Proteomics and Metabolomics in Pregnancy-An Overview.

IMPORTANCE: Pregnancy is getting more and more complex due to increasing number of complications that may affect fetal outcomes. The introduction of newer "proteomics and metabolomics" technologies in the field of obstetrics and gynecology may allow physicians to identify possible associated etiologies that affect the mother during pregnancy and lead to associated complications affecting the offspring. OBJECTIVE: The principal objective of this review article is to provide a comprehensive evaluation of the use of proteomics and metabolomics in complicated pregnancies. Future studies that incorporate data from multiple technologies may allow the development of an integrated biological system approach to maternal genomes, proteomes, and metabolomes in pregnancy. EVIDENCE ACQUISITION AND RESULTS: We conducted a substantial MEDLINE, EBSCOhost, and Cochrane database search for all the relevant articles containing use of "omics" technologies in pregnancy. We identified 197 relevant articles, following standardized systematic review process along with grading systems; 69 eligible articles were identified. CONCLUSION/RELEVANCE: We sought to provide a comprehensive review in this emerging field of "omics" in pregnancy and associated complications. This article focuses mainly on use of proteomics and metabolomics identification techniques and possible interventions for early pregnancy complications to improve neonatal outcomes.

Inflammation in Complicated Pregnancy and Its Outcome.

Background Normal pregnancy relies on a careful balance between immune tolerance and suppression. It is known that strict regulation of maternal immune function, in addition to components of inflammation, is paramount to successful pregnancy, and any imbalance between proinflammatory and anti-inflammatory cytokines and chemokines can lead to aberrant inflammation, often seen in complicated pregnancies. Inflammation in complicated pregnancies is directly associated with increased mortality and morbidity of the mother and offspring. Aberrant inflammatory reactions in complicated pregnancies often lead to adverse outcomes, such as spontaneous abortion, preterm labor, intrauterine growth restriction, and fetal demise. The role of inflammation in different stages of normal pregnancy is reviewed, compared, and contrasted with aberrant inflammation in complicated pregnancies. The complications addressed are preterm labor, pregnancy loss, infection, preeclampsia, maternal obesity, gestational diabetes mellitus, autoimmune diseases, and inflammatory bowel disease. Aim This article examines the role of various inflammatory factors contributing to aberrant inflammation in complicated pregnancies. By understanding the aberrant inflammatory process in complicated pregnancies, novel diagnostic tools and therapeutic interventions for modulating it appropriately can be identified.

Attenuation of hyperglycemia-induced apoptotic signaling and anti-angiogenic milieu in cultured cytotrophoblast cells.

OBJECTIVE: Preeclampsia (preE) is a hypertensive disorder that occurs 20% in diabetic pregnancy. We have shown that hyperglycemia impairs cytotrophoblast cell (CTB) function. In this study, we assess apoptotic and anti-angiogenic signaling in excess glucose-induced CTBs. STUDY DESIGN: Human extravillous CTBs (Sw. 71) were treated with 100, 150, 200, 300, or 400 mg/dL glucose for 48 h. Some cells were pretreated with a p38 inhibitor (SB203580) or a peroxisome proliferator-activated receptor gamma (PPARγ) ligand (rosiglitazone) or with D-mannitol. Cell lysates were utilized to measure p38 MAPK phosphorylation, PPARγ, Bcl-2-associated-X protein (Bax), anti-apoptotic Bcl-2, caspase-9, and cyclooxygenase-2 (Cox-2) expression by western blot. Levels of the vascular endothelial growth factor (VEGF), placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1), soluble endoglin (sEng), and interleukin 6 (IL-6) were measured in culture media using ELISA kits. Statistical comparisons were performed using analysis of variance with Duncan's post hoc test. RESULTS: p38 phosphorylation and PPARγ were upregulated (p < 0.05) in CTBs treated with ≥150 mg/dL glucose compared to basal (100 mg/dL). Expressions of Bax/Bcl-2, Cox-2, and caspase-9 were upregulated (p < 0.05) in CTBs treated with ≥150 mg/dL glucose. Secretion of sFlt-1, sEng, and IL-6 was increased while VEGF and PIGF were decreased in CTB-treated ≥150 mg/dl of glucose (*p < 0.01 for each). SB203580 or rosiglitazone pretreatment significantly attenuated hyperglycemia-induced apoptotic and anti-angiogenic signaling. D-Mannitol had no effect. CONCLUSION: Hyperglycemia induced apoptotic and anti-angiogenic signaling in CTBs. The observed diminution of hyperglycemia-induced signaling by SB203580 or rosiglitazone pretreatment suggests the involvement of apoptotic and anti-angiogenic signaling in CTB dysfunction.

Neonatal Thrombocytopenia as a Consequence of Maternal Preeclampsia.

Introduction Preeclampsia (preE) is pregnancy-induced hypertension affecting a significant proportion of pregnant women worldwide and can cause detrimental effects in the mother and newborn. Some of the effects in the newborn include neonatal thrombocytopenia. Pertaining specifically to neonatal thrombocytopenia, several questions remain unanswered. Discussion According to the current literature, neonatal thrombocytopenia due to maternal preE is highly prevalent in the general population and the incidence is reported to be around 30% worldwide. This review gives an insight into the syndrome and summarizes the possible pathological mechanisms, the diagnostic approach, complications, and therapeutic interventions of neonatal thrombocytopenia. It also identifies the involvement of other cell lines, apart from platelets in the newborns. Furthermore, we suggest a future prospective study to investigate the pathogenesis of preE and plan a study involving animal models to come up with a possible therapeutic intervention to prevent preE and its various consequences in neonates.

Hyperglycemia down-regulates cGMP-dependent protein kinase I expression in first trimester cytotrophoblast cells.

Diabetes in pregnancy is associated with microvascular complications and a higher incidence of preeclampsia. The regulatory signaling pathways involving nitric oxide, cGMP, and cGMP-dependent protein kinase (PKG) have been shown to be down-regulated under diabetic conditions and contribute to the pathogenesis of vascular complications in diabetes. The present study was undertaken to investigate how high glucose concentrations regulate PKG expression in cytotrophoblast cells (CTBs). Human CTBs (Sw. 71) were treated with 45, 135, 225, 495, or 945 mg/dL glucose for 48 h. Some cells were pretreated with a p38 inhibitor (10 µM SB203580) or 10 µM rosiglitazone. After treatment, the cell lysates were subjected to measure the expression of protein kinase G1α (PKG1α), protein kinase G1ß (PKG1ß), soluble guanylate cyclase 1α (sGC1α), and soluble guanylate cyclase 1 ß (sGC1ß) by Western blot. Statistical comparisons were performed using analysis of variance with Duncan's post hoc test. The expressions of PKG1α, PKG1ß, sGC1α, and sGC1ß were significantly down-regulated (p < 0.05) in CTBs treated with >135 mg/dL glucose compared to basal (45 mg/dL). The hyperglycemia-induced down-regulation of cGMP and cGMP-dependent PKG were attenuated by the SB203580 or rosiglitazone pretreatment. Exposure of CTBs to excess glucose down-regulates cGMP and cGMP-dependent PKG, contributing to the development of vascular complications in diabetic mothers during pregnancy. The attenuation of hyperglycemia-induced down-regulation of PKG proteins by SB203580 or rosiglitazone pretreatment further suggests the involvement of stress signaling mechanisms in this process.

Internalization of exogenous ADP-ribosylation factor 6 (Arf6) proteins into cells.

Endogenous Arf6 is a myristoylated protein mainly involved in endosomal membrane traffic and structural organization at the plasma membrane. It has been shown that Arf6 mediates cancer cell invasion and shedding of plasma membrane microvesicles derived from tumor cells. In this article, we determined that Arf6 proteins both in the GDP and GTPγS bound forms can enter cells when simply added in the cell culture medium without requiring the myristoyl group. The GTPγS bound can enter cells at a faster rate than the GDP-bound Arf6. Despite the role of the endogenous Arf6 in endocytosis and membrane trafficking, the internalization of exogenous Arf6 may involve non-endocytic processes. As protein therapeutics is becoming important in medicine, we examined the effect of the uptake of Arf6 proteins on cellular functions and determined that exogenous Arf6 inhibits proliferation, invasion, and migration of cells. Future studies of the internalization of Arf6 mutants will reveal key residues that play a role in the internalization of Arf6 and its interaction and possible structural conformations bound to the plasma membrane.

Marinobufagenin levels in preeclamptic patients: a preliminary report.

Preeclampsia is a disorder resulting in significant fetomaternal complications with no definitive pharmacological intervention. A bufadienolide, marinobufagenin, has been implicated in the etiology of preeclampsia. We investigated both the blood and urine levels of marinobufagenin in preeclamptic and control subjects. Preeclamptic and normotensive pregnant women were recruited at various gestational age periods. Blood and urine specimens were obtained and analyzed for marinobufagenin levels and creatinine. The former determination was performed utilizing a new, novel chemifluorescent enzyme-linked immunosorbent assay. The marinobufagenin levels were higher in preeclamptics than in the controls in both serum and urine at various gestational age periods. Additionally, the mean level of marinobufagenin in the preeclamptic group was significantly greater than in controls in both blood and urine specimens ( P < 0.05). These data are consistent with a role for marinobufagenin in the etiology of preeclampsia. This study demonstrated comparable results in blood and urine samples. This suggests that subsequent studies on levels of marinobufagenin as a screening test for preeclampsia could be done utilizing urine samples, which are easier to obtain, less invasive, more cost-effective, and as accurate as the serological tests.

Non-proteolytic activation of prorenin: activation by (pro)renin receptor and its inhibition by a prorenin prosegment, "decoy peptide".

Prorenin is the enzymatically inactive precursor of renin. Recent interest has focused on the nonproteolytic activation of prorenin by antibodies and renin/prorenin receptors since markedly increased levels of circulating prorenin have been associated with both physiological and pathological changes. Prorenin has been considered to be activated in vivo proteolytically and/or non-proteolytically. It has been demonstrated in vitro the "gate" and "handle" regions in the prorenin molecule is crucial for its non-proteolytic activation by a protein-protein interaction. Prorenin was also activated by the renin/prorenin receptors. Decapeptides (10P-19P) known as "decoy" peptide and pentapeptides (11P-15P) named as "handle" region peptide, were observed to inhibit the binding of both prorenins to receptors. The "handle" region plays an important role in prorenin binding to the receptor and its enzymatic activity by non-proteolytic activation. Prorenin receptors so far revealed by animal experiments have indicated that the decoy peptide prevented diabetes nephropathy and retinopathy. It was postulated the existence of novel regulative system that stimulated signal transduction as well as that of renin-angiotensin system. These findings help to find out the clue to design useful drug with greater benefit on the end-organ damage in diabetes and hypertension than those of conventional renin-angiotensin system inhibitors.

Slowly progressive, angiotensin II-independent glomerulosclerosis in human (pro)renin receptor-transgenic rats.

For defining the pathogenic effects of the (pro)renin receptor-transgenic rat, strains that overexpressed the human receptor were generated. Although transgenic rats were normotensive and euglycemic and had a renal angiotensin II (AngII) level that was comparable to that of wild-type rats, transgenic rats developed proteinuria with aging and significant glomerulosclerosis at 28 wk of age. In kidneys of 28-wk-old transgenic rats, mitogen-activated protein kinases (MAPK) were activated without recognizable tyrosine phosphorylation of the EGF receptor, and expression of TGF-beta1 was enhanced. In vivo infusion of the (pro)renin receptor blocker peptide (formerly handle region decoy peptide) significantly inhibited the development of glomerulosclerosis, proteinuria, MAPK activation, and TGF-beta1 expression in the kidneys, but the angiotensin-converting enzyme inhibitor did not attenuate these changes despite a significant decrease in the renal AngII level. In addition, recombinant rat prorenin stimulated MAPK activation in the human receptor-expressed cultured cells, but human receptor was unable to evoke the enzyme activity of rat prorenin. Thus, human (pro)renin receptor elicits slowly progressive nephropathy by AngII-independent MAPK activation in rats. This study clearly provided in vivo evidence for the AngII-independent MAPK activation by human (pro)renin receptor and induction of glomerulosclerosis with increased TGF-beta1 expression.

A simple spectrophotometric method for the determination of cobalt in industrial, environmental, biological and soil samples using bis(salicylaldehyde)orthophenylenediamine.

Bis(salicylaldehyde)orthophenylenediamine (BSOPD) has been proposed as new analytical reagent for the direct non-extractive spectrophotometric determination of cobalt. It reacts with cobalt in slightly acidic (0.0002-0.001 M H(2)SO(4)) 50% 1,4-dioxanic medium to form a red-orange chelate with a molar ratio 1:1. The reaction is instantaneous and the maximum absorbance was obtained at 458 nm and remains constant for over 24h. The average molar absorption coefficient and Sandell's sensitivity were found to be 1.109 x 10(4)l mol(-1)cm(-1) and 20 ng cm(-2) of Co(II), respectively. Linear calibration graph was obtained for 0.1-15 mg l(-1) of Co(II) with a correlation coefficient value of 0.995 for Co-BSOPD complex. Large excess of 44 cations, anions and complexing agents do not interfere in the determination. The method was successfully used in the determination of cobalt(II) from synthetic mixture and certified reference materials for the purpose of validating the method and the results of analyses were found to be excellent agreement with those of actual values. This developed method was also used for determination of cobalt in some environmental waters (potable and polluted), biological (blood and urine) and soil samples and solution containing both cobalt(II) and cobalt(III). The results of the proposed method for biological samples were comparable with AAS and were found to be in good agreement. The method has high precision and accuracy (s=+/-0.01 for 0.5 mg l(-1)).

Inhibition of diabetic nephropathy by a decoy peptide corresponding to the "handle" region for nonproteolytic activation of prorenin.

We found that when a site-specific binding protein interacts with the "handle" region of the prorenin prosegment, the prorenin molecule undergoes a conformational change to its enzymatically active state. This nonproteolytic activation is completely blocked by a decoy peptide with the handle region structure, which competitively binds to such a binding protein. Given increased plasma prorenin in diabetes, we examined the hypothesis that the nonproteolytic activation of prorenin plays a significant role in diabetic organ damage. Streptozotocin-induced diabetic rats were treated with subcutaneous administration of handle region peptide. Metabolic and renal histological changes and the renin-Ang system components in the plasma and kidneys were determined at 8, 16, and 24 weeks following streptozotocin treatment. Kidneys of diabetic rats contained increased Ang I and II without any changes in renin, Ang-converting enzyme, or angiotensinogen synthesis. Treatment with the handle region peptide decreased the renal content of Ang I and II, however, and completely inhibited the development of diabetic nephropathy without affecting hyperglycemia. We propose that the nonproteolytic activation of prorenin may be a significant mechanism of diabetic nephropathy. The mechanism and substances causing nonproteolytic activation of prorenin may serve as important therapeutic targets for the prevention of diabetic organ damage.

Short-term administration of conjugated linoleic acid reduces liver triglyceride concentration and phosphatidate phosphohydrolase activity in OLETF rats.

The present study explored the short-term effects of dietary conjugated-linoleic acid (CLA) on liver lipid metabolism in starved/refed Otsuka Long Evans Tokushima Fatty (OLETF) rats. Male OLETF rats (12 weeks old) were starved for 24 hours, then refed for 48 hours with either a CLA diet [7.5% CLA and 7.5% Safflower oil (SAF)] or a SAF control diet (15% SAF). The results demonstrated a 30% reduction of hepatic triglyceride (TG) concentration in the CLA group when compared to the control group. Liver cholesterol concentration was also 26% lower in the CLA fed rats. The activity of mitochondrial carnitine palmitoyltransferase, the rate-limiting enzyme of fatty acid oxidation, was moderately elevated by 1.2-fold in the livers of the CLA group when compared to the control. In contrast, phosphatidate phosphohydrolase, the rate-limiting enzyme for TG synthesis, was found to be 20% lower in the livers of the CLA-fed rats. Therefore, dietary CLA evidently lowers liver lipid concentrations through a reduced TG synthesis and enhanced fatty acid oxidation in starved/refed OLETF rats.