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      Expression of mRNA for Phospholipase A 2, Cyclooxygenases, and Lipoxygenases in Cultured Human Umbilical Vascular Endothelial and Smooth Muscle Cells and in Biopsies from Umbilical Arteries and Veins


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          Arachidonic acid (AA) is released by phospholipase A<sub>2</sub> (PLA<sub>2</sub>) and then converted into vasoactive and inflammatory eicosanoids by cyclooxygenases (COX) and lipoxygenases (LOX). These eicosanoids are important paracrine regulators of vascular permeability, blood flow, local pro- and anticoagulant activity and they play a major role in the local inflammatory response. We have investigated the presence of mRNAs for PLA<sub>2</sub> and for isoforms of COX and LOX in both human endothelial cells (EC) and in human smooth muscle cells (SMC) in culture and in vascular biopsies of human umbilical veins (HUVB) and arteries (HUAB) by using the reversed transcription-polymerase chain reaction (RT-PCR) technique. Results show detectable levels of PLA<sub>2</sub> type IV (cPLA<sub>2</sub>) in cultured EC and SMC and in vascular wall biopsies from HUAB and HUVB. The cultured EC and SMC demonstrate higher levels of both COX-1 and COX-2 with PCR analyses than do vascular wall biopsies from HUAB and HUVB. This indicates a difference in the native expression of COX-1 and COX-2 in cultures of EC and SMC compared to that in biopsies from intact vessel walls. The EC and SMC in culture do not express mRNA for 5-LOX, that was, however, expressed in the vascular wall biopsies. This speaks in favour of a constitutive, i.e. in vivo expression of 5-LOX in SMC in the vascular wall of both umbilical vein and arteries. Thus results from in vitro studies of constitutive COX and LOX expression in EC and vascular SMC in culture cannot simply be extrapolated to represent in vivo conditions.

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          Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.

          A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
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            Extracellular phospholipase A2 secretion is a common effector pathway of interleukin-1 and tumour necrosis factor action.

            Inflammatory processes are characterized by increased levels of extracellular phospholipase A2 (PLA2) and cytokines such as interleukin 1 (IL-1) and tumour necrosis factor (TNF). IL-1, TNF and PLA2 share a number of proinflammatory, arthritogenic effects. The sequential induction, first of the cytokines followed by PLA2, suggests that these cytokines may regulate synthesis and secretion of PLA2. To test this postulate, foetal rat calvarial bone-forming cells (FRCC) were treated with recombinant human IL-1 and TNF and extracellular PLA2 release was quantitated. Both IL-1 and TNF induced the de novo synthesis of PLA2 in a concentration-dependent manner. Continuous exposure of FRCC in primary culture to IL-1 (50 units/ml) over 15 days resulted in as much as 100-fold increase in PLA2 secretion. IL-1 (50 units/ml) added to post-confluent cultures for a 48-h pulse increased PLA2 activity 9.4-fold. The combination of IL-1 (50 units/ml) and TNF (500 units/ml) was synergistic with an observed increase in extracellular PLA2 secretion of 146-fold following a 48-h pulse. Interleukin-6, alone or in combination with IL-1 or TNF, did not further enhance PLA2 synthesis of secretion. Cytokine-induced synthesis of PLA2 was inhibited 80% by 10 microM cycloheximide but not by dexamethasone over the range of 10(-6) to 10(-8) M. FRCC-derived PLA2 was neutral-active with a pH optimum of 6-7.5 and was calcium-dependent with optimal activity in the presence of 2-7 mM calcium. It had absolute 2-acyl specificity using micellar phosphatidylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)
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              Group II phospholipase A2 mRNA synthesis is stimulated by two distinct mechanisms in rat vascular smooth muscle cells.

              Two potent inflammatory mediators, interleukin 1 (IL-1) and tumor necrosis factor (TNF) as well as lipopolysaccharide (LPS) increased group II phospholipase A2 (PLA2) mRNA levels, which resulted in enhanced secretion of the PLA2 enzyme from rat smooth muscle cells. cAMP-elevating agents also stimulated the release of PLA2 and increased the mRNA, but IL-1, TNF and LPS did not affect cAMP levels. Furthermore, the effects of TNF and cAMP-elevating agents were not additive but synergistic. Therefore, we concluded that the level of rat group II PLA2 mRNA is controlled at least by two distinct mechanisms, one involves cAMP and the other is mediated by TNF, IL-1 and LPS. This study also suggests important roles of group II PLA2 in pathogenesis of vascular inflammation.

                Author and article information

                J Vasc Res
                Journal of Vascular Research
                S. Karger AG
                June 1998
                09 June 1998
                : 35
                : 3
                : 150-155
                a Clinical Research Center, and b Department of Occupational and Environmental Medicine, Faculty of Health Sciences, University of Linköping, Linköping, Sweden; c Institute for Surgical Research, Ludwig Maximilians University, Munich, Germany
                25578 J Vasc Res 1998;35:150–155
                © 1998 S. Karger AG, Basel

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                Page count
                Figures: 3, References: 41, Pages: 6
                Research Paper

                General medicine,Neurology,Cardiovascular Medicine,Internal medicine,Nephrology
                Endothelium,Phospholipase A2 ,Veins,Smooth muscle cells,Lipoxygenase,Arteries,Cyclooxygenase


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