Influence of subretinal fluid in advanced stage retinopathy of prematurity on proangiogenic response and cell proliferation

Purpose The clinical phenotype of advanced stage retinopathy of prematurity (ROP, stages 4 and 5) cannot be replicated in an animal model. To dissect the molecular events that can lead up to advanced ROP, we examined subretinal fluid (SRF) and surgically dissected retrolental membranes from patients with advanced ROP to evaluate its influences on cell proliferation, angiogenic properties, and macrophage polarity. Methods: We compared our findings to SRF collected from patients with uncomplicated rhegmatogenous retinal detachment (RD) without proliferative vitreoretinopathy and surgically dissected epiretinal membrane from eyes with macular pucker. All subretinal fluid samples were equalized for protein. The angiogenic potential of SRF from ROP eyes was measured using a combination of capillary cord formation in a fibrin clot assay, and its proliferative effect was tested with a DNA synthesis of human retinal microvascular endothelial cells. Findings were compared with SRF collected from participants with uncomplicated rhegmatogenous RD without proliferative vitreoretinopathy. The ability of SRF to induce nitric oxide production was measured in vitro using murine J774A.1 macrophages. Cytokine profiles of SRF from ROP and RD eyes were measured using a multienzyme-linked immunosorbent assay (ELISA). Fluorescent immunohistochemistry of retrolental membranes from ROP was performed to detect the presence of leukocytes and the composition of tissue macrophages using markers for M1 and M2 differentiation. Results: The cytokine composition in SRF revealed that in ROP, not only were several proangiogenic factors were preferentially elevated but also the profile of proinflammatory factors was also increased compared to the RD eyes. SRF from ROP eyes supported cell proliferation and endothelial cord formation while SRF from RD eyes had inhibitory effects. SRF from eyes with ROP but not RD robustly induced nitric oxide production in macrophages. Furthermore, fluorescent immunostaining revealed a preponderance of M1 over M2 macrophages in retrolental fibrous membranes from ROP eyes. The cytokine profile and biologic properties of SRF in ROP promote a proangiogenic environment, which supports the maintenance and proliferation of fibrous membranes associated with advanced stages of ROP. In contrast, SRF from RD eyes exhibits a suppressive environment for endothelial cell proliferation and angiogenesis. Conclusions: Our investigation demonstrates that the microenvironment in advanced ROP eyes is proangiogenic and proinflammatory. These findings suggest that management of advanced ROP should not be limited to the surgical removal of the fibrovascular membranes and antiangiogenic therapy but also directed to anti-inflammatory therapy and to promote M2 activation over M1 activity

Isolation and characterization of specific antigens from cysticercus cellulosae

published_or_final_versionZoologyMasterMaster of Philosoph

Stimulating the Melanocortin System in Uveitis and Diabetes Preserves the Structure and Anti-Inflammatory Activity of the Retina

The endogenous neuropeptide α-Melanocyte Stimulating Hormone (α-MSH) is a potent suppressor of inflammation and has an essential role in maintaining the normal anti-inflammatory microenvironment of the retina. While the therapeutic use of α-MSH peptide in uveitis and diabetic retinopathy models has been demonstrated, its short half-life and instability limit its use as a therapeutic drug. A comparable analog, PL-8331, which has a stronger affinity to melanocortin receptors, longer half-life, and, so far, is functionally identical to α-MSH, has the potential to deliver melanocortin-based therapy. We examined the effects of PL-8331 on two mouse models of retinal disease, Experimental Autoimmune Uveoretinitis (EAU) and Diabetic Retinopathy (DR). PL-8331 therapy applied to mice with EAU suppressed EAU and preserved retinal structures. In diabetic mice, PL-8331 enhanced the survival of retinal cells and suppressed VEGF production in the retina. In addition, retinal pigment epithelial cells (RPE) from PL-8331-treated diabetic mice retained normal anti-inflammatory activity. The results demonstrated that the pan-melanocortin receptor agonist PL-8331 is a potent therapeutic drug to suppress inflammation, prevent retinal degeneration, and preserve the normal anti-inflammatory activity of RPE

Molecular basis for regeneration of CNS: a possible regulatory role of growth associated protein-43

published_or_final_versionAnatomyDoctoralDoctor of Philosoph

Immunology and Microbiology Retinal Pigment Epithelial Cells Suppress Phagolysosome Activation in Macrophages

PURPOSE. The eye is an immune-privileged microenvironment that has adapted several mechanisms of immune regulation to prevent inflammation. One of these potential mechanisms is retinal pigment epithelial cells (RPE) altering phagocytosis in macrophages. METHODS. The conditioned media of RPE eyecups from eyes of healthy mice and mice with experimental autoimmune uveitis (EAU) were used to treat primary macrophage phagocytizing pHrodo bacterial bioparticles. In addition, the neuropeptides were depleted from the conditioned media of healthy RPE eyecups and used to treat phagocytizing macrophages. The conditioned media from healthy and EAU RPE eyecups were assayed for IL-6, and IL-6 was added to the healthy conditioned media, and neutralized in the EAU conditioned media. The macrophages were treated with the conditioned media and assayed for fluorescence. The macrophages were imaged, and the fluorescence intensity, relative to active phagolysosomes, was measured. Also, the macrophages were assayed using fluorescent viability dye staining. RESULTS. The conditioned media from healthy, but not from EAU RPE eyecups suppressed phagolysosome activation. Depletion of the neuropeptides alpha-melanocyte-stimulating hormone and neuropeptide Y from the healthy RPE eyecup conditioned media resulted in macrophage death. In the EAU RPE eyecup conditioned media was 0.96 6 0.18 ng/mL of IL-6, and when neutralized the conditioned media suppressed phagolysosome activation. CONCLUSIONS. The healthy RPE through soluble molecules, including alpha-melanocytestimulating hormone and neuropeptide Y, suppresses the activation of the phagolysosome in macrophages. In EAU, the IL-6 produced by the RPE promotes the activation of phagolysosomes in macrophages. These results demonstrate that under healthy conditions, RPE promotes an altered pathway of phagocytized material in macrophages with implications on antigen processing and clearance

Prediction of mRNA targets of miR-101-3p in diabetic kidney disease by bioinformatics tools

Introduction: Diabetic kidney disease (DKD) remains the leading cause of chronic kidney disease (CKD) world- wide. Current biomarkers and treatment still fall short at preventing its progression. In search for a better diagnostic or therapeutic target, much interest in microRNAs, which act as post-translational regulators of gene expression has emerged. An upregulation of miR-101-3p was identified in the sera of type 2 diabetic patients with macroalbu- minuria in a selected Malaysian population by profiler RT-PCR array. Using bioinformatics tools, this study aimed to predict the mRNA targets of miR-101-3p. Given the scarcity of bioinformatics studies in DKD, this study also attempted to fill the gap. Methods: The mRNA targets were identified from two experimentally validated databases, namely Tarbase and MirTarBase. The commonly identified mRNA targets were submitted to Metascape and Enrichr bioinformatic tools. Results: A total of 2630 and 342 mRNA targets of miR-101-3p were identified by Tarbase and miRTarbase, respectively. One-hundred ninety-seven (197) mRNA targets were submitted for functional enrichment analysis. Our bioinformatics and bibliographical analyses suggested that ras-related C3 botulinum toxin substrate 1 (RAC1) and Ras-associated protein-1 b (RAP1b) were the most promising putative mRNA targets of miR-101-3p. The most enriched Gene Ontology term and pathway associated with these putative mRNA targets included Ras protein signal transduction and focal adhesion, respectively. Based on these analyses, their molecular mechanisms were proposed. Conclusion: Given the structural heterogeneity of the kidneys and cell type-dependent miRNA modula- tion, an in-silico target prediction of miR-101-3p increases the probability of a successful future in-vitro experimental verification