Key words
permeability - tight junction - adherens junction - corpus luteum - hCG
Schlüsselwörter
Permeabilität - Tight Junction - Adherens Junction - Corpus luteum - hCG
Introduction
The corpus luteum (yellow body) is an intermediary, endocrine-active gland that
alternately undergoes generation and degeneration in the course of the cycle. Hereby
the corpus luteum plays a central role in maintaining pregnancy. The pre-ovulatory
LH peak at first triggers ovulation and then induces the very rapid transformation
of the ruptured follicle into a corpus luteum. This process proceeds mainly under
the control of progesterone that is synthesised by the corpus luteum and is
essential for implantation and the maintenance of pregnancy [1]. If a pregnancy does not occur, the corpus luteum degenerates 14 days
after ovulation into a corpus albicans with transformed connective tissue. However,
if the egg cell is fertilised the persistence of the corpus luteum is ensured both
directly and indirectly by the joint effects of LH and hCG [2], [3], [4], [5], [6], [7]. This observation is supported by the fact that the corpus luteum can
be maintained in the absence of a pregnancy by exogenous administration of hCG [8].
The corpus luteum consists of various types of cells including endothelial cells,
luteinised granulosa cells and luteinised theca cells. The endothelial cells are
responsible for controlling vascular permeability which represents an indispensable
prerequisite for the development of the corpus luteum. The permeability itself is
controlled by the strictly regulated opening and closing of the cell-to-cell
contacts between the endothelial cells [9], [10], [11]. For these reasons,
any impairment of the expression of AJ and TJ can lead to a perturbed endothelial
cell function with consecutive functional consequences such as, e.g., oedemas or
ascites within the framework of an ovarian hyper-stimulation syndrome (OHSS) [12], [13], [14]. In such cases, the iatrogenous administration of hCG
to support the luteal phase is effective; this increases luteal function but also
increases the risk for the occurrence of OHSS [15]. Such
a syndrome is characterised by an increased permeability of the capillaries which
leads to a fluid shift from intravasal to extravasal spaces and thus to ascites
[16]. In the following paragraphs the exact molecular
mechanisms that participate in the regulation of endothelial function and thus the
fluid barriers of the vessels are described. A better understanding of these
mechanisms will hopefully aid the development of novel therapeutic options in the
future and thus the ability to limit tissue damage by influencing vascular
permeability.
Molecular Regulation of Endothelial Permeability
Molecular Regulation of Endothelial Permeability
The paracellular endothelial permeability is controlled by at least two different
types of cell-to-cell contacts, the AJ and the TJ. These are formed from various
transmembrane proteins that promote homophilic cell-to-cell contacts and transmit
intracellular signals [17]. It has been shown several
times that these cell-to-cell contacts are dynamically transformed not only during
embryonic development but also in resting cells [18].
Adhesion molecules such as AJ and TJ thereby form complexes that regulate the
permeability in a zipper-like manner, i.e., the transmissibility of the gaps between
neighbouring, mutually interacting cells [19], [20], [21], [22].
Endothelial cells express tissue-specific transmembrane proteins: the AJ protein VE
(vascular endothelial) cadherin and the TJ protein claudin 5 [9], [23]. Claudin 5-deficient knockout mice
show a normal embryonic development but die shortly after birth due to a defect of
the blood-brain barrier [23]. In comparison,
VE-cadherin-deficient mice suffer from numerous severe lethal defects in the course
of embryonic angiogenesis [24]. This indicates that
adhesion molecules not only have a structural but also a major functional
relevance.
Distribution of Cell Adhesion Proteins in the Human Corpus Luteum
Distribution of Cell Adhesion Proteins in the Human Corpus Luteum
Widely differing cell adhesion molecules can be localised in the corpus luteum. These
include the TJ proteins occludin, claudin 1 and claudin 5 as well as the AJ protein
VE-cadherin. The distribution of these cell-to-cell contacts naturally differs in
the various compartments of the corpus luteum. In the human corpus luteum occludin
can be detected ubiquitously in the membranes of endothelial cells in the vicinity
of granulosa and theca capillaries as well as in the membranes of luteinising
granulosa cells themselves. In contrast, however, occludin is not expressed in the
domain of luteinising theca cells [25]. The detection of
occludin in epithelial and endothelial cells has also been described for other types
of tissue such as, e.g., rat lung, human liver, mouse brain, etc. [26], [27], [28], [29], [30].
Claudin 1 is exclusively expressed in the domain of the membrane of luteinising
granulosa cells. In contrast to occluding, claudin 1 can only be discontinuously
detected and not band-like. This distribution pattern is comparable with that which
can be observed on the surface of human ovarian epithelium [31]. In contrast to the endothelial cells of the brain and salivary
glands, claudin 1 is not expressed in the domain of luteal vessels [32], [33]. Claudin 5, on the
other hand, is exclusively and specifically localised in the endothelium of the
human corpus luteum. Thereby it can be detected above all in the capillaries of the
granulosa compartment and in the larger vessels of the theca [25]. VE-cadherin, on the other hand, can be detected not only in
capillary endothelium of the granulosa but also in that of the theca. There is no
unambiguous explanation for the so widely differing tissue-specific occurrence of
these proteins, but it is apparent that in certain cell compartments a combination
of various AJ and TJ is responsible for the mediation of cell-to-cell communication
and adhesion [25].
Functional Relevance of hCG with Respect to Cell Adhesion Proteins in the Human
Corpus Luteum
Functional Relevance of hCG with Respect to Cell Adhesion Proteins in the Human
Corpus Luteum
The cyclic growth and development of the corpus luteum is regulated by gonadotropins.
During the normal cycle its life span is limited to 14 days under the influence of
LH. In the case of a pregnancy the corpus luteum survives for several months due to
the effect of hCG, this is known as “luteal rescue”. It has been shown for the human
corpus luteum that “luteal rescue” is accompanied by an expansion of the luteal
vessels [34], which presumably involves a re-arrangement
of cell adhesion proteins. Thus, it is of particular interest to examine the effects
of hCG on AJ and TJ in the human corpus luteum.
In the “rescued” human corpus luteum of an in vivo pregnancy simulated by hCG,
claudin 1 and occludin are significantly down-regulated in the luteinising granulosa
cell compartment [25]. In addition, there is a decrease
of occludin, claudin 5 and VE-cadherin in the endothelial cell compartment. It may
be assumed that this hCG-dependent regulation of AJ and TJ proteins supported by the
function of the corpus luteum is of exceptional importance for the maintenance of
early pregnancy. The decrease in the expression of cell adhesion molecules in the
luteinising granulosa cell compartment appears thereby to be a structural
prerequisite for the liberation of steroidogenic molecules such as progesterone or
vascular endothelial growth factor (VEGF) [35]. The
development of the corpus luteum following “luteal rescue” is characterised by a
structural new orientation. The down-regulation of cell adhesion molecules thereby
leads to an increase of the intercellular gaps which, on the one hand, favours the
expansion and invasion of new vessels and, on the other hand, increases the
transmissibility of the endothelium. Both facilitate the distribution or,
respectively, release of hormones into the bloodstream, which are necessary for the
maintenance of the early pregnancy. This is additionally supported by the fact that
hCG increases the permeability of vessels [36], [37]. In rats this increase is associated with a lowered
level of claudin 5 [37].
Influence of VEGF on Cell Adhesion Molecules in the Corpus Luteum of
Primates
Influence of VEGF on Cell Adhesion Molecules in the Corpus Luteum of
Primates
As described above in detail, it has been shown that hCG reduces the expression of
cell adhesion molecules and thus affects luteal angiogenesis and vascular
permeability. In addition it has been demonstrated that the inhibition of VEGF has
an inhibitory effect on ovarian angiogenesis, development and function [38], [39], [40]. Furthermore, it was shown that VEGF controls cell
adhesion molecules in the marmoset monkey.
The amount of occludin localised in the domain of the membranes of luteinising
granulosa cells decreases continuously in the course of the ovulation phase until
it
can no longer be detected [41]. This loss of occludin
seems to be involved in the formation of the antrum [42], [43]. In contrast, in vivo inhibition of
VEGF in marmosets by a VEGF antagonist (VEGF trap) leads to a significant increase
in the amount of occludin [41], above all in the
cytoplasmic domains where occludin itself has no function [44].
In the marmoset the TJ protein claudin 5 is exclusively detected in theca vessels
and
its amount increases in the course of follicle maturation. In the corpus luteum,
claudin 5 is also expressed in the domain of the vessels. In this case, inhibition
of VEGF leads to an increase of claudin 5. Apparently, claudin 5 plays an important
part in the contact inhibition of endothelial cells. In this way the rate of
proliferation of the cells is reduced and the vessels stabilise themselves [41]. As soon as individual endothelial cells come into
contact with neighbouring cells, the adhesion molecules combine to form complexes
and the endothelium is less impaired by the pro-angiogenic effect of VEGF. This cell
adhesion is essential with regard to the regulation of angiogenesis [45].
For VE-cadherin it has been proven that this inhibition triggers a decline in the
rate of angiogenesis and thus of vessel development [45].
Here VEGF acts as the key molecule for regulation of angiogenesis in the ovary [34], [35], [38], [39], [46]. Furthermore, a close relationship between VEGF and
the signal transduction of cellular adhesion molecules has been demonstrated from
functional considerations.
Cell Adhesion Proteins and Permeability
Cell Adhesion Proteins and Permeability
Some time ago it was demonstrated in an in vitro endothelial cell model that hCG
apparently has a direct effect on the expression of VE-cadherin and on endothelial
permeability [47]. However, the significance and the
signal transduction of an assumed LH/hCG receptor are still not clear. It is
proposed that the receptor exerts a hormonal transcytosis function and in this way
transports gonadotropins directly to the target cell [48]. In order to study the molecular regulation of endothelial cells in the
corpus luteum an in vitro co-culture model was developed. This consists of a
two-chamber model of endothelial and luteinising granulosa cells in which their
interactions can be studied as reactions on stimulating substances [49]. It was shown in vivo in the corpus luteum that the
endothelial cell adhesion protein claudin 5 decreases after hCG treatment in the
sense of a simulated pregnancy [25]; this was confirmed
in the cell culture model described above. In addition, by means of hCG treatment
in
co-cultured luteinising granulosa cells an effect also on endothelial permeability
was demonstrated for the first time [49]. Although a
possible direct effect of hCG on endothelial cells cannot be excluded [47], it is apparent that the effects of hCG on claudin 5
and on permeability are of an indirect nature because they are only observed in the
presence of luteinising granulosa cells. For this reason an hCG-dependent factor
that is synthesised by the luteinising granulosa cells was assumed – and this factor
is VEGF. The hCG treatment of luteinising granulosa cells does indeed lead to an
increase of VEGF [49], [50], [51] and the action of VEGF on
endothelial permeability has often been demonstrated in vitro [36], [47]. Thus, VEGF is considered to be an
important paracrine factor that controls the regulation of endothelial cell
permeability via an influence on adhesion proteins [52], [53]. This has found support in
observations in which the inhibition of VEGF in vivo suppresses not only
angiogenesis but also permeability [45]. Conversely, VEGF
in endothelial cells triggers the liberation of VE-cadherin and this, in turn,
results in an increased endothelial permeability [49].
This fact does indeed point to a direct relationship between hCG, VEGF, cell
adhesion proteins and increased permeability.
Interaction of Cell Adhesion Proteins in Endothelial Cells
Interaction of Cell Adhesion Proteins in Endothelial Cells
Functional interactions of cell adhesion proteins that act as regulators of vascular
permeability have been demonstrated for widely differing systems. In spite of the
facts that the corpus belongs by far to the most highly vascularised type of tissue
and that the control of permeability is of major significance for its function,
these relationships have mostly remained unknown.
Cell adhesion proteins as dynamic complexes change their conformation not only during
the embryonic development but also in resting cells of the adult vascular system
[54]. They interact thereby also with one another in
the sense of a response to extracellular signals [55], [56]. Accordingly, the co-existence of
widely varying cell adhesion proteins for completely different tissue types in
various species has been demonstrated. For the vascular system of the human corpus
luteum it has been shown that the AJ protein VE-cadherin as well as the TJ proteins
nectin 2 and claudin 5 are co-localised in the middle part of the luteal phase [57]. Treatment of luteinising granulosa cells with hCG in
vitro thereby leads via VEGF-mediation to a reduced expression of these proteins.
Beyond this, there is a functional relationship since VE-cadherin, nectin 2 and
claudin 5 mutually regulate each other. Elimination of VE-cadherin or claudin 5
triggers a down-regulation of the respective other proteins whereas nectin 2 does
not have a regulatory influence on VE-cadherin and claudin 5. These interactions are
not only of a structural nature but also have a functional impact on the
permeability of the endothelium. The hCG-induced down-regulation of the
above-mentioned proteins leads to an increased permeability. Moreover, the separate
elimination of VE cadherin, claudin 5 and nectin 2 in vitro also respectively leads
to an increase in the permeability.
Taken together, this suggests that VE-cadherin and claudin 5 apparently play a
significant part with regard to the regulation of permeability via nectin 2. In
addition, nectin 2 itself has a direct influence on the permeability.
Since the three proteins discussed above are co-localised in the vascular system of
the corpus luteum and can be down-regulated via VEGF by treatment with hCG, it can
be assumed that hCG triggers a chain reaction via VE-cadherin and/or claudin 5, in
order to control nectin 2 and concomitantly the luteal permeability ([Fig. 1]) [57]. These findings
may be of therapeutic relevance in situations with pathologically increased
endothelial permeability such as in, e.g. OHSS.
Fig. 1 Model for regulation of the vessel system in the corpus luteum.
Conclusion for Clinical Practice
Conclusion for Clinical Practice
The occurrence of an hCG-induced increase of endothelial permeability is apparently
mediated by VEGF. In cases of OHSS this mechanism may be responsible for the
development of ascites and represent a possible therapeutic option in the sense of
VEGF antagonism.
