Carolina Pustovrh

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.

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.

15-Deoxy-Δ12,14-prostaglandin J2 and peroxisome proliferator-activated receptor γ (PPARγ) levels in term placental tissues from control and diabetic rats: modulatory effects of a PPARγ agonist on nitridergic and lipid placental metabolism

15-Deoxy-Δ12,14-prostaglandin J2 (15dPGJ2) is a peroxisome proliferator-activated receptor γ (PPARγ) ligand that regulates lipid homeostasis and has anti-inflammatory properties in many cell types. We postulated that 15dPGJ2 may regulate lipid homeostasis and nitric oxide (NO) levels in term placental tissues and that alterations in these pathways may be involved in diabetes-induced placental derangements. In the present study, we observed that, in term placental tissues from streptozotocin-induced diabetic rats, 15dPGJ2 concentrations were decreased (83%) and immunostaining for nitrotyrosine, indicating peroxynitrite-induced damage, was increased. In the presence of 15dPGJ2, concentrations of nitrates/nitrites (an index of NO production) were diminished (40%) in both control and diabetic rats, an effect that seems to be both dependent on and independent of PPARγ activation. Exogenous 15dPGJ2 did not modify lipid mass, but decreased the incorporation of 14C-acetate into triacylglycerol (35%), cholesteryl ester (55%) and phospholipid (32%) in placenta from control rats, an effect that appears to be dependent on PPARγ activation. In contrast, the addition of 15dPGJ2 did not alter de novo lipid synthesis in diabetic rat placenta, which showed decreased levels of PPARγ. We conclude that 15dPGJ2 modulates placental lipid metabolism and NO production. The concentration and function of 15dPGJ2 and concentrations of PPARγ were altered in placentas from diabetic rats, anomalies probably involved in diabetes-induced placental dysfunction.

Oxidative stress and altered prostanoid production in the placenta of streptozotocin-induced diabetic rats

The oxidative stress in placental tissues during late pregnancy, as well as the relationship between reactive oxygen species (ROS) and the arachidonic acid (AA) pathway was evaluated in a neonatal streptozotocin (STZ)-induced diabetic rat model. Lipoperoxide levels are increased in diabetic tissues compared with control tissues (P<0.001) and they seem to increase throughout the development of gestation both in control (P<0.05) and STZ-induced diabetic (P<0.001) rats. Superoxide dismutase (SOD) activity is not modified on different days of pregnancy, but enzymatic activity is lower in diabetic tissues than in control tissues (P<0.01). Labour is preceded by an increase in placental 14C-prostaglandin conversion from 14C-AA in control and diabetic animals (P<0.05) and the thromboxane B2 (TXB2)/6-keto-prostaglandin F1alpha (PGF1alpha) ratio is higher in diabetic placental tissues than in controls. The addition of SOD and glutathione to the incubation medium does not modify prostanoid levels in control rats, but does decrease the AA conversion to PGF2alpha, PGE2 and TXB2 (P<0.05) in diabetic placenta. Superoxide radical generation (hypoxanthine/xanthine oxidase or hydrogen peroxide added to the incubation medium) produces a decrease in 6-keto-PGF1alpha (P<0.05) in control and diabetic tissues, whereas PGF2alpha, PGE2 and TXB2 levels, and PGF2alpha and TXB2 production are increased in control and diabetic animals respectively (P<0.05). Diabetic pregnant rats supplemented with a diet containing 400 mg day(-1) of alpha-tocopherol (vitamin E) have diminished placental PGF2alpha and TXB2 production and lipoperoxide levels. The results show a higher TXB2 and a decreased 6-keto-PGF1alpha placental production that may be linked to increased oxidative stress and to a reduced antioxidant capacity in STZ-induced diabetic rats. These imbalances, probably involved in abnormal placental structure and function, may potentially be corrected with dietary supplementation of alpha-tocopherol in diabetic pregnancies.

Modulatory effect of leptin on nitric oxide production and lipid metabolism in term placental tissues from control and streptozotocin-induced diabetic rats.

Leptin production by placental tissues contributes to its circulating levels and functions. The diabetic pathology induces alterations in leptin levels. In the present study, leptin levels were evaluated in placental tissue from control and neonatal streptozotocin-induced (n-STZ) diabetic rats during late gestation. The effects of leptin levels on the generation of nitric oxide (NO), prostaglandin (PG) E(2) production and lipid metabolism were examined. Leptin levels were diminished in placentas from n-STZ diabetic rats compared with controls (P < 0.01). These differences were also evident when leptin was evaluated immunohistochemically. Addition of leptin (1 nM) in vitro enhanced NO production in control (66%) and diabetic placentas (134%) by stimulating NO synthase activity (by 38% and 54%, respectively). The addition of leptin increased PGE(2) production in placentas from control (173%) and diabetic rats (83%) and produced a 50% decrease in placental lipid levels (phospholipids, triacylglycerides, cholesterol and cholesteryl ester) without involving a reduction in de novo lipid synthesis. These data indicate that leptin enhances the production of placental NO and PGE(2), vasoactive agents that modify placental blood flow, and that leptin stimulates placental lipid metabolism, probably generating more lipids for transfer to the fetus. In the diabetic rat, placental leptin was reduced, probably as a response to the maternal environment to locally regulate the transfer of nutrients to the developing fetus.

Diminished PGE2 content, enhanced PGE2 release and defects in 3H-PGE2 transport in embryos from overtly diabetic rats.

Diminished PGE2 levels in diabetic embryos are related to the development of malformations, and thus the aim of the present study was to determine whether PGE2 levels are modified in rat embryos cultured in diabetic serum during organogenesis, and if PGE2 content and release, and 3H-PGE2 uptake and release, are altered in incubated diabetic embryos. Rats were made diabetic by steptozotocin (60 mg kg(-1)) before mating. Control rat embryos cultured for 24 h (explantation Day 9) in the presence of diabetic serum showed diminished PGE2 levels. When Day 10 diabetic embryos were incubated, embryo PGE2 levels were lower, but the PGE2 released to the incubation media was much higher than in controls. Uptake of 3H-PGE2 by diabetic embryos was initially enhanced (5-10 min), then reached similar levels to controls (20-100 min). Release of 3H-PGE2 previously incorporated during a 60-min incubation was greater in diabetic embryos than in controls. These results show diminished PGE2 content in both diabetic and normal embryos cultured in the presence of diabetic serum, but suggest that diabetic embryos have the capability to produce and release high levels of PGE2. The enhanced release of PGE2 is probably the result of transport abnormalities, and leads to the elevated PGE2 concentrations found in the incubating medium and to the diminished intraembryonic PGE2 levels that alter embryonic development.

Elevated levels of endothelin-1 and prostaglandin E2 and their effect on nitric oxide generation in placental tissue from neonatal streptozotocin-induced diabetic rats

Endothelin-1 (ET-1), nitric oxide (NO) and prostaglandin E(2) (PGE(2)) are regulators of feto-placental hemodynamics. In this study we explore the inter-regulatory pathways that modulate the levels of these vasoactive agents in control and neonatal streptozotocin-induced (n-stz) diabetic rat placenta. ET-1 levels are increased in diabetic placenta when compared to controls (P<0.001), and are strongly reduced by an NO synthase inhibitor (P<0.001). PGE(2) production is increased in diabetic placenta when compared to controls (P<0.01), but these levels are not modulated by ET-1. NO levels, similar in control and in diabetic placenta, are not influenced by PGE(2), but they are negatively modulated by ET-1 in both control (P<0.05) and diabetic (P<0.01) placenta. We conclude that rat placental ET-1 inhibits NO levels but does not modify PGE(2) concentrations. The elevated levels of ET-1 and PGE(2) in diabetic placenta, potent vasoconstrictors of placental vasculature, are probably related to the induction of placental insufficiency and fetal hypoxia in this pathology.

Metalloproteinase 2 activity and modulation in uterus from neonatal streptozotocin-induced diabetic rats during embryo implantation.

Matrix metalloproteinases (MMPs) are responsible for the remodelling of the uterine extracellular matrix during embryo implantation. Nitric oxide (NO) production is increased at the time when implantation begins. Abnormal tissue levels of MMPs are present in diabetes; elevated NO levels in tissues and an increased oxidative stress are also found. The present work evaluates the uterine MMP2 activity and levels during embryo implantation, as well as the influence of nitridergic compounds and reactive oxygen species (ROS) on the MMP2 enzymatic activity in a model of neonatal streptozotocin-induced diabetic rat. Metalloproteinase 2 activity and levels are increased in diabetic tissues compared with controls (P < 0.05 and P < 0.002 respectively). The uterine enzymatic activity in diabetic animals decreases in the presence of the NOS inhibitor NG-nitro-L-arginine methyl ester (P < 0.01) and is enhanced (P < 0.005) when a generating ROS system (xanthine/xanthine oxidase) is added to the incubating medium. It was also found that uterine superoxide dismutase activity is higher in diabetic rats than in control rats on the day of implantation (P < 0.001), suggesting a compensatory antioxidant ability. In conclusion, the results show that the uterine MMP2 activity, which is higher in diabetic animals than in control animals, is modulated positively by NO and ROS during embryo implantation in a model of streptozotocin-induced diabetic rats.