Pregnancy poses a serious challenge for maintaining normal blood pressure. Pregnancy-induced hypertension, a major cause of maternal and fetal deaths, occurs in ~10% of pregnancies5, 6
. During pregnancy, the uterus undergoes profound morphological changes, including trophoblast invasion and spiral artery remodeling. In preeclampsia, impaired spiral artery remodeling is common, but the underlying mechanisms are unclear1, 2, 7-9
. Studies indicate that vascular growth factor receptors, angiotensin and estradiol are involved in the disease10-14
Corin is a cardiac protease that activates ANP, a cardiac hormone regulating blood pressure and sodium homeostasis15
. In mice, lack of corin prevents ANP generation and causes hypertension16
. In humans, corin variants are associated with hypertension17
. Interestingly, corin expression was detected in the pregnant mouse4
() and human uterus (Supplementary Fig. 1). As a transmembrane protein, corin is expected to act at the expression sites, suggesting a possible function in the pregnant uterus.
Hypertension, proteinuria and renal pathology in pregnant corin ko and ko/Tg mice
To understand the role of corin in pregnancy, we created a mouse model, in which a corin transgene was expressed under a cardiac promoter (). The transgenic (Tg) and corin knockout (ko) mice were crossed to generate ko/Tg mice expressing corin only in the heart (). In ko/Tg mice, transgenic corin expression restored pro-ANP processing in the heart (Supplementary Fig. 2) and normalized blood pressure (), indicating that cardiac corin was sufficient to maintain normal blood pressure in non-pregnant (np) mice.
In pregnant corin ko mice, blood pressure increased at ~17 days post-coitus and rose further before returning to the np level after delivery (), which resembled late gestational hypertension in preeclamptic women. In corin ko/Tg mice, which were normotensive, blood pressure increased similarly during pregnancy (), indicating that cardiac corin expression did not prevent pregnancy-induced hypertension. The data also show that in these mice hypertension in pregnancy was not due to preexisting high blood pressure. In addition to in the uterus, corin mRNA was detected in the umbilical cord and placenta (Supplementary Fig. 3). To distinguish the role of maternal corin from that of placental or other fetal organs, corin ko females were mated with either wt or ko males. The resulting fetuses carried one or no copy of the functional corin gene. Normally, enzymes encoded by one gene copy are sufficient for their function. As shown in , pregnant corin ko females, mated with either wt or ko males, had similarly increased blood pressure, indicating that lack of maternal, but not fetal, corin caused hypertension in pregnancy.
Proteinuria is a hallmark of preeclampsia. WT, corin ko and ko/Tg mice had similar urinary protein levels before pregnancy and at mid gestation. The levels, however, increased in corin ko and ko/Tg mice at late gestation (), consistent with reported proteinuria in mouse models of preeclampsia18
. Ischemic glomeruli, indicated by fewer red blood cells, were found in pregnant corin ko and ko/Tg mice () but not in np mice (Supplementary Fig. 4). PAS staining revealed increased extracellular matrixes and collapsed glomerular capillaries in pregnant corin ko and ko/Tg mice (). Electron microscopy showed narrow glomerular capillary lumens and thick basement membranes (), suggesting endotheliosis and increased extracellular matrixes. Additional pathological features such as necrotic cells and calcium deposits in the placental labyrinth also existed in these mice (Supplementary Fig. 5), indicating insufficient uteroplacental perfusion. Consistently, corin ko and ko/Tg mice had smaller litters (7.1 ± 2.3 (n=28) and 6.8 ± 2.7 (n=28), respectively, vs
. wt 9.1 ± 1.2 (n=21) pups/litter; p
We examined embryos of E12.5 day, an early time point before blood pressure increase in corin ko and ko/Tg mice, and E18.5 day before delivery. WT E12.5 embryos exhibited obvious trophoblast invasion, shown by cytokeratin (cytok) staining (), and large vessels mostly in the deep decidua, shown by smooth muscle actin (SMA) staining (), indicating that smooth muscles in the superficial decidua were replaced by invading trophoblasts. In contrast, trophoblast invasion in corin ko and ko/Tg embryos was markedly reduced () and smaller arteries were found in both superficial and deep decidua (). In E18.5 wt embryos, more abundant trophoblasts were found in the decidua and myometrium compared with those in corin ko and ko/Tg mice (). By H&E staining, larger and more abundant decidual spiral arteries were observed in wt than corin ko or ko/Tg mice (). showed strong cytok (trophoblasts) staining but weak von Willebrand factor (vWF) (endothelial) and SMA (smooth muscles) staining in wt decidual and myometrial arteries. These data indicate that trophoblast invasion and spiral artery remodeling were impaired in corin ko and ko/Tg mice and that this defect occurred before blood pressure increased in these mice.
Impaired trophoblast invasion and spiral artery remodeling in corin ko and ko/Tg mice
Corin activates ANP in the heart15
. It was unknown if the corin function in pregnancy also was mediated by ANP. Pro-ANP is expressed in the np and pregnant uterus (Supplementary Fig. 6). If corin acts on pro-ANP to promote trophoblast invasion and spiral artery remodeling, thereby preventing hypertension in pregnancy, ANP and corin ko mice should have similar phenotypes. ANP ko mice are hypertensive () but their blood pressure was not monitored during pregnancy19
. We found similarly increased blood pressure in pregnant ANP ko mice (). The mice also had late gestational proteinuria () and smaller litters (4.4 ± 1.7 (n=25) vs
. wt 9.1 ± 1.2 (n=21) pups/litter, p
<0.001). By immunostaining, impaired trophoblast invasion and smaller spiral arteries were observed in E12.5 embryos (). In E18.5 embryos, ANP ko mice had far fewer trophoblasts () and smaller arteries () in the decidua and myometrium than those in wt. Consistently, weak cytok-staining but strong vWF-staining were found in arteries in ANP ko mice (). Thus, ANP and corin ko mice had very similar phenotypes, indicating that the role of corin in pregnancy is most likely mediated by ANP.
Hypertension, proteinuria and uteroplacental pathology in pregnant ANP ko mice
In the heart, corin produces ANP, which in turn regulates blood pressure by promoting natriuresis and vasodilation3
. Here we found that lack of corin and ANP impaired trophoblast invasion and spiral artery remodeling, which was not rescued by cardiac corin expression in corin ko/Tg mice. ANP is known to relax vascular smooth muscles. Recently, ANP and its downstream cGMP-dependent protein kinase were shown to be important in angiogenic processes by promoting endothelial regeneration20, 21
. Thus, ANP may function locally to remodel uterine arteries. Our results also suggest that ANP may directly promote trophoblast invasion (). This hypothesis was tested. We found that ANP markedly stimulated human trophoblasts to invade Matrigels () (Supplementary Fig. 7a). In these cells, ANP receptor mRNA expression was confirmed (Supplementary Fig. 7b) and ANP-stimulated intracellular cGMP production was detected () (Supplementary Fig. 7c).
ANP-stimulated human trophoblast invasion, and impaired uterine corin expression and corin mutations in preeclamptic patients
Our findings underscore the importance of locally produced ANP by corin, which acts on trophoblasts and vascular cells in the uterus. Because heart-derived ANP circulates inside the vessel, our model may explain why cardiac corin failed to promote trophoblast invasion and uterine artery remodeling, as shown in corin ko/Tg mice. To verify this hypothesis, we quantified corin mRNA and protein in human uteruses by RT-PCR and ELISA. The levels were low in np women but increased in pregnant women (). In preeclamptic women, the levels were significantly lower than in normal pregnancies. Similar results were found by immunostaining (Supplementary Fig. 8). Consistently, pro-ANP levels in uterine tissues were significantly higher in preeclamptic women than normal pregnant women (), indicating that reduced uterine corin expression impaired pro-ANP processing in these patients. Corin is a membrane-bound protein4, 15
. Recent studies showed that corin can be shed from cardiomyocytes and that soluble corin was detected in human plasma22, 23
. We found that plasma corin levels were higher in preeclamptic patients than np or normal pregnant women (). Thus, plasma corin levels did not reflect that in tissues, indicating that plasma corin was likely derived from the heart, where corin expression was increased in response to high blood volume and pressure in pregnancy. These results further support a local corin function in the pregnant uterus.
We then sequenced the CORIN
in preeclamptic patients and identified a mutation altering Lys to Glu at position 317 in LDL receptor repeat 2 in one woman () and another mutation altering Ser to Gly at position 472 in frizzled 2 domain in two women from the same family who had preeclampsia (). In functional studies, K317E and S472G mutations did not affect corin expression in HEK293 cells but markedly reduced corin activity in processing pro-ANP (). The data was consistent with previous findings that LDL receptor repeats and frizzled domains are critical for corin activity25
, suggesting that the mutations may impair corin function in the patients, thereby contributing to preeclampsia. Interestingly, corin variants in frizzled 2 domain that impaired corin function have been reported in African Americans17, 26
, a high risk population for preeclampisa.
Previously, high levels of plasma pro-ANP/ANP were detected in preeclamptic patients27, 28
. As shown with our plasma soluble corin data, plasma protein levels may not reflect those in tissues. Together, we have identified a novel local function of corin and ANP in promoting trophoblast invasion and spiral artery remodeling to prevent hypertension in pregnancy. Our data suggest that impaired corin expression or function in the pregnant uterus may represent an important mechanism underlying preeclampsia. Studies to further understand impaired uterine corin expression in preeclamptic patients may help to develop new strategies to enhance the corin/ANP pathway to prevent or treat this life-threatening disease.