PubMed · 8092152
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L A Trissel. 1994-06-15. Compounding our problems.. https://pubmed.ncbi.nlm.nih.gov/8092152/
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Accumulation of lipids in atherosclerotic plaques causes progressive narrowing of the arterial lumen, often followed by thrombosis and ischemia. Currently several different methods, most requiring disruption of the plaque, are used to study the physical properties of lipids accumulated in plaques, and lipid composition is typically determined by chemical analysis of completely disrupted plaques. In this study, 13C magic angle spinning NMR spectroscopy (MAS NMR) was used to determine in situ the lipid composition and molecular organization of all lipid phases in human carotid artery plaques (ex vivo). Protocols were developed to observe signals from one lipid phase without interference from other phases. In addition, 31P MAS NMR detected calcification in plaques by the signals from inorganic phosphate complexed to calcium. Together, 13C and 31P MAS NMR comprise a powerful nondisruptive approach for determining the quantity and phase state of components in arterial plaques.
Calcium deposition diseases caused by calcium pyrophosphate dihydrate (CPPD) and basic calcium phosphate (BCP) crystals are a significant source of morbidity in the elderly. We have shown previously that both types of crystals can induce mitogenesis, as well as metalloproteinase synthesis and secretion by fibroblasts and chondrocytes. These responses may promote degradation of articular tissues. We have also shown previously that both CPPD and BCP crystals activate expression of the c-fos and c-jun proto-oncogenes. Phosphocitrate (PC) can specifically block mitogenesis and proto-oncogene expression induced by either BCP or CPPD crystals in 3T3 cells and human fibroblasts, suggesting that PC may be an effective therapy for calcium deposition diseases. To understand how PC inhibits BCP and CPPD-mediated cellular effects, we have investigated the mechanism by which BCP and CPPD transduce signals to the nucleus. Here we demonstrate that BCP and CPPD crystals activate a protein kinase signal transduction pathway involving p42 and p44 mitogen-activated protein (MAP) kinases (ERK 2 and ERK 1). BCP and CPPD also cause phosphorylation of a nuclear transcription factor, cyclic AMP response element-binding protein (CREB), on serine 133, a residue essential for CREB's ability to transactivate. Treatment of cells with PC at concentrations of 10(-3) to 10(-5) M blocked both the activation of p42/p44 MAP kinases, and CREB serine 133 phosphorylation, in a dose-dependent fashion. At 10(-3) M, a PC analogue, n-sulfo-2-aminotricarballylate and citrate also modulate this signal transduction pathway. Inhibition by PC is specific for BCP- and CPPD-mediated signaling, since all three compounds had no effect on serum-induced p42/P44 or interleukin-1beta induced p38 MAP kinase activities. Treatment of cells with an inhibitor of MEK1, an upstream activator of MAPKs, significantly inhibited crystal-induced cell proliferation, suggesting that the MAPK pathway is a significant mediator of crystal-induced signals.
Although the control elements which regulate the transcriptional activity of promoter sequences are largely determined by the use of reporter plasmids in transient transfection analyses, controlling variability in these experiments can often be a vexing problem. Problems arise when the promoter of the internal control plasmid, used to correct for transfection efficiency, either affects test promoter strength or is itself regulated by trans-acting factors or inducing agents used to study the test promoter. Here we report the use of beta-galactosidase protein as an unbiased standard of transfection efficiency. beta-Galactosidase protein is readily internalized by adherent cell lines when incorporated into a calcium phosphate precipitate; significant enzyme activity can be recovered up to 72 h after transfection. Use of beta-galactosidase protein as a control obviates the concerns associated with promoter-dependent reporter plasmids as controls.