Porcine Pulmonary Artery Research Articles

Experimental surgery as part of the development of degradable biomaterials for cardiovascular surgery

Introduction: Cardiovascular diseases are responsible for significant morbidity and mortality in the population. Artificial vascular grafts are often essential for surgical procedures in radical or palliative treatment. Many new biodegradable materials are currently under development. Preclinical testing of each new material is imperative, both in vitro and in vivo, and therefore animal experiments are still a necessary part of the testing process before any clinical use. The aim of this paper is to present the options of using various experimental animal models in the field of cardiovascular surgery including their extrapolation to clinical medicine. Methods: The authors present their general experience in the field of experimental surgery. They discuss the selection process of an optimal experimental animal model to test foreign materials for cardiovascular surgery and of an optimal region for implantation. Results: The authors present rat, rabbit and porcine models as optimal experimental animals for material hemocompatibility and degradability testing. Intraperitoneal implantation in the rat is a simple and feasible procedure, as well as aortic banding in the rabbit or pig. The carotid arteries can also be used, as well. Porcine pulmonary artery banding is slightly more difficult with potential complications. The banded vessels, explanted after a defined time period, are suitable for further mechanical testing using biomechanical analyses, for example, the inflation-extension test. Conclusion: An in vivo experiment cannot be avoided in the last phases of preclinical research of new materials. However, we try to strictly observe the 3R concept – Replacement, Reduction and Refinement; in line with this concept, the potential of each animal should be used as much as possible to reduce the number of animals.

Percutaneous delivery and degradation of a shape memory elastomer poly(glycerol dodecanedioate) in porcine pulmonary arteries

Shape memory biodegradable elastomers are an emergent class of biomaterials well-suited for percutaneous cardiovascular repair requiring nonlinear elastic materials with facile handling. We have previously developed a chemically crosslinked shape memory elastomer, poly (glycerol dodecanedioate) (PGD), exhibiting tunable transition temperatures around body temperature (34–38 °C), exhibiting nonlinear elastic properties approximating cardiac tissues, and favorable degradation rates in vitro. Degree of tissue coverage, degradation and consequent changes in polymer thermomechanical properties, and inflammatory response in preclinical animal models are unknown material attributes required for translating this material into cardiovascular devices. This study investigates changes in the polymer structure, tissue coverage, endothelialization, and inflammation of percutaneously implanted PGD patches (20 mm × 9 mm x 0.5 mm) into the branch pulmonary arteries of Yorkshire pigs for three months. After three months in vivo, 5/8 samples exhibited (100%) tissue coverage, 2/8 samples exhibited 85–95% tissue coverage, and 1/8 samples exhibited limited (<20%) tissue coverage with mild-moderate inflammation. PGD explants showed a (60–70%) volume loss and (25–30%) mass loss, and a reduction in polymer crosslinks. Lumenal and mural surfaces and the cross-section of the explant demonstrated evidence of degradation. This study validates PGD as an appropriate cardiovascular engineering material due to its propensity for rapid tissue coverage and uneventful inflammatory response in a preclinical animal model, establishing a precedent for consideration in cardiovascular repair applications.

Abstract 15870: Integrated Transcriptomic and Proteomics Analyses Identify Lipid Metabolism as a Key Regulator of RV Size and Function in a Porcine Pulmonary Artery Banded Model

Introduction: Right ventricular (RV) dysfunction is the leading cause of death in pulmonary arterial hypertension (PAH), but the molecular determinants of RV dysfunction are not well defined. In addition, most molecular evaluations of RV dysfunction use small animal models, and data from large animal models is sparse. Methods: Control and pulmonary artery banded (PAB) male piglets were subjected to cardiac MRI to quantify RV ejection fraction (EF), mass, and end systolic volume (ESV). Total RNA was isolated from RV free wall specimens and subjected to Next Gene RNAsequencing. RV mitochondrial enrichments were analyzed with quantitative mass spectrometry to define RV proteome changes. Bioinformatics analyses integrated transcriptomics, proteomics, and MRI data to identified pathways associated with RV dysfunction, hypertrophy, and volume. Results: Both transcriptomic and proteomic profiling revealed significantly different molecular signatures when comparing control to PAB piglets. Both analyses identified metabolism of lipids, lysosome, and mitochondrial fatty acid beta-oxidation pathways were associated with RVEF, while dilated cardiomyopathy, and arrhythmogenic RV cardiomyopathy pathways correlated with RV mass, and metabolism, metabolic pathways, citric acid cycle, and respiratory electron transport pathways were associated with RVESV. Fatty acid metabolism was identified on our analyses, and ferroptosis, a nonapoptotic form of cell death initiated by lipid peroxidation, was associated with RVESV and RV mass. Finally, in addition to coding genes we identified 72 lncRNAs that were strongly (r&gt;|0.85|) associated with RVEF. Conclusions: A combined proteomics and transcriptomics analysis identified multiple lncRNAs and lipid metabolism pathways associated with pathological RV remodeling and RV function. Future studies are needed to define how modulation of these pathways could impact modulate RV size and function in large animal models.

Effect of ethanol washing on porcine pulmonary artery wall decellularization using sodium dodecyl sulfate.

To determine the effectiveness of ethanol (EtOH) washing on porcine pulmonary artery (PA) wall decellularization using sodium dodecyl sulfate (SDS), we compared three different washing methods (phosphate-buffered saline [PBS], pH 9 alkali, and EtOH washing). Fresh porcine PA walls were decellularized using 0.5% SDS and 0.5% sodium deoxycholate (SDC). The decellularized tissues were rinsed using three different washing techniques. Histological, biochemical, and mechanical analyses were conducted. Implantation into the subcutaneous tissue of rats and patch implantation into the carotid artery of dogs were performed as preliminary in vivo studies. The decellularization protocol based on SDS and SDC effectively removed the cells. The major extracellular matrix (ECM) structures (collagen, elastic fiber, and glycosaminoglycan) were properly preserved with the 75% EtOH-washing method. Significantly reduced residual SDS content was identified in EtOH-washed tissues compared to that in the other methods. No significant difference in the mechanical strength test was observed between the washing methods, and the EtOH-washing method showed better results in the metabolic activity test compared to the PBS-washing method. In the rat study model, no acute rejection or massive calcification was observed. The in vivo preliminary canine study showed better cell repopulation in the EtOH-washed group. EtOH washing of SDS-based decellularized porcine PA wall can reduce the residual SDS content and preserve ECM structures, especially the elastin content, and could also enhance cell repopulation after re-implantation.

Potential of Autologous Progenitor Cells and Decellularized Porcine Artery Matrix in Construction of Tissue-engineered Vascular Grafts

ABSTRACT To develop a tissue-engineered vascular graft, we used pericardial effusion-derived progenitor cells (PEPCs) collected from drained fluid after open-heart surgery in children with congenital heart diseases to repopulate a decellularized porcine pulmonary artery. The PEPCs were compared with human fibroblasts (HS68) and human umbilical vein endothelial cells (HUVECs) in cell growth and migration. They were cultured with the matrices via an inner approach (intima), lateral approach (media), and outer approach (adventitia). PEPCs grew and migrated better than the other two cells 14 days after seeding in the decellularized vessel. In immunofluorescence assays, PEPCs expressed CD90 and CD105 indicating a vascular differentiation. PEPCs grew in a decellularized porcine pulmonary artery matrix may have the potential for producing tissue-engineered vascular grafts.

Ex Vivo Regional Mechanical Characterization of Porcine Pulmonary Arteries.

Regional mechanical characterization of pulmonary arteries can be useful in the development of computational models of pulmonary arterial mechanics. We performed a biomechanical and microstructural characterization study of porcine pulmonary arteries, inclusive of the main, left, and right pulmonary arteries (MPA, LPA, and RPA, respectively). The specimens were initially stored at -20°C and allowed to thaw for 12-24 hours prior to testing. Each artery was further subdivided into proximal, middle, and distal regions, leading to ten location-based experimental groups. Planar equibiaxial tensile testing was performed to evaluate the mechanical behavior of the specimens, from which we calculated the stress at the maximum strain (S 55), tensile modulus (TM), anisotropy index (AI), and strain energy in terms of area under the stress-strain curve (AUC). Histological quantification was performed to evaluate the area fraction of elastin and collagen content, intima-media thickness (IMT), and adventitial thickness (AT). The constitutive material behavior of each group was represented by a five-constant Holzapfel-Gasser-Ogden model. The specimens exhibited non-linear stress-strain characteristics across all groups. The MPA exhibited the highest mean wall stress and TM in the longitudinal and circumferential directions, while the bifurcation region yielded the highest values of AI and AUC. All regions revealed a higher stiffness in the longitudinal direction compared to the circumferential direction, suggesting a degree of anisotropy that is believed to be within the margin of experimental uncertainty. Collagen content was found to be the highest in the MPA and decreased significantly at the bifurcation, LPA and RPA. Elastin content did not yield such significant differences amongst the ten groups. The MPA had the highest IMT, which decreased concomitantly to the distal LPA and RPA. No significant differences were found in the AT amongst the ten groups. The mechanical properties of porcine pulmonary arteries exhibit strong regional dissimilarities, which can be used to inform future studies of high fidelity finite element models.

Vascular Lipidomic Profiling of Potential Endogenous Fatty Acid PPAR Ligands Reveals the Coronary Artery as Major Producer of CYP450-Derived Epoxy Fatty Acids.

A number of oxylipins have been described as endogenous PPAR ligands. The very short biological half-lives of oxylipins suggest roles as autocrine or paracrine signaling molecules. While coronary arterial atherosclerosis is the root of myocardial infarction, aortic atherosclerotic plaque formation is a common readout of in vivo atherosclerosis studies in mice. Improved understanding of the compartmentalized sources of oxylipin PPAR ligands will increase our knowledge of the roles of PPAR signaling in diverse vascular tissues. Here, we performed a targeted lipidomic analysis of ex vivo-generated oxylipins from porcine aorta, coronary artery, pulmonary artery and perivascular adipose. Cyclooxygenase (COX)-derived prostanoids were the most abundant detectable oxylipin from all tissues. By contrast, the coronary artery produced significantly higher levels of oxylipins from CYP450 pathways than other tissues. The TLR4 ligand LPS induced prostanoid formation in all vascular tissue tested. The 11-HETE, 15-HETE, and 9-HODE were also induced by LPS from the aorta and pulmonary artery but not coronary artery. Epoxy fatty acid (EpFA) formation was largely unaffected by LPS. The pig CYP2J homologue CYP2J34 was expressed in porcine vascular tissue and primary coronary artery smooth muscle cells (pCASMCs) in culture. Treatment of pCASMCs with LPS induced a robust profile of pro-inflammatory target genes: TNFα, ICAM-1, VCAM-1, MCP-1 and CD40L. The soluble epoxide hydrolase inhibitor TPPU, which prevents the breakdown of endogenous CYP-derived EpFAs, significantly suppressed LPS-induced inflammatory target genes. In conclusion, PPAR-activating oxylipins are produced and regulated in a vascular site-specific manner. The CYP450 pathway is highly active in the coronary artery and capable of providing anti-inflammatory oxylipins that prevent processes of inflammatory vascular disease progression.

Pressurized carbon dioxide as a potential tool for decellularization of pulmonary arteries for transplant purposes

Vascular bio-scaffolds produced from decellularized tissue offer a promising material for treatment of several types of cardiovascular diseases. These materials have the potential to maintain the functional properties of the extracellular matrix (ECM), and allow for growth and remodeling in vivo. The most commonly used methods for decellularization are based on chemicals and enzymes combinations, which often damage the ECM and cause cytotoxic effects in vivo. Mild methods involving pressurized CO2-ethanol (EtOH)-based fluids, in a supercritical or near supercritical state, have been studied for decellularization of cardiovascular tissue, but results are controversial. Moreover, data are lacking on the amount and type of lipids remaining in the tissue. Here we show that pressurized CO2-EtOH-H2O fluids (average molar composition, ΧCO2 0.91) yielded close to complete removal of lipids from porcine pulmonary arteries, including a notably decrease of pro-inflammatory fatty acids. Pressurized CO2-limonene fluids (ΧCO2 0.88) and neat supercritical CO2 (scCO2) achieved the removal of 90% of triacylglycerides. Moreover, treatment of tissue with pressurized CO2-limonene followed by enzyme treatment, resulted in efficient DNA removal. The structure of elastic fibers was preserved after pressurized treatment, regardless solvent composition. In conclusion, pressurized CO2-ethanol fluids offer an efficient tool for delipidation in bio-scaffold production, while pressurized CO2-limonene fluids facilitate subsequent enzymatic removal of DNA.

Effect of Betanin, a Beetroot Component, on Vascular Tone in Isolated Porcine Arteries.

Beetroot has attracted much attention because of its blood pressure-lowering properties. Although beetroot contains various nutritional compounds, including inorganic nitrate, some of their physiological properties are not fully understood. In this study, we examined whether betanin, a beetroot component, has a regulatory effect on vascular tone. Mechanical responses of isolated porcine coronary, mesenteric, and pulmonary arteries were assessed by organ chamber technique. In some cases, the vascular reactivity was observed in the presence of a physiological concentration of betanin (10 µM). Betanin did not induce vasorelaxation at physiological concentrations both in endothelium-intact and -denuded coronary, mesenteric, and pulmonary arteries. The endothelium-dependent agonists, bradykinin and A23187 induced vasorelaxation of endothelium-intact coronary arteries, both of which were not affected by exposure to betanin. Likewise, endothelium-independent vasorelaxation induced by sodium nitrite and sodium nitroprusside was also not affected by the presence of betanin. In addition, exposure of endothelium-intact coronary arteries to betanin did not attenuate prostaglandin F2α- and endothelin-1-induced vasocontraction. These findings suggest that betanin does not have a vasorelaxant activity. It is unlikely that betanin is a component directly responsible for the beetroot-induced acute blood pressure-lowering effect in a nitrate-independent manner.

Effects of dexmedetomidine on porcine pulmonary artery vascular smooth muscle.

BackgroundThe α2-receptor agonist dexmedetomidine (Dex) has been shown to produce sedative and analgesic effects not only with systemic administration but also when administered in the extradural space and around peripheral nerves. The effects and mechanism of action of Dex on pulmonary arteries, however, have not been determined. This study therefore aimed to investigate the effect of Dex on pulmonary arterial vascular smooth muscle by evaluating changes in isometric contraction tension. We then attempted to determine the effects of Dex on depolarization stimulation and receptor stimulation.MethodsEndothelium-denuded porcine pulmonary arteries were sliced into 2- to 3-mm rings. We then exposed them to certain substances at various concentrations under different conditions of baseline stimulation (with KCl, adrenaline, caffeine, or histamine) and to α2-receptor stimulants or antagonists, or α1-receptor antagonists (imidazoline, yohimbine, rauwolscine, prazosin), and different conditions of Ca2+ depletion of the intracellular reservoir or extracellular stores. We measured the changes in isometric contraction tension with each addition or change in conditions.ResultsDex enhanced the contraction induced by high-concentration KCl stimulation. Dex-induced enhancement of contraction induced by high-concentration KCl was completely suppressed by yohimbine and rauwolscine, which are α2-receptor antagonists, but not by prazosin. Dex, imidazoline, yohimbine, and rauwolscine reduced the increases in contraction tension induced by the receptor stimulant adrenaline. Dex suppressed the adrenaline-induced increases in contraction tension after depletion of the Ca2+ reservoir. In the absence of extracellular Ca2+, Dex suppressed the adrenaline- and histamine-induced increases in contraction tension but did not affect caffeine-induced increases.ConclusionsDex-enhanced, high-concentration KCl-induced contraction was mediated by α2-receptors. Adrenaline-induced contraction was suppressed by the α2-receptor stimulant Dex and α2-receptor antagonists yohimbine and rauwolscine, suggesting that the effect of Dex on adrenaline-induced contraction is attributable to its α2-receptor-blocking action. Dex inhibited receptor-activated Ca2+ channels and phosphatidylinositol-1,4,5-triphosphate-induced Ca2+ release but not Ca2+-induced Ca2+ release.

Urapidil, but not dihydropyridine calcium channel inhibitors, preserves the hypoxic pulmonary vasoconstriction: an experimental study in pig arteries.

Hypoxic pulmonary vasoconstriction (HPV) is a protective mechanism maintaining blood oxygenation by redirecting blood flow from poorly ventilated to well-ventilated areas in the lung. Such a beneficial effect is blunted by antihypertensive treatment with dihydropyridine calcium channel inhibitors. The aim of the present study was to evaluate the effect of urapidil, an antihypertensive agent acting as an α1 adrenergic antagonist and a partial 5-HT1A agonist, on HPV in porcine proximal and distal pulmonary artery rings, and to characterize underlying mechanisms. Rings from proximal and distal porcine pulmonary artery were suspended in organ chambers and aerated with a 95% O2 + 5% CO2 gas mixture. HPV was induced by changing the gas to a 95% N2 + 5% CO2 mixture following a low level of pre-contraction with U46619. Hypoxia induced a contractile response in both proximal and distal pulmonary artery rings. This effect is observed in the presence of a functional endothelium and is inhibited by a soluble guanylyl cyclase inhibitor (ODQ), a NO scavenger (carboxy-PTIO), and by catalase in proximal pulmonary artery rings. The endothelium-dependent HPV is prevented by nicardipine and clevidipine but remained unaffected by urapidil in both proximal and distal pulmonary artery rings. These findings indicate that urapidil, in contrast to nicardipine and clevidipine, preserves the hypoxia-triggered vasoconstriction in isolated pulmonary arteries. They further indicate the involvement of the NO-guanylyl cyclase pathway and H2 O2 in HPV. Further research is warranted to determine the potential clinical relevance of the preserved hypoxia-induced pulmonary vasoconstriction by urapidil.

Efficient methodology for the extraction and analysis of lipids from porcine pulmonary artery by supercritical fluid chromatography coupled to mass spectrometry

Pulmonary artery grafts are needed as cardiovascular bioprosthetics. For successful tissue recellularization after transplantation, lipids have to be removed from the donor artery. Developing a selective process to remove lipids without damaging the extracellular matrix greatly depends on knowing the amount and type of lipid compounds in the specific tissue. Here we present an efficient methodology for the study of lipids present in porcine pulmonary arteries. The performance of six extraction methods to recover lipids from artery was evaluated. For this purpose, a supercritical fluid chromatography method coupled to quadrupole time-of-flight mass spectrometry detection (UHPSFC/QTOF-MS) was adapted. The method enabled separation of lipids of a wide range of polarity according to lipid class in less than 7 min. One dichloromethane-based extraction method was shown to be the most efficient one for the recovery of lipids from pulmonary artery. However, one MTBE-based extraction method was able to show the highest fatty acid extraction yields (to the expense of longer extraction times). Lipids were relative quantified according to class, and the major species within each class were identified. Triacylglycerols and glycerophospholipids were the most abundant classes, followed by sphingomyelins, monoacylglycerols and fatty acyls. The matrix effect exerted no interference on the analytical method, except for some few combinations of extraction method and lipid class. These results are of relevance for lipidomic studies from solid tissue, in particular for studies on pulmonary and cardiovascular diseases. Finally, our work sets the basis for the further development of a selective processes to remove lipids from pulmonary artery without damaging the tissue prior to transplantation.

２．ブタ舌動脈・肺動脈血管平滑筋に対するデクスメデトミジン塩酸塩の作用機序

２．ブタ舌動脈・肺動脈血管平滑筋に対するデクスメデトミジン塩酸塩の作用機序

Quantitative Real Time PCR (qRT‐PCR) Evaluation of Pig Mitochondrial DNA Damage

Mitochondrial DNA (MtDNA) damage has generated much interest in recent years as a potential biomarker after ischemia/reperfusion and traumatic injury and in critical illness. MtDNA is particularly susceptible to damage due to is close proximity to the source of generation of reactive oxygen species during metabolism and its relatively low ability to repair DNA damage compared to nuclear repair mechanisms. Reduction in MtDNA damage appears to be a good indicator of a successful intervention. QRT‐PCR has been shown to be the most sensitive and accurate method to evaluate mtDNA damage requiring only nanogram amounts of DNA. While validated primer pairs for mtDNA damage for human and rodent mtDNA damage are available, comparable sets for pig have not previously been described, yet many studies in trauma and critical illness have utilized swine models. To select primer pairs for pigs, we aligned the pig mitochondrial DNA sequence to the pig genome in the UCSC Genome Browser and primers for regions not exhibiting overlap with the nuclear genome were selected with NCBI Primer3. Using the bleomycin injury model, porcine pulmonary artery endothelial cells obtained from Cell Biologics and cultivated in MCDB 131 medium were treated for 1 hr with bleomycin (10 μg/ml). Short and long sequences were tested with pig total DNA and demonstrated that one set detected specific induction of DNA lesions. The assay was able to detect 11 lesions per 10,000 base pairs. The short primer pairs also accurately predicted mitochondrial copy number DNA as corroborated for mtDNA by published results for the pig ND4 gene. We are currently employing this assay to detect mtDNA damage in a swine model of hemorrhagic shock.Support or Funding InformationFunded by US Army Medical Research Materiel Command

A comparison of vasodilation mode among selexipag (NS-304; [2-{4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}-N-(methylsulfonyl)acetamide]), its active metabolite MRE-269 and various prostacyclin receptor agonists in rat, porcine and human pulmonary arteries

Selexipag (NS-304; [2-{4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}-N- (methylsulfonyl)acetamide]) is a novel, orally available non-prostanoid prostacyclin receptor (IP receptor) agonist that has recently been approved for the treatment of pulmonary arterial hypertension (PAH). We examined the effect of the active metabolite of selexipag, MRE-269, and IP receptor agonists that are currently available as PAH therapeutic drugs on the relaxation of rat, porcine and human pulmonary artery. cAMP formation in human pulmonary artery smooth muscle cells was induced by all test compounds (MRE-269, epoprostenol, iloprost, treprostinil and beraprost sodium) and suppressed by IP receptor antagonists (CAY10441 and 2-[4-(1H-indol-4-yloxymethyl)-benzyloxycarbonylamino]−3-phenyl-propionic acid). MRE-269 induced endothelium-independent vasodilation of rat extralobar pulmonary artery (EPA). In contrast, endothelial denudation or the addition of a nitric oxide synthase inhibitor markedly attenuated the vasodilation of EPA induced by epoprostenol, treprostinil and beraprost sodium but not iloprost. The vasorelaxant effects of MRE-269 on rat small intralobar pulmonary artery (SIPA) and EPA were the same, while the other IP receptor agonists induced less vasodilation in SIPA than in EPA. Furthermore, a prostaglandin E receptor 3 antagonist enhanced the vasodilation induced by all IP receptor agonists tested except MRE-269. We also investigated the relaxation induced by IP receptor agonists in pulmonary arteries from non-rodent species and found similar vasodilation modes in porcine and human as in rat preparations. These results suggest that MRE-269, in contrast to other IP receptor agonists, works as a selective IP receptor agonist, thus leading to pronounced vasorelaxation of rat, porcine and human pulmonary artery.

Human immune-cell response towards diverse xenogeneic and allogeneic decellularized biomaterials

BackgroundImmunological knowledge on processed biological implants and mesh-prostheses is still mainly based on animal models, lacking information on the species-specific human immune response. We hypothesized that in contrast to human tissue even decellularized xenogenic specimens would lead to significant and tissue source dependent human immune reactions. MethodsSpecimens from processed allogenic and xenogenic pulmonary arteries, pericardium or dermis, were co-cultured with human peripheral blood mononuclear cells (PBMNC). Proliferative responses were measured in tritiated thymidine incorporation assays (n = 10). Stimulation indices (SI), calculated as counts-per-minute of co-cultured PBMNC divided by the cpm of basic cell proliferation, were compared. ResultsCompared to native porcine pulmonary artery tissue decellularization significantly reduced human PBMNC proliferation (mean SI: 48.7 vs. 18.0, p < 0.01), which was still higher compared to the human equivalent (SI: 0.7 vs. 1.7). Also the processed human dermal implant did not elicit immune response (SI: 1.5), whereas the decellularized and cross-linked porcine dermis lead to a significant human cell-proliferation (SI: 8.4, p < 0.01). Interestingly, both the processed human (SI: 15.2) and bovine pericardial patches (SI: 15.1) led to higher immune cell proliferation. ConclusionEven decellularized or cross-linked xenogenic cardiovascular and reconstructive biomaterials elicit increased human immune responses not seen in the majority of allogenic specimens tested.

The effect of urapidil, an alpha-1 adrenoceptor antagonist and a 5-HT1A agonist, on the vascular tone of the porcine coronary and pulmonary arteries, the rat aorta and the human pulmonary artery

Urapidil (Eupressyl®) an antihypertensive drug acting as an α1 antagonist and a 5-HT1A agonist, may be of special interest in the treatment of hypertension associated with preeclamptic toxaemia and hypoxia-induced pulmonary arterial vasoconstriction. However, the effect of urapidil on vascular tone has been poorly investigated. Vascular reactivity was evaluated using pulmonary and coronary arteries from 36 pigs, aortae from 22 rats and 9 human pulmonary artery samples suspended in organ chambers. Concentration-relaxation curves either to urapidil, 5-HT, or the 5-HT1A receptor agonist 8-OH-DPAT were constructed after pre-contraction of rings. Pig pulmonary and coronary artery rings were contracted with U46619, a thromboxane mimetic, rat aortic rings with either endothelin-1 or phenylephrine, and human pulmonary artery rings with U46619 or phenylephrine. Urapidil markedly inhibited phenylephrine-induced contractions in rat aortic rings with and without endothelium with a more pronounced effect observed in rings without endothelium. Both 5-HT and 8-OH-DPAT failed to induce relaxation in rat aortic rings with an intact endothelium. 5-HT, but not urapidil and 8-OH-DPAT, induced a concentration-dependent relaxation in the porcine coronary and pulmonary artery rings with an intact endothelium (P<0.05). 5-HT and phenylephrine but not urapidil caused concentration-dependent contractions in human pulmonary artery rings. The present findings, while confirming that urapidil is a potent inhibitor of α1-adrenoceptor-induced contraction, do not support the role of 5-HT1A receptor activation in the control of the vascular tone of the different types of arteries tested in response to urapidil. In addition, they indicate that urapidil seems to preferentially target arteries with endothelial dysfunction.

Thromboxane promotes smooth muscle phenotype commitment but not remodeling of hypoxic neonatal pulmonary artery.

BackgroundPersistent pulmonary hypertension of the newborn (PPHN) is characterized by vasoconstriction and pulmonary vascular remodeling. Remodeling is believed to be a response to physical or chemical stimuli including pro-mitotic inflammatory mediators such as thromboxane. Our objective was to examine the effects of hypoxia and thromboxane signaling ex vivo and in vitro on phenotype commitment, cell cycle entry, and proliferation of PPHN and control neonatal pulmonary artery (PA) myocytes in tissue culture.MethodsTo examine concurrent effects of hypoxia and thromboxane on myocyte growth, serum-fed first-passage newborn porcine PA myocytes were randomized into normoxic (21 % O2) or hypoxic (10 % O2) culture for 3 days, with daily addition of thromboxane mimetic U46619 (10−9 to 10−5 M) or diluent. Cell survival was detected by MTT assay. To determine the effect of chronic thromboxane exposure (versus whole serum) on activation of arterial remodeling, PPHN was induced in newborn piglets by a 3-day hypoxic exposure (FiO2 0.10); controls were 3 day-old normoxic and day 0 piglets. Third-generation PA were segmented and cultured for 3 days in physiologic buffer, Ham’s F-12 media (in the presence or absence of 10 % fetal calf serum), or media with 10−6 M U46619. DNA synthesis was measured by 3H-thymidine uptake, protein synthesis by 3H-leucine uptake, and proliferation by immunostaining for Ki67. Cell cycle entry was studied by laser scanning cytometry of nuclei in arterial tunica media after propidium iodide staining. Phenotype commitment was determined by immunostaining tunica media for myosin heavy chain and desmin, quantified by laser scanning cytometry.ResultsContractile and synthetic myocyte subpopulations had differing responses to thromboxane challenge. U46619 decreased proliferation of synthetic and contractile myocytes. PPHN arteries exhibited decreased protein synthesis under all culture conditions. Serum-supplemented PA treated with U46619 had decreased G1/G0 phase myocytes and an increase in S and G2/M. When serum-deprived, PPHN PA incubated with U46619 showed arrested cell cycle entry (increased G0/G1, decreased S and G2/M) and increased abundance of contractile phenotype markers.ConclusionsWe conclude that thromboxane does not initiate phenotypic dedifferentiation and proliferative activation in PPHN PA. Exposure to thromboxane triggers cell cycle exit and myocyte commitment to contractile phenotype.

Reducing Risk of Cardiovascular Disease: Exploring the Effects of Quercetin on the Contractile Force of Vascular Smooth Muscle

Calcium plays an important role in regulating the contraction of smooth muscles. Membrane potential of the smooth muscles regulates the smooth muscle contractility through variation in Ca2+ influx with the assistance of the voltage-operated Ca2+ channel. Using the porcine pulmonary arteries (PPA) as the model system, the effect of the antioxidant quercetin on the regulation of pulmonary arteries was investigated in the presence and absence of calcium. It was hypothesized that quercetin reduces the contractile force in PPA by limiting Ca2+ release from the sarcoplasmic reticulum (SR) without imposing any effect on the voltage-operated channel (VOC). However, the biochemical pathway of quercetin is still unknown. In this study, it is shown that PPA contracts both in the presence and absence of Ca2+. Further investigations are required to study the exact effects of KCl in 0 Ca2+ to completely break down the biochemical pathway of quercetin and reassure its effect on the SR. Since quercetin demonstrates reduction in contractile force, it has high potential to provide a solution for the side effects of hypertension, which is a complex heterogeneous disorder that can coexist with other cardiovascular abnormalities.

To be EndMT or not to be, that is the question in pulmonary hypertension.

To be EndMT or not to be, that is the question in pulmonary hypertension.