| Abstract |
An inverse relationship between the levels of high density lipoprotein (HDL)-
cholesterol and the risk of developing cardiovascular disease has been well
established. As the major protein component of HDL, apolipoprotein A-I (apoA-I)
possesses a critical role in biogenesis, structure and function of this lipoprotein. The
atheroprotective properties of apoA-I are mediated through its key-interactions with
other factors participating in HDL metabolism.
Recent studies have demonstrated that myeloperoxidase (MPO)-dependent
oxidation of apoA-I can convert the cardioprotective HDL into dysfunctional forms
through targeting of specific methionine and tyrosine residues of apoA-I, such as
Met148 and Tyr192. In order to investigate the mechanisms resulting in MPOmediated
impediment of normal apoA-I function, the Met148Ala and Tyr192Ala
mutations in the apoA-I gene were generated using the overlapping PCR method. The
apoA-I(Met148Ala) and apoA-I(Tyr192Ala) sequences were subsequently cloned into
the appropriate shuttle vector and the respective recombinant adenoviruses were
generated using the AdEasy method. The properties of each of the Ad-GFP-apoA-I
mutants will be studied both in vitro and in vivo through adenovirus-mediated gene
transfer in apoA-I knockout mice. Finally, co-infection of the mice with adenoviruses
expressing either of the two mutants and human MPO will also be performed to assess
the in vivo effect of MPO on plasma lipids, size and shape of HDL.
The present study also addressed another aspect of the role of apoA-I in HDL
biogenesis and function; the effect of two naturally occurring apoA-I mutations,
apoA-I(Leu141Arg)Pisa and apoA-I(Leu159Arg)Finland, on HDL metabolism.
Heterozygous subjects for either mutation exhibit very low plasma HDL-cholesterol
levels attributed to apoA-I’s reduced capacity of LCAT activation. Previous studies in
our laboratory using adenovirus-mediated gene transfer in apoA-I deficient mice have
demonstrated that both mutations fail to form discoidal or spherical HDL particles and
that treatment with LCAT can restore the aberrant HDL phenotype present in these
cases. In order to further study the properties of these structural mutations in apoA-I
in vivo, transgenic mice carrying these naturally occurring variants of human apoA-I,
apoA-I(Leu141Arg)Pisa and apoA-I(Leu159Arg)Finland, were generated. In this context,
wild-type hapoA-I, hapoA-I(Leu141Arg) and hapoA-I(Leu159Arg) were subcloned
into the pBluescript-TTR1 vector, downstream of the TTR1 promoter which was used
to drive the liver-specific expression of the transgenes. Following preparation of the
TTR1-apoA-I injection fragments and transgenesis procedures, the founders for each
line were identified by genotyping using both PCR and Southern Blot. Next, one
founder of each line will be selected according to the expression levels of each
transgene in a way that all three founders will exhibit similar protein levels of human
apoA-I. The selected founders will be subsequently crossed with apoA-I -/- mice in
order to transfer the transgenic lines in an apoA-I deficient background. Finally, these
mice will be used for studying the effect of these mutations on the interactions
between apoA-I and other factors involved in key-steps of HDL metabolism.
Moreover, the contribution of these mutants in the molecular mechanisms affecting
HDL biogenesis and lipoprotein homeostasis in the plasma will also be evaluated.
Overall, the generation of hapoA-I(Leu141Arg)Pisa and hapoA-I(Leu159Arg)Finland
transgenic mice will provide long-term animal models that will facilitate in-depth
investigation of their abnormal phenotype and could possibly uncover the etiology of
genetically determined low levels of HDL, offering a new perspective in diagnosis,
prognosis or even therapy.
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Δημιουργία αδενοϊών και διαγονιδιακών ποντικιών που φέρουν μεταλλαγμένες μορφές της απολιποπρωτεϊνης Α-Ι για τη μελέτη των επιπτώσεών τους στο μεταβολισμό της HDL in vivo
