E.T. Gonzalez

Regulation of metalloproteinases by nitric oxide in human trophoblast cells in culture

The process of embryo implantation requires extensive remodelling of the endometrial extracellular matrix, a function largely performed by matrix-degrading metalloproteinases (MMPs). In the present study, we used trophoblast cells isolated from human term placentas to study the regulation of MMPs by nitric oxide (NO). Using a combination of zymography, Western blot and indirect immunofluorescence, we showed that MMP-2 and MMP-9 are increased during the conversion from low-motile cytotrophoblast cells to the highly motile and differentiated syncytiotrophoblast multinucleated cells. We also observed an increase in NO production and NO synthase (NOS) expression during this cellular differentiation process. In addition, we demonstrated a positive regulatory role of NO on the activity and protein expression of MMP-2 and MMP-9, because NO donors (NOC-18 and spermine-NONOate) or the NOS substrate (L-arginine) stimulate, whereas NOS inhibitors (N(G)-nitro-L-arginine methyl ester and N(G)-monomethyl-L-arginine) reduce the expression and gelatinolytic activity of MMP-2 and MMP-9 in isolated trophoblast cells. Taken together, these results suggest that, in differentiating trophoblasts, NO regulates the induction of matrix-degrading proteases required for invasion during embryo implantation.

Interaction between uterine PGE and PGF2α production and the nitridergic system during embryonic implantation in the rat

Embryonic implantation is a complex process in which both maternal and embryonic signals are involved. In the present study, we evaluated changes in uterine prostaglandins production and nitric oxide synthase (NOS) activity during the course of early pregnancy and their interaction during implantation in rats. Uterine phospholipase A2 (PLA2) activity is increased on days 5 (day of ovoimplantation) and 6, compared to preimplantation days (3 and 4). This enhanced activity might be responsible for the observed increase in uterine PGE and PGF2 alpha production observed on day 5 of pregnancy, which induces endometrial vascular permeability and decidualization. When embryo access to the uterus is impaired, the increase of PG production is suppressed. During postimplantation, PGE levels return to preimplantation values, while PGF2 alpha decreased with respect to preimplantation values. Uterine NOS activity is also increased on day 4 and reaches a maximum on day 5, with a profile similar to PGE and PGF2 alpha. Dexamethasone administered in vivo decreased uterine NOS activity on day 4 of pregnancy but not on day 5, suggesting the presence of at least two types of NOS enzymes in the early days of pregnancy. A competitive inhibitor of NOS, L-NAME (600 and 1000 microM) induced a decrease in PGE and PGF2 alpha production in uterine tissue on day 5 of pregnancy. These results suggest the existence of a physiologically relevant nitridergic system which modulates prostaglandin production in the rat uterus during embryonic implantation.

Oxidative stress promotes the increase of matrix metalloproteinases-2 and -9 activities in the feto-placental unit of diabetic rats

Maternal diabetes increases the risk of congenital malformations, placental dysfunction and diseases in both the neonate and the offsprings later life. Oxidative stress has been involved in the etiology of these abnormalities. Matrix metalloproteases (MMPs), involved in multiple developmental pathways, are increased in the fetus and placenta from diabetic experimental models. As oxidants could be involved in the activation of latent MMPs, we investigated a putative relationship between MMPs activities and oxidative stress in the feto-placental unit of diabetic rats at midgestation. We found that H2O2 enhanced and that superoxide dismutase (SOD) reduced MMPs activities in the maternal side of the placenta and in the fetuses from control and diabetic rats. MMPs were not modified by oxidative status in the fetal side of the placenta. Lipid peroxidation was enhanced in the maternal and fetal sides of the placenta and in the fetus from diabetic rats when compared to controls, and gradually decreased from the maternal placental side to the fetus in diabetic animals. The activities of the antioxidant enzymes SOD and catalase were decreased in the maternal placental side, catalase activity was enhanced in the fetal placental side and both enzymes were increased in the fetuses from diabetic rats when compared to controls. Our data demonstrate changes in the oxidative balance and capability of oxidants to upregulate MMPs activity in the feto-placental unit from diabetic rats, a basis to elucidate links between oxidative stress and alterations in the developmental pathways in which MMPs are involved.

Membrane-type matrix metalloproteinase-9 activity in placental tissue from patients with pre-existing and gestational diabetes mellitus.

The activity of matrix metalloproteinase (MMP)-9 was evaluated in placental tissue from healthy subjects (controls) and from patients with gestational and pre-existing diabetes mellitus (GDM and PDM, respectively). Compared with controls, MMP-9 activity was greater in placental tissue from patients with PDM and lower in placental tissue from patients with GDM. The modulatory role of nitric oxide (NO) and reactive oxygen species (ROS) on MMP-9 activity in placental tissue was evaluated. In healthy placenta, NO synthase inhibitors diminished MMP-9 activity, whereas NO donors enhanced it. The addition of xanthine/xanthine oxidase or hydrogen peroxide to placental incubates enhanced MMP-9 activity, while the addition of superoxide dismutase (SOD) diminished it. In placental tissue from patients with PDM, MMP-9 activity was stimulated by NO and by ROS. In placental tissue from patients with PDM, concentrations of nitrates/nitrites and thiobarbituric acid-reactive substances (TBARS) were enhanced, whereas SOD activity was decreased, suggesting that elevated concentrations of NO and ROS may be related to the enhanced MMP-9 concentrations found in these tissues. In placenta from GDM patients, in which a diminished concentration of MMP-9 were detected, nitrate/nitrite concentrations were increased, but placental MMP-9 activity did not change in the presence of either NO donors or inhibitors. The activity of MMP-9 in placental tissue from patients with GDM was stimulated by ROS donor systems and was inhibited by the addition of SOD; however, TBARS and SOD concentrations were unchanged in these tissues compared with controls. These findings demonstrate that placental MMP-9 activity is modulated by NO and ROS and that, in diabetic pathology, NO and ROS may determine changes in MMP-9 activity, which are probably involved in the structural and functional abnormalities of diabetic placental tissue.

Pancreatic nitric oxide and oxygen free radicals in the early stages of streptozotocin-induced diabetes mellitus in the rat

The objective of the present study was to explore the regulatory mechanisms of free radicals during streptozotocin (STZ)-induced pancreatic damage, which may involve nitric oxide (NO) production as a modulator of cellular oxidative stress. Removal of oxygen species by incubating pancreatic tissues in the presence of polyethylene glycol-conjugated superoxide dismutase (PEG-SOD) (1 U/ml) produced a decrease in nitrite levels (42%) and NO synthase (NOS) activity (50%) in diabetic but not in control samples. When NO production was blocked by N(G)-monomethyl-L-arginine (L-NMMA) (600 microM), SOD activity increased (15.21 +/- 1.23 vs 24.40 +/- 2.01 U/mg dry weight). The increase was abolished when the NO donor, spermine nonoate, was added to the incubating medium (13.2 +/- 1.32). Lipid peroxidation was lower in diabetic tissues when PEG-SOD was added (0.40 +/- 0.02 vs 0.20 +/- 0.03 nmol/mg protein), and when L-NMMA blocked NOS activity in the incubating medium (0.28 +/- 0.05); spermine nonoate (100 microM) abolished the decrease in lipoperoxide level (0.70 +/- 0.02). We conclude that removal of oxygen species produces a decrease in pancreatic NO and NOS levels in STZ-treated rats. Moreover, inhibition of NOS activity produces an increase in SOD activity and a decrease in lipoperoxidation in diabetic pancreatic tissues. Oxidative stress and NO pathway are related and seem to modulate each other in acute STZ-induced diabetic pancreas in the rat.

Nitric oxide induces gelatinase A (matrix metalloproteinase 2) during rat embryo implantation

OBJECTIVE To evaluate a reciprocal signaling interaction initiated by embryo-derived nitric oxide (NO) to facilitate implantation by increased production of gelatinase A (matrix metalloproteinase 2, MMP2) in uterine stroma. DESIGN Experimental animal studies. SETTING Reproductive-physiology research laboratory. ANIMAL(S) Female syngeneic Wistar rats aged 14 weeks. INTERVENTION(S) Vaginal smears to confirm pregnancy. Oviductal ligature to avoid the descent of blastocysts to the uterine lumen. Plasma exudation assays to locate uterine blastocyst implantation sites. Organ cultures treated with NO donors and nitric oxide synthase (NOS) inhibitors. MAIN OUTCOME MEASURE(S) Expression of MMP2 and NO was assessed by Western blot and zymography of tissue extracts and by immunofluorescence of tissue sections. RESULT(S) An increase in MMP2 activity was found in uterine extracts in early pregnant rats and was concentrated at implantation sites. Immunolocalization experiments showed that inducible NOS was expressed on the surface of the implanting blastocyst adjacent to the uterine epithelium at the sites of increased MMP2 expression. In organ culture experiments, NO donors were found to increase, whereas NOS inhibitors were found to decrease MMP2 activity in uterine tissue sections. CONCLUSION(S) Blastocyst-derived NO contributes to the production of uterine-derived MMP2, an essential component of implantation and initiation of placentation.

Prostaglandin E2 alters the metabolism of labelled glucose in uteri isolated from ovariectomized rats. Effects of 17-beta estradiol and indomethacin

The effects of prostaglandins (PGs) E1, E2 and F2 alpha on the oxidation of labelled glucose in uterine strips from ovariectomized rats, were explored. Moreover, the influences of in vivo estrogenization of spayed rats and the presence in vitro of indomethacin (10(-6)M) on labelled glucose metabolism by isolated uteri, were also determined. PGE2 (10(-7)M), but not PGE1 or PGF2 alpha, enhanced significantly the formation of 14CO2 from U14C-glucose in uteri from spayed rats. After the injection of 17-beta estradiol to castrated animals prior to sacrifice the three PGs augmentated in vitro uterine glucose metabolism. In uterine strips from spayed non-estrogenized rats incubated with indomethacin, only PGE2 (10(-7) and 10(-6) M) incremented the formation of 14CO2 from labelled glucose, whereas PGE1 and PGF2 alpha were devoid of action, whereas in uteri from ovariectomized-estrogenized animals also incubated with indomethacin, PGE1, PGE2 and PGF2 alpha enhanced significantly the metabolism of labelled glucose, as was observed in the absence of indomethacin. However, in the presence of indomethacin the magnitude of the stimulation evoked by PGs in vitro was 3 to 4 times greater than without the inhibitor of the cyclo-oxygenase. The role of estradiol modulating the actions of PGE2 and PGF2 alpha on uterine glucose metabolism and possible reasons subserving the influence of indomethacin incrementing the effects of PGs on glucose oxidation in uteri from spayed-estrogenized rats, are discussed.

Norepinephrine alters PGE2/PGE1 output ratio in isolated uterus from ovariectomized rats.

The rat uterus generates and releases prostaglandins (PGs) of the series 2 as well as PGs of the series 1. The main purposes of the present study are to compare the effects of norepinephrine (NE) on the production and outputs of PGE1, PGE2 and PGF2 alpha by the uterus isolated from ovariectomized rats, treated or not with 17-beta-estradiol and to explore also, whether the effects of NE on PG synthesis are mediated through alpha, beta or both types of adrenoreceptors. Segments of control uterine horns obtained from ovariectomized rats generated and released into the incubating solution, equal amounts of PGE1, PGE2 and PGF2 alpha and propranolol (10(-6) M) or phentolamine (10(-6) M) failed to alter this basal production of PGs. Norepinephrine (3 X 10(-6) M) significantly depressed the outputs of PGE1 and PGF2 alpha but enhanced, also significantly, the release of PGE2. In the presence of the beta-adrenoreceptor blocker, propranolol, the reduction induced by NE on the output of PGE1 was not altered, but the stimulatory influence of NE on the release of PGE2 as well as the depression on the output of PGF2 alpha, were abolished. On the other hand the diminution evoked by NE on the release of PGF1 and PGF2 alpha as well as the increment induced on PGE2 output, were inhibited by the presence of phentolamine in the incubating solution. Uterine horns from ovariectomized rats treated with 17-beta-estradiol released into the incubating solution significantly more PGF2 alpha than PGE1 or PGE2. NE, either alone of in the presence of alpha 0 or beta-adrenoceptor blockers, did not modify this pattern of PG production. A possible mechanism(s) of action for NE on PG synthesis is discussed.

Effects of experimental diabetes on spontaneous contractions, on the output of prostaglandins and on the metabolism of labelled arachidonic acid, in uteri isolated from ovariectomized rats. Influences of estradiol

Spontaneous changes in isometric developed tension (IDT) as a function of time after isolation (contractile constancy) in uteri from control-castrated and castrated chronic streptozotocin-diabetic rats, were explored. The effects of injecting 17-beta estradiol (Eo) were also studied. No differences in the minor changes of contractile constancy, between control and diabetic preparations, during a period of 60 min, were detected, whereas uteri from non-diabetic Eo injected animals (0.5 + 1.0 ug, prior to sacrifice), exhibited a profound reduction of IDT, significantly greater than in tissues obtained from Eo injected-diabetic rats. Moreover, basal generation and outputs into the suspending solution of prostaglandins (PGs) E1, E2 and F2 alpha, were explored in the same groups, at 60 min following tissue isolation. The basal outputs of these three PGs were similar in castrated control rats, but preparations from castrated-diabetics released significantly more PGE1. The administration of Eo to castrated-diabetics, failed to alter the releases of the three PGs explored. In addition, the metabolism of labelled arachidonic acid (AA) into different prostanoids (6-keto-PGF1, PGF2, PGE2 and thromboxane B2-TXB2), was also investigated. The non-diabetic spayed rat uterus converted AA into these four prostanoids, the transformation into 6-keto-PGF1 alpha (as an index of PGI2 formation) being the most prominent. In preparations from diabetic rats the formation) being the most prominent. In preparations from diabetic rats the formation of 6-keto-PGF1 alpha, PGF2 alpha and PGE2, was significantly smaller than in controls, whereas a greater % of TXB2 formation (as an index of TXA2), was detected. On the other hand uterine preparations from non-diabetic spayed rats injected with Eo formed less 6-keto-PGF1 alpha and PGE2 and similar amounts of PGF2 alpha or of TXB2 from AA, than Eo injected controls, whereas uteri from castrated diabetic animals injected with Eo, formed a similar % of 6-keto-PGF1 alpha, PGF2 alpha and PGE2 from AA, than tissue preparations from non-estrogenized controls. However, the enhanced transformation of the labelled fatty acid precursor (AA) into TXB2 in the diabetic group, was significantly reduced by the steroid. The role of the augmented generation and release of PGE1 in uteri from diabetic rats is discussed in terms of precedents indicating the relevance of PGs type E supporting rat uterine motility. In addition the influence of Eo is attractive, because its reducing effect on TX production, in diabetes, a disease known to be accompanied by enhanced synthesis of vasoconstrictor and platelet aggregation TXA2, and by frequent obstructive circulat

Nitric oxide modulates placental prostanoid production from late pregnant non-insulin-dependent diabetic rat

Severe reproductive dysfunction has been described in non-insulin-dependent diabetes mellitus (NIDDM), correlated with high glucose levels in the plasma. We have characterized an abnormal prostanoid profile in tissues from NIDDM rats, and a tight correlation between nitric oxide (NO) levels and prostaglandin production. Likewise, we have determined that parturition is delayed in NIDDM rats compared to control animals. In order to characterize the events which precede delayed parturition in NIDDM rats, we evaluate (a) the arachidonic acid (AA) conversion in placental tissue obtained from control (day 21 and 22) and NIDDM (day 21, 22 and 23) late pregnant rats into prostaglandin E2 (PGE2) and F2alpha (PGF2alpha), thromboxane B2 (TXB2) and 6-keto-prostaglandin F1alpha (6-keto-PGF1alpha), and (b) NO synthase (NOS) activity in control and NIDDM late pregnant animals. Placental arachidonate conversion from control rats into different prostanoids, namely PGE2, PGF2alpha, and TXB2, is higher in day 22 than in day 21, and radioconversion from diabetic rats into PGE2, PGF22, TXB2 and 6-keto-PGF1alpha on day 23 is higher than in day 21 and 22. 6-keto-PGF1alpha is lower and TXB2 is higher in diabetic tissues than in control. Placental AA conversion of control diabetic tissues on the day of delivery is decreased by N(G) monomethyl-L-arginine (LNMMA) (600 mM), a well known NOS inhibitor, while prostanoid production remains unaltered on previous days. NOS activity is higher in control on day 22 when compared to day 21, and in diabetic on day 23 when compared to day 22 of pregnancy. We conclude that elevated NO placental levels are observed in control (day 22) and NIDDM (day 23) rats, and may increase placental prostaglandin production on the day of delivery.