BackgroundFetal hepatic glycogen is essential for intrauterine development and postnatal metabolic adaptation. This study aimed to explore the impact of intrauterine exposure to angiotensin II type 1 receptor autoantibodies (AT1-AA) on fetal hepatic glycogen homeostasis and underlying mechanisms.MethodsAT1-AA-positive pregnant rat models were established by intravenous administration of AT1-AA on gestational days 13 and 15, with angiotensin II- and L-NAME-treated groups serving as controls. Serum levels of AT1-AA in pregnant rats were quantified using ELISA. Fetal hepatic glycogen content was evaluated through PAS staining and an anthrone-sulfuric acid assay kit. On gestational day 18, fetal livers were harvested for RNA sequencing. Alterations in signaling molecules in hepatocytes were analyzed by Western blot.ResultsUnlike the angiotensin II- and L-NAME-treated groups, which similarly induced placental ischemia and growth restriction, intrauterine exposure of AT1-AA uniquely reduced fetal hepatic glycogen content at gestational day 18. This reduction was attributed to impaired glycogen synthesis, as characterized by decreased GYS2 expression and glycogen synthase activity. Transcriptomic profiling revealed that suppression of the PI3K/AKT pathway was the predominant mechanism involved. Both in vivo and in vitro studies confirmed that AT1-AA-mediated PI3K/AKT inhibition occurs through AT1R activation. Either stimulation of the PI3K/AKT pathway or blockade of AT1R significantly restored glycogen synthesis.ConclusionsOur study identifies AT1-AA as a unique maternal factor that selectively impairs fetal hepatic glycogen storage via AT1R-mediated suppression of PI3K/AKT pathway.

Abstract Autoantibodies against angiotensin II type 1 receptor (AT1-AA) are prevalent in preeclampsia. They induce fetal cardiac structural remodeling in late pregnancy via still unknown mechanisms, the subject of this study’s investigation. MicroRNA array revealed differential expression of microRNAs in fetal hearts exposed to AT1-AA. miR-124-3p and miR-181a-5p, the most significantly altered microRNAs, were significantly enriched in the myocardium. Gain- and loss-of-function experiments demonstrated that miR-124-3p and miR-181a-5p governed cardiomyocyte phenotype. Overexpression of miR-124-3p or inhibition of miR-181a-5p activated neonatal rat cardiomyocyte proliferation and size (the converse also being true). Moreover, miR-124-3p overexpression and miR-181a-5p inhibition acted in concert to exacerbate the effects of AT1-AA (increasing the size and proliferation of neonatal rat cardiomyocytes). Through TMT™ quantitative proteomics and luciferase reporter gene assay, miR-181a-5p and miR-124-3p respectively target the 3’ UTR and 5’UTR of VCAN, and oppositely regulate its expression. VCAN knockdown in neonatal rat cardiomyocytes blocked the pathological effect of AT1-AA. Furthermore, ELISA demonstrated positive correlation between AT1-AA expression and VCAN level in preeclamptic patient umbilical cord blood. Our study demonstrates that upregulation of miR-124-3p promotes VCAN expression, and miR-181a-5p downregulation decreases VCAN inhibition, thereby co-mediating AT1-AA induced fetal cardiac remodeling.

The abnormality of large-conductance calcium-activated potassium channels (BK channels) is an important factor in inducing vascular inflammation. BK channel agonists can readily recover BK channel function and improve vascular inflammation. However, it is not clear how to improve BK dysfunction caused by downregulation of BK channel protein expression. This study found that angiotensin II-1 receptor autoantibodies (AT1-AA), which are widely present in the body of various types of cardiovascular diseases, can down-regulate the expression of BK channel protein and induce vascular inflammation. Further research found that the elevated neural precursor cells expressed developmentally downregulated 4-like (NEDD4L) protein level is involved in the down-regulation of BK channel α subunit (BKα) protein level by AT1-AA. Bioinformatics analysis and experiments have confirmed that miR-339-3p plays an irreplaceable role in the high expression of NEDD4L and the low expression of BKα, and aggravates the vascular inflammation induced by AT1-AA. Overall, AT1-AA increased miR-339-3p expression (targeting BKα via the miR-339-3p/NEDD4L axis or miR-339-3p alone), reduced BKα protein expression in VSMCs, and induced vascular inflammation. The results of the study indicate that miR-339-3p may become a new target for reversing vascular inflammation in AT1-AA-positive patients.

Vascular remodeling can be caused by angiotensin II type 1 receptor (AT1R) autoantibody (AT1-AA), although the related mechanism remains unknown. Angiotensin II type 2 receptor (AT2R) plays multiple roles in vascular remodeling through cross-talk with AT1R in the cytoplasm. Here, we aimed to explore the role and mechanism of AT2R in AT1-AA-induced vascular smooth muscle cell (VSMC) migration, which is a key event in vascular remodeling. In vitro and in vivo, we found that AT2R can promote VSMC migration in AT1-AA-induced vascular remodeling. Moreover, AT2R expression was upregulated via Klf-5/IRF-1-mediated transcriptional and circErbB4/miR-29a-5p-mediated posttranscriptional mechanisms in response to AT1-AA. Our data provide a molecular basis for AT1-AA-induced AT2R expression by transcription factors, namely, a circular RNA and a microRNA, and showed that AT2R participated in AT1-AA-induced VSMC migration during the development of vascular remodeling. AT2R may be a potential target for the treatment of AT1-AA-induced vascular diseases.

Exposure to adverse factors in utero may lead to adaptive changes in cardiac structure and metabolism, which increases the risk of chronic cardiovascular disease later in life. Studies showed that the angiotensin II type 1 receptor autoantibodies (AT1-AAs) are able to cross the placenta into the circulation of pregnant rodents' embryo, which adversely affects embryogenesis. However, the effects of AT1-AA exposure on the fetal heart in utero are still unknown. In this study, we investigated whether intrauterine AT1-AA exposure has adverse effects on fetal heart structure, function and metabolism. AT1-AA-positive pregnant mouse models were successfully established by passive immunity, evidenced by increased AT1-AA content. Morphological and ultrasonic results showed that the fetal mice on embryonic day 18 (E18) of AT1-AA group have loose and disordered myocardial structure, and decreased left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS), compared with control groups. The myocardium of AT1-AA group fetal mice on E18 exhibited increased expression of the key molecules in the glycolytic pathway, pyruvate and lactic acid content and ATP production, suggesting that the glycolysis rate was enhanced. Furthermore, the enhanced effect of glycolysis caused by AT1-AA is mainly through the PPARβ/δ pathway. These data confirmed that fetus exposure to AT1-AA in utero developed left ventricular dysfunction, myocardial structural arrangement disorders, and enhanced glycolysis on E18. Our results support AT1-AA being a potentially harmful factor for cardiovascular disease in fetal mice.

Blood glucose is of great importance to development and metabolic homeostasis in fetuses. Stimulation of harmful factors during gestation induces pathoglycemia. Angiotensin II type 1 receptor autoantibody (AT1-AA), a newly discovered gestational harmful factor, has been shown to induce intrauterine growth restriction in fetuses and glucose disorders in adults. However, whether and how AT1-AA influences the blood glucose level of fetuses during gestation is not yet clear. The purpose of the current study was to observe the fetal blood glucose level of AT1-AA-positive pregnant rats during late pregnancy and to determine the roles that hepatic glucose transporters play in this process. We established AT1-AA-positive pregnant rats by injecting AT1-AA into the caudal veins of rats in the 2nd trimester of gestation. Although the fetal blood glucose level in the 3rd trimester of gestation decreased, hepatic glucose uptake increased detected. Through separating membrane and cytosolic proteins, we demonstrated that both the expression and membrane transport ratio of glucose transporter 1 (GLUT1), which is responsible for glucose transport in fetal hepatocytes, were upregulated, accompanied by increased expression of N-glycosyltransferase STT3A, which contributes to the N-glycosylation of GLUT1. In vitro, we identified that AT1-AA increased glucose uptake, the expression and membrane transport ratio of GLUT1 and the expression of STT3A in HepG2 cell lines via separating membrane and cytosolic proteins and immunofluorescence, resulting in the decreased glucose content in the medium. The GLUT1 inhibitor WZB117 reversed the decreases in glucose content in the medium, the increases in glucose uptake, the increases in the expression and membrane transport ratio of GLUT1 caused by AT1-AA. The N-glycosyltransferase inhibitor NGI as well as si-STT3A reversed the AT1-AA-induced upregulation of the STT3A-GLUT1-glucose uptake effect. This study demonstrates that AT1-AA lowers the blood glucose level of fetuses via the STT3A-GLUT1-glucose uptake axis in liver.

Angiotensin II (Ang II) is known to promote proliferation of vascular smooth muscle cells (VSMCs) in vascular remodeling, but whether it has an anti-apoptotic effect needs to be explored. Neuregulin-1 (NRG-1) as a member of the epidermal growth factor family was reported to suppress the proliferation of VSMCs by activating ErbB receptors, and therefore we hypothesized that there might be a cross talk between the anti-apoptotic effect of Ang II and the anti-proliferative effect of NRG-1 in VSMCs. The aim of the present study was to observe the expression and role of NRG-1 underlying the inhibitory effect of Ang II on apoptosis of mouse aortic smooth muscle cells (MASMCs). It was found that NRG-1 expression was down-regulated via the circNRG-1/miR-193b-5p-mediated post-transcriptional mechanism in response to Ang II. In addition, NRG-1 overexpression reversed the inhibitory effect of Ang II on apoptosis in MASMCs. Our data may provide a molecular basis for further understanding the mechanism of Ang II in suppressing the apoptosis of MASMCs by decreasing NRG-1 expression at circular RNA and micro RNA levels. The circNRG-1/miR-193b-5p/NRG-1 axis may prove to be a potential target for Ang II to inhibit the apoptosis of VSMCs and lead to vascular remodeling.

The internalization of angiotensin II type 1 receptor (AT1R) plays an important role in maintaining cardiovascular homeostasis. Decreased receptor internalization is closely related to cardiovascular diseases induced by the abnormal activation of AT1R, such as hypertension. However, the mechanism behind reduced AT1R internalization is not fully understood. This review focuses on four parts of the receptor internalization process (the combination of agonists and receptors, receptor phosphorylation, endocytosis, and recycling) and summarizes the possible mechanisms by which AT1R internalization is reduced based on these four parts of the process. (1) The agonist has a large molecular weight or a stronger ability to hydrolyze phosphatidylinositol 4,5-bisphosphate (PtdIns (4,5) P2), which can increase the consumption of PtdIns (4,5) P2. (2) AT1R phosphorylation is weakened because of an abnormal function of phosphorylated kinase or changes in phospho-barcoding and GPCR–β-arrestin complex conformation. (3) The abnormal formation of vesicles or AT1R heterodimers with fewer endocytic receptors results in less AT1R endocytosis. (4) The enhanced activity and upregulated expression of small GTP-binding protein 4 (Rab4) and 11 (Rab11), which regulate receptor recycling, and phosphatidylinositol 3-kinase increase AT1R recycling. In addition, lower expression of AT1R-associated protein (ATRAP) or higher expression of AT1R-associated protein 1 (ARAP1) can reduce receptor internalization.

ObjectiveInsulin resistance is highly associated with an adverse intrauterine environment. We previously reported that fetal rats exposed to angiotensin II type 1 receptor (AT(1)R) autoantibody (AT1-AA) displayed increased susceptibility to metabolic diseases during middle age. However, the timing of the onset of insulin resistance remains unknown. In this study, we examined the offspring of AT1-AA-positive rats, tracking the development of insulin resistance.MethodsPregnant rats were intravenously injected with AT1-AA. Afterwards, we collected serum samples and liver tissues of the offspring at various stages, including gestation day 18, 3 weeks (weaning period), 18 weeks (young adulthood), and 48 weeks (middle age) after birth.ResultsCompared with saline control group, hepatic vacuolar degeneration was visible in AT1-AA offspring rats as early as 3 weeks; hyperinsulinemia and impaired glucose tolerance occurred at 18 weeks of age, however, insulin resistance was not observed until 48 weeks. At 18 weeks we detected suppressed protein levels of insulin receptor (IR) but increased levels of IR substrate 1 (IRS1) in the liver of AT1-AA group rats. Interestingly, both IR and IRS1/2 were significantly decreased at 48 weeks. Liver proteomic analysis indicated that the differences in protein expression between the AT1-AA and control rats became more pronounced with age, particularly in terms of mitochondrial energy metabolism.ConclusionRats exposed to AT1-AA in utero developed hyperinsulinemia from young adulthood which subsequently progressed to insulin resistance, and was linked with abnormal hepatic structure and impaired IR signaling. Additionally, dysregulation of energy metabolism may play a fundamental role in predisposing offspring to insulin resistance.