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Beta 2 glycoprotein I (β2GPI) is the major auto antigen in the antiphospholipid syndrome but also interacts with fibrinolytic and angiogenic proteins. The aim of this study was to examine the angiogenic potential of β2GPI in vivo in β2GPI deficient mice utilizing angiogenic assays. β2GPI deficient mice show increased microvessel formation in comparison to β2GPI replete controls when injected with growth factor free-matrigel implants. However, microvessel formation in matrigel plugs of β2GPI deficient mice was less than in β2GPI replete mice when basic fibroblast growth factor (bFGF) was included in the matrigel. Hemoglobin content was higher in vascular endothelial growth factor (VEGF) containing-matrigel plugs in the β2GPI deficient mouse demonstrating that the lack of β2GPI led to increased extravasation by VEGF. Melanoma B16F10 tumour growth was enhanced in β2GPI deficient mice. Melanoma microvessel density was increased in β2GPI deficient mice but the proliferation rate of tumour cells (determined by Ki67 immunohistochemistry) was unaffected by the presence or absence of β2GPI. Subcutaneous delivery of native human β2GPI by the ALZET osmotic pump did not affect melanoma tumour growth in β2GPI deficient mice. We conclude that the in vivo unopposed action of β2GPI is anti-angiogenic however this function is modified in the presence of a strong angiogenic stimulus into stabilization of vessel formation. Although the presence of β2GPI attenuates vessel sprouting in certain tumours, no survival benefit is conferred to tumour bearing animals. This does not preclude the potential benefit of modified or fragments of β2GPI in anti-angiogenesis research., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Background: Beta-2 glycoprotein I (beta(2)GPI) is a plasma glycoprotein which interacts with various proteins of the coagulation and fibrinolysis system. beta(2)GPI has recently been shown to have anti-angiogenic properties., Objectives: We undertook this study to investigate the specific domain of beta(2)GPI involved in the anti-angiogenic function and its effect on downstream signaling of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF)., Methods: Various preparations of beta(2)GPI were used on human umbilical vein endothelial cells (HUVECs) in the absence or presence of VEGF and bFGF. The effect on HUVECs' proliferation, migration and tubule formation in Matrigel matrix was investigated. The effect of beta(2)GPI on the mRNA expression of VEGF receptors and phosphorylation of signaling molecules was also studied., Results: beta(2)GPI is shown in this study to be an anti-angiogenic molecule in vitro by inhibiting VEGF and bFGF-induced proliferation, migration and papillary-like tubule formation of HUVECs. This inhibition was achieved by native, proteolytically clipped and domain deletion mutants, domain I-IV (DI-IV) but not domain II-V (DII-V) of beta(2)GPI. Native beta(2)GPI was found to downregulate the expression of the VEGF receptor KDR/Flk-1 on endothelial cells and to block the phosphorylation of VEGF's downstream effector molecules in the MAPK/ERK and PI3K/Akt/GSK3beta pathways., Conclusions: These results indicate that beta(2)GPI has anti-angiogenic functions which depend on the presence of domain I. This anti-angiogenic activity may have important implications for the therapeutic manipulation of angiogenesis in various disease states.

Recent studies have documented that angiogenesis plays a significant role in haematological malignancies, including mylodysplastic syndromes (MDS). Basic fibroblast growth factor (b-FGF), Hepatocyte growth factor (HGF) and Tumor necrosis factor-alpha (TNF-alpha) are multifunctional cytokines that potently stimulate angiogenesis. The aim of the present study was to evaluate the microvascular density (MVD) and the serum levels of these angiogenic factors in patients with myelodysplastic syndromes (MDS). In 61 patients with MDS, MVD was measured in bone marrow biopsies and b-FGF, HGF and TNF-alpha were determined in the serum of the same patients by enzyme-linked immunosorbent assay (ELISA). Serum levels of b-FGF, HGF and TNF-alpha as well as MVD in the bone marrow were increased in MDS patients compared to healthy controls (p<0.0001). Levels of b-FGF, HGF and TNF-alpha were also significantly higher in high-risk for leukemic transformation MDS than in low-risk (p<0.0001). Significant differences were also found regarding MVD in high and low risk patients (p<0.001). Both b-FGF and HGF levels were significant predictors of survival (p<0.0005, log-rank test). The present study showed that serum levels of b-FGF, HGF and TNF-alpha are significantly increased and dependent on the severity of MDS suggesting that the determination of these parameters may offer considerable information regarding disease progression and prognosis.

Several prognostic factors for patients with myelodysplastic syndromes (MDS) have been identified in previous years. In order to determine prognostic factors characterizing haematopoietic cell kinetics, bone marrow proliferative activity and serum TNF-a levels were measured in 51 cases of MDS. Cell proliferation was evaluated by employing a monoclonal antibody directed against the proliferating cell nuclear antigen (PCNA). The PCNA proliferating index (PCNA PI) and serum TNF-a levels showed significant differences between patients with MDS and normal controls (p<0.0001). PCNA PI and serum TNF-a were significantly higher in the high risk for leukemic transformation FAB subgroups (RAEB, RAEB-t and CMML) in comparison to the low risk group (RA and RARS) (p<0.001). PCNA PI and TNF-a also increased with increasing IPSS score (p<0.05). A positive correlation was noted between TNF-a concentrations and PCNA PI (r:0.36, p<0.008). Univariate analysis using the log-rank test showed that a higher PCNA PI was associated with a significantly shorter survival (p<0.001). We conclude that elevated PCNA PI and TNF-a serum levels are increased in high risk myelodysplastic disease and that a high PCNA PI is predictive of a shorter survival in this group of patients.

Interleukin-18 (IL-18) plays a role in the host's response to tumours and angiogenesis. We determined serum levels of IL-18, vascular endothelial growth factor (VEGF), angiogenin (ANG), tumor necrosis factor (TNF-alpha) and CRP in 65 newly diagnosed myeloma patients. IL-18, VEGF, angiogenin, TNF-alpha and CRP were significantly higher at stage III in comparison to stages II and I. These cytokines (measured in 27 patients) significantly decreased after treatment. In survival analysis, higher levels of IL-18 were associated with a poorer prognosis. We conclude that increased serum IL-18 in myeloma patients correlates with advanced disease, increased levels of angiogenic cytokines and worse survival.

The expression of proliferating cell nuclear antigen (PCNA) was studied in plasma cells in bone marrow biopsies from patients with multiple myeloma (MM) using a double immunostaining method. In the same samples, microvessel density (MVD), after staining with anti-CD34 antibodies, was determined before and after chemotherapy. The correlation of PCNA expression and MVD with other myeloma parameters (clinical stage, bone marrow plasma cell infiltration and serum interleukin-6 (IL-6)) was also investigated. The study population included 51 newly diagnosed MM patients, 15 patients in plateau phase after treatment and 15 normal controls. Pretreatment mean +/- SE values of PCNA, MVD, plasma cell infiltration and serum IL-6 were significantly higher than post treatment values and controls. Pretreatment PCNA expression correlated significantly with bone marrow MVD (p<0.05) plasma cell infiltration (p<0.01) and IL-6 (p<0.01). These findings show that the proliferative activity of plasma cells is related to the angiogenic activity in the bone marrow of multiple myeloma patients. Both PCNA and MVD correlate with markers of disease activity thus may provide additional information when included in the initial evaluation of myeloma bone marrow biopsies.

Hepatocyte growth factor (HGF) has been shown to be involved in angiogenesis, epithelial cell proliferation, and osteoclast activation. HGF and its receptor are expressed on myeloma cell lines and could be involved in the pathogenesis of bone destruction in multiple myeloma (MM). The aim of this study was to examine serum levels of HGF in untreated MM patients and its correlation with bone turnover indices and markers of disease activity. Forty-seven newly diagnosed MM patients and 25 controls were included: 12 patients were of stage I, 13 of stage II, and 22 of stage III (Durie-Salmon classification). Bone lesions were scored from 0 to 3, according to X-ray findings. Serum osteocalcin (OC), interleukin-6 (IL-6), TNF-alpha, beta(2)-microglobulin (beta(2)M), CRP, calcium, and 24-hr urine N-telopeptide cross-links of collagen breakdown (NTx) were determined. HGF levels were significantly higher at stage III compared to stages II and I (medians: 1,990.4 vs. 1,743.8 and 1,432.4 pg/mL, respectively, P < 0.05). Similarly, NTx, IL-6, TNF-alpha, CRP, beta(2)M, and calcium increased significantly with advancing stage (P < 0.01). OC was higher at stage I in comparison to stages II and III (P < 0.01). All parameters were significantly higher in patients than controls. HGF showed a strong correlation with IL-6 and TNF-alpha and less with beta(2)M, CRP, NTx, and OC. We conclude that serum HGF levels are increased in advanced stages of MM disease and extended bone lesions. HGF correlates with IL-6 and TNF-alpha, which are cytokines involved in osteoclast stimulation in MM. However, an independent association of HGF with bone turnover markers was not shown in this study, thus its role in MM bone disease needs to be further clarified., (Copyright 2003 Wiley-Liss, Inc.)

Proinflammatory cytokines Interleukin-1 beta (IL-1 beta) and Interleukin-6 (IL-6) play a significant role in the pathogenetic processes related to various malignant and inflammatory conditions. Leukocytosis, thrombocytosis and increased acute phase protein levels are part of a systemic inflammatory response. In this study, we measured the concentrations of IL-1 beta, IL-6 and ferritin as well as hemoglobin, lactate dehydrogenase (LDH), C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) in 23 patients (male 15, female 8, median age 68 years) with lung cancer and reactive thrombocytosis (LCRT), in 27 (male 18, female 9, median age 64 years) with benign inflammatory lung disorder (BILD) and 18 (male 10, female 8, median age 62 years) lung cancer patients with a normal platelet count (LCNP). IL-1 beta levels were significantly higher in the three patient groups in comparison with control subjects (P < 0.001) but without significant difference among the three patient groups. IL-6 was higher in all three patients groups but only in the BILD group it was significantly higher than the control group (P < 0.05). However, no significant difference in IL-6 serum levels was found between the two lung cancer groups. CRP and LDH were significantly higher in the LCRT group in comparison with the other two patient groups (P < 0.01 and 0.001, respectively), while ferritin was higher in both lung cancer groups in comparison with the BILD group (P < 0.001). Our data suggest that in lung cancer patients, reactive thrombocytosis is part of the systemic inflammatory reaction for which IL-1 beta and IL-6 may be intermediate but not independent mediators.

Technetium 99m-2-methoxyisobutil-isonitrile (Tc-99m-MIBI), also called sestaMIBI, has been used successfully to detect malignant tumours at diagnosis. Recently, it has been proposed as a safe and effective tracer in patients with multiple myeloma (MM). The purpose of this study was to demonstrate the value of the Tc-99m-MIBI uptake in disease detection and to assess the correlation between the uptake of this scintigraphy agent and prognostic factors in newly diagnosed MM patients. Thirty-five untreated patients were enrolled in the study. Tc-99m-MIBI scanning was performed in 33 patients after intravenous injection of 7.4 MBq/kg. Whole-body anterior and posterior scans were obtained after 30 min, 60 min, 2 and 4 h. The correlation between known prognostic factors of MM and the intensity of Tc-99m-MIBI uptake was assessed. Our results showed seven patients with an intensity score of I0, 12 patients with I1, eight patients with I2 and six patients with a score of I3. There was a positive correlation between Tc-99m-MIBI intensity and C-reactive protein (CRP; r=0.506, P < 0.01), erythrocyte sedimentation rate (ESR; r=0.368, P < 0.05), beta2- microglobulin (beta2M; r=0.749, P < 0.001), interleukin-6 (IL-6; r=0.823, P < 0.001), soluble Interleukin-6 receptor (sIL-6r; r=0.806, P < 0.001), serum calcium (r=0.578, P < 0.001) and bone alkaline phosphatase (BAP; r=0.472, P < 0.01). An inverse correlation was found between Tc-99m-MIBI intensity and osteocalcin (OC) and type I procollagen carboxyterminal propeptide (PICP). In conclusion, the results of this study suggest that more extensive disease activity, as determined by high levels of CRP, beta2M, IL-6 and sIL-6r correlated with a higher uptake of the radiotracer.