Gene and Protein Profiling of the Preeclamptic Placenta

Aims State-of-the-art methodology was used to screen and profile the placenta, gene and protein expression, for changes related to preeclampsia (PE) and cases with increased resistance in the uterine arteries. Women with increased resistance in the uterine arteries have increased risk of developing PE. Since not all of them develop PE, this group, identified by Doppler ultrasound, was included to search for genes and/or proteins that may protect them from developing PE. Results The PE placenta showed increased gene expression of fetal hemoglobin (Hb). Protein expression analysis confirmed the accumulation of free Hb, particularly the gamma chain was detected in the vascular lumen. Patients with increased resistance in the uterine arteries, expressed as a notch in blood velocity tracings recorded with Doppler ultrasound. Notching without PE, showed increased expression of genes related to apoptosis and antigen presentation in their placentas. In the notch placentas that later developed PE, an increased expression of genes related to inflammatory cell movement was seen. Antibody microarray screening of maternal plasma showed that late and early onset PE as well as PE with notching and IUGR showed different inflammatory responses. Conclusions The changes in gene expression suggested that PE may be a three-stage disease with notch as a reversible middle stage. Accumulation of inflammatory cells in the notch placenta may cause inflammation that drives the pathophysiology into PE. Increased expression of antigen presenting genes may protect the notch placenta from pro-inflammatory damage thereby preventing progression into PE. Free fetal Hb was identified as a possible placental factor that further induces inflammation and tissue damage. Increased maternal plasma levels of free fetal Hb may be used as a prognostic and diagnostic marker for PE. The maternal immune reaction and inflammatory response may be important factors that further determine the severity and the clinical manifestations of PE

Differential Proteome Analysis of the Preeclamptic Placenta Using Optimized Protein Extraction

The human placenta is a difficult tissue to work with using proteomic technology since it contains large amounts of lipids and glycogen. Both lipids and glycogen are known to interfere with the first step in the two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), the isoelectric focusing. In order to gain the best possible protein separation on 2D-PAGE, an optimized sample preparation protocol for placental proteins was developed. Two different buffers, urea/CHAPS and Hepes, were used for solubilization in combination with six different precipitation methods. The removal of glycogen from the samples by centrifugation was crucial for the final proteome maps. Solubilization with urea/CHAPS in combination with dichloromethane/methanol or acidified acetone proved to be the best precipitation procedures. When applied to clinical placenta samples apolipoprotein A1 was found to be accumulated in the preeclamptic placenta, where it may either have a nutritional effect or act as a modifier of signal transduction

Gene expression profiling of placentae from women with early- and late-onset pre-eclampsia: down-regulation of the angiogenesis-related genes ACVRL1 and EGFL7 in early-onset disease

The underlying mechanisms behind the obstetric condition pre-eclampsia (PE) are still unclear. Manifestation of PE is heterogeneous and it has therefore been proposed to be a syndrome with different causes rather than one disease with a specific aetiology. Recently, we showed differences in circulating angiogenic factors between two subgroupsearly- and late-onset PE. To further elucidate the differences between the two, we investigated placental gene expression profiles. Whole genome microarray technology and bioinformatic analysis were used to evaluate gene expression profiles in placentae from early- (2432 gestational weeks, n 8) and late-onset (3641 gestational weeks, n 7) PE. The results were verified by using quantitative real-time (qRT)PCR. We found significant differences in the expression of 196 genes in early- compared with late-onset PE, 45 of these genes showing a fold change above 2. Bioinformatic analysis revealed alterations in angiogenesis and regulation of cell motility. Two angiogenesis-associated transcripts (Egfl7 and Acvrl1) showed lower expression in early-onset PE versus late-onset PE (P 0.037 and P 0.003) and versus gestational age-matched controls (P 0.007 and P 0.011). We conclude that angiogenesis-associated genes are regulated in a different manner in the two subgroups, and that the gene expression profiles of early- and late-onset PE diverge, supporting the hypothesis of early- and late-onset PE being at least partly two separate entities

Placental expression profiling in preeclampsia: local overproduction of hemoglobin may drive pathological changes.

OBJECTIVE: To create a library enriched in cDNAs from preeclamptic placentas to print onto microarrays for placental profiling of preeclampsia (PE) and high risk pregnancies. DESIGN: Prospective study. SETTING: University women's clinic and academic research laboratory. PATIENT(S): Ten patients with PE, 5 with PE and bilateral notching, 5 with bilateral notching without PE, and 15 normotensive patients were recruited. INTERVENTION(S): Placenta and placenta bed biopsies were collected after delivery. MAIN OUTCOME MEASURE(S): Subtracted libraries of PE transcripts were produced, and cDNAs from these libraries were used to make PE-specific cDNA arrays. Results were verified quantitatively using real-time polymerase chain reaction (PCR) and histologically using in situ hybridization and immunohistochemistry. RESULT(S): Thirty genes were significantly altered in at least one group comparison. Differences in two candidate genes were confirmed using quantitative real-time PCR. Hemoglobin alpha2 and gamma transcripts were significantly overexpressed in the PE placenta. Scattered cells in the placenta and placental blood vessels were shown to express genes encoding these hemoglobin chains. CONCLUSION(S): We demonstrate increased hemoglobin production in the PE placenta. The hemoglobin may be released into the placenta blood vessel lumen. Free heme and hemoglobin are potent toxins that cause endothelial damage and inflammation

Fetal hemoglobin and alpha(1)-microglobulin as first- and early second-trimester predictive biomarkers for preeclampsia

OBJECTIVE: The aim of this study was to evaluate fetal hemoglobin (HbF) and alpha(1)-microglobulin (A1M) in maternal serum as first-trimester biomarkers for preeclampsia (PE). STUDY DESIGN: The design was a case-control study. We included 96 patients in the first trimester of pregnancy (60 with PE and 36 controls). Venous serum samples were analyzed for HbF and total hemoglobin (Hb) by enzyme-linked immunosorbent assay and for A1M by radioimmunoassay. Sensitivity and specificity was calculated by logistic regression and receiver operating characteristic curve analysis. RESULTS: The HbF/Hb ratio and A1M concentration were significantly elevated in serum from women with subsequent development of PE (P < .0001). The optimal sensitivity and specificity was obtained using the biomarkers in combination; 69% sensitivity for a 5% screen positive rate and 90% sensitivity for a 23% screen positive rate. CONCLUSION: The study suggests that HbF/Hb ratio in combination with A1M is predictive biomarkers for PE

Increased levels of cell-free hemoglobin, oxidation markers, and the antioxidative heme scavenger alpha(1)-microglobulin in preeclampsia.

Preeclampsia is a major cause of morbidity and mortality during pregnancy. To date, the pathogenesis of the disease is not fully understood. Recent studies show that preeclampsia is associated with overexpression of the hemoglobin genes alpha2 and gamma and accumulation of the protein in the vascular lumen of the placenta. Hypothesizing that cell-free hemoglobin leaks from the placenta into the maternal circulation and contributes to the endothelial damage and symptoms by inducing oxidative stress, we analyzed fetal and adult hemoglobin (HbF, HbA), haptoglobin, oxidation markers, and the heme scavenger and antioxidant alpha(1)-microglobulin in plasma, urine, and placenta in preeclamptic women (n=28) and women with normal pregnancy (n=27). The mean plasma concentrations of HbF, HbA, protein carbonyl groups, membrane peroxidation capacity, and alpha(1)-microglobulin were significantly increased in preeclamptic women. The levels of total plasma Hb correlated strongly with the systolic blood pressure. The plasma haptoglobin concentrations of women with preeclampsia were significantly depressed. Increased amounts of alpha(1)-microglobulin mRNA and protein were found in placenta from preeclamptic women, and the levels of plasma and placenta alpha(1)-microglobulin correlated with the plasma Hb concentrations. The heme-degrading form t-alpha(1)-microglobulin was significantly increased in urine in preeclampsia. These results support the idea that hemoglobin-induced oxidative stress is a pathogenic factor in preeclampsia

Perfusion Of Human Placenta With Hemoglobin, Introduces Preeclampsia-Like Injuries That Are Prevented By Alpha 1-Microglobulin

Preeclamptic women have increased plasma levels of free fetal hemoglobin (HbF), increased gene expression of placental HbF and accumulation of free HbF in the placental vascular lumen. Free hemoglobin (Hb) is pro-inflammatory, and causes oxidative stress and tissue damage