The differential placental expression of ERp44 and pre-eclampsia; association with placental zinc, the ERAP1 and the renin-angiotensin-system

Introduction Endoplasmic reticulum resident protein 44 (ERp44) is a zinc-metalloprotein, regulating Endoplasmic reticulum aminopeptidase 1 (ERAP1) and Angiotensin II (Ang II). We explored placental ERp44 expression and components of the renin-angiotensin-system (RAS) in pre-eclampsia (PE), correlating these to ERAP1 expression and placental zinc concentrations. Methods Placental tissue, taken at time of delivery in normotensive women or women with PE (n = 12/group), were analysed for ERp44, AT1R, AT2R and AT4R by qPCR. Protein ERp44 expression was measured by immunohistochemistry and compared to previously measured ERAP1 expression. Placental zinc was measured by inductively-coupled-mass-spectrometry. Results ERp44 gene/protein expression were increased in PE (P < 0.05). AT1R expression was increased (P = 0.02) but AT4R decreased (P = 0.01) in PE, compared to normotensive controls. A positive association between ERp44 and AT2R expression was observed in all groups. ERp44 was negatively correlated with ERAP1 protein expression in all samples. Placental zinc concentrations were lower in women with PE (P = 0.001) and negatively associated with ERp44 gene expression. Discussion Increased placental ERp44 could further reduce ERAP1 release in PE, potentially preventing release of Ang IV and thus lowering levels of Ang IV which consequently diminishes the possibility of counterbalancing the activity of vasoconstrictive, Ang II. The lower placental zinc may contribute to dysfunction of the ERp44/ERAP1 complex, exacerbating the hypertension in PE.


Introduction
Pre-eclampsia (PE) is a hypertensive disorder of pregnancy that occurs in 2-4% of all pregnancies; it is associated with the greatest risk of maternal and fetal morbidity and mortality of all pregnancy-related hypertensive disorders [1].PE presents with new-onset hypertension and proteinuria in the mother after the 20th week of gestation.It can progress to multi-organ dysfunction, including hepatic, renal and cerebral disease, if the fetus and placenta are not delivered [2].The hallmark of PE is maternal endothelial dysfunction due to circulating factors of fetal origin from the placenta [3].PE has lifelong consequences for both the mother and her child, such as increased risks of cardiovascular, renal, and metabolic health problems [4].
ERp44 is a chaperone of the protein disulfide isomerase (PDI) family and an endoplasmic reticulum thioredoxin (TRX)-like motif-containing protein.It plays key roles in thiol-mediated retention and maturation of several secretory proteins within the endoplasmic reticulum (ER) [5].ERp44 cycles between the ER and cis-Golgi compartments to patrol proteins and control the traffic and oligomeric assembly of various secretory proteins, including IgM and adiponectin [6].
ERAP1 is expressed ubiquitously in all tissues including placenta [7].The enzyme plays an essential role in multiple biological processes which require cleavage of N-terminal amino acid residues.Such processes include the regulation of blood pressure, angiogenesis, the shedding of cytokine receptors, and immune recognition.
ERp44 also contributes to the control of blood pressure by regulating circulating Angiotensin II (Ang II) levels, which acts differentially through type 1 (AT1R) and type 2 (AT2R) receptors to regulate blood pressure and fluid homeostasis via the renin-angiotensin system (RAS).The balance between the vasoconstrictive and vasodilatory arms of the RAS is disrupted in PE [8,9].
ER aminopeptidase 1 (ERAP1) cleaves Ang II to Ang III and the vasodilatory Ang IV, the latter acting through its Angiotensin type 4 receptor (AT4R) [10].We have previously reported lower expression of ERAP1 placental protein expression in women PE [7], as well as alterations in the placental RAS in PE [8,11,12].ERp44 also complexes with several ER-resident enzymes that lack an ER-retention motif, such as ERAP1, with ERp44 controlling the release of ERAP1 in a redox-dependent manner [5,13].This ERp44-ERAP1 interaction has been shown to regulate blood pressure, as ERp44 suppresses the release of ERAP1.Thus, dysregulation of ERp44 leading to ERAP1 suppression will act downstream to prevent the cleavage of Ang II, contributing to RAS-induced hypertension [5].
Zinc and zinc ions (Zn 2+ ) are crucial elements of metalloenzymes and vital for successful embryogenesis and essential antioxidant activity [14,15].Zinc is retained during pregnancy with estimated concentrations of around 100 mg total [16], with 57% accrued in the fetus, 6.5% in the placenta, <1% in the amniotic fluid, 24% in the uterus, 5% in mammary tissue, and 6.5% in the expanded maternal blood volume [17].Zinc concentrations are increased approximately 2-fold during the third trimester when compared to non-pregnant women [18].Zinc deficiency has been associated with PE [14,15], with some data suggesting zinc level could be a useful clinical marker for severity of PE [19].The zinc metal ion also serves as a cofactor for stabilizing the three-dimensional structures of proteins [20][21][22].including ERp44.Zn 2+ binds ERp44 with sub-micromolar affinity, regulating its traffic and activity [13].Alterations in Zn 2+ have been shown to disrupt ERp44 function and its complex with ERAP1 [13], but this relationship has not yet been investigated in the placenta or in relation to PE.Thus, we hypothesize that in PE the decreased placental expression of ERAP1 will be due to increased ERp44 expression induced by zinc dysregulation.This increased ERp44 expression will prevent the conversion of Ang II to its vasodilatory metabolite products, contributing to RAS hyperactivity and maternal hypertension.Furthermore, due to the known association with the RAS, we expect to observe a correlation between ERp44 expression and Ang II receptor expression, further exacerbating blood pressure dysregulation.Hence, in this study we firstly investigated the mRNA abundance and protein expression of ERp44 in normotensive pregnant women compared to those with PE: secondly, we explored the relationship between this expression to components of the RAS, as well as to ERAP1 and placental zinc concentrations.

Participants
This study was carried out under the HRA-REC approval gained by the University of Nottingham (REF: 15/EM/0523).All methods were carried out in accordance with relevant guidelines and regulations.Fully informed, signed consent was obtained from all participating women.PE (n = 12) was defined as systolic blood pressure ≥140 mm Hg and diastolic blood pressure ≥90 mmHg, determined on 2 occasions >4 h apart and arising after 20 weeks of gestation in a previously normotensive woman and de novo proteinuria (protein: creatinine ratio (PCR) > 30; urine protein concentration >3 g/L in 2 random clean-catch midstream specimens collected >4 h apart) with no evidence of urinary tract infection [23].No women had any underlying renal or hypertensive disease before 20 weeks gestation.Healthy normotensive pregnant women matched for age (n = 12) who had no prior pregnancy complications, and no evidence of any urinary tract infections were recruited as comparative controls.
Medical and obstetric histories, including delivery data, were obtained from each woman.A summary of the demographic and pregnancy outcome of the women recruited in this study are presented in Table 1.Full-depth placental tissue biopsies were collected within 10 min of the placenta being delivered.Samples were collected from a standardized location midway between the cord insertion and placental border and were either snap frozen for gene expression or processed for immunohistochemistry as previously described [24].

Immunohistochemical staining
Placental protein expression was assessed by immunohistochemistry as previously described [25], using antibody to ERp44 (Sigma-Prestige, rabbit monoclonal: HPA001318; 30 μg/ml).As with gene expression, protein expression of ERAP1 was previously measured in the same samples [7].All slides were assessed by the same observer, blinded to the participant groups.Quantification was performed as described previously [25,27], using the Positive Pixel Algorithm of Aperio Image-Scope software; a visual check was also performed.

Placental zinc concentrations
Placental tissue concentrations of Zn 2+ (μg/kg of dry matter (DM) were also measured by ICP-MS (intra-assay variability <2%), after prior digestion of ~400 mg of freeze-dried tissue, to determine the percentage of water content, and subsequent digestion using 2% nitric acid as previously described [28,29].Certified reference material (NIST SRM bovine liver, 1577c) was used to validate elemental recovery and to correct for any batch variation.

Statistical analysis
All tests were performed using SPSS version 26 and GraphPad Prism version 8. Summary data are presented as means ± standard deviation (SD) or median and interquartile range (IQR) as appropriate.The Student's t-test or Mann-Whitney U-tests were applied depending on whether the data distribution was normal or skewed, as indicated by the Kolmogorov-Smirnov test.Spearman's Rank correlations tests were used to establish associations between continuous variables.The null hypothesis was rejected where P < 0.05.

Results
Baseline demographic and pregnancy outcome data are presented in Table 1.
Further detailed data regarding the whole cohort have been previously published [24].Overall, the groups were matched for maternal age, BMI, and parity.By definition, women who had PE had significantly higher blood pressures (P < 0.05) and significant proteinuria.In addition, birth weights were also lower in the PE diagnostic group.

ERp44 protein expression
Placental ERp44 protein expression was confirmed in placental tissue, with strong immunohistochemical staining localised to the syncytiotrophoblast (Fig. 2).

Discussion
These novel data report that women who suffer from PE display higher ERp44 placental expression and lower placental zinc concentrations when compared to their normotensive controls.Additional, potentially important, associations with the RAS and ERAP1 were also observed.We speculate these data suggest that dysregulation of placental expression of ERp44 contributes to the hypertension which is characteristic of PE.Our data supports previous reports of higher ERp44 mRNA expression in placentae from women with PE [30,31].The full function of ERp44 in PE is still unclear, but increased ERp44 in the placentae of women with PE may suggest that the trophoblasts need the ERp44 activity as previously hypothesized [31].ERp44 can be induced by agents that cause the accumulation of unfolded proteins in the ER upregulating ER-resistance proteins during ER stress [32], which is a characteristic pathological finding of PE [33].
The negative association found between ERp44 and ERAP1 indicates that higher levels of ERp44 correlate to decreased ERAP1 expression.ERp44 is a binding partner of ERAP1 through disulfide bonds in the endoplasmic reticulum.It also controls the intracellular and extracellular localization of ERAP1 [5] In the early secretory pathway of ERp44, zinc ions regulate the ability of ERp44 to localize ERAP1, all of which occurs in the trophoblast cells of the placenta [13].Our data suggest that even though ERp44 is increased, the fact that zinc levels are decreased results in diminished ERAP1 expression in PE patients.These observations may additionally contribute to the pathophysiological mechanism underlying hypertension.Future work is required to establish whether ERp44 and ERAP1 are co-localised and to determine if zinc is bound to ERp44 in the different groups.
The differences in the placental expression of RAS components in PE, confirm our previous data in a different sample cohort [8,12].A negative correlation between AT1R and AT4R indicates an imbalance towards vasoconstriction in PE.Moreover, when ERp44 complexes with the ERAP1 protein, as noted in previous studies [7], it may result in less protein available to complete the conversion of Ang II.Hisatsune et al. have reported, in a sepsis study carried out in mice, that in ERp44 +/− mice (which express half the amount of ERp44) a much greater drop in blood pressure was observed compared to the ERp44 +/+ mice with sepsis [7].Thus, in PE, where there is systemic inflammation, the ERp44-ERAP1 association is strengthened leading to lower availability of ERAP1.This ERp44-ERAP1 complex contributes to reduced generation of Ang IV.Moreover, since AT4R is unique in that it is not a GPCR, but an insulin-regulated aminopeptidase, the AT4R that is secreted into the maternal circulation may also cleave AngIII to AngIV [34,35].Thus with the reduced AT4R, this may provide less potential to counterbalance the activity of vasoconstrictive Ang II, raising blood pressure [5].
Zinc deficiency has increased over the last decade due to a trend towards a zinc-poor diet, based on processed foods and soy-based substitutes as well as food grown in zinc-poor soil [15].In this study, the lower placental zinc concentrations from women who had PE concurs with the findings of a previous study [36], as well as several reports of lower maternal zinc concentrations in PE [ [37][38][39]].Zinc binds with high affinity to ERp44, modulating its localization and ability to retrieve proteins such as ERAP1 [13].Therefore, the lower placental zinc concentration may also contribute to the dysfunction of the ERp44/ERAP1 complex, further exacerbating the hypertension in PE.Fig. 5 illustrates a schematic summarizing our proposed mechanism.
Other factors that have been reported to affect placental zinc concentrations are the expression and function of the zinc transporters ZnT 5, 6, 7 and 10, all of which have been shown to be upregulated in the Golgi in conditions of zinc deficiency or ER stress [40].This is consistent with the physiological stress encountered in PE.Future work is required to fully elucidate the mechanistic relationship between the transporter with zinc and ERp44.
In summary, the increased expression in ERp44 combined with lower placental zinc concentrations prevents an adequate release of the ERAP1 protein.This ERAP1 deficiency decreases the conversion of Ang II to Ang IV, contributing to the hypertension observed in PE.

Fig. 5 .
Fig. 5.A summary diagram of the Renin-AngiotensinSystem (RAS) pathway and the ERp44/ERAP1 complex illustrating how changes in zinc, can influence the ERp44-ERAP1 complex, results in disruption in components of the RAS and blood pressure regulation.Angiotensinogen is converted into angiotensin I (Ang 1), through renin, which is then converted to the hypertension-promoting angiotensin II (Ang II) by angiotensin converting enzyme (ACE) or into Angiotensin 1-9 (Ang 1-9) through angiotensin converting enzyme 2 (ACE2), which promotes hypotensive effects.Both Ang II and Ang 1-9 convert to angiotensin 1-7 (Ang 1-7), which advances hypotension, through ACE2 and ACE, respectively.ERAP1 is required to modulate the conversion of Ang II to angiotensin III (Ang III) and angiotensin IV (Ang IV).Ang III and Ang IV activate the Ang II type 1 and type 4 receptors (AT2R and AT4R), respectively, resulting in vasodilation.Due the negative association with ERp44 and ERAP1, the higher levels of ERp44 implicate lower levels of ERAP1, and this loss of function prevents Ang III and Ang IV from being formed thus disrupting the hypotensive effects.Instead, hypertension is exacerbated through Ang II, which activates AT1R and leads to increased blood pressure.Thus, we suggest that hypertension in pre-eclampsia results via the RAS and impaired ERAP1.Red functions indicate hypertensive effects and green functions indicate hypotensive effects.Gray functions delineate a lack of presence; LOF -Loss of Function.

Table 1
Demographic, clinical and biochemical data of participants.