Histone deacetylase 9 promotes endothelial-mesenchymal transition and an unfavorable atherosclerotic plaque phenotype.

Endothelial-mesenchymal transition (EndMT) is associated with various cardiovascular diseases and in particular with atherosclerosis and plaque instability. However, the molecular pathways that govern EndMT are poorly defined. Specifically, the role of epigenetic factors and histone deacetylases (HDACs) in controlling EndMT and the atherosclerotic plaque phenotype remains unclear. Here, we identified histone deacetylation, specifically that mediated by HDAC9 (a class IIa HDAC), as playing an important role in both EndMT and atherosclerosis. Using in vitro models, we found class IIa HDAC inhibition sustained the expression of endothelial proteins and mitigated the increase in mesenchymal proteins, effectively blocking EndMT. Similarly, ex vivo genetic knockout of Hdac9 in endothelial cells prevented EndMT and preserved a more endothelial-like phenotype. In vivo, atherosclerosis-prone mice with endothelial-specific Hdac9 knockout showed reduced EndMT and significantly reduced plaque area. Furthermore, these mice displayed a more favorable plaque phenotype, with reduced plaque lipid content and increased fibrous cap thickness. Together, these findings indicate that HDAC9 contributes to vascular pathology by promoting EndMT. Our study provides evidence for a pathological link among EndMT, HDAC9, and atherosclerosis and suggests that targeting of HDAC9 may be beneficial for plaque stabilization or slowing the progression of atherosclerotic disease.

Androgens Stimulate EPC-Mediated Neovascularization and Are Associated with Increased Coronary Collateralization.

Endothelial progenitor cells (EPCs) play a key role in neovascularization and have been linked to improved cardiovascular outcomes. Although there is a well-established inverse relationship between androgen levels and cardiovascular mortality in men, the role of androgens in EPC function is not fully understood. In this study, we investigated the effects of androgens on 2 subpopulations of EPCs, early EPCs (EEPCs) and late outgrowth EPCs (OECs), and their relationships with coronary collateralization. Early EPCs and OECs were isolated from the peripheral blood of young healthy men and treated with dihydrotestosterone (DHT) with or without androgen receptor (AR) antagonist, hydroxyflutamide, in vitro. Dihydrotestosterone treatment enhanced AR-mediated proliferation, migration, and tubulogenesis of EEPCs and OECs in a dose-dependent manner. Furthermore, DHT augmented EPC sensitivity to extracellular stimulation by vascular endothelial growth factor (VEGF) via increased surface VEGF receptor expression and AKT activation. In vivo, xenotransplantation of DHT pretreated human EPCs augmented blood flow recovery and angiogenesis in BALB/c nude male mice, compared to mice receiving untreated EPCs, following hindlimb ischemia. In particular, DHT pretreated human OECs exhibited higher reparative potential than EEPCs in augmenting postischemic blood flow recovery in mice. Furthermore, whole blood was collected from the coronary sinus of men with single vessel coronary artery disease (CAD) who underwent elective percutaneous intervention (n = 23). Coronary collateralization was assessed using the collateral flow index. Serum testosterone and EPC levels were measured. In men with CAD, circulating testosterone was positively associated with the extent of coronary collateralization and the levels of OECs. In conclusion, androgens enhance EPC function and promote neovascularization after ischemia in mice and are associated with coronary collateralization in men.

Androgens Ameliorate Impaired Ischemia-Induced Neovascularization Due to Aging in Male Mice.

There is abundant evidence that low circulating testosterone levels in older men are associated with adverse cardiovascular outcomes; however, the direction of causality is unclear. Although there is burgeoning interest in the potential of androgen therapy in older men, the effect of androgens on cardiovascular regeneration in aging males remains poorly defined. We investigated the role of androgens in age-related impairment in ischemia-induced neovascularization. Castrated young (2 months) and old (24 months) male mice were subjected to unilateral hindlimb ischemia and treated with subdermal DHT or placebo Silastic implants. Blood flow recovery was enhanced by DHT treatment in young and old mice compared with age-matched placebo controls. DHT augmented angiogenesis in young mice and ameliorated age-related impairment in neovascularization in old mice. Administration of DHT was associated with increased hypoxia inducible factor-1α (HIF-1α) and stromal cell‒derived factor-1 expression in young mice, but not in old mice. In vitro, DHT-induced HIF-1α transcriptional activation was attenuated in fibroblasts from old mice. Interaction between androgen receptor (AR) and importins, key proteins that facilitate nuclear translocation of AR, was impaired with age. In contrast, DHT treatment stimulated the production and mobilization of Sca1+/CXCR4+ circulating progenitor cells in both young and old mice. DHT stimulated the migration and proangiogenic paracrine effect of ex vivo cultured bone marrow‒derived angiogenic cells from young and old mice. In conclusion, androgens ameliorated age-related impairment in ischemia-induced neovascularization. Although age-dependent dysfunction in androgen signaling attenuated some androgen effects on angiogenesis, provasculogenic effects of androgens were partially preserved with age.

Fenofibrate Rescues Diabetes-Related Impairment of Ischemia-Mediated Angiogenesis by PPARα-Independent Modulation of Thioredoxin-Interacting Protein.

Fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist, reduces lower limb amputations in patients with type 2 diabetes. The mechanism is, however, unknown. In this study, we demonstrate that fenofibrate markedly attenuates diabetes-related impairment of ischemia-mediated angiogenesis. In a murine model of hindlimb ischemia, daily oral fenofibrate treatment restored diabetes-impaired blood flow recovery, foot movement, hindlimb capillary density, vessel diameter, and vascular endothelial growth factor signaling to nondiabetic levels in both wild-type and PPARα-knockout mice, indicating that these fenofibrate effects are largely PPARα independent. In vitro, fenofibric acid (FFA) rescued high glucose-induced (25 mmol/L) impairment of endothelial cell migration, tubulogenesis, and survival in a PPARα-independent manner. Interestingly, fenofibrate in vivo and FFA in vitro reversed high glucose-induced expression of thioredoxin-interacting protein (TXNIP), an exquisitely glucose-inducible gene previously identified as a critical mediator of diabetes-related impairment in neovascularization. Conversely, adenoviral overexpression of TXNIP abrogated the restorative effects of FFA on high glucose-impaired endothelial cell function in vitro, indicating that the effects of FFA are mediated by TXNIP. We conclude that fenofibrate rescues diabetic impairment in ischemia-mediated angiogenesis, in large part, by PPARα-independent regulation of TXNIP. These findings may therefore explain the reduction in amputations seen in patients with diabetes treated with fenofibrate.

Androgen action augments ischemia-induced, bone marrow progenitor cell-mediated vasculogenesis.

Bone marrow-derived progenitor cell-mediated vasculogenesis is a key process for vascular repair and regeneration. However, the role of androgens in the mechanism of ischemia-induced vasculogenesis remains unclear. In this study, a gender-mismatch murine bone marrow transplant model was used to allow tissue tracking of transplanted cells. Bone marrow from 2-month-old male mice was transplanted into irradiated age-matched female recipients. Following the transplantation, ovariectomized female recipients were subjected to unilateral hindlimb ischemia and immediately implanted with either dihydrotestosterone (DHT) or placebo pellets. Laser Doppler perfusion imaging revealed that DHT significantly augmented blood flow recovery, with increased capillary density compared to placebo-treated female recipient controls. Flow cytometry analysis showed that DHT modulated vasculogenesis by increasing Sca1+/CXC4+ progenitor cell production in bone marrow and spleen and enhancing cell mobilization in circulating blood following hindlimb ischemia. Bone marrow cell homing was examined by detecting expression levels of male-specific SRY gene in the ischemic female tissues. DHT treatment promoted bone marrow cell homing to ischemic tissue shown by significantly higher SRY expression compared to placebo-treated females as well as reduced apoptotic features in DHT-treated females, including increased Bcl-2 expression, reduced Bax levels and decreased TUNEL staining. In conclusion, the gender-mismatched bone marrow transplant study shows that androgens directly enhance bone marrow cell-mediated vasculogenesis that contributes to ischemia-induced neovascularization.

CD90 Identifies Adventitial Mesenchymal Progenitor Cells in Adult Human Medium- and Large-Sized Arteries.

Mesenchymal stem cells (MSCs) reportedly exist in a vascular niche occupying the outer adventitial layer. However, these cells have not been well characterized in vivo in medium- and large-sized arteries in humans, and their potential pathological role is unknown. To address this, healthy and diseased arterial tissues were obtained as surplus surgical specimens and freshly processed. We identified that CD90 marks a rare adventitial population that co-expresses MSC markers including PDGFRα, CD44, CD73, and CD105. However, unlike CD90, these additional markers were widely expressed by other cells. Human adventitial CD90+ cells fulfilled standard MSC criteria, including plastic adherence, spindle morphology, passage ability, colony formation, and differentiation into adipocytes, osteoblasts, and chondrocytes. Phenotypic and transcriptomic profiling, as well as adoptive transfer experiments, revealed a potential role in vascular disease pathogenesis, with the transcriptomic disease signature of these cells being represented in an aortic regulatory gene network that is operative in atherosclerosis.

CXCR4 and CXCR7 play distinct roles in cardiac lineage specification and pharmacologic ß-adrenergic response.

CXCR4 and CXCR7 are prominent G protein-coupled receptors (GPCRs) for chemokine stromal cell-derived factor-1 (SDF-1/CXCL12). This study demonstrates that CXCR4 and CXCR7 induce differential effects during cardiac lineage differentiation and ß-adrenergic response in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Using lentiviral vectors to ablate CXCR4 and/or CXCR7 expression, hiPSC-CMs were tested for phenotypic and functional properties due to gene knockdown. Gene expression and flow cytometry confirmed the pluripotent and cardiomyocyte phenotype of undifferentiated and differentiated hiPSCs, respectively. Although reduction of CXCR4 and CXCR7 expression resulted in a delayed cardiac phenotype, only knockdown of CXCR4 delayed the spontaneous beating of hiPSC-CMs. Knockdown of CXCR4 and CXCR7 differentially altered calcium transients and ß-adrenergic response in hiPSC-CMs. In engineered cardiac tissues, depletion of CXCR4 or CXCR7 had opposing effects on developed force and chronotropic response to ß-agonists. This work demonstrates distinct roles for the SDF-1/CXCR4 or CXCR7 network in hiPSC-derived ventricular cardiomyocyte specification, maturation and function.

Androgen Receptor-Mediated Genomic Androgen Action Augments Ischemia-Induced Neovascularization.

Increasing evidence indicates that androgens regulate ischemia-induced neovascularization. However, the role of genomic androgen action mediated by androgen receptor (AR), a ligand-activated nuclear transcription factor, remains poorly understood. Using an AR knockout (KO) mouse strain that contains a transcriptionally inactive AR (ARΔex3KO), we examined the role of AR genomic function in modulating androgen-mediated augmentation of ischemia-induced neovascularization. Castrated wild-type (ARWT) and ARΔex3KO mice were implanted with 5α-dihydrotestosterone (DHT) or placebo pellets after hindlimb ischemia (HLI). DHT modulation of angiogenesis and vasculogenesis, key processes for vascular repair and regeneration, was examined. Laser Doppler perfusion imaging revealed that DHT enhanced blood flow recovery in ARWT mice post-HLI. In ARWT mice, DHT enhanced angiogenesis by down-regulating prolyl hydroxylase 2 and augmenting hypoxia-inducible factor-1α (HIF-1α) levels in the ischemic tissues post-HLI. DHT also enhanced the production and mobilization of Sca1+/CXCR4+ progenitor cells in the bone marrow (BM) and circulating blood, respectively, in ARWT mice. By contrast, DHT-mediated enhancement of blood flow recovery was abrogated in ARΔex3KO mice. DHT modulation of HIF-1α expression was attenuated in ARΔex3KO mice. DHT-induced HIF-1α transcriptional activity and DHT-augmented paracrine-mediated endothelial cell tubule formation were attenuated in fibroblasts isolated from ARΔex3KO mice in vitro. Furthermore, DHT-induced augmentation of Sca1+/CXCR4+ progenitor cell production and mobilization was absent in ARΔex3KO mice post-HLI. BM transplantation revealed that ischemia-induced mobilization of circulating progenitor cells was abolished in recipients of ARΔex3KO BM. Together, these results indicate that androgen-mediated augmentation of ischemia-induced neovascularization is dependent on genomic AR transcriptional activation.

Endothelial to mesenchymal transition is common in atherosclerotic lesions and is associated with plaque instability.

Endothelial to mesenchymal transition (EndMT) plays a major role during development, and also contributes to several adult cardiovascular diseases. Importantly, mesenchymal cells including fibroblasts are prominent in atherosclerosis, with key functions including regulation of: inflammation, matrix and collagen production, and plaque structural integrity. However, little is known about the origins of atherosclerosis-associated fibroblasts. Here we show using endothelial-specific lineage-tracking that EndMT-derived fibroblast-like cells are common in atherosclerotic lesions, with EndMT-derived cells expressing a range of fibroblast-specific markers. In vitro modelling confirms that EndMT is driven by TGF-ß signalling, oxidative stress and hypoxia; all hallmarks of atherosclerosis. 'Transitioning' cells are readily detected in human plaques co-expressing endothelial and fibroblast/mesenchymal proteins, indicative of EndMT. The extent of EndMT correlates with an unstable plaque phenotype, which appears driven by altered collagen-MMP production in EndMT-derived cells. We conclude that EndMT contributes to atherosclerotic patho-biology and is associated with complex plaques that may be related to clinical events.

High-Density Lipoproteins Rescue Diabetes-Impaired Angiogenesis via Scavenger Receptor Class B Type I.

Disordered neovascularization and impaired wound healing are important contributors to diabetic vascular complications. We recently showed that high-density lipoproteins (HDLs) enhance ischemia-mediated neovascularization, and mounting evidence suggests HDL have antidiabetic properties. We therefore hypothesized that HDL rescue diabetes-impaired neovascularization. Streptozotocin-induced diabetic mice had reduced blood flow recovery and neovessel formation in a hindlimb ischemia model compared with nondiabetic mice. Reconstituted HDL (rHDL) infusions in diabetic mice restored blood flow recovery and capillary density to nondiabetic levels. Topical rHDL application rescued diabetes-impaired wound closure, wound angiogenesis, and capillary density. In vitro, rHDL increased key mediators involved in hypoxia-inducible factor-1α (HIF-1α) stabilization, including the phosphoinositide 3-kinase/Akt pathway, Siah1, and Siah2, and suppressed the prolyl hydroxylases (PHD) 2 and PHD3. rHDL rescued high glucose-induced impairment of tubulogenesis and vascular endothelial growth factor (VEGF) A protein production, a finding associated with enhanced phosphorylation of proangiogenic mediators VEGF receptor 2 and endothelial nitric oxide synthase. Siah1/2 small interfering RNA knockdown confirmed the importance of HIF-1α stability in mediating rHDL action. Lentiviral short hairpin RNA knockdown of scavenger receptor class B type I (SR-BI) in vitro and SR-BI(-/-) diabetic mice in vivo attenuated rHDL rescue of diabetes-impaired angiogenesis, indicating a key role for SR-BI. These findings provide a greater understanding of the vascular biological effects of HDL, with potential therapeutic implications for diabetic vascular complications.

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.

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.

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.

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.

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.

ß(1) and ß(3) integrins disassemble from basal focal adhesions and ß(3) 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 ß(1) and ß(3) integrins in rat uterine luminal epithelial cells during early pregnancy. The ß(1) and ß(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, ß(1) and ß(3) integrins disassembled from the site of focal adhesions, facilitating the removal of uterine luminal epithelial cells for embryo invasion. Also at this time, ß(3) integrin markedly increased along the apical membrane, suggesting a role in embryo attachment. This distributional change in ß(1) and ß(3) integrins seen at the time of implantation was predominantly under the influence of progesterone. Taken together, ß(1) and ß(3) integrin disassembly from focal adhesions and the increase in ß(3) integrin apically are key components of hormonally regulated endometrial receptivity.

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.

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.