Inhibition of Pterygium Fibroblast Migration and Outgrowth by Bevacizumab and Cyclosporine A Involves Down-Regulation of Matrix Metalloproteinases-3 and -13

<div><p>We examined the connection between matrix metalloproteinase (MMP) expression/activity and pterygium fibroblast migration, and how these were affected by bevacizumab and/or cyclosporine A (CsA). Fibroblasts were obtained from 20 pterygia and 6 normal conjunctival specimens. Expression levels of MMP-3 and MMP-13 were examined after bevacizumab administration. Immunofluorescence staining was used to examine expression of both MMPs in fibroblasts migrating out from explanted pterygium tissues. Rates of cell migration from explant-cultured pterygia tissues and scratch-wounded confluent pterygium fibroblasts were examined in the presence of MMP-3 or MMP-13 inhibitors, as well as bevacizumab and/or CsA. A scratch wound healing migration assay was performed to determine the effects of bevacizumab and/or CsA. Protein expression of both MMPs in pterygium tissues and in cells migrating from organ-cultured pterygium tissues was greater than that observed in normal cells. Inhibition of the activities of both MMPs decreased their expression levels; these were also significantly reduced in bevacizumab-injected pterygium tissues. Bevacizumab significantly reduced the expression of both MMPs and cell migration. Pretreatment with CsA prior to bevacizumab exposure markedly inhibited cell migration and the expression of both MMPs. CsA enhanced the inhibitory effects of bevacizumab on pterygium fibroblast migration <i>in vitro</i>, possibly by inhibiting expression of both MMPs. These findings suggest that combined CsA and bevacizumab treatment may provide a potential therapeutic strategy for reducing the rate of pterygium recurrence.</p></div

Inhibition of MMP-3 or MMP-13 activity reduced pterygium-derived fibroblast migration.

<p>Pterygium fibroblasts were treated with an MMP-3 (1 μM) or MMP-13 (1 μM) inhibitor in serum-free medium, directly after the scratch wound. (A) Representative phase contrast microscopy images taken at 24 h and 48 h after a single uniform-width scratch wound of confluent pterygium fibroblasts. Dashed lines indicate the precise location of the scratch wound. Scale bar: 50 μm. (B) Pooled quantitative data of pterygium-derived fibroblast migration. Data were summarized as the mean ± standard deviation of 3 separate experiments. ***P < 0.001 vs control.</p

Subconjunctival administration of bevacizumab into pterygium tissues inhibited MMP-3 and MMP-13 protein expression.

<p>After injection of bevacizumab (2.5 mg/0.1 ml) for 7 days, pterygium tissues were surgically harvested. (A) Immunohistochemistry of paraffin-embedded sections (5 μm) was performed to identify MMP-3 and MMP-13 expression in bevacizumab-injected pterygium tissue (left panels) and untreated pterygium tissue (right panels). No staining was observed in negative control sections that were incubated with the secondary antibodies, but not the primary antibodies (middle panels). All tissue sections were counterstained with hematoxylin. White scale bar: 50 μm. (B) The expression of MMP-3 and MMP-13 proteins in equal amounts of cell lysate protein (30 μg) was analyzed by a western blot. β-actin was used as the loading control. (C) MMP-3 and MMP-13 band densities were quantified using ImageJ, relative to the relevant β-actin band. (B, C) Data represent the mean ± standard deviation of 3 separate experiments. ***P < 0.001 vs conjunctiva tissue; ^###P < 0.001 vs pterygium tissue.</p

Combined bevacizumab- and CsA-induced inhibition of Cell migration from pterygium explants.

<p>(A) Cell outgrowth from explanted pterygium tissues (T) was examined for 48 h in the presence of bevacizumab (1 μg/ml) and/or following pretreatment with CsA (0.01% for 3 min). The arrow direction indicates cells migrating out from the cultured explant. White scale bar: 200 μm. (B) Migration distance quantification showing the mean ± standard deviation of 3 separate experiments. **P < 0.01, ***P < 0.001 vs control; ^###P < 0.001 vs bevacizumab; ^†P < 0.05 vs CsA.</p

Inhibition of MMP-3 or MMP-13 activity reduced cell migration from pterygium explants.

<p>The effect of MMP inhibition on cell migration was determined by measuring the length of migration from the explanted tissue. When cells began to migrate from the explant, they were exposed to MMP-3 inhibitor VII and an MMP-13 inhibitor. (A) Cell migration was captured using a phase contrast microscope after treatment with the indicated inhibitor for 0 h, 24 h, and 48 h. White scale bar: 50 μm. Cell outgrowth from the explanted tissue in the control group increased continuously during the culture period. In contrast, the rate of cell migration was greatly reduced by treatment with an MMP-3 or MMP-13 inhibitor. (B) Migration rates under the indicated conditions. Migration was quantified under 0 h control condition. ***P < 0.001 vs the corresponding control value.</p

Bevacizumab-induced inhibition of scratch wound pterygium fibroblast migration and down-regulation of MMP-3 and MMP-13 expression.

<p>(A) Effects of bevacizumab (1 μg/ml) on cell migration were determined using a scratch wound assay and phase-contrast microscopy. Dashed lines indicate the precise location of the scratch wound. White scale bar: 50 μm. (B) MMP expression in these cells was determined by western blot analysis using equal amounts of cell lysate protein (30 μg), harvested at 24 h. Note that levels of both MMP-3 and MMP-13 expression were dramatically down-regulated by bevacizumab treatment. β-actin was used as the loading control. (C) Band densities were measured using ImageJ, relative to the relevant β-actin band. Data represent the mean ± standard deviation of 3 separate experiments. ***P < 0.001 vs control.</p

Combined Bevacizumab- and CsA-induced inhibition of scratch wound pterygium fibroblast migration and down-regulation of MMP-3 and MMP-13 expression.

<p>(A) Phase contrast microscopy of pterygium fibroblasts 24 h after scratch wounding. Cell migration in the scratched area was greatly reduced in the presence of CsA and/or bevacizumab. Dashed lines indicate the precise location of the scratch wound. White scale bar: 50 μm. (B) The migration rates were quantified in cells pretreated with CsA for 3 min or 10 min prior to incubation with bevacizumab for 24 h. Data represent the mean ± standard deviation of 3 separate experiments. ***P < 0.001 vs CsA. (C) Western blot analysis showed that expression of both MMPs was markedly reduced by combined bevacizumab and CsA treatment. (D) Band densities were analyzed using ImageJ, relative to the relevant β-actin band. Data represent the mean ± standard deviation of 3 separate experiments. ***P < 0.001 vs control; ^#P < 0.05, ^### P < 0.001 vs CsA; ^††P < 0.01, ^†††P < 0.001 vs bevacizumab.</p

Manganese Complex of Ethylene­diamine­tetraacetic Acid (EDTA)–Benzothiazole Aniline (BTA) Conjugate as a Potential Liver-Targeting MRI Contrast Agent

A novel manganese­(II) complex based on an ethylene­diamine­tetraacetic acid (EDTA) coordination cage bearing a benzothiazole aniline (BTA) moiety (Mn-EDTA-BTA) was designed and synthesized for use as a liver-specific MRI contrast agent with high chelation stability. In addition to forming a hydrophilic, stable complex with Mn²⁺, this new Mn chelate was rapidly taken up by liver hepatocytes and excreted by the kidneys and biliary system. The kinetic inertness and <i>R</i>₁ relaxivity of the complex were much higher than those of mangafodipir trisodium (MnDPDP), a clinically approved liver-specific MRI contrast agent. The diagnostic utility of this new Mn complex in MRI was demonstrated by high-sensitivity tumor detection in an animal model of liver cancer

Gadolinium Complex of 1,4,7,10-Tetraazacyclo­dodecane-1,4,7-trisacetic Acid (DO3A)–Ethoxybenzyl (EOB) Conjugate as a New Macrocyclic Hepatobiliary MRI Contrast Agent

We report the synthesis of a macrocyclic Gd chelate based on a 1,4,7,10-tetraazacyclo­dodecane-1,4,7-trisacetic acid (DO3A) coordinationn cage bearing an ethoxybenzyl (EOB) moiety and discuss its use as a <i>T</i>₁ hepatobiliary magnetic resonance imaging (MRI) contrast agent. The new macrocyclic liver agent shows high chelation stability and high <i>r</i>₁ relaxivity compared with linear-type Gd chelates, which are the current clinically approved liver agents. Our macrocyclic, liver-specific Gd chelate was evaluated in vivo through biodistribution analysis and liver MRI, which demonstrated its high tumor detection sensitivity and suggested that the new Gd complex is a promising contrast agent for liver cancer imaging