Sirolimus inhibits key events of restenosis in vitro/ex vivo: evaluation of the clinical relevance of the data by SI/MPL- and SI/DES-ratio's

<p>Abstract</p> <p>Background</p> <p>Sirolimus (SRL, Rapamycin) has been used successfully to inhibit restenosis both in drug eluting stents (DES) and after systemic application. The current study reports on the effects of SRL in various human in vitro/ex vivo models and evaluates the theoretical clinical relevance of the data by SI/MPL- and SI/DES-ratio's.</p> <p>Methods</p> <p>Definition of the SI/MPL-ratio: relation between <b>s</b>ignificant <b>i</b>nhibitory effects in vitro/ex vivo and the <b>m</b>aximal <b>p</b>lasma <b>l</b>evel after systemic administration in vivo (6.4 ng/ml for SRL). Definition of the SI/DES-ratio: relation between <b>s</b>ignificant <b>i</b>nhibitory effects in vitro/ex vivo and the drug concentration in <b>DES </b>(7.5 mg/ml in the ISAR drug-eluting stent platform). Part I of the study investigated in cytoflow studies the effect of SRL (0.01–1000 ng/ml) on TNF-α induced expression of intercellular adhesion molecule 1 (ICAM-1) in human coronary endothelial cells (HCAEC) and human coronary smooth muscle cells (HCMSMC). Part II of the study analysed the effect of SRL (0.01–1000 ng/ml) on cell migration of HCMSMC. In part III, IV, and V of the study ex vivo angioplasty (9 bar) was carried out in a human organ culture model (HOC-model). SRL (50 ng/ml) was added for a period of 21 days, after 21 and 56 days cell proliferation, apoptosis, and neointimal hyperplasia was studied.</p> <p>Results</p> <p>Expression of ICAM-1 was significantly inhibited both in HCAEC (SRL ≥ 0.01 ng/ml) and HCMSMC (SRL ≥ 10 ng/ml). SRL in concentrations ≥ 0.1 ng/ml significantly inhibited migration of HCMSMC. Cell proliferation and neointimal hyperplasia was inhibited at day 21 and day 56, significance (p < 0.01) was achieved for the inhibitory effect on cell proliferation in the media at day 21. The number of apoptotic cells was always below 1%.</p> <p>Conclusion</p> <p>SI/MPL-ratio's ≤ 1 (ICAM-1 expression, cell migration) characterize inhibitory effects of SRL that can be theoretically expected both after systemic and local high dose administration, a SI/MPL-ratio of 7.81 (cell proliferation) represents an effect that was achieved with drug concentrations 7.81-times the MPL. SI/DES-ratio's between 10^-6and 10^-8indicate that the described inhibitory effects of SRL have been detected with micro to nano parts of the SRL concentration in the ISAR drug-eluting stent platform. Drug concentrations in DES will be a central issue in the future.</p

Simultaneous intra/extravascular administration of antiproliferative agents as a new strategy to inhibit restenosis: The peak of reactive cell proliferation as a hallmark for the duration of the treatment

BACKGROUND: Strictly intravascular approaches for the treatment of postangioplasty restenosis are effective in the intima and the inner parts of the media but may be insufficient to control redundant pathways in the more outer parts of the media and the adventitia. An inverse situation may occur subsequently to a strictly extravascular approach, like the recently suggested pericardial approach in pigs. We hypothesized that simultaneous intra/extravascular administration of anti-restenotic agents inhibits restenosis by blocking all stimulatory pathways in the entire arterial wall. METHODS: Fresh hearts of 25 domestic pigs were obtained from a local slaughterhouse. Left anterior descending coronary arteries (LAD) were harvested, cut into cylindric 5 mm segments, and cultured as ex vivo porcine organ cultures (POCs). After 9 bar ballooning simultaneous intra/extravascular administration of high dose diltiazem (50 μg/mL) was carried out for a period of 1, 2, 3, 4, 5, 6, and 7 days. At day 7 and 28 proliferative activity (BrdU), neointimal thickening, and staining against smooth muscle α-actin and vWF was analysed. RESULTS: 7 days after ballooning administration of diltiazem for 4, 5, 6, and 7 days inhibited reactive cell proliferation by more than 50% (n.s.) as compared to control, 28 days after ballooning administration for 6 and 7 days inhibited neointimal thickening by more than 75% (p < 0.05). Simultaneous intra/extravascular administration of high dose diltiazem did not affect the expression of vWF in endothelial cells or smooth muscle α-actin in smooth muscle cells. CONCLUSIONS: Simultaneous intra/extravascular administration of high dose diltiazem (50 μg/mL) has to be maintained for at least 6 days to achieve a significant inhibition of neointimal thickening. The data demonstrate the importance of the maximal reactive cell proliferation (= day 7 in the POC-model) for the calculation of the duration of the treatment period

Effects of mycophenolate mofetil on key pattern of coronary restenosis: a cascade of in vitro and ex vivo models

BACKGROUND: Mycophenolate mofetil (MMF), the prodrug of mycophenolic acid (MPA), is a rationally designed immunosuppressive drug. The current study investigates the effect of MMF on key pattern of restenosis in a cascade of in vitro and ex vivo models. METHODS: Part I of the study investigated in northern blot and cytoflow studies the effect of MMF (50, 100, 150, 200, 250, and 300 μg/mL) on TNF-α induced expression of intercellular adhesion molecule 1 (ICAM-1) in human coronary endothelial cells (HCAEC) and human coronary medial smooth muscle cells (HCMSMC). Part II of the study applied a human coronary 3D model of leukocyte attack, the 3DLA-model. HCAEC and HCMSMC were cultured on both sides of a polycarbonate filters, mimicking the internal elastic membrane. Leukocyte attack (LA) was carried out by adding human monocytes (MC) on the endothelial side. The effect of MMF (50 μg/mL) on adhesion and chemotaxis (0.5, 1, 2, 3, 4, 6, and 24 h after LA) and the effect on proliferation of co-cultured HCMSMC (24 h after LA) was studied. In part III of the study a porcine coronary organ culture model of restenosis (POC-model) was used. After ex vivo ballooning MMF (50 μg/mL) was added to the cultures for a period of 1, 2, 3, 4, 5, 6, and 7 days. The effect on reactive cell proliferation and neointimal thickening was studied at day 7 and day 28 after ballooning. RESULTS: Expression of ICAM-1 in northern blot and cytoflow studies was neither clearly inhibited nor stimulated after administration of MMF in the clinical relevant concentration of 50 μg/mL. In the 3DLA-model 50 μg/mL of MMF caused a significant antiproliferative effect (p < 0.001) in co-cultured HCMSMC but had no effect on MC-adhesion and MC-chemotaxis. In the ex vivo POC-model neighter reactive cell proliferation at day 7 nor neointimal hyperplasia at day 28 were significantly inhibited by MMF (50 μg/mL). CONCLUSION: Thus, the data demonstrate a significant antiproliferative effect of clinical relevant levels of MMF (50 μg/mL) in the 3DLA-model. The antiproliferative effect was a direct antiproliferative effect that was not triggered via reduced expression of ICAM-1 or via an inhibition of MC-adhesion and chemotaxis. Probably due to technical limitations (as e.g. the missing of perfusion) the antiproliferative effect of MMF (50 μg/mL) could not be reproduced in the coronary organ culture model. A cascade of focused in vitro and ex vivo models may help to gather informations on drug effects before large experimental studies are initiated

HCMV-infection in a human arterial organ culture model: effects on cell proliferation and neointimal hyperplasia

<p>Abstract</p> <p>Background</p> <p>The impact of infections with the human cytomegalovirus (HCMV) for the development of atherosclerosis and restenosis is still unclear. Both a clear correlation and no correlation at all have been reported in clinical, mostly serological studies. In our study we employed a human non-injury ex vivo organ culture model to investigate the effect of an in vitro permissive HCMV-infection on cell proliferation and neointimal hyperplasia for a period of 56 days.</p> <p>Results</p> <p>During routine-nephrectomies parts of renal arteries from 71 patients were obtained and prepared as human organ cultures. Cell free HCMV infection was performed with the fibroblast adapted HCMV strain AD169, the endotheliotropic strain TB40E, and a clinical isolate (AN 365). After 3, 7, 14, 21, 28, 35, and 56 days in culture staining of HCMV-antigens was carried out and reactive cell proliferation and neointimal thickening were analysed. Successful HCMV-infection was accomplished with all three virus strains studied. During the first 21 days in organ culture no cell proliferation or neointimal hyperplasia was detected. At day 35 and day 56 moderate cell proliferation and neointimal hyperplasia was found both in HCMV-infected segments and mock infected controls. Neointimal hyperplasia in productively HCMV-infected segments was lower than in non infected at day 35 and day 56, but relatively higher after infection with the endotheliotropic TB40E in comparison with the two other strains.</p> <p>Conclusion</p> <p>The data do not support the hypothesis that HCMV-infection triggers restenosis via a stimulatory effect on cell proliferation and neointimal hyperplasia in comparison to non infected controls. Interestingly however, even after lytic infection, a virus strain specific difference was observed.</p

Effects of abciximab on key pattern of human coronary restenosis in vitro: impact of the SI/MPL-ratio

BACKGROUND: The significant reduction of angiographic restenosis rates in the ISAR-SWEET study (intracoronary stenting and antithrombotic regimen: is abciximab a superior way to eliminate elevated thrombotic risk in diabetes) raises the question of whether abciximab acts on clopidogrel-independent mechanisms in suppressing neointimal hyperplasia. The current study investigates the direct effect of abciximab on ICAM-1 expression, migration and proliferation. METHODS: ICAM-1: Part I of the study investigates in cytoflow studies the effect of abciximab (0.0002, 0.002, 0.02, 0.2, 2.0, and 20.0 μg/ml) on TNF-α induced expression of intercellular adhesion molecule 1 (ICAM-1). Migration: Part II of the study explored the effect of abciximab (0.0002, 0.002, 0.02, 0.2, 2.0, and 20.0 μg/ml) on migration of HCMSMC over a period of 24 h. Proliferation: Part III of the study investigated the effect of abciximab (0.0002, 0.002, 0.02, 0.2, 2.0, and 20.0 μg/ml) on proliferation of HUVEC, HCAEC, and HCMSMC after an incubation period of 5 days. RESULTS: ICAM-1: In human venous endothelial cells (HUVEC), human coronary endothelial cells (HCAEC) and human coronary medial smooth muscle cells (HCMSMC) no inhibitory or stimulatory effect on expression of ICAM-1 was detected. Migration: After incubation of HCMSMC with abciximab in concentrations of 0.0002 – 2 μg/ml a stimulatory effect on cell migration was detected, statistical significance was achieved after incubation with 0.002 μg/ml (p < 0.05), 0.002 μg/ml (p < 0.001), and 0.2 μg/ml (p < 0.05). Proliferation: Small but statistically significant antiproliferative effects of abciximab were detected after incubation of HUVEC (0.02 and 2.0 μg/ml; p = 0.01 and p < 0.01), HCAEC (2.0 and 20.0 μg/ml; p < 0.05 and p < 0,01), and HCMSMC (2.0 and 20.0 μg/ml; p < 0.05 and p < 0.05). The significant inhibition (SI) of cell proliferation found in HCAEC and HCMSMC was achieved with drug concentrations more than 10 times beyond the maximal plasma level (MPL), resulting in a SI/MPL-ratio > 1. CONCLUSION: Thus, the anti-restenotic effects of systemically administered abciximab reported in the ISAR-SWEET-study were not caused by a direct inhibitory effect on ICAM-1 expression, migration or proliferation

Edge restenosis: impact of low dose irradiation on cell proliferation and ICAM-1 expression

BACKGROUND: Low dose irradiation (LDI) of uninjured segments is the consequence of the suggestion of many authors to extend the irradiation area in vascular brachytherapy to minimize the edge effect. Atherosclerosis is a general disease and the uninjured segment close to the intervention area is often atherosclerotic as well, consisting of neointimal smooth muscle cells (SMC) and quiescent monocytes (MC). The current study imitates this complex situation in vitro and investigates the effect of LDI on proliferation of SMC and expression of intercellular adhesion molecule-1 (ICAM-1) in MC. METHODS: Plaque tissue from advanced primary stenosing lesions of human coronary arteries (9 patients, age: 61 ± 7 years) was extracted by local or extensive thrombendarterectomy. SMC were isolated and identified by positive reaction with smooth muscle α-actin. MC were isolated from buffy coat leukocytes using the MACS cell isolation kit. For identification of MC flow-cytometry analysis of FITC-conjugated CD68 and CD14 (FACScan) was applied. SMC and MC were irradiated using megavoltage photon irradiation (CLINAC2300 C/D, VARIAN, USA) of 6 mV at a focus-surface distance of 100 cm and a dose rate of 6 Gy min(-1 )with single doses of 1 Gy, 4 Gy, and 10 Gy. The effect on proliferation of SMC was analysed at day 10, 15, and 20. Secondly, total RNA of MC was isolated 1 h, 2 h, 3 h, and 4 h after irradiation and 5 μg of RNA was used in standard Northern blot analysis with ICAM-1 cDNA-probes. RESULTS: Both inhibitory and stimulatory effects were detected after irradiation of SMC with a dose of 1 Gy. At day 10 and 15 a significant antiproliferative effect was found; at day 20 after irradiation cell proliferation was significantly stimulated. Irradiation with 4 Gy and 10 Gy caused dose dependent inhibitory effects at day 10, 15, and 20. Expression of ICAM-1 in human MC was neihter inhibited nor stimulated by LDI. CONCLUSION: Thus, the stimulatory effect of LDI on SMC proliferation at day 20 days after irradiation may be the in vitro equivalent of a beginning edge effect. Extending the irradiation area in vascular brachytherapy in vivo may therefore merely postpone and not inhibit the edge effect. The data do not indicate that expression of ICAM-1 in quiescent MC is involved in the process

Cultivation of cells from human atherosclerotic lesions : inhibitory and stimulatory effects on cell proliferation

1. Kultivierung von Plaquezellen des Menschen Die Migration und Proliferation von glatten Muskelzellen aus der Media in den subendothelialen Raum wird als Schlüsselereignis bei der Entstehung der Atherogenese angesehen. Um weitere Informationen über das Wachstumsverhalten dieser Zellen zu erhalten, wurden aus atherosklerotischem Plaquematerial Zellen isoliert und ihre Wachstumscharakteristika in Zellkulturen untersucht. Die Entnahme des Plaquematerials erfolgte perkutan durch einen Atherektomie-Katheter aus der A. femoralis superficialis, der A. poplitea und der A. renalis und im Rahmen von operativen Eingriffen aus der A. coronaria, der A. carotis und der Aorta abdominalis. Die Mehrzahl dieser Zellen konne durch positive Reaktion mit Antikörpern gegen glattmuskuläres alpha-Aktin als glatte Muskelzellen identifiziert werden. Hierbei zeigten Zellen aus re-stenosierendem Plaquematerial ein extrem gesteigertes Wachstum im Vergleich zu Zellen aus primär-stenosierendem Plaquematreial. Diese unterschiedlichen Wachstumsraten wurden unabhängig von der Entnahme-Technik und -Lokalisation registriert. Das gesteigerte Wachstum der glatten Muskelzellen aus restenosierendem Plaquematerial könnte das in vitro-Äquivalent zu dem oftmals rapide verlaufendem Re-stenosierungsprozeß nach Angioplastie in vivo sein. 2. Einflüsse auf das Wachstumsverhalten In unseren Versuchen zeigte sich eine zunehmende proliferative Aktivität der Zellen durch steigende Serumkonzentrationen. Plaquezellen aus Restenosen proliferierten bei geringeren Konzentrationen und in stärkerem Ausmaß als Plaquezellen aus primär-stenosierenden Läsionen. Durch das Kulturmedium von re-stenosierenden glatten Muskelzellen konnte das Wachstum von primär-stenosierenden glatten Muskelzellen um 60% gesteigert werden. Der Wachstumsfaktor PDGF steigert die Proliferation von glatten Muskelzellen um das 2.7-fache, ECGF sogar um das 4-fache. Glatte Muskelzellen aus primär-stenosierendem Plaquematerial ändern ihr Wachstum unter der Wirkung von PDGF und ECGF nicht. 3. Medikamenten-Testungen Acetylsalizylsäure und Dipyridamol werden zur Verhinderung von Thrombosen als Vor- und Nachbehandlung bei Angioplastien angewendet. Die in vitro-Untersuchungen ergaben weder bei der Einzelgabe, noch bei der Kombination der Substanzen einen Hinweis für eine Inhibition der Migration und Proliferation von Plaquezellen. Fibrinolytika werden in der klinische Routine bei akutem Myokardinfarkt zur Lysetherapie verwendet. In vitro zeigte sich im getesteten Konzentrationsbereich weder eine Stimulation noch eine Inhibition der Proliferation. In der Literatur wurde berichtet, dass beta-Blocker das Wachstum von Plaquezellen stimulieren. In unseren Untersuchungen wurden im therapeutischen Bereich die glatten Muskelzellen aus der unveränderten Media und aus koronarem Plaquematerial nicht in ihrem Wachstum verändert. Die Austestung des Calziumantagonisten Diltiazem an Plaquezellen aus peripheren und koronaren Arterien des Menschen erbrachte eine signifikante Inhibition der Zellproliferation im therapeutisch relevanten Bereich. Da eine Inhibition der Zellmigration und Zellproliferation von klinischem Interesse ist, könnte der schwerpunktmäßige Einsatz der Zellkultur als „Prescreening-Modell“ erfolgen, um aus der Vielzahl der zur Verfügung stehenden Substanzen diejenigen mit antimigratorischen und antiproliferativen Eigenschaften herauszufiltern. Ausgewählte Substanzen könnten dann in Transfilter-Co-Kulturen und im Tierversuch weiteruntersucht werden, um abschließend Empfehlungen für klinische Studien aussprechen zu können.1) Plaque cells of human primary stenosing and restenosing tissue of peripheral and coronary arteries have been cultured and identified as smooth muscle cells (SMC). Proliferation of SMC derived of restenosing tissue was significantly increased in comparison to SMC of primary stenosing lesions. 2) Growth of primary stenosing SMC was stimulated by 1) culture media of restenosing SMC, 2) PDGF and 3) ECGF. 3) Drug testing: No effect was detected after adding of aspirin, dypiridamole, betablocker (propranolol), and fibrinolytic agents (streptokinase, urokinase, plasminogen tissue activator, t-PA) in concentrations of clinical relevance. Therapeutic concentrations of diltiazem caused a significant inhibition of SMC proliferation. Conclusion: Cell culture studies may be used as prescreening model to investigate the effects of potential antiproliferative effects in vitro, before large experimental and clinical studies are initiated

Humane Restenose-Modelle: Annäherungen an die klinische Situation - [kumulative Habilitationsschrift auf der Grundlage von 11 verschiedenen Zeitschriftenaufsätzen]

Die makellose Erfolgsbilanz der koronaren Ballonangioplastie wird von dem Umstand getrübt, dass auch mehr als 20 Jahre seit ihrer Einführung das Problem der Restenose ungelöst ist. Zum aktuellen Zeitpunkt zeichnet sich ein Fehlschlagen der bislang propagierten systemischen Therapieansätze ab, obwohl diese in zahlreichen in vitro und experimentellen Ansätzen signifikante Erfolge erzielt hatten und als vielversprechenden klinische Therapieansätze gehandelt wurden. Es ist dringend erforderlich, den Ursachen dieser Fehleinschätzung auf den Grund zu gehen. Anderenfalls besteht die Gefahr, dass in 10 Jahren wiederum das klinische Fehlschlagen der momentan favorisierten Therapieansätze festgestellt werden muss. Obwohl die Ursachen zweifelsohne komplexester Natur sind, lassen sich drei Kernprobleme ansprechen: 1) Unterschiede von 1 : 100 und mehr zwischen den experimentell erfolgreichen Konzentrationen und den Konzentrationen, welche klinisch erreicht werden konnten; 2) direkte Rückschlüsse von tierexperimentellen in vivo Erfolgen auf die humane Situation; 3) Wechselwirkungen benachbarter Stimulations-/Inhibitionswege im Sinne eines "crosstalks", welche zwar bei isolierter Betrachtung einen Effekt suggerieren, die jedoch netto in vivo neutralisiert oder sogar ins Gegenteil verkehrt werden können. Ziel der vorliegenden Arbeit ist es Wege zu finden, eine bessere Einschätzung des klinischen Potentials verschiedener Anti-Restenose Therapiestrategien zu ermöglichen. Um dieses Ziel zu erreichen, wurde den drei angesprochenen Hauptproblemen wie folgt begegnet: 1) eine durchgehende Angabe der in vitro/in vivo Relation der verschiedenen Effekte; 2) die Verwendung humaner in vitro/ex vivo Modelle; 3) der gestaffelte Einsatz von humanen Prescreening Modellen, komplexen Transfilter-Co-Kultur-Modellen und ex vivo Organkultur-Modellen