Dariusz Biały

Inhibition of MMP-2 expression affects metabolic enzyme expression levels: Proteomic analysis of rat cardiomyocytes

UNLABELLED In this study we examined the effect of inhibition of MMP-2 expression, using siRNA, on the cardiomyocyte proteome. Isolated cardiomyocytes were transfected with MMP-2 siRNA and incubated for 24h. Control cardiomyocytes from the same heart were transfected with scrambled siRNA following the same protocol. Comparison of control cardiomyocyte proteomes with proteomes from MMP-2 suppressed cardiomyocytes revealed 13 protein spots of interest (9 protein spots increased; 4 decreased). Seven protein spots were identified as mitochondrial enzymes involved in energy production and represent: ATP synthase beta subunit, dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex, cytochrome c oxidase subunit 5A, electron transfer flavoprotein subunit beta, NADH dehydrogenase (ubiquinone) 1 alpha subcomplex subunit 5 and a fragment of mitochondrial precursor of long-chain specific acyl-CoA dehydrogenase. Furthermore, precursor of heat shock protein 60 and Cu-Zn superoxide dismutase were identified. Two protein spots corresponding to MLC1 were also detected. In addition, ATP synthase activity was measured and was increased by approximately 30%. Together, these results indicate that MMP-2 inhibition represents a novel cardioprotective therapy by promoting alterations in the levels of mitochondrial enzymes for improved energy metabolism and by preventing degradation of contractile proteins needed for normal excitation-contraction coupling. BIOLOGICAL SIGNIFICANCE During ischemia and reperfusion of cardiomyocytes, abnormality in excitation-contraction coupling and decreased energy metabolism often lead to myocardial infarction, but the cellular mechanisms are not fully elucidated. We show for the first time that intracellular inhibition of MMP-2 in cardiomyocytes increases contractility of aerobically perfused myocytes, which was accompanied by increased expression of contractile proteins (e.g., MLC-1). We also showed that MMP-2 inhibition produced a cardiomyocyte proteome that is consistent with improved mitochondrial energy metabolism (e.g., increased expression and activity of mitochondrial beta ATP synthase). Thus, MMP-2 appears to be involved in homeostatic regulation of protein turnover. Our results are significant since they point to targeting MMP-2 activity as a novel therapeutic option to limit myocardial damage by decreasing proteolytic degradation of mitochondrial metabolic enzymes and myocardial contractile proteins during ischemia. In addition, the development of novel pharmacological agents that selectively targets cardiac MMP-2 represents a novel approach to treat and prevent other heart diseases.

Biventricular Response of the Heart to Endurance Exercise Training in Previously Untrained Subjects

Functional adaptation of the heart to regular strenuous exercise has not been fully elucidated yet, with different patterns of alterations being reported. We evaluated the effect of endurance exercise training (EET) on left (LV) and right ventricular (RV) mechanics in amateur individuals preparing for triathlon competitions.

Intravascular low-power laser light illumination: a new method in restenosis prevention

The procedure of percutaneous coronary intervention is associated with the 30% risk of restenosis in the dilatated coronary artery. in order to minimize its occurrence we developed the method of intracoronary low power laser irradiation and called it the photoremodling. We developed and constructed at total set-up for the intravascular illumination. It consists of the laser diode connected with a multimode step-index silica fiber 200/270 μm terminated with a special fiber diffuser, which allows to irradiate homogeneously a coronary vessel in the place of dilatation. The diffuser is inserted into the coronary vessel by a modificated angioplasty catheter. Till now PTCA plus photoremodeling procedures have been carried out in 40 patients (28 with stent implantation and 12 with balloon angioplasty). We did not observed any side effects and complications of the procedure. All patients were qualified for 6 months follow-up, which was terminated in 19 cases with a control coronarography. We did not find any case of restenosis in the stent group. In the group of patients after balloon angioplasty restenosis rate was 25%. The new method of treatment is safe. The preliminary results seem to be beneficial especially in the case of stent implantation.

In vitro human atherosclerotic plaque recognition by photosensitizer mono-L-aspartyl chlorin e6 assisted light induced fluorescence (PALIF)

The main aim of the presented experiments was to assess in vitro capabilities of Photosensitizer Assisted Light Induced Fluorescence (PALIF) to recognise different stages of atherosclerosis. Mono-L-asparyl-chlorin e-6 was used as a photosensitising agent and ultraviolet light (440 nm) as an excitation source to obtain spectra map of artery wall. The luminescence spectra were collected and analised. The specimans were histologically examined and classified into three groups: normal artery wall, atherosclerotic noncalcified plaque and calcified plaque. The ratio of green fluorescence (coming from collagen) to red fluorescence (coming from chlorin e6 bonded with lipid reach, noncalcified tissue) gies a chance to distinguish between normal artery or calcified plaque and noncalcified, lipid reach plaque. Further trials must be performed to evaluate in vitro athermoa detection algorithm as a feedback system for photoangioplasty.

In vitro experimental photodynamic diagnosis of artery atherosclerosis

Background: Although there are several methods for atherosclerosis detection available, none of them seems to be accurate enough to identify the vulnerable atheroscleroitc plaque. Photodynamic diagnosis (PDD) and therapy (PDT) -- a new method evaluated for neoplasms treatment is a modern approach to detecting and treating atherosclerosis. Aim: The purpose of this study was to assess in vitro the capability of PDD with use of chlorin e6 to recognize atherosclerotic plaque and its usefulness as a feedback system for photoangioplasty treatment. Methods: 30 specimens of human aorta. The samples were soaked with chlorin e6 and then washed out. The luminescence spectra were then collected. All samples were examined with light microscopy. Results: Tissue fluorescence is seen as green light. We noted a very strong red fluorescence of chlorin e6 originating from lipid reach plaque. We established a quantitative factor which would be the ratio R of chlorin e6 red intensity in its 660 nm maximum compared to the area of green luminescence centered at 515 nm. The highest value of the ratio was reached at atheromatous samples, then calcified and normal ones R2 = 3.51 ± 0.62, R3 = 1.63 ± 0.31, R1 = 1.51 ± 0.15 respectively. Statistically significant difference was noted between group two and one and between group two and three R2 = 3.51 ± 0.62 vs R3 = 1.63 ± 0.31 (p < 0,05); R2 = 3.51 ± 0.62 vs. R1 = 1.51 ± 0.15 (p<0.05) respectively. Conclusions: the following in vitro study confirms that photosensitizer chlorin e6 accumulates within atheromatous plaque. It may be a specific tool for atheromatous and normal or calcified segments discrimination. The advantage of the above method is a possibility of a real time imaging followed by targeted therapy of various forms and stages of atherosclerosis.

Optical device for intravascular low-power laser illumination

The treatment method presented in this paper is an adjunct to coronary angioplasty. It consists in irradiating a previously dilated artery with laser light which stimulates endothelium proliferation and reduces local inflammation. The influence of 808 nm laser light on the endothelium was studied in vitro. Because of the location of atherosclerotic plaques, illumination of the endothelium poses a problem. To overcome it, we have designed and built a laser set-up for homogeneous intravascular illumination in vivo.