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1.  Measurement of intraocular pressure after epikeratophakia 
AIMS—To assess the accuracy of three commonly used tonometers in eyes after epikeratophakia.
METHODS—Five eye bank eyes with sutured epikeratophakia buttons were connected to a manometer and a pressure transducer. Intraocular pressure was adjusted in 5 mm Hg increments from 0 to 50 mm Hg. The intraocular pressure was measured at each increment using a Goldmann tonometer, a pneumatonometer, and a Tono-pen.
RESULTS—The difference between the manometer (actual pressure) and the Goldmann tonometer ranged from −19 to + 9 mm Hg (mean (SD) overestimation 2.6 (5.8) mm Hg). The pneumatonometer error ranged from −27.5 to + 5.5 mm Hg (mean (SD) overestimation 4.7 (6.1) mm Hg), and for the Tono-pen the range was −18 to + 11 mm Hg (mean (SD) overestimation 0.05 (7.9) mm Hg). The correlation coefficients for the three tonometers were 0.94, 0.92, and 0.87 for the Goldmann tonometer, pneumatonometer, and Tono-pen respectively.
CONCLUSION—The Goldmann tonometer had the best correlation with the manometer in eye bank eyes with epikeratophakia (correlation coefficient 0.94), but none of the tonometers was accurate over the entire range of pressures tested. Detection of glaucoma in eyes with epikeratophakia cannot rely on tonometry alone, but requires examination of the optic nerve and visual field.

PMCID: PMC1722219  PMID: 9274407
2.  Improved preservation of human corneal basement membrane following freezing of donor tissue for epikeratophakia. 
Current methods for the production of lenticules for epikeratophakia involve rapid freezing, cryolathing, and slow warming of the donor cornea. We have found that this procedure causes structural damage to the epithelial basement membrane in the donor cornea which may subsequently contribute to poor postoperative re-epithelialisation of the implant, leading to graft failure. Endeavouring to overcome these problems, the effects of cryoprotection of donor cornea were investigated, using dimethyl sulphoxide, in conjunction with different cooling and warming rates as part of the protocol for cryolathing. The structural integrity of the epithelial basement membrane zone (BMZ) was then assessed by electron microscopy and by immunofluorescence microscopy using antibodies to types IV and VII collagen, components of the basal lamina and anchoring fibrils respectively, and an antibody to a component of the anchoring filaments. No differences in the pattern of immunostaining for these components were detected, indicating that the composition of the BMZ was unaltered by the different treatment regimens applied. However, electron microscopy showed that preservation of basement membrane ultrastructure was markedly improved when cornea was warmed rapidly rather than slowly, both in cryoprotected and non-cryoprotected tissue. Epithelial cell retention and preservation of stromal architecture appeared superior in cryoprotected samples, while keratocyte structure was heterogeneous throughout the experimental groups. Further work is in progress to assess the efficacy of these protocols in the preservation of keratocyte viability in association with improved basement membrane structure in donor tissue for epikeratophakia.
PMCID: PMC504974  PMID: 7848985
3.  Experimental epikeratophakia using tissue lathed at room temperature. 
This report presents for the first time the results of carrying out epikeratophakia with tissue lathed at room temperature. Using an experimental model of epikeratophakia in the rabbit, we evaluated tissue handling techniques for the preparation of donor lenticules. Details of the technique are described and the in-vivo and histopathological findings reported.
PMCID: PMC1041453  PMID: 3293653
4.  Viability of keratocytes in epikeratophakia lenticules. 
AIM: To study the influence of cryoprotectant, cooling rate, and warming rate on recovery and viability of keratocytes from corneas for cryolathing. METHODS: Corneas were frozen at -50 degrees C for 2 minutes either after exposure to 10% dimethyl sulphoxide in Eagle's MEM for 15 minutes at room temperature (about 22 degrees C), or without earlier exposure to the cryoprotectant. Corneas were cooled either rapidly (20 degrees C/min) or slowly (1 degree C/min), and they were warmed either rapidly (> 50 degrees C/min) by direct transfer into medium at 22 degrees C or slowly (< 20 degrees C/min) in air at 22 degrees C. The cryoprotectant was removed by dilution in medium containing 0.5 mol/l sucrose. Recovery of keratocytes was determined by using collagenase digestion to release the cells from the stroma and trypan blue staining. Viability was assessed by the outgrowth of cells from stromal explants in primary tissue culture. RESULTS: The use of a cryoprotectant before freezing was beneficial, irrespective of the different cooling and warming regimens. Both collagenase digestion and tissue culture revealed that keratocyte survival was improved when corneas were warmed rapidly rather than slowly. The collagenase digestion assay showed an apparently higher recovery of keratocytes after slow cooling (54.3%) than after rapid cooling (34.1%), but no differences in cell viability could be demonstrated by primary tissue culture. CONCLUSION: Although in these experiments slow cooling apparently provided the best recovery of keratocyte numbers (though not viability), previous work had revealed some disruption of the epithelial basement membrane after slow cooling. If viable keratocytes and good preservation of epithelial basement membrane are considered to be prerequisites for epikeratophakia lenticules then it is suggested that corneas should be prepared for cryolathing by freezing rapidly after exposure to 10% dimethyl sulphoxide and, following cryolathing, they should be warmed rapidly.
PMCID: PMC505467  PMID: 8703892
5.  Epikeratophakia for aphakia, keratoconus, and myopia. 
A series of 67 cases of epikeratophakia is presented with an average time from surgery of 12.2 months. For aphakia there was a delay in the recovery of vision, but by nine months 83% of 57 patients achieved an acuity equal to, or within 1 line of, the preoperative value. 57% were corrected to within 3 dioptres of emmetropia, but in the latter part of the series 75% were within this range. Astigmatism and reduced contrast sensitivity, especially in the presence of glare, were important complications. For keratoconus, 86% of seven patients with over two months of follow-up achieved a spectacle corrected acuity of 6/9 or better. One patient had surgery for myopia and obtained the desired refractive correction.
PMCID: PMC1041992  PMID: 2310729
6.  Expression and potential role of major inflammatory cytokines in experimental keratomycosis 
Molecular Vision  2009;15:1303-1311.
The aim of this study was to investigate the expression and regulation of the four major inflammatory cytokines in fungal keratitis (FK) with the goal of further understanding its pathogenesis in order to develop more effective therapeutic approaches.
Aspergillus fumigatus and Candida albicans were the corneal pathogens selected for this study to establish murine FK using epikeratophakia with the aid of corneal epithelium erasion. One, three, five, and seven days post-infection, the corneal lesions and inflammatory responses were observed by slit-lamp and histopathology, and the expressions of the four inflammatory cytokines, macrophage inflammatory protein-2 (MIP-2), cytokine-induced neutrophil chemoattractant (KC), interleukin-1β (IL-1β), and interleukin-6 (IL-6), in the infected corneas were determined using reverse transcription polymerase chain reaction (RT–PCR) and enzyme-linked immunosorbent assay (ELISA). For the intervention experiment with neutralizing antibodies, the experimental mice were then injected subconjunctivally with 5 μl (2 ng/μl) MIP-2 or IL-1β polyclonal antibody 1 h before and 24 h after surgery. Reestablishment of the FK murine model was performed following injection. Effects of MIP-2 or IL-1β polyclonal antibody on the corneal diseases were observed by slit-lamp microscopy, histopathology, and ELISA.
Expression of MIP-2, KC, IL-1β, and IL-6 was upregulated significantly in the infected group one, three, five, and seven days after surgery. Following treatment with an MIP-2 polyclonal antibody, the corneal clinical scores and inflammatory responses decreased, the MIP-2 protein levels were downregulated significantly (p<0.01), and the KC protein levels decreased slightly (p>0.05). Upon administration of IL-1β polyclonal antibodies, the decrease in clinical scores, inflammatory responses, and protein levels of MIP-2 and KC was apparent at 1 and 3 days after infection (p<0.01).
A persistent, high level expression of MIP-2 and IL-1β is an important and even major factor in the corneal pathogenesis of FK. Specific polyclonal neutralizing antibodies may be administered to inhibit the major chemokines and cytokines responsible for corneal damage thus effectively relieving the injury caused by FK.
PMCID: PMC2707360  PMID: 19590756
7.  Rehabilitation of children with cataracts. 
Over a period of 10 years, 160 children with cataracts underwent operation at the University of Tennessee Medical Center, Memphis. The surgical, optical, and psychosocial rehabilitation of these patients was analyzed and studied. The optical rehabilitation included patients with glasses, intraocular lens implants, epikeratophakia, and contact lenses. Seventy three of these patients were chosen at random and reevaluated as to visual outcome, and 46 were subjected to a psychosocial test to evaluate their quality of life and their rehabilitation. Eighteen of these were also given a psychosocial test to evaluate the quality of life enjoyed by these children at an older age following treatment for the cataract. Surgical, optical, and psychosocial rehabilitation of such children is also discussed. This is the first report of the psychological evaluation of such children. The further needs of these children as they approach adulthood are discussed in detail.
PMCID: PMC1298408  PMID: 10360302
8.  Refractive Surgery: New Options for Visual Correction 
Canadian Family Physician  1986;32:1505-1510.
Refractive surgery is now at the forefront of ophthalmological development and has proved to be a safe and effective method of correcting refractive errors. The incisions performed during radial keratotomy flatten the cornea to reduce or eliminate myopia. In keratomileusis, resecting and reshaping a portion of the cornea can correct either myopia or hyperopia when the cornea is sutured back into position. In epikeratophakia, donor corneas become “living contact lenses”. They have the potential for correcting high degrees of myopia and hyperopia. Although there are controversial aspects to refractive surgery, most informed people consider that the benefits outweigh the risks for suitable candidates.
PMCID: PMC2327443  PMID: 21267102
ophthalmology; eye; refractive surgery
9.  Nudel and FAK as Antagonizing Strength Modulators of Nascent Adhesions through Paxillin 
PLoS Biology  2009;7(5):e1000116.
Competition for binding to the cellular protein paxillin by the proteins Nudel and focal adhesion kinase is important for the proper regulation of cell adhesion and migration.
Adhesion and detachment are coordinated critical steps during cell migration. Conceptually, efficient migration requires both effective stabilization of membrane protrusions at the leading edge via nascent adhesions and their successful persistence during retraction of the trailing side via disruption of focal adhesions. As nascent adhesions are much smaller in size than focal adhesions, they are expected to exhibit a stronger adhesivity in order to achieve the coordination between cell front and back. Here, we show that Nudel knockdown by interference RNA (RNAi) resulted in cell edge shrinkage due to poor adhesions of membrane protrusions. Nudel bound to paxillin, a scaffold protein of focal contacts, and colocalized with it in areas of active membrane protrusions, presumably at nascent adhesions. The Nudel-paxillin interaction was disrupted by focal adhesion kinase (FAK) in a paxillin-binding–dependent manner. Forced localization of Nudel in all focal contacts by fusing it to paxillin markedly strengthened their adhesivity, whereas overexpression of structurally activated FAK or any paxillin-binding FAK mutant lacking the N-terminal autoinhibitory domain caused cell edge shrinkage. These results suggest a novel mechanism for selective reinforcement of nascent adhesions via interplays of Nudel and FAK with paxillin to facilitate cell migration.
Author Summary
Cell migration is an essential process in both single-cell and multicellular organisms. In higher animals, cell migration is important for many biological processes, including embryonic development, the immune response, and wound healing. Cancer cell invasion into healthy tissues occurs as a result of inappropriate cell migration. As can be easily visualized when cultured in the lab, mammalian cells attach to surfaces through focal adhesions, cellular structures characterized by complexes of the transmembrane protein integrin and intracellular proteins including paxillin and focal adhesion kinase (FAK). In order for cells to move, they must coordinate two processes: extension of the front edge of the cell and retraction of the back edge. To accomplish this, a cell first protrudes membranous structures from the front edge and then establishes adhesion structures known as nascent adhesions to hold the extensions in place. At the same time, the focal adhesions that hold a cell in place must be disrupted in order for the back edge of the cell to retract. Here, we show that a protein called Nudel is enriched at the front edge of moving cells, where it interacts with paxillin but is not detected in focal adhesions. We further show that the focal adhesion protein FAK is able to abolish the Nudel-paxillin interaction, leading to repression of the formation of nascent adhesions and to the loss of cell extensions. We therefore propose a model in which modulation of paxillin interactions in nascent adhesions and in focal adhesions is critical for coordinated cell movement: the Nudel-paxillin interaction enhances the strength of nascent adhesions to promote the attachment of membrane protrusions at the front edge of the cell, whereas FAK prevents the Nudel-paxillin interaction in focal adhesions in order to facilitate retraction of the back edge of the cell.
PMCID: PMC2684528  PMID: 19492042
10.  Caries-resistant bonding layer in dentin 
Scientific Reports  2016;6:32740.
The present study examined the mechanism for caries resistance and the pulp responses in vital teeth following the use of the augmented-pressure adhesive displacement technique. Dentin adhesives were applied to the surface of sound dentin disks in 4 experimental groups: non-antibacterial adhesive and gentle adhesive displacement (N-G), non-antibacterial adhesive and augmented-pressure adhesive displacement (N-H), antibacterial adhesive and gentle adhesive displacement (A-G), antibacterial adhesive and augmented-pressure adhesive displacement (A-H). The depth of demineralization induced by biological or chemical demineralization models was measured using confocal laser scanning microscopy and analyzed with two-way ANOVA. Pulp responses of vital dog’s teeth to the augmented-pressure adhesive displacement technique were evaluated using light microscopy. Depth of demineralization was significantly affected by “adhesive type” and “intensity of adhesive displacement” for biological demineralization. For chemical demineralization, only “intensity of adhesive displacement” showed significant influence on lesion depth. Pulp response of 0.1, 0.2 and 0.3 MPa groups showed only moderate disorganization of the odontoblast layer at 24 hours that completely re-organized after 3 weeks. Augmented-pressure adhesive displacement improves the caries resistance property of bonded dentin and does not cause irreversible pulpal damage to vital teeth when the air pressure employed is equal or smaller than 0.3 MPa.
PMCID: PMC5013435  PMID: 27599621
11.  An Adhesion-Dependent Switch between Mechanisms That Determine Motile Cell Shape 
PLoS Biology  2011;9(5):e1001059.
Keratocytes are fast-moving cells in which adhesion dynamics are tightly coupled to the actin polymerization motor that drives migration, resulting in highly coordinated cell movement. We have found that modifying the adhesive properties of the underlying substrate has a dramatic effect on keratocyte morphology. Cells crawling at intermediate adhesion strengths resembled stereotypical keratocytes, characterized by a broad, fan-shaped lamellipodium, clearly defined leading and trailing edges, and persistent rates of protrusion and retraction. Cells at low adhesion strength were small and round with highly variable protrusion and retraction rates, and cells at high adhesion strength were large and asymmetrical and, strikingly, exhibited traveling waves of protrusion. To elucidate the mechanisms by which adhesion strength determines cell behavior, we examined the organization of adhesions, myosin II, and the actin network in keratocytes migrating on substrates with different adhesion strengths. On the whole, our results are consistent with a quantitative physical model in which keratocyte shape and migratory behavior emerge from the self-organization of actin, adhesions, and myosin, and quantitative changes in either adhesion strength or myosin contraction can switch keratocytes among qualitatively distinct migration regimes.
Author Summary
Cell migration is important for many biological processes: white blood cells chase down and kill bacteria to guard against infection, epithelial cells crawl across open wounds to promote healing, and embryonic cells move collectively to form organs and tissues during embryogenesis. In all of these cases, migration depends on the spatial and temporal organization of multiple forces, including actin-driven protrusion of the cell membrane, membrane tension, cell-substrate adhesion, and myosin-mediated contraction of the actin network. In this work, we have used a simple cell type, the fish epithelial keratocyte, as a model system to investigate the manner in which these forces are integrated to give rise to large-scale emergent properties such as cell shape and movement. Keratocytes are normally fan-shaped and fast-moving, but we have found that keratocytes migrate more slowly and assume round or asymmetric shapes when cell-substrate adhesion strength is too high or too low. By correlating measurements of adhesion-dependent changes in cell shape and speed with measurements of adhesion and myosin localization patterns and actin network organization, we have developed a mechanical model in which keratocyte shape and movement emerge from adhesion and myosin-dependent regulation of the dynamic actin cytoskeleton.
PMCID: PMC3086868  PMID: 21559321
12.  A plastic relationship between vinculin-mediated tension and adhesion complex area defines adhesion size and lifetime 
Nature Communications  2015;6:7524.
Cell-matrix adhesions are central mediators of mechanotransduction, yet the interplay between force and adhesion regulation remains unclear. Here we use live cell imaging to map time-dependent cross-correlations between vinculin-mediated tension and adhesion complex area, revealing a plastic, context-dependent relationship. Interestingly, while an expected positive cross-correlation dominated in mid-sized adhesions, small and large adhesions display negative cross-correlation. Furthermore, although large changes in adhesion complex area follow vinculin-mediated tension alterations, small increases in area precede vinculin-mediated tension dynamics. Modelling based on this mapping of the vinculin-mediated tension-adhesion complex area relationship confirms its biological validity, and indicates that this relationship explains adhesion size and lifetime limits, keeping adhesions focal and transient. We also identify a subpopulation of steady-state adhesions whose size and vinculin-mediated tension become stabilized, and whose disassembly may be selectively microtubule-mediated. In conclusion, we define a plastic relationship between vinculin-mediated tension and adhesion complex area that controls fundamental cell-matrix adhesion properties.
Cell-matrix adhesions may increase or decrease in size in response to tension; however, the factors determining which of these responses predominates remain unclear. Hernández-Varas et al. quantify the plastic relationship between adhesion size and tension and use modelling to explain this behaviour.
PMCID: PMC4491829  PMID: 26109125
13.  Identifying the Rules of Engagement Enabling Leukocyte Rolling, Activation, and Adhesion 
PLoS Computational Biology  2010;6(2):e1000681.
The LFA-1 integrin plays a pivotal role in sustained leukocyte adhesion to the endothelial surface, which is a precondition for leukocyte recruitment into inflammation sites. Strong correlative evidence implicates LFA-1 clustering as being essential for sustained adhesion, and it may also facilitate rebinding events with its ligand ICAM-1. We cannot challenge those hypotheses directly because it is infeasible to measure either process during leukocyte adhesion following rolling. The alternative approach undertaken was to challenge the hypothesized mechanisms by experimenting on validated, working counterparts: simulations in which diffusible, LFA1 objects on the surfaces of quasi-autonomous leukocytes interact with simulated, diffusible, ICAM1 objects on endothelial surfaces during simulated adhesion following rolling. We used object-oriented, agent-based methods to build and execute multi-level, multi-attribute analogues of leukocytes and endothelial surfaces. Validation was achieved across different experimental conditions, in vitro, ex vivo, and in vivo, at both the individual cell and population levels. Because those mechanisms exhibit all of the characteristics of biological mechanisms, they can stand as a concrete, working theory about detailed events occurring at the leukocyte–surface interface during leukocyte rolling and adhesion experiments. We challenged mechanistic hypotheses by conducting experiments in which the consequences of multiple mechanistic events were tracked. We quantified rebinding events between individual components under different conditions, and the role of LFA1 clustering in sustaining leukocyte–surface adhesion and in improving adhesion efficiency. Early during simulations ICAM1 rebinding (to LFA1) but not LFA1 rebinding (to ICAM1) was enhanced by clustering. Later, clustering caused both types of rebinding events to increase. We discovered that clustering was not necessary to achieve adhesion as long as LFA1 and ICAM1 object densities were above a critical level. Importantly, at low densities LFA1 clustering enabled improved efficiency: adhesion exhibited measurable, cell level positive cooperativity.
Author Summary
To gain access to sites of inflammation, leukocytes must first adhere to the blood vessel wall using integrin molecules. It has been hypothesized that integrin clustering is essential for sustaining adhesion prior to transmigration into the inflamed tissue. We cannot challenge such hypotheses directly because it is infeasible to measure molecular level events during the leukocyte adhesion process. At best correlative relationships have been made. The alternative approach undertaken was to experimentally challenge the hypothesized mechanisms in silico. We used object-oriented, software engineering methods to build and execute multi-level, multi-attribute analogues of leukocytes and binding surfaces. The simulated leukocytes contained diffusible objects (representing integrins) on their surface that were allowed to interact with binding partners on simulated endothelial surfaces. Validation was achieved across different experimental conditions, in vitro, ex vivo, and in vivo, at both the individual cell and population levels. Consequently, the finalized virtual mechanisms stand as a concrete, working theory about detailed events occurring at the leukocyte-surface interface during adhesion. We challenged mechanistic hypotheses by conducting experiments in which the consequences of multiple mechanistic events were tracked. We discovered that integrin clustering was not necessary to achieve adhesion as long as integrin and binding partner object densities were above a critical level. Importantly, at low densities integrin clustering enabled adhesion that exhibited measurable, cell level positive cooperativity.
PMCID: PMC2824748  PMID: 20174606
14.  Effectiveness and biocompatibility of a novel biological adhesive application for repair of meniscal tear on the avascular zone 
We have investigated the effectiveness and safety of a newly developed biological adhesive for repair of meniscal tear. The adhesive was composed of disuccinimidyl tartrate (DST) as a crosslinker and human serum albumin (HSA) as a hardener. To determine adequate concentration, bonding strength was measured using a tensiometer 5 min after applying the adhesive on the avascular zone tear of porcine meniscus; it was compared with the strengths of commercially available cyanoacrylate-based and fibrin-based adhesives. In vivo examination was performed using Japanese white rabbits, creating longitudinal tears on the avascular zone of meniscus and applying DST–HSA adhesive. Three months after operation the rabbits were sacrificed and tension test and histological evaluation were performed. Bonding strength was measured in three porcine meniscus groups: (i) only suturing, (ii) suturing after applying the adhesive on surface and (iii) suturing using an adhesive-soaked suture. The optimum concentrations were 0.1 mmol of DST and 42 w/v% of HAS. Bonding strength was greatest with cyanoacrylate-based adhesive, followed by DST–HSA adhesive, and fibrin-based adhesive. No inflammation was observed in the synovium or surrounding tissues 3 months after using the DST–HSA adhesive. Bonding strength was greatest with DST–HSA adhesive-soaked suturing group (77 ± 6 N), followed by suturing only group (61 ± 5 N) and surface adhesive application group (60 ± 8 N). The newly developed DST-HSA adhesive is considered safe and may be effective in enforcement of bonding of avascular zone tear of the meniscus.
PMCID: PMC5099779  PMID: 27877546
biological adhesive; meniscal tear; disuccinimidyl tartrate; human serum albumin
15.  Biological adhesion of the flatworm Macrostomum lignano relies on a duo-gland system and is mediated by a cell type-specific intermediate filament protein 
Frontiers in Zoology  2014;11:12.
Free-living flatworms, in both marine and freshwater environments, are able to adhere to and release from a substrate several times within a second. This reversible adhesion relies on adhesive organs comprised of three cell types: an adhesive gland cell, a releasing gland cell, and an anchor cell, which is a modified epidermal cell responsible for structural support. However, nothing is currently known about the molecules that are involved in this adhesion process.
In this study we present the detailed morphology of the adhesive organs of the free-living marine flatworm Macrostomum lignano. About 130 adhesive organs are located in a horse-shoe-shaped arc along the ventral side of the tail plate. Each organ consists of exactly three cells, an adhesive gland cell, a releasing gland cell, and an anchor cell. The necks of the two gland cells penetrate the anchor cell through a common pore. Modified microvilli of the anchor cell form a collar surrounding the necks of the adhesive- and releasing glands, jointly forming the papilla, the outer visible part of the adhesive organs. Next, we identified an intermediate filament (IF) gene, macif1, which is expressed in the anchor cells. RNA interference mediated knock-down resulted in the first experimentally induced non-adhesion phenotype in any marine animal. Specifically, the absence of intermediate filaments in the anchor cells led to papillae with open tips, a reduction of the cytoskeleton network, a decline in hemidesmosomal connections, and to shortened microvilli containing less actin.
Our findings reveal an elaborate biological adhesion system in a free-living flatworm, which permits impressively rapid temporary adhesion-release performance in the marine environment. We demonstrate that the structural integrity of the supportive cell, the anchor cell, is essential for this adhesion process: the knock-down of the anchor cell-specific intermediate filament gene resulted in the inability of the animals to adhere. The RNAi mediated changes of the anchor cell morphology are comparable to situations observed in human gut epithelia. Therefore, our current findings and future investigations using this powerful flatworm model system might contribute to a better understanding of the function of intermediate filaments and their associated human diseases.
PMCID: PMC4016567  PMID: 24520881
Flatworm; Intermediate filaments; Duo-gland system; Attachment; Bioadhesion
16.  Sialylation and glycosylation modulate cell adhesion and invasion to extracellular matrix in human malignant lymphoma: Dependency on integrin and the Rho GTPase family 
International Journal of Oncology  2015;47(6):2091-2099.
To determine the biological roles of cell surface glycosylation, we modified the surface glycosylation of human malignant lymphoma cell lines using glycosylation inhibitors. The O-glycosylation inhibitor, benzyl-α-GalNAc (BZ) enhanced the fibronectin adhesion of HBL-8 cells, a human Burkitt's lymphoma cell line, and of H-ALCL cells, a human anaplastic large cell lymphoma cell line, both of which were established in our laboratory. The N-glycosylation inhibitor, tunicamycin (TM) inhibited the surface expression of Phaseolus vulgaris leukoagglutinating (L-PHA) lectin- and Canavalia ensiformis (ConA) lectin-reactive oligosaccharides in the HBL-8 cell line. Assay of the adhesion of HBL-8 cells to fibronectin showed that fibronectin adhesion is mediated by the integrin very late antigen (VLA)-4 and that not only BZ but also TM treatment enhanced HBL-8 cell adhesion to fibronectin. Furthermore, although BZ treatment also enhanced H-ALCL cell adhesion to fibronectin, this effect was not mediated by VLA-5 or the RGD sequence of fibronectin. We also showed that H-ALCL cell adhesion to galectin-3 was enhanced by pre-treatment with neuraminidase, which cleaves cell surface sialic acid. Additionally, H-ALCL cell adhesion to galectin-3 was inhibited by pre-treatment with the RGD peptide suggesting that cell adhesion to galectin-3 is mediated by integrin (VLA-5). Furthermore, H-ALCL cell invasion of galectin-1 and galectin-3 was inhibited by pre-treatment with the RGD peptide. Therefore, cell adhesion to and invasion of galectin-1 and galectin-3 are integrin-dependent. In addition to these findings, cell adhesion to galectin-3 was markedly inhibited by treatment with β-lactose compared to treatment with sucrose. Therefore, interactions between integrins and galectin-3 may be mediated through β-galactose that is linked to glycans of integrins. AZA1, an inhibitor of Ras homolog oncoprotein (Rho) GTPase family proteins, RAS-related C3 botulinus toxin substrate 1 (Rac 1) and Cell division control protein 42 homolog (Cdc42) markedly inhibited cell invasion of galectin-1 and galectin-3 suggesting that Rac 1 and Cdc42 may be involved in the regulation of H-ALCL cell invasion of galectins. In conclusion, artificial modification of cell surface glycosylation revealed the biological roles of glycosylation in the adhesion to and invasion of the extracellular matrix (ECM) by human malignant lymphoma cell lines. These findings will provide new insight into the glycobiology of human malignant lymphoma.
PMCID: PMC4665765  PMID: 26497328
glycosylation inhibitors; N-linked glycosylation; O-linked glycosylation; sialic acid; cell adhesion; extracellular matrix; integrin; RGD peptide; Rac 1; Cdc42; Rho
17.  Effect of pH on the Rate of Curing and Bioadhesive Properties of Dopamine Functionalized Poly(ethylene glycol) Hydrogels 
Biomacromolecules  2014;15(8):2861-2869.
The remarkable underwater adhesion strategy employed by mussels has inspired bioadhesives that have demonstrated promise in connective tissue repair, wound closure, and local delivery of therapeutic cells and drugs. While the pH of oxygenated blood and internal tissues is typically around 7.4, skin and tumor tissues are significantly more acidic. Additionally, blood loss during surgery and ischemia can lead to dysoxia, which lowers pH levels of internal tissues and organs. Using 4-armed PEG end-capped with dopamine (PEG-D) as a model adhesive polymer, the effect of pH on the rate of intermolecular cross-linking and adhesion to biological substrates of catechol-containing adhesives was determined. Adhesive formulated at an acidic pH (pH 5.7–6.7) demonstrated reduced curing rate, mechanical properties, and adhesive performance to pericardium tissues. Although a faster curing rate was observed at pH 8, these adhesives also demonstrated reduced mechanical and bioadhesive properties when compared to adhesives buffered at pH 7.4. Adhesives formulated at pH 7.4 demonstrated a good balance of fast curing rate, elevated mechanical properties and interfacial binding ability. UV–vis spectroscopy evaluation revealed that the stability of the transient oxidation intermediate of dopamine was increased under acidic conditions, which likely reduced the rate of intermolecular cross-linking and bulk cohesive properties for hydrogels formulated at these pH levels. At pH 8, competing cross-linking reaction mechanisms and reduced concentration of dopamine catechol due to auto-oxidation likely reduced the degree of dopamine polymerization and adhesive strength for these hydrogels. pH plays an important role in the adhesive performance of mussel-inspired bioadhesives and the pH of the adhesive formulation needs to be adjusted for the intended application.
PMCID: PMC4130238  PMID: 25010812
18.  Modulation of monocyte-endothelial cell interactions by platelet microparticles. 
Journal of Clinical Investigation  1998;102(1):136-144.
Platelets, activated by various agonists, produce microparticles (MP) from the plasma membrane, which are released into the extracellular space. Although the mechanism of MP formation has been clarified, their biological importance remains ill defined. We have recently shown that platelet-derived MP influence platelet and endothelial cell function. In this study, we have further examined the mechanism of cellular activation by platelet MP. To address the possibility that they may influence monocyte-endothelial interactions, we used an in vitro assay to examine their effects on the adhesion of monocytes to human umbilical vein endothelial cells (HUVEC). Platelet MP increased the adhesion of monocytes to HUVEC in a time- and dose-dependent manner. Maximal adhesion of monocytes to resting HUVEC was observed after 24 h of stimulation with MP. Similar kinetics were observed with U-937 (human promonocytic leukemia) cells, used as a model for the blood-borne monocyte. Maximal adhesion of resting monocytes to MP-stimulated HUVEC was observed after 5 h of stimulation with MP. The EC50s for MP-induced increases in HUVEC, monocyte, and U-937 cell adhesion is 8.74, 43.41, and 10.83 microg/ml of MP protein, respectively. The induction of monocyte-endothelial adhesion was mimicked by arachidonic acid isolated from MP. The observed increased cellular adhesiveness correlated with MP-induced upregulation of cell adhesion molecules. MP-stimulated HUVEC increased intracellular cell adhesion molecule-1 (ICAM-1) but not vascular cell adhesion molecule-1 (VCAM-1), P-, or E-selectin expression. Monocyte and U-937 lymphocyte function-associated antigen-1 (CD11a/CD18) and macrophage antigen-1 (CD11b/ CD18, alpham/beta2) were both upregulated upon MP stimulation, but an increase in p150,95 (CD11c/CD18), very late antigen-1, or ICAM-1 expression was not observed. The functional importance of these changes was demonstrated with blocking antibodies. MP also induced the chemotaxis of U-937 cells in a dose-dependent manner with an EC50 of 4.40 microg/ml of MP protein. Similarly, arachidonic acid isolated from MP mimicked the chemotactic response. A role for PKC was implicated in both adhesion and chemotaxis. GF 109203X, a specific inhibitor of PKC, significantly reduced monocyte-endothelial adhesion, as well as U-937 chemotaxis. The demonstration that platelet MP may modulate important aspects of endothelial and monocyte function provides a novel mechanism by which platelets may interact with such cells in human atherosclerosis and inflammation.
PMCID: PMC509075  PMID: 9649567
19.  Investigation of Cell-Substrate Adhesion Properties of Living Chondrocyte by Measuring Adhesive Shear Force and Detachment Using AFM and Inverse FEA 
Scientific Reports  2016;6:38059.
It is well-known that cell adhesion is important in many biological processes such as cell migration and proliferation. A better understanding of the cell adhesion process will shed insight into these cellular biological responses as well as cell adhesion-related diseases treatment. However, there is little research which has attempted to investigate the process of cell adhesion and its mechanism. Thus, this paper aims to study the time-dependent adhesion properties of single living chondrocytes using an advanced coupled experimental-numerical approach. Atomic Force Microscopy (AFM) tips will be used to apply lateral forces to detach chondrocytes that are seeded for three different periods. An advanced Finite Element Analysis (FEA) model combining porohyperelastic (PHE) constitutive model and cohesive zone formulation is developed to explore the mechanism of adhesion. The results revealed that the cells can resist normal traction better than tangential traction in the beginning of adhesion. This is when the cell adhesion molecules establish early attachment to the substrates. After that when the cells are spreading, stress fiber bundles generate tangential traction on the substrate to form strong adhesion. Both simulation and experimental results agree well with each other, providing a powerful tool to study the cellular adhesion process.
PMCID: PMC5125162  PMID: 27892536
20.  Automated Analysis of Cell-Matrix Adhesions in 2D and 3D Environments 
Scientific Reports  2015;5:8124.
Cell-matrix adhesions are of great interest because of their contribution to numerous biological processes, including cell migration, differentiation, proliferation, survival, tissue morphogenesis, wound healing, and tumorigenesis. Adhesions are dynamic structures that are classically defined on two-dimensional (2D) substrates, though the need to analyze adhesions in more physiologic three-dimensional (3D) environments is being increasingly recognized. However, progress has been greatly hampered by the lack of available tools to analyze adhesions in 3D environments. To address this need, we have developed a platform for the automated analysis, segmentation, and tracking of adhesions (PAASTA) based on an open source MATLAB framework, CellAnimation. PAASTA enables the rapid analysis of adhesion dynamics and many other adhesion characteristics, such as lifetime, size, and location, in 3D environments and on traditional 2D substrates. We manually validate PAASTA and utilize it to quantify rate constants for adhesion assembly and disassembly as well as adhesion lifetime and size in 3D matrices. PAASTA will be a valuable tool for characterizing adhesions and for deciphering the molecular mechanisms that regulate adhesion dynamics in 3D environments.
PMCID: PMC4309964  PMID: 25630460
21.  Entactin stimulates neutrophil adhesion and chemotaxis through interactions between its Arg-Gly-Asp (RGD) domain and the leukocyte response integrin. 
Journal of Clinical Investigation  1992;90(6):2251-2257.
Entactin is an integral component of basement membranes that plays a major role in basement membrane assembly through its ability to bind avidly to both laminin and type IV collagen. Because neutrophil (PMN) interactions with entactin have not been examined, we investigated the ability of natural and recombinant entactin to mediate PMN adhesion and chemotaxis. With both forms of entactin, we observed that entactin-coated surfaces promoted PMN adhesion and that entactin stimulated PMN chemotaxis. The increase in adhesion to entactin over control was two to threefold whereas the chemotactic response to 15 ng/ml (1 x 10(-10) M) entactin was equivalent to the chemotactic response elicited with 1 x 10(-8) M formyl-methionyl-leucyl-phenylalanine (fMLP). HL-60 cells, after differentiation with dimethylsulfoxide, also demonstrated adhesion and chemotaxis to entactin. A synthetic peptide of the Arg-Gly-Asp (RGD) domain in entactin, SIGFRGDGQTC (S-RGD), mediated PMN adhesion and chemotaxis, and preexposure of PMN to S-RGD blocked PMN adhesion and chemotaxis induced by entactin without diminishing the adhesive and chemotactic activities of fMLP. In contrast, preexposure to peptides SIGFRGEGQTCA or SIGFKGDGQTCA had no effect. The findings with synthetic peptides were confirmed with a recombinant entactin mutant in which aspartic acid at residue 674 was replaced with glutamic acid, thus converting the RGD sequence of entactin to RGE. RGE-entactin was neither adhesive nor chemotactic for neutrophils. Monoclonal antibodies to the leukocyte response integrin (LRI) and the integrin-associated protein blocked entactin-mediated adhesion and chemotaxis whereas monoclonal antibodies to beta 1 and beta 2 integrins had no effect and PMN from an individual with leukocyte-adhesion deficiency adhered normally to entactin-coated surfaces. These data demonstrate that entactin mediates biologically and pathologically important functions of PMN through its RGD domain and that LRI, which has been shown previously to mediate RGD-stimulated phagocytosis, is also capable of mediating RGD-stimulated PMN adhesion and chemotaxis.
PMCID: PMC443376  PMID: 1469085
22.  Coordinate role for cell surface chondroitin sulfate proteoglycan and alpha 4 beta 1 integrin in mediating melanoma cell adhesion to fibronectin 
The Journal of Cell Biology  1992;118(2):431-444.
Cellular recognition and adhesion to the extracellular matrix (ECM) has a complex molecular basis, involving both integrins and cell surface proteoglycans (PG). The current studies have used specific inhibitors of chondroitin sulfate proteoglycan (CSPG) synthesis along with anti- alpha 4 integrin subunit monoclonal antibodies to demonstrate that human melanoma cell adhesion to an A-chain derived, 33-kD carboxyl- terminal heparin binding fragment of human plasma fibronectin (FN) involves both cell surface CSPG and alpha 4 beta 1 integrin. A direct role for cell surface CSPG in mediating melanoma cell adhesion to this FN fragment was demonstrated by the identification of a cationic synthetic peptide, termed FN-C/H-III, within the fragment. FN-C/H-III is located close to the amino terminal end of the fragment, representing residues #1721-1736 of intact FN. FN-C/H-III binds CSPG directly, can inhibit CSPG binding to the fragment, and promotes melanoma cell adhesion by a CSPG-dependent, alpha 4 beta 1 integrin- independent mechanism. A scrambled version of FN-C/H-III does not inhibit CSPG binding or cell adhesion to the fragment or to FN-C/H-III, indicating that the primary sequence of FN-C/H-III is important for its biological properties. Previous studies have identified three other synthetic peptides from within this 33-kD FN fragment that promote cell adhesion by an arginyl-glycyl-aspartic acid (RGD) independent mechanism. Two of these synthetic peptides (FN-C/H-I and FN-C/H-II) bind heparin and promote cell adhesion, implicating cell surface PG in mediating cellular recognition of these two peptides. Additionally, a third synthetic peptide, CS1, is located in close proximity to FN-C/H-I and FN-C/H-II and it promotes cell adhesion by an alpha 4 beta 1 integrin-dependent mechanism. In contrast to FN-C/H-III, cellular recognition of these three peptides involved contributions from both CSPG and alpha 4 integrin subunits. Of particular importance are observations demonstrating that CS1-mediated melanoma cell adhesion could be inhibited by interfering with CSPG synthesis or expression. Since CS1 does not bind CSPG, the results suggest that CSPG may modify the function and/or activity of alpha 4 beta 1 integrin on the surface of human melanoma cells. Together, these results support a model in which the PG and integrin binding sites within the 33-kD fragment may act in concert to focus these two cell adhesion receptors into close proximity on the cell surface, thereby influencing initial cellular recognition events that contribute to melanoma cell adhesion on this fragment.
PMCID: PMC2290058  PMID: 1629241
23.  Biomechanical properties of Achilles tendon repair augmented with a bioadhesive-coated scaffold 
The Achilles tendon is the most frequently ruptured tendon. Both acute and chronic (neglected) tendon ruptures can dramatically affect a patient’s quality of life, and require a prolonged period of recovery before return to pre-injury activity levels. This paper describes the use of an adhesive-coated biologic scaffold to augment primary suture repair of transected Achilles tendons. The adhesive portion consisted of a synthetic mimic of mussel adhesive proteins that can adhere to various surfaces in a wet environment, including biologic tissues. When combined with biologic scaffolds such as bovine pericardium or porcine dermal tissues, these adhesive constructs demonstrated lap shear adhesive strengths significantly greater than that of fibrin glue, while reaching up to 60% of the strength of a cyanoacrylate-based adhesive. These adhesive constructs were wrapped around transected cadaveric porcine Achilles tendons repaired with a combination of parallel and three-loop suture patterns. Tensile mechanical testing of the augmented repairs exhibited significantly higher stiffness (22–34%), failure load (24–44%), and energy to failure (27–63%) when compared to control tendons with suture repair alone. Potential clinical implications of this novel adhesive biomaterial are discussed.
PMCID: PMC3046464  PMID: 21266745
24.  Antithrombin significantly influences platelet adhesion onto immobilized fibrinogen in an in-vitro system simulating low flow 
Thrombosis Journal  2006;4:19.
Adhesion of platelets onto immobilized fibrinogen is of importance in initiation and development of thrombosis. According to a recent increase in evidence of a multiple biological property of antithrombin, we evaluated the influence of antithrombin on platelet adhesion onto immobilized fibrinogen using an in-vitro flow system.
Platelets in anticoagulated whole blood (29 healthy blood donors) were labelled with fluorescence dye and perfused through a rectangular flow chamber (shear rates of 13 s-1 to 1500 s-1). Platelet adhesion onto fibrinogen-coated slips was assessed using a fluorescence laser-scan microscope and compared to the plasma antithrombin activity. Additionally the effect of supraphysiological AT supplementation on platelets adhesion rate was evaluated.
Within a first minute of perfusion, an inverse correlation between platelet adhesion and plasma antithrombin were observed at 13 s-1 and 50 s-1 (r = -0.48 and r = -0.7, p < 0.05, respectively). Significant differences in platelet adhesion related to low (92 ± 3.3%) and high (117 ± 4.1%) antithrombin activity (1786 ± 516 U vs. 823 ± 331 U, p < 0.05) at low flow rate (13 s-1, within first minute) have been found. An in-vitro supplementation of whole blood with antithrombin increased the antithrombin activity up to 280% and platelet adhesion rate reached about 65% related to the adhesion rate in a non-supplemented blood (1.25 ± 0.17 vs. 1.95 ± 0.4 p = 0.008, respectively).
It appears that antithrombin in a low flow system suppresses platelet adhesion onto immobilized fibrinogen independently from its antithrombin activity. A supraphysiological substitution of blood with antithrombin significantly reduces platelet adhesion rate. This inhibitory effect might be of clinical relevance.
PMCID: PMC1618384  PMID: 17040572
25.  Galectin-1-mediated cell adhesion, invasion and cell death in human anaplastic large cell lymphoma: Regulatory roles of cell surface glycans 
International Journal of Oncology  2014;44(5):1433-1442.
Galectin-1 is known to be one of the extracellular matrix proteins. To elucidate the biological roles of galectin-1 in cell adhesion and invasion of human anaplastic large cell lymphoma, we performed cell adhesion and invasion assays using the anaplastic large cell lymphoma cell line H-ALCL, which was previously established in our laboratory. From the cell surface lectin array, treatment with neuraminidase from Arthrobacter ureafaciens which cleaves all linkage types of cell surface sialic acid enhanced Arachis hypogaea (PNA), Helix pomatia (HPA) and Phaseolus vulgaris-L (L-PHA) lectin binding reactivity to cell surface of lymphoma cells suggesting that neuraminidase removes cell surface sialic acid. In cell adhesion and invasion assays treatment with neuraminidase markedly enhanced cell adhesion to galectin-1 and decreased cell invasive capacity through galectin-1. α2,6-linked sialic acid may be involved in masking the effect of the interaction between galectin-1 and cell surface glycans. H-ALCL cells expressed the β-galactoside-α2,6-sialyltransferase ST6Gal1. On resialylation assay by recombinant ST6Gal1 with CMP-Neu5Ac, α2,6-resialylation of L-PHA reactive oligosaccharide by ST6Gal1 resulted in inhibition of H-ALCL cell adhesion to galectin-1 compared to the desialylated H-ALCL cells. On knockdown experiments, knockdown of ST6Gal1 dramatically enhanced cell adhesion to galectin-1. N-glycosylation inhibitor swainsonine treatment resulted in enhancement of cell adhesion to galectin-1. In glycomic analysis using the lectin blocking assay treatment with PNA, Artocarpus integrifolia (Jacalin), Glycine max (SBA), Helix pomatia (HPA), Vicia villosa (VVA), Ulex europaeus (UEA-1), Triticum vulgaris (WGA), Canavalia ensiformis (ConA), Phaseolus vulgaris-L (L-PHA), Phaseolus vulgaris-E4 (E-PHA), Datura stramonium (DSA) lectins resulted in modulation of lymphoma cell to galectin-1 suggesting that several types of glycans may regulate cell adhesion to galectin-1 by steric hindrance. The adhesive capacity of H-ALCL cells is regulated by phosphatidylinositol 3 phosphate kinase (PI3K) and actin cytoskeleton, and the invasive capacity of H-ALCL cells is regulated by PI3K, mitogen-activated protein kinase (MAPK), Rho and actin cytoskeleton. Furthermore, galectin-1-induced cell death in H-ALCL cells was accompanied by inhibition of CD45 protein tyrosine phosphatase (PTP) activity. In conclusion, cell adhesion and invasion to galectin-1 appeared to be regulated by cell surface sialylation and N-glycosylation, and galectin-1 regulates cell death through inhibition of CD45 PTP activity of H-ALCL.
PMCID: PMC4027875  PMID: 24589677
galectin-1; glycosylation; sialic acid; sialyltransferase; cell adhesion; cell death; lectin array

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