Regine Baur

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.

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/mm2. 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.

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.

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.

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.

Expression of intercellular adhesion molecule-1 in human coronary endothelial and smooth muscle cells after stimulation with tumor necrosis factor-alpha.

BACKGROUND The intercellular adhesion molecule-1 (ICAM-1) is one of several human cell adhesion molecules that play a critical role in the early stages of postangioplasty restenosis. In this study, the in-vitro expression of ICAM-1 in human coronary endothelial cells and human coronary smooth muscle cells (SMC) after stimulation with tumor necrosis factor-alpha (TNF-alpha) was investigated. METHODS AND RESULTS SMC were isolated from the media of normal human coronary arteries (n = 26) up to 10 h post mortem (HCMSMC) and from human atherosclerotic coronary arteries (HCPSMC) that were extracted by thrombendarterectomy (n = 25). Endothelial cells of human coronary arteries (HCAEC) were purchased from Clonetics (Cell System, Remagen, Germany), and endothelial cells from human umbilical cord veins (HUVEC) were isolated after vaginal delivery. For investigations of the effect of TNF-alpha (2.5, 5, 10, and 20 ng/ml) on the proliferative activity of HUVEC, HCAEC, HCPSMC, and HCMSMC, serum-free media was used. After 24 h cell number and cell size distribution were measured in a cell analyzer system. The proliferation of HCPSMC and HCMSMC was increased by TNF-alpha; however, significant differences compared with controls were not reached. The proliferation of HUVEC and HCAEC was significantly reduced by TNF-alpha. For investigations of the effect of TNF-alpha (2.5, 5, 10, and 20 ng/ml) on the surface expression of ICAM-1, monoclonal anti-ICAM-1 antibodies (84H10) were used. The expression of ICAM-1 was analyzed using an immunofluorescence microscope. For flow cytometry analysis, 5 x 10(3) cells (100% gated) were analyzed using a fluorescence-activated cell sorter. In control cultures with no stimulation, the expression of ICAM-1 was positive in HCAEC, HCPSMC, HCMSMC, and HUVEC. TNF-alpha stimulated the expression of ICAM-1 in a time- and dose-dependent manner. After maximal stimulation with TNF-alpha (20 ng/ml for 24 h), the expression of ICAM-1 was stronger in HCMSMC than in HCPSMC. CONCLUSIONS These results suggest that the cytokine TNF-alpha regulates the expression of ICAM-1 in both human coronary endothelial cells and SMC, and could therefore play an important role in the pathophysiology of inflammatory and immune processes in restenosis after angioplasty.

Different effects of antisense RelA p65 and NF-κB1 p50 oligonucleotides on the nuclear factor-κB mediated expression of ICAM-1 in human coronary endothelial and smooth muscle cells

BackgroundActivation of nuclear factor-κB (NF-κB) is one of the key events in early atherosclerosis and restenosis. We hypothesized that tumor necrosis factor-α (TNF-α) induced and NF-κB mediated expression of intercellular adhesion molecule-1 (ICAM-1) can be inhibited by antisense RelA p65 and NF-κB1 p50 oligonucleotides (RelA p65 and NF-κB1 p50).ResultsSmooth muscle cells (SMC) from human coronary plaque material (HCPSMC, plaque material of 52 patients), SMC from the human coronary media (HCMSMC), human endothelial cells (EC) from umbilical veins (HUVEC), and human coronary EC (HCAEC) were successfully isolated (HCPSMC, HUVEC), identified and cultured (HCPSMC, HCMSMC, HUVEC, HCAEC). 12 hrs prior to TNF-α stimulus (20 ng/mL, 6 hrs) RelA p65 and NF-κB1 p50 (1, 2, 4, 10, 20, and 30 μM) and controls were added for a period of 18 hrs. In HUVEC and HCAEC there was a dose dependent inhibition of ICAM-1 expression after adding of both RelA p65 and NF-κB1 p50. No inhibitory effect was seen after incubation of HCMSMC with RelA p65 and NF-κB1 p50. A moderate inhibition of ICAM-1 expression was found after simultaneous addition of RelA p65 and NF-κB1 p50 to HCPSMC, no inhibitory effect was detected after individual addition of RelA p65 and NF-κB1 p50.ConclusionsThe data point out that differences exist in the NF-κB mediated expression of ICAM-1 between EC and SMC. Experimental antisense strategies directed against RelA p65 and NF-κB1 p50 in early atherosclerosis and restenosis are promising in HCAEC but will be confronted with redundant pathways in HCMSMC and HCPSMC.