Lipoprotein-X stimulates monocyte chemoattractant protein-1 expression in mesangial cells via nuclear factor-kappa B.

BACKGROUND: Lipoprotein-X (Lp-X) is an abnormal lipoprotein found in the plasma of patients with familial lecithin:cholesterol acyltransferase (LCAT) deficiency. The majority of patients with this disorder develop progressive glomerulosclerosis. One key event in the pathogenesis of glomerulosclerosis is the infiltration of monocytes into affected glomeruli. Mesangial cells can synthesize and secrete monocyte chemoattractant protein-1 (MCP-1), an important chemoattractant for monocytes. The objective of the present study was to examine the effect of Lp-X on MCP-1 expression in mesangial cells leading to an enhanced monocyte chemotaxis and to elucidate the mechanisms involved in this process. METHODS: Lp-X was isolated from the plasma of a patient with familial LCAT deficiency. After rat mesangial cells were incubated with Lp-X for four or six hours, the expression of MCP-1 mRNA was determined by nuclease protection assay, and MCP-1 protein was measured by Western immunoblotting analysis. Monocyte chemotaxis was determined by using a Micro Chemotaxis Chamber. RESULTS: Lp-X (50 to 100 nmol/mL) stimulated mesangial cell MCP-1 mRNA expression (137 to 220%) and MCP-1 protein levels (233 to 375%). Conditioned media collected from Lp-X-treated mesangial cells stimulated human acute monocytic leukemia (THP-1) monocyte chemotaxis (165 to 200%). The increase in MCP-1 expression in mesangial cells was associated with an elevation of intracellular diacylglycerol levels, and activation of protein kinase C (PKC) as well as nuclear factor-kappa B (NF-kappa B). CONCLUSION: These results suggest that Lp-X participates in the pathogenesis of glomerulosclerosis and subsequent renal failure in familial LCAT deficient patients by stimulating monocyte infiltration via a mechanism involving mesangial MCP-1 expression.

Role of lecithin:cholesterol acyltransferase and apolipoprotein A-I in cholesterol esterification in lipoprotein-X in vitro.

Lipoprotein-X (Lp-X) is an abnormal particle present in the plasma of patients with familial lecithin:cholesterol acyltransferase (LCAT) deficiency syndromes or cholestatic liver disease. Compared to other lipoproteins, Lp-X contains a high content of unesterified cholesterol (30%, w/w) to phosphatidylcholine (60%, w/w). The objective of this study was to evaluate the role of LCAT and apolipoprotein A-I (apoA-I) in Lp-X metabolism in vitro and to elucidate the regulation of cholesterol esterification in this unique lipoprotein. Lp-X isolated from sera of patients with obstructive jaundice had a high content of unesterified cholesterol and phosphatidylcholine and contained apolipoprotein E, apoCs, and albumin. Although human recombinant LCAT used as an enzyme source did bind to isolated Lp-X, no cholesterol esterification was detected. However, addition of human apoA-I in the presence of albumin resulted in significant cholesterol esterification in Lp-X (Vmax 0.25 +/- 0.04 nmol/h per microgram LCAT protein). Exogenous apoA-I did not change the size of Lp-X particle as determined by quasi-elastic light scattering analysis. A reduction in Lp-X size was observed when both apoA-I and LCAT were included in the reaction mixture (from 47 nm to 42 nm). Furthermore, addition of apoA-I (but not HDL) dramatically changed the electrophoretic mobility of Lp-X from cathodic to anodic migration. Such changes are not due to displacement of apoC or apoE proteins from Lp-X by apoA-I. While increasing apoA-I concentration (up to 35 micrograms/ml) in the reaction mixture stimulated cholesterol esterification in Lp-X, addition of apoA-I at the concentration of 8 micrograms/ml inhibited cholesterol esterification in VLDL, LDL, and HDL by more than 50%. Albumin was required for the LCAT reaction to Lp-X. Our results suggest that while LCAT binds to isolated Lp-X, apoA-I is needed for the LCAT reaction to proceed. The presence of apoA-I does not result in the displacement of apoCs and apoE from Lp-X and addition of apoA-I changes the electrophoretic mobility but not the size of Lp-X.

Purification of an alkaline phosphatase-lipoprotein-X complex.

An alkaline phosphatase isoenzyme was observed in an abnormal position in the agar-agarose gel pattern of sera from several patients suffering from intrahepatic and extrahepatic cholestasis. We purified this isoenzyme, by gel filtration and affinity chromatography, from the serum of a patient suffering from extrahepatic cholestasis. Analysis demonstrated an alkaline phosphatase-lipoprotein-X complex with a relative molecular mass of at least 669,000. We discuss the interpretation of alkaline phosphatase isoenzyme patterns produced by different techniques.

Lipoprotein-X.

Lipoprotein-X is an abnormal lipoprotein that appears in the sera of patients with obstructive jaundice, and thus is a sensitive indicator of cholestasis. In patients with familial plasma lecithin, Cholesterol acyltransferase (LCAT) deficiency, there is an inverse relationship between plasma Lp-X levels and LCAT activity. Ultracentrifugation procedures utilized for isolation of Lp-X have shown that it is associated with the low density lipoprotein fraction. Lp-X can be visualized by electrophoresis on either Agar or Agarose. The purity of Lp-X preparations has been documented by immunochemical procedures. The availability of highly purified antisera to Lp-X has served as a basis of one of the assay procedures for this lipoprotein. It's chemical composition has been established. Phospholipids and unesterified cholesterol constitute the bulk of the Lp-X molecule. Electron microscopic studies have demonstrated that Lp-X is a spherical particle which has strong aggregating properties. Membrane bound enzymes have been shown to aggregate with Lp-X. The fact that bile lipoprotein can be converted to Lp-X by the addition of albumin and that Lp-X can be converted to bile lipoprotein by the addition of bile salts offers a possible explanation for the origins of Lp-X. Phospholipases of plasma might play a role in the catabolism of Lp-X. The value and limitations of Lp-X determinations will also be addressed in this review.