Doctor of Philosophy

dissertationDiabetes affects 25.8 million people in the United States and its prevalence is expected to triple in the next 20 years. Diabetic retinopathy (DR) affects nearly 30% of people with diabetes and is the leading cause of blindness in the working-age population. Current treatments for DR improve vision in only a minority of patients, and carry significant risks. This work advances a new approach that works by reversing retinal vascular damage and restoring normal perfusion to improve vision in this condition. Chapter 1 reviews the epidemiology, pathophysiology, and current standards of therapy for diabetic retinopathy. The roles of vascular maturation factor Angiopoietin-1 (Ang1) and its receptor Tie2 are introduced. Chapter 2 describes the development of an improved method for visualization of leukocytes in the diabetic mouse retina, which was critical for studies in this dissertation and broadly applicable to fields studying leukocyte endothelial interaction and inflammation. Chapter 3 focuses on the studies describing prevention of neurovascular dysfunction in diabetic retinopathy achieved by treating diabetic mice with gene therapy expressing COMP-Ang1. This chapter further details the studies performed to reverse diabetic retinopathy with a combination therapy consisting of endothelial colony-forming cells (ECFCs) and COMP-Ang1. We demonstrated that COMP-Ang1 enhanced the vasculogenic capabilities of ECFCs leading to increased integration into the diabetic retina and preservation of visual function in mice with advanced diabetic retinopathy. Chapter 4 represents my contributions toward the understanding of how targeting alternative VEGF receptor 2 splicing can suppress hemangiogenesis and lymphangiogenesis in the retina and choroid. This work was published in the FASEB journal in 2013. Chapter 5 describes my work published in PLoS ONE describing suppression of both tumor and ocular neovascularization, wherein we used morpholinos to increased soluble VEGF receptor 1. Chapter 6 concludes this work by recapping how the work accomplished in this dissertation built off of previous discoveries. The Appendix describes studies initiated to test the effects of COMP-Ang1 in an acute model of retinal ischemia, central retinal artery occlusion

Morpholino-Mediated Increase in Soluble Flt-1 Expression Results in Decreased Ocular and Tumor Neovascularization

BACKGROUND: Angiogenesis is a key process in several ocular disorders and cancers. Soluble Flt-1 is an alternatively spliced form of the Flt-1 gene that retains the ligand-binding domain, but lacks the membrane-spanning and intracellular kinase domains of the full-length membrane bound Flt-1 (mbFlt-1) protein. Thus, sFlt-1 is an endogenous inhibitor of VEGF-A mediated angiogenesis. Synthetic mopholino oligomers directed against splice site targets can modulate splice variant expression. We hypothesize that morpholino-induced upregulation of sFlt-1 will suppress angiogenesis in clinically relevant models of macular degeneration and breast cancer. METHODS AND FINDINGS: In vivo morpholino constructs were designed to target murine exon/intron 13 junction of the Flt-1 transcript denoted VEGFR1_MOe13; standard nonspecific morpholino was used as control. After nucleofection of endothelial and breast adenocarcinoma cell lines, total RNA was extracted and real-time RT-PCR performed for sFlt-1 and mbFlt-1. Intravitreal injections of VEGFR1_MOe13 or control were done in a model of laser-induced choroidal neovascularization and intratumoral injections were performed in MBA-MD-231 xenografts in nude mice. VEGFR1_MOe13 elevated sFlt-1 mRNA expression and suppressed mbFlt-1 mRNA expression in vitro in multiple cellular backgrounds (p<0.001). VEGFR1_MOe13 also elevated sFlt/mbFlt-1 ratio in vivo after laser choroidal injury 5.5 fold (p<0.001) and suppressed laser-induced CNV by 50% (p = 0.0179). This latter effect was reversed by RNAi of sFlt-1, confirming specificity of morpholino activity through up-regulation of sFlt-1. In the xenograft model, VEGFR1_MOe13 regressed tumor volume by 88.9%, increased sFlt-1 mRNA expression, and reduced vascular density by 50% relative to control morpholino treatment (p<0.05). CONCLUSIONS: Morpholino oligomers targeting the VEGFR1 mRNA exon/intron 13 junction promote production of soluble FLT-1 over membrane bound FLT-1, resulting in suppression of lesional volume in laser induced CNV and breast adenocarcinoma. Thus, morpholino manipulation of alternative splicing offers translational potential for therapy of angiogenic disorders

Early Visual Changes Portend Visual Loss in a Case of Fludarabine Toxicity

Visual loss after fludarabine is a rare side effect. Here, we report a case of early visual changes associated with initiation of fludarabine treatment followed by rapid vision loss four weeks later

VEGFR1_MOe13 increases sFLT-1 and decreases mbFLT-1 mRNA in MCF-7 and MBA-MD-231 breast adenocarcinoma cell lines.

<p>MCF7 or MBA-MD-231 human breast adenocarcinoma cells were electroporated with VEGFR1_MOe13 and (a; c) sFLT-1 and (b; d) mbFLT-1 mRNA levels assessed at 72 hours using real time PCR (n = 3). Data were normalized to GAPDH mRNA levels and normal MCF7 or MBA-MD-231 cells were used a 1.0. *p<0.01.</p

VEGFR1_MOe13 localizes to the nucleus and increases sFLT-1 expression in human endothelial vein cells (HUVEC).

<p>(a) Fluorescently tagged VEGFR1_MOe13 (F-MO) or standard morpholino (std-MO) were electroporated into HUVECs. After 48 hours fluorescence was assessed using light microscopy. Colocalization with DAPI staining represents nuclear localization of morpholino constructs. HUVECs were electroporated with VEGFR1_MOe13, VEGFR1_MOi13, a combination of VEGFR1_MOe13 and VEGFR1_MOi13, Standard_MO. All morpholino sequences were designed to target the human VEGFR1 transcript. (b) mbFLT-1 mRNA (n = 6) or (c) sFLT-1 mRNA expression (n = 6) were assessed using real time PCR. Values were normalized to GAPDH mRNA and normal HUVEC was used as 1.0. (d) sFLT protein expression in culture medium was determined by ELISA (n = 3). Data shows sFLT protein at 96 h – 48 h. Error bar is S.E.M. Each p-value was calculated by two-tail student's t-test against normal HUVEC.</p

VEGFR1_MOe13 inhibits laser-induced CNV in vivo.

<p>a. sFlt/mFlt mRNA ratio in the retina treated with PBS, Standard_MO and VEGFR1_MOe13 (n = 4). Representative images of laser CNV injected with b PBS, c Standard_MO and d VEGFR1_MOe13 designed to target the murine VEGFR1 transcript. e The averages of laser CNV volumes (n = 11–14). Error bar is S.E.M. p-values were calculated by two-tail student's t test.</p

Intra-tumoral VEGFR1_MOe13 injection results in regression of established MBA-MD-231 human breast adenocarcinoma xenograft tumors and decreased tumor vascularity.

<p>a. MB-MDA-231 breast cancer cells were grown as xenografts in female nude mice for 2 weeks prior to beginning treatment with either a standard morpholino or VEGFR1_MOe13 designed to target the murine VEGFR1 transcript. Morpholino treated tumors demonstrate size regression after a 4 week treatment course as demonstrated by volume change analysis of 5 individual tumors within each treatment condition. p = 0.04 b–c. Following treatment course, RNA was extracted from xenograft tumors and soluble or membrane bound Flt1 levels were measured using real-time RT-PCR. Error bars indicate variation between individual PCR reactions per tumor sample. d. Following the treatment course, VEGFR1_MOe13 or standard morpholino xenograft tumors were sectioned and stained with isolectin as a measure of vascularity. Individual bars represent sections analyzed thoughout each tumor sample.</p

Acridine orange leukocyte fluorography in mice

Simultaneous non-invasive visualization of blood vessels and nerves in patients can be obtained in the eye. The retinal vasculature is a target of many retinopathies. Inflammation, readily manifest by leukocyte adhesion to the endothelial lining, is a key pathophysiological mechanism of many retinopathies, making it a valuable and ubiquitous target for disease research. Leukocyte fluorography has been extensively used in the past twenty years; however, fluorescent markers, visualization techniques, and recording methods have differed between studies. The lack of detailed protocol papers regarding leukocyte fluorography, coupled with lack of uniformity between studies, has led to a paucity of standards for leukocyte transit (velocity, adherence, extravasation) in the retina. Here, we give a detailed description of a convenient method using acridine orange (AO) and a commercially available scanning laser ophthalmoscope (SLO, HRA-OCT Spectralis) to view leukocyte behavior in the mouse retina. Normal mice are compared to mice with acute and chronic inflammation. This method can be readily adopted in many research labs