Functional binding of E-selectin to its ligands is enhanced by structural features beyond its lectin domain.

Selectins are key to mediating interactions involved in cellular adhesion and migration, underlying processes such as immune responses, metastasis, and transplantation. Selectins are composed of a lectin domain, an epidermal growth factor (EGF)-like domain, multiple short consensus repeats (SCRs), a transmembrane domain, and a cytoplasmic tail. It is well-established that the lectin and EGF domains are required to mediate interactions with ligands; however, the contributions of the other domains in mediating these interactions remain obscure. Using various E-selectin constructs produced in a newly developed silkworm-based expression system and several assays performed under both static and physiological flow conditions, including flow cytometry, glycan array analysis, surface plasmon resonance, and cell-rolling assays, we show here that a reduction in the number of SCR domains is correlated with a decline in functional E-selectin binding to hematopoietic cell E- and/or L-selectin ligand (HCELL) and P-selectin glycoprotein ligand-1 (PSGL-1). Moreover, the binding was significantly improved through E-selectin dimerization and by a substitution (A28H) that mimics an extended conformation of the lectin and EGF domains. Analyses of the association and dissociation rates indicated that the SCR domains, conformational extension, and dimerization collectively contribute to the association rate of E-selectin-ligand binding, whereas just the lectin and EGF domains contribute to the dissociation rate. These findings provide the first evidence of the critical role of the association rate in functional E-selectin-ligand interactions, and they highlight that the SCR domains have an important role that goes beyond the structural extension of the lectin and EGF domains.

Quantitative Characterization of E-selectin Interaction with Native CD44 and P-selectin Glycoprotein Ligand-1 (PSGL-1) Using a Real Time Immunoprecipitation-based Binding Assay.

Selectins (E-, P-, and L-selectins) interact with glycoprotein ligands to mediate the essential tethering/rolling step in cell transport and delivery that captures migrating cells from the circulating flow. In this work, we developed a real time immunoprecipitation assay on a surface plasmon resonance chip that captures native glycoforms of two well known E-selectin ligands (CD44/hematopoietic cell E-/L-selectin ligand and P-selectin glycoprotein ligand-1) from hematopoietic cell extracts. Here we present a comprehensive characterization of their binding to E-selectin. We show that both ligands bind recombinant monomeric E-selectin transiently with fast on- and fast off-rates, whereas they bind dimeric E-selectin with remarkably slow on- and off-rates. This binding requires the sialyl Lewis x sugar moiety to be placed on both O- and N-glycans, and its association, but not dissociation, is sensitive to the salt concentration. Our results suggest a mechanism through which monomeric selectins mediate initial fast on and fast off kinetics to help capture cells out of the circulating shear flow; subsequently, tight binding by dimeric/oligomeric selectins is enabled to significantly slow rolling.

Galectins Differentially Regulate the Surface Glycosylation of Human Monocytes.

Monocytes are circulating blood cells that rapidly mobilize to inflamed sites where they serve diverse effector functions shaped in part by microenvironmental cues. The establishment of specific glycosylation patterns on the immune cell glycocalyx is fundamental to direct the inflammatory response, but relatively little is known about the mechanisms whereby the microenvironment controls this process. Here, we report that galectins differentially participate in remodeling the surface glycosylation of human primary CD14+CD16- monocytes under proinflammatory conditions. Using a lectin array on biotinylated protein, we found that the prototypic galectin-1 negatively influenced the expression of galactose epitopes on the surface of monocytic cells. On the other hand, the tandem-repeat galectin-8 and, to a certain extent, the chimeric galectin-3 promoted the expression of these residues. Jacalin flow cytometry and pull-down experiments further demonstrated that galectin-8 causes a profound upregulation of mucin-type O-glycosylation in cell surface proteins from primary monocytes and THP-1 cells. Overall, these results highlight the emerging role of the galectin signature on inflamed tissues and provide new insights into the contribution of extracellular galectins to the composition of the glycocalyx in human monocytes.

Refining the migration and engraftment of short-term and long-term HSCs by enhancing homing-specific adhesion mechanisms.

In contrast to the short-term (ST) CD34+ stem cells, studies have suggested that long-term (LT) hematopoietic stem cells (HSCs) found in the CD34- stem cell pool have trouble migrating and engrafting when introduced through IV. To understand why these deficiencies exist, we set out to fully elucidate the adhesion mechanisms used by ST and LT-HSCs to migrate to the bone marrow(BM). Specifically focusing on murine ST-HSCs (Flk2-CD34+) and LT-HSCs (Flk2-CD34-), we observed a distinctive expression pattern of BM homing effectors necessary for the first step, namely sialyl Lewis-X (sLex) (ligand for E-selectin), and the second step, namely CXCR4 chemokine receptor (receptor for SDF-1). sLex expression was higher on Flk2-CD34+ ST-HSCs (>60%) compared with Flk2-CD34- LT-HSCs (<10%), which correlated to binding to E-selectin. Higher concentrations of CXCR4 were observed on Flk2-CD34+ ST-HSCs compared with Flk2-CD34- LT-HSCs. Interestingly, the expression of CD26, a peptidase known to deactivate chemokines (ie, SDF-1), was higher on Flk2-CD34- LT-HSCs. Given that both E-selectin-binding and CXCR4-mediated migration are compromised in Flk2-CD34- LT-HSCs, we aimed to enhance their ability to migrate using recombinant human fucosyltransferase 6 (rhFTVI) and the CD26 inhibitor, Dip A (diprotin A). To this end, we observed that although LT-HSCs expressed low concentrations of sLex, they were able to engraft when transplanted into recipient mice. Moreover, although both CD26 inhibition and fucosylation enhanced migration of both HSC populations in vitro, only pretreatment of LT-HSCs with Dip A enhanced engraftment in vivo after transplantation into recipient mice. Remarkably, fucosylation of Flk2-CD34+ ST-HSCs consistently led to their ability to transplant secondary recipients. These data suggest that using fucosylation and Dip A to overcome the molecular disparity in adhesion mechanisms among ST-HSCs and LT-HSCs differentially influences their abilities to migrate and engraft in vivo and promotes the ability of ST-HSCs to engraft secondary recipient mice, the gold standard for testing functionality of LT-HSCs.

Crosslinker-free collagen gelation for corneal regeneration.

Development of an artificial cornea can potentially fulfil the demand of donor corneas for transplantation as the number of donors is far less than needed to treat corneal blindness. Collagen-based artificial corneas stand out as a regenerative option, having promising clinical outcomes. Collagen crosslinked with chemical crosslinkers which modify the parent functional groups of collagen. However, crosslinkers are usually cytotoxic, so crosslinkers need to be removed from implants completely before application in humans. In addition, crosslinked products are mechanically weak and susceptible to enzymatic degradation. We developed a crosslinker free supramolecular gelation strategy using pyrene conjugated dipeptide amphiphile (PyKC) consisting of lysine and cysteine; in which collagen molecules are intertwined inside the PyKC network without any functional group modification of the collagen. The newly developed collagen implants (Coll-PyKC) are optically transparent and can effectively block UV light, are mechanically and enzymatically stable, and can be sutured. The Coll-PyKC implants support the growth and function of all corneal cells, trigger anti-inflammatory differentiation while suppressing the pro-inflammatory differentiation of human monocytes. Coll-PyKC implants can restrict human adenovirus propagation. Therefore, this crosslinker-free strategy can be used for the repair, healing, and regeneration of the cornea, and potentially other damaged organs of the body.

Electron-Beam Irradiated Recombinant Human Collagen-Phosphorylcholine Corneal Implants Retain Pro-Regeneration Capacity.

Sterilization of biodegradable, collagen-based implants is challenging as irradiation sterilization methods can alter their mechanical properties. Electron beam (EB) irradiation is a terminal sterilization method that has been used for biologically-derived implants. Here, recombinant human collagen type III-phosphorylcholine (RHCIII-MPC) hydrogels were irradiated with EB doses of 17, 19, or 21 kGy and their subsequent biocompatibility and ability to promote regeneration in rabbit corneas was evaluated. Unirradiated hydrogels stored in 1% chloroform in phosphate-buffered saline (C-PBS) were the controls. There were no significant differences between irradiated and non-irradiated samples in optical or physical properties (tensile strength, modulus, elasticity), or the ability to support cell growth. However, irradiated implants were more sensitive to high levels of collagenase than unirradiated controls and the C-PBS implants had increased cell growth compared to EB and controls at 72 h. Corneal implants e-beamed at 17 kGy or e-beamed and subsequently frozen (EB-F) to increase shelf-life showed no adverse biological effects of the irradiation. EB, EB-F, and C-PBS implanted corneas all rapidly re-epithelialized but showed mild neovascularization that resolved over 6 months. The regenerated neo-corneas were transparent at 6 months post-operation. In vivo confocal microscopy confirmed normal morphology for the epithelium, stroma, sub-basal nerves and unoperated endothelium. Histology showed that all the regenerated corneas were morphologically similar to the normal. Immunohistochemistry indicated the presence of a differentiated corneal epithelium and functional tear film. In conclusion, the e-beamed corneal implants performed as well as non-irradiated control implants, resulting in fully regenerated neo-corneas with new nerves and without blood vessels or inflammation that may impede vision or corneal function. Therefore, a complete validation study to establish EB irradiation as an effective means for corneal implant sterilization prior to clinical application is necessary as a next step.

The Epithelial Cell Glycocalyx in Ocular Surface Infection.

The glycocalyx is the main component of the transcellular barrier located at the interface between the ocular surface epithelia and the external environment. This barrier extends up to 500 nm from the plasma membrane and projects into the tear fluid bathing the surface of the eye. Under homeostatic conditions, defense molecules in the glycocalyx, such as transmembrane mucins, resist infection. However, many pathogenic microorganisms have evolved to exploit components of the glycocalyx in order to gain access to epithelial cells and consequently exert deleterious effects. This manuscript reviews the implications of the ocular surface epithelial glycocalyx to bacterial, viral, fungal and parasitic infection. Moreover, it presents some ongoing controversies surrounding the functional relevance of the epithelial glycocalyx to ocular infectious disease.

Induction of CXCL10-Mediated Cell Migration by Different Types of Galectins.

Chemokines are an extended group of chemoattractant cytokines responsible for the recruitment of leukocytes into tissues. Among them, interferon-γ-inducible protein 10 (CXCL10) is abundantly expressed following inflammatory stimuli and participates in the trafficking of monocytes and activated T cells into sites of injury. Here, we report that different members of the galectin family of carbohydrate-binding proteins promote the expression and synthesis of CXCL10 independently of interferon-γ. Interestingly, CXCL10 induction was observed when galectins came in contact with stromal fibroblasts isolated from human cornea but not other cell types such as epithelial, monocytic or endothelial cells. Induction of CXCL10 by the tandem repeat galectin-8 was primarily associated with the chemotactic migration of THP-1 monocytic cells, whereas the prototype galectin-1 promoted the CXCL10-dependent migration of Jurkat T cells. These results highlight the potential importance of the galectin signature in dictating the recruitment of specific leukocyte populations into precise tissue locations.

Galectin-3 Enhances Vascular Endothelial Growth Factor-A Receptor 2 Activity in the Presence of Vascular Endothelial Growth Factor.

Galectin-3 (Gal3) is a carbohydrate-binding protein reported to promote angiogenesis by influencing vascular endothelial growth factor-A receptor 2 (VEGFR2) signal transduction. Here we evaluated whether the ability of Gal3 to function as an angiogenic factor involved vascular endothelial growth factor (VEGF). To address this possibility we used human retinal microvascular endothelial cells (HRECs) to determine whether exogenous Gal3 requires VEGF to activate VEGFR2 signaling and if Gal3 is required for VEGF to activate VEGFR2. VEGFR2 phosphorylation and HREC migration assays, following either VEGF neutralization with ranibizumab or Gal3 silencing, revealed that VEGF endogenously produced by the HRECs was essential for the effect of exogenous Gal3 on VEGFR2 activation and cell migration, and that VEGF-induced VEGFR2 activation was not dependent on Gal3 in HRECs. Gal3 depletion led to no reduction in VEGF-induced cell function. Since Gal3 has been suggested to be a potential therapeutic target for VEGFR2-mediated angiogenesis, it is crucial to define the possible Gal3-mediated VEGFR2 signal transduction mechanism to aid the development of efficacious therapeutic strategies.

Optimization of Collagen Chemical Crosslinking to Restore Biocompatibility of Tissue-Engineered Scaffolds.

Collagen scaffolds, one of the most used biomaterials in corneal tissue engineering, are frequently crosslinked to improve mechanical properties, enzyme tolerance, and thermal stability. Crosslinkers such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) are compatible with tissues but provide low crosslinking density and reduced mechanical properties. Conversely, crosslinkers such as glutaraldehyde (GTA) can generate mechanically more robust scaffolds; however, they can also induce greater toxicity. Herein, we evaluated the effectivity of double-crosslinking with both EDC and GTA together with the capability of sodium metabisulfite (SM) and sodium borohydride (SB) to neutralize the toxicity and restore biocompatibility after crosslinking. The EDC-crosslinked collagen scaffolds were treated with different concentrations of GTA. To neutralize the free unreacted aldehyde groups, scaffolds were treated with SM or SB. The chemistry involved in these reactions together with the mechanical and functional properties of the collagen scaffolds was evaluated. The viability of the cells grown on the scaffolds was studied using different corneal cell types. The effect of each type of scaffold treatment on human monocyte differentiation was evaluated. One-way ANOVA was used for statistical analysis. The addition of GTA as a double-crosslinking agent significantly improved the mechanical properties and enzymatic stability of the EDC crosslinked collagen scaffold. GTA decreased cell biocompatibility but this effect was reversed by treatment with SB or SM. These agents did not affect the mechanical properties, enzymatic stability, or transparency of the double-crosslinked scaffold. Contact of monocytes with the different scaffolds did not trigger their differentiation into activated macrophages. Our results demonstrate that GTA improves the mechanical properties of EDC crosslinked scaffolds in a dose-dependent manner, and that subsequent treatment with SB or SM partially restores biocompatibility. This novel manufacturing approach would facilitate the translation of collagen-based artificial corneas to the clinical setting.

Strain-induced Differentiation of Mesenchymal Stem Cells.

Directing the fate of human mesenchymal stem/stromal cells (hMSCs) toward bone formation using mechanical strain is a promising approach in regenerative medicine related to bone diseases. Numerous studies have evaluated the effects of vibration or cyclic tensile strain on MSCs towards developing a mechanically-based method for stimulating differentiation. Here, we study the differentiation of hMSCs cultured on elastic polydimethylsiloxane (PDMS) membrane, which is magnetically actuated to induce periodically varying strain. The strain distribution across the membrane was calculated by finite-element modeling and demonstrates three main areas of different strain amplitudes. The strain effect on the hMSCs was evaluated by measuring the mineralization of differentiated hMSCs using Alizarin S red stain. The results indicate a strain-dependent differentiation of hMSCs, where the highest region of strain on the membrane resulted in the most accelerated differentiation. Osteogenic differentiation was achieved as early as two weeks, which is significantly sooner than control hMSCs treated with osteogenic media alone.

Golgi α1,2-mannosidase I induces clustering and compartmentalization of CD147 during epithelial cell migration.

CD147 is a widely expressed matrix metalloproteinase inducer involved in the regulation of cell migration. The high glycosylation and ability to undergo oligomerization have been linked to CD147 function, yet there is limited understanding on the molecular mechanisms behind these processes. The current study demonstrates that the expression of Golgi α1,2-mannosidase I is key to maintaining the cell surface organization of CD147 during cell migration. Using an in vitro model of stratified human corneal epithelial wound healing, we show that CD147 is clustered within lateral plasma membranes at the leading edge of adjacent migrating cells. This localization correlates with a surge in matrix metalloproteinase activity and an increase in the expression of α1,2-mannosidase subtype IC (MAN1C1). Global inhibition of α1,2-mannosidase I activity with deoxymannojirimycin markedly attenuates the glycosylation of CD147 and disrupts its surface distribution at the leading edge, concomitantly reducing the expression of matrix metalloproteinase-9. Likewise, treatment with deoxymannojirimycin or siRNA-mediated knockdown of MAN1C1 impairs the ability of the carbohydrate-binding protein galectin-3 to stimulate CD147 clustering in unwounded cells. We conclude that the mannose-trimming activity of α1,2-mannosidase I coordinates the clustering and compartmentalization of CD147 that follows an epithelial injury.

Galectin-3 initiates epithelial-stromal paracrine signaling to shape the proteolytic microenvironment during corneal repair.

Paracrine interactions between epithelial cells and stromal fibroblasts occur during tissue repair, development, and cancer. Crucial to these processes is the production of matrix metalloproteinases (MMPs) that modify the microenvironment. Here, we demonstrated that the carbohydrate-binding protein galectin-3 stimulated microenvironment remodeling in the cornea by promoting the paracrine action of secreted interleukin-1ß (IL-1ß). Through live cell imaging in vitro, we observed rapid activation of the MMP9 promoter in clusters of cultured human epithelial cells after direct heterotypic contact with single primary human fibroblasts. Soluble recombinant galectin-3 and endogenous galectin-3 of epithelial origin both stimulated MMP9 activity through the induction of IL-1ß secretion by fibroblasts. In vivo, mechanical disruption of the basement membrane in wounded corneas prompted an increase in the abundance of IL-1ß in the stroma and increased the amount of gelatinase activity in the epithelium. Moreover, corneas of galectin-3-deficient mice failed to stimulate IL-1ß after wounding. This mechanism of paracrine control has broad importance for our understanding of how the proteolytic microenvironment is modified in epithelial-stromal interactions.

Effects of gamma radiation sterilization on the structural and biological properties of decellularized corneal xenografts.

To address the shortcomings associated with corneal transplants, substantial efforts have been focused on developing new modalities such as xenotransplantion. Xenogeneic corneas are anatomically and biomechanically similar to the human cornea, yet their applications require prior decellularization to remove the antigenic components to avoid rejection. In the context of bringing decellularized corneas into clinical use, sterilization is a crucial step that determines the success of the transplantation. Well-standardized sterilization methods, such as gamma irradiation (GI), have been applied to decellularized porcine corneas (DPC) to avoid graft-associated infections in human recipients. However, little is known about the effect of GI on decellularized corneal xenografts. Here, we evaluated the radiation effect on the ultrastructure, optical, mechanical and biological properties of DPC. Transmission electron microscopy revealed that gamma irradiated decellularized porcine cornea (G-DPC) preserved its structural integrity. Moreover, the radiation did not reduce the optical properties of the tissue. Neither DPC nor G-DPC led to further activation of complement system compared to native porcine cornea when exposed to plasma. Although, DPC were mechanically comparable to the native tissue, GI increased the mechanical strength, tissue hydrophobicity and resistance to enzymatic degradation. Despite these changes, human corneal epithelial, stromal, endothelial and hybrid neuroblastoma cells grew and differentiated on DPC and G-DPC. Thus, GI may achieve effective tissue sterilization without affecting critical properties that are essential for corneal transplant survival.

Lectin-Glycan Interactions in Corneal Infection and Inflammation.

The cornea is an extraordinary component of vision that functions as the principal barrier to pathogens in the eye while allowing light transmission into the retina. Understanding the cellular and molecular mechanisms that maintain homeostasis in this tissue is the subject of intense scientific study given the high prevalence of corneal disease. Over the past decade, the interactions between lectins and glycans on plasma membranes have emerged as important regulatory factors in corneal biology. In particular, members of the galectin family have been shown to bind multiple ß-galactoside-containing receptors to regulate immunopathological processes associated with viral and bacterial infection, transplantation, wound healing, dry eye, angiogenesis, and lymphangiogenesis. In this review, we describe the current understanding of how these surface interactions intersect with different pathways to activate unique cellular responses in cornea as well as their potential therapeutic implications.

Colocalization of Galectin-3 With CD147 Is Associated With Increased Gelatinolytic Activity in Ulcerating Human Corneas.

Purpose: Galectin-3 is a carbohydrate-binding protein known to promote expression of matrix metalloproteinases, a hallmark of ulceration, through interaction with the extracellular matrix metalloproteinase inducer CD147. The aim of this study was to investigate the distribution of galectin-3 in corneas of patients with ulcerative keratitis and to determine its relationship to CD147 and the presence of gelatinolytic activity. Methods: This was an observational case series involving donor tissue from 13 patients with active corneal ulceration and 6 control corneas. Fixed-frozen sections of the corneas were processed to localize galectin-3 and CD147 by immunofluorescence microscopy. Gelatinolytic activity was detected by in situ zymography. Results: Tissue from patients with active corneal ulceration showed a greater galectin-3 immunoreactivity in basal epithelia and stroma compared with controls. Immunofluorescence grading scores revealed increased colocalization of galectin-3 and CD147 in corneal ulcers at the epithelial-stromal junction and within fibroblasts. Quantitative analysis using the Manders' colocalization coefficient demonstrated significant overlap in corneas from patients with ulcerative keratitis (M1 = 0.29; M2 = 0.22) as opposed to control corneas (M1 = 0.01, P < 0.01; M2 = 0.02, P < 0.05). In these experiments, there was a significant positive correlation between the degree of galectin-3 and CD147 colocalization and the presence of gelatinolytic activity. Conclusions: Our results indicate that concomitant stimulation and colocalization of galectin-3 with CD147 are associated with increased gelatinolytic activity in the actively ulcerating human cornea and suggest a mechanism by which galectin-3 may contribute to the degradation of extracellular matrix proteins during ulceration.

Not just a marker: CD34 on human hematopoietic stem/progenitor cells dominates vascular selectin binding along with CD44.

CD34 is routinely used to identify and isolate human hematopoietic stem/progenitor cells (HSPCs) for use clinically in bone marrow transplantation, but its function on these cells remains elusive. Glycoprotein ligands on HSPCs help guide their migration to specialized microvascular beds in the bone marrow that express vascular selectins (E- and P-selectin). Here, we show that HSPC-enriched fractions from human hematopoietic tissue expressing CD34 (CD34pos) bound selectins, whereas those lacking CD34 (CD34neg) did not. An unbiased proteomics screen identified potential glycoprotein ligands on CD34pos cells revealing CD34 itself as a major vascular selectin ligand. Biochemical and CD34 knockdown analyses highlight a key role for CD34 in the first prerequisite step of cell migration, suggesting that it is not just a marker on these cells. Our results also entice future potential strategies to investigate the glycoforms of CD34 that discriminate normal HSPCs from leukemic cells and to manipulate CD34neg HSPC-enriched bone marrow or cord blood populations as a source of stem cells for clinical use.