Biomedical subjects

Günther Schlunck

Publications and source records attributed to Günther Schlunck.

7 recordsLinked to original sources

PubMed2006

p38 inhibitors prevent TGF-beta-induced myofibroblast transdifferentiation in human tenon fibroblasts.

PURPOSE: The role of mitogen-activated protein kinase (MAPK) pathways in TGF-beta-induced myofibroblast transdifferentiation of human tenon fibroblasts (HTFs) was investigated to identify potential pharmacologic targets for the inhibition of scarring after glaucoma surgery. METHODS: TGF-beta-dependent activation of Smad2, p38, and Erk-1/2 was examined by Western blot analysis. TGF-beta-induced mRNA expression of collagen Ialpha1, fibronectin, and the myofibroblast transdifferentiation marker alpha smooth muscle actin (alpha-SMA) was analyzed by real-time RT-PCR. alpha-SMA protein expression and subcellular distribution were determined by Western blot analysis and immunofluorescence cytochemistry. Fibroblast contractility was assessed in three-dimensional collagen gel contraction assays, stress fiber assembly with rhodamine-phalloidin stains, and confocal microscopy. Cell proliferation was measured with an MTT assay. Specific pharmacologic kinase inhibitors were used to characterize the involvement of MAPK-dependent pathways. RESULTS: TGF-beta stimulation of HTF induced a rapid and transient activation of Smad2 and Erk, whereas p38 activation was biphasic and sustained. After 24 hours of TGF-beta stimulation, increased levels of collagen Ialpha1, fibronectin, and alpha-SMA transcripts were detected. After 3 days of stimulation, HTF displayed increased alpha-SMA protein levels, enhanced contractility, and assembly of actin stress fibers. TGF-beta also induced HTF proliferation. Specific p38 inhibitors prevented all these aspects of TGF-beta-induced myofibroblastic transdifferentiation. CONCLUSIONS: Pharmacologic inhibition of p38 abrogates TGF-beta-induced myofibroblast transdifferentiation, reduces extracellular matrix protein expression and HTF proliferation, and may therefore serve to inhibit scarring after glaucoma surgery.

Actins↗

PubMed2006

Contractility as a prerequisite for TGF-beta-induced myofibroblast transdifferentiation in human tenon fibroblasts.

PURPOSE: To assess the significance of Rho-kinase-dependent contractility in TGF-beta-induced myofibroblast transdifferentiation of human tenon fibroblasts to characterize possible pharmacological targets for the inhibition of postoperative scarring after glaucoma surgery. METHODS: Human tenon fibroblasts (HTFs) were grown in culture and stimulated with TGF-beta1. The effect of TGF-beta on Rho-GTPase activity was assessed by GST-rhotekin binding domain pulldown assay and detected by Western blot analysis. Contractility was evaluated in a silicone substrate wrinkling assay and in fibroblast-populated collagen gels. The actin cytoskeleton and focal adhesions were visualized by immunofluorescence microscopy. alpha-SMA transcripts were measured by real-time RT-PCR. TGF-beta-induced Smad- and p38-activation and expression of alpha-SMA were detected by Western blot analysis. Nuclear translocation of Smad2/3 was determined by confocal immunofluorescence microscopy. The influence of Rho-dependent kinase (ROCK) and myosin light chain kinase (MLCK) were studied by using specific kinase inhibitors (Y-27632, HA-1077, H-1152, and ML-7). RESULTS: Within 10 minutes of stimulation, TGF-beta induced Rho activation that was associated with an increase in cell tension and followed by actin stress fiber enhancement. ROCK inhibitors released cell tension and averted TGF-beta-induced cytoskeletal changes, p38 activation and subsequent alpha-SMA expression, whereas Smad2-phosphorylation and nuclear translocation were preserved. MLCK inhibition also blocked alpha-SMA expression. In fibroblast-populated collagen lattices, ROCK inhibitors prevented TGF-beta-induced stress fiber assembly and contraction. CONCLUSIONS: TGF-beta induces a rapid contractile response in HTFs that precedes myofibroblast transdifferentiation. ROCK inhibitors release this contraction and block subsequent TGF-beta-induced myofibroblast transdifferentiation and may therefore serve to modulate postoperative scarring after glaucoma filtering surgery.

Actins↗

PubMed2005

The peptides ADNF-9 and NAP increase survival and neurite outgrowth of rat retinal ganglion cells in vitro.

PURPOSE: Recent studies demonstrated that short peptides derived from activity-dependent neurotrophic factor (ADNF) and activity-dependent neuroprotective protein (ADNP) are neuroprotective at femtomolar concentrations. We evaluated these findings in cultures of purified rat retinal ganglion cells (RGCs) using two such peptides: ADNF-9 and NAP. In a second step, the influence of these peptides on neurite outgrowth in retinal explants was investigated. METHODS: Retinal ganglion cells (RGCs) were purified from newborn (postnatal day [P]0-P2) rat retina by immunopanning with antibodies against Thy1.1 and were cultured in serum-free N2 medium for 2 days. RGCs were treated with ADNF-9 and NAP at concentrations ranging from 10(-18) to 10(-10) M. Survival was quantified by counting viable cells by phase-contrast microscopy. Retinal explants from postnatal (P9-P11) rats were cultured in three-dimensional fibrin clots in serum-free medium for 3 days. Explants were treated with 1 microM NAP or 1 microM ADNF-9. Neurite outgrowth was visualized by staining with Sudan black and quantified by measuring axonal length. RESULTS: Both peptides enhanced survival of RGCs in a dose-dependent manner. ADNF-9 showed a maximum effect at 0.1 pM with an increase in survival to 177% (95% confidence interval: 149-204) of the control level. The EC(50) was 10.9 fM. NAP showed a maximum effect at 5 pM with an increase in survival to 167% (146-189) and an EC(50) of 6.1 fM. In the explants, 1 microM ADNF-9 enhanced axonal outgrowth to 126% (118-133) and 1 microM NAP to 117% (98-137) compared with the control. CONCLUSIONS: Both peptides, ADNF-9 and NAP, not only increase RGC survival in vitro but also support neurite outgrowth in retinal explants. These peptides deserve further attention as potential neuroprotective compounds in retinal and optic nerve diseases.

Animals↗

PubMed2004

Differential expression of angioregulatory factors in normal and CNV-derived human retinal pigment epithelium.

BACKGROUND: Choroidal neovascularization (CNV) causes loss of vision in age-related macular degeneration (AMD). In CNV, choroidal capillaries penetrate Bruch's membrane and the retinal pigment epithelium (RPE). Angiogenic factors produced by RPE cells are suspected as major contributors to CNV development. We therefore studied the differential expression of angioregulatory factors in normal and CNV-derived RPE. METHODS: Cultures of normal (ARPE-19) and CNV-derived RPE (CNV-RPE) were compared by quantitative PCR. Differential expression was verified on the protein level by immunohistochemistry in tissue samples. RESULTS: The angioregulatory factors VEGF-A, VEGF-B, VEGF-C, Angiopoietin-1 (Ang-1) and Angiopoietin-2, Semaphorin-3A, PEDF, HIF-1, FGF-2, and the receptors VEGF-R2, Neuropilin-1 and Neuropilin-2 were detected in both, ARPE-19 and CNV-RPE. Transcription of PEDF, FGF-2, Neuropilin-2, Ang-1 and Ang-2 was significantly upregulated in CNV-RPE. EphA7, VEGF-R1 and leptin were transcribed exclusively in CNV-RPE and Eph-A7 and VEGF-R1 proteins were present exclusively in CNV specimens. CONCLUSIONS: A set of common factors controlling angiogenesis was detected in both, ARPE-19 cells and CNV-RPE cells. Surprisingly, PEDF and other factors inhibiting angiogenesis are strongly upregulated in CNV-RPE; thus, at least in later stages, the RPE has a potential to control angiogenesis in age-related macular degeneration.

Angiogenic Proteins↗

PubMed2003

Modulation of Rac localization and function by dynamin.

The GTPase dynamin controls a variety of endocytic pathways, participates in the formation of phagosomes, podosomal adhesions, and invadopodia, and in regulation of the cytoskeleton and apoptosis. Rac, a member of the Rho family of small GTPases, controls formation of lamellipodia and focal complexes, which are critical in cell migration and phagocytosis. We now show that disruption of dynamin(-2) function alters Rac localization and inhibits cell spreading and lamellipodia formation even though Rac is activated. Dominant-negative K44A dynamin(-2) inhibited cell spreading and lamellipodia formation on fibronectin without blocking cell adhesion; dynamin(-2) depletion by specific small interfering RNA inhibited lamellipodia in a similar manner. Dyn2(K44A) induced Rac mislocalization away from cell edges, into abnormal dorsal ruffles, and led to increased total Rac activity. Fluorescence resonance energy transfer imaging of Rac activity confirmed its predominant localization to aberrant dorsal ruffles in the presence of dominant-negative dyn2(K44A). Dyn2(K44A) induced the accumulation of tubulated structures bearing membrane-bound Rac-GFP. Constitutively active but not wild-type GFP-Rac was found on macropinosomes and Rac-dependent, platelet-derived growth factor-induced macropinocytosis was abolished by Dyn2(K44A) expression. These data suggest an indispensable role of dynamin in Rac trafficking to allow for lamellipodia formation and cell spreading.

Animals↗

PubMed2002

Cultivation of retinal pigment epithelial cells from human choroidal neovascular membranes in age related macular degeneration.

A method is described for cultivating retinal pigment epithelial cells from choroidal neovascular membrane (CNV) specimens that were surgically removed in patients with age-related macular degeneration (AMD). CNV specimens of 43 patients were available for cultivation. They were incubated in supplemented DMEM/Ham's F12 cell culture medium on microporous semipermeable filter membranes. Thirty-four specimens gave rise to cell cultures, 28 of which could be subcultivated for up to 15 passages. The membrane type as classified by fluorescence angiography was compared with cellular growth in vitro. Immunocytochemistry revealed a uniform expression of cytokeratin 18 and vimentin, while factor 8, glial fibrillary acidic protein and alpha smooth muscle actin were absent in all 21 cultures stained. The expression of RPE markers cellular retinaldehyde binding protein (CRALBP) and RPE65 was detected by RT-PCR in all cultures tested. An epithelial character of the cultures was supported by the presence of apical microvilli as determined by electron microscopical studies. Therefore, the cell cultures from CNV in AMD bear characteristics of retinal pigment epithelial cells. For the first time, this cell culture system holds the potential to study human RPE cells in the context of neovascular AMD in vitro.

Aged↗

PubMed2001

Genotypes of JC virus in East, Central and Southwest Europe.

Distinctive genotypes of JC virus have been described for the major continental landmasses. Studies on European-Americans and small cohorts in Europe showed predominantly Type 1. Types 2 and 7 are found in Asia, and Types 3 and 6 in Africa. These genotypes differ in sequence by about 1--3%. Each genotype may have several subtypes which differ from each other by about 0.5--1%. The genotypes can be defined by a distinctive pattern of nucleotides in a typing region of the VP1 gene. This genotyping approach has been confirmed by phylogenetic reconstruction using the entire genome exclusive of the rearranging regulatory region. In this first large European study, we report on the urinary excretion of JCV DNA of 350 individuals from Poland, Hungary, Germany and Spain. We included Gypsy cohorts in Hungary (Roma), Germany (Sinti), and Spain (Gitano), as well as Basques in Spain. We show that while Type 1 predominates in Europe, the proportions of Type 1A and 1B may differ from East to Southwest Europe. Type 4, closely related to the Type 1 sequence (only approximately 1% difference) was a minor genotype in Germany, Poland and Spain, but represented the majority in Basques. The Gitanos in Spain showed a variant Type 4 sequence termed 'Rom-1'. Interestingly, neither the Gitanos in Spain, nor Sinti or Roma in Germany or Hungary showed the Type 2 or Type 7 genotype that might be expected if their origins were in an Asian population.

Adolescent↗