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.

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.

Summary 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 multivariate 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 patients 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.

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%