Inflammation plays an important role in pathological angiogenesis. Receptor-interacting protein 1 (RIP1) is highly expressed in inflammatory cells and is known to play an important role in the regulation of apoptosis, necroptosis, and inflammation; however, a comprehensive description of its role in angiogenesis remains elusive. Here, we show that RIP1 is abundantly expressed in infiltrating macrophages during angiogenesis, and genetic or pharmacological inhibition of RIP1 kinase activity using kinase-inactive RIP1K45A/K45A mice or necrostatin-1 attenuates angiogenesis in laser-induced choroidal neovascularization, Matrigel plug angiogenesis, and alkali injury-induced corneal neovascularization in mice. The inhibitory effect on angiogenesis is mediated by caspase activation through a kinase-independent function of RIP1 and RIP3. Mechanistically, infiltrating macrophages are the key target of RIP1 kinase inhibition to attenuate pathological angiogenesis. Inhibition of RIP1 kinase activity is associated with caspase activation in infiltrating macrophages and decreased expression of proangiogenic M2-like markers but not M1-like markers. Similarly, in vitro, catalytic inhibition of RIP1 down-regulates the expression of M2-like markers in interleukin-4–activated bone marrow-derived macrophages, and this effect is blocked by simultaneous caspase inhibition. Collectively, these results demonstrate a nonnecrotic function of RIP1 kinase activity and suggest that RIP1-mediated modulation of macrophage activation may be a therapeutic target of pathological angiogenesis.

KC progresses even after PK over the long term, requiring re-grafting in some cases. Risk factors for recurrent KC after PK are increasing Ks over time and a large CYL.

PurposeTo screen for and characterize compounds that protect corneal endothelial cells against unfolded protein response (UPR) and oxidative stress.MethodsBovine corneal endothelial cells (BCECs) were treated for 48 hours with 640 compounds from a Food and Drug Administration (FDA)-approved drug library and then challenged with thapsigargin or H2O2 to induce UPR or oxidative stress, respectively. Cell viability was measured using the CellTiter-Glo survival assay. Selected “hits” were subjected to further dose-response testing, and their ability to modulate expression of UPR and oxidative stress markers was assessed by RT-PCR, Western blot, and measurement of protein carbonyl and 8-hydroxydeoxyguanosine (8-OHdG) adducts in immortalized human corneal endothelial cells (iHCECs).ResultsForty-one drugs at 20 μM and 55 drugs at 100 μM increased survival of H2O2-challenged cells, and 8 drugs at 20 μM and 2 drugs at 100 μM increased survival of thapsigargin-challenged cells, compared with untreated control cells. Nicergoline, ergothioneine, nimesulide, oxotremorine, and mefenamic acid increased survival of both H2O2- and thapsigargin-challenged cells. Oxotremorine altered DNA damage inducible 3 (CHOP) gene expression, glucose-regulated protein 78 kDa (GRP78) and activating transcription factor 4 (ATF4) protein expression, and protein carbonyl and 8-OHdG levels. Mefenamic acid altered GRP78 protein expression and protein carbonyl and 8-OHdG levels.ConclusionsOxotremorine and mefenamic acid are potential survival factors for corneal endothelial cells under UPR and oxidative stress. The described assay can be further expanded to screen additional drugs for potential therapeutic effect in corneal endothelial diseases such as Fuchs' endothelial corneal dystrophy.

UMIN000009372.

Purpose MicroRNAs are small non-coding RNAs which regulate gene expression at the post-transcriptional level. We reported that levels of microRNA (miR)-29 family are decreased in Fuchs endothelial corneal dystrophy (FECD) patient corneas. The miR-29 family regulates production of extracellular matrix (ECM) proteins. Accumulation of ECM proteins in Descemet’s membrane is an important pathologic change in FECD. In this study, we transfected miR-29b into human corneal endothelial cells and tissues and evaluated ECM protein expression levels. Methods Immortalized Fuchs human corneal endothelial cell line (iFECD) was established by infection of FECD patient’s corneal endothelial cells with hTERT lentivirus. MiR-29b was transfected into iFECD and expression levels of ECMs (COL1A1, COL4A1, LAMC1) were evaluated with quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and Western blot. Expression level of LAMC1 protein in miR-29b transfected donor corneal endothelium was also evaluated by Western blot. Results Compared with control, miR-29b expression level after transfection of iFECD was increased to 335.6 (±91.0)% and ECM expression levels were significantly decreased. Compared with control, qRT-PCR demonstrated reduction of ECM to the following levels: COL1A1: 1.9 (±0.4)%; COL4A1: 7.1 (±1.7)%; LAMC1: 21.5 (±2.7)%. Western blot showed reduced protein expression: COL1A1: 4.8 (±3.2)%; COL4A1: 42.5 (±25.0)%; and LAMC1: 44.8 (±3.1)%. In miR-29b transfected corneal tissue, LAMC1 protein expression level was decreased to 14.4 (±20.5)%. Conclusions Over-expression of miR-29b decreased ECM protein production in human corneal endothelial cells. Thus, miR-29 replacement therapy might be a new treatment strategy for FECD aimed at reducing pathologic production of ECM proteins in Descemet’s membrane.

Granular corneal dystrophy (GCD) is an autosomal dominant hereditary disease in which multiple discrete and irregularly shaped granular opacities are deposited in the corneal stroma. GCD is caused by a point mutation in the transforming growth factor-β-induced (TGFBI) gene, located on chromosome 5q31. Here, we report the first successful application of CRISPR-Cas9-mediated genome editing for the correction of a TGFBI mutation in GCD patient-derived primary corneal keratocytes via homology-directed repair (HDR). To correct genetic defects in GCD patient cells, we designed a disease-specific guide RNA (gRNA) targeting the R124H mutation of TGFBI, which causes GCD type 2 (GCD2). An R124H mutation in primary human corneal keratocytes derived from a GCD2 patient was corrected by delivering a CRISPR plasmid expressing Cas9/gRNA and a single-stranded oligodeoxynucleotide HDR donor template in vitro. The gene correction efficiency was 20.6% in heterozygous cells and 41.3% in homozygous cells. No off-target effects were detected. These results reveal a new therapeutic strategy for GCD2; this method may also be applicable to other heredity corneal diseases.

PurposeWe sought to identify the anti-angiogenic molecule expressed in corneal keratocytes that is responsible for maintaining the avascularity of the cornea.MethodsHuman umbilical vein endothelial cells (HUVECs) were cultured with either human dermal fibroblasts or with human corneal keratocytes under serum-free conditions. The areas that exhibited blood vessel formation were estimated by immunostaining the cultures with an antitibody against CD31, a blood vessel marker. We also performed microarray gene-expression analysis and selected one molecule, angiopoietin-like 7 (ANGPTL7) for further functional studies conducted with the keratocytes and in vivo in mice.ResultsAreas showing blood vessel formation in normal serum-free medium were conditions were markedly smaller when HUVECs were co-cultured with corneal keratocytes than when they were co-cultured with the dermal fibroblasts under the same conditions. Microarray analysis revealed that ANGPTL7 expression was higher in keratocytes than in dermal fibroblasts. In vitro, inhibiting ANGPTL7 expression by using a specific siRNA led to greater tube formation than did the transfection of cells with a control siRNA, and this increase in tube formation was abolished when recombinant ANGPTL7 protein was added to the cultures. In vivo, intrastromal injections of an ANGPTL7 PshRNA into the avascular corneal stroma of mice resulted in the growth of blood vessels.Conclusions ANGPTL7, which is abundantly expressed in keratocytes, plays a major role in maintaining corneal avascularity and transparency.

HS-SM was similar to FBS-SM for HCEC culture when assessed by cell morphology, proliferation and protein/gene expression.