Direct inhibitory effects of Ganciclovir on ICAM-1 expression and proliferation in human coronary vascular cells (SI/MPL-ratio: >1).

BACKGROUND: Treatment of the human cytomegalovirus (HCMV) infection with ganciclovir has beneficial indirect effects on the complex interactions of HCMV with restenosis, atherosclerosis, and transplant vascular sclerosis. The current study reports on direct effects of ganciclovir on expression of ICAM-1 and cell proliferation, key events of coronary atherosclerosis/restenosis. A potential clinical relevance of the data will be evaluated with the help of SI/MPL-ratio's. MATERIAL/METHODS: Definition of the SI/MPL-ratio: relation between significant inhibitory effects in vitro/ex vivo and the maximal plasma level after systemic administration in vivo (ganciclovir: 9 µg/ml). Part I of the study investigated in cytoflow studies the effect of ganciclovir (0.05-5000 µg/mL) on TNF-a induced expression of intercellular adhesion molecule 1 (ICAM-1) in endothelial cells derived from umbilical veins (HUVEC), human coronary endothelial cells (HCAEC), and human coronary smooth muscle cells (HCMSMC). Part II of the study analysed the effect of ganciclovir (0.05-5000 µg/mL) on cell proliferation (HUVEC, HCAEC, and HCMSMC). In part III cytotoxic effects of ganciclovir (0.05-5000 µg/mL) were studied (HUVEC, HCAEC, and HCMSMC). RESULTS: Ganciclovir caused slight but significant inhibitory effects on expression of ICAM-1 in HUVEC, HCAEC, and HCMSMC. In all three cell types studied strong dose depending significant antiproliferative effects of ganciclovir were detected. Partially, the antiproliferative effects of ganciclovir were caused by cytotoxic effects. CONCLUSIONS: SI/MPL-ratio's >1 in HCAEC and HCMSMC indicate that the inhibitory effects of gancliclovir on ICAM-1-expression and cell proliferation may only be expected in vivo following local high dose administration e.g. in drug eluting stents (DES).

Pulsed perfusion in a venous human organ culture model with a Windkessel function (pulsed perfusion venous HOC-model).

BACKGROUND: The effectiveness of human saphenous vein grafting is limited by hyperplasia of the vessel wall. The current paper reports on a pulsed perfused venous human organ culture model (pp-venous HOC-model) with a Windkessel function. MATERIAL/METHODS: Saphenous vein grafts from 21 patients undergoing coronary bypass grafting were cultured either in venous or arterial hemodynamic conditions. Up to four vein segments were fixed in parallel connection and attached to a closed loop pulsed perfusion system consisting of large and small elastic tubes, mimicking the Windkessel function. RESULTS: First, after exposure to arterial blood pressure first signs of reactive cell proliferation (n.s.) were detected at day 4. Second, media thickness of the venous segments in the pulsed pressure group was decreased at day 4 (n.s.) and day 7 (n.s.). Third, staining against smooth muscle alpha actin and v. Willebrand factor was always positive at day 1, 4, and 7. CONCLUSIONS: Pulsed perfusion in a human venous organ culture model with a Windkessel function is an approach to better understand the events taking place during early arterial-vein grafting. First signs of reactive cell proliferation were detected at day four. A period of seven days as described in the current model is probably too short to detect reactive cell proliferation and medial thickening. If the device might be activated for a longer period of time, it should be a suitable model to characterize the effects of intra- and extravascular drug administration as treatment strategies of vein graft disease.

Valproic acid inhibits proliferation of human coronary vascular cells (SI/MPL-ratio: 0.5): a novel candidate for systemic and local therapy of postinterventional restenosis.

OBJECTIVES: The branched-chain fatty acid, valproic acid (VPA), is the most commonly used anti-epileptic drug for treating generalized epilepsy. Recently antiproliferative effects of VPA have been described in human cancer cells, and phase I trials for the treatment of solid tumors have been initiated. In cardiologic patients, increased cell proliferation and migration from the media into the subendothelial space are the key events causing restenosis after coronary angioplasty and stenting. This study investigates the effect of VPA on proliferation and migration in human coronary vascular cells. METHODS AND RESULTS: The theoretical clinical relevance of the data is estimated with a SI/MPL-ratio, which is defined as the relationship between a significant effect in vitro (SI) and the maximal plasma level in vivo (MPL). Dilution of VPA: Aqua dest, MPL in vivo: 100 microg/ml. Cell culture: HUVEC, human umbilical endothelial cells; HCAEC, human coronary artery endothelial cells; HCMSMC, human coronary media smooth muscle cells. Proliferation assay: HUVEC, HCAEC, and HCMSMC were seeded as described. At day 1, after seeding the cell number was calculated in a cell counter. VPA was added in six different concentrations ranging between 50 and 300 microg/ml. At day 3, the medium and agent were renewed, and after another 2 days, the cell number was calculated in relation with the cell number at day 1. Cell toxicity: Cytotoxic effects of VPA were studied in concentrations ranging from 50 to 300 microg/ml. Migration assay: migration of HCMSMC after incubation with VPA in concentrations ranging from 50 to 300 microg/ml was studied for a period of 24 h. Proliferation assay: strong dose-dependent antiproliferative effects were detected after 5 days of incubation with all the three tested cell types. In HUVEC, significant antiproliferative effects were found with VPA in concentrations of 100 microg/ml (P<0.05, SI/MPL-ratio: 1.0) and more. In HCAEC and HCMSMC, significant antiproliferative effects were detected after incubation with VPA in the concentrations of 50 microg/ml (HCAEC: P<0.01, SI/MPL ratio: 0.5; HCMSMC: P<0.001, SI/MPL-ratio: 0.5). Migration assay: no effect on cell migration was detected after incubation of HCMSMC for a period of 48 h with VPA in concentrations ranging from 50 to 300 microg/ml. Cell toxicity: in HUVEC, HCAEC, and HCMSMC significant toxic effects were detected in all the VPA concentrations studied. CONCLUSION: Significant dose-dependent antiproliferative effects of VPA with SI/MPL ratios of 0.5 identify the drug as a promising candidate for both systemic and local therapy of postinterventional restenosis. The partial cytotoxic effects, however, may restrict the use of VPA to local high-dose devices such as drug eluting stents.

Myocardial inflammation and non-ischaemic heart failure: is there a role for C-reactive protein?

Whereas C-reactive protein (CRP) is acknowledged as a cardiovascular risk marker, there is ongoing discussion about its role as a risk factor. Previous studies focused on the effects of CRP on ischaemic heart failure and atherosclerosis. In this study we investigated distribution of CRP, the Terminal Complement Complex (C5b-9) and macrophages (CD68) in the myocardium of patients suffering from non-ischaemic heart failure and their implication on clinical parameters. Endomyocardial biopsies were taken from 66 patients suffering from dilated cardiomyopathy (DCM). Biopsies were analysed by immunohistochemical and immunofluorescent staining for CRP, C5b-9 and CD68. Viral DNA/RNA for adenovirus, enterovirus, parvovirus B19 and human herpes virus 6 was detected by PCR and Southern blot analysis. Myocardial biopsy findings were correlated with plasma level of hsCRP and NT-proBNP as well as echocardiography, exercise test and NYHA class. In 18 (27%) patients, a positive staining for CRP and in 57 (86%) patients a positive staining for C5b-9 was detected. All patients showed myocardial infiltration with macrophages with an average of 39 cells/mm(2). CRP, C5b-9 and CD68 co-localised within the myocardium. No correlation was observed for inflammatory proteins and plasma level of hsCRP, NT-proBNP and clinical parameters. CRP is frequently present in the myocardium of patients suffering from DCM and co-localises with C5b-9 and macrophages. CRP may contribute to myocardial damage in DCM via activation of the complement system and chemotaxis of macrophages.

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

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

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

BACKGROUND: 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. METHODS: Definition of the SI/MPL-ratio: relation between significant inhibitory effects in vitro/ex vivo and the maximal plasma level after systemic administration in vivo (6.4 ng/ml for SRL). Definition of the SI/DES-ratio: relation between significant inhibitory effects in vitro/ex vivo and the drug concentration in DES (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-alpha 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. RESULTS: Expression of ICAM-1 was significantly inhibited both in HCAEC (SRL > or = 0.01 ng/ml) and HCMSMC (SRL > or = 10 ng/ml). SRL in concentrations > or = 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%. CONCLUSION: SI/MPL-ratio's < or = 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 10-6 and 10-8 indicate 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.

Low-dose irradiation stimulates TNF-alpha-induced ICAM-1 mRNA expression in human coronary vascular cells.

BACKGROUND: Low-dose irradiation (LDI) is employed to extend the irradiation area in vascular brachytherapy to minimize the edge effect. Stimulation of adhesion molecule 1 (ICAM-1) is one of many mechanisms important in the early stages of atherosclerosis and restenosis. This study investigates the effect of gamma-irradiation on mRNA expression of ICAM-1 in human coronary vascular cells. MATERIAL/METHODS: Human coronary endothelial cells (HCAECs), human umbilical endothelial cells (HUVECs), and human coronary smooth muscle cells (HCMSMCs) were cultured and identified. Twenty-four hours after seeding, gamma-irradiation at doses of 0.5 Gy, 1 Gy, 10 Gy, 20 Gy, and 30 Gy (Linac Philips FL 75/20) was carried out. Twenty hours after irradiation, ICAM-1 expression was stimulated for 6 h with TNF-alpha (20 ng/ml). In Northern blot assays, 10 microg of RNA were used. The relative band density of ICAM-1 mRNA of irradiated and stimulated cells were compared with that of non-irradiated cells after TNF-alpha stimulus only. RESULTS: In HCAECs and HCMSMCs, stimulation of ICAM-1 mRNA was detected after irradiation, no matter which dose was applied. After low-dose irradiation (0.5 Gy and 1 Gy) the stimulating effect was pronounced, significant differences being found in HCAECs after irradiation with 1 Gy. CONCLUSIONS: Stimulation of ICAM-1 mRNA expression after LDI of human coronary vascular cells may be one of the many mechanisms that trigger the edge effect in vivo. If these results are confirmed by further studies and if anti-inflammatory treatment strategies cannot inhibit the stimulatory effects, a major problem exists for the future of vascular brachytherapy.

A perfused renal human organ culture model: impact of monocyte attack.

BACKGROUND: Key processes of atherosclerosis and restenosis are triggered and/or modified by the contact of human monocytes (MCs) with the inner layers of the arterial vessel wall. This is the first report on monocyte attack in a perfused renal human organ culture model (perfused renal HOC-model). MATERIAL/METHODS: Parts of the renal arteries were extracted during routine nephrectomies. A closed loop system was established by fixing the segments between two hard plastic tubes and connecting the distal endings of the hard plastic tubes with soft plastic tubes. 5x10(5) human MCs were added to the culture medium for a period of 24 h. Immunohistological staining was carried out before adding the MCs and after 2, 24, 48, and 72 hours. RESULTS: Perfusion of the model with culture medium was performed with a steady flow of 1.6 mL/min. One, two, and three days after adding the intravascular MCs, almost no extravascular MCs were detected. Although the number of MCs was merely slightly increased on the endothelium, in the plaque-intima, and in the media, a large number of MCs was detected in the adventitia. During the three-day period of steady flow perfusion, no stimulation of smooth muscle cell proliferation in the artery wall was detected. CONCLUSION: Steady perfusion of the renal HOC-model is an important step in the attempt to adapt human ex vivo models to the various roles of inflammation in the pathophysiology of atherosclerosis and restenosis.

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 alpha-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 microg 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.

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 microg/ml) on TNF-alpha 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 microg/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 microg/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 microg/ml a stimulatory effect on cell migration was detected, statistical significance was achieved after incubation with 0.002 microg/ml (p < 0.05), 0.002 microg/ml (p < 0.001), and 0.2 microg/ml (p < 0.05). Proliferation: Small but statistically significant antiproliferative effects of abciximab were detected after incubation of HUVEC (0.02 and 2.0 microg/ml; p = 0.01 and p < 0.01), HCAEC (2.0 and 20.0 microg/ml; p < 0.05 and p < 0,01), and HCMSMC (2.0 and 20.0 microg/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.

Triple-coated stents (Hirudin/Iloprost/Paclitaxel): an in vitro approach for characterizing the antiproliferative potential of each individual compound.

BACKGROUND: Hirudin (H)/iloprost (I)/paclitaxel (P)-coated stents represent a multifactorial approach to reducing the proliferative response caused by ballooning and stenting. The study presented compares the net effect of each individual compound of HIP-coated stents with the summed effect of the compounds in the stent coating. METHODS AND RESULTS: For proliferation prescreening studies, human coronary smooth muscle cells were incubated with H (0.005-500 microg/ml), I (0.00001-1 microg/ml), and P (0.0001-10 microg/ml). After 5 days, cell number was studied in a cell analyzer system. Secondly, 8-mm stents were coated with (1) HI, (2) HIP-10 microg/20 microg/40 microg (HIP5%/10%/20%), (3) P-40 microg (P), (4) IP-40 microg (IP), and (5) HP-40 microg (HP). After 5 days, the effect on cell proliferation and cytoskeletal structures was studied. No antiproliferative effect was found after incubation with H; significant inhibition was seen after incubation with I (p<0.05) or lipophilically dissolved P (p<0.001). After 5 days incubation with HIP5%-, HIP10%-, HIP20%-, P20%-, IP20%-, and HP20%-coated stents, cell proliferation was inhibited by 55.5% (p<0.05), 61% (p<0.05), 57.9% (p<0.05), 59.5% (p<0.001), 59.8% (p<0.001), and 63.3% (p<0.001), respectively. HI- and HIP-coated stents caused a severe destruction of the cytoskeletal structures smooth muscle alpha-actin and alpha-tubulin; despite the destruction, vital cells could be identified with positive FDA staining. CONCLUSIONS: Although both lipophilically dissolved P and hydrophilically dissolved I contributed to the antiproliferative effect, no additive effect of the two compounds was detected. In vivo P can be released more easily from the coating material due to the permanent lipophilic contact of the stent struts with the vessel wall. The current study is the first report on a clear and uncomplicated technique to obtain information on the antiproliferative potential of coated stents before large experimental studies are initiated.

Antiproliferative profile of sirolimus and mycophenolate mofetil: impact of the SI/MPL ratio.

BACKGROUND: Recently, preliminary data of the ORBIT study have been presented; coronary restenosis after oral treatment with sirolimus (SRL) was merely 7.7%. The present study thought to investigate the antiproliferative profile of SRL and mycophenolate mofetil (MMF), both as individual compounds and as a combined therapy. METHODS AND RESULTS: Proliferation studies were carried out with smooth muscle cells of human coronary arteries (human coronary smooth muscle cells, HCMSMC). SRL (0.01-1000 ng/ml) and MMF (0.005-500 microg/ml) were added in six descending concentrations, cell proliferation was studied at day 5. To characterize the clinical relevance of the data, the authors calculated a SI/MPL ratio between a significant antiproliferative effect (SI) in vitro and the maximal systemic plasma level (MPL) in vivo. The SI/MPL ratios of SRL and MMF were 0.16 and 0.014, respectively. Second, SRL (1 and 0.1 ng/ml) was combined with four concentrations of MMF (0.5 and 0.05 microg/ml) and MMF was combined with four concentrations of SRL. Additive and overadditive antiproliferative effects were found, no destruction of alpha-tubulin was detected. CONCLUSIONS: Thus, SRL and MMF exhibit dose-dependent direct antiproliferative effects with SI/MPL ratios smaller than one. Both agents, as individual compounds or as combined therapy, are candidates for an oral therapy of human coronary restenosis.

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 microg/mL) on TNF-alpha 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 microg/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 microg/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 microg/mL. In the 3DLA-model 50 microg/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 microg/mL). CONCLUSION: Thus, the data demonstrate a significant antiproliferative effect of clinical relevant levels of MMF (50 microg/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 microg/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.

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-1beta, IL-8, beta-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.

Human cytomegalovirus infection in human renal arteries in vitro.

Studies with animal cytomegaloviruses, epidemiological data from humans as well as in vitro studies suggest the involvement of the human cytomegalovirus (HCMV) in the development of atherosclerosis. Cell culture systems are insufficient for examination of the entire pathogenetic process and a satisfactory animal model for HCMV is not available. An organ culture model was established for HCMV infection of human renal arteries in vitro. After infection with three representative HCMV strains, infectious virus was recovered from supernatants until 144 days post-infection with a peak around day 30 due to a long-lasting productive HCMV infection in still vital cells. Differences in cell tropism and kinetics of infection were identified between the HCMV strains. Specifically, differences in infecting endothelial cells and virus penetration into the lamina media were observed. In infected artery segments, but also in some non-infected arteries from seropositive donors, HCMV DNA could be localized by in situ PCR. Nevertheless, HCMV early antigen was detected by immunohistochemistry exclusively in artery segments infected in vitro. The new organ culture model will permit the study of functional and molecular consequences of HCMV infection in a more physiological micro-environment.

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 microg/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 alpha-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 alpha-actin in smooth muscle cells. CONCLUSIONS: Simultaneous intra/extravascular administration of high dose diltiazem (50 microg/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.