Secretoneurin facilitates endothelium-dependent relaxations in porcine coronary arteries

Secretoneurin enhances the adhesion and transendothelial migration properties of monocytes and is a part of the peptide family encoded by the secretogranin II gene. The expression of the secretogranin II gene is upregulated in senescent endothelium. The present study was designed to examine the effects of secretoneurin on endothelium-dependent responsiveness. Isometric tension was measured in rings (with or without endothelium) of porcine coronary arteries. Secretoneurin did not induce contraction of quiescent or contracted rings. In preparations contracted by U-46619, relaxation was observed with high concentrations of the peptide. This relaxation was endothelium dependent and reduced by the nitric oxide synthase inhibitor N(ω)-nitro-l-arginine methyl ester (l-NAME). It was abolished when the preparations were incubated with l-NAME in combination with the cyclooxygenase inhibitor indomethacin. The relaxation was not affected by the combination of 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34) and 6,12,19,20,25,26-hexahydro-5,27:13,18:21,24-trietheno-11,7-etheno-7H-dibenzo[b,m][1,5,12,16]tetraazacyclotricosine-5,13-diiumditrifluoroacetate hydrate (UCL 1684), which abrogates endothelium-dependent hyperpolarizations. These results indicate that secretoneurin acutely induces relaxation through the activation of endothelial nitric oxide synthase (eNOS) and cyclooxygenase, with nitric oxide playing the dominant role. Prolonged (24 h) incubation with physiological concentrations of secretoneurin enhanced the relaxations to bradykinin and to the calcium ionophore A-23187, but this difference was not observed in preparations incubated with l-NAME or the calmodulin antagonist calmidazolium. Under these conditions, the relaxation to sodium nitroprusside remained unchanged. Incubation with secretoneurin significantly augmented the expression of eNOS and calmodulin as well as the dimerization of eNOS in cultures of porcine coronary arterial endothelial cells. These observations suggest that secretoneurin not only acutely causes but also, upon prolonged exposure, enhances endothelium-dependent relaxations.     

A generic constitutive model for the passive porcine coronary artery

Constitutive models describing the arterial mechanical behavior are important in the development of catheterization products, to be used in arteries with a specific radius. To prove the possible existence of a constitutive model that, provided with a generic set of material and geometric parameters, is able to predict the radius-specific mechanical behavior of a coronary artery, the passive pressure–inner radius (P–r _i ) and pressure–axial force change (P–ΔF _z ) relations of seven porcine left anterior descending coronary arteries were measured in an in-vitro set-up and fitted with the model of Driessen et al. in J Biomech Eng 127(3):494–503 (2005), Biomech Model Mechanobiol 7(2):93–103 (2008). Additionally, the collagen volume fraction, physiological axial pre-stretch, and wall thickness to inner radius ratio at physiological loading were determined for each artery. From this, two generic parameter sets, each comprising four material and three geometric parameters, were obtained. These generic sets were used to compute the deformation of each tested artery using a single radius measurement at physiological loading as an artery-specific input. Artery-specific P–r _i and P–ΔF _z relations were predicted with an accuracy of 32 μm (2.3%) and 6 mN (29% relative to ΔF _z -range) on average compared to the relations measured in-vitro. It was concluded that the constitutive model provided with the generic parameters found in this study can well predict artery-specific mechanical behavior.     

Constitutive modeling of porcine coronary arteries using designed experiments

The development of new coronary artery constitutive models is of critical importance in the design and analysis of coronary replacement grafts. In this study, a two-parameter logarithmic complementary energy function, with normalized measured force and internal pressure as the independent variables and strains as the dependent variables, was developed for healthy porcine coronary arteries. Data was collected according to an experimental design with measured force ranging from 9.8 to 201 mN and internal pressure ranging from 0.1 to 16.1 kPa (1 to 121 mmHg). Comparisons of the estimated constitutive parameters showed statistically significant differences between the left anterior descending [LAD] and right coronary artery [RCA], but no differences between the LAD and left circumflex [LCX] or between the LCX and RCA. Point-by-point strain comparisons confirm the findings of the model parameter study and isolate the difference to the axial strain response. Average axial strains for the LAD, LCX, and RCA are 0.026 +/- 0.009, 0.015 +/- 0.005, and 0.011 +/- 0.009, respectively, at all physiologic loads, suggesting that the axial strains in the LAD are significantly higher than in the other regions.     

Diameter-dependent axial prestretch of porcine coronary arteries and veins

The pressure-diameter relation (PDR) and the wall strain of coronary blood vessels have important implications for coronary blood flow and arthrosclerosis, respectively. Previous studies have shown that these mechanical quantities are significantly affected by the axial stretch of the vessels. The objective of this study was to measure the physiological axial stretch in the coronary vasculature; i.e., from left anterior descending (LAD) artery tree to coronary sinus vein and to determine its effect on the PDR and hence wall stiffness. Silicone elastomer was perfused through the LAD artery and coronary sinus trees to cast the vessels at the physiologic pressure. The results show that the physiological axial stretch exists for orders 4 to 11 (> 24 μm in diameter) arteries and orders -4 to -12 (>38 μm in diameter) veins but vanishes for the smaller vessels. Statistically, the axial stretch is higher for larger vessels and is higher for arteries than veins. The axial stretch λ(z) shows a linear variation with the order number (n) as: λ(z) = 0.062n + 0.75 (R(2) = 0.99) for artery and λ(z) = -0.029n + 0.89 (R(2) = 0.99) for vein. The mechanical analysis shows that the axial stretch significantly affects the PDR of the larger vessels. The circumferential stretch/strain was found to be significantly higher for the epicardial arteries (orders 9-11), which are free of myocardium constraint, than the intramyocardial arteries (orders 4-8). These findings have fundamental implications for coronary blood vessel mechanics.     

Development of the adrenergic innervation in the myocardium and coronary arteries of the dog

The development of the adrenergic cardiac innervation was studied in premature dog fetuses, puppies and adult dogs by means of the formalin-induced fluorescence technique. A point-counting technique was used to evaluate the density of innervation. Two types of fluorescent profiles can be observed in the heart during development: (1) sprouting axons, and (2) beaded terminals. The axonal fluorescence disappears in adult neurons. A different morphology and a different time course of development enable to study separately the innervation of the myocardium (cardiomotor innervation) and that of the vessels (vasomotor innervation). The late prenatal innervation is very poor (0.1 hit). The first but very scant cardiomotor terminals appear in this period. A mature cardiomotor innervation is found in 4-month-old puppies [1.5 +/- 0.3 (SD) hits]. The vasomotor innervation is shifted to the right. The development of beaded vascular terminals begins and matures 1-2 weeks later. The growing fluorescent axons reveal that the myocardium is supplied by axons of the cardiac plexus and of the perivascular nerves; the vascular wall, on the other hand, is supplied by the perivascular nerves only. The developmental, spatial and morphological differences in innervation suggest that two different types of neurons exist in the sympathetic ganglia: (1) neurons innervating the vessels (coronaromotor neurons), and (2) neurons innervating the myocardium (cardiomotor neurons).     

Flow-induced shear strain in intima of porcine coronary arteries.

The in vivo circumferential strain has a small variation throughout the vascular system (aorta to arterioles). The axial strain has also been shown to be nearly the same as the circumferential strain under physiological loading. Since the endothelium is mechanically much softer than the media-adventitia in healthy arteries, the porcine intima was considered as a mechanically distinct layer from the media-adventitia in a two-layer computational model. Based on the simulation result, we hypothesize that the flow-induced shear strain in intima can be of similar value as the pressure-induced circumferential strain in healthy coronary arteries, even though the shear stress is orders of magnitude smaller than the circumferential stress. The nearly isotropic deformation (circumferential, axial, and shear strains) may have important implications for mechanical homeostasis of endothelial cells, mechanotransduction, growth, and remodeling of blood vessels.     

Effect of respiratory gases on compliance of intrapulmonary arteries and veins

A method was developed that permitted changes in the pressure-volume characteristics of large intrapulmonary vessels occurring with changes in the composition of alveolar gas to be studied in excised lungs. The capillary bed was emptied by keeping intravascular pressure well below alveolar pressure, and the relationship between changes in the volume of the pulmonary arteries or veins with changes in transpulmonary pressure was measured. The volume of the arteries and veins always decreased with a decrease in transpulmonary pressure, but when the alveoli contained carbon dioxide, the decrease in vascular volume was less, for the same decrease in transpulmonary pressure, than when the alveoli contained oxygen or nitrogen without carbon dioxide. This change with carbon dioxide was probably due to a decrease in the compliance of the larger intrapulmonary arteries and veins. Since there was no pathway for carbon dioxide to enter these vessels except by diffusion from the alveoli, it is concluded that carbon dioxide can act directly on the intrapulmonary arteries and veins to reduce their compliance, but it is not known whether this effect has physiological significance. No effect on the large pulmonary vessels was found with variations in alveolar concentrations of oxygen. blood vesselsblood volumecarbon dioxidediffusionlungspulmonary circulation     

Flow inhibits inward remodeling in cannulated porcine small coronary arteries

The mechanisms of flow-induced vascular remodeling are poorly understood, especially in the coronary microcirculation. We hypothesized that application of flow in small coronary arteries in organoid culture would cause a nitric oxide (NO)-mediated dilation and inhibit inward remodeling. We developed an organoid culture setup to drive a flow through cannulated arterioles at constant luminal pressure via a pressure gradient between the pipettes. Subepicardial porcine coronary arterioles with diameter at full dilation and 60 mmHg (D0) of 168 +/- 10 (SE) microm were cannulated. Vessels treated with Nomega-nitro-L-arginine (L-NNA) to block NO production and untreated vessels were pressurized at 60 mmHg for 3 days with and without flow. Endothelium-dependent dilation to 10(-7) M bradykinin was preserved in all groups. Tone was significantly less in vessels cultured under flow conditions in the last half of the culture period. Untreated and L-NNA-treated vessels regulated their diameter to yield shear stresses of 10.3 +/- 2.1 and 14.0 +/- 2.4 (SE) dyn/cm2, respectively (not significantly different). Without L-NNA, passive pressure-diameter curves at the end of the culture period revealed inward remodeling in the control group [to 92.3 +/- 1.3% of D0 (SE)] and no remodeling in the vessels cultured under flow conditions (100.2 +/- 1.3% of D0); with L-NNA, the group subjected to flow showed inward remodeling (92.1 +/- 2.5% of D0). We conclude that pressurized coronary resistance arteries could be maintained in culture for several days with flow. Vessels cultured under flow conditions remained more dilated when NO synthesis was blocked. Inward remodeling occurred in vessels cultured under no-flow conditions and was inhibited by flow-dependent NO synthesis.     

Improved protocol for processing stented porcine coronary arteries for immunostaining

Percutaneous coronary intervention has resulted in a paradigm shift in the treatment of coronary artery disease and myocardial infarction. However, neither bare-metal stents nor polymer-coated drug-eluting stents represent ideal therapies at this time due to the undesired in-stent stenosis or delayed thrombosis. Hence there is pressing clinical need for greater understanding of the cellular mechanisms involved. It is hoped that this in turn will provide insight into designing and developing the next generation of stents. Although immunohistochemistry and immunofluorescence are appropriate tools in understanding the molecular histology, performing these techniques on stented blood vessels is technically challenging because of poor permeability of antibodies into the stented blood vessels which are embedded in methacrylate-based resins and inadequate image resolution due to autofluorescence. Hence there is a need to develop techniques which can facilitate immunohistochemistry/immunofluorescence procedures on stented blood vessel cross-sections. In this study we describe an improved protocol for processing stented porcine coronary arteries for immunostaining with smooth muscle cell, endothelial cell, monocyte and macrophage markers. We first identified the optimal conditions for resin embedding of stented artery and cross sectioned the vessels using high speed precision wafering diamond blade. The sections were then ground using two levels of water sandpaper on a Metaserve 2000 grinder to achieve the desired thickness. For immunostaining, we developed a novel deplasticization protocol which favors optimal antibody permeabilization. Our protocol not only provides feasibility of improved immunostaining of stented artery sections but also results in high quality images.     

Differences in nitric oxide production in porcine resistance arteries and epicardial conduit coronary arteries

The purpose of this study was to test the hypothesis that endothelial cells from resistance arteries and epicardial conduit coronary arteries differ in their expression of nitric oxide synthase (NOS) and calcium metabolism, and that these differences contribute to the mechanism underlying disparate physiological vasodilator responses observed between the two populations of vessels. The functional vasodilator responses of isolated resistance arteries and epicardial conduit coronary arteries were compared in vitro using both the receptor-independent agonist A23187 ionophore to increase intracellular calcium and the receptor-dependent agonist bradykinin. Constitutive NOS (cNOS) activity in monocultures of endothelial cells derived from resistance arteries and conduit arteries was assayed using a fibroblast-reporter cell method. Intracellular calcium concentration was assessed using fura-2 microfluorometry. Nitric oxide production was determined using a chemiluminescence technique, while cNOS protein was quantitated by Western blot analysis. A23187 was a less potent vasodilator of resistance arteries studied in vitro, compared to epicardial conduit arteries (EC₅₀ = 1.6 μM, resistance artery vs. EC₅₀ = 0.03 μM, conduit artery); however, bradykinin was more potent in resistance arteries (EC₅₀ = 0.3 nM, resistance artery vs. EC₅₀ = 2 nM, conduit artery). In pure monocultures of endothelium, nitric oxide production measured by chemiluminescence both basally and in response to A23187 was significantly less in resistance arteries (6.1 ± 0.5, basal vs. 10.80 ± 0.55, stimulated nmol/μg protein), compared to conduit arteries (7.7 ± 0.5, basal vs. 17.00 ± 1.52, stimulated nmol/μg protein; P < 0.05 resistance artery endothelium vs. conduit artery endothelium). cNOS enzyme activity assessed by cGMP production in reporter cell fibroblasts was also lower in resistance arteries compared to conduit arteries (0.17 ± 0.03 vs. 0.33 ± 0.05 fmol cGMP/μg protein, respectively; P < 0.05 resistance artery endothelium vs. conduit artery endothelium). Conduit arteries expressed 2.1 × more cNOS protein than resistance arteries, as assessed by Western blotting of cellular homogenates. No significant differences were found with microfluorimetry in either basal or ionophore-stimulated intracellular calcium concentrations. The results signified that porcine resistance arteries expressed less NOS and produced less nitric oxide than epicardial conduit arteries both basally and in response to an increase in intracellular calcium. This difference was reflected functionally as a decreased vasodilatory response to increased intracellular calcium in resistance arteries that could not be explained on the basis of differences in the metabolism of intracellular calcium. In contrast, the functional vasodilator response of intact vessels to a receptor-mediated agonist was enhanced in resistance arteries compared to conduit arteries, suggesting an important role of signal transduction mechanisms in specific physiological responses. Thus, the ability of the endothelium to regulate on a regional basis the expression of NOS and integrate receptor-mediated responses with these differences may provide a mechanism for diverse vasomotor responses in different populations of vessels. © 1996 Wiley-Liss, Inc.     

Duration of no-load state affects opening angle of porcine coronary arteries

The zero-stress state of a blood vessel has been extensively studied because it is the reference state for which all calculations of intramural stress and strain must be based. It has also been found to reflect nonuniformity in growth and remodeling in response to chemical or physical changes. The zero-stress state can be characterized by an opening angle, defined as the angle subtended by two radii connecting the midpoint of the inner wall. All prior studies documented the zero-stress state or opening angle with no regard to duration of the no-load state. Our hypotheses were that, given the viscoelastic properties of blood vessels, the zero-stress state may have "memory" of prior circumferential and axial loading, i.e., duration of the no-load state influences opening angle. To test these hypotheses, we considered ring pairs of porcine coronary arteries to examine the effect of duration in the no-load state after circumferential distension. Our results show a significant reduction in opening angle as duration of the no-load state increases, i.e., vessels that are reduced to the zero-stress state directly from the loaded state attain much larger opening angles at 30 min after the radial cut than rings that are in the no-load state for various durations. To examine the effect of axial loading, we found similar reductions in opening angle with duration in the no-load from the in situ state, albeit the effect was significantly smaller than that of circumferential loading. Hence, we found that the zero-stress state has memory of both circumferential and axial loading. These results are important for understanding viscoelastic properties of coronary arteries, interpretation of the enormous data on the opening angle and strain in the literature, and standardization of future measurements on the zero-stress state.     

Action of agonists and antagonists on adrenergic receptors in isolated porcine coronary arteries

The adrenergic receptors of porcine coronary arteries were investigated in helically cut strips of small (less than or equal to 0.5 mm outer-diameter (od), medium (0.8-1.2 mm od), large (1.5-2.5 mm od), and very large (greater than 4 mm od) coronary arteries. Both the beta1 agonist dobutamine and the beta2 agonist terbutaline relaxed coronary arteries partially contracted by 25 mM of KCl. Dobutamine contracted small coronary arteries at 10(-5) M concentrations, then relaxed them at 10(-4) M. The beta1-adrenoceptor antagonist metoprolol contracted coronary arteries relaxed by either dobutamine or terbutaline, but the beta2 antagonist H35/25 did so only in high and probably nonselective concentrations. Alpha1-adrenoreceptor stimulating concentrations of phenylephrine did not contract any of the arteries. Metoprolol and high concentrations of H35/25 further contracted large coronary arteries partially contracted by 25 mM potassium. These contractions were blocked by verapamil and papaverine but not by atropine, phentolamine, yohimbine, mepyramine, or methysergide. This seems to indicate that beta-adrenergic receptors in porcine coronary arteries are beta1-receptors, or closely resemble beta1-receptors. They differ from many other beta1-receptors, however, in that they are stimulated by terbutaline. Alpha1 adrenoreceptors seem not to be present in these porcine coronary arteries to a significant extent. Metoprolol and high concentrations of H35/25 have a direct contractile effect in large porcine coronary artery that is not mediated by alpha-adrenergic, muscarinic, histaminergic, or serotonergic receptors but requires verapamil-sensitive calcium.     

Effects of vessel compliance on flow pattern in porcine epicardial right coronary arterial tree

The compliance of the vessel wall affects hemodynamic parameters which may alter the permeability of the vessel wall. Based on experimental measurements, the present study established a finite element (FE) model in the proximal elastic vessel segments of epicardial right coronary arterial (RCA) tree obtained from computed tomography. The motion of elastic vessel wall was measured by an impedance catheter and the inlet boundary condition was measured by an ultrasound flow probe. The Galerkin FE method was used to solve the Navier–Stokes and Continuity equations, where the convective term in the Navier–Stokes equation was changed in the arbitrary Lagrangian–Eulerian (ALE) framework to incorporate the motion due to vessel compliance. Various hemodynamic parameters (e.g., wall shear stress—WSS, WSS spatial gradient—WSSG, oscillatory shear index—OSI) were analyzed in the model. The motion due to vessel compliance affects the time-averaged WSSG more strongly than WSS at bifurcations. The decrease of WSSG at flow divider in elastic bifurcations, as compared to rigid bifurcations, implies that the vessel compliance decreases the permeability of vessel wall and may be atheroprotective. The model can be used to predict coronary flow pattern in subject-specific anatomy as determined by noninvasive imaging.     

Gene delivery from a DNA controlled-release stent in porcine coronary arteries

Expandable intra-arterial stents are widely used for treating coronary disease. We hypothesized that local gene delivery could be achieved with the controlled release of DNA from a polymer coating on an expandable stent. Our paper reports the first successful transfection in vivo using a DNA controlled-release stent. Green fluorescent protein (GFP) plasmid DNA within emulsion-coated stents was efficiently expressed in cell cultures (7.9% +/- 0.7% vs. 0.6% +/- 0.2% control, p < 0.001) of rat aortic smooth muscle cells. In a series of pig stent-angioplasty studies, GFP expression was observed in all coronary arteries (normal, nondiseased) in the DNA-treated group, but not in control arteries. GFP plasmid DNA in the arterial wall was confirmed by PCR, and GFP presence in the pig coronaries was confirmed by immunohistochemistry. Thus, DNA-eluting stents are capable of arterial transfection, and could be useful as delivery systems for candidate vectors for gene therapy of cardiovascular diseases.     

Melatonin potentiates contractile responses to serotonin in isolated porcine coronary arteries

The present study was designed to determine the effects of melatonin on coronary vasomotor tone. Porcine coronary arteries were suspended in organ chambers for isometric tension recording. Melatonin (10(-10)-10(-5) M) itself caused neither contraction nor relaxation of the tissues. Serotonin (10(-9)-10(-5) M) caused concentration-dependent contractions of coronary arteries, and in the presence of melatonin (10(-7) M) the maximal response to serotonin was increased in rings with but not without endothelium. In contrast, melatonin had no effect on contractions produced by the thromboxane A(2) analog U-46619 (10(-10)-10(-7) M). The melatonin-receptor antagonist S-20928 (10(-6) M) abolished the potentiating effect of melatonin on serotonin-induced contractions in endothelium-intact coronary arteries, as did treatment with 1H-[1, 2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10(-5) M), methylene blue (10(-5) M), or N(G)-nitro-L-arginine (3 x 10(-5) M). In tissues contracted with U-46619, serotonin caused endothelium-dependent relaxations that were inhibited by melatonin (10(-7) M). Melatonin also inhibited coronary artery relaxation induced by sodium nitroprusside (10(-9)-10(-5) M) but not by isoproterenol (10(-9)-10(-5) M). These results support the hypothesis that melatonin, by inhibiting the action of nitric oxide on coronary vascular smooth muscle, selectively potentiates the vasoconstrictor response to serotonin in coronary arteries with endothelium.