Normal Pregnant Research Articles

Angiotensin II Type I Receptor Agonistic Autoantibody Blockade Improves Cerebral Blood Flow Autoregulation, Blood Brain Barrier Permeability, and Hypertension in the Pre‐Clinical Rat Model of Preeclampsia

BackgroundPreeclampsia (PE) is a pregnancy disorder that is characterized by an increase in blood pressure, angiotensin II type1 receptor agonistic autoantibodies (AT1‐AA), and neurological complications. The pre‐clinical RUPP (reduced uterine perfusion pressure) rat model of PE displays many of the clinical pathologies associated with PE, such as increased blood pressure, AT1‐AAs, blood brain barrier (BBB) permeability, and impaired cerebral blood flow (CBF) autoregulation.HypothesisWe hypothesized that specific inhibition of AT1‐AA, using the epitope binding 7 amino acid peptide sequence (7AA), will improve blood pressure, BBB integrity, and CBF autoregulation in RUPP rats.MethodsSprague Dawley rats, were divided into 3 groups: normal pregnant (NP) (n=19), RUPP (n=19), and RUPP+AT1‐AA inhibition (7AA) (n=21). RUPP surgery was performed on gestational day (GD) 14 and 7AA (2μg/μl) was administered via osmotic minipumps. GD 19, mean arterial pressure (MAP) was determined and BBB permeability was assessed using the in‐vivo imaging system (IVIS). CBF was measured by laser Doppler flowmetry through a 4 mm x 4 mm cranial window under anesthesia. MAP was elevated step‐wise from 100–190 mmHg by infusion of phenylephrine to determine changes in CBF.ResultsMAP was increased in RUPP vs NP (124±3 vs 100±1 mmHg, p<0.05) and decreased in RUPP+7AA vs RUPP (105±2 vs 124±3 mmHg, p<0.05) rats. Using IVIS, Texas Red/FITC green fluorescent imaging was decreased in RUPP+7AA vs RUPP (0.2±0.001 vs 0.6±0.001, p<0.05), indicating improvement in BBB permeability with AT1‐AA inhibition. CBF autoregulation was impaired in RUPP vs NP rats at 140 mmHg (142±7 vs 119±4%, p<0.05) and 160 mmHg (157±10 vs 129±6%, p<0.05) (both pressures are within the normal CBF autoregulatory range). AT1‐AA inhibition prevented impaired CBF at 140mmHg (125±5 RUPP+7AA vs 142±7% RUPP, p<0.05) and 160 mmHg (125±9 RUPP+7AA vs 157±10% RUPP, p<0.05). Furthermore, the increases in CBF at pressures above the autoregulatory range, 180 mmHg (122±10 vs 198±17%, p<0.05) and 190 mmHg (123±11 vs 208±19%, p<0.05), was prevented in RUPP+7AA vs RUPP, respectively.ConclusionAT1‐AA inhibition improved blood pressure, BBB permeability, and CBF autoregulation in the pre‐clinical RUPP rat model of PE, indicating that AT1‐AA inhibition may be a potential therapy used to improve neurological complications during PE.Support or Funding InformationSupported: AHA18CDA34110264

Acute Kidney Injury During Pregnancy Leads to Memory Impairment but not Anxiety in Rats with a History of Severe Preeclampsia/HELLP Syndrome

OBJECTIVEWomen with hypertensive conditions of pregnancy such as severe PE (preeclampsia)/HELLP (hemolysis, elevated liver enzyme, and low platelets) syndrome are at an increased risk of developing Acute Kidney Injury (AKI). New reports suggest that AKI during pregnancy can lead to the progression of chronic kidney disease (CKD). Between 27–67% of patients with CKD have evidence of depression, cognitive impairment and dementia that is directly linked to renal damage. Among these deficits are impairments in memory, orientation, reasoning and executive function. Incidentally, women with a history of hypertensive disorders with and without AKI have an increased risk of developing mental health and cognitive disorders. Using an established animal model of severe PE/HELLP syndrome we sought to test the hypothesis that AKI in the presence of severe PE/HELLP exacerbates cognitive impairment and anxiety in the postpartum period.STUDY DESIGNOn gestational day (GD) 12, sFlt‐1 and sEng was infused via mini‐osmotic pump into the abdominal space of timed‐pregnant Sprague Dawley rats to induce severe PE/HELLP. A subset of HELLP and normal pregnant (NP) rats underwent bilateral renal ischemia‐reperfusion surgery for 45 minutes on GD18 to induce AKI. All rats delivered on GD21, underwent behavioral testing between postpartum weeks (PPW) 11–13 and were euthanized at PPW15. FITC‐sinistrin which was used to assess glomerular filtration (measure of kidney function), blood pressure via carotid catheter, and urinary creatinine were all measured during PPW15. N’s per group = 7 –10.RESULTSCompared to NP rats (p=0.01), HELLP, NP+AKI and HELLP+AKI rats had significant impairments in learning and memory. HELLP rats spent significantly less time in the open arms of the zero maze compared to NP (p=0.04) and NP+AKI (p=0.04). The time in open arms for HELLP+AKI rats were significantly less, as well, compared to NP (p=0.01) and NP+AKI (p=0.01) rats. Kidney function significantly declined in H+AKI (p= 0.001) and NP+AKI (p=0.009) rats compared to NP rats, as well as HELLP+AKI (p=0.03) rats compared to HELLP rats. Urinary creatinine was significantly increased in HELLP+AKI rats compared to NP (p=0.03) and HELLP (p=0.007) rats as well as NP+AKI (p=0.01) rats to HELLP rats. Blood pressure was significantly increased in NP+AKI (p=0.003), HELLP+AKI (p=0.0007), and HELLP (p=0.02) rats compared to NP rats.CONCLUSIONThe results from this study suggest that HELLP during pregnancy impairs learning and memory and is exacerbated by AKI. Anxiety was only impaired in rats with a history of severe PE/HELLP with/without AKI suggesting that renal injury may lead to selective psychological impairments. Hypertension persisted into the post‐partum period for rats with a history of severe PE/HELLP. Renal function wasn’t recovered into the post‐partum period for rats with history of AKI.Support or Funding InformationThis work was sponsored by P20GM121334 and the UMMC Office of Sponsored Program.

Stereological study of the placental structure in abortion-prone mice model (CBA/J×DBA/2J)

Recurrent spontaneous abortion (RSA) is an important reproductive health issue defined as the loss of two or more consecutive pregnancies before the 20th week of gestation, affecting 2–5% of couples. This study aimed to evaluate the volume, number of cells, and length of the vessels in the placenta in normal and abortion-prone (AP) pregnant mice on gestational day (gd) 13.5. Fetal and placental tissues of female CBA/J mated DBA/2J (AP group) and BALB/c (normal pregnant group) were collected and prepared for stereological assessments on gd13.5. The volumes of the placenta and its main layers decidua basalis (Db), junctional zone (Jz), and labyrinth zone (Lz) were investigated. The number of spongiotrophoblast cells, glycogen cells, giant cells, trophoblast cells, lymphocytes, and neutrophils were estimated as well. The AP group showed a reduction in the volume of the placenta (48.7%) and its components. Moreover, the number of spongiotrophoblast cells (66.7%), glycogen cells (76.2%), giant cells (73.3%), and trophoblast cells (81.4%) was decreased in AP compared to normal pregnant (NP) mice. Also, in AP group recognized a 10-fold increase in the number of lymphocytes and a four-fold increase in the number of neutrophils in comparison to the NP group (p < 0.05). Activation of different immune cell types might induce systemic inflammation at the feto-maternal interface, resulting in impaired placenta formation and abortion.

Analysis of the frequency of type 2 innate lymphoid cells and regulatory T cells in abortion-prone mice

Recurrent spontaneous abortion (RSA) is the most common pregnancy related complication, affecting 1–5 % of pregnancies. Despite hormonal, genetic and anatomical factors that result in abortion, impairment of immune response at the feto-maternal interface during the first trimester of pregnancy is also one of the main causes of RSA. In the present study, we evaluated the frequency of blood and uterine group 2 innate lymphoid cells (ILC2s), their subsets and regulatory T cells (Tregs) in CBA/J × DBA/2 J as an abortion-prone model compared to normal pregnant (NP) mice using immunophenotyping. Results indicated that the percentages of ILC2s were significantly decreased in the AP group compared to the NP group at mid-gestation (P ≤ 0.01). Moreover, the percentages of both blood and uterine nILC2s were increased in NP mice at mid-gestation (P ≤ 0.01, and P ≤ 0.05, respectively), while iILC2s significantly increased in AP mice at mid-gestation (P ≤ 0.01, and P ≤ 0.05, respectively). Tregs were reduced in AP mice at both early and mid-gestation stages (P ≤ 0.01). Overall, our findings suggest that the changes in blood and uterine ILC2s might be associated with abortion in mice.

24: NK cell-mediated mitochondrial oxidative stress and hypertension in response to CD4+T cells from RUPP rats

Preeclampsia (PE) is new onset hypertension associated with activated CD4+T cells, NK cells, and cytokines during pregnancy. We have previously shown that women with PE exhibit placental mitochondrial (mt) dysfunction and ROS compared to normal pregnant (NP) women. The Reduced Uterine Perfusion Pressure (RUPP) rat model produces many characteristics of PE including hypertension, increases in CD4+T cells and renal/placental NK cells, and mt ROS and dysfunction. We have previously demonstrated that RUPP CD4+T cells cause hypertension in NP rats, however the role of RUPP CD4+T cells in stimulating NK cells to cause mt dysfunction and ROS remains unclear. Therefore, we examined the effect of adoptive transfer of RUPP CD4+T cells to activate NK cells in NP rats. Splenic CD4+T cells were isolated from RUPP rats, cultured, and injected into NP rats on gestation day 13. On gestation day 19, MAP and tissue samples were collected, mt respiration and ROS were measured in isolated mitochondria using the Oxygraph 2K and fluorescent microplate reader, and results were compared with a student’s t-test. MAP increased to 110±2 in NP + RUPP CD4+T cells (n=12) compared to 101±2 mmHg in NP controls (n=7, p< 0.05). Circulating cytolytic NK cells increased to 3±0.6% in recipients of RUPP CD4+T cells (n=8) compared to 0.3±0.2% in NP (n=7, p< 0.05). Renal state 3 (231.9±127.7 vs 814.1±291.6 pmol/sec/mg, p=0.17) and maximal (213.3±117 vs 422.3±89.5 pmol/sec/mg, p=0.21) respiration rates, indicative of ATP production and mt efficacy, were reduced in NP+RUPP CD4+T cells (n=3) compared to NP (n=5). Renal mtROS increased from 249.2±45.6% in NP to 597.1±155.4% in NP+RUPP CD4+T cells (p< 0.05). These data indicate that CD4+T cells stimulated in response to placental ischemia activates NK cells and causes renal mt dysfunction in association with ROS and hypertension during pregnancy, all of which are important mechanisms in the pathophysiology of PE.

Abstract 017: Cytolytic Natural Killer Cells Play a Direct Role in Causing Hypertension and Intrauterine Growth Restriction in Pregnant Rats

The R educed U terine P erfusion P ressure (RUPP) rat model of placental ischemia mirrors many of the hallmark features of preeclampsia (PE) such as hypertension, intrauterine growth restriction (IUGR), and immune activation, which includes increased cytolytic natural killer cells (cNKs). We have previously demonstrated that depletion of NK cells in RUPP rats improves PE pathophysiology. In this study we tested the hypothesis that RUPP-stimulated NKs have increased cytotoxicity and play a direct role in inflammation, oxidative stress, hypertension, and intrauterine growth restriction in pregnant rats. NK cells were isolated from the placentas of normal pregnant (NP) and RUPP rats on gestation day (GD) 19 and assessed for cytotoxic activity. RUPP NK cells demonstrated a 5-fold increase in cytotoxic activity vs NP NK cells. On GD 12, 5x10 6 NK cells from RUPP rats were infused into a subset of NP rats. On GD 19, circulating and placental total NK cells and cNKs were quantified via flow cytometry in NP, RUPP, and NP+RUPP NK rats. MAP, fetal and placental weights, placental reactive oxygen species (ROS), and placental cytokines were also measured. Placental cNK cells (%gated) were significantly increased in RUPP (18.5±3.6%) and NP+RUPP NK (16.1±3.6%) compared to NP (4.1±0.8%) controls. Circulating cNKs followed a similar trend. MAP increased from 100 mmHg in NP to 126 mmHg in RUPP, and 119 mmHg in NP+RUPP NK (p&lt;0.05 vs NP). Fetal weight decreased from 2.4±0.03 g in NP to 2.0±0.04 g in RUPP and to 2.1±0.02 g in NP+RUPP NK (p&lt;0.05 vs NP). Placental weights followed a similar trend. Placental cNK cytokines were significantly increased in RUPP and in NP recipients of RUPP NK cells: TNF-α: NP- 23±3.1, RUPP- 62.3±7.8 pg/mg, NP+RUPP NK-43.9±5.7 pg/mg (p&lt;0.05 vs NP); IFNγ: NP- 3.3±0.7, RUPP- 7.6±1.2 pg/mg, NP+RUPP NK-6.6±1.9 pg/mg (p&lt;0.05 vs NP). Placental VEGF decreased from 6.5±1.3 pg/mg in NP to 2±0.8pg/mg in RUPP and 2.9±0.6 pg/mg in NP+RUPP NK. Circulating levels followed a similar trend. Placental ROS significantly increased from 27.3±4.6 RLUs/min/mg in NP to 63.1±5.7 RLUs/min/mg in RUPP and 84±17.6 RLUs/min/mg in NP+RUPP NK. These data demonstrate a direct role for cNKs in causing PE pathophysiology and identifies cNKs as a novel therapeutic target for treatment of PE.

Abstract P101: Interleukin 17 Signaling Mediates Cytolytic Natural Killer Cell Activation in Response to Placental Ischemia

The R educed U terine P erfusion P ressure (RUPP) rat model of placental ischemia mimics many characteristics of preeclampsia (PE) including hypertension, fetal growth restriction, and increased T H 17s, IL-17, and cytolytic natural killer cells (cNKs). IL-17 stimulates NK cytotoxicity in vitro . This study tested the hypothesis that blockade of IL-17 signaling would inhibit activation of cNKs to improve blood pressure and fetal growth in RUPP rats. On gestation day (GD) 14, osmotic pumps infusing IL-17 RC (100 pg/d), a soluble receptor for IL-17, were implanted into pregnant rats undergoing RUPP (RUPP+IL17 RC). On GD 19, circulating and placental T H 17s and cNKs were quantified via flow cytometry in normal pregnant (NP), RUPP, and RUPP+IL17 RC rats. cNK activation, placental ROS, fetal and placental weights, and MAP were also assessed. As we have previously shown, placental T H 17 populations (% gated) were significantly increased in RUPP (23.1±3.9%, n=9) compared to NP (8.5±3.9%, n=9) rats and were suppressed in RUPP+IL17 RC (8.3±3.1%, n=9). Circulating T H 17s followed the same trend. Placental cNKs, significantly increased from 7.2±2.8% in NP to 16.6±3.3% in RUPP, and were normalized to 7.2±2.8% after IL-17 blockade (p&lt;0.05 vs RUPP). Placental cNK proteins and circulating cNKs showed a similar trend. Placental levels of the cNK cytokine, TNFα, increased from 28.5±.01 pg/mg in NP to 46.2±6.0 pg/mg in RUPP and was normalized to 28.5±4.6 pg/mg in RUPP+IL-17 RC (p&lt;0.05 vs RUPP). Placental MIP3a, a cNK chemokine, had a similar trend. Assessment of in vitro cytotoxic activity of placental NK cells from rats in each group demonstrated a 5-fold increase in cytotoxicity by RUPP NK cells compared to NP NK cells. This was blunted in NK cells from RUPP+IL17 RC rats. Placental ROS was increased in RUPP compared to NP and significantly decreased with IL-17 blockade. Fetal weight decreased from 2.4±0.04 g in NP to 2.1±0.04 g in RUPP and increased to 2.3±0.05 g in RUPP+IL17 RC (p&lt;0.05 vs RUPP). Placental weights followed a similar trend. MAP increased from 93 mmHg in NP to 120 mmHg in RUPP, and was decreased to 105 mmHg in RUPP+IL17 RC (p&lt;0.05 vs RUPP). These data demonstrate a direct role for IL-17 signaling to mediate NK cytotoxic activation in response to placental ischemia during pregnancy.

Abstract P1103: Angiotensin II Type I Receptor Agonistic Autoantibody Blockade Improves Hypertension and Immune Activation in Postpartum Preeclamptic Rats

Women with preeclampsia (PE) have a risk of developing cardiovascular diseases (CVD) later in life. The angiotensin II type I receptor agonistic autoantibodies (AT1-AAs) are elevated in women with PE, PE women 2 years post-partum (PP), and the reduced uterine perfusion pressure (RUPP) rat model of PE. Blockade of the AT1-AA by using a specific binding seven amino acid peptide sequence (7AA) improves the pathology of PE in RUPP rats. The long-term effects of AT1-AA inhibition on blood pressure, NK cell activation, heart mitochondria (mt) proteins, and CVD in the RUPP model PP is unknown. Therefore, we hypothesized that PP RUPP rats have elevated blood pressure, NK cell activation, and changes in heart mt proteins, which will be prevented in RUPP rats administered the 7AA during pregnancy. Methods: Pregnant Sprague Dawley rats were divided into groups; normal pregnant (NP) (n=7), RUPP (n=10), and RUPP+7AA (n=9). Gestational day 14, RUPP surgery was performed and 7AA (2 μg/ml) administered via minipump. Results: At 8 and 10 weeks (wks) PP, blood pressure (MAP), blood, and hearts were collected. NK cells were quantified by flow cytometry. At 8 wks PP, MAP was elevated in RUPP vs. NP (130±2 vs. 123±4 mmHg, ns), and RUPP+7AA (124±4 mmHg) treatment prevented this increase. At 10 wks, MAP was elevated in RUPP vs NP (133±5 vs. 120±5 mmHg, p=0.08), with a significant decrease in MAP in RUPP+7AA (107±6 mmHg) vs. RUPP (p&lt;0.05). Total circulating NK cells were increased in RUPP vs NP (45±9. vs. 29± 8% gated cells, ns), which was prevented in RUPP+7AA (19±16 % gated cells) at 8 wks PP. Hearts were enlarged with RUPP vs NP (0.41±0.04 vs. 0.36±0.02g/100gBW, ns), which was normalized in RUPP+7AA (0.34±0.02g/100gBW). Previous studies show that during pregnancy complex IV is significantly lower along with a decrease mt function in RUPP vs NP. Complex IV mt proteins in the heart were elevated in RUPP+7AA vs. RUPP (5.5±1.7 vs. 3.0±0.2 AU, ns). Conclusion: In summary, PP PE rats have an increase in MAP, NK cells, and larger hearts. AT1-AA inhibition restores complex IV mt levels and improves HTN, immune activation, and cardiac hypertrophy PP. This study highlights the importance of AT1-AA inhibition during PE to prevent CVD later in life. Supported: AHA18CDA34110264

Abstract P1110: Tumor Necrosis Factor Alpha Blockade Improves Natural Killer Cell Activation, Hypertension, and Mitochondrial Oxidative Stress in a Preclinical Rat Model of Preeclampsia

Preeclamptic (PE) women have placental ischemia, hypertension (HTN), mitochondrial (mt) dysfunction, increased mt-reactive oxygen species (ROS), cytolytic natural killer (NK) cells, and pro-inflammatory cytokines, such as tumor necrosis factor (TNFα). The reduced uterine perfusion pressure (RUPP) preclinical rat model of PE, is a well-established model to study mechanisms of hypertension in response to placental ischemia during pregnancy. Previous studies from our lab have shown that TNFα is elevated in RUPP rats and that blockade of TNFα, via etanercept (ETAN) decreases TNFα and HTN. However, the effect of TNFα blockade on NK cell activation, mt protein/function, and mtROS is unknown. We hypothesized that ETAN would decrease blood pressure, NK cell activation, mtROS, and improve mt function in RUPP rats. Rats were divided into 4 groups: normal pregnant (NP) (n=41), RUPP (n=34), RUPP+low dose (LD) ETAN (0.4 mg/kg) (n=11), RUPP+high dose (HD) ETAN (0.8 mg/kg) (n=19). LD was used previously, while HD is clinically used to treat chronic inflammatory diseases. Gestational day 14, RUPP surgery was performed and ETAN administered subcutaneous on day 18. Day 19, conscious blood pressure (MAP), blood and tissues were collected, and mt were isolated. Flow cytometry was used for quantification of NK cells. MAP was elevated in RUPP vs. NP (119 ± 1 vs. 102± 1 mmHg, p&lt;0.05) which was reduced to 110 ± 2 in RUPP + LD ETAN (p&lt;0.05 vs. RUPP) and 116 ± 1 in RUPP + HD ETAN. Activated NK cells were increased in circulation (5.9 ± 1.8 vs. 4.0 ± 1.4 %), placenta (4.8 ± 1.3 vs. 1.9 ± 0.7 %), and kidneys (3.1 ± 0.5 vs. 0.8 ± 0.5%) of RUPP vs. NP (p&lt;0.05). Both LD (2.1 ± 0.7%, 1.7 ± 0.7%, 0.8 ± 0.5%) and HD (0.2 ± 0.1%, 0.4 ± 0.1%, 0.8 ± 0.2%) ETAN decreased activated NK cells in circulation, placenta, and kidney respectively (p&lt;0.05 vs RUPP). Placental (0.79± 0.02 vs 1±0.05 fold) and renal (0.67± 0.05 vs 1±0.06 fold) mtROS was reduced with LD ETAN vs RUPP (p&lt;0.05). ETAN treatment lowers blood pressure and reduces circulating, placental, and renal activated NK cells and mtROS in placental ischemic rats. Therefore ETAN could be a potential therapeutic for improving HTN, inflammation, and mitochondrial function during complicated pregnancies. Supported: AHA18CDA34110264, HL130456, R01HD067541, &amp; P20GM121334

The ameliorative effect of angiotensin 1-7 on experimentally induced-preeclampsia in rats: Targeting the role of peroxisome proliferator-activated receptors gamma expression & asymmetric dimethylarginine

This study was designed to explore the effect of angiotensin 1-7 (Ang 1–7) on experimentally induced-preeclampsia in Wistar rats targeting the role of peroxisome proliferator-activated receptors gamma expression (PPARs-γ) & asymmetric dimethylarginine (ADMA). 30 female Wistar rats were divided into three groups: Normal pregnant (NP), preeclampsia (PE), and preeclampsia treated with Ang 1–7 (PE + Ang 1–7) groups. Reduced uterine perfusion pressure (RUPP) model was induced on GD14. On GD18, protein in urine, urine volume and urinary sodium excretion were determined. On GD19, the systolic blood pressure (SBP) was measured, and the gene expression of PPARs-γ were determined. The serum samples were separated for determination of Ang 1–7, ADMA, soluble fms-like tyrosine kinase (sFlt-1), vascular endothelial growth factor (VEGF), nitric oxide (NO) products, endothelial nitric oxide synthase (eNOS) activity, interleukin-6 (IL-6), interleukin-10 (IL-10), malondialdehyde (MDA), and total anti-oxidant capacity (T-AOC). Compared to NP group, SBP, urine protein, serum levels of ADMA, sFlt-1, IL-6 and MDA significantly increased, while expression of PPARs-γ, serum levels of Ang 1–7, VEGF, NO products, eNOS, IL-10 and T-AOC significantly decreased in PE group, while treatment of Ang 1–7 significantly ameliorated all these studied parameters as compared to PE group. We concluded that Ang 1–7 attenuated the symptoms of preeclampsia, which might be via increasing the expression of PPARs-γ and reduction of ADMA levels which could explain its anti-hypertensive, anti-angiogenic, anti-inflammatory and antioxidant effects.

Hypertension Editors' Picks Preeclampsia.

Hypertension Editors' Picks Preeclampsia.

Circular RNA expression profiling identifies hsa_circ_0011460 as a novel molecule in severe preeclampsia.

To identify circRNA expression profiles in the placentae of severe preeclampsia (SPE) and normal pregnant (NP) women. Placental samples were collected from six paired SPE and NP women. CircRNA expression profiles were identified by RNA-Seq and validated in another 30 SPE and NP samples by qRT-PCR. Several bioinformatic tools were utilised to analyse the potential function of differentially expressed circRNAs (DE-circRNAs) and to predict target microRNAs (miRNAs) and proteins. Furthermore, immunohistochemistry, Western blotting and RNA binding protein immunoprecipitation (RIP) were conducted to confirm the interaction between the circRNA and protein. In total, 18,631 circRNAs were detected. Among them, 180 circRNAs were differentially expressed, including 94 upregulated and 86 downregulated circRNAs. Seven DE-circRNAs were selected for validation, and the results of six circRNAs (hsa_circ_0007611, hsa_circ_0011460, hsa_circ_0002888, and hsa_circ_0007445, hsa_circ_0017068, hsa_circ_0012737) were consistent with the sequencing results. Bioinformatics analyses of DE-circRNAs revealed that most of them are involved in vasodilation, regulation of blood vessel size, protein transport and localization, and pathways in cancer. In addition to the mRNA expression profile, it was interesting to find that the hsa_circ_0011460 target gene solute carrier organic anion transporter family member 2A1 (SLCO2A1, PGT) was also significantly increased in SPE placentae. Immunohistochemistry, Western blotting and qRT-PCR validated the expression and distribution of PGT. Finally, RIP of HTR-8/SVneo cells confirmed that hsa_circ_0011460 targets PGT directly. A total of 180 DE-circRNAs were detected. One crucial circRNA, hsa_circ_0011460, was shown to interact directly with its host gene PGT. These findings indicated that hsa_circ_0011460 may serve as a potential therapeutic target for patients with SPE.

Suppression of TH17 Cells Blunts Preeclampsia‐Associated Cytolytic Natural Killer Cells during Pregnancy

The Reduced Uterine Perfusion Pressure (RUPP) rat model of placental ischemia mimics many of the characteristics of preeclampsia (PE) including hypertension, intrauterine growth restriction (IUGR), and increases in T‐helper 17 cells, (TH17s), IL‐17, and placental cytolytic natural killer cells (cNKs). We have previously demonstrated that placental ischemia (PI)‐stimulated TH17s mediate cNK activation and cause hypertension and IUGR in pregnant rats similar to that seen in RUPP and human PE. In this study we tested the hypothesis that suppression of TH17s via blockade of IL‐17 signaling would inhibit activation of cNKs and decrease cytolytic proteins (perforin, granzymes) to improve blood pressure and fetal growth during placental ischemia. On gestation day (GD) 14 mini osmotic pumps infusing 100 pg/d of IL‐17 RC, a soluble receptor for IL‐17, were implanted into pregnant rats undergoing RUPP (RUPP+IL17 RC). On GD 19, circulating and placental TH17s and placental total NK cells and cNKs were quantified via flow cytometry in normal pregnant (NP), RUPP, and RUPP+IL17 RC rats. MAP, fetal and placental weights, placental reactive oxygen species (ROS), perforin, and granzymes were also measured. As we have previously shown, both circulating and placental TH17 populations were significantly increased in RUPP compared to NP rats. Treatment with IL‐17 RC suppressed TH17 populations in RUPP+IL17 RC rats. Circulating TH17s (% gated): NP (n=9)– 2.5±1.2%, RUPP (n=9)– 7.4±1.4%, RUPP+IL17 RC (n=10)– 0.9±0.4% (p&lt;0.05 vs RUPP). Placental TH17s: NP–8.5±3.8%, RUPP– 23.1±3.9%, RUPP+IL17 RC– 8.3±3.1% (p&lt;0.05 vs RUPP). Placental total NKs and cNKs, were significantly increased after RUPP and normalized with IL‐17 blockade. Total NKs (% gated): NP– 23.6±6.7%, RUPP– 46.8±5.1%, RUPP+IL17 RC‐23.5±4.6 % (p&lt;0.05 vs RUPP). cNKs: NP– 7.2±2.8%, RUPP– 16.6±3.3%, RUPP+IL17 RC–6.9±1.5% (p&lt;0.05 vs RUPP). MAP increased from 93 mmHg in NP to 119 mmHg in RUPP, and significantly decreased to 105 mmHg in RUPP+IL17 RC (p&lt;0.05 vs RUPP). Fetal weight decreased from 2.4±0.04 g in NP to 2.1±0.04 g in RUPP and increased to 2.3±0.05 g in RUPP+IL17 RC (p&lt;0.05 vs RUPP). Placental weight decreased from 0.55±0.01 g in NP to 0.43±0.01 g in RUPP and increased to 0.52±0.03 g in RUPP+IL17 RC (p&lt;0.05 vs RUPP). Placental ROS increased from 890.1±218.1 relative light units (RLUs) in NP to 2240±318.9 RLUs in RUPP and decreased to 11458±136.7 RLUs in RUPP+IL17 RC (p&lt;0.05 vs RUPP). Placental NK cytolytic proteins were significantly increased in response to RUPP and were not decreased after IL‐17 RC administration. Importantly in vitro cytotoxicity of RUPP NK cells increased 5‐fold compared to NP NK cells, and IL‐17 blockade blunted cytotoxic activity of RUPP NK. These data identify TH17/IL‐17 signaling as a mechanism of NK cytotoxic activation in response to placental ischemia during pregnancy. Therefore, targeting TH17s and IL‐17 may be a therapeutic strategy to normalize polarization of the NK population during PE and improve maternal and fetal outcomes associated with this maternal disorder.Support or Funding InformationFunding: National Institutes of Health/National Heart Lung and Blood Institute grant R00HL130456 to DCCThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Chronic infusion of interleukin-17 promotes hypertension, activation of cytolytic natural killer cells, and vascular dysfunction in pregnant rats.

Previous studies by our lab have established that placental‐ischemia stimulated T‐helper 17 cells (TH17s) cause increased cytolytic natural killer (cNK) cell proliferation and activation during pregnancy; however, the exact mechanism is unknown. The objective of this study was to investigate the role of interlukin 17 (IL‐17) in inducing cNK cell activation in pregnancy. We infused 150 pg/day of recombinant IL‐17 into a subset of normal pregnant (NP) Sprague Dawley rats from gestation day (GD) 12–19 (NP+IL‐17). On GD 19, mean arterial pressure (MAP), fetal and placental weights, cytokines, cNK cell activation, cytotoxic enzymes, and vascular reactivity were assessed. MAP significantly increased from 99 ± 3 mmHg in NP to 120 ± 1 mmHg in NP+IL‐17 (P < 0.05). Fetal weight significantly decreased from 2.52 ± 0.04 g in NP to 2.32 ± 0.03 g in NP+IL‐17 as did placental weight (NP: 0.65 ± 0.03 g; NP+IL‐17: 0.54 ± 0.01 g, P < 0.05). Plasma levels of TNF‐α increased to 281.4 ± 55.07 pg/mL in NP+IL‐17 from 145.3 ± 16.03 pg/mL in NP (P < 0.05) while placental levels of VEGF decreased from 74.2 ± 6.48 pg/mg in NP to 54.2 ± 3.19 pg/mg in NP+IL‐17. Total NK cells were increased in the placenta (NP: 14.3 ± 3.49%; NP+IL‐17: 29.33 ± 2.76%, P < 0.05) as were cytolytic NK cells (NP: 3.31 ± 1.25%; NP+IL‐17: 13.41 ± 1.81%, P < 0.05). A similar trend was observed in circulating NK cells. Plasma granzyme K increased from 3.55 ± 2.29 pg/mL in NP to 20.9 ± 7.76 pg/mL in NP+IL‐17 (P < 0.05), and plasma granzyme B increased from 10.95 ± 0.64 pg/mL in NP to 14.9 ± 0.98 pg/mL in NP+IL‐17(P < 0.05). In the placenta, both granzyme A (NP: 246.1 ± 16.7 pg/mg; NP+IL‐17: 324.3 ± 15.07 pg/mg, P < 0.05) and granzyme B (NP: 15.18 ± 3.79 pg/mg; NP+IL‐17: 27.25 ± 2.34 pg/mg, P < 0.05) increased in response to IL‐17 infusion. Finally, vascular reactivity of uterine arteries was significantly impaired in response to IL‐17 infusion. The results of this study suggest that IL‐17 plays a significant role in the activation of cNK cells during pregnancy.

Acute Kidney Injury during pregnancy decreases pup size and sensorimotor development

BACKGROUNDAcute kidney injury during pregnancy (NP+AKI) leads to growth restriction and a delay in neurodevelopment in the resulting babies. As the incidence of AKI during pregnancy has been increasing over the last few years it is important to examine the consequences of this disorder during pregnancy. We hypothesize that similar to what is seen in humans with intrauterine growth restriction, there will be a sex difference in sensorimotor development between NP and NP+AKI rat pups.METHODSTo induce AKI, on gestational day (GD) 18, normal pregnant (NP) rats (n=4) had their renal pedicles isolated and occluded with microvascular clamps for 45 minutes under anesthesia. NP control rats (n=4) that received no surgical interventions served as controls and all rats delivered on GD21. 2–3 pups/litter were placed with a separate set of SHAM rats. Beginning on postnatal day (PND) 4 rat pups underwent the standard Irwin battery of developmental milestones: Surface righting, negative geotaxis, cliff avoidance, open circle. All pups were sexed and euthanized between PND32‐35.RESULTSRat pups born to NP+AKI dams weighed significantly less compared to NP pups (p&lt;0.0001). AKI pups were significantly smaller compared to NP pups (p&lt;0.0001). This trend remained true for female AKI pups through PND9 (p&lt;0.05); after which time both NP and NP+AKI females were significantly smaller than male pups. On PND3 NP+AKI females took longer to complete surface righting (23.5±12.7sec) compared to rat pups in the groups. Similar results were seen in cliff avoidance between groups (30.5±16.5 vs. 48.7±11.3sec; respectively). On PND5 NP+AKI pups (55.7±4.3sec) take longer to complete the negative geotaxis task compared to NP pups (17±1sec). There was a statistically significant difference in kidney size between female and male NP rats (p=0.02). There was not a statistically significant sex differences between groups in brain size (p=0.06), however NP+AKI pups had significantly smaller brains compared to NP pups (p=0.03).CONCLUSIONNP+AKI pups are smaller compared to NP pups and have delays in surface righting. AKI during pregnancy did not affect kidney size in offspring, however both male and female NP+AKI pups had smaller brains more than 30 days post‐birth compared to NP pups. The results from this study suggest that AKI during pregnancy leads to a significant delay in growth and sensorimotor development.Support or Funding InformationResearch reported in this publication was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number P20GM121334. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Interleukin‐4 supplementation improves the proinflammatory cell ratios, autoantibodies and blood pressure in response to placental ischemia

Preeclampsia (PE), new onset hypertension, is associated with chronic inflammation, autoantibodies to the AT1 receptor (AT1‐AA) and placental ischemia. Currently, there is no effective treatment for PE except for early delivery, making PE the leading cause for premature births worldwide. Evidence indicates a shift towards proinflammatory NK cell, CD4+TH1 vs CD4+TH2 profile exist in PE and in response to placental ischemia in pregnant rats (RUPP). Therefore, we hypothesized that decreasing cytolytic NK and CD4+ TH1 cells and increasing CD4+TH2 cells would improve blood pressure during pregnancy. To do so we infused IL‐4, an anti‐inflammatory cytokine strongly associated with the proliferation of TH2 cells, in to normal pregnant (NP) and RUPP rats. Interleukin‐4, 600 ng/day, was administered via a mini‐osmotic pump on day 14 of gestation, carotid catheters were inserted on gestation day 18, and blood pressure (MAP), AT1‐AA, NK cells, TH2 and B cells were measured on GD 19. MAP in NP rats (n=10) was 96 ± 2, 101 ± 2 in NP+IL‐4 rats (n=14), 130 ± 4 in RUPP rats (n=6), and 108 ± 2 mmHg in RUPP+IL‐4 rats (n=15), p&lt; 0.05. Circulating TH2 cells were 17 ± 3 in NP rats (n=3), 9.0 ± 2.0 in RUPP rats (n=5), but improved to 13 ± 3 % gated in RUPP+IL‐4 rats (n=9). Total placental NK cells were 0.7±0.1 in NP rats (n=4), 0.7±0.3 in NP+IL‐4 rats (n=7), 5.0 ±2 in RUPP rats (n=3) which significantly decreased to 1.6±4 in RUPP±IL‐4 rats (n=7), p&lt;0.05. Placental cytolytic NK cells were 6±2 % in RUPP rats but reduced significantly to 2±0.8 in RUPP+IL‐4 rats. Importantly, circulating B cells were 2.5±0.3 in NP rats (n=3), which increased to 7.5±2 in RUPP rats (n=4) and reduced to 1.4±0.5 in RUPP+IL‐4 rats (n=3). Similarly, AT1‐AA were 0.3±0.4 in NP rats (n=8), 18±0.3 in RUPP rats (n=9), and were blunted to 4±1 bpm in RUPP+IL‐4 rats (n=14), p&lt;0.05. This study illustrates the importance of a balanced CD4+ TH1/TH2, B and NK cell profile during pregnancy and that this can be achieved by administration of IL‐4, which ultimately improved hypertension in response to placental ischemia during pregnancy.Support or Funding InformationNIH grants R01HD067541‐06, T32HL105324This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Natural killer cells contribute to mitochondrial dysfunction in response to placental ischemia in reduced uterine perfusion pressure rats.

Preeclampsia (PE) is characterized by new-onset hypertension during pregnancy and is associated with immune activation and placental oxidative stress. Mitochondrial dysfunction is a major source of oxidative stress and may play a role in the pathology of PE. We (Vaka VR, et al. Hypertension 72: 703-711, 2018. doi: 10.1161/HYPERTENSIONAHA.118.11290 .) have previously shown that placental ischemia is associated with mitochondrial oxidative stress in the reduced uterine perfusion pressure (RUPP) model of PE. Furthermore, we have also shown that placental ischemia induces natural killer (NK) cell activation in RUPP. Thus, we hypothesize that NK cell depletion could improve mitochondrial function associated with hypertension in the RUPP rat model of PE. Pregnant Sprague-Dawley rats were divided into three groups: normal pregnant (NP), RUPP, and RUPP+NK cell depletion rats (RUPP+NKD). On gestational day (GD)14, RUPP surgery was performed, and NK cells were depleted by administering anti-asialo GM1 antibodies (3.5 µg/100 µl ip) on GD15 and GD17. On GD19, mean arterial pressure (MAP) was measured, and placental mitochondria were isolated and used for mitochondrial assays. MAP was elevated in RUPP versus NP rats (119 ± 1 vs.104 ± 2 mmHg, P = 0.0004) and was normalized in RUPP+NKD rats (107 ± 2 mmHg, P = 0.002). Reduced complex IV activity and state 3 respiration rate were improved in RUPP+NKD rats. Human umbilical vein endothelial cells treated with RUPP+NKD serum restored respiration with reduced mitochondrial reactive oxygen species (ROS). The restored placental or endothelial mitochondrial function along with attenuated endothelial cell mitochondrial ROS with NK cell depletion indicate an important role of NK cells in mediating mitochondrial oxidative stress in the pathology of PE.

Spectral photoacoustic imaging to estimate in vivo placental oxygenation during preeclampsia

Preeclampsia is a pregnancy-related hypertensive disorder accounting for 14% of global maternal deaths annually. Preeclampsia — maternal hypertension and proteinuria — is promoted by placental ischemia resulting from reduced uteroplacental perfusion. Here, we assess longitudinal changes in placental oxygenation during preeclampsia using spectral photoacoustic imaging. Spectral photoacoustic images were acquired of the placenta of normal pregnant (NP) and preeclamptic reduced uterine perfusion pressure (RUPP) Sprague Dawley rats on gestational days (GD) 14, 16, and 18, corresponding to mid- to late gestation (n = 10 per cohort). Two days after implementation of the RUPP surgical model, placental oxygen saturation decreased 12% in comparison with NP. Proteinuria was determined from a 24-hour urine collection prior to imaging on GD18. Blood pressure measurements were obtained on GD18 after imaging. Placental hypoxia in the RUPP was confirmed with histological staining for hypoxia-inducible factor (HIF)-1α, a cellular transcription regulator which responds to local oxygen levels. Using in vivo, longitudinal imaging methods we determined that the placenta in the reduced uterine perfusion pressure rat model of preeclampsia is hypoxic, and that this hypoxia is maintained through late gestation. Future work will utilize these methods to assess the impact of novel therapeutics on placental ischemia and the progression of preeclampsia.

Interleukin-4 supplementation improves the pathophysiology of hypertension in response to placental ischemia in RUPP rats.

Preeclampsia (PE) is characterized by chronic inflammation and elevated agonistic autoantibodies to the angiotensin type 1 receptor (AT1-AA), endothelin-1, and uterine artery resistance index (UARI) during pregnancy. Previous studies report an imbalance among immune cells, with T-helper type 2 (Th2) cells being decreased during PE. We hypothesized that interleukin-4 (IL-4) would increase Th2 cells and improve the pathophysiology in response to placental ischemia during pregnancy. IL-4 (600 ng/day) was administered via osmotic minipump on gestational day 14 to normal pregnant (NP) and reduced uterine perfusion pressure (RUPP) rats. Carotid catheters were inserted, and Doppler ultrasound was performed on gestational day 18. Blood pressure (mean arterial pressure), TNF-α, IL-6, AT1-AA, natural killer cells, Th2 cells, and B cells were measured on gestational day 19. Mean arterial pressure was 97 ± 2 mmHg in NP ( n = 9), 101 ± 3 mmHg in IL-4-treated NP ( n = 14), and 137 ± 4 mmHg in RUPP ( n = 8) rats and improved to 108 ± 3 mmHg in IL-4-treated RUPP rats ( n = 17) ( P < 0.05). UARI was 0.5 ± 0.03 in NP and 0.8 in RUPP rats and normalized to 0.5 in IL-4-treated RUPP rats ( P < 0.05). Plasma nitrate-nitrite levels increased in IL-4-treated RUPP rats, while placental preproendothelin-1 expression, plasma TNF-α and IL-6, and AT1-AA decreased in IL-4-treated RUPP rats compared with untreated RUPP rats ( P < 0.05). Circulating B cells and placental cytolytic natural killer cells decreased after IL-4 administration, while Th2 cells increased in IL-4-treated RUPP compared with untreated RUPP rats. This study illustrates that IL-4 decreased inflammation and improved Th2 numbers in RUPP rats and, ultimately, improved hypertension in response to placental ischemia during pregnancy.

Lin28B Expression in Reduced Uterine Perfusion Pressure Rat Model

Objective: Preeclampsia (PE) is a serious complication of pregnancy. Placental ischemia could be an initiating event, but the molecular mechanisms underlying PE are unclear. Lin28B, a paralog of Lin28 RNA-binding protein, is predominantly expressed in human placenta, and decreased Lin28B expression may play a role in PE by reducing trophoblast invasion. The current study was intended to verify whether Lin28B plays a role in the pathogenesis of PE in rat model for reduced uterine perfusion pressure (RUPP). Methods: We used RUPP rat model. The changes in blood pressure, 24-h urine protein excretion, and fetal development in RUPP rats were recorded and compared to those of normal pregnant (NP) rats. Furthermore, the expression of Lin28B mRNA and protein in placenta was determined using quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry. Results: The blood pressure, 24-h urine protein excretion, and embryo absorption rate were significantly increased in RUPP rats on the 20th day of gestational period compared with the NP rats (P 0.05). The expression levels of Lin28B mRNA and protein in the placenta of RUPP rats were also significantly decreased in comparison to NP rats (P < 0.001). Conclusion: Our results show that the expression of Lin28B in the placenta of RUPP rats is different from that in NP rats, thus suggesting a role of Lin28B in the pathogenesis of preeclampsia. Key words: Lin28B; Placenta; Preeclampsia; Reduced Uterine Perfusion Pressure