Placenta Growth Factor Levels Research Articles

Neoadjuvant Chemotherapy Alters Neuropilin-1, PlGF, and SNAI1 Expression Levels and Predicts Breast Cancer Patients Response.

Circulating proteins hold a potential benefit as biomarkers for precision medicine. Previously, we showed that systemic levels of neuropilin-1 (NRP-1) and its associated molecules correlated with poor-prognosis breast cancer. To further identify the role of NRP-1 and its interacting molecules in correspondence with patients' response to neoadjuvant chemotherapy (NAC), we conducted a comparative study on blood and tissue samples collected from a cohort of locally advanced breast cancer patients, before and after neoadjuvant chemotherapy (NAC). From a panel of tested proteins and genes, we found that the levels of plasma NRP-1, placenta growth factor (PlGF) and immune cell expression of the transcription factor SNAI1 before and after NAC were significantly different. Paired t-test analysis of 22 locally advanced breast cancer patients showed that plasma NRP-1 levels were increased significantly (p = 0.018) post-NAC in patients with pathological partial response (pPR). Kaplan–Meier analysis indicated that patients who received NAC cycles and their excised tumors remained with high levels of NRP-1 had a lower overall survival compared with patients whose tissue NRP-1 decreased post-NAC (log-rank p = 0.049). In vitro validation of the former result showed an increase in the secreted and cellular NRP-1 levels in resistant MDA-MB-231 cells to the most common NAC regimen Adriyamicin/cyclophosphamide+Paclitaxel (AC+PAC). In addition, NRP-1 knockdown in MDA-MB-231 cells sensitized the cells to AC and more profoundly to PAC treatment and the cells sensitivity was proportional to the expressed levels of NRP-1. Unlike NRP-1, circulating PlGF was significantly increased (p = 0.014) in patients with a pathological complete response (pCR). SNAI1 expression in immune cells showed a significant increase (p = 0.018) in patients with pCR, consistent with its posited protective role. We conclude that increased plasma and tissue NRP-1 post-NAC correlate with pPR and shorter overall survival, respectively. These observations support the need to consider anti-NRP-1 as a potential targeted therapy for breast cancer patients who are identified with high NRP-1 levels. Meanwhile, the increase in both PlGF and SNAI1 in pCR patients potentially suggests their antitumorigenic role in breast cancer that paves the way for further mechanistic investigation to validate their role as potential predictive markers for pCR in breast cancer.

Differences in Levels of Placenta Growth Factor in The Vitreous After Administration of Aflibercept Compared to the Control Group of Proliferative Diabetic Retinopathy


 
 
 Introduction: to compare the levels of Placenta Growth Factor in the vitreous on patients with proliferative diabetic retinopathy at Aflibercept administration with control. Method: This is a prospective experimental study with post-test with control design. Subjects were taken from 24 eyes from 24 patients, who came to dr. Kariadi General Hospital and National Diponegoro Hospital between March until September 2016. Subjects were divided into the patient with Aflibercept administration and control. Study variables in this study are Placenta Growth Factor levels in the vitreous in the patient with diabetic proliferative retinopathy. Measurements of PlGF levels with ELISA methods are conducted at GAKI Laboratory, Medical Faculty, Diponegoro University. This data tested for normality used with Saphiro Wilk, and homogeneity variance with Lavene and test hypothesis used with Mann Whitney. 
 Results: Sample of this study are 24 patients proliferative diabetic retinopathy, twelve are the control group, and twelve are Aflibercept group. Average levels of PlGF in the control group are 263,97 pg/ml + 354,98 SD, with minimum value 12,19 pg/ml and maximum value 1002,00 pg/ml. Average levels of PlGF in the treatment group are 92,84 pg/ml + 9,54 SD, with minimum value 76,45 pg/ml and maximum value 112,30 pg/ml. Hypothesis test results obtained significance value 0,488. 
 Conclusion: There was no significant difference between PlGF levels in the control group and the treatment group. 
 
 

BASIC CLINICAL AND PATHOGENETIC ASPECTS OF DEVELOPING THE COMPLICATIONS DURING MULTIPLE PREGNANCIES

Introduction: In order to evaluate the value of the Placenta Growth Factor (PlGF) in the developing the gestational complications during multiple pregnancies, a study of this indicator in serum of 320 pregnant women with multiple pregnancies in the first trimester, as well as 40 pregnant women with single pregnancy, constituted a control group. The aim: of the study is to investigate the effect of the placental growth factors on gestational process during multiple pregnancies. Materials and methods: A prospective study of maternity pregnancy in 320 females with multiple pregnancies was conducted, which comprised the main group of the subjects and 40 healthy women with unipolar pregnancy. The level of PlGF in serum was determined by solid phase enzyme analysis using monoclonal antibody sets in the first trimester of pregnancy. Indicators of the hemostasis system (vascular thrombocyte and coagulation link) were evaluated according to generally accepted methods. Dopplerometry of placental and fetal blood flow was performed in uterine arteries, arteries and umbilical cord veins, middle cerebral artery of the fetus. Results: Women with multiple pregnancies are at the risk of gestational complications - premature births in 67.8% (p<0.01), feto placental dysfunction, pre eclampsia - in 17.5% (p <0.05) cases. The revealed violations of the vascular thrombocyte and coagulation homeostasis in the first trimester of pregnancy are the main risk factors for early premature abortion. It has been shown that the low level of placental growth factor in serum of pregnant women with multiple pregnancies in the case of premature labor, feto placental dysfunction and pre-eclampsia (111.23 ± 8.4, 203.24 ± 6.4 and 305.86 ± 7.4 pg / ml) compared with the corresponding indicators for single-pregnancy (418.2 ± 10.4 pg / ml) is a prognostic marker for the development of gestational complications. Conclusions: Timely medical correction of gestational complications during multiple pregnancies with the use of micronized progesterone, low molecular weight heparins, angio protectants allowed prolonging the pregnancy with mono choric type of placentation by 3.2 weeks (up to 34.2 ± 2.4 weeks), and in the case of dichoric twins - to full-term pregnancy.

CLINICAL AND PATHOGENETIC ASPECTS OF DEVELOPMENT OF GESTATIONAL COMPLICATIONS WITH MULTIPLE PREGNANCY

In order to evaluate the value of the placenta growth factor (PlGF) in the gestational complications during multiple pregnancy, a study of this indicator in serum in 320 pregnant women with multiple pregnancies in the first trimester and in 40 pregnant women with single pregnancy constituted a control group.Goal – The purpose of the study is to investigate the effect of the dynamics of placental growth factors on gestational process during multiple pregnancy.Material and methods of research. A prospective study of maternity pregnancy in 320 females with multiple pregnancy was conducted, which comprised the main group of the subjects and 40 healthy women with singleton pregnancy. The level of PlGF in serum was determined by solid-phase enzyme-linked immunosorbent assay using monoclonal antibody sets in the first trimester of pregnancy. Indicators of the hemostasis system (vascular trombocyte and coagulation link) were evaluated according to generally accepted methods. Dopplerometry of placental and fetal blood flow was performed in uterine arteries (MA), arteries (PA) and umbilical cord veins (PV), middle cerebral artery (CMA) fetuses.Results of the research and their discussion. Women with multiple pregnancy are at risk for developing gestational complications - premature births in 67.9% (p <0.01), fetoplacental dysfunction, preeclampsia - in 17.6% (p <0.05) cases. The revealed violations of the vascular thrombocyte and coagulation homeostasis in the first trimester of pregnancy are the main risk factors for early premature abortion. It has been shown that the low level of placental growth factor in serum of pregnant women with multiple pregnancies in the case of premature labor, fetoplacental dysfunction and preeclampsia (111.23 ± 8.4, 203.24 ± 6.4 and 305.86 ± 7.4 pg / ml) is a prognostic marker in the development of gestating complications, which is reliably confirmed by the conducted research (p <0.01) in comparison with the corresponding indices in single-pregnancy (418.2 ± 10.4 pg / ml).Conclusions. Timely medical correction of gestational complications during multiple pregnancy with the use of micronized progesterone, low molecular weight heparins, angioprotectants allowed prolonging the pregnancy with monochoric type of placentation by 3.2 weeks (up to 34.2 ± 2.4 weeks), and in the case of dichorionique placentation - to term full-term pregnancy

Linking systemic angiogenic markers to synovial vascularization in rheumatoid arthritis.

BackgroundNeoangiogenesis is a crucial event to promote the development of the hyperplasic proliferative pathologic synovium in Rheumatoid arthritis (RA). Ultrasound (US) is sensitive for detection of power Doppler (PD) vascularization.ObjectiveTo explore the associations between a set of complementary circulating angiogenic markers and a comprehensive US assessment in patients with RA.Patients and methodsSerum levels of eight angiogenic markers were measured by quantitative ELISAs in a total of 125 patients with RA, who were all systematically assessed in parallel by PDUS, performed on 32 joints.ResultsSerum levels of soluble Vascular Cell Adhesion Molecule-1 (sVCAM-1) and Tie-2 were more likely to be increased in patients with synovial hyperemia detected on at least one joint (Power Doppler grade ≥1). sVCAM-1, Tie-2 and Angiostatin concentrations gradually increased together with the grade of the semiquantitative PDUS scale and concentrations of these three markers were markedly increased in patients with moderate to marked hyperemia (Power Doppler grade 2 and 3). Levels of sVCAM-1, Tie-2, and Angiostatin correlated with a global arthritis sum score, defined by the sum of the semiquantitative PDUS scores for all joints examined. Levels of Tie-2 and Placenta Growth Factor (PlGF) were associated with PDUS features indicating residual disease activity.ConclusionOur results support the relevance of measuring serum levels of vascular markers to evaluate the intensity and extent of synovial vascularization. Angiogenic markers, and particularly Tie-2, could be a valuable surrogate of active synovitis and their place in relation to PDUS in clinical practice deserve further investigation.

Activation of brown adipocytes by placental growth factor

Gestational diabetes mellitus (GDM) is a type of diabetes and occurs during pregnancy. Brown adipose tissue (BAT) improves glucose homeostasis and mitigates insulin resistance, however, its activity is reduced in GDM. Placenta growth factor (PlGF) is an angiogenic factor produced by placental trophoblasts. Nevertheless, whether and how PlGF could affect BAT function in GDM are not defined. To investigate this question, 91 non-diabetic pregnant participants and 73 GDM patients were recruited to Gynaecology and Obstetrics Centre in Lu He hospital. Serum levels of PlGF were quantified by ELISA. Skin temperature was measured by far infrared thermography in the supraclavicular region where classical BATs were located. The direct effect of PlGF on BAT function was explored using the established human preadipocyte differentiation system. Thereby, we demonstrated that serum levels of PlGF were lower in GDM patients compared with controls, which was accompanied by decreased skin temperature in the supraclavicular region. By qPCR and western blot, mRNA and protein expression of UCP1 and OXPHOS were elevated in differentiated adipocytes treated with PlGF. PlGF stimulated mitochondrion transcription and increased copy number of mitochondrial. When subjected for respirometry, PlGF-treated differentiated adipocytes showed higher oxygen consumption rates than controls. PlGF induced AMPK phosphorylation and blockade of AMPK phosphorylation blunted UCP1 and OXPHOS expression in differentiated adipocytes. PlGF administration reduced cholesterol and triglyceride content in the liver and improved insulin sensitivity in db mice compared with control. In Conclusion, PlGF could activate BAT function. Downregulation of PlGF might contribute to the reduced BAT activity in GDM.

Activation of Browning Adipose Tissues by Placental Growth Factor

Background: Gestational diabetes mellitus (GDM) is a type of metabolic disorders. Browning adipose tissue (BAT) activity is reduced which strengthens insulin resistance in GDM. Placenta growth factor (PlGF) is one of the main factors produced by placenta during pregnancy. Once secreted into circulation, it participates in angiogenesis in peripheral tissues. However, whether and how PlGF modulates BAT function is yet unknown. Methods: We recruited 91 nondiabetic pregnant participants and 73 GDM patients. Serum levels of PlGF were quantified by ELISA. Skin temperature was measured by Far infrared thermography in the supraclavicular region where classical BAT was located. Primary human preadipocytes isolated from intercapsular BAT were differentiated under different concentrations of PlGF in vitro. Terminally differentiated adipocytes were characterized by qPCR, western blot and oxygen consumption rates by respirometry. Phosphorylation signaling pathways in differentiated cells were dissected using Bio-Plex target assay (Bio-Rad). Cells were exposed with 1 ng PlGF in the presence or absence of 1 nM AMPK inhibitor Compound C to assess UCP-1 expression. Results: Serum levels of PlGF were lower in GDM patients than non GDM controls, which was accompanied by decreased skin temperature in the supraclavicular region. By qPCR, PlGF dose-dependently induced BAT marker expression in differentiated brown adipocytes (BAC) and peaked at 1 ng as evidenced by the increased mRNA expression of UCP-1 and Oxphos. The increased BAT marker expression was further confirmed by western blot in differentiated BAC exposed to 1 ng PlGF. Accordingly, PlGF-exposed BAC showed higher oxygen consumption rates than control and stimulated AMPK phosphorylation. Blockade of AMPK phosphorylation abrogated UCP-1 and Oxphos expression in BAC induced by PlGF. Conclusions: Physically, PlGF improves preadipocyte differentiation into active BAC. Decreased PlGF levels might contribute to the reduced BAC activity in GDM. Disclosure R. Yin: None. J. Zhou: None. C. Zhang: None. C. Yan: None. W. Ma: None. N. Wu: None. D. Zhao: None. Y. Feng: None.

Celastrol attenuates symptoms of preeclampsia in rats by inhibiting matrix metalloproteinase-9

Preeclampsia is reported in pregnant women around the world and often causes maternal/fetal mortality and morbidity. In the current study, we assessed the efficacy of celastrol on a rat preeclampsia model induced by Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME). Pregnant rats were administered L-NAME to establish preeclampsia. A total of 48 animals were randomly assigned into 4 groups (n = 12 each): control, control plus celastrol treatment (control+celastrol), preeclampsia, and preeclampsia plus celastrol. Physiological parameters including total urine protein, urine volume and blood pressure were evaluated. Urinary messenger RNA (mRNA) levels of podocin and nephrin were determined using RT-PCR. Further, levels of serum placenta growth factor (PlGF), matrix metalloproteinase (MMP)-9 and renal renal soluble fms-like tyrosine kinase-1 (sFlt-1) were also measured. In rats with preeclampsia, there were robust increases in total urine protein, urine volume and blood pressure, which were significantly attenuated in rats treated with celastrol. Urinary mRNA levels of podocin and nephrin, as well as PlGF, MMP-9 and sFlt-1, were all reversed in preeclampsia plus celastrol group compared to rats in the preeclampsia group without celastrol treatments. MMP-9 overexpression in rats completely abolished the alleviating effect of celastrol. We hereby presented the first evidence that celastrol attenuated preeclampsia symptoms in an L-NAME-induced rat model of preeclampsia through inhibition of MMP-9 expression, supporting the potential therapeutic value of celastrol in the treatment of preeclampsia.

Angiogenic and inflammatory biomarkers for screening and follow-up in patients with pulmonary arterial hypertension

Objective: To identify circulating angiogenic and inflammatory biomarkers with potential in screening for pulmonary arterial hypertension (PAH) in systemic sclerosis (SSc), and in early diagnosis and determination of treatment response in PAH.Method: Plasma samples were taken at the time of PAH diagnosis and at treatment follow-up after a median (interquartile range) of 4 months (3–9.8 months) in idiopathic (n = 9) and SSc-associated PAH (n = 11). In patients with SSc-associated PAH, plasma samples had also been gathered a median of 2 years (0.8–3 years) before PAH diagnosis (n = 10). Additional plasma samples were retrieved at two time-points separated by a median of 12 years (10–13 years) from SSc patients who did not develop PAH (n = 10) and from controls (n = 8). Angiogenic and inflammatory biomarkers were analysed by multiplex immunoassays.Results: Plasma levels of placenta growth factor (PlGF), soluble vascular endothelial growth factor receptor-1 (sVEGFR-1), and tumour necrosis factor-α (TNF-α) were higher (p < 0.05) in SSc patients who later developed PAH than in those who did not. Plasma vascular endothelial growth factor (VEGF)-D increased (p < 0.05) in SSc patients as PAH developed. Plasma levels of PlGF, VEGF-A, VEGF-D, sVEGFR-1, interleukin-6, and TNF-α were higher (p < 0.05) in PAH than controls. There were no significant differences in circulating biomarkers between idiopathic and SSc-associated PAH. Plasma sVEGFR-1 decreased (p < 0.05) after initiating PAH-targeted treatments.Conclusions: Plasma levels of PlGF, sVEGFR-1, TNF-α, and VEGF-D have potential in screening for SSc-associated PAH. Plasma sVEGFR-1 may be a biomarker of treatment response.

Methacholine Challenge Test Reveals a High Prevalence of Subclinical Severe Airway Hyper-Reactivity in Children and Yound Adults with Sickle Cell Disease Which Correlates with Higher Sickle Acute Events and Acute Chest

▪Background: Although increase prevalence of airway hyper-reactivity (AHR) has been detected in children, and even adults with sickle cell disease (SCD) using methacholine provocative challenge test (MCT), AHR was not been found to be associated with clinical asthma.Hence, the clinical implications of AHR in SCD are unknown. Furthermore, AHR studies in SCD patients using MCT lack comparisons with a control population. We recently showed that increased placenta growth factor (PlGF) levels in sickle mice causes AHR by upregulating leukotriene synthesis and inflammation. We therefore designed a prospective study to determine the prevalence of AHR in children and young adults with SCD, and in unaffected siblings living in the same household as a control population, and also determine the biochemical and clinical implications of AHR in SCD.Methods: A total of 67 patients with SCD and 14 unaffected siblings of the patients enrolled on to the study for determining baseline AHR (ClinicalTrials.gov Identifier: NCT00448370). Chief eligibility criteria included a diagnosis of SCD (HbSS, HbSC or HbS b-thalassemia) confirmed by hemoglobin electrophoresis or gene sequencing, ages 5.5 years to 30 years who were able to complete pulmonary function testing and were at baseline/steady state (no acute sickle events 3 weeks prior or acute chest syndrome (ACS) 4 weeks prior). Excluded were patients taking anti-leukotriene medications within 30 days prior, although patients with SCD who had a known history of asthma but not on leukotriene inhibitors were eligible. Patients with chronic transfusions, congenital lung or heart condition, or other medical condition that could affect the pulmonary function testing were also ineligible. Unaffected siblings of the 67 enrolled SCD patients living in the same household were offered enrollment for MCT and those consenting were enrolled.Results: 64 SCD patients and 14 siblings underwent a MCT; 3 of 67 SCD patients had FEV1<70% of predicted at baseline, and therefore, for safety reasons, were not subjected to MCT. The degree of AHR was defined by the provocative concentration of methacholine that resulted in a 20% decline in FEV1 (PC20): no AHR=PC20 >16mg/mL; borderline AHR=PC20 4-16mg/mL; mild AHR=PC20 1-3mg/mL and moderate-severe AHR=PC20 <1mg/mL. All subjects were given a dose of albuterol after completion of MCT. MCT was safe in all subjects, and no AHR or acute sickle events occurred in any subject for three weeks after the MCT. Overall, 25 of 67 (37%) SCD patients had no AHR, 8 of 67 (12%) had borderline AHR (PC20 9±1.4mg/ml), 10 of 67 (15%) had mild AHR (PC20 1.72 ±0.16) and 24 of 67 (36%) had moderate-severe AHR (PC20 0.6 ±0.06). Of unaffected siblings, 10 of 14 (71%) had no AHR, 2 of 14 (14%) had borderline AHR (PC20 10±0.4) and 2 of 14 (14%) had mild AHR (PC20 1.7±0.4). Moderate to severe AHR was only seen in SCD patients and not sibling controls. Severe AHR prevalence was highest in the HbSS group. We analyzed the clinical characteristics of the SCD subjects, including acute sickle episodes, defined as admission for one of the following: pain crises, fever with or without a known source of infection, cough/wheezing/chest pain, impending acute chest syndrome, enlarged and splenic sequestration or priapism. SCD patients with AHR had significantly higher (P<0.05) admissions from acute sickle episodes (2.7±0.1) and acute chest syndrome (1.5±0.15). We next assessed PlGF and leukotriene levels in SCD subjects. PlGF levels were not quantifiable due to presence of hemolysis, which confounds PlGF ELISA. However, we found a trend towards higher (1.3-2 fold) expression of PlGF-induced target genes (VEGF, IL-1β, IL-8, CCL2) in the mononuclear cells of patients with severe AHR; Also, circulating cysteinyl leukotriene D4 levels in their plasma were significantly higher.Conclusions: There was a higher prevalence of mild to severe AHR (51%) in patients with SCD, compared to a 14% prevalence of mild AHR in siblings living in the same household. The higher degree of AHR was associated with increased SCD morbidity - increased rates of admission from ACS and pain crisis, when compared to those with no AHR. AHR was associated with higher leukotrienes and a trend towards higher PlGF target gene induction. Together, our data suggests that AHR may be a biomarker for SCD morbidity, and may also be a therapeutic target, since blockade of PlGF or leukotriene synthesis in patients with severe AHR may reduce SCD morbidity. DisclosuresNo relevant conflicts of interest to declare.

Maternal serum placental growth factor combined with second trimester aneuploidy screening to predict small-for-gestation neonates without preeclampsia

ObjectiveTo investigate the role of maternal serum placenta growth factor (PlGF) and quadruple test parameters in predicting the risk of small for gestational age (SGA) infants of mothers without preeclampsia. Materials and methodsWe prospectively enrolled 300 pregnant patients who underwent blood sampling at 15–18 weeks gestation and followed them until delivery. Cases with SGA neonate delivery (n = 100) were compared with matched AGA neonate controls (n = 200). The plasma PlGF and quadruple markers were measured by enzyme-linked immunosorbent assay. The results were analyzed with Mann–Whitney U tests, and regression analysis was used to develop a model for the prediction of SGA. ResultsWomen who delivered SGA neonates had decreased levels of PlGF (median 0.71 MoM versus 0.7 MoM; p < 0.01), hCG (median 0.97 MoM versus 1.06 MoM; p = 0.046) and uE3 (median 0.92 MoM versus 1.04 MoM) compared to the AGA group. AFP, hCG and inhibin-A levels did not differ significantly. A PlGF concentration <0.37 MoM had a sensitivity of 28.0% (95% CI: 19.5–37.9) and a specificity of 89.5% (95% CI: 84.4–93.4) for the prediction of SGA neonates without PE. ConclusionSGA neonates in the absence of PE could potentially be identified at 15–18 weeks of pregnancy.

Use of first-trimester placenta growth factor concentration to predict hypertensive disorders of pregnancy in a low-risk Asian population.

To determine the value of placenta growth factor (PlGF) for predicting hypertensive disorders of pregnancy (HDP) in a low-risk population when used either alone or in combination with other markers. A prospective observational cohort study was conducted among women with singleton pregnancy in the first trimester in New Delhi, India, between October 1, 2013, and September 30, 2016. First-trimester measures included maternal history, body mass index (BMI), mean arterial pressure (MAP), Doppler uterine artery pulsatility index, and serum levels of biomarkers (including PlGF). Among 1725 women, 208 (12.1%) developed HDP; 74 (35.6%) of these cases were early onset. Mean PlGF levels were significantly lower among cases than among controls (30.42±10.22 vs 37.31±13.28pg/mL; P<0.001). As a biomarker for prediction of HDP, PlGF fared better than pregnancy-associated plasma protein A (PAPP-A): area under the curve 0.685 (95% confidence interval [CI] 0.620-0.750; P<0.001) versus 0.659 (95% CI 0.593-0.726; P<0.001). The highest odds ratio was found for MAP (8.369, 95% CI 5.752-12.215). The combination of PlGF, PAPP-A, BMI, MAP, and Doppler uterine artery pulsatility index provided an area under the receiver operating characteristic curve of 0.876 (95% CI 0.833-0.919; P<0.001). Combining PlGF concentration with biochemical and biophysical markers provided a good screening test for HDP during the first trimester.

Differential hypoxic response of human choroidal and retinal endothelial cells proposes tissue heterogeneity of ocular angiogenesis

PurposeTo elaborate molecular differences between choroidal and retinal angiogenesis by generating and comparatively analyzing human primary choroidal and retinal endothelial cell (CEC and REC) lines.MethodsHuman CEC and REC were isolated by positive selection and cultured. Characterization was performed by immunostaining for endothelial cell (EC)‐specific markers. Total RNA and protein were extracted from normoxic or hypoxic CEC and REC cultures. qPCR arrays were used to comparatively analyze CEC and REC, and expression differences were calculated by ΔΔCt method. Angiogenesis‐related protein expression differences were investigated by western blot and proteome profiler, and were calculated by densitometry. Functional responses to hypoxia were analyzed by sprouting assay.ResultsPrimary human CEC and REC lines stained positively for all EC markers and demonstrated high purity with similar staining and morphology. Under normoxia, CEC showed significantly lower expression levels for cell proliferation and vessel maturation genes and higher expression levels for inflammation‐related genes when compared to REC. In response to hypoxia, CEC and REC displayed differential regulation for a multitude of angiogenesis‐related genes and proteins. Furthermore, within the vascular endothelial growth factor (VEGF) family, CEC showed preferential upregulation for vascular endothelial growth factor A (VEGFA) while REC upregulated placenta growth factor (PlGF) levels. Sprouting was observed in normoxia, and was significantly by hypoxia.ConclusionsDifferential normoxic and hypoxic regulation of angiogenesis‐related factors by CEC and REC outlines tissue heterogeneity of ocular angiogenesis and suggests that tissue specificity should be considered as a novel treatment modality for successfully overcoming choroidal and retinal angiogenic conditions in the clinic.

Ferulic acid alleviates symptoms of preeclampsia in rats by upregulating vascular endothelial growth factor.

Preeclampsia is a complication affecting pregnant women worldwide, which leads to maternal and fetal morbidity and mortality. In this study, we evaluated the efficacy of ferulic acid (FA) on an Nω -nitro-L-arginine methyl ester hydrochloride (L-NAME) induced rat model of preeclampsia. L-NAME was administered to pregnant rats to induce preeclampsia. 48 rats were divided into three experimental groups (n=16 each): control group, preeclampsia group and preeclampsia with FA treatment (preeclampsia+FA). Physiological characteristics such as urine volume, total urine protein and blood pressure were assessed. Expressions levels of urinary nephrin and podocin mRNAs were analyzed by RT-PCR. Levels of renal vascular endothelial growth factor (VEGF), renal soluble fms-like tyrosine kinase-1 (sFlt-1) and serum placenta growth factor (PlGF) were also examined. Urine volume, total urine protein and blood pressure were markedly increased in preeclampsia group rats compared to control (P<.05), which were then significantly reduced in preeclampsia+FA group (P<.05). Expressions of urinary nephrin and podocin mRNAs, levels of VEGF, sFlt-1 and PlGF were also reversed in preeclampsia+FA group compared to preeclampsia rats (P<.05). We hereby report for the first time, FA alleviates preeclampsia symptoms in a rat preeclampsia model, supporting its potential value in treating preeclampsia.

Expression of miR-126 and its potential function in coronary artery disease.

This study aimed to explore the role of miR-126 in coronary artery disease (CAD) patients and the potential gene targets of miR-126 in atherosclerosis. A total of 60 CAD patients and 25 healthy control subjects were recruited in this study. Among the 60 CAD patients, 18 cases were diagnosed of stable angina pectoris (SAP), 20 were diagnosed of unstable angina pectoris (UAP) and 22 were diagnosed of acute myocardial infarction (AMI). Plasma miR-126 levels from both groups of participants were analyzed by real-time quantitative PCR. ELISA was used to measure plasma level of placenta growth factor (PLGF). The results showed that the miR-126 expression was significantly down-regulated in the circulation of CAD patients compared with control subjects (P<0.01). Plasma PLGF level was significantly upregulated in patients with unstable angina pectoris and acute myocardial infarction (AMI) compared with controls (both P<0.01) the miR-126 expression in AMI was significantly associated with PLGF. miR-126 may serve as a novel biomarker for CAD.

Dysregulation of circulating autoantibodies against VEGF-A, VEGFR-1 and PlGF in preeclampsia - A role in placental and vascular health?

Preeclampsia is a state of antiangiogenesis, with high levels of maternal circulating sVEGFR-1 (soluble vascular endothelial growth factor receptor 1, also named sFlt1) and low levels of PlGF (placenta growth factor). Various autoantibodies have been detected in preeclamptic patients. We hypothesize that circulating autoantibodies against VEGF-A (AA-VEGF-A), VEGFR-1 (AA-VEGFR-1) and PlGF (AA-PlGF) are present in preeclamptic women, with different levels from pregnant women with normotensive pregnancies. Secondly, we wanted to analyze if autoantibody levels are associated to sFlt1 or PLGF levels. Retrospective cross sectional study of 88 women with singleton pregnancies who delivered at Oslo University Hospital of whom 46 had preeclampsia and 42 had uncomplicated normotensive pregnancies. Novel immunoassays for IgG-autoantibodies against VEGFA, VEGFR-1 and PlGF were developed and serum samples were assayed. AA-VEGF-A, AA-VEGF-R1 and AA-PlGF were significantly lower in preeclamptic pregnancies (n=42) compared to normotensive pregnancies (n=46) (p<0.05). On unadjusted analysis, only AA-VEGFA and AA-VEGFR-1 were predictors of PE, but none were independent predictors after adjusting for BMI (body mass index) and parity. In the subgroup of normotensive and PE women with overlapping sVEGFR-1/PlGF-ratios, AA-VEGF was a significant predictor of PE with AUC: 0.735. IgG autoantibodies against VEGF-A VEGFR-1 and PlGF can be found in pregnant women. They are dysregulated in preeclampsia. The roles of these autoantibodies are unknown, but this study suggests they play a protective role in pregnancy. The levels of AA against VEGF-A, VEGFR-1 and PlGF might be important factors contributing to anti-angiogenesis regulation.

Abstract P1-02-03: Circulating inflammatory markers, growth factors, and tumor associated antigens in women with early stage breast cancer receiving neoadjuvant metformin

Abstract Background: Numerous clinical studies have reported that diabetic patients receiving metformin exhibit decreased cancer incidence and cancer related mortality. Metformin's mechanism of anti-tumor action has been attributed to both direct effects on cancer cells and systemic changes in insulin metabolism. Indeed, metformin reduces circulating insulin levels, which may be integral to its effectiveness in the breast cancer (BC) setting where hyperinsulinemia is associated with both recurrence and death. While the impact of metformin on blood glucose and insulin is well documented, its effects on other systemic physiologic and inflammatory factors are unknown. We completed a neoadjuvant "window of opportunity" study of metformin in non-diabetic women with BC and a series of analyses were performed on plasma samples to assess the impact of metformin on circulating inflammatory markers, growth factors, and tumor associated antigens. Methods: Non-diabetic women with early stage, untreated BC were given metformin 500 mg tid for ≥2 weeks post diagnostic core biopsy until surgery. Fasting blood was collected at diagnosis and surgery to assess circulating markers pre- and post-metformin administration. Plasma was isolated from blood samples and evaluated for CRP, TNF-alpha, IL-6, IL-8, VEGF, EGF, PlGF (placenta growth factor), CA15-3, and SHBG (sex hormone binding globulin). Change scores (post-metformin minus pre-) were calculated and the degree of change characterized by the median change and the rank-biserial correlation. The Wilcoxon signed-rank test was used to test the null hypothesis that the change scores were symmetrically distributed around zero versus more positive or negative change. Results: A total of 39 patients (mean age 51 years) completed the study and received metformin for a median of 18 days (range 13-40). Metformin was associated with changes in the levels of growth factors, with increases seen in EGF (median increase 1.1 pg/mL, r=0.42, p=0.027) and VEGF (1.7 pg/mL, r=0.31, p=0.09). A reduction in PlGF levels (-0.18 pg/mL, r=-0.6, p=0.0028) was also observed. The tumor associated antigen CA15-3 was significantly reduced after metformin treatment (-0.4 pg/mL, r=-0.56, p=0.0024) and a marker of sex hormone bioavailability (SHBG) was increased (2 nM, r=0.30, p=0.1). For circulating inflammatory markers, a significant increase in the levels of IL-8 (0.8 pg/mL, r=0.36, p=0.048) was observed, but changes in TNF-alpha and IL-6 were minimal (TNF-alpha 0.2 pg/mL, r=0.20, p=0.29; IL-6 0.1 pg/mL, r=0.14, p=0.46) and no change was seen in CRP (0 mg/L, r=-0.05, p=0.93). Conclusions: Short-term metformin administration was associated with alterations in systemic physiologic and inflammatory factors. Such increases in circulating cytokines and growth factors indicate possible alterations in the inflammatory state of the host and/or tumor. Of note, the reduction seen in the tumor antigen CA15-3 may reflect a disease-modifying effect of metformin in BC. The authors wish to acknowledge the generous support of the Hold'Em For Life Charity Challenge and the Breast Cancer Research Foundation. Citation Format: Dowling RJ, Niraula S, Chang MC, Ennis M, Stambolic V, Goodwin PJ. Circulating inflammatory markers, growth factors, and tumor associated antigens in women with early stage breast cancer receiving neoadjuvant metformin [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-02-03.

Kaempferol inhibited VEGF and PGF expression and in vitro angiogenesis of HRECs under diabetic-like environment.

Diabetic retinopathy (DR) is one of the common and specific microvascular complications of diabetes. This study aimed to investigate the anti-angiogenic effect of kaempferol and explore its underlying molecular mechanisms. The mRNA expression level of vascular endothelial growth factor (VEGF) and placenta growth factor (PGF) and the concentrations of secreted VEGF and PGF were measured by qTR-PCR and ELISA assay, respectively. Human retinal endothelial cells (HRECs) proliferation, migration, and sprouting were measured by CCK-8 and transwell, scratching wound, and tube formation assays, respectively. Protein levels were determined by western blot. High glucose (25 mM) increased the mRNA expression levels of VEGF and PGF as well as the concentrations of secreted VEGF and PGF in HRECs, which can be antagonized by kaempferol (25 µM). Kaempferol (5-25 µM) significantly suppressed cell proliferation, migration, migration distance and sprouting of HRECs under high glucose condition. The anti-angiogenic effect of kaempferol was mediated via downregulating the expression of PI3K and inhibiting the activation of Erk1/2, Src, and Akt1. This study indicates that kaempferol suppressed angiogenesis of HRECs via targeting VEGF and PGF to inhibit the activation of Src-Akt1-Erk1/2 signaling pathway. The results suggest that kaempferol may be a potential drug for better management of DR.

The effects of sildenafil citrate on urinary podocin and nephrin mRNA expression in an L-NAME model of pre-eclampsia.

We investigated the effects of sildenafil citrate (SC) on podocyturia in N ω-nitro-L-arginine methyl ester hydrochloride (L-NAME) model of pre-eclampsia (PE). One hundred and twenty Sprague-Dawley rats (SDR) were divided into five groups like pregnant control (PC), early-onset PE (EOPE), late-onset PE(LOPE), early and late-onset PE with SC-treated groups [EOPE (SC); LOPE (SC)]. PE was induced in SDR by oral administration of L-NAME in drinking water for 4-8days for EOPE and 8-14day for LOPE. The blood pressure, urine volume and total urine protein were increased in EOPE and LOPE groups when compared to PC, and all the above parameters decreased in EOPE (SC) and LOPE (SC) groups when compared to EOPE and LOPE groups, respectively. The EOPE and LOPE groups showed an increase in urinary nephrin mRNA and podocin mRNA levels compared to PC group. Increases in serum and renal soluble fms-like tyrosine kinase-1 (sFlt-1) expression levels and decreases in renal vascular endothelial growth factor (VEGF) expression and serum placenta growth factor (PlGF) levels were observed in EOPE and LOPE groups when compared to PC group. In addition, decreases in serum and renal sFlt-1 expression levels and increases in renal VEGF expression and serum PlGF levels were observed in EOPE (SC) and LOPE (SC) groups when compared to EOPE and LOPE groups, respectively. The light microscopy showed that the renal tissue of L-NAME-treated rats had extensive glomerular damage, tubular damage and infiltration by mononuclear cells when compared to PC group. Therefore, SC ameliorated podocyturia through its effects on the antiangiogenic/angiogenic status in this animal model.

The trend and the disease prediction of vascular endothelial growth factor and placenta growth factor in nontuberculous mycobacterial lung disease

Nontuberculous mycobacteria (NTM)-lung disease (LD) is an increasing health problem worldwide. The diagnosis of this disease remains difficult, however the application of placenta growth factor (PlGF) and vascular endothelial growth factor (VEGF) has not yet been studied. We screened patients with Mycobacterium avium complex or M. abscessus isolated from sputum, and enrolled 32 patients with NTM-LD and 93 with NTM pulmonary colonization. The NTM-LD group had a lower body mass index, higher proportion of bronchiectasis, more respiratory symptoms and pulmonary lesions, and higher titers of sputum acid-fast stain than the NTM pulmonary colonization group. The plasma level of PlGF was lower in the NTM-LD group than in the NTM colonization group, whereas the level of VEGF was higher in the NTM-LD group. In multivariable logistic regression analysis excluding NTM cultures, the predictive model for NTM-LD included sputum AFS titer, a nodular-bronchiectasis radiographic pattern, plasma VEGF/PlGF ratio, and chest radiographic score (VEGF/P1GF ratio became not significant as a factor in multivariable generalized linear model). The four-factor predictive index had good positive likelihood ratio and negative likelihood ratio for predicting NTM-LD in the patients with NTM in their sputum.