A series of trans-tetrahydro-4-hydroxy-6-[2-(2,3,4,5-substituted-1H-pyrrol-1-yl) ethyl]-2H-pyran-2-ones and their dihydroxy acids were prepared and tested for their ability to inhibit the enzyme HMG-CoA reductase in vitro. Inhibitory potency was found to increase substantially when substituents were introduced into positions three and four of the pyrrole ring. A systematic exploration of structure-activity relationships at these two positions led to the identification of a compound ((+)-33,(+)-(4R)-trans-2-(4-fluororphenyl)-5-(1-methylethyl)-N,3- diphenyl-1- [(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)ethyl]-1H-pyrrole-4- carboxamide) with five times the inhibitory potency of the fungal metabolite compactin.

In the present studies, we examined the effect of flavonoids on the endothelial cell expression of adhesion molecules, an early step in inflammation and atherogenesis. Addition of tumor necrosis factor-alpha (TNF) to human aortic endothelial cells (HAECs) led to the induction of vascular cell adhesion molecule-1 (VCAM-1) expression and enhancement in expression of intercellular adhesion molecule-1 (ICAM-1). A flavonoid, 2-(3-amino-phenyl)-8-methoxy-chromene-4-one (PD 098063), markedly inhibited TNF-induced VCAM-1 cell-surface expression in a concentration-dependent fashion with half-maximal inhibition at 19 mumol/L but had no effect on ICAM-1 expression. Another structurally distinct flavonoid, 2-phenyl-chromene-4-one, similarly selectively decreased VCAM-1 expression. The inhibition in cell-surface expression of VCAM-1 by PD 098063 correlated with decreases in steady-state mRNA levels, but there was no effect on ICAM-1 mRNA levels. The decrease in VCAM-1 mRNA levels was not due to changes in mRNA stability but rather resulted from a reduction in the rate of transcription of the gene. However, electrophoretic mobility shift assays using nuclear extracts from TNF-induced HAECs treated with PD 098063 failed to show a decrease in the activation of NF-kappa B, indicating that inhibition of activation of this transcription factor may not be its mode of action. Similarly, PD 098063 did not affect chloramphenicol acetyltransferase reporter gene activity in TNF-inducible minimal VCAM-1 promoter constructs containing two NF-kappa B sites, suggesting that the compound does not affect the transactivation driven by these sites. We conclude that this compound selectively blocks agonist-induced VCAM-1 protein and gene expression in HAECs by NF-kappa B-independent mechanism(s).

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Lipoprotein accumulation in the subendothelial matrix is an important step in atherogenesis. We have previously shown that addition of lipoprotein lipase (LPL) markedly increased binding of apolipoprotein B (apoB)-containing lipoproteins to an endothelial cell-derived matrix, and this enhanced lipoprotein binding was inhibited by apoE. In the present studies we examined the role of various regions of apoB in the binding of LDL to LPL-containing endothelial cell matrix and the ability of various apoE domains to decrease lipoprotein retention. We studied three apoB epitope-specific monoclonal antibodies for their ability to block the binding of 125I-LDL to LPL-containing matrix. Of these, monoclonal antibody 4G3, which recognizes an arginine-containing epitope in apoB, was the most effective in reducing LDL binding. Chemical modification of LDL apoB lysines or arginines markedly reduced the ability of the lipoprotein to block the binding of 125I-LDL to LPL-containing matrix, suggesting that apoB positively charged amino acids are involved in the interaction. Furthermore, polyarginine or polylysine markedly decreased 125I-LDL binding to LPL-containing matrix, whereas polyleucine was ineffective. These data suggest that apoB positively charged regions are important in LDL binding. To explore the role of charge modifications on apoE by single arginine-cysteine interchanges, we examined the effects of the three major human apoE isoforms (apoE2, apoE3, and apoE4). ApoE3 was the most effective in decreasing 125I-LDL retention, followed by apoE4; apoE2 was the least effective. Similarly, apoE2-containing HDL was much less effective than apoE3-containing HDL in decreasing 125I-LDL retention.(ABSTRACT TRUNCATED AT 250 WORDS)