Nicholas F. Parchim

Elevated Placental Adenosine Signaling Contributes to the Pathogenesis of Preeclampsia

Background— Preeclampsia is a prevalent hypertensive disorder of pregnancy and a leading cause of maternal and neonatal morbidity and mortality worldwide. This pathogenic condition is speculated to be caused by placental abnormalities that contribute to the maternal syndrome. However, the specific factors and signaling pathways that lead to impaired placentas and maternal disease development remain elusive. Methods and Results— Using 2 independent animal models of preeclampsia (genetically engineered pregnant mice with elevated adenosine exclusively in placentas and a pathogenic autoantibody-induced preeclampsia mouse model), we demonstrated that chronically elevated placental adenosine was sufficient to induce hallmark features of preeclampsia, including hypertension, proteinuria, small fetuses, and impaired placental vasculature. Genetic and pharmacological approaches revealed that elevated placental adenosine coupled with excessive A2B adenosine receptor (ADORA2B) signaling contributed to the development of these features of preeclampsia. Mechanistically, we provided both human and mouse evidence that elevated placental CD73 is a key enzyme causing increased placental adenosine, thereby contributing to preeclampsia. Conclusions— We determined that elevated placental adenosine signaling is a previously unrecognized pathogenic factor for preeclampsia. Moreover, our findings revealed the molecular basis underlying the elevation of placental adenosine and the detrimental role of excess placental adenosine in the pathophysiology of preeclampsia, and thereby, we highlight novel therapeutic targets.

Hypoxia-Independent Upregulation of Placental Hypoxia Inducible Factor-1α Gene Expression Contributes to the Pathogenesis of Preeclampsia

Accumulation of hypoxia inducible factor-1&agr; (HIF-1&agr;) is commonly an acute and beneficial response to hypoxia, whereas chronically elevated HIF-1&agr; is associated with multiple disease conditions, including preeclampsia, a serious hypertensive disease of pregnancy. However, the molecular basis underlying the persistent elevation of placental HIF-1&agr; in preeclampsia and its role in the pathogenesis of preeclampsia are poorly understood. Here we report that Hif-1&agr; mRNA and HIF-1&agr; protein were elevated in the placentas of pregnant mice infused with angiotensin II type I receptor agonistic autoantibody, a pathogenic factor in preeclampsia. Knockdown of placental Hif-1&agr; mRNA by specific siRNA significantly attenuated hallmark features of preeclampsia induced by angiotensin II type I receptor agonistic autoantibody in pregnant mice, including hypertension, proteinuria, kidney damage, impaired placental vasculature, and elevated maternal circulating soluble fms-like tyrosine kinase-1 levels. Next, we discovered that Hif-1&agr; mRNA levels and HIF-1&agr; protein levels were induced in an independent preeclampsia model with infusion of the inflammatory cytokine tumor necrosis factor superfamily member 14 (LIGHT). SiRNA knockdown experiments also demonstrated that elevated HIF-1&agr; contributed to LIGHT-induced preeclampsia features. Translational studies with human placentas showed that angiotensin II type I receptor agonistic autoantibody or LIGHT is capable of inducing HIF-1&agr; in a hypoxia-independent manner. Moreover, increased HIF-1&agr; was found to be responsible for angiotensin II type I receptor agonistic autoantibody or LIGHT-induced elevation of Flt-1 gene expression and production of soluble fms-like tyrosine kinase-1 in human villous explants. Overall, we demonstrated that hypoxia-independent stimulation of HIF-1&agr; gene expression in the placenta is a common pathogenic mechanism promoting disease progression. Our findings reveal new insight to preeclampsia and highlight novel therapeutic possibilities for the disease.

Excess LIGHT Contributes to Placental Impairment, Increased Secretion of Vasoactive Factors, Hypertension, and Proteinuria in Preeclampsia

Preeclampsia, a prevalent hypertensive disorder of pregnancy, is believed to be secondary to uteroplacental ischemia. Accumulating evidence indicates that hypoxia-independent mediators, including inflammatory cytokines and growth factors, are associated with preeclampsia, but it is unclear whether these signals directly contribute to placental damage and disease development in vivo. We report that LIGHT, a novel tumor necrosis factor superfamily member, is significantly elevated in the circulation and placentas of preeclamptic women compared with normotensive pregnant women. Injection of LIGHT into pregnant mice induced placental apoptosis, small fetuses, and key features of preeclampsia, hypertension and proteinuria. Mechanistically, using neutralizing antibodies specific for LIGHT receptors, we found that LIGHT receptors herpes virus entry mediator and lymphotoxin &bgr; receptor are required for LIGHT-induced placental impairment, small fetuses, and preeclampsia features in pregnant mice. Accordingly, we further revealed that LIGHT functions through these 2 receptors to induce secretion of soluble fms-like tyrosine kinase-1 and endothelin-1, 2 well-accepted pathogenic factors in preeclampsia, and thereby plays an important role in hypertension and proteinuria in pregnant mice. Lastly, we extended our animal findings to human studies and demonstrated that activation of LIGHT receptors resulted in increased apoptosis and elevation of soluble fms-like tyrosine kinase-1 secretion in human placental villous explants. Overall, our human and mouse studies show that LIGHT signaling is a previously unrecognized pathway responsible for placental apoptosis, elevated secretion of vasoactive factors, and subsequent maternal features of preeclampsia, and reveal new therapeutic opportunities for the management of the disease.

Tissue Transglutaminase Contributes to the Pathogenesis of Preeclampsia and Stabilizes Placental Angiotensin Receptor Type 1 by Ubiquitination-Preventing Isopeptide Modification

Preeclampsia is a life-threatening pregnancy disorder that is widely thought to be triggered by impaired placental development. However, the placenta-related pathogenic factors are not fully identified, and their underlying mechanisms in disease development remain unclear. Here, we report that the protein level and enzyme activity of tissue transglutaminase (TG2 or tTG), the most ubiquitous member of a family of enzymes that conducts post-translational modification of proteins by forming &egr;-(&ggr;-glutamyl)-lysine isopeptide bonds, are significantly elevated in placentas of preeclamptic women. TG2 is localized in the placental syncytiotrophoblasts of patients with preeclampsia where it catalyzes the isopeptide modification of the angiotensin receptor type 1 (AT1). To determine the role of elevated TG2 in preeclampsia, we used a mouse model of preeclampsia based on injection of AT1-agonistic autoantibody. A pathogenic role for TG2 in preeclampsia is suggested by in vivo experiments in which cystamine, a potent transglutaminase inhibitor, or small interfering RNA–mediated TG2 knockdown significantly attenuated autoantibody-induced hypertension and proteinuria in pregnant mice. Cystamine treatment also prevented isopeptide modification of placental AT1 receptors in preeclamptic mice. Mechanistically, we revealed that AT1-agonistic autoantibody stimulation enhances the interaction between AT1 receptor and TG2 and results in increased AT1 receptor stabilization via transglutaminase-mediated isopeptide modification in trophoblasts. Mutagenesis studies further demonstrated that TG2-mediated isopeptide modification of AT1 receptors prevents ubiquitination-dependent receptor degradation. Taken together, our studies not only identify a novel pathogenic involvement of TG2 in preeclampsia but also suggest a previously unrecognized role of TG2 in the regulation of G protein–coupled receptor stabilization by inhibiting ubiquitination-dependent degradation.

Neurokinin 3 Receptor and Phosphocholine Transferase Missing Factors for Pathogenesis of C-Reactive Protein in Preeclampsia

C-reactive protein (CRP), an innate immune mediator, is elevated in the circulation before symptoms in patients with preeclampsia, a severe hypertensive pregnancy disorder with high mortality and morbidity. However, the specific sources underlying increased CRP and the role of elevated CRP in preeclampsia are undefined. Here, we report that circulating CRP levels are significantly increased in a large cohort of normotensive pregnant individuals when compared with nulligravid women and is further increased in patients with preeclampsia. These findings led us to discover further that placental syncytiotrophoblasts are previously unrecognized cellular sources of CRP and underlie elevated CRP in normotensive pregnant women and the additional increase in patients with preeclampsia. Next, we demonstrated that injection of CRP induces preeclampsia features, including hypertension (157 mm Hg CRP treated versus 119 mm Hg control), proteinuria (35.0 mg/μg CRP treated versus 14.1 mg/μg control), kidney, and placental damage and increased levels of sFlt-1 in pregnant mice but not in nonpregnant mice. Our study implicates that phosphocholine transferase, a placental-specific enzyme post-translationally modifying neurokinin B, is essential for the pathogenic role of CRP in preeclampsia through activation of the neurokinin 3 receptor. Overall, our studies have provided significant new insight on the pathogenic role of CRP in preeclampsia and highlighted innovative therapeutic strategies. # Novelty and Significance {#article-title-34}C-reactive protein (CRP), an innate immune mediator, is elevated in the circulation before symptoms in patients with preeclampsia, a severe hypertensive pregnancy disorder with high mortality and morbidity. However, the specific sources underlying increased CRP and the role of elevated CRP in preeclampsia are undefined. Here, we report that circulating CRP levels are significantly increased in a large cohort of normotensive pregnant individuals when compared with nulligravid women and is further increased in patients with preeclampsia. These findings led us to discover further that placental syncytiotrophoblasts are previously unrecognized cellular sources of CRP and underlie elevated CRP in normotensive pregnant women and the additional increase in patients with preeclampsia. Next, we demonstrated that injection of CRP induces preeclampsia features, including hypertension (157 mm Hg CRP treated versus 119 mm Hg control), proteinuria (35.0 mg/&mgr;g CRP treated versus 14.1 mg/&mgr;g control), kidney, and placental damage and increased levels of sFlt-1 in pregnant mice but not in nonpregnant mice. Our study implicates that phosphocholine transferase, a placental-specific enzyme post-translationally modifying neurokinin B, is essential for the pathogenic role of CRP in preeclampsia through activation of the neurokinin 3 receptor. Overall, our studies have provided significant new insight on the pathogenic role of CRP in preeclampsia and highlighted innovative therapeutic strategies.

Hypoxia-mediated impaired erythrocyte Lands’ Cycle is pathogenic for sickle cell disease

Although Lands’ cycle was discovered in 1958, its function and cellular regulation in membrane homeostasis under physiological and pathological conditions remain largely unknown. Nonbiased high throughput metabolomic profiling revealed that Lands’ cycle was impaired leading to significantly elevated erythrocyte membrane lysophosphatidylcholine (LysoPC) content and circulating and erythrocyte arachidonic acid (AA) in mice with sickle cell disease (SCD), a prevalent hemolytic genetic disorder. Correcting imbalanced Lands’ cycle by knockdown of phospholipase 2 (cPLA2) or overexpression of lysophosphatidycholine acyltransferase 1 (LPCAT1), two key enzymes of Lands’ cycle in hematopoietic stem cells, reduced elevated erythrocyte membrane LysoPC content and circulating AA levels and attenuated sickling, inflammation and tissue damage in SCD chimeras. Human translational studies validated SCD mouse findings and further demonstrated that imbalanced Lands’ cycle induced LysoPC production directly promotes sickling in cultured mouse and human SCD erythrocytes. Mechanistically, we revealed that hypoxia-mediated ERK activation underlies imbalanced Lands’ cycle by preferentially inducing the activity of PLA2 but not LPCAT in human and mouse SCD erythrocytes. Overall, our studies have identified a pathological role of imbalanced Lands’ cycle in SCD erythrocytes, novel molecular basis regulating Lands’ cycle and therapeutic opportunities for the disease.

Characterization of Antibody Specificities Associated With Preeclampsia

The presence of maternal autoantibodies has been previously associated with preeclampsia, although the composition of the antibody repertoire in preeclampsia has not been well characterized. Given this, we applied a bacterial display peptide library to identify peptides that preferentially react with plasma antibodies from patients with preeclampsia (n=15) versus healthy-outcome pregnancies (n=18). Screening using fluorescence-activated cell sorting identified 38 peptides that preferentially bind to antibodies from individuals with preeclampsia. These preeclampsia-specific peptides possessed similar motifs of RG/S G/-WWG/S, RWWG/S, or WGWGXXR/K distinct from the angiotensin II type 1 receptor epitope AFHYESQ. Seven library-isolated peptides and a cell surface–displayed angiotensin II type 1 receptor epitope were used to construct a diagnostic algorithm with a training set of 18 new preeclamptic and 22 healthy-outcome samples from geographically distinct cohorts. Cross-validation within the training group resulted in averaged areas underneath a receiver operating characteristic curve of 0.78 and 0.72 with and without the known receptor epitope, respectively. In a small validation set (12 preeclamptic; 8 healthy), the algorithm consisting only of library-isolated peptides correctly classified 10 preeclamptic and 6 healthy samples using a predefined cutoff that achieved 61% sensitivity (95% confidence interval, 36%–83%) at 95% specificity (95% confidence interval, 77%–100%) in training set (n=40) cross-validation. Our results indicate that antibodies with specificities other than anti–angiotensin II type 1 receptor are prevalent in preeclampsia patients and may be useful as diagnostic biomarkers.

Elevated Transglutaminase Activity Triggers Angiotensin Receptor Activating Autoantibody Production and Pathophysiology of Preeclampsia

Background Preeclampsia (PE) is a life‐threatening hypertensive disorder of pregnancy associated with autoantibodies, termed AT 1‐AA, that activate the AT 1 angiotensin receptor. Although the pathogenic nature of these autoantibodies has been extensively studied, little is known about the molecular cause of their generation. Methods and Results Here we show that tissue transglutaminase (TG2), an enzyme that conducts posttranslational modification of target proteins, directly modified the 7‐amino acid (7‐aa) epitope peptide that localizes to the second extracellular loop of the AT 1 receptor. These findings led us to further discover that plasma transglutaminase activity was induced and contributed to the production of AT 1‐AA and disease development in an experimental model of PE induced by injection of LIGHT, a tumor necrosis factor superfamily member. Key features of PE were regenerated by adoptive transfer of purified IgG from LIGHT‐injected pregnant mice and blocked by the 7‐amino acid epitope peptide. Translating our mouse research to humans, we found that plasma transglutaminase activity was significantly elevated in PE patients and was positively correlated with AT 1‐AA levels and PE features. Conclusions Overall, we provide compelling mouse and human evidence that elevated transglutaminase underlies AT 1‐AA production in PE and highlight novel pathogenic biomarkers and innovative therapeutic possibilities for the disease.

Transglutaminase is a Critical Link Between Inflammation and Hypertension

Background The pathogenesis of essential hypertension is multifactorial with different underlying mechanisms contributing to disease. We have recently shown that TNF superfamily member 14 LIGHT (an acronym for homologous to lymphotoxins, exhibits inducible expression, and competes with herpes simplex virus glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes, also known as TNFSF14) induces hypertension when injected into mice. Research reported here was undertaken to examine the role of transglutaminase (TGase) in LIGHT‐induced hypertension. Methods and Results Initial experiments showed that plasma and kidney TGase activity was induced by LIGHT infusion (13.91±2.92 versus 6.75±1.92 mU/mL and 19.86±3.55 versus 12.00±0.97 mU/10 μg) and was accompanied with hypertension (169±7.16 versus 117.17±11.57 mm Hg at day 14) and renal impairment (proteinuria, 61.33±23.21 versus 20.38±9.01 μg/mg; osmolality, 879.57±93.02 versus 1407.2±308.04 mmol/kg). The increase in renal TGase activity corresponded to an increase in RNA for the tissue TGase isoform, termed TG2. Pharmacologically, we showed that LIGHT‐induced hypertension and renal impairment did not occur in the presence of cystamine, a well‐known competitive inhibitor of TGase activity. Genetically, we showed that LIGHT‐mediated induction of TGase, along with hypertension and renal impairment, was dependent on interleukin‐6 and endothelial hypoxia inducible factor‐1α. We also demonstrated that interleukin‐6, endothelial hypoxia inducible factor‐1α, and TGase are required for LIGHT‐induced production of angiotensin receptor agonistic autoantibodies. Conclusions Thus, LIGHT‐induced hypertension, renal impairment, and production of angiotensin receptor agonistic autoantibodies require TGase, most likely the TG2 isoform. Our findings establish TGase as a critical link between inflammation, hypertension, and autoimmunity.

A pre-eclampsia-associated Epstein-Barr virus antibody cross-reacts with placental GPR50

To characterize antibody specificities associated with pre-eclampsia (PE), bacterial displayed peptide library screening and evolution was applied to identify peptide epitopes recognized by plasma antibodies present in women with PE near the time of delivery. Pre-eclamptic women exhibited elevated IgG1 titers towards a peptide epitope KRPSCIGCK within the Epstein-Barr virus nuclear antigen 1 (EBNA-1). EBNA-1 epitope antibodies cross-reacted with a similar epitope within the extracellular N-terminus of the human G protein-coupled receptor, GPR50, expressed in human placental tissue and immortalized placental trophoblast cells. We observed increased antibody binding activity to epitopes from EBNA-1 and GPR50 among women with PE (n=42) compared to healthy-outcome pregnancies (n=43) and nulligravid samples (n=21). The EBNA-1 peptide potently blocked binding of the PE-associated antibody to the GPR50 epitope (IC50=58-81pM). These results reveal the existence of molecular mimicry between EBNA-1 and placental GPR50, supporting a mechanism for IgG1 deposition in the pre-eclamptic placenta.