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Stephan Hoffmann

Publications and source records attributed to Stephan Hoffmann.

11 recordsLinked to original sources
PubMed2006

MMP-2 and MMP-9 secretion by rpe is stimulated by angiogenic molecules found in choroidal neovascular membranes.

PURPOSE: Matrix metalloproteinases (MMP)-2 and -9 play an important role in the pathogenesis of choroidal neovascularization (CNV). Retinal pigment epithelial cells (RPE) are an important source of MMPs in the outer retinal environment, however little is known about the local factors that modulate MMP secretion in these cells. The purpose of this study was to determine the effects of CNV involved growth factors and the extracellular matrix molecule fibronectin on MMP-2 and -9 secretion by cultured human RPE. METHODS: MMP-2 and -9 secretion was studied using gelatin zymography, Western blot, and ELISA assay of RPE culture supernatants. The effects of stimulating the cells for 36 hours with vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bGFG), tumor necrosis factor-alpha (TNF-alpha), or fibronectin (FN), all angiogenic factors found in CNV membranes, was determined. RESULTS: Resting RPE cells secreted MMP-2 but not MMP-9. Stimulation with TNF-alpha induced secretion of MMP-9 and increased the secretion of MMP-2. MMP-2 secretion was also increased by stimulation with FN and VEGF, but not bFGF. CONCLUSION: The results indicated that the angiogenic molecules VEGF, FN, and TNF-alpha stimulate MMP-2 and -9 secretion from RPE and thus further promote CNV.

Angiogenic Proteins↗
PubMed2005

Sequential induction of angiogenic growth factors by TNF-alpha in choroidal endothelial cells.

Inflammatory mediators have been proposed to play a critical role in the pathogenesis of choroidal neovascularization, a blinding complication of age-related macular degeneration. We evaluated the expression of TNF-alpha in human choroidal neovascular membranes and found that it colocalized with cells expressing VEGF, angiopoietin (Ang)-1 and Ang2. In cultured choroidal endothelial cells we found that TNF-alpha increased Ang2 mRNA (increased transcription) and protein levels prior to those of Ang1 and VEGF. The results raise the possibility that during neovascularization, TNF-alpha may modulate endothelial plasticity and survival by sequential inactivation of Tie2 followed by activation of Tie2 and VEGF receptors.

Angiopoietin-1↗
PubMed2005

Inhibitory effects of verapamil isomers on the proliferation of choroidal endothelial cells.

PURPOSE: To evaluate the effects of verapamil isomers on in vitro proliferation of bovine choroidal endothelial cells (CECs). MATERIALS AND METHODS: CECs were isolated from bovine eyes and cultured in endothelial growth medium (EGM). For the proliferation assays, CECs were exposed to verapamil isomers (0.1-100 microM) in EGM with 2% fetal bovine serum or basic fibroblast growth factor (bFGF) (10 ng/ml). After 72 h of incubation with the desired drug, the cellular proliferation was determined by an MTT assay and a BrdU assay. In addition, the drug toxicity on CECs stimulated with EGM was evaluated by cell counting with trypan blue. RESULTS: All verapamil isomers inhibited the bFGF- or medium-stimulated growth significantly in a concentration range of 10-40 microM without toxicity. No significant differences were seen between the inhibitory effects of the various isomers. Cell toxicity was detected at a concentration of 100 microM verapamil isomers on EGM-stimulated CECs. CONCLUSION: The results demonstrate the efficacy of all verapamil isomers in inhibiting CEC proliferation involved in the process of choroidal neovascularization. D: -(+)-Verapamil may be recommended for further in vivo evaluation in an animal model of exudative AMD; it has fewer systemic and local side effects because calcium channels are not blocked.

Animals↗
PubMed2005

Simultaneous multiple immunoassays in a compact disc-shaped microfluidic device based on centrifugal force.

BACKGROUND: We explored the potential of a microfluidic device based on centrifugal force as an immunoassay platform by examining the imprecision of assays carried out with 200 nL of sample. METHODS: Biotinylated antibodies against alpha-fetoprotein (AFP), interleukin-6 (IL-6), and carcinoembryonic antigen [(CEA); 0.1 g/L each in 15 mmol/L phosphate-buffered saline (PBS) containing 0.1 mL/L Tween 20] were attached to a microcolumn packed with streptavidin-coated particles. A 200-nL sample was then allowed to pass through the microcolumn for 240 s, followed by Alexa 647-labeled detection antibody (7.5 mg/L in 15 mmol/L PBS containing 10 g/L bovine serum albumin). The flow rate was controlled by altering the rotational speed. Up to 104 sandwich type immunoassays were completed within 50 min. RESULTS: For AFP, IL-6, and CEA the detection limits were, respectively, 0.15, 1.25, and 1.31 pmol/L. Inter- and intraassay imprecisions (CVs) were <10% and <20%, respectively, for analyte concentrations >5 pmol/L. The CEA antibody had the lowest affinity according to fluorescence image analysis of the microcolumn region. The result was confirmed in a comparative study using BIAcore 3000. CONCLUSIONS: Day-to-day (total) imprecision (CV) of immunoassays on the compact disc-shaped device are <20%. Analysis of fluorescence images allows rapid ranking of antibodies according to their affinities.

Animals↗
PubMed2005

A selective cyclic integrin antagonist blocks the integrin receptors alphavbeta3 and alphavbeta5 and inhibits retinal pigment epithelium cell attachment, migration and invasion.

BACKGROUND: Proliferative vitreoretinopathy (PVR) is a leading cause of blindness after failed retinal reattachment surgery. PVR is characterized by the proliferation, migration and contraction of retinal pigmented epithelial cells (RPE), and these cellular responses are influenced by the expression and function of integrin receptors. The effect of a cyclic integrin antagonist containing the amino acid sequence Arg-Gly-Asp-D-Phe-Val (RGDfV), specific for the integrin receptors alphavbeta3 and alphavbeta5, was investigated on basic fibroblast growth factor (bFGF), platelet derived growth factor-BB (PDGF-BB), and serum induced human RPE proliferation, migration, invasion and attachment to the extracellular matrix. Furthermore, the effects of bFGF and PDGF-BB regulated expression of integrins alphavbeta3 and alphavbeta5 on RPE cells was examined. METHODS: The effect of a cyclic integrin antagonist and a control peptide (0.01 microg/ml to 300 microg/ml) was investigated on serum or cytokine (bFGF or PDGF-BB pretreatment) induced human fetal RPE cell proliferation by H3-thymidine uptake. The effect of the cyclic integrin antagonist on RPE cell attachment onto different extracellular matrices (laminin, collagen IV, fibronectin), RPE cell invasion stimulated by PDGF-BB or serum, and migration stimulated by PDGF-BB, vascular endothelial growth factor (VEGF) or serum was explored. PDGF-BB and bFGF modulation of the integrin receptors alphavbeta3 and alphavbeta5 was evaluated by flow cytometry. RESULTS: The integrin antagonist did not inhibit DNA synthesis stimulated by serum, bFGF, or PDGF-BB treatment. RPE attachment onto fibronectin was inhibited in a concentration range of 1-10 microg/ml (p < 0.05). Attachment of the RPE cells onto collagen IV and laminin was inhibited in a range of 3-10 microg/ml (p < 0.05). Serum and PDGF-BB stimulated migration was inhibited by the cyclic integrin antagonist in a concentration range of 1-10 microg/ml (p < 0.05). Furthermore, the cyclic integrin antagonist inhibited PDGF-BB stimulated RPE cell invasion through fibronectin (3 microg/ml: 66% inhibition, p < 0.001). In each of these experiments, the control peptides had no significant effects. PDGF-BB and bFGF pretreatment of RPE cells increased the expression of integrin receptors alphavbeta3 (bFGF: 1.9 fold, PDGF-BB: 2.3 fold) and alphavbeta5 (bFGF: 2.9 fold, PDGF-BB: 1.5 fold). CONCLUSION: A selective inhibition of the integrin receptors alphavbeta3 and alphavbeta5 through a cyclic integrin antagonist is able to inhibit RPE cell attachment, migration and invasion. Since these steps are of importance for the progression of PVR, a cyclic integrin antagonist should be further evaluated for the treatment of this disease.

Becaplermin↗
PubMed2005

Carboxyamido-triazole modulates retinal pigment epithelial and choroidal endothelial cell attachment, migration, proliferation, and MMP-2 secretion of choroidal endothelial cells.

PURPOSE: To determine the effect of the calcium signaling modulating drug carboxyamido-triazole (CAI) on substeps of exudative age-related macular degeneration (AMD) in vitro. MATERIALS AND METHODS: Zymography and ELISA determined the effect of CAI on MMP-2 production of choroidal endothelial cells (CECs) stimulated by bFGF and VEGF. The effects of CAI on attachment of retinal pigment endothelial (RPE) cells/CECs onto fibronectin, laminin, collagen IV, and migration toward fibronectin were investigated. Proliferation induced by serum and bFGF (10 microg/ml) with and without CAI (0.1-10 microM) was measured by cell counting and 3H-uptake. Viability and apoptosis of the exposed cells was assessed by an MTT and an apoptosis assay. RESULTS: CAI inhibited serum- and bFGF-induced proliferation, cell attachment onto fibronectin and collagen IV, but only CEC attachment onto laminin. Inhibition of MMP-2 production was observed (10 microM CAI). CAI reduced the cellular viability by apoptosis induction. CONCLUSIONS: CAI inhibits substeps of exudative macular degeneration and may be of value for the treatment of the disease.

Animals↗
PubMed2005

Impact of endostatin on bFGF-induced proliferation, migration, and matrix metalloproteinase-2 expression/secretion of bovine choroidal endothelial cells.

PURPOSE: To investigate the potential role of endostatin, an endogenous angiogenesis inhibitor, in the prevention of choroidal angiogenesis-related disorders. METHODS: Bovine choroidal endothelial cells (CEC) were cultured and treated with basic fibroblast growth factor (bFGF) alone or combined with endostatin at concentrations ranging from 0.1 to 10 microg/ml. The proliferation and migration of CECs were evaluated by using 3, (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay and modified Boyden chamber assay, respectively. For evaluating expression and secretion of matrix metalloproteinase-2 (MMP-2), CEC-conditioned media were subjected to zymography and/or Western blot analysis, and the cells were used for semiquantitative reverse transcription polymerase chain reaction (RT-PCR) analysis. RESULTS: Endostatin did not inhibit bFGF-induced or nonstimulated CEC proliferation (p > 0.05). The bFGF-induced migration was significantly inhibited by endostatin at concentrations of 1 and 10 microg/ml (p < 0.05). The bFGF-upregulated expression of mRNA in CECs and the secretion of MMP-2 protein of CECs were both suppressed by endostatin. CONCLUSIONS: Inhibitory effect of endostatin on expression and secretion of MMP-2 and cell migration, but not on proliferation of CECs, could respond to its therapeutic action for choroidal neovascularization-dependent disorders.

Angiogenesis Inhibitors↗
PubMed2002

Advanced glycation end products quench nitric oxide in vitro.

BACKGROUND: Nitric oxide (NO) is known as an important mediator of endothelial function. The aim of this investigation was to evaluate the influence of mediators of retinal pathology - vascular endothelial growth factor (VEGF) and advanced glycation end products (AGEs) - on NO release from choroidal endothelial cells (CEC) and retinal pigment epithelial (RPE) cells to elucidate the complex role of NO. METHODS: Bovine CEC were stimulated using VEGF (1, 10, and 100 ng/ml), and RPE cells were exposed to interferon-gamma (IFN-gamma 100 U/ml) and lipopolysaccharide (LPS 1 micro g/ml). NO release into the media was assessed by an amperometric NO sensor. The influence of AGEs (10 and 100 micro g/ml) on NO release from CEC and RPE cells was investigated. The competitive NO synthase inhibitor L(omega)-nitro-L-arginine methyl ester (L-NAME 2 nmol) was used to pretreat the cells 2 h before NO measurement. RESULTS: Unstimulated CEC produced low basal levels of NO in vitro (39.1+/-13.9 nmol), and unstimulated RPE cells produced minimal basal levels of NO (15.7+/-7.0 nmol). Exposure of CEC to VEGF for 30 min resulted in a dose-dependent rise of NO in the medium, which was significantly inhibited by L-NAME. Stimulation of RPE cells with IFN-gamma and LPS resulted in a rise of NO in the bath to 125.9+/-18.5% of basal values. Basal NO release from CEC, and stimulated NO release from RPE cells, was significantly reduced by AGE treatment and L-NAME. CONCLUSIONS: These data demonstrate that AGEs formed from the nonenzymatic glycation of proteins with reducing sugars quench NO activities in vitro. The results implicate AGEs as important modulators of NO activity and may be relevant to the impairment of endothelial functions observed in diabetes and aging.

Animals↗
PubMed2002

Inhibitory effects of triamcinolone acetonide on bFGF-induced migration and tube formation in choroidal microvascular endothelial cells.

BACKGROUND: Angiostatic drugs might provide desirable modulation of choroidal angiogenesis-related diseases, including histoplasmosis and the exudative form of age-related macular degeneration. However, the precise effects of this class of compounds in the choroidal neovascularization are still unclear. In the present study, we investigated the effects of triamcinolone acetonide (TA), an angiostatic steroid, on choroidal angiogenesis in vitro. METHODS: Bovine choroidal endothelial cells (CEC), which are the critical cellular component of choroidal angiogenesis in vivo, were isolated with Lycopersicon esculentum agglutinin-coated Dynabeads and cultured in EGM medium. CEC were treated with basic fibroblast growth factor (bFGF) and TA at various concentrations ranging from 50 to 300 mg/l. The capacities for CEC migration and tube formation were evaluated with the modified Boyden chamber and the Vitrogen collagen assay, respectively. The activities of matrix metalloproteinases (MMP)-2 and -9 were examined using gelatin zymography. RESULTS: The stimulation of CEC with 50 ng/ml bFGF resulted in an increase of about 100% in migration activity (P<0.01). Preincubation of CEC with TA at the indicated concentrations for 20 min inhibited the bFGF-stimulated migration in a dose-dependent manner (P<0.01). After 5 days, the bFGF-stimulated tube formation in CEC was inhibited by TA at the concentrations 100, 150 and 300 mg/l (P<0.01). Gelatin zymography of the culture media of CEC showed that the bFGF-induced activation of MMP-2 was attenuated by 300 mg/l TA (P<0.05). CONCLUSION: Downregulation of the activation of MMPs in CEC could be one of the mechanisms by which angiostatic steroids inhibit choroidal angiogenesis.

Animals↗
PubMed2002

Role and function of the 26S proteasome in proliferation and apoptosis.

The 26S proteasome constitutes the central proteolytic machinery of the highly conserved ubiquitin/proteasome system, the cell's major tool for extralysosomal protein degradation. Recently, a plethora of cell proteins implicated in the regulation of basic cellular processes, such as proliferation, differentiation, cell cycling, and apoptosis have been discovered to undergo processing and functional limitation by entering the ubiquitin/proteasome pathway with the final destination to be proteolytically degraded by the 26S proteasome. Because both negative and positive regulators of proliferation and apoptosis undergo proteasomal degradation in a tightly regulated and temporally controlled fashion, the 26S proteasome can play opposite roles in the regulation of proliferation and apoptosis. These roles are apparently defined by the cell's environment and proliferative state. Finally, proteasomal protein degradation is deregulated in a number of human diseases, including cancer and neurodegenerative and myodegenerative diseases, which all exhibit an imbalance of proliferation and apoptosis. An improved understanding of the modes of proteasomal action should lead to the development of beneficial therapeutic and diagnostic strategies in the future.

Animals↗