Jing Z. Cui

Stage specificity of novel growth factor expression during development of proliferative vitreoretinopathy.

ObjectiveTo compare the relative levels of connective tissue growth factor (CTGF), platelet-derived growth factor alpha (PDGF-AA), and hepatocyte growth factor (HGF) in glial and retinal pigment epithelial (RPE) cells of epiretinal membranes from proliferative vitreoretinopathy (PVR).MethodsA total of 37 PVR membranes, of various stages, underwent fluorescent immunohistochemisty and confocal laser scanning microscopy to localize CTGF, HGF, and PDGF-AA in RPE and glial cells.ResultsNumerous RPE, and relatively fewer glial cells, were found in all stages of PVR. CTGF immunoreactivity increased from early to late stage PVR and was principally expressed by RPE cells in early stage, and by glial cells in late stage PVR. HGF, expressed by both RPE and glial cells, was principally expressed in mid-stage PVR. PDGF-AA, expressed by both cell types, demonstrated a uniform level of staining throughout all stages of PVR.ConclusionsRPE and glial cells contribute to the expression of CTGF, HGF, and PDGF-AA during PVR, but with specific developmental patterns. PDGF-AA is expressed uniformly throughout all stages of PVR, while HGF expression peaks during mid stage, and CTGF expression is highest during late stage PVR. These results allow for the development of stage-specific therapeutics for PVR that may allow targeting of the early proliferative and/or the late tractional stages of PVR.

Microarray Analysis Identifies Changes in Inflammatory Gene Expression in Response to Amyloid-β Stimulation of Cultured Human Retinal Pigment Epithelial Cells

PURPOSE Age-related macular degeneration (AMD) is a common cause of irreversible vision loss in the elderly. The hypothesis was that in vitro stimulation of RPE cells with Abeta(1-40), a constituent of drusen, promotes changes in gene expression and cellular pathways associated with the pathogenesis of AMD, including oxidative stress, inflammation, and angiogenesis. METHODS Confluent human RPE cells were stimulated with Abeta(1-40), or the reverse peptide Abeta(40-1), and genome wide changes in gene expression were studied with gene microarrays. Selected genes were verified by qRT-PCR and ELISA. Pathway analysis with gene set enrichment analysis (GSEA) and ingenuity revealed top functional pathways in RPE after Abeta(1-40) stimulation. RESULTS RPE cells stimulated with Abeta(1-40) (0.3 microM) for 24 hours resulted in 63 upregulated and 22 downregulated previously known genes. The upregulated genes were predominantly in inflammatory and immune response categories, but other categories were also represented, including apoptosis, cell signaling, cell proliferation, and signal transduction. Categories of downregulated genes included immune response, transporters, metabolic functions and transcription factors. ELISA confirmed that secreted levels of IL-8 were two times higher than control levels. GSEA and ingenuity analysis confirmed that the top affected pathways in RPE cells after Abeta(1-40) stimulation were inflammation and immune response related. Surprisingly, few angiogenic pathways were activated at the doses and exposure times studied. CONCLUSIONS Abeta(1-40) promotes RPE gene expression changes in pathways associated with immune response, inflammation, and cytokine and interferon signaling pathways. Results may relate to in vivo mechanisms associated with the pathogenesis of AMD.

Inflammatory mediators induced by amyloid-beta in the retina and RPE in vivo: implications for inflammasome activation in age-related macular degeneration.

PURPOSE Drusen are hallmarks of age-related macular degeneration (AMD). Amyloid-beta 1-40 (Aβ 1-40), a constituent of drusen, is known to stimulate inflammatory pathways in RPE; however, its effect in vivo is not known. The purpose of this study was to examine the effect of Aβ 1-40 on cytokine expression and inflammasome activation relevant to AMD in an animal model. METHODS Wild-type rats received intravitreal injections of Aβ 1-40, and eyes were taken at days 1, 4, 14, and 49 postinjection. The RPE, neuroretina, and vitreous were analyzed for cytokine expression, inflammasome activation, and microglial response via RT-PCR, immunohistochemistry, and suspension array assay. Retinal cell loss was assessed via apoptotic markers and retinal thickness. RESULTS Aβ 1-40 stimulated upregulation of IL-6, TNF-α, IL-1β, IL-18, caspase-1, NLRP3, and XAF1 genes in the RPE/choroid and the neuroretina. Increased IL-1β and IL-6 immunoreactivity was found in retinal sections, and elevated levels of IL-1β and IL-18 were found in the vitreous of Aβ-injected eyes. Aβ 1-40 induced a moderate increase in CD11b/c-reactive cells on day 1 postinjection only. No evidence of the proapoptotic XAF1 protein, p53, TUNEL immunoreactivity, or retinal thinning was observed. CONCLUSIONS These results confirm earlier in vitro work and support the proinflammatory role of drusen component Aβ 1-40 in the RPE and retina. Inflammasome activation may be responsible for this effect in vivo. This model is useful for understanding cellular triggers of inflammasome activation and proposed early inflammatory events in the outer retina associated with the etiology of AMD.

Effect of N-acetylcysteine on the early expression of inflammatory markers in the retina and plasma of diabetic rats

Purpose:  The aim of this study is to investigate markers of inflammation and oxidative stress in an early model of diabetic retinopathy, correlate retinal and plasma results and evaluate the influence of treatment by N‐acetylcysteine (NAC), a free radical scavenger.

NLRP3 Inflammasome: Activation and Regulation in Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is the leading cause of legal blindness in the elderly in industrialized countries. AMD is a multifactorial disease influenced by both genetic and environmental risk factors. Progression of AMD is characterized by an increase in the number and size of drusen, extracellular deposits, which accumulate between the retinal pigment epithelium (RPE) and Bruchs membrane (BM) in outer retina. The major pathways associated with its pathogenesis include oxidative stress and inflammation in the early stages of AMD. Little is known about the interactions among these mechanisms that drive the transition from early to late stages of AMD, such as geographic atrophy (GA) or choroidal neovascularization (CNV). As part of the innate immune system, inflammasome activation has been identified in RPE cells and proposed to be a causal factor for RPE dysfunction and degeneration. Here, we will first review the classic model of inflammasome activation, then discuss the potentials of AMD-related factors to activate the inflammasome in both nonocular immune cells and RPE cells, and finally introduce several novel mechanisms for regulating the inflammasome activity.

Parainflammation associated with advanced glycation endproduct stimulation of RPE in vitro: Implications for age-related degenerative diseases of the eye

Age related macular degeneration (AMD) is one of the leading causes of blindness in Western society. A hallmark of early stage AMD are drusen, extracellular deposits that accumulate in the outer retina. Advanced glycation endproducts (AGE) accumulate with aging and are linked to several age-related diseases such as Alzheimers disease, osteoarthritis, atherosclerosis and AMD. AGE deposits are found in drusen and in Bruchs membrane of the eye and several studies have suggested its role in promoting oxidative stress, apoptosis and lipofuscin accumulation. Recently, complement activation and chronic inflammation have been implicated in the pathogenesis of AMD. While AGEs have been shown to promote inflammation in other diseases, whether it plays a similar role in AMD is not known. This study investigates the effects of AGE stimulation on pro- and anti-inflammatory pathways in primary culture of human retinal pigment epithelial cells (RPE). Differential gene expression studies revealed a total of 41 up- and 18 down-regulated RPE genes in response to AGE stimulation. These genes fell into three categories as assessed by gene set enrichment analysis (GSEA). The main categories were inflammation (interferon-induced, immune response) and proteasome degradation, followed by caspase signaling. Using suspension array technology, protein levels of secreted cytokines and growth factors were also examined. Anti-inflammatory cytokines including IL10, IL1ra and IL9 were all overexpressed. Pro-inflammatory cytokines including IL4, IL15 and IFN-γ were overexpressed, while other pro-inflammatory cytokines including IL8, MCP1, IP10 were underexpressed after AGE stimulation, suggesting a para-inflammation state of the RPE under these conditions. Levels of mRNA of chemokine, CXCL11, and viperin, RSAD2, were up-regulated and may play a role in driving the inflammatory response via the NF-kB and JAK-STAT pathways. CXCL11 was strongly immunoreactive and associated with drusen in the AMD eye. The pathways and novel genes identified here highlight inflammation as a key response to AGE stimulation in primary culture of human RPE, and identify chemokine CXCL11 as putative novel agent associated with the pathogenesis of AMD.

Vitreous leptin levels in retinal disease.

Background/aimsThe purpose of this study was to investigate the relationship between vitreous leptin levels and retinal diseases.MethodsLevels of vitreous leptin were evaluated in proliferative diabetic retinopathy (PDR) and a variety of other retinopathies including: macular disease, neovascular maculopathies, primary retinal detachments, and vascular occlusive disease.ResultsIn patients with PDR (N=7), the average vitreous level of leptin (37.4 ng/ml) was significantly higher than that in patients with PVR (<1.0 ng/ml, P<0.05). Vitreous leptin level in patients with PVR or macular disease (N=18), with or without diabetes, was not significantly different from the control subjects who had retinal detachment only (N=7).ConclusionThe results show that the leptin level in vitreous taps is elevated in PDR. We suggest that leptin plays an active role in PDR.

Induction of proliferative vitreoretinopathy by a unique line of human retinal pigment epithelial cells

BACKGROUND The most widely used models of proliferative vitreoretinopathy (PVR) rely on injection of cells into the vitreous of animals. Using retinal pigment epithelial (RPE) cells from human PVR membranes may produce a more accurate model of human PVR. We performed a study to determine whether human RPE cells derived from a single epiretinal membrane (ERM) are capable of inducing the same disease in the rabbit eye, and whether the induced ERMs had cellular components similar to those of human PVR membranes. METHODS Cells were harvested from a human ERM obtained at surgery for PVR. RPE cells were cultured from the membrane and injected into the right eye of 24 New Zealand albino rabbits. The left eyes served as controls. The eyes were examined by indirect ophthalmoscopy over 4 weeks. The enucleated eyes were then examined by means of microscopy and histochemical analysis. RESULTS By day 7, PVR had developed in all but 1 of the 24 experimental eyes, with 8 progressing to localized tractional retinal detachment. By day 21, localized tractional retinal detachment had developed in 17 eyes; 1 eye progressed to extensive tractional retinal detachment by day 28. Immunostaining showed that mostly RPE cells, but also myofibroblasts, glial cells and collagen, were present in the newly formed rabbit PVR membranes. INTERPRETATION Human RPE cells cultured from a PVR membrane appear to be capable of inducing PVR in rabbits. The resultant ERMs are similar to those formed in human PVR and consist mainly of RPE cells.

The role of leptin in choroidal neovascularization

PURPOSE The purpose of this study was to investigate the role of leptin in choroidal neovascularization. METHODS We examined the localization of leptin by immunohistochemistry in nine choroidal neovascular membranes surgically excised from patients with age-related macular degeneration, idiopathic choroidal neovascularization, and ocular histoplasmosis. Controls included omission of primary antibody, use of an irrelevant primary antibody and leptin staining of posterior segment of four normal donor eyes. RESULTS Leptin was present in eight membranes and appeared vesicular, within the cytoplasm. The more vascular membranes and those consisting of a larger number of retinal pigment epithelium cells were associated with greater leptin staining. Leptin was not seen in the posterior segment of the four normal eyes. CONCLUSION We suggest that leptin plays an active role in choroidal neovascularization, although further experiments are necessary to establish a causal relationship.

CFH Y402H polymorphism and the complement activation product C5a: effects on NF-κB activation and inflammasome gene regulation

Background/aims The Y402H polymorphism in the complement factor H (CFH) gene is an important risk factor for age-related macular degeneration (AMD). Complement activation products and proinflammatory cytokines are associated with this polymorphism at the systemic level, but less is known of the associations in the outer retina of the genotyped eye. Here we investigate complement activation products and their role in nuclear factor (NF)-κB activation and gene expression of the NLRP3 inflammasome pathway. Methods Postmortem donor eyes were genotyped for the CFH Y402H polymorphism and assessed for complement C3a, C5a, interleukin (IL)-18 and tumour necrosis factor (TNF)-α. ARPE19 cells were stimulated basolaterally with C5a or TNF-α in polarised cultures. NF-κB activation was assessed with a reporter cell line. Gene expression of inflammasome-related (NLRP3, caspase-1, IL-1β and IL-18) and classic inflammatory (IL-6 and IL-8) genes was studied. The distribution of inflammasome products, IL-1β and IL-18, was studied in postmortem donor eyes with AMD pathologies. Results Eyes with the homozygous at-risk variant demonstrated higher levels of C5a, IL-18 and TNF-α in Bruchs membrane and choroid. C5a promoted NF-κB activation and upregulation of IL-18 in polarised ARPE19. TNF-α promoted NF-κB activation and gene expression of caspase-1, IL-1β, IL-18, IL-6 and IL-8, but downregulated NLRP3. In eyes with geographic atrophy, strong immunoreactivity was observed for inflammasome products IL-1β and IL-18 compared with age-matched controls. Conclusion The at-risk polymorphism of the CFH Y402H may contribute to AMD disease process through increased complement and NF-κB activation, and the upregulation of IL-18, a product of inflammasome activation.