Mice lacking phosphatase PP2A subunit PR61/B'delta (Ppp2r5d) develop spatially restricted tauopathy by deregulation of CDK5 and GSK3beta.

Functional diversity of protein phosphatase 2A (PP2A) enzymes mainly results from their association with distinct regulatory subunits. To analyze the functions of one such holoenzyme in vivo, we generated mice lacking PR61/B'δ (B56δ), a subunit highly expressed in neural tissues. In PR61/B'δ-null mice the microtubule-associated protein tau becomes progressively phosphorylated at pathological epitopes in restricted brain areas, with marked immunoreactivity for the misfolded MC1-conformation but without neurofibrillary tangle formation. Behavioral tests indicated impaired sensorimotor but normal cognitive functions. These phenotypical characteristics were further underscored in PR61/B'δ-null mice mildly overexpressing human tau. PR61/B'δ-containing PP2A (PP2A(T61δ)) poorly dephosphorylates tau in vitro, arguing against a direct dephosphorylation defect. Rather, the activity of glycogen synthase kinase-3ß, a major tau kinase, was found increased, with decreased phosphorylation of Ser-9, a putative cyclin-dependent kinase 5 (CDK5) target. Accordingly, CDK5 activity is decreased, and its cellular activator p35, strikingly absent in the affected brain areas. As opposed to tau, p35 is an excellent PP2A(T61δ) substrate. Our data imply a nonredundant function for PR61/B'δ in phospho-tau homeostasis via an unexpected spatially restricted mechanism preventing p35 hyperphosphorylation and its subsequent degradation.

Pre-eclampsia.

Pre-eclampsia remains a leading cause of maternal and perinatal mortality and morbidity. It is a pregnancy-specific disease characterised by de-novo development of concurrent hypertension and proteinuria, sometimes progressing into a multiorgan cluster of varying clinical features. Poor early placentation is especially associated with early onset disease. Predisposing cardiovascular or metabolic risks for endothelial dysfunction, as part of an exaggerated systemic inflammatory response, might dominate in the origins of late onset pre-eclampsia. Because the multifactorial pathogenesis of different pre-eclampsia phenotypes has not been fully elucidated, prevention and prediction are still not possible, and symptomatic clinical management should be mainly directed to prevent maternal morbidity (eg, eclampsia) and mortality. Expectant management of women with early onset disease to improve perinatal outcome should not preclude timely delivery-the only definitive cure. Pre-eclampsia foretells raised rates of cardiovascular and metabolic disease in later life, which could be reason for subsequent lifestyle education and intervention.

The "Great Obstetrical Syndromes" are associated with disorders of deep placentation.

Defective deep placentation has been associated with a spectrum of complications of pregnancy including preeclampsia, intrauterine growth restriction, preterm labor, preterm premature rupture of membranes, late spontaneous abortion, and abruptio placentae. The disease of the placental vascular bed that underpins these complications is commonly investigated with targeted biopsies. In this review, we critically evaluate the biopsy technique to summarize the salient types of defective deep placentation, and propose criteria for the classification of defective deep placentation into 3 types based on the degree of restriction of remodeling and the presence of obstructive lesions in the myometrial segment of the spiral arteries.

The enigmatic uterine junctional zone: the missing link between reproductive disorders and major obstetrical disorders?

While there is a growing realization that the origins of major obstetrical complications associated with defective deep placentation, such as pre-term labour, fetal growth restriction and pre-eclampsia, may lie in the very early pregnancy events, the underlying mechanisms are not understood. Impaired deep placentation is foremost a vascular pathology, characterized by a lack of endovascular trophoblast invasion and remodelling of a segment of the spiral arteries embedded within the inner myometrium of the uterus. Outside pregnancy, the inner myometrium represents a highly specialized, hormone-dependent structure, termed the junctional zone (JZ), which plays an integral part in the implantation process. The JZ changes with age and is disrupted in several reproductive disorders, such as endometriosis and adenomyosis, which in turn may account for the increased risk of adverse pregnancy outcome. Unlike the endometrium, the myometrial JZ is not readily accessible to biochemical or molecular studies, yet its structure and function can be assessed using imaging techniques, such as high-resolution ultrasound and magnetic resonance imaging. Thus, non-invasive assessment of the JZ prior to conception may turn out to be useful in identifying those women at risk of major obstetrical complications.

A role for menstruation in preconditioning the uterus for successful pregnancy.

Menstruation is widely viewed as serving no purpose other than to reinitiate the endometrial cycle in the absence of pregnancy. Yet, it is striking that cyclic endometrial decidualization followed by menstrual shedding is confined to the few species, including human beings, where placenta formation entails deep trophoblast invasion of maternal tissues and its vasculature. Both menstruation and pregnancy are inflammatory conditions that cause a degree of physiological ischemia-reperfusion tissue injury, albeit much more so in pregnancy. Thus, the emergence of cyclic menstruation may not have been an evolutionary coincidence but serves to protect uterine tissues from the profound hyperinflammation and oxidative stress associated with deep placentation, a process known as preconditioning. The concept of menstrual preconditioning provides a novel paradigm for understanding how reproductive disorders impact on pregnancy outcome. For example, endometriosis could be viewed as a disorder of exaggerated menstrual preconditioning that confers protection against placentation-related disorders, such as preeclampsia.

In vivo analysis of trophoblast cell invasion in the human.

In vivo analysis of trophoblast cell invasion is highly dependent on histological techniques, which are amply described in standard textbooks. The emphasis of this chapter therefore lies on material collection and interpretation of tissue sections, rather than on histological techniques per se. Proper identification of vascular structures on placental bed histological sections is important, the more because invading trophoblastic cells induce significant structural changes in uterine blood vessels, which may be disturbed in complicated pregnancies. Guidelines for distinguishing several vascular structures are provided, and different approaches for qualitative and quantitative assessment of spiral artery changes are discussed. The purpose of such studies is not only to obtain a better insight into mechanisms of trophoblast invasion and associated maternal tissue changes, but also to understand placental bed defects in various pregnancy complications.

Natural Killer Cell Reduction and Uteroplacental Vasculopathy.

Uterine natural killer cells are important for uteroplacental development and pregnancy maintenance. Their role in pregnancy disorders, such as preeclampsia, is unknown. We reduced the number of natural killer cells by administering rabbit anti-asialo GM1 antiserum in an established rat preeclamptic model (female human angiotensinogen×male human renin) and evaluated the effects at the end of pregnancy (day 21), compared with preeclamptic control rats receiving normal rabbit serum. In 100% of the antiserum-treated, preeclamptic rats (7/7), we observed highly degenerated vessel cross sections in the mesometrial triangle at the end of pregnancy. This maternal uterine vasculopathy was characterized by a total absence of nucleated/living cells in the vessel wall and perivascularly and prominent presence of fibrosis. Furthermore, there were no endovascular trophoblast cells within the vessel lumen. In the control, normal rabbit serum-treated, preeclamptic rats, only 20% (1/5) of the animals displayed such vasculopathy. We confirmed the results in healthy pregnant wild-type rats: after anti-asialo GM1 treatment, 67% of maternal rats displayed vasculopathy at the end of pregnancy compared with 0% in rabbit serum-treated control rats. This vasculopathy was associated with a significantly lower fetal weight in wild-type rats and deterioration of fetal brain/liver weight ratio in preeclamptic rats. Anti-asialo GM1 application had no influence on maternal hypertension and albuminuria during pregnancy. Our results show a new role of natural killer cells during hypertensive pregnancy in maintaining vascular integrity. In normotensive pregnancy, this integrity seems important for fetal growth.

The role of invasive trophoblast in implantation and placentation of primates.

We here review the evolution of invasive placentation in primates towards the deep penetration of the endometrium and its arteries in hominoids. The strepsirrhine primates (lemurs and lorises) have non-invasive, epitheliochorial placentation, although this is thought to be derived from a more invasive type. In haplorhine primates, there is differentiation of trophoblast at the blastocyst stage into syncytial and cellular trophoblast. Implantation involves syncytiotrophoblast that first removes the uterine epithelium then consolidates at the basal lamina before continuing into the stroma. In later stages of pregnancy, especially in Old World monkeys and apes, cytotrophoblast plays a greater role in the invasive process. Columns of trophoblast cells advance to the base of the implantation site where they spread out to form a cytotrophoblastic shell. In addition, cytotrophoblasts advance into the lumen of the spiral arteries. They are responsible for remodelling these vessels to form wide, low-resistance conduits. In human and great apes, there is additional invasion of the endometrium and its vessels by trophoblasts originating from the base of the anchoring villi. Deep trophoblast invasion that extends remodelling of the spiral arteries to segments in the inner myometrium evolved in the common ancestor of gorilla, chimp and human.

Risk of obstetrical complications in organ transplant recipient pregnancies.

Pregnancy after kidney and liver transplantation is becoming relatively common, although, in both groups, maternal complications are higher than in the general population. Both mean gestational age and mean birthweight seems significantly greater for liver transplant versus kidney transplant recipients and the risk of hypertension during pregnancy seems also lower for liver transplant than kidney transplant recipients. Thus, sequelae of chronic kidney diseases have stronger adverse effects on pregnancy, leading to a higher occurrence of adverse neonatal complications. Also, gestation in heart recipients may be complicated and preeclampsia seems to occur more frequently. However, the transplanted heart seems to adapt well to changes caused by pregnancy, such as increased cardiac workload and output, and elevated maternal oxygen consumption. More problematic is pregnancy in lung transplant recipients. Spontaneous pregnancy and healthy childbirth after bone marrow grafting is relatively rare due to irradiation, but, if gestation occurs, no specific problems have been identified. Obstetrical syndromes associated with transplantation reflect the pathology of defective deep placentation, where conversion of uterine spiral arteries remains largely restricted to the decidual segment. The myometrial segments of the uteroplacental arteries have a unique vascular memory and are at great risk to develop obstructive, atherosclerotic lesions. A similar increased risk of complications already existed in pregnancies during the years before transplantation. The effect of immunosuppressive therapy remains speculative. Therefore, the main target for improving the outcome of pregnancy in women at risk is the strict antihypertensive treatment from the earliest stage of pregnancy.

Evolution of invasive placentation with special reference to non-human primates.

It is now possible to view human placentation in an evolutionary context because advances in molecular phylogenetics provide a reliable scenario for the evolution of mammals. Perhaps the most striking finding is the uniqueness of human placenta. The lower primates have non-invasive placentae and even tarsiers and New World monkeys show restricted trophoblast invasion. Moreover, a truly villous placenta occurs only in Old World monkeys and great apes. The two latter groups of haplorhine primates show varying degrees of trophoblast-uterine interaction, including differences in the extent of decidualization, formation and disintegration of a cytotrophoblastic shell, degree of interstitial trophoblast invasion and depth of trophoblast invasion into spiral arteries. Recently, the occurrence of human-like deep invasion was confirmed in gorillas and chimpanzees. As the still enigmatic disease of pre-eclampsia also occurs in these species, such information may reveal the evolutionary roots of this disease of impaired maternal-fetal interaction.

Endovascular trophoblast and preeclampsia: A reassessment.

Since the earliest report on impaired spiral artery remodelling in preeclamptic human pregnancies, numerous studies have been devoted to possible mechanisms of impaired trophoblast invasion. A better knowledge of early uteroplacental blood flow has provided a physiological context for the processes of spiral artery invasion and associated remodelling, revealing a closely timed relationship between increasing flow and early steps in vascular remodelling. Concerning the impaired trophoblast invasion in preeclampsia, it has also to be considered that impaired invasion not only concerns invasion depth per se, but also the extension of this deep invasion from the central towards the more lateral spiral arteries of the placental bed. Since also in preeclampsia the very central spiral arteries may be normally invaded, the existence of such spatial gradient provides a further dimension to the problem. A practical consequence is that frequently used rodent models, which show invasion of two or three spiral arteries only, may be less useful for studying this particular aspect of the disease. Amongst non-human primates, baboons and rhesus monkeys are 'shallow invaders', and only in some of the great apes deep trophoblast invasion and associated spiral artery remodelling occurs. A better knowledge of the evolutionary history of deep invasion and its possible selective benefit might ultimately improve our understanding of failed deep invasion and impaired spiral artery remodelling in preeclampsia.

Angiotensin II type 1 receptor antibodies and increased angiotensin II sensitivity in pregnant rats.

Pregnant women who subsequently develop preeclampsia are highly sensitive to infused angiotensin (Ang) II; the sensitivity persists postpartum. Activating autoantibodies against the Ang II type 1 (AT(1)) receptor are present in preeclampsia. In vitro and in vivo data suggest that they could be involved in the disease process. We generated and purified activating antibodies against the AT(1) receptor (AT(1)-AB) by immunizing rabbits against the AFHYESQ epitope of the second extracellular loop, which is the binding epitope of endogenous activating autoantibodies against AT(1) from patients with preeclampsia. We then purified AT(1)-AB using affinity chromatography with the AFHYESQ peptide. We were able to detect AT(1)-AB both by ELISA and a functional bioassay. We then passively transferred AT(1)-AB into pregnant rats, alone or combined with Ang II. AT(1)-AB activated protein kinase C-α and extracellular-related kinase 1/2. Passive transfer of AT(1)-AB alone or Ang II (435 ng/kg per minute) infused alone did not induce a preeclampsia-like syndrome in pregnant rats. However, the combination (AT(1)-AB plus Ang II) induced hypertension, proteinuria, intrauterine growth retardation, and arteriolosclerosis in the uteroplacental unit. We next performed gene-array profiling of the uteroplacental unit and found that hypoxia-inducible factor 1α was upregulated by Ang II plus AT(1)-AB, which we then confirmed by Western blotting in villous explants. Furthermore, endothelin 1 was upregulated in endothelial cells by Ang II plus AT(1)-AB. We show that AT(1)-AB induces Ang II sensitivity. Our mechanistic study supports the existence of an "autoimmune-activating receptor" that could contribute to Ang II sensitivity and possible to preeclampsia.

Learning from the placenta: acute atherosis and vascular remodeling in preeclampsia-novel aspects for atherosclerosis and future cardiovascular health.

Preeclampsia is a common and potentially lethal pregnancy complication for women and offspring. Women who develop preeclampsia also run a long-term augmented risk of cardiovascular disease and premature death, and two theories are discussed. Women developing preeclampsia and persons developing cardiovascular disease may have common risk factors, which are unmasked by the "stress" of pregnancy. Alternatively, a new risk factor might occur de novo during the preeclamptic pregnancy. In preeclampsia, lipid deposition in walls of the maternal uterine arteries leading to the placenta, named spiral arteries, regularly occurs. These vascular lesions resemble early stages of atherosclerosis and are named "acute atherosis" and is thought to regress after delivery. The mechanisms that contribute to acute atherosis in preeclampsia are largely unknown, but are related to the impaired vascular remodeling of the spiral arteries in the first half of pregnancy. One striking feature is that the development of these "atherosclerosis-like" lesions requires a few months in pregnancy and may be partly linked to invasion of trophoblasts (specialized fetally derived placenta cells). We summarize normal and pathological vessel remodeling in pregnancy and discuss similarities and differences between preeclampsia and arteriosclerosis. The transient appearance of acute atherosis of uterine wall spiral arteries seen in pregnancy complications and the molecular interaction between trophoblast, smooth muscle and vascular cells could add important elements to explain arteriosclerosis and stenosis in cardiovascular disease. Further understanding of the process underlying spiral artery atherosis in the months of pregnancy may cast light on development of cardiovascular disease later in life.

Inhibition of trophoblast-induced spiral artery remodeling reduces placental perfusion in rat pregnancy.

Rats harboring the human angiotensinogen and human renin genes develop preeclamptic features in pregnancy. The preeclamptic rats exhibit a deeper trophoblast invasion associated with a reduced resistance index by uterine Doppler. Doxycycline inhibits matrix metalloproteinase activity. We tested the hypothesis that matrix metalloproteinase inhibition reduces trophoblast invasion with subsequent changes in placental perfusion. Preeclamptic and pregnant control Sprague-Dawley rats were treated with doxycycline (30 mg/kg of body weight orally) from gestational day 12 until day 18. Placental perfusion was assessed using a micromarker contrast agent. The animals were euthanized on day 18 of pregnancy; biometric data were acquired, and trophoblast invasion was analyzed. Doxycycline resulted in intrauterine growth retardation and lighter placentas in both groups. Maternal body weight was not affected. As shown earlier, preeclamptic rats exhibited a deeper endovascular trophoblast invasion. However, doxycycline treatment reduced trophoblast invasion in the preeclamptic rats. The physiological spiral artery remodeling, as assessed by the deposition of fibrinoid and alpha-actin in the spiral artery contour, was significantly reduced by doxycycline. The vascularity index, as assessed by perfusion measurement of the placenta, was reduced after doxycycline treatment in preeclamptic rats. Thus, matrix metalloproteinase inhibition with doxycycline leads to reduced trophoblast invasion and associated reduced placental perfusion. These studies are the first to show that reducing trophoblast-induced vascular remodeling decreases subsequent placental perfusion. Our model allows the study of dysregulated trophoblast invasion and vascular remodeling in vivo to gain important insights into preeclampsia-related mechanisms.

Effects of circulating and local uteroplacental angiotensin II in rat pregnancy.

The renin-angiotensin (Ang) system is important during placental development. Dysregulation of the renin-Ang system is important in preeclampsia (PE). Female rats transgenic for the human angiotensinogen gene crossed with males transgenic for the human renin gene develop the PE syndrome, whereas those of the opposite cross do not. We used this model to study the role of Ang II in trophoblast invasion, which is shallow in human PE but deeper in this model. We investigated the following groups: PE rats, opposite-cross rats, Ang II-infused rats (1000 ng/kg per day), and control rats. Ang II infusion increased only circulating Ang II levels (267.82 pg/mL), opposite cross influenced only uteroplacental Ang II (13.52 fmol/mg of protein), and PE increased both circulating (251.09 pg/mL) and uteroplacental (19.24 fmol/mg of protein) Ang II. Blood pressure and albuminuria occurred in the models with high circulating Ang II but not in the other models. Trophoblast invasion increased in PE and opposite-cross rats but not in Ang II-infused rats. Correspondingly, uterine artery resistance index increased in Ang II-infused rats but decreased in PE rats. We then studied human trophoblasts and villous explants from first-trimester pregnancies with time-lapse microscopy. Local Ang II dose-dependently increased migration by 75%, invasion by 58%, and motility by 282%. The data suggest that local tissue Ang II stimulates trophoblast invasion in vivo in the rat and in vitro in human cells, a hitherto fore unrecognized function. Conceivably, upregulation of tissue Ang II in the maternal part of the placenta represents an important growth factor for trophoblast invasion and migration.

Fetal-maternal conflict, trophoblast invasion, preeclampsia, and the red queen.

The much publicized conflict hypothesis for understanding fetal-maternal interaction during pregnancy often invokes a 'battle' metaphor, rather than a well orchestrated interplay occurring as a series of well controlled moves and counter-moves as happens in a game of chess. Such stepwise interaction is particularly obvious in the spiral artery remodelling process, and it would be interesting to trace the history of the successive steps in histological adaptation throughout primate phylogeny. The restricted invasion observed in a few species on a 'lower' evolutionary scale suggests a tendency of progressive deeper invasion during primate evolution. Unfortunately, our knowledge of invasive processes in the placental bed in nonhuman primates is highly inadequate. A paradigm underscoring the stepwise interaction between mother and fetus may be provided by the Red Queen hypothesis, which is a useful model to explain co-evolutionary processes between different species. The apparent association between preeclampsia and restricted endovascular trophoblast invasion, combined with the absence of the disease in primate species showing shallow invasion, suggests that preeclampsia may result from a failure in one or more interactive steps necessary for deeper invasion. Evidence for a genetic component invokes the puzzling question as to why "preeclampsia genes" are not eliminated from human populations. As in other fields of medicine, a proper understanding of Darwinian selection processes may throw some light on the causes of preeclampsia.

Uterine vascular function in a transgenic preeclampsia rat model.

We investigated intrauterine growth restriction, endothelial function, and uterine artery blood flow characteristics in a transgenic preeclampsia rat model with an activated renin-angiotensin system. We compared preeclamptic Sprague-Dawley (SD-PE) rats with normal pregnant Sprague-Dawley and nonpregnant Sprague-Dawley rats. We used transabdominal ultrasound and found that SD-PE rat embryos developed intrauterine growth restriction. Isolated uterine arteries from SD-PE rats incubated with phenylephrine exhibited an increased contractile response, whereas a single high dose of acetylcholine resulted in an impaired vasorelaxation compared with controls. Incremental acetylcholine doses increased relaxation of SD-PE vessels at low acetylcholine doses but caused a paradoxical contraction at higher acetylcholine doses. Indomethacin and a thromboxane-receptor antagonist (SQ 29,548) blocked this effect, suggesting maternal prostanoid-dependent endothelial dysfunction. SD-PE rats had a decreased prostacyclin (6-keto-prostaglandin F1alpha):thromboxane ratio in the serum compared with normal pregnant Sprague-Dawley rats or nonpregnant Sprague-Dawley. Surprisingly, the Doppler resistance index decreased during pregnancy in SD-PE compared with normal pregnant Sprague-Dawley rats, suggesting unimpaired uteroplacental flow in the uterine artery. Umbilical flow was unchanged with absent end-diastolic flow in all of the groups. Renin-angiotensin system activation-induced preeclampsia is associated with altered placentation, modified resistance index, and endothelial dysfunction. A disturbed prostacyclin:thromboxane ratio could be an important mediator.

Trophoblasts reduce the vascular smooth muscle cell proatherogenic response.

Maternal spiral artery remodeling is the consequence of controlled trophoblast invasive interaction with the maternal cellular environment and is fundamentally important for successful placentation. In preeclampsia, trophoblast invasion is shallow, remodeling is incomplete, and vessels develop an inflammatory appearance, termed "acute atherosis." We noted that, in our preeclampsia, human renin-human angiotensinogen transgenic rat model, complement component 3 (C3), and tumor necrosis factor-alpha were upregulated and heavily expressed in atherotic uteroplacental vessels. We next used coculture involving human trophoblasts, rat vascular smooth muscle cells (VSMCs), and human VSMCs to observe VSMC-trophoblast regulatory interactions. Tumor necrosis factor-alpha induced complement C3 and interleukin-6 expression in VSMCs. We found that trophoblasts were able to reduce VSMC C3 and interleukin-6 expression after the VSMCs were stimulated with tumor necrosis factor-alpha. However, a direct VSMC-trophoblast cell-cell contact was necessary for this anti-inflammatory response. We also studied double-transgenic VSMCs that express inflammatory components and exhibit accelerated proliferation ("synthetic" phenotype). Trophoblasts could not downregulate C3 in these cells. We then examined uteroplacental tissues from preeclamptic and control patients. In control deciduas, only traces of C3 staining were observed, and vessels were thin walled without thrombus formation. In preeclampsia, the decidual vessels showed atherosis, thrombus formation, and C3 expression. Our data suggest that fetally derived trophoblasts require direct cell-cell contact with maternally derived VSMCs to downregulate VSMC C3 and interleukin-6 expression and to avoid atherosis. The findings also implicate C3 in the placental vasculopathy observed in preeclampsia.