The intracellular balance between un-esterified and esterified cholesterol is regulated by two enzyme activities, cholesterol ester hydrolases, which drive the balance in favor of un-esterified cholesterol, and acyl-CoA:cholesterol acyl transferase (ACAT) which acts in the opposite direction. During acute inflammation apo-serum amyloid A (apoSAA) isoforms 1.1 and 2.1 become major constituents of high density lipoprotein and this complex is internalized by macrophages. Mixtures of the two isoforms have been shown to enhance cholesterol esterase activity. Using a purified form of the pancreatic enzyme we have explored the mechanism by which apoSAA may accomplish this stimulation. The pancreatic esterase cleaves cholesteryl-oleate with a Km of 0.255 mM, releasing both cholesterol and oleate. Cholesterol exhibits a product inhibition which is relieved by isoform 2.1 but not 1.1 nor apolipoprotein A-I. The NH2-terminal 16 residues of isoform 2.1 had no effect on the esterase, but the 80 residue peptide constituting its COOH-terminus possessed the stimulatory property. Purified isoforms 1.1, 2.1, 2.2, apolipoprotein A-I, the NH2-terminal 16 residues and COOH-terminal 80 residues of isoform 2.1 were also examined for their effects on macrophage ACAT activity. Isoforms 2.1 and 2.2 produced dose dependent inhibitions of up to 50%, (p<0.001). Isoform 1.1, and apoA-I had no effect on ACAT activity. The NH2-terminal 16 residue peptide of isoform 2.1 reduced the ACAT activity in a dose dependent manner by 74% (p<0.001), whereas the COOH-terminal 80 residues, in contrast to its enhancing effect on the esterase, had no inhibitory effect on ACAT. Such complementary but opposite effects of isoform 2.1 on ACAT and the esterase are consistent with a role for this protein in shifting the balance between unesterified (transportable) and esterified (storage) forms of cholesterol in favor of the latter. They suggest that apoSAA2.1 may mediate cholesterol mobilization at sites of tissue injury.

Cholesterol esterification by acyl-CoA:cholesterol acyltransferase (ACAT) and proliferation of vascular smooth muscle cells (VSMC) are key events in vascular proliferative diseases. Here we performed experiments to ascertain the role of cholesterol ester pathway in the control of human aortic VSMC cycle progression. Results showed that serum-induced VSMC proliferation was preceded by an increased ability of the cells to esterify cholesterol as well as by an increased expression of ACAT and multidrug resistance (MDR1) mRNAs and extracellular related kinases 1/2 (ERK1/2), whereas caveolin-1 levels were markedly decreased. Cell cycle analyses performed in the presence of two inhibitors of cholesterol esterification, directly inhibiting ACAT (Sandoz 58-035) or the transport of cholesterol substrate from plasma membrane to endoplasmic reticulum (progesterone), indicate that each inhibitor suppressed the serum-induced DNA synthesis by accumulation of VSMCs in the G1 phase. The effect was associated with a rapid inhibition of ERK1/2 mitogenic signaling pathway; a down-regulation of cyclin D1, ACAT, and MDR1 mRNA; and an up-regulation of caveolin-1. These data provide a plausible link between cholesterol esterification and control of cell cycle G1/S transition, supporting the hypothesis that cholesterol esterification may accelerate the progression of human vascular proliferative diseases by modulating the rate of the VSMC proliferation.

CEM and MOLT4 are human T-cell lines isolated from patients with acute cell leukaemia. In culture they show important differences in cholesterol metabolism, CEM being less efficient at synthesizing cholesterol and having a lower activity of 3-hydroxy-3-methylglutaryl-CoA (HMGCoA) reductase. To investigate further the relationship between regulation of intracellular cholesterol metabolism at various steps and rate of cell growth, cholesterol synthesis, esterification and efflux were evaluated in CEM and MOLT4 cells at different times during exponential and stationary growth in vitro. It was shown that, although CEM cells have a lower rate of cholesterol synthesis, they grow at a faster rate than MOLT4 cells. However, CEM cells exhibit an increased capacity to esterify cholesterol associated with a decreased efflux of newly synthesized cholesterol into the medium. These results provide evidence for an association between the capability to synthesize and retain cell cholesterol esters and the growth rate potential.