Abrogation of galectin-3 impairs barrier function in mouse corneas.

<p>Numerical scoring for the intensity of staining with rose bengal revealed a higher incidence of epithelial defects in corneas of galectin-3 null mice (Gal-3^−/−) as compared to wild-type (Gal-3^+/+) controls. Representative images are shown in the left panel. *P<0.05.</p

Cellobiose glycopolymers impair glycocalyx barrier function in human corneal epithelial cells.

<p>As determined using the rose bengal penetration assay, treatment of stratified corneal epithelial cells for 1 hour with cellobiose glycopolymers, containing either 240 or 640 repeating units, significantly impaired glycocalyx barrier function. No effect on rose bengal uptake was observed when lactose-containing glycopolymers were used. Images were obtained using a 10× objective lens. All the experiments were performed in triplicate and represent the mean ±SD. ns, not significant; ***P<0.001.</p

Disruption of galectin-3 binding and multimerization impairs glycocalyx barrier function.

<p>(A) One-hour preincubation of stratified human corneal epithelial cell cultures with β-lactose—but not with the non-inhibitory controls of galectin binding, sucrose and maltose—resulted in a significant and transient increase in rose bengal uptake. (B) Incubation with rhGal3 after treatment with β-lactose allowed recovery of barrier function in corneal epithelial cells. On the other hand, addition of rhGal3C resulted in sustained rose bengal uptake by the cell culture. Representative images are shown in the left panel. Images were obtained using a 10× objective lens. All the experiments were performed in triplicate and represent the mean ±SD. ns, not significant, **P<0.01, ***P<0.001.</p

Synthetic glycopolymers incorporate into stratified cultures of human corneal epithelial cells.

<p>(A) Schematic structure and properties of Alexa Fluor 488 cellobiose- and lactose-containing glycopolymers functionalized with a phospholipid end group. (B) Fluorescence microscopy images demonstrated that, following a 1-hour incubation, glycopolymers (green) with 240 and 640 repeating units incorporated into islands of stratified corneal epithelial cells. DAPI was included in the mounting medium to localize the position of the nuclei (blue) in the cell culture. Images were obtained using a 40× objective lens. (C) By pull-down assay, synthetic glycopolymers with lactose-decorated backbones, but not cellobiose derivatives, bound to an rhGal3 affinity column.</p

Topical CLU ameliorates pre-existing ocular surface barrier disruption caused by desiccating stress.

<p><b>(Left).</b> The standard desiccating stress (DS) protocol was applied for 5-days to create ocular surface disruption. Non-stressed (NS) mice housed under normal ambient conditions served as a baseline control. <b>(Left)</b> After the indicated time period, barrier disruption was confirmed by measuring corneal epithelial uptake of fluorescein (FU = Fluorescence Units at 521 nm) in a subset of mice. Values are expressed as the mean ± SD. *p<0.0001 (n = 4). <b>(Right)</b> The same desiccating stress (DS) protocol was continued for another 5 days while eyes with desiccating stress were treated topically with 1 uL of recombinant human CLU (rhCLU) formulated in PBS at 2 ug/mL, or with PBS control, 4 times/day. The fluorescein uptake test was then performed on these remaining mice. Values are expressed as the mean ± SD. *p<0.0001(n = 4).</p

Topical CLU protects the ocular surface barrier against proteolytic damage due to desiccating stress.

<p><b>(A)</b> The standard desiccating stress (DS) protocol was applied, while eyes were left untreated (UT) or treated topically, 4 times/day, with 1 uL of recombinant human CLU (rhCLU) formulated in PBS, or with 1 uL of PBS control. Non-stressed (NS) mice housed under normal ambient conditions were included as a control for PBS treatment. At the end of the experiment, eyes were removed and embedded for frozen sectioning at 10-um thickness. TUNEL staining was performed and nuclei were counterstained with DAPI. Images were taken at 20X magnification. Arrows indicate apoptotic cells in the apical ocular surface epithelium of DS+PBS eyes. <b>(B)</b> The standard desiccating stress (DS) protocol was applied, while eyes were left untreated (UT) or treated topically, 4 times/day, with 1 uL of recombinant human CLU (rhCLU) formulated in PBS, or with 1 uL of PBS control. Non-stressed (NS) mice housed under normal ambient conditions were included as a control for PBS treatment. Desiccating stress was applied to 7 mice per treatment group for 5 days (OCLN) or 9 days (LGALS3) while treated with PBS or CLU at 1 ug/mL. Then total proteins were extracted from the ocular surface epithelia using TRIzol, pooled among the same treatment groups, and subjected to Western blotting with anti-LGALS3 and anti-OCLN antibodies. The protein band image was obtained by Fuji Doc digital camera. “F” indicates full length LGALS3 protein, and “C” is the cleaved product of LGALS3. A digital image analyzer built into the camera was used to quantify the density of individual protein bands. The relative cleavage of LGALS3 was calculated by ratio of the C over the total (F+C) LGALS3 protein. The relative amount of OCLN was normalized to the loading control (ACTB) in each gel lane. <b>(C)</b> Stratified HCLE cells were treated with TNFA (5 ng/mL), alone or with recombinant human CLU (rhCLU) (4 ug/mL) or BSA (40 ug/mL) for 24 h. the conditioned media were subject to gelatin zymography and the developed MMP9 image were analyzed by Image J software. *P<0.05 (n = 3, student’s t-test)</p

Topical CLU directly seals the ocular surface barrier disrupted by desiccating stress.

<p>The standard desiccating stress (DS) protocol was applied for 5-days to create ocular surface disruption. Non-stressed (NS) mice housed under normal ambient conditions served as a baseline control. Eyes with desiccating stress were then treated topically, a single time, with 1 uL of CLU formulated in PBS, 1 uL of BSA formulated in PBS for comparison, or 1 uL of PBS control. Barrier disruption was assayed by measuring corneal epithelial uptake of fluorescein (FU = Fluorescence Units at 521 nm). Values are expressed as the mean ± SD. <b>(A)</b> Eyes were treated a single time with recombinant human CLU (rhCLU) at 1, 3, 6 or 10 ug/mL, BSA at 10 ug/mL, or PBS. Fifteen minutes later, the fluorescein uptake test was performed, before there was time for barrier repair to occur. *P<0.0001 (n = 4). <b>(B)</b> Images of central cornea from the experiment shown in (A), obtained using laser scanning confocal microscopy at 10X magnification. One representative image out of two independent experiments is shown. Scale bar = 100 um. <b>(C)</b> Eyes were treated a single time with recombinant human CLU (rhCLU) at 10 ug/mL (right eyes) or PBS (left eyes). Then the mice were kept further for 2 h or 16 h while continuing with the same desiccating stress protocol. The fluorescein uptake test was performed following the indicated time period to assess the time length of CLU treatment effect. *p<0.0001 (n = 4)</p

Topical CLU protects the ocular surface barrier via an all-or-none mechanism.

<p>The standard desiccating stress (DS) protocol was applied, while eyes were left untreated (UT) or treated topically 4 times/day with 1 uL of CLU formulated in PBS, or with PBS control. Non-stressed (NS) mice housed under normal ambient conditions served as a baseline control. After the indicated time period, barrier integrity was assayed by measuring corneal epithelial uptake of fluorescein (FU = Fluorescence Units at 521 nm). Values are expressed as the mean ± SD. <b>(A) Dose response experiment.</b> The desiccating stress (DS) protocol was applied for 5 days while also treating with <b>(Left)</b> recombinant human CLU (rhCLU) at the indicated 10-fold dilutions (n = 6), <b>(Middle)</b> recombinant human CLU (rhCLU) at 0.1, 0.3, 0.6, or 1 ug/mL (n = 6), or <b>(Right)</b> recombinant mouse CLU (rmCLU) at 0.3, 0.6, and 1 ug/mL (n = 4). *P<0.0001. <b>(B) Experiment comparing CLU with BSA.</b> The desiccating stress (DS) protocol was applied for 5 days while also treating with recombinant human CLU (rhCLU) and BSA, individually or in combination, as indicated. *P<0.0001 (n = 4)<b>. (C) Stress reduction experiment.</b> The standard desiccating stress (DS) protocol was applied for 5 days while eyes were also treated with recombinant human CLU (rhCLU) at 0.01, 0.1, and 1 ug/mL. Using a subset (n = 4) of each treatment group the effect of each rhCLU dose on integrity of the ocular surface barrier was confirmed by the fluorescein uptake test at day 5. Then the rest of the mice in each treatment group were subjected for two more days to a more moderate desiccating stress by continuing with the air draft and heat, but omitting scopolamine and CLU treatments. The fluorescein uptake test was then performed on these remaining mice. *P = 0.004 (n = 4); **P = 0.05 (n = 4)</p

Causal association between endogenous CLU concentration in tears and ocular surface barrier vulnerability.

<p><b>(A)</b> Tears were collected from mice housed under normal ambient conditions or after application of the standard desiccating stress (DS) protocol for 5-days, and ELISA was used to measure CLU concentration (*P = 5x10^-8 n = 6, student’s t-test). <b>(B)</b> Representative transmission electron microscopy comparing images of anterior cornea from wild type C57BL6/J mice (A and C) and mice with homozygous CLU^-/- knockout on the C57BL6/J background (B and D). In low power (4000x) magnifications (A and B), five layers of epithelial cells divided into squamous, wing, and basal cell regions are visualized along with an intact basement membrane and Bowman's layer in both types of animals. Higher power images (C and D, 20,000x) of similar regions to those boxed in panels A and B show numerous surface microplicae (fat arrows) in both genotypes. Desmosomes (thin arrows) are similar in both frequency and structure. Higher power images (not shown) demonstrate intact adherens junctions in both genotypes. <b>(C)</b> Tears from wild type or heterozygous CLU^+/- knockout mice kept at ambient conditions were collected and ELISA was used to measure CLU concentration (p = 2.1x10^-5; n = 7, student’s t-test). <b>(D)</b> Wild type mice or heterozygous CLU^+/- knockout mice were subjected to the standard desiccating stress protocol, but without scopolamine injection for four weeks and then ocular surface barrier integrity was measured by fluorescein uptake (**p<0.0001, n = 4).</p

Topical CLU protects the ocular surface barrier against functional disruption by desiccating stress.

<p>The standard desiccating stress (DS) protocol was applied, while eyes were left untreated (UT) or treated topically 4 times/day with 1 uL of CLU formulated in PBS, or with PBS control. Non-stressed (NS) mice housed under normal ambient conditions served as a baseline control. After the indicated time period, barrier integrity was assayed by measuring corneal epithelial uptake of fluorescein (FU = Fluorescence Units at 521 nm). Values are expressed as the mean ± SD. <b>(A)</b> The desiccating stress (DS) protocol was applied for 5 days while also treating with rhCLU at 10 or 100 ug/mL. *P<0.0001 (n = 9). <b>(B)</b> The desiccating stress (DS) protocol was applied for 7 days while also treating with rhCLU at 1 or 10 ug/mL. *P<0.0001 (n = 4). <b>(C)</b> The desiccating stress (DS) protocol was applied for 5 days while also treating with human plasma CLU (pCLU) at 2 ug/mL *P<0.0001 (n = 4). (D) The desiccating stress (DS) protocol was applied for 5 days while also treating with recombinant mouse CLU (rmCLU) at 2 ug/mL. *P<0.0001 (n = 4)</p