Eucommia granules activate Wnt/ß-catenin pathway, and improve oxidative stress, inflammation, and endothelial injury in preeclampsia rats.

PURPOSE: Pre-eclampsia (PE) is a pregnancy-related complication. Eucommia is effective in the treatment of hypertensive disorders in pregnancy, but the specific effects and possible mechanisms of Eucommia granules (EG) in PE remain unknown. The aim of this study was to investigate the effects and possible mechanisms of EG in PE rats. METHODS: Pregnant Sprague Dawley rats were divided into five groups (n = 6): the control group, the model group, the low-dose group, the medium-dose group, and the high-dose group of EG. The PE model was established by subcutaneous injection of levonitroarginine methyl ester. Saline was given to the blank and model groups, and the Eucommia granules were given by gavage to the remaining groups. Blood pressure and urinary protein were detected. The body length and weight of the pups and the weight of the placenta were recorded. Superoxide dismutase (SOD) activity and levels of malondialdehyde (MDA), placental growth factor (PIGF), and soluble vascular endothelial growth factor receptor-1 (sFIt-1) were measured in the placenta. Pathological changes were observed by hematoxylin-eosin staining. Wnt/ß-catenin pathway-related protein expression was detected using Western blot. RESULTS: Compared with the model group, the PE rats treated with EG had lower blood pressure and urinary protein. The length and weight of the pups and placental weight were increased. Inflammation and necrosis in the placental tissue was improved. SOD level increased, MDA content and sFIt-1/PIGF ratio decreased, and Wnt/ß-catenin pathway-related protein expression level increased. Moreover, the results of EG on PE rats increased with higher doses of EG. CONCLUSIONS: EG may activate the Wnt/ß-catenin pathway and inhibit oxidative stress, inflammation, and vascular endothelial injury in PE rats, thereby improving the perinatal prognosis of preeclamptic rats. EG may inhibit oxidative stress, inflammation, and vascular endothelial injury through activation of the Wnt/ß-catenin pathway in preeclampsia rats, thereby improving perinatal outcomes in PE rats.

Feeding rumen-protected lysine prepartum alters placental metabolism at a transcriptional level.

Rumen-protected Lys (RPL) fed to Holstein cows prepartum resulted in a greater intake and improved health of their calves during the first 6 wk of life. However, whether increased supply of Lys in late gestation can influence placental tissue and, if so, which pathways are affected remain to be investigated. Therefore, we hypothesize that feeding RPL during late gestation could modulate placental metabolism, allowing for improved passage of nutrients to the fetus and thus influencing the offspring development. Therefore, we aimed to determine the effects of feeding RPL (AjiPro-L Generation 3, Ajinomoto Health and Nutrition North America) prepartum (0.54% DM of TMR) on mRNA gene expression profiles of placental samples of Holstein cows. Seventy multiparous Holstein cows were randomly assigned to 1 of 2 dietary treatments, consisting of TMR top-dressed with RPL (PRE-L) or without (control, CON), fed from 27 ± 5 d prepartum until calving. After natural delivery (6.87 ± 3.32 h), placentas were rinsed with physiological saline (0.9% sodium chloride solution) to clean any dirtiness from the environment and weighed. Then, 3 placentomes were collected, one from each placental region (cranial, central, and caudal), combined and flash-frozen in liquid nitrogen to evaluate the expression of transcripts and proteins related to protein metabolism and inflammation. Placental weights did not differ from cows in PRE-L (15.5 ± 4.03 kg) and cows in CON (14.5 ± 4.03 kg). Feeding RPL prepartum downregulated the expression of NOS3 (nitric oxide synthase 3), involved in vasodilation processes, and SOD1, which encodes the enzyme superoxide dismutase, involved in oxidative stress processes. Additionally, feeding RPL prepartum upregulated the expression of transcripts involved in energy metabolism (SLC2A3, glucose transporter 3; and PCK1, phosphoenolpyruvate carboxykinase 1), placental metabolism and cell proliferation (FGF2, fibroblast growth factor 2; FGF2R, fibroblast growth factor 2 receptor; and PGF, placental growth factor), Met metabolism (MAT2A, methionine adenosyltransferase 2-α), and tended to upregulate IGF2R (insulin-like growth factor 2 receptor). Placental FGF2 and LRP1 (low-density lipoprotein receptor-related protein 1) protein abundance were greater for cows that received RPL prepartum than cows in CON. In conclusion, feeding RPL to prepartum dairy cows altered uteroplacental expression of genes and proteins involved in cell proliferation, and in metabolism and transport of glucose. Such changes are illustrated by increased expression of SLC2A3 and PCK1 and increased protein abundance of FGF2 and LRP1 in uteroplacental tissue of cows consuming RPL.

Pathophysiology of Preeclampsia and L-Arginine/L-Citrulline Supplementation as a Potential Strategy to Improve Birth Outcomes.

In preeclampsia, the shallow invasion of cytotrophoblast cells to uterine spiral arteries, leading to a reduction in placental blood flow, is associated with an imbalance of proangiogenic/antiangiogenic factors to impaired nitric oxide (NO) production. Proangiogenic factors, such as vascular endothelial growth factor (VEGF) and placental growth factor (PlGF), require NO to induce angiogenesis through antioxidant regulation mechanisms. At the same time, there are increases in antiangiogenic factors in preeclampsia, such as soluble fms-like tyrosine kinase type 1 receptor (sFIt1) and toll-like receptor 9 (TLR9), which are mechanism derivates in the reduction of NO bioavailability and oxidative stress in placenta.Different strategies have been proposed to prevent or alleviate the detrimental effects of preeclampsia. However, the only intervention to avoid the severe consequences of the disease is the interruption of pregnancy. In this scenario, different approaches have been analysed to treat preeclamptic pregnant women safely. The supplementation with amino acids is one of them, especially those associated with NO synthesis. In this review, we discuss emerging concepts in the pathogenesis of preeclampsia to highlight L-arginine and L-citrulline supplementation as potential strategies to improve birth outcomes. Clinical and experimental data concerning L-arginine and L-citrulline supplementation have shown benefits in improving NO availability in the placenta and uterine-placental circulation, prolonging pregnancy in patients with gestational hypertension and decreasing maternal blood pressure.

Zinc promotes cell proliferation via regulating metal-regulatory transcription factor 1 expression and transcriptional activity in pulmonary arterial hypertension.

Metal responsive transcription factor 1 (MTF-1) is a zinc-dependent transcription factor involved in the development of pulmonary arterial hypertension (PAH), which is a life-threatening disease characterized by elevated pulmonary artery pressure and pulmonary vascular remodeling. However, little is known about the role and regulatory signaling of MTF-1 in PAH. This study aimed to investigate the effect and mechanism of MTF-1 on the proliferation of pulmonary arterial smooth muscle cells (PASMCs). Several techniques including intracellular-free zinc detected by fluorescent indicator-fluozinc-3-AM, western blot, luciferase reporter, and cell proliferation assay were conducted to perform a comprehensive analysis of MTF-1 in proliferation of PASMCs in PAH. Increased cytosolic zinc was shown in monocrotaline (MCT)-PASMCs and ZnSO4-treated PASMCs, which led to overexpression and overactivation of MTF-1, followed by the up-regulation of placental growth factor (PlGF). Elevated MTF-1 and PlGF were observed in western blot, and high transcriptional activity of MTF-1 was confirmed by luciferase reporter in ZnSO4-treated cells. Further investigation of cell proliferation revealed a favorable impact of zinc ions on PASMCs proliferation, with the deletion of Mtf-1/Plgf attenuating ZnSO4-induced proliferation. Flow cytometry analysis showed that blockade of PKC signaling inhibited the cell cycle of MCT-PASMCs and ZnSO4-treated PASMCs. The Zinc/PKC/MTF-1/PlGF pathway is involved in the up-regulatory effect on the PASMCs proliferation in the process of PAH. This study provided novel insight into zinc homeostasis in the pathogenesis of PAHs, and the regulation of MTF-1 might be a potential target for therapeutic intervention in PAH.

Empagliflozin Induced Ketosis, Upregulated IGF-1/Insulin Receptors and the Canonical Insulin Signaling Pathway in Neurons, and Decreased the Excitatory Neurotransmitter Glutamate in the Brain of Non-Diabetics.

Sodium-glucose cotransporter-2 inhibitors (SGLT2is), such as empagliflozin, lower blood glucose in type 2 diabetes mellitus and improve cardiorenal outcomes regardless of diabetes presence. Whether SGLT2is exert any effects on the brain's metabolism has not been studied. We conducted a single-arm clinical trial to investigate the effects of once daily administration of oral empagliflozin (25 mg) for 14 days on systemic and brain metabolism in 21 non-diabetics aged 55 years old or older. Empagliflozin lowered circulating insulin and elevated ß-hydroxybutyrate over 34-h periods, both following its first administration and after 14 days of daily administration, with minor alterations in glucose homeostasis. Levels of phosphorylated insulin-like growth factor-1 receptor (pIGF-1R), phosphorylated insulin receptor (pIR), phosphorylated-in-tyrosine insulin receptor substrate-1 (pY-IRS-1), and phosphorylated protein kinase B or AKT (pAKT) were increased in extracellular vesicles enriched for neuronal origin (NEVs) following the first empagliflozin administration, but not after 14 days. Our finding of IGF-1R upregulation in NEVs is promising because several post-mortem and epidemiological studies support the idea that upregulation of IGF signaling may protect against Alzheimer's disease (AD). Moreover, our finding showing activation of insulin signaling and, in particular, the canonical pathway (pIR, pY-IRS-1, pAKT) in NEVs is important because such changes have been repeatedly associated with neuronal survival. Using brain magnetic resonance spectroscopy (MRS), we detected decreased concentrations of the excitatory neurotransmitter glutamate and its precursor glutamine after empagliflozin administration. This finding is also encouraging since glutamatergic excitotoxicity has long been implicated in AD pathology. Overall, our findings may motivate the repurposing of SGLT2is for use in AD and other, related diseases that are characterized by downregulation of IGF-1/insulin signaling in neurons and excitotoxicity.

The Potential for Placental Activation of PPARγ to Improve the Angiogenic Profile in Preeclampsia.

Preeclampsia (PE) is one of the most common causes of maternal-fetal morbidity and mortality world-wide. While the underlying causes of PE remain elusive, aberrant trophoblast differentiation and function are thought to cause an imbalance of secreted angiogenic proteins resulting in systemic endothelial dysfunction and organ damage in the mother. The placental dysfunction is also characterized by a reduction of the transcription factor, peroxisome proliferator activated receptor γ (PPARγ) which normally promotes trophoblast differentiation and healthy placental function. This study aimed to understand how placental activation of PPARγ effects the secretion of angiogenic proteins and subsequently endothelial function. To study this, healthy and PE placental tissues were cultured with or without the PPARγ agonist, Rosiglitazone, and a Luminex assay was performed to measure secreted proteins from the placenta. To assess the angiogenic effects of placental activation of PPARγ, human umbilical vein endothelial cells (HUVECs) were cultured with the placental conditioned media and the net angiogenic potential of these cells was measured by a tube formation assay. This is the first study to show PPARγ's beneficial effect on the angiogenic profile in the human preeclamptic placenta through the reduction of anti-angiogenic angiopoietin-2 and soluble endoglin and the upregulation of pro-angiogenic placental growth factor, fibroblast growth factor-2, heparin-binding epidermal growth factor, and follistatin. The changes in the angiogenic profile were supported by the increased angiogenic potential observed in the HUVECs when cultured with conditioned media from rosiglitazone-treated preeclamptic placentas. The restoration of these disrupted pathways by activation of PPARγ in the preeclamptic placenta offers potential to improve placental and endothelial function in PE.

Endothelium dysfunction in hind limb arteries of male Zucker Diabetic-Sprague Dawley rats.

Endothelium dysfunction produces peripheral vascular disease comorbidities in type 2 diabetes, including hypertension, and critical limb ischemia. In this study we aimed to test endothelial dysfunction, the vasodilator effects of a proteinase-activated receptor 2 (PAR2) agonist (2fLIGRLO), and thromboxane A2 synthase inhibitor (ozagrel) on PAR2 vasodilation in hind limb arteries ex vivo, using Zucker Diabetic-Sprague Dawley (ZDSD) rats, a model of type 2 diabetes. Male Sprague Dawley rats (SD) and ZDSD were fed a high-fat content 'Western diet' from 16 to 20 weeks of age (wks) then fed a standard laboratory diet. We identified diabetic ZDSD rats by two consecutive blood glucose measurements > 12.5 mM, based on weekly monitoring. We used acetylcholine, 2fLIGRLO, and nitroprusside with wire-myograph methods to compare relaxations of femoral, and saphenous arteries from diabetic ZDSD (21-23 wks) to age-matched normoglycemic SD. All arteries showed evidence of endothelium dysfunction using acetylcholine (reduced maximum relaxations, reduced sensitivity), and higher sensitivities to 2fLIGRLO, and nitroprusside in ZDSD vs SD. Ozagrel treatment of ZDSD distal segments, and end-branches of saphenous arteries decreased their sensitivities to 2fLIGRLO. We tested aortas for altered expression of endothelium-specific gene targets using PCR array and qPCR. PAR2, and placental growth factor gene transcripts were 1.5, and 4-times higher in ZDSD than SD aortas. Hind limb arteries of ZDSD exhibit endothelium dysfunction having less GPCR agonist induced vasodilation by endothelial NO-release. Different expression of several endothelial genes in ZDSD vs SD aortas, including PAR2, suggests altered inflammatory, and angiogenesis signaling pathways in the endothelium of ZDSD.

The effect of genistein on IGF-1, PlGF, sFLT-1 and fetoplacental development.

The mechanisms by which genistein, a phytoestrogen, affects fetoplacental development adversely are still poorly understood. It is reported that genistein ingestion modulates thyroid functions, leptin hormone, C-reactive protein, and thyroxin kinase activities. In this study, we evaluated changes in serum and placental insulin-like growth factor-I (IGF-1), placental growth factor (PIGF), and soluble fms-like tyrosine kinase-1 (sFLT-1) in pregnant rats exposed to genistein using ELISA. According to the treatments, Rats were divided into control, 2 mg genistein, and 4 mg genistein groups. Genistein groups were administered with the doses orally from gestational day (GD) one onwards until sacrifice, while the control group received an equal volume of distilled water the vehicle. At GD-12, GD-16, and GD-20, serum samples and placenta homogenates were prepared from maternal blood samples and the placenta and were analysed to determine the concentration of IGF-1, sFLT-1, and PIGF. Serum IGF-1 and PIGF were both increased in all genistein groups at GD-12 and GD-16, and at GD-20 in the 4 mg group. However, serum IGF-1and PIGF levels were decreased in the placenta from all genistein groups at GD-20. Placenta sFLT-1 levels increased at both GD-16 and GD-20 in genistein-treated rat serum. An initial decrease in placental sFLT-1 at GD-12 was followed by an increase at GD-16 and finally a decrease at GD-20 in all genistein-treated rats. The sFL-1/PlGF ratio in placenta samples of genistein-exposed rats was decreased at GD-16 and increased at GD-20, while the reverse was recorded in the serum sample at the same gestational periods. The fetoplacental growth disruption mechanism of genistein can be partly explained by its interference with placental growth factor signalling.

Maternal cadmium exposure impairs placental angiogenesis in preeclampsia through disturbing thyroid hormone receptor signaling.

Cadmium is a ubiquitous environmental pollutant, which can increase the risk of preeclampsia. This study was designed to determine the mechanism of cadmium exposure during pregnancy impaired placental angiogenesis that was associated with the occurrence of preeclampsia. The effects of cadmium exposure on placental thyroid hormone receptor signaling were explored. JEG3 cells were treated with CdCl2 (20 µM) and the Dio2 inhibitor, IOP (100 µM). Cadmium levels in maternal blood and placentae were increased in preeclampsia group. Placental angiogenesis of preeclampsia was decreased with decreased expression of PLGF and VEGF and increased expression of sFlt1. Meanwhile, the expression and nuclear translocation of thyroid hormone receptor α were decreased in preeclampsia placenta, as well as the expression of Dio2, but not the expression and nuclear translocation of thyroid hormone receptor ß. Furthermore, we found that cadmium exposure downregulated the expression of thyroid hormone receptor α and Dio2, but not the expression of thyroid hormone receptor ß in JEG3 cells. Also, we found that cadmium exposure decreased the expression of PLGF and VEGF and increased the expression of sFlt1 in JEG3 cells. IOP pretreatment decreased the expression of PLGF and increased the expression of sFlt1. In conclusion, our results elucidated that cadmium exposure would impair placental angiogenesis in preeclampsia through disturbing thyroid hormone receptor signaling.

CYT387 Inhibits the Hyperproliferative Potential of Fibroblast-like Synoviocytes via Modulation of IL-6/JAK1/STAT3 Signaling in Rheumatoid Arthritis.

Fibroblast-like synoviocytes (FLS) are the critical effector cells primarily involved in rheumatoid arthritis (RA) disease pathogenesis. Interleukin (IL)-6, a proinflammatory cytokine most abundantly expressed in the rheumatoid synovium, promotes Janus kinase (JAK)/signal transducer and transcriptional activator (STAT) signaling cascade activation in RA-FLS, thus leading to its aggressive phenotype, invasiveness, and joint destruction. Momelotinib (CYT387) is a selective small-molecule inhibitor of JAK1/2 and is clinically approved to treat myelofibrosis. However, the therapeutic efficacy of CYT387 in FLS mediated RA pathogenesis is less known. In the present study, we investigated the modulatory effect of CYT387 on IL6/JAK/STAT signaling cascade in FLS induced RA pathogenesis. CYT387 treatment inhibited IL-6 induced high proliferative and migratory potential of FLS cells isolated from adjuvant-induced arthritic (AA) rats. CYT387 reduced the expression of PRMT5, survivin, and HIF-1α mediated by IL-6/sIL-6R in AA-FLS in a dose-dependent manner. The IL-6/sIL-6R induced expression of angiogenic factors such as VEGF and PIGF in AA-FLS cells was found downregulated by CYT387 treatment. Importantly, CYT387 significantly reduced IL-6/sIL-6R dependent activation of JAK1 and STAT3 and increased SOCS3 expression in AA-FLS cells. Next, the S3I-201 mediated blockade of STAT3 activation supported the inhibitory effect of CYT387 on IL-6/JAK1/STAT3 signaling cascade in AA-FLS. Overall, this study proves that CYT387 inhibits proliferation, migration, and pathogenic disease potential of FLS isolated from adjuvant-induced arthritic (AA) rats via targeting IL-6/JAK1/STAT3 signaling cascade.

In-silico Screening of Phytoconstituents on Wound Healing Targets - Approaches and Current Status.

Over recent years, there has been tremendous research focused on the effective utilization of natural products in wound management. Natural or herbal products contain several phytoconstituents that may act on various stages in wound healing and thereby provide a multi-targeted approach especially in the treatment of chronic wounds. Currently, attempts have been made to screen the phytoconstituents present in herbs on various targets involved in wound healing. This review includes a systematic evaluation of scientific reports by various groups of researchers on the herbals evaluated for wound management, their phytochemical profiling, pre-clinical studies, and molecular modeling studies. Various wound targets discussed include Interleukin-1, Interleukin-6, Tumor necrosis factor-α (TNF-α), Thymosin beta-4 (Tß-4) that regulate the early inflammatory stage and the novel T cell immune response cDNA 7(TIRC7) that regulates angiogenesis. Also, neuropeptides P and Y act on the inflammatory, migratory, and proliferation phases, and growth factors like vascular endothelial growth factor family (VEGF) and placental growth factor family (PGF) are involved in angiogenesis, while the role of Fibroblast growth factor in tissue remodeling is discussed. As many of the natural products include polyherbal systems, this approach can help in the judicious selection of a combination of herbs that will act on multiple targets in the wound healing process and provide a multi-factorial approach in wound management.

PlGF Reduction Compromises Angiogenesis in Diabetic Foot Disease Through Macrophages.

Diabetic foot disease (DFD) is a common and serious complication for diabetes and is characterized with impaired angiogenesis. In addition to the well-defined role of vascular endothelial growth factor (VEGF) -A and its defect in the pathogenesis of DFD, another VEGF family member, placental growth factor (PlGF), was also recently found to alter expression pattern in the DFD patients with undetermined mechanisms. This question was thus addressed in the current study. We detected attenuated PlGF upregulation in a mouse DFD model. In addition, the major cell types at the wound to express the unique PlGF receptor, VEGF receptor 1 (VEGFR1), were macrophages and endothelial cells. To assess how PlGF regulates DFD-associated angiogenesis, we injected recombinant PlGF and depleted VEGF1R specifically in macrophages by local injection of an adeno-associated virus (AAV) carrying siRNA for VEGFR1 under a macrophage-specific CD68 promoter. We found that the angiogenesis and recovery of the DFD were both improved by PlGF injection. The PlGF-induced improvement in angiogenesis and the recovery of skin injury were largely attenuated by macrophage-specific depletion of VEGF1R, likely resulting from reduced macrophage number and reduced M2 polarization. Together, our data suggest that reduced PlGF compromises angiogenesis in DFD at least partially through macrophages.

RNA-Seq reveals placental growth factor regulates the human retinal endothelial cell barrier integrity by transforming growth factor (TGF-ß) signaling.

Placental growth factor (PlGF or PGF) is a member of the VEGF (vascular endothelial growth factor) family. It plays a pathological role in inflammation, vascular permeability, and pathological angiogenesis. The molecular signaling by which PlGF mediates its effects in non-proliferative diabetic retinopathy (DR) remains elusive. This study aims to characterize the transcriptome changes of human retinal endothelial cells (HRECs) with the presence and the absence of PlGF signaling. Primary HRECs were treated with the PlGF antibody (ab) to block its activity. The total RNA was isolated and subjected to deep sequencing to quantify the transcripts and their changes in both groups. We performed transcriptome-wide analysis, gene ontology, pathway enrichment, and gene-gene network analyses. The results showed that a total of 3760 genes were significantly differentially expressed and were categorized into cell adhesion molecules, cell junction proteins, chaperone, calcium-binding proteins, and membrane traffic proteins. Functional pathway analyses revealed that the TGF-ß pathway, pentose phosphate pathway, and cell adhesion pathway play pivotal roles in the blood-retina barrier and antioxidant defense system. Collectively, the data provide new insights into the molecular mechanisms of PlGF's biological functions in HRECs relevant to DR and diabetic macular edema (DME). The newly identified genes and pathways may act as disease markers and target molecules for therapeutic interventions for the patients with DR and DME refractory to the current anti-VEGF therapy.

Administration of recombinant human placental growth factor decreases blood pressure in obese hypertensive pregnant rats.

OBJECTIVES: Although epidemiological studies have shown that obesity is associated with increased incidence of hypertension during pregnancy, the mechanisms linking these two comorbidities are not as well studied. Previous investigations detected lower levels of the anti-hypertensive and pregnancy-related factor, placental growth factor (PlGF), in obese hypertensive pregnancies. Therefore, we examined whether obese hypertensive pregnant rats have reduced PlGF and whether increasing its levels by administering recombinant human (rh)PlGF reduces their blood pressure. METHODS: We utilized a genetic model of obesity characterized to be heavier, hypertensive and fertile, namely rats having heterozygous deficiency of the melanocortin-4 receptor (MC4R-def). RESULTS: MC4R-def obese rats had lower circulating levels of PlGF than wild-type lean controls at gestational day 19. Also, assessment of the PlGF receptor, Flt-1, in the vasculature showed that its levels were reduced in aorta and kidney glomeruli but increased in small mesenteric arteries. Chronic intraperitoneal administration of rhPlGF from gestational day 13-19 significantly increased circulating PlGF levels in both obese and lean rats, but reduced blood pressure only in the obese pregnant group. The rhPlGF treatment did not alter maternal body and fat masses or circulating levels of the adipokines, leptin and adiponectin. In addition, this treatment did not impact average foetal weights but increased placental weights regardless of obese or lean pregnancy. CONCLUSION: PlGF is reduced in MC4R-def obese hypertensive pregnant rats, which is similar to findings in obese hypertensive pregnant women, while increasing its levels with exogenous rhPlGF reduces their blood pressure.

Effect of Placental Growth Factor on Trophoblast-Endothelial Cell Interactions In Vitro.

Placental growth factor (PlGF) is an important angiogenic factor which has an emerging role in the clinical management of suspected preeclampsia. The role of PlGF in normal placental development is not completely understood and it is uncertain whether PlGF influences trophoblast and endothelial cell interactions central to uterine spiral artery remodelling, especially in variable oxygen conditions. A two-cell model of endovascular invasion was used. Tissue culture plates were coated with Matrigel™, on which fluorescent-labelled uterine microvascular endothelial cells (1 × 105/well) and HTR8/SVNeo cells were co-cultured (1 × 105/well) for 20 h. Co-cultures were treated with recombinant human PlGF (rhPlGF) (10 or 100 ng/mL) and incubated at either 21% O2 or 2% O2. Images were captured by fluorescence microscopy and analysed using ImageJ (n = 7). Data was analysed using SPSSv24. Treatment with rhPlGF did not improve integration in co-cultures irrespective of oxygen conditions but increased proliferation in 2% O2 of both trophoblast and endothelial cells. Expression of angiogenic factors VEGF, sFLT-1, PlGF and CXCL12 in both co-cultures and in isolated trophoblast cells was not altered by rhPlGF treatment. Expression of TLR-3 mRNA in co-cultures was increased by rhPlGF 100 ng/mL at 21% O2 (p = 0.03). PlGF contributes to trophoblast and endothelial cell proliferation in the setting of physiological hypoxia but does not influence trophoblast and endothelial cell interactions in an in vitro model of spiral artery remodelling. Upregulation of TLR-3 expression in co-cultures may indicate a role for PlGF in the placental inflammatory response.

The novel axis of YAP1, transcription enhancer factor 3 and Down Syndrome Candidate Region 1 isoform 1L is a common signaling pathway downstream of several angiogenic factors.

Angiogenesis is a hallmark of many diseases. Previously, we found that Down Syndrome Candidate Region 1 Isoform 1L (DSCR1-1L) was expressed in human tumor vessels, but was not detectable in normal tissues, and played important roles in angiogenesis induced by vascular endothelial growth factor (VEGF-A165). The expressions of DSCR1-1L mRNA and protein induced by VEGF-A165 were regulated via the direct interaction of transcription enhancer factor 3 (TEF3) with DSCR1-1L promoter. However, the function and the regulation of DSCR1-1L in angiogenesis had not been completely understood. In this study, we found that the expressions of DSCR1-1L mRNA and proteins were upregulated by other angiogenic factors, including VEGF-A121, VEGF-E, histamine, PAF, the endothelial cell (EC) growth medium, and the conditional medium obtained from cancer cells, but not by PlGF, bFGF, PDGF, and serotonin. The EC proliferation, migration and elongation induced by histamine and EC growth medium were inhibited by knocking down the mRNA and protein expressions of DSCR1-1L and TEF3. The TEF3 activation was regulated by its interaction with YAP1, and translocation from cytosol to nuclei, but not by increase of protein expression, after the stimulation of VEGF, histamine and EC growth medium. YAP1 regulated the protein expression of DSCR1-1L, the proliferation, migration and elongation of ECs induced by VEGF, histamine and EC growth medium. Taken together, this study identified a novel axis of YAP1, TEF3 and DSCR1-1L that was a common signaling pathway downstream of several angiogenic factors to regulate angiogenesis, suggesting that this pathway is an excellent therapeutic target for angiogenic diseases and cancers. Our results contribute significantly to the field of mechanistic studies.

Treatment with placental growth factor attenuates myocardial ischemia/reperfusion injury.

Studies have established that oxidative stress plays an important role in the pathology of myocardial ischemia/reperfusion injury (MIRI). Vascular endothelial growth factor receptor 1 (VEGFR1) activation was reported to reduce oxidative stress and apoptosis. In the present study, we tested the hypothesis that the activation of VEGFR1 by placental growth factor (PlGF) could reduce MIRI by regulating oxidative stress. Mouse hearts and neonatal mouse cardiomyocytes were subjected to ischemia/reperfusion (I/R) and oxygen glucose deprivation (OGD), respectively. PlGF pretreatment markedly ameliorated I/R injury, as demonstrated by reduced infarct size and improved cardiac function. The protection was associated with a reduction of cardiomyocyte apoptosis. Similarly, our in vitro study showed that PlGF treatment improved cell viability and reduced cardiomyocyte apoptosis. Also, activation of VEGFR1 by PlGF suppressed intracellular and mitochondrial reactive oxygen species (ROS) generation. However, VEGFR1 neutralizing monoclonal antibody, which preventing PlGF binding, totally blocked this protective effect. In conclusion, activation of VEGFR1 could protect heart from I/R injury by suppression of oxidative stress and apoptosis.

Placental growth factor promotes epithelial-mesenchymal transition-like changes in ARPE-19 cells under hypoxia.

Purpose: To investigate the role of placental growth factor (PGF) in the epithelial-mesenchymal transition (EMT) of ARPE-19 cells under hypoxia, and whether the NF-κB signaling pathway is involved in this process. Methods: ARPE-19 cells were treated in five groups: a control group, hypoxia group, PGF group, hypoxia+PGF group, and NF-κB-blocked group. A chemical hypoxia model was established in the ARPE-19 cells by adding CoCl2 to the culture medium. The morphological changes after treatment were observed. The proliferation rates were measured with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The migration abilities were measured with scratch assay. The EMT biomarkers were measured with quantitative real-time PCR (qRT-PCR), western blotting, and immunofluorescence. The relative protein expression of components of the NF-κB signaling pathway was measured with western blotting and immunofluorescence. Results: Cells treated with PGF under hypoxia exhibited morphological changes consistent with the transition from an epithelial to a mesenchymal phenotype. In the ARPE-19 cells, exogenous PGF under hypoxia increased the proliferation rate compared to the rate under hypoxia alone (p<0.05) and increased the migration rate (p<0.05). Treatment of hypoxia-exposed cells with PGF caused decreased expression of the epithelial biomarkers E-cadherin and ZO-1 (both p<0.05) and increased expression of the mesenchymal marker α-SMA (p<0.05) by enhancing the phosphorylation of NF-κB p65 of the total protein, promoting the translocation of p65 to the nucleus, and inducing the degradation of IκB-α (a negative regulator of the NF-κB pathway) in the ARPE-19 cells. Additionally, the effect of PGF-induced EMT in the ARPE-19 cells under hypoxia was counteracted with BAY 11-7082 (a selective NF-κB inhibitor). Conclusions: Exogenous PGF promotes EMT-like changes in ARPE-19 cells under hypoxia by activating the NF-κB signaling pathway. The study results suggest that PGF may play a role in scar formation in neovascular age-related macular degeneration (AMD) and that the inhibition of PGF may be a promising target for the prevention and treatment of AMD.

Placental growth factor reverses decreased vascular and uteroplacental MMP-2 and MMP-9 and increased MMP-1 and MMP-7 and collagen types I and IV in hypertensive pregnancy.

Preeclampsia is a complication of pregnancy manifested as maternal hypertension (HTN) and fetal intrauterine growth restriction, with unclear mechanisms. Placental ischemia increases antiangiogenic soluble fms-like tyrosine kinase-1 (sFlt-1) relative to angiogenic placental growth factor (PlGF); however, the molecular targets are unclear. To test the hypothesis that placental ischemia-induced changes in sFlt-1 and PlGF target vascular and uteroplacental matrix metalloproteinases (MMPs), we tested whether raising the sFlt-1-to-PlGF ratio by infusing sFlt-1 (10 µg·kg-1·day-1) in pregnant (Preg) rats increases blood pressure (BP) and alters MMPs and whether correcting sFlt-1/PlGF by infusing PlGF (20 µg·kg-1·day-1) in Preg rats with reduced uterine perfusion pressure (RUPP) improves BP and reverses the changes in MMPs. On gestational day 19, BP was higher and the litter size and uterine, placenta, and pup weight were less in Preg + sFlt-1 and RUPP than Preg rats and restored in RUPP + PlGF versus RUPP rats. Gelatin and casein zymography and Western blots revealed decreases in MMP-2 and MMP-9 and increases in MMP-1 and MMP-7 in the aorta, uterine artery, uterus, and placenta of Preg + sFlt-1 and RUPP versus Preg rats, which were reversed in RUPP + PlGF versus RUPP rats. Collagen types I and IV were more abundant in Preg + sFlt-1 and RUPP versus Preg rats and were reversed in RUPP + PlGF versus RUPP rats. Thus, PlGF reverses decreased vascular and uteroplacental MMP-2 and MMP-9 and increased MMP-1, MMP-7, and collagen types I and IV induced by placental ischemia and sFlt-1 in HTN in pregnancy. Angiogenic factors and MMP modulators could rectify changes in MMPs and collagen, restore vascular and uteroplacental remodeling, and improve HTN and intrauterine growth restriction in preeclampsia. NEW & NOTEWORTHY Understanding the mechanisms of preeclampsia could help in its prevention and management. This study shows that correcting soluble fms-like tyrosine kinase-1 (sFlt-1)/placental growth factor (PlGF) imbalance by infusing PlGF reverses the decreases in vascular and uteroplacental matrix metalloproteinase (MMP)-2 and MMP-9 and the increases in MMP-1, MMP-7, and collagen types I and IV induced by placental ischemia and antiangiogenic sFlt-1 in hypertension in pregnancy. Angiogenic factors and MMP modulators could rectify changes in vascular and uteroplacental MMPs and collagen content and ameliorate hypertension and intrauterine growth restriction in preeclampsia.

Poly(ADP-ribose) polymerase inhibitor olaparib hampers placental growth factor-driven activation of myelomonocytic cells.

The vascular endothelial growth factor receptor-1 (VEGFR-1) is a tyrosine kinase receptor activated by the angiogenic factors VEGF­A and placental growth factor (PlGF). While VEGF­A binds to both VEGFR­1 and VEGFR­2, PlGF interacts exclusively with VEGFR­1 triggering a signaling pathway involved in: i) tumor­associated angiogenesis; ii) chemotaxis and invasion of the extracellular matrix (ECM) by cancer cells; and iii) mobilization of bone marrow­derived myeloid cells that generate tumor­associated macrophages. By using a novel anti­VEGFR­1 monoclonal antibody (D16F7 mAb), which hampers receptor activation without avoiding ligand binding, we recently demonstrated that VEGFR­1 blockade reduced myeloid progenitor mobilization and monocyte/macrophage cell infiltration of tumor grafts in vivo. Since poly(ADP­ribose) polymerase (PARP)­1 exerts a pro­inflammatory role favoring monocyte activation, in the present study we investigated whether the PARP inhibitor (PARPi) olaparib hampers PlGF­induced activation of human myelomonocytic cells. HL­60 cells induced to differentiate towards the monocytic/macrophage lineage were tested and the results were confirmed in freshly isolated monocytes obtained from healthy donors. Cells were treated with olaparib, at clinically achievable concentrations, before exposure to PlGF and were analyzed for migration and ECM invasion in response to PlGF. Olaparib effects were compared to those obtained with D16F7 mAb used as single agent or in combination with the PARPi. The results indicate that differentiated HL­60 cells and monocytes expressed VEGFR­1 and migrated in response to PlGF. Moreover, olaparib and D16F7 inhibited PlGF­induced chemotaxis and ECM invasion in a dose­dependent manner and with similar efficacy. However, in combination studies the PARPi and D16F7 did not exert synergistic effects. Olaparib also hampered PlGF­induced monocyte adhesion to fibronectin, while it did not affect NF­κB activation in response to the angiogenic factor. These data suggest that olaparib likely interferes with the same pathway affected by the anti­VEGFR­1 mAb and that inhibition of PlGF-induced monocyte activation may contribute to PARPi antitumor activity.