Intravitreal pharmacokinetic study of the anti-angiogenic glycoprotein opticin

Opticin is an endogenous vitreous glycoprotein that may have therapeutic potential as it has been shown that supra-normal concentrations supress preretinal neovascularisation. Herein we investigated the pharmacokinetics of opticin following intravitreal injection in rabbits. To measure simultaneously concentrations of human and rabbit opticin, a selected reaction monitoring mass spectrometry assay was developed. The mean concentration of endogenous rabbit opticin in 7 uninjected eyes was measured and found to be 19.2 nM or 0.62 µg/ml. When the vitreous was separated by centrifugation into a supernatant and collagen-containing pellet, 94 % of the rabbit opticin was in the supernatant. Intravitreal injection of human opticin (40 µg) into both eyes of rabbits was followed by enucleation at 5 h, 24 h, 72 h, 7 days, 14 days and 28 days post-injection (n=6 at each time point) and measurement of vitreous human and rabbit opticin concentrations in the supernatant and collagen-containing pellet following centrifugation. The volume of distribution of human opticin was calculated to be 3.31 ml and the vitreous half-life 4.2 days. Assuming that rabbit and human opticin are cleared from rabbit vitreous at the same rate, opticin is secreted into the vitreous at a rate of 0.14 µg/day. We conclude that intravitreally injected opticin has a vitreous half-life that is similar to currently available anti-angiogenic therapeutics. Whilst opticin was first identified bound to vitreous collagen fibrils, here we demonstrate that >90% of endogenous opticin is not bound to collagen. Endogenous opticin is secreted by the non-pigmented ciliary epithelium into the rabbit vitreous at a remarkably high rate and the turnover in vitreous is approximately 15% per day

Intravitreal administration of recombinant human opticin protects against hyperoxia-induced pre-retinal neovascularization

Opticin is an extracellular glycoprotein present in the vitreous. Its antiangiogenic properties offer the potential for therapeutic intervention in conditions such as proliferative diabetic retinopathy and retinopathy of prematurity. Here, we investigated the hypothesis that intravitreal administration of recombinant human opticin can safely protect against the development of pathological angiogenesis and promote its regression. We generated and purified recombinant human opticin and investigated its impact on the development and regression of pathological retinal neovascularization following intravitreal administration in murine oxygen-induced retinopathy. We also investigated its effect on normal retinal vascular development and function, following intravitreal injection in neonatal mice, by histological examination and electroretinography. In oxygen-induced retinopathy, intravitreal administration of human recombinant opticin protected against the development of retinal neovascularization to similar extent as aflibercept, which targets VEGF. Opticin also accelerated regression of established retinal neovascularization, though the effect at 18 h was less than that of aflibercept. Intravitreal administration of human recombinant opticin in neonatal mice caused no detectable perturbation of subsequent retinal vascular development or function. In summary we found that intraocular administration of recombinant human opticin protects against the development of pathological angiogenesis in mice and promotes its regression

PP1 loss impairs electrotaxis in HeLa cells.

<p><b>A.</b> Treatment of parental HeLa Tet-Off (HTO) cells with siRNA strongly depletes PP1 levels 48 h post transfection. Endogenous PP1 levels were visualized with PP1 antibodies that recognize all isoforms. <b>B.</b> Plot diagrams show that loss of PP1 impairs the ability of cells to migrate towards the cathode. Each line represents the migration trajectory of a single cell. The starting point for each cell migration track is at the origin. Cell tracks with end positions to the right appear in red (“C”, cathode) and those to the left appear in black (“A”, anode). EF-untreated cells were assayed as controls. Control siRNA cells migrate strongly towards the cathode; PP1 siRNA treated cells are unable to migrate in response to a DC EF. Scales show distance migrated in µm. <b>C.</b> PP1 depletion strongly reduces distance migrated, speed, and directedness in response to physiological DC EF. Error bars are S.E.M. <i>p</i> values for significant differences in distance, speed and directedness are shown. <b>D.</b> Localization of endogenous PP1 and distribution of filamentous-actin in control and PP1 depleted cells treated with DC EF. Endogenous PP1 levels were visualized with PP1 antibodies that recognize all isoforms (green) and polymerised actin was detected using rhodamine phalloidin (red). The nuclei have been stained with DAPI (blue). Arrows mark cells with a strong decrease in PP1 levels which correlate with defects in the formation of actin rich protrusions. Representative images are shown. Scale bar is 50 µm. <b>E.</b> Numbers of cells with filopodia were quantified by counting 100 cells. Error bars are S.E.M. <i>p</i> values for significant differences are shown. Images show a detail of cell protrusions in control siRNA and PP1 siRNA cells. Arrows mark numerous filopodia in control cells and outline areas with a major lack of filopodia at the cell edges in PP1 siRNA cells.</p

Cartoon showing the basic organization of the cervical epithelium and a mechanistic model to explain how PP1/NIPP1 may contribute to invasiveness of tumour cells.

<p>Cervical and vaginal epithelia have lumen potentials of about −25 to −50 mV <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040769#pone.0040769-Boskey1" target="_blank">[65]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040769#pone.0040769-Szatkowski1" target="_blank">[66]</a>. Such a lumen potential would correspond to a transepithelial voltage gradients of 1.7 V/cm (170 mV/mm). In these electrophysiological conditions cervical epithelial cells would migrate towards the lumen as they turn over the epithelial lining layer (green arrow). Upregulation of NIPP1 and its recruitment to PP1 would reverse migration into the lumen, encouraging invasion of the surrounding tissue (red arrow).</p

Effect of pharmacological inhibition of Cdc42-GTPase on the HTO cells.

<p><b>A.</b> Effect of ML141 on Cdc42 GTPase activity in unstimulated cells cultured in complete medium and in EF-stimulated HTO cells overexpressing the FLAG-NIPP1 protein variants. Levels of Cdc42-GTP determined by G-LISA in parental, W.T-NIPP1, ΔC-NIPP1 and mRATA cells in the absence or presence of DC EF and in cells pre-treated with 10 µM of ML141 before electrical stimulation. <i>p</i> values parental to W.T-NIPP1 and parental to ΔC-NIPP1 in complete medium were 0.1 and 0.01, respectively; <i>p</i> values comparing samples in the absence and presence of ML141 were in all cases <0.01. <b>B.</b> Cdc42 inhibition rescues cathodal polarisation and this correlates with centrosome positioning. Directedness values for the migration of EF-treated cells incubated with ML141. Cdc42 inhibition rescues the positive cell directedness decreased by W.T-NIPP1 overexpression. The strongly negative directedness value displayed by ΔC-NIPP1 cells becomes closer to 0 when cells are pretreated with Cdc42 inhibitor. For simplification directedness values in the absence of EF of the parental, W.T-NIPP1, ΔC-NIPP1, and mNIPP1 with and without ML141 have not been included in the diagram. These were, without ML141, −0.07±0.04; 0.05±0.09; −0.08±0.05 and −0.01±0.04, respectively; with ML141 were −0.07±0.04; 0.09±0.05; −0.07±0.05 and −0.01±0.04, respectively. In the absence of EF values were in all cases very close to 0 and differences between the four lines were not statistically significant in any of the cases. Data was quantified from at least three experiments. Error bars are S.E.M. <i>p</i> values for significant differences in directedness are shown. Polarisation index of centrosomes calculated as explained in materials and methods. Polarisation index of W.T-NIPP1 and ΔC-NIPP1 cells becomes similar to the polarisation index of parental cells when cells are treated with the Cdc42 inhibitor ML141.</p

List of genes from the Cdc42 pathway that are significantly upregulated by the overexpression of W.T-NIPP1 (WT) or ΔC-NIPP1 (ΔC), but not by mNIPP1 (m), in the HTO cells and compared to parental HTO cells.

<p>List of genes from the Cdc42 pathway that are significantly upregulated by the overexpression of W.T-NIPP1 (WT) or ΔC-NIPP1 (ΔC), but not by mNIPP1 (m), in the HTO cells and compared to parental HTO cells.</p

NIPP1 expression in HTO cells and control of EF-induced directional migration via its binding to PP1.

<p><b>A.</b> Cartoon of endogenous NIPP1 and the different FLAG-tagged NIPP1 variants expressed after doxycyclin removal in HeLa Tet-Off (HTO) cell lines. All three NIPP1 variants have a forkhead associated domain (FHA). The consensus PP1-binding sequence, RVTF in W.T-NIPP1 has been mutated to RATA in the FLAG-mNIPP1 variant. The C-terminal auto-inhibitory (ID) domain is not included in the FLAG tagged ΔC-NIPP1 protein, resulting in the expression of a constitutively active PP1/NIPP1 holoenzyme. <b>B.</b> Expression of NIPP1 variants confirmed by Western blotting after removal of doxycyclin. Cell lysates were analysed by SDS/PAGE and immunoblotting. Bands corresponding to the PP1 isoforms were detected and GAPDH was used as loading control. <b>C.</b> NIPP1 expression and localization in the HTO cells was confirmed by ICC in EF-treated and untreated HTO cells. Anti-FLAG antibody and rhodamine phalloidin have been used to detect the FLAG-tagged NIPP1 variants (green) and F-actin (red). The nuclei were stained with DAPI. Overexpressed NIPP1 localizes to the nucleus in EF-treated and untreated cells. Scale bar is 50 µm. Plot diagrams show that an EF of physiological strength (200 mV/mm) induced distinct migratory responses in the HTO cells expressing different NIPP1 variants. EF-untreated HTO cells are shown as controls. Migration trajectories were tracked for three hours in the absence and presence of EF. Each cell’s position at 0 h is positioned at the origin (0, 0). Cells whose end position is to the right are coloured red and those to the left appear in black. Cathode is marked as “C” and anode is marked as “A” when DC EF is applied to cells. Scales show distance migrated in µm. Note that scales are different among diagrams in order to include the tracks of every cell assayed.</p

Loss of the PP1 interactor NIPP1 impairs the electrotactic response of PC-3-M cells.

<p><b>A.</b> Treatment of PC-3-M cells with IPTG induces NIPP1 depletion. Cell lysates were analysed by SDS/PAGE and immunoblotting. Bands corresponding to the PP1 isoforms were detected and GAPDH was used as loading control. <b>B.</b> Plot diagrams show that loss of NIPP1 impairs the ability of PC-3-M cells to migrate anodally. Migration trajectories were tracked for three hours. The starting point for each cell migration track is at the origin. Cell tracks with end positions to the right appear in red and those to the left appear in black. Cathode is marked as “C” and anode as “A” when a DC EF is applied to cells. Control scrambled PC-3-M cells migrate strongly anodally (negative directedness value); cells expressing shRNA targeting NIPP1 show a much reduced anodal response. Scales show distance migrated in µm. Scales are different between diagrams in order to include the tracks of every cell assayed. <b>C.</b> NIPP1 depletion strongly reduced distance migrated and directedness in response to physiological DC EF. Data are from at least three experiments. Error bars are S.E.M. <i>p</i> values for significant differences in distance, speed and directedness are shown.</p