Angiogenesis and angioregression gene expression analyses in swine corpus luteum
Creators: name: De Andrea Ribeiro, Luciana <1971>
Publisher: name: Alma Mater Studiorum - Università di Bologna
Year: 2007
Subjects: VET/02 Fisiologia veterinaria

description: The corpus luteum (CL) lifespan is characterized by a rapid growth,
differentiation and controlled regression of the luteal tissue, accompanied by
an intense angiogenesis and angioregression. Indeed, the CL is one of the
most highly vascularised tissue in the body with a proliferation rate of the
endothelial cells 4- to 20-fold more intense than in some of the most
malignant human tumours. This angiogenic process should be rigorously
controlled to allow the repeated opportunities of fertilization. After a first
period of rapid growth, the tissue becomes stably organized and prepares
itself to switch to the phenotype required for its next apoptotic regression. In
pregnant swine, the lifespan of the CLs must be extended to support
embryonic and foetal development and vascularisation is necessary for the
maintenance of luteal function. Among the molecules involved in the
angiogenesis, Vascular Endothelial Growth Factor (VEGF) is the main
regulator, promoting endothelial cells proliferation, differentiation and survival
as well as vascular permeability and vessel lumen formation. During vascular
invasion and apoptosis process, the remodelling of the extracellular matrix is
essential for the correct evolution of the CL, particularly by the action of
specific class of proteolytic enzymes known as matrix metalloproteinases
(MMPs). Another important factor that plays a role in the processes of
angiogenesis and angioregression during the CL formation and luteolysis is the isopeptide Endothelin-1 (ET-1), which is well-known to be a potent
vasoconstrictor and mitogen for endothelial cells. The goal of the present
thesis was to study the role and regulation of vascularisation in an adult
vascular bed. For this purpose, using a precisely controlled in vivo model of
swine CL development and regression, we determined the levels of
expression of the members of VEGF system (VEGF total and specific
isoforms; VEGF receptor-1, VEGFR-1; VEGF receptor-2, VEGFR-2) and ET-
1 system (ET-1; endothelin converting enzyme-1, ECE-1; endothelin receptor
type A, ET-A) as well as the activity of the Ca++/Mg++-dependent
endonucleases and gelatinases (MMP-2 and MMP-9). Three experiments
were conducted to reach such objectives in CLs isolated from ovaries of
cyclic, pregnant or fasted gilts.
In the Experiment I, we evaluated the influence of acute fasting on VEGF
production and VEGF, VEGFR-2, ET-1, ECE-1 and ET-A mRNA expressions
in CLs collected on day 6 after ovulation (midluteal phase). The results
indicated a down-regulation of VEGF, VEGFR-2, ET-1 and ECE-1 mRNA
expression, although no change was observed for VEGF protein.
Furthermore, we observed that fasting stimulated steroidogenesis by luteal
cells. On the basis of the main effects of VEGF (stimulation of vessel growth
and endothelial permeability) and ET-1 (stimulation of endothelial cell
proliferation and vasoconstriction, as well as VEGF stimulation), we
concluded that feed restriction possibly inhibited luteal vessel development.
This could be, at least in part, compensated by a decrease of vasal tone due
to a diminution of ET-1, thus ensuring an adequate blood flow and the
production of steroids by the luteal cells.
In the Experiment II, we investigated the relationship between VEGF,
gelatinases and Ca++/Mg++-dependent endonucleases activities with the
functional CL stage throughout the oestrous cycle and at pregnancy. The
results demonstrated differential patterns of expression of those molecules in
correspondence to the different phases of the oestrous cycle. Immediately
after ovulation, VEGF mRNA/protein levels and MMP-9 activity are maximal.
On days 5–14 after ovulation, VEGF expression and MMP-2 and -9 activities
are at basal levels, while Ca++/Mg++-dependent endonuclease levels
increased significantly in relation to day 1. Only at luteolysis (day 17),
Ca++/Mg++-dependent endonuclease and MMP-2 spontaneous activity
increased significantly. At pregnancy, high levels of MMP-9 and VEGF were
observed. These results suggested that during the very early luteal phase,
high MMPs activities coupled with high VEGF levels drive the tissue to an
angiogenic phenotype, allowing CL growth under LH (Luteinising Hormone)
stimulus, while during the late luteal phase, low VEGF and elevate MMPs
levels may play a role in the apoptotic tissue and extracellular matrix
remodelling during structural luteolysis.
In the Experiment III, we described the expression patterns of all distinct
VEGF isoforms throughout the oestrous cycle. Furthermore, the mRNA expression and protein levels of both VEGF receptors were also evaluated.
Four novel VEGF isoforms (VEGF144, VEGF147, VEGF182, and
VEGF164b) were found for the first time in swine and the seven identified
isoforms presented four different patterns of expression. All isoforms showed
their highest mRNA levels in newly formed CLs (day 1), followed by a
decrease during mid-late luteal phase (days 10–17), except for VEGF182,
VEGF188 and VEGF144 that showed a differential regulation during late
luteal phase (day 14) or at luteolysis (day 17). VEGF protein levels paralleled
the most expressed and secreted VEGF120 and VEGF164 isoforms. The
VEGF receptors mRNAs showed a different pattern of expression in relation
to their ligands, increasing between day 1 and 3 and gradually decreasing
during the mid-late luteal phase. The differential regulation of some VEGF
isoforms principally during the late luteal phase and luteolysis suggested a
specific role of VEGF during tissue remodelling process that occurs either for
CL maintenance in case of pregnancy or for noncapillary vessel development
essential for tissue removal during structural luteolysis.
In summary, our findings allow us to determine relationships among factors
involved in the angiogenesis and angioregression mechanisms that take
place during the formation and regression of the CL. Thus, CL provides a
very interesting model for studying such factors in different fields of the basic
research.