Cell growth and cholesterol metabolism in human glucose-6-phosphate dehydrogenase deficient lymphomononuclear cells.

Atherosclerosis is an inflammatory-fibroproliferative response of the arterial wall involving a complex set of interconnected events where cell proliferation (lymphomonocytes, and endothelial and smooth-muscle cells) and substantial perturbations of intracellular cholesterol metabolism are considered to be among the main features. Glucose-6-phosphate dehydrogenase (G6PD), the key enzyme of the hexose-monophosphate shunt pathway, is an essential enzyme involved in both cell growth and cholesterol metabolism, raising the question as to whether G6PD deficiency may have metabolic and growth implications in a deficient population. In the present study, we investigated cell growth and cholesterol metabolism in peripheral blood lymphomononuclear cells (PBMC) from G6PD-normal (n = 5) and -deficient (n = 5) subjects stimulated with lectins (phytohaemoagglutinin and Concanavalin A). G6PD activity, DNA ([3H]-thymidine incorporation) cholesterol synthesis and esterification ([14C]-acetate and [14C]-oleate incorporation), and G6PD, HMGCoA reductase and low density lipoprotein (LDL) receptor mRNA levels (RT-PCR) all increased following lectin stimulation in both normal and G6PD-deficient cells. However, these parameters were significantly lower in G6PD-deficient cells (P < 0.05). It is of interest that G6PD-deficient PBMC, which showed lower expression of G6PD and higher expression of the LDL receptor gene than normal PBMC under basal conditions, exhibited an opposite pattern after stimulation: G6PD and HMGCoA reductase being expressed at significantly higher levels in deficient than in normal cells (P < 0.05). We conclude that the reduced capability of G6PD-deficient cells to respond to mitogenic stimuli and to synthesize cholesterol esters may represent favourable conditions for reducing the risk of cardiovascular diseases.

The in-vitro influence of serum amyloid A isoforms on enzymes that regulate the balance between esterified and un-esterified cholesterol.

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.

Opposite pattern of MDR1 and caveolin-1 gene expression in human atherosclerotic lesions and proliferating human smooth muscle cells.

Cholesterol esterification and smooth muscle cell (SMC) proliferation are the crucial events in the development of atherosclerotic lesions. The objective of this study was to analyse cholesterol esterification and the expression of MDR1 (multidrug resistance), ACAT (acyl-CoA:cholesterol acyltransferase) and caveolin-1 genes in atherosclerotic and healthy vascular walls, in SMCs obtained from atherosclerotic lesions and saphenous veins. Results demonstrated higher levels of cholesterol esters, ACAT and MDR1 mRNAs and lower levels of caveolin-1 mRNA in atherosclerotic segments compared to adjacent serial sections of the same artery and the corresponding non-atherosclerotic arteries from cadaveric donors. SMCs isolated from atherosclerotic plaques manifested an increased capacity to esterify cholesterol and to grow at a faster rate than SMCs isolated from saphenous veins. In addition, when SMCs from atherosclerotic plaques were cultured in the presence of progesterone, a potent inhibitor of cholesterol esterification, significant growth suppression was observed. An increase in ACAT and MDR1 expression and a concomitant decrease in caveolin-1 expression were also observed in SMCs isolated from atherosclerotic arteries as early as 12 h after serum stimulation. An opposite pattern was found when SMCs were treated with progesterone. These findings support the idea that cholesterol esterification plays a role both in early atherogenesis and in clinical progression of advanced lesions and raise the possibility that the cholesterol ester pathway might directly modulate the proliferation of SMCs.

Lipid metabolism and molecular changes in normal and atherosclerotic vessels.

OBJECTIVES: a positive correlation between cholesterol esterification, acyl-CoA:cholesterol acyltransferase (ACAT), multidrug resistance (MDR1) gene expression and atherosclerotic lesions has been shown in human arteries. The objective of this study was to map the expression of MDR1, ACAT genes and the cholesteryl ester content in normal, atherosclerotic and varicose human vessels. MATERIALS: vascular segments were obtained from seven cadaveric donors, 27 patients undergoing vascular surgery for severe atherosclerotic disease and 11 patients with saphenous vein varicosities. METHODS: lipid analysis and RT-PCR of MDR1 and ACAT mRNAs were performed. RESULTS: an increase in cholesteryl ester content and in ACAT and MDR1 expression was demonstrated in relation to the age in the arteries prone to atherosclerosis; this expression was maximal in arteries from symptomatic patients. In resistant arteries and in veins cholesteryl ester accumulation was rare and light, while ACAT and MDR1 expression was not related to the age of the subjects. CONCLUSIONS: the results showed that an increase in MDR1 and ACAT expression may be responsible for the accumulation of cholesteryl esters as well as for cell growth rate acceleration in vessel sites prone to atherosclerosis.

G6PD activity and gene expression in leukemic cells from G6PD-deficient subjects.

In the present study we examined gene expression and glucose-6-phosphate dehydrogenase (G6PD) activity in leukemic cells isolated from G6PD normal and deficient subjects. The results have shown that G6PD activity strongly increases in G6PD normal leukemic cells as well as in G6PD deficient leukemic cells when compared to peripheral blood mononuclear cells (PBMC). Higher levels of G6PD gene expression were observed in leukemic cells from G6PD deficient patients compared to G6PD normal. A similar pattern of gene expression was also observed for 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase. These results support the hypothesis that G6PD deficient cell, in order to sustain their growth, must respond to the low activity of their mutant enzyme with an increase in quantity through an induction of gene expression.

Correlation between cholesterol esterification, MDR1 gene expression and rate of cell proliferation in CEM and MOLT4 cell lines.

A positive correlation between cholesterol esterification and growth rate potential was previously found in our laboratory during the growth of CEM and MOLT4 lymphoblastic cells. In the current study, we investigated whether the rates of cholesterol esters synthesis correlate with changes of acyl-CoAcholesterol acyltransferase (ACAT) mRNA levels and of other genes implied in cholesterol biosynthesis and uptake, such as 3-hydroxy-3-methylglutaryl-CoA (HMGCoA) reductase and low density lipoprotein (LDL) receptor. The results showed that the more rapid growing CEM cells had lower levels of expression of HMGCoA-reductase and LDL receptors compared to MOLT4. By contrast, ACAT mRNA levels were higher in CEM cells, further supporting the concept of a possible involvement of cholesterol esters in the regulation of cell growth and division. In this study, high levels of cholesterol esterification and of expression of ACAT gene were also associated with a markedly increased expression of multidrug resistance (MDR1) gene, suggesting that MDR1 activity might contribute to regulate the rate of cell growth and division by modulating intracellular cholesterol ester levels.

Role of cholesterol synthesis and esterification in the growth of CEM and MOLT4 lymphoblastic cells.

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.