Canine Pulmonary Artery Research Articles

Sympathetic innervation of canine pulmonary artery and morphometric and functional analysis in dehydromonocrotaline-induced models after pulmonary artery denervation.

We aimed to describe the anatomic distribution of periarterial pulmonary sympathetic nerves and to observe the long-term morphometric and functional changes after pulmonary artery denervation (PADN), a novel therapy for pulmonary arterial hypertension (PAH). A total of 45 beagles were divided into a sympathetic innervation group (n = 3, 33.3% were females), a PAH group (n = 35, 34.3% were females) and a control group (n = 7, 28.5% were females). The PAH group was randomly divided into no-PADN (n = 7), instant-PADN (n = 7), 1M-PADN (n = 7), 2M-PADN (n = 7) and 3M-PADN (n = 7) subgroups. The sympathetic innervation group was sacrificed to reveal the sympathetic innervation of pulmonary arteries. PAH was induced by injecting dehydromonocrotaline (DHMCT) through the right atrium. The pulmonary capillary wedge pressure, right ventricular systolic pressure, right ventricular mean pressure, pulmonary artery systolic pressure and pulmonary artery mean pressure of each group were continuously measured. The cardiac output was detected to calculate the pulmonary vascular resistance. PAH and control groups were subjected to immunofluorescence assay, sympathetic nerve conduction velocity measurement and transmission electron microscopy. The no-PADN group had significantly higher PVSP, PVMP, pulmonary artery systolic pressure, pulmonary artery mean pressure and pulmonary vascular resistance but lower cardiac output than those of the control group (P < 0.05). Instant-PADN, 1M-PADN, 2M-PADN and 3M-PADN groups had significantly lower PVSP, PVMP, pulmonary artery systolic pressure, pulmonary artery mean pressure and pulmonary vascular resistance but higher cardiac output than those of the no-PADN group (P < 0.05). Most sympathetic nerves were located within 2.5 mm of the intimae of the bifurcation and proximal trunk, mainly in the left trunk. The diameter and cross-sectional area of myelinated fibres in the PAH group were significantly larger than those of the control group. Sympathetic nerve conduction velocity of the PAH group gradually decreased, and nerve fibres were almost demyelinated 3 months after PADN. PADN effectively relieved dehydromonocrotaline-induced canine PAH and decreased sympathetic nerve conduction velocity.

Selection of the appropriate reference genes for relative quantitative reverse transcription polymerase chain reaction (qRT-PCR) in canine pulmonary arteries

Selection of the appropriate reference genes for relative quantitative reverse transcription polymerase chain reaction (qRT-PCR) in canine pulmonary arteries

Comparison of two ultrasonic coagulating shears in sealing pulmonary vessels

Ultrasonic cutting and coagulating devices have been used successfully in thoracic applications such as pulmonary resection or artery harvesting, but few studies have evaluated their use in sealing pulmonary vessels. In this study we compared two commercially available devices, Harmonic Ace+ (HAR, Ethicon Endo-Surgery, Inc., Cincinnati OH, USA) and Sono- Surg (SS, Olympus America, Center Valley, PA, USA), in a canine preclinical model. There were three sections to the study: acute, survival, and ex vivo (burst pressure). Hemostasis of sealed pulmonary arteries and veins was assessed for the initial application and during a simu- lated hypertensive crisis, both immediately after vessel sealing and after a survival period of 30 days. Other intraoperative measures were also evaluated, including transection time, tissue sticking, tissue tags, and char on the seal. Histological evaluation was performed both after initial sealing and after the survival period. Burst pressure of sealed vessels was measured ex vivo. For both devices, hemostasis was excellent, including those measurements made under simulated hypertensive crisis. There were no differences in any of the intraoperative measures or thermal damage evaluated histologically. Wound healing was normal. The burst pressures for ex vivo vessels sealed by HAR (median 619.2 mmHg) were significantly higher than those of SS (350.3 mmHg, P = 0.022). Both devices displayed acceptable characteristics in sealing canine pulmonary arteries and veins. The only difference observed was that HAR produced burst pressures 76.8% greater than SS, which may lead to a lower percentage of failures in the region of physiological interest. Use of ultrasonic devices in thoracic applications provides a high rate of initial hemostasis, supraphysiological burst pressures, and durable seals that heal normally.

Effect of contrast medium injection duration on peak enhancement of canine pulmonary arteries

Our goal was to investigate the effect of contrast medium injection duration on pulmonary artery peak enhancement and time to peak enhancement. Fourteen dogs were allocated into one of seven predefined weight categories, each category contained two dogs. Dogs in each weight category were assigned to group A or B. Animals in each group received a different contrast medium injection protocol. In group A, a fixed injection rate of 5ml/s was used. In group B, the contrast injection rate was calculated as follows: flow rate1⁄4contrast volume/scan durationþ10s. Time to peak enhancement and peak enhancement of the main left and right pulmonary arteries were measured on single-level, dynamic CT images for a fixed time of 30 s. Rank correlation (Spearman’s) coefficients between injection duration and time to peak enhancement and between body weight and peak enhancement were calculated. For group A, there was a significant negative correlation between peak enhancement and weight (r1⁄4

Cell culture alters Ca2+ entry pathways activated by store-depletion or hypoxia in canine pulmonary arterial smooth muscle cells

Previous studies have shown that, in acutely dispersed canine pulmonary artery smooth muscle cells (PASMCs), depletion of both functionally independent inositol 1,4,5-trisphosphate (IP(3))- and ryanodine-sensitive Ca(2+) stores activates capacitative Ca(2+) entry (CCE). The present study aimed to determine if cell culture modifies intracellular Ca(2+) stores and alters Ca(2+) entry pathways caused by store depletion and hypoxia in canine PASMCs. Intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured in fura 2-loaded cells. Mn(2+) quench of fura 2 signal was performed to study divalent cation entry, and the effects of hypoxia were examined under oxygen tension of 15-18 mmHg. In acutely isolated PASMCs, depletion of IP(3)-sensitive Ca(2+) stores with cyclopiazonic acid (CPA) did not affect initial caffeine-induced intracellular Ca(2+) transients but abolished 5-HT-induced Ca(2+) transients. In contrast, CPA significantly reduced caffeine- and 5-HT-induced Ca(2+) transients in cultured PASMCs. In cultured PASMCs, store depletion or hypoxia caused a transient followed by a sustained rise in [Ca(2+)](i). The transient rise in [Ca(2+)](i) was partially inhibited by nifedipine, whereas the nifedipine-insensitive transient rise in [Ca(2+)](i) was inhibited by KB-R7943, a selective inhibitor of reverse mode Na(+)/Ca(2+) exchanger (NCX). The nifedipine-insensitive sustained rise in [Ca(2+)](i) was inhibited by SKF-96365, Ni(2+), La(3+), and Gd(3+). In addition, store depletion or hypoxia increased the rate of Mn(2+) quench of fura 2 fluorescence that was also inhibited by these blockers, exhibiting pharmacological properties characteristic of CCE. We conclude that cell culture of canine PASMCs reorganizes IP(3) and ryanodine receptors into a common intracellular Ca(2+) compartment, and depletion of this store or hypoxia activates voltage-operated Ca(2+) entry, reverse mode NCX, and CCE.

Role of InsP3 and ryanodine receptors in the activation of capacitative Ca2+ entry by store depletion or hypoxia in canine pulmonary arterial smooth muscle cells

Experiments were performed to determine if capacitative Ca(2+) entry (CCE) in canine pulmonary arterial smooth muscle cells (PASMCs) is dependent on InsP(3) receptors or ryanodine receptors as induction of CCE is dependent on simultaneous depletion of the functionally separate InsP(3)- and ryanodine-sensitive sarcoplasmic reticulum (SR) Ca(2+) stores in these cells. Myocytes were isolated from canine pulmonary arteries using enzymatic procedures and were used within 8 h of preparation. Measurements of cytosolic Ca(2+) were made by imaging fura-2 loaded individual myocytes that were perfused with physiological buffered saline solution with or without Ca(2+). Treating myocytes with 10 microM cyclopiazonic acid (CPA), removing extracellular Ca(2+), and briefly applying 10 mM caffeine and 10 microM 5-hydroxytryptamine (5-HT) depleted SR Ca(2+) stores. Extracellular Ca(2+) reintroduction caused cytosolic [Ca(2+)] to elevate above baseline signifying CCE. The InsP(3) receptor inhibitors 2-aminobiphenylborate (50-75 microM; 2-APB) and xestospongin-C (20 microM; XeC) abolished CCE. Yet, CCE was unaffected by 10 microM or 300 microM ryanodine or 10 microM dantrolene, which modify ryanodine receptor activity. Higher dantrolene concentrations (50 microM), however, can inhibit both ryanodine receptors and InsP(3) receptors, did reduce CCE. In contrast, CCE activated by hypoxia was unaffected by XeC (20 microM). The results provide evidence that CCE activated by depletion of both InsP(3) and ryanodine SR Ca(2+) stores in canine PASMCs is dependent on functional InsP(3) receptors, whereas the activation of CCE by hypoxia appears to be independent of functional InsP(3) receptors.

Reorganization of Intracellular Ca 2+ Stores Caused by Cell Culture Does Not Prevent Hypoxic‐Induced Rise in [Ca 2+ ] i in Canine Pulmonary Arterial Smooth Muscle Cells

We previously showed in acutely isolated canine pulmonary artery smooth muscle cells (PASMCs) that the IP3-sensitive and ryanodine-sensitive Ca2+ stores are functionally independent and hypoxia causes rise in [Ca2+]i through mobilization of the intracellular Ca2+ stores leads to consequent activation of voltage–operated Ca2+ entry and capacitative Ca2+ entry. The aim of the present study was to determine if cell culture changes organization of intracellular Ca2+ stores and whether the change could affect hypoxic-induced rise in [Ca2+]i in canine PASMCs. In acutely isolated PASMCs loaded with fura-2, CPA abolished 5HT-induced intracellular Ca2+ transient but had no effect on the initial caffeine-induced intracellular Ca2+ transient. In contrast, CPA significantly reduced 5HT- and caffeine-induced intracellular Ca2+ transient in cultured PASMCs. In 2mM Ca2+-solution, hypoxia caused a rise in [Ca2+]i but it was abolished when extracellular Ca2+ was removed. Subsequent addition of 2mM Ca2+ elicited a transient rise in [Ca2+]i that was partially inhibited by 10μM nifedipine, leaving a nifedipine-insensitive transient and nifedipine-insensitive sustained rise in [Ca2+]i. These results suggest that cell culture of PASMCs results in reorganization of IP3-sensitive and ryanodine-sensitive stores and this does not prevent hypoxic-induced rise in [Ca2+]i. (Supported by HL49254 and NCRR P20RR15581)

Prolonged relaxation consistent with persistent soluble guanylyl cyclase activation in canine pulmonary artery following brief treatment with nitric oxide donors

Recent work has indicated that prolonged treatment with nitric oxide (NO) donors results in tissue storage of NO as S-nitrosothiols and N-nitrosamines. The possibility thus exists that NO treatment may result in the development of tissue stores of NO with functionally significant effects following removal of the original NO source. In these studies, the effects of 10 min treatment with two chemically distinct NO sources, S-nitrosoglutathione (GSNO) and ( Z)-1-( N, N-diethylamino)diazen-1-ium-1,2-diolate (DEA-NO) were determined in canine pulmonary artery using a superfusion system that permitted continuous isometric force recording during addition and removal of the NO donors. Relaxation that persisted for up to 1 h after removal of the NO source, was demonstrated for both NO sources, but at lower concentrations relative to the relaxant EC 50 for GSNO versus DEA-NO. Persistent relaxation with both NO sources was fully reversed by both the sGC inhibitor, ODQ, and an inhibitor of cGMP-dependent protein kinase, Rp-8-Br-PET-cGMPS, indicating that persistent relaxation was consistent with persistent activation of the sGC-cGMP signaling pathway. In separate measurements, a GSNO-induced persistent increase in both tissue cGMP ([cGMP] i) and relaxation were fully reversed by both ODQ and the thiol reducing agent dithiothreitol (DTT). The results indicate that vascular smooth muscle is capable of converting short-lived NO responses following short term exposure to NO donors by a mechanism consistent with prolonged sGC activation, resulting in persistent relaxation. Reversal of this cGMP-dependent process with DTT suggests that it occurs via mechanisms that are thiol redox sensitive.

Serotonin-Induced Inhibition of K V Current

See related article, pages 931–938 Pulmonary vascular tone is largely determined by potassium and calcium currents in the smooth muscle cells (SMCs) of the small resistance arteries. Under normoxic circumstances an outward potassium current ( I K) passes through voltage-gated (Kv), calcium-activated (KCa), and background tandem-pore domain (K2P) potassium channels. The latter (coded by the KCNK family of genes) include the twik-related acid sensitive K+ channel TASK and TASK-like channels. This current keeps the resting membrane potential in the range of −50 to −60 mV and inhibits calcium entry through L-type calcium channels.1 Vasodilator substances such as nitric oxide and prostacyclin, released from the endothelium, increase I K, one of several actions that lead to further vasodilatation.2,3 Vasoconstrictor substances, such as endothelin, can inhibit I K4 and/or release calcium from the sarcoplasmic reticulum.5 In addition to vasoactive effects, an increase in cytosolic potassium inhibits smooth muscle cell apoptosis,6 and an increase in cytosolic calcium promotes cellular proliferation.7 Consequently, the inhibition of I K is important not only in causing membrane depolarization and calcium entry but also in stimulating vascular remodeling and in the development of chronic pulmonary hypertension. Agents that can cause pulmonary hypertension, such as the anorectic drugs and hypoxia, inhibit I K8,9 and also enhance calcium release from the sarcoplasmic reticulum, leading to subsequent repletion of calcium through store-operated channels.10,11,12 The smooth muscle cells in the resistance pulmonary arteries of patients with pulmonary arterial hypertension (PAH) exhibit both reduced I K and decreased expression of Kv channels,13 and also increased expression of the TRPC ( t ransient r eceptor p otential, c anonical) genes that code for store-operated and receptor-operated calcium channels.14 The decreased expression and function of the Kv channels may …

Immobilization of biomacromolecules on poly-L-lactide surface via a layer-by-layer method for the improving of its cytocompatibility to bone marrow stromal cells

Hyaluronic acid (HA) and chitosan (CS) were immobilized on the surface of poly-L-lactide (PLLA) by the following procedure: Firstly, PLLA was aminolyzed with 1, 6-hexanediamine, and part of the PLLA surface ester groups were converted to free amino groups. Then negatively charged hyaluronic acid and positively charged chitosan were deposited onto the surface of aminolyzed PLLA film in a layer-by-layer assembly manner. The effect of the layer-by-layer deposition was evaluated by ATR-FTIR spectroscopy, Raman spectroscopy and static contact angle measurements. The cytocompatibility of PLLA sample to bone marrow stromal cells (BMSCs) was improved after modification with chitosan and HA. The cell attachment, activity, and proliferation on CS/HA modified PLLA films were enhanced comparing with the control. The cells cultured on the modified PLLA samples excreted abundant cytoplasm and can differentiate to vascular smooth muscle (SM)-like (SM-like) cells. A macroporous three-dimensional PLLA scaffold was prepared by integrating both the technique of freeze-drying and particle leaching. Layer-by-layer modification by HA/CS and cell culture was also applied on this scaffold. The scaffold cultured with BMSCs for 2 weeks has been tested successfully in vivo as a patch for repairing the artificial incision on canine pulmonary artery.

NO responsiveness in pulmonary artery and airway smooth muscle: the role of cGMP regulation

The purpose of this study was to assess intrinsic smooth muscle mechanisms contributing to greater nitric oxide (NO) responsiveness in pulmonary vascular vs. airway smooth muscle. Canine pulmonary artery smooth muscle (PASM) and tracheal smooth muscle (TSM) strips were used to perform concentration response studies to an NO donor, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO). PASM exhibited a greater NO responsiveness whether PASM and TSM were contracted with receptor agonists, phenylephrine and acetylcholine, respectively, or with KCl. The >10-fold difference in NO sensitivity in PASM was observed with both submaximal and maximal contractions. This difference in NO responsiveness was not due to differences in endothelial or epithelial barriers, since these were removed, nor was it due to the presence of cGMP-independent NO-mediated relaxation in either tissue. At equal concentrations of NO, the intracellular cGMP concentration ([cGMP]i) was also greater in PASM than in TSM. Phosphodiesterase (PDE) inhibition using isobutylmethylxanthine indicated that the greater [cGMP]i in PASM was not due to greater PDE activity in TSM. Expression of soluble guanylate cyclase (sGC) subunit mRNA (2 +/- 0.2 and 1.3 +/- 0.2 attomol/microg total RNA, respectively) and protein (47.4 +/- 2 and 27.8 +/- 3.9 ng/mg soluble homogenate protein, respectively) was greater in PASM than in TSM. sGCalpha1 and sGCbeta1 mRNA expression was equal in PASM but was significantly different in TSM, suggesting independent regulation of their expression. An intrinsic smooth muscle mechanism accounting for greater NO responsiveness in PASM vs. TSM is greater sGC activity.

Fentanyl Attenuates α1B-Adrenoceptor–Mediated Pulmonary Artery Contraction

The authors tested the hypothesis that the intravenous anesthetic fentanyl would attenuate the pulmonary vasoconstrictor response to alpha1-adrenoceptor activation. They also investigated the alpha1-adrenoceptor subtypes that could potentially mediate this effect of fentanyl. Endothelium-denuded canine pulmonary arterial rings were suspended for isometric tension recording. Dose-response curves for the alpha1-adrenoceptor agonist phenylephrine were generated in the absence and presence of fentanyl. The effects of inhibiting alpha2 (rauwolscine), alpha1 (prazosin), alpha1A (5-methylurapidil), alpha1B (chloroethylclonidine), and alpha1D (BMY 7378) adrenoceptors on phenylephrine contraction were also investigated. Receptor "protection" studies were performed to investigate the specific role of alpha1B adrenoceptors in mediating fentanyl-induced changes in phenylephrine contraction. Finally, competition binding studies were performed in rat-1 fibroblasts stably transfected with human alpha1-adrenoceptor complementary DNAs corresponding to the alpha1A-, alpha1B-, or alpha1D-adrenoceptor subtypes to directly assess whether fentanyl can compete for the alpha1-adrenoceptor activation pocket. Fentanyl attenuated phenylephrine contraction in a dose-dependent fashion. Rauwolscine had no effect on phenylephrine contraction. Phenylephrine contraction was inhibited by prazosin and abolished by chloroethylclonidine but was relatively resistant to inhibition by 5-methylurapidil and BMY 7378. Pretreatment with fentanyl before exposure to chloroethylclonidine increased the maximal contractile response to phenylephrine compared to chloroethylclonidine pretreatment alone. Competition binding studies revealed that fentanyl binds to all three alpha1-adrenoceptor subtypes, with a fivefold greater affinity for the alpha1B-adrenoceptor compared with the alpha1D-adrenoceptor subtype. Phenylephrine-induced contraction is primarily mediated by alpha1B-adrenoceptor activation in canine pulmonary artery. Fentanyl attenuates phenylephrine contraction by binding to alpha1B adrenoceptors.

ClC‐3 chloride channel is upregulated by hypertrophy and inflammation in rat and canine pulmonary artery

Cl- channels have been implicated in essential cellular functions including volume regulation, progression of cell cycle, cell proliferation and contraction, but the physiological functions of the ClC-3 channel are controversial. We tested the hypothesis that the ClC-3 gene (ClCn-3) is upregulated in hypertensive pulmonary arteries of monocrotaline-treated rats, and upregulated ClC-3 channel aids viability of pulmonary artery smooth muscle cells (PASMCs). Experimental pulmonary hypertension was induced in rats by a single subcutaneous administration of monocrotaline (60 mg kg(-1)). Injected animals developed characteristic features of pulmonary hypertension including medial hypertrophy of pulmonary arteries and right ventricular hypertrophy. Reverse transcriptase-polymerase chain reaction (RT-PCR), immunohistochemistry and Western immunoblot analysis indicated that histopathological alterations were associated with upregulation of the ClC-3 mRNA and protein expression in both smooth muscle cells of hypertensive pulmonary arteries and in cardiac myocytes. RT-PCR analysis of mRNA, extracted from canine cultured PASMCs, indicated that incubation with the inflammatory mediators endothelin-1 (ET-1), platelet-derived growth factor (PDGF), interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF alpha), but not transforming growth factor beta (TGFbeta), upregulated ClC-3 mRNA. Adenovirus-mediated delivery and overexpression of ClC-3 in canine PASMCs improved cell viability against increasing concentrations of hydrogen peroxide (H2O2, range 50-250 microM). In conclusion, upregulation of ClC-3 in rat hypertensive lung and heart is a novel observation. Our functional data suggest that upregulation of ClC-3 is an adaptive response of inflamed pulmonary artery, which enhances the viability of PASMCs against reactive oxygen species.

Mobilization of sarcoplasmic reticulum stores by hypoxia leads to consequent activation of capacitative Ca2+ entry in isolated canine pulmonary arterial smooth muscle cells

Capacitative Ca2+ entry (CCE) has been speculated to contribute to Ca2+ influx during hypoxic pulmonary vasoconstriction (HPV). The aim of the present study was to directly test if acute hypoxia causes intracellular Ca2+ concentration ([Ca2+]i) rises through CCE in canine pulmonary artery smooth muscle cells (PASMCs). In PASMCs loaded with fura-2, hypoxia produced a transient rise in [Ca2+]i in Ca2+-free solution, indicating Ca2+ release from the intracellular Ca2+ stores. Subsequent addition of 2 mm Ca2+ in hypoxia elicited a sustained rise in [Ca2+]i, which was partially inhibited by 10 microm nisoldipine. The dihydropyridine-insensitive rise in [Ca2+]i was due to increased Ca2+ influx, because it was abolished in Ca2+-free solution and hypoxia was shown to significantly enhance the rate of Mn2+ quench of fura-2 fluorescence. The dihyropyridine-insensitive rise in [Ca2+]i and the increased rate of Mn2+ quench of fura-2 fluorescence were inhibited by 50 microm SKF 96365 and 500 microm Ni2+, but not by 100 microm La3+ or 100 microm Gd3+, exhibiting pharmacological properties characteristic of CCE. In addition, predepletion of the intracellular Ca2+ stores inhibited the rise in [Ca2+]i induced by hypoxia. These results provide the first direct evidence that acute hypoxia, by causing Ca2+ release from the intracellular stores, activates CCE in isolated canine PASMCs, which may contribute to HPV.

Role of basal extracellular Ca2+ entry during 5‐HT‐induced vasoconstriction of canine pulmonary arteries

1. Measurements of artery contraction, cytosolic [Ca(2+)], and Ca(2+) permeability were made to examine contractile and cytosolic [Ca(2+)] responses of canine pulmonary arteries and isolated cells to 5-hydroxytryptamine (5-HT), and to determine the roles of intracellular Ca(2+) release and extracellular Ca(2+) entry in 5-HT responses. 2. The EC(50) for 5-HT-mediated contractions and cytosolic [Ca(2+)] increases was approximately 10(-7) M and responses were inhibited by ketanserin, a 5-HT(2A)-receptor antagonist. 3. 5-HT induced cytosolic [Ca(2+)] increases were blocked by 20 microM Xestospongin-C and by 2-APB (IC(50)=32 microM inhibitors of InsP(3) receptor activation. 4. 5-HT-mediated contractions were reliant on release of InsP(3) but not ryanodine-sensitive Ca(2+) stores. 5. 5-HT-mediated contractions and cytosolic [Ca(2+)] increases were partially inhibited by 10 microM nisoldipine, a voltage-dependent Ca(2+) channel blocker. 6. Extracellular Ca(2+) removal reduced 5-HT-mediated contractions further than nisoldipine and ablated cytosolic [Ca(2+)] increases and [Ca(2+)] oscillations. Similar to Ca(2+) removal, Ni(2+) reduced cytosolic [Ca(2+)] and [Ca(2+)] oscillations. 7. Mn(2+) quench of fura-2 and voltage-clamp experiments showed that 5-HT failed to activate any significant voltage-independent Ca(2+) entry pathways, including store-operated and receptor-activated nonselective cation channels. Ni(2+) but not nisoldipine or Gd(3+) blocked basal Mn(2+) entry. 8. Voltage-clamp experiments showed that simultaneous depletion of both InsP(3) and ryanodine-sensitive intracellular Ca(2+) stores activates a current with linear voltage dependence and a reversal potential consistent with it being a nonselective cation channel. 5-HT did not activate this current. 9. Basal Ca(2+) entry, rather than CCE, is important to maintain 5-HT-induced cytosolic [Ca(2+)] responses and contraction in canine pulmonary artery.

PDGF and IL-1β Upregulate Cofilin and LIMK2 in Canine Cultured Pulmonary Artery Smooth Muscle Cells

Actin cytoskeleton reorganization is regulated by various actin-binding proteins. Cofilin is the principal filament-depolymerizing protein, whose activity is reduced upon phosphorylation by LIMK. Thus, LIMK and cofilin comprise a signal transduction module regulating actin turnover and myogenic tone in healthy vasculature. Novel functions of smooth muscle cells (SMCs) in the hypertensive pulmonary artery, such as increased motility and proliferation, are supported by the actin cytoskeleton. We therefore hypothesized that bioactive peptides that affect these SMC functions may also result in an upregulation of LIMK and cofilin expression. Semiquantitative RT-PCR and immunoblotting indicated that LIMK2 and cofilin mRNA and protein expression is upregulated in canine pulmonary artery SMCs (PASMCs) exposed to PDGF or IL-1β (10 ng/ml). Inhibition of ERK MAPKs (U-0126, 10 µM) or p38 MAPK (PD-169316, 10 µM), but not PI3Ks (LY-294002, 50 µM), reduced LIMK2 and cofilin gene expression stimulated by PDGF or IL-1β. Inhibition of ROCK (Y-27632, 10 µM) reduced only the IL-1β-stimulated LIMK2 and cofilin expression. These novel observations in PASMCs indicate that LIMK2 and cofilin expression can be induced by PDGF or IL-1β. This parallel upregulation of LIMK2 and cofilin may have potentially broad functional significance for the progress of pulmonary artery disease.

Biodegradable polymer with collagen microsponge serves as a new bioengineered cardiovascular prosthesis

ObjectiveBiodegradable materials with autologous cell seeding have attracted much interest as potential cardiovascular grafts. However, pretreatment of these materials requires a complicated and invasive procedure that carries the risk of infection. To avoid these problems, we sought to develop a biodegradable graft material containing collagen microsponge that would permit the regeneration of autologous vessel tissue. The ability of this material to accelerate in situ cellularization with autologous endothelial and smooth muscle cells was tested with and without precellularization. MethodsPoly(lactic-co-glycolic acid) as a biodegradable scaffold was compounded with collagen microsponge to form a vascular patch material. These poly(lactic-co-glycolic acid)–collagen patches with (n = 10) or without (n = 10) autologous vessel cellularization were used to patch the canine pulmonary artery trunk. Histologic and biochemical assessments were performed 2 and 6 months after the implantation. ResultsThere was no thrombus formation in either group, and the poly(lactic-co-glycolic acid) scaffold was almost completely absorbed in both groups. Histologic results showed the formation of an endothelial cell monolayer, a parallel alignment of smooth muscle cells, and reconstructed vessel wall with elastin and collagen fibers. The cellular and extracellular components in the patch had increased to levels similar to those in native tissue at 6 months. ConclusionsThe poly(lactic-co-glycolic acid)–collagen microsponge patch with and without precellularization showed good histologic findings and durability. This patch shows promise as a bioengineered material for promoting in situ cellularization and the regeneration of autologous tissue in cardiovascular surgery.

Implante em aorta abdominal de suíno de artéria pulmonar heteróloga conservada em glicerina

A correção cirúrgica de várias doenças vasculares requer a implantação de próteses. Neste estudo, foi avaliado o comportamento da artéria pulmonar de cão conservada em glicerina a 98% implantada na aorta abdominal de dois suínos, acompanhados por cinco meses de pós-operatório. Ao exame macroscópico, um dos implantes apresentou dilatação e o outro estenose, em relação à aorta adjacente. Microscopicamente, o tecido hospedeiro e o implante estavam íntegros e a cicatrização se apresentou adequada. Apesar das diferenças de diâmetro encontradas (estenose e dilatação), ambos os animais tiveram desenvolvimento normal e não demonstraram problemas clínicos.

Endothelium-dependent relaxation of canine pulmonary artery after prolonged lung graft preservation in University of Wisconsin solution: role of l-arginine supplementation

Background The University of Wisconsin (UW) solution has been demonstrated to enhance pulmonary allograft preservation. Endothelial nitric oxide (NO) production has been shown to be significantly impaired after ischemia and reperfusion (I/R) injury. The present experiments aimed to determine the protective effects of pulmonary endothelium-dependent function by using supplemental NO in University of Wisconsin (UW) solution following prolonged lung graft preservation. Methods Thirty-six healthy mongrel dogs underwent thoracotomy to expose the left lung. In addition to a group given UW solution ( n = 4), 100 μmol/liter l-arginine, ( n = 7), 100 μmol/liter N G-monomethyl- l-arginine ( l-NMMA n = 7) and 1.0 μmol/liter 3-morpholinosydnonimine (SIN-1, n = 18 respectively, were added to UW solution, and infused from the aortic root and pulmonary artery to the pulmonary vein. The perfused lung was then allowed to inflate to its maximum volume for 24-hour oxygenated preservation in each supplemented condition of UW solution at 4°C. In the SIN-1 group, the preservation period was further divided into 8 hours and 16 hours, respectively. Rings of the third-order pulmonary artery of the inflated lung were then suspended in organ chambers to measure isometric force. Results Endothelium-dependent relaxation (EDR) to acetylcholine, adenosine diphosphate and sodium fluoride of the pulmonary rings in the l-arginine group was significantly preserved compared with UW-solution-only group. The l-NMMA group showed significant EDR impairment after 24-hour preservation compared with the UW solution group. Similar to the l-arginine group, the SIN-1 group showed significant EDR protection with 8-hour preservation, but not with 24-hour preservation. In contrast, EDR to calcium ionophore A23187 showed no EDR changes after 24-hour preservation in any of the supplemented groups. Conclusions Supplemental l-arginine in UW solution ameliorates impaired pulmonary EDR following prolonged lung preservation of up to 24 hours.

Hypotonic activation of volume-sensitive outwardly rectifying chloride channels in cultured PASMCs is modulated by SGK.

The serum- and glucocorticoid-inducible kinase (SGK) is a serine/threonine protein kinase (PK) transcriptionally regulated by corticoids, serum, and cell volume. SGK regulates cell volume of various cells by effects on Na(+) and K(+) transport through membrane channels. We hypothesized a role for SGK in the activation of volume-sensitive osmolyte and anion channels (VSOACs) in cultured canine pulmonary artery smooth muscle cells (PASMCs). Intracellular dialysis through the patch electrode of recombinant active SGK, but not kinase-dead Delta60-SGK-K127M, heat-inactivated SGK, or active Akt1, partially activated VSOACs under isotonic conditions. Dialysis of active SGK before cell exposure to hypotonic medium significantly accelerated the activation kinetics and increased the maximal density of VSOAC current. Exposure of PASMCs to hypotonic medium (230 mosM) activated phosphatidylinositol 3-kinases (PI3Ks) and their downstream targets Akt/PKB and SGK but not PKC-epsilon. Inhibition of PI3Ks with wortmannin reduced the activation rate and maximal amplitude of VSOACs. Immunoprecipitated ClC-3 channels were phosphorylated by PKC-epsilon but not by SGK in vitro, suggesting that SGK may activate VSOACs indirectly. These data indicate that the PI3K-SGK cascade is activated on hypotonic swelling of PASMCs and, in turn, affects downstream signaling molecules linked to activation of VSOACs.