Ioakim Spyridopoulos

All-trans retinoic acid regulates proliferation, migration, differentiation, and extracellular matrix turnover of human arterial smooth muscle cells

OBJECTIVE The vitamin-A derivative all-trans retinoic acid (atRA) is a potent regulator of cell growth, differentiation, and matrix formation of various cell types and plays an important role in embryogenesis. However, sparse data are available about its effects on human vessel diseases. Thus, we studied the effects of atRA on human arterial smooth muscle cell (haSMC) and endothelial cell (haEC) proliferation, migration, differentiation and extracellular matrix (ECM) turnover in mono- and transfilter cocultures. METHODS Effects of atRA on human arterial cells in monocultures were determined using cell counting assays, BrdU-ELISA and MTT-tests. In transfilter cocultures haSMC-growth was studied under the stimulatory effect of proliferating haEC. Using Northern blot analysis, effects of atRA on mRNA expression of ECM-proteins were examined while protein expression and activity of matrix metalloproteinases were determined by Western blotting and zymography. RESULTS atRA caused a dose dependent inhibition of haSMC-growth in monocultures (IC(50) at 0.022 microM) whereas haEC-growth was inhibited less potently (IC(50) at 97 microM). In addition, proliferation and migration of haSMC through a porous membrane were inhibited dose dependently by micromolar atRA-doses after non-stop and single dose application of atRA on the endothelial side of the complex transfilter coculture system. Immunostainings and Northern blotting demonstrated an enhanced alpha-smooth muscle actin and heavy chain myosin expression in haSMC after atRA-treatment. Whereas mRNA-expression of the glycoproteins thrombospondin-1 and fibronectin were decreased, collagen-1 mRNA expression was even slightly stimulated. Transcription of biglycan and TGF-beta1 were not influenced in a specific manner. Finally, protein expression and activity of the matrix metalloproteinases MMP-2 and MMP-9 were inhibited significantly by atRA. CONCLUSIONS atRA was found to be a potent inhibitor of both haSMC-proliferation and -migration, even in coculture with haEC releasing growth factors. In addition, redifferentiation, ECM synthesis and ECM degradation were regulated by atRA which also influence haSMC migration and intima formation. Thus, atRA-treatment seems to be a promising strategy for the inhibition of processes involved both in atherosclerosis and restenosis.

Toxicity, uptake kinetics and efficacy of new transfection reagents: increase of oligonucleotide uptake.

Human arterial smooth muscle cell (haSMC) proliferation is stimulated by platelet-derived growth factor (PDGF) release of human arterial endothelial cells (haEC) whereas transforming growth factor-β1 (TGF-β1) secretion by haSMC promotes extracellular matrix formation. Inhibitory concepts with antisense oligonucleotides (ASO) against those growth factors might be promising, requiring, however, sufficient transfection efficacy. Thus, toxicity and efficacy of new transfection reagents were examined. MTT tests showed that high doses >1.6 μg/ml of the liposome Cytofectin GSV® (CF) and the dendrimer SuperFect® (SF) reduced mitochondrial activity of haEC after ≥4 h transfection whereas viability of haSMC was not influenced. DAC-30® showed significant toxic effects on haEC and haSMC at each dose after ≥4 h and Lipofectin® (LF) caused complete detachment of haEC and haSMC in medium containing 10% serum. Uptake studies demonstrated that ‘naked’ ASO were not incorporated intracellularly whereas transfection within CF or SF resulted in a strong cytoplasmic and nuclear labeling after 2–5 h. With DAC-30®, only a slight cytoplasmic fluorescence was found. SF caused an unexpected stimulation of endothelial PDGF-AB synthesis. Thus, CF was favored for inhibition studies. ELISA, Western and Northern blotting showed a significant inhibition of endothelial PDGF-B and smooth muscle TGF-β1 mRNA expression and synthesis after transfection for 3–5 h using 0.1–1.0 μM ASO versus control oligonucleotides. We conclude that Cytofectin GSV® is superior to the other transfection reagents, predominantly at haEC, showing an improved efficacy and less toxicity than the classical liposome Lipofectin®. Cytofectin GSV® might offer a promising tool for antisense strategies in the treatment of vascular disorders.

Late Outcome of Survivors of Idiopathic Ventricular Fibrillation

This report describes clinical, hemodynamic, and electrophysiologic characteristics of 18 consecutive survivors of sudden cardiac arrest due to idiopathic ventricular fibrillation (VF) between 1986 and 1996. Long-term data in relation to the prescribed therapy are presented. The mean age of the 18 patients was 48 +/- 14 years (median 49). Electrophysiologic studies showed a low inducibility of sustained ventricular tachyarrhythmias in 4 patients (22%). Treatment consisted of class III agents, beta blockers, or implantable cardioverter-defibrillators. Two patients were discharged without any therapy. Therapy control was undertaken either by serial drug testing or by the empirical approach. Serious complications of therapy occurred in 2 patients: 1 patient experienced a proarrhythmic effect of antiarrhythmic drug therapy, and the other patient received multiple inadequate defibrillator discharges due to a defect in the transvenous lead. All but 1 patient (94%) remained free of recurrences of sudden cardiac arrest during a follow-up time of 45 +/- 29 months (median 41). One patient died 2 weeks after surviving cardiac arrest due to intractable VF while receiving sotalol treatment. Therapy guided by electrophysiologic studies did not have any impact on survival. Adverse effects or noncompliance led to discontinuation of drug therapy in 7 patients after a mean period of 31 +/- 30 months. Without any treatment 9 patients remained without recurrences over 45 +/- 33 months. Because of the absence of risk factors for arrhythmia recurrence and criteria to select therapy, randomized prospective studies are warranted to assess the optimal therapies in these young, ostensibly healthy patients.

Initial clinical experience with a modified excimer laser for coronary angioplasty

A clinical study was conducted in 32 patients to evaluate the efficacy and safety of a modified excimer laser system for percutaneous transluminal coronary angioplasty. In this system, the laser beam is scanned and transmitted into eight fibre bundles of the catheter device, consisting of 20 50 μm fibres, each. Twenty-eight patients were treated with 1.5 mm laser catheters, four patients with 1.8 mm laser catheters. Mean time of energy delivery was 82±39 s using a mean energy fluence of 49±2 mJ mm−2. In all 32 patients in whom laser angioplasty was attempted, laser irradiation resulted in a stenosis reduction from 85±10% (mean±s.d.) before to 57±20% after laser treatment. In 16 patients, additional balloon angioplasty had to be performed, either due to an insufficient angiographic result in 11 patients or due to abrupt vessel closure in five patients. In these 16 patients, percent stenosis decreased after balloon angioplasty to 35±14%, corresponding to a luminal diameter of 1.6±0.5 mm. In 10 patients, dissection was observed. In one of these patients, the dissection resulted in a reduction in antegrade flow, necessitating balloon dilatation. One perforation occurred which did not require surgery. There were no deaths, bypass surgery or myocardial infarction. During the time of follow-up, restenosis occurred in 14 patients; in two of these patients bypass surgery was performed and five patients were treated with conventional balloon angioplasty.These results suggest that this form of modified excimer energy delivery provides effective therapy for patients with coronary artery disease. Due to the small catheter sizes, however, one-half of the patients still required additional balloon dilatation. To increase the number of stand-alone laser procedures and to address the issue of restenosis in this patient population appropriately, larger catheter devices will be necessary.

Apoptosis and cell cycle in endothelial cells

Apoptosis is an active process of cell death that occurs both under physiologic and pathophysiologic conditions. Specifically, apoptosis has been involved in vasculogenesis and atherosclerosis, inflammation, as well as wound healing, respectively. Its hallmark is the cleavage of genomic DNA into nucleosomal fragments of 180 bp (1). Although it has not been established to what extent endothelial cell death occurs in vivo, there is increasing evidence that endothelium may become apoptotic in various disease states.

Magnetic resonance imaging in hypertrophied left ventricular myocardium due to amyloidosis

The certain diagnosis of cardiac amyloidosis is only possible with myocardial biopsy, even if echocardiographic studies often show a typical sparkling pattern. Using magnetic resonance imaging (MRI) we examined if there is a specific morphological pattern in patients with amyloidosis compared with patients with hypertrophic cardiomyopathy (LVH). With a 1.0 T magnetom and FISP 2D sequences two patients with biopsy-proven cardiac amyloidosis (AL), two patients with generalized AL and suspected cardiac AL, and five patients with LVH were examined and data were compared with echocardiography. In two cases with cardiac AL, contrast medium (Gd-DTPA) was given and dynamic-turbo-flash-sequences were obtained. In patients with AL, both ventricles were hypertrophied, whereas in the cases of hypertrophy due to other reasons only the left ventricle was hypertrophied. The systolic wall thickening was in all cases of amyloidosis below 30%. In contrast to echocardiography, a myocardial sparkling pattern in amyloidosis was not found with MRI. Even with additional contrast examination we could not differentiate the types of hypertrophy by imaging solely the left ventricular wall. There is no specific myocardial pattern in cardiac amyloidosis neither in standard MRI nor after examination with additional contrast medium, but concomitant right ventricular (RV) and left ventricular (LV) hypertrophy is a typical observation in these patients.