Sijia Cao

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.

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.

Optical coherence tomography-based correlation between choroidal thickness and drusen load in dry age-related macular degeneration.

Purpose: Spectral domain optical coherence tomography can be used to measure both choroidal thickness and drusen load. The authors conducted an exploratory study using spectral domain optical coherence tomography to determine if a correlation between choroidal thickness and drusen load exists in patients with dry age-related macular degeneration. Methods: Forty-four patients with dry age-related macular degeneration were recruited. The drusen area and volume were determined using the automated software algorithm of the spectral domain optical coherence tomography device, and choroidal thickness was measured using enhanced depth imaging. Correlations were determined using multivariable and univariable analyses. Results: The authors found an inverse correlation between choroidal thickness and drusen load (r = −0.35, P = 0.04). Drusen load was also correlated with visual acuity (r = 0.32, P = 0.04). A correlation between choroidal thickness and visual acuity was suggested (r = −0.22, P = 0.21). Conclusion: Spectral domain optical coherence tomography can be used to assess the correlation between drusen load and choroidal thickness, both of which show a relationship with visual acuity. The measurement of these outcomes may serve as important outcome parameters in routine clinical care and in clinical trials for patients with dry age-related macular degeneration.

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.

The enigma of autoimmune retinopathy.

Autoimmune retinopathy (AIR) refers to an immunologic process whereby retinal antigens are aberrantly recognized as autoantigens, leading to retinal degeneration. AIR was initially described in 1976 as a paraneoplastic syndrome, termed cancer-associated retinopathy (CAR), secondary to a remote malignancy.1 Laboratory investigations revealed that the serum of patients with CAR contained autoantibodies against the photoreceptor protein recoverin, which are believed to represent the underlying etiology of CAR.2,3 A related syndrome, termed melanoma-associated retinopathy (MAR), was reported in patients with cutaneous malignant melanoma who were found to have autoantibodies to an unknown antigen on bipolar cells.4,5 Since that time, AIR in the absence of neoplasm, termed nonparaneoplastic AIR (npAIR), has been described as well.6 Along with this has come the identification of several novel putative antiretinal autoantibodies. Although all these novel discoveries have expanded our knowledge of AIR, they have also brought with them some confusion. There are numerous excellent reviews of AIR that have recently been published in the literature.7–13 The purpose of this current article is not to provide another review of this topic, but rather to highlight some of the ambiguity and uncertainty that exists in this field. For the purposes of this review, the term AIR will be used to refer to CAR, npAIR, and MAR, as the ocular features and proposed pathogenesis of these entities are essentially the same.

Immuno-modulatory Effect of IFN-gamma in AMD and its Role as a Possible Target for Therapy

Age-related macular degeneration (AMD) is a neurodegenerative disease characterized by retinal cell atrophy, and/or choroidal neovascularization in the macula and constitutes the most common cause of blindness among the elderly in industrialized countries. The management of AMD is constrained by our insufficient knowledge of its underlying mechanisms. Recent studies point towards an emerging involvement of interferon-gamma (IFN-γ), a soluble cytokine associated with innate and adaptive immunity. IFN-γ promotes proinflammatory responses by activating proinflammatory cytokines and chemokines, thereby recruiting immune cells such as macrophages and T cells. On the other hand, IFN-γ modulates inflammatory response by upregulating anti-inflammatory factors or inhibiting development of immune cells related to autoimmune response. The complex role of IFN-γ in AMD pathogenesis is intriguing and worth further investigation in terms of therapeutic development.

Drusen and Pro-inflammatory Mediators in the Post-Mortem Human Eye

With age and drusen accumulation, the environment of the eye tends to shift more towards a pro-inflammatory state (Figure 6) through either IL-1β and/or possibly IFN pathways. Little change is induced in the complement inhibitor, CFI, in association with drusen, suggesting a failure to activate protective mechanisms against complement activation. Alternatively, the lack of change in CFI accumulation level could indicate that that the pro-inflammatory actions of drusen may not yet be enough to trigger inhibition of the complement cascade in healthy eye tissue. Here, we identified three inflammatory molecules in the postmortem human eye, p-STAT3, CXCL-10, and CXCL-11, to be strongly correlated with the presence of drusen within a population controlled for age (≤57 years old, Figure 6). This pattern of accumulation suggests that these molecules are closely involved with the presence of drusen rather than with aging, and may represent a predisposition toward AMD pathogenesis. A recent microarray study on mouse RPE cells reported expression changes in over 315 genes associated with advanced age [48]. Many pathways they identified were also found in our prior microarray study (unpublished observation) [16]. Similarly, Chen et al. [49] found that the mouse neuroretina displayed age-related upregulation of the complement cascade and activation of retinal microglia [49]. Curiously, neither study found expression changes in any of the gene products examined in this study. It is important to note that drusen-like deposits have not been documented in wild-type-mouse eyes [50]. Thus, one interpretation for this discrepancy may be that the genes identified in our microarray studies, and specifically the gene products studied here reflect a drusen-specific response rather than a general response to aging. Our studies now set the stage for future experiments testing the function of each of these potential mediators in the pathogenesis of AMD. One approach might involve assessing the response of each mediator following Aβ and/or AGE injection into young wild-type mice to determine degree of mimicry to AMD pathology. Figure 6 Summary diagram depicting the distribution of IL-1β, RSAD2, p-STAT3, and CXCL-10/11 immunoreactivity in post-mortem eye tissue with respect to aging and present of drusen. Icons denote the relative immunostaining of each molecule in a given retinallocation. ... Acknowledgments We thank Jonathan Tang, Jonathan Coleman, and Dr. Ian Clark for discussion and editing of the manuscript. This work was funded by Canadian Institutes of Health Research (CIHR-MOP-97806) to J.A.M. Grant Support Supported by Canadian Institutes of Health Research (CIHR) Grant# MOP-97806 (to JAM).

Long-term in vitro functional stability of compounded ranibizumab and aflibercept

OBJECTIVE To determine the long-term in vitro functional stability of compounded ranibizumab and aflibercept. DESIGN Laboratory study. METHODS Ranibizumab and aflibercept were compounded into plastic syringes and stored under refrigerated conditions for up to 4 weeks. Half maximal inhibitory concentrations (IC50) from dose-response curves generated by using drugs and their respective targets in enzyme-linked immunosorbent assays were calculated. The functional activity of the drugs stored under these conditions was then compared with that of drug from a fresh glass vial obtained from the manufacturer. RESULTS There was no statistically significant change in IC50 between ranibizumab stored in plastic syringes for 4 weeks compared with drug obtained from a fresh glass vial (p = 0.4883). Similarly, there was no statistically significant change in IC50 between aflibercept stored in plastic syringes for 4 weeks compared with drug obtained from a fresh glass vial (p = 0.6202). CONCLUSION Compounding of ranibizumab and aflibercept in plastic syringes with storage for up to 4 weeks does not appear to have a detrimental effect on the in vitro functional activity of these medications. Because the cost of these medications can be prohibitive, compounding may be considered as a method of reducing cost.

Alterations in intraocular cytokine levels following intravitreal ranibizumab

OBJECTIVE Our previous work has shown that, after intravitreal bevacizumab (IVB) administration, decreases in the levels of vascular endothelial growth factor (VEGF)-A and placental growth factor (PlGF), along with increases in the levels of interleukin (IL)-8 and transforming growth factor (TGF)-β2, can be observed. It is not yet known if similar changes occur after intravitreal ranibizumab (IVR). The purpose of this study was to examine intraocular cytokine changes after IVR. DESIGN Prospective clinical study. PARTICIPANTS Subjects with proliferative diabetic retinopathy requiring pars plana vitrectomy (PPV) were recruited. METHODS Participants received IVR as pre-treatment before PPV. Aqueous humour levels of IL-8, VEGF-A, PlGF, and TGF-β2 were measured at time of pre-treatment and PPV. Results were analyzed using univariate statistical models. RESULTS A total of 14 participants were recruited. After IVR administration, we observed a decrease in the levels of VEGF-A and PlGF, and an increase in the levels of IL-8 and TGF-β2. These results were statistically significant only for VEGF-A (p = 0.0001) and IL-8 (p = 0.0002). CONCLUSIONS The changes in cytokine levels after IVR mirror the changes seen after IVB. Further studies are warranted in order to determine if there are any differences between IVB and IVR in this regard.