*4.1. Animals and Diets*

This study was carried out following the recommendations of the National Institute of Health (NIH) *Guide for the Care and Use of Laboratory Animals* and the Directive 2010/63/EU. All procedures and protocols were governmentally approved by the corresponding board (Regierung von Mittelfranken, AZ #54-2531.31-31/09 (10 November 2010) and AZ #55.2-2532-2-820 (17 January 2019)). Surgical procedures were performed under isoflurane anesthesia and all e fforts were made to minimize su ffering.

#### 4.1.1. Alimentary Rat Model with IUGR-Like Features

Animal procedures and the dietary regimen were carried out as previously described by us in detail [36]. Wistar rats were ordered from Charles River (Sulzfeld, Germany). Weighing 240–260 g, rats were mated, and the beginning of gestation was determined via assessment of vaginal plug expulsion. Subsequently, dams were randomly assigned into two groups consisting of six animals each and received semi-purified diets (Altromin Spezialfutter GmbH & Co. KG, Lage, Germany) of either low protein diet (LP group, 25 g/d of Altromin C1003, 8% protein) or an isocaloric diet of normal protein content (NP group, 25 g/d of Altromin C1000, 17% protein) from day 1 of gestation. This results in reduced birth weight and increased placental-to-fetal weight ratio, indicating preserved placental efficiency [63]. Rat placentas were obtained at E18.5. Animal characteristics are displayed in Table 1. Sex verification was carried out via sex-determining region Y (Sry) gene PCR, as previously described by us in detail [36].

### 4.1.2. Mouse Model with PE/IUGR-Like Features

The eNOS-knockout (eNOS−/−) mice came from Jackson Laboratories (Bar Harbor, Maine, USA). The recommended wild-type (WT) C57BL/6 mice were ordered from Charles River (Sulzfeld, Germany). A homozygous breeding strategy was followed. Both strains were kept over ten generations in our animal facility before being utilized in experiments. Mice were housed at 22 ± 2 ◦C and a 12 h light/dark cycle in our animal facilities. Animals had unlimited access to standard chow (SSNIFF V1534, ssni ff Spezialdiäten GmbH, Soest, Germany) and tap water. The animal model of eNOS−/− mice was previously described in detail by others [28,64,65]. The placental dysfunction in eNOS−/− mice [26,64,65] is caused with an impaired systemic vascularization of the dam [29]: eNOS deficiency significantly reduces the essential maternal cardiovascular adaptive capacity via reduction of circulating nitric oxide [28]. Thus, maintenance of constant uterine and feto-placental blood flow and of low feto-placental vascular resistance via modulation of smooth muscle myogenic tone is disabled [19,27,30]. Moreover, eNOS deficiency seems to be associated with an increased inflammatory state [31–34]. We chose eNOS−/− mice over various other rodent models of PE (reviewed by [18]) as placentas of this model lack gross anatomic alterations [28] similar to our low protein rat model [66]. Each group of mice was mated, and the presence of a copulation plug was denoted as day 0.5 of pregnancy. Mouse placentas were obtained from these mice at day E15 and E18.5. Animal characteristics are displayed in Table 1. Based on our finding that sex seemed to have no influence on *Rarres1*/*2* expression in the rat placenta, we did not include it as a variable in the mouse model analysis. The placental *eNOS* mRNA expression was well detectable in control mice but was below the detection limit in eNOS−/− mice (data not shown).

All rodent placentas were fixed in methyl Carnoy's solution (Roth, Karlsruhe, Germany) for embedding in para ffin or were snap frozen and stored at −80 ◦C for messenger RNA (mRNA) preparation and protein extraction.

#### *4.2. RNA Extraction, RT-PCR, and Real-Time Quantitative PCR*

Gene expression analysis has previously been described by us in detail [36]. PCR was performed in *n* = 5 pups (mean) per litter from 4 NP/LP dams, respectively. In our mouse model, *n* = 2 pups per litter from *n* = 6 eNOS−/− dams and *n* = 5 C57BL/6 wild type controls, respectively, were examined at two di fferent time points (E15; E18.5). Snap-frozen placental tissues were minced using a Mikro-Dismembrator (Sartorius Stedim Biotech GmbH, Göttingen, Germany). RNA purification of our rat placentas was achieved with peqGold TriFast reagen<sup>t</sup> (Peqlab, Erlangen, Germany), and RNA pretreatment with DNase I (Sigma-Aldrich, Darmstadt, Germany) was used. For mouse samples, the frozen tissue was homogenized by grinding with a T10 basic ULTRA TURRAX disperser (IKA, Staufen im Breisgau, Germany), and total RNA was extracted using the RNeasy Mini Kit with DNase treatment (Qiagen, Hilden, Germany) according to the manufacturer's instructions. RNA concentration was determined by NanoDrop spectrophotometry (Peqlab, Erlangen, Germany) and adjusted to 100 ng/ mL for all rodent placenta samples. Complementary DNA (cDNA) synthesis was conducted using TaqMan Reverse Transcription (Applied Biosystems, Waltham, MA, USA) in a Biometra Trio thermal cycler (Analytik Jena, Jena, Germany). Quantification of *Rarres1*, *Rarres2*, *CmlkR1*, and *IL11* mRNA expression was achieved by qRT PCR analysis using the Fast SYBR Green Master Mix and Sequence Detector StepOnePlus (Applied Biosystems, Waltham, MA, USA) with *r18s* RNA as a reference gene. Measurements were performed in duplicate. Primers were designed using Primer Express software (version 3.0.1, Applied Biosystems, Waltham, MA, USA) or Primer-BLAST (NCBI, NIH). Primers were ordered from Eurofins (Eurofins Genomics Germany GmbH, Ebersberg, Germany) and sequences are listed in Table 4.


