sFlt-1 Protein Levels Research Articles

Mesenchymal stem cell therapy attenuates complement C3 deposition and improves the delicate equilibrium between angiogenic and anti-angiogenic factors in abortion-prone mice

Immunological disorders are one of the main causes of recurrent spontaneous abortions (RSA). A rapidly expanding body of evidence indicates that excessive activation of the complement system is critically involved in the development of miscarriages. In the CBA/J × DBA/2 murine model of recurrent miscarriage, exaggerated and unrestrained complement activation is reported to be the underlying cause of angiogenic factor imbalance and persistent inflammation. We have previously shown that mesenchymal stem cell (MSC) therapy can significantly reduce the abortion rate in abortion-prone mice through regulating the feto-maternal immune response. In the present study, we hypothesized that MSCs might improve the balance of angiogenic factors at the feto-maternal unit of CBA/J × DBA/2 mice by restraining complement activation and deposition.To explore this hypothesis, autologous adipose tissue-derived mesenchymal stem cells (AD-MSCs) were administered intra-peritoneally to abortion-prone mice on the 4.5th day of gestation. Control mice received PBS as vehicle. On day 13.5 of pregnancy, deposition of the complement component C3 and expression levels of Crry, CFD (adipsin), VEGF, PlGF and FLT-1 were measured at the feto-maternal interface by immunohistochemistry and real-time PCR, respectively. Decidual cells were also cultured in RPMI 1640 medium for 48 h and VEGF and sFLT-1 protein levels were quantified in supernatants using enzyme-linked immunosorbent assay (ELISA).Our results indicated that MSC therapy significantly reduced C3 deposition and adipsin transcription in the fetal-maternal interface of abortion-prone mice. Furthermore, administration of MSCs robustly upregulated the mRNA expression levels of Crry, VEGF, PlGF and FLT-1 in the placenta and decidua of CBA/J × DBA/2 mice. Consistently, the in vitro results demonstrated that decidual cells obtained from MSC-treated dams produced increased concentrations of VEGF in culture supernatants, with concomitant decreased levels of sFLT-1 protein.Here, we show for the first time that adoptive transfer of MSCs rectifies the disturbed balance of angiogenic factors observed at the feto-maternal unit of CBA/J × DBA/2 mice, in part at least, through inhibiting excessive complement activation and promoting the production of angiogenic factors. Collectively, these alterations seem to play a pivotal role in reducing the abortion rate and improving the intrauterine condition for the benefit of the fetus.

Non-Viral Gene Delivery Utilizing RALA Modulates sFlt-1 Secretion, Important for Preeclampsia

Background: Overexpression of sFlt-1 or modulation of FKBPL, key antiangiogenic proteins, are important in the pathogenesis of preeclampsia. Methods: A newly developed nonviral gene-delivery system, RALA, capable of overexpressing sFlt-1 (e15a isoform) was delivered in vivo in transgenic haploinsufficient (Fkbpl+/-) mice. RALA was also used in vitro to deliver human Flt1 (hFlt1) in trophoblast cells. Results: Serumstable and nontoxic RALA/DNA-based nanoparticles induced an increase in sFlt-1 protein levels in the blood and total protein in the urine; the effect was more pronounced in Fkbpl+/- mice. In vitro, RALA-hFlt nanoparticles significantly reduced secretion of sFlt-1 in trophoblast cells. Conclusion: The RALA-based genetic nanodelivery system can be safely and effectively applied to emulate preeclampsia-like features or reduce sFlt-1 levels in vitro.

Inhibiting trophoblast PAR-1 overexpression suppresses sFlt-1-induced anti-angiogenesis and abnormal vascular remodeling: a possible therapeutic approach for preeclampsia.

Is it possible to improve vascular remodeling by inhibiting the excessive expression of protease-activated receptor 1 (PAR-1) in trophoblast of abnormal placenta? Inhibition of trophoblast PAR-1 overexpression may promote placental angiogenesis and vascular remodeling, offering an alternative therapeutic approach for preeclampsia. PAR-1 is high-affinity receptor of thrombin. Thrombin increases sFlt-1 secretion in trophoblast via the activation of PAR-1. It is reported that the expression of both thrombin and PAR-1 expression are increased in placentas of preeclampsia patients compared with normal placentas. Trophoblast cells were transfected with PAR-1 short hairpin RNA (shRNA) or PAR-1 overexpression plasmids in vitro. Tube formation assays and a villus-decidua co-culture system were used to study the effect of PAR-1 inhibition on placental angiogenesis and vascular remodeling, respectively. Placentas from rats with preeclampsia were transfected with PAR-1 shRNA to confirm the effect of inhibiting PAR-1 overexpression in placenta. The trophoblast cell line HTR-8/SVneo was transfected with PAR-1 shRNA or PAR-1 overexpression plasmids. After 48 h, supernatant was collected and the level of sFlt-1 secretion was measured by ELISA. Human umbilical cord epithelial cells and a villus-decidua co-culture system were treated with conditioned media to study the effect of PAR-1 inhibition on tube formation and villi vascular remodeling. A preeclampsia rat model was established by intraperitoneal injection of L-NAME. Plasmids were injected into the placenta of the preeclampsia rats and systolic blood pressure was measured on Days 15 and 19. The effect of different treatments was evaluated by proteinuria, placental weights, fetal weights and fetal numbers in study and control groups. The level of serum sFlt-1 in rats with preeclampsia was also measured. Changes in the placenta microvessels were studied by histopathological staining. PAR-1 shRNA inhibited PAR-1 expression and significantly suppressed sFlt-1 expression in trophoblasts. Soluble Flt-1 level in the supernatant was suppressed by PAR-1 inhibition plasmid transfection and increased by PAR-1 overexpression plasmids (46.93 ± 5.22 vs. 25.21 ± 4.18 vs. 67.84 ± 3.58 ng/ml, P < 0.01). Tube formation assays showed that conditioned media from shPAR-1 transfected cells resulted in an increase in the total number of branching points compared with that of blank controls (P < 0.05). The villus-decidua co-culture system confirmed down-regulation of PAR-1 was conducive to angiogenesis and vascular remodeling. Transfecting placenta with PAR-1 shRNA plasmids improved placental vascular development and ameliorated the symptoms of preeclampsia in rats. After treatment with shRNA, blood pressure was controlled (140.83 ± 1.08 vs. 123.6 ± 1.47 mmHg, P < 0.001) and proteinuria levels were decreased (4.48 ± 0.36 vs. 2.64 ± 0.25 μg/μl, P < 0.01). sFlt-1 protein levels were significantly higher in preeclampsia group than in the control group (1.44 ± 0.33 vs. 2.92 ± 0.85 ng/ml, P < 0.001), but was reduced (0.92 ± 0.06 ng/ml, vs. PE, P < 0.001) in the treatment group. The histopathological changes of the placental microvessels showed that in the preeclampsia group, the number of blood vessels was reduced, while in treatment group, the placental microvasculature was improved (P < 0.001). N/A. Despite our promising results, the evaluation of kidney damage was studied only by proteinuria measurement. Histochemistry of kidney damage will be supplemented in a further study. The data showed that inhibition of trophoblast PAR-1 overexpression may promote placental angiogenesis and vascular remodeling, potentially offering an alternative therapeutic approach for preeclampsia. This work was supported by grants from the National Natural Science Foundation of China (Grant Nos. 81100442 and 81771605 for Y.Z. and 81179584 for L.Z.) and the Hubei Province Health and Family Planning Scientific Research Project (Grant No. WJ2017 M093 for Y.Z.). The authors declare that there is no conflict of interest.

Chymotrypsin Enhances Soluble Fms-Like Tyrosine Kinase 1 Production Through Protease-Activated Receptor 2 in Placenta-Derived Immortalized Human Trophoblast Cells

The production of soluble fms-like tyrosine kinase 1 (sFLT1) by exogenous chymotrypsin in trophoblast cells through protease-activated receptor (PAR) 2 was investigated to identify the role of a chymotrypsin-like serine protease in preeclampsia (PE) pathogenesis. We evaluated the expression of chymotrypsin, FLT1, and sFLT1 in monolayers of immortalized human trophoblast cells derived from placenta (TCL-1 cells). To investigate whether chymotrypsin enhances the production and release of sFLT1 through PAR-2, we examined changes in sFLT1 protein levels in conditioned medium by enzyme-linked immunosorbent assay and sFLT1 messenger RNA (mRNA) levels by real-time polymerase chain reaction in TCL-1 cells treated with exogenous chymotrypsin in the presence or absence of a PAR-2 antagonist or a chymotrypsin inhibitor (TPCK). We also examined changes in PAR-2 expression in TCL-1 cells treated with tumor necrosis factor (TNF) α in the presence or absence of a polyclonal anti-TNF-α antibody. Western blot analysis showed that TCL-1 trophoblast cells expressed chymotrypsin, FLT1, and sFLT1. Compared with the control cells, the sFLT1 level in the conditioned medium and sFLT1 mRNA level in cells were both significantly enhanced when treated with a PAR-2 agonist or chymotrypsin for 6 hours. In contrast, the sFLT1 level in the medium and sFLT1 mRNA level in cells treated with a PAR-2 agonist or chymotrypsin were suppressed in the presence of a PAR-2 antagonist or a chymotrypsin inhibitor. The PAR-2 expression was upregulated by TNF-α, which was suppressed in the presence of TNF-α antibodies. These results indicate that chymotrypsin-like serine protease enhances sFLT1 production through PAR-2 in trophoblast cells and thus plays an important additional role in PE pathogenesis.

63 Transcriptome wide profiling of mRNA 3′-ends in preeclampsia using PAS-SEQ

Maternal symptoms of preeclampsia (PE) are a primary consequence of excessive levels of sFlt1 protein in the maternal blood supply 1. sFlt1 is encoded by the gene Fms-related tyrosine kinase 1 (FLT1), which produces multiple protein isoforms: (1) transmembrane bound Flt1 (mFlt1), a tyrosine kinase receptor participating in vascular endothelial growth factor (VEGF) signaling; and (2) truncated and blood circulating (soluble) forms, sFlt1s, which lack the transmembrane anchoring domain and act as antagonists to mFlt1. Relative production of these isoforms results from polyadenylation sites within exon 30 (mFlt1) or within introns 13 and 14 (sFlt1s). The goals of this project are three-fold: (1) Determine whether increased sFlt1 production in PE results from an overall increase in FLT1 gene expression, from preferential use of intronic polyadenylation sites or both; (2) assess general gene expression changes in PE versus non-PE placentae; (3) determine if there is a global change in APA (alternative polyadenylation) site usage in PE. To address these goals we used a next-generation sequencing specifically targeting the 3′-ends of mRNAs: PolyAdenylation Site Sequencing (PAS-Seq). PAS-Seq allows for quantitative gene expression measurements as well as transcriptome-wide assessment of APA 2,3. Using total RNA extracted from the villous tissue of 6 normal (N) and 11 PE patient placentae, we generated and sequenced 17 PAS-Seq libraries. Among the PE patients, 8 were early onset (eoPE = delivery <34 weeks). By combining all counts from individual mRNA isoforms into a single measure (reads per million, RPM) for each gene and performing differential expression analysis (via DESeq2), we identified 288 up-regulated genes and 170 down-regulated genes in PE compared to N (padj ⩽0.05). Of these 458 differentially expressed genes, 200 (44%) overlap with the 1295 differentially expressed genes previously identified for severe PE upon meta-analysis of microarray data 4. However, only 15 (3%) of our set overlap with the 297 differentially expressed genes recently determined by RNA-Seq 5.Given the extreme heterogeneity of this disease [i.e., eoPE vs loPE; intrauterine growth restriction (IUGR) PE versus non-IUGR-PE] such a high degree of variability is perhaps unsurprising. Nonetheless, FLT1 was among the small set of differentially expressed genes common to all three datasets. In our PAS-Seq data, FLT1 was the most up-regulated gene (2.8-fold in all PE samples vs. N; 4.2-fold in eoPE vs N). Among all detectable APA isoforms, the four most abundant were: sFlt1-e15a > sFlt1-i13 long > sFlt1-i13 short > m-Flt1. All four isoforms were significantly higher (p ⩽0.05; t-test) in PE versus N; this suggests an overall increase in Flt1 gene transcription in PE. Along with this increased transcription, there also appears to be a switch toward more promoter-proximal sites. We are currently working to determine whether this switch to more promoter-proximal APA sites is a general phenomenon in PE placentae, or is solely a feature of the FLT1 gene. Our PAS-Seq data indicate that the FLT1 gene is subject to both transcriptional upregulation and a shift toward promoter-proximal polyadenylation in PE. Further data analysis regarding global APA switches and pathway analysis will be presented at the conference. R.J. Levine, et al., N. Engl. J. Med. 350, (2004) 672–683. P.J. Shepard, et al., RNA 17 (2011), 761–772. A. Derti et al., Gen. Res. 22 (2012), 1173–1183. K. Leavey, et al., PLoS One 10 (2015), e0116508. S. Sõber, et al., Sci. Rep. (2015), 1–17.

394. Baseline Data for Patients Participating in the Phase 2a rAAV.sFlt-1 Gene Therapy Trial for Exudative Age-Related Macular Degeneration

rAAV.sFlt-1 has the potential to provide long-term treatment of retinal neovascular diseases following a single injection by driving expression of sFlt-1, a potent (Kd 10 pM), naturally-occurring VEGF inhibitor. Here we present the baseline data for subjects with exudative age-related macular degeneration (AMD) enrolled in an investigator-sponsored, single center, Phase 2a safety study of rAAV.sFlt-1 gene therapy. Of the 32 randomized subjects, 21 were randomized to the rAAV.sFlt-1 group and 11 to the ranibizumab control group. All subjects were treated with two initial intravitreal injections of ranibizumab and then followed monthly for the need for rescue ranibizumab injections based on pre-specified criteria for evidence of active exudative AMD. Subjects underwent a thorough ophthalmic examination at baseline, and clinical laboratory assessments included blood biochemistry, complete blood count, and T-cell response. Anti-AAV antibodies (both total and neutralizing), AAV capsid protein, and sFlt-1 protein levels were also measured. Baseline demographics were 100% Caucasian, 59% female, and average age 79+7 years. All subjects had active subfoveal choroidal neovascularization secondary to exudative AMD, and the average visual acuity was 59+15 EDTRS letters (Snellen equivalent 20/63). Subjects had received an average of 10 anti-VEGF injections prior to enrolment. Anti-AAV antibody results indicated that 25% of subjects serum contained neutralizing antibody titres 1/100-1/500 while 19% had titres 1/20-1/100. All subjects tested negative for presence of AAV capsid in fluids at baseline. sFlt-1 protein averaged 13.9+5.4 pg/ml in urine, 116+5.9 pg/ml in serum, and 229+12.7 pg/ml in saliva. One patient had IgM deficiency and 6/32 patients had lymphopenia or T cell deficiency and/or CD8 lymphopenia. No other immune defects that might pose a hazard to the use of the rAAV vector were identified. In conclusion, the study subjects had characteristics representative of a population with exudative AMD and are appropriate for enrollment in a safety study for rAAV.sFlt-1.

Preclinical safety evaluation of subretinal AAV2.sFlt-1 in non-human primates

We report on the long-term safety of AAV2.sFlt-1 (a recombinant adeno-associated virus serotype 2 carrying the soluble form of the Flt-1 receptor) injection into the subretinal space of non-human primates. Levels of sFlt-1 protein were significantly higher (P<0.05) in the vitreous of four out of five AAV2.sFlt-1-injected eyes. There was no evidence of damage to the eyes of animals that received subretinal injections of AAV2.sFlt-1; ocular examination showed no anterior chamber flare, normal fundus and electroretinography responses equivalent to those observed before treatment. Notably, immunological analysis demonstrated that gene therapy involving subretinal injection of AAV2.sFlt-1 does not elicit cell-mediated immunity. Biodistribution analysis showed that AAV2.sFlt-1 could be detected only in the eye and not in the other organs tested. These data indicate that gene therapy with subretinal AAV2.sFlt-1 is safe and well tolerated, and therefore promising for the long-term treatment of neovascular diseases of the eye.

Expression of Placental FLT1 Transcript Variants Relates to Both Gestational Hypertensive Disease and Fetal Growth

The recent discovery of additional alternative spliced FLT1 transcripts encoding novel soluble (s)FLT1 protein isoforms complicates both the predictive value and functional implications of sFLT1 in preeclampsia. We investigated FLT1 expression levels and splicing patterns in placentas of normotensive and preeclamptic women, and established the tissue specificity of all FLT1 transcript variants. mRNA levels of sFLT1 splice variants were determined by real-time polymerase chain reaction in 21 normal human tissues and placental biopsies from 91 normotensive and 55 preeclamptic women. Cellular localization of placental FLT1 expression was established by RNA in situ hybridization. Of all tissues investigated, placenta has by far the highest FLT1 mRNA expression level, mainly localized in the syncytiotrophoblast layer. More than 80% of placental transcripts correspond to sFLT1_v2. Compared with normotensive placenta, preeclamptic placenta has ≈3-fold higher expression of all FLT1 transcript variants (P<0.001), with a slight shift in favor of sFLT1_v1. Although to a lesser degree, transcript levels are also increased in placenta from normotensive women that deliver a small for gestational age neonate. We conclude that sFLT isoform-specific assays could potentially improve the accuracy of current sFLT1 assays for the prediction of preeclampsia. However, placental FLT1 transcript levels are increased not only in preeclampsia but also in normotensive pregnancy with a small for gestational age fetus. This may indicate a common pathway involved in the development of both conditions but complicates the use of circulating sFLT1 protein levels for the prediction or diagnosis of preeclampsia alone.

RAAV.sFlt-1 Gene Therapy Achieves Lasting Reversal of Retinal Neovascularization in the Absence of a Strong Immune Response to the Viral Vector

To determine the efficacy of rAAV.sFlt-1-mediated gene therapy in a transgenic mouse model of retinal neovascularization (trVEGF029) and to assess whether rAAV.sFlt-1 administration generated any deleterious, long-lasting immune response that could affect efficacy. trVEGF029 mice were injected subretinally with rAAV.sFlt-1 or phosphate-buffered saline. Fluorescein angiography and electroretinography were used to compare the extent of fluorescein leakage from retinal vessels and retinal function, respectively. A group of eyes was enucleated, and the retinal vasculature and morphology were studied by confocal and light microscopy. Cells were isolated from the posterior eyecups and spleens of a further group, and immune cell subset populations were investigated by flow cytometry. sFlt-1 protein levels in the eyes were evaluated by ELISA. After a single rAAV.sFlt-1 injection, sFlt-1 protein levels were upregulated, and there was a reduction in fluorescein leakage from the retinal vessels and an improvement in retinal function. Confocal microscopy of isolectin-IB4-labeled retinal wholemounts showed more normal-appearing capillary beds in rAAV.sFlt-1-injected than in PBS-injected trVEGF029 mouse eyes. Light microscopy demonstrated retinal morphology preservation, with fewer aberrant vessels invading the outer nuclear layer of rAAV.sFlt-1-injected eyes. Furthermore, the immune response to subretinal injection of rAAV.sFlt-1 was limited to a transient increase in CD45(+) leukocytes that disappeared by 4 weeks after injection. This transient increase was localized to the eye and did not affect long-term therapeutic efficacy. The data support the notion that rAAV.sFlt-1 gene therapy is safe and effective for the long-term inhibition of deleterious blood vessel growth in the eye.

Extra-placental Expression of Vascular Endothelial Growth Factor Receptor-1, (Flt-1) and Soluble Flt-1 (sFlt-1), by Peripheral Blood Mononuclear Cells (PBMCs) in Normotensive and Preeclamptic Pregnant Women

The soluble VEGF receptor, sFlt-1 (otherwise referred to as sVEGFR-1), has been implicated in the pathogenesis of preeclampsia. The preeclamptic placenta has been previously demonstrated to produce high levels of the soluble VEGF receptor. Here we tested the hypothesis that peripheral blood mononuclear cells (PBMCs) may also represent an additional source for circulating sFlt-1 during normal and preeclamptic pregnancies. We first demonstrate that preeclamptic placentae show five-fold increased Flt-1 and sFlt-1 mRNA levels. We also show that the Flt-1 and sFlt-1 levels are eight-fold higher in preeclamptic placentae if we collect biopsies without rinsing them in saline to remove excess blood. Cultured villous explants from women with preeclampsia failed to show the increased amount of Flt-1 and sFlt-1 mRNA that was observed in the placental biopsies of normal pregnancy and preeclampsia. Under normoxic conditions the Flt-1 and sFlt-1 mRNA levels in the explants were 3.11+/-0.6 fold in normal pregnancy and 3.6+/-0.4 fold in women with preeclampsia (p = NS by ANOVA). However, the same villous explants showed hypoxic induction of Flt-1 mRNA (NP 3.96+/-0.4 fold, p = NS and PE 5.24+/-0.6 fold, p < 0.05 by ANOVA). We analyzed Flt-1 and sFlt-1 protein levels in the peripheral blood mononuclear cells (PBMCs) to analyze the possibility of an extra-placental sFlt-1 source. Our results indicate that PBMCs of pregnant women are capable of expressing variable amounts of Flt-1 proteins. PBMCs from pregnant women exposed to hypoxia show up-regulation of HIF-1alpha and Flt-1 proteins. PBMCs obtained from women with preeclampsia (n = 9) produced significantly higher amounts of sFlt-1 under normal tissue culture conditions (104.6+/-14.3 pg/ml vs. 46.23+/-5.03 pg/ml, p < 0.05 by ANOVA) and much higher concentrations under hypoxia (196.74+/-26.3pg/ml vs. 83.3+/-13.6pg/ml, p < 0.05 by ANOVA) than PBMCs from normal pregnant women (n = 11). Moreover, analysis of PBMCs from a different group of women with a history of preeclampsia showed persistent abnormality of Flt-1 women one year post-partum. The present study indicates that Flt-1 dysregulation in PBMCs of pregnant women resulting in over-expression of sFlt-1 could be an additional (extra-placental) source of sFlt-1 that contributes to the pathogenesis of preeclampsia.