ABSTRACT
BACKGROUND: An imbalance between the activity of matrix metalloproteinases (MMPs), particularly gelatinases, and tissue inhibitors of metalloproteinases (TIMPs) is considered as one of the mechanisms leading to aortocoronary graft failure. AIM: We aimed to assess the variability in gelatinase expression in the walls of aortocoronary conduits and to evaluate its impact on coronary artery bypass grafting (CABG) outcomes. METHODS: The study included 101 consecutive patients (61 men and 40 women) who underwent CABG. An immunohisto-chemical analysis of MMP-2, MMP-9, TIMP-1, and TIMP-2 expression was performed on the cross-sections of the internal thoracic artery (ITA), radial artery (RA), and saphenous vein (SV). The histological findings were compared between patients with SV graft disease (SVGD[+] group) and those without occlusions in the SV (SVGD[-] group). RESULTS: The median MMP and TIMP expression was the weakest in the ITA wall. MMP expression was comparable between the RA and SV cross-sections, whereas TIMP expression was stronger in the RA than in the SV wall (p < 0.05). In most SV segments, but not in the arteries, immunostaining intensity for MMP was comparable to or stronger than for TIMPs. In the veins harvested from the SVGD(+) group, MMP-2 and MMP-9 tissue expression was more pronounced than in the SVGD(-) group. TIMP levels were comparable between groups. CONCLUSIONS: Imbalance in the metalloproteinase-to-inhibitor tissue expression in the vessel wall might predispose to graft failure. A stronger expression of TIMPs than MMPs in the arterial grafts might explain favourable long-term outcomes.
Subject(s)
Blood Vessels/enzymology , Coronary Artery Bypass , Coronary Disease/enzymology , Gelatinases/genetics , Tissue Inhibitor of Metalloproteinases/genetics , Adult , Aged , Aged, 80 and over , Blood Vessels/metabolism , Coronary Disease/genetics , Coronary Disease/metabolism , Coronary Disease/surgery , Female , Gene Expression Regulation , Humans , Male , Middle Aged , Radial Artery/enzymology , Radial Artery/metabolism , Saphenous Vein/enzymology , Saphenous Vein/metabolism , Thoracic Arteries/enzymology , Thoracic Arteries/metabolism , Tissue Inhibitor of Metalloproteinase-1/genetics , Tissue Inhibitor of Metalloproteinase-2/genetics , Treatment OutcomeABSTRACT
Neointimal hyperplasia is a product of VSMC replication and consequent accumulation within the blood vessel wall. In this study, we determined whether inhibition of protein kinase CK2 and the resultant stabilisation of proline-rich homeodomain (PRH) could suppress VSMC proliferation. Both silencing and pharmacological inhibition of CK2 with K66 antagonised replication of isolated VSMCs. SiRNA-induced knockdown as well as ectopic overexpression of proline-rich homeodomain indicated that PRH disrupts cell cycle progression. Mutation of CK2 phosphorylation sites Ser163 and Ser177 within the PRH homeodomain enabled prolonged cell cycle arrest by PRH. Concomitant knockdown of PRH and inhibition of CK2 with K66 indicated that the anti-proliferative action of K66 required the presence of PRH. Both K66 and adenovirus-mediated gene transfer of S163C:S177C PRH impaired neointima formation in human saphenous vein organ cultures. Importantly, neither intervention had notable effects on cell cycle progression, cell survival or migration in cultured endothelial cells.
Subject(s)
Cell Proliferation/drug effects , Homeodomain Proteins/metabolism , Muscle, Smooth, Vascular/drug effects , Myocytes, Smooth Muscle/drug effects , Neointima , Protein Kinase Inhibitors/pharmacology , Transcription Factors/metabolism , Animals , Casein Kinase II/antagonists & inhibitors , Casein Kinase II/genetics , Casein Kinase II/metabolism , Cell Cycle Checkpoints/drug effects , Cells, Cultured , Homeodomain Proteins/genetics , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/enzymology , Humans , Hyperplasia , Muscle, Smooth, Vascular/enzymology , Muscle, Smooth, Vascular/pathology , Mutation , Myocytes, Smooth Muscle/enzymology , Myocytes, Smooth Muscle/pathology , Phosphorylation , Proline-Rich Protein Domains , RNA Interference , Rats , Saphenous Vein/drug effects , Saphenous Vein/enzymology , Saphenous Vein/pathology , Signal Transduction/drug effects , Tissue Culture Techniques , Transcription Factors/genetics , TransfectionABSTRACT
BACKGROUND: Arterial blood pressure-induced shear stress causes endothelial cell apoptosis and inflammation in vein grafts after coronary artery bypass grafting. As the inflammatory protein type IIA secretory phospholipase A2 (sPLA2-IIA) has been shown to progress atherosclerosis, we hypothesized a role for sPLA2-IIA herein. METHODS: The effects of PX-18, an inhibitor of both sPLA2-IIA and apoptosis, on residual endothelium and the presence of sPLA2-IIA were studied in human saphenous vein segments (n = 6) perfused at arterial blood pressure with autologous blood for 6 hrs. RESULTS: The presence of PX-18 in the perfusion blood induced a significant 20% reduction in endothelial cell loss compared to veins perfused without PX18, coinciding with significantly reduced sPLA2-IIA levels in the media of the vein graft wall. In addition, PX-18 significantly attenuated caspase-3 activation in human umbilical vein endothelial cells subjected to shear stress via mechanical stretch independent of sPLA2-IIA. CONCLUSIONS: In conclusion, PX-18 protects saphenous vein endothelial cells from arterial blood pressure-induced death, possibly also independent of sPLA2-IIA inhibition.
Subject(s)
Alkanesulfonic Acids/pharmacology , Arterial Pressure , Endothelial Cells/drug effects , Group II Phospholipases A2/antagonists & inhibitors , Mechanotransduction, Cellular/drug effects , Oleic Acids/pharmacology , Phospholipase A2 Inhibitors/pharmacology , Saphenous Vein/drug effects , Apoptosis/drug effects , Cells, Cultured , Endothelial Cells/enzymology , Endothelial Cells/pathology , Group II Phospholipases A2/metabolism , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/enzymology , Human Umbilical Vein Endothelial Cells/pathology , Humans , Saphenous Vein/enzymology , Saphenous Vein/pathology , Time FactorsABSTRACT
OBJECTIVE: Vein graft adaptation is characterized by loss of expression of the tyrosine kinase receptor Eph-B4, the embryonic determinant of venous identity, without increased expression of its ligand ephrin-B2, the embryonic determinant of arterial identity. Endothelial nitric oxide synthase (eNOS) is an important mediator of vessel remodeling. We hypothesized that the mechanism of action of Eph-B4 during vein graft adaptation might be through regulation of downstream eNOS activity. METHODS: Mouse lung endothelial cells were stimulated with ephrin-B2/Fc, without and with preclustering, without and with the eNOS inhibitor Nω-nitro-l-arginine methyl ester hydrochloride or the Eph-B4 inhibitor NVP-BHG712, and assessed by Western blot and immunofluorescence for eNOS and Eph-B4 phosphorylation. Nitric oxide (NO) production was assessed using an NO-specific chemiluminescence analyzer. Cell migration was assessed using a Transwell assay. Human and mouse vein graft specimens were examined for eNOS activity by Western blot, and vessel remodeling was assessed in vein grafts in wild-type or eNOS knockout mice. RESULTS: Ephrin-B2/Fc stimulated both Eph-B4 and eNOS phosphorylation in a bimodal temporal distribution (n = 4; P < .05), with preclustered ephrin-B2/Fc causing prolonged peak Eph-B4 and eNOS phosphorylation as well as altered subcellular localization (n = 4; P < .05). Ephrin-B2/Fc increased NO release (n = 3; P < .01) as well as increased endothelial cell migration (n = 6; P < .05) in an eNOS-dependent fashion. Both human and mouse vein grafts showed increased eNOS phosphorylation compared with normal veins (n = 3; P < .05). Vein grafts from eNOS knockout mice showed less dilation and less wall thickening compared with wild-type vein grafts (n = 7; P < .05). CONCLUSIONS: eNOS is a mediator of vein graft adaptation to the arterial environment. Eph-B4 stimulates eNOS phosphorylation in vitro and may mediate vein graft adaptation by regulation of eNOS activity in vivo.
Subject(s)
Nitric Oxide Synthase Type III/metabolism , Receptor, EphB4/metabolism , Saphenous Vein/transplantation , Vascular Remodeling , Vena Cava, Inferior/transplantation , Adaptation, Physiological , Animals , Cell Movement , Cells, Cultured , Enzyme Inhibitors/pharmacology , Ephrin-B2/pharmacology , Genotype , Humans , Mice, Inbred C57BL , Mice, Knockout , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide/metabolism , Nitric Oxide Synthase Type III/antagonists & inhibitors , Nitric Oxide Synthase Type III/deficiency , Nitric Oxide Synthase Type III/genetics , Phenotype , Phosphorylation , Saphenous Vein/enzymology , Saphenous Vein/pathology , Signal Transduction , Time Factors , Vena Cava, Inferior/drug effects , Vena Cava, Inferior/enzymology , Vena Cava, Inferior/pathologyABSTRACT
Hypoxia is known to promote vasodilation of coronary vessels through several mediators including cardiac-derived adenosine and endothelium-derived prostanoids and nitric oxide. To date, the impact of endogenous glycogen depletion in vascular smooth muscle and the resultant alterations in cellular energy state (e.g., AMP-activated protein kinase, AMPK) on the contractile response to G protein-coupled receptor agonists (e.g., serotonin, 5-HT) has not yet been studied. In the present study, ex vivo exposure of endothelium-denuded human saphenous vein rings to hypoxic and glucose-deprived conditions during KCl-induced contractions for 30 min resulted in a marked depletion of endogenous glycogen by â¼80% (from â¼1.78 µmol/g under normoxia to â¼0.36 µmol/g under hypoxia). Importantly, glycogen-depleted HSV rings, which were maintained under hypoxia/reoxygenation and glucose-deprived conditions, exhibited significant increases in basal AMPK phosphorylation (â¼6-fold ↑) and 5-HT-induced AMPK phosphorylation (â¼19-fold ↑) with an accompanying suppression of 5-HT-induced maximal contractile response (â¼68% ↓), compared with respective controls. Exposure of glycogen-depleted HSV rings to exogenous D-glucose, but not the inactive glucose analogs, prevented the exaggerated increase in 5-HT-induced AMPK phosphorylation and restored 5-HT-induced maximal contractile response. In addition, the ability of exogenous D-glucose to rescue cellular stress and impaired contractile function occurred through GLUT1-mediated but insulin/GLUT4-independent mechanisms. Together, the present findings from clinically-relevant human saphenous vein suggest that the loss of endogenous glycogen in vascular smooth muscle and the resultant accentuation of AMPK phosphorylation by GPCR agonists may constitute a yet another mechanism of metabolic vasodilation of coronary vessels in ischemic heart disease.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Allostasis , Glucose/metabolism , Glycogen/metabolism , Muscle, Smooth, Vascular/metabolism , Myocardial Ischemia/metabolism , Saphenous Vein/metabolism , Aged , Animals , Aorta, Thoracic/metabolism , Biological Transport , Cell Hypoxia , Enzyme Activation , Female , Glucose/analogs & derivatives , Glycogenolysis , Humans , In Vitro Techniques , Male , Middle Aged , Muscle, Smooth, Vascular/enzymology , Myocardial Ischemia/enzymology , Oxidative Stress , Phosphorylation , Protein Processing, Post-Translational , Rats, Wistar , Saphenous Vein/enzymology , VasoconstrictionABSTRACT
AIM: On the average, 15% to 25% of peripheral grafts and 10% to 30% of coronary grafts fail within 5 years. Changes in mechanical forces to which the vein is subjected could be an explanation for this phenomenon. We submitted human saphenous vein segments to non-pulsatile ex vivo perfusion with crescent pressures and evaluated morphology, nitric oxide synthase immunohistochemical expression; tissue levels of nitrite/nitrate and oxidative stress products. METHODS: Intact segments of human saphenous veins were obtained from 30 patients submitted to elective coronary artery bypass graft surgery. Ex vivo perfusion was performed during 3 hours, using oxygenated Krebs solution, flow of 100 mL/min and pressures of 0, 50, 100, 200 and 300 mmHg, defining five groups. RESULTS: Optical microscopy showed that veins of groups perfused with 200 and 300 mmHg presented increased luminal area and endothelial denuding. Electron microscopy transmission showed alterations in veins perfused with 200 and 300 mmHg. Immunohistochemical expression of the three nitric oxide synthase isoforms was observed in all vein layers, without significant difference among groups. Tissue levels of nitrite/nitrate were not significantly different among distinctive perfusion. Nitrotyrosine was not immunohistochemically expressed in all veins and malondialdehyde tissue levels were not different among groups. CONCLUSION: Non-pulsatile ex vivo perfusion during 3h caused morphological alterations in human saphenous veins (HSVs), which were not accompanied by immunohistochemical and biochemical alterations. Even with mechanical lesions, HSVs maintained the ability of express nitric oxide synthase (NOS) and release nitric oxide.
Subject(s)
Nitric Oxide Synthase/analysis , Perfusion/methods , Saphenous Vein/enzymology , Saphenous Vein/ultrastructure , Aged , Female , Humans , Immunohistochemistry , In Vitro Techniques , Male , Malondialdehyde/analysis , Microscopy, Electron, Transmission , Middle Aged , Nitrates/analysis , Nitric Oxide Synthase Type I/analysis , Nitric Oxide Synthase Type II/analysis , Nitric Oxide Synthase Type III/analysis , Nitrites/analysis , Oxidative Stress , Pressure , Stress, Mechanical , Tyrosine/analogs & derivatives , Tyrosine/analysisABSTRACT
OBJECTIVE: Glucocorticoid-induced leucine zipper (GILZ) represents an anti-inflammatory mediator, whose downregulation has been described in various inflammatory processes. Aim of our study was to decipher the regulation of GILZ in vascular inflammation. APPROACH AND RESULTS: Degenerated aortocoronary saphenous vein bypass grafts (n = 15), which exhibited inflammatory cell activation as determined by enhanced monocyte chemoattractrant protein 1 (MCP-1, CCL2) and Toll-like receptor 2 (TLR2) expression, showed significantly diminished GILZ protein and mRNA levels compared to healthy veins (n = 23). GILZ was also downregulated in human umbilical vein endothelial cells (HUVEC) and macrophages upon treatment with the inflammatory cytokine TNF-α in a tristetraprolin (ZFP36, TTP)- and p38 MAPK-dependent manner. To assess the functional implications of decreased GILZ expression, we determined NF-κB activation after GILZ knockdown by siRNA and found that NF-κB activity and inflammatory gene expression were significantly enhanced. Importantly, ZFP36 is induced in TNF-α-activated HUVEC as well as in degenerated vein bypasses. When atheroprotective laminar shear stress was employed, GILZ levels in HUVEC increased on mRNA and protein level. Laminar flow also counteracted TNF-α-induced ZFP36 expression and GILZ downregulation. MAP kinase phosphatase 1 (MKP-1, DUSP1), a negative regulator of ZFP36 expression, was distinctly upregulated under laminar shear stress conditions and downregulated in degenerated vein bypasses. CONCLUSION: Our data show a diminished expression of the anti-inflammatory mediator GILZ in the inflamed vasculature and indicate that GILZ downregulation requires the mRNA binding protein ZFP36. We suggest that reduced GILZ levels play a role in cardiovascular disease.
Subject(s)
Human Umbilical Vein Endothelial Cells/enzymology , Saphenous Vein/enzymology , Transcription Factors/metabolism , Vasculitis/enzymology , Case-Control Studies , Cells, Cultured , Chemokine CCL2/metabolism , Coronary Artery Bypass/adverse effects , Down-Regulation , Gene Expression Regulation, Enzymologic , Human Umbilical Vein Endothelial Cells/drug effects , Humans , Macrophages/enzymology , NF-kappa B/metabolism , RNA Interference , Signal Transduction , Stress, Mechanical , Time Factors , Toll-Like Receptor 2/metabolism , Transcription Factors/genetics , Transfection , Tristetraprolin/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Vasculitis/etiology , Vasculitis/genetics , Vasculitis/prevention & control , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
About 8% of the adult population is taking angiotensin-converting enzyme (ACE) inhibitors to treat cardiovascular disease including hypertension, myocardial infarction and heart failure. These drugs decrease mortality by up to one-fifth in these patients. We and others have reported previously that endogenous inhibitory substances suppress serum ACE activity, in vivo, similarly to the ACE inhibitor drugs. Here we have made an effort to identify this endogenous ACE inhibitor substance. ACE was crosslinked with interacting proteins in human sera. The crosslinked products were immunoprecipitated and subjected to Western blot. One of the crosslinked products was recognized by both anti-ACE and anti-HSA (human serum albumin) antibodies. Direct ACE-HSA interaction was confirmed by binding assays using purified ACE and HSA. HSA inhibited human purified (circulating) and human recombinant ACE with potencies (IC50) of 5.7 ± 0.7 and 9.5 ± 1.1 mg/mL, respectively. Effects of HSA on the tissue bound native ACE were tested on human saphenous vein samples. Angiotensin I evoked vasoconstriction was inhibited by HSA in this vascular tissue (maximal force with HSA: 6.14 ± 1.34 mN, without HSA: 13.54 ± 2.63 mN), while HSA was without effects on angiotensin II mediated constrictions (maximal force with HSA: 18.73 ± 2.17 mN, without HSA: 19.22 ± 3.50 mN). The main finding of this study is that HSA was identified as a potent physiological inhibitor of the ACE. The enzymatic activity of ACE appears to be almost completely suppressed by HSA when it is present in its physiological concentration. These data suggest that angiotensin I conversion is limited by low physiological ACE activities, in vivo.
Subject(s)
Peptidyl-Dipeptidase A/blood , Renin-Angiotensin System/drug effects , Serum Albumin/pharmacology , Angiotensin I/pharmacology , Angiotensin-Converting Enzyme Inhibitors/pharmacology , Biomechanical Phenomena/drug effects , Catalytic Domain , Humans , Kinetics , Molecular Weight , Recombinant Proteins/metabolism , Saphenous Vein/drug effects , Saphenous Vein/enzymologyABSTRACT
BACKGROUND/PURPOSE: Coronary heart disease is the leading cause of morbidity in patients with type 2 diabetes mellitus (T2DM), frequently resulting in a requirement for coronary revascularization using the internal mammary artery (IMA) or saphenous vein (SV). Patency rates of SV grafts are inferior to IMA and further impaired by T2DM whilst IMA patencies appear similar in both populations. Smooth muscle cells (SMC) play a pivotal role in graft integration; we therefore examined the phenotype and proliferative function of IMA- and SV-SMC isolated from non-diabetic (ND) patients or those diagnosed with T2DM. METHODS/MATERIALS: SMC were cultured from fragments of SV or IMA. Morphology was analyzed under light microscopy (spread cell area measurements) and confocal microscopy (F-actin staining). Proliferation was analyzed by cell counting. Levels of RhoA mRNA, protein and activity were measured by real-time RT-PCR, western blotting and G-LISA respectively. RESULTS: IMA-SMC from T2DM and ND patients were indistinguishable in both morphology and function. By comparison, SV-SMC from T2DM patients exhibited significantly larger spread cell areas (1.5-fold increase, P<0.05), truncated F-actin fibers and reduced proliferation (33% reduction, P<0.05). Furthermore, lower expression and activity of RhoA were observed in SV-SMC of T2DM patients (37% reduction in expression, P<0.05 and 43% reduction in activity, P<0.01). CONCLUSIONS: IMA-SMC appear impervious to phenotypic modulation by T2DM. In contrast, SV-SMC from T2DM patients exhibit phenotypic and functional changes accompanied by reduced RhoA activity. These aberrancies may be epigenetic in nature, compromising SMC plasticity and SV graft adaptation in T2DM patients. SUMMARY: The internal mammary artery (IMA) is the conduit of choice for bypass grafting and is generally successful in all patients, including those with type 2 diabetes (T2DM). By contrast, saphenous vein (SV) is inferior to IMA and furthermore patients with T2DM suffer strikingly poorer outcomes than their non-diabetic (ND) counterparts. We discovered that SV-SMC from T2DM patients exhibit altered persistent morphology and function compared to ND SV-SMC, with differential expression and activity of the small GTPase RhoA, yet ND and T2DM IMA-SMC were indistinguishable. These data offer an explanation for the superior patency of IMA grafting independent of the presence of diabetes.
Subject(s)
Diabetes Mellitus, Type 2/complications , Diabetic Angiopathies/etiology , Muscle, Smooth, Vascular/enzymology , Myocytes, Smooth Muscle/enzymology , rhoA GTP-Binding Protein/metabolism , Adult , Aged , Aged, 80 and over , Case-Control Studies , Cell Proliferation , Cell Shape , Cells, Cultured , Diabetes Mellitus, Type 2/enzymology , Diabetes Mellitus, Type 2/pathology , Diabetic Angiopathies/enzymology , Diabetic Angiopathies/pathology , Down-Regulation , Female , Humans , Male , Mammary Arteries/enzymology , Mammary Arteries/pathology , Middle Aged , Muscle, Smooth, Vascular/pathology , Phenotype , Saphenous Vein/enzymology , Saphenous Vein/pathology , Time FactorsABSTRACT
The saphenous vein is the conduit of choice in bypass graft procedures. Haemodynamic factors play a major role in the development of intimal hyperplasia (IH), and subsequent bypass failure. To evaluate the potential protective effect of external reinforcement on such a failure, we developed an ex vivo model for the perfusion of segments of human saphenous veins under arterial shear stress. In veins submitted to pulsatile high pressure (mean pressure at 100 mmHg) for 3 or 7 days, the use of an external macroporous polyester mesh 1) prevented the dilatation of the vessel, 2) decreased the development of IH, 3) reduced the apoptosis of smooth muscle cells, and the subsequent fibrosis of the media layer, 4) prevented the remodelling of extracellular matrix through the up-regulation of matrix metalloproteinases (MMP-2, MMP-9) and plasminogen activator type I. The data show that, in an experimental ex vivo setting, an external scaffold decreases IH and maintains the integrity of veins exposed to arterial pressure, via increase in shear stress and decrease wall tension, that likely contribute to trigger selective molecular and cellular changes.
Subject(s)
Saphenous Vein/pathology , Tissue Scaffolds/chemistry , Tunica Intima/pathology , Aged , Aged, 80 and over , Caspase 3/metabolism , Down-Regulation , Ephrin-B2/genetics , Ephrin-B2/metabolism , Female , Heme Oxygenase (Decyclizing)/metabolism , Humans , Hyperplasia , In Vitro Techniques , Male , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Middle Aged , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/pathology , Nitric Oxide Synthase Type III/metabolism , Perfusion , Plasminogen Activator Inhibitor 1/metabolism , Pressure , Receptor, EphB4/genetics , Receptor, EphB4/metabolism , Saphenous Vein/enzymology , Stress, Mechanical , Transforming Growth Factor beta/metabolismABSTRACT
Coronary artery disease is the major cause of mortalilty in the West with coronary artery bypass surgery (CABG) being a means of restoring blood supply to ischaemic myocardium. The long saphenous vein is the most commonly used bypass conduit but its patency is inferior to the internal thoracic artery, the 'gold standard' graft. In conventional procedures the saphenous vein is harvested in such a manner that considerable vascular damage is inflicted. The structures mainly affected by this vascular trauma are the endothelium, autonomic nerves and vascular smooth muscle all containing cells with the potential to release nitric oxide (NO). While the majority of studies into the potential role of NO in vein graft performance have focussed on the involvement of endothelial nitric oxide synthase (eNOS) less information is available regarding the role of the inducible isoform of nitric oxide synthase (iNOS). While the effects of eNOS-derived NO are principally beneficial, iNOS is generally associated with pathological conditions. While potential pathophysiological roles of iNOS are discussed in this review we also outline many studies suggesting that this isoenzyme plays an important role in maintaing vein graft patency in patients undergoing CABG, particularly when the saphenous vein is harvested with minimal surgical trauma.
Subject(s)
Coronary Artery Bypass , Coronary Artery Disease/surgery , Nitric Oxide Synthase Type II/metabolism , Saphenous Vein/enzymology , Tissue and Organ Harvesting/methods , Transplants/enzymology , Animals , Coronary Artery Disease/enzymology , Coronary Artery Disease/pathology , Endothelium, Vascular/enzymology , Humans , Nitric Oxide/metabolism , Saphenous Vein/pathology , Saphenous Vein/physiopathology , Saphenous Vein/transplantation , Transplants/pathology , Transplants/physiopathology , Vascular PatencyABSTRACT
Varicose veins are a major chronic venous disease characterised by extensive remodelling of the extracellular matrix architecture in the vascular wall. Although matrix metalloproteinases have been implicated in these pathologic events, little is known about the functional relevance of other protease family members. Here, we studied the distribution of lysosomal cysteine proteases, cathepsins B, L, K, and S, and their endogenous inhibitor, cystatin C, in long saphenous vein specimens from nine normal donors and 18 patients with varicose veins (VVs). Immunohistochemical analysis demonstrated increased levels of cathepsins L, K, B, and S and reduced levels of cystatin C in VVs. This imbalance between cysteinyl cathepsins and cystatin C may favour VV remodelling. To investigate the inflammatory mechanism of their expression, we examined a detailed inflammatory cell profile in VVs, including macrophages, T lymphocytes, and mast cells. Increased numbers of CD3-positive T cells and tryptase-positive mast cells were found in VVs, and enhanced levels of cysteinyl cathepsins were detected from lesion CD3-positive T cells, chymase-positive mast cells, endothelial cells, and smooth-muscle cells. Elevated cathepsins, and their co-localisation to infiltrated inflammatory cells and to vascular cells, suggest that these proteases participate in extracellular matrix degradation in response to inflammation during VV pathogenesis.
Subject(s)
Cathepsins/analysis , Inflammation/enzymology , Saphenous Vein/enzymology , Varicose Veins/enzymology , Adult , Biomarkers/analysis , Case-Control Studies , Cystatin C/analysis , Endothelial Cells/enzymology , Female , Humans , Immunohistochemistry , Inflammation/immunology , Inflammation/pathology , Male , Mast Cells/enzymology , Middle Aged , Muscle, Smooth, Vascular/enzymology , Myocytes, Smooth Muscle/enzymology , Saphenous Vein/immunology , Saphenous Vein/pathology , T-Lymphocytes/enzymology , Up-Regulation , Varicose Veins/immunology , Varicose Veins/pathologyABSTRACT
BACKGROUND: Varicocele, inguinal hernia, and clinical manifestations related to chronic venous disorders are often associated, and collagen metabolism together with metalloproteinases (MMPs) alterations may be implicated. The aim of this study was to analyze the relationship between these factors. METHODS: We evaluated tissue and plasma samples from patients with varicocele, inguinal hernia, and great saphenous vein reflux, who underwent surgical treatment for their conditions. We then analyzed and correlated these findings with MMP levels. RESULTS: Significantly higher levels of MMP-1, -2, -12, and -13 were found in patients with inguinal hernia. MMP-9 levels were higher in patients with at least two of the conditions indicated. CONCLUSION: MMP-9 seems to be the common thread in various clinical conditions and is related to a more general and progressive disorder of collagen metabolism.
Subject(s)
Hernia, Inguinal/enzymology , Matrix Metalloproteinase 9/blood , Saphenous Vein/enzymology , Varicocele/enzymology , Venous Insufficiency/enzymology , Adult , Biomarkers/blood , Chronic Disease , Female , Hernia, Inguinal/blood , Hernia, Inguinal/diagnosis , Humans , Male , Middle Aged , Up-Regulation , Varicocele/blood , Varicocele/diagnosis , Venous Insufficiency/blood , Venous Insufficiency/diagnosisABSTRACT
OBJECTIVE: Following bypass surgery vein grafts undergo a remodelling process that can lead to restenosis and ultimately vein graft failure. Signalling through mitogen activated protein kinases (MAPKs) is a key mechanism involved in vein graft failure. Here, we investigated whether CBS3830 (c-a-i-r biosciences GmbH, Tübingen, Germany), a new highly selectively inhibitor of p38 MAPK, has a significant effect on inhibiting intimal, medial and adventitial hyperplasia. METHODS: Sixty specific pathogen free Sprague Dawley male rats were randomly divided into three groups. The control group with a reversed right jugular vein, which is common to carotid artery interposition graft, was compared with sham-operated, and CBS3830 treated animals. Intimal, medial and adventitia morphometric examinations and expression of proliferating cell nuclear antigen (PCNA) were analysed after one, two and four weeks for vein grafts. RESULTS: Intimal, medial and adventitia thickening in CBS3830 group were significantly lower than in the control group at each time point. Moreover, CBS3830 significantly reduced the phosphorylation of p38 MAPK and PCNA expression compared to the control. CONCLUSION: On the basis of the present work, intima, media and adventitia of saphenous vein grafts undergo vascular remodelling after surgery. The new, highly selective p38 MAPK inhibitor, CBS3830, ameliorates intimal, medial, and adventitial remodelling by varying degrees.
Subject(s)
Coronary Artery Bypass , Graft Occlusion, Vascular/prevention & control , Protein Kinase Inhibitors/pharmacology , Saphenous Vein/enzymology , Tunica Intima/enzymology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Adventitia/enzymology , Adventitia/pathology , Adventitia/physiopathology , Animals , Gene Expression Regulation/drug effects , Graft Occlusion, Vascular/enzymology , Graft Occlusion, Vascular/pathology , Graft Occlusion, Vascular/physiopathology , Male , Phosphorylation/drug effects , Proliferating Cell Nuclear Antigen/biosynthesis , Rats , Rats, Sprague-Dawley , Saphenous Vein/pathology , Saphenous Vein/physiopathology , Tunica Intima/pathology , Tunica Intima/physiopathology , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
No-touch (NT) saphenous vein (SV) grafts are superior to SVs harvested by the conventional technique (CT), with a patency comparable with the internal thoracic artery (ITA). Preservation of the vasa vasorum is implicated in the success of NT harvesting. We compared the vasa vasorum and endothelial nitric oxide synthase (eNOS) in NT SV with ITA and radial artery (RA) grafts. Skeletonized SV (SSV) was also analyzed. The NT SV had a higher number and larger vasa vasorum compared with ITA (P = .0001) and RA (P = .0004) that correlated with eNOS protein. Activity of eNOS in SSV grafts was significantly lower than NT SV grafts (P = 004). Since a high proportion of the vasa vasorum are removed in SSV using the CT, we suggest that preservation of the vasa vasorum and eNOS-derived NO contributes to the high patency for NT as compared with SSV grafts.
Subject(s)
Coronary Artery Bypass , Mammary Arteries/enzymology , Nitric Oxide Synthase Type III/metabolism , Radial Artery/enzymology , Saphenous Vein/enzymology , Tissue and Organ Harvesting/methods , Vasa Vasorum/enzymology , Aged , Blotting, Western , Coronary Artery Bypass/adverse effects , Graft Occlusion, Vascular/enzymology , Graft Occlusion, Vascular/etiology , Graft Occlusion, Vascular/physiopathology , Humans , Immunohistochemistry , Male , Mammary Arteries/physiopathology , Mammary Arteries/transplantation , Middle Aged , Nitric Oxide/metabolism , Radial Artery/physiopathology , Radial Artery/transplantation , Randomized Controlled Trials as Topic , Saphenous Vein/physiopathology , Saphenous Vein/transplantation , Tissue and Organ Harvesting/adverse effects , Vasa Vasorum/physiopathology , Vasa Vasorum/transplantation , Vascular PatencyABSTRACT
OBJECTIVE: To investigate the abnormal expressions of Tie1 on the valves of great saphenous varicose vein, and to discuss the relationship between the phenomenon and pathogenesis of varicose vein of lower extremity. METHODS: Varicose veins group 18 samples, normal control group 14 samples. Immunohistochemistry staining has investigated the expression of CD31 and Tie1 in the first valves of great saphenous veins. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) has checked mRNA expression of Tie1. Western blot has checked the expression of Tie1 protein in venous valves. RESULTS: In normal control group valves, there was no difference between proximal and distal sides endothelium, which expressing CD31 in both valvar basement and valve cusp (positive endothelial cells [ECs] percentage: P > 0.05, P > 0.05). However, the endothelium of the proximal side demonstrates Tie1 stronger than distal side in valvar basement (positive ECs percentage: P < 0.05), which was not found at valve cusp (positive ECs percentage: P > 0.05). In varicose veins group, the endothelium of proximal side cells expresses CD31 weaker than distal side at both valvar basement and valve cusp (positive ECs percentage: P < 0.05, P < 0.05) besides the morphological alteration of valves. Moreover, it expresses Tie1 much weaker than diatal side (positive ECs percentage: P < 0.01). Semi-quantitative RT-PCR showed that valves of varicose veins group expressed Tie1 much weaker than the normal control group (P < 0.01). Western blot could not detect the expression of Tie1 in venous valves. CONCLUSION: The decreasing expression of Tie1 may play an important role in the pathogenesis of primary lower extremity varicose veins.
Subject(s)
Endothelium, Vascular/enzymology , Gene Expression Regulation, Enzymologic , Receptor, TIE-1/biosynthesis , Saphenous Vein/enzymology , Varicose Veins/enzymology , Adult , Aged , Endothelium, Vascular/pathology , Female , Humans , Male , Middle Aged , Platelet Endothelial Cell Adhesion Molecule-1/biosynthesis , RNA, Messenger/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction , Saphenous Vein/pathology , Varicose Veins/pathologyABSTRACT
BACKGROUND: Smoking has numerous effects that may promote atherosclerosis, but the pathogenesis of smoking-related vein graft disease after coronary artery bypass grafting (CABG) remains incompletely understood. Matrix metalloproteinase (MMP) subtypes MMP-2 and MMP-9 have been identified as the key components in vascular remodeling processes. However little is known about the native MMP2 and MMP9 gene expression in saphenous vein (SV) conduits of heavy smokers undergoing CABG. METHODS: Two hundred eight patients were divided into 6 groups: nonsmokers, heavy smokers, 3-month quitters, 6-month quitters, 12-month quitters, and long-term quitters. mRNA and protein levels of MMP-2, MMP-9, and tissue inhibitors of metalloproteinases (TIMPs) 1 and TIMP-2 were analyzed. In a clinical study, SV graft patency after surgical procedures was followed up. RESULTS: Compared with the nonsmoker group, MMP2 and MMP9 gene expression was significantly increased in the other 5 groups (p < 0.05). In contrast to MMP response, TIMP1 and TIMP2 gene expression was significantly decreased (p < 0.05). An association of increased MMP2 and MMP9 gene expression with poor SV graft patency could be found in the clinical data from follow-up. CONCLUSIONS: Heavy smoking noticeably increases native MMP2 and MMP9 gene expression in the SV before CABG. Even after long-term cessation of smoking, the dysregulated MMP2 and MMP9 gene expression cannot recover to normal levels. With the elevated native MMP2 and MMP9 gene expression in the SV induced by heavy smoking, more vein graft disease can be found on long-term follow-up.
Subject(s)
Coronary Artery Bypass , Gene Expression Regulation , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 9/genetics , RNA, Messenger/genetics , Saphenous Vein/enzymology , Smoking/genetics , Aged , Blotting, Western , Coronary Artery Disease/enzymology , Coronary Artery Disease/genetics , Coronary Artery Disease/surgery , Female , Graft Survival , Humans , Immunohistochemistry , Male , Matrix Metalloproteinase 2/biosynthesis , Matrix Metalloproteinase 9/biosynthesis , Middle Aged , Preoperative Period , RNA, Messenger/biosynthesis , Real-Time Polymerase Chain Reaction , Saphenous Vein/transplantation , Severity of Illness Index , Smoking/metabolism , Tomography, Spiral ComputedABSTRACT
AIMS: The goal of this study was to investigate the importance of the vascular angiotensin convertase enzyme (ACE) in coronary artery bypass graft surgery (CABG) patients. METHODS: Vascular tissue (distal saphenous vein [n= 163] and/or radial artery [n= 120] segments) and blood samples were collected from CABG patients (n= 81). We studied (i) the potency of angiotensin I (AngI) and angiotensin II (AngII) to evoke vascular contractions; (ii) vascular and plasma ACE concentrations; and (iii) ACE genotype of the patients enrolled. RESULTS: The ratio of the potencies (EC(50) ) of AngII and AngI was significantly lower in radial artery compared to the saphenous vein (0.17 ± 0.03 nM and 0.51 ± 0.14 nM, respectively, P= 0.003), suggesting a 3-fold more effective AngI conversion in saphenous vein samples. Angiotensin constrictions were inhibited with telmisartan and captopril in both saphenous veins and radial arteries. Vascular ACE expression was significantly higher in saphenous vein compared to radial artery (9.7 ± 1.0 ng/mg and 5.3 ± 0.7 ng/mg, respectively, P= 0.01). Serum but no tissue ACE concentration was determined by ACE insertion/deletion polymorphism. Accordingly, no relation was found between serum and tissue ACE expression. CONCLUSION: ACE-inhibitor therapy targeting tissue located ACE may be beneficial to patients with saphenous vein grafts after CABG surgery.
Subject(s)
Coronary Artery Bypass/adverse effects , Peptidyl-Dipeptidase A/metabolism , Postoperative Complications/etiology , Saphenous Vein/transplantation , Aged , Angiotensin II Type 1 Receptor Blockers/pharmacology , Angiotensin-Converting Enzyme Inhibitors/pharmacology , Dose-Response Relationship, Drug , Female , Humans , Hungary , Male , Middle Aged , Peptidyl-Dipeptidase A/blood , Peptidyl-Dipeptidase A/genetics , Postoperative Complications/blood , Postoperative Complications/enzymology , Prospective Studies , Radial Artery/drug effects , Radial Artery/enzymology , Risk Assessment , Risk Factors , Saphenous Vein/drug effects , Saphenous Vein/enzymology , Time Factors , Treatment Outcome , Vasoconstrictor Agents/pharmacologyABSTRACT
We examined the contractile reactivity to 5-hydroxytryptamine (5-HT) in isolated human saphenous vein (SV), as a vascular conduit in coronary artery bypass grafting (CABG), harvested from patients with diabetes mellitus (DM) and non-DM (NDM). Vascular rings of endothelium-denuded SV were used for functional and biochemical experiments. The vasoconstrictions caused by 5-HT were significantly greater (hyperreactivity) in the DM group than in the NDM group. RhoA/ROCK pathway is activated by various G-protein-coupled receptor agonists and consequently induces phosphorylation of myosin phosphatase target subunit 1 (MYPT1), a subunit of myosin light chain phosphatase (MLCP), which inhibits MLCP activity. In the resting state of the vessels, total tissue protein levels of 5-HT(2A) receptor, 5-HT(1B) receptor, RhoA, ROCK1, and ROCK2 did not differ between NDM and DM groups. However, the total protein level of MYPT1 was significantly lower in the DM group than in the NDM group. Furthermore, the ratio of P(Thr(696))-MYPT1 to total MYPT1 was significantly higher in the DM group than in the NDM group. These results suggest that the hyperreactivity to 5-HT in the SV smooth muscle of patients with DM is due to not only enhanced phosphorylation of MLCP but also defective protein level of MLCP. Thus, we reveal for the first time that the defective protein level of MLCP in the DM group can partially explain the poor patency of SV graft harvested from patients with DM.