title: Short Pulse of Clinical Concentration of Bevacizumab Modulates Human Retinal Pigment Epithelial Functionality.
abstract: OBJECTIVE
  Cross-talk between Notch signaling and vascular endothelial growth factor (VEGF) is a major driver of angiogenesis. Here we investigated the temporal effect of bevacizumab (BEV) on Notch signaling and the functional features of cultured primary retinal pigment epithelial (PRPE) cells.
  
  
  METHODS
  Human (cadaver) PRPE cells were treated with clinical concentrations of BEV (0.25 mg/mL). Notch signaling pathway receptors, ligands, and downstream target genes were analyzed with quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation along with phagocytosis and transmembrane potential was analyzed by fluorescent activated cell sorter (FACS) and immunofluorescence.
  
  
  RESULTS
  Bevacizumab-treated PRPE cultures revealed a significant temporal downregulation of notch4 (P < 0.05) and Delta-like-4 (P < 0.005) gene (16% reduced) and protein (29.7% reduced) expression only at the 2-hour exposure, though secreted VEGF levels were significantly blocked (P < 0.005) at all the time points (2, 4, 6 hours). Further, a significant downregulation (P < 0.005) in cell cycle (reduced by 34.1%) and a concurrent (P < 0.005) upregulation of F-actin staining (increased by 2.5-fold) could be detected. Bevacizumab-treated PRPE cells revealed an elevated transmembrane potential (by 63%) and significant decrease (P < 0.01) in phagocytosis (by 19.25%) in comparison to untreated controls.
  
  
  CONCLUSIONS
  There is temporal interaction between BEV and the Notch signaling pathway, specifically with Notch4 and Delta-like-ligand-4 in PRPE cultures. This transient decrease in Notch signaling can impact the functionality of RPE cells. These findings can help to provide a better understanding of the effect of long-term usage of anti-VEGF agents in the treatment of retinal degenerative and vitreoretinopathy diseases.
authors.affiliation: Stem Cell Research Laboratory GROW Laboratory, Narayana Nethralaya Foundation, Bangalore, Karnataka, India 2Post Graduate Department of Biotechnology, Jamal Mohamed College (Autonomous), Tiruchirappalli, Tamil Nadu, India.
authors.forename: Murali
authors.initials: M
authors.lastname: Subramani
authors.affiliation: Stem Cell Research Laboratory GROW Laboratory, Narayana Nethralaya Foundation, Bangalore, Karnataka, India.
authors.forename: Ponnalagu
authors.initials: P
authors.lastname: Murugeswari
authors.affiliation: Stem Cell Research Laboratory GROW Laboratory, Narayana Nethralaya Foundation, Bangalore, Karnataka, India 3School of Biosciences and Technology, Vellore Institute of Technology University, Vellore, Tamil Nadu, India.
authors.forename: Kamesh
authors.initials: K
authors.lastname: Dhamodaran
authors.affiliation: GROW Laboratory, Narayana Nethralaya Foundation, Bangalore, Karnataka, India.
authors.forename: Priyanka
authors.initials: P
authors.lastname: Chevour
authors.affiliation: Stem Cell Research Laboratory GROW Laboratory, Narayana Nethralaya Foundation, Bangalore, Karnataka, India.
authors.forename: Sindhu
authors.initials: S
authors.lastname: Gunasekaran
authors.affiliation: Department of Glaucoma Service, Narayana Nethralaya Eye Hospital, Rajaji Nagar, Bangalore, Karnataka, India.
authors.forename: Rajesh S
authors.initials: RS
authors.lastname: Kumar
authors.affiliation: Department of Vitreo-Retinal Services, Narayana Nethralaya Eye Hospital, Rajaji Nagar, Bangalore, Karnataka, India.
authors.forename: Chaitra
authors.initials: C
authors.lastname: Jayadev
authors.affiliation: Department of Cornea and Refractive Surgery, Narayana Nethralaya Eye Hospital, Rajaji Nagar, Bangalore, Karnataka, India.
authors.forename: Rohit
authors.initials: R
authors.lastname: Shetty
authors.affiliation: Post Graduate Department of Biotechnology, Jamal Mohamed College (Autonomous), Tiruchirappalli, Tamil Nadu, India.
authors.forename: Nargis
authors.initials: N
authors.lastname: Begum
authors.affiliation: Stem Cell Research Laboratory GROW Laboratory, Narayana Nethralaya Foundation, Bangalore, Karnataka, India.
authors.forename: Debashish
authors.initials: D
authors.lastname: Das
mesh_terms: D000328:Adult; D000368:Aged; D020533:Angiogenesis Inhibitors / Q000008:administration & dosage; D000068258:Bevacizumab / Q000008:administration & dosage*; D015153:Blotting, Western; D002102:Cadaver; D049109:Cell Proliferation; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D008297:Male; D008875:Middle Aged; D012313:RNA / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D055213:Retinal Pigment Epithelium / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D012168:Retinal Pigments / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D042461:Vascular Endothelial Growth Factor A / Q000187:drug effects / Q000378:metabolism*; D018630:Vitreoretinopathy, Proliferative / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D055815:Young Adult
chemical_list: D020533:Angiogenesis Inhibitors; D012168:Retinal Pigments; D042461:Vascular Endothelial Growth Factor A; D000068258:Bevacizumab; D012313:RNA