PEG-cross-linked heparin is an affinity hydrogel for sustained release of vascular endothelial growth factor

An affinity-based controlled release system for growth factors having heparin-binding domains was prepared using a cross-linked heparin gel. The heparin gel was made by reacting hydrazide-functionalized heparin (Hep-ADH) with the N-hydroxysuccinimidyl ester of poly(ethylene glycol)-bis-butanoic acid (SBA-PEG-SBA). The degree of cross-linking could be controlled by defining the stoichiometry of hydrazide modification and the PEG cross-linker addition. The release of vascular endothelial growth factor (VEGF) was characterized as a heparin-binding growth factor. VEGF was directly injected into the heparin gel and the loaded VEGF displayed a slow, controlled release over 3 weeks with little initial burst phase. The biological activity of the released VEGF was measured with a proliferation assay utilizing human umbilical vein endothelial cells. The released VEGF maintained its biological activity at all time points investigated. The heparin gel with loaded VEGF was implanted sub-cutaneously in the dorsal region of mice. A significantly increased density of the endothelial cell marker platelet endothelial adhesion molecule (PECAM-1) was observed in histological specimens of the tissues surrounding the implanted gel.     

Controlled release of vascular endothelial growth factor by use of collagen hydrogels

In vivo profile of vascular endothelial growth factor (VEGF) release from collagen hydrogels was investigated comparing that of hydrogel degradation while angiogenesis induced by the released VEGF was assessed. Collagen sponges were chemically cross-linked with different amounts of glutaraldehyde for various time periods. When 125I-labeled collagen hydrogels incorporating VEGF were subcutaneously implanted into the back subcutis of mice, the hydrogel radioactivity decreased with time, the decrement profile depending on the cross-linking conditions. The radioactivity was retained for longer time periods as the glutaraldehyde concentration and cross-linking time increased. Implantation study of collagen hydrogels incorporating 125I-labeled VEGF revealed that the remaining VEGF radioactivity decreased with time and the retention period was prolonged with the decreased hydrogel biodegradation. The slower the hydrogel degradation, the longer the period of VEGF retention. The collagen hydrogel incorporating VEGF induced significant angiogenesis around the implanted hydrogel, in marked contrast to VEGF in the solution form and VEGF-free empty hydrogel. The retention period of angiogenesis became longer with a decrease of the in vivo degradation rate of hydrogels. It is possible that the slower degraded hydrogel achieves a longer period of VEGF release, resulting in prolonged angiogenetic effect. We concluded that in our hydrogel system, biologically-active VEGF was released as a result of in vivo degradation of the hydrogel.     

Controlled release of growth factors based on biodegradation of gelatin hydrogel

To develop a carrier for the controlled release of biologically-active growth factors, biodegradable hydrogels were prepared through glutaraldehyde cross-linking of gelatin with isoelectric points (IEP) of 5.0 and 9.0, i.e. 'acidic' and 'basic' gelatins, respectively. Radioiodinated growth factors were used to investigate their sorption and desorption from the hydrogel of both types of gelatin. Basic fibroblast growth factor (bFGF) and transforming growth factor-β1 (TGF-β1) were well sorbed with time to the acidic gelatin hydrogel, while less sorption was observed for the basic gelatin hydrogel. This could be explained in terms of the electrostatic interaction between the growth factors and the acidic gelatin. However, bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF), though their IEPs are higher than 7.0, were sorbed to the acidic gelatin hydrogel to a smaller extent than the two other growth factors. Under in vitro non-degradation conditions, approximately 20% of the incorporated bFGF and TGF-β1 was desorbed from the hydrogels within the initial 40 min, followed by no further substantial desorption, whereas large initial desorption was observed for BMP-2 and VEGF. When implanted in the back subcutis of mice, gelatin hydrogels were degraded over time. Each growth factor was retained in vivo being incorporated in the acidic gelatin hydrogel: the smaller the in vitro desorption amount from the hydrogel, the longer the in vivo retention time. The in vivo profile of bFGF and TGF-β1 retention was in good accordance with that of the hydrogel. These findings indicate that the growth factor immobilized to the acidic gelatin hydrogel through ionic interaction was released in vivo as a result of hydrogel degradation.     

Controlled Release of Stromal-Cell-Derived Factor-1 from Gelatin Hydrogels Enhances Angiogenesis

Controlled release of a chemokine, stromal-cell-derived factor-1 (SDF-1), could be achieved with gelatin hydrogels of release carrier. Gelatin was chemically derivatized to give it different electric charge and hydrophobicity. Among the derivatives, succinylated gelatin (Succ) of an anionic charge was the most suitable for preparation of the hydrogel in terms of SDF-1 release. The time profile of SDF-1 release from the hydrogel of succinylated gelatin could be controlled by changing the water content of hydrogel which could be modified by changing the conditions of hydrogel preparation. When evaluated after the subcutaneous implantation of Succ hydrogels incorporating SDF-1 or injection of SDF-1 solution, significantly stronger angiogenesis by the hydrogel was observed. The hydrogel implantation also enhanced the mRNA level of SDF-1 receptor at the site implanted. It is possible that the gelatin hydrogel enabled SDF-1 to be released locally, resulting in an enhanced angiogenesis at the site implanted.     

Photo-cross-linkable and thermo-responsive hydrogels containing chitosan and Pluronic for sustained release of human growth hormone (hGH)

A Pluronic/chitosan hydrogel was prepared by employing di-acrylated Pluronic and acrylated chitosan for thermo-responsive and photo-cross-linkable in situ gelation. Mixtures of diacrylated Pluronic and acrylated chitosan were transformed to physical gels at elevated temperatures and the gelation temperature of the hydrogels gradually increased by increasing chitosan content in the hydrogels from 0% to 15%. Photo-cross-linked Pluronic/chitosan hydrogels were prepared by UV irradiation of the physical gels above their gelation temperatures. Hydrogels with a long photo-cross-linking time showed low degradation rates and chitosan contents in the hydrogels also impeded the degradation rates of the hydrogels, which was caused by a high degree of inter-connected polymer networks between acrylated Pluronic and acrylated chitosan. Human growth hormone (hGH), mixed with the mixture of Pluronic and chitosan, was photo-cross-linked to prepare biodegradable hGH hydrogels. The hydrogels containing hGH showed sustained release profiles for those with long photo-cross-linking times and high chitosan contents in the hydrogel. The hydrogels with a long cross-linking time showed impeded release of the protein and high content of chitosan in the hydrogels also decreased burst release of hGH from the hydrogels while hGH was rapidly released out for the hydrogels with low content of chitosan.     

Affinity evaluation of gelatin for hepatocyte growth factor of different types to design the release carrier

The objective of this study was to investigate the physicochemical interaction of hepatocyte growth factor (HGF) and its variant with 5 amino-acid residues deleted (dHGF) with an acidic gelatin for the design of factors release from the gelatin hydrogel. When the interaction of HGF or dHGF with gelatin-immobilized agarose beads was evaluated by Scatchard binding assay, the dissociation constant of dHGF was higher than that of HGF, although the two proteins had a similar binding ratio. dHGF was released more rapidly from the hydrogel of acidic gelatin than HGF. In vivo release study with ¹²⁵I-labeled HGF or dHGF in mice subcutis showed that HGF was released from the gelatin hydrogel as a result of hydrogel degradation. In contrast, dHGF was rapidly released by a simple diffusion from the gelatin hydrogel. From electrophoresis experiments, mixing with the acidic gelatin enabled HGF to complex and suppressing the trypsin-digested molecular weight loss, in marked contrast to that of dHGF. In addition, the percentage of HGF recognized by the antibody was reduced by the gelatin complexation, but that of dHGF was not. We conclude that unlike dHGF, HGF has a strong affinity for the acidic gelatin, resulting in the controlled release of HGF accompanied with hydrogel degradation of the release carrier.     

Controlled release of vascular endothelial growth factor from alginate hydrogels nano-coated with polyelectrolyte multilayer films

Alginate (ALG) hydrogels incorporating vascular endothelial growth factor (VEGF) were nano-coated with polyelectrolyte multilayer (PEM) films composed of chitosan (CT) and dextran sulfate (DEX) in order to control the VEGF release from the hydrogels. When non- and nano-coated ALG hydrogels containing VEGF were incubated in phosphate-buffered saline (PBS) at 37 degrees C for the prescribed times, the nano-coated hydrogels were stable, even after incubation for a week, whereas the non-coated hydrogels collapsed after 6 h. The release profile of VEGF from the non- and nano-coated ALG hydrogels was evaluated by an enzyme-linked immunosorbent assay (ELISA). Although all of the VEGF incorporated into the non-coated ALG hydrogels was released within 6 h by the collapse, only a few percent of the VEGF incorporated in the nano-coated hydrogels was released. Furthermore, the incorporated VEGF in the nano-coated hydrogels was released continuously, even after a month, without any initial burst release. The release percentage of VEGF was easily controlled by the PEM film thickness on the surface of the ALG hydrogels. The VEGF released from the nano-coated hydrogels retained its activity without denaturation. Consequently, the nano-coating of hydrogel surfaces with PEM films may be useful for controlled and sustained drug-delivery systems.     

血管内皮生长因子水平与糖尿病患者微血管病变的观察

血管内皮生长因子 (VEGF)是促进新生血管形成的主要细胞因子 [1 ] 。糖尿病视网膜病变是全身微血管病变的一部分 ,也是糖尿病患者重要的慢性并发症。我们通过测定不同视网膜病变期患者血清中 VEGF水平 ,观察 VEGF与糖尿病患者微血管病变的关系。1　材料与方法1.1　实验对象　糖尿病患者 5 2例 ,诊断依照 1985年 WHO制定糖尿病诊断标准和全国眼底病学组制定的糖尿病视网膜病变诊断标准分为 :单纯型视网膜病变组 (SDR) 4 1例 ,年龄 6 1.1± 10 .9岁 ;增殖型视网膜病变 (PDR)组 11例 ,年龄6 3.1± 18.9岁 ;无视网膜病变 (NDR) 33例 ,…     

Modulation of placental vascular endothelial growth factor by leptin and hCG

Vascular endothelial growth factor (VEGF) has been identified as an endothelium-specific mitogen and inducer of angiogenesis and endothelial cell survival. Leptin and hCG have also been suggested as possible regulators of angiogenesis in various models. In-vivo and in-vitro assays revealed that leptin has an angiogenic activity and that the vascular endothelium is a target for leptin. Thus, we hypothesized that products of cytotrophoblastic cells may play a role in placental angiogenesis and we therefore investigated the effects of leptin and hCG on cytotrophoblast VEGF secretion. We incubated cytotrophoblastic cells (CTB) with recombinant human leptin (rhLept) (0-4 pg/ml) or hCG (0-30000 IU/ml) for 4 h. rhLept significantly stimulated hCG (P = 0.0045) and decreased VEGF release (P = 0.0008) by CTB in a concentration-dependent manner. On the other hand, increasing concentrations of hCG (0-30000 IU/ml), induced a significant inhibition of leptin secretion (P = 0.0028) and a marked dose-dependent stimulation of VEGF(165) secretion (P < 0.0001). We observed an increase of >1000-fold in basal trophoblastic VEGF secretion with physiological concentrations of hCG in vitro. An inhibitory effect of hCG on trophoblastic leptin secretion was also observed, suggesting that hCG might exert a possible negative feedback on trophoblastic release of leptin. We hypothesize that trophoblastic products such as hCG and leptin are probably involved in the control of VEGF secretion at the maternal-fetal interface.     

血管内皮细胞生长因子表位肽体外抗肿瘤作用研究

目的:研究筛选血管内皮细胞生长因子(VEGF)不同表位肽抗肿瘤及抗血管新生效用。方法:构建4种不同的pCANTAB5-E/VEGF表位肽载体,将VEGF表位肽通过噬菌体展示技术展示于噬菌体表面,MTT法检测VEGF噬菌体表位肽对黑色素瘤细胞(B16)、人脐静脉血管内皮细胞(HUVEC)增殖的影响,Transwell小室检测其对B16F10细胞迁移的影响,ECMgel检测其对HUVEC成管的影响。结果:筛选出VEGF的4种(VSB、VNB、VEB、VTB)表位肽中,VNB对HUVEC细胞增殖的抑制率达到64.8%,VEB、VTB对B16的增殖抑制率分别为75.7%和66.5%。4株表位肽对高转移细胞B16F10迁移抑制率均超过80%。结论:筛选得到的4株VEGF表位肽对黑色素瘤细胞、血管内皮细胞的增殖、迁移和成管有抑制作用,通过分析其表位区段后可制备成小分子多肽,为今后抗肿瘤多肽药物和疫苗的研发奠定基础。     

Effects of peritoneal fluid from endometriosis patients on the release of vascular endothelial growth factor by neutrophils and monocytes

BACKGROUND: An increase in the level of the vascular endothelial growth factor (VEGF) production has been reported in the peritoneal fluid (PF) of endometriosis patients. This suggests that changes in the vascular permeability and angiogenesis play an important role in the pathophysiology of this disease. This study examined the effects of the PF obtained from endometriosis patients on the release of VEGF by neutrophils and monocytes. METHODS: Neutrophils and monocytes were obtained from young healthy volunteers and cultured with the PF obtained from either endometriosis patients (EPF) (n=18) or a control group (CPF) (n=4). A human monocyte/macrophage cell line, THP-1, was cultured with either 10% EPF or 10% CPF. The PF and culture supernatants were assayed for VEGF using ELISA. Real-time PCR and Western blotting were used to measure the VEGF mRNA and protein expression level, respectively. RESULTS: The VEGF levels were higher in the EPF than in the CPF (591+/-75 versus 185+/-31 pg/ml, P<0.05). However, the level of VEGF released by THP-1 cells in CPF and EPF was similar. The EPF induced the release of VEGF by neutrophils, but no VEGF was released by monocytes. The VEGF mRNA expression levels in the neutrophils were higher in the EPF, which was abrogated by cycloheximide, suggesting that the EPF induces the production of VEGF in neutrophils. Neutralizing antibodies against IL-8 and TNF-alpha did not completely prevent the EPF-induced release of VEGF by the neutrophils, even though these growth factors stimulated the release of VEGF by neutrophils. There was a positive correlation between the VEGF and IL-10 concentrations in the EPF (correlation coefficient=0.549, P=0.012, n=18), but the neutralizing antibody of IL-10 did not affect the release of VEGF by the EPF-treated neutrophils. CONCLUSION: The EPF induced the production and release of VEGF by neutrophils, suggesting that neutrophils may be a source of peritoneal VEGF. In addition, neutrophil-derived VEGF might be a marker for diagnosing endometriosis.     

Expression of vascular endothelial growth factor and basic fibroblast growth factor in extramammary Paget disease

Extramammary Paget’s disease (EMPD) is a special type of cancers. The etiology of the disease is still unclear. We aimed to study the expression differences of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in EMPD tissues and corresponding adjacent normal tissues. The mRNA expression was detected by RT-PCR and the protein expression was explored by immunohistochemistry. Higher immunostaining signal scores of bFGF and VEGF in EMPD tissues had been found (z = -3.827, P < 0.001, z = -3.729, P < 0.001, respectively). In addition, the mRNA expression of bFGF and VEGF was higher in EMPD tissues, which had been validated by RT-PCR (t = 5.771, P < 0.001, t = 3.304, P = 0.004, respectively). The VEGF and bFGF might be the key signaling proteins in angiogenesis of EMPD. How to block the VEGF and bFGF in EMPD and to destroy the blood supply of the tumor cells becomes the focus of our future research.     

紫草素对人鼠嵌合内异位症模型血管内皮生长因子表达的影响

目的通过研究紫草素对内异症小鼠模型血管内皮生长因子（VEGF）表达的影响,探究其治疗子宫内膜异位症（EMs）的可能机制。方法建立人鼠嵌合内异症动物模型,随机分为紫草素大、中、小剂量组,另设PBS阴性对照组及达菲林阳性对照组,采用免疫组化法,比较用药前后移植人子宫内膜VEGF表达变化情况。结果各剂量紫草素均抑制VEGF表达,大、中剂量组与阴性对照组相比差异有统计学意义（P〈0.05）,与达菲林组比较无显著性差异（P〉0.05）。结论紫草素可能通过抑制异位内膜血管新生,阻止其种植和生长而治疗EMs。     

Capture of endothelial cells under flow using immobilized vascular endothelial growth factor

We demonstrate the ability of immobilized vascular endothelial growth factor (VEGF) to capture endothelial cells (EC) with high specificity under fluid flow. To this end, we engineered a surface consisting of heparin bound to poly-l-lysine to permit immobilization of VEGF through the C-terminal heparin-binding domain. The immobilized growth factor retained its biological activity as shown by proliferation of EC and prolonged activation of KDR signaling. Using a microfluidic device we assessed the ability to capture EC under a range of shear stresses from low (0.5 dyne/cm²) to physiological (15 dyne/cm²). Capture was significant for all shear stresses tested. Immobilized VEGF was highly selective for EC as evidenced by significant capture of human umbilical vein and ovine pulmonary artery EC but no capture of human dermal fibroblasts, human hair follicle derived mesenchymal stem cells, or mouse fibroblasts. Further, VEGF could capture EC from mixtures with non-EC under low and high shear conditions as well as from complex fluids like whole human blood under high shear. Our findings may have far reaching implications, as they suggest that VEGF could be used to promote endothelialization of vascular grafts or neovascularization of implanted tissues by rare but continuously circulating EC.     

Modulation of angiogenesis by human gliomaxenograft models that differentially express vascular endothelial growth factor

To evaluate the potential actions of vascular endothelial growth factor (VEGF) on capillary permeabilityand drug transport, tumorigenic human glioma cell lines were developed that expressed different levels ofVEGF. Three human glioma cell lines (i.e. U373, SF126, SF188) were screened for VEGF under normoxicand hypoxic (i.e. induced by CoCl 2 ) conditions by Western blot analysis. Subsequent to these results, senseand antisense VEGF 164 cDNA transfections were conducted. It was found that parental SF188 (SF188/V-)and SF188/V+ (sense transfected) cells could serve as an appropriate in vivo model based on their diver-gentlevels of VEGF expression. Media derived from SF188/V+ cells stimulated endothelial cell growth by30-60%, and enhanced endothelial cell clonogenicity by 5-10-fold compared to SF188/V- or empty vectortransfected cells. Nude rats implanted with either SF188/V- or SF188/V+ cells subcutaneously and intrac-erebrallyformed tumors, with those derived from SF188/V+ cells growing at a faster rate.Immunohistochemistry analysis indicated that the expression of VEGF and number of capillaries (factorVIII assay) were approximately 3-fold greater in SF188/V+ tumors compared to SF188/V- tumors.Pharmacological assays, such as measurements of cytotoxicity and DNA adducts, in SF188/V- and SF188/V+cells treated with carmustine or temozolomide were similar. Therefore, other than differences in VEGFexpression and growth in vivo, SF188/V- and SF188/V+ cells possess a similar phenotype, and can serve asmodels to evaluate the influence of VEGF on drug transport.© Kluwer Academic Publishers     

Genetic polymorphisms of vascular endothelial growth factor in severe pre-eclampsia

Several lines of evidence support the hypothesis that vascular endothelial growth factor (VEGF) plays an important role in the pathogenesis of pre-eclampsia (PE). VEGF is a key component in the regulation of vascular remodelling and the survival of cytotrophoblasts in the placenta. In this case-control study, we aimed to test whether VEGF genetic polymorphisms are associated with the risk of severe PE. We enrolled 84 nulliparous pregnant women with severe PE (PE group). Their VEGF G(+405)C and VEGF C(-2578)A genotypes were determined by PCR-restriction fragment length polymorphism (PCR-RFLP) from venous blood samples and were compared with the corresponding VEGF genotypes of 96 nulliparous patients with uncomplicated pregnancies (control group). Carriers of the VEGF(+405)G allele occurred less frequently in PE than in the control group [P = 0.039; adjusted odds ratio (aOR) = 0.28, range: 0.08-0.93]. Hypertension and proteinuria were diagnosed earlier (by 1.6 weeks and 1.9 weeks, respectively) in PE patients with VEGF(-2578)A only after adjustment of this association for risk factors of PE. Our results suggest that carriers of VEGF(+405)G allele have a decreased susceptibility to PE and that the progression of PE may be modified by the presence of VEGF(-2578)A allele. Nevertheless, the clinical significance of these findings remains to be determined.     

High serum vascular endothelial growth factor correlates with disease activity of spondylarthropathies

Angiogenesis is involved in chronic inflammatory joint diseases such as rheumatoid arthritis (RA). Vascular endothelial growth factor (VEGF) plays a crucial role in angiogenesis. The spondylarthropathies (SpA) are characterized by enthesitis and synovitis, in which blood vessels participate. The objective of this study was to investigate serum VEGF levels and their potential associations with disease activity markers for SpA. Sera were collected from 105 patients with SpA (72 with ankylosing spondylitis (AS), four with psoriatic arthritis (PsA), six with reactive arthritis (ReA), eight with enteropathic arthropathy and 15 with undifferentiated SpA), 50 patients with rheumatoid arthritis (RA) and 64 healthy controls. Disease activity in SpA patients was assessed using the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and laboratory parameters of inflammation [erythrocyte sedimentation rate (ESR) and C-reactive protein level (CRP)]. Serum VEGF levels were significantly higher in SpA patients (316.4 +/- 215.6 pg/ml) and RA patients (405.2 +/- 366.5) than in controls (217.3 +/- 145.2) (P = 0.003). In SpA patients, serum VEGF levels correlated with disease activity indices (BASDAI: r = 0.22, P = 0.04; ESR: r = 0.3, P = 0.003; and CRP: r = 0.23, P = 0.02). Serum VEGF levels were not associated with presence of extra-articular manifestations or syndesmophytes or with the grade of sacroiliitis. These results suggest that VEGF and therefore angiogenesis may play a role in SpA pathogenesis and may serve as a disease activity marker in SpAs.     

Functional porous hydrogels to study angiogenesis under the effect of controlled release of vascular endothelial growth factor

Angiogenesis occurs through a cascade of events controlled by complex multiple signals that are orchestrated according to specific spatial patterns and temporal sequences. Vascularization is a central issue in most tissue engineering applications. However, only a better insight into spatio-temporal signal presentation can help in controlling and guiding angiogenesis in vivo. To this end, versatile and accessible material platforms are required in order to study angiogenic events in a systematic way. In this work we report a three-dimensional porous polyethylene glycol (PEG) diacrylate hydrogel bioactivated with heparin that is able to deliver vascular endothelial growth factor (VEGF) in a sustained and controlled manner. The efficiency of the material has been tested both in vitro and in vivo. In particular, the VEGF released from the hydrogel induces cell proliferation when tested on HUVECs, retains its bioactivity up to 21days, as demonstrated by Matrigel assay, and, when implanted on a chorion allantoic membrane, the hydrogel shows superior angiogenic potential in stimulating new vessel formation compared with unfunctionalized hydrogels. Moreover, in the light of potential tissue regeneration studies, the proposed hydrogel has been modified with adhesion peptides (RGD) to enable cell colonization. The porous hydrogel reported here can be used as a valid tool to characterize angiogenesis, and, possibly, other biological processes, in different experimental set-ups.     

A hybrid thermo-sensitive chitosan gel for sustained release of Meloxicam

The purpose of this work was to develop a multi-phase gel system for sustained release drug delivery. A thermo-sensitive hydrogel composed of chitosan and glycerol was prepared, and then an o/w emulsion was introduced to the thermo-sensitive gel in order to modulate the gelation behavior. Meloxicam was chosen as a model drug in this study and its release profile was investigated. This study revealed that the factors such as pH, chitosan molecular mass and glycerol concentration could significantly influence the gel formation. Chitosan with a molecular mass of 950 kDa and glycerol proportion ranging from 30 to 60% can form a pH-dependent thermo-sensitive gel system. Both the chitosan–glycerol gel and chitosan–glycerol-emulsion gel systems were applied in delivering drugs. The drug release from the two gels was both in Higuchi mode. Higuchi moduli were 3.04 × 10^?3 mg · h^½ in the chitosan–glycerol-emulsion gel and 1.28 mg · h^½ in the chitosan– glycerol gel. The former was significantly slower in sustained release. The in vivo investigation on the chitosan–glycerol gel indicated that the gel may be useful in sustained drug release in situ. Thermosensitive hydrogels composed of chitosan and glycerol were well formed and could act as a sustained release drug carrier in the work, it showed that this hybrid thermo-sensitive hydrogel system may be a promising sustained release drug carrier.     

Prognostic significance of epidermal growth factor receptor and vascular endothelial growth factor receptor in colorectal adenocarcinoma

The purpose of this study was to evaluate the association between the expression of growth factors and the clinicopathological variables of colorectal adenocarcinoma. Immunohistochemistry and fluorescence in situ hybridization (FISH) were used to evaluate the amplification and expression of epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), VEGF-D, VEGF receptor (VEGFR)-2, VEGFR-3, transforming growth factor (TGF)-β1, and insulin-like growth factor-1 receptor (IGF-1R) in a tissue microarray of 292 colorectal adenocarcinomas. The expression of EGFR, VEGF, VEGF-D, VEGFR-2 and VEGFR-3 was detected in 5.1%, 10.0%, 6.8%, 5.2%, and 57.2%. EGFR expression was associated with angioinvasion (p < 0.05) and lymph node metastasis (p < 0.005). VEGFR-3 expression was higher in the rectum than in the colon (p < 0.05). VEGF expression correlated with VEGF-D (p < 0.05) and VEGFR-3 (p < 0.005) expression, while VEGF-D expression showed no significant association with VEGFR-2 or VEGFR-3. EGFR amplification was present in 10.6% and was not associated with EGFR protein expression. VEGFR-2 and VEGFR-3 expression levels were related to poor patient survival. Stage, perineural invasion, and lymph node metastasis were independent prognostic factors based on a Cox analysis. VEGFR-2 and VEGFR-3 expression are markers of a poor prognosis in patients with surgically resected colorectal adenocarcinoma, whereas EGFR has a minor influence.