Targeting Atypical Protein Kinase C Isoforms for the Prevention of Blood-Retinal Barrier Dysfunction in Ophthalmic Disease
Open Access
- Author:
- Titchenell, Paul Michael
- Graduate Program:
- Physiology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- December 07, 2012
- Committee Members:
- Charles H Lang, Dissertation Advisor/Co-Advisor
Charles H Lang, Committee Chair/Co-Chair
Thomas E Spratt, Committee Member
David Antonetti, Special Member
Yuguang Shi, Committee Member
Christopher Martin Yengo, Committee Member - Keywords:
- vegf
drug discovery
PKC
blood-retinal barrier
vascular permeability
macular edema
phosphoproteomics
kinase inhibitor
TNF - Abstract:
- Blood-Retinal Barrier (BRB) dysfunction and subsequent macular edema (ME) are hallmarks of several ocular diseases including diabetic retinopathy (DR), age-related macular degeneration (AMD), uveitis, and retinal vein occlusions (RVO). Vascular endothelial growth factor (VEGF) is a main pathological driver of the vascular permeability and BRB abnormalities observed in these insults. Importantly, agents targeting VEGF are robust medical therapies that have revolutionized eye care. However, only 35% of patients improve vision while 60% have further loss vision loss prevented when treated with anti-VEGF therapies suggesting alternative factors are also able to induce BRB dysfunction. Tumor necrosis factor (TNF) contributes to retinal inflammation and is a candidate to mediate some of the deleterious effects observed in these diseases. Therefore, elucidating the downstream commonalities of VEGF and TNF signaling may provide an ideal point of therapeutic intervention. This dissertation tests the hypothesis that the atypical protein kinase C (aPKC) isoforms represent a common permeabilizing signaling node downstream of VEGF and TNF. Previously data from our laboratory defines an essential role for aPKC in TNF-induced retinal endothelial permeability. Building upon this observation, the contribution of aPKC to VEGF-induced retinal endothelial permeability was assessed. Here, VEGF treatment activated aPKC in both primary retinal endothelial cells and in the rodent retina. Furthermore, pharmacological and genetic manipulation of aPKC demonstrated that aPKC not only contributed to VEGF-induced permeability but also was sufficient to increase endothelial permeability. Novel small molecule inhibitors of aPKC were identified and were shown to prevent VEGF-induced retinal permeability in primary culture and in the rodent retina. Data in chapter 3 of the dissertation provides evidence that aPKC isoforms mediate VEGF-induced permeability and identifies novel small molecules inhibitors of aPKC isoforms and are effective anti- permeabilizing agents that warrant further investigation. Further characterization of the novel small molecule aPKC inhibitors was performed to define the structural requirements necessary for inhibitory activity. Detailed structure-activity relationships (SAR) were performed which delineated a novel pharmacophore required for inhibitory activity in two aPKC-dependent cell based assays that measure inflammation and vascular permeability. Data in chapter 4 provides the structural framework necessary for future medicinal chemistry efforts to solidify this chemotype as a clinically efficacious compound and identified a lead compound for further in vivo study (aPKC-I-diMeO). The contribution of aPKC isoforms to VEGF and TNF-induced BRB dysfunction and retinal edema was assessed using these newly identified small molecule inhibitors of aPKC. Importantly, both terminal and non-terminal assays to measure the extent VEGF/TNF-induced BRB dysfunction and retinal edema were utilized. Here, a nanomolar potent aPKC inhibitor (aPKC-I-diMeO) significantly prevented the combined permeabilizing effects of VEGF and TNF in primary endothelial culture and in vivo. The combined treatment of VEGF and TNF-induced diffuse leakage of fluorescently labeled albumin and increased retinal edema by 20% within 24 h in anesthetized rats. Strikingly, co-administration of aPKC-I-diMeO significantly prevented the VEGF/TNF-induced tight junction alterations and retinal vascular permeability in the rodent retina. Furthermore, aPKC inhibition prevented albumin leakage from retinal vessels and retinal edema as measured by fluorescent angiography and optical coherence tomography (OCT). Chapter 5 suggests targeting aPKC with a potent and specific small molecule inhibitor effectively prevents BRB dysfunction induced by VEGF/TNF in clinically relevant models. Finally, preliminary studies were performed to elucidate the VEGF-induced phosphoproteome and to identify novel substrates downstream of aPKC isoforms. Multiple VEGF-induced phosphosites were identified using phosphopeptide enrichment strategies coupled with isobaric relative and absolute quantitation (iTRAQ) tags. Furthermore, several of these phosphosites were dependent upon aPKC kinase activity and specifically associated with cytoskeletal and endocytosis regulation. The data described in this dissertation, taken together with published reports, strongly implicates aPKC in the regulation of both VEGF and TNF-induced BRB dysfunction and provides a novel point of therapeutic intervention. Multiple small molecule inhibitors of aPKC isoforms were identified and represent novel agents to prevent alterations in BRB integrity in ocular disease. Furthermore, these compounds should be applied to disease models of metabolic disease, cancer, and inflammation which all have implicated some degree of aberrant aPKC activity.