Cathy M. Holt

Interleukin-1β in Coronary Arteries of Patients With Ischemic Heart Disease

Interleukin-1 beta (IL-1 beta) is known to have a number of effects on the different cell types present within coronary arteries. In this study we identified the location and phenotype of cells containing IL-1 beta in human coronary artery specimens from patients suffering from either coronary atherosclerosis or cardiomyopathy and correlated the presence of IL-1 beta with disease severity. Luminal endothelial cells, adventitial vessel wall cells, and macrophages were double labeled immunohistochemically for IL-1 beta protein and a cell type-specific monoclonal antibody for either endothelial cells or macrophages. In situ hybridization was performed to locate the presence of IL-1 beta mRNA within the coronary artery wall. In this study IL-1 beta protein was found to be increased in the adventitial vessel walls of atherosclerotic coronary arteries compared with coronary arteries from nonischemic cardiomyopathic hearts. This increase was directly proportional to the severity of coronary atherosclerosis. IL-1 beta protein was also detected in luminal endothelium and macrophages of atherosclerotic coronary arteries and coronary arteries from nonischemic cardiomyopathic hearts. IL-1 beta mRNA was found in luminal endothelial cells, adventitial vessel endothelial cells, and macrophages. We conclude that IL-1 beta is produced by endothelial cells and macrophages in coronary arteries from ischemic hearts and to a lesser extent from nonischemic cardiomyopathic hearts.

The Effect of Oligonucleotides to c-myb on Vascular Smooth Muscle Cell Proliferation and Neointima Formation After Porcine Coronary Angioplasty

Proto-oncogenes, including c-myb, are expressed early after vascular injury. The application of antisense oligodeoxynucleotides (AS-ODNs) against these genes inhibits cell proliferation and neointima formation in small animals and in peripheral arteries. The aim of this study was to investigate the specificity of action of AS-ODN-c-myb in vitro and to assess its effect, when delivered locally, on neointima formation after percutaneous transluminal coronary angioplasty (PTCA) in porcine coronary arteries. AS-ODN-c-myb inhibited the proliferation of vascular smooth muscle cells (VSMCs) in vitro in a dose-dependent manner. There was a corresponding reduction in steady state levels of c-myb mRNA and protein. Expression of another early gene, c-fos, was unaffected. S1 nuclease analysis demonstrated intact full-length AS-ODN-c-myb retrieved from VSMCs in culture after 12 hours. A range of ODNs, related and unrelated to c-myb, with and without a GGGG sequence, inhibited VSMC proliferation. Phosphorothioated AS-ODN-c-myb was 30 times less potent than unphosphorothioated AS-ODN-c-myb. PTCA induced porcine coronary artery neointima formation. c-myb mRNA was maximally induced 18 hours after injury. Unmodified AS-ODN-c-myb, sense-ODN-c-myb, saline, or nothing was delivered immediately after balloon dilatation via a double-skinned porous balloon (Transport, SciMed). Fluorescence-labeled AS-ODN-c-myb was deposited throughout the vessel wall. Mean maximum intima/media cross-sectional area 4 weeks after PTCA was reduced with AS-ODN-c-myb by 79% compared with saline (P < .05), 82% compared with sense-ODN-c-myb, and 63% compared with nothing (P < .10). Conclusions are as follows: (1) c-myb is expressed in VSMCs after vascular injury. (2) AS-ODN-c-myb is retained intact in VSMCs, reducing their proliferation in vitro in dose-dependent fashion, with reduction in c-myb mRNA and protein, whereas sense-ODN-c-myb is not. (3) A range of ODNs can reduce VSMC proliferation by a non-sequence-specific mechanism. (4) Phosphorothioate protection of antisense molecules may reduce their efficacy. (5) Local delivery of unmodified AS-ODN-c-myb via the Transport catheter reduces neointima formation after porcine PTCA. (6) Local delivery of fluid may exacerbate neointimal thickening.

Temporal and spatial distribution of interleukin-1β in balloon injured porcine coronary arteries

Objective: Interleukin-1β (IL-1β) is a proinflammatory cytokine with a wide range of biological activities. We determined the distribution of IL-1β following percutaneous transluminal coronary angioplasty (PTCA) of porcine arteries, and the presence of caspase-1 (required for the activation of IL-1β). Methods: Oversized balloon angioplasty was performed in Yorkshire White pigs and the vessels excised at selected intervals (1, 6, 18 h, 3, 7, and 14 days) post-PTCA. IL-1β and caspase-1 were then identified using reverse transcription polymerase chain reaction (RT-PCR), in situ RT-PCR, and immunohistochemistry. Results: IL-1β protein was detected in the inflammatory infiltrate up to 3 days after PTCA. Luminal endothelial cells contained IL-1β at 1 h, with peak expression at 3–7 days. Adventitial capillaries were IL-1β-positive at all timepoints. IL-1β was detected in adventitial cells at 3 days, with reduced levels at 7 and 14 days. At 7 days, neointimal cells were also IL-1β positive. No IL-1β was detected in non-PTCA control arteries. RT-PCR demonstrated IL-1β mRNA expression to be induced at 1 h, and absent at 3 days. In situ RT-PCR revealed this expression to be distributed throughout the arterial layers at 6 h, but localized to the adventitia at 18 h, with a baseline expression in the adventitial layer of non-PTCA controls. Caspase-1 was detected in luminal endothelial cells from 6 h, in adventitial cells from 3 days, and in neointimal cells from 7 days post-PTCA. This expression colocalized with IL-1β, indicating the potential for the IL-1β present to become activated. Conclusions: We conclude that IL-1β is synthesised, in conjunction with caspase-1, by endothelial, inflammatory, and adventitial cells early (within 3 days) after PTCA, with decreased levels at later timepoints, suggesting that it has a key role to play in the early stages of healing following PTCA.

Comparison of response to injury in organ culture of human saphenous vein and internal mammary artery

Autologous saphenous vein grafts, unlike internal mammary artery grafts, suffer many late occlusions as a result of excessive proliferation of vascular smooth muscle cells and the superimposition of atheroma on the resulting thickened intima. We investigated the possible basis of this difference using organ cultures. Internal mammary artery segments and freshly isolated and surgically prepared saphenous vein segments were obtained from patients undergoing coronary artery bypass grafting. Internal mammary artery and freshly isolated vein segments showed a high degree of endothelial coverage and medial cell viability that were maintained during culture. Surgically prepared veins showed partial endothelial denudation and medial cell injury, both of which tended to be reversed during culture. Neointimal thickening was greater in surgically prepared vein (72 +/- 13 microns; n = 11) than in freshly isolated vein (44 +/- 8 microns; n = 10) or internal mammary artery (34 +/- 4 microns; n = 13) segments. The occurrence of proliferating cells in the medial layer was also significantly greater in surgically prepared vein (2.8 +/- 1.0/mm; n = 11) than in freshly isolated vein (0.8 +/- 0.3/mm; n = 9) or internal mammary artery (0.6 +/- 0.3/mm; n = 10) segments. The data show that although the smooth muscle proliferation was similar in undamaged saphenous vein and internal mammary artery, it was significantly greater in damaged vein. This implies that the greater intimal proliferation seen in saphenous vein grafts may arise not from intrinsic differences in arterial and venous smooth muscle cells but from a greater susceptibility to injury.

TGFβ is active, and correlates with activators of TGFβ, following porcine coronary angioplasty

Objective: Restenosis following angioplasty involves processes that may be influenced by local production of cytokines. We investigated the expression of active and total transforming growth factor β (TGFβ) following porcine coronary angioplasty (PTCA), and have correlated this with the expression of potential in vivo activators of TGFβ: mannose-6-phosphate/insulin-like growth factor-II (M6P/IGF-II) receptor and thrombospondin-1. Methods: Oversized porcine PTCA was performed and the arteries excised after selected intervals. Levels of in situ active and total (active plus latent) TGFβ were determined using a modified plasminogen activator–inhibitor/luciferase bioassay. Results: Levels of active TGFβ significantly increased 2 h to 7 days after angioplasty, compared to non-injured controls. Levels returned to baseline by 28 days. Active TGFβ in tissues adjacent to the injured artery did not change. Total TGFβ was significantly higher than controls 2–6 h after injury. M6P/IGF-II receptor mRNA was upregulated between 6 h and 3 days after injury, with protein detectable at 3–28 days. Thrombospondin-1 was detected between 1 h and 14 days. Conclusions: We conclude that balloon injury causes an early rapid increase in levels of active TGFβ, that correlates with the expression of TGFβ activators. Thus, TGFβ is a good potential target for anti-restenotic therapies.

Alterations in c-fos expression, cell proliferation and apoptosis in pressure distended human saphenous vein

OBJECTIVESnSaphenous vein graft failures, resulting from thrombosis and the abnormal proliferation, migration and apoptosis of vascular smooth muscle cells (VSMC) are major limitations of coronary artery bypass surgery. We investigated whether surgical trauma of human saphenous vein induces the early response gene c-fos and causes alterations in rates of proliferation and apoptosis.nnnMETHODSnSurgically prepared human vein consisted of distended (at 350 mmHg for 2 min) or non-distended segments of vein maintained in serum free RPMI at 37 degrees C and 5% CO2 for various time intervals. c-fos expression was detected by Northern analysis. Cell proliferation and apoptosis were determined by [3H]thymidine incorporation combined with proliferation cell nuclear antigen (PCNA) immunostaining and TUNEL, respectively. Labelling indices for proliferation and apoptosis were correlated with vessel was thicknesses.nnnRESULTSnControl, freshly isolated vein expressed no c-fos. Surgically prepared vein synthesized c-fos 1 h following harvesting. There was a significant increase in c-fos in distended compared to non-distended vein. c-Fos protein increased in surgically prepared vein 24 h after harvesting. There was a significant increase in vascular cell proliferation in the non-distended compared to the distended vein: mean (S.E.M.) 1279 (218) vs. 863 (155) dpm/microgram DNA, P < 0.05, n = 6. In addition, the apoptotic index was significantly lower in the media of non-distended vs. distended vein 0.82 (0.2) vs. 5.5 (1.5), P < 0.05, n = 5.nnnCONCLUSIONSnThese findings demonstrate that surgical preparation of human saphenous vein increases expression of c-fos mRNA and apoptosis and reduces proliferation when compared with non-distended vein. These changes may influence the failure of saphenous vein grafts.

In vitro evaluation of c7E3-Fab (ReoPro) eluting polymer-coated coronary stents.

OBJECTIVEnStent thrombosis and in-stent restenosis remain problematic in certain patient sub-groups. c7E3-Fab (ReoPro, abciximab) inhibits the platelet glycoprotein IIb/IIIa receptor as well as the smooth muscle cell alpha(v)beta3 receptor, and thus may influence both processes, especially if high local concentrations could be achieved. We have studied the adsorption and elution characteristics of c7E3-Fab on commercially available polymer-coated stents. We have also investigated the effect of such antibody binding on platelet deposition in vitro, and on antibody deposition into ex vivo human saphenous vein wall to assess whether such stents may influence stent thrombosis and restenosis.nnnMETHODS AND RESULTSnAdsorption was measured using a radioisotope technique after immersing segments of polymer-coated stents in c7E3-Fab solutions. Uptake was dependent on antibody concentration and duration of immersion of wire in the solution. After 22 h (at 5 mg ml(-1)), 1146+/-101 ng cm(-1) wire was adsorbed. In an in vitro perfusion circuit, the antibody eluted slowly, with 53% remaining after 12 days washing. To determine the value that such stents might have in clinical practise, adsorption to balloon-mounted stents was assessed at room temperature, using commercially available c7E3-Fab (2 mg ml(-1)). Efficacy of eluting c7E3-Fab was determined by measuring deposition of 111-Indium platelets. Immersing stents in c7E3-Fab for 20 min inhibited platelet deposition by 82.3% compared to controls (P=0.018). Deployment of treated stents in ex vivo saphenous vein resulted in the deposition of c7E3-Fab in the intima and media.nnnCONCLUSIONSnc7E3-Fab can be passively adsorbed onto polymer-coated stents. It elutes slowly and in a predictable manner, significantly inhibiting platelet deposition in vitro. These studies pave the way to developing stent-based delivery of a potent anti-platelet agent that may additionally affect smooth muscle cell activity.

eNOS inhibition of proliferation: a role for p21Sdi1/Cip1/Waf1 and p27kip1

See article by Sato et al. [12] (pages 697–706) in this issue. nnIt is over 10 years since NO donors were shown to reduce proliferation of vascular smooth muscle cells (VSMC) [1]. More recently, increase in vessel wall thickness and hyperplasia were shown to occur following ligation of the carotid artery in NO synthase (eNOS) knockout mice suggesting NO to be a negative regulator of vascular cell proliferation [2]. Due to the short half-life and limited solubility of NO, NO synthase (NOS) is often used as a way of enhancing local NO production within the vessel wall, with potential therapeutic effects in vascular proliferative disorders. In addition to inhibiting VSMC proliferation, NOS also causes vasodilation, reduces platelet aggregation and adhesion as well as inhibiting leucocyte adhesion to the endothelium [3]. Thus it has been proposed as a useful inhibitor of neointimal hyperplasia, the hallmark of restenosis following angioplasty and stenting. Indeed, the last few years have seen a plethora of studies investigating gene transfer of NOS to various vascular systems for potential therapeutic benefit. One of the early studies showed that eNOS gene transfer using HVJ virus–liposome complexes delivered … nn* Corresponding author. Tel.: +44-114-271-4973; fax: +44-114-261-9587 c.holt{at}sheffield.ac.uk

793-4 Local Delivery of C-myb Antisense attenuates Neo-Intimal Thickening in a Porcine Model of Coronary Angioplasty

Intimal smooth muscle cell proliferation and extracellular matrix production compose the restenotic lesion. The proto-oncogene c-myb is up-regulated in proliferating cells and is a common ‘downstream’ link in several mitogenic pathways. We have recently shown that c-myb antisense specifically inhibits proliferation of several types of human vascular cells in vitro. The development of catheter-based local drug delivery systems allows us to test the feasibility and efficacy of local intra-mural delivery of c-myb antisense in a porcine model of coronary angioplasty. We first tested an unmodified HPLC-purified 18-mer antisense oligonucleotide directed against the human sequence of c-myb in porcine aortic smooth muscle cells in culture. More than 90% inhibition of cellular proliferation was observed at 5 μM with no adverse effect on cellular viability. This effect was not seen with sense oligonucleotides. The in vivo delivery system used was the Transport (CVD) double skinned balloon catheter, allowing high pressure oversized balloon dilatation followed by low pressure delivery of 500 μg antisense in 2 ml saline over 1–2xa0mins. Ex vivo and in vivo delivery of fluorescein labelled oligonucleotide using this system demonstrated successful intramural localisation. Wethen performed oligo delivery in vivo into a single porcine coronary artery (nxa0=xa010); the control group underwent angioplasty using a conventional balloon without drug delivery (nxa0=xa012). Animals were sacrificed at 4 weeks. Quantitative morphometric analysis of serial transverse histological sections demonstrated a consistent reduction in absolute and percent intimal thicknesses and areas together with a significant reduction in mean (SEM) intimal/medial thickness ratio from 0.37 (0.09) to 0.13 (0.03) (pxa0lxa00.05) in the treatment group.

Serum-Free organ culture of vascular tissues

Dear Editor: The aim of organ culture is to retain the original structural relationship of different and similar cells and their interactive function in order to study the effect of exogenous stimuli on further development (4). However, most organ culture systems described in the literature have a high requirement for serum and other undefined biological fluids (6,7) which can create problems when assessing the role of growth factors in regulating tissue growth and differentiation. To overcome this we attempted to develop a serum-free organ culture of vascular tissue. This was then used to evaluate the role of growth factors and cytokines in pathological conditions such as coronary atherosclerosis, restonosis after angioplasty and coronary saphenous vein bypass graft failure. Growth factors released from cells intrinsic to the vessel wall (5) are believed to cause smooth muscle cells to proliferate and secrete extracellular matrix components which contribute to vessel wall thickening and luminal narrowing. Our hypothesis is that during short-term serum-free culture if vessel viability is maintained, soluble bioeffectors such as peptides may be released into the medium. Analysis of the conditioned media for mitogenic activity can then be performed. We report here our experience of short-term serum-free organ culture of human coronary arteries and porcine arteriovenous bypass grafts. To our knowledge, there is no previous evidence in the literature on organ culture of vascular tissue in a chemically defined media free from both serum and hormones. Human coronary arteries were obtained from the discarded hearts of transplant recipients (3) and saphenous vein grafts from a pig model of myointimal hyperplasia developed in our laboratory (1). Vessels were carefully dissected and immediately transported to the laboratory (23 ° C) in sterile RPMI 1640 culture media containing 20 mmol/liter HEPES buffer (Flow Labs, High Wycombe, UK) supplemented with penicillin (100/xg/ml), streptomycin (100 U/ml), gentamycin (2.5/,tg/ml), amphotericin (2.5 ttg/ml), glutamine (2 mM), all from Flow Labs, High Wycombe, UK and 4 U/ml whole heparin (CP Pharmaceuticals, Wrexham, UK). Vessels were then washed several times in RPMI 1640 medium, dissected free from excess fatty tissue and adventitia, cut open with scissors along their upper aspect and pinned open endothelial surface uppermost using minuten pins (Watkins and Doncaster, Kent) on top of a square of polyester gauze (2 cm X 1 cm) supported on sylgard resin in the base of a glass petri dish (3,7). Artery and graft segments were then cultured for 24 hours in 6 ml of RPMI 1640 medium containing 2 g/liter sodium bicarbonate (Flow Labs, High Wycombe, UK) supplemented with antibiotics as above. Cultures were maintained at 37 ° C in a humidified atmosphere of 5% v/v CO2 in air and labeled with [3H]thymidine [1 #Ci/ml (80 Ci/mmol), Amersham International, UK] at the start of the culture period in order to assess and locate cell proliferation. In a separate experiment arteries and grafts were cultured in medium containing 30% fetal bovine serum (J-Bio Chemicals, France). Segments of artery and graft were snap frozen in liquid nitrogen (following preparation for culture and also after culture) for measurement of adenosine nucleotide concentrations using high performance liquid chromatography (2). Tissue ATP concentrations and ATP/ADP ratios which have previously been used to quantify the viability of vascular tissue (2,3,7) were unchanged in arteries and grafts following culture in serumfree medium or in medium containing 30% fetal bovine serum (Table 1). Transmission electron microscopy of ultra-thin sections confirmed these findings. An internal elastic lamina separated a monolayer of endothelial cells from several layers of medial smooth muscle cells and extracellular matrix. No necrotic areas were seen. Cell proliferation (DPM/ttgDNA) occurred to a similar extent in vessels cultured in the presence and absence of serum (human coronary arteries; 596 + 80 (n = 14) and 450 + 50 (n = 9), pig grafts; 892.8 _+ 113 (n -17)and 948.7 + 339 (n = 6), both n.s. Autoradiography of cultured arteries and grafts showed dividing cells predominantly in the intima with only occasional dividing cells in the media. Conditioned media from the serum-free experiments was stored at 8 0 ° C and its mitogenic potential investigated using a Swiss 3T3 fibroblast bioassay by determining the incorporation of 1/.tCi/ ml [aH]tbymidine (80 Ci/mmol) into a trichloroacetic acid insoluble cell fraction. Briefly, cells (ATCC CCL 92, Flow Labs, High Wy-