Various lines of evidence indicate that oxidative stress resulting in lipid peroxidation and protein modification is involved in the pathogenesis of atherosclerosis and coronary heart disease. We have investigated the effect of modified (oxidized) low-density lipoproteins (oxLDL) on collagen and fibronectin synthesis in cultured human coronary artery smooth muscle cells (HCA-SMC). As shown by immunofluorescence microscopy and time-resolved fluorescence immunoassay, oxLDL dose-dependently stimulated collagen type I and fibronectin synthesis in cultured HCA-SMC. The effect on matrix synthesis was biphasic, with a maximum effect at concentrations between 1 and 10 microg/ml oxLDL. Higher oxLDL concentrations (>25 microg/ml) were cytotoxic. Beside oxLDL, malondialdehyde-modified LDL also stimulated extracellular matrix synthesis. In the presence of 100 microg/ml ascorbic acid, 25, 50 and 100 microg/ml oxLDL induced apoptosis within 6-8 hours (demonstrated by TUNEL-reaction, annexin-V binding and APO-2.7-expression). Apoptosis was not induced by normal (unmodified) LDL and malondialdehyde-modified LDL. The radical scavengers and antioxidants TROLOX and probucol and the hydrogen peroxide eliminator catalase significantly reduced oxLDL-induced apoptosis. Our results demonstrate that low concentrations of oxLDL are profibrogenic by stimulating extracellular matrix synthesis, whereas higher oxLDL concentrations induce oxidative stress and apoptosis in coronary artery smooth muscle cells. The profibrogenic effect might be relevant in the formation of atherosclerotic plaques, and the proapoptotic effect might contribute to an increased plaque vulnerability.

No abstract

Low-density lipoproteins (LDL) are known to cause endothelial injury and to promote the development of atherosclerotic lesions. This study demonstrates a significant concentration-dependent stimulatory effect of LDL on hepatocyte growth factor (HGF) synthesis (maximum release: 423 +/- 16% of control) and HGF receptor mRNA expression in cultured human coronary artery endothelial cells (HCAEC). HGF is a potent mitogen for endothelial cells but does not affect smooth muscle cell proliferation. In contrast, endothelin-1 (ET-1) acts as a mitogen on vascular smooth muscle cells and seems to be upregulated in coronary atherosclerosis. In this study, the basal ET-1 synthesis in HCAEC was concentration-dependently reduced by HGF (minimum: 54 +/- 3% of control). This inhibitory effect seems to be mediated via the tyrosine kinase activity of the HGF receptor c-met, since it was antagonized by the tyrosine kinase inhibitor lavendustin A. In addition, HGF also significantly reduced the LDL-stimulated ET-1 release. The LDL-induced upregulation of HGF synthesis in HCAEC and the inhibitory effect of HGF on ET-1 synthesis suggest a protective role of HGF in coronary atherosclerosis.

Combining specific enzymatic derivatization of cholesterol or triglycerides with capillary isotachophoresis (CITP), human serum lipoproteins are separated into 14 lipoprotein subfractions, monitored and quantitated by direct capillary UV detection. By comparing the separation patterns of human serum with the patterns of lipoprotein particles isolated by sequential ultracentrifugation it became evident that peaks 1-5 represent lipoproteins of the high density lipoprotein (HDL) fraction, peaks 6-8 embody the very low density lipoprotein (VLDL) fraction and chylomicrons, and peaks 7-14 represent the low density lipoprotein (LDL) fraction. Peaks 7 and 8 were found in the VLDL as well as in the LDL fraction. Using triglyceride-specific staining peaks 6-8 occurred prominently; and with cholesterol-specific staining, peaks 1-5 and 7-14 were prominent. The coefficient of variation, for the sum of the peak heights of a pooled serum, was 3.94 for triglyceride-specific staining and 2.32 for cholesterol-specific staining. A linearity range between 0.23 and 2.29 mM/L was found for triglyceride-specific staining and between 0.043 and 4.33 mM/L for cholesterol-specific staining. The practicability of the method was evaluated (i) using blood of humans before and 45 min after an oral fat load. Triglyceride-specific staining revealed a prominent increase in the VLDL fraction and chylomicrones containing peaks 6 and 7, and a minor increase in the HDL fraction containing peaks 3 and 4, and (ii) in patients with manifest hypothyroidism before and after thyroxine therapy. Cholesterol-specific staining demonstrated a massive decrease in the first peak of the HDL fraction and in peaks 9 and 11 of the LDL fraction regarding the hypo versus hyperthyroid state.

Oxidative modification of low density lipoproteins (LDLs) is an important pathogenetic factor in atherosclerosis. The various steps in oxidative modifications of LDL can be monitored using different methodologies with varying degrees of complexity. In this study, we propose capillary isotachophoresis (CITP) as a suitable tool to detect and measure the degree of oxidation of LDL. LDL was isolated from pooled plasma of healthy volunteers by sequential ultracentrifugation, and oxidation was performed in vitro as well as in cell culture experiments. Native LDL and oxidatively modified LDL were characterized by apo B-100 fluorescence and conjugated diene formation. Samples were separated by CITP combined with sudan black B staining. To underline the inherent advantages of this approach, CITP was compared with classical lipoprotein electrophoresis using agarose gel. We demonstrate the CITP method to be highly sensitive, as changes in peak area of the separated LDL subfractions were detected after only 2 h of oxidation. The leading LDL peaks increased, while the terminating LDL peaks decreased in parallel throughout the duration of oxidation. The LDL samples, oxidized for 4-24 h, also exhibited an increased migration velocity of the fractions. In summary, we present the first study investigating LDL-subfractions separated by CITP and the alterations of these LDL-subfractions after gradual in vitro oxidation and after oxidative modification by monocyte-derived macrophages and vascular smooth muscle cells.