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plasma and serum VEGF and D Arterio venous gradients of IL 6, dimers in human cancer R Salgado1,2, I Benoy1,2, R Weytjens2, D Van Bockstaele3, E Van Marck2, Ph Huget1, M Hoylaerts4, P Vermeulen1,2 and L Y Dirix1Received 12 June 2002; Revised 16 September 2002; Accepted 19 September 2002 Top of pageAbstractMaterials and methods Elisa Results Discussion References Acknowledgements Figures and TablesThe 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. 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. in wound healing and deregulated in cancer development (Carmeliet and Jain, 2000). growth, invasion and metastasis (Carmeliet and Collen, 1998). Vascular Endothelial Growth Factor A (VEGF A) is one of the most important positive mediators of angiogenesis. Tumour cells as well as stromal cells express VEGF A (Leung et al, 1989; Veikkola et al, 2000). VEGF A not only regulates endothelial cell migration, proliferation and survival, but is also known as Vascular Permeability Factor (VPF) because it enhances endothelial cell permeability even more potently than histamine (Dvorak et al, 1995). VEGF A also induces tissue factor on endothelium and tumour cells, activating coagulation and fibrin formation (Mechtcheriakova et al, 1999). This is of primary importance for angiogenesis and tumour growth (Nagy et al, 1989; 1995). High serum levels of VEGF A are predictive for tumour doubling time in patients with colorectal cancer. Elevated levels nearly normalise after resection of the primary tumour (Dirix et al, 1997). These observations indicate that circulating levels of VEGF A mirror the ongoing growth process in tumours. A nearly 10 fold higher serum than plasma VEGF A level is found in both cancer patients as in healthy persons (Banks et al, 1998), indicating that circulating VEGF A is mainly blood cell associated, in particular in platelets (Verheul et al, 1997; Salgado et al, 1999; Gunsilius et al, 2000). High serum VEGF A levels are frequently encountered in cancer patients, the platelets of whom indeed have a higher VEGF A content compared with healthy individuals; a prognostic significance is attributed to these levels (Salven et al, 1999; O'Byrne et al, 2000). How VEGF A is regulated in platelets or in megakaryocytes in cancer patients is unclear, but VEGF A in epithelial cells is regulated by IL 6 (Cohen et al, 1996). Platelet production is also regulated by IL 6 (Ishibashi et al, 1989). The interstitial fibrin matrix, formed by VEGF A mediated tissue factor expression on tumour and endothelial cells, or by shedding of cancer pro coagulant proteins (Mielicki et al, 1990), may further serve as a scaffold for endothelial cell migration and proliferation (Contrino et al, 1997). In addition, fibrin (ogen) degradation products have pro angiogenic properties (Thompson et al, 1985; Stirk et al, 2000). Remarkably, high fibrinolytic derivatives are one of the most frequent coagulation test abnormalities encountered in the oncological setting (Edwards et al, 1987). In the present work we measured the circulating factors VEGF and IL pandora carms 6 in veins draining the tumour in order to provide direct evidence of tumour secretion of these factors. We further investigated how platelets of cancer patients acquire a higher VEGF A content. We then analysed whether circulating fibrin degradation products, D dimers, are derived from intra tumoural activation of haemostasis. We more specifically focused on the role IL 6 could have in both increasing the VEGF content in platelets as well as in mediating fibrin formation in tumours. Top of pageMaterials pandora rings price and methodsMaterials and methods Elisa Results Discussion References Acknowledgements Figures and TablesPatientsTwo patients with cervical carcinoma (FIGO Ib), five with ovarian carcinoma (FIGO III), two of them with extensive disease (FIGO IIIc), and 14 with colorectal carcinoma (Dukes A of which four had extensive disease at the moment of surgery (Dukes D), were scheduled for elective abdominal surgery. Exclusion criteria were: previous, concurrent or non resectable cancer, local or systemic inflammatory disease. The following staging procedures were performed: chest X ray, bone scintigraphy, CT scan and echography of the liver. All patients received 0.3 fraxiparin subcutaneously for prevention of deep venous thrombosis (DVT). All routine precautions for prevention of DVT were taken. TissueAs part of the normal surgical resection procedure, of the main tumour mass, a representative, full cross section of the tumour sample surrounded by adjacent mucosa was taken from all patients involved. The tissue was fixed in buffered formalin and paraffin embedded. Five sections were cut and mounted on poly L lysine coated slides. Successive sections were used for immunostaining. Blood coagulation testsDuring the surgical procedure, blood sampling occurred simultaneously in the mesenteric or uterine vein as well as in the brachial vein and brachial artery respectively. Plasma collection and measurement of prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen, platelet counts and D dimer levels were performed as mentioned previously (Dirix et al, 2002). Top of pageElisaMaterials and methods Elisa Results Discussion References Acknowledgements Figures and TablesAngiogenic cytokines Blood sampling was performed as mentioned above. Serum collection and VEGF A165 and IL 6 measurements were performed as mentioned previously (Salgado et al, 1999). Plasma VEGF A165 was measured with an ELISA kit of R systems (R systems, Minneapolis, MN, USA) on trisodium citrate anticoagulated collected blood. For the ELISA assay of VEGF A165, no cross reactivity with other members of the VEGF A family is documented. Samples were assayed in duplicate. Within assay variability has been tested before (Salgado et al, 1999). ImmunohistochemistryVEGF Immunohistochemistry was performed on colorectal cancer tissue as described previously with the anti VEGF A VG 1 antibody that stains VEGF A efficiently on paraffin sections (Turley et al, 1998). This antibody recognises the 121, 165 and 189 isoforms of the VEGF A protein. IL 6 Deparaffinisation and quenching of endogenous peroxidase was performed. The primary 1/10 diluted polyclonal anti IL 6 antibody (Genzyme Co., Cambridge, MA, USA) was then applied on sections of the colorectal carcinomas followed by a biotinylated secondary antibody. Streptavidin conjugated with peroxidase was subsequently applied on the sections (DAKO, Glostrup, Denmark). Finally, amino ethylcarbamazimine (AEC) was used as a substrate reagent. Negative controls included the omission of the primary antibody and the use of an irrelevant primary antibody. Fibrin After deparaffinisation and quenching of endogenous peroxidase, protein digestion was performed with protease. Slides were placed on a Ventana NexES (Ventana Medical Systems, Inc.) automated immunostainer and stained with a 1/100 diluted monoclonal T2 G1 antibody (Acc. Chem. Sci. Corp., USA) overnight at 4 This antibody is specific for the peptide B 15 42 of fibrin and for fibrin II. Fibrin II is obtained after cleavage of fibrinogen by thrombin at the bond A 16 17 and B 14 15. Fibrinopeptides pandora bracelet for kids A and B are released after cleavage by thrombin. Consequently, intact fibrinogen was not stained (Kudryk and Bini, 2000). The staining was completed with a Vector Basic DAB detection kit (Ventana Medical Systems, Inc.). Negative controls included the omission of the primary antibody. Paraffin embedded placental tissue with peri villous fibrin deposits was used as a positive control. Platelets Immunohistochemistry for platelet glycocalicin was performed to demonstrate adherence and aggregation of platelets on endothelium in colorectal tumours. Glycocalicin is a fragment of the platelet membrane glycoprotein Ib (Steinberg et al, 1987). Deparaffinisation and quenching of endogenous peroxidase was performed. Normal rabbit serum (NRS), diluted 1/5, was applied, followed by 1/20000 diluted primary mouse anti human glycocalicin (clone: G28E5) antibody (kind gift from Dr Hoylaerts). Subsequently, the 1/400 diluted, secondary biotinylated rabbit anti mouse antibody (DAKO, Glostrup, Denmark) was applied. The immune reaction was completed with ABC streptavidin detection system and DAB (diaminobenzidine) as chromogen. The slides were counterstained with haematoxylin and mounted. Negative controls included the omission of the primary antibody, the addition of an irrelevant primary antibody directed against glucose oxidase from Aspergillus Niger species (this enzyme is not inducible in mammalian cells) and paraffin embedded normal endometrium tissue. Positive controls included staining of platelets in paraffin embedded thrombus and placenta with prominent fibrin deposits as well as in a platelet pellet. Cell culture experimentsWe used the human megakaryoblastic cell line MEG 01 to evaluate whether IL 6 is able to modulate the expression of VEGF in megakarycocytes. Ogura and colleagues established the MEG 01 cell line from a patient in a megakaryoblastic crisis of chronic myelogenous leukaemia. This cell line possesses many megakaryocytic specific markers and does not posses any marker of B cells, T cells and myeloid cells (Ogura et al, 1985). The MEG 01 cell line has the receptor for IL 6 and blocking IL 6 with anti IL 6 antibodies did not have any significant effects on cell growth (Kellar et al, 1995). The cells were cultured in Iscove's modified Dulbecco's medium (IMDM), with 10 foetal calf serum and antibiotics. Fluorescence intensities, as a measure of reduction of the Alamar blue medium by proliferating cells, was measured every 6 with a Cytofluor (Cytofluor 2300, Millipore). Cells without antibody served as negative controls. Cell viability was assessed using Trypan blue exclusion dye. Conditioned medium was collected and ELISA for VEGF A was performed. Experiments were performed in triplicate. Ethical committeeThe local Ethical Committee approved this study and informed consent was obtained from all patients involved. Statistical analysisStatistical analysis was performed with Graphpad Prism, version 2.0 (Graphpad Software, Inc.). Half the detection limit value of the patient samples was used for statistical analysis in case the measured values did not reach the detection limit of the assay. Comparisons of continuous variables were performed with the Mann U test. A paired students t test was used to evaluate observed differences in the cell culture experiments. The relation between continuous variables was analysed with a Spearman rank correlation analysis. Means deviations are given. A P value was considered to be significant. Top of pageResultsMaterials and methods Elisa Results Discussion References Acknowledgements Figures and TablesAnalysis of VEGF, IL 6 and D dimers in patients without distant metastasisIn order to verify whether there was a gradient for VEGF, IL 6 and D dimers between the afferent and efferent blood flow in a tumour we explicitely used samples from patients without distant metastasis (n as VEGF, IL 6 and D dimers produced by metastases could mask observed gradients. We also assumed that the values in the brachial artery samples are comparable with mesenteric and uterine artery samples. Mean age of all patients involved is 65.71 years. VEGF A, serum and plasma VEGF A Seventy five per cent of all patients involved with colorectal cancer (n had tumour cell expression of VEGF A (Figure 1A). We analysed in patients without distant disease (n whether tumour cell produced VEGF A is secreted in the main blood stream, accounting herewith for the high serum VEGF A levels found in cancer patients. None of the patients had serum or plasma values below the detection limit of the assay. We compared plasma VEGF between arterial and mesenteric/uterine vein samples. Figure 1.(A) VEGF immunoreactivity in colorectal tumour cells (arrows 400). (B) Interleukin 6 immunoreactivity in colorectal tumour cells (arrow 400). (C) Adherence, aggregation and extravasation of platelets (thin arrows) in the vicinity of colorectal tumour cells (thick arrows 400).

(D) Intra vascular immunostaining of fibrin on endothelial cells (arrow 400). (E) Extra vascular stromal staining (thin arrows) of pandora uk delivery fibrin near tumour cells (thick arrow 400).


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