Laura Lecce

β1 and β3 integrins disassemble from basal focal adhesions and β3 integrin is later localised to the apical plasma membrane of rat uterine luminal epithelial cells at the time of implantation

The present study investigated the expression of integrin subunits that are known to be associated with focal adhesions, namely β(1) and β(3) integrins in rat uterine luminal epithelial cells during early pregnancy. The β(1) and β(3) integrins were concentrated along the basal cell surface and were colocalised and structurally interacted with talin, a principal focal adhesion protein, on Day 1 of pregnancy. At the time of implantation, β(1) and β(3) integrins disassembled from the site of focal adhesions, facilitating the removal of uterine luminal epithelial cells for embryo invasion. Also at this time, β(3) integrin markedly increased along the apical membrane, suggesting a role in embryo attachment. This distributional change in β(1) and β(3) integrins seen at the time of implantation was predominantly under the influence of progesterone. Taken together, β(1) and β(3) integrin disassembly from focal adhesions and the increase in β(3) integrin apically are key components of hormonally regulated endometrial receptivity.

A Critical Role for Thioredoxin-Interacting Protein in Diabetes-Related Impairment of Angiogenesis

Impaired angiogenesis in ischemic tissue is a hallmark of diabetes. Thioredoxin-interacting protein (TXNIP) is an exquisitely glucose-sensitive gene that is overexpressed in diabetes. As TXNIP modulates the activity of the key angiogenic cytokine vascular endothelial growth factor (VEGF), we hypothesized that hyperglycemia-induced dysregulation of TXNIP may play a role in the pathogenesis of impaired angiogenesis in diabetes. In the current study, we report that high glucose–mediated overexpression of TXNIP induces a widespread impairment in endothelial cell (EC) function and survival by reducing VEGF production and sensitivity to VEGF action, findings that are rescued by silencing TXNIP with small interfering RNA. High glucose–induced EC dysfunction was recapitulated in normal glucose conditions by overexpressing either TXNIP or a TXNIP C247S mutant unable to bind thioredoxin, suggesting that TXNIP effects are largely independent of thioredoxin activity. In streptozotocin-induced diabetic mice, TXNIP knockdown to nondiabetic levels rescued diabetes-related impairment of angiogenesis, arteriogenesis, blood flow, and functional recovery in an ischemic hindlimb. These findings were associated with in vivo restoration of VEGF production to nondiabetic levels. These data implicate a critical role for TXNIP in diabetes-related impairment of ischemia-mediated angiogenesis and identify TXNIP as a potential therapeutic target for the vascular complications of diabetes.

Ovarian hormones regulate expression of the focal adhesion proteins, talin and paxillin, in rat uterine luminal but not glandular epithelial cells

During early pregnancy in the rat, focal adhesions disassemble in uterine luminal epithelial cells at the time of implantation to facilitate their removal so that the implanting blastocyst can invade into the underlying endometrial decidual cells. This study investigated the effect of ovarian hormones on the distribution and protein expression of two focal adhesion proteins, talin and paxillin, in rat uterine luminal and glandular epithelial cells under various hormone regimes. Talin and paxillin showed a major distributional change between different hormone regimes. Talin and paxillin were highly concentrated along the basal cell surface of uterine luminal epithelial cells in response to oestrogen treatment. However, this prominent staining of talin and paxillin was absent and also a corresponding reduction of paxillin expression was demonstrated in response to progesterone alone or progesterone in combination with oestrogen, which is also observed at the time of implantation. In contrast, the distribution of talin and paxillin in uterine glandular epithelial cells was localised on the basal cell surface and remained unchanged in all hormone regimes. Thus, not all focal adhesions are hormonally dependent in the rat uterus; however, the dynamics of focal adhesion in uterine luminal epithelial cells is tightly regulated by ovarian hormones. In particular, focal adhesion disassembly in uterine luminal epithelial cells, a key component to establish successful implantation, is predominantly under the influence of progesterone.

Focal adhesion kinase localizes to sites of cell-to-cell contact in vivo and increases apically in rat uterine luminal epithelium and the blastocyst at the time of implantation

Focal adhesions play an important role in promoting embryo invasion; in particular, focal adhesions disassemble at the time of implantation in the rat, facilitating the detachment of the uterine luminal epithelium to allow the embryo to invade the endometrium. This study investigated focal adhesion protein, focal adhesion kinase (FAK) in the rat uterine luminal, and glandular epithelial cells to understand the dynamics of focal adhesions during early pregnancy. FAK undergoes extensive distributional change during early pregnancy, and surprisingly, FAK was not localized at the site of focal adhesions, instead being localized to the site of cell‐to‐cell contact and colocalizing with ZO‐1 on day 1 of pregnancy. At the time of implantation, FAK increases in the apical region of the uterine luminal epithelial cells which was regulated by progesterone. Using an in vitro co‐culture model of rat blastocysts attached to Ishikawa cells, FAK was present apically both in the rat blastocyst and the Ishikawa cells, suggesting a role in attachment andin mediating signal transduction between these two genetically different cell types. J. Morphol., 2012.

Aging Impairs VEGF-Mediated, Androgen-Dependent Regulation of Angiogenesis

There is a progressive impairment of vascular repair mechanisms with advancing age concomitant with a steady decline in circulating androgen levels in men. Emerging evidence indicates androgens regulate angiogenesis; however, little research has focused on the impact of age upon androgen-mediated regulation of angiogenic mechanisms. Human dermal fibroblasts from young (<30 years) and older (>65 years) men were incubated with DHT, with or without androgen receptor antagonist hydroxyflutamide, or phosphoinositide 3-kinase inhibitor. Fibroblast-conditioned medium was used to stimulate angiogenic functions in human umbilical vein endothelial cells. Nuclear fractionation and fluorescence microscopy were used to study androgen receptor (AR) distribution. Conditioned medium from fibroblasts of young men, but not old men, treated with DHT produced a 3-fold increase in human umbilical vein endothelial cell tubulogenesis and 2-fold increase in migration via increased vascular endothelial growth factor (VEGF) expression and secretion, predominantly of VEGF145. DHT-induced VEGF secretion from fibroblasts of young men was AR-dependent and increased AKT phosphorylation, which was abrogated by phosphoinositide 3-kinase inhibition. By contrast, fibroblasts from older men were unresponsive to DHT and lacked androgen-mediated enhancement in VEGF production. These findings were associated with reduced AR nuclear translocation in old fibroblasts. The failure of DHT-induced paracrine stimulation of angiogenesis in fibroblasts from older men is likely due to defective nuclear translocation of AR. This first demonstration of androgen resistance (or insensitivity) acquired by human fibroblasts with aging suggests that pharmacological testosterone therapy for old men may be less effective in enhancing angiogenesis and facilitating tissue regeneration mechanisms reliant on paracrine release of VEGF.

Ezrin and EBP50 redistribute apically in rat uterine epithelial cells at the time of implantation and in response to cell contact

Uterine epithelial cells (UECs) undergo extensive morphological remodelling in preparation for an implanting blastocyst. This remodelling involves changes in the actin cytoskeleton and surface structures including microvilli. Ezrin and ezrin-radixin-moesin-binding protein-50-kDa (EBP50) link actin filaments to intra-membranous adhesion molecules and are important molecules in polarised epithelia. The current study is the first to describe the colocalisation and molecular association of ezrin and EBP50 in rat UECs by using immunofluorescence microscopy and immunoprecipitation techniques. These proteins have also been localised in relation to uterine epithelial cytoskeletal rearrangement during early pregnancy in the rat and to the effect of apical surface contact between opposing epithelial cells, blastocyst contact and contact with a silicon filament. Immunofluorescence microscopy has revealed that ezrin and EBP50 respond to contact between opposing epithelial cells and increase apically on day 6 of pregnancy. This apical distribution is also observed in UECs in contact with a silicon filament. Ezrin and EBP50 are however absent within the implantation chamber itself, seemingly mimicking the events that take place in leucocyte-endothelium binding. Thus, ezrin and EBP50 occur apically in UECs at the time of implantation in the rat and in response to a substitute blastocyst (filament) suggesting a role for these proteins in the cytoskeletal rearrangements that facilitate uterine receptivity and blastocyst-epithelial adhesion. Their loss within the implantation chamber possibly allows the subsequent invasion of the embryo.

ICAM1 and fibrinogen-γ are increased in uterine epithelial cells at the time of implantation in rats

Uterine epithelial cells transform into a receptive state to adhere to an implanting blastocyst. Part of this transformation includes the apical concentration of cell adhesion molecules at the time of implantation. This study, for the first time, investigates the expression of ICAM1 and fibrinogen‐γ (FGG) in uterine epithelial cells during normal pregnancy, pseudopregnancy and in hormone‐treated rats. An increase (P < 0.05) in ICAM1 was seen at the apical membrane of uterine epithelial cells at the time of implantation compared with day 1 of pregnancy. ICAM1 was also increased (P < 0.05) on day 6 of pseudopregnancy as well as in ovariectomized rats treated with progesterone plus oestrogen. These results show that ICAM1 up‐regulation at the time of implantation is under the control of progesterone, and is not dependent on cytokine release from the blastocyst or in semen. FGG dimerization increased (P < 0.05) on day 6 of pregnancy compared with day 1, and was not up‐regulated in day 6 pseudopregnant animals, suggesting this increase is dependent on a developing blastocyst. The presence of ICAM1 and FGG in the uterine epithelium at the time of implantation in the rat is similar to that seen in lymphocyte–endothelium adhesion, and we suggest a similar mechanism in embryo–uterine epithelium adhesion is utilized. Mol. Reprod. Dev. 78:318–327, 2011.

ICAM-2 and lipid rafts disappear from the basal plasma membrane of uterine epithelial cells during early pregnancy in rats

Adhesion molecules are redistributed in rat uterine epithelial cells (UECs) during early pregnancy for endometrial receptivity and implantation. Intercellular adhesion molecule-2 (ICAM-2) is located as an oligomer on the basal plasma membrane of non-receptive UECs on day 1 of pregnancy and colocalizes with the lipid raft marker flotillin-2. At the time of implantation in rats and in ovariectomized rats primed with progesterone, ICAM-2 disappears from the basal plasma membrane and lipid rafts redistribute to the apical membrane. The loss of ICAM-2 might render UECs less adherent to the underlying basal lamina and more prone to apoptosis. Flotillin-2 in the apical plasma membrane at the time of implantation might provide an anchoring point for several adhesion molecules that are known to localize to this region at this time. We suggest that flotillin-2 is involved with adhesion between UECs and the implanting blastocyst, whereas ICAM-2 is associated with the ability for UECs to be removed at the time of implantation.

CD43 is relocated from the basal to the apical plasma membrane of rat uterine epithelial cells by progesterone

Adhesion molecules play an important part in preparing uterine epithelial cells for receptivity to the implanting embryo, and their rearrangement is crucial in allowing successful implantation. CD43 is an adhesion molecule which has previously been suggested to take part in implantation in mice. Indirect immunofluorescence microscopy localising CD43 was performed on uterine tissue during early pregnancy, and tissue obtained from ovariectomised rats administered with ovarian hormones. Western blotting was performed during early pregnancy on isolated epithelial cells and ovariectomised rats for comparison of the amount of CD43. Immunofluorescence microscopy showed CD43 was situated basally in uterine luminal epithelial cells on day 1 of pregnancy and during oestrogen administration, corresponding to a 95-kDa band of CD43 seen in western blotting. At the time of implantation, and during progesterone or progesterone plus oestrogen combined treatment, CD43 is apical in uterine luminal epithelial cells, resulting in an 85-kDa form of CD43. We suggest that a de-glycosylated form of CD43 moves from basally to apically at the time of implantation, thus facilitating blastocyst attachment to uterine epithelial cells as well as their removal.

Mucin 15 is lost but mucin 13 remains in uterine luminal epithelial cells and the blastocyst at the time of implantation in the rat

The glycocalyx of the uterine luminal epithelium in the rat undergoes considerable reduction before implantation. In particular, the reduction of some mucins is necessary to facilitate blastocyst adhesion and subsequent implantation. The present study investigated the localisation, abundance and hormonal control of two mucin proteins, Muc13 and Muc15, in rat uterine epithelial cells during early pregnancy to determine whether they are likely to play a role in uterine receptivity for implantation. Muc13 and Muc15 are localised to the uterine luminal epithelium but show a presence and an absence, respectively, at the apical cell surface at the time of implantation. This localisation corresponds to changes in the molecular weights of Muc13 and Muc15, as shown with western blotting analysis. Furthermore, the localisation of Muc13 and Muc15 was shown to be controlled by the ovarian hormones, oestrogen and progesterone, and they were also localised in preimplantation rat blastocysts. Our results suggest that Muc15 may operate in an anti-adhesive capacity to prevent implantation while Muc13 potentially functions in either an adhesive or cell-signalling role in the events of implantation.