Platelet number and interleukin-6 correlate with VEGF but not with bFGF serum levels of advanced cancer patients.

We have compared the platelet number and the serum concentration of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and interleukin-6 (IL-6) in 80 blood samples of 50 patients with advanced cancer. We have also measured the mitogenic effect of patient sera on endothelial cells in vitro in order to estimate the biological activity of serum VEGF. Serum VEGF concentration correlated with platelet number (r = 0.61; P < 10(-4)). Serum IL-6 levels correlated with platelet count (r = 0.36; P < 10(-3)), with serum VEGF levels (r = 0.55; P < 10(-4)) and with the calculated load of VEGF per platelet (r = 0.4; P = 3 x 10(-4)). Patients with thrombocytosis had a median VEGF serum concentration which was 3.2 times higher (P < 10(-4)) and a median IL-6 serum level which was 5.8 times higher (P = 0.03) than in other patients. Serum bFGF did not show an association with any of the other parameters. Patient sera with high VEGF and bFGF content stimulated endothelial cell proliferation significantly more than other sera (P = 4 x 10(-3)). These results support the role of platelets in the storage of biologically active VEGF. Platelets seem to prevent circulating VEGF from inducing the development of new blood vessels except at sites where coagulation takes place. IL-6, besides its thrombopoietic effect, also seems to affect the amount of VEGF stored in the platelets. This is in accordance with the indirect angiogenic action of IL-6 reported previously. The interaction of IL-6 with the angiogenic pathways in cancer might explain the stimulation of tumour growth occasionally observed during IL-6 administration. It also conforms to the worse outcome associated with high IL-6 levels and with thrombocytosis in several tumour types and benign angiogenic diseases.

Platelets and vascular endothelial growth factor (VEGF): a morphological and functional study.

The growth of primary tumours beyond a critical mass is dependent on angiogenesis. The switch to the angiogenic phenotype involves changes in the local equilibrium of cytokines with either pro- or anti-angiogenic properties. Vascular Endothelial Growth Factor (VEGF) is one of the major positive regulators of tumour angiogenesis. Serum VEGF is, in cancer patients, correlated with worse prognosis. Recent evidence suggests that platelets are the main contributors of serum VEGF. We demonstrate, ultrastructurally and with immunofluorescence techniques, the alpha granule and membranous localisation of VEGF and provide further evidence for the role of platelets, both in healthy individuals as in patients with locally and advanced breast cancer, in the storage of circulating VEGF. We also demonstrate that, linear with tumoural progression, platelets accumulate more VEGF. Enhanced production in bone marrow platelet progenitors as well as endocytosis of circulating VEGF by platelets and/or megakaryocytes could explain the higher VEGF load in platelets from advanced cancer patients. This study provides further evidence for a role of platelets in transporting VEGF.

Arterio-venous gradients of IL-6, plasma and serum VEGF and D-dimers in human cancer.

The circulating angiogenic factors vascular endothelial growth factor-A, interleukin-6 and the fibrin D-dimer fragment were measured in the mesenteric vein, the uterine vein, as well as in peripheral venous and arterial samples in 21 randomly selected patients with operable colorectal, ovarian and cervical carcinoma. In addition, immunohistochemistry for vascular endothelial growth factor-A and interleukin-6 was performed on colorectal tumours of such patients. Serum and plasma vascular endothelial growth factor-A were not significantly elevated in the vein draining the tumours, despite tumour cell expression of vascular endothelial growth factor-A. Serum vascular endothelial growth factor-A is therefore not all tumour-derived. In contrast, serum interleukin-6 was highly elevated in the draining veins in agreement with expression of interleukin-6 in the cytoplasm of tumour cells. In the megakaryoblastic cell line MEG-01, the expression of vascular endothelial growth factor-A was found to be regulated by interleukin-6. Thus, the higher platelet vascular endothelial growth factor-A load resulting in higher serum vascular endothelial growth factor levels in cancer patients may partly result from an interleukin-6 mediated up-regulation of the expression of vascular endothelial growth factor-A in the precursor of the platelet, i.e. the megakaryocyte. We also confirmed by immunohistochemistry that platelets adhere and aggregate on tumour endothelium. We propose that interleukin-6 indirectly promotes tumour angiogenesis through its up-regulation of the vascular endothelial growth factor-A load in platelets. In addition, the correlations found between peripheral venous interleukin-6 and peripheral venous fibrinogen and D-dimers levels, and the high D-dimer levels found in the draining vein of the tumour, in agreement with fibrin deposits found in the tumour stroma, suggest an important role for interleukin-6 in extra-vascular fibrinogen metabolism. Our results suggest a pivotal role for interleukin-6 in the intrinsic link between haemostasis and angiogenesis. This might be of importance in the development of anti-angiogenic agents based on interference with haemostasis.

Plasma fibrin D-dimer levels correlate with tumour volume, progression rate and survival in patients with metastatic breast cancer.

Plasma levels of D-dimer are elevated in cancer patients. Activation of the extrinsic coagulation system and the fibrinolytic cascade within a tumour is thought to be related with growth, invasion and metastasis. We have investigated the relationship between these markers of fibrin metabolism, standard clinicopathological variables and serum levels of angiogenic cytokines in three cohorts: group A (n=30) consisted of 30 healthy female volunteers, group B (n=23) of consecutive patients with operable breast cancer and group C (n=84) of patients with untreated or progressive metastatic breast cancer. Plasma D-dimers, fibrinogen, IL-6, vascular endothelial growth factor and calculated vascular endothelial growth factor load in platelets are clearly increased in patients with breast cancer. D-dimers were increased in nearly 89% of patients with progressive metastatic disease. The level of D-dimers was positively correlated with tumour load (P<0.0001), number of metastatic sites (P=0.002), progression kinetics (P<0.0001) and the cytokines related to angiogenesis: serum vascular endothelial growth factor (P=0.0016, Spearman correlation=0.285), calculated vascular endothelial growth factor load in platelets (P<0.0001, Spearman correlation=0.37) and serum interleukin-6 (P<0.0001, Spearman correlation=0.59). Similarly increased D-dimer levels were positively correlated with increased fibrinogen levels (P<0.0001, Spearman correlation=0.38). The association between markers of fibrin degradation in patients with progressive breast cancer suggests that the D-dimer level is a clinically important marker for progression and points towards a relation between haemostasis and tumour progression. A role of interleukin-6, by influencing both angiogenesis and haemostasis, is suggested by these observations.