Didier Rouy

Apolipoprotein(a) Yeast Artificial Chromosome Transgenic Rabbits LIPOPROTEIN(a) ASSEMBLY WITH HUMAN AND RABBIT APOLIPOPROTEIN B

The in vivo analysis of lipoprotein(a) (Lp(a)), an independent atherosclerosis risk factor in humans, has been limited in part by its restricted distribution among mammals. Although transgenic mice have been created containing Lp(a), the relatively small size of the mouse has precluded some studies. To examine the properties of this molecule in a significantly larger mammal, we have used a 270-kilobase yeast artificial chromosome clone containing the human apolipoprotein(a) (apo(a)) gene and a 90-kilobase P1 phagemid clone containing the human apolipoprotein B (apoB) gene to create transgenic rabbits that express either or both transgenes. Expression of both transgenes was tissue specific and localized predominantly to the liver. Average apolipoprotein plasma levels in the rabbits were 2.5 mg/dl for apo(a) and 17.6 mg/dl for human apoB. In contrast to observations in apo(a) transgenic mice, we found that apo(a) plasma levels in the rabbits were stable throughout sexual maturity. Also, apo(a) formed a covalent association with the endogenous rabbit apoB albeit with a lower efficiency than its association with human apoB. The analysis of Lp(a) transgenic rabbits has provided new insights into apo(a) expression and Lp(a) assembly. In addition, these transgenic rabbits potentially will provide an improved experimental model for the in vivo analysis of Lp(a) and its role in promoting atherosclerosis and restenosis.

HDL3 binds to glycosylphosphatidylinositol-anchored proteins to activate signalling pathways

Previous studies have indicated that in HepG2 cells HDL3-signalling involves glycosylphosphatidylinositol (GPI) anchored proteins. HDL3-binding to HepG2 cells was found to be enhanced by cellular preincubation with PI-PLC inhibitors and sensitive to a cellular preincubation with exogenous PI-PLC, suggesting that HDL3 binds directly on GPI-anchored proteins to initiate signaling. Moreover HDL3-binding was found to be partly inhibited by antibodies against the HDL-binding protein (AbHBP). HDL3, when binding to HepG2 cells, promoted the release in the culture medium of a 110 kDa protein that binds AbHBP, while a cellular preincubation with antibodies against the inositol-phosphoglycan (IPG) moiety of GPI-anchor (AbIPG), used to block lipolytic cleavage of the GPI-anchor, inhibits HDL3-induced release of the 110 kDa protein in the culture medium. In [3H]-PC prelabeled HepG2 cells, AbHBP were found to stimulate PC-hydrolysis and DAG generation within 5 min as did HDL3 stimulation. Cellular preincubation with AbIPG was found to inhibit only the HDL3-signal and not the AbHBP-signal, while a prior cellular pretreatment with PI-PLC from Bacillus cereus was found to inhibit the HDL3-and AbHBP-signal. Moreover cellular preincubation with AbHBP for 1 h at 37 degrees C was found to inhibit HDL3-signalling pathways. Our results suggest that in HepG2 cells a 110 kDa protein, which could be HBP, can be anchored to the membrane via GPI, and can function in HDL3-signalling pathways as binding sites.