Still taboo subjects. 

Alterations in mucosal transudation in vulvovaginal atrophy: what mechanisms are involved? Influence of hyaluronic acid.

Denis COUCHOUREL, PhD,

Medical and Scientific Director of Vivacy Laboratories

Denis Couchourel, PhD

Introduction

In 2014, the foundation for the definition of genitourinary menopausal syndrome was laid (Portman et al, 2014).
In essence, any disturbance in the metabolism of sex hormones can induce a series of symptoms grouped under the name of MGS (Figure 1).
Thus, this syndrome can occur obviously at menopause (Nappi et al, 2014) but also in younger patients, particularly in women who are breast cancer survivors.
In fact, some of the treatments offered to them result in an estrogen deficiency leading to permanent or non-permanent amenorrhea (Rosenberg et al, 2013; Falk et al, 2016).
Patients undergoing chemotherapy or radiotherapy protocols may also be concerned.
MUMS therefore greatly affects the quality of life of patients, and not only physically.
Indeed, the sexual and intimate health of women as well as their self-esteem are also largely impacted, leading to deleterious consequences on their sleep and the quality of their daily life (Nappi et al, 2019).
So this is a very important issue at the population level.

Vulvovaginal atrophy (VVA) is one of the main components of MUMS (Shifren et al, 2018).

The very structure of the tissues of the urogenital tract is in fact directly dependent on sexual hormones and in particular on estrogens.
The symptomatology related to VVA is also well characterized and includes sensations of vaginal dryness, burning, and irritation evoked by the MUMS scale (McBride et al, 2010).
Therefore, it is interesting to focus on this VVVA because it implies an alteration in the synthesis and diffusion of mucus used for lubrication of the vaginal walls and introitus.
This is a very specific phenomenon involving the process of transudation that we will detail below and which depends largely on the capacity of the submucosal tissues to transport water to the surface.

Synthesis and regulation of endogenous vaginal moisturizing fluid.

The importance of hormonal support on the proper functioning of the female reproductive tract is illustrated by the figure 2 :

Figure 2.Regulation of cellular functions of vaginal tissues by sex steroid hormones (I). (Traish et al, 2004).

Estrogens, androgens and progestins influence specific cellular processes depending on the anatomical plane considered within the vaginal tissue.

  • In particular, these actions can lead to
  • (1) the synthesis, secretion and reuptake of neurotransmitters;
  • (2) the contraction of the smooth muscles present in the vascular walls;
  • (3) permeation of the vaginal epithelium;
  • (4) autocrine or paracrine production of vasoactive growth factors;
  • (5) regulation of the synthesis/degradation balance of the main components of the extracellular matrices of the epithelium and the lamina propria. 

The sum of these different actions determines the global physiological responses which will result in a particular blood flow, a hydration / lubrication of the epithelial surface which may or may not be effective, or a capacity for muscle contraction which may or may not be effective. 

Therefore, any change (physiological or pathological) in the hormonal support described in this figure will lead to varying degrees of alterations in neurotransmitter function, tissue structure and composition, and smooth muscle contractility.
The vaginal blood flow will therefore tend to decrease.
However, the efficiency of lubrication depends closely on vascular flow.

Mechanism of vaginal fluid synthesis

Vaginal fluid is therefore produced by the vaginal wall and is not strictly speaking a secretion. It is a transudate.
Indeed, under the influence of arterial pressure, an ultrafiltrate is likely to form directly from the capillary network which is particularly dense at the level of the lamina propria.
These small vessels are fenestrated allowing a certain level of 'leakage' and therefore the formation of a transudate.
(Figure 3, subtypes C and D).

Figure 3: Classification of capillaries according to their degree of permeability.

  • (A) continuous capillaries (brain).
  • (B) continuous capillaries (muscles). The transit of pinocytosis vesicles allows limited exchanges with surrounding tissues.
  • (C) Fenestrated capillaries allowing wider exchanges.
  • (D) Sinusoidal capillaries allowing maximum exchange. (Kobayashi et al, 2005)
The fluid thus formed then literally 'percolates' to the superficial epithelial layers and then reaches the surface.
Interestingly, the membrane of the vaginal epithelium limits the reabsorption of Na+ present in the transudate.
This feature creates an additional osmotic force that further increases the call for water to the epithelial surface (Levin et al, 1997).
K+, Ca2+ and Cl- ions are also particularly concentrated there.
Vaginal transudate also contains a large number of sialoproteins, albumin, and small molecules such as lactate and urea amino acids (Levin et al, 1977; Levin R et al, 1978).
The amount produced is sufficient to moisten the vaginal wall and allow for the comfort of daily life.

Hormonal regulation of vaginal transudate production

Hormonal support of vaginal tissue homeostasis (Figure 2) is particularly important for the transudation mechanism mentioned in the previous paragraph.
In particular, the binding of sex steroids to their respective receptors induces a complex signaling cascade leading to lubrication of the epithelium. (Figure 4) 

Figure 4: Regulation of cellular functions of vaginal tissues by sex steroid hormones (II). (Traish et al, 2004).

The binding of the hormonal ligand (estrogens, progesterone and testosterone in particular) to their specific receptors (whether they are classically intra-cellular or membrane-based) triggers a cellular response.

It can take various forms: activation of ion channels, mitotic activity, induction of the synthesis of growth factors or neurotransmitters.

In any case, these hormone-dependent mechanisms allow the vagina to adapt the intensity of transudation, especially during phases of sexual excitement.

Note that the transduction pathways described can be non-genomic, i.e. they can be activated via membrane receptors.
This concept is relatively new for steroid hormones since the authors have long thought that their actions could only be envisaged through their binding to a cytoplasmic receptor which necessarily induces protein synthesis.
However, this system is by definition incompatible with a rapid reaction to a stimulus since the steps of transcription, translation, maturation and excretion of proteins take time.
However, rapid effects of steroid hormones have been increasingly demonstrated since the early 2000s and these functions must now be taken into account in the spectrum of expected hormonal actions (Schmidt et al, 2000; Falkenstein E et al, 2000; Couchourel et al, 2004).
It is therefore logical to observe a significant decrease in transudate production in ovariectomized animals.
The phenomenon is fully reversible if estrogenic supplementation is subsequently applied (Min et al, 2003).

Transport of transudate through the vaginal epithelium

The transport of aqueous fluids through the mucosal wall is not so obvious.
Indeed, the transudate must find its way through a relatively thick non-keratinized pluristratified epithelium.
This tissue is of course made up of successive layers of cells linked together by focal adhesion plates of the desmosome type.
It is therefore a globally hydrophobic environment and not very conducive to baso-apical aqueous fluid movements.
In this context, the transudate will use dedicated structures to be able to cross this cellular barrier.
Aquaporins (AQPs) are a family of transmembrane proteins with 4 members identified in mammals (AQP1 to AQP4) (Ishibashi K, 2009).
Hundreds of other isoforms have also been identified in other organisms, including plants (Maurel C et al, 2015).
They all share a number of characteristics.
For example, they weigh on average 30 KDa, they have 6 transmembrane segments and they form a tetramer within the lipid bilayer of the membrane and they are often associated with ion channels (Figure 5)

Figure 5:

  1. (A)a tetramer of 4 subunits delimit a central water-permeable channel.
  2. (B) Each subunit itself allows the passage of water molecules.
  3. (C) PQAs are very often associated with ion channels (here TRPV4, permeable to Ca2+ and Mg2+ ions) (Verkman et al, 2011)
In a tissue such as the epithelium of the vaginal mucosa, PQAs are strictly necessary for the transport of transudate from the capillaries of the lamina propria to the surface.
Numerous studies in animals have highlighted this crucial role.
For example, in 1999, Ma et al demonstrated that deletion of the gene encoding AQP5 in mice suppressed fluid secretion from the salivary glands (Ma et al, 1999).
The same demonstration could be made for the mucous membrane of the trachea.
In this particular case, the recovered fluid (which was significantly reduced in quantity) also exhibited hyperosmolarity (Song et al, 2001).
When the knockout involved the gene coding for AQP1, the synthesis of cerebrospinal fluid in the choroid plexus as well as the production of aqueous humor by the ciliary epithelium of the eye were decreased.
Thus, the authors concluded that AQP is strongly involved in the regulation of intracranial and intraocular pressures (Zhang et al, 2002).
As in the previous work, the osmolarity of the obtained fluids was also strongly disturbed.
At the vaginal level, AQP3, AQP5 and AQP6 could be identified in rats. (Park et al, 2008).
In addition, the amount of PQAs present in the tissues is sensitive to the estrogenic hormonal support (Figure 6), confirming some of the assumptions made in the model presented in the figures 3 and 4. 

Figure 6: Expression levels of identified PQAs in the vaginal wall.

The measurement is done by quantifying the optical density after immunocytochemical labeling.

The measured amounts of PQA decrease compared to the healthy control when the model (rat) is ovariectomized.

When ovariectomized rats are supplemented with estrogen, the PQA level rises and becomes comparable to the control (Zhu et al, 2105)

Park K et al have also suggested that nerve stimulation at the pelvic level could also induce translocation of AQP1 and AQP2 from the cell cytosol to the membranes.
Thus, the role of PQAs in vaginal lubrication could also be of great importance during the sexual arousal phase (Park et al, 2008) by allowing a drastic increase in the quantity of transudate available on the surface of the vaginal epithelium.
The transcellular transport just described is therefore an important aspect of epithelial crossing.
It is necessary, but not sufficient to fully optimize the lubrication of the vaginal wall.
Indeed, the extracellular matrix present between the cells can be a facilitator depending on its structure and composition. (Figure7) 

Figure 7: Composition of the extracellular matrix between the submucosal capillaries and the basal lamina supporting the epithelium.

This basal lamina is mainly composed of highly organized repetitive patterns of collagen, laminin, fibronectin and proteoglycans.

As mentioned above, the transudate (green downward arrow) originates from the windows separating the endothelial cells from the capillaries and tends to flow passively (thanks to hydrostatic pressure) towards the epithelial cells.
Nevertheless, for the system to be effective, the basal lamina must not block this aqueous flow.
That is why it must necessarily show a certain hygroscopicity.
This essential property is held by the molecules of the glycosaminoglycan group, and more particularly by hyaluronic acid (HA). 
Indeed, this long sweet polymer is charged because of the presence of carboxyl groups, giving it a very important affinity for water.
Moreover, when these molecules are in solution, electrostatic bonds are created, defining subdomains within which water can also be trapped and attracted.
Hyaluronic acid is synthesized by 3 isoforms of the 'hyaluronan synthase': HAS1, HAS2 and HAS3.
However, it was shown in 2013 that HAS2 (responsible for the formation of the longest chains of endogenous hyaluronic acid) could modulate its effectiveness depending on the hormones present.
Specifically, when the hormonal environment is estrogen-dominant, HAS2 increases its synthesis of high molecular weight HA.
At the same time, the expression of CD44 on the surface of the surrounding cell membranes tends to decrease, as if the organism wanted to favour a mode of action of HA based on its intrinsic chemical properties rather than its functions depending on a receptor-ligand interaction.
On the other hand, in a progestin-dominant environment, it is HAS3 that sees its activity increase strongly.
Yet this isoform is specialized in the synthesis of small molecules of HA that have a high affinity for HA membrane receptors (including CD44) (Raheem et al, 2013).
These results further illustrate that in addition to the intensity of transudate synthesis (which is highly dependent on vasodilation and intra capillary pressure, figure 3), the amount of fluid available at the mucosal surface is also tightly regulated by many parameters active during the epithelial crossing stage.
AQP and AH are 2 essential actors of this regulation.

Conclusion

Vaginal lubrication is a complex phenomenon whose effectiveness can vary according to a large number of regulatory parameters:
  • The vascular origin of the ultrafiltrate induces a dependence on the density of the subepithelial capillary network but also on its vasodilatation and on the blood pressure prevailing in these vessels.
  • Once formed, the transudate must then pass through the vaginal epithelium via AQPs in the function is subject to complex hormonal control.
  • In addition to transcellular fluid transport, the HA content of the extracellular matrix is also a facilitating parameter for lubrication. Here again, the hormonal environment will direct the profile of actions that HA can perform in this context.
Finally, the factors influencing vaginal lubrication presented in this summary are by no means exhaustive. There are of course other regulatory loops.
As an example and to open the reflection, let's just note that in 2015 a paper published in "Nature" demonstrated the ability of HA from the extracellular matrix to act on a cationic transporter named TRPV1.
TRPV1 was previously studied in the context of pain transmission at nociceptive nerve endings (Caires R et al, 2015).
We could therefore consider an additional and much more direct means of action of HA on pain related to the absence of transudate on the surface of the vaginal epithelium.
In addition to its moisturizing properties, HA may also act directly on the nerve signal. This hypothesis remains unconfirmed to date, but it illustrates the need to refine our knowledge of this type of molecular interaction.

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