Estrogen stimulates fetal vascular endothelial growth factor expression and microvascularization.

We recently showed that the ratio of capillaries to myofibers in skeletal muscle, which accounts for 80% of insulin-directed glucose uptake and metabolism, was reduced in baboon fetuses in which estrogen was suppressed by maternal letrozole administration. Since vascular endothelial growth factor (VEGF) promotes angiogenesis, the present study determined the impact of estrogen deprivation on fetal skeletal muscle VEGF expression, capillary development, and long-term vascular and metabolic function in 4- to 8-year-old adult offspring. Maternal baboons were untreated or treated with letrozole or letrozole plus estradiol on days 100-164 of gestation (term = 184 days). Skeletal muscle VEGF protein expression was suppressed by 45% (P < 0.05) and correlated (P = 0.01) with a 47% reduction (P < 0.05) in the number of capillaries per myofiber area in fetuses of baboons in which serum estradiol levels were suppressed 95% (P < 0.01) by letrozole administration. The reduction in fetal skeletal muscle microvascularization was associated with a 52% decline (P = 0.02) in acetylcholine-induced brachial artery dilation and a 23% increase (P = 0.01) in mean arterial blood pressure in adult progeny of letrozole-treated baboons, which was restored to normal by letrozole plus estradiol. The present study indicates that estrogen upregulates skeletal muscle VEGF expression and systemic microvessel development within the fetus as an essential programming event critical for ontogenesis of systemic vascular function and insulin sensitivity/glucose homeostasis after birth in primate offspring.

B-Flow/Spatiotemporal Image Correlation M-Mode and Contrast-Enhanced/Microbubble Ultrasonography Quantification of Spiral Artery Distensibility and Placental Intervillous Perfusion in the First Trimester in a Primate Model of Impaired Spiral Artery Remodeling.

OBJECTIVE: During early human pregnancy, placental trophoblasts remodel spiral arteries into distensible low-resistance vessels to promote placental perfusion. We have established a model of impaired spiral artery remodeling (SAR) by elevating estradiol levels in the first trimester of baboon pregnancy. In the present study, B-flow/spatiotemporal image correlation (STIC) M-mode ultrasonography, a non-Doppler technology for sharp rendering of vessel dimensions, was used to determine whether spiral artery distensibility was altered in SAR-suppressed baboons. Contrast-enhanced ultrasound/microbubble imaging was also performed to determine whether it detected changes in placenta intervillous space perfusion in SAR-suppressed baboons. METHODS: The two imaging procedures were performed in the first trimester in baboons not treated or treated with estradiol to suppress SAR. RESULTS: Spiral artery distensibility, that is, luminal diameter at systole minus diameter at diastole, and volume flow as quantified by B-flow/STIC M-mode were 26% (p = 0.03) and 55% (p = 0.059) lower, respectively, in SAR-suppressed baboons. However, placental intervillous space flow rate and video intensity plateau levels reflecting blood perfusion, quantified by contrast-enhanced ultrasound/microbubble imaging, were unaltered in SAR-suppressed baboons. CONCLUSION: The results indicate that B-flow/STIC M-mode ultrasonography provides a non-invasive method to detect reduced distensibility and, thus, function of spiral arteries across the cardiac cycle in the first trimester in a primate model of impaired SAR. This study represents a first step in determining whether B-flow/STIC M-mode detects a similar defect in SAR early in adverse human pregnancy. This would provide an avenue to develop therapeutic modalities to prevent the devastating consequences of impaired SAR.

Estrogen Promotes Microvascularization in the Fetus and Thus Vascular Function and Insulin Sensitivity in Offspring.

We have shown that normal weight offspring born to estrogen-deprived baboons exhibited insulin resistance, although liver and adipose function and insulin receptor and glucose transporter expression were unaltered. The blood microvessels have an important role in insulin action by delivering insulin and glucose to target cells. Although little is known about the regulation of microvessel development during fetal life, estrogen promotes capillary proliferation and vascular function in the adult. Therefore, we tested the hypothesis that estrogen promotes fetal microvessel development and thus vascular function and insulin sensitivity in offspring. Capillary/myofiber ratio was decreased 75% (P < 0.05) in skeletal muscle, a major insulin target tissue, of fetal baboons in which estradiol levels were depleted by administration of aromatase inhibitor letrozole. This was sustained after birth, resulting in a 50% reduction (P < 0.01) in microvessel expansion; 65% decrease (P < 0.01) in arterial flow-mediated dilation, indicative of vascular endothelial dysfunction; and 35% increase (P < 0.01) in blood pressure in offspring from estrogen-deprived baboons, changes prevented by letrozole and estradiol administration. Along with vascular dysfunction, peak insulin and glucose levels during a glucose tolerance test were greater (P < 0.05 to P < 0.01) and the homeostasis model of insulin resistance 2-fold higher (P < 0.01) in offspring of letrozole-treated than untreated animals, indicative of insulin resistance. This study makes the novel discovery that estrogen promotes microvascularization in the fetus and thus normal vascular development and function required for eliciting insulin sensitivity in offspring and that placental hormonal secretions, independent from improper fetal growth, are an important determinant of risk of developing insulin resistance.

Quantification of Protein Expression by Proximity Ligation Assay in the Nonhuman Primate in Response to Estrogen.

In the field of protein biology, immunology-based techniques are continuously evolving for the detection and quantification of individual protein levels, protein-protein interaction, and protein modifications in cells and tissues. The proximity ligation assay (PLA), a method of detection that combines immunologic and PCR-based approaches, was developed to overcome some of the drawbacks that are inherent with other detection methods. The PLA allows for very sensitive and discretely quantifiable measures of unmodified, native protein levels and protein-protein interaction/modification complexes in situ in both fixed tissues and cultured cells. We describe herein the PLA method and its applicability to quantify the effects of estrogen on expression of angioregulatory factors, e.g., endothelial nitric oxide synthase (eNOS) in the vasculature, vascular endothelial growth factor (VEGF) in the placenta, and melanocortin 2 receptor (MC2R)/accessory protein (MRAP) in the fetal adrenal of the nonhuman primate.

Placental sFlt-1 Gene Delivery in Early Primate Pregnancy Suppresses Uterine Spiral Artery Remodeling.

Uterine spiral artery remodeling (SAR) is essential for promoting placental perfusion and fetal development. A defect in SAR results in placental ischemia and increase in placental expression and serum levels of the soluble fms-like tyrosine kinase-1 (sFlt-1) receptor that binds to and suppresses vascular endothelial growth factor (VEGF) bioavailability, thereby leading to maternal vascular dysfunction. We have established a nonhuman primate model of impaired SAR and maternal vascular dysfunction by prematurely elevating estradiol levels in early baboon pregnancy. However, it is unknown whether this primate model of defective SAR involves an increase in placental expression of sFlt-1, which may suppress VEGF bioavailability and thus SAR in the first trimester. Therefore, to establish the role of sFlt-1 in early pregnancy, SAR was quantified in baboons treated on days 25 through 59 of gestation (term = 184 days) with estradiol or with the sFlt-1 gene targeted selectively to the placental basal plate by ultrasound-mediated/microbubble-facilitated gene delivery technology. Placental basal plate sFlt-1 protein expression was 2-fold higher (P < 0.038) and the level of SAR for vessels > 25 µm in diameter was 72% and 63% lower (P < 0.01), respectively, in estradiol-treated and sFlt-1 gene-treated baboons than in untreated animals. In summary, prematurely elevating estradiol levels or sFlt-1 gene delivery increased placental basal plate sFlt-1 protein expression and suppressed SAR in early baboon pregnancy. This study makes the novel discovery that in elevated levels sFlt-1 has a role both in suppressing SAR in early primate pregnancy and maternal vascular endothelial function in late gestation.

Maternal systemic vascular dysfunction in a primate model of defective uterine spiral artery remodeling.

Uterine spiral artery remodeling (UAR) is essential for placental perfusion and fetal development. A defect in UAR underpins placental ischemia disorders, e.g., preeclampsia, that result in maternal systemic vascular endothelial dysfunction and hypertension. We have established a model of impaired UAR by prematurely elevating maternal serum estradiol levels during the first trimester of baboon pregnancy. However, it is unknown whether this experimental paradigm is associated with maternal vascular endothelial dysfunction. Therefore, in the present study baboons were administered estradiol on days 25-59 of gestation to suppress UAR and maternal vascular function determined on day 165 (term = 184 days) peripherally and in skeletal muscle, which accounts for over 40% of body mass and 25% of resting systemic vascular resistance. Maternal serum sFlt-1 levels were 2.5-fold higher (P < 0.05), and skeletal muscle arteriolar endothelial nitric oxide synthase (eNOS) protein expression and luminal area, and skeletal muscle capillary density were 30-50% lower (P < 0.05) in UAR suppressed baboons. Coinciding with these changes in eNOS expression, luminal area, and capillary density, maternal brachial artery flow-mediated dilation and volume flow were 70% and 55% lower (P < 0.05), respectively, and mean arterial blood pressure 29% higher (P < 0.01) in UAR defective baboons. In summary, maternal vascular function was disrupted in a baboon model of impaired UAR. These results highlight the translational impact of this primate model and relevance to adverse conditions of human pregnancy underpinned by improper uterine artery transformation.NEW & NOTEWORTHY Maternal vascular dysfunction is a hallmark of abnormal human pregnancy, particularly early-onset preeclampsia, elicited by impaired UAR. The present study makes the novel discovery that maternal systemic vascular dysfunction was induced in a baboon experimental model of impaired UAR. This study highlights the translational relevance of this nonhuman primate model to adverse conditions of human pregnancy underpinned by defective UAR.

Vascular Endothelial Growth Factor Delivery to Placental Basal Plate Promotes Uterine Artery Remodeling in the Primate.

Extravillous trophoblast (EVT) uterine artery remodeling (UAR) promotes placental blood flow, but UAR regulation is unproven. Elevating estradiol (E2) in early baboon pregnancy suppressed UAR and EVT vascular endothelial growth factor (VEGF) expression, but this did not prove that VEGF mediated this process. Therefore, our primate model of prematurely elevating E2 and contrast-enhanced ultrasound cavitation of microbubble (MB) carriers was used to deliver VEGF DNA to the placental basal plate (PBP) to establish the role of VEGF in UAR. Baboons were treated on days 25 to 59 of gestation (term, 184 days) with E2 alone or with E2 plus VEGF DNA-conjugated MBs briefly infused via a maternal peripheral vein on days 25, 35, 45, and 55. At each of these times an ultrasound beam was directed to the PBP to collapse the MBs and release VEGF DNA. VEGF DNA-labeled MBs per contrast agent was localized in the PBP but not the fetus. Remodeling of uterine arteries >25 µm in diameter on day 60 was 75% lower (P < 0.001) in E2-treated (7% ± 2%) than in untreated baboons (30% ± 4%) and was restored to normal by E2/VEGF. VEGF protein levels (signals/nuclear area) within the PBP were twofold lower (P < 0.01) in E2-treated (4.2 ± 0.9) than in untreated (9.8 ± 2.8) baboons and restored to normal by E2/VEGF (11.9 ± 1.6), substantiating VEGF transfection. Thus, VEGF gene delivery selectively to the PBP prevented the decrease in UAR elicited by prematurely elevating E2 levels, establishing the role of VEGF in regulating UAR in vivo during primate pregnancy.

Estrogen Suppresses Interaction of Melanocortin 2 Receptor and Its Accessory Protein in the Primate Fetal Adrenal Cortex.

We have shown that fetal adrenal fetal zone (FZ) volume and serum dehydroepiandrosterone sulfate (DHAS) levels were increased, whereas definitive and transitional zone (DZ/TZ) volume was unaltered, in baboons in which estrogen levels were suppressed by the administration of the aromatase inhibitor letrozole. The interaction of the melanocortin 2 receptor (MC2R) with its accessory protein (MRAP) is essential for trafficking MC2R to the adrenal cell surface for binding to ACTH. The present study determined whether the estrogen-dependent regulation of fetal adrenocortical development is mediated by ACTH and/or expression/interaction of MC2R and MRAP. Fetal pituitary proopiomelanocortin mRNA and plasma ACTH levels and fetal adrenal MC2R-MRAP interaction were assessed in baboons in which estrogen was suppressed/restored by letrozole/letrozole plus estradiol administration during the second half of gestation. Although fetal pituitary proopiomelanocortin and plasma ACTH levels and fetal adrenal MC2R and MRAP protein levels were unaltered, MC2R-MRAP interaction was 2-fold greater (P < .05) in the DZ/TZ in letrozole-treated baboons than in untreated animals and restored by letrozole plus estradiol treatment. We propose that the increasing levels of estradiol with advancing pregnancy suppress interaction of MC2R with MRAP, thereby diminishing MC2R movement to the cell membrane in the DZ/TZ. This would be expected to reduce progenitor cell proliferation in the DZ and migration to the FZ, thereby restraining FZ growth and DHAS production to maintain fetal adrenal DHAS and placental estradiol levels in a physiological range late in gestation.

Assessment of Protein Expression by Proximity Ligation Assay in the Nonhuman Primate Endometrium, Placenta, and Fetal Adrenal in Response to Estrogen.

In the field of protein biology, immunology-based techniques have been evolving for detection and quantification of protein levels, protein-protein interaction, and protein modifications in cells and tissues. The proximity ligation assay (PLA), a method of detection that combines immunologic and PCR-based approaches, was developed to overcome some of the drawbacks that are inherent to other detection methods. The PLA allows for very sensitive and discretely quantifiable measures of unmodified, native protein levels, and protein-protein interaction/modification complexes in situ in both fixed tissues and cultured cells. We describe herein the PLA method and its applicability to quantify the effects of estrogen on expression of angioregulatory factors, e.g., angiopoietin-1 (Ang-1) in the endometrium, vascular endothelial growth factor (VEGF) in the placenta, and melanocortin 2 receptor (MC2R)/accessory protein (MRAP) in the fetal adrenal of the nonhuman primate.

Prematurely elevating estradiol in early baboon pregnancy suppresses uterine artery remodeling and expression of extravillous placental vascular endothelial growth factor and α1ß1 and α5ß1 integrins.

We previously showed that advancing the increase in estradiol levels from the second to the first third of baboon pregnancy suppressed placental extravillous trophoblast (EVT) invasion and remodeling of the uterine spiral arteries. Cell culture studies show that vascular endothelial cell growth factor (VEGF) plays a central role in regulating EVT migration and remodeling of the uterine spiral arteries by increasing the expression/action of certain integrins that control extracellular matrix remodeling. To test the hypothesis that the estradiol-induced reduction in vessel remodeling in baboons is associated with an alteration in VEGF and integrin expression, extravillous placental VEGF and integrin expression was determined on d 60 of gestation (term is 184 d) in baboons in which uterine artery transformation was suppressed by maternal estradiol administration on d 25-59. EVT uterine spiral artery invasion was 5-fold lower (P < 0.01), and VEGF protein expression, quantified by in situ proximity ligation assay, was 50% lower (P < 0.05) in the placenta anchoring villi of estradiol-treated than in untreated baboons. α1ß1 and α5ß1 mRNA levels in cells isolated by laser capture microdissection from the anchoring villi and cytotrophoblastic shell of estradiol-treated baboons were over 2-fold (P < 0.01) and 40% (P < 0.05) lower, respectively, than in untreated animals. In contrast, placental extravillous αvß3 mRNA expression was unaltered by estradiol treatment. In summary, extravillous placental expression of VEGF and α1ß1 and α5ß1 integrins was decreased in a cell- and integrin-specific manner in baboons in which EVT invasion and remodeling of the uterine spiral arteries were suppressed by prematurely elevating estradiol levels in early pregnancy. We propose that estrogen normally controls the extent to which the uterine arteries are transformed by placental EVT in primate pregnancy by regulating expression of VEGF and particular integrin extracellular remodeling molecules that mediate this process.

Expression of P-450 aromatase, estrogen receptor α and ß, and α-inhibin in the fetal baboon testis after estrogen suppression during the second half of gestation.

Expression of the molecules that modulate the synthesis and action of estrogen in, or reflect function of, Sertoli cells was determined in the fetal testis of baboons in which estrogen levels were suppressed in the second half of gestation to determine whether this may account for the previously reported alteration in fetal testis germ cell development. P-450 aromatase, estrogen receptor (ER) ß, and α-inhibin protein assessed by immunocytochemistry was abundantly expressed in Sertoli cells of the fetal baboon testis, but unaltered in baboons in which estrogen levels were suppressed by letrozole administration. Moreover, P-450 aromatase and ERα and ß mRNA levels, assessed by real-time RT-PCR, were similar in germ/Sertoli cells and interstitial cells isolated from the fetal testis of untreated and letrozole-treated baboons. These results indicate that expression of the proteins that modulate the formation and action of estrogen in, and function of, Sertoli cells is not responsible for the changes in germ cell development in the fetal testis of estrogen-deprived baboons.

Divergent regulation of angiopoietin-1 and -2, Tie-2, and thrombospondin-1 expression by estrogen in the baboon endometrium.

Estrogen has an important role in the reconstruction of a new vascular network in the endometrium during each menstrual cycle; however, the underlying mechanisms are incompletely understood. Angiopoietin-1 (Ang-1) promotes vessel assembly, whereas Ang-2 and thrombospondin-1 (TSP-1) cause vessel breakdown. To determine the potential effect of estrogen on the expression of these angioregulatory factors in the endometrium, Ang-1, Ang-2, TSP-1, and Tie-2 receptor mRNA levels were assessed by real-time reverse transcriptase polymerase chain reaction in glandular epithelial and stromal cells isolated from the endometrium of ovariectomized baboons treated acutely with estradiol. Corresponding protein expression was assessed by immunocytochemistry and the proximity ligation assay (PLA) during advancing stages of the baboon menstrual cycle. Serum estradiol levels in ovariectomized baboons were 400 pg/ml within 4-6 hr of estradiol treatment. Ang-1 mRNA levels in glandular epithelial cells increased threefold (P < 0.01) within 4 hr of estradiol administration. In contrast, TSP-1 mRNA levels decreased four- to fivefold (P < 0.01) in endometrial glandular epithelial and stromal cells 4-6 hr after estradiol, whereas Ang-2 and Tie-2 expression was unaltered. Immunostaining for Ang-1 increased, TSP-1 decreased, and Ang-2 and Tie-2 were unaltered in the endometrium during the secretory compared with the proliferative phase of the cycle. Endometrial Ang-1 protein expression, quantified by PLA, increased 10-fold (P < 0.05) between the early proliferative and late proliferative/mid-secretory phases of the menstrual cycle in association with the rise in estrogen. In summary, estrogen induced a rapid, divergent, and cell-specific change in expression of angiostimulatory and angioinhibitory growth factors in the endometrium of the nonhuman primate.

Estrogen stimulates the human endometrium to express a factor(s) that promotes vascular smooth muscle cell migration as an early step in microvessel remodeling.

Vascular smooth muscle cell (VSMC) migration is a pivotal early step in blood vessel remodeling; however, very little is known about the regulation of this process in the human endometrium during the menstrual cycle. In this study, explants of human endometrium were incubated with estradiol and/or progesterone and the conditioned medium (CM) applied to cultures of VSMC to test the hypothesis that estrogen and progesterone stimulate endometrial cells to secrete a factor(s) that promotes VSMC migration. Endometrial explants were composed of highly organized glands and stroma. VSMC migration (cells migrated in 21 h/mm(2) fibronectin-coated semipermeable membrane) in the presence of CM from human endometrial explants obtained in the proliferative phase of the menstrual cycle and incubated for 24 h with estradiol was approximately threefold greater (P < 0.001) than with medium alone and greater (P < 0.05) than with CM from explants treated with estradiol plus progesterone or progesterone. It is concluded, therefore, that estrogen stimulates endometrial secretion of a factor(s) that promotes VSMC migration as an early step in vessel remodeling within the endometrium.

Regulation of placental villous angiopoietin-1 and -2 expression by estrogen during baboon pregnancy.

We recently showed an increase in vascular endothelial growth factor (VEGF), decrease in angiopoietin-1 (Ang-1) and unaltered Ang-2 expression by the villous placenta with advancing baboon pregnancy. Moreover, placental VEGF expression was increased by estrogen in early pregnancy. In the present study, we determined whether placental Ang-1 and Ang-2 are regulated by estrogen. Ang-1 and Ang-2 mRNA and protein were determined by RT-PCR and immunocytochemistry in the placenta of baboons on Day 60 of gestation (term is 184 days) after administration of estrogen precursor androstenedione on Days 25-59 or on Day 54 after acute estradiol administration. Chronic androstenedione treatment increased serum estradiol levels three-fold (P < 0.001) and decreased (P < 0.05) villous cytotrophoblast Ang-1 mRNA to a level (0.36 +/- 0.08 relative to 18S rRNA) that was one-third of that in untreated animals (0.98 +/- 0.26). Within 2 hr of estradiol administration, cytotrophoblast Ang-1 mRNA was decreased to a level (0.24 +/- 0.05) one-fifth (P < 0.05) of that in untreated animals (1.14 +/- 0.23). However, Ang-2 mRNA levels were unaltered. Ang-1, Ang-2 and estrogen receptors alpha and beta protein were localized within villous cytotrophoblasts providing a mechanism for estrogen action at this site. In summary, estrogen increased VEGF, decreased Ang-1, and had no effect on Ang-2 expression within placental cytotrophoblasts during early baboon pregnancy. We propose that the estrogen-dependent differential regulation of these angioregulatory factors underpins the unique pattern of neovascularization established within the villous placenta during primate pregnancy.

Expression of estrogen receptors alpha and beta in the fetal baboon testis and epididymis.

Although studies in transgenic mice suggest that estrogen is important for development of the testis, very little is known about the potential role of estrogen in maturation of the primate fetal testis. Therefore, as a first step to determine whether estrogen regulates maturation of the fetal primate testis, we used immunocytochemistry to determine estrogen receptor (ER) alpha and beta expression in the fetal baboon testis. Second, we established methods to quantify ERbeta mRNA levels by competitive reverse transcription-polymerase chain reaction in Sertoli cells isolated by laser capture microdissection (LCM) from the fetal baboon testis. ERbeta protein expression was abundant in the nuclei of Sertoli, peritubular, and interstitial cells in baboon fetuses at mid (Day 100) and late (Day 165) gestation (term is 184 days). ERbeta mRNA level was 0.03 attomole/femtomole 18S rRNA in Sertoli cell nuclei and associated cytoplasm isolated by LCM. ERalpha was expressed in low level in seminiferous tubules and in moderate level in peritubular cells on Day 165. Germ cells expressed very little ERalpha or ERbeta protein, whereas the baboon fetal epididymis exhibited extensive ERalpha and ERbeta immunostaining at mid- and late gestation. In contrast to the robust expression of ERbeta, androgen receptor protein was not demonstrable within the cells of the seminiferous cords but was abundantly expressed in epididymal epithelial cells of the fetal baboon. In summary, the results of this study show that the fetal baboon testis and epididymis expressed the ERalpha and ERbeta, and we suggest that our nonhuman primate baboon model can be used to study the potential role of estrogen on maturation of the fetal testis.

Effect of estrogen on angiogenesis in co-cultures of human endometrial cells and microvascular endothelial cells.

BACKGROUND: We recently showed that vascular endothelial growth factor (VEGF) expression by endometrial glandular epithelial and stromal cells, and endometrial microvascular endothelial cell permeability, an early step in angiogenesis, were rapidly increased by estradiol (E(2)) administration to ovariectomized baboons. We proposed that estrogen promotes endometrial angiogenesis by regulating VEGF expression by glandular epithelial and stromal cells. In the present study, we developed a co-culture of human endometrial cells and microvascular endothelial cells to determine whether the regulatory role shown for estrogen on endometrial angiogenesis in vivo in the non-human primate would be demonstrable in vitro in the human. METHODS AND RESULTS: Human endometrial glandular epithelial and stromal cells were co-cultured with human myometrial microvascular endothelial cells (HMMECs) and E(2). HMMEC tube formation (means +/- SEM, % endothelial tube area/total endothelial cell area), an index of angiogenesis, was 65% (P < 0.05) and 2-fold (P < 0.01) greater in cells co-cultured with human glandular epithelial cells (54 +/- 7%) and glandular epithelial cells plus E(2) (66 +/- 11%), respectively, compared with medium (33 +/- 4%). In contrast, endothelial tube formation was not altered in HMMECs incubated with endometrial stromal cells (32 +/- 4%), stromal cells plus E(2) (36 +/- 2%) or E(2) (39 +/- 3%). CONCLUSIONS: We propose that estrogen, by regulating expression and secretion of angiogenic factors such as VEGF by glandular epithelial cells of the endometrium, regulates endometrial angiogenesis.

Acute temporal regulation of vascular endothelial growth/permeability factor expression and endothelial morphology in the baboon endometrium by ovarian steroids.

We recently showed that endometrial vascular endothelial growth/permeability factor (VEG/PF) mRNA expression was decreased by ovariectomy of baboons and restored by chronic administration of estrogen. However, it remains to be determined whether this effect of estrogen reflects genomic up-regulation of VEG/PF and leads to an increase in microvascular permeability, an early physiological event in angiogenesis. Therefore, we determined the temporal expression of VEG/PF mRNA in glandular epithelial and stromal cells isolated by laser capture microdissection from and width of microvascular paracellular clefts that regulate vessel permeability in the endometrium of ovariectomized baboons after acute estradiol and/or progesterone administration. Endometrial VEG/PF mRNA levels were increased in five of five animals within 2 h of estradiol administration and remained elevated at 4 and 6 h. The net increase in glandular epithelial (7.31 +/- 2.72 attomol/fmol 18S ribosomal rRNA) and stromal (3.13 +/- 0.36) cell VEG/PF mRNA levels after estradiol administration was over 8-fold (P < 0.05) and 2.6-fold (P < 0.01) greater, respectively, than after vehicle (0.90 +/- 0.30, glands and 1.20 +/- 0.33, stroma). In contrast, endometrial VEG/PF mRNA expression was unaltered by progesterone. After estradiol treatment, endometrial paracellular cleft width was increased (P < 0.01) from a mean (+/-SE) of 71.6 +/- 4.6 nm at 0 h to 101.1 +/- 6.4 nm at 6 h, whereas vehicle or progesterone had no effect. We suggest that estrogen has a major role in regulating VEG/PF synthesis and early events in angiogenesis in the primate endometrium.

Effect of estrogen on vascular endothelial growth/permeability factor expression by glandular epithelial and stromal cells in the baboon endometrium.

The ovarian steroid hormones, estrogen and progesterone, have important roles in establishing the new vascular bed within the endometrium during each menstrual cycle; however, little is known about the mechanisms underlying this process. We recently showed that mRNA and protein levels for the angiogenic factor vascular endothelial growth/permeability factor (VEG/PF) in endometrial glandular epithelial and stromal cells of baboons were decreased to very low levels by ovariectomy, and we proposed that the levels of estrogen and progesterone exhibited during the menstrual cycle regulate endometrial VEG/PF expression in the primate. To test this hypothesis, VEG/PF mRNA levels were determined by reverse transcription-polymerase chain reaction in glandular epithelial and stromal cells isolated by laser-capture microdissection from, and VEG/PF protein was determined by immunocytochemistry in the endometrium of baboons after ovariectomy and chronic administration of estradiol and progesterone in levels designed to replicate the hormonal profiles that are characteristic of the proliferative and secretory phases of the menstrual cycle. Administration of estradiol to ovariectomized baboons in levels that replicated the late-proliferative phase of the menstrual cycle (209 +/- 40 pg/ml serum) increased/restored VEG/PF mRNA to levels in the glands (5.57 +/- 1.53 amol/fmol 18S rRNA, P < 0.01) and stroma (2.61 +/- 1.57 amol/fmol 18S rRNA, P < 0.02) that were approximately 10-fold greater than those observed after ovariectomy alone (0.52 +/- 0.21 and 0.22 +/- 0.11 amol/fmol 18S rRNA, respectively) and were similar to those previously shown in intact baboons. Concomitant administration of estradiol and progesterone to ovariectomized baboons in levels that replicated the midsecretory phase of the menstrual cycle (44 +/- 15 pg/ml serum and 9.8 +/- 2.2 ng/ml serum, respectively) resulted in glandular epithelial (3.65 +/- 1.42 amol/fmol 18S rRNA) and stromal (1.25 +/- 0.77 amol/fmol 18S rRNA) VEG/PF mRNA levels that were not significantly different from those exhibited after ovariectomy or ovariectomy and estradiol treatment. Comparable results were obtained for VEG/PF mRNA expression in whole-endometrial tissue, although the relative 2-fold increase (P < 0.03) in VEG/PF mRNA levels induced by estrogen in mixed endometrial cells of ovariectomized baboons appeared to be less marked than that in isolated glandular epithelial and stromal cells. After ovariectomy, endometrial width (0.98 +/- 0.09 mm) was approximately one-third of that in intact baboons (3.58 +/- 0.32 mm), and endometrial VEG/PF protein expression was low. Estradiol restored endometrial width (3.00 +/- 0.12 mm, P < 0.01) and VEG/PF protein expression to normal. In summary, estrogen has a significant role in regulating and maintaining VEG/PF expression by glandular epithelial and stromal cells of the endometrium during the menstrual cycle.

Expression of vascular endothelial growth/permeability factor by endometrial glandular epithelial and stromal cells in baboons during the menstrual cycle and after ovariectomy.

Vascular endothelial growth/permeability factor (VEG/PF) has a crucial role in angiogenesis, and neovascularization is essential in preparing the uterine endometrium for implantation. However, the regulation of VEG/PF synthesis by particular cell types of the endometrium during the human menstrual cycle is not well understood. Therefore, in the present study the baboon was used as a nonhuman primate to determine the role of the ovary in vivo in endometrial VEG/PF expression. VEG/PF mRNA levels were quantified by competitive RT-PCR in whole uterine endometrium and in glandular epithelial and stromal cells isolated from the endometrium by laser capture microdissection of baboons during the normal menstrual cycle and after ovariectomy, which decreased serum estradiol and progesterone to undetectable levels. Mean (+/-SE) levels (attomoles per micrograms of total RNA) of the 323-bp VEG/PF mRNA product, which reflected collective expression of all VEG/PF isoforms, in whole endometrium were 785 and 727 +/- 158 during the mid and late follicular phases, respectively, and 1108 +/- 320 during the midcycle surge in serum estradiol. VEG/PF mRNA levels then declined briefly before increasing to 1029 +/- 365 attomoles/ micro g RNA during the late luteal phase of the menstrual cycle. VEG/PF mRNA levels (attomoles per femtomole of 18S rRNA) were similar in glandular epithelial (2.27 +/- 1.11) and stromal (2.54 +/- 0.70) cells at the midcycle estradiol peak and the midluteal phase of the menstrual cycle (2.34 +/- 1.30 and 1.49 +/- 0.53, respectively). Immunocytochemical expression of VEG/PF protein was abundant in glandular and luminal epithelium, stroma, and vascular endothelium. Endometrial vessel density and percent vascularized area, determined by morphometric image analysis, were similar during the various stages of the baboon menstrual cycle. After ovariectomy, VEG/PF mRNA levels (attomoles per femtomole of 18S rRNA) in the endometrial glands (0.52 +/- 0.21) and stroma (0.22 +/- 0.11) were decreased to values that were approximately 20% and 10% (P < 0.05), respectively, of those in intact baboons during the midcycle estrogen surge. Moreover, there was relatively little VEG/PF protein immunostaining in the endometrial glands, stroma, and vascular endothelium after ovariectomy. In summary, VEG/PF mRNA and protein expression in glandular epithelial and stromal cells were markedly suppressed after ovariectomy, indicating that synthesis of this angiogenic factor in these endometrial cells is dependent upon a product(s) secreted by the ovary. Moreover, endometrial VEG/PF expression remained relatively constant and thus was available as a component of the angiogenic system throughout the menstrual cycle, presumably to progressively promote vascular reconstruction of the endometrium.