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

Abstract

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.

Figure 1. Heatmap of significant genes

Expression profile of genes found significantly changed in at least one group comparison. After filtering genes for signal to noise ratio above 2.0 and fold change above 1.4, a total of 30 unique genes were obtained. In the figure genes are sorted on mean expression ratio from high to low in the PE group. A cut off q-value at 0.005 was selected.

Figure 2. Real-time PCR quantification of Hbα2, Hbγ, Hbβ and HLA-DPA1 in placenta and blood samples

All values are normalized against the amount of β-actin and presented as scatter plots. (A) Hbα mRNA expression in the placenta. Significant changes were found between PE vs. controls (p=0.004) and between PE\Notch (PE with notching) vs. controls (p=0.03). (B) Hbγ relative mRNA values showing significant changes between PE vs. controls (p=0.003) and between PE\Notch vs. controls (p=0.03). (C) Hbβ showed significant overexpression in PE vs. controls (p=0.02) and in PE\Notch vs. controls (p=0.04). (D) HLA-DPA1 mRNA was overexpressed in the notch group vs. all other groups: vs. controls (p=0.01), vs. PE (p=0.01), and vs. PE with notching (p=0.02). (E) Hbα mRNA expression in blood samples. No changes were seen between the groups. (F) Hbγ mRNA expression was very low in blood samples. No changes were seen between PE and controls.

Figure 3. In situ hybridizations from placenta and placenta bed samples

Schematic image of the human placenta showing the villous section (V) of the placenta and below a placenta bed section (M) with spiral arteries (S) in between. (A) A light field image of Hbα mRNA-expression in a representative preeclamptic placenta sample. Hbα expression was especially seen in and around blood vessels. However, several scattered cells in the villous section are also seen. (B) Darkfield image of the same section. (C) A darkfield image of Hbα mRNA expression from a representative control placenta. Compared with PE placentas, the control placentas show fewer Hbα-expressing cells in the villous section. (D) Light field image from a representative myometrial sample from a PE patient. Hbα expression is only seen in the spiral arteries, no expression is seen in the myometrial tissue. (E) The same myometrial section in dark field. (F) A myometrial sample from a control placenta. Hbα mRNA expression is similar to the expression seen in PE myometrium. mRNA expression for Hbγ and Hbβ were similar to that for Hbα (data not shown). Scale-bar is 100μm.

Figure 4. A representative image of Hbγ protein expression in the placenta

Protein expression is shown with a red fluorescent marker. In the PE placenta there is a strong expression of Hbγ in the vascular lumen (lu), but Hbγ is also expressed in the vascular endothelium (arrow) as well as in the extra-vascular section of the villous stroma (A). The placenta from normotensive controls however, showed no expression of Hbγ in the vascular lumen (B) but Hbγ is expressed in the vascular endothelium (arrow). Scale bar is 25 µm.