A.L. García-Otín

Human Apolipoprotein A-IV Reduces Secretion of Proinflammatory Cytokines and Atherosclerotic Effects of a Chronic Infection Mimicked by Lipopolysaccharide

Objective—Expression of human apolipoprotein (h-apo) A-IV in apoE-deficient (apoE0) mice (h-apoA-IV/E0) reduces susceptibility to atherosclerosis. Chronic infection mimicked by exposure to lipopolysaccharide (LPS) increases the size of atherosclerosis lesions in apoE0 mice. Thus, we used h-apoA-IV/E0 mice to determine whether h-apoA-IV plays a protective role after LPS administration. Methods and Results—We injected apoE0, h-apoA-IV/E0, and C57Bl/6 (wild-type) mice intraperitoneally with either LPS or phosphate-buffered saline (PBS) every week for 10 weeks. Atherosclerotic lesions were significantly smaller in h-apoA-IV/E0 mice treated with LPS than in their apoE0 counterparts. The titers of IgG2a and IgG2b autoantibodies to oxidized low-density lipoprotein (LDL) were higher in the LPS-group of h-apoA-IV/E0 mice than in apoE0 mice, suggesting that the Th1 response is stronger in the presence of h-apoA-IV. Lymphocytes from the blood, liver, spleen, and thymus of h-apoA-IV/E0 mice treated with LPS produced less IL-4, INF-&ggr;, and TNF-&agr; proinflammatory cytokines than their apoE0 counterparts. Furthermore, we demonstrated that recombinant h-apoA-IV blocks the LPS-induced stimulation of monocytes. Conclusions—The expression of h-apoA-IV in apoE0 mice reduces the susceptibility to atherogenesis and decreases the secretion of proinflammatory cytokines after LPS administration.

Allelic polymorphism −491A/T in apo E gene modulates the lipid‐lowering response in combined hyperlipidemia treatment

Background Combined hyperlipidemia (CHL) is one of the dyslipidemias more frequently found in clinical practice, and lipid‐lowering drugs are often necessary in its management. Some genetic loci have been associated with CHL expression, and some studies have shown modulation of drugs efficiency in the treatment of dyslipidemias by genetic polymorphisms. We have investigated whether common polymorphisms and mutations in the apolipoprotein (apo) E, lipoprotein lipase (LPL), and apo CIII genes influence atorvastatin or bezafibrate responses in patients with CHL.

Expression and purification of recombinant apolipoprotein A-I Zaragoza (L144R) and formation of reconstituted HDL particles.

Apolipoprotein A-I Zaragoza (L144R) (apo A-I Z), has been associated with severe hypoalphalipoproteinemia and an enhanced effect of high density lipoprotein (HDL) reverse cholesterol transport. In order to perform further studies with this protein we have optimized an expression and purification method of recombinant wild-type apo A-I and apo A-I Z and produced mimetic HDL particles with each protein. An pET-45 expression system was used to produce N-terminal His-tagged apo A-I, wild-type or mutant, in Escherichia coli BL21 (DE3) which was subsequently purified by affinity chromatography in non-denaturing conditions. HDL particles were generated via a modified sodium cholate method. Expression and purification of both proteins was verified by SDS-PAGE, MALDI-TOF MS and immunochemical procedures. Yield was 30mg of purified protein (94% purity) per liter of culture. The reconstituted HDL particles checked via non-denaturing PAGE showed high homogeneity in their size when reconstituted both with wild-type apo A-I and apo A-I Z. An optimized system for the expression and purification of wild-type apo A-I and apo A-I Z with high yield and purity grade has been achieved, in addition to their use in reconstituted HDL particles, as a basis for further studies.

Análisis de las regiones reguladoras de la expresión de apo A-I en sujetos con hipoalfalipoproteinemia

Introduccion/Objetivos La concentracion de apolipoproteina (apo) A-I esta directamente relacionada con la concentracion de colesterol ligado a HDL (cHDL) e inversamente relacionada con el riesgo de padecer enfermedad coronaria. El gen de apo A-I forma parte de la agrupacion genica A-I/C-III/A-IV, localizada en el cromosoma 11. La expresion de la proteina se encuentra regulada por una region promotora y una region exaltadora, situadas en 5’ de los genes de apo A-I y apo C-III, respectivamente. Se han descrito varios sitios polimorficos, que podrian afectar la expresion de la proteina: g.(–78)G>A, en 5’ del gen de apo A-I, g.(–636)C>A, g.(–625)G>A, g.(–620)T>del, g.(–479)C>T y g.(–452)T>C, en 5’ del gen de apo C-III; SstI, en 3’ de apo C-III Metodos Hemos analizado el genotipo de los polimorfismos y la presencia de nuevas mutaciones en las regiones reguladoras del gen de apo A-I en un grupo de 66 sujetos espanoles no relacionados con hipoalfalipoproteinemia (HALP) (cHDL Resultados Las frecuencias alelicas observadas para los polimorfismos fueron similares a las descritas en sujetos normolipemicos. Solamente se observo una asociacion significativa entre el alelo menor del polimorfismo g.(–452)T>C con concentraciones bajas de cLDL (p Conclusiones Nuestros resultados sugieren que la variabilidad genetica del promotor y exaltador del gen de apo A-I no justifica un porcentaje significativo de casos de HALP en poblacion espanola