Proteomic Analysis of Mitochondria Unravels the Pathophysiology of Pre-Eclampsia

Researchers are reporting the use of quantitative mitochondrial proteomic analysis to demonstrate that pre-eclampsia is primarily a disorder of placental mitochondria involving an altered mitochondrial proteome.

Previous studies have identified changes in some placental mitochondrial proteins such as peroxiredoxin III (PRDX3)¹ and HSPA4/HSP70.² Increased cytochrome C from mitochondria correlates with increased caspase 3 in pre-eclamptic placentae.³ Other studies suggest that a comparison between healthy mitochondrial proteome and diseased tissues enables the identification of biomarkers for early diagnosis and management of mitochondrial dysfunction.⁴

Zhonghua Shi and colleagues of the State Key Laboratory of Reproductive Medicine, Nanjing Maternity and Child Health Care Hospital (China) sought to determine the protein expression changes of placental mitochondria in pre-eclampsia.⁵ The researchers used isobaric tags for relative and absolute quantitation (iTRAQ) analysis combined with liquid chromatography–tandem mass spectrometry (LC-MS/MS) on differentially expressed placental mitochondrial proteins from four normal and four pre-eclamptic pregnancies. For the nano LC-MS/MS experiments, the researchers used the LTQ-Orbitrap MS (Thermo Scientific) equipped with a nanoelectrospray ion source and operated in positive ion mode. The identified proteins were further analyzed using the GeneOntology and MitoCarta-human databases, to avoid cross-contamination by other cellular organelles.

The results showed degenerative and apoptotic changes in the mitochondria of pre-eclamptic placentae. Mitochondria in the pre-eclamptic placentae were in a state of intermittent anoxia. The investigators found increased expression of four proteins and decreased expression of 22 proteins in pre-eclamptic placentae compared with normal placentae. Bioinformatics analysis revealed the critical role of these proteins in apoptosis, fatty acid oxidation (FAO), the respiratory chain, reactive oxygen species generation (ROS), the tricarboxylic acid cycle, and oxidative stress in the development of pre-eclampsia. The LC-MS/MS data showed a decreased expression of the three proteins TFRC, PRDX3 and HSPE1 in the pre-eclamptic group as compared with normal individuals; this decreased expression was significantly lower in the pre-term pre-eclamptic group (as opposed to the term pre-eclamptic group). The data were confirmed by western blot and immunohistochemical analysis.

Reduced mitochondrial levels of respiratory chain and FAO enzymes lead to abnormal energy production, disrupting the function of the feto-placental unit. Because impaired mitochondria generate large amounts of cellular ROS, a cascade of events was observed, including secondary mtDNA mutations and aggravated mitochondrial respiratory defects, which increased ROS production and led to oxidative stress. Other studies have shown increased apoptosis, particularly in the syncytiotrophoblast of placentae with pre-eclampsia.⁶ “The increased placental apoptosis may be a primary pathologic event or, alternatively, a secondary effect of altered placental oxygenation in preeclampsia,” the authors write.

The study results are consistent with previous studies showing that decreased placental mitochondrial FAO is a critical process in the pathophysiology of pre-eclampsia.^7,8 The proteins HADHA, HADHB and ACADVL, the critical acyl-CoA dehydrogenases that catalyze the initial step in the FAO pathway, were all reduced in pre-eclampsia.

The researchers used the Pathway Studio software⁹ to map several proteins such as TFRC, PRDX5, OGDH and TGM2 to the processes of apoptosis, oxidative stress, ROS generation and mitochondrial damage. The results indicated that mitochondrial oxidative stress and apoptosis play key roles in the development of pre-eclampsia. The researchers also show that the mitochondrial proteins involved in the tricarboxylic acid cycle and the respiratory chain — such as ATP5B, OGDH, DLST, MDH2 and ACADVL — were downregulated in pre-eclamptic placentae in the third trimester of pregnancy.

These results suggest that insufficient energy production in the pre-eclamptic placenta prevents invasion of the trophoblast, causing pre-eclampsia. However, additional in-vitro functional studies using trophoblast cells will need to confirm this theory.

References

1. Shibata, E., Nanri, H., Ejima, K., Araki, M., Fukuda, J., et al. (2003) “Enhancement of mitochondrial oxidative stress and up-regulation of antioxidant protein peroxiredoxin III/SP-22 in the mitochondria of human pre-eclamptic placentae,” Placenta, 24:698–705, doi: 10.1016/S0143-4004(03)00083-3.

2. Padmini, E., Lavanya, S., and Uthra, V. (2009) “Preeclamptic placental stress and over expression of mitochondrial HSP70,” Clin Chem Lab Med, 47:1073–80, doi: 10.1515/CCLM.2009.247.

3. Hung, T., Skepper, J.N., Charnock-Jones, D.S., and Burton, G.J. (2002) “A potent inducer of apoptotic changes in the human placenta and possible etiological factor in preeclampsia,” Circulation Research, 90:1274–81, doi: 10.1161/01.RES.0000024411.22110.AA.

4. Jiang, Y., and Wang, X. (2012, Mar 18) “Comparative mitochondrial proteomics: Perspective in human diseases,” J Hematol Oncol, 5:11.

5. Shi, Z., Long, W., Zhao, C., Guo, X., Shen, R., and Ding, H. (2013, May 9) “Comparative Proteomics Analysis Suggests that Placental Mitochondria are Involved in the Development of Pre-Eclampsia,” PLoS One, 8(5):e64351, doi: 10.1371/journal.pone.0064351.

6. Leung, D.N., Smith, S.C., To, K.F., Sahota, D.S., and Baker, P.N. (2001) “Increased placental apoptosis in pregnancies complicated by preeclampsia,” Am J Obstet Gynecol, 184:1249–50, doi: 10.1067/mob.2001.112906.

7. Rakheja, D., Bennett, M.J., Foster, B.M., Domiati-Saad, R., and Rogers, B.B. (2002) “Evidence for fatty acid oxidation in human placenta, and the relationship of fatty acid oxidation enzyme activities with gestational age,” Placenta, 23:447–50, doi: 10.1053/plac.2002.0808.

8. Shekhawat, P.S., Matern, D., and Strauss, A.W. (2005) “Fetal fatty acid oxidation disorders, their effect on maternal health and neonatal outcome: Impact of expanded newborn screening on their diagnosis and management,” Pediatr Res, 57:78R–86R, doi:10.1203/01.PDR.0000159631.63843.3E.

9. Nikitin, A., Egorov, S., and Daraselia, N. (2003) “Pathway Studio — The analysis and navigation of molecular networks,” Bioinformatics, 19:2155–7, doi: 10.1093/bioinformatics/btg290.