A Tenon's capsule/bulbar conjunctiva interface biomimetic to model fibrosis and local drug delivery.

Glaucoma filtration surgery is one of the most effective methods for lowering intraocular pressure in glaucoma. The surgery efficiently reduces intra-ocular pressure but the most common cause of failure is scarring at the incision site. This occurs in the conjunctiva/Tenon's capsule layer overlying the scleral coat of the eye. Currently used antimetabolite treatments to prevent post-surgical scarring are non-selective and are associated with potentially blinding side effects. Developing new treatments to target scarring requires both a better understanding of wound healing and scarring in the conjunctiva, and new means of delivering anti-scarring drugs locally and sustainably. By combining plastic compression of collagen gels with a soft collagen-based layer, we have developed a physiologically relevant model of the sub-epithelial bulbar conjunctiva/Tenon's capsule interface, which allows a more holistic approach to the understanding of subconjunctival tissue behaviour and local drug delivery. The biomimetic tissue hosts both primary human conjunctival fibroblasts and an immune component in the form of macrophages, morphologically and structurally mimicking the mechanical proprieties and contraction kinetics of ex vivo porcine conjunctiva. We show that our model is suitable for the screening of drugs targeting scarring and/or inflammation, and amenable to the study of local drug delivery devices that can be inserted in between the two layers of the biomimetic. We propose that this multicellular-bilayer engineered tissue will be useful to study complex biological aspects of scarring and fibrosis, including the role of inflammation, with potentially significant implications for the management of scarring following glaucoma filtration surgery and other anterior ocular segment scarring conditions. Crucially, it uniquely allows the evaluation of new means of local drug delivery within a physiologically relevant tissue mimetic, mimicking intraoperative drug delivery in vivo.

Rab27a co-ordinates actin-dependent transport by controlling organelle-associated motors and track assembly proteins.

Cell biologists generally consider that microtubules and actin play complementary roles in long- and short-distance transport in animal cells. On the contrary, using melanosomes of melanocytes as a model, we recently discovered that the motor protein myosin-Va works with dynamic actin tracks to drive long-range organelle dispersion in opposition to microtubules. This suggests that in animals, as in yeast and plants, myosin/actin can drive long-range transport. Here, we show that the SPIRE-type actin nucleators (predominantly SPIRE1) are Rab27a effectors that co-operate with formin-1 to generate actin tracks required for myosin-Va-dependent transport in melanocytes. Thus, in addition to melanophilin/myosin-Va, Rab27a can recruit SPIREs to melanosomes, thereby integrating motor and track assembly activity at the organelle membrane. Based on this, we suggest a model in which organelles and force generators (motors and track assemblers) are linked, forming an organelle-based, cell-wide network that allows their collective activity to rapidly disperse the population of organelles long-distance throughout the cytoplasm.

Macrophages promote a profibrotic phenotype in orbital fibroblasts through increased hyaluronic acid production and cell contractility.

Graves' orbitopathy (GO) is an autoimmune inflammatory disease affecting the orbit. Orbital fibroblasts are a key component in GO pathogenesis, which includes inflammation, adipogenesis, hyaluronic acid (HA) secretion, and fibrosis. Macrophages are thought to participate in the immunological stage of GO, but whether they can directly affect the fibroblasts phenotype and modulate disease progression is unknown. We previously showed that GO adipogenic and fibrotic phenotypes could be modelled in a pseudo-physiological 3D environment in vitro. Here, we introduced macrophages in this 3D culture model to investigate role for macrophages in modulating adipogenesis, HA production, and contractility in orbital fibroblasts. Macrophages had a minimal effect on lipid droplet formation in fibroblasts, but significantly increased HA production and cell contractility, suggesting that they may promote the fibrotic phenotype. This effect was found to be mediated at least in part through phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) activation and linked to an increase in actin polymerization and protrusive activity in fibroblasts. Overall our work shows for the first time a direct role for macrophages in modulating the fibroblasts' phenotype in GO, supporting a role for macrophages in the progression of the fibrotic phenotype through induction of HA production and stimulation of the contractile phenotype in orbital fibroblasts.

An Ilomastat-CD Eye Drop Formulation to Treat Ocular Scarring.

Purpose: The purpose of this study was to develop a topical matrix metalloproteinase inhibitor preparation for antiscarring therapy. Methods: The broad spectrum matrix metalloproteinase inhibitor ilomastat was formulated using 2-hydroxypropyl-ß-cyclodextrin in aqueous solution. In vitro activity of ilomastat-cyclodextrin (ilomastat-CD) was examined using fibroblasts seeded in collagen. Permeation of ilomastat-CD eye drop through pig eye conjunctiva was confirmed using Franz diffusion cells. Ilomastat-CD eye drop was applied to rabbit eyes in vivo, and the distribution of ilomastat in ocular tissues and fluids was determined by liquid chromatography-mass spectroscopy. Results: The aqueous solubility of ilomastat-CD was ∼1000 µg/mL in water and 1400 µg/mL in PBS (pH 7.4), which is greater than ilomastat alone (140 and 160 µg/mL in water and PBS, respectively). The in vitro activity of ilomastat-CD to inhibit collagen contraction in the presence of human Tenon fibroblast cells was unchanged compared to uncomplexed ilomastat. Topically administered ilomastat-CD in vivo to rabbit eyes resulted in a therapeutic concentration of ilomastat being present in the sclera and conjunctiva and within the aqueous humor. Conclusions: Ilomastat-CD has the potential to be formulated as an eye drop for use as an antifibrotic, which may have implications for the prevention of scarring in many settings, for example glaucoma filtration surgery.

Assessment of solid microneedle rollers to enhance transmembrane delivery of doxycycline and inhibition of MMP activity.

Many chronic wounds exhibit high matrix metalloproteinase (MMP) activity that impedes the normal wound healing process. Intradermal delivery (IDD) of sub-antimicrobial concentrations of doxycycline, as an MMP inhibitor, could target early stages of chronic wound development and inhibit further wound progression. To deliver doxycycline intradermally, the skin barrier must be disrupted. Microneedle rollers offer a minimally invasive technique to penetrate the skin by creating multiple microchannels that act as temporary conduits for drugs to diffuse through. In this study, an innovative and facile approach for delivery of doxycycline across Strat-MTM membrane was investigated using microneedle rollers. The quantity and rate of doxycycline diffusing through the micropores directly correlated with increasing microneedle lengths (250, 500 and 750 µm). Treatment of Strat-MTM with microneedle rollers resulted in a reduction in fibroblast-mediated collagen gel contraction and MMP activity compared with untreated Strat-MTM. Our results show that treatment of an epidermal mimetic with microneedle rollers provides sufficient permeabilization for doxycycline diffusion and inhibition of MMP activity. We conclude that microneedle rollers are a promising, clinically ready tool suitable for delivery of doxycycline intradermally to treat chronic wounds.

Local delivery of novel MRTF/SRF inhibitors prevents scar tissue formation in a preclinical model of fibrosis.

The myocardin-related transcription factor/serum response factor (MRTF/SRF) pathway represents a promising therapeutic target to prevent fibrosis. We have tested the effects of new pharmacological inhibitors of MRTF/SRF signalling in a preclinical model of fibrosis. CCG-222740, a novel MRTF/SRF inhibitor, markedly decreased SRF reporter gene activity and showed a greater inhibitory effect on MRTF/SRF target genes than the previously described MRTF-A inhibitor CCG-203971. CCG-222740 was also five times more potent, with an IC50 of 5 µM, in a fibroblast-mediated collagen contraction assay, was less cytotoxic, and a more potent inhibitor of alpha-smooth muscle actin protein expression than CCG-203971. Local delivery of CCG-222740 and CCG-203971 in a validated and clinically relevant rabbit model of scar tissue formation after glaucoma filtration surgery increased the long-term success of the surgery by 67% (P < 0.0005) and 33% (P < 0.01), respectively, and significantly decreased fibrosis and scarring histologically. Unlike mitomycin-C, neither CCG-222740 nor CCG-203971 caused any detectable epithelial toxicity or systemic side effects with very low drug levels measured in the aqueous, vitreous, and serum. We conclude that inhibitors of MRTF/SRF-regulated gene transcription such as CCG-222740, potentially represent a new therapeutic strategy to prevent scar tissue formation in the eye and other tissues.

Fibroblasts profiling in scarring trachoma identifies IL-6 as a functional component of a fibroblast-macrophage pro-fibrotic and pro-inflammatory feedback loop.

Trachoma is a conjunctiva scarring disease, which is the leading infectious cause of blindness worldwide. Yet, the molecular mechanisms underlying progressive fibrosis in trachoma are unknown. To investigate the contribution of local resident fibroblasts to disease progression, we isolated conjunctival fibroblasts from patients with scarring trachoma and matching control individuals, and compared their gene expression profiles and functional properties in vitro. We show that scarring trachoma fibroblasts substantially differ from control counterparts, displaying pro-fibrotic and pro-inflammatory features matched by an altered gene expression profile. This pro-inflammatory signature was exemplified by increased IL-6 expression and secretion, and a stronger response to macrophage-mediated stimulation of contraction. We further demonstrate that scarring trachoma fibroblasts can promote Akt phosphorylation in macrophages in an IL-6 -dependent manner. Overall this work has uncovered a distinctive molecular fingerprint for scarring trachoma fibroblasts, and identified IL-6- as a potential contributor to the chronic conjunctival fibrosis, mediating reciprocal pro-fibrotic/pro-inflammatory interactions between macrophages and fibroblasts.

Rod disc renewal occurs by evagination of the ciliary plasma membrane that makes cadherin-based contacts with the inner segment.

The outer segments of vertebrate rod photoreceptors are renewed every 10 d. Outer segment components are transported from the site of synthesis in the inner segment through the connecting cilium, followed by assembly of the highly ordered discs. Two models of assembly of discrete discs involving either successive fusion events between intracellular rhodopsin-bearing vesicles or the evagination of the plasma membrane followed by fusion of adjacent evaginations have been proposed. Here we use immuno-electron microscopy and electron tomography to show that rhodopsin is transported from the inner to the outer segment via the ciliary plasma membrane, subsequently forming successive evaginations that "zipper" up proximally, but at their leading edges are free to make junctions containing the protocadherin, PCDH21, with the inner segment plasma membrane. Given the physical dimensions of the evaginations, coupled with likely instability of the membrane cortex at the distal end of the connecting cilium, we propose that the evagination occurs via a process akin to blebbing and is not driven by actin polymerization. Disassembly of these junctions is accompanied by fusion of the leading edges of successive evaginations to form discrete discs. This fusion is topologically different to that mediated by the membrane fusion proteins, SNAREs, as initial fusion is between exoplasmic leaflets, and is accompanied by gain of the tetraspanin rim protein, peripherin.

Mesenchymal Stem Cell-Like Properties of Orbital Fibroblasts in Graves' Orbitopathy.

PURPOSE: Graves' orbitopathy (GO) is a sight-threatening autoimmune disorder causing extraocular muscle fibrosis, upper lid retraction and eye bulging due to orbital fat expansion. These clinical features are mediated by aspects of orbital fibroblasts differentiation, including adipogenesis and fibrosis. Our previous work suggested that this dual phenotype might be a manifestation of mixed cell populations, partially linked to the expression of mesenchymal stem cell (MSC) marker CD90. Thus, we set out to determine whether GO orbital fibroblasts displayed MSC properties. METHODS: Control and GO orbital fibroblasts previously characterized for CD90 and CD45 expression were analyzed by flow cytometry for classical MSC positive (CD73, CD105) and negative (CD14, CD19, HLA-DR, and CD34) markers. Graves' orbitopathy fibroblasts were tested further for their ability to undergo lineage specific differentiation following standard protocols. RESULTS: Control and GO fibroblasts strongly expressed CD73 and CD105, with a higher percentage of positive cells and stronger expression levels in GO. Neither cell type expresses CD14, CD19, and HLA-DR. Protein CD34 was expressed at low levels by 45% to 70% of the cells, with its expression significantly lower in GO cells. Graves' orbitopathy fibroblasts displayed features of osteogenesis (calcium deposits, and osteocalcin [BGLAP] and osteonectin [SPARC] expression), chondrogenesis (glycosaminoglycan production; SOX9 and aggrecan [ACAN] expression), myogenesis (α-smooth muscle actin expression), and neurogenesis (ß-III tubulin expression) upon differentiation. CONCLUSIONS: Our findings suggest that orbital fibroblasts contain a population of cells that fulfil the criteria defining MSC. This subpopulation may be increased in GO, possibly underlying the complex differentiation phenotype of the disease.

Eyelid and Sternum Fibroblasts Differ in Their Contraction Potential and Responses to Inflammatory Cytokines.

BACKGROUND: Adverse skin scarring varies by anatomical site with, for example, presternal skin showing a greater hypertrophic response when compared with eyelid; such differences have traditionally been attributed to regional variations in skin tension, thickness, and Langer's lines. Fibroblasts are the main cell implicated in fibrosis, and they too are known to show anatomical variation in their expression, differentiation, and intercellular interactions. We, therefore, investigated whether intrinsic differences in skin fibroblasts derived from separate locations might contribute to the observed discrepancies in clinical scarring. METHODS: Primary in vitro cultures were established using matched eyelid and presternal skin from 3 healthy donors undergoing blepharoplasty surgery. We used an in vitro collagen gel model of fibroblast-mediated tissue contraction to compare the properties of the dermal fibroblasts from each site. Cell contractile force and matrix stiffness were assessed in 3-dimensional tissue constructs using an automated high-throughput device. RESULTS: Dermal fibroblasts isolated from eyelid and sternum differ both in their ability to contract a gel matrix and in their response to cytokine stimulation; despite having lower intrinsic contractile force (P < 0.01) and resting stiffness (P < 0.02), the presternal cells were more contractile (P < 0.001) following stimulation with serum, or inflammatory cytokines transforming growth factor-ß (P < 0.01) and interleukin-1ß (P < 0.05). CONCLUSIONS: The propensity to cutaneous scarring may, at least in part, result from intrinsic differences in the local fibroblasts' ability to contract and their sensitivity to inflammatory cytokines. Improved understanding of the underlying molecular pathways should prove useful in identifying new therapeutic targets for altering surgical and other scarring.

The role of the MRTF-A/SRF pathway in ocular fibrosis.

Tissue contraction and fibrosis are major causes of morbidity in the human body. In the eye in particular, fibrosis and scarring are responsible for the pathogenesis or failure of treatment of all major blinding diseases, with postoperative wound healing responses posing a major problem for most ocular surgery on a worldwide scale. This is one of the largest areas of unmet need in ophthalmology, with currently no antifibrotic treatments available clinically. This review focuses on the ubiquitous myocardin-related transcription factor/serum response factor (MRTF-A/SRF) transcription pathway as a potential novel therapeutic target in fibrotic eye diseases. It describes how the MRTF-A/SRF pathway is intricately linked to all the key regulators and pathways in ocular fibrosis, and how it could potentially lead to a new avenue of antifibrotic therapies in the future.

Independent adipogenic and contractile properties of fibroblasts in Graves' orbitopathy: an in vitro model for the evaluation of treatments.

Graves' orbitopathy (GO) is a disfiguring and sometimes blinding disease, characterised by inflammation and swelling of orbital tissues, with fibrosis and adipogenesis being predominant features. Little is known about the disease aetiology and the molecular mechanisms driving the phenotypic changes in orbital fibroblasts are unknown. Using fibroblasts isolated from the orbital fat of undiseased individuals or GO patients, we have established a novel in vitro model to evaluate the dual profile of GO cells in a three-dimensional collagen matrix; this pseudo-physiological 3D environment allows measurement of their contractile and adipogenic properties. GO cells contracted collagen matrices more efficiently than control cells following serum or TGFß1 stimulation, and showed a slightly increased ability to proliferate in the 3D matrix, in accordance with a fibro-proliferative phenotype. GO cells, unlike controls, also spontaneously differentiated into adipocytes in 3D cultures - confirming an intrinsic adipogenic profile. However, both control and GO cells underwent adipogenesis when cultured under pathological pressure levels. We further demonstrate that a Thy-1-low population of GO cells underlies the adipogenic - but not the contractile - phenotype and, using inhibitors, confirm that the contractile and adipogenic phenotypes are regulated by separate pathways. In view of the current lack of suitable treatment for GO, we propose that this new model testing the duality of the GO phenotype could be useful as a preclinical evaluation for the efficacy of potential treatments.

Doxycycline prevents matrix remodeling and contraction by trichiasis-derived conjunctival fibroblasts.

PURPOSE: Trachoma is a conjunctival scarring disease, which is the leading infectious cause of blindness worldwide. Elimination of blinding trachoma is being held back by the high rate of trichiasis recurrence following surgery. There is currently no treatment available to suppress the profibrotic state and reduce recurrence. Although the mechanisms underlying trichiasis development are unknown, the profibrotic phenotype has been linked to matrix metalloproteinase (MMP) expression. Doxycycline, a well-known tetracycline antibiotic, can act as a broad MMP inhibitor and has showed some success in preventing fibrosis in various clinical contexts. The purpose of this work was to assess the antiscarring properties of doxycycline in an in vitro model of trichiasis fibroblast-mediated tissue contraction. METHODS: Primary cultures of fibroblasts were established from conjunctival samples obtained from normal donors or during surgery for trachomatous trichiasis. The effect of doxycycline on matrix contraction was investigated in our standard collagen gel contraction model. Cell morphology and matrix integrity were assessed using confocal reflection microscopy. Quantitative real time polymerase chain reaction and a FRET-based assay were used to measure MMP expression and activity, respectively. RESULTS: Doxycycline treatment successfully suppressed the contractile phenotype of trichiasis fibroblasts, matrix degradation, and significantly altered the expression of MMP1, MMP9, and MMP12 associated with the profibrotic phenotype. CONCLUSIONS: In view of the results presented here and the wider use of doxycycline in clinical settings, we propose that doxycycline might be useful as a treatment to prevent recurrence following trichiasis surgery.

Stimulation of cortical myosin phosphorylation by p114RhoGEF drives cell migration and tumor cell invasion.

Actinomyosin activity is an important driver of cell locomotion and has been shown to promote collective cell migration of epithelial sheets as well as single cell migration and tumor cell invasion. However, the molecular mechanisms underlying activation of cortical myosin to stimulate single cell movement, and the relationship between the mechanisms that drive single cell locomotion and those that mediate collective cell migration of epithelial sheets are incompletely understood. Here, we demonstrate that p114RhoGEF, an activator of RhoA that associates with non-muscle myosin IIA, regulates collective cell migration of epithelial sheets and tumor cell invasion. Depletion of p114RhoGEF resulted in specific spatial inhibition of myosin activation at cell-cell contacts in migrating epithelial sheets and the cortex of migrating single cells, but only affected double and not single phosphorylation of myosin light chain. In agreement, overall elasticity and contractility of the cells, processes that rely on persistent and more constant forces, were not affected, suggesting that p114RhoGEF mediates process-specific myosin activation. Locomotion was p114RhoGEF-dependent on Matrigel, which favors more roundish cells and amoeboid-like actinomyosin-driven movement, but not on fibronectin, which stimulates flatter cells and lamellipodia-driven, mesenchymal-like migration. Accordingly, depletion of p114RhoGEF led to reduced RhoA, but increased Rac activity. Invasion of 3D matrices was p114RhoGEF-dependent under conditions that do not require metalloproteinase activity, supporting a role of p114RhoGEF in myosin-dependent, amoeboid-like locomotion. Our data demonstrate that p114RhoGEF drives cortical myosin activation by stimulating myosin light chain double phosphorylation and, thereby, collective cell migration of epithelial sheets and amoeboid-like motility of tumor cells.

Rac1 inhibition prevents tissue contraction and MMP mediated matrix remodeling in the conjunctiva.

PURPOSE: To evaluate the efficiency of Rac1 inhibition in preventing matrix contraction by Tenon's capsule fibroblasts. METHODS: The involvement of Rac1 in serum-stimulated matrix contraction by human Tenon's fibroblasts (HTFs) was investigated in a classic collagen contraction model and our ex vivo model of tissue contraction using immunocytochemistry, chemical inhibitors, and small interfering RNA (siRNA) technology. Matrix integrity was assessed using confocal reflection microscopy and Coomassie blue staining. Quantitative real-time polymerase chain reaction (QRT-PCR) and Western blot analysis were used to assess matrix metalloproteinase (MMP) expression. RESULTS: Serum induced Rac1 activation in HTF-populated collagen gels and stimulated HTFs to contract collagen matrices down to ~90% of their original size. Rac1 inhibition using NSC23766 or depletion using siRNA both significantly reduced HTF-mediated contraction. Early brief exposure to NSC23766 reduced HTF-mediated gel contraction by 70%, while transient treatment with the Rac1 inhibitor once a week decreased ex vivo tissue contraction down to serum-free levels. Transient exposure to NSC23766 prevented early cell protrusions, fiber alignment, and matrix degradation, as seen upon continuous exposure to broad-spectrum MMP inhibitor. However, unlike MMP inhibition, transient treatment with NSC23766 led to a significant reduction in MMP1 mRNA and protein expression during contraction, without increasing MMP2 and MMP14 expression. CONCLUSIONS: Rac1 inhibition efficiently prevents conjunctival tissue and collagen matrix contraction and prevents matrix degradation.

Hyaluronan hydration generates three-dimensional meso-scale structure in engineered collagen tissues.

Here, we show that the local incorporation of osmotically active hyaluronan into previously compressed collagen constructs results in further rapid dehydration/compression of collagen layers, channel formation and generation of new interfaces; these novel structures, at the nano-micro (i.e. meso-scale) were formed within native collagen gels, in a highly predictable spatial manner and offer important new methods of fabricating scaffolds (e.g. tubes and open-spirals) with potential for use in tissue regeneration such as in peripheral nerves and small vessels. This paper tests the possibility that the local fluid content of a dense collagen network can be controlled by incorporation of an osmotically active (native) macromolecule--hyluronan. This is an exemplar physiological, osmotic swelling agent. Hyaluronan is commonly secreted by cells deep in connective tissues, so is a good candidate for this role in a cell-driven system balancing mechanical compaction of bulk tissue collagen. These constructs may have potential as functional in vitro models representing developmental and pathological processes.

Transcriptome-level microarray expression profiling implicates IGF-1 and Wnt signalling dysregulation in the pathogenesis of thyroid-associated orbitopathy.

AIMS: The pathogenesis of thyroid-associated orbitopathy (TAO) remains unclear. The aim of this study is to elucidate the gene expression profile of orbital fat from patients with active, but untreated, TAO. METHODS: A case-control gene expression study was conducted using test samples of orbital fat from TAO patients and control orbital fat specimens; apart from drugs to control thyrotoxicosis, the TAO patients had received no treatment for orbital disease. cDNA expression analysis was performed using the Affymetrix GeneChip Human Genome U133 Plus 2.0 platform and validated using quantitative PCR. RESULTS: The highest-ranked differentially expressed genes were dominated by IGF-1 signalling genes. These include IGF-1, IGF-1 receptor binding/signalling genes, such as SOCS3 and IRS2, and downstream signalling and transcriptional regulators, such as SGK (PDK/Akt signalling) and c-JUN. Our microarray data also demonstrate dysregulation of wingless-type MMTV (Wnt) signalling gene expression, including Wnt5a, sFRPs and DKK. CONCLUSION: Altered Wnt signalling confirms previous array findings. Further investigation of the role of Wnt signalling in TAO pathogenesis is warranted. These data also provide the first evidence of dysregulation of IGF-1 pathway genes in TAO tissue, further strengthening the evidence for the role of IGF-1 signalling in the pathogenesis and potential treatment of TAO.

Ajuba is required for Rac activation and maintenance of E-cadherin adhesion.

Maintenance of stable E-cadherin-dependent adhesion is essential for epithelial function. The small GTPase Rac is activated by initial cadherin clustering, but the precise mechanisms underlying Rac-dependent junction stabilization are not well understood. Ajuba, a LIM domain protein, colocalizes with cadherins, yet Ajuba function at junctions is unknown. We show that, in Ajuba-depleted cells, Rac activation and actin accumulation at cadherin receptors was impaired, and junctions did not sustain mechanical stress. The Rac effector PAK1 was also transiently activated upon cell-cell adhesion and directly phosphorylated Ajuba (Thr172). Interestingly, similar to Ajuba depletion, blocking PAK1 activation perturbed junction maintenance and actin recruitment. Expression of phosphomimetic Ajuba rescued the effects of PAK1 inhibition. Ajuba bound directly to Rac·GDP or Rac·GTP, but phosphorylated Ajuba interacted preferentially with active Rac. Rather than facilitating Rac recruitment to junctions, Ajuba modulated Rac dynamics at contacts depending on its phosphorylation status. Thus, a Rac-PAK1-Ajuba feedback loop integrates spatiotemporal signaling with actin remodeling at cell-cell contacts and stabilizes preassembled cadherin complexes.

Changes in tarsal plate fibrillar collagens and elastic fibre phenotype in floppy eyelid syndrome.

BACKGROUND: The aims of this study are to investigate the expression of the main structural components of the tarsal extracellular matrix (ECM) in floppy eyelid syndrome (FES) focusing on elastic fibres and collagen types I and III, and also to identify possible cell-mediated inflammatory mechanisms in the pathogenesis of this condition. METHODS: A histopathological case control study was conducted using 30 upper lid specimens from patients with FES and 15 undiseased upper lid control specimens. Structural ECM components were assessed using a combination of immunctorial ataining ohistochemical and techniques including antibodies to collagens I and III, Verhöeff's iron haematoxylin, Gomori's aldehyde fuchsin and Lillie's oxidised aldehyde fuchsin. The contribution of different cellular components of the inflammatory response was investigated by immunohistochemical techniques using antibodies to CD3, CD20, CD68. Slide scoring was performed using a semiquantitative technique on an ordinal scale. Statistical analysis was performed using matched ordinal regression analysis. RESULTS: FES tarsal plate tissue demonstrated a decreased abundance of mature elastic fibres (P ≤ 0.001) and an increased abundance of oxytalan fibres (P = 0.006). Intensity of staining for collagens I (P = 0.012) and III (P < 0.001) was increased. No significant difference in the abundance of CD3, CD20 and CD68 expressing cells was identified. CONCLUSIONS: The findings of altered elastic fibre phenotype and collagen accumulation are consistent with an adaptive response to cyclic mechanical loading of the tarsal plate, rather than an aetiological feature. These findings are important in understanding how the tarsal ECM responds to mechanical loading.