Glucose transport, transporters and metabolism in diabetic retinopathy.

Diabetic retinopathy (DR) is the most common reason for blindness in working-age individuals globally. Prolonged high blood glucose is a main causative factor for DR development, and glucose transport is prerequisite for the disturbances in DR caused by hyperglycemia. Glucose transport is mediated by its transporters, including the facilitated transporters (glucose transporter, GLUTs), the "active" glucose transporters (sodium-dependent glucose transporters, SGLTs), and the SLC50 family of uniporters (sugars will eventually be exported transporters, SWEETs). Glucose transport across the blood-retinal barrier (BRB) is crucial for nourishing the neuronal retina in the context of retinal physiology. This physiological process primarily relies on GLUTs and SGLTs, which mediate the glucose transportation across both the cell membrane of retinal capillary endothelial cells and the retinal pigment epithelium (RPE). Under diabetic conditions, increased accumulation of extracellular glucose enhances the retinal cellular glucose uptake and metabolism via both glycolysis and glycolytic side branches, which activates several biochemical pathways, including the protein kinase C (PKC), advanced glycation end-products (AGEs), polyol pathway and hexosamine biosynthetic pathway (HBP). These activated biochemical pathways further increase the production of reactive oxygen species (ROS), leading to oxidative stress and activation of Poly (ADP-ribose) polymerase (PARP). The activated PARP further affects all the cellular components in the retina, and finally resulting in microangiopathy, neurodegeneration and low-to-moderate grade inflammation in DR. This review aims to discuss the changes of glucose transport, glucose transporters, as well as its metabolism in DR, which influences the retinal neurovascular unit (NVU) and implies the possible therapeutic strategies for treating DR.

Anti-HIV Drugs Reduce Risk of Prediabetes and Progression to Type 2 Diabetes in HIV-Infected Patients.

The aim of this study was to investigate whether the use of nucleoside reverse transcriptase inhibitors (NRTIs) impacts the incidence of prediabetes or type 2 diabetes mellitus (T2DM) or the progression from prediabetes to T2DM in people living with HIV (PLWH). We conducted a retrospective cohort study using the U.S. Veterans Health Administration database among adult patients with an HIV diagnosis from the year 2000 until 2021 to determine the incidence of prediabetes and further progression to T2DM among NRTI exposed and unexposed patients. A multistate model was used to evaluate progression from normoglycemia to prediabetes and then to T2DM, and covariate adjustment with the Cox proportional hazards model was used to estimate the hazard ratios. Among 32,240 veterans diagnosed with HIV, prediabetes and T2DM were observed among 20.2% and 20.7% of patients, respectively. Among those diagnosed with prediabetes, 31.8% progressed to T2DM. Patients exposed to NRTIs at any time (86.6%), had a reduced risk of prediabetes [HR 0.50 (0.47-0.53 95% CI)] and among prediabetics, a lower risk of progression to T2DM [HR 0.73 (0.63-0.85 95% CI)] when compared to patients who never used NRTIs. In summary, NRTIs may reduce the risk of developing prediabetes and the progression from prediabetes to T2DM in PLWH.

Kamuvudine-9 Protects Retinal Structure and Function in a Novel Model of Experimental Rhegmatogenous Retinal Detachment.

Purpose: Rhegmatogenous retinal detachment (RRD) is a vision-threatening event that benefits from surgical intervention. While awaiting surgical reattachment, irreversible hypoxic and inflammatory damage to the retina often occurs. An interim therapy protecting photoreceptors could improve functional outcomes. We sought to determine whether Kamuvudine-9 (K-9), a derivative of nucleoside reverse transcriptase inhibitors (NRTIs) that inhibits inflammasome activation, and the NRTIs lamivudine (3TC) and azidothymidine (AZT) could protect the retina following RRD. Methods: RRD was induced in mice via subretinal injection (SRI) of 1% carboxymethylcellulose (CMC). To simulate outcomes following the clinical management of RRD, we determined the optimal conditions by which SRI of CMC induced spontaneous retinal reattachment (SRR) occurs over 10 days (RRD/SRR). K-9, 3TC, or AZT was administered via intraperitoneal injection. Inflammasome activation pathways were monitored by abundance of cleaved caspase-1, IL-18, and cleaved caspase-8, and photoreceptor death was assessed by TUNEL staining. Retinal function was assessed by full-field scotopic electroretinography. Results: RRD induced retinal inflammasome activation and photoreceptor death in mice. Systemic administration of K-9, 3TC, or AZT inhibited retinal inflammasome activation and photoreceptor death. In the RRD/SRR model, K-9 protected retinal electrical function during the time of RRD and induced an improvement following retinal reattachment. Conclusions: K-9 and NRTIs exhibit anti-inflammatory and neuroprotective activities in experimental RRD. Given its capacity to protect photoreceptor function during the period of RRD and enhance retinal function following reattachment, K-9 shows promise as a retinal neuroprotectant and warrants study in RRD. Further, this novel RRD/SRR model may facilitate experimental evaluation of functional outcomes relevant to RRD.

METTL3-mediated m6A modification of HMGA2 mRNA promotes subretinal fibrosis and epithelial-mesenchymal transition.

Subretinal fibrosis is a major cause of the poor visual prognosis for patients with neovascular age-related macular degeneration (nAMD). Myofibroblasts originated from retinal pigment epithelial (RPE) cells through epithelial-mesenchymal transition (EMT) contribute to the fibrosis formation. N6-Methyladenosine (m6A) modification has been implicated in the EMT process and multiple fibrotic diseases. The role of m6A modification in EMT-related subretinal fibrosis has not yet been elucidated. In this study, we found that during subretinal fibrosis in the mouse model of laser-induced choroidal neovascularization, METTL3 was upregulated in RPE cells. Through m6A epitranscriptomic microarray and further verification, high-mobility group AT-hook 2 (HMGA2) was identified as the key downstream target of METTL3, subsequently activating potent EMT-inducing transcription factor SNAIL. Finally, by subretinal injections of adeno-associated virus vectors, we confirmed that METTL3 deficiency in RPE cells could efficiently attenuate subretinal fibrosis in vivo. In conclusion, our present research identified an epigenetic mechanism of METTL3-m6A-HMGA2 in subretinal fibrosis and EMT of RPE cells, providing a novel therapeutic target for subretinal fibrosis secondary to nAMD.

Fat mass and obesity-associated protein alleviates Aß1-40 induced retinal pigment epithelial cells degeneration via PKA/CREB signaling pathway.

Amyloid-ß (Aß) is thought to be a critical pathologic factor of retinal pigment epithelium (RPE) degeneration in age-related macular degeneration (AMD). Aß induces inflammatory responses in RPE cells and recent studies demonstrate the N6-methyladenosine (m6A) regulatory role in RPE cell inflammation. m6A is a reversible epigenetic posttranslational modification, but its relationship with Aß-induced RPE degeneration is yet to be thoroughly investigated. The present study explored the role and mechanism of m6A in Aß-induced RPE degeneration model. This model was induced via intravitreally injecting oligomeric Aß and the morphology of its retina was analyzed. One of m6A demethylases, the fat mass and obesity-associated (FTO) gene expression, was assessed. An m6A-messenger RNA (mRNA) epitranscriptomic microarray was employed for further bioinformatic analyses. It was confirmed that Aß induced FTO upregulation within the RPE. Hypopigmentation alterations and structural disorganization were observed in Aß-treated eyes, and inhibition of FTO exacerbated retinal degeneration and RPE impairment. Moreover, the m6A-mRNA epitranscriptomic microarray suggested that protein kinase A (PKA) was a target of FTO, and the PKA/cyclic AMP-responsive element binding (CREB) signaling pathway was involved in Aß-induced RPE degeneration. m6A-RNA binding protein immunoprecipitation confirmed that FTO demethylated PKA within the RPE cells of Aß-treated eyes. Altered expression of PKA and its downstream targets (CREB and brain-derived neurotrophic factor) was confirmed by quantitative reverse-transcription polymerase chain reaction and Western blot analyses. Hence, this study's findings shed light on FTO-mediated m6A modification in Aß-induced RPE degeneration and indicate potential therapeutic targets for AMD.

Growth of nonexudative macular neovascularization in age-related macular degeneration: an indicator of biological lesion activity.

PURPOSE: To investigate the growth of nonexudative macular neovascularization (MNV) in age-related macular degeneration (AMD) using swept-source optical coherence tomography angiography (SS-OCTA). METHODS: Patients with treatment-naïve nonexudative AMD in one eye and exudative AMD in the fellow eye who underwent SS-OCTA imaging for at least 12 months were retrospectively reviewed. The MNV area measurement was quantified in eyes with treatment-naïve nonexudative MNV using ImageJ for analysing the correlation between MNV growth and the onset of exudation, as well as evaluating the consistency of the MNV growth rate during the subclinical and exudative stages. Kaplan-Meier survival analysis and logistic regression analyses were used. RESULTS: In total, 45 eyes with treatment-naïve nonexudative AMD from 45 patients were enrolled. Treatment-naïve nonexudative MNV was identified in 21 eyes (46.67%) at baseline. The development of exudative findings was noted in eight eyes (17.78%), including six eyes with previously noted nonexudative MNV. Eyes with growing MNV (increase in area ≥50% within 12 months) had an increased risk of exudation and developed exudation earlier than eyes with stable MNV (13.60 [6.43-20.77] months versus 31.11 [26.61-35.62] months, P < 0.0001, Log-rank test). Consistent growth pattern of MNV lesions was further identified in eyes with growing MNV during anti-VEGF treatment. CONCLUSION: SS-OCTA allows to qualitatively and quantitatively evaluate nonexudative MNV in AMD patients. Growing MNV involved higher probabilities and a faster onset of exudation compared to stable MNV. Identifying the growth of MNV on OCTA might be helpful for establishing treatment strategies and follow-up planning.

Progression of Polypoidal Lesions Associated with Exudative Recurrence in Polypoidal Choroidal Vasculopathy.

PURPOSE: To investigate the characteristics of the branching vascular network (BVN) and polypoidal lesions in polypoidal choroidal vasculopathy (PCV) to determine near-term indicators that may predict exudative recurrence. DESIGN: Retrospective cohort study. PARTICIPANTS: Patients with PCV receiving anti-vascular endothelial growth factor (VEGF) monotherapy or anti-VEGF plus photodynamic therapy were followed for at least 1 year using swept-source OCT angiography (SS-OCTA) imaging. METHODS: Patients were divided into 2 groups based on whether exudative recurrence occurred during follow-up. Multiple parameters were collected and compared between the 2 groups, such as age, gender, visual acuity, number of polypoidal lesions, lesion area at the first SS-OCTA visit, and total lesion area change from the first SS-OCTA visit to the last SS-OCTA visit. To evaluate the association between SS-OCTA imaging-based risk factors and the exudative recurrences, imaging features associated with PCV such as BVN growth and polypoidal lesion progression (enlargement, new appearance, and reappearance) at each follow-up visit were analyzed. The time intervals from the nonexudative visit with lesion progression to the corresponding exudative recurrence visit were documented to explore their association with exudative recurrences. Cox regression and logistic regression analyses were used. MAIN OUTCOME MEASURES: Association between BVN growth and polypoidal lesion progression with exudative recurrence. RESULTS: Thirty-one eyes of 31 patients (61% men) were included. Sixteen eyes had no recurrence of exudation, and 15 eyes had recurrence during follow-up. The average follow-up duration was 20.55 ± 6.86 months (range, 12-36 months). Overall, the recurrence group had worse best-corrected visual acuity (P = 0.019) and a greater increase in lesion area (P = 0.010). Logistical regression analysis showed that polypoidal lesion progression, including new appearance, enlargement, and reappearance of polypoidal lesions, was associated with exudative recurrences within 3 months (odds ratio, 26.67, 95% confidence interval, 3.77-188.54, P = 0.001). CONCLUSIONS: Growth of nonexudative BVN and progression of polypoidal lesions were found to be lesion characteristics associated with exudative recurrences, and progression of polypoidal lesions might serve as a stand-alone indicator for the near-term onset of exudation. In PCV, more frequent follow-up visits are recommended when polypoidal lesions show progression.

Subretinal injection in mice to study retinal physiology and disease.

Subretinal injection (SRI) is a widely used technique in retinal research and can be used to deliver nucleic acids, small molecules, macromolecules, viruses, cells or biomaterials such as nanobeads. Here we describe how to undertake SRI of mice. This protocol was adapted from a technique initially described for larger animals. Although SRI is a common procedure in eye research laboratories, there is no published guidance on the best practices for determining what constitutes a 'successful' SRI. Optimal injections are required for reproducibility of the procedure and, when carried out suboptimally, can lead to erroneous conclusions. To address this issue, we propose a standardized protocol for SRI with 'procedure success' defined by follow-up examination of the retina and the retinal pigmented epithelium rather than solely via intraoperative endpoints. This protocol takes 7-14 d to complete, depending on the reagent delivered. We have found, by instituting a standardized training program, that trained ophthalmologists achieve reliable proficiency in this technique after ~350 practice injections. This technique can be used to gain insights into retinal physiology and disease pathogenesis and to test the efficacy of experimental compounds in the retina or retinal pigmented epithelium.

The Learning Curve of Murine Subretinal Injection Among Clinically Trained Ophthalmic Surgeons.

Purpose: Subretinal injection (SRI) in mice is widely used in retinal research, yet the learning curve (LC) of this surgically challenging technique is unknown. Methods: To evaluate the LC for SRI in a murine model, we analyzed training data from three clinically trained ophthalmic surgeons from 2018 to 2020. Successful SRI was defined as either the absence of retinal pigment epithelium (RPE) degeneration after phosphate buffered saline injection or the presence of RPE degeneration after Alu RNA injection. Multivariable survival-time regression models were used to evaluate the association between surgeon experience and success rate, with adjustment for injection agents, and to calculate an approximate case number to achieve a 95% success rate. Cumulative sum (CUSUM) analyses were performed and plotted individually to monitor each surgeon's simultaneous performance. Results: Despite prior microsurgery experience, the combined average success rate of the first 50 cases in mice was only 27%. The predicted SRI success rate did not reach a plateau above 95% until approximately 364 prior cases. Using the 364 training cases as a cutoff point, the predicted probability of success for cases 1 to 364 was 65.38%, and for cases 365 to 455 it was 99.32% (P < 0.0001). CUSUM analysis showed an initial upward slope and then remained within the decision intervals with an acceptable success rate set at 95% in the late stage. Conclusions: This study demonstrates the complexity and substantial LC for successful SRI in mice with high confidence. A systematic training system could improve the reliability and reproducibility of SRI-related experiments and improve the interpretation of experimental results using this technique. Translational Relevance: Our prediction model and monitor system allow objective quantification of technical proficiency in the field of subretinal drug delivery and gene therapy for the first time, to the best of our knowledge.

Small dome-shaped pigment epithelium detachment in polypoidal choroidal vasculopathy: an under-recognized sign of polypoidal lesions on optical coherence tomography?

PURPOSE: To evaluate the diagnostic accuracy of spectral-domain optical coherence tomography (SD-OCT) and swept-source optical coherence tomographic angiography (SS-OCTA) to identify polypoidal lesions in serous or serosanguinous maculopathy. MATERIALS AND METHODS: A retrospective review of patients presenting pigment epithelial detachments (PEDs) with the diagnosis of polypoidal choroidal vasculopathy (PCV), neovascular age-related macular degeneration (nAMD), and central serous chorioretinopathy (CSC), all of which underwent SD-OCT, SS-OCTA, and indocyanine green angiography (ICGA). Typical features of polypoidal lesions on SD-OCT included sharply peaked PED, notched PED, and hyperreflective ring underneath PED. SS-OCTA feature was vascularized PEDs on cross-sectional images corresponding to cluster-like structures on en face images. The parameters of PEDs were measured for analysis. RESULTS: Of 72 eyes, 30 had PCV, 22 had nAMD, and 20 had CSC. A total of 128 localized PEDs were detected on SD-OCT. Typical features on SD-OCT had a high specificity (94.0%) but a limited sensitivity (73.8%). SS-OCTA features provided a higher sensitivity (96.7%). PEDs of the polypoidal lesions unrecognized by SD-OCT were dome-shaped, with smaller ratio of height to base diameter and less area, and almost had heterogeneous internal reflectivity and a connected double-layer sign. Some lesions misidentified by SS-OCTA developed into ICGA-proven polypoidal lesions at follow-up visits. CONCLUSION: A small dome-shaped PED with heterogeneous internal reflectivity and a connected double-layer sign on SD-OCT may suggest a polypoidal lesion of PCV. SS-OCTA may be a helpful tool to investigate preclinical PCV and observe the formation of polypoidal lesions.

Association of Microvasculature and Macular Sensitivity in Idiopathic Macular Epiretinal Membrane: Using OCT Angiography and Microperimetry.

Purpose: To investigate the correlation between retinal capillary structure and macular function in patients with idiopathic epiretinal membrane (iERM) by using optical coherence tomography angiography (OCTA) and microperimetry. Methods: This retrospective and observational study included 30 idiopathic ERM eyes of 30 consecutive patients. OCTA was performed to evaluate macular microvasculature including the superficial capillary plexus, deep capillary plexus, and foveal avascular zone. Best corrected visual acuity (BCVA) and microperimetry were measured at baseline and 3 months after surgery. Associations between macular microvasculature and visual function were assessed. Results: Visual function including BCVA and macular sensitivity improved significantly at 3 months post-operatively (p < 0.001). At baseline, BCVA was positively correlated with foveal or parafoveal sensitivities and negatively correlated with central foveal thickness (p < 0.05). Pre-operative foveal sensitivity was significantly correlated with the vessel density of foveal or parafoveal superficial capillary plexus (p < 0.05). A multiple regression model revealed that pre-operative vessel density of foveal deep capillary plexus was an independent positive prognostic factor for post-operative BCVA (B = -0.020 ± 0.006, p = 0.006) and macular sensitivity (B = 0.200 ± 0.081, p = 0.027). Conclusion: Integrated evaluation of iERM by using OCTA and microperimetry shows an association between microvasculature and macular sensitivity. Pre-operative vessel density of foveal deep capillary plexus assessed by OCTA may be a potentially valuable prognostic factor for iERM surgery.

DDX17 is an essential mediator of sterile NLRC4 inflammasome activation by retrotransposon RNAs.

Detection of microbial products by multiprotein complexes known as inflammasomes is pivotal to host defense against pathogens. Nucleotide-binding domain leucine-rich repeat (NLR) CARD domain containing 4 (NLRC4) forms an inflammasome in response to bacterial products; this requires their detection by NLR family apoptosis inhibitory proteins (NAIPs), with which NLRC4 physically associates. However, the mechanisms underlying sterile NLRC4 inflammasome activation, which is implicated in chronic noninfectious diseases, remain unknown. Here, we report that endogenous short interspersed nuclear element (SINE) RNAs, which promote atrophic macular degeneration (AMD) and systemic lupus erythematosus (SLE), induce NLRC4 inflammasome activation independent of NAIPs. We identify DDX17, a DExD/H box RNA helicase, as the sensor of SINE RNAs that licenses assembly of an inflammasome comprising NLRC4, NLR pyrin domain­containing protein 3, and apoptosis-associated speck-like protein­containing CARD and induces caspase-1 activation and cytokine release. Inhibiting DDX17-mediated NLRC4 inflammasome activation decreased interleukin-18 release in peripheral blood mononuclear cells of patients with SLE and prevented retinal degeneration in an animal model of AMD. Our findings uncover a previously unrecognized noncanonical NLRC4 inflammasome activated by endogenous retrotransposons and provide potential therapeutic targets for SINE RNA­driven diseases.

Identification of fluoxetine as a direct NLRP3 inhibitor to treat atrophic macular degeneration.

The atrophic form of age-related macular degeneration (dry AMD) affects nearly 200 million people worldwide. There is no Food and Drug Administration (FDA)-approved therapy for this disease, which is the leading cause of irreversible blindness among people over 50 y of age. Vision loss in dry AMD results from degeneration of the retinal pigmented epithelium (RPE). RPE cell death is driven in part by accumulation of Alu RNAs, which are noncoding transcripts of a human retrotransposon. Alu RNA induces RPE degeneration by activating the NLRP3-ASC inflammasome. We report that fluoxetine, an FDA-approved drug for treating clinical depression, binds NLRP3 in silico, in vitro, and in vivo and inhibits activation of the NLRP3-ASC inflammasome and inflammatory cytokine release in RPE cells and macrophages, two critical cell types in dry AMD. We also demonstrate that fluoxetine, unlike several other antidepressant drugs, reduces Alu RNA-induced RPE degeneration in mice. Finally, by analyzing two health insurance databases comprising more than 100 million Americans, we report a reduced hazard of developing dry AMD among patients with depression who were treated with fluoxetine. Collectively, these studies identify fluoxetine as a potential drug-repurposing candidate for dry AMD.

Alu complementary DNA is enriched in atrophic macular degeneration and triggers retinal pigmented epithelium toxicity via cytosolic innate immunity.

Long interspersed nuclear element-1 (L1)­mediated reverse transcription (RT) of Alu RNA into cytoplasmic Alu complementary DNA (cDNA) has been implicated in retinal pigmented epithelium (RPE) degeneration. The mechanism of Alu cDNA­induced cytotoxicity and its relevance to human disease are unknown. Here we report that Alu cDNA is highly enriched in the RPE of human eyes with geographic atrophy, an untreatable form of age-related macular degeneration. We demonstrate that the DNA sensor cGAS engages Alu cDNA to induce cytosolic mitochondrial DNA escape, which amplifies cGAS activation, triggering RPE degeneration via the inflammasome. The L1-extinct rice rat was resistant to Alu RNA­induced Alu cDNA synthesis and RPE degeneration, which were enabled upon L1-RT overexpression. Nucleoside RT inhibitors (NRTIs), which inhibit both L1-RT and inflammasome activity, and NRTI derivatives (Kamuvudines) that inhibit inflammasome, but not RT, both block Alu cDNA toxicity, identifying inflammasome activation as the terminal effector of RPE degeneration.

Thymopentin alleviates premature ovarian failure in mice by activating YY2/Lin28A and inhibiting the expression of let-7 family microRNAs.

OBJECTIVE: Thymopentin (5TP) significantly improved typical murine premature ovarian failure (POF) symptoms induced by a high-fat and high-sugar (HFHS) diet. However, its effect and mechanism remain unclear. MATERIALS AND METHODS: RNA-Seq was used to detect the differentially expressed genes among each group. HFHS-induced POF mouse model was generated and injected with siRNA using Poly (lactic-co-glycolic acid) (PLGA) as a carrier. RESULTS: RNA-Seq suggested that 5TP promoted the expression of Yin Yang 2 (YY2) in mouse ovarian granulosa cell (mOGC) of HFHS-POF mice. Luciferase reporter assay indicated that 5TP promoted the binding of YY2 to the specific sequence C(C/T)AT(G/C)(G/T) on the Lin28A promoter and promoted Lin28A transcription and expression. We continuously injected PLGA-cross-linked siRNA nanoparticles targeting YY2 into HFHS-POF mice (siYY2@PLGA), which significantly reduced the therapeutic effect of 5TP. siYY2@PLGA injection also significantly attenuated the upregulation of Lin28a expression in mOGCs induced by 5TP and enhanced the expression of let-7 family microRNAs, thereby inhibiting the proliferation and division of mOGCs. qPCR results showed that there was a significant difference in the expression levels of exosome-derived Yy2 mRNAs between POF patients and normal women, and that there was a specific correlation between the expression level of exosome-derived Yy2 and the peripheral concentrations of the blood hormones pregnenolone, progesterone and oestradiol. CONCLUSIONS: Thymopentin promotes the transcriptional activation of Lin28A via stimulating transcription factor YY2 expression, inhibits the activity of let-7 family microRNAs and alleviates the ageing of ovarian granulosa cells, ultimately achieving a therapeutic effect on POF in mice.

Untargeted metabolomics for uncovering plasma biological markers of wet age-related macular degeneration.

Wet age-related macular degeneration (wAMD) causes central vision loss and represents a major health problem in elderly people. Here we have used untargeted metabolomics using UHPLC-MS to profile plasma from 127 patients with wAMD (67 choroidal neovascularization (CNV) and 60 polypoidal choroidal vasculopathy (PCV)) and 50 controls. A total of 545 biochemicals were detected. Among them, 17 metabolites presented difference between patients with wAMD and controls. Most of them were oxidized lipids (N=6, 35.29%). Comparing to controls, 28 and 18 differential metabolites were identified in patients with CNV and PCV, respectively. Two metabolites, hyodeoxycholic acid and L-tryptophanamide, were differently distributed between PCV and CNV. We first investigated the genetic association with metabolites in wet AMD (CFH rs800292 and HTRA1 rs10490924). We identified six differential metabolites between the GG and AA genotypes of CFH rs800292, five differential metabolites between the GG and AA genotypes of HTRA1 rs10490924, and four differential metabolites between the GG and GA genotypes of rs10490924. We selected four metabolites (cyclamic acid, hyodeoxycholic acid, L-tryptophanamide and O-phosphorylethanolamine) for in vitro experiments. Among them, cyclamic acid reduced the activity, inhibited the proliferation, increased the apoptosis and necrosis in human retinal pigment epithelial cells (HRPECs). L-tryptophanamide affected the proliferation, apoptosis and necrosis in HRPECs, and promoted the tube formation and migration in primary human retinal endothelial cells (HRECs). Hyodeoxycholic acid and O-phosphorylethanolamine inhibited the tube formation and migration in HRECs. The results suggested that differential metabolites have certain effects on wAMD pathogenesis-related HRPECs and HRECs.

Nucleoside reverse transcriptase inhibitors and Kamuvudines inhibit amyloid-ß induced retinal pigmented epithelium degeneration.

Nonfibrillar amyloid-ß oligomers (AßOs) are a major component of drusen, the sub-retinal pigmented epithelium (RPE) extracellular deposits characteristic of age-related macular degeneration (AMD), a common cause of global blindness. We report that AßOs induce RPE degeneration, a clinical hallmark of geographic atrophy (GA), a vision-threatening late stage of AMD that is currently untreatable. We demonstrate that AßOs induce activation of the NLRP3 inflammasome in the mouse RPE in vivo and that RPE expression of the purinergic ATP receptor P2RX7, an upstream mediator of NLRP3 inflammasome activation, is required for AßO-induced RPE degeneration. Two classes of small molecule inflammasome inhibitors-nucleoside reverse transcriptase inhibitors (NRTIs) and their antiretrovirally inert modified analog Kamuvudines-both inhibit AßOs-induced RPE degeneration. These findings crystallize the importance of P2RX7 and NLRP3 in a disease-relevant model of AMD and identify inflammasome inhibitors as potential treatments for GA.

Cytoplasmic synthesis of endogenous Alu complementary DNA via reverse transcription and implications in age-related macular degeneration.

Alu retroelements propagate via retrotransposition by hijacking long interspersed nuclear element-1 (L1) reverse transcriptase (RT) and endonuclease activities. Reverse transcription of Alu RNA into complementary DNA (cDNA) is presumed to occur exclusively in the nucleus at the genomic integration site. Whether Alu cDNA is synthesized independently of genomic integration is unknown. Alu RNA promotes retinal pigmented epithelium (RPE) death in geographic atrophy, an untreatable type of age-related macular degeneration. We report that Alu RNA-induced RPE degeneration is mediated via cytoplasmic L1-reverse-transcribed Alu cDNA independently of retrotransposition. Alu RNA did not induce cDNA production or RPE degeneration in L1-inhibited animals or human cells. Alu reverse transcription can be initiated in the cytoplasm via self-priming of Alu RNA. In four health insurance databases, use of nucleoside RT inhibitors was associated with reduced risk of developing atrophic macular degeneration (pooled adjusted hazard ratio, 0.616; 95% confidence interval, 0.493-0.770), thus identifying inhibitors of this Alu replication cycle shunt as potential therapies for a major cause of blindness.

Physical attributes of salivary calcium particles and their interaction with gingival epithelium.

BACKGROUND: The formation of dental plaque and its involvement in the pathogenesis of periodontitis is a topic of intense interest given the high prevalence of periodontitis in humans. Even though calcium-based particles play an active role in both dental plaque formation and periodontitis, few publications describe the physical-chemical properties of these particles. METHODS: Saliva samples were collected from healthy volunteers. From these samples, saliva-derived particles were isolated and stained for calcium using calcein or Fluo-4. The salivary particles were also subjected to characterization by flow cytometry and immunoblotting. Internalization of calcein-labeled salivary particles by gingival epithelial cells was visualized by confocal microscopy. RESULTS: We found that calcium-based salivary particles from healthy volunteers varied greatly in size but were enriched in particles of sizes at or greater than 1.5 µm. Immunoblotting analysis of the salivary particles identified several proteins including albumin, fetuin-A, and statherin, which have been found in calcium phosphate particles from other tissues or are known to modulate calcium homeostasis in saliva. In addition, calcium particles were internalized by both gingival epithelial cells and monocyte-derived macrophages. CONCLUSION: Salivary calcium particles were enriched in the micrometer range, internalized by gingival epithelial cells, and contain albumin, fetuin-A and statherin, regulators of particle formation. These characteristics of the calcium-based salivary particles and their biological activities provide a basis for further studies to understand the molecular basis for pathogenesis of periodontitis.

ROS production and mitochondrial dysfunction driven by PU.1-regulated NOX4-p22phox activation in Aß-induced retinal pigment epithelial cell injury.

Rationale: Amyloid ß (Aß) deposition, an essential pathological process in age-related macular degeneration (AMD), causes retinal pigment epithelium (RPE) degeneration driven mostly by oxidative stress. However, despite intense investigations, the extent to which overoxidation contributes to Aß-mediated RPE damage and its potential mechanism has not been fully elucidated. Methods: We performed tandem mass-tagged (TMT) mass spectrometry (MS) and bioinformatic analysis of the RPE-choroid complex in an Aß1-40-induced mouse model of retinal degeneration to obtain a comprehensive proteomic profile. Key regulators in this model were confirmed by reactive oxygen species (ROS) detection, mitochondrial ROS assay, oxygen consumption rate (OCR) measurement, gene knockout experiment, chromatin immunoprecipitation (ChIP), and luciferase assay. Results: A total of 4243 proteins were identified, 1069 of which were significantly affected by Aß1-40 and found to be enriched in oxidation-related pathways by bioinformatic analysis. Moreover, NADPH oxidases were identified as hub proteins in Aß1-40-mediated oxidative stress, as evidenced by mitochondrial dysfunction and reactive oxygen species overproduction. By motif and binding site analyses, we found that the transcription factor PU.1/Spi1 acted as a master regulator of the activation of NADPH oxidases, especially the NOX4-p22phox complex. Also, PU.1 silencing impeded RPE oxidative stress and mitochondrial dysfunction and rescued the retinal structure and function. Conclusion: Our study suggests that PU.1 is a novel therapeutic target for AMD, and the regulation of PU.1 expression represents a potentially novel approach against excessive oxidative stress in Aß-driven RPE injury.