Ang-1 Ratio Research Articles

Angiogenic growth factors in rheumatoid arthritis

We investigated whether the angiogenic profile, which is based on the local expression and systemic levels of angiogenic growth factors (VEGF, Ang-1, Ang-2, and the corresponding receptors), differs between rheumatoid arthritis (RA) and osteoarthritis (OA) patients. We determined the expression of VEGF, Ang-1, and Ang-2 together with its receptors (VEGFR-1/-2 and Tie2) in synovium tissue (ST) and muscular tissue (MT) from patients with RA and OA using quantitative PCR. Tissue samples were obtained from 15 RA and 19 OA patients during total knee arthroplasty. Control MT samples (n = 10) were obtained during spinal surgery. Results are correlated to VEGF and angiopoietin serum levels via ELISA measurements. The VEGF expressions in ST and serum levels were significantly higher in RA patients than in OA patients (P < 0.05). Furthermore, the VEGFR-1 and VEGFR-2 expression in ST from RA patients were significantly higher than in OA patients (P < 0.001 and P < 0.05). The relative concentration of angiopoietins (Ang-1/Ang-2 ratio) was significantly increased in RA (P < 0.01). Serum levels for Ang-2 showed no significant differences. Statistical analysis showed a significant higher level of Tie2 in RA patients (P < 0.001). Analysis of local levels of VEGF, VEGFR-1, VEGFR-2, Ang-1, Ang-2, and Tie2 in the muscular tissue showed no significant difference between RA and OA patients. These results underline the importance of pro-angiogenic growth factor levels for RA corroborating the assumption that VEGF and angiopoietins play an important role in the pathogenesis of RA.

Changes and significance of Serum Angiopoietin-1 and Angiopoietin-2 levels in Patients with coronary heart disease

ObjectiveAngiopoietin-1 (Ang-1) and Angiopoietin-2 (Ang-2) are the members of angiopoietin families. The regulation between Ang-1 and Ang-2 plays an important role in accommodating endothelial cell function and inflammation. Several recent...

Circulating angiopoietin‐1 to angiopoietin‐2 ratio is an independent prognostic factor for survival in newly diagnosed patients with multiple myeloma who received therapy with novel antimyeloma agents

The circulating levels of several angiogenic cytokines [angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), vascular endothelial growth factor (VEGF), angiogenin and basic fibroblast growth factor (bFGF)] were evaluated in 174 consecutive patients with newly diagnosed, symptomatic, multiple myeloma (MM). Circulating levels of Ang-1/Ang-2 were reduced in myeloma patients compared to controls, whereas VEGF and angiogenin levels were increased. Reduced angiopoietin-1/angiopoietin-2 ratio correlated with advanced disease features including international staging system (ISS)-3 stage, renal impairment and extensive bone disease. Based on immunohistochemical results in 20 patients (10 with the higher and 10 with the lower values of circulating angiopoietin-2) we found that angiopoietin-2 is expressed by myeloma cells and correlates with increased microvessel density in subsets of patients. Furthermore, Ang-1/Ang-2 ratio correlated with survival. Patients with circulating Ang-1/Ang-2 below or equal to the median value (6.03) had a median survival of 26.3 months compared to 53 months of all others (p = 0.002). Interestingly, this was mainly observed in patients who received first-line therapy with novel agent-based regimens (65% of our patients). Furthermore, a subset of ISS-3 patients with serum Ang-1/Ang-2 above the median value had favourable prognosis (median survival: 45 months versus 17 months of all others; p = 0.0001). The multivariate analysis revealed that low Ang-1/Ang-2 ratio could independently predict for inferior survival in our cohort of patients (relative risk (RR) 2.07, 95% CI 1.50-2.42; p < 0.001). These results highlight the role of angiopoietins pathway in the biology of MM and reveal novel targets for the development of antimyeloma agents.

Tangiopoietins-1 and -2 are differentially regulated and make distinct contributions to synovial Tie2 activation in patients with rheumatoid arthritis and psoriatic arthritis

Background and objectivesThe angiogenic factors angiopoietin-1 (Ang-1) and Ang-2 are differentially expressed in the serum and synovial fluid of patients with different forms of arthritis. Ang-1 and Ang-2 contribute to...

204: Regional variation in placental expression of angiopoietin-1 and angiopoietin-2 in normal pregnancies

Fetal growth and development are dependent on the adequacy of placental angiogenesis. Angiopoietin 1 (Ang-1) and angiopoietin 2 (Ang-2) are known to be involved in this process. However, there is a dearth of information on the regional variations in Ang-1 and Ang-2 activity in human placenta. Such information would elucidate molecular regulation of placental angiogenesis in health and disease. The aim of this study was to test the hypothesis that there are regional variations in Ang-1 and Ang-2 expression, and the Ang-1/Ang-2 ratio in human placenta.

Imbalances in serum angiopoietin concentrations are early predictors of septic shock development in patients with post chemotherapy febrile neutropenia

BackgroundFebrile neutropenia carries a high risk of sepsis complications, and the identification of biomarkers capable to identify high risk patients is a great challenge. Angiopoietins (Ang -) are cytokines involved in the control microvascular permeability. It is accepted that Ang-1 expression maintains endothelial barrier integrity, and that Ang-2 acts as an antagonizing cytokine with barrier-disrupting functions in inflammatory situations. Ang-2 levels have been recently correlated with sepsis mortality in intensive care units.MethodsWe prospectively evaluated concentrations of Ang-1 and Ang-2 at different time-points during febrile neutropenia, and explored the diagnostic accuracy of these mediators as potential predictors of poor outcome in this clinical setting before the development of sepsis complications.ResultsPatients that evolved with septic shock (n = 10) presented higher levels of Ang-2 measured 48 hours after fever onset, and of the Ang-2/Ang-1 ratio at the time of fever onset compared to patients with non-complicated sepsis (n = 31). These levels correlated with sepsis severity scores.ConclusionsOur data suggest that imbalances in the concentrations of Ang-1 and Ang-2 are independent and early markers of the risk of developing septic shock and of sepsis mortality in febrile neutropenia, and larger studies are warranted to validate their clinical usefulness. Therapeutic strategies that manipulate this Ang-2/Ang-1 imbalance can potentially offer new and promising treatments for sepsis in febrile neutropenia.

Amniotic Fluid Angiopoietin-1, Angiopoietin-2, and Soluble Receptor Tunica Interna Endothelial Cell Kinase-2 Levels and Regulation in Normal Pregnancy and Intraamniotic Inflammation-Induced Preterm Birth

Angiopoietin-1 (Ang-1) and Ang-2 act selectively on endothelial cells by engaging the Tunica interna endothelial cell kinase-2 (Tie2) receptor. A soluble form of Tie2 (sTie2) blocks angiopoietin bioactivity. The aim of the study was to characterize changes and expression patterns of Ang-1, Ang-2, and sTie2 in amniotic fluid (AF) and placenta during human pregnancy and intraamniotic inflammation (IAI)-induced preterm birth. We conducted a cross-sectional study at a tertiary university hospital. AF levels of Ang-1, Ang-2, and sTie2 were evaluated in 176 women during second trimester (n = 40), third trimester (n = 37), and preterm labor (positive IAI, n = 50; negative IAI, n = 49). Placenta and cord blood of select women were analyzed. Ang-1, Ang-2, sTie2, and IL-6 were evaluated by ELISA. Real-time PCR measured Ang-1, Ang-2, and Tie2 placental mRNA levels. Placenta was immunostained for Ang-1 and Ang-2. Placental explant cultures were stimulated with lipopolysaccharide, Pam3Cys, and modulators of protein synthesis/secretion (cycloheximide, monensin, and brefeldin A). In normal pregnancy, the levels and ratios of AF Ang-1, Ang-2, and sTie2 varied with gestational age (GA) (P < 0.001). PCR revealed corresponding changes in placental Ang-1 and Ang-2, but not Tie2, mRNA. IAI raised AF Ang-1, Ang-2, and sTie2 above the expected level for GA without affecting their placental mRNA. Ang-2 immunoreactivity appeared enhanced in areas of villous edema. AF Ang-2/Ang-1 ratio was an important determinant of cord blood IL-6 (P < 0.001). Ex-vivo, sTie2 release was increased by Golgi disrupting but not bacterial mimic agents. Ang-1, Ang-2, and sTie2 are physiological constituents of AF that are GA and IAI regulated. Ang-2/Ang-1 ratio may play a role in modulating the fetal inflammatory response to IAI. Placental sTie2 shedding likely involves a Golgi-mediated mechanism.

Dysregulation of Angiopoietins Is Associated with Placental Malaria and Low Birth Weight

BackgroundPlacental malaria (PM) is associated with adverse pregnancy outcomes including low birth weight (LBW). However, the precise mechanisms by which PM induces LBW are poorly defined. Based on the essential role of angiopoietin (ANG)-1 and -2 in normal placental vascular development, we hypothesized that PM may result in the dysregulation of angiopoietins and thereby contribute to LBW outcomes.Methods and FindingsIn a mouse model of PM, we show that Plasmodium berghei ANKA infection of pregnant mice resulted in dysregulated angiopoietin levels and fetal growth restriction. PM lead to decreased ANG-1, increased ANG-2, and an elevated ratio of ANG-2/ANG-1 in the placenta and the serum. These observations were extended to malaria-exposed pregnant women: In a study of primigravid women prospectively followed over the course of pregnancy, Plasmodium falciparum infection was associated with a decrease in maternal plasma ANG-1 levels (P = 0.031) and an increase in the ANG-2:ANG-1 ratio (P = 0.048). ANG-1 levels recovered with successful treatment of peripheral parasitemia (P = 0.010). In a cross-sectional study of primigravidae at delivery, angiopoietin dysregulation was associated with PM (P = 0.002) and LBW (P = 0.041). Women with PM who delivered LBW infants had increased ANG-2:ANG-1 ratios (P = 0.002) compared to uninfected women delivering normal birth weight infants.ConclusionsThese data support the hypothesis that dysregulation of angiopoietins is associated with PM and LBW outcomes, and suggest that ANG-1 and ANG-2 levels may be clinically informative biomarkers to identify P. falciparum-infected mothers at risk of LBW deliveries.

Angiopoietin-1/Angiopoietin-2 Ratio for Prediction of Preeclampsia

A number of different biophysical and biochemical markers have been proposed as predictors of preeclampsia. Factors involved in the angiogenic balance are suggested as candidate markers. The purpose of this prospective, longitudinal cohort study was to determine whether a ratio between Angiopoietin-1 (Ang-1) and Angiopoietin-2 (Ang-2) can be used to predict preeclampsia in a low-risk population. A cohort of healthy pregnant women (n = 469) were enrolled at gestational weeks 8-12. Plasma samples were collected at gestational weeks 10, 25, 28, 33, and 37. By using commercially available enzyme-linked immunosorbent assay kits Ang-1 and Ang-2 were analyzed. The median Ang-1/Ang-2 ratio increased during pregnancy in all women, but the ratios were significantly lower at gestational weeks 25 and 28 in women who later developed preeclampsia than in normal pregnant women (1.49 compared to 2.19 and 2.12 compared to 3.54, P < 0.05 and P < 0.05). Our data indicate that in a low-risk population of women the Ang-1/Ang-2 ratio in plasma constitutes a possible biomarker for prediction of later onset of preeclampsia.

Implications Of Angiogenic Factors And Thrombospondin-1 Underlying Capillary Regression In Chronically Unloaded Rat Soleus Muscle

Capillary regression is induced by chronic unloading of skeletal muscles. The mechanisms involved, however, are not fully understood. At least two independent mechanisms can be considered, i.e., adaptations in the levels of specific angiogenic factors such as vascular endothelial growth factor (VEGF) and angiopoietin, and of thrombospondin-1 (TSP-1), an inhibitor of angiogenesis that induces apoptosis in endothelial cells. PURPOSE: The aim of present study was to determine the responses of pro- and anti-angiogenic factors in rat soleus capillaries after 2 weeks of hindlimb unloading (HU). METHODS: Male Wistar rats (8-9 weeks of age) were assigned randomly into a control or HU group. Frozen transverse sections from the soleus mid-belly were stained for alkaline phosphatase which is present in the capillary endothelium. Apoptotic endothelial cells were identified by von Willebrand factor and DNA fragmentation (TUNEL) using immunofluorescent staining. VEGF, KDR, Flt-1, angiopoietin-1 (Ang-1), Ang-2, Tie-2, HIF 1 alpha, and TSP-1 mRNAs were determined by TaqMan probe-based real-time PCR reactions. RESULTS: The capillary-to-fiber ratio was 23% lower in HU than control rats. TUNEL-positive endothelial cells were observed only in HU rats and TSP levels were higher in HU than control rats, suggesting capillary regression with vascular endothelial cell apoptosis. mRNA levels of angiogenic factors VEGF and Ang-1, their receptors, and HIF 1 alpha were lower in HU than control rats whereas Ang-2 levels were unaffected. As a result, the Ang-2/Ang-1 ratio increased, suggesting that the vasculature was destabilized. Ang-2 expressed in the absence of VEGF also results in vessel regression. CONCLUSIONS: These data indicate that a decrease in angiogenic factors and an increase in TSP-1 expression may play an important role in capillary regression associated with chronic unloading. Supported by Grants-in-Aid for Science Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology, 19650148, and 19300196.

Angiopoietin 2 and Cardiovascular Disease in Dialysis and Kidney Transplantation

Accelerated atherosclerosis in patients with chronic kidney disease (CKD) is still incompletely understood. Angiopoietin 1 (Ang-1) and Ang-2 are 55-kDa antagonistic nonredundant gatekeepers of endothelial activation and thus are potential important factors in accelerated atherosclerosis. We aimed to study: (1) angiopoietin levels in patients treated by means of dialysis and kidney transplantation, (2) the association of altered angiopoietin levels with atherosclerosis, and (3) changes in altered levels after renal transplantation. Cross-sectional and longitudinal observational study. 117 patients with CKD (61 hemodialysis [HD] patients, 24 peritoneal dialysis [PD] patients, and 32 renal transplant recipients) and 22 healthy controls. Treatment by means of HD or PD or renal transplantation versus healthy controls. Serum Ang-1 and Ang-2 levels and ratio and changes in levels before and 3 months after transplantation. Correlations of angiopoietin levels with the presence and severity of coronary heart disease and peripheral arterial disease. Ang-1 and Ang-2 were measured in sera by using an immunoradiometric sandwich assay and enzyme-linked immunosorbent assay, respectively. Coronary heart disease was scored by using coronary angiography, and peripheral arterial disease, by using ultrasonography. Ang-1 level was decreased in HD patients compared with controls (29.1 +/- 12 versus 45.3 +/- 11.5 ng/mL; P < 0.001). In contrast, Ang-2 level was increased (HD, 8.7 +/- 0.64; PD, 6.48 +/- 8.1 ng/mL versus controls, 0.88 +/- 0.43 ng/mL; P < 0.001). Ang levels in renal transplant recipients were not different from healthy controls. Longitudinally, individual Ang-2 levels decreased after kidney transplantation (P = 0.01). In addition, in patients with CKD, Ang-2 level correlated significantly with scores of coronary heart disease (r = 0.486; P < 0.001) and peripheral arterial disease (r = 0.648; P < 0.001). Cross-sectional study design. Circulating Ang-2 level was increased in patients treated with dialysis, although the mechanism is unknown. Kidney transplantation normalized circulating Ang-2 levels after 3 months. In addition, Ang-2 might be a mediator (and thus a marker) that accounts for accelerated atherosclerosis in dialysis patients.

The absence of angiopoietin-2 leads to abnormal vascular maturation and persistent proliferative retinopathy

Angiopoietin-2 (Ang-2) antagonises the maturing effect of angiopoietin-1 (Ang-1) on blood vessels, and cooperates with VEGF to induce neovascularisation. In knockout mice, Ang-2 displayed a specific role in postnatal angiogenic remodelling. Here, we demonstrate that mice deficient in Ang-2 fail to form a proper spatial retinal vascular network. The retinal vasculature was characterised by reduced large vessel numbers and defects forming the superficial periphery mostly on the arteriolar site, and the secondary and tertiary deep capillary network. Hypoxia in the retinal periphery induced a four-fold VEGF upregulation and active endothelial proliferation for up to 60 days. Concomitantly, retinal digest preparations showed increased arteriolar (+33%) and capillary diameters (+90%), and fluorescein angiograms revealed leakiness of neovascular front. At one year of age, persistent preretinal vessels were non-leaky in accordance with a relative increase in the ratio of Ang-1 to VEGF. Taken together, the data suggest that Ang-2 has an important function in the spatial configuration of the three-dimensional retinal vasculature. Secondarily, prolonged VEGF activity results in a model of persistent proliferative retinopathy.

Differential regulation of angiogenic factors by radiation in rat brain

It has been hypothesized that radiation‐induced brain injury is a consequence of rarefaction of cerebral microvasculature. In the present study, we investigated the molecular regulatory mechanisms of radiation‐induced vessel rarefaction in the brain. Four month old F344×BN rats received either whole brain irradiation with a single dose of 10 Gy γ‐rays or sham‐irradiation, and were maintained for 4, 8, and 24 h following irradiation. The expression levels of a variety of angiogenic factors such as angiopoietin‐1 (Ang‐1), angiopoietin‐2 (Ang‐2), endothelial receptor tyrosine kinase (Tie‐2), and vascular endothelial growth factor (VEGF) were analyzed by quantitative real‐time RT‐PCR. A significant down‐regulation of Ang‐1 and Tie‐2 was observed in hippocampal and cortical regions isolated from irradiated animals. In addition, irradiation significantly attenuated the mRNA and protein expression levels of VEGF. In contrast, Ang‐2 expression was significantly and dramatically up‐regulated in the irradiated brain, demonstrating that irradiation markedly reduces the ratio of Ang‐1/Ang‐2 and increases the ratio of Ang‐2/VEGF. These data provide the first evidence that vascular plasticity may be impaired in irradiated animals though differential regulation of Ang‐1, Tie‐2, Ang‐2 and VEGF in the brain, and this may contribute to cerebrovascular rarefaction. (This work was supported by NINDS R01NS056218)

Aberrant expression of angiopoietins-1 and -2 and vascular endothelial growth factor-A in peri-implantation endometrium after gonadotrophin stimulation

Ovarian stimulation affects normal endometrial development. The expression of angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2) and vascular endothelial growth factor-A (VEGF-A) and the vascular state in the peri-implantation endometrium in women with natural and gonadotrophin-stimulated cycles were compared. The expression of these angiogenesis-associated molecules in endometrial biopsies, collected on Day 7 after human chorionic gonadotrophin injection or luteinizing hormone surge in stimulated or natural cycles respectively, or at mid-luteal phase of women undergoing diagnositic laparoscopy, were analysed. Women with gonadotrophin-stimulation had lower Ang-1, but higher Ang-2, mRNA and protein expression (P < 0.05), and increased concentrations of von Willebrand factor (vWF) and blood vessel density than those with natural cycles (P < 0.05). Although stimulated cycles had higher VEGF-A mRNA expression (P = 0.023), VEGF-A protein expression was similar between the groups. Lower Ang-1/Ang-2 but higher Ang-2/VEGF-A mRNA ratios (P = 0.025) were found after gonadotrophin-stimulation. The ratios were negatively (P < 0.001) and positively correlated (P < 0.001) with estradiol levels, respectively. Cyclical changes in Ang-1 and Ang-2, but not in VEGF-A expression were noted. The decreased Ang-1 concentration and Ang-1/Ang-2 ratio and the increased Ang-2 concentration, with the increased vWF concentration and blood vessel density, in stimulated cycles suggests advanced endometrial angiogenesis after gonadotrophin-stimulation.

The Ratio of Angiopoietin-1 to Angiopoietin-2 Is Reduced and Predicts Independently for Survival in Newly Diagnosed Patients with Multiple Myeloma.

Angiogenesis represents an essential step of disease progression in several hematological malignancies, including multiple myeloma (MM). Angiopoietin-1 (Ang-1) and its natural antagonist angiopoietin-2 (Ang-2), both ligands for the receptor tyrosine kinase Tie-2, are essential cytokines for angiogenesis process. Maturation and stabilization of the vascular wall are critically regulated by Ang-1 binding to Tie-2 receptor, while Ang2- antagonizes Tie-2 binding and induces vessel destabilization, which leads to the angiogenic sprouting. Vascular endothelial growth factor (VEGF), angiogenin and basic fibroblast growth factor (bFGF) are also potent stimulators of both physiological and pathological angiogenesis. The aim of this study was to evaluate the serum levels of the above angiogenesis cytokines and explore possible correlations with clinical data, including survival, in 143 newly diagnosed, untreated, MM patients (75M/68F; median age: 68 years, range: 40–94 years). According to ISS, 49 patients had stage 1, 46 stage 2 and 48 stage 3 disease. Serum levels of Ang-1, Ang-2, VEGF, VEGF-A (the major angiogenesis component of VEGF), angiogenin, and bFGF were evaluated using ELISA methodology (R&D Systems, Minneapolis, MN, USA, for all, except VEGF-A: Diaclone, Bensancon, France). MM patients had increased serum levels of Ang-2 (p<0.0001), angiogenin (p<0.0001), VEGF (p=0.033) and VEGF-A (p=0.010) compared with 25 controls of similar age and gender. Ang-1 levels were not different between patients and controls; thus the ratio of Ang-1/Ang-2 was reduced in MM (p<0.0001). Ang-1/Ang-2 ratio correlated with ISS (p=0.022) and beta2-microglobulin (p=0.03), while angiogenin showed strong correlations with ISS (p<0.0001), bone disease status (p=0.01) and hemoglobin (p=0.01). Interestingly, VEGF levels correlated with both Ang-1 (p<0.0001) and Ang-2 (p<0.0001) as well as with bFGF (p<0.0001) and LDH (p=0.01) serum levels. The median survival of all patients was 47 months and the median follow-up was 20 months. The univariate analysis revealed that ISS stage (p=0.001), serum LDH (p=0.001), serum beta2-microglobulin (p=0.0002), bone disease status (p=0.0007), serum creatinine (p=0.045), and the ratio of Ang-1/Ang-2 predicted for survival. Patients with serum Ang-1/Ang-2 of below or equal to the median value (4.8) had a median survival of 25 months, while patients with Ang-1/Ang-2 values of above the median value of 4.8 had a median survival of 53 months (p=0.0065). The multivariate analysis revealed that only serum LDH (p=0.003), Ang-1/Ang-2 ratio (p=0.005) and bone disease status (p=0.015) could independently predict for survival. These results reveal for the first time in MM patients the correlation of reduced Ang-1/Ang-2 ratio with advanced disease and highlight the role of Ang-1/Ang-2 pathway in the biology of plasma cell growth as reflected by its influence on survival. These observations reveal Ang-1/Ang-2/Tie-2 system as a possible target for the development of novel anti-myeloma agents.

High Serum Levels of Angiogenic Cytokines in AL Amyloidosis Correlates with Disease Features.

Angiogenesis is a crucial step for disease progression in several hematological malignancies, including plasma cell dyscrasias like multiple myeloma (MM). Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) are antagonistic ligands for the receptor tyrosine kinase Tie-2. These cytokines are crucial for maturation and stabilization of the vascular wall: Ang-1 binds to Tie-2 and stabilizes the vascular wall, while Ang2- antagonizes Tie-2 binding and induces vessel destabilization, which leads to the angiogenic sprouting in conjunction with onset of angiogenesis. Vascular endothelial growth factor (VEGF), angiogenin and basic fibroblast growth factor (bFGF) are also potent stimulators of both physiological and pathological angiogenesis. However little is known about the role of these molecules in AL amyloidosis and their possible correlations with the features of the disease. Serum levels of Ang-1, Ang-2, VEGF, and VEGF-A (the major angiogenesis component of VEGF), angiogenin, and bFGF were evaluated using ELISA methodology (R&D Systems, Minneapolis, MN, USA, for all, except VEGF-A: Diaclone, Bensancon, France). Serum samples were collected from 62 previously untreated AL amyloidosis patients as well as from 35 age-matched healthy controls and 35 newly diagnosed, untreated, myeloma patients. Microvascular Density (MVD) of the bone marrow (BM) was also measured in 15 AL patients who had both serum and trephine biopsies available. Definition of organ involvement was based on established criteria (Gerrtz et al Am J Hematol 2005). Serum levels of VEGF (p<0.001), angiogenin (p<0.001), Ang-1(p=0.002) and Ang-2 (p<0.001) were significantly higher in AL patients than in controls; however the ang1/ang2 ratio was not different. The levels of Ang-2 (r=0.776, p=0.005), angiogenin (r=0.746, p=0.008) and bFGF ((r=0.743, p=0.009) correlated with MVD in BM biopsies of AL patients while VEGF, VEGF-A and Ang-1 were not. When compared to MM patients, AL patients had significantly higher VEGF levels (p=0.007), angiogenin (p=0.003) and Ang-1(p<0.001) levels but lower angiopoietin-2 levels (p=0.09); thus the Ang-1/Ang-2 ratio was higher in AL compared to MM (p=0.08). Both VEGF and VEGF-A were increased in patients with symptoms of peripheral neuropathy (p=0.016 and 0.029 respectively), while Ang-2 was increased in patients with heart involvement (p=0.03). There were no differences between AL patients with a creatinine ≥2 mg/dl and those with creatinine<2 mg/dl. In patients with BM plasma cells (PCs)>10%, Ang-1 was lower than in those with PCs<10% (p=0.013), while Ang-2 was not different (p=0.170); thus the Ang-1/Ang-2 ratio was lower in patients with BMPCs >10% (p=0.021). There was no correlation among the number of involved organs, a significant predictor of survival in AL amyloidosis, and levels of angiogenic cytokines. In conclusion, serum markers of angiogenesis are significantly higher in AL patients compared to both healthy individuals and MM patients indicating a possible pathogenetic role in some features of the disease and also a possible therapeutic target.

The Combination of Bortezomib, Doxorubicin, and Dexamethasone (PAD) Is an Effective Regimen for High Risk, Newly Diagnosed, Patients with Multiple Myeloma, Reduces Bone Resorption and Normalizes Angiopoietin-1 to Angiopoietin-2 Ratio.

Bortezomib has significant activity in multiple myeloma (MM). Its efficacy is increased with the addition of dexamethasone and doxorubicin in vitro, thus providing the rationale for combination regimens with these agents. The aim of this study was to evaluate the efficacy and safety of PAD regimen (bortezomib, doxorubicin, dexamethasone) in high-risk, newly diagnosed, MM patients and evaluate its effect on bone remodeling and angiogenesis. The inclusion criteria included newly diagnosed MM, ISS 2/3 disease or del13q detected by FISH. Patients received four 21-day cycles of PAD: bortezomib 1.3 mg/m2 on days 1, 4, 8 and 11; dexamethasone 40 mg on days 1–4 and 8–11; bolus doxorubicin 9 mg/m2 on days 1–4. All patients received monthly zoledronic acid and prophylactic dose of co-trimoxazole and acyclovir. Following peripheral blood stem cell (PBSC) collection, eligible patients received high-dose melphalan with PBSC transplantation. Effect of PAD on angiogenesis was evaluated by measuring serum levels of VEGF, VEGF-A, angiogenin, angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), and basic fibroblast growth factor at baseline and on day 21 of cycle 4. Bone remodeling was studied by the measurement of serum indices:i.osteoclast stimulators [soluble RANKL, and osteoprotegerin (OPG)],ii.bone resorption markers [C-telopeptide of collagen type-I (CTX), and tartrate resistant acid phosphatase-5b (TRACP-5b)], andiii.bone formation markers [bone alkaline phosphatase (bALP), and osteocalcin] at baseline and on day 21 of cycle 4.All above molecules were also measured in 22 healthy controls of similar age and gender. To-date, 23 patients (14M/9F, median age 60 years) completed 4 cycles of therapy: 12 (52%) had ISS stage 2 and 11 (47%) stage 3 disease. Del13q was detected in 12 patients. The majority of patients (n=12) had more than 3 lytic lesions and/or a pathological fracture in the plain radiography of the skeleton. The objective response rate was 95% (22/23 patients): CR 26%, vgPR 13% and PR 56%. Median time to response was 35 days. Grade 3/4 adverse events included infections (7 patients-30%; one died due to septicemia), lymphopenia (6-26%), thrombocytopenia (6–26%), neutropenia (4–17%), peripheral neuropathy (3–13%), fatigue (2–8%), and hyponatremia (2–8%). At baseline, MM patients had increased serum levels of CTX, TRACP-5b, OPG, angiogenin, and Ang-2 compared with controls (p<0.01), while the ratio of Ang-1/Ang-2 was reduced. The administration of PAD resulted in a dramatic reduction of bone resorption markers (p<0.01) and a borderline increase in bALP (p=0.09). PAD also produced a significant increase of Ang-1/Ang-2 ratio (p=0.006), which was normalized. No patient developed a skeletal related event during 4 cycles of therapy. Eight patients (34%) had a PBSC collection; the median number of CD34+ cells was 6.45x106/kg (range: 2.3-13x106cells/kg). In conclusion, PAD has significant activity in high-risk, newly diagnosed patients with MM, overriding del13q. This regimen reduces bone resorption and normalizes Ang-1/Ang-2 balance which is crucial for the process of angiogenesis in MM.

High Bone Turnover and Increased Angiogenesis Cytokines in Patients with POEMS Syndrome.

POEMS syndrome is defined by the presence of peripheral neuropathy (P), monoclonal plasma cell disorder (M), organomegaly (O), endocrinopathy (E), and skin changes (S). Virtually all patients will have sclerotic bone lesions, although the mechanisms of their development have not been clarified to-date. Increased plasma level of VEGF is another characteristic of the disease. However, there is very little information for the role of other angiogenic molecules in POEMS pathogenesis. The aim of this study was to evaluate bone metabolism and angiogenic cytokines in POEMS and compare the results with multiple myeloma (MM) and osteosclerotic patients of other etiology. Seven patients with POEMS at diagnosis (6M/1F; median age: 49 years) were evaluated. A series of bone remodeling indices were measured: i) bone resorption markers [C- and N- telopeptide of type-I collagen (CTX and NTX), TRACP-5b], ii) bone formation markers [bone-alkaline phosphatase, osteocalcin (OC), and C-terminal propeptide of type-I collagen (CICP)], and iii) osteoclast regulators [soluble RANKL, osteoprotegerin (OPG), MIP-1α, and TNF-α]. Furthermore, the following angiogenic cytokines were measured in the plasma of POEMS patients: VEGF, VEGF-A, angiogenin, angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), and bFGF. All above molecules were also measured in 25 newly diagnosed, untreated MM patients (14M/11F, median age: 64 years) and 20 healthy controls. Bone markers were also evaluated in 24 patients with HbS/beta-thalassemia (10M/14F, median age: 43 years) who presented with osteosclerosis, i.e. osteosclerotic bone lesions in plain radiography and a median T-score of lumbar spine Bone Mineral Density of +3.6 (range: +2 to +7.9). All POEMS patients had P, O, E, S, edema and thombocytosis; 6 had E and 2 Castleman's disease. Six patients had sclerotic bone lesions, while 4 had also a lytic component in their bone lesions. POEMS patients had increased levels of sRANKL (p=0.003), OPG (p=0.008), TNF-α (p=0.001), NTX (p<0.001), CTX (p=0.001) and CICP (p=0.001) compared with controls. Interestingly, NTX levels were even higher in POEMS than in MM patients (p<0.01). NTX and CTX were elevated in POEMS compared with osteosclerotic HbS/β-thal patients (p<0.01), who had reduced bone resorption compared with controls. POEMS patients had increased levels of OC and CICP compared with MM (p=0.02 and p=0.003, respectively), and increased levels of OC compared with osteosclerotic HbS/β-thal patients (p<0.01). In terms of angiogenic cytokines, POEMS patients had increased levels of all studied cytokines compared with controls (p<0.01). All POEMS patients had at least 1.8-fold higher value of VEGF-A (the major angiogenic component of VEGF) compared with the higher value observed in controls. Furthermore, VEGF (p=0.03), VEGF-A (p<0.001), angiogenin (p<0.001), and bFGF (p=0.01) were higher in POEMS than in MM. The ratio of Ang-1/Ang-2 was very low in POEMS compared with both controls and MM. These results suggest that POEMS patients have increased bone turnover in contrast to MM patients who have reduced bone formation and osteosclerotic HbS/β-thal patients who have diminished bone resorption. Furthermore the imbalance of Ang-1/Ang-2 pathway and the increased levels of angiogenic cytokines, even compared with MM, may indicate possible targets for the development of novel agents against POEMS.

Serum concentrations of angiogenic cytokines in Waldenstrom macroglobulinaemia: the ratio of angiopoietin‐1 to angiopoietin‐2 and angiogenin correlate with disease severity

Angiogenesis represents an essential step of disease progression in several haematological malignancies. Microvessel density is increased in 30% of patients with Waldenstrom macroglobulinaemia (WM), but there is very limited information regarding the role of angiogenic cytokines in this disease. Serum levels of vascular endothelial growth factor (VEGF), VEGF-A, angiogenin, angiopoietin (Ang)-1 and -2, and basic fibroblast growth factor (bFGF) were evaluated in 56 WM patients at different disease phases (24 untreated, 20 relapsed/refractory and 12 patients at remission) and 11 patients with immunoglobulin M type monoclonal gammopathy of undetermined significance (IgM-MGUS). All patients had increased levels of angiogenin, VEGF, VEGF-A, and bFGF compared with controls. The Ang-1/Ang-2 ratio was reduced in WM but not in IgM-MGUS patients. Angiogenin levels correlated with disease status: when compared with healthy subjects, patients with IgM-MGUS and untreated WM patients had increased angiogenin serum levels, which were higher in untreated WM patients than in MGUS. WM patients at remission had lower angiogenin serum levels compared with untreated patients, but these levels were increased again in active disease post-therapy. Angiogenin also correlated with albumin levels, while VEGF-A correlated with beta(2)-microglobulin (beta2M). Ang-1/Ang-2 ratio showed a strong, negative correlation with beta2M, and positive correlation with albumin, haemoglobin and lymphadenopathy. Our results indicate a potential use of angiogenin levels for follow-up in WM and angiogenic molecules as targets for the development of novel anti-WM agents.

Elevated Serum sFlt-1/Ang-2 Ratio in Women with Preeclampsia

Background: An imbalance of angiogenesis-associated factors may predispose to preeclampsia. Here, we determined the ratio of serum concentration of soluble fms-like tyrosine kinase 1 (sFlt-1), a natural inhibitor of pro-angiogenic vascular endothelial growth factor (VEGF) relative to angiopoietin-2 (Ang-2), a natural antagonist of angiopoietin-1 (Ang-1) involved in promoting angiogenesis in the presence of VEGF, in women with preeclampsia. Methods: The levels of serum sFlt-1 and Ang-2 were measured by enzyme-linked immunosorbent assay. Results: Significant decrease of serum Ang-2 and the increase of sFlt-1 were observed in women with preeclampsia as compared to healthy pregnant women. The serum sFlt-1/Ang-2 ratio was strikingly increased in preeclamptic women in contrast to healthy pregnant women exhibiting lower value similar to non-pregnant women. The serum sFlt-1 concentrations tended to positively correlate with mean blood pressure (BP) in preeclamptic women, but not in healthy pregnant women. A cut-off value >0.25 in the serum sFlt-1/Ang-2 ratio showed 87.1% sensitivity and 82.8% specificity in differentiating preeclamptic women from healthy pregnant women. Conclusion: The serum sFlt-1/Ang-2 ratio is significantly elevated in preeclamptic women as compared to healthy pregnant women. Remarkable difference of sFlt-1/Ang-2 ratio between these two groups with excellent specificity and sensitivity suggests the clinical usefulness of the serum sFlt-1/Ang-2 ratio in diagnosing and potentially predicting the onset of preeclampsia.