Novel insight into the inflammatory and cellular responses following experimental glaucoma surgery: a roadmap for inhibiting fibrosis.

Failure after glaucoma filtration surgery is attributed to fibrosis at the operated site. To understand the wound healing process after glaucoma filtration surgery, we have developed a mouse model for glaucoma filtration surgery which closely mimics the clinical response. In this study, we describe a systematic analysis of the wound healing response in vivo. Our data revealed that the post-surgical tissue response was separable into two distinguishable phases. The early "acute inflammatory" phase was characterized by significantly increased transcript expression of Vegfa, Cxcl1, Cxcl5, Ccl2, Ccl3, Ccl4, Gmcsf and specific Mmps as well as greater infiltration of monocytes/macrophages and T cells. The late "fibrotic" phase was characterized by an increased expression of Tgfb2 and extracellular matrix genes as well as a notable reduction of infiltrating inflammatory cells. Significantly, more mitotic cells were observed at both time points post-surgery. Subconjunctival fibroblasts may be involved in both phases since they have the capacity to reiterate the in vivo gene expression profiles upon either pro-inflammatory or pro-fibrotic cytokine stimulation. Given that the cellular and molecular targets that govern the early and late phases of wound healing are distinct and time sensitive, a multi-targeted therapeutic approach to sequentially inhibit inflammation and fibrogenesis at the critical time point may lead to improved surgical outcomes in glaucoma filtration surgery.

The Phox homology (PX) domain, a new player in phosphoinositide signalling.

Phosphoinositides are key regulators of diverse cellular processes. The pleckstrin homology (PH) domain mediates the action of PtdIns(3,4)P(2), PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3), while the FYVE domain relays the pulse of PtdIns3P. The recent establishment that the Phox homology (PX) domain interacts with PtdIns3P and other phosphoinositides suggests another mechanism by which phosphoinositides can regulate/integrate multiple cellular events via a spectrum of PX domain-containing proteins. Together with the recent discovery that the epsin N-terminal homologue (ENTH) domain interacts with PtdIns(4,5)P(2), it is becoming clear that phosphoinositides regulate diverse cellular events through interactions with several distinct structural motifs present in many different proteins.

Endofin, an endosomal FYVE domain protein.

KIAA0305 is an uncharacterized member of the FYVE domain protein family. It is closely related to SARA, with about 50% identity in the carboxyl-terminal 800-amino acid region. Indirect immunofluorescence microscopy using polyclonal antibodies raised against KIAA0305 revealed that it is enriched in early endosomes. The Myc-tagged version is also faithfully targeted to the early endosome. We have tentatively called KIAA0305 endofin (for endosome-associated FYVE-domain protein). The association of endofin with endosomes is mediated by its FYVE domain because deletion mutants lacking the central FYVE finger motif are distributed in the cytoplasm. In addition, a single point mutation in the FYVE finger motif at cysteine residue 753 (C753S) is sufficient to abolish its endosomal association. Its endosomal localization is also sensitive to the phosphatidylinositol 3-kinase inhibitor, wortmannin. Using in vitro liposome binding assays, we demonstrate that Myc-tagged endofin associates preferentially with phosphatidylinositol 3-phosphate, whereas the C753S point mutant was unable to do so. We also show that endofin co-localizes with SARA but that they are not associated in a common complex because they failed to co-immunoprecipitate in co-expressing cells. Endofin also does not associate with Smad2 nor behave like SARA in affecting transforming growth factor-beta signaling. At high levels of expression, both endofin and SARA can cause an endosome aggregation/fusion effect. In COS7 cells, which can support high levels of exogenous protein expression, both proteins can also cause other structural anomalies in the endocytic pathway, as represented by enlarged vesicular structures. These endosomal aggregates/fusions accumulated endocytosed epidermal growth factor. Taken together, this report provides evidence to suggest that endofin and the highly related SARA are endosomal proteins with potential roles in regulating membrane traffic.

Molecular cloning of multiple isoforms of synaptojanin 2 and assignment of the gene to mouse chromosome 17A2-3.1.

Synaptojanin 2 is an inositol polyphosphate 5'-phosphatase that appears to be regulated by alternative splicing. By screening mouse cDNA libraries derived from either mouse day 16 embryo or adult liver, we have identified additional synaptojanin 2 cDNAs that represent six new isoforms of the protein. This finding, together with other reports, indicates the presence of eight isoforms of synaptojanin 2. Sequence analysis of our cDNA clones suggests that there are at least two putative initiation sites and at least six different sequences coding for the carboxyl-terminus of the molecule. In addition, we have mapped synaptojanin 2 to mouse chromosome 17 band A2-3.1 by fluorescence in situ hybridization.