K. Wunderlich

Regulation of connective tissue growth factor gene expression in retinal vascular endothelial cells by angiogenic growth factors

Abstract Background: Connective tissue growth factor (CTGF) is a novel, cysteine-rich secreted protein, which is implicated in fibrotic disorders and atherosclerosis. To elucidate the role of CTGF in fibrovascular proliferative retinopathy, we investigated the regulation of CTGF gene expression in a cell line of retinal vascular endothelial cells (RVEC) stimulated with fetal calf serum (FCS) and angiogenic growth factors, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor-BB (PDGF-BB), endothelial growth factor (EGF), transforming growth factor-β1 and -β3 (TGF-β1, TGF-β3), and insulin-like growth factor-I (IGF-I). Methods: RVEC derived from Macaca mulatta (CRL-1780; ATCC) were stimulated with 10% FCS as well as with VEGF, bFGF, PDGF-BB, TGF-β1, TGF-β3, EGF, or IGF-I. Time-dependent CTGF gene expression was assessed by northern blot analysis. Results: FCS, TGF-β1, TGF-β3, bFGF, and EGF induced an upregulation of CTGF gene expression in RVEC in a time-dependent manner. Highest expression was induced with TGF-β1. No response on CTGF gene ex- pression could be detected to VEGF, PDGF-BB, or IGF-I. Conclusion: The present study demonstrates for the first time that CTGF mRNA is expressed at high levels in RVEC, and that the level of the temporal pattern of its expression is differentially regulated by angiogenic growth factors, indicating a significant role of CTGF in the pathological course of uncontrolled retinal angiogenesis.

Human Connective Tissue Growth Factor mRNA Expression of Epiretinal and Subretinal Fibrovascular Membranes: A Report of Three Cases

Purpose: To investigate the correlation between connective tissue growth factor (CTGF) mRNA expression and immunohistochemical characteristics (expression of type I collagen and tenascin) of fibrovascular membranes of proliferative retinal diseases under in vivo conditions. Methods: CTGF mRNA expression was investigated using in situ hybridization. Expression of type I collagen and tenascin was detected by immunohistochemical staining. Results: CTGF mRNA is produced in transformed retinal pigment epithelial cells and appears also in fibroblast-like cells, which are embedded in epiretinal and subretinal membranes of proliferative retinal diseases as well as in surgically removed subretinal membranes. In all examined membranes, expression of human CTGF mRNA appears in concurrence with the expression of type I collagen and tenascin. Conclusions: The predominant expression of CTGF mRNA in the development of fibrovascular membranes of proliferative retinal diseases suggests a significant role of CTGF in the pathological course of these ocular disorders.

Lens epithelial cell response to isoforms of platelet-derived growth factor

It has been reported previously that platelet derived growth factor (PDGF) may play an important role in the regulation of lens growth and differentiation. To evaluate PDGF-induced effects at the cellular level, we investigated the response of cultured bovine lens epithelial cells (BLEC) to PDGF-AB, -AA, and -BB isoforms at the cellular level. Stimulation of BLEC with PDGF isoforms showed no increase in cell proliferation under the culture conditions of this study. In contrast, measurement of cytosolic free calcium concentration ([Ca2+]i), which has been shown to be an important second messenger for controlling multiple cellular processes in the lens, revealed a dose-dependent rise in [Ca2+]i upon stimulation with PDGF-AB and -BB isoforms. PDGF-AA used in similar concentrations was not effective. Our data suggest that PDGF-AB and -BB may play a role in the regulation of cellular functions in BLEC via modulation of intracellular calcium homeostasis.

Disease proteomics reveals altered basic gene expression regulation in leukocytes of Normal-Tension and Primary Open-Angle glaucoma patients.

Glaucomatous damage is a neurodegenerative eye disease and one of the leading causes of blindness with 67 million patients worldwide. Major currently challenging questions include early diagnosis, risk evaluation, and follow‐up. Circulating leukocytes have been demonstrated as potentially important source of disease specific markers. The relevance of expression alterations in leukocytes for glaucomatous damage needs to be clarified. Noteworthy, gene expression patterns of trabecular meshwork and Schlemms canal, which are anatomically and functionally highly relevant for glaucoma pathology, were shown to be similar to those of circulating leukocytes. Here, we report extensive alterations in characteristic protein expression patterns of circulating leukocytes for Normal‐Tension and Primary Open‐Angle Glaucoma, as revealed by analysis of 2‐D PAGE images. Among most conservative alterations we found the protein spot identified by MALDI‐TOF as basic transcription factor activating protein‐2beta (AP‐2β). Western‐blot analysis demonstrated significantly increased protein expression rates of AP‐2β in both Normal‐Tension and Primary Open‐Angle Glaucoma versus controls. AP proteins are essential factors of the basic transcription regulation; AP‐2 proteins play a decisive role, particularly, in morphogenesis of eye. Conservative AP‐2 up‐regulation is of special importance in terms of basic transcriptional dysregulation that might be specific for glaucoma disease.

Activated STAT3 in Choroidal Neovascular Membranes of Patients with Age-Related Macular Degeneration

Purpose: The Jak/STAT (Janus tyrosine kinase/signal transducers and activators of transcription) pathway is critical for growth control, developmental regulation and homeostasis. Here we studied the expression of STAT proteins in the proliferative disease of age-related macular degeneration (AMD) with choroidal neovascular membranes (CNVM). The STATs are cytoplasmic proteins with roles as signal messengers and transcription factors that participate in normal cellular responses to cytokines and growth factors. Abnormal activity of certain STAT family members, particularly STAT3 and STAT5, is associated with a wide variety of human malignancies and other diseases. Here were studied STAT activation in CNVM of patients with AMD. Methods: Sections of formalin-fixed, paraffin-embedded samples from 8 eyes with AMD and 5 controls were included in this study. Immunohistochemical staining was performed using antibodies against activated STAT1, STAT3 and STAT5 proteins, and tenascin. Results: In CNVM, we observed a strong positive staining for tenascin and STAT3 in retinal pigmented epithelial (RPE) cells restricted to areas of developing scars. In contrast, STAT3 immunoreactivity failed in areas completely composed of fibrovascular disciform scar material. In addition, no immunoreactivity for both STAT1 and STAT5 was detected in all CNVM and in all control samples. Conclusion: In CNVM, activation of STAT3 appears in RPE cells simultaneously with the formation of scars.

Proliferative response of cultured human tenon's capsule fibroblasts to platelet-derived growth factor isoforms

Abstract• Background: Although platelet-derived growth factor (PDGF) has been thought to be critical in the wound-healing response of Tenons capsule fibroblasts after glaucoma filtration surgery, no information is currently available concerning the proliferative effect of PDGF isoforms on this cell type. The aim of the present study was to evaluate the proliferative effect of PDGF-AB heterodimer and PDGF-AA and -BB homodimers on cultured human Tenons capsule fibroblasts. • Methods: Human Tenons capsule fibroblasts, cultured under serum-free conditions, were stimulated with PDGF-AA, -AB and -BB isoforms in concentrations ranging from 1 to 100 ng/ml. Cell numbers were determined on days 1, 3, 5 and 7, using a cell counter. • Results: Addition of PDGF-AB and -BB led to a dosedependent increase in cell proliferation. A maximal response (79.9% over control) was obtained after 7 days with 30 ng/ml of PDGF-BB, with an EC50 of 8.9 ng/ml. The maximal increase in cell proliferation caused by PDGF-AB (30 ng/ml) was 54.9%, with an EC50 of 12.5 ng/ml. Stimulation with PDGF-AA revealed a significant effect only with concentrations higher than 30 ng/ml. • Conclusion: Our results indicate that PDGF-AB and -BB isoforms are potent stimulators of proliferation of human Tenons capsule fibroblasts, suggesting that PDGF-AB and -BB isoforms play an important role in the wound-healing response after glaucoma filtration surgery.

Serum-free cultivation of bovine lens epithelial cells

Mammalian cells in culture have individual nutritional requirements which are mainly fulfilled by the addition of fetal calf serum to the basic culture medium. Since many of the serum components are as yet poorly understood or even completely unknown, a number of difficulties arise in the evaluation of the effect of exogenously added factors or drugs on the growth of the cells. This paper presents data demonstrating the successful adaptation, routine cultivation and cryopreservation of bovine lens epithelial cells (BLEC) under serum-free culture conditions by use of the commercially available serum substitutes BMS, BM 86 Wissler and Ultroser G. Moreover, a culture medium especially designed for cell quiescence, named LR-1, is presented. Cells cultivated in culture medium containing 10% fetal calf serum served as controls. While BM-86 Wissler caused significantly reduced growth rates within 2 days and, finally, cell death after 12 days of incubation, the use of BMS resulted in growth rates which did not differ from the corresponding controls. Ultroser G resulted in a significant increase of proliferative activity of BLEC. LR-1 medium caused cell quiescence and kept the cells alive for a number of days. Thus, LR-1 allowed evaluation of the response of the cells to a mitogenic mixture from bovine brain mainly containing endothelial cell growth factor. The results demonstrate that cultivation of BLEC is possible under serum-free culture conditions. Moreover, the medium LR-1, which causes cell quiescence, is useful for the evaluation of growth factor-induced effects in vitro.

Influence of a lymphocyte-conditioned medium on the expression of smooth-muscle α-aktin in lens epithelial cells in situ

SummaryPurpose: Contraction of the capsule of the ocular lens is based upon proliferation and contraction of transformed lens epithelial cells. It is assumed that these processes can be assisted by postoperative intraocular inflammation. Previously, we reported that lens epithelial cell proliferation is enhanced by lymphocyte-conditioned medium (LCM). In this study we investigated the effect of LCM as well as of a culture medium conditioned by pigmented ciliary epithelial cells (CBCM) on the expression of the smooth-muscle alpha-actin of the contractile cytoskeletal elements. Methods: Explants of the anterior lens capsule of freshly enucleated bovine eyes were cultured in serum-free LCM and CBCM for 3 days, followed by fixation. Smooth-muscle alpha-actin was identified by indirect immunofluorescence. Explants cultured in serum-free bFGF-containing and TGF-β-containing medium served as control. Results: Lens epithelial cells expressed smooth-muscle alpha-actin under the influence of LCM or TGF-β. No smooth muscle alpha-actin could be detected under the influence of CBCM or bFGF. Conclusion: Our results demonstrate that secreted molecules of activated lymphocytes are able to induce the transformation of lens epithelial cells into contractile myofibroblasts and may be involved in the postoperative contraction of lens capsules.ZusammenfassungHintergrund: Zellbiologische Grundlage der posterioren Kapselfältelung sowie der sog. vorderen Kapselschrumpfung nach Kataraktextraktion ist die Proliferation und Kontraktion der postoperativ im Kapselsack verbliebenen Linsenepithelzellen. Vermutet wird, daß postoperativ persistierende Entzündungen diese Veränderungen begünstigen. Nachdem wir früher zeigen konnten, daß ein Lymphozyten konditioniertes Medium (LKM) die Proliferation von Linsenepithelzellen stark stimuliert, wurde in der vorliegenden Arbeit untersucht, inwieweit die Expression des kontraktilen Zytoskelettelements alpha-Aktin durch das LKM begünstigt wird. Zusätzlich wurde der Einfluß eines durch pigmentierte Ziliarkörperepithelzellen konditionierten Mediums (ZKKM) untersucht. Material und Methode: Anteriore Kapselexplantate schlachtfrischer Rinderaugen wurden 3 Tage in serumfreiem LKM bzw. ZKKM kultiviert. Im Anschluß daran erfolgte an fixierten Kapselexplantaten der Nachweis glattmuskulären alpha-Aktins mittels indirekter Immunfluoreszenz. Als Kontrollen dienten in serumfreiem, bFGF-haltigen oder TGF-β-haltigem, aber unkonditioniertem Medium kultivierte Explantate. Ergebnisse: Unter dem Einfluß des LKM bzw. TGF-β exprimierten Linsenepithelzellen glattmuskuläres alpha-Aktin. Der Nachweis dieser Zytoskelettkomponente fiel dagegen bei Linsenepithelien, die in bFGF-haltigem, unkonditioniertem Medium oder in ZKKM inkubiert worden waren, negativ aus. Schlußfolgerung: Die vorliegenden Untersuchungen zeigen, daß Sekretionsprodukte aktivierter Lymphozyten die Expression glattmuskulären alpha-Aktins als zelluläres Zeichen einer myofibrillären Transformation stimulieren und somit an der Induktion einer Linsenkapselschrumpfung beteiligt sein können.