Placental Vesicles Carry Active Endothelial Nitric Oxide Synthase and Their Activity is Reduced in Preeclampsia

Abstract

Preeclampsia, a multisystem hypertensive disorder of pregnancy, is associated with increased systemic vascular resistance. Placentae from patients with preeclampsia have reduced levels of endothelial nitric oxide synthase (eNOS) and, thus, less nitric oxide (NO). Syncytiotrophoblast extracellular vesicles (STBEV), comprising microvesicles (STBMV) and exosomes, carry signals from the syncytiotrophoblast to the mother. We hypothesized that STBEV-bound eNOS (STBEV-eNOS), capable of producing NO, are released into the maternal circulation. Dual-lobe ex vivo placental perfusion and differential centrifugation was used to isolate STBEV from preeclampsia (n=8) and normal pregnancies (NP; n=11). Plasma samples of gestational age-matched preeclampsia and NP (n=6) were used to isolate circulating STBMV. STBEV expressed placental alkaline phosphatase, confirming placental origin. STBEV coexpressed eNOS, but not inducible nitric oxide synthase, confirmed using Western blot, flow cytometry, and immunodepletion. STBEV-eNOS produced NO, which was significantly inhibited by N ^G-nitro-l-arginine methyl ester (eNOS inhibitor; P<0.05) but not by N-(3-(aminomethyl) bezyl) acetamidine) (inducible nitric oxide synthase inhibitor). STBEV-eNOS catalytic activity was confirmed by visualizing eNOS dimerization. STBEV-eNOS was more abundant in uterine vein compared with peripheral blood, indicating placental origin. STBEV isolated from preeclampsia-perfused placentae had lower levels of STBEV-eNOS (STBMV; P<0.05) and overall lower NO activity (STBMV, not significant; syncytiotrophoblast extracellular exosomes, P<0.05) compared with those from NP. Circulating plasma STBMV from preeclampsia women had lower STBEV-eNOS expression compared with that from NP women (P<0.01). This is the first observation of functional eNOS expressed on STBEV from NP and preeclampsia placentae, as well as in plasma. The lower STBEV-eNOS NO production seen in preeclampsia may contribute to the decreased NO bioavailability in this disease.

Keywords: endothelial nitric oxide synthase; hypertension; nitric oxide; preeclampsia; syncytiotrophoblast extracellular vesicles.

Figure 1.

Immunohistochemical staining of normal pregnancy (NP) placenta tissue and Western blot of NP and preeclampsia (PE)-derived placental lysate (PL), syncytiotrophoblast extracellular microvesicles (STBMV) and syncytiotrophoblast extracellular exosomes (STBEX; n=3). A, Placental tissue showing endothelial nitric oxide synthase (eNOS) staining (brown; left) on syncytiotrophoblast (STB) layer and IgG2a-negative staining (right). Immunoblot showing eNOS (140 kDa) and placental alkaline phosphatase (PlAP; 60 kDa) expression in PL, STBMV, and STBEX derived from NP (B) and PE (C) similar to expression of positive control, human umbilical vein endothelial cells (HUVEC). D, Placental tissue demonstrating lack of inducible nitric oxide synthases (iNOS) staining (left) and isotype control IgG1-negative staining (right). Immunoblot showing no expression for iNOS (131 kDa) though positive control, RAW, is expressed and PlAP (60 kDa) is also expressed in PL, STBMV, and STBEX derived from NP (E) and PE (F). Scale bar set at 100 mm.

Figure 2.

Flow cytometry analysis of ex vivo syncytiotrophoblast extracellular microvesicles (STBMV) derived from normal pregnancy (NP) and preeclampsia (PE) placentae (n=6). A, STBMV-bound endothelial nitric oxide synthase (eNOS) expression were significantly lower in PE compared with NP (*P<0.05). Flow cytometry analysis of circulating in vivo STBMV derived from paired peripheral vein blood (PB) and uterine vein blood (UV) plasma (n=8). B, Double-positive eNOS and placental alkaline phosphatase (PlAP) events per milliliter were higher in UV compared with PB (**P<0.01). Flow cytometry analysis of circulating in vivo syncytiotrophoblast extracellular vesicles (STBEV) derived from matched PB plasma of NP and PE patients (n=6). C, PlAP and eNOS double-positive STBMV events per milliliter were significantly lower in plasma from PE compared with NP (**P<0.01). PFP indicates platelet-free plasma.

Figure 3.

Immunobead depletion and nanoparticle tracking analysis (NTA) profiles of syncytiotrophoblast extracellular microvesicles (STBMV) and syncytiotrophoblast extracellular exosomes (STBEX) pools from normal pregnancy (NP; n=3). A, Representative immunoblot showing endothelial nitric oxide synthase (eNOS) and placental alkaline phosphatase (PlAP) coexpression on STBMV pool (Total) and STBMV pulled out (PO) with anti-eNOS Dynabeads, anti-PlAP Dynabeads, anti-IgG2a Dynabeads (isotype control for eNOS), and anti-IgG1 Dynabeads (isotype control for PlAP). B, Representative NTA size vs number profiles of STBMV pool (solid line), supernatant from post-incubation with anti-eNOS Dynabeads (dashed line) and anti-PlAP Dynabeads (dotted line). C, STBMV pool (solid line), supernatant of post incubation with anti-IgG2a Dynabeads (dashed line) and anti-IgG1 Dynabeads (dotted line). D, Representative immunoblot showing eNOS and PlAP coexpression on STBEX pool (Total) and STBEX PO with anti-eNOS, anti-PlAP, anti-IgG2a, and anti-IgG1 Dynabeads. E, Representative NTA size vs number profiles of STBEX pool alone (solid line), supernatant from post-incubation with anti-eNOS (dashed line) and anti-PlAP (dotted line) Dynabeads. F, STBEX pool (solid line), supernatant of post incubation with anti-IgG2a (dashed line) and anti-IgG1 (dotted line) Dynabeads.

Figure 4.

Syncytiotrophoblast extracellular microvesicles (STBMV) and syncytiotrophoblast extracellular exosomes (STBEX) express functional endothelial nitric oxide synthase (eNOS) and produce nitric oxide (NO). A, Dimerization of syncytiotrophoblast extracellular vesicles (STBEV) immunoblot image showing eNOS dimer (260 kDa) and eNOS monomer (140 kDa) expressed in human umbilical vein endothelial cells (HUVEC), STBMV, and STBEX (n=3). B, STBMV (***P<0.001) and STBEX (***P<0.001) pools showed NO production in a dose-dependent manner (n=6). C, 25 μg pool of STBMV and STBEX preincubated for 1 hour with 1 mmol/L of N^G-nitro-l-arginine methyl ester (L-NAME), NOS inhibitor, showed significant reductions in NO production compared with controls (both *P<0.05; n=3). D, 25 μg pool of STBMV and STBEX preincubated for 1 hour with 2 μmol/L N-(3-(aminomethyl) bezyl) acetamidine) (1400W), inducible nitric oxide synthases (iNOS)–specific inhibitor, showed no changes in NO production compared with controls (both ns; n=3).

Figure 5.

Nitric oxide synthase (NOS) activity of syncytiotrophoblast extracellular microvesicles (STBMV) and syncytiotrophoblast extracellular exosomes (STBEX) isolated from perfused normal pregnancy (NP) and preeclampsia (PE) placentae (n=11 and n=8, respectively). STBMV (A) from PE placentae showed a no overall reduction in NO production compared with NP (ns; P=0.2416). Analysis of the same data as a function of the gestational age of the samples (B) shows a decrease in NO production in samples closest in gestational age to controls (PE>34 weeks vs NP<40 weeks; *P<0.05). While STBEX (C) from PE showed a significant reduction in NO production compared with NP (*P<0.05). This decrease was maintained in samples closest in gestational age to controls (D).