ABSTRACT
Although saphenous veins (SVs) are commonly used as conduits for coronary artery bypass grafting (CABG), internal thoracic artery (ITA) grafts have significantly higher long-term patency. As SVs and ITA endothelial cells (ECs) have a considerable level of heterogeneity, we suggested that synergistic paracrine interactions between CA and ITA ECs (HCAECs and HITAECs, respectively) may explain the increased resistance of ITA grafts and adjacent CAs to atherosclerosis and restenosis. In this study, we measured the gene and protein expression of the molecules responsible for endothelial homeostasis, pro-inflammatory response, and endothelial-to-mesenchymal transition in HCAECs co-cultured with either HITAECs or SV ECs (HSaVECs) for an ascending duration. Upon the co-culture, HCAECs and HITAECs showed augmented expression of endothelial nitric oxide synthase (eNOS) and reduced expression of endothelial-to-mesenchymal transition transcription factors Snail and Slug when compared to the HCAEC-HSaVEC model. HCAECs co-cultured with HITAECs demonstrated an upregulation of HES1, a master regulator of arterial specification, of which the expression was also exclusively induced in HSaVECs co-cultured with HCAECs, suggestive of their arterialisation. In addition, co-culture of HCAECs and HITAECs promoted the release of pro-angiogenic molecules. To conclude, co-culture of HCAECs and HITAECs results in reciprocal and beneficial paracrine interactions that might contribute to the better performance of ITA grafts upon CABG.
Subject(s)
Coronary Vessels/cytology , Endothelium, Vascular/cytology , Mammary Arteries/cytology , Paracrine Communication , Vascular Patency , Cells, Cultured , Coculture Techniques , Coronary Artery Bypass , Coronary Vessels/metabolism , Endothelium, Vascular/metabolism , Humans , Mammary Arteries/metabolismABSTRACT
Voltage-gated Kv7 (or KCNQ) channels control activity of excitable cells, including vascular smooth muscle cells (VSMCs), by setting their resting membrane potential and controlling other excitability parameters. Excitation-contraction coupling in muscle cells is mediated by Ca2+ but until now, the exact role of Kv7 channels in cytosolic Ca2+ dynamics in VSMCs has not been fully elucidated. We utilised microfluorimetry to investigate the impact of Kv7 channel activity on intracellular Ca2+ levels and electrical activity of rat A7r5 VSMCs and primary human internal mammary artery (IMA) SMCs. Both, direct (XE991) and G protein coupled receptor mediated (vasopressin, AVP) Kv7 channel inhibition induced robust Ca2+ oscillations, which were significantly reduced in the presence of Kv7 channel activator, retigabine, L-type Ca2+ channel inhibitor, nifedipine, or T-type Ca2+ channel inhibitor, NNC 55-0396, in A7r5 cells. Membrane potential measured using FluoVolt exhibited a slow depolarisation followed by a burst of sharp spikes in response to XE991; spikes were temporally correlated with Ca2+ oscillations. Phospholipase C inhibitor (edelfosine) reduced AVP-induced, but not XE991-induced Ca2+ oscillations. AVP and XE991 induced a large increase of [Ca2+]i in human IMA, which was also attenuated with retigabine, nifedipine and NNC 55-0396. RT-PCR, immunohistochemistry and electrophysiology suggested that Kv7.5 was the predominant Kv7 subunit in both rat and human arterial SMCs; CACNA1C (Cav1.2; L-type) and CACNA1â¯G (Cav3.1; T-type) were the most abundant voltage-gated Ca2+ channel gene transcripts in both types of VSMCs. This study establishes Kv7 channels as key regulators of Ca2+ signalling in VSMCs with Kv7.5 playing a dominant role.
Subject(s)
Calcium/metabolism , Intracellular Space/metabolism , KCNQ Potassium Channels/metabolism , Muscle, Smooth, Vascular/metabolism , Animals , Calcium Channel Blockers/pharmacology , Calcium Channels/metabolism , Calcium Signaling/drug effects , Cells, Cultured , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/metabolism , Gene Expression Regulation/drug effects , HEK293 Cells , Humans , KCNQ Potassium Channels/genetics , Mammary Arteries/cytology , Muscle, Smooth, Vascular/drug effects , Rats , Type C Phospholipases/metabolism , Vasopressins/pharmacologyABSTRACT
This study investigates the extent of cell seeding as well as lumen patency in acellular Internal mammary artery (IMA) scaffolds that have been re-cellularized by omentum as a natural bioreactor. Sixteen Virgin female Wistar rats were selected for implantation of acellular scaffold in omentum. Following laparotomy omentum was retracted to the outside of the abdomen and the more vascularized portion of it was selected for the experiment. The scaffold was wrapped by omentum and placed between two layers of rectus muscles that have been previously dissected. Samples were taken from scaffolds at 2 and 3 months for histopathological evaluation. All the grafts were explanted at 2 and 3 months and the lumens were completely patent compared to the native scaffold. The histology of implanted IMA after 2 and 3 months showed progressive recellularization especially by smooth muscle cells over the media layer. CD 34 staining was positive adjacent to the grafts which showed well angiogenesis. Trichrome and Movat's Pentachrome staining showed normal collagen formation in the inner media layer. Promising results obtained from the introduced protocol for in vivo recellularization, including patent lumen and proper cell seeding, encourages application of the mentioned technique for experimental vascular graft application. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2685-2693, 2017.
Subject(s)
Mammary Arteries/chemistry , Mammary Arteries/transplantation , Neovascularization, Physiologic , Omentum/blood supply , Tissue Scaffolds/chemistry , Animals , Bioreactors , Blood Vessel Prosthesis , Female , Humans , Mammary Arteries/cytology , Mammary Arteries/physiology , Rats, Wistar , Regeneration , Tissue Engineering/instrumentationABSTRACT
The aim of the present study was to investigate and characterize vasorelaxant effect of procyanidin B2 on human internal mammary artery (HIMA) as one of the mechanisms of its protective effect against vascular risk. Procyanidin B2 induced strong concentration-dependent relaxation of HIMA rings pre-contracted by phenylephrine. Pretreatment with L-NAME, a NO synthase inhibitor, hydroxocobalamin, a NO scavenger, and ODQ, an inhibitor of soluble guanylate cyclase, significantly inhibited procyanidin B2-induced relaxation of HIMA, while indomethacin, a cyclooxygenase inhibitor, considerably reduced effects of low concentrations. Among K+ channel blockers, iberiotoxin, a selective blocker of large conductance Ca2+-activated K+ channels (BKCa), abolished procyanidin B2-induced relaxation, glibenclamide, a selective ATP-sensitive K+(KATP) channels blocker, induced partial inhibition, while 4-aminopyridine, a blocker of voltage-gated K+(KV) channels, and TRAM-34, an inhibitor of intermediate-conductance Ca2+-activated K+(IKCa) channels, slightly reduced maximal relaxation of HIMA. Further, procyanidin B2 relaxed contraction induced by phenylephrine in Ca2+-free Krebs solution, but had no effect on contraction induced by caffeine. Finally, thapsigargin, a sarcoplasmic reticulum Ca2+-ATPase inhibitor, significantly reduced relaxation of HIMA produced by procyanidin B2. These results demonstrate that procyanidin B2 produces endothelium-dependent relaxation of HIMA pre-contracted by phenylephrine. This effect is primarily the result of an increased NO synthesis and secretion by endothelial cells and partially of prostacyclin, although it involves activation of BKCa and KATP, as well as KV and IKCa channels in high concentrations of procyanidin B2.
Subject(s)
Biflavonoids/pharmacology , Catechin/pharmacology , Endothelium, Vascular/drug effects , Mammary Arteries/drug effects , Mammary Arteries/physiology , Proanthocyanidins/pharmacology , Vasodilator Agents/pharmacology , Calcium/metabolism , Endothelium, Vascular/metabolism , Humans , Intracellular Space/drug effects , Intracellular Space/metabolism , Mammary Arteries/cytology , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/drug effects , Nitric Oxide/metabolism , Phenylephrine/pharmacology , Potassium Channel Blockers/pharmacology , Vasoconstriction/drug effectsABSTRACT
Evidences have suggested that flavanol compound (-)-epicatechin is associated with reduced risk of cardiovascular diseases. One of the mechanisms of its cardioprotective effect is vasodilation. However, the exact mechanisms by which (-)-epicatechin causes vasodilation are not yet clearly defined. The aims of the present study were to investigate relaxant effect of flavanol (-)-epicatechin on the isolated human internal mammary artery (HIMA) and to determine the mechanisms underlying its vasorelaxation. Our results showed that (-)-epicatechin induced a concentration-dependent relaxation of HIMA rings pre-contracted by phenylephrine. Among the K(+) channel blockers, 4-aminopyridine (4-AP) and margatoxin, blockers of voltage-gated K(+) (KV) channels, and glibenclamide, a selective ATP-sensitive K(+) (KATP) channels blocker, partly inhibited the (-)-epicatechin-induced relaxation of HIMA, while iberiotoxin, a most selective blocker of large conductance Ca(2+)-activated K(+) channels (BKCa), almost completely inhibited the relaxation. In rings pre-contracted by 80mM K(+), (-)-epicatechin induced partial relaxation of HIMA, whereas in Ca(2+)-free medium, (-)-epicatechin completely relaxed HIMA rings pre-contracted by phenylephrine and caffeine. Finally, thapsigargin, a sarcoplasmic reticulum Ca(2+)-ATPase inhibitor, slightly antagonized (-)-epicatechin-induced relaxation of HIMA pre-contracted by phenylephrine. These results suggest that (-)-epicatechin induces strong endothelium-independent relaxation of HIMA pre-contracted by phenylephrine whilst 4-AP- and margatoxin-sensitive KV channels, as well as BKCa and KATP channels, located in vascular smooth muscle, mediate this relaxation. In addition, it seems that (-)-epicatechin could inhibit influx of extracellular Ca(2+), interfere with intracellular Ca(2+) release and re-uptake by the sarcoplasmic reticulum.
Subject(s)
Catechin/pharmacology , Mammary Arteries/drug effects , Mammary Arteries/physiology , Vasodilation/drug effects , Calcium/metabolism , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Extracellular Space/drug effects , Extracellular Space/metabolism , Humans , Intracellular Space/drug effects , Intracellular Space/metabolism , Male , Mammary Arteries/cytology , Mammary Arteries/metabolism , Middle Aged , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/drug effects , Phenylephrine/pharmacology , Potassium Channel Blockers/pharmacology , Potassium Channels/metabolism , Vasoconstriction/drug effectsSubject(s)
Mammary Arteries/cytology , Mediastinitis/therapy , Mesenchymal Stem Cell Transplantation/methods , Mesenchymal Stem Cells/cytology , Sternotomy/adverse effects , Surgical Wound Infection/therapy , Aged, 80 and over , Cell Differentiation , Cells, Cultured , Follow-Up Studies , Humans , MaleABSTRACT
The aim of this study was to generate extended length, small diameter vascular scaffolds that could serve as potential grafts for treatment of acute ischemia. Biological tissues are considered excellent scaffolds, which exhibit adequate biological, mechanical, and handling properties; however, they tend to degenerate, dilate, and calcify after implantation. We hypothesized that chemically stabilized acellular arteries would be ideal scaffolds for development of vascular grafts for peripheral surgery applications. Based on promising historical data from our laboratory and others, we chose to decellularize bovine mammary and femoral arteries and test them as scaffolds for vascular grafting. Decellularization of such long structures required development of a novel "bioprocessing" system and a sequence of detergents and enzymes that generated completely acellular, galactose-(α1,3)-galactose (α-Gal) xenoantigen-free scaffolds with preserved collagen, elastin, and basement membrane components. Acellular arteries exhibited excellent mechanical properties, including burst pressure, suture holding strength, and elastic recoil. To reduce elastin degeneration, we treated the scaffolds with penta-galloyl glucose and then revitalized them in vitro using a tunic-specific cell approach. A novel atraumatic endothelialization protocol using an external stent was also developed for the long grafts and cell-seeded constructs were conditioned in a flow bioreactor. Both decellularization and revitalization are feasible but cell retention in vitro continues to pose challenges. These studies support further efforts toward clinical use of small diameter acellular arteries as vascular grafts.
Subject(s)
Blood Vessel Prosthesis , Femoral Artery/cytology , Femoral Artery/physiology , Mammary Arteries/cytology , Mammary Arteries/physiology , Tissue Engineering/methods , Animals , Biomechanical Phenomena , Cattle , Endothelium/physiology , Female , Humans , Perfusion , Tissue ScaffoldsABSTRACT
The Na(+)-H(+) exchanger (NHE) and the Na(+)-HCO3(-) co-transporter (NBC) have been confirmed as two major active acid extruders in many mammalian cells. Whether the NHE and NBC functional co-exist in human internal mammary artery smooth muscle cells (HIMASMCs) remains unclear. The aims of the present study were to investigate the acid-extruding mechanisms and to explore the effects of urotensin-II (U-II), a powerful vasoconstrictor, on pHi regulators in HIMASMCs. We investigated the changes of pHi by BCECF-fluorescence in HIMASMCs. We found that (a) two Na(+)-dependent acid extruders, i.e. NHE and NBC, functionally co-exist; (b) U-II (3-100 nM) induced a concentration-dependent intracellular acidosis; and (c) U-II (3-100 nM) caused a concentration-dependent increase on NHE activity, while decrease on NBC activity. In summary, we demonstrate for the first time that two acid-extruders, NHE and NBC, functionally co-exist in HIMASMCs. Moreover, U-II induces a concentration-dependent intracellular acidosis through the balanced effect of its effect on increasing NHE activity and decreasing NBC activity.
Subject(s)
Mammary Arteries/cytology , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/metabolism , Urotensins/pharmacology , Cells, Cultured , Humans , Hydrogen-Ion ConcentrationABSTRACT
BACKGROUND: Phenotypic modulation or switching of vascular smooth muscle cells from a contractile/quiescent to a proliferative/synthetic phenotype plays a key role in vascular proliferative disorders such as atherosclerosis and restenosis. Although several calcium handling proteins that control differentiation of smooth muscle cells have been identified, the role of protein phosphatase inhibitor 1 (I-1) in the acquisition or maintenance of the contractile phenotype modulation remains unknown. METHODS AND RESULTS: In human coronary arteries, I-1 and sarco/endoplasmic reticulum Ca2+ -ATPase expression is specific to contractile vascular smooth muscle cells. In synthetic cultured human coronary artery smooth muscle cells, protein phosphatase inhibitor 1 (I-1 target) is highly expressed, leading to a decrease in phospholamban phosphorylation, sarco/endoplasmic reticulum Ca2+ -ATPase, and cAMP-responsive element binding activity. I-1 knockout mice lack phospholamban phosphorylation and exhibit vascular smooth muscle cell arrest in the synthetic state with excessive neointimal proliferation after carotid injury, as well as significant modifications of contractile properties and relaxant response to acetylcholine of femoral artery in vivo. Constitutively active I-1 gene transfer decreased neointimal formation in an angioplasty rat model by preventing vascular smooth muscle cell contractile to synthetic phenotype change. CONCLUSIONS: I-1 and sarco/endoplasmic reticulum Ca2+ -ATPase synergistically induce the vascular smooth muscle cell contractile phenotype. Gene transfer of constitutively active I-1 is a promising therapeutic strategy for preventing vascular proliferative disorders.
Subject(s)
Intracellular Signaling Peptides and Proteins/metabolism , Muscle, Smooth, Vascular/metabolism , Protein Phosphatase 1/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Vasoconstriction/physiology , Animals , Aorta, Thoracic/cytology , Aorta, Thoracic/physiology , Calcium Signaling/physiology , Coronary Vessels/cytology , Coronary Vessels/physiology , Femoral Artery/cytology , Femoral Artery/physiology , Humans , Intracellular Signaling Peptides and Proteins/genetics , Male , Mammary Arteries/cytology , Mammary Arteries/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscle, Smooth, Vascular/cytology , Phenotype , Protein Phosphatase 1/genetics , Proteins/genetics , Proteins/metabolism , Rats , Rats, Sprague-Dawley , Sarcoplasmic Reticulum Calcium-Transporting ATPases/genetics , Vascular Diseases/genetics , Vascular Diseases/metabolismABSTRACT
Despite recent advances in medical procedures, cardiovascular disease remains a clinical challenge and the leading cause of mortality in the western world. The condition causes progressive smooth muscle cell (SMC) dedifferentiation, proliferation, and migration that contribute to vascular restenosis. The incidence of disease of the internal mammary artery (IMA), however, is much lower than in nearly all other arteries. The etiology of this IMA disease resistance is not well understood. Here, using paired primary IMA and coronary artery SMCs, serum stimulation, siRNA knockdowns, and verifications in porcine vessels in vivo, we investigate the molecular mechanisms that could account for this increased disease resistance of internal mammary SMCs. We show that the residue-specific phosphorylation profile of the retinoblastoma tumor suppressor protein (Rb) appears to differ significantly between IMA and coronary artery SMCs in cultured human cells. We also report that the differential profile of Rb phosphorylation may follow as a consequence of differences in the content of cyclin-dependent kinase 2 (CDK2) and the CDK4 phosphorylation inhibitor p15. Finally, we present evidence that siRNA-mediated CDK2 knockdown alters the profile of Rb phosphorylation in coronary artery SMCs, as well as the proliferative response of these cells to mitogenic stimulation. The intrinsic functional and protein composition specificity of the SMCs population in the coronary artery may contribute to the increased prevalence of restenosis and atherosclerosis in the coronary arteries as compared with the internal mammary arteries.
Subject(s)
Cyclin-Dependent Kinase 2/metabolism , Mitogens/metabolism , Myocytes, Smooth Muscle/metabolism , Retinoblastoma Protein/metabolism , Animals , Cell Movement , Cell Proliferation , Coronary Vessels/cytology , Coronary Vessels/metabolism , Culture Media, Serum-Free , Cyclin-Dependent Kinase 2/genetics , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase Inhibitor p15/metabolism , Gene Knockdown Techniques , Humans , Male , Mammary Arteries/cytology , Mammary Arteries/metabolism , Phosphorylation , Primary Cell Culture , Serum , Swine , YY1 Transcription Factor/metabolismABSTRACT
Preclinical in vivo research models to investigate pathobiological and pathophysiological processes in the development of intimal hyperplasia after vessel stenting are crucial for translational approaches (1,2). The commonly used animal models include mice, rats, rabbits, and pigs (3-5). However, the translation of these models into clinical settings remains difficult, since those biological processes are already studied in animal vessels but never performed before in human research models (6,7). In this video we demonstrate a new humanized model to overcome this translational gap. The shown procedure is reproducible, easy, and fast to perform and is suitable to study the development of intimal hyperplasia and the applicability of diverse stents. This video shows how to perform the stent technique in human vessels followed by transplantation into immunodeficient rats, and identifies the origin of proliferating cells as human.
Subject(s)
Graft Occlusion, Vascular/etiology , Mammary Arteries/transplantation , Stents , Vascular Grafting/methods , Animals , Cell Growth Processes/physiology , Graft Occlusion, Vascular/pathology , Humans , Mammary Arteries/cytology , Rats , Rats, Nude , Transplantation, HeterologousABSTRACT
Coronary artery disease (CAD) is the leading cause of death worldwide. It has been established that internal mammary arteries (IMA) are resistant to the development of atherosclerosis, whereas left anterior descending (LAD) coronary arteries are athero-prone. The contrasting properties of these two arteries provide an innovative strategy to identify the genes that play important roles in the development of atherosclerosis. We carried out microarray analysis to identify genes differentially expressed between IMA and LAD. Twenty-nine genes showed significant differences in their expression levels between IMA and LAD, which included the TES gene encoding Testin. The role of TES in the cardiovascular system is unknown. Here we show that TES is involved in endothelial cell (EC) functions relevant to atherosclerosis. Western blot analysis showed higher TES expression in IMA than in LAD. Reverse transcription polymerase chain reaction and western blot analyses showed that TES was consistently and markedly down-regulated by more than 6-fold at both mRNA and protein levels in patients with CAD compared with controls without CAD (P= 0.000049). The data suggest that reduced TES expression is associated with the development of CAD. Knockdown of TES expression by small-interfering RNA promoted oxidized-LDL-mediated monocyte adhesion to ECs, EC migration and the transendothelial migration of monocytes, while the over-expression of TES in ECs blunted these processes. These results demonstrate association between reduced TES expression and CAD, establish a novel role for TES in EC functions and raise the possibility that reduced TES expression increases susceptibility to the development of CAD.
Subject(s)
Biomarkers/metabolism , Coronary Artery Disease/genetics , Coronary Artery Disease/pathology , Coronary Vessels/metabolism , Cytoskeletal Proteins/metabolism , Endothelium, Vascular/metabolism , LIM Domain Proteins/metabolism , Mammary Arteries/metabolism , Apoptosis , Blotting, Western , Cell Adhesion , Cell Movement , Cell Proliferation , Cells, Cultured , Coronary Artery Disease/metabolism , Coronary Vessels/cytology , Cytoskeletal Proteins/antagonists & inhibitors , Cytoskeletal Proteins/genetics , Endothelium, Vascular/cytology , Gene Expression Profiling , Humans , LIM Domain Proteins/antagonists & inhibitors , LIM Domain Proteins/genetics , Mammary Arteries/cytology , Monocytes/cytology , Monocytes/metabolism , Oligonucleotide Array Sequence Analysis , RNA, Messenger/genetics , RNA, Small Interfering/genetics , RNA-Binding Proteins , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Here, we identify CD44(+)CD90(+)CD73(+)CD34(-)CD45(-) cells within the adult human arterial adventitia with properties of multipotency which were named vascular wall-resident multipotent stem cells (VW-MPSCs). VW-MPSCs exhibit typical mesenchymal stem cell characteristics including cell surface markers in immunostaining and flow cytometric analyses, and differentiation into adipocytes, chondrocytes and osteocytes under culture conditions. Particularly, TGFß1 stimulation up-regulates smooth muscle cell markers in VW-MPSCs. Using fluorescent cell labelling and co-localisation studies we show that VW-MPSCs differentiate to pericytes/smooth muscle cells which cover the wall of newly formed endothelial capillary-like structures in vitro. Co-implantation of EGFP-labelled VW-MPSCs and human umbilical vein endothelial cells into SCID mice subcutaneously via Matrigel results in new vessels formation which were covered by pericyte- or smooth muscle-like cells generated from implanted VW-MPSCs. Our results suggest that VW-MPSCs are of relevance for vascular morphogenesis, repair and self-renewal of vascular wall cells and for local capacity of neovascularization in disease processes.
Subject(s)
Hyaluronan Receptors/metabolism , Mammary Arteries/cytology , Morphogenesis , Multipotent Stem Cells/cytology , Myocytes, Smooth Muscle/cytology , Neovascularization, Physiologic , Pericytes/cytology , Adult , Animals , Biomarkers/metabolism , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Endothelium, Vascular/ultrastructure , Gels , Gene Expression Regulation/drug effects , Humans , Mice , Mice, SCID , Morphogenesis/drug effects , Multipotent Stem Cells/drug effects , Multipotent Stem Cells/metabolism , Multipotent Stem Cells/ultrastructure , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/metabolism , Neovascularization, Physiologic/drug effects , Pericytes/drug effects , Pericytes/ultrastructure , Protein Transport/drug effects , Transforming Growth Factor beta1/pharmacologyABSTRACT
INTRODUCTION: Immunological studies of giant cell arteritis (GCA) suggest that a triggering antigen of unknown nature could generate a specific immune response. We thus decided to detect autoantibodies directed against endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) in the serum of GCA patients and to identify their target antigens. METHODS: Sera from 15 GCA patients were tested in 5 pools of 3 patients' sera and compared to a sera pool from 12 healthy controls (HCs). Serum immunoglobulin G (IgG) reactivity was analysed by 2-D electrophoresis and immunoblotting with antigens from human umbilical vein ECs (HUVECs) and mammary artery VSMCs. Target antigens were identified by mass spectrometry. RESULTS: Serum IgG from GCA patients recognised 162 ± 3 (mean ± SD) and 100 ± 17 (mean ± SD) protein spots from HUVECs and VSMCs, respectively, and that from HCs recognised 79 and 94 protein spots, respectively. In total, 30 spots from HUVECs and 19 from VSMCs were recognised by at least two-thirds and three-fifths, respectively, of the pools of sera from GCA patients and not by sera from HCs. Among identified proteins, we found vinculin, lamin A/C, voltage-dependent anion-selective channel protein 2, annexin V and other proteins involved in cell energy metabolism and key cellular pathways. Ingenuity pathway analysis revealed that most identified target antigens interacted with growth factor receptor-bound protein 2. CONCLUSIONS: IgG antibodies to proteins in the proteome of ECs and VSMCs are present in the sera of GCA patients and recognise cellular targets that play key roles in cell biology and maintenance of homeostasis. Their potential pathogenic role remains to be determined.
Subject(s)
Autoantibodies/immunology , Autoantigens/immunology , Endothelial Cells/immunology , Giant Cell Arteritis/immunology , Muscle, Smooth, Vascular/immunology , Proteomics/methods , Aged , Aged, 80 and over , Cell Line, Transformed , Cells, Cultured , Cytokines/immunology , Electrophoresis, Gel, Two-Dimensional/methods , Endothelial Cells/cytology , Female , Human Umbilical Vein Endothelial Cells , Humans , Immunoblotting/methods , Immunoglobulin G/immunology , Male , Mammary Arteries/cytology , Mammary Arteries/immunology , Mass Spectrometry/methods , Muscle, Smooth, Vascular/cytology , Receptors, Growth Factor/immunology , Vinculin/immunologyABSTRACT
While the salutary effects of exercise training on conduit artery endothelial cells have been reported in animals and humans with cardiovascular risk factors or disease, whether a healthy endothelium is alterable with exercise training is less certain. The purpose of this study was to evaluate the impact of exercise training on transcriptional profiles in normal endothelial cells using a genome-wide microarray analysis. Brachial and internal mammary endothelial gene expression was compared between a group of healthy pigs that exercise trained for 16-20 wk (n = 8) and a group that remained sedentary (n = 8). We found that a total of 130 genes were upregulated and 84 genes downregulated in brachial artery endothelial cells with exercise training (>1.5-fold and false discovery rate <15%). In contrast, a total of 113 genes were upregulated and 31 genes downregulated in internal mammary artery endothelial cells using the same criteria. Although there was an overlap of 66 genes (59 upregulated and 7 downregulated with exercise training) between the brachial and internal mammary arteries, the identified endothelial gene networks and biological processes influenced by exercise training were distinctly different between the brachial and internal mammary arteries. These data indicate that a healthy endothelium is indeed responsive to exercise training and support the concept that the influence of physical activity on endothelial gene expression is not homogenously distributed throughout the vasculature.
Subject(s)
Brachial Artery/metabolism , Endothelial Cells/metabolism , Gene Expression Profiling/methods , Mammary Arteries/metabolism , Oligonucleotide Array Sequence Analysis , Physical Exertion , RNA, Messenger/metabolism , Animals , Bayes Theorem , Brachial Artery/cytology , Gene Expression Regulation , Gene Regulatory Networks , Linear Models , Male , Mammary Arteries/cytology , Reverse Transcriptase Polymerase Chain Reaction , Swine , Swine, Miniature , Time FactorsABSTRACT
Because adrenergic contractions can contribute to the development of life-threatening spasm of coronary artery bypass graft, this study was performed to investigate the effect of adenosine 3-phosphate (ATP)-sensitive K channel (KATP) opener P1075 on contractions of isolated human saphenous vein (HSV) and human internal mammary artery (HIMA). Phasic contractions were evoked by electric field stimulation (20 Hz) and noradrenaline. The sustained contractions were evoked by phenylephrine. The presence of pore-forming Kir6.1 and Kir6.2 subunits of the KATP channels in the HIMA and only Kir6.2 in the HSV was confirmed immunomorphologically. P1075 inhibited in the HSV only, the electrical field stimulation contractions more strongly than noradrenaline contractions. In addition, the phenylephrine contractions of HSV were more sensitive to P1075 in comparison to those of HIMA. Glibenclamide, a KATP channel blocker antagonized the vasodilatation produced by P1075 in both grafts differently, because its effect was more prominent on the P1075-induced inhibition of contractions of HSV than of HIMA. We conclude that P1075 has a vasorelaxant effect and inhibited adrenergic contractions of the tested grafts. This effect is graft and vasoconstrictor selective and seems to be mediated by Kir6.1- and/or Kir6.2-containing KATP channels. Thus, P1075 can be considered as a potential drug in the prevention of graft spasm.
Subject(s)
Guanidines/pharmacology , KATP Channels/agonists , Mammary Arteries/drug effects , Pyridines/pharmacology , Saphenous Vein/drug effects , Vasodilation/drug effects , Vasodilator Agents/pharmacology , Aged , Electric Stimulation , Glyburide/pharmacology , Guanidines/antagonists & inhibitors , Humans , KATP Channels/metabolism , Male , Mammary Arteries/cytology , Mammary Arteries/metabolism , Middle Aged , Organ Culture Techniques , Organ Specificity , Osmolar Concentration , Potassium Channel Blockers/pharmacology , Potassium Channels, Inwardly Rectifying/metabolism , Protein Subunits/metabolism , Pyridines/antagonists & inhibitors , Saphenous Vein/cytology , Saphenous Vein/metabolism , Vasoconstriction/drug effects , Vasodilator Agents/antagonists & inhibitorsABSTRACT
BACKGROUND: Graft spasm remains challenging in CABG (coronary artery bypass grafting) surgery. We investigated the inhibitory effect of a dihydropyridine calcium antagonist amlodipine on the vasoconstriction mediated by potassium chloride (KCl), human urotensin-II (hU-II), and U46619 in human internal mammary artery (IMA) from patients undergoing CABG. METHODS: Isolated IMA rings (n = 78, taken from 42 patients) were studied in organ baths in two ways: the relaxing effect of amlodipine on vasoconstrictor-induced precontraction by KCl, hU-II, and U46619 and the depressing effect of amlodipine on the contraction. RESULTS: Amlodipine caused full relaxation in KCl-contracted (98.0% ± 2.1%), in hU-II-contracted (98.5% ± 2.4%), and in U46619-contracted (96.3% ± 1.3%) IMA rings (n = 8) with 15.5-fold higher potency to KCl than to hU-II (effective concentration causing 50% of maximal response [EC(50)]: -8.17 ± 0.28 vs -6.98 ± 0.01 log M, p < 0.001) and 19.5-fold that to U46619 (EC(50): -8.17 ± 0.28 vs -6.88 ± 0.08 log M, p < 0.001). Pretreatment of IMA with plasma concentrations of amlodipine (-6.6 log M) significantly depressed subsequent contraction to KCl (from 20.8 ± 2.5 mN to 7.6 ± 3.0 mN, p = 0.004) and hU-II (from 14.1 ± 4.2 mN to 3.8 ± 2.0 mN, p = 0.026), but did not significantly affect the contraction to U46619. CONCLUSIONS: We conclude that in human IMA amlodipine has a potent inhibitory effect on the vasoconstriction mediated by a variety of vasoconstrictors. Thus, use of amlodipine in CABG patients is favored in treating and preventing graft spasm.
Subject(s)
Amlodipine/pharmacology , Calcium Channel Blockers/pharmacology , Mammary Arteries/drug effects , Vasodilation/physiology , 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid/pharmacology , Humans , Mammary Arteries/cytology , Mammary Arteries/physiology , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/physiology , Potassium Chloride/pharmacology , Urotensins/pharmacology , Vasoconstrictor Agents/pharmacology , Vasodilation/drug effectsSubject(s)
Amlodipine/pharmacology , Calcium Channel Blockers/pharmacology , Mammary Arteries/drug effects , Vasodilation/physiology , Humans , Mammary Arteries/cytology , Mammary Arteries/physiology , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/physiology , Vasoconstrictor Agents/pharmacology , Vasodilation/drug effectsABSTRACT
Coronary revascularization by coronary artery bypass grafting (CABG) is recommended in patients with recurrent myocardial ischemia. However, the long-term results of CABG using saphenous vein (SV) graft, compared to internal mammary artery (IMA) graft, have not been satisfactory. The SV graft failure is due to the development of intimal hyperplasia, a process characterized by abnormal migration and proliferation of smooth muscle cells (SMCs) in the intimal layer of the vein graft. Insulin growth factor 1 (IGF-1) is a major mitogenic growth factor released at the site of the shear stress-induced graft injury. This study, for the first time, compares the extent of IGF-1-PI3K-Akt activation in isolated human bypass graft conduits. Human SV and IMA vessels were collected and SMCs isolated and cultured. In cultured SMCs, effect of IGF-1 was examined on total and phosphorylated PI3K, Akt and IGF-1R by Western blot analysis. Cell proliferation was measured using BrdU ELISA. There was no significant difference in the basal expression of phosphorylated PI3K, Akt and IGF-1R in SV and IMA SMCs from human bypass conduits. However, we observed an upregulation of IGF-1 receptors in the SV SMCs in response to IGF-1 stimulation with no effect in IMA SMCs. Furthermore, the immunoblotting and cellular activation of signaling ELISA (CASE) assay demonstrated a significantly higher activity of both PI3K and Akt in IGF-1-stimulated SV SMCs than IMA. This was inhibited by an IGF-1R blocking antibody. IGF-1 induced proliferation in both SV and IMA SMCs was inhibited by a PI3K inhibitor, wortmannin. These data demonstrate differential activity of IGF-1-induced PI3K-Akt activation, which was quantitatively and temporally greater in SV SMCs than in the IMA. This, at least in part, could explain the greater propensity of the SV conduits than the IMA to undergo intimal hyperplasia following CABG.
Subject(s)
Cell Proliferation/drug effects , Insulin-Like Growth Factor I/pharmacology , Muscle, Smooth, Vascular/drug effects , Phosphatidylinositol 3-Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , 3-Phosphoinositide-Dependent Protein Kinases , Aged , Cells, Cultured , Enzyme-Linked Immunosorbent Assay , Female , Humans , Immunoblotting , Male , Mammary Arteries/cytology , Mammary Arteries/metabolism , Middle Aged , Muscle, Smooth, Vascular/metabolism , Phosphorylation , Receptor, IGF Type 1/metabolism , Saphenous Vein/cytology , Saphenous Vein/metabolismABSTRACT
OBJECTIVES: The beta-2 adrenergic receptor is involved in mediating vasodilatation via neurohumoral and sympathetic nervous system pathways. Alterations in beta-2 adrenergic receptor gene expression (mRNA transcription) may contribute to the hypertensive phenotype. Human gene expression in clinical phenotypes remains largely unexplored due to ethical constraints involved in obtaining human tissue. We devised a method to obtain normally discarded internal mammary artery tissue from coronary artery bypass graft patients. We then investigated differences in hypertensive and normotensive participants' beta-2 adrenergic receptor gene expression in this tissue. METHODS: We collected arterial tissue samples from 46 coronary artery bypass patients in a surgical setting. Using 41 of the samples, we performed TaqMan real-time polymerase chain reaction (RT-PCR) and used the delta delta cycle threshold (DeltaDeltaCt) relative quantitation method for determination of fold-differences in gene expression between normotensive and hypertensive participants. The beta-2 adrenergic receptor target was normalized to glyceraldehyde-phosphate dehydrogenase. RESULTS: Participants with hypertension had significantly less-expressed beta-2 adrenoceptor gene (2.76-fold, p<.05) compared to normotensive participants. After Bonferroni correction, gene expression did not differ by race, gender, type/dose of beta-blocker prescribed, positive family history of hypertension, or diagnosis of diabetes mellitus type 2. CONCLUSIONS: These data support the possibility of a molecular basis for impaired adrenoceptor-mediated vascular tone in hypertension. Modification and extension of this research is required.