Porcine Coronary Artery Smooth Muscle Research Articles

In vitro biological responses of plasma nanocoatings for coronary stent applications.

In-stent restenosis and thrombosis remain to be long-term challenges in coronary stenting procedures. The objective of this study was to evaluate the in vitro biological responses of trimethylsilane (TMS) plasma nanocoatings modified with NH3 /O2 (2:1 molar ratio) plasma post-treatment (TMS + NH3 /O2 nanocoatings) on cobalt chromium (CoCr) alloy L605 coupons, L605 stents, and 316L stainless steel (SS) stents. Surface properties of the plasma nanocoatings with up to 2-year aging time were characterized by wettability assessment and x-ray photoelectron spectroscopy (XPS). It was found that TMS + NH3 /O2 nanocoatings had a surface composition of 41.21 ± 1.06 at% oxygen, 31.90 ± 1.08 at% silicon, and 24.12 ± 1.7 at% carbon, and very small but essential amount of 2.77 ± 0.18 at% nitrogen. Surface chemical stability of the plasma coatings was noted with persistent O/Si atomic ratio of 1.292-1.413 and N/Si atomic ratio of ~0.087 through 2 years. The in vitro biological responses of plasma nanocoatings were studied by evaluating the cell proliferation and migration of porcine coronary artery endothelial cells (PCAECs) and smooth muscle cells (PCASMCs). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay results revealed that, after 7-day incubation, TMS + NH3 /O2 nanocoatings maintained a similar level of PCAEC proliferation while showing a decrease in the viability of PCASMCs by 73 ± 19% as compared with uncoated L605 surfaces. Cell co-culture of PCAECs and PCASMCs results showed that, the cell ratio of PCAEC/PCASMC on TMS + NH3 /O2 nanocoating surfaces was 1.5-fold higher than that on uncoated L605 surfaces, indicating enhanced selectivity for promoting PCAEC growth. Migration test showed comparable PCAEC migration distance for uncoated L605 and TMS + NH3 /O2 nanocoatings. In contrast, PCASMC migration distance was reduced nearly 8.5-fold on TMS + NH3 /O2 nanocoating surfaces as compared to the uncoated L605 surfaces. Platelet adhesion test using porcine whole blood showed lower adhered platelets distribution (by 70 ± 16%), reduced clotting attachment (by 54 ± 12%), and less platelet activation on TMS + NH3 /O2 nanocoating surfaces as compared with the uncoated L605 controls. It was further found that, under shear stress conditions of simulated blood flow, TMS + NH3 /O2 nanocoating significantly inhibited platelet adhesion compared to the uncoated 316L SS stents and TMS nanocoated 316L SS stents. These results indicate that TMS + NH3 /O2 nanocoatings are very promising in preventing both restenosis and thrombosis for coronary stent applications.

A Biocompatibility Study of Plasma Nanocoatings onto Cobalt Chromium L605 Alloy for Cardiovascular Stent Applications.

The objective of this study was to evaluate the biocompatibility of trimethylsilane (TMS) plasma nanocoatings modified with NH3/O2 (2:1 molar ratio) plasma post-treatment onto cobalt chromium (CoCr) L605 alloy coupons and stents for cardiovascular stent applications. Biocompatibility of plasma nanocoatings was evaluated by coating adhesion, corrosion behavior, ion releasing, cytotoxicity, and cell proliferation. Surface chemistry and wettability were studied to understand effects of surface properties on biocompatibility. Results show that NH3/O2 post-treated TMS plasma nanocoatings are hydrophilic with water contact angle of 48.5° and have a typical surface composition of O (39.39 at.%), Si (31.92 at.%), C (24.12 at.%), and N (2.77 at.%). The plasma nanocoatings were conformal to substrate surface topography and had excellent adhesion to the alloy substrates, as assessed by tape test (ASTM D3359), and showed no cracking or peeling off L605 stent surfaces after dilation. The plasma nanocoatings also improve the corrosion resistance of CoCr L605 alloy by increasing corrosion potential and decreasing corrosion rates with no pitting corrosion and no mineral adsorption layer. Ion releasing test revealed that Co, Cr, and Ni ion concentrations were reduced by 64–79%, 67–69%, and 57–72%, respectively, in the plasma-nanocoated L605 samples as compared to uncoated L605 control samples. The plasma nanocoatings showed no sign of cytotoxicity from the test results according to ISO 10993-05 and 10993-12. Seven-day cell culture demonstrated that, in comparison with the uncoated L605 control surfaces, the plasma nanocoating surfaces showed 62 ± 7.3% decrease in porcine coronary artery smooth muscle cells (PCASMCs) density and had comparable density of porcine coronary artery endothelial cells (PCAECs). These results suggest that TMS plasma nanocoatings with NH3/O2 plasma post-treatment possess the desired biocompatibility for stent applications and support the hypothesis that nanocoated stents could be very effective for in-stent restenosis prevention.

Hesperetin Inhibits Sphingosylphosphorylcholine-Induced Vascular Smooth Muscle Contraction by Regulating the Fyn/Rho-Kinase Pathway.

Cardiovascular diseases are the leading cause of mortality and disability worldwide. We have previously found that sphingosylphosphorylcholine (SPC) is the key molecule leading to vasospasm. We have also identified the SPC/Src family protein tyrosine kinase Fyn/Rho-kinase (ROK) pathway as a novel signaling pathway for Ca2+ sensitization of vascular smooth muscle (VSM) contraction. This study aimed to investigate whether hesperetin can inhibit the SPC-induced contraction with little effect on 40 mM K+-induced Ca2+-dependent contraction and to elucidate the underlying mechanisms. Hesperetin significantly inhibited the SPC-induced contraction of porcine coronary artery smooth muscle strips with little effect on 40 mM K+-induced contraction. Hesperetin blocked the SPC-induced translocation of Fyn and ROK from the cytosol to the membrane in human coronary artery smooth muscle cells (HCASMCs). SPC decreased the phosphorylation level of Fyn at Y531 in both VSMs and HCASMCs and increased the phosphorylation levels of Fyn at Y420, myosin phosphatase target subunit 1 at T853, and myosin light chain (MLC) at S19 in both VSMs and HCASMCs, which were significantly suppressed by hesperetin. Our results indicate that hesperetin inhibits the SPC-induced contraction at least in part by suppressing the Fyn/ROK pathway, suggesting that hesperetin can be a novel drug to prevent and treat vasospasm.

Protection of dilator function of coronary arteries from homocysteine by tetramethylpyrazine: Role of ER stress in modulation of BKCa channels

ObjectivesWe recently reported the involvement of ER stress-mediated BKCa channel inhibition in homocysteine-induced coronary dilator dysfunction. In another study, we demonstrated that tetramethylpyrazine (TMP), an active ingredient of the Chinese herb Chuanxiong, possesses potent anti-ER stress capacity. The present study investigated whether TMP protects BKCa channels from homocysteine-induced inhibition and whether suppression of ER stress is a mechanism contributing to the protection. Furthermore, we explored the signaling transduction involved in TMP-conferred protection on BKCa channels. MethodsBKCa channel-mediated relaxation was studied in porcine small coronary arteries. Expressions of BKCa channel subunits, ER stress molecules, and E3 ubiquitin ligases, as well as BKCa ubiquitination were determined in porcine coronary arterial smooth muscle cells (PCASMCs). Whole-cell BKCa currents were recorded. ResultsExposure of PCASMCs to homocysteine or the chemical ER stressor tunicamycin increased the expression of ER stress molecules, which was significantly inhibited by TMP. Suppression of ER stress by TMP preserved the BKCa β1 protein level and restored the BKCa current in PCASMCs, concomitant with an improved BKCa-mediated dilatation in coronary arteries. TMP attenuated homocysteine-induced BKCa β1 protein ubiquitination, in which inhibition of ER stress-mediated FoxO3a activation and FoxO3a-dependent atrogin-1 and Murf-1 was involved. ConclusionsReversal of BKCa channel inhibition via suppressing ER stress-mediated loss of β1 subunits contributes to the protective effect of TMP against homocysteine on coronary dilator function. Inhibition of FoxO3a-dependent ubiquitin ligases is involved in TMP-conferred normalization of BKCa β1 protein level. These results provide new mechanistic insights into the cardiovascular benefits of TMP.

Arsenic Trioxide-Coated Stent Is an Endothelium-Friendly Drug Eluting Stent.

An ideal vascular stent would both inhibit in-stent restenosis (ISR) and promote rapid re-endothelialization. In the current study, the performance of arsenic trioxide (ATO)-drug eluting stent (AES) is compared with the bare metal stent, poly-lactic-co-glycolic acid-coating metal stent, and rapamycin-drug eluting stent (RES). In vivo AES is shown to prevent neointimal hyperplasia more efficiently than the others when implanted into the carotid arteries of rabbits. Moreover, AES promotes endothelial cells proliferation and re-endothelialization more quickly than RES. In vitro ATO exposure significantly increases the viability, proliferation, adhesion, and spreading of primary porcine coronary artery endothelial cells (PCAECs), which are critical for endothelialization. However, ATO exposure reduces the viability of porcine coronary artery smooth muscle cells (PCASMCs). The evaluation of mitochondrial morphology, membrane potential, and function demonstrates that ATO at 2 µmol L-1 causes enlargement of the mitochondrion, enhancement of mitochondrial membrane potential, and adenosine triphosphate (ATP) production in PCAECs but not in PCASMCs. Thus, both in vivo and in vitro studies demonstrate that AES is an effective strategy for rapid re-endothelialization and inhibition of ISR.

The activation of G protein-coupled estrogen receptor induces relaxation via cAMP as well as potentiates contraction via EGFR transactivation in porcine coronary arteries.

Estrogen exerts protective effects against cardiovascular diseases in premenopausal women, but is associated with an increased risk of both coronary heart disease and stroke in older postmenopausal women. Studies have shown that activation of the G-protein-coupled estrogen receptor 1 (GPER) can cause either relaxation or contraction of arteries. It is highly likely that these dual actions of GPER may contribute to the seemingly paradoxical effects of estrogen in regulating coronary artery function. The objective of this study was to test the hypothesis that activation of GPER enhances agonist-stimulated porcine coronary artery contraction via epidermal growth factor receptor (EGFR) transactivation and its downstream extracellular signal-regulated kinases (ERK1/2) pathway. Isometric tension studies and western blot were performed to determine the effect of GPER activation on coronary artery contraction. Our findings demonstrated that G-1 caused concentration-dependent relaxation of ET-1-induced contraction, while pretreatment of arterial rings with G-1 significantly enhanced ET-1-induced contraction. GPER antagonist, G-36, significantly inhibited both the G-1-induced relaxation effect and G-1-enhanced ET-1 contraction. Gallein, a Gβγ inhibitor, significantly increased G-1-induced relaxation, yet inhibited G-1-enhanced ET-1-mediated contraction. Similarly, inhibition of EGFR with AG1478 or inhibition of Src with phosphatase 2 further increased G-1-induced relaxation responses in coronary arteries, but decreased G-1-enhanced ET-1-induced contraction. Western blot experiments in porcine coronary artery smooth muscle cells (PCASMC) showed that G-1 increased tyrosine phosphorylation of EGFR, which was inhibited by AG-1478. Furthermore, enzyme-linked immunosorbent assays showed that the level of heparin-binding EGF (HB-EGF) released by ET-1 treatment increased two-fold; whereas pre-incubation with G-1 further increased ET-1-induced HB-EGF release to four-fold over control conditions. Lastly, the role of ERK1/2 was determined by applying the MEK inhibitor, PD98059, in isometric tension studies and detecting phospho-ERK1/2 in immunoblotting. PD98059 potentiated G-1-induced relaxation response, but blocked G-1-enhanced ET-1-induced contraction. By western blot, G-1 treatment decreased phospho-ERK1/2, however, in the presence of the adenylyl cyclase inhibitor, SQ22536, G-1 significantly increased ERK1/2 phosphorylation in PCASMC. These data demonstrate that activation of GPER induces relaxation via cAMP as well as contraction via a mechanism involving transactivation of EGFR and the phosphorylation of ERK1/2 in porcine coronary arteries.

Activation of PERK branch of ER stress mediates homocysteine-induced BKCa channel dysfunction in coronary artery via FoxO3a-dependent regulation of atrogin-1.

The molecular mechanism of endoplasmic reticulum (ER) stress in vascular pathophysiology remains inadequately understood. We studied the role of ER stress in homocysteine-induced impairment of coronary dilator function, with uncovering the molecular basis of the effect of ER stress on smooth muscle large-conductance Ca2+-activated K+ (BKCa) channels. The vasodilatory function of BKCa channels was studied in a myograph using endothelium-denuded porcine small coronary arteries. Primary cultured porcine coronary artery smooth muscle cells were used for mRNA and protein measurements and current recording of BKCa channels. Homocysteine inhibited vasorelaxant response to the BKCachannel opener NS1619, lowered BKCa β1 subunit protein level and suppressed BKCa current. Inhibition of ER stress restored BKCa β1 protein level and NS1619-evoked vasorelaxation. Selective blockade of the PKR-like ER kinase (PERK) yielded similarly efficient restoration of BKCa β1, preserving BKCa current and BKCa-mediated vasorelaxation. The restoration of BKCa β1 by PERK inhibition was associated with reduced atrogin-1 expression and decreased nuclear localization of forkhead box O transcription factor 3a (FoxO3a). Silencing of atrogin-1 prevented homocysteine-induced BKCa β1 loss and silencing of FoxO3a prevented atrogin-1 upregulation induced by homocysteine, accompanied by preservation of BKCa β1 protein level and BKCa current. ER stress mediates homocysteine-induced BKCa channel inhibition in coronary arteries. Activation of FoxO3a by PERK branch underlies the ER stress-mediated BKCa inhibition through a mechanism involving ubiquitin ligase-enhanced degradation of the channel β1 subunit.

Activation of G protein-coupled estrogen receptor 1 induces coronary artery relaxation via Epac/Rap1-mediated inhibition of RhoA/Rho kinase pathway in parallel with PKA.

Previously, we reported that cAMP/PKA signaling is involved in GPER-mediated coronary relaxation by activating MLCP via inhibition of RhoA pathway. In the current study, we tested the hypothesis that activation of GPER induces coronary artery relaxation via inhibition of RhoA/Rho kinase pathway by cAMP downstream targets, exchange proteins directly activated by cAMP (Epac) as well as PKA. Our results show that Epac inhibitors, brefeldin A (BFA, 50 μM), or ESI-09 (20 μM), or CE3F4 (100 μM), all partially inhibited porcine coronary artery relaxation response to the selective GPER agonist, G-1 (0.3–3 μM); while concurrent administration of BFA and PKI (5 μM), a PKA inhibitor, almost completely blocked the relaxation effect of G-1. The Epac specific agonist, 8-CPT-2Me-cAMP (007, 1–100 μM), induced a concentration-dependent relaxation response. Furthermore, the activity of Ras-related protein 1 (Rap1) was up regulated by G-1 (1 μM) treatment of porcine coronary artery smooth muscle cells (CASMCs). Phosphorylation of vasodilator-stimulated phosphoprotein (p-VASP) was elevated by G-1 (1 μM) treatment, but not by 007 (50 μM); and the effect of G-1 on p-VASP was blocked by PKI, but not by ESI-09, an Epac antagonist. RhoA activity was similarly down regulated by G-1 and 007, whereas ESI-09 restored most of the reduced RhoA activity by G-1 treatment. Furthermore, G-1 decreased PGF2α-induced p-MYPT1, which was partially reversed with either ESI-09 or PKI; whereas, concurrent administration of ESI-09 and PKI totally prevented the inhibitory effect of G-1. The inhibitory effects of G-1 on p- MLC levels in CASMCs were mostly restored by either ESI-09 or PKI. These results demonstrate that activation of GPER induces coronary artery relaxation via concurrent inhibition of RhoA/Rho kinase by Epac/Rap1 and PKA. GPER could be a potential drug target for preventing and treating cardiovascular diseases.

Scavenging of nitric oxide by hemoglobin in the tunica media of porcine coronary arteries

Scavenging of nitric oxide (NO) often interferes with studies on NO signaling in cell-free preparations. We observed that formation of cGMP by NO-stimulated purified soluble guanylate cyclase (sGC) was virtually abolished in the presence of cytosolic preparations of porcine coronary arteries, with the scavenging activity localized in the tunica media (smooth muscle layer). Electrochemical measurement of NO release from a donor compound and light absorbance spectroscopy showed that cytosolic preparations contained a reduced heme protein that scavenged NO. This protein, which reacted with anti-human hemoglobin antibodies, was efficiently removed from the preparations by haptoglobin affinity chromatography. The cleared cytosols showed only minor scavenging of NO according to electrochemical measurements and did not decrease cGMP formation by NO-stimulated sGC. In contrast, the column flow-through caused a nearly 2-fold increase of maximal sGC activity (from 33.1 ± 1.6 to 54.9 ± 2.2 μmol×min−1×mg−1). The proteins retained on the affinity column were identified as hemoglobin α and β subunits. The results indicate that hemoglobin, presumably derived from vasa vasorum erythrocytes, is present and scavenges NO in preparations of porcine coronary artery smooth muscle. Selective removal of hemoglobin-mediated scavenging unmasked stimulation of maximal NO-stimulated sGC activity by a soluble factor expressed in vascular tissue.

Kaempferol enhances endothelium‐dependent relaxation in the porcine coronary artery through activation of large‐conductance Ca2+‐activated K+ channels

Kaempferol, a plant flavonoid present in normal human diet, can modulate vasomotor tone. The present study aimed to elucidate the signalling pathway through which this flavonoid enhanced relaxation of vascular smooth muscle. The effect of kaempferol on the relaxation of porcine coronary arteries to endothelium-dependent (bradykinin) and -independent (sodium nitroprusside) relaxing agents was studied in an in vitro organ chamber setup. The whole-cell patch-clamp technique was used to determine the effect of kaempferol on potassium channels in porcine coronary artery smooth muscle cells (PCASMCs). At a concentration without direct effect on vascular tone, kaempferol (3 × 10(-6) M) enhanced relaxations produced by bradykinin and sodium nitroprusside. The potentiation by kaempferol of the bradykinin-induced relaxation was not affected by N(ω)-nitro-L-arginine methyl ester, an inhibitor of NO synthase (10(-4) M) or TRAM-34 plus UCL 1684, inhibitors of intermediate- and small-conductance calcium-activated potassium channels, respectively (10(-6) M each), but was abolished by tetraethylammonium chloride, a non-selective inhibitor of calcium-activated potassium channels (10(-3) M), and iberiotoxin, a selective inhibitor of large-conductance calcium-activated potassium channel (KCa 1.1; 10(-7) M). Iberiotoxin also inhibited the potentiation by kaempferol of sodium nitroprusside-induced relaxations. Kaempferol stimulated an outward-rectifying current in PCASMCs, which was abolished by iberiotoxin. The present results suggest that, in smooth muscle cells of the porcine coronary artery, kaempferol enhanced relaxations caused by endothelium-derived and exogenous NO as well as those due to endothelium-dependent hyperpolarization. This vascular effect of kaempferol involved the activation of KCa 1.1 channels.

Vanillins relax isolated porcine coronary arterial smooth muscle by inhibiting L‐type Ca2+ channels (LB574)

Vanillin (VA) and vanillyl alcohol (VAA) are major components of natural vanilla, while ethyl vanillin (EtVA) is a synthetic vanillin analog with antioxidant and anti‐inflammatory properties. This study compared the relaxing effect of vanillins (VA, VAA, EtVA) and poly(vanillin) oxalate (PVO) nanoparticles (Ø 260nm) containing VA in porcine coronary artery rings by measuring isometric tension. Vanillins but not PVO caused concentration‐dependent, endothelium‐independent and complete relaxations during contractions to U46619, KCl or endothelin‐1 with an order of potency of VAA<VA<EtVA. The relaxations of U46619 contracted preparations were unaffected by inhibitors of NOS (L‐NAME), cyclooxygenases (indomethacin), soluble guanylyl cyclase (ODQ), KCa (TRAM‐34, UCL‐1684, iberiotoxin), KATP (glibenclamide), Kir (BaCl2), and TRPV3 channels (camphor) or by antioxidants (catalase, apocynin, tempol, N‐acetylcysteine, tiron). Vanillins inhibited contractions induced by both Ca2+ reintroduction in 40mM KCl solution and BayK8644. Both VA and PVOs caused partial relaxation of XO/XOD contracted rings. In conclusion, relaxation to vanillin(s) is solely due to the inhibition of L‐type Ca2+ channels in porcine coronary arteries. Both VA and PVO can prevent contractions induced by superoxide anions, and thus could be used to relieve coronary vasospasm associated with elevated oxidative stress.Grant Funding Source: Supported by Brain Korea 21 PLUS Project, NRF and NRF‐2012M3A9C6050204

IP3 decreases coronary artery tone via activating the BKCa channel of coronary artery smooth muscle cells in pigs

Large conductance Ca2+-activated K+ channel (BKCa) is a potential target for coronary artery-relaxing medication, but its functional regulation is largely unknown. Here, we report that inositol trisphosphate (IP3) activated BKCa channels in isolated porcine coronary artery smooth muscle cells and by which decreased the coronary artery tone. Both endogenous and exogenous IP3 increased the spontaneous transient outward K+ currents (STOC, a component pattern of BKCa currents) in perforated and regular whole-cell recordings, which was dependent on the activity of IP3 receptors. IP3 also increased the macroscopic currents (MC, another component pattern of BKCa currents) via an IP3 receptor- and sarcoplasmic Ca2+ mobilization-independent pathway. In inside-out patch recordings, direct application of IP3 to the cytosolic side increased the open probability of single BKCa channel in an IP3 receptor-independent manner. We conclude that IP3 is an activator of BKCa channels in porcine coronary smooth muscle cells and exerts a coronary artery-relaxing effect. The activation of BKCa channels by IP3 involves the enhancement of STOCs via IP3 receptors and stimulation of MC by increasing the Ca2+ sensitivity of the channels.

ASSA13-03-19 Function Role on Coronary Artery Tone of IP3 by Activating IP3R and BKCa of Porcine Coronary Artery Smooth Muscle Cells

<h3>Background and Objective</h3> The large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel mediates coronary relaxation. However, endogenous factors that regulate BK_Ca channel activities are not well defined. The present study sought to test the hypothesis that inositol trisphosphate (IP3) is an activator of BK_Ca channels in coronary smooth muscle cells (SMCs) and therefore would relax the coronary tone. <h3>Methods and Results</h3> Porcine coronary artery rings were prepared to perform the vasomotor assay. The whole-cell voltage clamp and single-channel patch clamp experiments were performed in freshly isolated coronary SMCs to test the effect of IP3 on BK_Ca channels and to figure out the underlying mechanisms. The results showed that blocking IP3 receptor (IP3R) with xestospongin-C (XeC) increased the tension of coronary rings. After pre-constricting with 0.1 μM PGF2α, the use of XeC (2 μM) induced a further increase of LAD tension by 26.10 ± 5.73% (n = 4) of that induced by PGF2α, and this effect of XeC could be abolished by pretreatment with IbTX, a specific blocker of BK_Ca. In the perforated whole-cell recordings, in which intracellular IP3 leak cannot occur, our data confirmed that IP3 had a physiological contribution to STOC generation (approximately 30% of the STOCs are XeC-sensitivive). Under traditional whole-cell recording, introduction of exogenous IP3 (0.5–50 μM) from the pipette solution into the cytoplasm increased the XeC-sensitive STOC roughly by 30% to 50% and this effect could be abolished by XeC, suggesting that exogenous IP3 stimulates the STOC via IP3R. However, it is likely that IP3 impacts the macroscopic current (MC, another component of BKCa currents) by a differential signalling pathway. Although IP3 increased the MC, this effect could be abolished neither by XeC, nor by pretreating with 1μM thapsigargin (a depletor of SR Ca2 + store), indicating an IP3R-independent way was involved. Interestingly, in the inside-out single channel recordings, application of IP3 (10–50 μM) to the cytosolic side of the membrane increased the single BK_Ca channel currents likely by enhancing the Ca²⁺ sensitivity of BK_Ca channel and as a result, changed the channel kinetics. Further, we identified that these effects were IP3R-independent. <h3>Conclusions</h3> We conclude that IP3 is an activator of BK_Ca channels in the porcine coronary SMCs and can relax coronary tone.

Decreased Expression of Vitamin D Receptors in Neointimal Lesions following Coronary Artery Angioplasty in Atherosclerotic Swine

BackgroundInflammatory cytokines, such as TNF-α, play a key role in the pathogenesis of occlusive vascular diseases. Activation of vitamin D receptors (VDR) elicits both growth-inhibitory and anti-inflammatory effects. Here, we investigated the expression of TNF-α and VDR in post-angioplasty coronary artery neointimal lesions of hypercholesterolemic swine and examined the effect of vitamin D deficiency on the development of coronary restenosis. We also examined the effect of calcitriol on cell proliferation and effect of TNF-α on VDR activity and expression in porcine coronary artery smooth muscle cells (PCASMCs) in-vitro.Methodology/Principal FindingsExpression of VDR and TNF-α and the effect of vitamin D deficiency in post-angioplasty coronary arteries were analyzed by immunohistochemistry and histomorphometry. Cell proliferation was examined by thymidine and BrdU incorporation assays in cultured PCASMCs. Effect of TNF-α-stimulation on the activity and expression of VDR was analyzed by luciferase assay, immunoblotting and immunocytochemistry. In-vivo, morphometric analysis of the tissues revealed typical lesions with significant neointimal proliferation. Histological evaluation showed expression of smooth muscle α-actin and significantly increased expression of TNF-α in neointimal lesions. Interestingly, there was significantly decreased expression of VDR in PCASMCs of neointimal region compared to normal media. Indeed, post-balloon angioplasty restenosis was significantly higher in vitamin D-deficient hypercholesterolemic swine compared to vitamin D-sufficient group. In-vitro, calcitriol inhibited both serum- and PDGF-BB-induced proliferation in PCASMCs and TNF-α-stimulation significantly decreased the expression and activity of VDR in PCASMCs.Conclusions/SignificanceThese data suggest that significant downregulation of VDR in proliferating smooth muscle cells in neointimal lesions could be due to atherogenic cytokines, including TNF-α. Vitamin D deficiency potentiates the development of coronary restenosis. Calcitriol has anti-proliferative properties in PCASMCs and these actions are mediated through VDR. This could be a potential mechanism for uncontrolled growth of neointimal cells in injured arteries leading to restenosis.

Involvement of Inositol 1,4,5-Trisphosphate Formation in the Voltage-Dependent Regulation of the Ca2+Concentration in Porcine Coronary Arterial Smooth Muscle Cells

The involvement of inositol 1,4,5-trisphosphate (IP(3)) formation in the voltage-dependent regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) was examined in smooth muscle cells of the porcine coronary artery. Slow ramp depolarization from -90 to 0 mV induced progressive [Ca(2+)](i) increase. The slope was reduced or increased in the presence of Cd(2+) or (±)-1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-[trifluoromethyl]-phenyl)pyridine-3-carboxlic acid methyl ester (Bay K 8644), respectively. The decrease in [Ca(2+)](i) via the membrane hyperpolarization induced by K(+) channel openers (levcromakalim and Evans blue) under current clamp was identical to that under voltage clamp. The step hyperpolarization from -40 to -80 mV reduced [Ca(2+)](i) uniformly over the whole-cell area with a time constant of ∼10 s. The [Ca(2+)](i) at either potential was unaffected by heparin, an inhibitor of IP(3) receptors. Alternatively, [Ca(2+)](i) rapidly increased in the peripheral regions by depolarization from -80 to 0 mV and stayed at that level (∼400 nM) during a 60-s pulse. When the pipette solution contained IP(3) pathway blockers [heparin, 2-aminoethoxydiphenylborate, xestospongin C, or 1-[6-[((17β)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione (U73122)], the peak [Ca(2+)](i) was unchanged, but the sustained [Ca(2+)](i) was gradually reduced by ∼250 nM within 30 s. In the presence of Cd(2+), a long depolarization period slightly increased the [Ca(2+)](i), which was lower than that in the presence of heparin alone. In coronary arterial myocytes, the sustained increase in the [Ca(2+)](i) during depolarization was partly caused by the Ca(2+) release mediated by the enhanced formation of IP(3). The initial [Ca(2+)](i) elevation triggered by the Ca(2+) influx though voltage-dependent Ca(2+) channels may be predominantly responsible for the activation of phospholipase C for IP(3) formation.

Melatonin inhibits NO‐dependent activation of large conductance, calcium‐activated K channels (BKCa) in porcine coronary artery smooth muscle cells

We recently demonstrated that melatonin inhibits NO‐induced relaxation of porcine coronary arteries by activating MT2 melatonin‐receptors (JPET 336: 127, 2011). We tested the hypothesis that melatonin inhibits NO/cGMP‐dependent activation of BKCa channels in coronary artery smooth muscle cells. Melatonin (10−7M) inhibited sodium nitroprusside (SNP)‐induced relaxation of isolated coronary arteries but had no effect on relaxation induced by 8‐Br‐cGMP, a stable, cell permeable cGMP analog. ODQ (10−5 M) completely inhibited SNP‐induced relaxation, as well as the increase in intracellular cGMP levels in response to SNP. In patch clamp studies, SNP (10−5 M)‐induced increases in BKCa currents were abolished in the presence of ODQ (10−5 M). Melatonin (10−7M) inhibited both the voltage‐dependent and SNP‐induced increase in BKCa currents, but had no effect on BKCa currents elicited by 8‐Br‐cGMP (10−3M). The inhibitory effect of melatonin on the voltage‐dependent and SNP‐induced increase in BKCa currents was abolished by either the PDE inhibitor, zaprinast (10−5M), or the MT2‐receptor antagonist, 4P‐PDOT (10−7 M). Coronary artery relaxation in response to NO involves the cGMP‐dependent activation of BKCa channels. Melatonin impairs NO‐induced relaxation of porcine coronary arteries by inhibiting the cGMP‐dependent activation of BKCa channels in coronary smooth muscle cells. (Supported by NIH HL77204)

Abstract 16932: Melatonin Inhibits Large Conductance, Calcium-Activated K Channel Activity in Porcine Coronary Artery Smooth Muscle Cells by Activating MT 2 -Receptors

Introduction: Increasing evidence indicates that melatonin plays a role in cardiovascular homeostasis by regulating blood vessel diameter, thereby impacting arterial blood pressure and local blood flow to organs and tissues. Alterations in circulating melatonin levels are associated with several cardiovascular disorders, including hypertension, ischemic heart disease, myocardial infarction, and heart failure. We have shown previously that melatonin impairs nitric oxide (NO)-induced relaxations in porcine coronary arteries by activating melatonin type-2 (MT 2 )-receptors (JPET 336: 127-33, 2011). Hypothesis: Melatonin impairs NO-induced relaxation of coronary arteries by inhibiting BK Ca channel activity in coronary artery smooth muscle cells (SMC). Methods and Results: Sodium nitroprusside (SNP), an exogenous NO-donor, caused relaxation of isolated porcine coronary arteries (-logEC 50 = 7.49 + 0.1; n=5). In the presence of either melatonin (10 -7 M) or iberiotoxin (IbTx; 10 -7 M), a selective BK Ca -channel blocker, the SNP dose-response was shifted to the right by approximately 6-fold (p&lt;0.05). The presence of BK Ca α- and β-subunits was identified by immunoblot analysis in intact porcine coronary arteries. Immunocytochemistry studies demonstrated that both subunits co-localized with α-actin in isolated single coronary SMC. Whole-cell K currents (10-mV steps from -70mV to +60mV, 200 ms duration) were obtained in freshly isolated coronary SMC in the absence or presence of IbTx (10 -7 M) or SNP (10 -5 M) (n=5-6). IbTx markedly inhibited (p&lt;0.05) both the voltage-dependent activation of whole-cell K currents and the SNP-induced increase in BK Ca currents in coronary SMC. Treatment of coronary SMC with melatonin (10 -7 M) inhibited the activation of BK Ca currents (56.8 ± 2.9% inhibition of peak current density at +60 mV; n=12; p&lt;0.05) and shifted the I-V curve to the right, as compared to control. The inhibitory effect of melatonin (10 -7 M) on BK Ca currents was abolished in the presence of the selective MT 2 -receptor antagonist, 4P-PDOT (10 -7 M). Conclusions: Melatonin acts via MT 2 -receptors to inhibit BK Ca activity in porcine coronary SMC, suggesting a mechanistic basis for the inhibitory effect of melatonin on NO-induced relaxation.

Suppressor of cytokine signaling-3 and intimal hyperplasia in porcine coronary arteries following coronary intervention

Aims The growth and differentiation of cells is regulated by cytokines by binding to cell-surface receptors and activating intracellular signal transduction cascade. Suppressor of cytokine signaling (SOCS)-3 is a negative regulator of cytokines. In this study we examined the expression of SOCS-3 in porcine coronary artery smooth muscle cells (PCASMCs) in vitro and in proliferating smooth muscle cells of neointimal lesions after coronary artery intervention in a swine model. Methods and results PCASMCs were cultured and stimulated with TNF-α and/or IGF-1 individually or in combination. Protein expression of SOCS-3 was examined using Western blot. For in vivo studies, six female Yucatan miniswine were fed with special high cholesterol diet for 8 months. At 4 months of high cholesterol diet, animals underwent coronary balloon angioplasty. At the end of 8 months animals were euthanized, coronary arteries were isolated and morphological and histological studies were performed. Western blot data revealed significantly high SOCS-3 expression in PCASMCs in the presence of either TNF-α or IGF-1 (5–6 fold) alone. However, in the presence of both TNF-α and IGF-1 the SOCS-3 expression was significantly decreased (4–5 fold). Results from morphological studies including, H&E and Masson's trichrome stain showed typical lesions with significant neointimal proliferation. Histological evaluation showed expression of smooth muscle α-actin and significantly increased proliferating cell nuclear antigen (PCNA) in neointimal lesion. Interestingly, there was significantly decreased expression of SOCS-3 in smooth muscle cells of neointima as compared to control. Conclusions These data suggest that SOCS-3 expression is decreased in proliferating smooth muscle cells of neointimal lesions. This leads to uncontrolled growth of vascular smooth muscle cells in injured arteries leading to restenosis. Therefore, local delivery of SOCS-3 gene at the site of injury after coronary artery intervention could regulate the proliferation of vascular smooth muscle cells and help in preventing the neointimal hyperplasia and restenosis.

Intrinsic circadian oscillation of myosin light chain phosphorylation in vascular smooth muscle cells

The onset of coronary artery disease, such as myocardial infarction, shows an apparent circadian variation. However, its molecular mechanism still remains to be elucidated. This study investigated the role of intrinsic vascular clock in the circadian regulation of vascular contractility. The porcine coronary artery smooth muscle cells in culture were exposed to 100 nM dexamethosone for 2 hr (Dex shock) to induce a circadian rhythm. The response of the myosin light chain (MLC) phosphorylation to the contractile stimuli was evaluated as an index of vascular contractility. A real‐time PCR analysis revealed a circadian oscillation of the expression of clock genes, such as bmal1 and per1. The resting level of MLC phosphorylation did not significantly change after DEX shock. In contrast, the level of MLC phosphorylation induced by thrombin exhibited a circadian oscillation with a 25.3‐hr cycle, reaching peaks at 36 hr and 60 hr. MLC phosphorylation changed from 0.85 at a nadir to 1.05 PO4 mol/MLC mol at a peak. However, there was no change in the level of thrombin receptor. The thrombin‐induced contraction of smooth muscle cells also exhibited a circadian oscillation after DEX shock. The results demonstrated the existence of an intrinsic circadian oscillation of the response of MLC phosphorylation. The present study thus suggests the vascular clock to contribute to the circadian regulation of vascular contractility.

Endothelium-Derived Nitric Oxide Inhibits the Relaxation of the Porcine Coronary Artery to Natriuretic Peptides by Desensitizing Big Conductance Calcium-Activated Potassium Channels of Vascular Smooth Muscle

The present experiments investigated whether endothelium-derived mediators modulate the effect of natriuretic peptides in porcine coronary arteries. Rings with and without endothelium were suspended in organ chambers for isometric tension recording. Concentration-relaxation curves to C-type natriuretic peptide (CNP) and atrial natriuretic peptide (ANP) were obtained during contractions to endothelin-1. Removal of the endothelium potentiated relaxations to both CNP and ANP. N(omega)-nitro-L-arginine methyl ester potentiated relaxations to natriuretic peptides only in arteries with endothelium. Sodium nitroprusside (SNP) inhibited the response to the natriuretic peptides only in the absence of the endothelium. In rings with endothelium, 1H-[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ) and 4H-8-bromo-1,2,4-oxadiazolo[3,4-d]benz[b][1,4]oxazin-1-one (NS2028) potentiated CNP-mediated relaxations. Iberiotoxin (IBTX) reduced the response only in rings without endothelium. Glybenclamide inhibited the relaxations in both the presence and absence of endothelium. CNP-induced relaxations were reduced by 8-bromoguanosine 3',5'-cGMP (8-bromo-cGMP) to the same extent in rings with and without endothelium. There was no significant difference between the increased cGMP content caused by CNP in porcine coronary arteries with or without endothelium. In patch-clamp studies in porcine coronary arterial smooth muscle cells, the natriuretic peptide-mediated enhancement of the IBTX-sensitive big conductance calcium-activated potassium channel (BK(Ca)) amplitude was reversed by SNP and 8-bromo-cGMP. These findings demonstrate that, in the porcine coronary artery, the opening of BK(Ca) and ATP-dependent potassium channels of the vascular smooth muscle contributes to CNP-mediated relaxations. Endothelium-derived and exogenous NO inhibit the direct relaxing effect of natriuretic peptides by desensitizing the response of the BK(Ca)s of the vascular smooth muscle to the generation of cGMP.