Biomedical subjects

Peter A W Rogers

Publications and source records attributed to Peter A W Rogers.

16 recordsLinked to original sources

PubMed2006

In vitro culture significantly alters gene expression profiles and reduces differences between myometrial and fibroid smooth muscle cells.

Cultured myometrial (M) and fibroid (F) smooth muscle cells (SMCs) have been widely used as a model for the study of F growth. The aim of this study was to compare gene expression profiles using microarrays between six paired M and F tissues from hysterectomy specimens, as well as cells isolated from the same tissues and cultured for up to three passages. A total of 2055 genes were differentially expressed by ANOVA between all experimental groups. Among them, 128 genes were found to be statistically different between M and F tissues. More than 1100 genes were significantly changed between tissues and cultured cells, with 648 genes common between both M and F cells at P0 and P3. Expression profiles of six genes including estrogen receptor-alpha (ERalpha) and progesterone receptor (PR) were also validated using real-time PCR. These data demonstrate that large changes occur in SMC gene expression in culture, reducing differences between M and F cells. They also show that ERalpha and PR levels are reduced in cells compared with whole tissue. These results indicate that although M and F cell cultures provide an important tool to study these tumours, in vitro studies must be carefully planned and evaluated to provide meaningful results.

Cells, Cultured↗

PubMed2006

Molecular profiling of human endometrium during the menstrual cycle.

This summary of the findings of investigations of the changing transcriptional profile of human endometrium during the menstrual cycle shows that it is possible to classify the menstrual cycle based on the global gene expression profile, and identifies groups of known and novel genes that may be associated with different biological processes that occur in the endometrium such as implantation and menstruation.

Adult↗

PubMed2005

Recent advances in endometrial angiogenesis research.

This review summarises recent research into the mechanisms and regulation of endometrial angiogenesis. Understanding of when and by what mechanisms angiogenesis occurs during the menstrual cycle is limited, as is knowledge of how it is regulated. Significant endometrial endothelial cell proliferation occurs at all stages of the menstrual cycle in humans, unlike most animal models where a more precise spatial relationship exists between endothelial cell proliferation and circulating levels of oestrogen and progesterone. Recent stereological data has identified vessel elongation as a major endometrial angiogenic mechanism in the mid-late proliferative phase of the cycle. In contrast, the mechanisms that contribute to post-menstrual repair and secretory phase remodelling have not yet been determined. Both oestrogen and progesterone/progestins appear to have paradoxical actions, with recent studies showing that under different circumstances both can promote as well as inhibit endometrial angiogenesis. The relative contribution of direct versus indirect effects of these hormones on the vasculature may help to explain their pro- or anti-angiogenic activities. Recent work has also identified the hormone relaxin as a player in the regulation of endometrial angiogenesis. While vascular endothelial growth factor (VEGF) is fundamental to endometrial angiogenesis, details of how and when different endometrial cell types produce VEGF, and how production and activity is controlled by oestrogen and progesterone, remains to be elucidated. Evidence is emerging that the different splice variants of VEGF play a major role in regulating endometrial angiogenesis at a local level. Intravascular neutrophils containing VEGF have been identified as having a role in stimulating endometrial angiogenesis, although other currently unidentified mechanisms must also exist. Future studies to clarify how endometrial angiogenesis is regulated in the human, as well as in relevant animal models, will be important for a better understanding of diseases such as breakthrough bleeding, menorrhagia, endometriosis and endometrial cancer.

Angiogenesis Inducing Agents↗

PubMed2005

Increased expression of the relaxin receptor (LGR7) in human endometrium during the secretory phase of the menstrual cycle.

This study investigated localization and expression of relaxin and its receptor, LGR7, in the human endometrium during the proliferative and secretory phases of the menstrual cycle. H2 relaxin binding was identified in endometrium, but not myometrium, and particularly in the epithelium of the endometrial glands and uterine lumen. Binding sites increased in the early secretory phase of the menstrual cycle and were paralleled by similar increases in LGR7 mRNA measured by Q-PCR. The increase in LGR7 expression and H2 relaxin binding in the secretory phase of the menstrual cycle suggests a specific role for relaxin after ovulation in the human uterus.

Endometrium↗

PubMed2005

The role of progesterone in endometrial angiogenesis in pregnant and ovariectomised mice.

The role of progesterone (and oestrogen) in endometrial angiogenesis remains controversial. The aims of this study were to quantify endometrial angiogenesis in pregnant mice and to investigate the role of progesterone in promoting endothelial cell proliferation in ovariectomized mice. Uteri were collected on days 1 to 4 of pregnancy when circulating progesterone concentrations were increasing, prior to implantation. Before dissection, mice were injected with bromodeoxyuridine (BrdU) enabling proliferating endothelial cells to be quantified with CD31/BrdU double-immunohistochemistry. There was a significant increase in proliferating endothelial cells on day 3 of pregnancy when plasma progesterone also increased. To determine if this endothelial cell proliferation was due to progesterone, an experiment was performed on ovariectomised mice. One group was treated with a single oestradiol injection on day 8 after ovariectomy, followed by a no-treatment day and three consecutive daily injections of progesterone. Other groups were treated with either the vehicle, oestradiol or progesterone injections only; all were dissected on day 13 following ovariectomy. Unexpectedly, mice treated with progesterone-only had the highest amount of endothelial cell proliferation and oestrogen priming was found to significantly reduce this progesterone-induced endothelial cell proliferation. To determine if this proliferation is mediated by vascular endothelial growth factor (VEGF), a further experiment in which VEGF anti-serum was administered concurrently with the progesterone injections was performed. Endothelial cell proliferation was reduced but not abolished suggesting progesterone-induced endometrial angiogenesis is only partly mediated by VEGF. Results indicate that oestrogen priming is not required for progesterone to stimulate endometrial endothelial cell proliferation and that oestrogen inhibits progesterone-induced angiogenesis in ovariectomised mice.

Animals↗

PubMed2004

Endometrial endothelial cell steroid receptor expression and steroid effects on gene expression.

Controversy exists regarding the expression of specific steroid receptor proteins and mRNA in human microvascular endometrial endothelial cells (HEECs). Thus, we studied steroid receptor expression in early passaged HEEC cultures and freshly isolated HEECs. Analysis of estrogen receptor (ER) and progesterone receptor (PR) mRNA levels was carried out with real-time quantitative RT-PCR, and the repertoire of genes activated by their respective steroid ligands was assessed by mRNA microarray analyses of 18,400 genes and expressed sequence tags. We observed that cultured and freshly isolated HEECs each express ER-beta mRNA but not ER-alpha. In addition, PR mRNA was also detectable in both HEEC sources. Microarray analysis demonstrated that treatment of HEEC cultures with either estradiol or medroxyprogesterone acetate produced differential effects on a wide variety of genes, and cluster analysis demonstrated that many of the genes are involved in intracellular signaling and enzymatic pathways. Thus, quantitative RT-PCR and microarray analyses demonstrate that HEECs express ER-beta and PR mRNA and that gene expression by HEECs is differentially regulated by treatment with estrogen or progestin.

Cells, Cultured↗

PubMed2004

Molecular classification of human endometrial cycle stages by transcriptional profiling.

Endometrium is a dynamic tissue that undergoes cyclic changes each month, under the overall control of estrogen and progesterone. The aims of this study were to investigate the changing global gene expression profile of human endometrium during the menstrual cycle using microarray technology and to determine the correlation between histopathological evaluation and molecular profile of the samples. Standard two-colour cDNA microarrays were performed on the 43 samples against a common reference, using a 10.5 K cDNA glass slide microarray. The results were validated using real-time PCR. Analysis of expression data was carried out using parametric analysis of variance with Benjamini-Hochberg correction. Hierarchical clustering reveals a strong relationship between histopathology and transcriptional profile of the samples. The study identified 1452 genes that showed significant changes in expression (P< or =0.05) across the menstrual cycle, with 425 genes having changes that are at least 2-fold. The data were also independently analysed by a CSIRO algorithm called GeneRaVE that identified a small subset of genes whose expression profiles could be used to classify nearly all the biopsies into their correct cycle stage. We also identified and validated three genes [(natural cytotoxicity triggering receptor (NCR)3, fucosyl transferase (FUT)4 and Fyn-binding protein (FYB)] that had not been shown to have significant cyclic changes in the human endometrium, previously. We have shown for the first time that endometrial cycle stage prediction is possible based on global gene expression profile.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Estrogen receptor-alpha agonists promote angiogenesis in human myometrial microvascular endothelial cells.

OBJECTIVE: The relative role of the two estrogen receptors, ERalpha and ERbeta, in mediating angiogenic responses in adult human endothelium is unknown. The aim of this study was to determine whether novel ERalpha-selective agonists, propyl pyrazole triol (PPT) and the tetrahydrochrysene (R,R-THC), up-regulate the expression of vascular endothelial growth factor receptor-2 (VEGFR-2), and promote VEGF-stimulated endothelial cell proliferation in primary cultures of adult female microvascular endothelial cells co-expressing endogenous ERalpha and ERbeta. METHODS: Confluent primary cultures of microvascular endothelial cells isolated from human myometrium were incubated with 17beta-estradiol (1 and 10 nM), PPT (10 nM to 3 microM), or R,R-THC (10 nM to 3 microM) for 18 hours and VEGFR-2 expression measured by biotin-VEGF165 binding and flow cytometry. Endothelial cell proliferation was assessed in microvascular endothelial cells after incubation with 17beta-estradiol (10 nM), PPT (100 nM), and R,R-THC (100 nM) for 6 days using a tetrazolium-based bioassay. RESULTS: Both PPT and R,R-THC increased VEGFR-2 expression on myometrial microvascular endothelial cells in a dose-dependent manner, reaching a maximum at 1 microM. Approximately 40% of myometrial microvascular endothelial cell isolates only express ERbeta and do not express ERalpha, and in these neither PPT, R,R-THC, nor 17beta-estradiol increased VEGF binding. PPT- or R,R-THC-stimulated increase in VEGF binding was significantly different between ERalpha+ and ERalpha- microvascular endothelial cell samples (P < .001 and P < .05, respectively). PPT, R,R-THC, and 17beta-estradiol significantly augmented VEGF-stimulated microvascular endothelial cell proliferation in ERalpha+ (P < .05), but not in ERalpha- samples. CONCLUSIONS: This angiogenic effect of 17beta-estradiol on adult female microvascular endothelial cells is mediated by ERalpha, rather than ERbeta.

Cell Division↗

PubMed2004

Increased expression of the relaxin receptor (LGR7) in human endometrium during the secretory phase of the menstrual cycle.

Relaxin (RLX) is a structural homolog of insulin that is the ligand for the LGR7 receptor. Although the 6k peptide is produced by the ovaries to cause connective tissue remodeling of the rodent and pig reproductive tracts to facilitate parturition, in human reproduction, the role of RLX is less well understood. Binding of human gene 2 (H2) [(33)P]-RLX, expression of RLX peptides and the LGR7 receptor was examined in the human uterus at different stages of the menstrual cycle. A significant increase in RLX receptor binding in endometrium was identified by quantitative autoradiography in the secretory compared with the proliferative phase. H2RLX competed with [(33)P]-H2RLX binding with higher affinity than porcine RLX during both the proliferative and secretory phases. Increased LGR7 receptor gene expression during the secretory phase paralleled the changes in [(33)P]-H2RLX binding. Human gene 1 RLX transcripts were not detected in the uterus, and H2RLX gene expression was low and not influenced by the stage of the menstrual cycle. The studies show that binding to and gene expression of the LGR7 RLX receptor changes markedly with the phases of the menstrual cycle, suggesting a specific role for the hormone in the physiology of the human uterus.

Adult↗

PubMed2004

Coexpression of fractalkine and its receptor in normal human endometrium and in endometrium from users of progestin-only contraception supports a role for fractalkine in leukocyte recruitment and endometrial remodeling.

Leukocytes are critical mediators of endometrial remodeling, but the mechanisms by which leukocyte subpopulations enter the uterus are currently unknown. Endometrial leukocytes have no genomic progesterone receptors; thus, we hypothesized that leukocyte migration is induced indirectly by progesterone-regulated chemokines. Fractalkine (CX3CL1), a chemotactic membrane-bound adhesion factor, and its receptor (CX3CR1) were assessed by immunohistochemistry in endometrial samples across the menstrual cycle, in early pregnancy, and in women using progestin-only contraceptives. Fractalkine was localized predominantly to glandular epithelial and decidualized stromal cells, with the highest staining intensity in the secretory phase and early pregnancy. It was also detected in subpopulations of endometrial leukocytes (macrophages and uterine NK cells), with maximal numbers during the proliferative phase and early pregnancy. CX3CR1 was similarly colocalized to the glandular epithelium and decidualized stromal cells, with the highest expression in the secretory phase. CX3CR1-positive leukocytes (macrophages and neutrophils) were in greatest abundance during the menstrual phase. In the endometrium of women using progestin-only contraceptives, immunoreactive fractalkine was markedly reduced in the glandular epithelium, but was increased in decidualized stroma and infiltrating leukocytes. These findings support a number of roles for fractalkine in the endometrium, in the secretory phase, in early pregnancy, and when influenced by progestin-only contraceptives.

CX3C Chemokine Receptor 1↗

PubMed2003

Endometrial arteriogenesis: vascular smooth muscle cell proliferation and differentiation during the menstrual cycle and changes associated with endometrial bleeding disorders.

The cyclical regrowth of the human endometrial vasculature every 28 days following menstruation provides an excellent opportunity for studying arteriogenesis in a human tissue. Despite this, very little has been published to date about temporal or spatial changes in endometrial arteriolar structure or function. Immunohistochemical studies using vascular smooth muscle cell (VSMC) differentiation markers have identified spatially organised differences in VSMC phenotype. However, these have not significantly increased our understanding of how or when arteriogenesis occurs. Endometrial VSMC proliferation continues at a steady rate throughout the cycle, increasing in the specialised spiral arterioles in the mid and late secretory phases. Although estrogen and progesterone play a dominant role in regulating endometrial growth and regression, their direct involvement in arteriogenesis is less clear. Estrogen and progesterone receptors have been reported in endometrial VSMC, although heterogeneity of expression occurs. The angiopoietin/Tie axis has been investigated in endometrium, although the results of 3 studies published to date are contradictory, making conclusions about a role in arteriogenesis problematic. Abnormalities of arteriogenesis may play a role in disorders such as menorrhagia and breakthrough bleeding in contraceptive and hormone replacement therapy users. There is evidence that VSMC proliferation is reduced in spiral arterioles of women with menorrhagia, along with reduced pulsatility index of the uterine artery as shown by Doppler ultrasound. In women taking progestin-only contraception, endometrial perivascular alpha smooth muscle actin is reduced in those who suffer from breakthrough bleeding compared to those that don't. Considerable work is still required to elucidate when in the cycle, where in the tissue, and what mechanisms regulate endometrial arteriogenesis.

Arterioles↗

PubMed2003

Genomics in obstetrics and gynaecology.

With the Human Genome Project complete, and microarray technology progressing rapidly, the study of whole genomes has become a reality. The emerging field of genomics is full of promise, has become a cornerstone of commercial drug development, and looks certain to make a major contribution to clinical practice in the future. There is an increasing number of genomic studies concerned with obstetric and gynaecological conditions. Despite this, clinicians in their busy practices often lack a basic understanding of genomics and the tools involved in generating genome-based information. In the present review, we aim to provide the clinician with a basic overview of genomics--what it is, what tools it uses, and how it may benefit our patients. The existing published reports on genomic studies in the reproductive field is reviewed.

DNA Fingerprinting↗

PubMed2003

Fibroids display an anti-angiogenic gene expression profile when compared with adjacent myometrium.

The aetiology of uterine fibroids remains unknown, despite causing significant gynaecological morbidity. Fibroids have a reduced microvascular density when compared with adjacent myometrial tissue. The aim of this study was to identify genes with differential expression between fibroid and adjacent normal myometrium, particularly genes with a role in angiogenesis. Total RNA was extracted from fibroid/myometrium pairs from 12 hysterectomy specimens, and used to perform 24 cDNA microarrays. There were 10,500 genes screened on each microarray for differential expression. Analysis of expression data was carried out using multiple t-tests, as well as a novel class prediction algorithm (GeneRaVE). The differential gene expression of selected genes was confirmed by quantitative 'real time' RT-PCR. Selected genes with a role in angiogenesis were further analysed for expression in isolated cell populations of endothelial cells (fibroid and myometrium) and smooth muscle cells (fibroid and myometrium), to see if their expression was confined to particular cell types. Twenty-five genes with differential gene expression between fibroid and myometrium were identified. Insulin-like growth factor-2, endothelin A receptor, connective tissue growth factor (CTGF), cysteine-rich angiogenic inducer 61 (CYR61) and collagen 4alpha2 (COL4A2) were confirmed by RT-PCR. CTGF and CYR61, both angiogenesis promoters, were reduced in expression relative to myometrium. COL4A2, the precursor for an angiogenesis inhibitor, canstatin, was increased relative to myometrium. These three genes display an anti-angiogenic expression profile in fibroids relative to myometrium. These findings may explain the reduced microvascular density seen in fibroids relative to myometrium.

Adult↗

PubMed2002

Angiogenesis occurs by vessel elongation in proliferative phase human endometrium.

BACKGROUND: Angiogenesis occurs by at least three mechanisms: sprouting, intussusception and elongation. Studies to date have failed to identify the mechanisms or timing of endometrial angiogenesis during the menstrual cycle. The aim of this study was to determine if vessel elongation plays a role in human endometrial angiogenesis. METHODS: Forty-nine full thickness endometrial sections from 27 hysterectomy samples were immunostained for CD34 to identify blood vessels, and analysed using an interactive computerized stereological program. Based on counts from 9746 individual microscope fields, blood vessel length density (L(v)), branch point density (N(v)) and mean vessel length per branch point (L(v)/N(v)) were calculated for three endometrial zones during five phases of the menstrual cycle. RESULTS: There was an increase in L(v)/N(v) in the mid-late proliferative compared with early proliferative, early-mid secretory and late secretory phases of the menstrual cycle in the functionalis (mean +/- SEM: 174.5 +/- 20.1 versus 76.6 +/- 8.4, 118.6 +/- 9.4 and 104.2 +/- 4.1 microm respectively, P < 0.001) and between the mid-late proliferative and the menstrual phases in the basalis (158.0 +/- 18.2 versus 95.4 +/- 10.0 microm, P = 0.025). An increase in L(v) occurred in the subepithelial capillary plexus in the mid-late proliferative and early-mid secretory phases compared with the early proliferative phase (316.7 +/- 32.4 and 338.8 +/- 45.3 versus 178.5 +/- 8.9 mm/mm(3), P = 0.027). CONCLUSIONS: These data are the first evidence that vessel elongation is a major angiogenic mechanism in mid-late proliferative phase human endometrium.

Adult↗

PubMed2002

Estrogen receptor-alpha and -beta expression in microvascular endothelial cells and smooth muscle cells of myometrium and leiomyoma.

The two estrogen receptors, ERalpha and ERbeta, are likely to have roles in the pathophysiology of fibroid development. They have been detected in myometrial and leiomyoma (fibroid) tissue, but the cell types expressing ERalpha and ERbeta have not been determined. ERs have also been detected in human endothelial cells. The aims of the present study were to determine whether pure populations of myometrial microvascular endothelial cells (MEC) express ERalpha and ERbeta, to compare MEC ERalpha/ERbeta expression with that of pure populations of myometrial smooth muscle cells (SMC) and to determine if ERalpha/ERbeta are differentially expressed in MEC and SMC of myometrium and fibroids from nine paired samples. Using RT-PCR (for ERalpha and ERbeta) and Western blotting (for ERalpha only), we demonstrated that all cultures of early passage myometrial and fibroid SMC (>99% pure) expressed ERalpha but not ERbeta, while myometrial and fibroid MEC (>99% CD31+) constitutively expressed ERbeta. However, both myometrial and fibroid MEC showed variable expression of ERalpha, with approximately 60% of MEC samples expressing ERalpha. While the majority (6/9) of MEC from myometrial and fibroid pairs demonstrated the same pattern of ERalpha expression, 3/9 pairs showed discordant ERalpha expression. These results show that ERalpha and ERbeta are differentially expressed in SMC and MEC of human myometrium and fibroids. Since ERalpha and ERbeta mediate opposing transcriptional activities, any effect of estrogen on the growth and development of fibroids is likely to be complex and may involve both SMC and MEC.

Adult↗

PubMed2002

17Beta-estradiol up-regulates vascular endothelial growth factor receptor-2 expression in human myometrial microvascular endothelial cells: role of estrogen receptor-alpha and -beta.

Estrogen has a cardiovascular protective role in women due in part to its effect on the vasculature. The roles of the two estrogen receptors (ERs), ERalpha and ERbeta, in the vascular actions of estrogen are unclear, as are effects of estrogen on microvascular endothelial cells (MEC) derived from sex steroid-responsive tissues. The present study demonstrates that 17beta-estradiol, but not progesterone, increases vascular endothelial growth factor (VEGF) receptor (VEGFR) expression on human myometrial MEC measured using biotin-recombinant human (rh) VEGF(165) and flow cytometry. This response occurred in a time- and dose-dependent manner, with significantly increased rhVEGF(165) binding at 3 h and maximal responses between 0.1 and 10 nmol/liter 17beta-estradiol, which was blocked by the antiestrogen ICI 182,780. Approximately 60% of samples demonstrated this response to 17beta-estradiol. All samples of myometrial MEC expressed both ERbeta mRNA and protein demonstrated by semiquantitative RT-PCR and Western blotting. However, ERalpha mRNA and protein were expressed in only 13 of 21 MEC samples. There was a significant association between ERalpha expression in myometrial MEC and their ability to respond to 17beta-estradiol by increasing rhVEGF(165) binding. 17beta-estradiol increased VEGFR-2 expression in ERalpha-expressing MEC isolates, which also demonstrated increased rhVEGF(165) binding, but failed to have these effects on ERalpha negative samples. Similarly, 17beta-estradiol augmented VEGF-induced MEC proliferation in ERalpha-expressing MEC samples, which was blocked by ICI 182,780. These observations suggest that 17beta-estradiol increases VEGFR-2 expression on human myometrial MEC promoting endothelial cell proliferation, an effect that varies between subjects and appears to be mediated primarily by ERalpha.

Adult↗