Margaret K. McLaughlin

Nitric Oxide Produced by Endothelial Cells Increases Production of Eicosanoids Through Activation of Prostaglandin H Synthase

The endothelium serves many functional roles, including the modulation of vascular smooth muscle tone through the release of vasoactive agents such as nitric oxide (NO) and the eicosanoids. We proposed that NO produced by endothelial cells would increase the production of eicosanoids through enhanced expression and/or activation of prostaglandin H synthase. NO and eicosanoid synthesis were stimulated in a bovine coronary microvessel endothelial cell line with the calcium ionophore A23187 (1 mumol/L). Our data demonstrated the following: (1) A23187 stimulated NO synthesis along with prostacyclin and thromboxane production. (2) Inhibition of NO synthesis with NG-nitro-L-arginine methyl ester (0.1 mmol/L) significantly diminished both prostacyclin and thromboxane production. (3) Cells incubated with hemoglobin (2 micrograms/mL), which inactivates NO, decreased A23187-stimulated prostacyclin production, whereas cells incubated with superoxide dismutase (20 U/mL), which protects NO from superoxide anions, enhanced prostacyclin production. (4) Exogenous NO stimulated prostacyclin production. (5) The interaction of NO with prostacyclin persisted in the presence of excess exogenous arachidonic acid (100 mumol/L). (6) Cyclooxygenase activity in cell lysates increased in the first hour of NO stimulation. (7) NO stimulation of prostacyclin occurred within 1 hour and continued for 8 hours. (8) Neither constitutive nor inducible prostaglandin H synthase enzyme expression was altered by NO. (9) Cycloheximide (10 mumol/L) had no effect on A23187 stimulation of prostacyclin production. (10) Exogenous cGMP (10 mumol/L) or a phosphodiesterase inhibitor (1 mmol/L) did not affect prostacyclin production. These data indicate that stimulating synthesis of endogenous NO in cultured endothelial cells increased eicosanoid production through activation of prostaglandin H synthase.

Increased ascorbate radical formation and ascorbate depletion in plasma from women with preeclampsia: implications for oxidative stress.

There is evidence that oxidative stress accompanies preeclampsia and plasma ascorbate concentrations are reported to be decreased in the disorder. We tested the hypothesis that an ascorbate-oxidizing activity is increased in plasma from women with preeclampsia relative to normal pregnancy. Electron paramagnetic resonance (EPR) spectroscopy was used to determine (1) plasma functional reserves of ascorbate and total thiols, (2) temporal changes in ascorbate and thiol concentrations during incubation of whole blood in vitro, and (3) ascorbate radical signal kinetics in plasma after equalization of ascorbate concentrations. High-pressure liquid chromatography (HPLC) was used to measure plasma alpha-tocopherol. Ascorbate concentrations were 50% lower in preeclampsia relative to normal pregnancy plasma but thiols and alpha-tocopherol did not differ. The elapsed time prior to half-consumption of plasma ascorbate was decreased approximately three-fold during incubation of whole blood from preeclamptics. No concomitant decrease in thiols was evident. The initial ascorbate radical signal amplitude was greater in preeclampsia plasma and then, in contrast to normal pregnancy plasma, decreased progressively. The iron chelator, deferoxamine had no effect on plasma ascorbate radical formation. We conclude that an ascorbate-oxidizing activity is increased in preeclampsia plasma which might contribute to vascular dysfunction in the disorder.

Uteroplacental insufficiency lowers the threshold towards hypoxia-induced cerebral apoptosis in growth-retarded fetal rats.

Infants suffering uteroplacental insufficiency and hypoxic ischemic injury often demonstrate cerebral apoptosis. Our objective was to determine the global effects of uteroplacental insufficiency upon cerebral gene expression of the apoptosis related proteins Bcl-2 and Bax and their role in increasing vulnerability to hypoxia-induced cerebral apoptosis. We therefore caused uteroplacental insufficiency and growth retardation by performing bilateral uterine artery ligation upon pregnant rats 2 days prior to term delivery and elicited further perinatal fetal hypoxia by placing maternal rats in 14% FiO(2) 3 h prior to delivery. We quantified cerebral levels of Bcl-2 and Bax mRNA, lipid peroxidation, caspase-3 activity, and cAMP in control and growth retarded term rat pups that experienced either normoxia or hypoxia. Uteroplacental insufficiency alone caused a significant decrease in cerebral Bcl-2 mRNA levels without altering cerebral Bax mRNA levels, malondialdehyde levels, or caspase-3 activity. In contrast, uteroplacental insufficiency and subsequent fetal hypoxia significantly increased cerebral Bax mRNA levels, lipid peroxidation and caspase-3 activity; Bcl-2 mRNA levels continued to be decreased. Hypoxia alone increased cerebral cAMP levels, whereas uteroplacental insufficiency and subsequent hypoxia decreased cerebral cAMP levels. We speculate that the decrease in Bcl-2 gene expression increases the vulnerability towards cerebral apoptosis in fetal rats exposed initially to uteroplacental insufficiency and subsequent hypoxic stress.

NITRIC OXIDE BIOSYNTHESIS DURING PREGNANCY: IMPLICATIONS FOR CIRCULATORY CHANGES

1. The biosynthesis of NO and its second messenger, cGMP, increases from pre‐pregnant levels during rat gestation. An increase in plasma level and urinary excretion of cGMP is also evident during human pregnancy. However, the relative contribution of the maternal vasculature and other tissues to increased NO and cGMP biosynthesis during gestation is uncertain. Consensus is lacking about the contribution of NO to reduced maternal vascular tone and reactivity during gestation in various organ beds; clearly, further investigation is still needed. That NO may also regulate vascular smooth muscle behaviour during pregnancy by altering membrane potential is another intriguing possibility.

Using women's health research to develop women leaders in academic health sciences: the National Centers of Excellence in Women's Health.

While the number of women entering U.S. medical schools has risen substantially in the past 25 years, the number of women in leadership positions in academic medicine is disproportionately small. The traditional pathway to academic leadership is through research. Womens health research is an ideal venue to fill the pipeline with talented women physicians and scientists who may become academic leaders in positions where they can promote positive change in womens health as well as mentor other women. The Office on Womens Health (OWH) in the U.S. Department of Health and Human Services has contracted with 18 academic medical centers to develop National Centers of Excellence in Womens Health. Emphasizing the integral link between womens health and women leaders, each of the Centers of Excellence must develop a leadership plan for women in academic medicine as part of the contract requirements. This paper describes the training programs in womens health research that have developed at five of the academic medical centers: the University of Wisconsin, Magee Womens Hospital, the University of Maryland, Medical College of Pennsylvania Hahnemann University, and the University of Illinois at Chicago. We discuss some of the challenges faced for both initiation and future viability of these programs as well as criteria by which these programs will be evaluated for success.

Mishandling of copper by albumin: Role in redox-cycling and oxidative stress in preeclampsia plasma

Objective. To test the hypothesis that enhanced oxidative stress during pregnancies complicated by preeclampsia is associated with improper copper (Cu) binding by plasma albumin, resulting in enhanced Cu redox-cycling activity and that altered Cu binding, in turn, is caused by interactions of excessive amounts of free fatty acids with albumin. Study Design. We studied binding and redox-cycling activity of Cu in 17 normal pregnancy and 17 preeclampsia plasma samples. Binding of exogenous Cu in plasma samples was quantified indirectly using spectrophotometric measurements of its complex with a specific chelator of Cu(I), bathocuproine disulfonate. Redox-cycling activity of Cu in plasma samples was estimated by electron paramagnetic resonance (EPR) spectroscopy of ascorbate radicals formed during one-electron oxidation of ascorbate by redox-active catalytic Cu. Formation of Cu/albumin complexes in model systems in the presence and absence of fatty acids was studied using EPR spectroscopy of Cu(II)/albumin. Results. We found that preeclampsia plasma (as compared to normal pregnancy plasma) (1) displays elevated endogenous ascorbate redox-cycling that is normalized by a Cu(II) chelator, cuprizone I, (2) has lowered capacity to bind and redox-regulate exogenously added Cu, and (3) responds to treatment with fatty-acid-free albumin by diminished ascorbate oxidizing activity. Conversely, addition of free fatty acid (oleic acid) to normal pregnancy plasma sample yields increased ascorbate redox-cycling activity. Our model experiments showed that Cu-dependent redox-cycling activity of purified human serum albumin is significantly increased by excess free fatty acids. Conclusion. Mishandling of Cu by albumin contributes to oxidative stress in preeclampsia. Cu chelators may represent promising mechanism-based antioxidants to attenuate oxidative stress in preeclampsia.

Biphasic stimulation of prostacyclin by endogenous nitric oxide (NO) in endothelial cells transfected with inducible NO synthase

Nitric oxide (NO) regulates prostaglandin H synthase (PGHS) activity in various cell types, but reports conflict in regard to its stimulatory versus inhibitory role. Murine lung endothelial cells infected with a retroviral vector expressing the human inducible NO synthase gene were used to prevent ambiguous effects of NO from either exogenous chemical donors or cytokine-stimulated cells. Low concentrations of endogenous NO led to a dose-dependent increase in 6-keto PGF1alpha production (p < 0.05), whereas the highest production of NO resulted in lower 6-keto PGF1alpha production. These data demonstrate a complex regulation of PGHS activity by NO that needs to be considered when proposing a physiological or pathophysiological role for NO.

tert-butyl hydroperoxide/hemoglobin-induced oxidative stress and damage to vascular smooth muscle cells: Different effects of nitric oxide and nitrosothiols

The goal of the present work was to determine whether nitric oxide (NO) released from different donors (NONOates and nitrosothiols) can act as a protective antioxidant against oxidative stress and cytotoxicity induced by extracellular hemoglobin/tert-butyl hydroperoxide (Hb/tert-BuOOH) in vascular smooth muscle cells (VSMCs). No changes in phospholipid composition were found in VSMCs incubated with oxyhemoglobin (oxyHb)/tert-BuOOH. Using our newly developed HPLC-fluorescence technique for measurement of site-specific oxidative stress in membrane phospholipids, we produced VSMCs in which endogenous phospholipids were metabolically labeled with an oxidation-sensitive fluorescent fatty acid, cis-parinaric acid. In these cells, we were able to reliably quantitate oxidative stress in major phospholipid classes-phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylinositol-induced by tert-BuOOH in the presence of oxyHb or methemoglobin (metHb). The oxidative stress was accompanied by cytotoxic effects of oxyHb/tert-BuOOH and metHb/tert-BuOOH on VSMCs. We further found that an NO donor, (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen 1-ium-1,2-diolate (PAPANONO), but not nitrosothiols, protected VSMCs against oxidative stress and cytotoxicity induced by Hb/tert-BuOOH. The protective effect of PAPANONO was most likely due to its ability to form NO-heme Hb (detectable by low temperature EPR spectroscopy and visible spectrophotometry). These findings are important for further understanding the physiological antioxidant role of NO against oxidative stress induced by hemoproteins as well as for pathological hypertensive events induced by extracellular Hb via NO depletion.

Altered active but not passive properties of mesenteric resistance arteries from the vitamin E-deprived rat

We tested the hypothesis that lowering antioxidant protection through dietary vitamin E deprivation would alter active and passive mechanical properties in resistance arteries of the rat. Specifically, we hypothesized that vascular tone in isolated mesenteric arteries of the vitamin E‐deprived rats would be altered due to impaired endothelial influences of nitric oxide and/or prostaglandins. Lumen diameter and wall thickness were measured in pressurized arteries (≈amp;250 μm diameter) from control (n=9) and vitamin E deprived (n=9) Sprague‐Dawley female rats by use of a dimension analysing system. Treatment with a cyclo‐oxygenase inhibitor (meclofenamate) did not affect the basal vascular tone in either group. Treatment with a nitric oxide synthase inhibitor (NG‐methyl‐L‐arginine) caused a significant increase in basal tone only in the vitamin E‐deprived rats (% tone: 6.2±1.1 vs 1.2±0.3%; P<0.05). When tone was induced to 25% of the initial diameter with phenylephrine, treatment with the nitric oxide synthase inhibitor resulted in a greater potentiated tone in the vitamin E‐deprived rats compared to the controls (26.5±2.7 vs 16.4±3.4%; P<0.05); suggesting a greater nitric oxide affect in the vessels from the vitamin E‐deprived rats. Meclofenamate treatment in the induced tone arteries significantly relaxed (−17.4±4.0%; P<0.05) only the arteries from the vitamin E‐deprived rats, indicating that a vasoconstrictor was modifying tone. The passive characteristics of distensibility and stress‐strain relationship were not different between the two groups of rats. In summary, vitamin E deprivation in the rat enhanced the modulation of vascular tone by both the nitric oxide and cyclo‐oxygenase pathways but did not alter passive characteristics of mesenteric arteries.

Human Chorionic Plate Arteries Lack an Endothelium-Dependent Relaxation Response

This study was designed to demonstrate that human chorionic plate arteries exhibit endothelium-dependent relaxation. Vessel ring segments (2mm long) were studied in a wire myograph system in which force and vessel dimensions were measured. The calcium ionophore A23187 (n = 10), or histamine (n = 2) was added to precontracted arteries in concentrations known to stimulate release of endothelium-derived relaxing factor(s) in umbilical vessels (10−8 M-10−6 M). A23187 did not cause relaxation of these arteries, while 10−5 M histamine enhanced contraction. The vessels exhibited concentration-dependent relaxation in response to the addition of nitric oxide (n = 3), and relaxed completely upon addition of 10−6 M sodium nitroprusside (n = 8), indicating a functional guanylate cyclase in the smooth muscle.