Platelet and mitochondrial RNA is decreased in plasma-derived extracellular vesicles in women with preeclampsia-an exploratory study

Abstract

Background: Circulating extracellular vesicles (EVs) are increased in preeclampsia (PE) and are associated with severity and progression. We examined in this exploratory cohort study if the mRNAs and long noncoding RNAs (lncRNAs) in plasma-derived EVs were dysregulated in PE compared to normal pregnancy and display different temporal patterns during gestation.

Methods: We isolated EVs from plasma at weeks 22-24 and 36-38 in women with and without PE (n=7 in each group) and performed RNA-seq, focusing on mRNAs and lncRNAs. We validated highly expressed mitochondrial and platelet-derived RNAs discovered from central pathways in 60 women with/without PE. We examined further one of the regulated RNAs, noncoding mitochondrially encoded tRNA alanine (MT-TA), in leukocytes and plasma to investigate its biomarker potential and association with clinical markers of PE.

Results: We found abundant levels of platelet-derived and mitochondrial RNAs in EVs. Expression of these RNAs were decreased and lncRNAs increased in EVs from PE compared to without PE. These findings were further validated by qPCR for mitochondrial RNAs MT-TA, MT-ND2, MT-CYB and platelet-derived RNAs PPBP, PF4, CLU in EVs. Decreased expression of mitochondrial tRNA MT-TA in leukocytes at 22-24 weeks was strongly associated with the subsequent development of PE.

Conclusions: Platelet-derived and mitochondrial RNA were highly expressed in plasma EVs and were decreased in EVs isolated from women with PE compared to without PE. LncRNAs were mostly increased in PE. The MT-TA in leukocytes may be a useful biomarker for prediction and/or early detection of PE.

Keywords: Extracellular vesicles; Preeclampsia; lncRNA.

Fig. 1

A The 100 most abundant RNAs in plasma EVs in normotensive controls (n=7) from the RNA-seq analysis at 22–24 weeks and 36–38 weeks gestation. B Volcano plot and distribution of differentially expressed RNAs from plasma extracellular vesicles are shown between women with preeclampsia (PE) (n=7) and normotensive controls (n=7) at gestational weeks 36–38 (B), and between gestational weeks 22–24 and 36–38 in women who subsequently developed PE (D). The distribution (number and percentage) of the different classes of differentially regulated RNA and lncRNA are shown between PE and normotensive pregnancies (C) and between gestational weeks 22–24 and 36–38 in women who subsequently developed PE (E). *divergent/overlapping/intronic

Fig. 2

Differentially regulated RNAs at 36–38 weeks between women with PE and controls from platelet-derived extracellular vesicles (A) and mitochondria (B) as characterized by the RNA-seq analysis. Numbers are log-2 fold change (adjusted p-value) in PE vs. normal pregnancy samples. Created with BioRender.com

Fig. 3

PCR validation of data from the EV RNA-seq analysis and their association with uterine artery doppler pulsatility index (PI). Samples from women with preeclampsia (PE)/normal pregnancy (NP), n=28/n=32 at 22–24 weeks and n=20/n=32 at 36–38 weeks, respectively, were used. A Validation of the mitochondrial RNAs MT-TA, MT-ND2 and MT-CYB at weeks 22–24 and 36–38 between women who subsequently developed PE and controls (NP), adjusted for age and BMI. B Scatter plot showing the correlation between mitochondrial RNAs and uterine artery PI at weeks 22–24 and 36–38 between women who subsequently developed PE and controls (NP). C Validation of the platelet-derived RNAs PF4, PPBP and CLU at weeks 22–24 and 36–38 between women who subsequently developed PE and controls (NP), adjusted for age and BMI. r=correlation coefficient, *p-value<0.05, ** p<0.01, ***p<0.001

Fig. 4

A ROC analysis to identify the best predictor of preeclampsia (PE) at 22–24 weeks using RNAs from EVs MT-TA, MT-ND2, MT-CYB, CLU, PF4 and PPBP (35 PE, 35 normal pregnancy (NP)) and MT-TA^PBMC from leukocytes (peripheral blood mononuclear cells, PBMC) (38 PE, 215 NP). B Expression of mitochondrial tRNA MT-TA in leukocytes (PBMC) and plasma between women who subsequently developed PE and those that did not (NP) at weeks 22–24 and 36–38, adjusted for age and BMI. C Scatter plot showing correlation between MT-TA in leukocytes (PBMC) and EVs at weeks 22–24 and 36–38 between women who developed PE and those that did not (NP). r=correlation coefficient, *p<*, ** p<0.01, ***p<0.001. D Logistic regression analysis of predictors of PE at 22–24 weeks. The top part shows MT-TA^PBMC and established risk predictors of PE in univariate analysis. The bottom part shows MT-TA^PBMC when adjusted for each of the established PE risk predictors. MT-TA^PBMC , MAP, Uterine artery PI (artUP) and sFlt-1/PlGF ratio were dichotomized according to Youden’s index: MT-TA^PBMC cut-off: 0.13, likelihood ratio (LHR): 3.2; MAP cut-off: 85.8, LHR: 2,1; artUP cut-off: 0.93, LHR: 1.9; sFlt-1/PlGF ratio cut-off: 9.3, LHR: 1.7

Fig. 5

Average normalized counts for RNAs coding for protein used as EV markers (from MISEV2018 standards of EV research) in the controls from the RNA-seq analysis. Category 1, use for all EVs: Transmembrane or GPI-anchored proteins associated to plasma membrane and/or endosomes, 1a non-tissue specific, 1b tissue specific; Category 2, use for all EVs: Cytosolic proteins recovered in EVs, 2a with lipid or membrane protein-binding ability, 2b promiscuous incorporation in EVs (and possibly exomeres); Category 3, use for all EVs as purity control, major components of non-EV co-isolated structures: 3a lipoproteins (produced by liver, abundant in plasma, serum), 3b protein and protein/nucleic acid aggregates; Category 4, use for subtypes of EVs (e.g. large oncosomes, large EVs) and/or pathologic/atypical state, Transmembrane, lipid-bound and soluble proteins associated to other intracellular compartments than PM/endosomes: 4a nucleus, 4b mitochondrial, 4c secretory pathway (endoplasmic reticulum, Golgi apparatus), 4d others (autophagosomes, cytoskeleton); Category 5, use for functional component of EVs: need to determine the mode of association with EVs: 5a Cytokines and growth factors, 5b adhesion and extracellular matrix proteins