Lack of Involvement of CEP Adducts in TLR Activation and in Angiogenesis

<div><p>Proteins that are post-translationally adducted with 2-(ω-carboxyethyl)pyrrole (CEP) have been proposed to play a pathogenic role in age-related macular degeneration, by inducing angiogenesis in a Toll Like Receptor 2 (TLR2)-dependent manner. We have investigated the involvement of CEP adducts in angiogenesis and TLR activation, to assess the therapeutic potential of inhibiting CEP adducts and TLR2 for ocular angiogenesis. As tool reagents, several CEP-adducted proteins and peptides were synthetically generated by published methodology and adduction was confirmed by NMR and LC-MS/MS analyses. Structural studies showed significant changes in secondary structure in CEP-adducted proteins but not the untreated proteins. Similar structural changes were also observed in the treated unadducted proteins, which were treated by the same adduction method except for one critical step required to form the CEP group. Thus some structural changes were unrelated to CEP groups and were artificially induced by the synthesis method. In biological studies, the CEP-adducted proteins and peptides failed to activate TLR2 in cell-based assays and in an <i>in vivo</i> TLR2-mediated retinal leukocyte infiltration model. Neither CEP adducts nor TLR agonists were able to induce angiogenesis in a tube formation assay. <i>In vivo</i>, treatment of animals with CEP-adducted protein had no effect on laser-induced choroidal neovascularization. Furthermore, <i>in vivo</i> inactivation of TLR2 by deficiency in Myeloid Differentiation factor 88 (Myd88) had no effect on abrasion-induced corneal neovascularization. Thus the CEP-TLR2 axis, which is implicated in other wound angiogenesis models, does not appear to play a pathological role in a corneal wound angiogenesis model. Collectively, our data do not support the mechanism of action of CEP adducts in TLR2-mediated angiogenesis proposed by others.</p></div

Retinal Leukocyte Infiltration Assay.

<p>A) Mice were injected intraperitoneally with either PBS, Pam3CSK4 (25 µg per animal, in PBS), or dipeptide-CEP (400 µg per animal, in PBS) and the retinas were analyzed 8 hours later. Retinal infiltration by neutrophils (Gr1+ cells) or macrophages (F4/80+ cells) was assessed by immunostaining with the respective markers and quantitated with Axiovision, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111472#s4" target="_blank">Materials and Methods</a>. Statistical analysis was performed using the Student t-test. Only statistically significant differences are indicated in the graph. B) Shown are representative images of the experiment in <b>Figure 3A</b>. Arrows indicate examples of macrophages or neutrophils in the corresponding images. <i>CEP</i>, Dipeptide-CEP; <i>Pam3</i>, Pam3CSK4.</p

Structural Analyses of CEP Adducts.

<p>A) <i>SDS-PAGE analysis</i>. Aliquots of HSA-CTL1 (untreated), HSA-CTL2 (treated but unadducted) and HSA-CEP were subjected to reducing SDS-PAGE on 4–10% gels. Compared to HSA-CTL1, both HSA-CTL2 and HSA-CEP showed an increase in high-MW bands. B) <i>Size exclusion chromatography</i>. SEC under non-denaturing conditions indicated an increase in faster-eluting peaks in HSA-CTL2 and HSA-CEP compared to HSA-CTL1. C) <i>Circular dichroism</i>. CD analysis revealed a loss of secondary structure in HSA-CTL2 and HSA-CEP compared to HSA-CTL1.</p

Tube Formation Assay.

<p>Shown are representative images of the experiments in presented numerically in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111472#pone-0111472-t001" target="_blank"><b>Table 1</b></a><b>.</b> The figure shows images for untreated negative control (untreated), positive control (VEGF 165, 4 ng/mL), HSA-CEP (2 µg/mL), Pam3CSK4 (500 nM), LPS (10 ng/mL), poly (I:C) (10 µg/mL). The arrow in the untreated image shows an example of an island of unmigrated HUVEC cells, which is also seen in other images.</p

Evaluation of CEP Adducts and TLR Agonists in the Tube Formation Assay.

<p>Evaluation of CEP Adducts and TLR Agonists in the Tube Formation Assay.</p

Mouse Laser-Induced CNV Assay.

<p>(A) Subretinal injection of MSA-CEP does not increase CNV area compared to mice injected with saline or MSA-CTL2. Bar graph shows mean area of CNV +/− SEM from first experiment evaluating the effect of subretinal injection of saline, 0.5 µg of rhVEGF165, 3.8 µg of MSA-CTL2, 3.8 µg of MSA-CEP, or 6.6 µg of 4G3 (an anti-mVEGF antibody) on laser-induced CNV in C57BL/6J mice. The number above each bar is the percentage inhibition relative to average CNV area in mice injected with MSA-CTL2. Subretinal injection of VEGF increases CNV area and subretinal injection of an anti-mVEGF antibody inhibits CNV area. * p<0.05, **** p<0.0001 by ANOVA with a Dunnett’s post hoc analysis. (B) Representative fluorescent images of CNV lesions 7 days after laser from mice injected in the subretinal space with MSA-CEP, MSA-CTL2, VEGF or a VEGF Antibody as described above. Scale bar = 100 microns. <i>CTL2</i>, MSA-CTL2; <i>CEP</i>, MSA-CEP.</p

The Discovery of <i>N</i>‑(1-Methyl-5-(trifluoromethyl)‑1<i>H</i>‑pyrazol-3-yl)-5-((6- ((methylamino)methyl)pyrimidin-4-yl)oxy)‑1<i>H</i>‑indole-1-carboxamide (Acrizanib), a VEGFR‑2 Inhibitor Specifically Designed for Topical Ocular Delivery, as a Therapy for Neovascular Age-Related Macular Degeneration

A noninvasive topical ocular therapy for the treatment of neovascular or “wet” age-related macular degeneration would provide a patient administered alternative to the current standard of care, which requires physician administered intravitreal injections. This manuscript describes a novel strategy for the use of in vivo models of choroidal neovascularization (CNV) as the primary means of developing SAR related to efficacy from topical administration. Ultimately, this effort led to the discovery of acrizanib (LHA510), a small-molecule VEGFR-2 inhibitor with potency and efficacy in rodent CNV models, limited systemic exposure after topical ocular administration, multiple formulation options, and an acceptable rabbit ocular PK profile