Biochemistry/Molecular Biology - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologysequencing. Changes in mRNA expression in human microvascularendothelial cells (HMECs) following direct transfection withmicroRNA over-expression vectors were analysed by microarray.Expression of specific genes was measured by RT-qPCR.Results: The global profile of microRNAs within vesicles releasedfrom EPCs is distinct from that inside the cells. MicroRNAs highlyenriched in extracellular vesicles included miR-486, miR-4792, miR-216a and miR-143. Vesicles from EPCs transfected with severalvectors driving expression of microRNAs, including miR-146a andmiR-451, were highly enriched in the over-expressed microRNA.When EPC-derived vesicles enriched with miR-146a were incubatedwith human microvascular endothelial cells they causeddownregulation of miR-146a target genes.Conclusions: Endogenous microRNAs are selectively packaged intoextracellular vesicles. EPC vesicles can be manipulated to deliverspecific microRNAs to endothelial cells. Such vesicles carryingcandidate anti- or pro-angiogenic microRNAs may have therapeuticapplications in multiple ocular diseases.Commercial Relationships: David A. Simpson, None; EoinBrown, None; Christina L. O'Neill, None; Reinhold J. Medina,None; Jasenka Guduric-Fuchs, NoneSupport: Biotechnology and Biological Sciences Research Council(BBSRC), grant no. BB/H005498/1Program Number: 1587 Poster Board Number: D0014Presentation Time: 8:30 AM - 10:15 AMP2X7 Expression in Vitrectomy SamplesCheryl Chi 1 , Deeba Husain 1 , Celeste B. Rich 2 , Nicole H. Siegel 1 ,Vickery E. Trinkaus-Randall 1, 2 . 1 Ophthalmology, Boston UniversitySchool of Medicine, Boston, MA; 2 Biochemistry, Boston UniversitySchool of Medicine, boston, MA.Purpose: P2X7 is an ionotropic receptor activated by ATP releasedin stress conditions. The full-length form of P2X7 (P2X7A) isassociated with the induction of apoptosis in many cell types, but atruncated variant, P2X7j, has also recently been found in transitionalstates that require cell proliferation. Altered regulation of these formshas been observed in diabetic patients, but specific relationshipsbetween the disease and receptor have yet to be elucidated. Our goalis to demonstrate the expression of P2X7A and P2X7j and tocorrelate this with sulfated glycosaminoglycan (GAG).Methods: Vitreous samples from 46 patients were centrifuged andsupernatant collected. Real-time PCR and delta delta Ct analysis wereperformed to determine expression of P2X7 mRNA. Each form wasnormalized to 1 using a control sample. Sulfated GAG in thesupernatant were measured by Dimethylmethylene Blue.Measurements were averaged and standard statistical tests wereperformed.Results: We observed an increased trend in the ratio of P2X7j toP2X7A in diabetic patients over control. The level of P2X7j did notchange between diabetic or control patients, but the level of P2X7Adecreased in diabetics, indicative of a less apoptotic phenotype. Thiswas supported by lower expression of P2X7A in patients withproliferative diabetic retinopathy. Hypertensive control patientsshowed a large increase in ratio over non-hypertensive controlpatients, but no such trend was seen in diabetic patients. Conversely,diabetic patients with retained lens fragments showed larger ratiosthan those without retained lens fragments, but this trend was notseen in the control population. Diabetic patients overall showdecreased sulfated GAG compared to control. Diabetic patients withretained lens fragments show a decrease, but those with hypertensionshow an increase, when compared to the corresponding control.Conclusions: Expression of P2X7j or pro-proliferative form, alongwith a decrease in P2X7A, were reflected in the phenotypicexpectations of pathological states. The inflammation associated withhypertensive states is suggested by the increased ratio, and thedecreased pro-apoptotic form illustrates cell proliferation in PDR.Changes in P2X7 receptor expression and sulfated GAG reflect theinfluence of multiple underlying pathologies.Commercial Relationships: Cheryl Chi, None; Deeba Husain,None; Celeste B. Rich, None; Nicole H. Siegel, None; Vickery E.Trinkaus-Randall, NoneSupport: Boston University Department of Ophthalmology ResearchGrant, Massachusetts Lions Eye Research Fund, Inc.Program Number: 1588 Poster Board Number: D0015Presentation Time: 8:30 AM - 10:15 AMPro-inflammatory cytokines induce apoptosis of human retinalendothelial cells by downregulating Hsp27Ram H. Nagaraj, Allison Palmer, Rooban B. Nahomi.Ophthalmology and Visual Sciences, Case Western ReserveUniversity, Cleveland, OH.Purpose: The biochemical mechanisms for retinal capillary celldeath in diabetes are not clear. In the diabetic retina of humans androdents, pro-inflammatory cytokines are upregulated. In this study,we have investigated the effect if pro-inflammatory cytokines on asmall heat shock protein, Hsp27 in primary human retinal endothelialcells (HREC).Methods: HREC were cultured in the presence of pro-inflammatorycytokines, interferon -γ (IFN-γ, 50 and 100 units/ml), interleukin-1β(IL-1β, 10 and 20 ng/ml) and tumor necrosis factor-α (TNF-α, 10 and20 ng/ml) for 48 hrs and in the presence or absence of high glucose(25 mM, HG). The roles of the kynurenine pathway and NOS weredetermined by adding 20 μM 1-methyl tryptophan (MT) and 500 μML-Nω-nitroarginine methyl ester hydrochloride (L-NAME),respectively to the culture medium. The mRNA and protein levels ofHsp27 and heat shock factor-1 (HSF-1) were determined by PCR andWestern blotting. Apoptosis was assessed by Hoechst staining.Results: HREC cultured in the presence of mixed cytokines showeda significant downregulation of Hsp27 at the protein and mRNAlevels. This downregulation was not due to downregulation of thetranscription factor, HSF-1. The presence of high glucose (25 mM)further increased the effect of mixed cytokines. Mixed cytokinesactivated indoleamine 2,3-dioxygenase (IDO), enhanced kynurenineproduction and increased the ROS content in HREC. An inhibitor ofIDO (MT) inhibited the effects of mixed cytokines on Hsp27. Mixedcytokines upregulated NOS2 and consequently increased levels ofnitric oxide. A peroxynitrite donor and exogenous peroxynitritedrastically reduced Hsp27 in HREC. The cytokine and high glucosemediateddownregulation of Hsp27 was accompanied by increasedapoptosis of HREC, which was largely inhibited by treatment withMT or a NOS inhibitor, L-NAME. Downregulation of Hsp27 by asiRNA promoted apoptosis in HREC.Conclusions: Together our data suggest that pro-inflammatorycytokines induce formation of ROS through the IDO-mediatedkynurenine pathway, which through peroxynitrite production reducesHsp27 and brings about apoptosis of capillary endothelial cells. Ourresults suggest a novel mechanism for capillary cell death in diabeticretinopathy.Commercial Relationships: Ram H. Nagaraj, None; AllisonPalmer, None; Rooban B. Nahomi, NoneSupport: International Retina Research Foundation, RPB, OhioLions Eye Research Foundation, P30EY-11373Program Number: 1589 Poster Board Number: D0016Presentation Time: 8:30 AM - 10:15 AM©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.