Corneal neovascularization is a sight-threatening condition caused by angiogenesis in the normally avascular cornea. Neovascularization of the cornea is often associated with an inflammatory response, thus targeting VEGF-A alone yields only a limited efficacy. The NF-κB signaling pathway plays important roles in inflammation and angiogenesis. Here, we study consequences of the inhibition of NF-κB activation through selective blockade of the IKK complex IκB kinase β (IKK2) using the compound IMD0354, focusing on the effects of inflammation and pathological angiogenesis in the cornea. In vitro, IMD0354 treatment diminished HUVEC migration and tube formation without an increase in cell death and arrested rat aortic ring sprouting. In HUVEC, the IMD0354 treatment caused a dose-dependent reduction in VEGF-A expression, suppressed TNFα-stimulated expression of chemokines CCL2 and CXCL5, and diminished actin filament fibers and cell filopodia formation. In developing zebrafish embryos, IMD0354 treatment reduced expression of Vegf-a and disrupted retinal angiogenesis. In inflammation-induced angiogenesis in the rat cornea, systemic selective IKK2 inhibition decreased inflammatory cell invasion, suppressed CCL2, CXCL5, Cxcr2, and TNF-α expression and exhibited anti-angiogenic effects such as reduced limbal vessel dilation, reduced VEGF-A expression and reduced angiogenic sprouting, without noticeable toxic effect. In summary, targeting NF-κB by selective IKK2 inhibition dampened the inflammatory and angiogenic responses in vivo by modulating the endothelial cell expression profile and motility, thus indicating an important role of NF-κB signaling in the development of pathologic corneal neovascularization.

Blinding eye diseases such as corneal neovascularization, proliferative diabetic retinopathy, and age-related macular degeneration are driven by pathological angiogenesis. In cancer, angiogenesis is key for tumor growth and metastasis. Current antiangiogenic treatments applied clinically interfere with the VEGF signaling pathway—the main angiogenic pathway—to inhibit angiogenesis. These treatments are, however, only partially effective in regressing new pathologic vessels, and the disease relapses following cessation of treatment. Moreover, the relapse of pathological angiogenesis can be rapid, aggressive and more difficult to treat than angiogenesis in the initial phase. The manner in which relapse occurs is poorly understood; however, recent studies have begun to shed light on the mechanisms underlying the revascularization process. Hypotheses have been generated to explain the rapid angiogenic relapse and increased resistance of relapsed disease to treatment. In this context, the present review summarizes knowledge of the various mechanisms of disease relapse gained from different experimental models of pathological angiogenesis. In addition, the basement membrane—a remnant of regressed vessels—is examined in detail to discuss its potential role in disease relapse. Finally, approaches for gaining a better understanding of the relapse process are discussed, including prospects for the management of relapse in the context of disease.

To evaluate conjunctival cell microRNA (miRNAs) and mRNA expression in relation to observed phenotype of progressive limbal stem cell deficiency in a cohort of subjects with congenital aniridia with known genetic status. Using impression cytology, bulbar conjunctival cells were sampled from 20 subjects with congenital aniridia and 20 age and sex-matched healthy control subjects. RNA was extracted and miRNA and mRNA analyses were performed using microarrays. Results were related to severity of keratopathy and genetic cause of aniridia. Of 2549 miRNAs, 21 were differentially expressed in aniridia relative to controls (fold change ≤ -1.5 or ≥ +1.5). Among these miR-204-5p, an inhibitor of corneal neovascularization, was downregulated 26.8-fold in severely vascularized corneas. At the mRNA level, 539 transcripts were differentially expressed (fold change ≤ -2 or ≥ +2), among these FOSB and FOS were upregulated 17.5 and 9.7-fold respectively, and JUN by 2.9-fold, all being components of the AP-1 transcription factor complex. Pathway analysis revealed enrichment of PI3K-Akt, MAPK, and Ras signaling pathways in aniridia. For several miRNAs and transcripts regulating retinoic acid metabolism, expression levels correlated with keratopathy severity and genetic status. Strong dysregulation of key factors at the miRNA and mRNA level suggests that the conjunctiva in aniridia is abnormally maintained in a pro-angiogenic and proliferative state, and these changes are expressed in a PAX6 mutation-dependent manner. Additionally, retinoic acid metabolism is disrupted in severe, but not mild forms of the limbal stem cell deficiency in aniridia.

Digital holographic microscopy (DHM) has emerged as a powerful non-invasive tool for cell analysis. It has the capacity to analyse multiple parameters simultaneously, such as cell- number, confluence and phase volume. This is done while cells are still adhered and growing in their culture flask. The aim of this study was to investigate whether DHM was able to monitor drug-induced cell cycle arrest in cultured cells and thus provide a non-disruptive alternative to flow cytometry. DHM parameters from G1 and G2/M cell cycle arrested L929 mouse fibroblast cells were collected. Cell cycle arrest was verified with flow cytometry. This study shows that DHM is able to monitor phase volume changes corresponding to either a G1 or G2/M cell cycle arrest. G1-phase arrest with staurosporine correlated with a decrease in the average cell phase volume and G2/M-phase arrest with colcemid and etoposide correlated with an increase in the average cell phase volume. Importantly, DHM analysis of average cell phase volume was of comparable accuracy to flow cytometric measurement of cell cycle phase distribution as recorded following dose-dependent treatment with etoposide. Average cell phase volume changes in response to treatment with cell cycle arresting compounds could therefore be used as a DHM marker for monitoring cell cycle arrest in cultured mammalian cells.

Age-related macular degeneration (AMD) is the most common, progressive, and polygenic cause of irreversible visual impairment in the world. The molecular pathogenesis of the primary events of AMD is poorly understood. We have investigated a transcriptome-wide analysis of differential gene expression, single-nucleotide polymorphisms (SNPs), indels, and simple sequence repeats (SSRs) in datasets of the human peripheral retina and RPE-choroid-sclera control and AMD. Adaptors and unbiased components were removed and checked to ensure the quality of the data sets. Molecular function, biological process, cellular component, and pathway analyses were performed on differentially expressed genes. Analysis of the gene expression datasets identified 5011 upregulated genes, 11,800 downregulated genes, 42,016 SNPs, 1141 indels, and 6668 SRRs between healthy controls and AMD donor material. Enrichment categories for gene ontology included chemokine activity, cytokine activity, cytokine receptor binding, immune system process, and signal transduction respectively. A functional pathways analysis identified that chemokine receptors bind chemokines, complement cascade genes, and create cytokine signaling in immune system pathway genes (p value < 0.001). Finally, allele-specific expression was found to be significant for Chemokine (C-C motif) ligand (CCL) 2, 3, 4, 13, 19, 21; C-C chemokine receptor (CCR) 1, 5; chemokine (C-X-C motif) ligand (CXCL) 9, 10, 16; C-X-C chemokine receptor type (CXCR) 6; as well as atypical chemokine receptor (ACKR) 3,4 and pro-platelet basic protein (PPBP). Our results improve our overall understanding of the chemokine receptors’ signaling pathway in AMD conditions, which may lead to potential new diagnostic and therapeutic targets.