Retinal pericytes and cytomegalovirus infectivity: implications for HCMV-induced retinopathy and congenital ocular disease.

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. CONCLUSION: 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.

Transcriptional profiling reveals ductus arteriosus-specific genes that regulate vascular tone.

Failure of the ductus arteriosus (DA) to close at birth can lead to serious complications. Conversely, certain profound congenital cardiac malformations require the DA to be patent until corrective surgery can be performed. In each instance, clinicians have a very limited repertoire of therapeutic options at their disposal - indomethacin or ibuprofen to close a patent DA (PDA) and prostaglandin E1 to maintain patency of the DA. Neither treatment is specific to the DA and both may have deleterious off-target effects. Therefore, more therapeutic options specifically targeted to the DA should be considered. We hypothesized the DA possesses a unique genetic signature that would set it apart from other vessels. A microarray was used to compare the genetic profiles of the murine DA and ascending aorta (AO). Over 4,000 genes were differentially expressed between these vessels including a subset of ion channel-related genes. Specifically, the alpha and beta subunits of large-conductance calcium-activated potassium (BKCa) channels are enriched in the DA. Gain- and loss-of-function studies showed inhibition of BKCa channels caused the DA to constrict, while activation caused DA relaxation even in the presence of O2. This study identifies subsets of genes that are enriched in the DA that may be used to develop DA-specific drugs. Ion channels that regulate DA tone, including BKCa channels, are promising targets. Specifically, BKCa channel agonists like NS1619 maintain DA patency even in the presence of O2 and may be clinically useful.