title: Retinal pericytes and cytomegalovirus infectivity: implications for HCMV-induced retinopathy and congenital ocular disease.
abstract: BACKGROUND
  Human cytomegalovirus (HCMV) is the leading infectious cause of vision loss among congenitally infected children. Retinal pericytes play an essential role in maintaining retinal vascular and endothelial cell proliferation. However, the role of retinal pericytes in ocular HCMV pathogenesis is unknown.
  
  
  METHODS
  Retinal pericytes were exposed to clinical (SBCMV) and lab strains of HCMV; infectivity was analyzed by microscopy, immunofluorescence and qRT-PCR (reverse transcription polymerase chain reaction). Cytokine expression was examined by Luminex assay. Recombinant HCMV-GPF was used to examine viral replication kinetics. A Tricell culture model of the inner blood-retinal barrier (IBRB) was examined for cell type infectivity using immunohistochemistry.
  
  
  RESULTS
  Retinal pericytes expressed the biomarker neuron-glial antigen 2. Antigenic expression profiles for several cytoskeletal, cell adhesion and inflammatory proteins were shared by both retinal and brain pericytes. Infected pericytes showed cytomegalic cytopathology and expressed mRNAs for the major immediate protein (MIE) and HCMV phosphorylated envelop protein 65. qRT-PCR analysis showed full lytic replication of HCMV in retinal pericytes. Pericytes exposed to SBCMV for 9 days expressed higher levels of vascular endothelial cell growth factor mRNA compared to controls. Luminex analysis of supernatants from SBCMV-infected retinal pericytes had increased levels of macrophage inflammatory protein-1α, beta-2 microglobulin (B2-m), matrix metalloproteinase-3 and -9 (MMP3/9), and lower levels of IL-6 and IL-8 compared to controls. At 24 hours post infection, pericytes expressed higher levels of IL-8, TIMP-1 (tissue inhibitor of metalloproteinase-1), and RANTES (regulated upon activation normal T cell-expressed and presumably secreted) but lower levels of MMP9. Time course analysis showed that both brain and retinal pericytes were more permissive for HCMV infection than other cellular components of the BBB (blood-brain barrier) and IBRB. Using a Tricell culture model of the IBRB (retinal endothelial, pericytes, Müller cells), retinal pericytes were most permissive for SBCMV infection. SBCMV infection of this IBRB Tricell mixture for 96 hours resulted in increased levels of IL-6, MMP9, and stem cell factor with a concomitant decrease in granulocyte-macrophage colony-stimulating factor and TNF-alpha.
  
  
  CONCLUSIONS
  In retinal pericytes, HCMV induces proinflammatory and angiogenic cytokines. In the IBRB, pericytes likely serve as an amplification reservoir which contributes to retinal inflammation and angiogenesis.
authors.affiliation: Department of Microbiology and Immunology, Center for AIDS Health Disparities Research, Meharry Medical College, School of Medicine, 1005 Dr DB Todd Jr Blvd, Nashville, TN, 37208, USA. imw0001@tigermail.auburn.edu.
authors.forename: Irene
authors.initials: I
authors.lastname: Wilkerson
authors.affiliation: Department of Microbiology and Immunology, Center for AIDS Health Disparities Research, Meharry Medical College, School of Medicine, 1005 Dr DB Todd Jr Blvd, Nashville, TN, 37208, USA. jlaban12@email.mmc.edu.
authors.forename: Joshua
authors.initials: J
authors.lastname: Laban
authors.affiliation: Department of Microbiology and Immunology, Center for AIDS Health Disparities Research, Meharry Medical College, School of Medicine, 1005 Dr DB Todd Jr Blvd, Nashville, TN, 37208, USA. mitchell155@live.marshall.edu.
authors.forename: Johnathan M
authors.initials: JM
authors.lastname: Mitchell
authors.affiliation: Department of Ophthalmology and Visual Sciences, University of Wisconsin School of Medicine and Public Health, Madison, WI, 53792, USA. nsheibanikar@wisc.edu.
authors.forename: Nader
authors.initials: N
authors.lastname: Sheibani
authors.affiliation: Department of Microbiology and Immunology, Center for AIDS Health Disparities Research, Meharry Medical College, School of Medicine, 1005 Dr DB Todd Jr Blvd, Nashville, TN, 37208, USA. dalcendor@mmc.edu.
authors.forename: Donald J
authors.initials: DJ
authors.lastname: Alcendor
mesh_terms: D000941:Antigens / Q000378:metabolism; D001921:Brain / Q000166:cytology; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002478:Cells, Cultured; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D003587:Cytomegalovirus / Q000502:physiology*; D005353:Fibronectins / Q000378:metabolism; D015967:Gene Expression Regulation, Viral / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009389:Neovascularization, Pathologic; D009457:Neuroglia / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D020286:Pericytes / Q000821:virology*; D011509:Proteoglycans / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012160:Retina / Q000166:cytology*; D014746:Vimentin / Q000378:metabolism; D014795:Visual Pathways / Q000378:metabolism; D000099216:Chondroitin Sulfate Proteoglycan 4
chemical_list: D000941:Antigens; D015815:Cell Adhesion Molecules; D016207:Cytokines; D005353:Fibronectins; D049452:Green Fluorescent Proteins; D011509:Proteoglycans; D012333:RNA, Messenger; D014746:Vimentin; D000099216:Chondroitin Sulfate Proteoglycan 4; C013065:cell aggregation factors