomethacin, and only Nomega-nitro-L-arginine. Polysorbate 80 caused a small degree of nonendothelium-dependent relaxation. Cordarone, amiodarone dissolved in water, and amiodarone dissolved in polysorbate 80 caused endothelium-dependent relaxation, which was greater for amiodarone dissolved in polysorbate and for Cordarone. Only the association of indomethacin and Nomega-nitro-L-arginine could eliminate the endothelium-dependent relaxation caused by amiodarone dissolved in polysorbate 80. The results obtained indicate that vasodilation promoted by amiodarone in canine coronary arteries is mainly caused by stimulation of the release of nitric oxide and cyclooxygenase-dependent relaxing endothelial factors.

Prolonged exposure of canine coronary arteries to a nitric oxide donor desensitizes soluble guanylate cyclase

This study investigated the role of soluble guanylate cyclase desensitization in the development of tolerance to organic nitrates. In organ baths, canine coronary arteries were exposed to either sodium nitroprusside (SNP) (experimental group) or papaverine (control group) at various concentrations (10(-9), 10(-7), or 10(-5) M) for 3 h. Arteries were then compared for response to vascular agonists and for inducible cyclic guanine monophosphate (cGMP) formation. KCl (5 to 50 mM) and prostaglandin F(2alpha) (10(-9) to 10(-5) M) induced similar vascular contractions (n = 7, P > 0.05). Vascular relaxation in response to calcium ionophore A23187 (10(-9) to 10(-6) M) and to authentic nitric oxide (NO) (3 x 10(-9) to 10(-5) M) was attenuated in arteries exposed to SNP at 10(-7) and 10(-5) M concentrations but not at a 10(-9) M concentration (n = 7 each, P < 0.05 versus the respective papaverine group). Pretreatment of arteries with methylene blue (10(-5) M) abolished the responses to authentic NO (n = 4). In cGMP determinations, control arteries demonstrated an increase in cGMP from 364 +/- 89 to 778 +/- 175 pg/mg of protein with A23187 (3 x 10(-5) M) stimulation (n = 5). Conversely, arteries exposed to SNP (10(-5) M) demonstrated similar levels of cGMP before (562 +/- 126 pg/mg of protein) and after (641 +/- 98 pg/mg of protein) A23187 stimulation. Prolonged exposure of coronary arteries to SNP did not alter vascular smooth muscle function, but it markedly attenuated the relaxation in response to both A23187 and authentic NO at concentrations above 10(-9) M in a concentration-dependent fashion. The constant levels of cGMP in response to an NO donor suggest that the attenuation of relaxation is due to desensitization of soluble guanylate cyclase. Thus, this study supports the role of soluble guanylate cyclase desensitization in the development of tolerance to organic nitrates.

Circumferential vascular deformation after stent implantation alters wall shear stress evaluated with time-dependent 3D computational fluid dynamics models

The success of vascular stents in the restoration of blood flow is limited by restenosis. Recent data generated from computational fluid dynamics (CFD) models suggest that stent geometry may cause local alterations in wall shear stress (WSS) that have been associated with neointimal hyperplasia and subsequent restenosis. However, previous CFD studies have ignored histological evidence of vascular straightening between circumferential stent struts. We tested the hypothesis that consideration of stent-induced vascular deformation may more accurately predict alterations in indexes of WSS that may subsequently account for histological findings after stenting. We further tested the hypothesis that the severity of these alterations in WSS varies with the degree of vascular deformation after implantation. Steady-state and time-dependent simulations of three-dimensional CFD arteries based on canine coronary artery measurements of diameter and blood flow were conducted, and WSS and WSS gradients were calculated. Circumferential straightening introduced areas of high WSS between stent struts that were absent in stented vessels of circular cross section. The area of vessel exposed to low WSS was dependent on the degree of circumferential vascular deformation and axial location within the stent. Stents with four vs. eight struts increased the intrastrut area of low WSS in vessels, regardless of cross-sectional geometry. Elevated WSS gradients were also observed between struts in vessels with polygonal cross sections. The results obtained using three-dimensional CFD models suggest that changes in vascular geometry after stent implantation are important determinants of WSS distributions that may be associated with subsequent neointimal hyperplasia.

Role of calcium-activated potassium channels in the regulation of basal and agonist-elevated tones in isolated conduit arteries

Functional role of calcium-activated potassium (KCa) channels on the basal and agonist-elevated arterial tones was investigated in isolated rabbit aorta, porcine and canine coronary arteries as well as in human internal mammary artery. The vascular tones enhanced by contractile agents were increased further by preincubation of these conduit blood vessels with selective (charybdotoxin or iberiotoxin) or nonselective (tetraethylammonium) inhibitors of KCa channels. The basal tone (without an agonist) was increased only in the canine coronary artery. The results indicate a feed-back regulatory role of KCa channels counteracting the vasospasm of conduit arteries.

Protective effect of N-acetylcysteine against oxygen radical-mediated coronary artery injury

The present study investigated the protective effect of N-acetylcysteine (NAC) against oxygen radical-mediated coronary artery injury. Vascular contraction and relaxation were determined in canine coronary arteries immersed in Kreb's solution (95% O2-5% CO2), incubated or not with NAC (10 mM), and exposed to free radicals (FR) generated by xanthine oxidase (100 mU/ml) plus xanthine (0.1 mM). Rings not exposed to FR or NAC were used as controls. The arteries were contracted with 2.5 microM prostaglandin F2alpha. Subsequently, concentration-response curves for acetylcholine, calcium ionophore and sodium fluoride were obtained in the presence of 20 microM indomethacin. Concentration-response curves for bradykinin, calcium ionophore, sodium nitroprusside, and pinacidil were obtained in the presence of indomethacin plus Nomega-nitro-L-arginine (0.2 mM). The oxidative stress reduced the vascular contraction of arteries not exposed to NAC (3.93 +/- 3.42 g), compared to control (8.56 +/- 3.16 g) and to NAC group (9.07 +/- 4.0 g). Additionally, in arteries not exposed to NAC the endothelium-dependent nitric oxide (NO)-dependent relaxation promoted by acetylcholine (1 nM to 10 microM) was also reduced (maximal relaxation of 52.1 +/- 43.2%), compared to control (100%) and NAC group (97.0 +/- 4.3%), as well as the NO/cyclooxygenase-independent receptor-dependent relaxation provoked by bradykinin (1 nM to 10 microM; maximal relaxation of 20.0 +/- 21.2%), compared to control (100%) and NAC group (70.8 +/- 20.0%). The endothelium-independent relaxation elicited by sodium nitroprusside (1 nM to 1 microM) and pinacidil (1 nM to 10 microM) was not affected. In conclusion, the vascular dysfunction caused by the oxidative stress, expressed as reduction of the endothelium-dependent relaxation and of the vascular smooth muscle contraction, was prevented by NAC.

Hirudin (desulfated, 54–65) contracts canine coronary arteries: Extracellular calcium influx mediates hirudin-induced contractions

Objective Although the anticoagulatory properties of hirudin are well known, its direct vasoactive effects have not been investigated extensively. Hirudin stimulates nitric oxide and prostacyclin production in noncoronary vascular beds, but its actions on coronary arteries are unknown. Materials and methods Five-millimeter segments of canine left circumflex coronary arteries were obtained for organ chamber experiments. Some segments were denuded of endothelium before study. Segments were exposed to hirudin (10 −10–10 −6 mol/L) following precontraction with prostaglandin F 2α with or without pretreatment with indomethacin or calcium channel blockers (verapamil and nifedipine). Results Hirudin stimulated endothelium-independent contraction in coronary arterial segments. Maximum tension (hirudin 10 −6 mol/L) above precontraction baseline was 33.6 ± 9.0% ( n = 10, P < 0.05) for endothelium-intact and 31.8 ± 11.5% ( n = 8, P < 0.05) for endothelium-denuded arterial segments. Differences between endothelium-intact and endothelium-denuded segments were not significant. Contractile responses to hirudin were unaffected by the presence of indomethacin. Pretreatment with either verapamil or nifedipine (10 −4 mol/L) for 1 h attenuated these contractions. The maximal increase in tension above baseline (hirudin 10 −6 mol/L) for verapamil and nifedipine was only 6.2 ± 12.4 and 3.8 ± 7.0% ( n = 6, P < 0.05 versus endothelium-intact control), respectively. Conclusions Hirudin stimulates endothelium-independent contractions of canine coronary arteries in vitro. Pretreatment with calcium channel blockers attenuates this response, suggesting that extracellular influx of calcium has an important mechanistic role in hirudin-mediated coronary artery constriction.

Stent design properties and deployment ratio influence indexes of wall shear stress: a three-dimensional computational fluid dynamics investigation within a normal artery.

Restenosis limits the effectiveness of stents, but the mechanisms responsible for this phenomenon remain incompletely described. Stent geometry and expansion during deployment produce alterations in vascular anatomy that may adversely affect wall shear stress (WSS) and correlate with neointimal hyperplasia. These considerations have been neglected in previous computational fluid dynamics models of stent hemodynamics. Thus we tested the hypothesis that deployment diameter and stent strut properties (e.g., number, width, and thickness) influence indexes of WSS predicted with three-dimensional computational fluid dynamics. Simulations were based on canine coronary artery diameter measurements. Stent-to-artery ratios of 1.1 or 1.2:1 were modeled, and computational vessels containing four or eight struts of two widths (0.197 or 0.329 mm) and two thicknesses (0.096 or 0.056 mm) subjected to an inlet velocity of 0.105 m/s were examined. WSS and spatial WSS gradients were calculated and expressed as a percentage of the stent and vessel area. Reducing strut thickness caused regions subjected to low WSS (<5 dyn/cm(2)) to decrease by approximately 87%. Increasing the number of struts produced a 2.75-fold increase in exposure to low WSS. Reducing strut width also caused a modest increase in the area of the vessel experiencing low WSS. Use of a 1.2:1 deployment ratio increased exposure to low WSS by 12-fold compared with stents implanted in a 1.1:1 stent-to-vessel ratio. Thinner struts caused a modest reduction in the area of the vessel subjected to elevated WSS gradients, but values were similar for the other simulations. The results suggest that stent designs that reduce strut number and thickness are less likely to subject the vessel to distributions of WSS associated with neointimal hyperplasia.

Myocardial protection with the non-selective endothelin receptor antagonist L-753,037 following acute coronary artery occlusion in the dog.

Efficacy of a new, potent non-selective endothelin antagonist, l-753037, was examined in a model of canine coronary artery occlusion and reperfusion to assess whether blockade of both ETA and ETB receptors would enhance or reduce myocardial ischemic injury. Instrumented dogs were randomized to receive vehicle (n = 9) or l-753037 (0.1 microg/kg/min, n = 9) by intracoronary infusion 30 minutes before a 90-minute LCx coronary artery occlusion and through 4 hours of reperfusion. After 4 hours of reperfusion, plasma ET-1 levels rose significantly in both groups: 24 +/- 3 fmol/ml in vehicle animals (P < 0.01) versus 42 +/- 5 fmol/ml with l-753037 (P < 0.05). Treatment with l-753037 normalized total LCx flow and regional myocardial flow after 4 hours of reperfusion in all regions. LCx flow was reduced 16% from pre-occlusion baseline (P = 0.45) with treatment compared with 35% with vehicle (P < 0.01). Endocardial flow in the risk region returned to baseline values with l-753037 treatment but was reduced approximately 50% in vehicle animals. l-753037 treatment was associated with a 38% reduction in infarct size (24.1 +/- 3.9% AAR with l-753037 treatment versus 38.7 +/- 3.1% with vehicle, P < 0.01). Thus, a non-selective endothelin antagonist provides significant myocardial protection primarily by improving regional myocardial flow distribution following reperfusion and demonstrated no detrimental effects associated with blockade of the ETB receptor.

Coronary vascular dysfunction associated with direct current shock injury.

To determine if cardiac injury following DC shocks includes impairment of coronary vascular reactivity. 36 dogs (18-32 kg) were anesthetized and a thoracotomy was performed. Either antioxidant enzymes, superoxide dismutase (SOD, 15,000 U/kg) plus catalase (55,000 U/kg) or the NO synthase inhibitor N(G)-nitro-L-arginine (L-NNA, 5 mg/kg) was administered IV prior to sham (no shocks) or DC shock treatment, and the results were compared to dogs which did not receive SOD/catalase or L-NNA. In sham dogs, electrodes cradled the heart, but no shocks were delivered. In shock dogs, three 20 Joule DC shocks were delivered to the epicardium using hand-held paddles. Other dogs were allowed a 3-hour recovery period after the shocks. Epicardial microvessels and conduit rings were studied in vitro. Antagonists were not added to the bath of the study vessel. Internal diameter was measured in microvessels after constriction with endothelin. Tension of conduit arteries was measured after constriction with PGF(2alpha). Responses to acetylcholine (Ach, 10(-10)-10(-4) M), bradykinin (10(-14)-10(-6) M), the calcium ionophore A23187 (A23187, 10(-12)-10(-4) M) or nitroprusside (SNP, 10(-10)-10(-4) M) were measured. Bradykinin, A23187 and SNP dependent dilation was not different between vessels from sham and shocked animals. Dilation to Ach was attenuated in vessels from shocked dogs. Superoxide production probably contributed to the impaired dilation to Ach since treatment with SOD/catalase improved dilation. Treatment with L-NNA also improved vascular function after DC shock. DC shocks cause endothelial dysfunction, as demonstrated by impaired dilation to acetylcholine, in both canine coronary microvascular and conduit arteries. Since pretreatment with either SOD/catalase or L-NNA protects against this damage, a free radical mechanism, possibly involving eNOS, may contribute to endothelial dysfunction.DC shocks for cardioversion and defibrillation cause myocardial injury that may be free radical mediated.

Endothelium-dependent relaxation in response to poly-L-arginine in canine coronary arteries: implications about hyperpolarization as a mechanism of vasodilatation.

To study the mechanism by which poly-L-arginine mediates endothelium-dependent relaxation. Vascular segments with and without endothelium were suspended in organ chambers filled with control solution maintained at 37 C and bubbled with 95% O2 / 5% CO2. Used drugs: indomethacin, acetycholine, EGTA, glybenclamide, ouabain, poly-L-arginine, methylene blue, N G-nitro-L-arginine, and verapamil and N G-monomethyl-L-arginine. Prostaglandin F2 and potassium chloride were used to contract the vascular rings. Poly-L-arginine (10-11 to 10-7 M) induced concentration-dependent relaxation in coronary artery segments with endothelium. The relaxation to poly-L-arginine was attenuated by ouabain, but was unaffected by glybenclamide. L-NOARG and oxyhemoglobin caused attenuation, but did not abolish this relaxation. Also, the relaxations was unaffected by methylene blue, verapamil, or the presence of a calcium-free bathing medium. The endothelium-dependent to poly-L-arginine relaxation was abolished only in vessels contracted with potassium chloride (40 mM) in the presence of L-NOARG and indomethacin. These experiments indicate that poly-L-arginine induces relaxation independent of nitric oxide.

Inhibition of arterial thrombosis by polyamines in a canine coronary artery injury model

Polyamines are polycations present in all living organisms and have been shown to play an important role in various physiological functions. Previous studies have shown that various amines including polyamines inhibit platelet activation. Among the amines tested tetra-amine, spermine is the potent inhibitor of platelet aggregation. In spite of vast literature on the anti-aggregatory effect of amines, there are no definitive studies testing their efficacy in an in vivo thrombosis model. In the present study, we investigated if polyamines could inhibit in-vivo thrombosis. A partially occlusive thrombus was generated by application of electric current in canine coronary artery. In control animals, the artery was completely in 76±14 min after the current was discontinued. When 40 mg/kg (1.44 mM) spermine was given immediately after stopping the current blood flow remained patent for >240 min. At equimolar concentration, triamine, spermidine and diamine putrescine are also equally effective in preventing thrombus development. The anti thrombic effect of polyamines was not associated with increased bleeding tendency, as judged by the amount of blood adsorbed by a gauge pad placed in a surgical incision extending to the muscle tissue and by a standard template bleeding. These results indicate that apart from inhibiting in-vitro platelet aggregation polyamines can also inhibit in-vivo thrombus formation.

Endothelium-dependent relaxation to poly-L-arginine in canine coronary arteries.

The endothelium-dependent relaxation elicited by poly-L-arginine was investigated in canine coronary arteries. Poly-L-arginine (10(-11) to 10(-7) M) induced concentration-dependent endothelium-dependent relaxation in segments of canine coronary arteries incubated with indomethacin. In the presence of indomethacin, arteries contracted with prostaglandin F2alpha and exposed also to ouabain, oxyhemoglobin, or N(omega)-nitro-L-arginine, as well as those contracted with 20 mM of KCl, presented reduced endothelium-dependent relaxation to poly-L-arginine. The combination of N(omega)-nitro-L-arginine with indomethacin produced further reduction of endothelium-dependent relaxation in arteries contracted with prostaglandin F2alpha as well as in arteries contracted with 20 mM of KCl. However, only quinacrine or calmidazolium, both in presence of N(omega)-nitro-L-arginine plus indomethacin, abolished the endothelium-dependent relaxation elicited by poly-L-arginine. The relaxation to poly-L-arginine was not significantly affected by glybenclamide. The present study demonstrates that in addition to the prostacyclin and nitric oxide-dependent mechanisms, poly-L-arginine also elicits endothelium-dependent relaxation in canine coronary arteries, stimulating the release of endothelial relaxing factor(s) produced by the phospholipase A2 pathway, but independent of the cyclooxygenase pathway, which may cause hyperpolarization of the vascular smooth muscle.

The role of magnesium in the endothelial dysfunction caused by global ischemia followed by reperfusion: in vitro study of canine coronary arteries

Objective—To study the role of magnesium in the endothelial dysfunction of canine coronary arteries caused by cardiopulmonary bypass (CPB) global ischemia followed by reperfusion.Design—Segments of canine coronary arteries were suspended in organ chambers to measure isometric contraction by prostaglandin F2α, and relaxed by acetylcholine (ACh), sodium fluoride (NaF), calcium ionophore (A23187) and sodium nitroprusside (SNP) in crescent concentrations. The investigation protocol had groups with six dogs: CONTROL group (without CPB), CPB group (105 min of CPB without aortic cross‐clamping), ISCH group (45 min of CPB with aortic cross‐clamping), ISCH/REP group (45 min of aortic cross‐clamping followed by 60 min of reperfusion). The coronary relaxations were evaluated with (phase I), without (phase II) and restored magnesium (phase III) to the organ bath.Results—The presence of magnesium in the organ bath was associated with the greater relaxation in response to agonists of the nitric oxide production. The removal of magnesium from the organ bath was associated with the reduction in the intensity of vessel relaxation. The magnesium restoration to the organ bath was associated with the additional reduction in the intensity of relaxation with the exception of NaF that allowed re‐acquisition of the relaxation observed in the presence of magnesium.Conclusion—This in vitro study demonstrates that magnesium ion favorably influences the nitric oxide production by the coronary endothelium, attenuating the endothelial dysfunction caused by global ischemia followed by reperfusion.

A novel antioxidant, EPC-K1, stimulates endothelial nitric oxide production and scavenges hydroxyl radicals.

EPC-K1, a hydroxyl radical scavenger synthesized by phosphate linkage of vitamin E and vitamin C, prevents myocardial reperfusion injury in vivo; however, the direct effects of EPC-K1 on coronary arteries are unknown. These experiments were undertaken to define possible mechanisms through which EPC-K1 imparts its protective action on the coronary vasculature. EPC-K1 (10(-5) to 10(-1) mg/ml) induced concentration-dependent relaxation in contracted canine coronary artery segments with endothelium, but no change in tension of arterial segments without endothelium (p<0.05, ANOVA). Endothelium-dependent relaxation to EPC-K1 was inhibited by N(G)-monomethyl-(L)-arginine ((L)-NMMA) (10(-5) mol/L). Inhibition of relaxation by (L)-NMMA was reversed by the addition of (L)-arginine (10(-4) mol/L), but not by (D)-arginine (10 (-4) mol/L). Subsequent exposure of canine coronary artery segments with intact endothelium to hydroxyl radicals for 30 min (generated by FeSO(4) [0.56 mmol/L] + H(2)O(2) [0.56 mmol/L]) impaired endothelium-dependent relaxation. However, pretreating the vascular segments with EPC-K1 (10(-4) mg/ml) prevented hydroxyl radical-mediated endothelial cell injury and maintained endothelium-dependent relaxation. These experiments indicate that EPC-K1 stimulates the release of endothelium-derived nitric oxide, an endogenous vasodilator and inhibitor of platelet and leukocyte activation and adhesion, from the coronary artery endothelium. Additionally, EPC-K1 scavenges hydroxyl radicals that mediate endothelial cell injury. These 2 independent and important actions are possible mechanisms by which EPC-K1 prevents reperfusion injury in the ischemic heart.

A stepwise method for the isolation of endothelial cells and smooth muscle cells from individual canine coronary arteries.

Methods for the stepwise isolation of endothelial cells and smooth muscle cells from individual canine coronary arteries are described. Both cell types can be isolated in pure culture with high yields. Dogs are a common species used in the study of atherosclerosis and coronary artery disease. Capacity to isolate endothelial cells and smooth muscle cells from individual canine coronary arteries should prove useful in the study of coronary artery disease.

Large animal models of congestive heart failure: A critical step in translating basic observations into clinical applications

Congestive heart failure (CHF) is a clinical syndrome in which pathophysiologic underpinnings include left ventricular (LV) dysfunction, remodeling, and increased neurohormonal activation. Accordingly, large animal constructs must be developed that mimic this disease process in order to define new pharmacologic and surgical treatment strategies. Multiple large animal species have been used for these purposes. For instance, canine coronary artery microembolization has been used to generate ischemia-induced LV dilation and dysfunction. Sheep have been subjected to total acute coronary artery occlusion to evaluate ischemia-induced mitral valve insufficiency. Rapid ventricular pacing has been used in both dogs and pigs to reproduce the characteristics of dilated cardiomyopathy. Each model is associated with advantages and disadvantages. Therefore findings derived from the study of large animal models of LV failure must be carefully evaluated. With proper interpretation, important insights into the pathogenesis of CHF may be realized. Furthermore, these models may be used in conjunction with imaging modalities such as magnetic resonance imaging, single photon emission computed tomography, and positron emission tomography to elucidate the identification of cellular and extracellular alterations associated with LV failure. Thus large animal models of CHF are critical components in the effort to translate basic observations into beneficial clinical applications. (J Nucl Cardiol 2003;10:77-86)

Gene transfer to coronary artery bypass conduits

Background. Gene therapy is a rational approach to prevention of stenosis in saphenous vein grafts used as conduits for coronary artery bypass grafting. To explore this possibility we developed methods for adenoviral-mediated gene transfer to canine saphenous veins. Methods. During a single procedure, autogenous canine saphenous vein segments were transduced ex vivo and used as coronary artery bypass grafts. The proximal end of each vein was ligated, adenovirus containing the Escherichia coli β-galactosidase gene (Ad.CMVLacZ) was delivered at titers of 2.5 × 10 9 or 5 × 10 9 plaque-forming units (pfu)/mL to the lumen through a distal heparin lock, and the segment was immersed in the viral solution for 1 hour at 37°C. Control segments were exposed to diluent alone in an identical manner. Aortocoronary anastomoses were made using cardiopulmonary bypass. Transgene expression was assessed qualitatively and quantitatively after 3 days. Results. β-Galactosidase levels showed a dose-dependent increase: 0.00 ± 0.00 ng/mg total protein for controls; 5.60 ± 2.27 ng/mg total protein for a viral titer of 2.5 × 10 9 pfu/mL and 11.97 ± 6.14 ng/mg for 5 × 10 9 pfu/mL. The two dosage groups differed significantly from each other ( p = 0.035) and from controls ( p = 0.003). X-gal staining demonstrated mostly endothelial and scattered adventitial transgene expression. Conclusions. Transgene expression after ex vivo gene transfer into saphenous vein grafts in a canine coronary artery bypass model indicates that this method may be useful for delivery of therapeutic genes to prevent or retard vein graft arteriosclerosis.

Propofol Potentiates U46619-Induced Vasoconstriction in Isolated Canine Coronary Artery: Role of Endothelium, PKC and Rho-Kinase

Propofol Potentiates U46619-Induced Vasoconstriction in Isolated Canine Coronary Artery: Role of Endothelium, PKC and Rho-Kinase

Disfunção endotelial após isquemia global e reperfusão em cirurgia cardíaca com circulação extracorpórea: estudo do papel do magnésio em artérias coronarianas caninas

OBJETIVO: Estudar a disfuncao endotelial consequente a lesao provocada por isquemia global seguida de reperfusao e a potencial influencia protetora do magnesio sobre a integridade funcional do endotelio em coronarias isoladas de caes. METODO: Segmentos de arterias coronarias caninas foram suspensos em banhos orgânicos para medida de forca isometrica. A disfuncao endotelial foi avaliada pela capacidade destes segmentos produzirem oxido nitrico, alterando a forca isometrica inicial. Foram selecionados quatro grupos com seis caes em cada um: SEM CEC (controle), CEC (110 minutos de perfusao sem isquemia), ISQ (45 minutos de isquemia), ISQ/REP (45 minutos de isquemia seguidos de 60 minutos de reperfusao). A acao do magnesio foi avaliada em tres fases: I (banho orgânico com magnesio), II (banho orgânico sem magnesio) e III (banho orgânico com magnesio restaurado). Foram utilizados tres agonistas farmacologicos que representam os principais passos da via de producao do oxido nitrico: receptor de membrana da celula endotelial - acetilcolina (ACh); transducao do sinal entre o receptor e os processos intracelulares atraves da G-proteina - fluoreto de sodio (NaF); liberacao dos estoques intracelulares de calcio - calcio ionoforo (A23187). Ao estudo da funcao endotelial associou-se a avaliacao da atividade da musculatura lisa dependente de GMPc - nitroprussiato de sodio (NPS). RESULTADOS: Os principais achados desta investigacao foram: 1) a presenca de magnesio no perfusato pareceu atenuar a disfuncao endotelial causada por isquemia global seguida de reperfusao; 2) a presenca de magnesio no banho orgânico (fase I) associou-se a maior relaxamento em resposta aos agonistas da producao de oxido nitrico; 3) a remocao de magnesio do banho orgânico (fase II) esteve associada a reducao na intensidade do relaxamento em resposta aos agonistas da producao de oxido nitrico; 4) a restauracao de magnesio ao banho orgânico (fase III) permitiu recuperacao do relaxamento observado na fase I, apenas em resposta a estimulacao direta das G-proteinas. Para os demais agonistas a restauracao esteve associada a reducao adicional na intensidade do relaxamento; 5) a musculatura lisa recebeu influencia da concentracao de magnesio no banho orgânico. CONCLUSAO: O magnesio influencia favoravelmente na producao de oxido nitrico pelo endotelio coronariano, atenuando a disfuncao endotelial causada por isquemia global seguida de reperfusao.