The development and assessment of a self-perceived quality of vision questionnaire to test pseudophakic patients.

PURPOSE: To develop and test a questionnaire to assess quality of vision in pseudophakic patients. METHODS: The problems with existing health measurement scales and the need for a new questionnaire are first discussed. Development of the new questionnaire from concept to completion is presented and all stages discussed. The questionnaire is then tested for internal consistency, reliability and validity. RESULTS: The Cronbach Alpha for internal consistency was 0.92. British Standards Institution repeatability coefficient was satisfactory at 6.6. Discriminant construct validity testing by extreme groups demonstrated excellent discrimination between patients with functionally significant posterior capsule opacification (PCO) and no significant PCO (Mann-Whitney U test, p = 0.001). CONCLUSION: The questionnaire is validated as a robust, stable measure of pseudophakic visual symptoms with a high degree of clinical utility. It should be invaluable for the many studies that compare outcomes from different forms of modern cataract surgery with implantation of different intraocular lenses.

The alpha4 laminin subunit regulates endothelial cell survival.

The alpha4 laminin subunit is a major structural component of assembling basement membranes of endothelial cells. We have been investigating its functions with regard to endothelial cell survival. An anti-laminin alpha4 antibody (2A3), directed against the G domain of the alpha4 laminin subunit of laminins-8 and -9, inhibits proliferation and enhances apoptosis of endothelial cells when cells are maintained in vitro. Activation of caspases-9 and -3 plays a role in 2A3 antibody-induced apoptosis, since inhibitors specific for these caspases and overexpression of the anti-apoptotic protein Bcl-X(L), but not c-FLIP, inhibit 2A3 antibody-triggered endothelial cell death. Extracellular matrix is known to play a role in regulating programmed cell death in an integrin-dependent fashion. The alpha4 laminin subunit conforms to this idea since activation of beta1 integrin subunits on endothelial cells blocks the ability of 2A3 antibody to induce endothelial cell death. In summary, our data indicate that complexes composed of alpha4 laminin/beta1 subunit-containing integrins at the cell surface support endothelial cell survival. Furthermore, we propose that antagonists of alpha4 laminin function, including antibody 2A3, have value as angiogenesis inhibitors in a clinical setting where blocking aberrant growth of blood vessel by triggering apoptosis of endothelial cells may be therapeutic.

Structure and function of a vimentin-associated matrix adhesion in endothelial cells.

The alpha4 laminin subunit is a component of endothelial cell basement membranes. An antibody (2A3) against the alpha4 laminin G domain stains focal contact-like structures in transformed and primary microvascular endothelial cells (TrHBMECs and HMVECs, respectively), provided the latter cells are activated with growth factors. The 2A3 antibody staining colocalizes with that generated by alphav and beta3 integrin antibodies and, consistent with this localization, TrHBMECs and HMVECs adhere to the alpha4 laminin subunit G domain in an alphavbeta3-integrin-dependent manner. The alphavbeta3 integrin/2A3 antibody positively stained focal contacts are recognized by vinculin antibodies as well as by antibodies against plectin. Unusually, vimentin intermediate filaments, in addition to microfilament bundles, interact with many of the alphavbeta3 integrin-positive focal contacts. We have investigated the function of alpha4-laminin and alphavbeta3-integrin, which are at the core of these focal contacts, in cultured endothelial cells. Antibodies against these proteins inhibit branching morphogenesis of TrHBMECs and HMVECs in vitro, as well as their ability to repopulate in vitro wounds. Thus, we have characterized an endothelial cell matrix adhesion, which shows complex cytoskeletal interactions and whose assembly is regulated by growth factors. Our data indicate that this adhesion structure may play a role in angiogenesis.

Spatial regulation and activity modulation of plasmin by high affinity binding to the G domain of the alpha 3 subunit of laminin-5.

Cells in complex tissues contact extracellular matrix that interacts with integrin receptors to influence gene expression, proliferation, apoptosis, adhesion, and motility. During development, tissue remodeling, and tumorigenesis, matrix components are modified by enzymatic digestion with subsequent effects on integrin binding and signaling. We are interested in understanding the mechanisms by which broad spectrum proteinases such as plasmin are targeted to their extracellular matrix protein substrates. We have utilized plasmin-mediated cleavage of the epithelial basement membrane glycoprotein laminin-5 as a model to evaluate molecular events that direct plasmin activity to specific structural domains. We report that plasminogen and tissue plasminogen activator (tPA) exhibit high affinity, specific binding to the G(1) subdomain of the N terminus of the laminin-5 alpha(3) subunit, with equilibrium dissociation constants of 50 nm for plasminogen and 80 nm for tPA. No high affinity binding to the G(2), G(3), and G(4) subdomains was observed. As a result of binding to the G(1) subdomain, the catalytic efficiency of tPA-catalyzed plasminogen activation is enhanced 32-fold, leading to increased matrix-associated plasmin that is positioned favorably for cleavage within the G(4) subdomain as we have reported previously (Goldfinger, L. E., Stack, M. S., and Jones, J. C. R. (1998) J. Cell Biol. 141, 255-265). Thus, physical constraints dictated by interaction of proteinase and matrix macromolecule control not only enzymatic activity but may regulate substrate targeting of proteinases.

The N terminus of the transmembrane protein BP180 interacts with the N-terminal domain of BP230, thereby mediating keratin cytoskeleton anchorage to the cell surface at the site of the hemidesmosome.

In epidermal cells, the keratin cytoskeleton interacts with the elements in the basement membrane via a multimolecular junction called the hemidesmosome. A major component of the hemidesmosome plaque is the 230-kDa bullous pemphigoid autoantigen (BP230/BPAG1), which connects directly to the keratin-containing intermediate filaments of the cytoskeleton via its C terminus. A second bullous pemphigoid antigen of 180 kDa (BP180/BPAG2) is a type II transmembrane component of the hemidesmosome. Using yeast two-hybrid technology and recombinant proteins, we show that an N-terminal fragment of BP230 can bind directly to an N-terminal fragment of BP180. We have also explored the consequences of expression of the BP230 N terminus in 804G cells that assemble hemidesmosomes in vitro. Unexpectedly, this fragment disrupts the distribution of BP180 in transfected cells but has no apparent impact on the organization of endogenous BP230 and alpha6beta4 integrin. We propose that the BP230 N terminus competes with endogenous BP230 protein for BP180 binding and inhibits incorporation of BP180 into the cell surface at the site of the hemidesmosome. These data provide new insight into those interactions of the molecules of the hemidesmosome that are necessary for its function in integrating epithelial and connective tissue types.

The alpha3 laminin subunit, alpha6beta4 and alpha3beta1 integrin coordinately regulate wound healing in cultured epithelial cells and in the skin.

Previously, we demonstrated that proteolytic processing within the globular domain of the alpha3 subunit of laminin-5 (LN5) converts LN5 from a cell motility-inducing factor to a protein complex that can trigger the formation of hemidesmosomes, certain cell-matrix attachment sites found in epithelial cells. We have prepared a monoclonal antibody (12C4) whose epitope is located toward the carboxy terminus of the globular domain of the alpha3 laminin subunit. This epitope is lost from the alpha3 subunit as a consequence of proteolytic processing. Antibody 12C4 stains throughout the matrix of cells that fail to process the alpha3 laminin subunit, but does not recognize the matrix of confluent cultures of MCF-10A cells, which efficiently process their alpha3 laminin chain. In subconfluent populations of MCF-10A cells, 12C4 only stains matrix deposited at the outer edges of cell colonies. In these cells, integrin alpha3beta1 occasionally colocalizes with the staining generated by the 12C4 antibody but alpha6beta4 integrin does not. In wounded MCF-10A cell cultures, the 12C4 antibody stains the extracellular matrix beneath those cells at the very edge of the cellular sheet that moves to cover the wound site. A similar phenomenon is observed in human skin wounds, since we also detect expression of the unprocessed alpha3 laminin subunit at the leading tip of the sheet of epidermal cells that epithelializes skin wounds in vivo. In addition, using alpha3 laminin subunit and integrin function-inhibiting antibodies, we provide evidence that LN5 and its two integrin receptors (alpha6beta4 and alpha3beta1) appear necessary for wound healing to occur in MCF-10A cell culture wounds. We propose a model for healing of wounded epithelial tissues based on these results.

Interaction of BP180 (type XVII collagen) and alpha6 integrin is necessary for stabilization of hemidesmosome structure.

The hemidesmosome is a multimolecular complex that integrates the extracellular matrix with the keratin cytoskeleton and that stabilizes epithelial attachment to connective tissue. A 180 kDa protein (BP180, type XVII collagen), first identified by its reactivity with autoantibodies in the serum of patients with a blistering skin disease called bullous pemphigoid (BP), is a transmembrane component of the hemidesmosome with a collagen-like extracellular domain. Here, using recombinantly expressed molecules and the yeast two-hybrid assay, we have identified alpha6 integrin as a BP180-binding partner. The association between specific domains of the BP180 and alpha6 integrin molecules is inhibited by a 14 mer peptide, whose sequence is identical to amino acid residues 506-519 in the noncollagenous region of the ectodomain of the BP180 molecule, as well as by antibodies raised against this peptide. The 14 mer peptide sequence is part of an epitope recognized by autoantibodies that are pathogenic in BP. In vivo, when 804G cells are plated into medium containing the same peptide, they fail to assemble hemidesmosomes. Furthermore, although BP180 and certain cytoplasmic components of the hemidesmosome colocalize in the peptide-treated cells, they are aberrantly distributed and fail to show extensive association with (alpha6beta4 integrin. Taken together, our results indicate that BP180 is a novel transmembrane ligand of the alpha6beta4 integrin heterodimer. In addition, our data provide support for the possibility that BP180 and alpha6 integrin interaction is not only mediated by the BP epitope but is necessary for hemidesmosome formation.

Structure and assembly of hemidesmosomes.

The hemidesmosome is a complex junction containing many proteins. The keratin cytoskeleton attaches to its cytoplasmic plaque, while its transmembrane elements interact with components of the extracellular matrix. Hemidesmosome assembly involves recruitment of alpha 6 beta 4 integrin heterodimers, as well as cytoskeletal elements and cytoskeleton-associated proteins to the cell surface. In our cell culture models, these phenomena appear to be triggered by laminin-5 in the extracellular matrix. Cell interaction with laminin-5 apparently induces both phosphorylation and dephosphorylation of subunits of alpha 6 beta 4 integrin. There is emerging evidence that such events are necessary for subsequent cytoskeleton anchorage to the hemidesmosome cytoplasmic plaque. Once assembled, the hemidesmosome plays an essential role in maintaining firm epithelial adhesion to the basement membrane, with hemidesmosome disruption being a hallmark of certain devastating blistering diseases. However, the hemidesmosome is more than just a stable anchor, as it may also be the site of signal transduction, mediated by its alpha 6 beta 4 integrin component. This review discusses our current knowledge of the structure and assembly of the hemidesmosome.

Laminin-5 and hemidesmosomes: role of the alpha 3 chain subunit in hemidesmosome stability and assembly.

Hemidesmosomes are complex macromolecular structures which integrate elements of the extracellular matrix and the cytoskeleton of epithelial cells. To characterize cell-matrix interactions in the hemidesmosome, we have made use of 804G cells which possess the unusual ability to assemble hemidesmosomes in vitro. During the course of our studies, we have raised a set of monoclonal antibodies against rat laminin-5, the major structural element comprising 804G matrix. One of these, termed CM6, recognizes the 150 kDa alpha chain of rat laminin-5 and binds the globular (G) domain of intact laminin-5 molecules as determined by rotary shadowing. CM6 antibodies perturb formed hemidesmosomes in 804G cells. In particular, within 1 hour of incubation of 804G cells with CM6 antibodies, colocalization of laminin-5 and alpha 6 beta 4 integrin is lost and by 2 hours, staining generated by hemidesmosomal antibodies appears primarily cytoplasmic in the perinuclear zone. Ultrastructurally, CM6 antibodies first appear to induce detachment of hemidesmosomes from the underlying matrix. Next, portions of the basal cell surface invaginate to form vesicles whose cytoplasmic-facing surface is coated with hemidesmosomes still associated with keratin intermediate filaments. Anchoring filaments extend into the inside compartment of the vesicles. We have also studied the impact of CM6 antibodies on a model system in which the matrix of 804G cells induces de novo assembly of hemidesmosomes in human keratinocytes. This process involves the plasma membrane reorganization of the hemidesmosome associated integrin alpha 6 beta 4 as well as a redistribution of other hemidesmosome components such as the 230 kDa bullous pemphigoid antigen. Pretreatment of 804G matrix with CM6 antibodies blocks such plasma membrane reorganization of hemidesmosome components and inhibits hemidesmosome formation. Our studies indicate a crucial role for the G domain of the alpha chain of laminin-5 in both nucleation of hemidesmosome assembly as well as maintenance of hemidesmosome structural integrity.

Molecular genetic studies of a human epidermal autoantigen (the 180-kD bullous pemphigoid antigen/BP180): identification of functionally important sequences within the BP180 molecule and evidence for an interaction between BP180 and alpha 6 integrin.

The 180-kD bullous pemphigoid autoantigen (BP180) is a component of the hemidesmosome, a cell-matrix connector. This protein is oriented in a type II fashion in the membrane of the hemidesmosome and is a hybrid collagen (classified as type XVII). We have analyzed the fate of various mutant BP180 molecules transfected into several different cell types. A protein, D1, lacking the collagen-like extracellular domains of BP180 polarizes normally in 804G epithelial cells and colocalizes with other hemidesmosomal components in the plane of the basal cell surface. However, deletion of a stretch of 36 amino acids located at the NH2 terminus of D1 induces an apical polarization of the protein (D1-36N) in the cell surface of 804G cells. Deletion of the 27-amino acid noncollagenous extracellular domain that is located immediately after the membrane spanning domain of BP180 results in a failure of D1-27C protein to codistribute with other hemidesmosomal components despite its basal localization in transfected 804G cells. In FG cells, which lack their own BP180, transfected D1 protein localizes with the alpha 6 beta 4 integrin heterodimer. In HT1080 cells, which do not possess BP180 or beta 4 integrin, D1 protein localizes with alpha 6 beta 1 integrin while both the D1-27C and D1-36N proteins do not. Moreover, D1 protein coprecipitates with alpha 6 integrin from extracts of HT1080 transfectants. Taken together, these results suggest that the NH2-terminal domain of BP180 determines polarization of BP180 while the noncollagenous extracellular domain of BP180 stabilizes its interactions with other hemidesmosomal components, such as alpha 6 integrin. Perturbation of this latter domain by human bullous pemphigoid autoantibodies may explain the loss of epidermal cell-dermis attachment that characterizes the BP disease.

Identification of a second protein product of the gene encoding a human epidermal autoantigen.

A 230 kDa polypeptide component of the hemidesmosome, an epithelial-cell-connective-tissue attachment device, is thought to be involved in cytoskeleton-cell-surface anchorage. This 230 kDa polypeptide is recognized by bullous pemphigoid auto-antibodies and for this reason is generally termed the bullous pemphigoid antigen (BPA). We have identified two distinct mRNA products of the single BPA gene by RACE (rapid amplification of cDNA ends)/PCR techniques. The first of these mRNAs encodes the 230 kDa protein component of the hemidesmosome. A second mRNA lacks over 1800 bases that encode the C-terminus of the 230 kDa protein. We have raised antibodies against a peptide specific to the predicted protein product of this second mRNA. To our surprise this antibody recognizes a protein that migrates at 280 kDa on SDS/PAGE of extracts of a variety of human epidermal cell lines that also express the 230 kDa BPA. Moreover, we have confirmed the co-expression of the 230 and 280 kDa polypeptides in these cells by immunoblotting analyses using a monoclonal antibody preparation directed against a polypeptide encoded by sequence common to both mRNAs transcribed from the BPA gene. Intriguingly, in one non-epidermal tumour line (a pancreatic cell line termed FG), the 280 kDa polypeptide appears to be the only product of the BPA gene. Furthermore, in FG cells the 280 kDa protein is found in association with the intermediate filament cytoskeleton. We discuss our results in relation to control of BPA gene expression and with regard to potential functions of the domains of the protein products of the BPA gene.

The matrix secreted by 804G cells contains laminin-related components that participate in hemidesmosome assembly in vitro.

Hemidesmosomes are important adhesion devices found in epithelial cells. They connect the intermediate filament cytoskeleton network with components of the basement membrane zone. 804G cells are an unusual epithelial cell line, since they form bona fide hemidesmosomes when plated on glass or plastic. In this study we tested an hypothesis: that this ability is a consequence of an extracellular component produced by the 804G cells. As probes for our study we generated a rabbit antiserum (J18) and monoclonal antibodies against components of urea-solubilized 804G matrix. Antibodies in the J18 serum recognize major lectin-binding polypeptides of 150, 140 and 135 kDa in the 804G matrix. A monoclonal antibody (5C5) that shows reactivity with the 150 and 135 kDa polypeptides in western immunoblots immunoprecipitates all three molecular mass species, indicating that these polypeptides are part of a matrix complex. Moreover, one, at least, of these matrix elements is immunologically related to laminin, since J18 antibodies selected on fusion protein fragments of a newly characterized laminin variant, laminin B2t (Kallunki et al., J. Cell Biol., 119, 679-694, 1992), react with the 140 kDa polypeptide component of the 804G cell matrix. To undertake functional analyses of 804G matrix, cells of the human epidermal carcinoma line SCC12, which do not assemble bona fide hemidesmosomes in vitro, were cultured on 804G matrix for 24 h and then analysed by confocal immunofluorescence and electron microscopy. In SCC12 cells maintained on 804G cell matrix, hemidesmosomal antigens localize in a distinctive leopard spot pattern that mirrors the distribution of 804G matrix elements. Furthermore, ultrastructural analysis reveals that the 804G cell matrix supports the formation of 'mature' hemidesmosomes by SCC12 cells. Thus 804G cell matrix is a remarkable tool for hemidesmosome studies and it will now be of great importance to determine the exact composition of the 804G matrix, especially its structural and antigenic relationship to laminins.

Hemidesmosomes in the epithelial cell line 804G: their fate during wound closure, mitosis and drug induced reorganization of the cytoskeleton.

Recently, we identified a novel epithelial cell line, 804G, derived from rat bladder, which readily forms hemidesmosomes in vitro. One of the major structural components of the plaques of 804G cell hemidesmosomes is a 230 kDa antigen recognized by autoantibodies in the sera of patients with bullous pemphigoid (BP). An additional polypeptide of 180 kDa also localizes to the hemidesmosome plaque of 804G cells as determined by immunoelectron microscopy. Using confocal fluorescence/phase microscopy, we have employed both 230 kDa and 180 kDa antibody probes to monitor the fate of hemidesmosomes following closure of in vitro wounds, during mitosis, and following drug induced disruption of the cytoskeleton. The punctate cell-substratum associated staining generated by the hemidesmosomal antibodies in stationary unwounded 804G cell cultures is greatly diminished or even lost in cells which enter wound sites, presumably in response to enhanced cell motility. Few, if any hemidesmosomes are observed at the ultrastructural level in cells which have migrated into the wound area. However, as closure of the wound becomes complete, staining along the substratum attached surface of cells returns. During mitosis, there is no obvious loss of hemidesmosomal antigens along the basal surface of 804G cells, and formed hemidesmosomes can be observed in mitotic cells at the ultrastructural level. In 804G cells treated with colchicine, the typical subnuclear pattern of distribution of hemidesmosomal antigens is unaffected. In contrast, following treatment of 804G cells with cytochalasin D, hemidesmosomal antigens become concentrated at the cell periphery and no longer appear in the subnuclear region. Furthermore, formed hemidesmosomes are observed at the cell periphery of cytochalasin D-treated cells by electron microscopy. We suggest that hemidesmosomal plaques are mobile within the plasma membrane. We speculate that hemidesmosomal interactions with extracellular ligands are dynamic and we discuss a possible mechanism by which cytochalasin D induces reorganization of hemidesmosomes along the basal surface of 804G cells.

Cytoplasmic domain of the 180-kD bullous pemphigoid antigen, a hemidesmosomal component: molecular and cell biologic characterization.

Using a serum sample of a bullous pemphigoid (BP) patient we have isolated a cDNA clone encoding a portion of a 180-kD polypeptide component of the hemidesmosome, the "BP180 autoantigen." The identity of the clone was confirmed by the generation of a fusion protein antibody that recognizes BP180 in both a basal epithelial cell extract of bovine tongue and extracts of human epidermal cells. Immunoelectron microscopy indicates that the 588-bp cDNA encodes a cytoplasmic fragment of BP180. Furthermore, the wide species reactivity of the fusion protein suggests that this portion of BP180 is highly conserved. In cultured human epidermal cells processed for confocal immunofluorescence microscopy, the fusion protein antibody generates a punctate cell substrate-associated staining pattern that is similar to that seen using BP230 antibodies. Using the original BP180 cDNA we have now isolated additional cDNA clones encoding approximately 1800bp of BP180 the 3' sequence of which overlaps with the sequence detailed in Guidice et al (J Clin Invest 87:734-738, 1991). Secondary structural analyses have been undertaken on the predicted amino acids encoded by the 1800bp. These suggest that the collagen-like sequences of BP180 described by Guidice et al (ibid.) are separated by a putative transmembrane region from the domain of BP180 recognized by our fusion protein antibody. Indeed, BP180 appears to belong to a relatively rare group of proteins in which the N-terminus is located in the cytoplasm and the C-terminus is extracellular. We detail some preliminary biochemical experiments in support of this hypothesis. We discuss possible functions of BP180 and BP230 in the hemidesmosome.