Angiogenic factors and the lectin pathway of complement in women with secondary recurrent pregnancy loss.

The poor remodeling of placental spiral arteries seen in preeclampsia is also discussed to contribute to recurrent pregnancy loss (RPL) preceded by abnormal angiogenesis and excessive complement activation. Low levels of Mannose-binding-lectin (MBL), a pattern recognition molecule (PRM) of the lectin pathway, have been found in women with RPL. We propose that pregnancy loss is connected to defective angiogenesis with reperfusion damage in the placenta and decreased levels of PRM in the lectin pathway in women with RPL. In this cohort study, we investigate the angiogenic factors and the lectin complement pathway in early pregnancy and their time-dependent relationship with pregnancy outcomes in 76 women with secondary RPL (sRPL) who have at least four prior pregnancy losses and a live birth. We evaluated levels of Angiopoietin-1 (Ang-1), Angiopoietin-2 (Ang-2), Vascular Endothelial Growth Factor (VEGF), soluble fms-like tyrosine kinase-1 (sFlt-1), and the PRMs, MBL, ficolin-1, -2, -3 and an additional soluble PRM, Pentraxin-3, during the 5th, 6th, and 7th gestational weeks. Our results showed that, compared to live births, pregnancies that ended in loss were associated with elevated VEGF levels and decreased levels of the Ang-2/Ang-1 ratio. Also, increasing levels of ficolin-2 were significantly associated with pregnancy loss, with MBL showing no association. Our research suggests that women with sRPL may have inadequate placentation with impaired angiogenesis in pregnancies ending in a loss.

Myxomatous mitral valve disease and associated pulmonary hypertension might increase serum angiopoietin-2 in dogs.

OBJECTIVE: To evaluate the relationships between the severity of myxomatous mitral valve disease (MMVD) and pulmonary hypertension (PH) and serum angiopoietin (Ang)-1 and Ang-2 concentrations in dogs with MMVD. ANIMALS: 74 dogs (control, n = 12; MMVD, n = 62) were included. METHODS: Serum Ang-1 and Ang-2 concentrations were estimated using the canine-specific ELISA kit. The concentrations were compared between dogs with MMVD and healthy dogs, and they were analyzed according to the severity of MMVD and PH. RESULTS: The median serum Ang-1 concentration did not differ among the study groups. The median serum Ang-2 concentration was higher in dogs with stage B2 MMVD (P = .041) and acute congestive heart failure (P = .002) than in control dogs. In addition, the median serum Ang-2 concentration was higher in MMVD dogs with PH than in those without PH (P = .031). Serum Ang-2 concentration was correlated with vertebral heart score (rs = 0.36, P = .004) and vertebral left atrial score (r = 0.50, P < .001) in dogs with MMVD, and correlated with vertebral heart score (r = 0.63, P = .01), maximum E wave amplitude of the diastolic transmitral flow (rs = 0.61, P = .018), ejection fraction (rs = -0.77, P < .001) and fractional shortening (rs = -0.56, P = .032) in dogs with acute congestive heart failure. CLINICAL RELEVANCE: Circulating Ang-2 levels increase in dogs with the severity of MMVD and the presence of PH.

Proximal heat stress up-regulates angiopoietin-1 in fingers and reduces the severity of Raynaud's phenomenon in systemic sclerosis: a single-centre pilot study.

OBJECTIVES: Raynaud's phenomenon (RP) is a peripheral vascular disorder that frequently occurs in systemic sclerosis (SSc). Although therapeutic heating seems reasonable given that RP is elicited by cold stimuli, the effects of heating are still unclear. We examined the effects of heating applied on various body parts in SSc patients with RP of fingers. METHODS: Fourteen SSc patients heated their neck, elbows, and wrists with disposable heating pads for 1 week each. The visual analogue scale (VAS) for RP during each heating period was compared with that of each 1-week pre-treatment interval. On the day after the expiration of each heating period, their finger temperature, the finger blood flow, and angiogenesis-related factors (vascular endothelial growth factor, endostatin, angiopoietin-1, and angiopoietin-2) obtained from the cubital vein and fingertip were measured. RESULTS: The mean VAS was significantly reduced during the heating of the neck and elbows. Fingertip blood samples showed significantly increased angiopoietin-1 after each of the heating periods and increased endostatin after wrist heating. After the termination of heating, changes in finger temperature or blood flow could not be detected. CONCLUSIONS: Heating the neck or elbows can alleviate RP in SSc. The heat up-regulates angiopoietin-1 in the fingers.

Diagnostic and prognostic significance of serum angiopoietin-1 and -2 concentrations in patients with pulmonary hypertension.

Several biomarkers for detecting pulmonary hypertension (PH) have been reported. However, these biomarkers are deemed insufficient to detect PH in its early stages. We evaluated the utility of serum angiopoietin (ANGP), a glycoprotein related to angiogenesis, as a diagnostic and prognostic biomarker of PH. Patients with PH who underwent right-heart catheterization, were retrospectively studied. Serum concentrations of ANGP-1 and ANGP-2 were measured using an enzyme-linked immunosorbent assay in patients with PH (n = 32), those with idiopathic pulmonary fibrosis (IPF) without PH (as a disease control, n = 75), and age-matched healthy controls (HC, n = 60). Nineteen patients (59.4%) with PH had World Health Organization group 3 PH. Serum ANGP-2 concentration, but not ANGP-1, in patients with PH was significantly higher compared with that in HC (p = 0.025) and in patients with IPF without PH (p = 0.008). Serum ANGP-2 concentration in patients with PH positively and significantly correlated with N-terminal pro-B-type natriuretic peptide (r = 0.769, p < 0.001), right ventricular diameter on echocardiography (r = 0.565, p = 0.035), and mean pulmonary arterial pressure (r = 0.449, p = 0.032) and pulmonary vascular resistance (r = 0.451, p = 0.031) on right-heart catheterization. ANGP-1 and ANGP-2 were expressed on lung vascular endothelial cells, as shown by immunohistochemistry. Patients with PH with higher ANGP-2 concentration (≥ 2.48 ng/mL) had significantly worse survival (p = 0.022). Higher ANGP-2 concentration was a significant worse prognostic factor (hazard ratio = 6.063, p = 0.037), while serum ANGP-1 concentration was not. In conclusion, serum ANGP-2 may be a useful diagnostic and prognostic biomarker in patients with PH, especially in patients with group 3 PH.

Circulating lncRNAs HIF1A-AS2 and LINLK-A: Role and Relation to Hypoxia-Inducible Factor-1α in Cerebral Stroke Patients.

Long noncoding RNAs (lncRNAs) have been recently recognized as key players of gene expression in cerebral pathogenesis. Thus, their potential use in stroke diagnosis, prognosis, and therapy is actively pursued. Due to the complexity of the disease, identifying stroke-specific lncRNAs remains a challenge. This study investigated the expression of lncRNAs HIF1A-AS2 and LINK-A, and their target gene hypoxia-inducible factor-1 (HIF-1) in Egyptian stroke patients. It also aimed to determine the molecular mechanism implicated in the disease. A total of 75 stroke patients were divided into three clinical subgroups, besides 25 healthy controls of age-matched and sex-matched. Remarkable upregulation of lncRNA HIF1A-AS2 and HIF1-α along with a downregulation of lncRNA LINK-A was noticed in all stroke groups relative to controls. Serum levels of phosphatidylinositol 3-kinase (PI3K), phosphorylated-Akt (p-Akt), vascular endothelial growth factor (VEGF), and angiopoietin-1 (ANG1) as well as their receptors, malondialdehyde (MDA), and total antioxidant capacity (TAC) were significantly increased, whereas brain-derived neurotrophic factor (BDNF) levels were significantly decreased particularly in hemorrhagic stroke versus ischemic groups. Eventually, these findings support the role of lncRNAs HIF1A-AS2 and LINK-A as well as HIF1-α in activation of angiogenesis, neovascularization, and better prognosis of stroke, especially the hemorrhagic type.

Angiopoietin-2 as a Prognostic Factor in Patients with Incurable Stage IV Colorectal Cancer.

PURPOSE: Angiopoietin (Ang), a ligand of the endothelium-specific receptor Tie-2 system, is associated with tumor growth and progression that depend on angiogenesis. The present study aimed to investigate the predictive potential of angiopoietin factors in incurable stage IV colorectal cancer (CRC) patients who have undergone primary tumor resection. METHODS: The study included 40 consecutive patients with incurable stage IV CRC who underwent primary tumor resection at our hospital between 2011 and 2015. Patients were divided into subgroups of low and high Ang-1, Ang-2, and Tie-2. Patient age and sex, tumor location, TNM stages, vascular invasion, chemotherapy, and overall survival were assessed. RESULTS: The cut-off values of Ang-1, Ang-2, and Tie-2 were 0.4, 1.8, and 15.0 ng/mL, respectively. Overall survival was significantly longer in the low Ang-2 group than in the high Ang-2 group. High Ang-2 levels were associated with age, N stage, and chemotherapy. Immunofluorescent staining of Ang-2 revealed that endothelial cells and cancer cells expressed Ang-2 in each case. CONCLUSIONS: Our findings suggest that the serum Ang-2 level is associated with disease progression and is an important predictor of mortality in incurable stage IV CRC patients. Thus, it may be a useful prognostic biomarker in these patients.

The ratio of plasma angiopoietin-2 to angiopoietin-1 as a prognostic biomarker in patients with sepsis.

BACKGROUND: We aimed to investigate the role of angiopoietin (Angpt) as a predictive biomarker for sepsis by evaluating associations between plasma Angpt and various inflammatory cytokines and mortality in critically ill patients with sepsis. METHODS: This study was a retrospective cohort study of the prospectively collected samples and clinical data of 145 patients with sepsis who were admitted to the medical intensive care unit (ICU) of a 2000-bed university tertiary referral hospital in South Korea. We collected plasma within 24 h of medical ICU admission, and several biomarkers (Angpt-1 and -2, Tie2, vascular endothelial growth factor, interleukin (IL)-1ß, IL-10, IL-18, IL-6, interferon gamma-induced protein-10, and tumor necrosis factor-α) were measured using a Human Magnetic Luminex Screening Assay kit. RESULTS: Plasma Angpt-2 was correlated with IL-6 (rs = 0.555) and tumor necrosis factor-α (rs = 0.559). Plasma Angpt-2 (rs = 0.530) and Angpt-2/1 (rs = 0.562) were correlated with the Sequential Organ Failure Assessment (SOFA) score. The area under the curve (AUC) for the 28-day mortality prediction for the plasma Angpt-2/1 ratio was 0.736; AUCs for the Acute Physiology and Chronic Health Evaluation II (APACHE II) and SOFA scores were 0.659 and 0.745, respectively. Using multivariate Cox proportional hazard regression analysis for 28-day mortality, we found that acute respiratory distress syndrome (hazard ratio (HR) = 2.235, 95% CI = 1.163-4.296,p = 0.016), APACHE II score (HR = 1.127, 95% CI = 1.037-1.224,p = 0.005), and Angpt-2/1 > 3.2 (HR = 2.522, 95% CI = 1.205-5.278,p = 0.014) were risk factors for 28-day mortality. CONCLUSIONS: Plasma Angpt-2 was related to cytokines, but Angpt-2/1 ratio was a good predictor of 28-day mortality in patients with sepsis.

Hereditary angioedema attack: what happens to vasoactive mediators?

Hereditary angioedema is a disabling, life-threatening condition caused by deficiency (type I) or dysfunction (type II) of the C1 inhibitor protein (C1-INH-HAE) leading to bradykinin accumulation and recurrent episodes of edema attack. Vascular leakage is a complex process sustained by the coordinated production of several permeabilizing factors including vascular endothelial growth factors (VEGFs), angiopoietins (ANGPTs) and phospholipase A2 enzymes (PLA2). We previously reported that patients with C1-INH-HAE in remission have increased plasma levels of VEGFs, ANGPTs and secreted PLA2. In this study, we sought to analyze plasma levels of these mediators in 15 patients with C1-INH-HAE during the acute attack compared to remission. Plasma concentrations of VEGF-A, VEGF-C and VEGF-D were not altered during attack compared to remission. Moreover, VEGF-D concentrations were not altered also in remission phase compared to controls. Concentrations of ANGPT1, a vascular stabilizer, were increased during attacks compared to symptoms-free periods, whereas ANGPT2 levels were not altered. The ANGPT2/ANGPT1 ratio was decreased during angioedema attacks. Platelet activating factor acetylhydrolase activity was increased in patients with C1-INH-HAE in remission compared to controls and was decreased during angioedema attacks. Our results emphasize the complexity by which several vasoactive mediators are involved not only in the pathophysiology of C1-INH-HAE, but also during angioedema attacks and its resolution.

Angiotensin-Converting Enzyme Inhibitor-Associated Angioedema: From Bed to Bench.

BACKGROUND AND OBJECTIVE: Angiotensin-converting enzyme inhibitor-associated angioedema (ACEI-AAE) affects 0.1%-0.7% of patients treated with ACEIs. While previous research suggests that angioedema attacks result from increased vascular permeability, the pathogenesis is not completely understood. Objective: This study aimed to describe the clinical, genetic, and laboratory parameters of ACEI-AAE patients and to investigate the role of vascular endothelial growth factors A and C (VEGF-A and VEGF-C), angiopoietins 1 and 2 (Ang1/Ang2), and secretory phospholipase A2 (sPLA2) in the pathogenesis of ACEI-AAE. METHODS: The clinical and laboratory data of ACEI-AAE patients were collected from 2 angioedema reference centers. Healthy volunteers and ACEI-treated patients without angioedema were enrolled to compare laboratory parameters. Genetic analyses to detect mutations in the genes SERPING1, ANGPT1, PLG, and F12 were performed in a subset of patients. RESULTS: A total of 51 patients (57% male) were diagnosed with ACEI-AAE. The average time to onset of symptoms from the start of ACEI therapy was 3 years (range, 30 days-20 years). The most commonly affected sites were the lips (74.5%), tongue (51.9%), and face (41.2%). Switching from ACEIs to sartans was not associated with an increased risk of angioedema in patients with a history of ACEIAAE. VEGF-A, VEGF-C, and sPLA2 plasma levels were higher in ACEI-AAE patients than in the controls. Ang1/2 concentrations remained unchanged. No mutations were detected in the genes analyzed. CONCLUSIONS: Our data suggest that sartans are a safe therapeutic alternative in ACEI-AAE patients. Increased concentrations of VEGF-A, VEGF-C, and sPLA2 in ACEI-AAE patients suggest a possible role of these mediators in the pathogenesis of ACEI-AAE.

Identification of Astrocytoma Blood Serum Protein Profile.

High-grade astrocytomas are some of the most common and aggressive brain cancers, whose signs and symptoms are initially non-specific. Up to the present date, there are no diagnostic tools to observe the early onset of the disease. Here, we analyzed the combination of blood serum proteins, which may play key roles in the tumorigenesis and the progression of glial tumors. Fifty-nine astrocytoma patients and 43 control serums were analyzed using Custom Human Protein Antibody Arrays, including ten targets: ANGPT1, AREG, IGF1, IP10, MMP2, NCAM1, OPN, PAI1, TGFß1, and TIMP1. The decision tree analysis indicates that serums ANGPT1, TIMP1, IP10, and TGFß1 are promising combinations of targets for glioma diagnostic applications. The accuracy of the decision tree algorithm was 73.5% (75/102), which correctly classified 79.7% (47/59) astrocytomas and 65.1% (28/43) healthy controls. The analysis revealed that the relative value of osteopontin (OPN) protein level alone predicted the 12-month survival of glioblastoma (GBM) patients with the specificity of 84%, while the inclusion of the IP10 protein increased model predictability to 92.3%. In conclusion, the serum protein profiles of ANGPT1, TIMP1, IP10, and TGFß1 were associated with the presence of astrocytoma independent of its malignancy grade, while OPN and IP10 were associated with GBM patient survival.

The contribution of dietary and plasma folate and cobalamin to levels of angiopoietin-1, angiopoietin-2 and Tie-2 receptors depend on vascular endothelial growth factor status of primary breast cancer patients.

The aim of this study was to determine the association of dietary folate and cobalamin with plasma levels of Angiopoietins (ANG), vascular endothelial growth factor-C (VEGF-C) and tyrosine kinase receptor-2 (Tie-2) of primary breast cancer patients. Women (n = 177), aged 30 to 75 years diagnosed with breast cancer were recruited from an ongoing case series study. Dietary intake of nutrients was estimated by using a validated food frequency questionnaire. Enzyme-linked immunosorbent assay was applied to measure biomarkers. MCF-7 cell cultures were supplemented with folic acid (0-40 µM) for 24 h to measure cell viability and fold change of expression by the real-time reverse transcriptase-polymerase chain reaction. Structural equation modeling was applied to analyze the structural relationships between the measured variables of nutrients and Angiopoietins. Dietary intake of folate and cobalamin showed a significant inverse correlation with plasma ANG-1 and ANG-2 (P < 0.05), particularly in subjects with estrogen-receptor positive tumors or low plasma VEGF-C. Plasma folate was positively associated with the ratio of ANG-1/ANG-2 (P < 0.05). Residual intake levels of total cobalamin were inversely associated with plasma ANG-1 when plasma stratum of VEGF-C was high (P < 0.05). Structural equation modeling identified a significant inverse contribution of folate profiles on the latent variable of Angiopoietins (coefficient ß = -0.99, P < 0.05). Folic acid treatment resulted in dose-dependent down-regulations on ANGPT1 and ANGPT1/ANGPT2 ratio but VEGF and ANGPT2/VEGF were upregulated at folic acid >20 µM. Studying the contributing role of dietary folate to pro-angiogenic biomarkers in breast cancer patients can infer the preventive role of folate in the ANGs/VEGF-C-dependent cascade of tumor metastasis. By contrast, high concentrations of folic acid in vitro supported VEGF-C-dependent ANGPT2 overexpression might potentiate micro-lymphatic vessel development to support malignant cell dissemination.

Biomarkers associating endothelial Dysregulation in pediatric-onset systemic lupus erythematous.

BACKGROUND/PURPOSE: Endothelium is a key element in the regulation of vascular homeostasis and its alteration can lead to the development of vascular diseases. Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with potential extensive vascular lesions, involving skin vessels, renal glomeruli, cardiovascular system, brain, lung alveoli, gastrointestinal tract vessels and more. We aimed to assess endothelial dysregulation related biomarkers in pediatric-onset SLE (pSLE) patient serum and elucidate its correlation with their clinical features, laboratory parameters, and the overall disease activity. METHODS: Disease activities were evaluated by SLE disease activity index (SLEDAI). Patient characteristics were obtained by retrospective chart review. Six biomarkers associated with endothelial dysregulation, including Angiopoietin-1 (Ang-1), Angiopoietin-2 (Ang-2), Tie2, Vascular endothelial growth factor (VEGF), thrombomodulin, and a disintegrin-like and metalloprotease with thrombospondin type 1 motif (ADAMTS13) were tested through enzyme-linked immunosorbent assay (ELISA) measurement. RESULTS: This study comprised 118 pSLE patients. Data from 40 age-matched healthy controls were also obtained. The mean diagnostic age was 13 ± 4.12 years-old and 90.7% are females. Serum levels of VEGF, Tie2, thrombomodulin were significantly higher while serum ADAMTS13 was lower in active pSLE patients when compared to those with inactive diseases (all p < 0.05). In organ specific association, serum thrombomodulin level was higher in pSLE patient with renal involvement, and serum ADAMTS13 levels was negatively associated with neurological involvement (p < 0.05). A cutoff of thrombomodulin at 3333.6 pg/ml best correlated renal involvement. (AUC = 0.752, p < 0.01). CONCLUSION: Endothelial dysregulation associating proteins seems to be potent biomarkers for pSLE activity as well as organ involvement in pSLE patients. These biomarkers may be beneficial in understanding of the vascular pathogenesis and disease monitoring.

Physician Judgment and Circulating Biomarkers Predict 28-Day Mortality in Emergency Department Patients.

OBJECTIVES: To determine whether biomarkers of endothelial activation and inflammation provide added value for prediction of in-hospital mortality within 28 days when combined with physician judgment in critically ill emergency department patients. DESIGN: Prospective, observational study. SETTING: Two urban, academic emergency departments, with ≈80,000 combined annual visits, between June 2016 and December 2017. PATIENTS: Admitted patients, greater than 17 years old, with two systemic inflammatory response syndrome criteria and organ dysfunction, systolic blood pressure less than 90 mm Hg, or lactate greater than 4.0 mmol/L. Patients with trauma, intracranial hemorrhage known prior to arrival, or without available blood samples were excluded. INTERVENTIONS: Emergency department physicians reported likelihood of in-hospital mortality (0-100%) by survey at hospital admission. Remnant EDTA blood samples, drawn during the emergency department stay, were used to measure angiopoietin-1, angiopoietin-2, tumor necrosis factor receptor-1, interleukin-6, and interleukin-8. MEASUREMENTS AND MAIN RESULTS: We screened 421 patients and enrolled 314. The primary outcome of in-hospital mortality within 28 days occurred in 31 (9.9%). When predicting the primary outcome, the best biomarker model included angiopoietin-2 and interleukin-6 and performed moderately well (area under the curve, 0.72; 95% CI, 0.69-0.75), as did physician judgment (area under the curve, 0.78; 95% CI, 0.74-0.82). Combining physician judgment and biomarker models improved performance (area under the curve, 0.85; 95% CI, 0.82-0.87), with area under the curve change of 0.06 (95% CI, 0.04-0.09; p < 0.01) compared with physician judgment alone. CONCLUSIONS: Predicting in-hospital mortality within 28 days among critically ill emergency department patients may be improved by including biomarkers of endothelial activation and inflammation in combination with emergency department physician judgment.

Imbalanced serum levels of Ang1, Ang2 and VEGF in systemic sclerosis: Integrated effects on microvascular reactivity.

INTRODUCTION AND AIM: Microangiopathy is a hallmark of systemic sclerosis (SSc). It is a progressive process from an early inflammatory and proangiogenic environment to insufficient microvascular repair with loss of microvessels. The exact underlying mechanisms remain ill-defined. Aim of the study was to investigate whether imbalanced angiopoietins/VEGF serum profile should be related in SSc to the altered microvascular reactivity characterized by aberrant angiogenesis and avascularity. MATERIALS AND METHODS: Serum levels of Angiopoietin-1 (Ang1), Angiopoietin-2 (Ang2) and VEGF were measured by ELISA in 47 SSc patients and 27 healthy controls. Microvascular alterations were assessed by nailfold videocapillaroscopy (NVC). RESULTS: Serum concentrations of Ang1 were significantly lower [mean (S.D.): 21516.04 (11,441.035) pg/ml], and Ang2 significantly increased [25,89.55 (934.225) pg/ml] in SSc as compared with the control group [Ang1: 28,457.08 (10,431.905) pg/ml; Ang2: 1556.23 (481.255) pg/ml, p < 0.01, respectively], whereas VEGF did not differ significantly. The ratios of Ang1/Ang2 and Ang1/VEGF were significantly lower in SSc patients (8.346 ±â€¯4.523 and 95.17 ±â€¯75.0, respectively) than in healthy subjects (17.612 ±â€¯6.731 p < 0.000001 and 183.11 ±â€¯137.73; p = 0.004]. Formation of giant capillaries with vascular leakage and collapse was associated with significant increase in VEGF and concomitant Ang1 deficiency. Capillary loss was related to significant increase in VEGF with respect to those with preserved capillary number (395.12 ±â€¯256.27 pg/mL vs. 254.80 ±â€¯213.61 pg/mL) whereas elevated Ang2 levels induced more advanced capillary damage as indicated by the presence of the "Late" NVC pattern. CONCLUSIONS: We found that serum levels of Ang1, Ang2 and VEGF are differentially expressed in SSc and altered Ang1/Ang2 profile might underlay the aberrant angiogenesis in SSc despite increase in VEGF. For the first time we identified that significant deficiency of Ang1 might be involved in early capillary enlargement, followed by collapse and lack of stable newly-formed vessels in VEGF-enriched environment, whereas Ang2 levels seem to increase later in disease progression and advanced microvascular damage ("Late" NVC pattern).

Microcirculatory perfusion disturbances following cardiac surgery with cardiopulmonary bypass are associated with in vitro endothelial hyperpermeability and increased angiopoietin-2 levels.

BACKGROUND: Endothelial hyperpermeability following cardiopulmonary bypass (CPB) contributes to microcirculatory perfusion disturbances and postoperative complications after cardiac surgery. We investigated the postoperative course of renal and pulmonary endothelial barrier function and the association with microcirculatory perfusion and angiopoietin-2 levels in patients after CPB. METHODS: Clinical data, sublingual microcirculatory data, and plasma samples were collected from patients undergoing coronary artery bypass graft surgery with CPB (n = 17) before and at several time points up to 72 h after CPB. Renal and pulmonary microvascular endothelial cells were incubated with patient plasma, and in vitro endothelial barrier function was assessed using electric cell-substrate impedance sensing. Plasma levels of angiopoietin-1,-2, and soluble Tie2 were measured, and the association with in vitro endothelial barrier function and in vivo microcirculatory perfusion was determined. RESULTS: A plasma-induced reduction of renal and pulmonary endothelial barrier function was observed in all samples taken within the first three postoperative days (P < 0.001 for all time points vs. pre-CPB). Angiopoietin-2 and soluble Tie2 levels increased within 72 h after CPB (5.7 ± 4.4 vs. 1.7 ± 0.4 ng/ml, P < 0.0001; 16.3 ± 4.7 vs. 11.9 ± 1.9 ng/ml, P = 0.018, vs. pre-CPB), whereas angiopoietin-1 remained stable. Interestingly, reduced in vitro renal and pulmonary endothelial barrier moderately correlated with reduced in vivo microcirculatory perfusion after CPB (r = 0.47, P = 0.005; r = 0.79, P < 0.001). In addition, increased angiopoietin-2 levels moderately correlated with reduced in vitro renal and pulmonary endothelial barrier (r = - 0.46, P < 0.001; r = - 0.40, P = 0.005) and reduced in vivo microcirculatory perfusion (r = - 0.43, P = 0.01; r = - 0.41, P = 0.03). CONCLUSIONS: CPB is associated with an impairment of in vitro endothelial barrier function that continues in the first postoperative days and correlates with reduced postoperative microcirculatory perfusion and increased circulating angiopoietin-2 levels. These results suggest that angiopoietin-2 is a biomarker for postoperative endothelial hyperpermeability, which may contribute to delayed recovery of microcirculatory perfusion after CPB. TRIAL REGISTRATION: NTR4212 .

Biomarkers Associated With Response to Regorafenib in Patients With Hepatocellular Carcinoma.

BACKGROUND & AIMS: In a phase 3 trial (RESORCE), regorafenib increased overall survival compared with placebo in patients with hepatocellular carcinoma (HCC) previously treated with sorafenib. In an exploratory study, we analyzed plasma and tumor samples from study participants to identify genetic, microRNA (miRNA), and protein biomarkers associated with response to regorafenib. METHODS: We obtained archived tumor tissues and baseline plasma samples from patients with HCC given regorafenib in the RESORCE trial. Baseline plasma samples from 499 patients were analyzed for expression of 294 proteins (DiscoveryMAP) and plasma samples from 349 patients were analyzed for levels of 750 miRNAs (miRCURY miRNA PCR). Tumor tissues from 7 responders and 10 patients who did not respond (progressors) were analyzed by next-generation sequencing (FoundationOne). Forty-six tumor tissues were analyzed for expression patterns of 770 genes involved in oncogenic and inflammatory pathways (PanCancer Immune Profiling). Associations between plasma levels of proteins and miRNAs and response to treatment (overall survival and time to progression) were evaluated using a Cox proportional hazards model. RESULTS: Decreased baseline plasma concentrations of 5 of 266 evaluable proteins (angiopoietin 1, cystatin B, the latency-associated peptide of transforming growth factor beta 1, oxidized low-density lipoprotein receptor 1, and C-C motif chemokine ligand 3; adjusted P ≤ .05) were significantly associated with increased overall survival time after regorafenib treatment. Levels of these 5 proteins, which have roles in inflammation and/or HCC pathogenesis, were not associated with survival independently of treatment. Only 20 of 499 patients had high levels and a reduced survival time. Plasma levels of α-fetoprotein and c-MET were associated with poor outcome (overall survival) independently of regorafenib treatment only. We identified 9 plasma miRNAs (MIR30A, MIR122, MIR125B, MIR200A, MIR374B, MIR15B, MIR107, MIR320, and MIR645) whose levels significantly associated with overall survival time with regorafenib (adjusted P ≤ .05). Functional analyses of these miRNAs indicated that their expression level associated with increased overall survival of patients with tumors of the Hoshida S3 subtype. Next-generation sequencing analyses of tumor tissues revealed 49 variants in 27 oncogenes or tumor suppressor genes. Mutations in CTNNB1 were detected in 3 of 10 progressors and VEGFA amplification in 1 of 7 responders. CONCLUSION: We identified expression patterns of plasma proteins and miRNAs that associated with increased overall survival times of patients with HCC following treatment with regorafenib in the RESORCE trial. Levels of these circulating biomarkers and genetic features of tumors might be used to identify patients with HCC most likely to respond to regorafenib. ClinicalTrials.gov number NCT01774344. NCBI GEO accession numbers: mRNA data (NanoString): GSE119220; miRNA data (Exiqon): GSE119221.

CD34+ enriched cell products intended for autologous transendocardial CD34+ cell transplantation release significant amounts of angiopoietin-1.

OBJECTIVES: Cell-based therapy has emerged as a promising strategy for the treatment of patients with heart failure. Increasing evidence supports the hypothesis that paracrine mechanisms mediated by soluble factors released by the cells play a predominate role in reparative processes. The aim of our study was to analyze which cytokines are released by CD34+ enriched cell products intended for autologous transendocardial CD34+ cell transplantation in patients with cardiomyopathy. MATERIAL AND METHODS: The peripheral blood CD34+ cells from 12 patients were mobilized with granulocyte colony-stimulating factor, collected via apheresis and enriched by immunoselection. RESULTS: In CD34+ enriched cell population, hematopoietic, but not mesenchymal or endothelial, progenitors were detected. Except for angiopoietin-1, other measured cytokines (FGF1, FGF2, VEGF, PDGF, IL-6, HGH, SDF-1α/CXCL12, NRG1) were not released by CD34+ cells. The average concentration of angiopoietin-1 released by 5×106 CD34+ cells grown in neutral DMEM medium was 213.6±130.0pg/mL (range: 74-448pg/mL). Angiopoietin-1 secretion correlated well with CD34+ cell's capacity for generating colonies derived from hematopoietic progenitors (Pearson's correlation=0.964; P<0.001). CONCLUSION: Our study presents angiopoietin-1 as an interesting candidate and suggests future studies to explore how its release by CD34+ cells might impact the success of autologous CD34+ cell transplantation.

Angiopoietin Level Trajectories in Toddlers With Severe Sepsis and Septic Shock and Their Effect on Capillary Endothelium.

OBJECTIVE: Angiopoietins are postulated diagnostic biomarkers in children and adults with severe sepsis and septic shock. The diagnostic value of angiopoietins in children less than 5 years old has not been established, nor has their effect on permeability in the capillary microvasculature. We aim to determine if levels of angiopoietin-1 or -2 (angpt-1, -2) are diagnostic for severe sepsis/shock in young children and whether they affect the permeability of cultured human dermal microvascular endothelial cells (HDMEC). DESIGN: Prospective observational study of children < 5 years old. Patients were classified as non-systemic inflammatory response syndrome (SIRS), SIRS/sepsis and severe sepsis/septic shock. SETTING: Tertiary care pediatric hospitals. PATIENTS: Critically ill children. INTERVENTIONS: None. MEASUREMENTS: Plasma angpt-1 and -2 levels were measured with enzyme-linked immunoassays. Expression of angpt-2 in endothelial cells was assessed with quantitative polymerase chain reaction. Permeability changes in cultured HDMECs were assessed with transendothelial electrical resistance measurements. RESULTS: Angpt-1 levels were significantly higher in younger children compared with levels found in previous study of older children across disease severity (all P < 0.001). Angpt-2 was significantly higher in this cohort with severe sepsis/septic shock compared with children without SIRS and SIRS/sepsis (all P < 0.003). Angpt-2/1 ratio was also elevated in children with severe sepsis/septic shock but an order of magnitude less than older children (P < 0.02, P = 0.002). Angpt-1 and -2 did not affect basal HDMEC permeability or modulate leak in isolation or in the presence of tumor necrosis factor (TNF). CONCLUSIONS: Angpt-2 levels and the angpt-2/1 ratio are appropriate diagnostic biomarkers of severe sepsis/septic shock in children less than 5 years old. Neither angpt-1 nor -2 affects basal HDMEC permeability alone or modulates TNF induced capillary leak.

First trimester placental vascularization and angiogenetic factors are associated with adverse pregnancy outcome.

BACKGROUND: Hypertensive disorders, fetal growth restriction and preterm birth are major obstetrical complications and are related to impaired placentation. Early identification of impaired placentation can advance clinical care by preventing or postpone adverse pregnancy outcome. OBJECTIVES: Determine whether sonographic assessed placental vascular development and concomitant changes in inflammation- and/or angiogenesis-related serumproteins differ in the first trimester between uncomplicated pregnancies and pregnancies with adverse outcome. STUDY DESIGN: This prospective longitudinal study defines adverse pregnancy outcome as conditions associated with impaired placentation; fetal growth restriction, hypertensive disorder, preterm birth and placental abruption. The vascularization index, flow index, vascularization flow index and placental volume were determined at 8, 10 and 12 weeks pregnancy from 64 women using 3D power Doppler. Serum levels were analyzed for Angiopoetin-1 and -2, Leptin, VEGF-R, VEGF, and EGF. RESULTS: The vascularization index and vascular flow index increased in uneventful pregnancies with almost 50% between 8 and 12 weeks, resulting in a ∼50% higher vascularization index at 12 weeks compared to women with an adverse pregnancy outcome. Women with an adverse pregnancy outcome (n = 13) had significantly lower indices and placental volumes at all time points measured and these indices did not increase between 8 and 12 weeks. Reduced vascular development was associated with increased Angiopoietin-1 levels at 8 and 12 weeks and increased Leptin levels at 8 weeks. CONCLUSIONS: Pregnancies with an adverse outcome caused by conditions associated with impaired placentation differ from uneventful pregnancies in having reduced placental vascularization accompanied by elevated circulating levels of Angiopoietin-1 and Leptin already in the first trimester.

Fluid resuscitation associated with elevated angiopoietin-2 and length of mechanical ventilation after cardiac surgery.

BACKGROUND: Fluid restriction in patients with acute respiratory distress syndrome increases ventilator-free days while lowering plasma angiopoietin-2 (Ang-2), a marker of pulmonary endothelial injury. We hypothesised that fluid resuscitation may lead to endothelial injury after cardiac surgery and analysed Ang-2, angiopoietin-1 (Ang-1) and phospholipase A2 (PLA2) levels and the impact of fluid management on ventilation time. METHODS: Patients enrolled in a single-centre, prospectively randomised interventional study of liberal or conservative fluid resuscitation strategy had plasma Ang-2, Ang-1 and PLA2 levels measured at baseline (pre-operative), 6 and 24 hours after commencement of cardiopulmonary bypass, and analysed by linear mixed models as liberal v conservative (intention to treat) or high v low fluid group (actual treatment, ≥ 3250 mL of fluid administered), and further subclassified as EuroSCORE (European System for Cardiac Operative Risk Evaluation) II ≥ 0.9 or < 0.9. RESULTS: Over 9 months, 144 patients were randomly allocated to either liberal (n =74) or conservative (n =70) fluid. Patients in the liberal fluid arm tended to an increased Ang-2 (P =0.12) and had higher PLA2 levels (P =0.03). Based on actual fluid administered, Ang-2 levels were higher, the Ang-1/Ang-2 ratio lower, and the length of mechanical ventilation and intensive care unit (ICU) stay was longer in the high fluid group (P < 0.001). The highest levels of Ang- 2 and corresponding lowest Ang-1/Ang-2 ratio, along with longest length of mechanical ventilation and ICU stay, were found with both the liberal and high fluid groups in patients with a EuroSCORE II ≥ 0.9 (P < 0.01). CONCLUSION: Liberal fluid resuscitation after cardiac surgery was associated with both pulmonary endothelial injury and prolonged length of mechanical ventilation. CLINICAL TRIAL REGISTRATION: ACTRN12612000754842.