Biomedical subjects

Jan Galle

Publications and source records attributed to Jan Galle.

23 records · Page 2Linked to original sources

PubMed2002

Nitric oxide prevents oxidised LDL-induced p53 accumulation, cytochrome c translocation, and apoptosis in macrophages via guanylate cyclase stimulation.

BACKGROUND: Oxidatively modified low density lipoprotein (OxLDL) induces apoptosis in vascular cells including macrophages, while NO exerts antiapoptotic effects. Here we studied the impact of nitric oxide (NO) on OxLDL-induced cytochrome c release, apoptosis, and expression of the proapoptotic p53 in macrophages. METHODS: Human LDL was oxidised by Cu(2+), and monocytes were prepared from human buffy coats. Differentiation to macrophages was achieved by culturing cells in the presence of human serum and was followed by detecting monocyte chemoattractant protein 1 (MCP-1) expression (RT-PCR). Cytochrome c release and p53 expression of macrophages were detected by immunoblotting, and apoptosis by visualisation of nuclear condensation. RESULTS: OxLDL dose-dependently (50-200 microg/ml) induced cytochrome c release that was prevented by preincubation with the NO-donor S-nitrosoglutathione (GSNO) (100 microM) or with the cGMP analogue 8-br-cGMP (100 microM) for 15 h. In cells co-treated with GSNO and the soluble guanylate cyclase (sGC) inhibitor oxadialoquinoxalione (ODQ, 10 microM, 15 h), OxLDL-evoked cytochrome c release remained effective, indicating that NO acted via sGC-dependent cGMP formation. Parallel incubation of macrophages with 8-br-cGMP (100 microM) and ODQ (10 microM) for 15 h left the protective effect of 8-br-cGMP unaltered. Short pre-incubation (30 min) with GSNO or 8-br-cGMP was ineffective in preventing OxLDL-elicited cytochrome c release. Initiation of cytochrome c release in macrophages was paralleled by a dose-dependent accumulation of the proapoptotic factor p53, and by enhanced rate of nuclear condensation. Stabilisation of p53 was prevented by preincubation with the NO-donor GSNO or 8-br-cGMP, thus implying a downmodulatory effect of cGMP on pathways that upregulate the tumor suppressor p53. CONCLUSIONS: OxLDL induces cytochrome c release and apoptosis in human macrophages in close association with p53 accumulation. NO attenuates OxLDL-induced cytochrome c release and p53 accumulation via activation of sGC and cGMP formation. These effects may be of particular importance in arterial tissue with reduced NO activity.

Apoptosis↗

PubMed2002

Augmented endothelial uptake of oxidized low-density lipoprotein in response to endothelin-1.

Endothelin-1 (ET-1) may be involved in the development and progression of atherosclerosis. Furthermore, endothelin receptor blockade was shown to reduce the formation of atherosclerotic lesions in experimental studies. Another potent pro-atherosclerotic risk factor is oxidized low-density lipoprotein (oxLDL). Endothelial cells mediate the uptake of oxLDL by the recently identified lectin-like oxLDL receptor-1 (LOX-1), which accumulates in atherosclerotic lesions. In the present study, we analysed the effects of ET-1 on oxLDL uptake and LOX-1 expression in primary cultures of human umbilical vein endothelial cells (HUVEC). ET-1 stimulated uptake of oxLDL in HUVEC, which reached a maximum after 1 h. In further studies, we found a similar induction of LOX-1 mRNA and protein expression in response to ET-1. The augmented oxLDL uptake and the increased LOX-1 expression in response to ET-1 are mediated by the endothelin receptor B. Our data support a new pathophysiological mechanism by which locally and systemically increased ET-1 levels, e.g. in hypertensive patients, could promote LOX-1-mediated oxLDL uptake in human endothelial cells. This mechanism could promote the development and progression of endothelial dysfunction and atherosclerosis. In addition, endothelin receptor blockade could be considered as a new anti-atherosclerotic therapeutic principle.

Arteriosclerosis↗

PubMed2002

Phosphorylation of blood vessel vasodilator-stimulated phosphoprotein at serine 239 as a functional biochemical marker of endothelial nitric oxide/cyclic GMP signaling.

The endothelium-derived relaxing factors nitric oxide (NO) and prostacyclin (PGI(2)) are important antithrombotic, relaxant, and antiproliferative agents of the blood vessel wall that exert their intracellular effects primarily via cGMP- and cAMP-dependent protein kinases (cGK, cAK). However, no biochemical marker for their activity in the intact blood vessel is available except for transient increases in the concentration of cGMP and cAMP. Using Western blot analysis and specific antibodies, we show here that phosphorylation of the vasodilator-stimulated phosphoprotein (VASP) at Ser239 (P(Ser239)-VASP) in rabbit aorta was detectable only in segments with an intact endothelium, although at least one third of VASP is contained in the remaining vascular wall. In endothelium-denuded aorta, VASP phosphorylation was increased by the NO donor sodium nitroprusside (SNP). Levels of P(Ser239)-VASP, in the presence of endothelium and either SNP or 8-bromo-cAMP, were maximal. VASP phosphorylation elicited by 8-bromo-cAMP was inhibited significantly by the cGK inhibitor Rp-8-Br-PET-cGMPS. Stimulated P(Ser239)-VASP formation was fully reversible, reaching basal levels after 10 min of repeated washouts. Consistent with the important role that the NO/cGMP pathway plays in the formation of P(Ser239)-VASP in rabbit aorta, inhibition of NO synthase by N(omega)-nitro-L-arginine methyl ester (L-NAME; 1 mM) or of soluble guanylyl cyclase by 1H-[1,2,4]oxadiazolo[3,4-a]quinoxalin-1-one (ODQ; 50 microM) almost completely abolished P(Ser239)-VASP formation in endothelium intact blood vessels. These data suggest that vascular P(Ser239)-VASP is primarily regulated by the NO/cGMP pathway and may thus serve as a biochemical marker for the activity state of this essential pathway in endothelial function.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed2001

Oxidized LDL suppresses NF-kappaB and overcomes protection from apoptosis in activated endothelial cells.

Atherosclerosis is a chronic inflammatory disease associated with enhanced apoptotic cell death in vascular cells, partly induced by oxidized low-density lipoprotein (OxLDL). However, proinflammatory stimuli such as lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-alpha) activate endothelial cells (EC) and inhibit apoptosis through induction of nuclear factor kappaB (NF-kappaB)-dependent genes. This study therefore investigated whether OxLDL or its component, lysophosphatidylcholine (LPC), interacts with the effect of LPS or TNF-alpha on cell survival. Human EC were incubated with LPS, TNF-alpha, OxLDL, or LPC alone or in combinations. OxLDL (100 to 200 microg/ml) and LPC (100 to 300 microM) induced apoptosis dose-dependently. LPS and TNF-alpha had no effect on cell survival in the presence or absence of OxLDL or LPC. LPS and TNF-alpha both induced the antiapoptotic gene A20, whereas OxLDL and LPC suppressed its induction. Expression of A20 is regulated by NF-kappaB. OxLDL and LPC dose-dependently suppressed NF-kappaB activity. For functional analysis, bovine EC were transfected with A20 encoding expression constructs in sense and antisense orientation. Bovine EC that overexpressed A20 were protected against OxLDL-induced apoptosis, whereas expression of antisense A20 rendered cells more sensitive to OxLDL. These results suggest that OxLDL not only induces cell death, as has been shown before, but also compromises antiapoptotic protection of activated EC. OxLDL sensitizes EC to apoptotic triggers by interfering with the induction of A20 during the inflammatory response seen in atherosclerotic lesions. This inhibition is based on repression of NF-kappaB activation. The effect may be caused by the OxLDL component LPC.

Animals↗

PubMed2000

Stimulation of NADPH oxidase by oxidized low-density lipoprotein induces proliferation of human vascular endothelial cells.

Oxidized low-density lipoprotein (OxLDL) exerts proliferation and apoptosis in vascular cells, depending on its concentration and the duration of exposure. Recent studies indicate that [O(2)](-) is involved in cell cycle regulation and that OxLDL stimulates endothelial cells to produce [O(2)](-). This study examined the role of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase as a potential source for [O(2)](-) in the proliferation-inducing activity of OxLDL in cultured human umbilical vein endothelial cells (HUVEC). Human LDL was oxidized by Cu(++), and proliferation of HUVEC was detected by 3H-thymidine incorporation. OxLDL (5 microg/ml) caused an increase in proliferation of HUVEC of 250 to 300%. OxLDL-induced proliferation was blocked by addition of the antioxidants superoxide dismutase and catalase, suggesting that enhanced [O(2)](-) formation was involved. Diphenylene iodonium (DPI, 1 microM), an inhibitor of NADPH oxidase, also prevented OxLDL-induced proliferation of HUVEC, indicating that NADPH oxidase was the source for enhanced [O(2)](-) formation. The OxLDL effect was mimicked by lysophosphatidylcholine (LPC, 10 microM), a compound formed during oxidation of LDL. LPC-induced proliferation was also prevented by coincubation with DPI. Treatment of HUVEC with [O(2)](-) generated by the xanthine/xanthine oxidase reaction resulted in proliferation as did treatment with OxLDL. As expected, this stimulation could not be blocked by DPI. With the use of the cytochrome c-assay, it was demonstrated that OxLDL and LPC enhanced [O(2)](-) formation in HUVEC (by factor 3.2 and by factor 3.5, respectively). Supporting the assumption that NADPH oxidase was the enzyme responsible for [O(2)](-) formation, cells transfected with antisense oligonucleotides for NADPH oxidase showed a significantly reduced [O(2)](-) formation after stimulation with OxLDL and LPC. OxLDL and its compound LPC induce proliferation of HUVEC through activation of NADPH oxidase. The active NADPH oxidase generates [O(2)](-), which mediates the proliferative effects.

Catalase↗