Granulosa cell proliferation is inhibited by PGE2 in the primate ovulatory follicle.

Prostaglandin E2 (PGE2) is a key paracrine mediator of ovulation. Few specific PGE2-regulated gene products have been identified, so we hypothesized that PGE2 may regulate the expression and/or activity of a network of proteins to promote ovulation. To test this concept, Ingenuity Pathway Analysis (IPA) was used to predict PGE2-regulated functionalities in the primate ovulatory follicle. Cynomolgus macaques underwent ovarian stimulation. Follicular granulosa cells were obtained before (0 h) or 36 h after an ovulatory dose of human chorionic gonadotropin (hCG), with ovulation anticipated 37-40 h after hCG. Granulosa cells were obtained from additional monkeys 36 h after treatment with hCG and the PTGS2 inhibitor celecoxib, which significantly reduced hCG-stimulated follicular prostaglandin synthesis. Granulosa cell RNA expression was determined by microarray and analyzed using IPA. No granulosa cell mRNAs were identified as being significantly up-regulated or down-regulated by hCG + celecoxib compared with hCG only. However, IPA predicted that prostaglandin depletion significantly regulated several functional pathways. Cell cycle/cell proliferation was selected for further study because decreased granulosa cell proliferation is known to be necessary for ovulation and formation of a fully-functional corpus luteum. Prospective in vivo and in vitro experiments confirmed the prediction that hCG-stimulated cessation of granulosa cell proliferation is mediated via PGE2. Our studies indicate that PGE2 provides critical regulation of granulosa cell proliferation through mechanisms that do not involve significant regulation of mRNA levels of key cell cycle regulators. Pathway analysis correctly predicted that PGE2 serves as a paracrine mediator of this important transition in ovarian structure and function.

Placental Growth Factor Is Required for Ovulation, Luteinization, and Angiogenesis in Primate Ovulatory Follicles.

Placental growth factor (PGF) is member of the vascular endothelial growth factor (VEGF) family of angiogenesis regulators. VEGFA is an established regulator of ovulation and formation of the corpus luteum. To determine whether PGF also mediates aspects of ovulation and luteinization, macaques received gonadotropins to stimulate multiple follicular development. Ovarian biopsies and whole ovaries were collected before (0 hours) and up to 36 hours after human chorionic gonadotropin (hCG) administration to span the ovulatory interval. PGF and VEGFA were expressed by both granulosa cells and theca cells. In follicular fluid, PGF and VEGFA levels were lowest before hCG. PGF levels remained low until 36 hours after hCG administration, when PGF increased sevenfold to reach peak levels. Follicular fluid VEGFA increased threefold to reach peak levels at 12 hours after hCG, then dropped to intermediate levels. To explore the roles of PGF and VEGFA in ovulation, luteinization, and follicular angiogenesis in vivo, antibodies were injected into the follicular fluid of naturally developed monkey follicles; ovariectomy was performed 48 hours after hCG, with ovulation expected about 40 hours after hCG. Intrafollicular injection of control immunoglobulin G resulted in no retained oocytes, follicle rupture, and structural luteinization, including granulosa cell hypertrophy and capillary formation in the granulosa cell layer. PGF antibody injection resulted in oocyte retention, abnormal rupture, and incomplete luteinization, with limited and disorganized angiogenesis. Injection of a VEGFA antibody resulted in oocyte retention and very limited follicle rupture or structural luteinization. These studies demonstrate that PGF, in addition to VEGFA, is required for ovulation, luteinization, and follicular angiogenesis in primates.

Vascular endothelial growth factors C and D may promote angiogenesis in the primate ovulatory follicle.

Angiogenesis in the ovary occurs rapidly as the ovarian follicle transforms into a mature corpus luteum. Granulosa cells produce vascular endothelial growth factor A (VEGFA) in response to the ovulatory gonadotropin surge. VEGFA is established as a key mediator of angiogenesis in the primate ovulatory follicle. To determine if additional VEGF family members may be involved in angiogenesis within the ovulatory follicle, cynomolgus monkeys (Macaca fascicularis) received gonadotropins to stimulate multiple follicular development, and human chorionic gonadotropin (hCG) substituted for the luteinizing hormone surge to initiate ovulatory events. Granulosa cells of monkey ovulatory follicles contained mRNA and protein for VEGFC and VEGFD before and after hCG administration. VEGFC and VEGFD were detected in monkey follicular fluid and granulosa cell-conditioned culture media, suggesting that granulosa cells of ovulatory follicles secrete both VEGFC and VEGFD. To determine if these VEGF family members can stimulate angiogenic events, monkey ovarian microvascular endothelial cells (mOMECs) were obtained from monkey ovulatory follicles and treated in vitro with VEGFC and VEGFD. Angiogenic events are mediated via three VEGF receptors; mOMECs express all three VEGF receptors in vivo and in vitro. Exposure of mOMECs to VEGFC increased phosphorylation of AKT, while VEGFD treatment increased phosphorylation of both AKT and CREB. VEGFC and VEGFD increased mOMEC migration and the formation of endothelial cell sprouts in vitro. However, only VEGFD increased mOMEC proliferation. These findings suggest that VEGFC and VEGFD may work in conjunction with VEGFA to stimulate early events in angiogenesis of the primate ovulatory follicle.

Temporal and spatial requirements for Hoxa3 in mouse embryonic development.

Hoxa3(null) mice have severe defects in the development of pharyngeal organs including athymia, aparathyroidism, thyroid hypoplasia, and ultimobranchial body persistence, in addition to defects of the throat cartilages and cranial nerves. Some of the structures altered in the Hoxa3(null) mutant embryos are anterior to the described Hoxa3 gene expression boundary: the thyroid, soft palate, and lesser hyoid horn. All of these structures develop over time and through the interactions of multiple cell types. To investigate the specific cellular targets for HOXA3 function in these structures across developmental time, we performed a comprehensive analysis of the temporal and tissue-specific requirements for Hoxa3, including a lineage analysis using Hoxa3(Cre). The combination of these approaches showed that HOXA3 functions in both a cell autonomous and non-cell autonomous manner during development of the 3rd and 4th arch derivatives, and functions in a neural crest cell (NCC)-specific, non-cell autonomous manner for structures that were Hoxa3-negative by lineage tracing. Our data indicate that HOXA3 is required for tissue organization and organ differentiation in endodermal cells (in the tracheal epithelium, thymus, and parathyroid), and contributes to organ migration and morphogenesis in NCCs. These data provide a detailed picture of where and when HOXA3 acts to promote the development of the diverse structures that are altered in the Hoxa3(null) mutant. Data presented here, combined with our previous studies, indicate that the regionally restricted defects in Hoxa3 mutants do not reflect a role in positional identity (establishment of cell or tissue fate), but instead indicate a wider variety of functions including controlling distinct genetic programs for differentiation and morphogenesis in different cell types during development.

Prostaglandin E2 and vascular endothelial growth factor A mediate angiogenesis of human ovarian follicular endothelial cells.

STUDY QUESTION: Which receptors for prostaglandin E2 (PGE2) and vascular endothelial growth factor A (VEGFA) mediate angiogenesis in the human follicle around the time of ovulation? SUMMARY ANSWER: PGE2 and VEGFA act via multiple PGE2 receptors (PTGERs) and VEGF receptors (VEGFRs) to play complementary roles in follicular angiogenesis. WHAT IS KNOWN ALREADY: Production of PGE2 and VEGFA by the follicle are prerequisites for ovulation. PGE2 is an emerging regulator of angiogenesis and has not been examined in the context of the human ovulatory follicle. VEGFA is an established regulator of follicular angiogenesis. STUDY DESIGN, SIZE, DURATION: Ovarian biopsies containing the ovulatory follicle were obtained from 11 women of reproductive age (30-45 years) undergoing surgery for laparoscopic sterilization. In some cases, women received hCG to substitute for the ovulatory LH surge before ovarian biopsy. In addition, aspirates from four women of reproductive age (18-31 years) undergoing gonadotrophin stimulation for oocyte donation were obtained for isolation of human ovarian microvascular endothelial cells (hOMECs). PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian biopsies were utilized for immunocytochemical detection of von Willebrand factor to identify endothelial cells. hOMECs were cultured with PGE2, PTGER receptor selective agonists, VEGFA, or VEGFR selective agonists. hOMECs were assessed for proliferation by Ki67 immunocytochemistry. hOMEC migration was determined by counting cells which migrated through a porous membrane in vitro. Sprout formation was quantified by determining sprout number and length from photographs take after culture of hOMECs in a 3-dimensional matrix. MAIN RESULTS AND THE ROLE OF CHANCE: Endothelial cells were not observed within the granulosa cell layer of human ovulatory follicles prior to an ovulatory dose of hCG and were first seen amongst granulosa cells 18-34 h after hCG. In vitro, PGE2 enhanced migration and sprout formation but did not alter hOMEC proliferation. Agonists selective for each PTGER increased migration with no change in proliferation. PTGER1 and PTGER2 agonists increased the number of sprouts, while only PTGER1 affected sprout length. VEGFA increased hOMEC proliferation, migration, and formation of structures resembling capillary sprouts. Signaling through VEGFR1 promoted hOMEC migration, proliferation, and the formation of few, long endothelial cell sprouts, while VEGFR2 stimulation promoted hOMEC migration and the formation of many, short sprouts. All effects of treatments in vitro were considered significant at P < 0.05. LIMITATIONS, REASONS FOR CAUTION: While primary cultures of hOMECs respond to PGE2 and VEGFA differently than other cultured endothelial cells, hOMECs may not respond to PGE2 and VEGFA in vivo as they do in vitro. WIDER IMPLICATIONS OF THE FINDINGS: Agonists and antagonists selective for PTGER1, PTGER2, VEGFR1, or VEGFR2 may have therapeutic value to promote or prevent ovulation in women. STUDY FUNDING/COMPETING INTERESTS: This research was supported by grant funding from the Eunice Kennedy Shriver National Institutes of Child Health and Human Development (HD071875 to D.M.D., T.E.C., M.B.). The authors have no conflicts of interest to disclose.

Angiogenesis in the primate ovulatory follicle is stimulated by luteinizing hormone via prostaglandin E2.

Rapid angiogenesis occurs as the ovulatory follicle is transformed into the corpus luteum. To determine if luteinizing hormone (LH)-stimulated prostaglandin E2 (PGE2) regulates angiogenesis in the ovulatory follicle, cynomolgus macaques received gonadotropins to stimulate multiple follicular development and chorionic gonadotropin (hCG) substituted for the LH surge to initiate ovulatory events. Before hCG, vascular endothelial cells were present in the perifollicular stroma but not amongst granulosa cells. Endothelial cells entered the granulosa cell layer 24-36 h after hCG, concomitant with the rise in follicular PGE2 and prior to ovulation, which occurs about 40 h after hCG. Intrafollicular administration of the PG synthesis inhibitor indomethacin was coupled with PGE2 replacement to demonstrate that indomethacin blocked and PGE2 restored follicular angiogenesis in a single, naturally developed monkey follicle in vivo. Intrafollicular administration of indomethacin plus an agonist selective for a single PGE2 receptor showed that PTGER1 and PTGER2 agonists most effectively stimulated angiogenesis within the granulosa cell layer. Endothelial cell tracing and three-dimensional reconstruction indicated that these capillary networks form via branching angiogenesis. To further explore how PGE2 mediates follicular angiogenesis, monkey ovarian microvascular endothelial cells (mOMECs) were isolated from ovulatory follicles. The mOMECs expressed all four PGE2 receptors in vitro. PGE2 and all PTGER agonists increased mOMEC migration. PTGER1 and PTGER2 agonists promoted sprout formation while the PTGER3 agonist inhibited sprouting in vitro. While PTGER1 and PTGER2 likely promote the formation of new capillaries, each PGE2 receptor may mediate aspects of PGE2's actions and, therefore, LH's ability to regulate angiogenesis in the primate ovulatory follicle.

Multiple roles for HOXA3 in regulating thymus and parathyroid differentiation and morphogenesis in mouse.

Hoxa3 was the first Hox gene to be mutated by gene targeting in mice and is required for the development of multiple endoderm and neural crest cell (NCC)-derived structures in the pharyngeal region. Previous studies have shown that the Hoxa3 null mutant lacks third pharyngeal pouch derivatives, the thymus and parathyroids by E18.5, and organ-specific markers are absent or downregulated during initial organogenesis. Our current analysis of the Hoxa3 null mutant shows that organ-specific domains did undergo initial patterning, but the location and timing of key regional markers within the pouch, including Tbx1, Bmp4 and Fgf8, were altered. Expression of the parathyroid marker Gcm2 was initiated but was quickly downregulated and differentiation failed; by contrast, thymus markers were delayed but achieved normal levels, concurrent with complete loss through apoptosis. To determine the cell type-specific roles of Hoxa3 in third pharyngeal pouch development, we analyzed tissue-specific mutants using endoderm and/or NCC-specific Cre drivers. Simultaneous deletion with both drivers resulted in athymia at E18.5, similar to the null. By contrast, the individual tissue-specific Hoxa3 deletions resulted in small, ectopic thymi, although each had a unique phenotype. Hoxa3 was primarily required in NCCs for morphogenesis. In endoderm, Hoxa3 temporally regulated initiation of the thymus program and was required in a cell-autonomous manner for parathyroid differentiation. Furthermore, Hoxa3 was required for survival of third pharyngeal pouch-derived organs, but expression in either tissue was sufficient for this function. These data show that Hoxa3 has multiple complex and tissue-specific functions during patterning, differentiation and morphogenesis of the thymus and parathyroids.

Magnetic resonance angiography-defined intracranial vasculopathy is associated with silent cerebral infarcts and glucose-6-phosphate dehydrogenase mutation in children with sickle cell anaemia.

Silent cerebral infarct (SCI) is the most commonly recognized cause of neurological injury in sickle cell anaemia (SCA). We tested the hypothesis that magnetic resonance angiography (MRA)-defined vasculopathy is associated with SCI. Furthermore, we examined genetic variations in glucose-6-phosphate dehydrogenase (G6PD) and HBA (α-globin) genes to determine their association with intracranial vasculopathy in children with SCA. Magnetic resonance imaging (MRI) of the brain and MRA of the cerebral vasculature were available in 516 paediatric patients with SCA, enrolled in the Silent Infarct Transfusion (SIT) Trial. All patients were screened for G6PD mutations and HBA deletions. SCI were present in 41·5% (214 of 516) of SIT Trial children. The frequency of intracranial vasculopathy with and without SCI was 15·9% and 6·3%, respectively (P < 0·001). Using a multivariable logistic regression model, only the presence of a SCI was associated with increased odds of vasculopathy (P = 0·0007, odds ratio (OR) 2·84; 95% Confidence Interval (CI) = 1·55-5·21). Among male children with SCA, G6PD status was associated with vasculopathy (P = 0·04, OR 2·78; 95% CI = 1·04-7·42), while no significant association was noted for HBA deletions. Intracranial vasculopathy was observed in a minority of children with SCA, and when present, was associated with G6PD status in males and SCI.

Increased risk of venous thromboembolism is associated with genetic variation in heme oxygenase-1 in Blacks.

BACKGROUND: Venous thromboembolism (VTE) affects as many as 1 in 1000 individuals in the United States. Although Blacks are disproportionately affected by VTE, few genetic risk factors have been identified in this population. The inducible heme oxygenase-1 (HMOX1) gene encodes a key cytoprotective enzyme with anti-inflammatory, antioxidant and anticoagulant activity acting in the vascular system. A (GT)(n) microsatellite located in the promoter of the HMOX1 gene influences the level of response. METHODS AND RESULTS: Using the Genetic Attributes and Thrombosis Epidemiology (GATE) study, we examined the association between HMOX1 repeat length and VTE events in 883 Black and 927 White patients and matched controls. We found no association between HMOX1 genotypes and VTE in Whites. However, in Black patients, carrying two long (L) alleles (≥ 34 repeats) was significantly associated with provoked (odds ratio (OR) 1.86, 95% confidence interval (CI): 1.19-2.90) or recurrent (OR 3.13, 95% CI: 1.77-5.53) VTE events. CONCLUSIONS: We have demonstrated for the first time an association between genetic variation in HMOX1, and VTE in Blacks. Our results support a key role for the heme oxygenase system in protecting patients at increased risk for thrombosis and suggest a potential mechanism for targeted screening and intervention.

Heme oxygenase-1 gene promoter polymorphism is associated with reduced incidence of acute chest syndrome among children with sickle cell disease.

Sickle cell disease is a common hemolytic disorder with a broad range of complications, including vaso-occlusive episodes, acute chest syndrome (ACS), pain, and stroke. Heme oxygenase-1 (gene HMOX1; protein HO-1) is the inducible, rate-limiting enzyme in the catabolism of heme and might attenuate the severity of outcomes from vaso-occlusive and hemolytic crises. A (GT)(n) dinucleotide repeat located in the promoter region of the HMOX1 gene is highly polymorphic, with long repeat lengths linked to decreased activity and inducibility. We examined this polymorphism to test the hypothesis that short alleles are associated with a decreased risk of adverse outcomes (hospitalization for pain or ACS) among a cohort of 942 children with sickle cell disease. Allele lengths varied from 13 to 45 repeats and showed a trimodal distribution. Compared with children with longer allele lengths, children with 2 shorter alleles (4%; ≤ 25 repeats) had lower rates of hospitalization for ACS (incidence rate ratio 0.28, 95% confidence interval, 0.10-0.81), after adjusting for sex, age, asthma, percentage of fetal hemoglobin, and α-globin gene deletion. No relationship was identified between allele lengths and pain rate. We provide evidence that genetic variation in HMOX1 is associated with decreased rates of hospitalization for ACS, but not pain. This study is registered at www.clinicaltrials.gov as #NCT00072761.