Terrance M. Phernetton

Endothelial vasodilator production by uterine and systemic arteries. III. Ovarian and estrogen effects on NO synthase

During the follicular phase of the ovarian cycle, when the local estrogen-to-progesterone ratio is elevated, uterine blood flow is elevated. This vasodilatory response is reproduced by exogenous 17beta-estradiol (E2beta) administration via a nitric oxide (NO)-mediated mechanism. We hypothesized that endogenous ovarian estrogen and exogenous E2beta treatment elevate expression of endothelial cell-derived NO synthase (eNOS) in uterine, but not in systemic, arteries. Uterine, mammary, and systemic (renal and/or omental) arteries were collected from 1) ewes synchronized to the follicular (day -1 to day 0) or luteal (day 10) phases of the ovarian cycle (n = 4 per phase), 2) ovariectomized ewes 120 min after systemic vehicle or E2beta (5 micrograms/kg iv) treatment, and 3) ovariectomized ewes on days 0, 3, 6, 8, and 10 of E2beta (5 micrograms/kg iv, followed by 6 micrograms/kg per day) treatment. Expression of eNOS was localized primarily to the endothelium rather than vascular smooth muscle (VSM) in all arteries examined by immunohistochemistry and Western analysis; inducible NOS was not detected in either endothelium or VSM. Expression of eNOS protein was greater (P < 0.05) in uterine, but not in systemic, artery endothelium-isolated protein collected from follicular versus luteal phase ewes. Acute systemic E2beta treatment of ovariectomized ewes increased (P < 0.05) eNOS protein levels in uterine artery endothelium. Prolonged E2beta administration progressively increased uterine, but not systemic, artery endothelial eNOS protein expression. Therefore, the increased local estrogen-to-progesterone ratio during the follicular phase locally elevates eNOS expression, which possibly elevates uterine blood flow. These responses can be partly reproduced with E2beta administration.During the follicular phase of the ovarian cycle, when the local estrogen-to-progesterone ratio is elevated, uterine blood flow is elevated. This vasodilatory response is reproduced by exogenous 17β-estradiol (E2β) administration via a nitric oxide (NO)-mediated mechanism. We hypothesized that endogenous ovarian estrogen and exogenous E2β treatment elevate expression of endothelial cell-derived NO synthase (eNOS) in uterine, but not in systemic, arteries. Uterine, mammary, and systemic (renal and/or omental) arteries were collected from 1) ewes synchronized to the follicular ( day -1 to day 0) or luteal ( day 10) phases of the ovarian cycle ( n = 4 per phase), 2) ovariectomized ewes 120 min after systemic vehicle or E2β (5 μg/kg iv) treatment, and 3) ovariectomized ewes on days 0, 3, 6, 8, and 10 of E2β (5 μg/kg iv, followed by 6 μg/kg per day) treatment. Expression of eNOS was localized primarily to the endothelium rather than vascular smooth muscle (VSM) in all arteries examined by immunohistochemistry and Western analysis; inducible NOS was not detected in either endothelium or VSM. Expression of eNOS protein was greater ( P < 0.05) in uterine, but not in systemic, artery endothelium-isolated protein collected from follicular versus luteal phase ewes. Acute systemic E2β treatment of ovariectomized ewes increased ( P < 0.05) eNOS protein levels in uterine artery endothelium. Prolonged E2β administration progressively increased uterine, but not systemic, artery endothelial eNOS protein expression. Therefore, the increased local estrogen-to-progesterone ratio during the follicular phase locally elevates eNOS expression, which possibly elevates uterine blood flow. These responses can be partly reproduced with E2β administration.

Systemic and uterine blood flow distribution during prolonged infusion of 17β-estradiol

Prolonged 17beta-estradiol (E2beta) infusion decreases mean arterial pressure (MAP) and systemic vascular resistance (SVR) while increasing heart rate (HR) and cardiac output (CO). It is unclear, however, which systemic vascular beds show increases in perfusion. The purpose of this study was to determine which reproductive and nonreproductive vascular beds exhibit alterations in vascular resistance and blood flow during prolonged E2beta infusion. Nonpregnant, ovariectomized sheep received either vehicle (n = 6) or E2beta (5 microg/kg iv bolus followed by 6 microg/kg over 24 h for 10 days; n = 9), and blood flow distribution was evaluated using radiolabeled microspheres at control and 120 min and 3, 6, 8, and 10 days of infusion. During E2beta infusion MAP (87 +/- 5 mmHg; mean +/- SE) decreased 3-9% and HR (83 +/- 5 beats/min) increased 4-31%. The combined baseline (control) perfusion to the uterus, broad ligament, oviducts, cervix, vagina, and mammary gland (reproductive blood flows) was 49 +/- 9 ml/min; at 120 min, E2beta increased flow (P < 0.001) to 605 +/- 74 ml/min (1,263%) and it remained elevated, but at a reduced rate, on day 3 (218 +/- 44 ml/min; 399%), day 6 (144 +/- 23; 217%), day 8 (181 +/- 19; 321%), and day 10 (204 +/- 48; 454%), accounting for only 3-17% of the E2beta-induced increase in CO. During this E2beta treatment, there also were significant decreases in vascular resistances leading to increases (P < 0.05) in blood flows to several nonreproductive (systemic) vascular beds including skin (32-113%), coronary (32-190%), skeletal muscle (25-133%), brain (21-292%), bladder (128-524%), spleen (87-180%), and pancreas (35-137%) vascular beds. Responses of these combined nonreproductive blood flows represent the major percentage (21-67%) of the E2beta-induced increase in CO. Vehicle infusion was without effect. We conclude that prolonged E2beta infusion increases reproductive and nonreproductive tissue blood flows. The latter appears to principally be responsible for the observed rise in CO and decrease in SVR.Prolonged 17β-estradiol (E2β) infusion decreases mean arterial pressure (MAP) and systemic vascular resistance (SVR) while increasing heart rate (HR) and cardiac output (CO). It is unclear, however, which systemic vascular beds show increases in perfusion. The purpose of this study was to determine which reproductive and nonreproductive vascular beds exhibit alterations in vascular resistance and blood flow during prolonged E2β infusion. Nonpregnant, ovariectomized sheep received either vehicle ( n = 6) or E2β (5 μg/kg iv bolus followed by 6 μg/kg over 24 h for 10 days; n= 9), and blood flow distribution was evaluated using radiolabeled microspheres at control and 120 min and 3, 6, 8, and 10 days of infusion. During E2β infusion MAP (87 ± 5 mmHg; mean ± SE) decreased 3-9% and HR (83 ± 5 beats/min) increased 4-31%. The combined baseline (control) perfusion to the uterus, broad ligament, oviducts, cervix, vagina, and mammary gland (reproductive blood flows) was 49 ± 9 ml/min; at 120 min, E2β increased flow ( P < 0.001) to 605 ± 74 ml/min (1,263%) and it remained elevated, but at a reduced rate, on day 3 (218 ± 44 ml/min; 399%), day 6 (144 ± 23; 217%), day 8(181 ± 19; 321%), and day 10 (204 ± 48; 454%), accounting for only 3-17% of the E2β-induced increase in CO. During this E2β treatment, there also were significant decreases in vascular resistances leading to increases ( P < 0.05) in blood flows to several nonreproductive (systemic) vascular beds including skin (32-113%), coronary (32-190%), skeletal muscle (25-133%), brain (21-292%), bladder (128-524%), spleen (87-180%), and pancreas (35-137%) vascular beds. Responses of these combined nonreproductive blood flows represent the major percentage (21-67%) of the E2β-induced increase in CO. Vehicle infusion was without effect. We conclude that prolonged E2β infusion increases reproductive and nonreproductive tissue blood flows. The latter appears to principally be responsible for the observed rise in CO and decrease in SVR.

Uterine blood flow responses to ICI 182 780 in ovariectomized oestradiol-17β-treated, intact follicular and pregnant sheep

Oestrogen dramatically increases uterine blood flow (UBF) in ovariectomized (Ovx) ewes. Both the follicular phase and pregnancy are normal physiological states with elevated levels of circulating oestrogen. ICI 182 780 is a pure steroidal oestrogen receptor (ER) antagonist that blocks oestrogenic actions in oestrogen‐responsive tissue. We hypothesized that an ER‐mediated mechanism is responsible for in vivo rises in UBF in physiological states of high oestrogen. The purpose of the study was to examine the effect of an ER antagonist on exogenous and endogenous oestradiol‐17β (E2β)‐mediated elevations in UBF. Sheep were surgically instrumented with bilateral uterine artery blood flow transducers, and uterine and femoral artery catheters. Ovx animals (n= 8) were infused with vehicle (35% ethanol) or ICI 182 780 (0.1–3.0 μg min−1) into one uterine artery for 10 min before and 50 min after E2β was given (1 μg kg−1i.v. bolus) and UBF was recorded for an additional hour. Intact, cycling sheep were synchronized to the follicular phase using progesterone, prostaglandin F2α(PGF2α) and pregnant mare serum gonadotrophin (PMSG). When peri‐ovulatory rises in UBF reached near peak levels, ICI 182 780 (1 or 2 μg (ml uterine blood flow)−1) was infused unilaterally (n= 4 sheep). Ewes in the last stages of pregnancy (late pregnant ewes) were also given ICI 182 780 (0.23–2.0 μg (ml uterine blood flow)−1; 60 min infusion) into one uterine artery (n= 8 sheep). In Ovx sheep, local infusion of ICI 182 780 did not alter systemic cardiovascular parameters, such as mean arterial blood pressure or heart rate; however, it maximally decreased ipsilateral, but not contralateral, UBF vasodilatory responses to exogenous E2β by ∼55–60% (P < 0.01). In two models of elevated endogenous E2β, local ICI 182 780 infusion inhibited the elevated UBF seen in follicular phase and late pregnant ewes in a time‐dependent manner by ∼60% and 37%, respectively; ipsilateral ≫ contralateral effects (P < 0.01). In late pregnant sheep ICI 182 780 also mildly and acutely (for 5–30 min) elevated mean arterial pressure and heart rate (P < 0.05). We conclude that exogenous E2β‐induced increases in UBF in the Ovx animal and endogenous E2β‐mediated elevations of UBF during the follicular phase and late pregnancy are partially mediated by ER‐dependent mechanisms.

Endothelial vasodilator production by ovine uterine and systemic arteries: ovarian steroid and pregnancy control of ERα and ERβ levels

Pregnancy and the follicular phase are physiological states of elevated oestrogen levels and rises in uterine blood flow (UBF). The dramatic increase in utero‐placental blood flow during gestation is required for normal fetal growth and development. Oestrogen exerts its vasodilatory effect by binding to its specific oestrogen receptors (ER) in target cells, resulting in increased expression and activity of endothelial nitric oxide synthase (eNOS) to relax vascular smooth muscle (VSM). However, the regulation of endothelial versus VSM ERα and ERβ expression in uterine arteries (UAs) during the ovarian cycle, pregnancy and with exogenous hormone replacement therapy (HRT) are currently unknown. ER mRNA and protein localization was determined by in situ hybridization (ISH) using 35S‐labelled riboprobes and immunohistochemistry (IHC), respectively. UA endothelial (UAendo), UA VSM, omental artery endothelium (OA endo), and OA VSM proteins were isolated and ERα and ERβ protein expression was determined by Western analysis. We observed by ISH and IHC that ERα and ERβ mRNA and protein were localized in both UAendo and UA VSM. Immunoblot data demonstrated ovarian hormone specific regulation of ERα and ERβ protein in UAendo and UA VSM. Compared to luteal phase sheep, both ERα and ERβ levels in UAendo were elevated in follicular phase sheep. Whereas ERβ was elevated by pregnancy in UAendo and UA VSM, ERα was not appreciably altered. eNOS was increased in UAendo from follicular and pregnant sheep. Ovariectomized ewes (OVEX) had substantially reduced UAendo ERβ, but not UAendo ERα or OAendo ERα and ERβ. In contrast, OVEX increased UA VSM ERα and ERβ and decreased OA VSM ERα and ERβ. Treatment with oestradiol‐17β (E2β), but not progesterone or their combination, increased UAendo ERα levels. The reduced ERβ in UAendo from OVEX ewes was reversed by E2β and progesterone treatment. While ERα and eNOS were not elevated in any other reproductive or non‐reproductive endothelia tested, ERβ was augmented by pregnancy in uterine, mammary, placenta, and coronary artery endothelia. ERα and ERβ mRNA and protein are expressed in UA endothelium with expression levels depending on the endocrine status of the animal, indicating UA endothelium is a target for oestrogen action in vivo, and that the two receptors appear to be differentially regulated in a spatial and temporal fashion with regard to the reproductive status or HRT.

Circulating levels of nitric oxide and vascular endothelial growth factor throughout ovine pregnancy

Nitric oxide (NO) production has been shown to increase uterine blood flow and be elevated in ewes carrying multiple fetuses during late gestation. Vascular endothelial growth factor (VEGF) has been reported to increase eNOS expression and NO production in endothelial cell cultures. As angiogenesis and vasodilatation of the uterine and placental vascular beds are important at all stages of pregnancy, it is important to understand how VEGF and NO change throughout gestation in circulation. Therefore the objectives of the current study were to evaluate the systemic levels of VEGF and NO metabolite (NOx) throughout ovine gestation and to determine if there was an effect of sheep carrying singletons versus multiple fetuses. NOx and VEGF concentrations were analysed in systemic blood from pregnant ewes starting on day 27 of pregnancy and at multiple intermittent intervals throughout pregnancy until term. Blood samples from non‐pregnant and postpartum ewes were also analysed. NOx concentrations in maternal blood expressed a biphasic pattern with NOx concentrations increasing (P < 0.05) over non‐pregnant values on days 40–69 of gestation, returning to non‐pregnant concentrations from days 70–100, and again increasing (P < 0.05) until term. Postpartum NOx concentrations were similar to non‐pregnant values. While ewes carrying multiple fetuses had increased (P < 0.05) concentrations of NOx on days 60–69, there were no differences in NOx concentrations in ewes carrying singletons or multiples from day 70–99 of gestation. Starting on day 100 and continuing throughout the duration of pregnancy, ewes carrying multiple fetuses had increased (P < 0.05) concentrations of NOx compared to ewes carrying singletons. Concentrations of VEGF showed a different pattern from NOx with VEGF decreasing (P < 0.05) from day 20–69 of pregnancy compared to non‐pregnant ewes. Concentrations of VEGF returned to non‐pregnant levels by day 70 and remained constant throughout the duration of pregnancy. On days 20–39, ewes carrying singleton fetuses had an increased VEGF concentration (P < 0.05), whereas ewes carrying multiple fetuses demonstrated elevated VEGF concentrations from day 90–109 of gestation. Concentrations from non‐pregnant and postpartum ewes did not differ (P > 0.1). While there was no effect of fetal number on circulating VEGF concentrations, circulating levels of NOx were substantially increased (P < 0.05) in ewes carrying multiple fetuses, compared to ewes carrying singletons. The pattern of the rise in NOx in circulating plasma was not directly associated with changes in VEGF regardless of the number of fetuses present. However, circulating concentrations of NOx and VEGF appear to, respectively, follow patterns of uterine blood flow and angiogenesis of the uterus. An understanding of these circulatory patterns may have important implications for fetal size, birth weight and fetal/developmental origins of adult disease.

Effect of nifedipine on fetal and maternal hemodynamics and blood gases in the pregnant ewe

OBJECTIVE Our purpose was to determine whether the fetal acidosis and hypoxia previously demonstrated in animal models with maternal nifedipine infusion is the result of a decrease in uteroplacental or fetoplacental blood flow and whether this effect is exacerbated by a higher drug concentration and duration of infusion. STUDY DESIGN Ten chronically instrumented pregnant ewes (gestational age 0.9 term, term = 145 days) received nifedipine infusions (n = 7) or vehicle (95% ethanol/water, 3:7) (n = 3). Three 90-minute periods were evaluated: 5 microg/kg/min infusion (low-dose nifedipine), no infusion, and 10 microg/kg/min (high-dose nifedipine). Paired maternal and fetal blood gases, glucose, lactate, and nifedipine levels were obtained every 30 minutes while hemodynamic parameters were monitored. We determined maternal and fetal blood flows using the radioactive microsphere technique. RESULTS Although maternal placental blood flows decreased by 25% during low-dose nifedipine (p < 0.05), this was only transient and there were no other decreases in uteroplacental or fetoplacental blood flow. Fetal blood flow increased to the adrenals and diaphragm with high-dose nifedipine (p < 0.05). Maternal and fetal lactate levels increased with both doses (p < 0.05). In addition, fetuses exhibited significant hypoxia (oxygen content fell 0.46 mmol/L) and acidosis (pH fell 0.06 units) throughout the nifedipine infusion and recovery period. Maternal heart rate increased transiently with both doses (p < 0.05); however, there were no changes in either fetal or maternal mean arterial pressure. Infusion of the vehicle alone did not alter maternal or fetal hemodynamics. Maternal and fetal plasma nifedipine levels reached steady-state by 30 minutes, and maternal/fetal ratios were 0.4 to 0.55. The maternal metabolic clearance rates for low- and high-dose nifedipine were 80.0 and 79.8 ml/min/kg, respectively. Maternal half-life calculation revealed a two-compartment model with a calculated half-life of 2.87 +/- 3.15 and 63.57 +/- 154.03 (+/-SD) minutes for the alpha and beta components, respectively. CONCLUSIONS Maternal nifedipine infusion is associated with hypoxia and acidosis in the sheep fetus, without persistent decreases in uteroplacental or fetoplacental blood flows or blood pressures. These fetal blood gas changes are more severe with high-dose nifedipine and longer duration of infusion and continue to deteriorate even when recovery is allowed. The deterioration of fetal blood gases is out of proportion to the transient decreases in uteroplacental blood flow and demonstrates that another mechanism for this fetal acidosis and hypoxia exists during nifedipine infusion.

Endothelial Vasodilator Production by Uterine and Systemic Arteries. VIII. Estrogen and Progesterone Effects on cPLA2, COX-1, and PGIS Protein Expression

Abstract During ovine pregnancy, when both estrogen and progesterone are elevated, prostacyclin (PGI2) production by uterine arteries and the key enzymes for PGI2 production, phospholipase A2 (cPLA2), cyclooxygenase 1 (COX-1), and prostacyclin synthetase (PGIS), are increased. This study was conducted to determine whether exogenous estradiol-17β (E2β) with or without progesterone (P4) treatment would increase cPLA2, COX-1, and PGIS protein expression in ovine uterine, mammary, and systemic (renal, mental, and coronary) arteries. Nonpregnant ovariectomized sheep received vehicle (n = 10), P4 (0.9-g controlled internal drug release vaginal implants; n = 13), E2β (5 μg/kg bolus followed by 6 μg kg−1 day−1; n = 10), or P4 + E2β (n = 12). Arteries were procured on Day 10, and cPLA2, COX-1, and PGIS protein were measured by Western immunoblot analysis in endothelial isolated proteins and vascular smooth muscle (VSM). The levels of cPLA2 was increased in uterine artery endothelium in ewes treated with P4 + E2β but was not altered by any steroid treatment in renal, coronary, mammary, or omental artery endothelium or in VSM of any evaluated artery. Similarly, COX-1 was increased in uterine artery endothelium with P4 + E2β but was not significantly altered by treatment in other endothelium or VSM. E2β treatment increased PGIS protein in uterine and renal artery endothelium but did not alter PGIS in other endothelial tissue. P4 increased PGIS expression in the uterine, mammary, omental, and renal artery VSM, and E2β increased PGIS expression in the uterine and omental artery VSM. Both E2β and P4 treatments differentially alter protein expression of the key enzymes involved in PGI2 production in different artery types and may play an important role in the control of blood flow redistribution during hormone replacement therapy.

Development and Use of an Ovarian Synchronization Model to Study the Effects of Endogenous Estrogen and Nitric Oxide on Uterine Blood Flow During Ovarian Cycles in Sheep

Abstract The objective of the current study was to develop an ovine animal model for consistent study of uterine blood flow (UBF) changes during synchronized ovarian cycles regardless of season. Sheep were surgically bilaterally instrumented with uterine artery blood flow transducers and 5–7 days later implanted with a vaginal progesterone (P4)-controlled internal drug-releasing device (CIDR; 0.3 g) for 7 days. On Day 6 of P4, sheep were given two prostaglandin F2α injections (7.5 mg i.m. 4 h apart). At CIDR removal, Experimental Day 0, zero (n = 9), 500 IU (n = 8), or 1000 IU (n = 7) eCG was injected i.m.; UBF was monitored continuously for 55–75 h. Jugular blood was sampled every 8 h to evaluate levels of P4, estradiol-17β (E2β) and luteinizing hormone (LH). The inhibitor of nitric oxide synthase, l- nitro-arginine methyl ester (l-NAME) was infused in a stepwise fashion unilaterally into one uterine artery at 48–50 h after 500 IU eCG and the effects on UBF were examined (n = 7). The zero-eCG group gradually increased UBF from a baseline of 17.4 ± 3.9 to 80.5 ± 1.1 ml/min. The 500-IU-eCG group increased UBF between 10 and 15 h from a baseline of 11 ± 3.3 to 83.3 ± 1.0 ml/min, whereas UBF for the 1000-IU-eCG group was higher (100.1 ± 1.7 ml/min) than that seen in either of the other groups. Plasma P4 fell to baseline within 8 h of CIDR removal, while E2β rose gradually in association with elevations in UBF. LH surges occurred between 32 and 56 h after CIDR removal and the LH surge occurred earlier in the 1000-IU-eCG group than the other two groups (P < 0.01). l-NAME infusion dose dependently reduced maximum levels of UBF ipsilaterally by 54.6% ± 6.2%, but contralaterally only by 27.4% ± 8.5%. Regardless of season, either dose of eCG will result in analogous UBF responses. During the follicular phase, elevations in UBF are in part locally controlled by the de novo production of nitric oxide.

The pathophysiology of the anophthalmic socket. Part II. Analysis of orbital fat.

The pathophysiologic mechanisms responsible for the clinical features of the anophthalmic socket are poorly understood. Atrophy of orbital fat has been thought to be a major contributing cause of enophthalmos and the superior sulcus deformities that develop after enucle-ation, but it has never been demonstrated histopathologically or confirmed by scientific analysis. This study was undertaken to investigate the changes that occur in the orbital fat compartment of the anophthalmic socket in an animal model by measuring orbital soft tissue mass and evaluating adipocyte cell size. Instead of reduction in the tissue mass, a statistically significant greater weight of the fat and connective tissue compartment was found in the anophthalmic orbit by nearly 13% compared to the control orbit in the animals in the long-term group. No significant change in the mean maximal diameter of adipocytes developed 7 months after enucleation. These analyses do not support the concept that orbital fat atrophy or a reduction of metabolic activity occurs in the anophthalmic socket in this animal model. From these results and our previous findings that the circulation dynamics and blood flow to orbital tissues do not change after enucleation, we propose that the pathophysiologic basis of the problems associated with anophthalmos is a disturbance in the spatial architecture and interrelationships of the multiple tissue components of the orbit, not a change in the orbital blood flow or development of fat atrophy.

Anomalous responses of tumor vasculature to norepinephrine and prostaglandin E2 in the rabbit.

SUMMARY We used 25-/im microspheres to compare blood flow to the V-2 carcinoma in the awake, unanesthetized rabbit with blood flow to other organs. Injection of norepinephrine (50 jig) into the left ventricle caused a 41fold [95% confidence interval = (25-69)] increase in tumor vascular resistance (P < 0.01). This was more than one order of magnitude greater than the increase in resistance in any other organ. Prostaglandin E2 (50 fig) injected into the left ventricle caused a 7-fold (4-13) increase in tumor vascular resistance (P < 0.01) and no significant increase of the vascular resistance of other organs. The change in tumor vascular resistance was not completely due to an increased level of circulating catecholamines because a 2-fold (1.6-3.4) increase in the resistance (P < 0.01) was seen when prostaglandin E, was injected into the left ventricle of animals pretreated with phenoxybenzamine. The prostaglandin E2-induced tumor vasoconstriction was not due to an increased level of circulating angiotensin II because in animals in which a and angiotensin receptors were blocked, prostaglandin E2 increased the tumor vascular resistance by a factor of 3 (2.3-5.5) (P < 0.01). The tumor vasculature appears to be hypersensitive to a-receptor activation and responds to prostaglandin E2 with vasoconstriction which cannot be accounted for by an increased level of circulating catecholamines or angiotensin II. In these experiments, the vasculature of the tumor responded to pharmacological agents in a manner that was not displayed by the vasculature of other organs. It may be possible to selectively control tumor blood flow without adversely affecting the blood flow to other organs of the host.