Gene expression profiling of connective tissue growth factor (CTGF) stimulated primary human tenon fibroblasts reveals an inflammatory and wound healing response in vitro

Purpose: The biologic relevance of human connective tissue growth factor (hCTGF) for primary human tenon fibroblasts (HTFs) was investigated by RNA expression profiling using affymetrix (TM) oligonucleotide array technology to identify genes that are regulated by hCTGF. Methods: Recombinant hCTGF was expressed in HEK293T cells and purified by affinity and gel chromatography. Specificity and biologic activity of hCTGF was confirmed by biosensor interaction analysis and proliferation assays. For RNA expression profiling HTFs were stimulated with hCTGF for 48h and analyzed using affymetrix (TM) oligonucleotide array technology. Results were validated by real time RT-PCR. Results: hCTGF induces various groups of genes responsible for a wound healing and inflammatory response in HTFs. A new subset of CTGF inducible inflammatory genes was discovered (e.g., chemokine [C-X-C motif] ligand 1 [CXCL1], chemokine [C-X-C motif] ligand 6 [CXCL6], interleukin 6 [IL6], and interleukin 8 [IL8]). We also identified genes that can transmit the known biologic functions initiated by CTGF such as proliferation and extracellular matrix remodelling. Of special interest is a group of genes, e.g., osteoglycin (OGN) and osteomodulin (OMD), which are known to play a key role in osteoblast biology. Conclusions: This study specifies the important role of hCTGF for primary tenon fibroblast function. The RNA expression profile yields new insights into the relevance of hCTGF in influencing biologic processes like wound healing, inflammation, proliferation, and extracellular matrix remodelling in vitro via transcriptional regulation of specific genes. The results suggest that CTGF potentially acts as a modulating factor in inflammatory and wound healing response in fibroblasts of the human eye

Course of disease in multifocal choroiditis lacking sufficient immunosuppression: a case report

Background: Multifocal choroiditis with panuveitis is a rare disease. The educational merit of this case presentation results from the good documentation and the impressive ocular fundus pictures. Case presentation: We illustrate the 3-year course of disease in a 22-year-old myopic white woman with multifocal choroiditis with panuveitis and secondary choroidal neovascularization. The activity of the disease was evaluated clinically by optical coherence tomography and fluorescein angiography. Choroidal neovascularization was treated by intravitreal bevacizumab (2.5 mg/0.1 ml). Our patient lacked systemic therapy for the first 11 months because of noncompliance. Conclusions: The case is remarkable as the delayed onset of peripheral lesions and the additional existence of high myopia made diagnosis difficult. In addition, it demonstrates that full outbreak of disease with multiple central and peripheral fundus lesions and secondary choroidal neovascularization can develop without systemic treatment

Colocalization of TGF-β-RII and the fibroblast marker prolyl-4-hydroxylase

<p><b>Copyright information:</b></p><p>Taken from "Localization of TGF-β type II receptor and ED-A fibronectin in normal conjunctiva and failed filtering blebs"</p><p></p><p>Molecular Vision 2008;14():136-141.</p><p>Published online 25 Jan 2008</p><p>PMCID:PMC2255025.</p><p></p> Native conjunctiva () and scarred filtering bleb specimens (, ) were double-stained for TGF-β-RII (green) and prolyl-4-hydroxylase (red). is a close-up of a portion of as indicated by the frame. Arrows point to a TGF-β-RII positive fibroblast, which are positive for both TGF-β-RII and prolyl-4-hydroxylase. Scale bar represents 50 µm (,) and 10 µm ()

Localization of α-smooth muscle actin (SMA) and the absence of lymphatic endothelium in scarred filtering bleb tissue

<p><b>Copyright information:</b></p><p>Taken from "Localization of TGF-β type II receptor and ED-A fibronectin in normal conjunctiva and failed filtering blebs"</p><p></p><p>Molecular Vision 2008;14():136-141.</p><p>Published online 25 Jan 2008</p><p>PMCID:PMC2255025.</p><p></p> Serial sections were double labeled for TGF-β-RII (, , and green) and α-SMA (, ), O-linked sialoglycoprotein (, ), and PECAM-1 (, , red). α-SMA is colocalized to TGF-β-RII in stromal fibroblasts and perivascular cells (,,). Vascular structures expressing TGF-β-RII() were negative for markers of lymphatic endothelium ().Some collagen fiber autofluorescence is present in the red channel (, , ), Scale bar represents 50 µm

Substrate rigidity modulates cell matrix interactions and protein expression in human trabecular meshwork cells. Invest Ophthalmol Vis Sci.

PURPOSE. Extracellular matrix (ECM) composition, tension, and rigidity modulate cell-ECM interactions and have substantial impact on cell functions. The authors studied the effects of ECM rigidity on human trabecular meshwork (HTM) cells to assess ECM rigidity as a possible pathophysiologic factor in glaucoma. METHODS. Trabecular meshwork cells derived from donor cornea rings and passaged three to seven times were plated on collagen-coated tissue culture plastic or polyacrylamide gels of different rigidity. Cell spreading and focal adhesions were assessed by immunofluorescence microscopy. Expression of focal adhesion kinase (FAK), ␣-smooth muscle actin (␣-SMA), tubulin, ␣-B-crystallin and GAPDH, as well as phosphorylation of FAK and serum-induced activation of ERK, were studied by Western blot. The subcellular distributions of ␣-SMA and fibronectin were examined by confocal immunofluorescence microscopy. RESULTS. ECM rigidity modulated cell spreading and focal adhesion size. FAK activation and serum-induced ERK phosphorylation increased with rising substrate rigidity. Expression of ␣-SMA and recruitment of ␣-SMA to stress fibers were enhanced on rigid substrates, whereas myocilin and ␣-B-crystallin expression increased on soft substrates. The structure of fibronectin deposits differed on stiff and soft matrices. CONCLUSIONS. Extracellular matrix rigidity modulates cytoskeletal structures, protein expression patterns, signal transduction, and fibronectin deposition in HTM cells. ECM changes altering trabecular meshwork resiliency may therefore have significant effects on ocular outflow tract functions with implications in glaucoma. (Invest Ophthalmol Vis Sci. 2008;49: 262-269