Concentrations of hepatocyte growth factor, basic fibroblast growth factor, and vascular endothelial growth factor in pericardial fluid and plasma

Some angiogenic factors, including hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF), have been reported to promote angiogenesis and improve myocardial perfusion in experimental models of ischemic heart disease. These factors are produced in various tissues, including myocardium. We measured the concentrations of HGF, bFGF, and VEGF by enzyme-linked immunosorbent assay in plasma and in pericardial fluid sampled during open heart surgery (12 patients with ischemic heart disease and 17 with nonischemic heart disease). HGF levels were significantly higher in plasma than in pericardial fluid (12.0 +/- 1.8 versus 0.26 +/- 0.04 ng/mL, P < 0.0001). On the other hand, bFGF levels were significantly higher in pericardial fluid than in plasma (243.5 +/- 50.9 versus 49.6 +/- 7.8 pg/mL, P = 0.009). VEGF levels were not significantly different between pericardial fluid and plasma (47.2 +/- 17.6 versus 24.5 +/- 3.6 pg/mL, P = 0.23). Concentrations of angiogenic factors in pericardial fluid and in plasma were not significantly different between patients with ischemic and nonischemic heart disease. These results suggest that the production, secretion, and kinetics of HGF, bFGF, and VEGF are different. These angiogenic factors may have different pathophysiologic roles.     

Fibrin fragment E stimulates the proliferation,migration and differentiation of human microvascular endothelial cells in vitro

Various factors involved in haemostasis also regulate the development of new blood vessels by a process called angiogenesis. Enzymatic cleavage of fibrin yields a variety of fibrin degradation products, particularly in areas of intense angiogenesis such as in healing wounds and active atherosclerotic plaques. One of these, fibrin fragment E (FnE), is a potent angiogenic factor in the chick chorioallantoic membrane assay of angiogenesis. Here, we extend these studies to show that FnE stimulates the proliferation, migration and differentiation of human dermal microvascular endothelial cells (HuDMECs) in vitro, both in the absence and presence of such additional endothelial growth factors as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). We also show that these stimulatory effects occur at concentrations of the protein known to be present in angiogenic tissues in vivo. FnE enhanced the angiogenic effects of VEGF or bFGF, indicating a possible synergy between the signalling pathways used by these three angiogenic factors. This revised version was published online in June 2006 with corrections to the Cover Date.     

A Novel, Microcarrier-Based in Vitro Assay for Rapid and Reliable Quantification of Three-Dimensional Cell Migration and Angiogenesis

Angiogenesis in situ occurs within the interstitial extracellular matrix. The complexity of currently used three-dimensional in vitro angiogenesis systems makes it difficult to quantify cellular growth and neovessel formation. To overcome this problem we were interested to develop an angiogenesis system which allows rapid and reliable quantification of three-dimensional neovessel formation in vitro. Endothelial cells were seeded on gelatine coated microcarriers (MCs). Cell-coated MCs were suspended in a solution of fibrinogen which was then induced to polymerize by addition of thrombin. By this way, MCs were entrapped in a three-dimensional fibrin matrix. Within a few hours, endothelial cells began to leave their supporting microcarriers and to migrate into the fibrin gel. Without addition of stimulators of angiogenesis, endothelial cells showed incoherent migration into the matrix. In contrast, in response to fibronectin, basic fibroblast growth factor (bFGF), or vascular endothelial growth factor (VEGF), respectively, endothelial cells assembled to form multicellular capillary-like structures occasionally exceeding 1000 μm in length. Each MC gave rise to a limited number of capillaries. A single culture dish contained hundreds of MCs, ensuring that a sufficient number of random samples was present for a reliable statistical evaluation. The angiogenic response could be easily quantified by determination of the average number of capillary-like formations per MC (cap/MC). The capillary count for macrovascular endothelial cells from the bovine pulmonary artery was 0.14 cap/MC when no angiogenic stimulators were contained within the fibrin gel. Addition of 200 μg/ml fibronectin increased capillary formation to 0.63 cap/MC (P < 0.0001) at Day 6. Already after 3 days, addition of bFGF (30 ng/ml) yielded a capillary count of 1.05 and addition of VEGF (100 ng/ml) resulted in 0.91 cap/MC. In contrast, addition of hyaluronic acid stimulated migration of dispersed endothelial cells into the fibrin matrix without leading to significant capillary formation (0.09 cap/MC). Hydrocortisone alone or in combination with heparin led to a significant inhibition of bFGF-stimulated angiogenesis. We thus have developed a convenient angiogenesis in vitro system which allows reliable quantification of capillary formation in a three-dimensional environment. Based on this assay we conclude that apart from proliferation and migration of endothelial cells, angiogenesis additionally requires the assembly of cells to form multicellular capillaries. This process is strongly induced by the extracellular matrix protein fibronectin. Hyaluronic acid, on the other hand, promotes migration but not capillary formation (assembly).     

Vascular endothelial growth factor- and platelet-derived growth factor-angiogenesis depressed but fetal bovine serum-angiogenesis enhanced choroidal tissue cultures of streptozotocin-diabetic Wistar and GK rats.

BACKGROUND: Diabetic state-induced alterations of angiogenic activity of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) were compared with that of fetal bovine serum (FBS) in the cultured choroidal explants of streptozotocin (STZ)-diabetic Wistar and diabetic GK rats. METHODS: Choroidal explants (0.04-1.0 mm2) were isolated from rat eyeballs and cultured in fibrin gels with FBS-Dulbecco's modified Eagle's medium (0.5 mL) containing antibiotics and 300 microg/mL epsilon-amino caproic acid in the presence of recombinant mouse vascular endothelial growth factor and recombinant human platelet-derived growth factor BB at 37 degrees C under 5% CO2 and 95% air. Microvessels newly budded from these choroidal explants were photographed. The number and length of all microvessels per choroidal explant were counted and measured as indices of angiogenesis in vitro. RESULTS: Fetal bovine serum (5-10%) enhanced both angiogenic indices in the explants of STZ-diabetic Wistar and GK rats. The actions of the serum on both angiogenic indices in both diabetic rats were greater than those in age-matched normal rats. Vascular endothelial growth factor (3-30 ng/mL) with 1% fetal bovine serum increased the angiogenic indices in diabetic choroids, but was less pronounced than in normal choroids. The action of the growth factor (2.5 ng/mL) on angiogenesis was also less in diabetic choroids. CONCLUSIONS: Results suggest that the diabetic state may down-regulate the receptors for vascular endothelial and platelet-derived growth factors and/or desensitize their post-receptor signaling in the vascular endothelial cells of choroids, being inexplicable for the enhanced actions of fetal bovine serum on angiogenesis in diabetic choroids.     

Synergistic effect of basic fibroblast growth factor and vascular endothelial growth factor in murine hepatocellular carcinoma

The growth of any solid tumor depends on angiogenesis. Among the known angiogenic factors, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), are potent and representative factors involved in tumor development. It has been reported that bFGF and VEGF showed a synergistic effect in both in vitro and in vivo angiogenesis. However, the interaction of these factors on tumor development and angiogenesis, including hepatocellular carcinoma (HCC), has not yet been elucidated. In this study, we examined the combined effect of bFGF and VEGF overexpression by means of a combination of a retroviral tetracycline (tet)-regulated (Retro-Tet) gene expression system, which can manipulate the gene expression in vivo by providing tet in the drinking water, and a conventional plasmid gene expression system. In an allograft study, bFGF and VEGF overexpression synergistically increased tumor growth and angiogenesis in the murine HCC cells. This synergistic effect also was found in established tumors. VEGF messenger RNA (mRNA) expression in the tumor was increased 3.1-fold by bFGF-overexpression, and the bFGF-induced tumor development was significantly attenuated by treatment with KDR/Flk-1 neutralizing monoclonal antibody. In conclusion, these results suggest that bFGF synergistically augments VEGF-mediated HCC development and angiogenesis at least partly by induction of VEGF through KDR/Flk-1.     

Spider angiomas in patients with liver cirrhosis： Role of vascular endothelial growth factor and basic fibroblast growth factor

AIM: To investigate whether vascular endothelial growth factor (VEGF) and basic fibroblastic growth factor (bFGF) are associated with spider angiomas in patients with liver cirrhosis. METHODS: Eighty-six patients with liver cirrhosis were enrolled and the number and size of the spider angiomas were recorded. Fifty-three healthy subjects were selected as controls. Plasma levels of VEGF and bFGF were measured in both the cirrhotics and the controls. RESULTS: Plasma VEGF and bFGF were increased in cirrhotics compared with controls (122 +/- 13 vs. 71 +/- 11 pg/mL, P=0.003 for VEGF; 5.1 +/- 0.5 vs. 3.4 +/- 0.5 pg/mL, P=0.022 for bFGF). In cirrhotics, plasma VEGF and bFGF were also higher in patients with spider angiomas compared with patients without spider angiomas (185 +/- 28 vs. 90 +/- 10 pg/mL, P=0.003 for VEGF; 6.8 +/- 1.0 vs. 4.1 +/- 0.5 pg/mL, P=0.017 for bFGF). Multivariate logistic regression showed that young age and increased plasma levels of VEGF and bFGF were the most significant predictors for the presence of spider angiomas in cirrhotic patients (odds ratio [OR]=6.64, 95 % confidence interval [CI]=2.02-21.79, P=0.002; OR=4.35, 95% CI=1.35-14.01, P=0.014; OR=5.66, 95% CI=1.72-18.63, P=0.004, respectively). CONCLUSION: Plasma VEGF and bFGF are elevated in patients with liver cirrhosis. Age as well as plasma levels of VEGF and bFGF are significant predictors for spider angiomas in cirrhotic patients.     

Role of vascular endothelial growth factor and angiopoietin systems in serum of Crohn's disease patients

BACKGROUND: The purposes of this study were to determine soluble angiogenic factors in Crohn's disease (CD) patients and to compare these factors according to the pathological behavior of the disease in order to establish a possible relationship with its evolution in patients with CD. METHODS: Blood samples were collected from 70 patients with CD, grouped according to their phenotypic behavior, and from 30 healthy controls. Vascular endothelial growth factor (VEGF), placental growth factor (PlGF), angiopoietin 1 (Ang1), angiopoietin 2 (Ang2), and their cognate receptors [VEGFR1, VEGFR2, and angiopoietin receptor tyrosine kinase (Tie2)] were assayed by ELISA. RESULTS: Circulating levels of VEGF, PlGF, VEGFR1, Ang2, and Tie2 were significantly higher in CD patients than in healthy controls (489 +/- 271 versus 335 +/- 118 pg/mL, P < 0.001; 31 +/- 9 versus 23 +/- 9 pg/mL, P < 0.001; 1.7 +/- 0.4 versus 1.0 +/- 0.3 ng/mL, P < 0.001; 4.8 +/- 2.0 versus 3.9 +/- 2.0 ng/mL, P < 0.05; and 36 +/- 5 versus 22 +/- 7 ng/mL, P < 0.001, respectively). Conversely, CD patients showed significantly lower serum levels of Ang1 than healthy controls (40 +/- 12 versus 67 +/- 22 ng/mL; P < 0.001). No differences between the groups were found in VEGFR2 serum level. The circulating levels of the angiogenic factors did not differ significantly when the CD patients were classified according to pathological phenotype. CONCLUSIONS: In comparison with healthy controls, CD patients were found to have an active angiogenic profile, as detected by significant alterations in levels of angiogenesis soluble markers. These patients did not differ in serum levels of angiogenic factors according to phenotypic disease behavior.     

Vascular endothelial growth factor levels in active pulmonary tuberculosis

BACKGROUND: Vascular endothelial growth factor (VEGF) is a mediator with potent angiogenic, mitogenic, and vascular permeability-enhancing activities that are specific for endothelial cells. Intense angiogenesis has been found in active pulmonary tuberculosis lesions. OBJECTIVES: To determine whether active pulmonary tuberculosis is associated with increased serum levels of VEGF compared with inactive tuberculosis and VEGF levels in healthy subjects, and to assess the changes in serum VEGF levels before and after therapy. DESIGN: Prospective clinical study. SETTING: Chest clinic of a university hospital, Eskisehir, Turkey. PATIENTS AND MEASUREMENTS: Serum VEGF levels of 44 patients with active pulmonary tuberculosis, 24 patients with inactive pulmonary tuberculosis, and 20 healthy subjects were determined. RESULTS: VEGF levels were increased in active pulmonary tuberculosis patients (mean [+/- SD] VEGF level, 598.03 +/- 298.25 pg/mL) compared to both inactive pulmonary tuberculosis patients (mean VEGF level, 296.98 +/- 115.31 pg/mL) and control subjects (mean VEGF level, 339.67 +/- 74.65 pg/mL). The increase in VEGF level observed in patients with active tuberculosis was statistically significant when compared with levels in two other groups (p < 0.001 for both). Serum VEGF levels were statistically different before treatment and after treatment in 10 patients who were observed from diagnosis to the end of treatment (p < 0.01). CONCLUSIONS: Increased serum VEGF levels may be an indicator of active pulmonary tuberculosis, since levels were higher in patients with active pulmonary tuberculosis and were lower after successful treatment. The role of VEGF-mediated angiogenesis in the pathogenesis and progression of pulmonary tuberculosis lesions should be further elucidated.     

Pigment epithelium-derived factor inhibits leptin-induced angiogenesis by suppressing vascular endothelial growth factor gene expression through anti-oxidative properties

Leptin, a circulating hormone secreted mainly from adipose tissues, is involved in the control of body weight. Recently, leptin was found to be an angiogenic factor, and its vitreous levels are associated with angiogenic eye diseases such as proliferative diabetic retinopathy. However, the molecular mechanism for leptin-elicited angiogenesis remains to be elucidated. Pigment epithelium-derived factor (PEDF) has been shown to be the most potent natural inhibitor of angiogenesis in the mammalian eye, and its levels in the vitreous were decreased in angiogenic eye diseases. In this study, we investigated whether and how PEDF could inhibit the leptin-induced DNA synthesis in microvascular endothelial cells (EC), a key step of angiogenesis. Leptin significantly increased intracellular reactive oxygen species (ROS) generation in microvascular EC. PEDF was found to inhibit the leptin-induced ROS generation in EC. An anti-oxidant, N-acetylcysteine, or PEDF completely prevented the leptin-induced upregulation of vascular endothelial growth factor (VEGF) mRNA levels as well as any increase in DNA synthesis in microvascular EC. Polyclonal antibodies against human VEGF were also found to completely inhibit DNA synthesis in leptin-exposed EC. The present study suggests that leptin could elicit angiogenesis through autocrine VEGF production via intracellular ROS generation. PEDF may block the angiogenic effects of leptin through its anti-oxidative properties.     

Increased vascular endothelial growth factor expression in human hearts with microvascular fibrin

We have shown that microvascular changes that promote fibrin deposition in human cardiac allografts adversely affect clinical outcome. However, some allografts exhibit phenotypic changes in capillaries following the deposition of fibrin, which subsequently provide a significant survival advantage. The mechanism(s) involved in these capillary changes is(are) unknown. Similarly, although we have shown a significant temporal relationship between microvascular fibrin deposition and vascular endothelial growth factor (VEGF) immunoreactivity in cardiac allografts, the cellular source and relative changes in VEGF gene expression under these conditions are not known. Using immunocytochemical techniques, biopsies devoid of fibrin deposition lacked detectable VEGF immunoreactivity, whereas biopsies with fibrin deposition showed VEGF immunoreactivity in cardiocytes, interstitium, and some microvessels. By in situ hybridization, biopsies without microvascular fibrin deposition showed faint VEGF hybridization signals confined primarily to cardiocytes. In biopsies with fibrin deposition, strong VEGF hybridization signals were detected in cardiocytes, arteriolar smooth muscle cells were occasionally labeled, and endothelial cells were rarely labeled. By quantitative RT-PCR, biopsies with fibrin deposition (n=5) relatively expressed approximately three-fold more VEGF mRNA than biopsies without fibrin deposition (n=5 P=0.02). Serum VEGF titers also were greater (P=0.01) in recipients with fibrin deposition (372.9+/-66.7 pg/ml n=18) compared to recipients without fibrin deposition (172.1+/-25.0 pg/ml n=16). Collectively, these results support the hypothesis that increased myocyte-derived VEGF production following microvascular fibrin deposition in transplanted human hearts may act in a paracrine manner to promote activational and phenotypic changes in capillaries that provide a survival advantage for the allografts.     

Vascular endothelial growth factor-stimulated endothelial cells promote adhesion and activation of platelets

Coagulation abnormalities, including an increased platelet turnover, are frequently found in patients with cancer. Because platelets secrete angiogenic factors on activation, this study tested the hypothesis that platelets contribute to angiogenesis. Stimulation with vascular endothelial growth factor (VEGF, 25 ng/mL) of human umbilical vein endothelial cells (HUVECs) promoted adhesion of nonactivated platelets 2.5-fold. In contrast, stimulation of HUVECs with basic fibroblast growth factor (bFGF) did not promote platelet adhesion. By blocking tissue factor (TF) activity, platelet adhesion was prevented and antibodies against fibrin(ogen) and the platelet-specific integrin, alpha(IIb)beta(3), inhibited platelet adhesion for 70% to 90%. These results indicate that VEGF-induced platelet adhesion to endothelial cells is dependent on activation of TF. The involvement of fibrin(ogen) and the alpha(IIb)beta(3) integrin, which exposes a high-affinity binding site for fibrin(ogen) on platelet activation, indicates that these adhering platelets are activated. This was supported by the finding that the activity of thrombin, a product of TF-activated coagulation and a potent platelet activator, was required for platelet adhesion. Finally, platelets at physiologic concentrations stimulated proliferation of HUVECs, indicative of proangiogenic activity in vivo. These results support the hypothesis that platelets contribute to tumor-induced angiogenesis. In addition, they may explain the clinical observation of an increased platelet turnover in cancer patients. Platelets may also play an important role in other angiogenesis-dependent diseases in which VEGF is involved, such as diabetes and autoimmune diseases. (Blood. 2000;96:4216-4221)     

Statins differentially regulate vascular endothelial growth factor synthesis in endothelial and vascular smooth muscle cells

Objectives: HMG-CoA reductase inhibitors (statins) can modulate the formation of new blood vessels, but the reports on their contribution to angiogenesis are contradictory. Therefore, we investigated whether the effect of statins is dependent either on the concentration of the drug or on the cell type. Methods and results: Under basal conditions human vascular smooth muscle cells (HVSMC) and microvascular endothelial cells (HMEC-1) constitutively generate and release vascular endothelial growth factor (VEGF). In contrast, primary macrovascular endothelial cells (HUVEC) produce minute amounts of VEGF. Different statins (atorvastatin, simvastatin and lovastatin, 1–10 μmol/l) significantly reduced basal and cytokine-, nitric oxide- or lysophosphatidylcholine (LPC)-induced VEGF synthesis in HMEC-1 and HVSMC. Interestingly, at the same concentrations statins upregulated VEGF generation in HUVEC. Furthermore, statins exerted dual, concentration-dependent influence on angiogenic activities of HUVEC as determined by tube formation assay. At low concentrations (0.03–1 μmol/l) the pro-angiogenic activity of statins is prevalent, whereas at higher concentrations statins inhibit angiogenesis, despite increasing VEGF synthesis. Conclusion: Our data show that statins exert concentration- and cell type-dependent effects on angiogenic activity of endothelial cells and on VEGF synthesis. The data are of relevance for elucidating the differential activity of statins on angiogenesis in cardiovascular diseases and cancer.     

Imbalance in production between vascular endothelial growth factor and endostatin in patients with rheumatoid arthritis

OBJECTIVE: To clarify whether synovial cell proliferation indicates an imbalance in production between angiogenic growth factors and angiogenesis inhibitors in rheumatoid arthritis (RA), we investigated the production of basic fibroblast growth factor (b-FGF) and vascular endothelial growth factor (VEGF) as representative angiogenic growth factors and endostatin as a representative angiogenesis inhibitor. METHODS: The b-FGF, VEGF, and endostatin levels in 90 samples of peripheral blood (PB) and 15 samples of joint fluid obtained from patients with RA and 30 samples of PB and 10 samples of joint fluid from patients without RA, including 20 patients with inflammatory arthritis without purulent arthritis, and 10 patients with osteoarthritis were measured by ELISA. VEGF and endostatin levels in blood samples from 22 patients with RA were measured at 2 points: before and 4 or 5 months after the commencement of medication. RESULTS: The b-FGF and VEGF levels in the PB and joint fluid samples from patients with RA were markedly elevated compared to samples from patients without RA. In contrast, endostatin levels in PB and joint fluid samples from patients with RA were almost the same as in the samples from patients without RA. VEGF levels in blood samples obtained 4 or 5 months after the commencement of medication (combination of prednisolone 5 mg/day and disease modifying antirheumatic drugs: either bucillamine 100 mg/day or salazosulfapyridine 1,000 mg/day) were significantly decreased from 27.1 +/- 8.5 pg/ml in samples obtained before commencement of medication to 18.1 +/- 16.2 pg/ml. Endostatin levels in the corresponding samples were significantly increased, from 31.5 +/- 7.0 to 57.1 +/- 22.8 ng/ml [correction]. CONCLUSION: Our results reveal significant differences in b-FGF and VEGF levels in PB and joint fluid samples, but no difference in endostatin levels, between patients with RA and those without RA, suggesting that angiogenesis in RA occurs as a result of an imbalance in production between angiogenic growth factors and angiogenesis inhibitors.     

Vascular endothelial growth factor C induces angiogenesis in vivo

Vascular endothelial growth factor C (VEGF-C) recently has been described to be a relatively specific growth factor for the lymphatic vascular system. Here we report that ectopic application of recombinant VEGF-C also has potent angiogenic effects in vivo. VEGF-C is sufficiently potent to stimulate neovascularization from limbal vessels in the mouse cornea. Similar to VEGF, the angiogenic response of corneas induced by VEGF-C is intensive, with a high density of new capillaries. However, the outgrowth of microvessels stimulated by VEGF-C was significantly longer than that induced by VEGF. In the developing embryo, VEGF-C was able to induce branch sprouts from the established blood vessels. VEGF-C also induced an elongated, spindle-like cell shape change and actin reorganization in both VEGF receptor (VEGFR)-2 and VEGFR-3-overexpressing endothelial cells, but not in VEGFR-1-expressing cells. Further, both VEGFR-2 and VEGFR-3 could mediate proliferative and chemotactic responses in endothelial cells on VEGF-C stimulation. Thus, VEGF-C may regulate physiological angiogenesis and participate in the development and progression of angiogenic diseases in addition to lymphangiogenesis.     

Large amounts of vascular endothelial growth factor at the site of hemostatic plug formation in vivo.

Vascular endothelial growth factor (VEGF) is important for the proliferation, differentiation, and survival of microvascular endothelial cells. It is a potent angiogenic factor and a specific endothelial cell mitogen that increases fenestration and extravasation of plasma macromolecules. Recently, large quantities of VEGF were detected in human megakaryocytes. Incubation of human platelets with thrombin in vitro resulted in the release of large amounts of VEGF. To investigate whether VEGF is released from platelets during coagulation activation in vivo, we measured in human subjects VEGF at the site of plug formation, ie, in blood emerging from a standardized injury made to determine bleeding time (shed blood). VEGF was also determined in the same volunteers after treatment with the specific thrombin inhibitor recombinant hirudin (r-hirudin). In a double-blind, randomized, crossover study, 17 healthy male volunteers (aged 20 to 35 years) were investigated. VEGF concentrations were measured in venous blood and in shed blood by the use of an immunoassay 10 minutes after intravenous administration of r-hirudin (0.35 mg/kg of body weight) or physiological saline. Prothrombin fragment f1.2 (f1.2) and beta-thromboglobulin (beta-TG) were determined as indicators of coagulation and platelet activation, respectively. Concentrations of VEGF, f1.2, and beta-TG in shed blood 4 minutes after injury were significantly higher than in venous blood (VEGF, 55.8+/-9.2 versus <20 pg/mL, P<0.001; f1.2, 71.3+/-10.4 versus 0.78+/-0.03 nmol/L, P<0. 001; beta-TG, 2290+/-170 versus 53.2+/-14.0 ng/mL, P<0.001). Administration of r-hirudin caused a >50% inhibition of the beta-TG and f1.2 levels in shed blood. In a similar manner, much lower amounts of VEGF were detectable at the site of plug formation after r-hirudin treatment (69.0+/-9.5 versus 37.8+/-2.6 pg/mL per minute; P=0.0015). Our data indicate that substantial quantities of VEGF are released from platelets during the interaction with the injured vessel wall in vivo. This finding may be relevant with respect to wound healing and tissue repair, tumor vascularization, or arterial thrombus formation.     

Microvascular permeability of human melanoma xenografts to macromolecules: relationships to tumor volumetric growth rate, tumor angiogenesis, and VEGF expression

The effective microvascular permeability of human melanoma xenografts to albumin-Evans blue was measured and related to tumor volumetric growth rate, rate of tumor angiogenesis, and expression of vascular endothelial growth factor (VEGF) in an attempt to identify mechanisms regulating the microvascular permeability of tumors to macromolecules. Three melanoma lines (A-07, R-18, and U-25) were included in the study. Effective microvascular permeability was assessed by using the indicator diffusion method. Intradermal and intratumor angiogenesis assays were used to measure the rate of tumor angiogenesis. VEGF expression was studied by ELISA, immunohistochemistry, Western blotting, and measurement of tumor-induced formation of ascitic fluid. The effective microvascular permeabilities of albumin-Evans blue were determined to be (1.5 +/- 0.2) x 10(-6) cm/s (A-07), (1.1 +/- 0.4) x 10(-6) cm/s (R-18), and (0.9 +/- 0.3) x 10(-6) cm/s (U-25). These values are high compared with those measured for other tumor lines and are not significantly different. Correlations between the effective microvascular permeability of albumin-Evans blue and tumor volumetric growth rate, rate of tumor angiogenesis, or VEGF expression were not found. The three last-mentioned parameters differed significantly among the melanoma lines and covered a broad range of values relative to those of other experimental tumors. Our study suggests that the effective microvascular permeability of macromolecules can be high even in slowly growing tumors, poorly angiogenic tumors, and tumors showing low VEGF expression.     

Matrix metalloproteinases, tissue inhibitors of matrix metalloproteinases and angiogenic cytokines in peripheral blood of patients with thyroid cancer.

Stimulation of growth of endothelial cells from preexisting blood vessels, i.e., angiogenesis, is one of the essential elements necessary to create a permissive environment in which a tumor can grow. During angiogenesis, the matrix metalloproteinase (MMP) family of tissue enzymes contributes to normal (embriogenesis or wound repair) and pathologic tissue remodeling (chronic inflammation and tumor genesis). The proposed pathogenic roles of MMPs in cancer are tissue breakdown and remodeling during invasive tumor growth and tumor angiogenesis. Tissue inhibitors of metalloproteinases (TIMPs) form a complex with MMPs, which in turn inhibits active MMPs. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are unique among mediators of angiogenesis with synergistic effect, and both can also be secreted by thyroid cancer cells. The goal of the study was to evaluate the plasma blood concentration of VEGF, bFGF, MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, TIMP-1, and TIMP-2 in patients with cancer and in normal subjects. Twenty-two patients with thyroid cancers (papillary cancer, 11; partly papillary and partly follicular cancer, 3; anaplastic cancer, 5; medullary cancer, 3) and 16 healthy subjects (controls) were included in the study. VEGF, bFGF MMPs, and TIMPs were evaluated by enzyme-linked immunosorbent assay (ELISA). In patients with thyroid cancer, normal VEGF concentrations (74.29 +/- 13.38 vs. 84.85 +/- 21.71 pg/mL; p > 0.05) and increased bFGF (29.52 +/- 4.99 vs. 6.05 +/- 1.43 pg/mL; p < 0.001), MMP-2 (605.95 +/- 81.83 vs. 148.75 +/- 43.53 ng/mL; p < 0.001), TIMP-2 (114.19 +/- 6.62 vs. 60.75 +/- 9.18 ng/mL; p < 0.001), as well as lower MMP-1 (0.70 +/- 0.42 vs. 3.87 +/- 0.53; p < 0.001) levels have been noted. Increased plasma levels of MMP-3 and MMP-9 were also found in patients with medullary carcinoma. In conclusion, predominance of MMP-2 over TIMP-2 and TIMP-1 over MMP-1 as well as increased concentration of bFGF in peripheral blood are common features in patients with thyroid cancer.     

Insulin stimulates the growth and tube formation of human microvascular endothelial cells through autocrine vascular endothelial growth factor.

Insulin treatment is known epidemiologically as an independent risk factor for the progression of diabetic retinopathy. However, how insulin exacerbates the retinopathy is not yet fully understood. In this study, we investigate the effects of insulin on the growth and tube formation of microvascular endothelial cells (EC). When human skin microvascular EC were grown under various concentrations of insulin, DNA synthesis as well as tube formation of EC was found to be significantly stimulated. We obtained evidence that it is mainly vascular endothelial growth factor (VEGF) that mediates the angiogenic activity of insulin as follows. (1) Insulin upregulates the level of mRNA coding for secretory forms of VEGF, while the expression of the two VEGF receptor genes, kinase insert domain-containing receptor (kdr) and fms-like tyrosine kinase1 (flt1), was essentially unchanged by exposure to insulin. (2) A monoclonal antibody against human VEGF can completely neutralize both the proliferation and the tube formation of EC induced by insulin. The angiogenic effects of insulin were additive with those of hypoxia, a principal factor that causes angiogenesis. Further, insulin significantly stimulated plasminogen activator inhibitor-1 activity in EC. The results thus suggest that insulin not only elicits angiogenesis through the induction of autocrine VEGF but also is a predisposing factor for thrombogenesis, which may give rise to focal ischemia that could superdrive angiogenesis, thereby leading to the exacerbation of diabetic retinopathy.     

Therapeutic angiogenesis using genetic transfection. An in vitro quantitative and functional study after gene code transfer for vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) gene transfer is a new therapeutic approach in clinical situations where insufficient angiogenesis may impair processes that require vessel formation, such as myocardial ischemia or peripheral vascular disease. The feasibility of this strategy was addressed in vitro by (i) optimisation of DNA transfection into cultured cells using a cationic liposome, (ii) study of the transgene production and it's angiogenic properties. An expression plasmid encoding VEGF165 was used to transfect endothelial cells (EAhy-926) in vitro. Different amounts of the cationic liposome were complexed to the plasmid DNA in order to achieve increasing ratios (1/1, 2/1, 3/1, 4/1, 5/1). Maximal gene expression was obtained when the cationic liposome was mixed to plasmid DNA in a 3/1 ratio. Using this optimised liposome/plasmid DNA formulation, VEGF expression measured by ELISA, reached a maximum of 60 ng/mL 24 hours after gene transfer then rapidly decreased. Conditioned media from transfected cells (CMVEGF) significantly increased the proliferation rate of endothelial cells. The maximal mitogenic effect was observed when the cell media was supplemented with 25% of CMVEGF, corresponding to 10 ng/mL of recombinant VEGF. Addition of the VEGF165 peptide into the cell media showed a similar mitogenic effect. The angiogenic property of the transgene was assessed by the demonstration of its ability to stimulate capillary tubes formation in a tridimensional biologic gel.