Imbalance between angiogenic and anti-angiogenic factors in sera from patients with large-vessel vasculitis.

OBJECTIVES: To investigate serum levels of a panel of angiogenic inducers (VEGF, FGF-2, Angiopoietin 1, -2, soluble VCAM-1) and inhibitors (angiostatin, endostatin, pentraxin-3) in patients with giant cell arteritis (GCA) and Takayasu's arteritis (TAK), in order to gain further insights into the molecular mechanisms driving angiogenesis dysregulation in large-vessel vasculitis (LVV). METHODS: Sera were obtained from 33 TAK patients and 14 GCA patients and from two groups of age-matched normal controls (NC). Disease activity was assessed using 18F-FDG PET/CT and clinical indices including NIH/Kerr criteria and ITAS. Angiogenic and anti-angiogenic factor serum levels were evaluated using commercial ELISA kits. Pentraxin 3 (PTX3) serum levels were evaluated by non-commercial ELISA, as already described. RESULTS: Among the angiogenic factors, only VEGF serum levels were significantly higher in TAK patients compared to NC. No difference was found between angiogenic factor levels in GCA patients compared to those detected in NC. Anti-angiogenic factor (Angiostatin, Endostatin, PTX3) serum levels were significantly higher in both GCA and TAK patients compared to NC. Significant associations were observed between VEGF and PTX3 levels and disease activity evaluated using PET scan and clinical indices. Cluster analysis based on PET scan scores in TAK patients showed significant ordered differences in VEGF and angiostatin serum levels. Indeed, we noted a progressive increase of VEGF and angiostatin from NC to the cluster including patients with the highest and more diffuse scan positivity. CONCLUSIONS: Our overall results demonstrate a circulating molecular profile characterised by a prevailing expression of anti-angiogenic soluble factors.

Biomarkers in systemic sclerosis: Their potential to predict clinical courses.

The concept of a biomarker was defined as "a characteristic marker that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention" by the National Institutes of Health Biomarkers Definitions Working group in 2001. Clinical features, disease progress, therapeutic response and prognosis are heterogeneous among patients with systemic sclerosis (SSc). Therefore, biomarkers that can predict these matters are required for the progress of clinical practice. At present, SSc-specific autoantibodies are the most useful biomarkers for diagnosis and predicting clinical features. Otherwise, biomarkers specific only for SSc have not been identified yet. The glycoprotein krebs von den Lungen-6, surfactant protein-D and CCL18 are promising serum biomarkers of SSc-related interstitial lung diseases. Serum/plasma levels of brain natriuretic peptide and serum N-terminal pro-brain natriuretic peptide have been used as biomarkers for SSc-related pulmonary arterial hypertension. Other potential serum/plasma biomarkers for fibrosis and vascular involvement of SSc are connective tissue growth factor, interleukin-6, CCL2, CXCL4, intercellular adhesion molecule (ICAM)-1, P-selectin, vascular endothelial growth factor, von Willebrand factor, endostatin, endoglin and endothelin-1. In our multicenter prospective studies of Japanese early SSc, serum ICAM-1 levels were predictive for subsequent respiratory dysfunction and serum levels of CXCL8 and P-selectin were predictive for subsequent physical disability. Further large, multicenter, prospective, longitudinal studies will be needed to identify and validate critical biomarkers of SSc.

Bosentan blocks the antiangiogenic effects of sera from systemic sclerosis patients: an in vitro study.

OBJECTIVES: In systemic sclerosis (SSc), clinical evidence has shown that Bosentan may foster the regeneration of the peripheral microcirculatory network. The aim of this study was to verify in vitro the influence of Bosentan on the angiogenic performance of dermal microvascular endothelial cells (MVECs) and its possible capacity to counteract the antiangiogenic effects of SSc sera. METHODS: Healthy dermal MVECs were challenged with Bosentan at different concentrations (0.1 µM, 1 µM, 10 µM) or with sera from patients with diffuse cutaneous SSc (n=8) and healthy subjects (n=8), alone or in combination with Bosentan (10 µM). Cell viability and chemoinvasion were determined by WST-1 and Boyden chamber assays, respectively. Angiogenesis was evaluated by capillary morphogenesis on Matrigel. RESULTS: Challenge of dermal MVECs with SSc sera induced a significant reduction in angiogenesis (p<0.005 vs. basal condition; p<0.001 vs. healthy sera). The addition of Bosentan could significantly restore angiogenesis in the presence of SSc sera (p<0.01 vs. SSc sera alone). Healthy sera promoted cell viability which was, instead, significantly reduced with SSc sera (p<0.005 vs. healthy sera). The addition of Bosentan to MVECs challenged with SSc sera significantly increased cell viability (p<0.005 vs. SSc sera alone), reaching levels similar to MVECs treated with healthy sera. Co-incubation of MVECs with Bosentan and SSc sera significantly increased chemoinvasion (p<0.005 vs. SSc sera alone) which was inhibited by SSc sera (<0.001 vs. healthy sera). CONCLUSIONS: Bosentan effectively counteracts the antiangiogenic effects of SSc sera on dermal MVECs and fosters the restoration of a proangiogenic environment.

Increase in both angiogenic and angiostatic mediators in patients with idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is associated with a marked pulmonary vascular remodeling. The aim of this study was to investigate a potential imbalance between angiogenic and angiostatic factors in this disease. METHODS AND RESULTS: Sixty-four subjects with IPF and 10 healthy control subjects (60-70 years old) were prospectively included in this multicenter study. Plasma levels of vascular endothelial growth factor A (VEGF-A), thrombospondin-1 (TSP-1) and stem cell factor (SCF) were determined by Elisa. Comparisons between IPF and controls were made using the Mann-Whitney U test. We also analyzed these soluble mediators in relation with IPF severity (DLCO<40% or>40%) predicted or total lung capacity (TLC) and forced vital capacity (FVC) (both<55% or>55% predicted) using the same test. VEGF-A plasma levels were increased in IPF vs. controls (P=0.0008) as well as those of TSP-1 (P=0.008), irrespective of the severity of the disease as reflected by DLCO, TLC or FVC values. In contrast, SCF levels were similar in IPF and controls. CONCLUSIONS: Factors modulating angiogenic responses are dysregulated in patients with IPF with increases in VEGF-A and TSP-1. The serial assessment of VEGF-A and TSP-1 during the follow-up and the search for potential relationships with the outcome of the disease might give us hints to the clinical implication of these results.

Imbalanced levels of angiogenic and angiostatic factors in vitreous, plasma and postmortem retinal tissue of patients with proliferative diabetic retinopathy.

A role for vascular endothelial growth factor (VEGF) has been clearly implicated in the pathogenesis of proliferative diabetic retinopathy (PDR). However, other molecules and mechanisms may be operating independently, or in conjunction with VEGF in the pathogenesis of this disease. Therefore, we made an attempt to comparatively investigate the levels of angiogenic and angiostatic factors in vitreous, plasma and postmortem retinal tissue of subjects with Proliferative Diabetic Retinopathy (PDR) compared to control subjects. The vitreous and plasma concentrations of VEGF, EPO (Erythropoietin) and PEDF (Pigment Epithelium Derived Factor) were measured using Enzyme Linked Immunosorbent Assay (ELISA) and the postmortem retinal tissue was subjected to Western blot analysis. The mean vitreous and plasma levels of VEGF and EPO in patients with PDR were significantly (p<0.001) higher than those in subjects without diabetes. Conversely, the vitreous and plasma levels of PEDF were significantly (p<0.001) lower in the PDR patients compared to control subjects. Multivariate logistic-regression analyses indicated that EPO was more strongly associated with PDR than VEGF. The protein expression of the VEGF and EPO in the retinal tissue was significantly higher in PDR and diabetes without complication groups compared to controls. Compared to controls, the protein expression of PEDF was significantly lower in retinal tissues from diabetes patients without complications and in patients with PDR. The fact that the vitreous and plasma levels and the retinal tissue protein expression of EPO were strongly associated with PDR implies a definite role of 'hypererythropoietinemia' in neovascularization processes.

Do anti-angiogenic or angiogenic factors contribute to the protection of birth weight at high altitude afforded by Andean ancestry?

OBJECTIVE: This prospective study was designed to determine whether variation in angiogenic (placental growth factor [PlGF]) and/or anti-angiogenic (soluble fms-like tyrosine kinase [sFlt-1]) factors contribute to the protective effect of highland ancestry (Andean) from altitude-associated reductions in fetal growth. STUDY DESIGN: Plasma sFlt-1 and PlGF levels, uterine artery (UA) blood flow, and fetal biometry were determined in low-altitude (400 m; Andean n = 27, European n = 28) and high-altitude (3600 m; Andean n = 51, European n = 44) residents during pregnancy (20 and 36 weeks) and 4 months postpartum. RESULTS: High-altitude decreased sFlt-1 levels in both groups, Andeans had lower sFlt-1, comparable PlGF, lower sFlt-1/PlGF ratios, and higher UA blood flow throughout pregnancy relative to Europeans. Altitude decreased birth weight in Europeans but not Andeans. In high-altitude Europeans sFlt-1/PlGF and sFlt-1 levels were negatively associated with UA diameter and birth weight, respectively. CONCLUSIONS: Lower sFlt-1 and sFlt-1/PLGF ratio may contribute to or result from variations in maternal vascular adaptation to pregnancy between Andean and Europeans at high altitude. Subsequently, these effects could potentially influence ancestry-associated differences in birth weight.

Expression of angiostatin and its related factors in the plasma of newborn pigs with hypoxia and reoxygenation.

Little is known about angiostatin and its related factors in the hypoxia-reoxygenation of neonates. In this study we compared the effect of 21% and 100% reoxygenation on temporal changes in the plasma level of these factors in newborn piglets subjected to hypoxia. Newborn piglets were subjected to 2 h hypoxia followed by 1 h of reoxygenation with either 21% or 100% oxygen and observed for 4 days. On day 4 of recovery in 100% hypoxic-reoxygenated group, there were increases in total angiostatin, plasminogen/plasmin and MMP-2 levels, and decreases in VEGF levels (vs. respective baseline levels, all P <0.001), whereas no significant temporal changes were found in the 21% hypoxic-reoxygenated and sham-operated groups. Angiostatin levels correlated positively with the levels of MMP-2 and HIF-1alpha and negatively with VEGF levels in 100% hypoxic-reoxygenated group (all P <0.05). In comparison to 21% oxygen, neonatal resuscitation with 100% oxygen was found to increase the levels anti-angiogenic factors.