Anti-human Von Willebrand Factor Antibodies Research Articles

Von Willebrand Factor Multimer Analysis by Low Resolution SDS-Agarose Gel Electrophoresis.

Von Willebrand factor (VWF) is a complex glycoprotein found in plasma, composed of disulfide-bond-linked multimers with apparent molecular weights between 500 kDa and 20,000 kDa. After release of VWF from storage granules, it is cleaved in flowing blood by the specific metalloproteinase ADAMTS13, resulting in a highly characteristic cleavage pattern and structure. As the structure of VWF multimers determines diagnosis of von Willebrand disease, which has different sub-types with different multimer- and cleavage patterns, VWF analysis is performed using low-resolution horizontal SDS-agarose gel electrophoresis. However, almost every laboratory uses a different protocol, and all experimental details are rarely, if at all, described. Therefore, the results from similar methods may be substantially different. Here, we present a detailed description of a validated VWF multimer method that we have developed. It has been successfully used for over more than 20 years in quality control of recombinant and plasma-derived VWF drug products, and in preclinical and clinical studies with VWF drug candidates. As most of the published methods, it enables visualization of VWF multimers separated by electrophoresis by immunostaining with a polyclonal anti-human VWF antibody followed by a secondary antibody coupled to alkaline phosphatase. VWF appears as a series of regularly spaced bands on the low and middle molecular weight range of the gel, with an unresolved zone in the high molecular weight (HMW) range, where ultra-large multimers are found. An example is shown below. This low-resolution agarose gel electrophoresis allows the determination of the number of VWF multimers with high reproducibility. Graphical abstract: Example of electrophoretic analysis of multimer structure of four batches of a recombinant VWF drug substance.

Ultra-Low Von Willebrand Factor (VWF) Adsorption Under Shear with a Polymer Coating for Blood-Contacting Devices

Introduction. Many human diseases are treated with blood-contacting medical devices. However, they are often compromised because of blood proteins that accumulate on their surfaces. Such accumulation activates both platelets and blood coagulation, and patients supported by blood-contacting devices are at high risk of both bleeding and clotting. Blood passing through medical devices is often subjected to high shear stresses (especially near surfaces) and flow acceleration, conditions that we have shown promote rapid deposition of the large plasma glycoprotein von Willebrand factor (VWF) onto uncoated surfaces. Additionally, acquired Von Willebrand syndrome (AVWS) - a bleeding disorder caused by deficiency of high molecular weight VWF multimers - has been described in patients supported with ECMOs (extracorporeal membrane oxygenators) and LVADs (left ventricular assist devices). Blood in these patients experiences high shear stress when passing through the blood pump in ECMOs and LVADs. Thus, the adsorption of VWF to artificial surfaces under shear is likely to initiate platelet binding and the adsorption of other plasma proteins, including clotting proteins. Here, we show that polymers generated from the sulfobetaine methacrylate (SBMA) when coated onto surfaces are able to render these surfaces resistant to VWF binding (< 5 ng/cm2) under both static conditions and under shear stress.Preliminary results. We synthesized SBMA polymers (pSBMA) from SBMA according to methods described in the literature. Previously, we tested various coating conditions for pSBMA to achieve the lowest VWF adsorption. Under optimal conditions, VWF adsorption to pSBMA-coated polypropylene tubes was 4% of that to uncoated tubes (< 2.6 ng/cm2) after the tubes were exposed to a VWF solution (5 µg/mL) under shear applied with a vortexer (3000 rpm at RT for 90 min). In this work, we prepared a microfluidic device of polydimethylsiloxane that contained a tortuous flow channel to mimic shear stress conditions of blood vessels and tested the ability of pSBMA to prevent VWF deposition and platelet binding under different shear stress conditions.Methods. Purified plasma VWF (2.5 µg/mL) was perfused through the uncoated and pSBMA-coated devices for 1 h with a gravity-driven pump. To image the VWF structure, 5% BSA was perfused through the system to block nonspecific protein adsorption. Then FITC-labeled anti-human VWF antibody was perfused into the chamber system. After incubation at room temperature for 1 h, PBS was introduced to wash the system. The devices were fixed with 3.7% formaldehyde, incubated, and washed with PBS. We also perfused 1 mL of citrated human whole blood containing platelets stained with calcein AM through the devices at 0.05 mL/min with syringe pump to image platelet binding.Results. Under both conditions, VWF and platelet binding to the pSBMA-coated device was much lower than to the uncoated device. On the uncoated device, VWF formed aggregates which could not be washed away. By quantifying fluorescence intensity, we found that most VWF adsorbed along curves compared to the straight regions, the inlet, or the outlet of the chamber, consistent with shear stress simulation results that indicate that VWF deposits in the regions of highest shear and elongational flow. When whole blood was perfused through the chamber, the platelets bound to the entire device surface, with the inlet and outlet having relatively higher levels. In contrast, in the pSBMA-coated device, VWF bound loosely and could be removed almost completely with washing. We then perfused whole blood through uncoated or pSBMA-coated chambers without VWF pre-perfusion. Unlike the findings with VWF pre-perfusion, there was no apparent platelet binding in either device, indicating that the high level of platelet binding on the uncoated devices required time for VWF to deposit on the surface.Conclusions. PSBMA effectively prevented VWF adsorption under shear in two types of devices, and was stable during storage. We expect this coating, or a variation of it to significantly enhance the lifetime of blood-contacting devices and reduce both costs and medical complications of their use. DisclosuresNo relevant conflicts of interest to declare.

Severe Reduction of Free ADAMTS13, Unbound to Von Willebrand Factor, in Plasma Milieu Is a Unique Feature of HELLP Syndrome

IntroductionPlasma ADAMTS13 exists at least two forms, bound and unbound to vonWillebrandfactor (VWF), of which the former was assumed to be 3% of the total by the immunoprecipitation method (Feyset al., JTH 2009). We have shown that plasma ADAMTS13 consists of 3 group bands, by means of isoelectric focusing (IEF) analysis (Hori et al, Transfusion 2013). Band I (pI4.9-5.6) was assumed to be free ADAMTS13, unbound to VWF, and Band III (pI7.0-7.5) was a complex with high-molecular-weight VWFmultimers(HMW-VWFM). However, the feature of Band II (pI5.8-6.7) was undetermined.HELLP (hemolysis, elevated liver enzymes and low platelet counts) syndrome is a life-threatening thromboembolic complication during pregnancy, and its pathogenesis is not determined. The curative therapy of HELLP syndrome is a termination of pregnancy. It was reported that plasma VWF antigen (VWF: Ag) was remarkably increased (215-422% of the normal) with a mild reduction of ADAMTS13 activity (ADAMTS13: AC) (12-43 % of the normal), but without qualitative analysis of ADAMTS13 antigen (Lattuadaet al.,Haematologica2003).We here analyzed plasma ADAMTS13 antigen in 9 patients with HELLP syndrome using the IEF gel, and revealed a unique picture of severe reduction of Band I (free ADAMTS13), unbound to VWF, that was not seen in plasmas from normal pregnancy.Patients and MethodsOne hundred twenty-nine normal pregnant women and 9 patients with HELLP syndrome were analyzed under approval by the ethics committees of Nara Medical University. All individuals gave written informed consent to the study. Diagnosis of HELLP syndrome was made by all the following laboratory abnormalities: characteristics peripheral blood smear, serum LDH >600 IU/L (or total bilirubin >1.2 mg/dL), AST >70 IU/L, and platelet counts < 100,000/mm3 (Sibai et al., Am JObstetGynecol 1993). Plasma level of ADAMTS13: AC was determined by chromogenic ADAMTS13-act-ELISA, and VWF: Ag was measured by a sandwich ELISA using a rabbit polyclonal anti-human VWF antibody. As reported previously (Hori et al., Transfusion 2013), the IEF was performed using a large-pore agarose-acrylamide composing gel followed by detection with anti-ADAMTS13 monoclonal antibody.ResultsIn normal women, plasma levels of VWF: Agweremarkedly increased during pregnancy, and the values of third trimester (median 223%) were almost two times higher than those of first trimester (119%). Contrarily,plasma levels ofADAMTS13: AC were significantly decreased in second (68.4%) and third (66.2%) trimester compared with first trimester (84.3%, p<0.01 and p<0.001, respectively). Of note, a reduction in ADAMTS13: AC was significantly prolonged in postpartum period (50.2%, p<0.001), while increased VWF: Ag was rapidly recovered after delivery. Further, the IEF analysis of normal pregnant women showed that Band I was slightly decreased in accord with progression of pregnancy.In puerperium, there was no difference in ADAMTS13: AC between normal pregnant women and patients with HELLP syndrome. However, the values of VWF: Ag of patients with HELLP syndrome (352%) were significantly higher than those of normal pregnant women (178%, p<0.001), in agreement with a previous report. Interestingly, however, here we have shown that 7 out of 9 patients with HELLP syndrome showed severe reduction of Band I in puerperium period by the IEF analysis of ADAMTS13 (Figure).DiscussionIn normal pregnant women, the decreased ADAMTS13:AC was assumed to reflect a consumption of the enzyme for cleavage of increased VWF. Interestingly, both Band II and III were almost unchanged during normal pregnancy, but Band I slightly decreased in third trimester, concomitantly with a mild reduction of ADAMTS13:AC. In contrast, Band I was severely reduced during the acute phase of HELLP syndrome in 7 out of 9 patients.Our previous report indicated that under high-shear-stress Band I inhibited the VWF-dependent platelet aggregation in a dose-response manner from the initial phase, whereas Band III (+II) worked from the later phase. This result indicates that ADAMTS13 lacking Band I neither readily binds to VWF nor efficiently blocks the heightened high-shear-stress induced platelet aggregation generated by newly produced HMW-VWFM. Our data suggest that ADAMTS13 preparation might be used as a therapeutic option for the treatment of HELLP syndrome, before termination of pregnancy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Hemangiosarcoma with Widespread Metastasis that Originated on the Metatarsal Pad of a Java Sparrow (&lt;i&gt;Padda oryzivora&lt;/i&gt;)

A five-year-old male Java sparrow (Padda oryzivora) was examined because of the appearance of a purple spot approximately 1 mm in diameter on the right metatarsal pad. Seven months later, the spot spread to the entire right leg, and the bird died. At necropsy, multiple neoplastic masses on the right leg reached up to the pelvic cavity. There was one similar mass on the right kidney and multiple red spots on the liver. Histopathological examination revealed that neoplastic cells proliferated solidly or formed abundant irregular blood vessels. Watanabe silver staining revealed that neoplastic cells were surrounded by argentophil fibers. Immunohistochemically, the neoplastic cells were positive for anti-human von Willebrand factor antibodies. This is the first report of hemangiosarcoma in a finch.

Differential Effect of Extracellular Acidosis on the Release and Dispersal of Soluble and Membrane Proteins Secreted from the Weibel-Palade Body

Proteins secreted from Weibel-Palade bodies (WPBs) play important roles in regulating inflammatory and hemostatic responses. Inflammation is associated with the extracellular acidification of tissues and blood, conditions that can alter the behavior of secreted proteins. The effect of extracellular pH (pH(o)) on the release of von Willebrand factor (VWF), the VWF-propolypeptide (Proregion), interleukin-8, eotaxin-3, P-selectin, and CD63 from WPBs was investigated using biochemical approaches and by direct optical analysis of individual WPB fusion events in human endothelial cells expressing green or red fluorescent fusions of these different cargo proteins. Between pH(o) 7.4 and 7.0, ionomycin-evoked WPB exocytosis was characterized by the adhesion of VWF to the cell surface and the formation of long filamentous strands. The rapid dispersal of Proregion, interleukin-8, and eotaxin-3 into solution, and of P-selectin and CD63 into the plasma membrane, was unaltered over this pH(o) range. At pH(o) 6.8 or lower, Proregion remained associated with VWF, in many cases WPB failed to collapse fully and VWF failed to form filamentous strands. At pH(o) 6.5 dispersal of interleukin-8, eotaxin-3, and the membrane protein CD63 remained unaltered compared with that at pH(o) 7.4; however, P-selectin dispersal into the plasma membrane was significantly slowed. Thus, extracellular acidification to levels of pH(o) 6.8 or lower significantly alters the behavior of secreted VWF, Proregion, and P-selectin while rapid release of the small pro-inflammatory mediators IL-8 and eotaxin-3 is essentially unaltered. Together, these data suggest that WPB exocytosis during extracellular acidosis may favor the control of inflammatory processes.

Endometrial-Peritoneal Interactions during Endometriotic Lesion Establishment

The pathophysiology of endometriosis remains unclear but involves a complex interaction between ectopic endometrium and host peritoneal tissues. We hypothesized that disruption of this interaction would suppress endometriotic lesion formation. We hoped to delineate the molecular and cellular dialogue between ectopic human endometrium and peritoneal tissues in nude mice as a first step toward testing this hypothesis. Human endometrium was xenografted into nude mice, and the resulting lesions were analyzed using microarrays. A novel technique was developed that unambiguously determined whether RNA transcripts identified via microarray analyses originated from human cells (endometrium) or mouse cells (mesothelium). Four key pathways (ubiquitin/proteasome, inflammation, tissue remodeling/repair, and ras-mediated oncogenesis) were revealed, demonstrating communication between host mesothelial cells and ectopic endometrium. Morphometric analysis of nude mouse lesions confirmed that necrosis, inflammation, healing and repair, and cell proliferation occurred during xenograft development. These processes were entirely consistent with the molecular networks revealed by the microarray data. The transcripts detected in the xenografts overlapped with differentially expressed transcripts in a comparison between paired eutopic and ectopic endometria from human endometriotic patients. For the first time, components of the interaction between ectopic endometrium and peritoneal stromal tissues are revealed. Targeted disruption of this dialogue is likely to inhibit endometriotic tissue formation and may prove to be an effective therapeutic strategy for endometriosis.

Species-Dependent Variability of ADAMTS-13 Mediated Proteolysis of Human Recombinant von Willebrand Factor.

ADAMTS-13 is a plasma metalloprotease that regulates platelet adhesion and aggregation through cleavage of von Willebrand factor (VWF) multimers within the A2 domain between residues tyrosine 1605 and methionine 1606. Genetic (Upshaw-Schulman syndrome) and acquired severe functional defects of ADAMTS-13 are associated with the presence of non-proteolysed ultra-large VWF multimers. These multimers are the most thrombogenic form of this molecule and spontaneously bind to platelets causing thrombotic thrombocytopenic purpura (TTP), a syndrome characterized by microvascular thrombosis and platelet consumption. We investigated proteolysis of human rVWF by ADAMTS-13 present in plasma of different species typically used as preclinical animal models. Our substrate was a recombinant, Chinese hamster ovary (CHO) cell line-derived rVWF, processed to mature rVWF by exposure to rfurin and highly purified by a series of chromatography steps. Multimer analysis of rVWF shows absence of satellite bands consistent with intact subunits not cleaved by ADAMTS-13. rVWF was incubated with BaCl2-activated plasma samples from different species, 4 hours at 37°C with urea. ADAMTS-13-mediated degradation of VWF was measured by collagen binding (CB) assay and proteolysis visualized by high-resolution multimer analysis stained with a polyclonal anti-human VWF antibody and by non-reducing SDS-PAGE, followed by immunoblotting with a polyclonal anti-human VWF antibody. Incubation of rVWF with normal human plasma resulted in the loss of collagen binding activity and disappearance of high molecular weight multimers with simultaneous formation of the specific satellite bands leading to a multimeric pattern similar to pdVWF. In contrast, using the same amount of animal plasma (50 mU/ml human-equivalent concentration), differences were found in the ADAMTS-13 activity levels in terms of the degree of proteolytic degradation of rVWF multimers as follows as measured by CBA: Mouse plasma samples from wild type or from mice lacking FVIII and VWF, all with a C57BL/6J background, showed no ADAMTS-13 activity towards human rVWF. No activities were observed in rat and guinea pig plasma. Porcine plasma showed only minimal activity. Samples from monkey strains (baboon, rhesus and cynomolgus) and from beagle dogs showed medium activities with a residual CB of 60%. Rabbit plasma was as effective as human plasma in cleaving and degrading VWF resulting in a residual CB of 20%. The quantitative results were confirmed by multimer visualization. We conclude that animal plasma differs markedly in plasma ADAMTS-13 activity and capacity to cleave human VWF multimers. Our study confirms previous in vivo studies with human rVWF where no proteolysis was seen in mice, and little proteolysis was observed in dogs (Turecek et al., Blood 1997) and pigs (Roussi J et al. Blood.Coag.Fibrinol. 1998). The present study also suggests caution is needed in interpreting the (patho)physiology and metabolism of human VWF from animal models.

Cyclooxygenase-2 expression in ependymoma of the spinal cord

Cyclooxygenase-2 (COX-2), also known as prostaglandin endoperoxide synthase, has been reported to play an important role in the tumorigenicity of many types of tumors. The expression of COX-2 in spinal ependymomas, however, has not been studied. The authors evaluated COX-2 expression in ependymoma of the spinal cord. Sixteen ependymoma samples obtained in patients undergoing surgery between 1995 and 2004 were utilized for immunohistochemical studies to evaluate COX-2 and vascular endothelial growth factor (VEGF) expression. Intratumoral microvessels were also stained immunohistochemically using anti-human von Willebrand factor antibody and were quantified to determine the microvessel density (MVD). The clinical features were reviewed and recorded and the association with COX-2 expression was assessed. Seven (43.8%) of the 16 ependymoma specimens expressed COX-2. All three of the myxopapillary-type ependymomas exhibited COX-2-positive staining. Excluding the three myxopapillary-type cases, COX-2 expression was identified in four (30.8%) of 13 cellular-type ependymomas. The COX-2-positive samples exhibited a significant increase in VEGF-positive staining cells and MVD compared with COX-2-negative samples. The clinical features were not associated with COX-2 expression. The results of the present study indicate that COX-2 expression may promote angiogenesis through VEGF expression in ependymomas of the spinal cord. It is suggested that the use of selective COX-2 inhibitors may provide a new therapeutic strategy for spinal cord ependymomas due to their inhibition of the COX-2-mediated angiogenesis.

Measurement of von Willebrand factor binding to a recombinant fragment of glycoprotein Ibα in an enzyme‐linked immunosorbent assay‐based method: performances in patients with type 2B von Willebrand disease

Type 2B von Willebrand disease (VWD) is characterised by an increased affinity of von Willebrand factor (VWF) for its platelet receptor glycoprotein Ib (GPIb). This feature is usually studied in vitro by a ristocetin-dependent VWF platelet-binding assay, which has some limitations as it requires [e.g. (radio)-labelled anti-VWF antibodies and normal formaldehyde-fixed platelets]. We, here, extended the applicability of an enzyme-linked immunosorbent assay-based method previously described for the measurement of ristocetin co-factor activity that used a recombinant fragment of GPIb (rfGPIb alpha) and horseradish peroxidase-labelled rabbit anti-human VWF antibodies for measuring the captured ristocetin-VWF complexes on the rfGPIb alpha. Thirty-one type 2B VWD patients from 15 families with eight different known mutations were studied. VWF in plasma from 28 of these patients bound better than normal VWF at 0.2 mg/ml ristocetin, with the ratio, optical density (OD) patient/OD normal pool plasma, higher than 1.8. For two of the three other patients with no enhanced response of plasma VWF, the platelet lysate VWF showed an enhanced binding capacity; for the last patient, the results in other members of the family are unequivocal. We conclude that, this new method for measurement of plasma or platelet VWF-binding capacity offers great advantages for correct type 2B VWD diagnosis.

Microvessel quantification in benign and malignant ovarian tumors

Microvessel density (MVD) in 92 paraffin sections of ovarian samples of different histologic subtypes was correlated with microvessel counts from 58 corresponding frozen sections. Anti-human von Willebrand factor antibody was used as an endothelial marker. MVD was performed in neovascular hotspots using a Quantimet 500+ Image Analyzer. The highest vessel density (HVD) and average vessel density (AVD) of three fields at the x 200 and x 400 magnification were recorded. There was a strong correlation between the HVD and AVD at the x 200 and x 400 magnifications when comparing fixed with frozen sections (correlation coefficients at x 200 for the HVD was 0.37, P = 0.005 and AVD was 0.30, P = 0.02; correlation coefficients at x 400 for the HVD was 0.38, P = 0.003 and AVD was 0.37, P = 0.004). In the fixed tissue, the HVD and AVD at both these magnifications were significantly greater in the group containing functional cysts; this was also the case for the frozen sections. These findings are consistent with the development of a microcirculation necessary for the growth and maturation of such cysts, and this appears to be greater than that in tumors. The good correlation between MVD in fixed and frozen sections suggests that such observations represent a true reflection of ovarian angiogenesis in both physiologic and pathologic states.

Microvessel quantification in benign and malignant ovarian tumors

Microvessel density (MVD) in 92 paraffin sections of ovarian samples of different histologic subtypes was correlated with microvessel counts from 58 corresponding frozen sections. Anti-human von Willebrand factor antibody was used as an endothelial marker. MVD was performed in neovascular hotspots using a Quantimet 500+ Image Analyzer. The highest vessel density (HVD) and average vessel density (AVD) of three fields at the × 200 and × 400 magnification were recorded. There was a strong correlation between the HVD and AVD at the × 200 and × 400 magnifications when comparing fixed with frozen sections (correlation coefficients at × 200 for the HVD was 0.37, P = 0.005 and AVD was 0.30, P = 0.02; correlation coefficients at × 400 for the HVD was 0.38, P = 0.003 and AVD was 0.37, P = 0.004). In the fixed tissue, the HVD and AVD at both these magnifications were significantly greater in the group containing functional cysts; this was also the case for the frozen sections. These findings are consistent with the development of a microcirculation necessary for the growth and maturation of such cysts, and this appears to be greater than that in tumors. The good correlation between MVD in fixed and frozen sections suggests that such observations represent a true reflection of ovarian angiogenesis in both physiologic and pathologic states.

Expression of Vascular Endothelial Growth Factor as a Prognostic Factor in Node‐positive Squamous Cell Carcinoma in the Thoracic Esophagus: Long‐term Follow‐up Study

To clarify the clinical significance of the expression of vascular endothelial growth factor (VEGF) in squamous cell carcinoma in the thoracic esophagus, particularly as a prognostic factor, we have investigated the correlation between VEGF expression in tumor cells and microvessel density (MVD), pathologic factors, and survival. A total of 92 specimens, each from a thoracic esophageal squamous cell cancer patient who underwent transthoracic curative R0 esophagectomy between 1991 and 1994, were examined immunohistochemically using anti-human VEGF and anti-human von Willebrand factor antibodies. The incidence of VEGF expression in the tumor cells was relatively low, at 23.9% of all specimens. There was no significant correlation between VEGF expression and histopathologic factors. The MVD at the tumor margin in patients with VEGF-positive tumor cells was significantly greater than that in VEGF-negative cases (35.2 +/- 8.9 vs. 22.7 +/- 8.2). The survival rate for patients with VEGF expression was significantly lower than that of those without VEGF expression; the same situation was found in node-positive patients but not in node-negative patients. In addition, multivariate analysis revealed that VEGF expression was an independent prognostic factor in node-positive patients. VEGF may be implicated in the definition of the malignant phenotype of squamous cell esophageal cancer via tumor angiogenesis. Accordingly, VEGF expression in the tumor cells could be a useful prognostic factor for esophageal cancer, particularly in node-positive patients.

Expression of vascular endothelial growth factor and its receptor KDR (kinase domain-containing receptor)/Flk-1 (fetal liver kinase-1) as prognostic factors in human colorectal cancer.

To clarify the clinical significance of the expression of vascular endothelial growth factor (VEGF) and its receptor, kinase domain-containing receptor (KDR) in colorectal cancer, we evaluated the relationship between the expression of VEGF and KDR, and the microvessel counts and clinicopathological factors in colorectal cancer. A total of 259 specimens from sequential colorectal cancer patients who had undergone surgery were examined by the avidin-biotin peroxidase complex method, using anti-human VEGF, anti-human KDR, and anti-human von Willebrand factor antibodies. The incidence of VEGF expression in the tumor cells of the patients with liver metastasis was significantly higher than that in the tumor cells of the patients without liver metastasis (67% vs 44%). The microvessel count at the tumor invasive edge in the patients whose tumor cells were positive for VEGF was significantly higher than that in the patients whose tumor cells were negative for VEGF (33.0 +/- 7.8 vs 28.0 +/- 7.9); the significant difference in microvessel counts was greater when there was a combination of VEGF and KDR expression. The overall survival rate of patients positive for VEGF was significantly (P = 0.0276) lower than that of those who were negative for VEGF. Although there was no significant difference (P = 0.0743) in the survival rates after potentially curative resection according to VEGF expression, the survival rate of the patients positive for both VEGF in tumor cells and KDR in endothelial cells was significantly (P = 0.0026) lower than that in the patients who were negative for VEGF and/or KDR. In addition, multivariate analysis revealed that the expression of both VEGF and KDR was an independent prognostic factor even after potentially curative resection. VEGF may be implicated in the definition of the malignant phenotype of colorectal cancer via tumor angiogenesis. VEGF and its receptor KDR expression in tumorous tissues could be useful prognostic factors in colorectal cancer.