Rainer Voisard

Intracoronary β-Irradiation With a Liquid 188Re-Filled Balloon Six-Month Results From a Clinical Safety and Feasibility Study

Background—Coronary irradiation is a new concept to reduce restenosis. We evaluated the feasibility and safety of intracoronary irradiation with a balloon catheter filled with 188Re, a liquid, high-energy β-emitter. Methods and Results—Irradiation with 15 Gy at 0.5-mm tissue depth was performed in 28 lesions after balloon dilation (n=9) or stenting (n=19). Lesions included 19 de novo stenoses, 4 occlusions, and 5 restenoses. Irradiation time was 515±199 seconds in 1 to 4 fractions. There were no procedural complications. One patient died of noncardiac causes at day 23. One asymptomatic patient refused 6-month angiography. Quantitative angiography after intervention showed a reference diameter of 2.77±0.35 mm and a minimal lumen diameter of 2.36±0.43 mm. At 6-month follow-up, minimal lumen diameter was 1.45±0.88 mm (late loss index 0.57). Target lesion restenosis rate (>50% in diameter) was low (12%; 3 of 26). In addition, we observed 9 stenoses at the proximal or distal end of the irradiation zone, potent...

Corticosteroid agents inhibit proliferation of smooth muscle cells from human atherosclerotic arteries in vitro.

We studied the in vitro effect of steroid agents on smooth muscle cells from human atherosclerotic arteries. Recent advances in the understanding of the biology of restenosis indicate that restenosis is predominantly caused by a multifactorial stimulation of smooth muscle cell proliferation. Primary stenosing plaque material of 24 patients (aged 63 +/- 14 years) and restenosing plaque material of 7 patients (aged 65 +/- 9 years) was selectively extracted from femoral arteries by the Simpson atherectomy device. Cells were isolated by enzymatic disaggregation and identified as smooth muscle cells by positive reaction with smooth muscle alpha-actin. The steroid agents prednisolone (0.0075-750 micrograms/ml), hydrocortisone (0.0125-1250 micrograms/ml), and dexamethasone (0.0004-40 micrograms/ml) were added to the cultures. Six days after seeding the cells were trypsinized and the cell number was measured by a cell counter. All three steroid agents exhibited a significant antiproliferative effect on smooth muscle cell proliferation. At high concentrations of hydrocortisone, cytoskeletal elements of smooth muscle cells such as actin, microtubules, and vimentin, were largely altered. Our data indicate that the proliferation of smooth muscle cells from human atherosclerotic arteries in vitro can be inhibited by steroid agents and thus may open the way for local post-angioplasty treatment strategies.

Rapamycin attenuates vascular wall inflammation and progenitor cell promoters after angioplasty

Rapamycin combines antiproliferative and antiinflammatory properties and reduces neointima formation after angioplasty in patients. Its effect on transcriptional programs governing neointima formation has not yet been investigated. Here, we systematically analyzed the effect of rapamycin on gene expression during neointima formation in a human organ culture model. After angioplasty, renal artery segments were cultured for 21 or 56 days in absence or presence of 100 ng/ml rapamycin. Gene expression analysis of 2312 genes revealed 264 regulated genes with a peak alteration after 21 days. Many of those were associated with recruitment of blood cells and inflammatory reactions of the vessel wall. Likewise, chemokines and cytokines such as M‐CSF, IL‐1β, IL‐8, β‐thromboglobulin, and EMAP‐II were found up‐regulated in response to vessel injury. Markers indicative for a facilitated recruitment and stimulation of hematopoetic progenitor cells (HPC), including BST‐1 and SDF‐1, were also induced. In this setting, rapamycin suppressed the coordinated proadhesive and proinflammatory gene expression pattern next to down‐regulation of genes related to metabolism, proliferation, and apoptosis. Our study shows that mechanical injury leads to induction of a proinflammatory, proadhesive gene expression pattern in the vessel wall even in absence of leukocytes. These molecular events could provide a basis for the recruitment of leukocytes and HPC. By inhibiting the expression of such genes, rapamycin may lead to a reduced recruitment of leukocytes and HPC after vascular injury, an effect that may play a decisive role for its effectiveness in reducing restenosis.

Aspirin (5 mmol/L) inhibits leukocyte attack and triggered reactive cell proliferation in a 3D human coronary in vitro model

BackgroundLeukocyte attack (LA) and the triggered reactive proliferation of smooth muscle cells (SMCs) are key events for the development of early atherosclerosis and restenosis. In the present study, we used a 3D human coronary in vitro model of LA (3DLA model) to examine the effect of high-dose aspirin on the adhesion and chemotaxis of leukocytes and the reactive proliferative response of SMCs. Methods and ResultsFor dose-finding, the effect of aspirin (1, 2, 5, and 10 mmol/L) on the tumor necrosis factor-&agr;-induced upregulation of intercellular adhesion molecule-1 was analyzed in monocultures of human coronary endothelial cells (HCAEC) and the SMCs of the human coronary media (HCMSMC). In cytoflow and Northern blot experiments, the expression of intercellular adhesion molecule-1 was slightly reduced after incubation with 5 mmol/L aspirin, and strong inhibition was found after incubation with 10 mmol/L. In 3DLA models, HCAECs and HCMSMCs were cultured on both sides of a porous filter. For LA, human monocytes or CD4+ lymphocytes were seeded on the HCAEC side of the 3DLA unit. A dose of 5 mmol/L aspirin inhibited the adherence of monocytes or CD4+ lymphocytes by 50% (P <0.01) and the chemotaxis of monocytes by 90% (P <0.01). The reactive proliferative response of cocultured HCMSMCs after LA, as measured by the uptake of bromodeoxyuridine, was significantly reduced by 83% after selective monocyte attack (P <0.001) and by 42% after selective CD4+ lymphocyte attack (P <0.05). ConclusionsA local concentration of 5 mmol/L aspirin should be accepted as the lowest rational concentration for the beneficial in vitro effects of high-dose aspirin to be reproduced in clinical studies.

Increased endothelin release by cultured human smooth muscle cells from atherosclerotic coronary arteries

OBJECTIVES Endothelin, a 21-amino acid peptide initially purified from the medium of cultured endothelial cells, is a potent vasoconstrictor exerting its effects predominantly in a paracrine or autocrine manner. Recent data indicate that endothelin is also synthesized by cultured vascular smooth muscle cells and that endothelin is an effective stimulator of smooth muscle cell proliferation. This study aimed to investigate the endothelin release of cultured human smooth muscle cells, isolated from coronary plaques and from normal coronary tunica media, and to determine circulating endothelin concentrations in patients with coronary artery disease compared to control subjects. METHODS Coronary plaque material was extracted by thrombendarterectomy during aorto-coronary bypass grafting (n = 19). Segments of normal coronary arteries were obtained at autopsy (n = 33). Cells were isolated by enzymatic disaggregation and identified as smooth muscle cells with antibodies against smooth muscle alpha-actin. Venous blood samples were drawn from patients with coronary artery disease undergoing cardiac catheterization (n = 32) and from control subjects (n = 38). Endothelin concentrations in culture medium and in plasma samples were measured by radioimmunoassay after Sep Pak C18 extraction. RESULTS Cultured smooth muscle cells, isolated from coronary plaques, released a significantly (P < 0.001) higher amount of immunoreactive endothelin into the culture medium (39.2 +/- 3.9 pg/10(4) cells, mean +/- s.e.m., 31 supernatant samples) than smooth muscle cells from normal coronary tunica media (3.9 +/- 0.8 pg/10(4) cells, 28 samples). Circulating endothelin concentrations were slightly elevated (P < 0.01) in patients with coronary artery disease (3.8 +/- 0.2 pg/ml) compared to control subjects (3.0 +/- 0.2 pg/ml). CONCLUSIONS These data suggest that the endothelin production is markedly increased in smooth muscle cells of coronary atherosclerotic plaques. The enhanced endothelin release may stimulate smooth muscle cell proliferation in a paracrine or autocrine manner and thus may contribute to the development or progression of coronary artery disease.

A human arterial organ culture model of postangioplasty restenosis: results up to 56 days after ballooning

BACKGROUND Restenosis is a reparative process that is activated in response to injury induced by angioplasty. Despite numerous experimental models of restenosis the number of human arterial organ culture systems is very limited and long-term experiences do not exist. METHODS AND RESULTS During routine nephrectomies parts of the renal arteries of 88 patients were extracted, 47 were suitable for organ culture preparations. Sections were made at 3 mm intervals perpendicular to the vessel wall axis. The arterial segments were treated with 3 mm standard balloon-catheters (Medtronic 14K2030E) for 60 s with 3, 6, 9, and 12 bar. After angioplasty, the organ segments were cultured in a mixture of Waymouths MB 752/1 and Ham F-12, supplemented with 15% fetal calf serum. After 0, 4, 14, 21, 28, and 56 days the organ cultures were fixed in 4% para-formaldehyde and embedded in paraffin. After staining with a modified elastica-van Gieson technique the intimal wall thickening was analyzed with a computerized morphometric system. For the identification of smooth muscle cells (SMC) a monoclonal antibody against smooth muscle alpha-actin was used. Endothelial cells were identified using an anti-human von Willebrand factor. To determine the number of cells undergoing DNA synthesis, bromodeoxyuridine (BrdU), a thymidine analogue, was added to the culture media 18 h prior to fixation. BrdU was detected with a monoclonal antibody, as secondary antibody a biotinylated horse-anti-mouse antibody was used. After 14, 21, and 28 days in culture BrdU-positive cells were detected in the neointima of the organ cultures, indicating mitotic activity in this area. After 28 and 56 days in culture a clear increase of neointimal thickening was found in the morphometric analysis. By positive reaction with antibodies against smooth muscle alpha-actin these cells were partly identified as SMC. CONCLUSIONS The organ culture model offers opportunities for in vitro investigations of postangioplasty restenosis. The data emphasize the importance of a relatively late proliferative response of SMC in the human arterial organ culture model.

The in-vitro effect of antineoplastic agents on proliferative activity and cytoskeletal components of plaque-derived smooth-muscle cells from human coronary arteries.

Background:Restenosis after successful percutaneous transluminal coronary angioplasty remains the major clinical problem limiting the long-term efficacy of the treatment. Recent advances in the understanding of the biology of restenosis indicate that its cause is predominantly a multifactorial stimulation of smooth-muscle cell proliferation. The aim of this study was to investigate the in-vitro effect of antineoplastic agents on smooth-muscle cells isolated from human coronary plaque material. Methods:Atherosclerotic tissue from coronary arteries was extracted from 15 patients of both sexes by thrombendarterectomy. Cells were isolated using enzymatic disaggregation and identified to be smooth-muscle cells with fluorescent antibodies for smooth-muscle-specific α-actin. The antineoplastic agents cytarabine (500–0.005 μg/ml), doxorubicin (50–0.0005 μg/ml), and vincristine (10–0.0001 μg/ml) were added to the cultures. Six days after seeding, the cells were trypsinized and then counted. Results:All three antineoplastic agents had a strong dose-dependent antiproliferative effect on cultured smooth-muscle cells. After the application of cytostatic agents, cells either became rounded or underwent complete lysis. Cytoskeletal elements, such as actin. microtubules, and vimentin, were largely altered. Conclusion:This investigation examined the potential role of antineoplastic therapy in the prevention of restenosis after coronary angioplasty. The development of new intravascular delivery systems, such as coated stents, may open the way for local antiproliferative strategies in interventional cardiology.

A prescreening system for potential antiproliferative agents : implications for local treatment strategies of postangioplasty restenosis

BACKGROUND Recent advances in the understanding of the biology of restenosis indicate that it is predominantly caused by a multifactorial stimulation of smooth muscle cell proliferation. The aim of this study was to investigate the in vitro effect of five potential antiproliferative agents on smooth muscle cells from human atherosclerotic femoral arteries. METHODS AND RESULTS Primary stenosing plaque material of 24 patients (aged 63 +/- 14 years) and restenosing plaque material of 7 patients (aged 65 +/- 9 years) was selectively extracted from femoral arteries by the Simpson atherectomy device. Cells were isolated by enzymatic disaggregation and identified as smooth muscle cells by positive reaction with smooth muscle alpha-actin. Dalteparin sodium (0.001-100 anti-Xa units/ml), cyclosporine A (0.005-500 micrograms/ml), colchicine (0.00004-4 pg/ml), etoposide (0.002-200 micrograms/ml), and doxorubicin (0.0005-50 micrograms/ml) were added to the cultures. Six days after seeding, cells were trypsinized and cell number was measured by a cell counter. All five agents tested exhibited a significant inhibition of smooth muscle cell proliferation (P < 0.001). After an incubation time of 48 h, the cytoskeletal components, alpha-actin, vimentin, and microtubules were investigated. At peak concentrations, all five tested agents except dalteparin sodium caused severe damage to the cytoskeleton. CONCLUSIONS All five potential antiproliferative agents exhibited a significant inhibition of smooth muscle cell proliferation. The development of new intravascular delivery systems may open the way for local antiproliferative treatment strategies in interventional cardiology.

Ca2+-activated K+ channels in human smooth muscle cells of coronary atherosclerotic plaques and coronary media segments

The behavior of Ca2+-activated K+ channels of large conductance (BKCa) in smooth muscle cells, which were obtained from atherosclerotic plaque material (SMCP) and from media segments (SMCM) of human coronary arteries, were compared using the patch-clamp technique. Voltage-clamp protocols in cell-attached patches revealed the characteristic voltage-dependent activation of BKCa in both cell groups. Single-channel conduction was 216.4±16.7pS (n=6) in SMCP and 199.9±6.7pS (n=6) in SMCM in symmetrical 140 mMK+ solutions. Using outside-out patches, external perfusion with 500 μM tetraethylammonium ions caused a typical “flickery block” of the unitary current. The selective BKCa channel inhibitor iberiotoxin (50 nM) effectively blocked BKCa channel activity. Comparing BKCa open-state probabilities (P0) at +80 mV in cell-attached patches, a highly significant difference between SMCP (P0=0.1438±0.1301; n=15) and SMCM (P0=0.0093±0.0044; n=15; Kruskal-Wallis test, p<0.001) was found. In contrast to this finding, there was no significant difference in the open-state probability of BKCa between SMCP (P0=0.0542±0.0237; n=9) and SMCM (P0=0.0472±0.0218; n=10; p=n.s.) using inside-out patches. The results show an interesting difference in the behavior of large conductance Ca2+-activated K+ channel in SMCP compared to SMCM with a significantly higher channel activity in human smooth muscle cells obtained from coronary atherosclerotic plaque material. This finding may indicate an important functional role of BKCa channels in the development of atherosclerosis.

Cyclosporine A stimulates endothelin release.

Summary: Administration of cyclosporine A is often associated with the development of renal dysfunction and hypertension. Because recent data provide evidence that endothelin (ET) might be involved in mediating cyclo-sporine-associated cardiovascular and renal side effects, the present study aimed to investigate the influence of cyclosporine A on ET release from cultured smooth-muscle cells and whether ET plasma concentrations are elevated in cyclosporine-treated patients. Addition of cyclosporine A to the medium of cultured human smoothmuscle cells, isolated from atherosclerotic iliac arteries, induced a dose-dependent increase in ET release. ET plasma levels were significantly elevated in cyclosporine-treated patients after bone marrow transplantation, compared to control groups. These data suggest that enhanced ET release might be involved in mediating the cyclosporine-associated side effects.