Saphenous Vein Smooth Muscle Research Articles

620. SMC-Specific Transcription Elements for Improved Adenoviral-Mediated Transgene Expression in Saphenous Veins

Top of pageAbstract Coronary artery bypass grafting (CABG) using autologous saphenous veins (SV) is an essential procedure to restore blood supply to the heart. However, long-term patency rates remain poor (93%, 74% and 41% at 1, 5 and 10 years post-grafting1) as vascular damage and remodeling often leads to neointima formation and accelerated atherosclerosis. Pharmacological interventions are largely ineffective and since the saphenous vein is accessible for ex vivo modification prior to arterial grafting, this presents an ideal window of opportunity for gene therapy. Several candidate therapeutic genes predicted to counteract vein graft failure have shown success pre-clinically but clinical translation is yet to take place. One of the most important issues is optimisation of transgene expression locally in the vein graft following gene delivery. We therefore assessed the ability of first-generation adenoviral vectors harbouring different promoter and post-transcriptional regulatory elements (Figure 1, ref. 2) for their expression levels in saphenous vein cells in vitro, ex vivo and in vivo. Our results demonstrate that Ad-PREP [in which lacZ expression is driven by the murine CMV promoter (mCMV), the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and a fragment of the smooth muscle myosin heavy chain promoter (RE)] produces significantly higher lacZ expression levels in primary saphenous vein smooth muscle cells (20-fold vs. RAd35 at m.o.i. 250) and endothelial cells (8-fold), but not in hepatocytes (1.23-fold), compared to RAd35, a standard Ad5-hCMV-bgal vector. This was particularly evident after short virus:cell incubation times (of 30 minutes), clearly relevant to the clinical setting. High levels of lacZ expression were also achieved with this vector following infection of human saphenous vein segments ex vivo, and 7 days after grafting autologous saphenous veins into pig carotid arteries in vivo. These results demonstrate that high transgene expression levels in saphenous vein can be achieved by the mCMV promoter, in combination with smooth muscle cell-specific and post-transcriptional regulatory elements.

N-Cadherin–Dependent Cell–Cell Contacts Promote Human Saphenous Vein Smooth Muscle Cell Survival

Vascular smooth muscle cell (VSMC) apoptosis is thought to contribute to atherosclerotic plaque instability. Cadherin mediates calcium-dependent homophilic cell-cell contact. We studied the role of N-cadherin in VSMC apoptosis. Human saphenous vein VSMCs were grown in agarose-coated wells to allow cadherin-mediated aggregate formation. Cell death and apoptosis were determined after disruption of cadherins using several approaches (n> or =3 per approach). Calcium removal from culture medium or addition of nonspecific cadherin antagonist peptides significantly decreased aggregate formation and increased cell death by apoptosis (34+/-6% versus 75+/-1% and 19+/-1% versus 40+/-5%, respectively; P<0.05). Specific inhibition of N-cadherin using antagonists and neutralizing antibodies similarly increased apoptosis. Supporting this, overexpression of full-length N-cadherin significantly reduced VSMC apoptosis from 44+/-10% to 20+/-3% (P<0.05), whereas abolishing N-cadherin expression by overexpression of a dominant-negative N-cadherin significantly, even in the presence of cell-matrix contacts, increased apoptosis from 9+/-2% to 50+/-1% (P<0.05). Interestingly, cell-cell contacts provided a similar degree of protection from apoptosis to cell-matrix contacts. Finally, N-cadherin-mediated cell-cell contacts initiated anti-apoptotic signaling by increasing Akt and Bad phosphorylation. Our results indicate that VSMC survival is dependent on N-cadherin-mediated cell-cell contacts, which could be important in the context of plaque instability.

Simvastatin inhibits MMP‐9 secretion from human saphenous vein smooth muscle cells by inhibiting the RhoA/ROCK pathway and reducing MMP‐9 mRNA levels

Increased matrix metalloproteinase-9 (MMP-9) expression is associated with intimal hyperplasia in saphenous vein (SV) bypass grafts. Recent evidence suggests that HMG-CoA reductase inhibitors (statins) can prevent the progression of vein graft failure. Here we investigated whether statins inhibited MMP-9 secretion from cultured human SV smooth muscle cells (SMC) and examined the underlying mechanisms. SV-SMC from different patients were exposed to phorbol ester (TPA) or PDGF-BB plus interleukin-1alpha (IL-1). MMP-9 secretion and mRNA expression were analyzed using gelatin zymography and RT-PCR, respectively. Specific signal transduction pathways were investigated by immunoblotting and pharmacological inhibition. Simvastatin reduced TPA- and PDGF/IL-1-induced MMP-9 secretion and mRNA levels, effects reversed by geranylgeranyl pyrophosphate and mimicked by inhibiting Rho geranylgeranylation or Rho-kinase (ROCK). MMP-9 secretion induced by PDGF/IL-1 was mediated via the ERK, p38 MAPK, and NFkappaB pathways, whereas that induced by TPA was mediated specifically via the ERK pathway. Simvastatin failed to inhibit activation of these signaling pathways. Moreover, simvastatin did not affect MMP-9 mRNA stability. Together these data suggest that simvastatin reduces MMP-9 secretion from human SV-SMC by inhibiting the RhoA/ROCK pathway and decreasing MMP-9 mRNA levels independently of effects on signaling pathways required for MMP-9 gene expression.

TNF-alpha induces proliferation or apoptosis in human saphenous vein smooth muscle cells depending on phenotype.

Tumor necrosis factor (TNF)-alpha is implicated in development of restenotic and atherosclerotic vascular lesions, which are pathological processes involving both proliferation and apoptosis of vascular smooth muscle cells (VSMCs). Human VSMCs were recently found to contain heterogeneous subpopulations. We therefore examined whether TNF has different effects on distinct subpopulations of VSMCs. With the use of cloning techniques, two stable subpopulations of VSMCs were isolated from human saphenous vein: spindle- and epithelioid-shaped smooth muscle cells (Sp- and Ep-SMCs, respectively). We found that TNF stimulated growth in Sp-SMCs but had a toxic effect on Ep-SMCs. TNF did not induce apoptosis in Sp-SMCs as determined by nuclear staining and cellular DNA electrophoresis. In contrast, the reduction of viability in Ep-SMCs was associated with induction of apoptosis as characterized by cellular DNA fragmentation and nuclear condensation. Higher levels of the TNF-R1 receptor subtype were detected in membrane preparations from Ep-SMCs than in membranes from Sp-SMCs. Activation of caspase-3 was also selectively induced in Ep-SMCs but not in Sp-SMCs. Cycloheximide, an inhibitor of protein synthesis, enhanced the toxicity of TNF in Ep-SMCs. This effect of cycloheximide was not seen in Sp-SMCs. The data presented here demonstrate for the first time that TNF either promotes growth or induces apoptosis in human VSMCs depending on phenotype.

128. Use of Chimeric Adenoviral Vectors To Assess Capsid Neutralization Determinants

In order to target vascular tissues site-specifically in vivo following systemic delivery it is necessary to develop platform vectors that possess negligible hepatic tropism. For Adenoviruses (Ad) this can be achieved by capsid mutation, capsid modification or pseudotyping which exploits Ad diversity. There are 51 known human Ads, divided into subgroups A–F. Certain Ads belonging to subgroup B and D bind to CD46 (Gagger, 2003; Wu, 2004) but other Ads bind receptors other than CD46 or the previously identified coxsackie and adenovirus receptor (CAR) and may therefore show a more favourable infectivity profile. Using pseudotyping we have investigated the infectivity and biodistribution of candidate fibers from subgroups B, C and D for vascular cell and hepatocyte transduction in vitro and their tissue distribution in vivo following iv administration. In vitro evaluation revealed that the subgroup D vectors Ad5/19p and Ad5/37 did not support the transduction of HepG2 hepatocytes (Ad5/19p 0.22% ± 0.06% vs Ad5; Ad5/37 1.5% ± 0.2% vs Ad5) whereas the other vectors evaluated, Ad5/3 and Ad5/16 produced high levels of transduction (Ad5/3 113% ± 10% vs Ad5; Ad5/16 74% ± 6% vs Ad5). The lack of hepatic tropism of Ad5/19p and Ad5/37 was replicated in both rat and mouse hepatocytes (ARL-6 and CCL-2254). Infectivity studies on vascular cells showed that Ad5/19p and Ad5/37 could achieve similar or improved transduction compared to Ad5 [Human saphenous vein endothelial cells (HSVEC) Ad5/19p and Ad5/37 produced 87% ± 11% and 84% ± 8% respectively of Ad5 transduction]. Both subgroup D vectors produced an increased transduction of human saphenous vein smooth muscle cells when compared to Ad5 (Ad5/19p 248% ± 42, Ad5/37 502% ± 130% vs Ad5 transduction). Transduction was mediated through a CAR independent pathway for Ad5/19p and Ad5/37 since an anti CAR neutralizing antibody failed to inhibit infection. We also examined CD46 as a possible primary receptor for Ad5/19p and Ad5/37. We found that Ad5/19p could not transduce CHO cells expressing the BC1 isoform of CD46 but Ad5/37 did albeit at much lower levels than Ad5/16. In vivo studies with Ad5/19p and Ad5/37 vectors were performed by iv injection into 12 week old male WKY rats. Animals were sacrificed at 1 hour and both Ad5/19p and Ad5/37 showed a 45% reduction (p < 0.05) in virion liver accumulation compared to Ad5, assessed by realtime PCR. At 5 days post viral infusion only Ad5 DNA was detectable in the liver. Transgene expression in liver sections was significantly reduced for Ad5/19p and Ad5/37 compared to Ad5 (Ad5 95%, Ad5/19p 12% and Ad5/37 7%, p < 0.05).These findings indicate that Ad19p and Ad37 pseudotyped Ad5 vectors show reduced hepatocyte transduction in vitro and in vivo and are promising for the development of systemically-injectable vascular-targeted gene therapies.

F-actin Capping (CapZ) and Other Contractile Saphenous Vein Smooth Muscle Proteins Are Altered by Hemodynamic Stress: A PROTEOMIC APPROACH

Increased force generation and smooth muscle remodeling follow the implantation of saphenous vein as an arterial bypass graft. Previously, we characterized and mapped 129 proteins in human saphenous vein medial smooth muscle using two-dimensional (2-D) PAGE and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Here, we focus on actin filament remodeling in response to simulated arterial flow. Human saphenous vein was exposed to simulated venous or arterial flow for 90 min in vitro, and the contractile medial smooth muscle was dissected out and subjected to 2-D gel electrophoresis using a non-linear immobilized pH 3-10 gradient in the first dimension. Proteins were analyzed quantitatively using PDQuest 2-D software. The actin polymerization inhibitor cytochalasin B (1 microm) prevented increases in force generation after 90 min of simulated arterial flow. At this time point, there were several consistent changes in actin filament-associated protein expression (seven paired vein samples). The heat shock protein HSP27, identified as a three-spot charge train, showed a 1.6-fold increase in abundance (p = 0.01), but with reduced representation of the phosphorylated Ser(82) and Ser(15)Ser(82) isoforms (p = 0.018). The abundance of actin-capping protein alpha2 subunit CapZ had decreased 3-fold, p = 0.04. A 19-kDa proteolytic fragment of actin increased 2-fold, p = 0.04. For the four-spot charge train of gelsolin, there was reduced representation of the more acidic isoforms, p = 0.022. The abundance of other proteins associated with actin filaments, including cofilin and destrin, remained unchanged after arterial flow. Actin filament remodeling with differential expression and/or post-translational modification of proteins involved in capping the barbed end of actin filaments, HSP27 and CapZ, is an early response of contractile saphenous vein smooth muscle cells to hemodynamic stress. The observed changes would favor the generation of contractile stress fibers.

Mechanical stretch induces phosphorylation of p38-MAPK and apoptosis in human saphenous vein.

Failure of saphenous vein grafts remains a major limitation of coronary bypass surgery. The aims of the present study were to determine whether pressure distension of human saphenous vein induces the activation of p38-MAPK and to determine its role in apoptosis. Phosphorylated p38 was detected at basal levels in human saphenous vein obtained immediately after harvesting. Distended saphenous vein showed significantly higher levels of phosphorylated p38 compared with control vein (P<0.01) and nondistended saphenous vein maintained for 3 and 6 hours after harvesting (both P<0.01). Apoptosis in distended and nondistended vein was significantly higher at 24 hours compared with control vein, with distended vein showing increased apoptosis compared with nondistended saphenous vein at all time points investigated (P<0.001). Immunolocalization showed co-localization of phosphorylated p38 and apoptosis. Inhibition of p38 activity reduced the apoptotic index of cultured vascular smooth muscle cells by 72.1%+/-1.2% and cultured distended saphenous vein segments by 72.7%+/-0.9%. Pressure distension of intact human saphenous vein induces activation of p38, and this is associated with apoptosis. Inhibition of p38 kinase activity in saphenous vein smooth muscle cells and intact vein reduces apoptosis. These findings contribute to our understanding of the mechanisms of saphenous vein graft failure.

Improved gene delivery to human saphenous vein cells and tissue using a peptide-modified adenoviral vector

The establishment of efficient gene delivery to target human tissue is a major obstacle for transition of gene therapy from the pre-clinical phases to the clinic. The poor long-term patency rates for coronary artery bypass grafting (CABG) is a major clinical problem that lacks an effective and proven pharmacological intervention. Late vein graft failure occurs due to neointima formation and accelerated atherosclerosis. Since CABG allows a clinical window of opportunity to genetically modify vein ex vivo prior to grafting it represents an ideal opportunity to develop gene-based therapies. Adenoviral vectors have been frequently used for gene delivery to vein ex vivo and pre-clinical studies have shown effective blockade in neointima development by overexpression of candidate therapeutic genes. However, high titers of adenovirus are required to achieve sufficient gene delivery to provide therapeutic benefit. Improvement in the uptake of adenovirus into the vessel wall would therefore be of benefit. Here we determined the ability of an adenovirus serotype 5 vector genetically-engineered with the RGD-4C integrin targeting peptide inserted into the HI loop (Ad-RGD) to improve the transduction of human saphenous vein smooth muscle cells (HSVSMC), endothelial cells (HSVEC) and intact saphenous vein compared to a non-modified virus (Ad-CTL). We exposed each cell type to virus for 10, 30 or 60 mins and measured transgene at 24 h post infection. For both HSVSMC and HSVEC Ad-RGD mediated increased transduction, with the largest increases observed in HSVSMC. When the experiments were repeated with intact human saphenous vein (the ultimate clinical target for gene therapy), again Ad-RGD mediated higher levels of transduction, at all clinically relevant exposures times (10, 30 and 60 mins tissue:virus exposure). Our study demonstrates the ability of peptide-modified Ad vectors to improve transduction to human vein graft cells and tissue and has important implications for gene therapy for CABG.

Inhibitory activity of clinical thiazolidinedione peroxisome proliferator activating receptor-gamma ligands toward internal mammary artery, radial artery, and saphenous vein smooth muscle cell proliferation.

The proliferation of vascular smooth muscle cells (VSMCs) is a known response to arterial injury that is an important part of the process of restenosis and atherosclerosis. People with diabetes have an increased risk of cardiovascular disease resulting from accelerated coronary atherosclerosis. The newest drugs for Type 2 diabetes are thiazolidinediones, which are insulin-sensitizing peroxisome proliferator activating receptor-gamma (PPARgamma) ligands. We investigated the antiproliferative effects of troglitazone, rosiglitazone, and pioglitazone on VSMCs derived from the three vascular beds used for coronary artery by-pass grafting: the internal mammary and radial artery and saphenous veins. The three vessels yielded proliferating cells of slightly differing morphology. Inhibition of cell proliferation was assessed by cell counting and cell cycle studies by Western blotting for phosphorylated retinoblastoma protein. All three thiazolidinediones showed inhibitory potency toward cell proliferation with a potency troglitazone>rosiglitazone approximately pioglitazone, and this potency profile was maintained toward the growth factor and insulin-stimulated phosphorylation of the retinoblastoma protein, which controls cell cycle progression. The inhibitory potency of clinical thiazolidinediones toward different vascular sources is dependent on the individual thiazolidinedione and very little on the vascular source.

Heat Shock Protein (65 kDa) – Stimulated Proliferation of Human Saphenous Vein Smooth Muscle Cells is Inhibited by Thapsigargin

Aim: Heat shock protein (HSP) expression has been widely implicated in atherogenesis. Vein graft failure following coronary artery bypass graft surgery (CABG) involves medial thickening and neointima formation, a phenomenon mediated by vascular smooth muscle cell (VSMC) proliferation. Superimposed atherogenesis can then result in vein graft failure in as many as 50% of cases within ten years after surgery. In order to explore the possible role of HSP in mediating vein graft disease, the effect of HSP 65 kDA on the proliferation of VSMCS obtained from human saphenous vein was investigated. The role of calcium was also studied using thapsigargin which blocks the release of calcium from intracellular storage pools. Methods: VSMCs were grown from human saphenous vein using standard culture methods. Cells were grown to confluence using DMEM + 10% Foetal Calf serum (FCS). When confluent, cells were trypsinised and cultured onto 96-well plates and rendered quiescent with 0.4% FCS. HSP 65kDA, over a range of concentrations (with and without 10 nM thapsigargin) was added to the cells and proliferation stimulated with 10% FCS and further incubated for 48 hours. Proliferation was assessed by the uptake of 5-bromo-2′ deoxyurindine (Brdu) using colorimetric ELISA and cell counts from which% changes in proliferation were calculated. Results: HSP elicited a dose-dependent increase in the proliferation of VSMCs (table 1) an effect completely inhibited by the presence of 10 nM thapsigargin (table 1). This concentration of thapsigargin has previously been shown to inhibit VSMC proliferation through depletion of intracellular calcium pools. Table 1. Effect of HSP 65 kDa on VSMC Proliferation (% Change Relative to Zero) [± SEM;n = 6]: A) Without Thapsigargin and B) with Thapsigargin*p < 0.05, Compared to Zero;# p < 0.05, A vs. B [HSP].(μg/ml) 0 0.001 0.01 0.1 1 10 A 0 +46 ± 10* +61 ± 14* +65 ± 18* +60 ± 12* +55 ± 10* B 0 −2 ± 0.2# −4 ± 0.4# −10 ± 4# −18 ± 4# −15 ± 3# Conclusions: Since HSP 65 kDa stimulates the proliferation of VSMCs, they may play a role in graft thickening and neointima formation as well as superimposed atherogenesis. As this may result in late vein graft failure, further investigations into the role of HSP expression in vein graft disease is warranted. The inhibition of this HSP-mediated effect by thapsigargin consolidates the crucial role of calcium in mediating VSMC proliferation and that inhibition of this event represents a potential therapeutic strategy for the prevention of vein graft thickening.

Ca2+ mobilization in saphenous vein smooth muscle cells derived from patients with primary varicosity.

Human primary varicosity is associated with 'weakness' of the vein wall. We investigated whether the reduced responsiveness of varicose veins to physiological vasoconstrictors might result from impaired Ca2+ mobilization in venous smooth muscle. The hypothesis was tested in cells derived from phenotypically different vein segments that were obtained from the inguinal saphenous vein (tissue with incompetent valves), the distal portion of the long saphenous vein just above the medial ankle (clinically healthy tissue), and from a tributary to the long saphenous vein just below the knee (incompetent and overtly varicose tissue). Saphenous vein from patients undergoing cardiac surgery served as control. Cytosolic free Ca2+ levels ([Ca2+]i) were determined with the fura-2 method in cultured medial smooth muscle cells of third to sixth passage (21-23 measurements per tissue derived from five controls and seven patients). Angiotensin II (10 nmol L-1 to 10 mumol L-1) induced a significantly (P < 0.05) smaller rise in [Ca(2+)1i response in cells derived from incompetent or varicose segments (approximatley 70 nmol L-1) than in cells derived from clinically healthy vein (approximately 130 nmol L-1) or controls (approximately 170 nmol L-1). Likewise, the effect of endothelin-1 (100 nmol L-1) on [Ca2+]i was considerably less in cells derived from segments with incompetent valves or from varicose vessel segments than in cells derived from control patients (P < 0.05). In organ baths, endothelium-denuded strips of varicose vessels contracted significantly less in response to these agonists than clinically healthy segments from the same patient. The reduced contractility of diseased human varicose veins in response to angiotensin II and endothelin-1 involves impaired Ca2+ mobilization.

Circumferential stretching of saphenous vein smooth muscle enhances vasoconstrictor responses by Rho kinase-dependent pathways.

Surgical preparation and/or pulsatile arterial perfusion of saphenous vein increases the sensitivity of vein rings to calcium mobilising agonists such as phenylephrine. We have investigated the mechanism(s) underlying this effect. We have used an ex vivo flow circuit, with simulated arterial or venous flows (mean pressure 100 and 20 mmHg, respectively), to investigate the sensitivity of human saphenous vein to phenylephrine, 5-hydroxytryptamine (5-HT) and KCl, using organ chamber pharmacology. After 90 min of pulsatile arterial perfusion the mean maximum tension induced by KCl had increased from 4.7 to 11.1 g (n=5), by phenylephrine had increased from 4.4 to 10.2 g (n=8) and by 5-HT had increased from 4.4 to 6.7 g (n=10), all P<0.01. Phenylephrine did not augment the tension in vein rings maximally precontracted with KCl (n=4). The EC(50) for KCl was unchanged after pulsatile arterial perfusion (n=5), but for phenylephrine and 5-HT there were significant reductions from 14+/-5 to 2+/-1 microM (n=8) and from 1.0+/-0.4 to 0.20+/-0.06 microM (n=10), respectively. The rate of contraction (in response to 3 microM phenylephrine) increased from 0.11 g/min to 0.37 g/min, P<0.02, after arterial perfusion (n=4). These changes in contractile properties (to phenylephrine) were endothelium-independent, evident within 5 min of simulated arterial perfusion. The changes in contractile properties could be abrogated by external stenting of the vein (to attenuate circumferential deformation) or inclusion in the perfusate of a vasodilator, e.g., cromakalim (5 microM) or the selective Rho kinase inhibitor Y-27632 (20 microM). The heightened sensitivity and contractility to phenylephrine was maintained after inclusion of adenosine (100 microM), gadolinium (10 microM) or cycloheximide (10 microM) in the vein perfusate. The circumferential deformations imposed by simulated arterial perfusion alter the vasomotor responses of saphenous vein smooth muscle. These effects are independent of new protein synthesis or the activation of stretch activated cation channels. The Rho kinase pathway appears to mediate the signalling mechanisms leading to increased agonist-induced tension and the increased sensitivity to vasoconstrictors.

Action of an HMG CoA reductase inhibitor, lovastatin, on apoptosis of untransformed and ts-SV40 transformed human smooth muscle cells derived from saphenous vein.

The effect of lovastatin, an inhibitor of 3-hydroxymethyl-3-glutaryl coenzyme A (HMG CoA) reductase, was examined on human vascular smooth muscle cells (HVSMC). Untransformed HVSMC were obtained from saphenous vein and in addition an SV-40 transformed immortalized cell line (HVTs-SM1) derived from saphenous vein smooth muscle was also used. HVTs-SM1 cell proliferation and DNA synthesis were measured, and cell cycle analysis was performed by flow cytometry. Apoptosis in both cell types was assessed by a combination of flow cytometry, terminal deoxynucleotidyl transferase (TUNEL) reagent-based immunocytochemistry, DAPI staining, and DNA agarose gel electrophoresis. Lovastatin had no effect on apoptosis of HVSMC over 96 h in serum-free conditions or after stimulation with platelet-derived growth factor (PDGF-BB), although PDGF-BB increased apoptosis in HVSMC, and this was prevented by lovastatin. In HVTs-SM1 cells lovastatin inhibited cell proliferation and DNA synthesis and induced apoptosis in a time- and concentration-dependent manner. The effects of lovastatin on cell proliferation, DNA synthesis, and apoptosis were prevented by coincubation with mevalonate and geranylgeranyl pyrophosphate, but not by farnesyl pyrophosphate. Lovastatin does not induce apoptosis in saphenous vein HVSMC in culture and inhibits PDGF-BB-induced DNA synthesis and apoptosis. In contrast, in SV40 transformed immortalized HVTs-SM1 cells, lovastatin induces apoptosis and inhibits cell proliferation and DNA synthesis. The pro-apoptotic effects of lovastatin in SV40 transformed HVTs-SM1 cells may be related to the enhanced rate of proliferation or deregulation of the cell cycle in this cell line.

Impaired Contractility of Left-over Vein Grafts used for CABG and the Possible Trauma Caused by Air Exposure

Objective –The patency of vein grafts limits the long-term results after bypass surgery. Vein graft trauma may contribute to the clinical results. Design –Left-over pieces of vein after routine coronary bypass surgery were compared with references of the same vein that were stored with maximum care. Blood perfusion (20 h, 37°C) was also tested to evaluate possible recovery. Vessel rings were measured by isometric tone recordings. Results –Left-over veins showed a contractile dysfunction compared to reference ( p < 0.01-0.001, n = 13), and that did not recover after blood perfusion. Four trauma models were tested, using saphenous vein graft (SVG)-references ( n = 5). Chemical exposures (0-90 min) to NaCl-papaverin or cardioplegic medium affected the baseline ( p < 0.05-0.01). With mechanical distension, up to 450 mmHg leak testing, an increased norepinephrine (NE) reactivity appeared. Air contact, 20 min, reduced the NE reactivity to half maximum ( p < 0.05). Longer exposures (40-60 min) completely abolished the contractility. Conclusions –The saphenous vein smooth muscle function appeared permanently traumatized following routine harvesting and handling. Air exposure gave dramatic deterioration and could in theory contribute to hamper the longterm results after bypass surgery.

Action of angiotensin II on DNA synthesis by human saphenous vein in organ culture.

Angiotensin II (Ang II), an effector peptide of the renin-angiotensin system, has been reported to stimulate growth of blood vessels in vivo and smooth muscle cells in culture. In this study, the effect of Ang II on DNA synthesis was examined in deendothelialized human saphenous vein in organ culture. After 7 days' exposure to medium containing 0.4% fetal calf serum plus Ang II, there was a marked increase in DNA synthesis. The effect of Ang II was comparable to the response to platelet-derived growth factor. Responses to Ang II were partially inhibited by the AT(1) receptor antagonist candesartan. An AT(2) receptor antagonist, PD123319, had no effect on Ang II-induced DNA synthesis, either alone or in combination with candesartan. The Ang II peptide analogues [Sar(1), Ile(8)]-Ang II (saralasin) and [Sar(1),Thr(8)]-Ang II (sarthran) acted as agonists, increasing DNA synthesis. In the presence of saralasin, responses to Ang II were inhibited. Tyrphostin-23, a tyrosine kinase inhibitor, prevented Ang II-induced DNA synthesis and reduced DNA synthesis in tissues incubated in medium containing only 0.4% fetal calf serum. In conclusion, Ang II stimulates DNA synthesis in human saphenous vein in organ culture. The effect of Ang II was more marked than has been previously reported in isolated cultured saphenous vein smooth muscle cells, and this effect is mediated in part by an angiotensin type 1 receptor. It is possible that an undefined receptor for Ang II may also be involved in the stimulation of DNA synthesis in this preparation.

Essential roles of IkappaB kinases alpha and beta in serum- and IL-1-induced human VSMC proliferation.

Interleukin-1 (IL-1) is a potent vascular smooth muscle cell (VSMC) mitogen, which can stimulate cells via activation of nuclear factor-kappaB (NF-kappaB) following phosphorylation of its inhibitory subunit (IkappaB). Because the proliferative effect of IL-1 is additive with that of serum, the present studies assessed the role of IkappaB kinases (IKKs) and NF-kappaB in both IL-1- and serum-induced VSMC proliferation. IL-1beta (1 ng/ml) induced marked and persistent NF-kappaB activation in VSMC that was maximal at 1 h and persisted for 3 days. There was a 3-fold increase in DNA synthesis after acute IL-1 exposure (24-96 h) and a 12-fold increase after chronic IL-1 exposure (>7 days). Electrophoretic mobility shift assay and supershift analysis indicated that IL-1-induced NF-kappaB complexes consisted of p65/p50 heterodimers and p50 homodimers. Human saphenous vein smooth muscle cells (HSVSMC) were transiently cotransfected with expression plasmids encoding a dominant negative mutant form of either IKKalpha or IKKbeta, in which K(44) was mutated to A (K44A), and a green fluorescent protein expression plasmid that allows identification of transfected cells. IL-1 induced nuclear localization of p65 in 95% of cells transfected with vector alone but in only 69% and 26% of cells expressing IKKalpha (K44A) or IKKbeta (K44A), respectively. Likewise, proliferation increased 3.2-fold in IL-1-treated HSVSMC which had been transfected with vector alone, but only 2.2- and 1.5-fold proliferation in HSVSMC expressing IKKalpha (K44A) or IKKbeta (K44A), respectively. Although serum activated NF-kappaB transiently, serum-induced proliferation was markedly attenuated in HSVSMC expressing IKKalpha (K44A) and IKKbeta (K44A) compared with HSVSMC transfected with vector alone. The results support an essential role of IKKs in the proliferative response of HSVSMC to IL-1 and to serum.

Role of a Ca2+-activated K+ current in the maintenance of resting membrane potential of isolated, human, saphenous vein smooth muscle cells.

Calcium-activated potassium currents were studied in dissociated smooth muscle cells from human saphenous vein (HSV) using the patch-clamp technique in the whole-cell configuration. The average measured resting membrane potential (Vm) was -41+/-2 mV (n=39), when the cells were dialysed with an intracellular pipette solution (IPS) containing 0.1 mM ethyleneglycol-bis(beta-aminoethylether)-N,N,N', N'-tetraacetic acid (EGTA) (IPS-0.1 mM EGTA). When the EGTA concentration was increased to 10 mM (IPS-10 mM EGTA) Vm became significantly less negative: -13+/-2 mV (n=23, P<0.05). These results suggest that 10 mM EGTA reduces a calcium-dependent current involved in the maintenance of Vm. Depolarizing voltage steps up to +60 mV from holding potentials of -60 mV resulted in large (1-10 nA) time- and voltage-dependent outward currents. The amplitudes of total whole-cell current densities measured at voltages above -20 mV were significantly greater in the cells dialysed with IPS-0.1 mM EGTA than in those dialysed with IPS-10 mM EGTA. In the cells dialysed with IPS-0.1 mM EGTA, 0.1 mM tetraethylammonium chloride (TEA) and 50 nM iberiotoxin (IBTX), which selectively block large conductance Ca2+-activated potassium channels (BKCa), diminished the total current recorded at +60 mV by 45+/-14% (P<0.05, n=5) and 50+/-6% (n=8, P<0.05), respectively. These blockers at the same concentrations did not affect the total current in cells dialysed with IPS-10 mM EGTA. When tested on intact HSV rings, both 0.1 mM TEA and 50 nM IBTX elicited vessel contraction. We conclude that BKCa channels present in HSV smooth muscle cells contribute to the maintenance of the Vm and sustain a significant portion of the total voltage-activated, outward current. Finally, BKCa channels appear to play a significant role in the regulation of HSV smooth muscle contractile activity.

Role of isoprenylation in the inhibitory action of lovastatin on proliferation of SV40 immortalized human saphenous vein smooth muscle cells.

Lovastatin is a competitive inhibitor of HMG CoA reductase [I]. Inhibition of this enzyme blocks both cellular synthesis of cholesterol and other intermediates derived from mevalonate. These include a number of important intermediates such as the isoprenoids, famesol pyrophosphate and geranylgeraniol pyrophosphate [2]. The latter are usually involved in the lipid modification (isoprenylation) of a number of proteins including key signalling molecules such as members of the Ras superfamily [3]. Isoprenoid modification seems to be required for proper localization and functional activity of these proteins. Previous studies have shown that inhibition of HMG CoA reductase by statins decreases vascular smooth muscle cell proliferation despite the free availability of cholesterol and without gduction in cellular levels of cholesterol [4]. These effects were reversed by mevalonate suggesting that they may be due to inhibition of protein isoprenylation. In this study we examined the effect of lovastatin on proliferation and DNA synthesis in a cultured immortalized smooth muscle cell line. SV-40 large T antigen transformed SM 1 cells, an immortalized clonal smooth muscle cell line derived from cultured human saphenous vein cells isolated from a single individual were used for all experiments. SMI cells were routinely cultured in DMEM supplemented with 1OOIU/ml penicillin, IOOpg/ml streptomycin sulphate, geneticin ((3418; 0.2m ml), and 10% (v/v) foetal calf serum (FCS). SMI cells (-14x10 cells/cm*' were seeded onto 24 well plates and 9cm petri dishes for proliferation assays and flow cytometric (FACS) analysis respectively. All treatments were carried out either in serum-free medium or medium containing 10% (vlv) dialysed FCS. Cell cycle analysis and quantification of % hypodiploid (A& apoptotic cells was performed following harvesting [5]. Cells were processed using Cell Testm Plus DNA reagent kit (Becton Dickinson, CA USA). SMl proliferation was measured using a Coulter Counter. DNA synthesis was also assessed by [3Hmethyl] thymidine incorporation. For these studies cells (seeded at -6000 cells/well) were exposed to ['H-methyl] thymidine (5pCVml) for 4 hours after a 22-hour stimulation with 10% dialysed FCS containing lovastatin (1Op.M) or vehicle. Mevalonate (0. ImM), famesyl pyrophosphate (FPP; 1Op.M) and geranylgeranyl pyrophosphates (GPP; 1Op.M) were also coincubated with lovastatin in some studies. Cell proliferation was inhibited by lovastatin (Fig.]). This effect was prevented by co-incubation with mevalonate or GPP, but not with FP (0.OlmM) (Fig.l).FACS analysis indicated that in the presence of 10% FCS S-phase increased from 13i1% to 26d% over 48h. This increase was inhibited by lovastatin at 48h, but not at 24h. Similarly lovastatin inhibited [3H-methyl] thymidine incorporation at 48h and 72h in response to 10% FCS but had no significant effect at 24h (Fig. 3). The inhibitory effect of lovastatin on ['H-methyl] thymidine incorporation was prevented by coincubation with mevalonate, GPP, but not by FPP. Y Cell number (x 104

Endothelin-B receptors mediate intimal hyperplasia in an organ culture of human saphenous vein

Objective: Although a number of pharmacologic agents have been shown to reduce intimal hyperplasia in animal models of restenosis, to date no systemic agent has conclusively been shown to be effective in humans. Recently, considerable attention has been directed towards endothelin (ET), a potent vasoconstrictor and a powerful mitogen for vascular smooth muscle cells, as a mediator of intimal hyperplasia. Endothelin-1 has been shown to be mitogenic for human saphenous vein smooth muscle cells, and expression also is elevated in human vein graft stenosis. The aim of this study was the investigation of whether ET receptor antagonists can attenuate neointima formation in a laboratory model of vein graft intimal hyperplasia and the determination of whether the effects are mediated by a specific ET receptor subtype. Methods: We used an organ culture of human saphenous vein, a well-validated model of vein graft intimal hyperplasia. Paired segments of human long saphenous vein were cultured with and without the following antagonists: bosentan, a nonselective ET receptor antagonist; BQ 123, a specific endothelin-A antagonist; or BQ 788, a specific endothelin-B (ET B) antagonist. After 14 days in the culture, the segments were fixed and processed and the sections were immunostained to facilitate the measurements of neointimal thickness with a computerized image analysis system. Results: The nonselective antagonist bosentan and the ET B selective antagonist BQ 788 significantly reduced neointima formation by 70% ( P = .001) and 50% ( P = .03), respectively, but the ET A antagonist BQ 123 had no significant effect on the reduction of neointima formation ( P = 1.0). Conclusion: The results of this study imply an important role for ET as a mediator of human vein graft intimal hyperplasia and imply further that a specific ET B antagonist may have a therapeutic potential for the prevention of vein graft stenosis. (J Vasc Surg 1998;28:695-701.)

P2X7 receptor activation-induced contraction and lysis in human saphenous vein smooth muscle.

In cutaneous veins where purinergic neurotransmission is more prominent compared with in deep vessels, physiological and pathological roles of nerve-released ATP have been described. Neuronally released ATP has been reported to act through activation of unidentified ionotropic P2X receptor(s). This study analyzed P2X receptor subtypes expressed in human saphenous vein smooth muscle and their physiological functions. Transcripts for both hP2X1 receptors, already identified in other smooth muscles, and, surprisingly, hP2X7 receptors known to be responsible for the cytotoxic effect of ATP in macrophages were detected by Northern blot analysis in total RNA from saphenous vein smooth muscle. ATP and other P2X receptor agonists [alphabeta-methylene-ATP, 2-methylthio-ATP, and 2',3'-(4-benzoyl)benzoyl-ATP] dose-dependently contracted venous rings, but the contraction induced by 2-methylthio-ATP was more transient than that evoked by the other P2X agonists. The effect of hP2X1 agonists involved the activation of a rapidly desensitizing cation current recorded in freshly isolated myocytes. The action of hP2X7 receptor agonists was related to a maintained nondesensitizing cation current. In addition, hP2X7 receptor activation formed membrane pores that were permeable to large molecules. hP2X1 and hP2X7 receptors coexpressed in COS cells did not associate to form heteromultimers. Our data indicate that both hP2X1 and hP2X7 receptors are expressed as 2 separated populations of channels in human saphenous vein myocytes and are involved in ATP-induced tension. We suggest that cell lysis consequent to hP2X7 receptor-induced pore formation contributes to the disorganization and decrease in the amount of contractile myocytes in the media of varicose veins.