Fig 2: Conceptual summary of proposed cellular pathways affected by specific NF-?B blockade in the context of inflammation and angiogenesis, based on the current findings. a Endothelial cell (EC) is exposed to inflammatory stimulus TNF-a, which binds to TNFR1 and triggers a signal transduction resulting in phosphorylation of I?Ba by IKK2; I?Ba is ubiquitinated and degraded; this process enables the nuclear translocation of NF-?B. Upon nuclear translocation, NF-?B up-regulates a variety of pro-inflammatory and pro-angiogenic genes, including TNF-a, CXCL5, CCL2, and HIF-1a. TNF-a has auto- and paracrine effects and activates NF-kB through positive feedback, which amplifies the inflammatory response. CCL2 and CXCL5 have chemotactic effects on monocytes and neutrophils that in turn secrete a variety of cytokines, including TNF-a and VEGF. Nuclear NF-?B also up-regulates HIF-1a and by this mechanism can directly increase VEGF production. VEGF, in turn, acts on the EC, affecting actin polymerization, cytoskeleton composition, cell motility, tube formation and sprouting angiogenesis. b Selective IKK2 inhibition by IMD-0354 inhibits I?Ba phosphorylation by IKK2 disrupting NF-?B activation and nuclear translocation. The pro-inflammatory and pro-angiogenic cytokine and chemokine production are substantially diminished. With decreased levels of VEGF, actin cytoskeleton formation, EC motility and migration are all suppressed. The inhibition of NF-kB also reduces the inflammatory response through suppression of the transcription of pro-inflammatory genes

Fig 3: Histology and immunofluorescence in rat corneal sections. a Hematoxylin and Eosin (H&E), b VEGF-A (green); c CCL2 (green); d TNF-a (green); e CXCL5 (green); f CD45 (green) and g HIF-1a (green) staining in rat cornea tissue. Nuclear counterstaining by DAPI (blue) in fluorescent images

Fig 4: Confirmation of regulation of selected genes at 24 suture IN and OUT, by qPCR and immunohistochemistry.Gene expression confirmation by qPCR, and protein level expression by immunohistochemistry. Panel (a) indicates gene expression by qPCR, (b) indicates the microarray gene expression and (c) is the protein expression in the tissue for Vegfa, Cxcl5, Fgf7 and Rasa2, respectively. For qPCR, ANOVA p-values were 0.006, <0.001, <0.001 and 0.009, for Vegfa, Cxcl5, Fgf7 and Rasa2, respectively. Pairwise significance between groups with p < 0.05 is indicated by asterisks. For microarray, p-values between suture IN and suture OUT were 4.49E-04, 3.89E-04, 3.66E-04 and 8.56E-03, for Vegfa, Cxcl5, Fgf7 and Rasa2, respectively. The asterisks represent a significant difference p < 0.05 between groups. Error bars for qPCR and microarray represent standard deviation. Microarray data represents 4 corneas/group and qPCR data 3 corneas/group with 2 technical replicates/sample.

Fig 5: Quantitative qRT-PCR and Western blot analysis of inflammatory and neovascular factors in suture-stimulated rat corneas at 96 h. Quantitative qRT-PCR analysis of a Vegf-a, b Cxcl5, c Ccl2, and d Cxcr2 expression in rat cornea. (n = 5) One-way ANOVA test with Tukey multiple comparisons were used to determine statistical significance. e Western blot analysis of CD45, phospho-I?Ba and VEGF-A expression in rat cornea lysate. ß-Actin used as a loading control. n.s. p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001