Characterization of novel angiotensin IV analogues as tools to elucidate the regulation of the insulin-regulated aminopeptidase in macrophages

Scriptie/masterproef: Doctoral Thesis

Uittreksel

The renin-angiotensin system (RAS) constitutes the principal hormonal system that regulates
blood pressure and sodium balance. The RAS component angiotensin IV (Ang IV), the 3-8
fragment of the vasoactive hormone Ang II, is rather studied for its effects on memory
enhancement and protection against ischemic stroke. The high-affinity binding site for Ang
IV is the insulin-regulated aminopeptidase (IRAP, EC 3.4.11.3). Studying the mechanism of
Ang IV is however complicated by its instability and its low selectivity, implying the need for
stable and highly selective Ang IV-analogues.
To fulfill this need, novel Ang IV-analogues bearing local conformational constraints were
developed by Prof. Dirk Tourwé and coworkers (ORGC, VUB). We screened these compounds
for high affinity binding to IRAP, high selectivity compared to the Ang II receptor AT1R and
resistance against proteases. The selected Ang IV-analogue IVDE77 possesses substantial
advantages compared to Ang IV: (i) it has a 40 times higher affinity for IRAP (Ki 1.71 nM), (ii)
it does not activate AT1R, and (iii) it is resistant to proteolysis, even in human plasma.
Macrophages are tissue-resident white blood cells that regulate and actively participate in
inflammation. These cells typically express high levels of proteases and peptidases, and are
even able to generate angiotensins locally by expressing RAS components such as the
angiotensin-converting enzyme (ACE). No such data however exists about the presence of
IRAP in macrophages.
[3H]IVDE77 binding experiments clearly demonstrated IRAP in macrophages. This prompted
us to investigate the regulation and the function of (Ang IV binding to) IRAP in macrophages.
IRAP expression was increased by interferon-g (IFN-g) and lipopolysaccharide (LPS), but not
by anti-inflammatory cytokines. IFN-g augmented [³H]IVDE77 binding steadily over time,
while LPS quickly and transiently recruited IRAP to the cell surface. Latex particles also
induced a transient recruitment of IRAP to the cell surface, but no difference was observed
in phagocytic uptake between wild-type and IRAP-/- macrophages, suggesting that the
enzymatic activity of IRAP is not required for the ingestion of particles.
In summary, [3H]IVDE77 is a promising research tool for studying the regulation and
translocation of IRAP, which can contribute to elucidating the function(s) of IRAP in an
immunological context and beyond.
Datum Prijs7 aug 2013
TaalEnglish
BegeleiderPatrick Vanderheyden (Promotor), Jo Van Ginderachter (Co-promotor) & Steven Ballet (Jury)

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