Mitochondrial-targeted antioxidant attenuates preeclampsia-like phenotypes induced by syncytiotrophoblast-specific Gαq signaling

Abstract

Syncytiotrophoblast stress is theorized to drive development of preeclampsia, but its molecular causes and consequences remain largely undefined. Multiple hormones implicated in preeclampsia signal via the Gαq cascade, leading to the hypothesis that excess Gαq signaling within the syncytiotrophoblast may contribute. First, we present data supporting increased Gαq signaling and antioxidant responses within villous and syncytiotrophoblast samples of human preeclamptic placenta. Second, Gαq was activated in mouse placenta using Cre-lox and DREADD methodologies. Syncytiotrophoblast-restricted Gαq activation caused hypertension, kidney damage, proteinuria, elevated circulating proinflammatory factors, decreased placental vascularization, diminished spiral artery diameter, and augmented responses to mitochondrial-derived superoxide. Administration of the mitochondrial-targeted antioxidant Mitoquinone attenuated maternal proteinuria, lowered circulating inflammatory and anti-angiogenic mediators, and maintained placental vascularization. These data demonstrate a causal relationship between syncytiotrophoblast stress and the development of preeclampsia and identify elevated Gαq signaling and mitochondrial reactive oxygen species as a cause of this stress.

Fig. 1.. Augmented Gαq-related activity in human preeclamptic placenta and development of a mouse model of fetoplacental Gαq signaling.

(A) Overview of Gαq-mediated signaling pathway. (B) Gene ontology biological processes (Shiny GO 0.74.1) enriched within the differentially expressed gene set of preterm preeclamptic placenta compared to preterm control (microarray, GSE75010; preterm control n = 35, preterm PreE n = 49). (C) PLC activity in human villous placental samples. (D) Breeding paradigm for targeting hM3Dq expression to the placenta and schematic depicting selective activation of the Gαq cascade with the hM3Dq DREADD. (E) Schematic of Cag-FLEX-hM3Dq in the Rosa26 locus (top), adapted from (40). Placenta of hM3Dq dam x Actb-Cre sire pregnancy. Scale bars, 1000 μm (left) and 10 μm (middle, right). PreE, preeclampsia. TCA, tricarboxylic acid. GTPase, guanosine triphosphatase. JZ, junctional zone. Images (A, D, and E) were created using BioRender (www.biorender.com).

Fig. 2.. Exogenous fetoplacental Gαq activation leads to severe pregnancy impairments in mice (hM3Dq^F/F dam x Actb-Cre^+/+ sire, GD 14.5).

(A) Twenty-four–hour maternal urine protein excretion. (B) Maternal plasma sFLT1. (C) Placental VEGF protein levels. (D) Hematoxylin and eosin stain of glomeruli. Scale bars, 25 μm. (E) Representative labyrinth image of diaminobenzidine (DAB) immunostained for CD31. Scale bars, 50 μm. (F) Percentage area of CD31-positive placenta. (G) Hematoxylin and eosin stain of fetoplacental unit. Scale bars, 500 μm (left), 50 μm (middle), and 10 μm (right). Each datapoint represents a biological replicate. *P < 0.05, independent samples t test (two-tailed).

Fig. 3.. Evidence of increased Gαq signaling and an oxidative defense response in human syncytiotrophoblast cells during preeclampsia.

(A) Diagram of syncytiotrophoblast layer within human placenta. (B) Laser capture microdissection was performed to collect syncytiotrophoblast-enriched cellular fractions. Scale bars, 100 μm. (C) PLCβ1/PLCβ3 protein expression within syncytiotrophoblast. (D) Correlation between villous placental PLC activity and syncytiotrophoblast PLCβ1/PLCβ3 protein levels. (E) ITPR3 mRNA within syncytiotrophoblast. (F) Overview of superoxide buffering by SOD2 in the mitochondrial matrix and the cellular effects of excess superoxide, adapted from (75). (G) SOD2 mRNA and protein expression within syncytiotrophoblast. (H) MDA protein expression within syncytiotrophoblast; *P < 0.05, independent samples t test (two-tailed) (C, G, and H) and Mann-Whitney U test (E). Each datapoint represents a biological replicate. STB, syncytiotrophoblast. Images (A and F) were created using BioRender (www.biorender.com).

Fig. 4.. Mouse model of syncytiotrophoblast-specific Gαq stimulation with mitochondrial-targeted antioxidant administration.

(A) Breeding paradigm for selective activation of the Gαq pathway in only the syncytiotrophoblast II layer. (B) General anatomy of the mouse placenta. (C) Double-transgenic placenta of hM3Dq^F/F dam x Gcm1-Cre^+/− sire. Scale bars, 300 μm (left) and 10 μm (middle, right). (D) RNAscope-based in situ hybridization of double-transgenic placenta. Scale bars, 50 μm and 10 μm (inset on right). STB II, syncytiotrophoblast II layer. Images (A and B) were created using BioRender (www.biorender.com).

Fig. 5.. Placental effects of syncytiotrophoblast-localized Gαq (hM3Dq^F/F dam x Gcm1-Cre^+/− sire, GD 14.5).

(A) Average luminal diameter of decidual spiral arteries. (B) Percentage area of CD31-positive placenta. (C) Representative labyrinth image of DAB immunostain for CD31. Scale bars, 50 μm. (D) Thickness of each placental layer. (E) Placental mass, fetal mass, and fetal mass/placental mass. (F) Soluble cytoplasmic (normalized to ACTB) and nuclear HIF1α (normalized to HDAC1) protein abundance. (G to I) SOD2, catalase, and MDA protein expression in labyrinth-enriched dissections. (J) Transmission electron micrographs of placental mitochondria. Arrows indicate occasional swelling. White asterisk denotes amorphous electron-dense precipitate, and arrowhead highlights a condensed mitochondrion. Scale bars, 300 nm; *P < 0.05, one-way analysis of variance (ANOVA) with Bonferroni multiple comparisons procedure, independent samples t test (two-tailed), or paired samples t test (two-tailed). Each datapoint represents a biological replicate.

Fig. 6.. Maternal effects of syncytiotrophoblast-restricted Gαq activation (hM3Dq^F/F dam x Gcm1-Cre^+/− sire).

(A) Average daily systolic blood pressure, diastolic blood pressure, heart rate, and activity separated by light and dark cycles. Dotted lines indicate time of CNO or saline administration (to GD 16.5: saline n = 6, CNO n = 8; to GD 17: saline n = 1, CNO n = 4). (B) Average 24-hour heart rate and mean arterial pressure (MAP) from GD 13 to 16.5 (postinjection period). (C) Twenty-four–hour urine protein excretion at GD 14.5. (D) Transmission electron micrographs of renal capillary loops at GD 14.5 indicating slight endothelial swelling in CNO mice (arrows) and slight congestion in both groups (* denotes red blood cells). Capillary basement membranes are highlighted in blue. Symbol “#” represents endothelial cell nuclei. Arrowheads identify podocyte foot processes. Scale bars, 2 μm; *P < 0.05, independent samples t test (two-tailed) or one-way ANOVA with Bonferroni multiple comparisons procedure. Datapoints for (B) and (C) represent biological replicates. BP, blood pressure.

Fig. 7.. Maternal circulating factors altered in mouse model of syncytiotrophoblast-specific Gαq signaling (hM3Dq^F/F dam x Gcm1-Cre^+/− sire, GD 14.5) and human preeclampsia.

(A) Levels within mouse plasma (saline n = 5, CNO n = 11, CNO + MitoQ n = 3). (B) Levels within human plasma during pregnancy (control n = 10, PreE n = 10, severe PreE = 6, “nonsevere” PreE n = 4). (C) Mouse plasma sFLT1 and correlation to the load of Cre⁺ placentas within CNO-injected double-transgenic pregnancies. Each datapoint represents a biological replicate; *P < 0.05, multiple unpaired t tests (two-tailed) or one-way ANOVA with Bonferroni multiple comparisons procedure.

Fig. 8.. Working model.

Schematic highlights the major experimental findings and illustrates the relationship between excess syncytiotrophoblast Gαq signaling, placental stress, and preeclamptic phenotypes; AVP, vasopressin. ANG, angiotensin II; ET-1, endothelin-1. Image was created using BioRender (www.biorender.com).