Type-3 ryanodine receptors mediate hypoxia-, but not neurotransmitter-induced calcium release and contraction in pulmonary artery smooth muscle cells

In this study we examined the expression of RyR subtypes and the role of RyRs in neurotransmitter- and hypoxia-induced Ca2+ release and contraction in pulmonary artery smooth muscle cells (PASMCs). Under perforated patch clamp conditions, maximal activation of RyRs with caffeine or inositol triphosphate receptors (IP3Rs) with noradrenaline induced equivalent increases in [Ca2+]i and Ca2+-activated Cl- currents in freshly isolated rat PASMCs. Following maximal IP3-induced Ca2+ release, neither caffeine nor chloro-m-cresol induced a response, whereas prior application of caffeine or chloro-m-cresol blocked IP3-induced Ca2+ release. In cultured human PASMCs, which lack functional expression of RyRs, caffeine failed to affect ATP-induced increases in [Ca2+]i in the presence and absence of extracellular Ca2+. The RyR antagonists ruthenium red, ryanodine, tetracaine, and dantrolene greatly inhibited submaximal noradrenaline- and hypoxia-induced Ca2+ release and contraction in freshly isolated rat PASMCs, but did not affect ATP-induced Ca2+ release in cultured human PASMCs. Real-time quantitative RT-PCR and immunofluorescence staining indicated similar expression of all three RyR subtypes (RyR1, RyR2, and RyR3) in freshly isolated rat PASMCs. In freshly isolated PASMCs from RyR3 knockout (RyR3-/-) mice, hypoxia-induced, but not submaximal noradrenaline-induced, Ca2+ release and contraction were significantly reduced. Ruthenium red and tetracaine can further inhibit hypoxic increase in [Ca2+]i in RyR3-/- mouse PASMCs. Collectively, our data suggest that (a) RyRs play an important role in submaximal noradrenaline- and hypoxia-induced Ca2+ release and contraction; (b) all three subtype RyRs are expressed; and (c) RyR3 gene knockout significantly inhibits hypoxia-, but not submaximal noradrenaline-induced Ca2+ and contractile responses in PASMCs.     

Effects of C-reactive protein on atherogenic mediators and adrenomedullin in human coronary artery endothelial and smooth muscle cells

We examined the effects of recombinant human C-reactive protein (rhCRP) on atherosclerosis-related factors in cultured human coronary artery endothelial and smooth muscle cells (HCAECs and HCASMCs). After removing endotoxin from commercial rhCRP preparations using the appropriate column, the purified (P)-rhCRP retained the ability to Ca(2+)-dependently bind to phosphorylcholine, but did not augment the secretion of interleukin-6 and MCP-1 from HCAECs, as non-purified (NP)-rhCRP did. By contrast, P-rhCRP elicited 2- to 3-fold increases in the secretion of both hormones from HCASMCs, though the effect was smaller than that obtained with NP-rhCRP. Production of PAI-1 and endothelin-1 was little affected by either rhCRP preparation in either cell type. In addition, P-rhCRP dose-dependently diminished adrenomedullin release from both cell types, but did not affect adrenomedullin receptor expression or function. Our findings highlight the importance of removing endotoxin from commercial rCRP preparations and show that hCRP elicits atherogenic responses from HCASMCs, but not HCAECs.     

Numerical investigation of the non-Newtonian blood flow in a bifurcation model with a non-planar branch

The non-Newtonian fluid flow in a bifurcation model with a non-planar daughter branch is investigated by using finite element method to solve the three-dimensional Navier–Stokes equations coupled with a non-Newtonian constitutive model, in which the shear thinning behavior of the blood fluid is incorporated by the Carreau–Yasuda model. The objective of this study is to investigate the influence of the non-Newtonian property of fluid as well as of curvature and out-of-plane geometry in the non-planar daughter vessel on wall shear stress (WSS) and flow phenomena. In the non-planar daughter vessel, the flows are typified by the skewing of the velocity profile towards the outer wall, creating a relatively low WSS at the inner wall. In the downstream of the bifurcation, the velocity profiles are shifted towards the flow divider. The low WSS is found at the inner walls of the curvature and the lateral walls of the bifurcation. Secondary flow patterns that swirl fluid from the inner wall of curvature to the outer wall in the middle of the vessel are also well documented for the curved and bifurcating vessels. The numerical results for the non-Newtonian fluid and the Newtonian fluid with original Reynolds number and the corresponding rescaled Reynolds number are presented. Significant difference between the non-Newtonian flow and the Newtonian flow is revealed; however, reasonable agreement between the non-Newtonian flow and the rescaled Newtonian flow is found. Results of this study support the view that the non-planarity of blood vessels and the non-Newtonian properties of blood are an important factor in hemodynamics and may play a significant role in vascular biology and pathophysiology.     

An echocardiogram-based 16-segment model for predicting left ventricular ejection fraction improvement

An important goal of cardiac revascularization is to improve the left ventricular ejection fraction, which is an important clinical determinant of the long-term outcome for patients with coronary artery disease. Regional myocardium function improvement may be expected from revascularization when viable myocardium is detected using non-invasive cardiac imaging. However, the quantitative relation between regional myocardial function recovery and global heart function improvement has not been determined and there is no tool to predict the amount of ejection fraction improvement prior to revascularization. A 16 segment biomechanical model of the left ventricle is proposed to establish the relationship between the ejection fraction improvement and the viable segments detected by echocardiography. With the assumption that the viable segments would potentially improve contractility after revascularization, the ejection fraction improvement is estimated for all possible wall motion score improvement in viable segments. The model shows that the ejection fraction improvement is linearly related to the contractility in the normal segments and a weighted sum of the numbers of viable segments that recover to normal or hypokinetic contractility. The predictive value of the model is illustrated for a group of patients reported in the literature. The model predictions of the post-revascularization ejection fraction are very close to the follow-up data with a very strong correlation (R2 = 0.92). By predicting the ejection fraction improvement, the model may provide a tool for evaluating the efficacy of revascularization and for selecting patients who would benefit from revascularization.     

Cardiovascular pharmacological characterization of novel 2,3-dimethyl-2-butylamine derivatives in rats

The electrophysiological properties and pharmacological effects of newly synthesized 2,3-dimethyl-2-butylamine derivatives on the rat cardiovascular system were evaluated both in vitro and in vivo. In conventional whole-cell recordings, 2,3-dimethyl-2-butylamine derivatives with ethyl, isopropyl, allyl, butyl, 1-methyl-propyl or cyclopropylmethyl substituents on the amine side chain had potent effects on the outward potassium currents in isolated rat tail arterial smooth muscle cell membranes. In contrast, the compounds with hydrogen, methyl, propyl or benzyl substituents displayed very low activities, which were not statistically significant. The effects of compounds with pyridine ring substituents were also investigated and their order of potency was (14)>(12)>(13). In addition, the opening activities of these compounds (5), (6), (8) could be prevented by glibenclamide, a highly specific blocker of ATP-sensitive potassium channels. The influences of the compounds on cardiovascular hemodynamics parameters, including mean blood pressure (MBP), heat rate (HR), myocardial contractility and diastolic force, were examined in normotensive anesthetized rats. The derivatives with branched 2-4 carbon alkyl substituents induced long-duration hypotensive effects with modest inhibition of cardiac function. The hypotensive effects of the compound (5) could also be inhibited by glibenclamide. Collectively, these results indicate that 2,3-dimethyl-2-butylamine derivatives, which differ chemically from previously described potassium channel openers, have potassium channel opening activity, hypotensive and cardiac-modulating properties that depend on their amino group substituents.     

Relations of APOE promoter polymorphisms to LDL cholesterol and markers of subclinical atherosclerosis in young adults

The common apolipoprotein E (apoE) gene (APOE) epsilon2/epsilon3/epsilon4 polymorphism explains part of serum lipid variation, and polymorphisms in the APOE promoter region have been proposed to participate in the regulation of serum lipid levels within the most common APOE epsilon3/epsilon3 genotype group. We determined APOE -219G/T and +113G/C promoter genotypes and estimated APOE haplotypes in 525 participants of the Cardiovascular Risk in Young Finns Study. We studied the associations of the APOE promoter polymorphisms and their haplotypes with cross-sectional and longitudinal serum lipid and apolipoprotein concentrations as well as with flow-mediated dilatation (FMD), carotid artery compliance (CAC), and intima-media thickness (IMT) within the APOE epsilon3/epsilon3 carriers. We found no significant association between the APOE promoter genotypes and serum lipids [low density lipoprotein-cholesterol (LDL-C), HDL-C, and triglycerides], apolipoproteins (apoA-I and apoB), or brachial artery FMD, CAC, or carotid IMT in either men or women. In longitudinal analyses in males, the carriers of heterozygous genotypes (-219G/T or +113G/C) and, furthermore, carriers of the -219T/+113C/epsilon3 haplotype had significantly higher LDL-C and total cholesterol concentrations throughout the 21 year follow-up period compared with homozygous G allele carriers or noncarriers of the -219T/+113C/epsilon3 haplotype. Such associations were not found in females. In summary, the APOE promoter polymorphisms -219G/T and +113G/C as well as their haplotype are associated with longitudinal changes in LDL-C and total cholesterol concentrations in young Finnish males but do not seem to be major determinants for FMD, CAC, or carotid IMT in males or females.     

Phosphoinositide 3-kinase mediated signalling contributes to development of diabetes-induced abnormal vascular reactivity of rat carotid artery

Diabetes mellitus is associated with vascular complications, including an impairment of vascular function and alterations in the reactivity of blood vessels to vasoactive agents. Phosphatidylinositol 3-kinase (PI3K) is a signalling enzyme that plays key roles in vascular growth, proliferation and cellular apoptosis and is implicated in modulating vascular smooth muscle contractility. The aim of this study was to determine whether PI3K plays a role in development of diabetes-induced altered vascular reactivity to selected vasoconstrictors and vasodilators. The effect of 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), a selective PI3K inhibitor, on isolated segments of carotid arteries from streptozotocin (STZ)-diabetic rats was investigated. Ring segments of the isolated carotid arteries were mounted in organ baths to measure changes in isometric tension. Our results showed that STZ treatment produced an increase in the vasoconstrictor response to norepinephrine (NE), angiotensin II (Ang II) and endothelin-1 (ET-1) and an attenuated vasodilator response to carbachol and histamine in the isolated carotid arteries from STZ-diabetic animals. Diabetes-induced impaired vascular responsiveness to the vasoactive agonists was prevented by chronic inhibition of PI3K by LY294002 even though blood glucose levels remained high. This is the first study to show that selective inhibition of PI3K can attenuate the development of diabetes-induced abnormal vascular reactivity in the isolated carotid arteries of diabetic rats.     

Centrally Acting Imidazolines Stimulate Vascular Alpha 1A-Adrenergic Receptors in Rat-Tail Artery

  1. Centrally acting imidazoline antihypertensive agents clonidine and moxonidine also act peripherally to contract blood vessels. While these agents act at both I₁-imidazoline and alpha 2 adrenergic receptors centrally, the receptor types by which they mediate contraction require further definition. We therefore characterized the receptor subtype by which these agents mediate contraction of proximal rat-tail artery.2. Dose–response curves were determined for phenylephrine and for several imidazoline ligands, using endothelium denuded, isolated ring segments, of tail arteries from adult male Sprague-Dawley rats. Ring segments were mounted on a force transducer with platinum wires and immersed in a tissue bath containing Krebs solution, to which drugs could be added. Signals were digitized and recorded by a computer.3. Tail artery contractions expressed as a percent of contraction to 106 mM potassium were phenylephrine (96%), moxonidine (88%), clonidine (52%), and UK14304 (30%). Neither rilmenidine nor harmane caused contraction. Contraction of tail artery to moxonidine or clonidine could be blocked by alpha 1 antagonist urapidil or prazosin, and also by alpha 1A subtype selective antagonist WB4101. Schild plots were generated and a calculated pA2 value of 9.2 for prazosin in the presence of clonidine confirms clonidine as an agonist at alpha 1A receptors in proximal segments of rat-tail artery.4. Our work suggests that clonidine and moxonidine are promiscuous compounds at micromolar concentrations and that harmane and rilmenidine are more selective compounds for in vivo imidazoline research.*This work represents a portion of a dissertation to be submitted to the School of Graduate Studies, Loma Linda University, for the degree of Doctor of Philosophy.     

CLA isomers inhibit TNFα-induced eicosanoid release from human vascular smooth muscle cells via a PPARγ ligand-like action

Conjugated linoleic acids (CLAs) were reported to have anti-atherogenic properties in animal feeding experiments. In an attempt to elucidate the molecular mechanisms of these anti-atherogenic effects, the modulatory potential of CLA on cytokine-induced eicosanoid production from smooth muscle cells (SMCs), which contributes to the chronic inflammatory response associated with atherosclerosis, has been investigated in the present study. cis-9, trans-11 CLA and trans-10, cis-12 CLA were shown to reduce proportions of the eicosanoid precursor arachidonic acid in SMC total lipids and to inhibit cytokine-induced NF-κB DNA-binding activity, mRNA levels of inducible enzymes involved in eicosanoid formation (cPLA₂, COX-2, mPGES), and the production of the prostaglandins PGE₂ and PGI₂ by TNFα-stimulated SMCs in a dose-dependent manner. The effect of 50 μmol/L of either CLA isomer was as effective as 10 μmol/L of the PPARγ agonist troglitazone in terms of inhibiting the TNFα-stimulated eicosanoid production by SMCs. PPARγ DNA-binding activity was increased by both CLA isomers compared to control cells. Moreover, it was shown that the PPARγ antagonist T0070907 partially abrogated the inhibitory action of CLA isomers on cytokine-induced eicosanoid production and NF-κB DNA-binding activity by vascular SMCs suggesting that PPARγ signalling is at least partially involved in the action of CLA in human vascular SMCs. With respect to the effects of CLA on experimental atherosclerosis, our findings suggest that the anti-inflammatory effect of CLA is at least partially responsible for the anti-atherogenic effects of CLA observed in vivo.