Goldmann-Applanationstonometrie und dynamische Konturtonometrie

BACKGROUND: The aim of the study was to compare intraocular pressure (IOP) measurements between Goldmann applanation tonometry (GAT) and dynamic contour tonometry (DCT) during product certification according to the international requirements for ophthalmic instruments (tonometers, ISO 8612:2001). METHODS: The study included 160 eyes of 80 subjects. IOP measurements were performed four times consecutively on each instrument in randomized order. The difference of mean IOP measurements between GAT and DCT was analyzed. Furthermore, Bland and Altman analysis was performed to assess agreement between the instruments. RESULTS: The mean difference between DCT and GAT IOP measurements was 0.30+/-2.18 mmHg. At low to normal IOP values of 7-16 mmHg and higher IOP values of > or =23 mmHg, the difference between DCT IOP measurements and GAT IOP measurements increased in the opposite direction (1.44+/-1.59 mmHg and -1.47+/-2.57 mmHg). The Bland and Altman analysis revealed a fixed bias of -0.4+/-2.0 mmHg. CONCLUSIONS: The test tonometer DCT exceeds the requirements for the international standard for tonometers ISO 8612:2001. The results are valid for a central corneal thickness of 540+/-40 microm.     

Soluble VEGFR-1 secreted by endothelial cells and monocytes is present in human serum and plasma from healthy donors

It was shown before that the soluble form of VEGFR-1 (sVEGFR-1) is present in serum of pregnant women. The aim of the present study was to investigate the presence of this endogenous vascular endothelial growth factor-A (VEGF-A) antagonist in human serum in more detail. sVEGFR-1 was detected in human serum and plasma from normal healthy male and female donors by ELISA. sVEGFR-1 levels ranged from non-detectable up to 440 pg/ml, with no significant difference between male and female donors. In addition, vein endothelial cells (ECs) from an intact vascular bed, the umbilical cord, were shown to secrete sVEGFR-1. Furthermore, human peripheral blood monocytes, a non-EC type expressing VEGFR-1, were shown to contribute to the sVEGFR-1 detectable in human serum and plasma for the first time. EC- and monocyte-derived sVEGFR-1 proved capable of inhibiting the VEGF-induced proliferation and migration of ECs in vitro. Finally, secretion of sVEGFR-1 was increased by the angiogenic factor basic fibroblast growth factor (bFGF) in human ECs and was also enhanced in lipopolysaccharide-activated human monocytes. In human umbilical vein endothelial cells, both the membrane-bound and the sVEGFR-1 seem to be equally regulated on the mRNA as well as the protein level. The presence of an sVEGFR-1 in human serum and plasma of normal male and female donors strongly suggests that it plays an important role as a naturally occurring VEGF antagonist in the regulation and availability of VEGF-mediated biological activities in vivo. This revised version was published online in June 2006 with corrections to the Cover Date.     

Comparison of dynamic contour tonometry and goldmann applanation tonometry in glaucoma patients and healthy subjects

PURPOSE: To investigate the agreement in the measurement of intraocular pressure (IOP) obtained by dynamic contour tonometry PASCAL (DCT-PASCAL) and Goldmann applanation tonometry (GAT) in glaucoma eyes and healthy eyes with different central corneal thickness (CCT). DESIGN: Prospective cross-sectional study. METHODS: In a randomized order, three consecutive IOP measurements were performed on 197 eyes of 107 subjects by one examiner using both DCT-PASCAL and GAT on all eyes. Furthermore, ultrasonic pachymetry was performed. The Spearman correlation coefficient (r) was determined to compare IOP readings between DCT-PASCAL and GAT. Regression-based Bland and Altman analysis was used to evaluate agreement between the instruments. RESULTS: Mean IOP values obtained by both instruments were significantly correlated in healthy and glaucoma eyes (all healthy eyes [n = 66]: r = 0.8, P < .001, all glaucoma eyes [n = 131]: r = 0.96, P < .001). Neither GAT nor DCT-PASCAL showed a significant correlation with CCT (GAT: all eyes: r = 0.009, P = .9, DCT-PASCAL: all eyes: r = -0.05, P = .5). Bland and Altman analysis revealed the existence of proportional bias. Thus, 95% limits of agreement between the instruments varied with the actual IOP measurement. CONCLUSIONS: DCT-PASCAL and GAT revealed a strong correlation in IOP measurements between glaucoma and healthy eyes. However, the analysis of agreement indicated some discrepancies between the instruments. Measurements with both GAT and DCT-PASCAL were not correlated with central corneal thickness.     

Quantification of vascular endothelial growth factor-C (VEGF-C) by a novel ELISA

Lymphangiogenesis plays an important role in several normal and pathological conditions such as wound healing, inflammation or metastasis formation in several malignancies. VEGF-C and VEGF-D are important and specific regulatory factors for lymphatic endothelial proliferation and lymphangiogenesis. In order to develop a highly sensitive and specific detection system for VEGF-C, we produced soluble binding proteins and antibodies for a microtiterplate-based assay. Here we describe a specific enzyme-linked immunosorbent assay (ELISA) for the measurement of human, rat and murine VEGF-C. The different antibodies developed against human and rat VEGF-C could be combined to detect processed and partially processed VEGF-C in a specific way. The ELISA was able to detect human and rat VEGF-C with a minimum detection limit of 100 pg/ml. The assay did not show any cross-reactivity with the related protein VEGF-D. Furthermore, complex formation with its soluble receptors VEGFR-2 and VEGFR-3 did not restricted the sensitivity of the assay. Using this assay, VEGF-C was measured in supernatants and lysates of different cell types and in tumour tissue samples of murine, rat and human origin. Cell lines secrete VEGF-C in very low amounts (<1 ng/ml) whereas VEGF-C transfected cells can secrete up to 50 ng/ml VEGF-C into the supernatant. In human tumour tissue samples VEGF-C was detected in some carcinomas in the low protein range. This ELISA will be a useful tool for investigations concerning the physiological function of VEGF-C in lymphangiogenesis under normal and pathophysiological conditions.     

Differential gene expression in response to ventricular unloading in rat and human myocardium

Left ventricular unloading by mechanical assist devices induces myocardial atrophy. We aimed to systematically identify differentially expressed genes in a model of physiological atrophy (unloading of healthy rat myocardium) and compare these changes to those in a unloaded, failing human heart. METHODS: Atrophy in rat hearts was induced by heterotopic transplantation of a donor heart into the abdomen of an isogenic recipient. After one week, donor and recipient RNA was isolated. Differential gene expression was assessed by subtractive hybridization. Two screens with radioactive probes were performed to verify differentially expressed clones. Positive clones were sequenced and cDNA of genes of known homology were used as probes for hybridization with RNA from separate atrophied rat hearts and human tissue from a normal, failing or failing and unloaded left ventricle. RESULTS: We picked 1880 clones from the subtractive hybridization procedure (940/940: forward/reverse runs assessing up- or down-regulation, respectively). The first screen verified 465/140 and the second screen verified 67/30 clones. 24/23 clones were sequenced and 14/10 homologies to known genes were found. In the atrophied heart, respiratory chain and metabolic genes were down-regulated (NADH-DH, cytochrome c oxidase, acetyl-CoA synthetase, myoglobin) and cellular recognition and stress genes were up-regulated (MHC1 and 2, HSP70). In the human heart, cytochrome c oxidase, acetyl-CoA synthetase, and myoglobin expression was increased in the failing heart and returned to normal with unloading. Unloading also resulted in up-regulation of HSP70. CONCLUSIONS: The genetic responses of failing human and healthy rat myocardium to mechanical unloading show similarities that appear to be independent of species differences and/or underlying disease. Thus, heterotopic heart transplantation is a relevant model for investigating the mechanisms of mechanical unloading.     

Soluble Tie2 and Flt1 extracellular domains in serum of patients with renal cancer and response to antiangiogenic therapy.

Antiangiogenesis drugs can be difficult to evaluate because they produce disease stabilization rather than tumor regression. Markers of endothelial mass in tumors may be of value to monitor therapy and evaluate such drugs. Soluble domains of the endothelial receptor tyrosine kinases, sTie2 (angiopoietin receptor) and sFlt1 (vascular endothelial growth factor receptor-1) were analyzed by sandwich ELISA in serum samples from 43 patients with advanced renal cancer before and 1 month after antiangiogenic therapy with razoxane. Pretreatment sFlt1 levels were 0.77 ng/ml +/- 0.48 (SD) and sTie2 74.3 ng/ml +/- 15 (SD). Pretreatment sFlt1 levels above the median were associated with a lesser chance of stable disease (P = 0.04) and poorer survival (P = 0.01). Fall of sTie2 on treatment was associated with stable disease (P = 0.05) and improved survival (P = 0.04). The soluble receptors measured weeks before response were assessed and correlated with response and survival, showing they may be useful to monitor and develop antiangiogenic therapy.     

Release and complex formation of soluble VEGFR-1 from endothelial cells and biological fluids

One of the key molecules promoting angiogenesis is the endothelial cell-specific mitogen, vascular endothelial growth factor (VEGF or VEGF-A), which acts through two high-affinity receptor tyrosine kinases (VEGFR), VEGFR-1 (or Flt-1) and VEGFR-2 (or KDR/Flk-1). It was shown before that a soluble variant of VEGFR-1 (sVEGFR-1) can be generated by differential splicing of the flt-1 mRNA. This soluble receptor is an antagonist to VEGF action, reducing the level of free, active VEGF-A, and therefore, plays a pivotal role in the generation of vascular diseases like pre-eclampsia or intra-uterine growth retardation. Here we show that sVEGFR-1 is produced by cultured human microvascular and macrovascular endothelial cells and a human melanoma cell line. The soluble receptor is mainly complexed with ligands; only 5-10% remains detectable as free, uncomplexed receptor protein. Furthermore, we show the time course of total and free sVEGFR-1 release together with its putative ligands, VEGF-A and placenta growth factor (PIGF), from macrovascular endothelial cells. The release of sVEGFR-1 was quantitatively measured in two different ELISA types. The release of sVEGFR-1 was strongly enhanced by phorbol-ester (PMA); the cells produced up to 22 ng/ml of sVEGFR-1 after 48 hours. The expression of VEGF-A and PIGF was moderately influenced by PMA. We also show a hypoxia-induced increase of sVEGFR-1 expression in cells cultured from placenta, a tissue that has a high flt-1 gene expression. Moreover, we demonstrate that sVEGFR-1 in amniotic fluids acts as a sink for exogenous VEGF165 and PIGF-2. Here, for the first time, to what extent recombinant ligands have to be added to compensate for the sink function of amniotic fluids was analyzed. In conclusion, human endothelial cells produce high levels of sVEGFR-1, which influences the availability of VEGF-A or related ligands. Therefore, sVEGFR-1 may reduce the ligand binding to transmembrane receptors and interfere with their signal transduction.     

An antagonistic vascular endothelial growth factor (VEGF) variant inhibits VEGF-stimulated receptor autophosphorylation and proliferation of human endothelial cells

Vascular endothelial growth factor (VEGF) is a potent mitogen with a unique specificity for endothelial cells and a key mediator of aberrant endothelial cell proliferation and vascular permeability in a variety of human pathological situations, such as tumor angiogenesis, diabetic retinopathy, rheumatoid arthritis, or psoriasis. VEGF is a symmetric homodimeric molecule with two receptor binding interfaces lying on each pole of the molecule. Herein we report on the construction and recombinant expression of an asymmetric heterodimeric VEGF variant with an intact receptor binding interface at one pole and a mutant receptor binding interface at the second pole of the dimer. This VEGF variant binds to VEGF receptors but fails to induce receptor activation. In competition experiments, the heterodimeric VEGF variant antagonizes VEGF-stimulated receptor autophosphorylation and proliferation of endothelial cells. A 15-fold excess of the heterodimer was sufficient to inhibit VEGF-stimulated endothelial cell proliferation by 50%, and a 100-fold excess resulted in an almost complete inhibition. By using a rational approach that is based on the structure of VEGF, we have shown the feasibility to construct a VEGF variant that acts as an VEGF antagonist.     

Identification of a soluble form of the angiopoietin receptor TIE-2 released from endothelial cells and present in human blood

The transmembrane tyrosine kinase TIE-2, the receptor for the angiopoietins-1 and -2, has been shown to be involved in angiogenic processes. Investigating the regulation of TIE-2 expression on endothelial cells, we found that stimulators such as PMA induce a decrease of TIE-2 protein from the cell surface without affecting TIE-2 mRNA. In conditioned media of PMA stimulated endothelial cells, a soluble form of this receptor comprising parts of the extracellular domain can be detected. Using a sandwich ELISA, we were able to detect and quantify TIE-2 receptors in cell lysates (representing the whole transmembrane receptor) and in cell culture supernatants (representing a soluble form of this receptor, sTIE-2). Several factors influencing this shedding process e.g. basic FGF could be identified. Finally, the soluble form of TIE-2 could also be detected in human biological fluids such as sera and plasma from healthy controls. This revised version was published online in June 2006 with corrections to the Cover Date.     

Novel highly efficient intrabody mediates complete inhibition of cell surface expression of the human vascular endothelial growth factor receptor-2 (VEGFR-2/KDR)

The human vascular endothelial growth factor receptor-2 (VEGFR-2/KDR) and its ligand vascular endothelial growth factor (VEGF) play an essential role in tumor angiogenesis and in haematological malignancies. To inhibit VEGF induced signalling, intrabodies derived from two scFv fragments recognizing the VEGF receptor were generated. When these intrabodies were expressed in endothelial cells, they blocked the transport of KDR to the cell surface. We developed a cell culture model using porcine aortic endothelial cells overexpressing KDR for testing the efficiency of anti-KDR intrabodies. The two intrabodies were targeted to the ER and colocalised with the KDR receptor in an intracellular compartment. No degradation of the receptor was observed. An immature incomplete glycosylated protein of 195 kDa was detected, suggesting that the intrabodies affect the maturation of the receptor. Despite the presence of significant amounts of receptor protein, the inactivation by one of the two intrabodies was highly effective, resulting in complete functional inhibition of KDR and inhibition of in vitro angiogenesis. The new intrabody appears to be a powerful tool with which to inhibit KDR function.