Quantification of spiral artery remodelling using an Adobe Photoshop-based technique.

Uterine spiral arteries undergo remodelling in normal pregnancy, with replacement of the musculoelastic arterial media by fibrinoid containing extravillous trophoblast cells. Deficient spiral artery remodelling is associated with several adverse pregnancy outcomes. Although there are distinct components of spiral artery remodelling, assessment is subjective and often based on an overall impression of morphology. We aimed to develop a quantitative approach for assessment of uterine spiral artery remodelling. Placental bed biopsies were immunostained using smooth muscle markers, digital images of spiral arteries were captured and Adobe Photoshop was used to analyse positive immunostaining. The method was then used to investigate variation in the same vessel at different levels within a paraffin block, and the effect of parity, pre-eclampsia or miscarriage on vascular smooth muscle cell content. Results were also compared with a more subjective morphology-based assessment system. There was good intra- and interobserver agreement and the method correlated well with the more subjective assessment system. There was an overall reduction in vascular smooth muscle, as detected by caldesmon 1 (h-caldesmon) immunopositivity, with increasing gestational age from 8 weeks to term. A previous pregnancy did not affect the amount of spiral artery smooth muscle. Comparison of pre-eclampsia and late miscarriage samples with controls of the appropriate gestational age demonstrated increased medial smooth muscle in pathological samples. This technique provides a simple, rapid, reproducible and inexpensive approach to quantitative assessment of spiral artery remodelling in normal and pathological human pregnancy, a process which although fundamental for successful pregnancy, is still incompletely understood.

The urokinase plasminogen activator (uPA) system in uterine natural killer cells in the placental bed during early pregnancy.

The urokinase plasminogen activator (uPA) system plays pivotal roles in cell invasion, adhesion and migration. Roles for uterine natural killer (uNK) cells in regulating extravillous trophoblast (EVT) invasion and spiral artery remodeling have been proposed. Placental bed biopsies from early pregnancy were obtained from three gestational age groups (8-10, 12-14 and 15-20 weeks). Total caseinase activity in the placental bed was studied using casein in situ zymography. Localisation of uPA, uPA receptor (uPAR), plasminogen activator inhibitor (PAI)-1 and -2 in the placental bed was investigated by immunohistochemistry. CD56(+) uNK cells were separated from collagenase-digested decidual cells using an immunomagnetic technique, and uPA activity was measured in isolated cell culture supernatants by casein/plasminogen gel zymography (8-10 and 12-14 weeks' gestation, n=10 each group). uPAR in cell lysates and PAI-1 and -2 secretion in supernatants were measured by Western blotting. Caseinase activity was stronger in decidua than myometrium as shown by in situ zymography. uPA localised strongly to uNK cells, especially at 8-10 weeks. Moderate uPAR localisation on uNK cells also observed. There was very weak immunostaining of uNK cells for PAI-1 and PAI-2. In casein gel zymography, uPA activity was similar in uNK cell culture supernatant compared with total unseparated decidual cells. uPAR in uNK cell lysates was significantly stronger than in total decidual cell lysates. PAI-1 and PAI-2 were not detected in uNK cell culture supernatants by Western blot analysis. These results suggest that uNK cells may regulate EVT invasion and spiral artery remodeling via the uPA system.

Localization of angiogenic growth factors and their receptors in the human placental bed throughout normal human pregnancy.

During early human pregnancy invasion of uterine spiral arteries by extravillous trophoblast cells contributes to their remodelling characterised by loss of musculo-elastic media and replacement by fibrinoid containing trophoblast. Despite its importance for successful pregnancy, the mechanisms underlying 'transformation' of spiral arteries are not well understood. The aim of this study was to localize expression of members of the angiopoietin (Ang) family (Ang-1, Ang-2 and their receptor Tie-2) and the vascular endothelial growth factor (VEGF) family (VEGF-A, VEGF-C, VEGF-D and their receptors VEGF-R1, VEGF-R2 and VEGF-R3) in the placental bed throughout normal human pregnancy. Placental bed biopsies were obtained from women undergoing elective termination of pregnancy at 8-10, 12-14 and 16-20 weeks' gestation and elective caesarean section at term (n=6 each group). Paraffin-embedded sections were immunostained for Ang-1, Ang-2, Tie-2, VEGF-A, VEGF-C, VEGF-D, VEGF-R1, VEGF-R2 and VEGF-R3 using an avidin biotin peroxidase technique. Reactivity of endovascular, interstitial, intramural and multinucleate extravillous trophoblast populations in the placental bed was analysed semi-quantitatively. There was an increase in the level of immunostaining of intramural EVT for Tie-2 and VEGF-C with increasing gestational age. In addition, there was a reduction in Ang-1 and Ang-2 expression by multinucleate interstitial EVT and of VEGF-R1 and VEGF-R2 by endovascular EVT with increasing gestational age. At the earlier gestational ages studied, immunostaining for Ang-1, Ang-2, Tie-2, VEGF-C, VEGF-R1 and VEGF-R2 on intramural EVT was reduced compared to both mononuclear interstitial and endovascular EVT. These findings suggest that the Ang and VEGF families may play a role in the process of spiral artery remodelling in normal pregnancy.

The responsiveness of isolated human hand vein endothelial cells in normal pregnancy and in pre-eclampsia.

1. Human hand vein endothelial cells were isolated from blood obtained by traumatic venepuncture. Cells were identified as endothelial by staining with endothelium-specific antibodies. The subject groups studied were (i) non-pregnant, (ii) pregnant (mean, 35 weeks gestation) and (iii) pre-eclamptic women (mean, 36 weeks gestation). 2. Fura-2 was used to measure agonist-induced responses in intracellular Ca2+ in single endothelial cells isolated and maintained in vitro. All of the cells examined responded to adenosine triphosphate (ATP) with a large transient increase in Ca2+ followed by a sustained plateau. 3. The responses to ATP were significantly larger in the cells from pregnant women than in those from non-pregnant and pre-eclamptic women, but no other differences were observed. The amplitudes of the responses to ATP were (means +/- s.e.m.) 0.56 +/- 0.04, 1.42 +/- 0.24 and 0.65 +/- 0.09 fura-2 ratio units for cells from non-pregnant, pregnant and pre-eclamptic subjects, respectively. 4. In cells isolated from non-pregnant subjects, the amplitude of the responses to carbachol, histamine and bradykinin were all smaller than those activated by ATP: 5.1, 13.9 and 4.4 %, respectively. Not all cells responded to these agonists: 25 % responded to carbachol, 70.5 % responded to histamine and 12.5 % responded to bradykinin. Sixty-five per cent of the cells from normotensive pregnant subjects responded to bradykinin compared with 25 % in the non-pregnant and 13.9 % in the pre-eclamptic subjects. 5. These data suggest that there may be differences in the responsiveness of venous endothelial cells in pregnancy and that pre-eclamptic cells behave differently.

Interactions between inositol trisphosphate and Ca2+ dependent Ca2+ release mechanisms on the endoplasmic reticulum of permeabilised bovine aortic endothelial cells.

In this paper we describe data from cultured bovine aortic endothelial (BAE) cells demonstrating a Ca2+ induced Ca2+ release (CICR) process which appears to have pharmacological properties different from CICR mechanisms in other cell types. CICR was measured in saponin permeabilised cells in which the internal stores had been preloaded with 45Ca2+. Step increases in the free Ca2+ concentration of the bathing solution, from 10 nM up to 10 microM were found to increase 45Ca2+ loss. This process was completely inhibited by ruthenium red. Caffeine induced a small release of 45Ca2+ and the response to a subsequent stimulation with a Ca2+ step was reduced. In intact cells, ryanodine activated small oscillations in intracellular Ca2+ in the presence, but not the absence, of external Ca2+. However, in permeabilised cells, ryanodine had no effect on either basal efflux or the increased efflux of 45Ca2+ seen following a step increase in free Ca2+. These data suggest the operation of a ruthenium red sensitive but ryanodine insensitive CICR mechanism on the endoplasmic reticulum (ER) which may also be modulated by caffeine. An IP3 dependent 45Ca2+ release was also observed. In the presence of ruthenium red, the IP3 induced 45Ca2+ release was reduced suggesting that CICR may operate to amplify the magnitude of the IP3 response. The Ca2+ dependence of the IP3 induced release was also measured. Co-operativity between IP3 and Ca2+ could not be detected between 100-300 nM Ca2+. The results suggest that the regulation of IP3 induced Ca2+ release may be different in BAE cells, and point to the operation of a 'novel' CICR process and to complex interactions between Ca2+ release systems in BAE cells.

The actions of caffeine and 2,3-butanedione monoxime on calcium transients in human vascular smooth muscle.

Ca2+ mobilization by membrane depolarization or histamine application, was measured in isolated human uterine artery smooth muscle cells. Nifedipine, a Ca2+ channel blocker, was found to inhibit the depolarization-induced increase in [Ca2+]i but not the histamine-induced increase. This suggests the presence of functional voltage-dependent Ca2+ channels and agonist-induced mobilization of Ca2+ via a different mechanism. Caffeine inhibited both the depolarization- and histamine-induced increases in intracellular calcium. The mechanisms of inhibition do not involve cAMP and point to a complex action of caffeine at multiple sites. The dephosphorylating agent 2,3-butanedione monoxime (BDM) was found to block voltage-dependent Ca2+ changes but not agonist-induced changes suggesting a role for phosphorylation in the regulation of the Ca2+ channels in these cells.

Repetitive transients in intracellular Ca2+ in cultured human vascular smooth muscle cells.

Human uterine vascular smooth muscle cells have been isolated and maintained in culture. When these cells are exposed to bathing solutions with nominally zero sodium, using potassium, N-methyl-D-glucamine or Tris as substitutes, repetitive transient increases in intracellular calcium are observed. These transients are abolished when the calcium concentration of the bathing solution is reduced to nominally zero suggesting a role for extracellular calcium in the activation or maintenance of the transients. The hypothesis is proposed that the underlying mechanism involves a calcium influx through the reversed operation of a sodium-calcium exchange mechanism and the cyclical activation of calcium-induced calcium release from the sarcoplasmic reticulum. Noradrenaline (10(-6) M) and caffeine (20-30 mM) reversibly inhibited the transients. The inhibitory action of these agents could not be mimicked by dibutyryl cAMP suggesting that cAMP does not mediate the inhibition. Caffeine alone had no effect on resting calcium. Thimerosal (1-100 microM), an agent thought to activate a second type of calcium-induced calcium release mechanism activated repetitive transient increases in intracellular calcium which behave in a similar manner to those activated by sodium removal. These data are consistent with the presence of a thimerosal-activated calcium-induced calcium release mechanism in these cultured human cells. It is proposed that this mechanism is different from the calcium-induced calcium release mechanism, described in other cell types, which is activated by caffeine.

Transients in intracellular free calcium in subconfluent and confluent cultures of a rat smooth muscle cell line.

The Ca2+ mobilizing mechanisms in the smooth muscle cell line A7r5 were found to undergo changes related to the degree of confluence of the cultures. In sparse cultures resting calcium was stable and exposure to arginine vasopressin (AVP) resulted in a single transient increase in intracellular free calcium (Ca2+i). In confluent cultures the cells could be divided into two general groups, those with a stable resting Ca2+i and those which demonstrated spontaneous brief elevations in Ca2+i of variable frequency. Application of AVP elevated Ca2+i, induced oscillations in quiescent confluent cells, increased the frequency of oscillatory activity in cells which were already active and, in cells which exhibited high frequency spontaneous fluctuations, inhibited this activity. Isotonic K+ depolarizing solution and normal solutions containing Co2+ inhibited Ca2+ spikes. These data suggest that the mechanism underlying the transients involves cyclical electrical phenomena at the cell membrane possibly utilizing calcium channels. There is no indication that the mechanism involves cytoplasmic oscillators.

The effect of [Na+]i and [Na+]o on Ca2+ mobilization in the rat aortic smooth muscle cell line A7r5.

Measurements of intracellular calcium (Ca2+i) and sodium (Na+i) have been made in single smooth muscle cells in confluent cultures of the A7r5 cell line using the Ca(2+)- and Na(+)-sensitive dyes Fura-2 and sodium-binding benzofuran isophthalate (SBFI). Reversal of the Na+ gradient in control cells results in a small increase in Ca2+i and slows the rate of recovery in Ca2+i following agonist stimulation. This suggests that a Na(+)-Ca2+ exchange mechanism may be functioning in these cells. In ouabain-pretreated cells, Na+i is elevated and the recovery from agonist stimulation is significantly slowed. This suggests that the elevation of Na+i alters Ca2+ homeostasis. Reversal of the Na+ gradient in ouabain-pretreated cells results in a transient increase in Ca2+i which was larger than in control cells. This response is reduced during a second or third exposure to zero Na+o.NA+i, in Na(+)-loaded cells, falls in the absence of external Na+. This fall is slowed in the absence of external Ca2+ supporting the idea that the Na+ loss is via Na(+)-Ca2+ exchange. The possible modulation of the Na(+)-Ca2+ exchanger and Ca2+ mobilization by internal Na+ is discussed.

Evidence for Na(+)-Ca2+ exchange and Ca(2+)-induced Ca2+ release in a cultured vascular smooth muscle cell line from the rat.

Measurements of intracellular calcium (Cai2+) and sodium (Nai+) have been made in single smooth muscle cells from the rat aortic cell line (A10) using the Ca(2+)- and Na(+)-sensitive dyes Fura-2 and SBFI (sodium-binding benzofuran isophthalate). The effects of manipulation of intracellular and extracellular Na+ on Cai2+ have been investigated. Reversal of the Na+ gradient in control cells does not result in any measurable increase in Cai2+ or change in the rate of recovery of the cells from agonist stimulation, suggesting that there is little functional Na(+)-Ca2+ exchange. In ouabain-pre-treated cells however, the recovery from agonist stimulation is significantly slowed, suggesting that in the presence of an elevated intracellular Na+ concentration there is an alteration in the Ca(2+)-handling mechanisms. Reversal of the Na+ gradient in ouabain-pre-treated cells results in a transient increase in Cai2+ followed by a slow secondary rise. The transient component of this rise is absent on a second activation of the cell or by prior mobilization of the intracellular stores of Ca2+ by agonist. Data presented in this paper suggest the possibility that the transient component is due to a Ca(2+)-induced Ca(2+)-release mechanism triggered by an initial influx of Ca2+. The mechanism underlying this influx is not known but may involve the Na(+)-Ca2+ exchanger operating in reverse. The possible modulation of the Na(+)-Ca2+ exchanger and Ca(2+)-induced Ca2+ release by internal Na+ is discussed.

Calcium oscillations in single isolated human vascular smooth muscle cells.

When cultured human vascular smooth muscle cells are exposed to sodium-free solutions oscillations in intracellular calcium concentration are observed. In some cells the oscillations are maintained at constant amplitude throughout the exposure to Na(+)-free solution, in others the amplitude of the oscillations falls to a lower, steady-state level. The oscillations are also activated and maintained in isotonic, Na(+)-free, K+ solutions suggesting that the underlying mechanism does not involve the opening and closing of voltage-dependent channels. The possible mechanisms responsible for this behaviour are discussed.

Inhibition of Ca2+ mobilization by caffeine in a cultured vascular smooth muscle cell line (A7r5).

The effects of caffeine on the resting level and agonist-induced changes in intracellular calcium ([Ca2+]i) have been studied in the vascular smooth muscle cell line A7r5. Caffeine (1-30 mM) lowers the resting [Ca2+]i by reducing the entry of Ca2+ and inhibits completely the mobilization of Ca2+ by arginine vasopressin. Application of forskolin, to elevate cAMP, does not affect the resting level of Ca2+i but does abolish the agonist-induced rise. These data add to the complexity of caffeine-induced changes in [Ca2+]i and point to a possible interaction between cAMP and other second messenger systems mobilizing Ca2+i in this cell type.

Changes in serum uric acid concentrations during normal pregnancy.

Serial changes in serum uric acid concentrations have been studied in a group of healthy women before conception, at regular intervals throughout pregnancy and finally 12 weeks after delivery. Compared with pre-pregnancy values uric acid concentrations decreased significantly by 8 weeks gestation and this reduced level was maintained until about 24 weeks. Thereafter the concentrations increased such that by term they were greater than the pre-pregnancy values in the majority of patients and remained elevated until at least 12 weeks after delivery. If clinical management during the second half of pregnancy is to be based on increases in serum uric acid concentrations then such increases will have to be carefully interpreted against the background of rising concentrations which occur as part of the physiological response to normal pregnancy.