Human Platelets Take up Anti-VEGF Agents

Purpose. Growing evidence suggests different systemic exposure of anti-vascular endothelial growth factor (anti-VEGF) agents with repeated intravitreal application. Since the penetration of anti-VEGF agents through vascular barrier was reported, the interaction of anti-VEGF with nonresident platelets has become a topic of interest. The purpose of this study was to evaluate, with the help of visualization techniques, whether platelets take up the anti-VEGF agents ranibizumab, aflibercept, and bevacizumab. Methods. The uptake of anti-VEGF agents with or without VEGF treatment was investigated using immunofluorescence and immunogold staining in human platelets. The role of actin filaments and clathrin-coated vesicles in the transport of ranibizumab, aflibercept, and bevacizumab was evaluated by two pharmacologic inhibitors: staurosporine (protein kinase C inhibitor) and cytochalasin D. Results. All three anti-VEGF agents were taken up by platelets and colocalized with VEGF. Ranibizumab and aflibercept were mainly detected in alpha-granules; however, bevacizumab was equally localized in alpha-granules and in platelet vesicles. Both staurosporine and cytochalasin D completely inhibited the uptake of aflibercept into platelets. Both pharmacological inhibitors also decreased the transport of ranibizumab and bevacizumab into platelets. Bevacizumab was significantly more frequently colocalized within clathrin-coated vesicles than ranibizumab and aflibercept. Conclusion. All three anti-VEGF agents are taken up by platelets and internalized in alpha-granules, which may result in a higher local exposure of anti-VEGF after the activation of platelets, potentially contributing to arterial thromboembolic events. Clathrin-coated vesicles seem to be more prominent in the transport of bevacizumab than ranibizumab and aflibercept. Nevertheless, whether the different localization and transport of bevacizumab are truly related to specific differences of receptor-mediated endocytosis has to be revealed by further research.

Formulation of Liver-Specific PLGA-DY-635 Nanoparticles Loaded with the Protein Kinase C Inhibitor Bisindolylmaleimide I

Bisindolylmaleimide I (BIM-I) is a competitive pan protein kinase C inhibitor with anti-inflammatory and anti-metastatic properties, suggested to treat inflammatory diseases and various cancer entities. However, despite its therapeutic potential, BIM-I has two major drawbacks, i.e., it has a poor water solubility, and it binds the human ether-à-go-go-related gene (hERG) ion channels, potentially causing deadly arrhythmias. In this case, a targeted delivery of BIM-I is imperative to minimize peripheral side effects. To circumvent these drawbacks BIM-I was encapsulated into nanoparticles prepared from poly(lactic-co-glycolic acid) (PLGA) functionalized by the near-infrared dye DY-635. DY-635 served as an active targeting moiety since it selectively binds the OATP1B1 and OATP1B3 transporters that are highly expressed in liver and cancer cells. PLGA-DY-635 (BIM-I) nanoparticles were produced by nanoprecipitation and characterized using dynamic light scattering, analytical ultracentrifugation, and cryogenic transmission electron microscopy. Particle sizes were found to be in the range of 20 to 70 nm, while a difference in sizes between the drug-loaded and unloaded particles was observed by all analytical techniques. In vitro studies demonstrated that PLGA-DY-635 (BIM-I) NPs prevent the PKC activation efficiently, proving the efficacy of the inhibitor after its encapsulation, and suggesting that BIM-I is released from the PLGA-NPs. Ultimately, our results present a feasible formulation strategy that improved the cytotoxicity profile of BIM-I and showed a high cellular uptake in the liver as demonstrated in vivo by intravital microscopy investigations.

Abstract 17473: GRK2 in the Mitochondria: Uncovering Novel Mechanisms in Heart Failure

Introduction: During heart failure, levels and activity of G protein-coupled receptor kinase 2 (GRK2) increase. GRK2 is canonically studied in the phosphorylation of GPCRs and β-adrenergic desensitization. Noncanonical activities of GRK2 are being uncovered, however. Our lab has recently discovered that in cardiac myocytes, GRK2 translocates to the mitochondria ( mtGRK2 ) following injury and is associated with negative effects on metabolism and cell survival. Hypothesis: GRK2 plays a role in regulating mitochondrial function following cardiac stress and contributes to HF pathogenesis in a novel manner, by interacting with a novel group of mitochondrial proteins involved in pro-death signaling, bioenergetics and substrate utilization. Methods: Mitochondrial translocation of GRK2 was validated with either protein kinase C inhibitor (chelerythine) administration or hypoxia/reoxygenation stress in primary neonatal rat ventricular myocytes or a cardiac-like cell line. Immunoprecipitation of the GRK2 interactome basally and under stress conditions was conducted endogenously in vitro, in vivo , and with purified recombinant GRK2 peptides. Proteins were separated via SDS-PAGE and potential binding partners were identified by mass spectroscopy (LCMS) and proteomics analysis conducted with Ingenuity Pathway (IPA; Qiagen) software to determine which partners in the GRK2 interactome were potentially involved in mitochondrial dysfunction. Results: Subunits of Complexes I, II, IV and V of the electron transport chain were identified as potential mtGRK2 interacting partners. Several mtGRK2-ETC interactions were increased following oxidative stress-induced translocation of GRK2. Finally, mtGRK2 appears to phosphorylate some of the interactome partners identified in mitochondrial dysfunction. Conclusions: The phosphorylation of subunits of the ATP synthesis machinery by mtGRK2, or other mechanisms of interaction between these proteins, may be regulating some of the phenotypic effects of HF previously observed by our lab, such as increased ROS production and reduced fatty acid metabolism. Further research is essential to elucidate the novel role of GRK2 in regulating mitochondrial bioenergetics and cell death in failing hearts.

Role of Glycine Receptor α3 in the Phosphorylation of Extracellular Signal-Regulated Kinase by Prostaglandin E2

Background: Glycine receptors (GlyRs) play a key role in the processing of inflammatory pain. We used Adeno-associated virus (AAV) for GlyRα1/3 gene transfer in F11 neuron cells, and investigated the effects and roles of pAAV-GlyRα1/3 on cell cytotoxicity and the prostaglandin E2 (PGE2)-mediated inflammatory response. Methods: pAAV-GlyRα1 and pAAV-GlyRα3 recombinant vectors were constructed, and cell viability was measured following pAAV-GlyRα1/3 transfection. The activation of mitogen-activated protein kinase (MAPK) inflammatory signaling and neuronal injury marker activating transcription factor 3 (ATF-3) were evaluated by western blotting; the level of cytokine expression was measured by ELISA.Results: We found that pAAV/pAAV-GlyRα1/3 transfection slightly, but not significantly, increased cell viability and induced extracellular signal-regulated kinase (ERK1/2) phosphorylation and ATF-3 activation. However, the transfection reagent lipofectamine significantly increased cell death and induced ERK1/2 phosphorylation and ATF-3 activation. More importantly, the PGE2-induced ERK1/2 phosphorylation in F11 cells was repressed by the expression of pAAV-GlyRα3 and administration of an EP2 inhibitor (PF-04418948), GlyRαs antagonist (strychnine), and protein kinase C inhibitor (G06983).Conclusions: PGE2-induced ERK1/2 phosphorylation can be modulated by GlyRα3. In addition, no changes in the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, or IL-6 of the F11 cells were observed 6 hours after lipopolysaccharide (LPS) or complete Freund's adjuvant (CFA) treatment. These data suggest that delivering pAAV-GlyRα3 into neuron cells should be safe for downregulating PGE2-induced ERK1/2 phosphorylation and inflammation.

Circulating tumor DNA (ctDNA) in patients (pts) with metastatic uveal melanoma (UM) treated with protein kinase C inhibitor (PKCi).

e22054 Background: UM is the most common primary intraocular malignancy. About 50% of pts with UM will develop metastatic disease and currently there is no effective treatment in metastatic UM. Nearly 95% of UM harbour mutually exclusive activating mutations in GNAQ, GNA11, CYSTLR2 and PLCb4. We sought to evaluate ctDNA in metastatic UM pts receiving experimental early phase clinical trial of LXS196, a PKCi, using digital droplet PCR (ddPCR) and targeted ion torrent next generation sequencing (NGS). Methods: 17 pts with metastatic UM were identified from a single institution. Pt characteristics including mutation status, disease volume using sum of product of disease (SPOD), best response and clinical benefit defined as partial response (PR) or stable disease (SD) > 6 months were assessed. Plasma samples at baseline and early on treatment (EOT) (14 – 30 days on treatment) were analysed for ctDNA using mutation specific ddPCR. A custom made NGS panel covering 95% of UM mutations was used on plasma ctDNA samples at baseline and on treatment. The allele frequency (AF) of activating mutations identified using ion torrent NGS analysis were then validated using ddPCR. Results: Using ddPCR, 16/17 pts had a +ve ctDNA at baseline with baseline ctDNA copies correlating with LDH (n = 17, p = < 0.001, Spearman’s rank r = 0.8015) and SPOD (n = 17, p = 0.005, r = 0.6642). 16/17 pts had paired samples at baseline and EOT and only 4/16 pts were undetectable at EOT. These 4 pts were GNA11 Q209L positive, had below median SPOD (median 5986, range 200 – 16782), and low numbers of liver metastases (median 9, range 1 – 49). Of these 4 pts, one had PR and three had SD (two with SD > 6 months) as best response. A further 8 pts had +ve ctDNA at baseline and showed a reduction in EOT (reduction range 46 – 99%). Of these 8 pts, one had PR, four had SD (two with SD > 6 months) and three had progressive disease (PD) as best response. The remaining 4 pts showed increasing ctDNA from baseline to EOT and all had SD/PD. Using ROC analysis, EOT ctDNA predicted clinical benefit to PKCi (AUC 0.84, [95% confidence interval, 0.65-1.0, p = 0.026]). AF of ion torrent NGS correlated significantly with ddPCR AF (n = 30, p = < 0.001, r = 0.968). Ion torrent NGS was able to detect additional mutations implicated in UM prognosis including SF3B1 mutations in 4 pts. Conclusions: Baseline ctDNA correlates with baseline LDH level and disease volume. EOT ctDNA predicted clinical benefit to PKCi. The ctDNA AF derived from ddPCR and NGS was comparable and targeted ion torrent NGS was useful in detecting driver as well as additional mutations in UM.