sFlt-1 Expression Research Articles

DIFFERENCES IN PROTEASE ACTIVATED RECEPTOR-1 AND THROMBINE LEVELS IN PREECLAMPSIA AND NORMAL PREGNANCY

Introduction: Preeclampsia is a condition caused by alterations in endothelial function during pregnancy. Changes in endothelial function result in an increase in coagulation and microvascular fibrin accumulation, which results in impaired placental perfusion. Thrombin, which converts fibrin to fibrinogen, as well as platelet activity, the fibrinolytic system, and anticoagulants, are all procoagulant circumstances in preeclampsia. Thrombin contributes to the pathogenesis of preeclampsia by increasing the expression of sFlt-1 thereby providing an antiangiogenic response. Protease Activated Receptor-1 (PAR-1) is a mediator of thrombin for coagulation and inflammation in preeclampsia. Inhibition of Protease Activated Receptor-1 expression in trophoblasts can enhance placental angiogenesis and vascular remodeling. Recently, only few studies have assessed the levels of Protease Activated Receptor -7 and thrombin in preeclampsia.Objective: To determine the difference in levels of Protease Activated Receptor-1 and thrombin in preeclampsia and normal pregnancyMethods: This study is observational with a cross-sectional comparative study design. Sampling was conducted from March 2020 to March 2021. A total of 66 patients were investigated, with 33 samples of preeclampsia and 33 samples of normal pregnancy. The independent sample T-test was used for statistical analysis.Results: The mean levels of Protease Activated Receptor-1 in the preeclampsia group were higher at 28.56 ± 7.68 ng/mL while normal pregnancy was 21.67 ± 6.92 ng/mL. The results of statistical tests showed that there was a significant difference in levels of Protease Activated Receptor-1 between the preeclampsia and normal pregnancy groups (p<0.05). The mean thrombin level in the preeclampsia group was higher at 72.23 ± 7.99 ng/mL, while in normal pregnancy it was 63.70 ± 8.92 ng/mL. The difference in thrombin levels between the preeclampsia and normal pregnancy groups was statistically significant (p<0.05).Conclusion: Preeclampsia was associated with greater levels of Protease Activated Receptor-1 and thrombin than normal pregnancy. There was a significant difference in the mean levels of Protease Activated Receptor-1 and thrombin between preeclampsia and normal pregnancy.Keywords: Thrombin, Protease Activated Receptor-1(PAR-1), Preeclampsia

Nordihydroguaiaretic Acid-Cross-Linked Phenylboronic Acid-Functionalized Polyplex Micelles for Anti-angiogenic Gene Therapy of Orthotopic and Metastatic Tumors.

Polyplexes are required to be equipped with multiple functionalities to accomplish adequate structure stability and gene transfection efficacy for gene therapy. Herein, a 4-carboxy-3-fluorophenylboronic acid (FPBA)-functionalized block copolymer of PEG-b-PAsp(DET/FBA) and PAsp(DET/FBA) (abbreviated as PB and HB) was synthesized and applied for engineering functional polyplex micelles (PMs) through ionic complexation with pDNA followed by strategic cross-linking with nordihydroguaiaretic acid (NDGA) in respect to the potential linkage of polyphenol and FPBA moieties. In relation to polyplex micelles void of cross-linking, the engineered multifunctional polyplex micelles (PBHBN-PMs) were determined to possess improved structural tolerability against the exchange reaction with charged species. Besides, the FPBA/NDGA cross-linking appeared to be selectively cleaved in the acidic endosomal compartments but not the neutral milieu. Furthermore, the PBHB-PMs with the optimal FPBA/NDGA cross-linking degree were identified to possess appreciable cellular uptake and endosomal escape activities, eliciting a significantly high level of gene expression relative to P-PMs and PB-PMs. Eventually, in vivo antitumor therapy by our proposed multifunctional PMs appeared to be capable of facilitating expression of the antiangiogenic genomic payloads (sFlt-1 pDNA) via systemic administration. The enriched antiangiogenic sFlt-1 in the tumors could silence the activities of angiogenic cytokines for the inhibited neo-vasculature and the suppressed growth of orthotopic 4T1 tumors. Of note, the persistent expression of the antiangiogenic sFlt-1 is also presumed to migrate into the blood circulation, thereby accounting for an overall antiangiogenic environment in preventing the potential pulmonary metastasis. Hence, our elaborated multifaceted PMs inspired fascinating potential as an intriguing gene delivery system for the treatment of clinical solid tumors and metastasis.

A gene delivery system with autophagy blockade for enhanced anti-angiogenic therapy against Fusobacterium nucleatum-associated colorectal cancer

Anti-angiogenesis has emerged a promising strategy against colorectal cancer (CRC). However, the efficacy of anti-angiogenic therapy is greatly compromised by the up-regulated autophagy levels resulting from the evolutionary resistance mechanism and the presence of Fusobacterium nucleatum (F. nucleatum) in CRC. Herein, we report a cationic polymer capable of blocking autophagic flux to deliver plasmid DNA (pDNA) encoding soluble FMS-like tyrosine kinase-1 (sFlt-1) for enhanced anti-angiogenic therapy against F. nucleatum-associated CRC. The autophagy-inhibiting cationic polymer, referred to as PNHCQ, is synthesized by conjugating hydroxychloroquine (HCQ) into 3,3′-diaminodipropylamine-pendant poly(β-benzyl-L-aspartate) (PAsp(Nors)), which can be assembled and electrostatically interacted with sFlt-1 plasmid to form PNHCQ/sFlt-1 polyplexes. Hydrophobic HCQ modification not only boosts transfection efficiency but confers autophagy inhibition activity to the polymer. Hyaluronic acid (HA) coating is further introduced to afford PNHCQ/sFlt-1@HA for improved tumor targeting without compromising on transfection. Consequently, PNHCQ/sFlt-1@HA demonstrates significant anti-tumor efficacy in F. nucleatum-colocalized HT29 mouse xenograft model by simultaneously exerting anti-angiogenic effects through sFlt-1 expression and down-regulating autophagy levels exacerbated by F. nucleatum challenge. The combination of anti-angiogenic gene delivery and overall autophagy blockade effectively sensitizes CRC tumors to anti-angiogenesis, providing an innovative approach for enhanced anti-angiogenic therapy against F. nucleatum-resident CRC. Statement of significanceUp-regulated autophagy level within tumors is considered responsible for the impaired efficacy of clinic antiangiogenic therapy against CRC colonized with pathogenic F. nucleatum. To tackle this problem, an autophagy-inhibiting cationic polymer is developed to enable efficient intracellular delivery of plasmid DNA encoding soluble FMS-like tyrosine kinase-1 (sFlt-1) and enhance anti-angiogenic therapy against F. nucleatum-associated CRC. HA coating that can be degraded by tumor-enriching hyaluronidase is further introduced for improved tumor targeting without compromising transfection efficiency. The well-orchestrated polyplexes achieve considerable tumor accumulation, efficient in vivo transfection, and effectively reinforce the sensitivity of CRC to the sFlt-1-derived anti-angiogenic effects by significantly blocking overall autophagy flux exacerbated by F. nucleatum challenge, thus harvesting robust antitumor outcomes against F. nucleatum-resident CRC.

Gene expression of iron transporters, markers of vascularization and structural integrity in placenta of mothers with and without anemia.

To compare the mRNA expression of placental iron transporters (TfR-1 and FPN), markers of placental vascularization (VEGF and sFLT1) and marker of structural integrity (LMN-A) in term women with and without iron deficiency anemia. A total of 30 pregnant women were enrolled; 15 cases of iron deficiency anemia (Hb 7-10.9 gm/dL) and 15 gestational age matched healthy controls (Hb ≥ 11 gm/dL). Peripheral venous blood was collected for assessment of hemoglobin levels and serum iron profile. Placental tissue was used for assessing the mRNA expression of TfR-1, FPN, VEGF, sFLT-1 and LMN-A via real time PCR. Placental expression of TfR-1, VEGF and LMN-A was increased in pregnant women with anemia compared to healthy pregnant controls. Placental expression of sFLT-1 was decreased in pregnant women with anemia compared to healthy pregnant controls. There was no change in the placental expression of FPN. The increased expression of TfR-1, VEGF and LMN-A in cases of iron deficiency anemia are most likely to be compensatory in nature to help maintain adequate fetal iron delivery. Compensatory changes in the placenta aimed at buffering transport of iron to the fetus are seen in pregnant women with anemia compared to healthy pregnant controls.

Cadmium exposure activates mitophagy through downregulating thyroid hormone receptor/PGC1α signal in preeclampsia

Gestational cadmium exposure increases the risk of preeclampsia. Placenta mitophagy was activated in preeclampsia. The aim of present study was to explore the mechanism of cadmium-induced mitophagy activation and its association with preeclampsia. Mitophagy markers expression levels were detected by quantitative real-time PCR, Western blot, immunofluorescence and immunochemistry in preeclampsia placenta. JEG3 cells were treated with CdCl2, iopanoic acid (IOP), 3-methyladenine and PGC1α SiRNA to verify mechanism of cadmium-induced mitophagy. Mitophagy marker LC3BII/I and P62 expression were increased and mitochondrial membrane receptor protein TOM20 and FUNDC1 expression were decreased in preeclampsia placenta as compared with that in normotension control. Mitophagy marker LC3BII/I and P62 expression were increased and TOM20 and FUNDC1 expression was decreased in CdCl2-treated JEG3 cells. Meanwhile, mitochondrial biogenesis regulator, PGC1α expression was decreased in preeclampsia and CdCl2-treated JEG3 cells. The expressions of LC3B and P62 were increased and the expressions of TOM20, FUNDC1 and PGC1α were decreased in IOP-treated cell. PGC1α SiRNA transfection led to increased expression of LC3BII/I and P62 and decreased expression of TOM20 and FUNDC1. The expression of sFlt1 was increased in preeclampsia placenta, CdCl2-treated cells, in IOP-treated cells and in PGC1α SiRNA transfected cells. 3-methyladenine treatment protected the increased expression of sFlt1 in CdCl2-treated cells, in IOP-treated cells and in PGC1α SiRNA transfected cells. Meanwhile, co-treatment of cadmium and IOP or PGC1αSiRNA led to a reduce expressions of OPA1, MFN1, MFN2 and FUNDC1 as compared to cadmium-treated, IOP-treated and PGC1α SiRNA-treated cells. These results elucidated that maternal cadmium exposure activated placenta mitophagy through downregulation of thyroid hormone receptor signal mediated decreased expression of PGC1α and was associated with the occurrence of preeclampsia.

Soyghurt Potentially Controls the Level of sFlt1 and PLGF in Preeclampsia Maternal Serum-Induced Placental Trophoblast Cell in vitro.

To observe the effect of soya yoghurt (Soyghurt), which is high in flavonoid substance, on the expression of preeclampsia biomarkers (sFLT-1 and PLGF) on preeclampsia serum-induced trophoblast primary cell culture isolated from placental tissue. The trophoblast primary culture was induced by preeclampsia serum (10%). The Soyghurt treatment was performed with 2.5%, 5%, and 7.5% Soyghurt supernatant concentrations in culture media. The expression of preeclampsia markers, sFLT-1 and PLGF, were evaluated using ELISA. Expression of sFLT-1 on preeclampsia-induced cell culture treated with Soyghurt was significantly lowered compared to the untreated group (p<0.01). However, no significant difference was observed in the PLGF levels of all groups induced by preeclampsia serum (p>0.05). This study demonstrates the potential effect of Soyghurt's in balancing preeclampsia marker expression by inhibiting the expression of sFLT-1 in preeclampsia serum -induced trophoblast cells.

Fructose Consumption Affects Placental Production of H2S: Impact on Preeclampsia-Related Parameters.

H2S, a gasotransmitter that can be produced both via the transsulfuration pathway and non-enzymatically, plays a key role in vasodilation and angiogenesis during pregnancy. In fact, the involvement of H2S production on plasma levels of sFLT1, PGF, and other molecules related to preeclampsia has been demonstrated. Interestingly, we have found that maternal fructose intake (a common component of the Western diet) affects tissular H2S production. However, its consumption is allowed during pregnancy. Thus, (1) to study whether maternal fructose intake affects placental production of H2S in the offspring, when pregnant; and (2) to study if fructose consumption during pregnancy can increase the risk of preeclampsia, pregnant rats from fructose-fed mothers (10% w/v) subjected (FF) or not (FC) to a fructose supplementation were studied and compared to pregnant control rats (CC). Placental gene expression, H2S production, plasma sFLT1, and PGF were determined. Descendants of fructose-fed mothers (FC) presented an increase in H2S production. However, if they consumed fructose during their own gestation (FF), this effect was reversed so that the increase disappeared. Curiously, placental synthesis of H2S was mainly non-enzymatic. Related to this, placental expression of Cys dioxygenase, an enzyme involved in Cys catabolism (a molecule required for non-enzymatic H2S synthesis), was significantly decreased in FC rats. Related to preeclampsia, gene expression of sFLT1 (a molecule with antiangiogenic properties) was augmented in both FF and FC dams, although these differences were not reflected in their plasma levels. Furthermore, placental expression of PGF (a molecule with angiogenic properties) was decreased in both FC and FF dams, becoming significantly diminished in plasma of FC versus control dams. Both fructose consumption and maternal fructose intake induce changes in molecules that contribute to increasing the risk of preeclampsia, and these effects are not always mediated by changes in H2S production.

The Role of the Aryl Hydrocarbon Receptor in Vascular Factors Related to Preeclampsia in a Smoking Mouse Model.

Smoking cigarettes is known to lower the risk of preeclampsia. The objective of this study is to evaluate the effect of smoking on the expression of soluble FMS-like tyrosine kinase-1 (sFlt-1), vascular endothelial growth factor (VEGF), and endoglin (sEng)-1 and the role of the aryl hydrocarbon receptor (AhR) in pregnant mice. We developed a smoking mouse model using a gas-filling system. One or two cigarettes per day were exposed to each of the five pregnant mice for five days a week throughout pregnancy. AhR agonist and antagonist were injected. Serum levels and expression in the placenta of sFlt-1, VEGF, and sEng-1 were analyzed and compared among the cigarette smoke and no-exposure groups after delivery. Compared to the no-smoke exposure group, the serum level of sFlt-1 was significantly decreased in the two-cigarette-exposed group (p < 0.001). When the AhR antagonist was added to the two-cigarette-exposed group, sFlt-1 levels were significantly increased compared to the two-cigarette group (p = 0.002). The levels of sFlt-1 in the AhR antagonist group did not change regardless of two-cigarette exposure (p = 0.064). With the AhR agonist, sFlt-1 decreased significantly compared to the control (p = 0.001) and AhR antagonist group (p = 0.002). The sFlt-1 level was significantly decreased after the injection of the AhR agonist compared to the control group (p = 0.001). Serum levels of VEGF were significantly decreased in the one-cigarette-exposed group compared to the control group; however, there was no difference between the control and the two-cigarette-exposed groups. The placental expression of sFlt-1, VEGF, and sEng were inconsistent. This study offers insights into the potential role of AhR on antiangiogenic sFlt-1 associated with preeclampsia. It may support the invention of a new treatment strategy for preeclampsia using AhR activation.

An Examination of the Effect of Aspirin and Salicylic Acid on Soluble Fms-like Tyrosine Kinase-1 Release from Human Placental Trophoblasts.

Low-dose aspirin (LDA) is efficacious in preventing preeclampsia, but its mechanism of action is unclear. Conflicting evidence suggests that it may inhibit placental trophoblast release of soluble fms-like tyrosine kinase-1 (sFlt1), a key mediator of preeclampsia. We examined whether, and at what concentrations, aspirin and its principal metabolite, salicylic acid, modulate sFlt1 release and/or expression in trophoblasts. Human trophoblast lines BeWo and HTR-8/SVneo were cultured; BeWo cells were also treated with 1% oxygen vs. normoxia to mimic hypoxia in preeclamptic placentas. Cells were treated with aspirin or salicylic acid vs. vehicle for 24 h at concentrations relevant to LDA and at higher concentrations. Protein concentrations (ELISA) and mRNA expression (RT-PCR) of sFlt1 were determined. Under normoxia, LDA-relevant concentrations of aspirin (10-50 µmol/L) or salicylic acid (20-100 µmol/L) had no significant effect on sFlt1 protein release or mRNA expression in BeWo cells. However, inhibition was observed at higher concentrations (1 mmol/L for aspirin and ≥200 μmol/L for salicylic acid). Hypoxia enhanced sFlt1 protein release and mRNA expression in BeWo cells, but these responses were not significantly affected by either aspirin or salicylic acid at LDA concentrations. Similarly, neither drug altered sFlt1 protein secretion or mRNA expression in normoxic HTR-8/SVneo cells at LDA concentrations. We suggest that direct modulation of trophoblast release or expression of sFlt1 is unlikely to be a mechanism underlying the clinical efficacy of LDA in preeclampsia.

The effect of cold exposure on the levels of glucocorticoids, 11-hydroxysteroid dehydrogenase 2, and placental vascularization in a rat model.

Cold exposure (CE) before birth is one of the initial stressors that may impact mammalian pregnancy, changing placental and fetal development and affecting the health of the offspring. While glucocorticoids (GCs) participate in the body's response to the stress of CE, the specific mechanisms of their action are unclear. This study aims to determine the effect of CE stress on the placenta and to test whether stress, caused by cold exposure in pregnancy impairs fetal development by changing placental angiogenesis via excessive GC expression. CE rat model was created by exposing 30 SD rats to cold preconception, or during the first, second, and third weeks of pregnancy. Serum cortisol and soluble fms-like tyrosine kinase-1 (sFlt-1) expression levels, physiological index changes (food intake, body weight change and blood pressure), and pregnancy outcomes (fetal rat weight, number of live fetal rats, and placental weight) were collected at baseline and at different time points after the conception. Protein expression levels of 11 β-hydroxysteroid dehydrogenase 2 (11β-HSD2), glucocorticoid receptor, vascular endothelial growth factor A (VEGF-A), placental growth factor (PIGF), and sFlt-1 in placental tissues were measured by western blotting. Cytokeratin (CK) and laminin (LN) in trophoblasts, and α-actin in vascular smooth muscle of the spiral arteries of pregnant rats after the systemic cold treatment were assessed by immunofluorescence and visualized by fluorescent microscopy. To test the effect of 11β-HSD2 levels on the placental recasting, human first-trimester extravillous trophoblast cells (HTR8/SVneo) underwent knockdown using specific 11β-HSD2 siRNA constructs. Expression levels of 11β-HSD2 were analyzed by quantitative real-time PCR (qPCR) and into HTR8 cells, and the expression levels of the 11β-HSD2 gene in each group were measured using qPCR. Cell migration and invasion was assessed by Transwell migration assay, and sFlt-1 levels in HTR8 cells were measured by ELISA. CE pre-conception led to consistently increasing serum corticosterone and sFlt-1 levels throughout pregnancy, and persistently increased diastolic blood pressure (DBP) in rat CE model compared to control animals. CE during the second week of gestation (Gp.3) was associated with significantly lower placental weight (p=0.0003). Cold exposure in the third week (Gp.4) was associated with significantly (p=0.001) lower fetal weight. CE pre-conception was associated with significantly decreased placental levels of 11β-HSD2, glucocorticoid receptor, VEGF-A, PIGF, and sFlt-1 proteins and α-actin compared to the control group. Silencing 11β-HSD2 by siRNA led to reduced cell migrations and invasion, and markedly increased expression levels of sFlt-1 in HTR8/SVneo cells (p<0.05). Pre-conception cold exposure and during early pregnancy leads to increased GCs levels and impaired placental 11β-HSD2 activity. We suggest that the subsequent 11β-HSD2-induced increase in the sFlt-1expression during early pregnancy may affect placental vascular remodeling and change placental morphological structure and function.

Thyroid hormone transport and metabolism are disturbed in the placental villi of miscarriage

BackgroundIt has been long known that thyroid hormone regulates placental villi development, which is associated with the occurrence of miscarriage. However, whether abnormal thyroid hormone metabolism and transport in placental villi are involved in miscarriage is still to be verified.MethodsPlacental villi of elective terminations of pregnancies (ETPs) and miscarriage were collected. Proliferative activity and apoptosis of villi trophoblasts and angiogenesis were detected by TUNEL and immunochemistry. The expressions of thyroid hormone receptors (THRs), transthyretin (TTR), monocarboxylate transporter 8 (MCT8), organic anion transporting polypeptides 1A1 (OATP1A1), deiodinase 2 (Dio2) and Dio3 were examined by RT-PCR, Western blot, immunohistochemistry and immunofluorescence. JEG3 cell was treated with iopanoic acid (IOP), an inhibitor of Dio2 activity, the expressions of Dio2, placenta growth factor (PLGF) and sFlt1 were detected by RT-PCR and Western blot.ResultsCell proliferation was suppressed and apoptosis was increased in placental villi cytotrophoblasts of miscarriage. CD34+ vessel number and vascular endothelial growth factor (VEGF) protein abundance were decreased in miscarriage. In miscarriage group, the gene expression of Dio2, Dio3, TTR and THRα, but not THRβ, MCT8 and OATP1A1, were downregulated. The protein abundances of TTR and THRα were downregulated in miscarriage group, but not THRβ. The protein abundance of Dio2 in miscarriage villi was decreased compared with that in ETP. In JEG3 cells, the gene expression of PLGF was decreased and the expression of sFlt1 was increased in IOP treatment; The protein abundance of Dio2 was downregulated but the gene expression of Dio2 was unaffected in IOP treatment.ConclusionThyroid hormone transport and metabolism in miscarriage were disturbed and may impaired angiogenesis of placental villi, which was associated with the occurrence of miscarriage.

Effects of sirtuin 1 deficiency on trophoblasts and its implications in the pathogenesis of pre-eclampsia

Background Sirtuin 1 (SIRT1) is mainly localised in syncytiotrophoblasts and cytotrophoblasts, and is involved in pregnancy regulation. However, data on the association between SIRT1 and pre-eclampsia (PE) remains limited. This study aimed to investigate the role of SIRT1 in PE pathophysiology. Methods Placental SIRT1 expression, as well as serum SIRT1, placental growth factor (PlGF), and soluble FMS-like tyrosine kinase 1 (sFlt-1) levels, were measured using quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, and enzyme-linked immunosorbent assays in 40 healthy pregnant women (NP group) and 40 women with severe PE (PE group). Additionally, the effects of SIRT1 on the migration, invasion, PlGF, and sFlt-1 secretion of HTR-8/SVneo cells were analysed. Results SIRT1 expression was significantly reduced in the placenta of patients with severe PE compared with that in healthy pregnant women. Compared with the NP group, serum SIRT1 and PlGF expression was significantly lower in the PE group; however, the expression of serum sFlt-1 was significantly higher in the PE group. Correlation analysis showed that in the PE group, placental SIRT1 protein levels positively correlated with serum PlGF levels (r = 0.468, P = .002) and negatively correlated with serum sFlt-1 levels (r = −0.542, P < .001). Cells with downregulated SIRT1 had a significantly shorter migration distance and a prominently reduced number of invasive cells compared with the corresponding negative control group, suggesting that SIRT1 deficiency may inhibit the migration and invasive ability of HTR-8/SVneo cells. The opposite results were observed after transfection with lentivirus overexpressing SIRT1. Compared with the corresponding controls, cells with downregulated SIRT1 had significantly reduced PlGF levels and significantly increased sFlt-1 levels in the cell culture supernatants, whereas SIRT1 overexpression produced the opposite results. Conclusions SIRT1 deficiency may contribute to the pathogenesis of pre-eclampsia by reducing trophoblastic migration, invasion, and PlGF secretion and increasing sFlt-1 secretion.

Mesenchymal stem cell secretome ameliorates over-expression of soluble fms-like tyrosine kinase-1 (sFlt-1) and fetal growth restriction (FGR) in animal SLE model

Introduction In the near future, stem cell research may lead to several major therapeutic innovations in medical practice. Secretome, a “by-product” of stem cell line cultures, has many advantages. Its easiness of storage, usage, and fast direct effect are some of those to consider. Fetal growth restriction (FGR) remains one of the significant challenges in maternal-fetal and neonatal medicine. Placentation failure is one of the most profound causal and is often related to increasing sFlt-1 in early pregnancy. This study aimed to investigate hUC-MSC secretome in ameliorating sFlt-1 and how to improve outcomes in preventing FGR in an animal model. Materials and methods Pristane-induced systemic lupus erythematosus (SLE) in a mouse model was used to represent placentation failure and its consequences. Twenty-one mice were randomized into three groups: (I) normal pregnancy, (II) SLE, and (III) SLE with secretome treatment. Pristane was administered in all Groups four weeks prior mating period. Secretome was derived from human umbilical cord mesenchymal stem cells (hUC-MSC) conditioned medium on the 3rd and 4th passage, around day-21 until day-28 from the start of culturing process. Mesenchymal stem cell was characterized using flow cytometry for CD105+, CD90+, and CD73+ surface antigen markers. Immunohistochemistry anlysis by using Remmele’s Immunoreactive Score (IRS) was used to quantify the placental sFlt-1 expression in each group. Birth weight and length were analyzed as the secondary outcome. The number of fetuses obtained was also calculated for pregnancy loss comparison between Groups. Results The administration of secretome of hUC-MSC was found to lower the expression of the placental sFlt-1 significantly in the pristane SLE animal model (10.30 ± 1.40 vs. 4.98 ± 2.57; p < 0.001) to a level seen in normal mouse pregnancies in Group I (3.88 ± 0.49; p = 0.159). Secretome also had a significant effect on preventing fetal growth restriction in the pristane SLE mouse model (birth weight: 354.29 ± 80.76 mg vs. 550 ± 64.03 mg; p < 0.001 and birth length: 14.43 ± 1.27 mm vs. 19.00 ± 1.41 mm), comparable to the birth weight and length of the normal pregnancy in Group I (540.29 ± 75.47 mg and 18.14 ± 1.34 mm, p = 0.808 and = 0.719). Secretome administration also showed a potential action to prevent high number of pregnancy loss as the number of fetuses obtained could be similar to those of mice in the normal pregnant Group (7.71 ± 1.11 vs. 7.86 ± 1.06; p = 0.794). Conclusions Administration of secretome lowers sFlt-1 expression in placenta, improves fetal growth, and prevents pregnancy loss in a mouse SLE model.

Chimpanzee adenovirus-mediated multiple gene therapy for age-related macular degeneration

Neovascular age-related macular degeneration AMD (nAMD) is characterized by choroidal neovascularization (CNV) and could lead to irreversible blindness. However, anti-vascular endothelial growth factor (VEGF) therapy has limited efficacy. Therefore, we generated a chimpanzee adenoviral vector (AdC68-PFC) containing three genes, pigment endothelial-derived factor (PEDF), soluble fms-like tyrosine kinase-1 (sFlt-1), and soluble forms of CD59 (sCD59), to treat nAMD. The results showed that AdC68-PFC mediated a strong onset of PEDF, sFlt-1, and sCD59 expression both invivo and invitro. AdC68-PFC showed preventive and therapeutic effects following intravitreal (IVT) injection in the laser-induced CNV model and very low-density lipoprotein receptor-deficient (Vldlr-/-) mouse model. Invitro assessment indicated that AdC68-PFC had a strong inhibitory effect on endothelial cells. Importantly, the safety test showed no evidence of invivo toxicity of adenovirus in murine eyes. Our findings suggest that AdC68-PFC may be a long-acting and safe gene therapy vector for future nAMD treatments.

A Novel Dual-Function Redox Modulator Relieves Oxidative Stress and Anti-Angiogenic Response in Placental Villus Explant Exposed to Hypoxia-Relevance for Preeclampsia Therapy.

Preeclampsia (PE) is a severe, life-threatening complication during pregnancy (~5-7%), and no causative treatment is available. Early aberrant spiral artery remodeling is associated with placental stress and the release of oxygen radicals and other reactive oxygen species (ROS) in the placenta. This precedes the production of anti-angiogenic factors, which ultimately leads to endothelial and trophoblast damage and the key features of PE. We tested whether a novel dual-function redox modulator-AKT-1005-can effectively reduce placental oxidative stress and alleviate PE symptoms in vitro. Isolated human villous explants were exposed to hypoxia and assessed to determine whether improving cell-redox function with AKT-1005 diminished ROS production, mitochondrial stress, production of the transcription factor HIF1A, and downstream anti-angiogenic responses (i.e., sFLT1, sEng production). MitoTEMPO was used as a reference antioxidant. In our villous explant assays, pretreatment with AKT-1005 reduced mitochondrial-derived ROS production, reduced HIF-1A, sFLT1, and sEng protein expression, while increasing VEGF in hypoxia-exposed villous trophoblast cells, with better efficiency than MitoTEMPO. In addition, AKT-1005 improved mitochondrial electron chain enzyme activity in the stressed explant culture. The redox modulator AKT-1005 has the potential to intervene with oxidative stress and can be efficacious for PE therapy. Future studies are underway to assess the in vivo efficacy of HMP.

Abstract 023: Prior Sflt1 Induced Preeclampsia Exacerbates Post-partum Hypertension-mediated Aortic Stiffness And Hypercholesterolemia-induced Atherosclerotic Inflammation

Preeclampsia (PE) is new high blood pressure (BP) and organ damage occurring late in pregnancy in association with increased soluble VEGF receptor (sFlt1). PE survivors have increased risk of future hypertension and myocardial infarction/stroke. We hypothesized that exposure to high sFlt1 during pregnancy exacerbates hypertension and atherosclerosis post-partum. PE was induced by transient adenoviral expression of sFlt1 compared to control virus in pregnant mice. PE mice had elevated sFlt1, BP and kidney damage during pregnancy which resolve post partum. Two months post-partum, prior PE mice had significantly increased BP response to high salt diet. After high BP stimuli, we studied large and small artery function. Prior PE mice had diminished nitric oxide-mediated vasodilation and stiffer aortas with more fibrosis as measured by pulse wave velocity (Con:3.7 vs sFlt:5.9 mm/ms, p&lt;0.0001) and trichrome staining (Con:13.7% vs sFlt:33.9%, p=0.0009). Atherosclerosis studies exposed LDLR knockout mice to PE followed by high fat diet feeding for 8 weeks. Body weight, fasting glucose and cholesterol were not altered by prior PE. Aortic roots were histologically analyzed for plaque size and composition. Aortic root plaque size (Con:73.3 vs sFlt:78.0 AU, p=0.60) and necrotic core were not different between groups. Aortic arch plaque inflammation was quantified via flow cytometry showing significantly increased plaque CD45+ leukocytes (sFlt:1.6 fold, p=0.036) and T cells (sFlt:1.8 fold, p=0.031) from mice with prior PE. Atheroprone mice with prior PE had significantly increased aortic expression of CCL22 (sFlt:1.5 fold, p=0.047), providing a potential mechanism for increased T cell infiltration into atherosclerotic plaques. Experimental PE produces a state of enhanced sensitivity to hypertensive stimuli and increased vascular inflammation in atherosclerotic plaques supporting the concept that PE directly exacerbates hypertension, vascular stiffness, and atherosclerotic inflammation independent of pre-existing risk factors.

The immune and metabolic phenotype is changed in fetal livers upon systemic sFLT1 expression in preeclamptic mice which is linked to adverse offspring metabolic responses

The immune and metabolic phenotype is changed in fetal livers upon systemic sFLT1 expression in preeclamptic mice which is linked to adverse offspring metabolic responses

Nitroxide-HMP-Protects Human Trophoblast HTR-8/SVneo Cells from H2O2-Induced Oxidative Stress by Reducing the HIF1A Signaling Pathway.

Preeclampsia (PE) is a pregnancy-specific syndrome affecting 5-7% of patients. There is no effective treatment available. Early abnormal placental development is associated with oxidative stress (OS) and a release of reactive oxygen species (ROS) in the placenta. This phenomenon leads to downstream signaling, Hypoxia Inducible Factor 1A (HIF1A) stabilization and transcription of the anti-angiogenic factors soluble fms-like tyrosine kinase 1 (sFLT1) and soluble endoglin (sEng), which are known to cause endothelial and trophoblast dysfunction and cardinal features of PE: hypertension, proteinuria and, in severe cases, eclampsia. We tested whether 3-(Hydroxymethyl)-1-oxy-2,2,5,5-tetramethylpyrrolidine (HMP)-a nitroxide-type antioxidant molecule-can reduce placental OS and mitigate PE symptoms in vitro. We induced OS in human trophoblast (HTR-8/SVneo) cells with hydrogen peroxide (H2O2) and assessed whether modulating cell redox function with HMP reduces cell injury, mitochondrial stress and HIF1A and sFLT1 production. Pre-treatment with HMP reduced mitochondrial-derived ROS production, restored LC3B expression and reduced HIF1A and sFLT1 expression in H2O2-exposed HTR-8/SVneo trophoblast cells. HMP improved the mitochondrial electron chain enzyme activity, indicating that a reduction in OS alleviates mitochondrial stress and also reduces anti-angiogenic responses. In reducing placental trophoblast OS, HMP presents a potential novel therapeutic approach for the treatment of PE. Future investigation is warranted regarding the in vivo use of HMP.

Bioflavonoid luteolin prevents sFlt-1 release via HIF-1α inhibition in cultured human placenta.

Preeclampsia (PE) is a serious hypertensive complication of pregnancy and is a leading cause of maternal death and major contributor to maternal and perinatal morbidity, including establishment of long-term complications. The continued prevalence of PE stresses the need for identification of novel treatments which can target prohypertensive factors implicated in the disease pathophysiology, such as soluble fms-like tyrosine kinase 1 (sFlt-1). We set out to identify novel compounds to reduce placental sFlt-1 and determine whether this occurs via hypoxia-inducible factor (HIF)-1α inhibition. We utilized a commercially available library of natural compounds to assess their ability to reduce sFlt-1 release from primary human placental cytotrophoblast cells (CTBs). Human placental explants from normotensive (NT) and preeclamptic (PE) pregnancies were treated with varying concentrations of luteolin. Protein and mRNA expression of sFlt-1 and upstream mediators were evaluated using ELISA, western blot, and real-time PCR. Of the natural compounds examined, luteolin showed the most potent inhibition of sFlt-1 release, with >95% reduction compared to vehicle-treated. Luteolin significantly inhibited sFlt-1 in cultured placental explants compared to vehicle-treated in a dose- and time-dependent manner. Additionally, significant decreases in HIF-1α expression were observed in luteolin-treated explants, suggesting a mechanism for sFlt-1 downregulation. The ability of luteolin to inhibit HIF-1α may be mediated through the Akt pathway, as inhibitors to Akt and its upstream regulator phosphatidylinositol-3 kinase (PI3K) resulted in significant HIF-1α reduction. Luteolin reduces anti-angiogenic sFlt-1 through inhibition of HIF-1α, making it a novel candidate for the treatment of PE.

MIF Increases sFLT1 Expression in Early Uncomplicated Pregnancy and Preeclampsia.

Insufficient immune tolerance during pregnancy is associated with pathological conditions such as preeclampsia (PE). Soluble fms-like tyrosine kinase-1 (sFLT1), which exerts a role in the late stage of PE, has shown its beneficial anti-inflammatory effects in inflammation-associated diseases. Macrophage migration inhibitory factor (MIF) was reported to upregulate sFLT1 production in experimental congenital diaphragmatic hernia. However, the placental sFLT1 expression in early uncomplicated pregnancy and whether MIF can regulate sFLT1 expression in uncomplicated and preeclamptic pregnancy are unclear. We collected first-trimester placentas and term placentas from uncomplicated and preeclamptic pregnancies to investigate sFLT1 and MIF expression in vivo. Primary cytotrophoblasts (CTBs) and a human trophoblast cell line (Bewo) were used to study the regulation of MIF on sFLT1 expression in vitro. In placentas from first-trimester pregnancy, we observed a high expression of sFLT1, specifically in extravillous trophoblasts (EVTs) and syncytiotrophoblast (STB) cells. MIF mRNA levels strongly correlated with sFLT1 expression in term placentas from preeclamptic pregnancies. In in vitro experiments, sFLT1 and MIF levels increased significantly in CTBs during their differentiation to EVTs and STBs, and MIF inhibitor (ISO-1) significantly reduced sFLT1 expression in a dose-dependent manner during this process. sFLT1 showed significant upregulation with increasing doses of MIF in Bewo cells. Our results show that sFLT1 is highly expressed at the maternal-fetal interface during early pregnancy and that MIF can increase sFLT1 expression in early uncomplicated pregnancy and PE, which suggests that sFLT1 plays an essential role in the modulation of inflammation in pregnancy.