Sonal Sathe

Cytokines, matrix metalloproteases, angiogenic and growth factors in tears of normal subjects and vernal keratoconjunctivitis patients

Background:  To detect the presence of multiple mediators and growth factors in tears of vernal keratoconjunctivitis (VKC) patients with active disease using stationary phase antibody arrays.

Identification, origins and the diurnal role of the principal serine protease inhibitors in human tear fluid

PURPOSE Previous work identified polymorphonuclear leukocyte (PMN) elastase as the major caseinolytic entity in tears collected after overnight eye closure. This study was designed to identify the principal serine protease inhibitors (serpins) in tears and to determine their function in the regulation of PMN cell proteases on eye closure. METHODS Reflex and closed eye tear samples were collected by microcapillary tube and centrifuged. After reflex and closed eye supernatants (R and C) were fractionated by HPLC, samples were subjected to casein zymography and reverse zymography. Western blots were utilized to screen tears and HPLC fractions for elastase, cathepsin G and proteinase-3 and to obtain semi-quantitative data on alpha 1-protease inhibitor (alp1), alpha 1-antichymotrypsin (alpha 1-Achy), secretory leukocyte protease inhibitor (SLPI), elafin and alpha 2-macroglobulin (alpha 2-M) as well as associated complexes and products. To confirm specificity of reactivity, samples were immunoprecipitated for a given protease or serpin and screened for the coprecipitation of interacting species. RESULTS Although R fluid contains no caseinolytic activity, it contains low levels of serpin-like activity principally in the form of SLPI (5-10 ng/microliter). Lesser amounts of alpha 2-M, alpha 1-Achy and alp1 (approximately < 1-3 ng/microliter) are also evident. C fluid is associated with very high levels of PMN cell proteases along with a approximately 5-20-fold increase in the concentrations of all of the above inhibitors. Trace levels of elafin were also detected. The concentrations of rapid reacting inhibitors exceeded that of proteases, with SLPI, alpha 1-Achy and alp1 being the principal functional entities. In atypical samples, complexes of elastase and alpha 2-M were also encountered. CONCLUSIONS SLPI, a known antimicrobial agent and an elastase and cathepsin G inhibitor, is the principal serpin in R fluid. C fluid is associated with a marked increase in the concentrations of an array of rapid reacting serpins capable of inhibiting all known PMN cell serine proteases. In the normal closed eye, the concentration of rapid reacting inhibitors always exceeds that of proteases with C fluid also containing a functional reserve of the slow reacting inhibitor alpha 2-M.

The effect of eye closure on protein and complement deposition on Group IV hydrogel contact lenses: relationship to tear flow dynamics.

PURPOSE This study was designed to determine the effect of overnight eye closure on the rate and composition of protein deposition on high water content ionic matrix soft contact lenses (Group IV SCLs) and to extrapolate from this data information on the probable change in the rate of reflex-type tear secretion associated with eye closure. METHODS Group IV SCLs were temporally sampled after equivalent periods of wear under closed eye (C) or open eye (O) conditions. Lenses were rinsed in saline and the majority of the tightly bound protein extracted at 90 degrees C in 40% urea, containing 1% SDS, 1 mM DTT, 100 mM Tris-HCl (pH 8.00). Residual protein was determined by Coomassie staining of the extracted lenses and densitometric analysis. Extracted protein was quantitated and separated by SDS-PAGE. Gels were either stained with Coomassie blue or reversibly stained with imidazole-zinc and blotted. Blots were PAS stained, or lectin and antibody probed for glycoproteins, secretory IgA (sIgA), IgG, lysozyme and complement C3. Laboratory simulated deposition studies were carried out on unworn lenses exposed to HPLC purified lysozyme. RESULTS The protein in the saline rinse, to a large degree mirrored the composition of tear fluid in which the lens had been residing (O or C). This would suggest that the saline wash consists of residual tear fluid and loosely adherent protein. In contrast, the urea extracts were highly homogeneous consisting primarily of lysozyme and to lesser extent lysozyme dimer. This supports the contention that the Group IV SCL functions in the eye much as cationic exchange resin selectively absorbing lysozyme. C extracts also proved relatively enriched in trace amounts of sIgA, IgG and complement C3 and its breakdown products. High levels of C3 and C3 breakdown products were specifically recovered only in the C worn lens extracts from a subject experiencing unilateral contact lens associated corneal infiltrates from the affected eye. In all subjects, markedly less protein (lysozyme) was recovered in urea extracts of lenses exposed to 7-8 h of closed eye as compared to open eye wear (0.20 +/- .08 versus 0.79 +/- .15 mg/lens (n = 6)). Temporal studies further revealed that deposition was linearly related to duration of wear during the initial phase of conditioning film formation giving rise to rate constants for lysozyme deposition of 2.2 +/- 0.29 (n = 5) and 0.20 +/- 0.06 microgram/min (n = 4) under open and closed eye conditions respectively. With further wear, deposition eventually reached a steady state. Under laboratory conditions, lysozyme was much rapidly and quantitatively removed from solution in a manner following a hyperbolic plot. This suggests that during the initial phase of deposition the rate of deposition is limited by the capacity of the tear fluid to deliver lysozyme to the lens surface under these two extremes of conditions. CONCLUSIONS Eye closure profoundly affects the rate of lysozyme deposition on Group IV hydrogels and the composition of minor biofilm constituents in a manner that could affect biocompatibility. Findings support the contention that eye closure results in a > 90% reduction in the rate of reflex-type tear secretion.

Profile of Lipid and Protein Autacoids in Diabetic Vitreous Correlates with the Progression of Diabetic Retinopathy

OBJECTIVE This study was aimed at obtaining a profile of lipids and proteins with a paracrine function in normal and diabetic vitreous and exploring whether the profile correlates with retinal pathology. RESEARCH DESIGN AND METHODS Vitreous was recovered from 47 individuals undergoing vitreoretinal surgery: 16 had nonproliferative diabetic retinopathy (NPDR), 15 had proliferative diabetic retinopathy, 7 had retinal detachments, and 9 had epiretinal membranes. Protein and lipid autacoid profiles were determined by protein arrays and mass spectrometry–based lipidomics. RESULTS Vitreous lipids included lipoxygenase (LO)- and cytochrome P450 epoxygenase (CYP)-derived eicosanoids. The most prominent LO-derived eicosanoid was 5-hydroxyeicosate traenoic acid (HETE), which demonstrated a diabetes-specific increase (P = 0.027) with the highest increase in NPDR vitreous. Vitreous also contained CYP-derived epoxyeicosatrienoic acids; their levels were higher in nondiabetic than diabetic vitreous (P < 0.05). Among inflammatory, angiogenic, and angiostatic cytokines and chemokines, only vascular endothelial growth factor (VEGF) showed a significant diabetes-specific profile (P < 0.05), although a similar trend was noted for tumor necrosis factor (TNF)-α. Soluble VEGF receptors R1 and R2 were detected in all samples with lowest VEGF-R2 levels (P < 0.05) and higher ratio of VEGF to its receptors in NPDR and PDR vitreous. CONCLUSIONS This study is the first to demonstrate diabetes-specific changes in vitreous lipid autacoids including arachidonate and docosahexanoate-derived metabolites indicating an increase in inflammatory versus anti-inflammatory lipid mediators that correlated with increased levels of inflammatory and angiogenic proteins, further supporting the notion that inflammation plays a role the pathogenesis of this disease.

Antibody protein array analysis of the tear film cytokines.

Purpose. Many bioactive proteins including cytokines are reported to increase in dry eye disease although the specific profile and concentration of inflammatory mediators varies considerably from study to study. In part, this variability results from inherent difficulties in quantifying low abundance proteins in a limited sample volume using relatively low sensitivity dot ELISA methods. Additional complexity comes with the use of pooled samples collected using a variety of techniques and intrinsic variation in the diurnal pattern of individual tear proteins. The current study describes a recent advance in the area of proteomics that has allowed the identification of dozens of low abundance proteins in human tear samples. Methods. Commercially available stationary phase antibody protein arrays were adapted to improve suitability for use in small volume biological fluid analysis with particular emphasis on tear film proteomics. Arrays were adapted to allow simultaneous screening for a panel of inflammatory cytokines in low volume tear samples collected from individual eyes. Results. A preliminary study comparing tear array results in a small population of Sjögren’s syndrome patients was conducted. The multiplex microplate array assays of cytokines in tear fluid present an unanticipated challenge due to the unique nature of tear fluid. The presence of factors that exhibit an affinity for plastic, capture antibodies and IgG and create a complex series of matrix effects profoundly impacting the reliability of dot ELISA, including with elevated levels of background reactivity and reduction in capacity to bind targeted protein. Conclusions. Preliminary results using tears collected from patients with Sjögren’s syndrome reveal methodological advantages of protein array technology and support the concept that autoimmune-mediated dry eye disease has an inflammatory component. They also emphasize the inherent difficulties one can face when interpreting the results of micro-well arrays that result from blooming effects, matrix effects, image saturation and cross-talk between capture and probe antibodies that can greatly reduce signal-to-noise and limit the ability to obtain meaningful results.

Tear turnover and immune and inflammatory processes in the open-eye and closed-eye environments: Relationship to extended wear contact lens use

Defense of the cornea presents a unique challenge because integrity must be maintained against physical and microbial assault while minimizing the risk of scarring or neovascularization. It is our contention that these constraints have resulted in the evolution of a redundant fail-safe external ocular defense system in which inflammatory and immune processes play decidedly different roles in the openand closed-eye states. This diurnal difference may be of paramount importance in understanding the origin of adverse reactions associated with extended wear use of soft contact lenses. It is well established that the preocular tear film undergoes a profound change in its composition, origins, and turnover with prolonged eye closure. This can be readily illustrated by comparing the distribution of the major tear proteins (Table 1) and the cellular components (not shown) found in reflex(RTF), open-, and closed-eye tear fluids (CTF). This data provide strong support for the contention that: (1) The lacrimal and accessory gland secretions represent the confluence of two distinct secretory processes. These are consist with an inducible, neurologically controlled secretion composed primarily of lysozyme, lactoferrin, and tear-specific lipocalin (TSL) and a hypothetical slower, constitutive-type secretion composed almost entirely of secretory immunoglobulin (sIg)A and a smaller amount of free secretory component. (2) On-eye closure neurologically controlled secretion ceases, or nearly ceases, with ongoing flow continuing in the form of a much slower constitutive type-tear secretion. (3) The closedeye environment is relatively stagnant in nature. This is not meant to imply that the inducible lacrimal secretion is devoid of other important proteins. Newer methods of biochemical analysis such as the matrix-assisted laser desorption-ionization (MALDI) allows for the identification of numerous other RTF components. These include high molecular weight sialoglycoproteins, which can now be identified as isoforms of DMBT1, and the proline-rich proteins such as prohistatins and truncated forms of the lacrimal gland-specific proline-rich protein (Table 2). Consistent with the function of the open-eye preocular tear film, most of these proteins and glycoproteins are either antimicrobial, antiinflammatory, or surface active in nature. Overnight eye closure is also associated with a second phenomenon, the build up and conversion of complement and the subsequent recruitment of massive numbers of polymorphonuclear (PMN) leukocyte cells into the CTF. These cells can be shown to have undergone partial degranulation as evidenced by the presence of very high concentrations of the PMN cell-derived proteases elastase, proteinase-3, cathepsin-G, and proMMP-9 (combined in part with neutrophil gelatinase-associated lipocalin [NGAL]) in CTF. This data and related data have unequivocally established that prolonged eye closure results in the induction of a complement associated subclinical state of inflammation. Thus the accumulation of albumin (Table 1) and other serum proteins can be directly attributable to two factors: an inflammatory-induced increase in vascular permeability coupled with a decrease in tear turnover. The decrease in tear turnover also results in the accumulation in CTF of a wide range of ocular surface tissue products. Identified products include goblet cell-derived mucins, DMBT1. Possibly of epithelial origin, an array of pro-inflammatory mediators and leukochemokines also accumulate, the most notable of which are interleukin (IL)-8 and 12(R) HETrE. These changes are indicative of a fundamental shift in host defense strategies from a passive barrier defense to an active inflammatory, immune, and complement-mediated phagocyte process. This shift presumably is essential to protect the ocular surfaces from entrapped microorganisms. How homeostasis is maintained and autolytic damage to the ocular surfaces is avoided from repetitive exposure to highly reactive and static environment is of particular interest. It is clear that the extent of PMN recruitment and activation must be carefully regulated (Table 3). Excess recruitment may be avoided by the oxidative and proteolytic degradation of chemotactic factors and the accumulation in CTF of several proteins that are capable of serving as sinks that can complex with and thereby remove specific leukochemokines. Identified accumulating entities that have this capacity include free secretory component (which binds IL-8), NGAL (which binds n-formal methionine-linked bacterial peptides and lipid chemokines), and cystatin C (which blocks complement-mediated recruitment). Complement-mediated damage is avoided by the buildup in CTF of several complement inhibitors and the presence on the plasma membrane of the ocular epithelium of bound complement From the SUNY College of Optometry, Manhattan, NY. Address correspondence to: Dr. Robert A. Sack, SUNY College of Optometry, 33 East 42nd Street, Manhattan, NY 10036. Phone: (212) 780-5162; fax: (212) 780-5174; e-mail: RSACK25968@AOL.com. Accepted September 23, 2002.

Changes in the diurnal pattern of the distribution of gelatinases and associated proteins in normal and pathological tear fluids: evidence that the PMN cell is a major source of MMP activity in tear fluid.

The matrix metalloproteases (MMP) play critical roles in modulating apoptosis, angiogenesis, cell migration, wound healing, tissue remodeling and inflammation in all connective tissues. Two extracellular MMPs, both type IV gelatinases, MMP-2 and MMP-9 (gelatinase A and B), as well as their respective inhibitors TIMP-1, TIMP-2 and TIMP-3 have been detected in the human cornea. MMP-9 is localized in the epithelium while MMP-2 is expressed preferentially by stromal keratocytes, and to a lesser extent, in the epithelium with expression of both enzymes up-regulated during wound healing.1 MMPs, principally pro-MMP-9 and associated complexes, have been detected in human tear fluid in the open eye condition. Active MMP-9 is presumed to be derived principally from the epithelium. Increased levels of enzyme and a shift in the pattern of distribution from proenzyme to the active MMP-9 species, along with the emergence of pro-MMP-2 and active MMP-2, has been observed in tear fluid in a wide range of pathological conditions. These conditions include external ocular infections, corneal wound healing secondary to LASIK, sterile corneal ulcers, dry eye syndrome, active keratoconus, ocular rosacea and sterile corneal melts.2–4 This study was designed to examine the origins and the nature of gelatinolytic species and associated proteins present in normal and pathological tear fluid during open and closed eye phases of the diurnal cycle.

Effect of external ocular surgery and mode of post-operative care on plasminogen, plasmin, angiostatins and a2-macroglobulin in tears

Purpose. To determine whether corneal surgery and the mode of post-surgical treatment influence the distribution of plasminogen, plasmin, angiostatins and a 2 -macrogobulin in tear fluid. Methods. Subjects underwent either photorefractive keratectomy (PRK), insertion of intra-stromal corneal rings (ICR), or cataract ablation followed by insertion of an intra-ocular lens (IOL). Post-surgical treatment consisted of prophylactic use of antibiotic and anti-inflammatory agents followed either by patching for 24 hours, or covering the wounded cornea with a bandage soft contact lens. Open eye tear fluid (OTF) was obtained prior to surgery and 10 minutes after patch removal or 24 hours after surgery and thereafter with the bandage lens still in place. After centrifugation, supernatants and controls were western blot analyzed using a protocol designed to allow the simultaneous semi-quantitative detection of a 2 -macroglobulin, plasminogen, plasmin, angiostatins and interleukin-8 (IL-8). Results. No obvious differences were apparent in OTF recovered from contralateral control eyes compared to the surgical eyes in individuals who underwent PRK surgery and whose eyes were covered with a bandage contact lens. In contrast, OTF samples recovered 10 minutes after patch removal from all individuals contained elevated levels of a 2 -macroglobulin and a diverse mixture of elevated levels of plasminogen/plasmin, angiostatins and possibly a plasmin-aa 1 -antiplasmin complex. All of these changes were seen, albeit to a lesser extent, in the patched control OTF samples. IL-8 could not be detected in any sample. The composition of the tear film returned to near normal on subsequent sampling 24 hours after patch removal. Conclusions. Patching results in a marked increase in the concentration of various proteins which could modulate inflammation and wound healing.

Membrane array analysis of tear proteins in ocular cicatricial pemphigoid.

Purpose. To explore non-invasive, protein-based, membrane array technology as a means to evaluate the global immune and angiogenic profile of tear proteins in patients with active ocular cicatricial pemphigoid (OCP). Methods. Forty-three proteins consisting of cytokines, angiogenic/growth factors, and immunoinflammatory modulators were measured by membrane array in tear samples of four control patients and four OCP patients during active disease and after treatment. Results. Signals for several distinct and consistent molecular entities were upregulated in all four active OCP tear samples relative to controls. In particular, interleukin-8 and matrix metalloproteinase-9 were elevated during active disease and decreased after systemic immunomodulatory therapy. Conclusions. Protein array analysis may provide a well-tolerated assay to monitor levels of inflammatory markers in the tears of OCP patients in response to therapy.

Protein Array Characterization of Bioactive Proteins Secreted By Immortalized Human Corneal Epithelium in Response to Pseudomonas Constituents

Purpose: To use protein arrays to delineate the spectrum of angiogenic bioactive protein modulators that might be secreted and up-regulated by the corneal epithelium in response to killed bacterial products. Methods: Immortalized human corneal epithelial cells were grown in culture, serum starved, and exposed to heat-killed Pseudomonas aeruginosa in a dose-dependent manner. The resultant culture medium was screened by antibody arrays for 43 proteins that can modulate angiogenesis and immune and inflammatory processes. Parallel analysis was carried out on tears recovered in the open and closed eye phases (OTF and CTF) of the diurnal cycle. Results: Array analysis reveals that the immortalized cells constitutively secrete several proteins and up-regulate the secretion of IL-6, IL-8, and GRO in response to killed bacteria. Also evident was the emergence of a strong signal for GM-CSF and moderate/weak signals for MCP-1, MMP-9, Leptin, and INFγ in a dose-dependent manner. Several of these proteins, including IL-6, IL-8, GRO, MMP-9, TIMP-1, and MCP-1, accumulate in the CTF. Other proteins are unique to tear fluid. Conclusions: Nine proteins were identified that are secreted by epithelium in response to killed bacteria that contribute to the innate and adaptive defense system through potentiating PMN and macrophage recruitment, activation, and opsonization in a cooperative manner. The vast majority of these proteins are angiogenic modulators, perhaps contributing to the imbalance between angiogenic and angiostatic processes and risk of corneal vascularization.