Background-Ischemia in the placenta is considered the base of the pathogenesis of preeclampsia, a pregnancy-specific syndrome in which soluble endoglin (sEng) is a prognostic marker and plays a pathogenic role. Here, we investigated the effects of hypoxia and the downstream pathways in the release of sEng. Methods and Results-Under hypoxic conditions, the trophoblast-like cell line JAR showed an increase in sEng parallel to an elevated formation of reactive oxygen species. Because reactive oxygen species are related to the formation of oxysterols, we assessed the effect of 22-(R)-hydroxycholesterol, a natural ligand of the liver X receptor (LXR), and the LXR synthetic agonist T0901317. Treatment of JAR cells or human placental explants with 22-(R)-hydroxycholesterol or T0901317 resulted in a clear increase in sEng that was dependent on LXR. These LXR agonists induced an increased matrix metalloproteinase-14 expression and activity and a significant reduction of its endogenous inhibitor, tissue inhibitor of metalloproteinase-3. In addition, mice treated with LXR agonists underwent an increase in the plasma sEng levels, concomitant with an increase in arterial pressure. Moreover, transgenic mice overexpressing sEng displayed high blood pressure. Finally, administration of an endoglin peptide containing the consensus matrix metalloproteinase-14 cleavage site G-L prevented the oxysterol-dependent increase in arterial pressure and sEng levels in mice. Conclusions-These studies provide a clue to the involvement of the LXR pathway in sEng release and its pathogenic role in vascular disorders such as preeclampsia. (Circulation. 2012;126:2612-2624.)Key Words: cell hypoxia Ⅲ hypertension Ⅲ pre-eclampsia Ⅲ pregnancy Ⅲ peptides P reeclampsia is a pregnancy-specific syndrome characterized by systemic hypertension, proteinuria, and edema in the third trimester of pregnancy. 1,2 It affects both the fetus and the mother and occurs in Ϸ5% of pregnancies. Severe preeclampsia leads to the appearance of the HELLP syndrome (hemolysis, elevated liver enzymes, and low platelets), seizures, or fetal growth restriction and can result in fetal death. Preeclampsia is thought to be the consequence of impaired placentation resulting from inadequate trophoblastic invasion of the maternal spiral arteries. 3 Abnormal placentation is an important predisposing factor for preeclampsia, whereas endothelial dysfunction appears to be central to the pathophysiological changes, possibly indicative of a 2-stage disorder characterized by reduced placental perfusion and a maternal syndrome. Hypoxia, followed by oxidative stress, has been postulated as a critical signal that initiates the pathogenic process in preeclampsia. 4,5 Hypoxia and extracellular inflammatory signals can induce the intracellular accumulation of reactive oxygen species (ROS). 6,7 In turn, the imbalance between ROS production and antioxidant systems induces oxidative stress that negatively affects reproductive processes, including cyclic luteal and endometrial changes, follicular d...

Upon inflammation, monocyte-derived macrophages (MΦ) infiltrate blood vessels to regulate several processes involved in vascular pathophysiology. However, little is known about the mediators involved. Macrophage polarization is crucial for a fast and efficient initial response (GM-MΦ) and a good resolution (M-MΦ) of the inflammatory process. The functional activity of polarized MΦ is exerted mainly through their secretome, which can target other cell types, including endothelial cells. Endoglin (CD105) is a cell surface receptor expressed by endothelial cells and MΦ that is markedly upregulated in inflammation and critically involved in angiogenesis. In addition, a soluble form of endoglin with anti-angiogenic activity has been described in inflammation-associated pathologies. The aim of this work was to identify components of the MΦ secretome involved in the shedding of soluble endoglin. We find that the GM-MΦ secretome contains metalloprotease 12 (MMP-12), a GM-MΦ specific marker that may account for the anti-angiogenic activity of the GM-MΦ secretome. Cell surface endoglin is present in both GM-MΦ and M-MΦ, but soluble endoglin is only detected in GM-MΦ culture supernatants. Moreover, MMP-12 is responsible for the shedding of soluble endoglin in vitro and in vivo by targeting membrane-bound endoglin in both MΦ and endothelial cells. These data demonstrate a direct correlation between GM-MΦ polarization, MMP-12, and soluble endoglin expression and function. By targeting endothelial cells, MMP-12 may represent a novel mediator involved in vascular homeostasis.

Increased levels of soluble endoglin (Sol-Eng) correlate with poor outcome in human cancer. We have previously shown that shedding of membrane endoglin, and concomitant release of Sol-Eng is a late event in chemical mouse skin carcinogenesis associated with the development of undifferentiated spindle cell carcinomas (SpCCs). In this report, we show that mouse skin SpCCs exhibit a high expression of hepatocyte growth factor (HGF) and an elevated ratio of its active tyrosine kinase receptor Met versus total Met levels. We have evaluated the effect of Sol-Eng in spindle carcinoma cells by transfection of a cDNA encoding most of the endoglin ectodomain or by using purified recombinant Sol-Eng. We found that Sol-Eng inhibited both mitogen-activated protein kinase (MAPK) activity and cell growth in vitro and in vivo. Sol-Eng also blocked MAPK activation by transforming growth factor-β1 (TGF-β1) and impaired both basal and HGF-induced activation of Met and downstream MAPK. Moreover, Sol-Eng strongly reduced basal and HGF-stimulated spindle cell migration and invasion. Both Sol-Eng and full-length endoglin were shown to interact with Met by coimmunoprecipitation experiments. However, full-length endoglin expressed at the plasma membrane of spindle carcinoma cells had no effect on Met signaling activity, and was unable to inhibit HGF-induced cell migration/invasion. These results point to a paradoxical suppressor role for Sol-Eng in carcinogenesis.

Elevated levels of a circulating form of the transforming growth factor-β (TGF-β) coreceptor endoglin correlate with poor clinical outcome in different types of cancer. Soluble endoglin (Sol-Eng) is primarily produced by cleavage of cell-surface endoglin by the transmembrane metalloprotease MMP14 that releases most of its extracellular domain. Sol-Eng has been found to contribute to different cardiovascular pathologies, including preeclampsia, a severe hypertensive syndrome of pregnancy. While the anti-angiogenic and pro-hypertensive functions of Sol-Eng appear well established, its role in cancer has not been fully investigated. Recently, we reported that Sol-Eng strongly inhibits signaling through the hepatocyte growth factor (HGF) tyrosine kinase receptor Met in mouse skin spindle carcinoma cells. Sol-Eng also blocked basal and HGF-mediated stimulation of carcinoma cell proliferation, migration and invasion. Taken together, the above results and the anti-angiogenic function exerted by Sol-Eng suggest a suppressor role for Sol-Eng in cancer. This conclusion is discussed in the paradoxical context of Sol-Eng as a marker of poor prognosis and as a potential contributor to the decreased risk of preeclamptic mothers to develop breast cancer later in life.

Autophagy, a mechanism of cell survival during times of stress, plays an important role in normal placentation, protecting the fetus during fluctuations in conditions such as nutrient availability. In cases of placental insufficiency, often present in PE, autophagy may be defective. We have shown that BPH/5 mice, in addition to spontaneously developing a PE-like syndrome including late-gestational hypertension and proteinuria, exhibit abnormal placentation and poor feto-placental outcomes. Here we tested the hypothesis that there is increased autophagy in fetoplacental units (FPUs) of pregnant BPH/5 females. RNA was extracted from FPUs (e10.5) of BPH/5 and C57 control mice and analyzed for biomarkers of autophagy. BPH/5 showed a significant increase in both Beclin-1 and LC3-β compared to C57 (1.5-fold for each, n=7; p<0.05). Increased autophagy in e10.5 BPH/5 FPUs was corroborated at the protein level using ELISA to measure Beclin-1 (0.6±0.1 control vs. 2±0.17 ng/mL BPH/5; n=7; p<0.05). We also analyzed protein levels of p62, an autophagy biomarker which is degraded during autophagy. Consistent with these other findings, the levels of p62 were lower in BPH/5 mice (29.7±2.5 controls vs. 7.2±1.3 ng/mL BPH/5, p<0.05). Given that autophagy in trophoblasts plays a key role in the invasion of these cells and thus remodeling of uterine arteries, we examined the invasion capacity of e10.5 trophoblasts isolated from C57 and BPH/5 mice. Trophoblasts from BPH/5 mice were much less invasive when compared to C57 (2-fold decrease, n=7; p<0.05). Inhibition of autophagy with chloroquine diphosphate (50 μM) significantly improved invasion capacity of these cells (1.6-fold increase; n=7; p<0.05). Collectively, these data demonstrate that autophagy is defective in FPUs from BPH/5 mice, and suggest that this may play a causal role in PE in this model.

Autophagy, a mechanism of cell survival, plays an important role in normal placentation and protects the fetus during stress. Previously, we showed that autophagy was increased in fetoplacental units (FPUs) from pregnant BPH/5 mice, a model that develops a PE-like syndrome including late-gestational hypertension, proteinuria, abnormal placentation and poor feto-placental outcomes. We also showed that increased autophagy led to reduced invasion capacity of BPH/5 trophoblasts. Evidence suggests that hypoxia increase autophagy, so we measured the invasion capacity of trophoblasts isolated from C57 mice in normoxic/hypoxic conditions. Trophoblasts cultured in hypoxia exhibited defective invasion, effect partially blocked by autophagy inhibitor chloroquine diphosphate (CQ; 1.7-fold decrease, n=4 p<0.005 hypoxia vs. normoxia; 1.35-fold increase p<0.5 CQ vs. hypoxia). We also tested the effect of conditioned media from BPH/5 or C57 placental explants on the invasion capacity of JAR cells, a human choriocarcinoma trophoblast cell line. Conditioned media from BPH5 explants decreased JAR invasion, but only for 24h after placental collection (1.8-fold decrease, n=4 p<0.005 vs. C571.33-fold p<0.05 vs. BPH/5). However, when the explants were kept in hypoxia, JAR invasion capacity decreased following treatment with BPH5 explants conditioned media, even after 48h; effect partially inhibited by CQ (1.6-fold decrease, n=4 p<0.005 vs. CTR; 1.7-fold, n=4 p<0.05 vs. BPH/5). As autophagy plays a key role in the invasion of trophoblasts and thus remodeling of uterine arteries, we performed a tube formation assay with endothelial cells treated with conditioned media from BPH/5 or C57 placental explants. Tube formation decreased after treatment with conditioned media from BPH/5 explants (1.7-fold decrease, n=3 p<0.05). Co-culture of endothelial and trophoblast cells under hypoxia resulted in less efficient tube formation. Treatment with CQ partially blocked this effect (1.55-fold decrease, n=3 p<0.05 vs normoxia; 1.2-fold, n=3 p<0.05 vs hypoxia). These data demonstrate that invasion capacity and tube formation are defective in trophoblasts from BPH/5 mice, suggesting that hypoxia-dependent autophagy may play a causal role in PE in the BPH/5 model.