Peptain-1 blocks ischemia/reperfusion-induced retinal capillary degeneration in mice.

Introduction: Neurovascular degeneration results in vascular dysfunction, leakage, ischemia, and structural changes that can lead to significant visual impairment. We previously showed the protective effects of peptain-1, a 20 amino acid peptide derived from the αB-crystallin core domain, on retinal ganglion cells in two animal models of glaucoma. Here, we evaluated the ability of peptain-1 to block apoptosis of human retinal endothelial cells (HRECs) in vitro and retinal capillary degeneration in mice subjected to retinal ischemia/reperfusion (I/R) injury. Methods: HRECs were treated with either peptain-1 or scrambled peptides (200 µg/mL) for 3 h and a combination of proinflammatory cytokines (IFN-γ 20 ng/mL + TNF-α 20 ng/mL+ IL-1ß 20 ng/mL) for additional 48 h. Apoptosis was measured with cleaved caspase-3 formation via western blot, and by TUNEL assay. C57BL/6J mice (12 weeks old) were subjected to I/R injury by elevating the intraocular pressure to 120 mmHg for 60 min, followed by reperfusion. Peptain-1 or scrambled peptide (0.5 µg) was intravitreally injected immediately after I/R injury and 7 days later. One microliter of PBS was injected as vehicle control, and animals were euthanized on day 14 post-I/R injury. Retinal capillary degeneration was assessed after enzyme digestion followed by periodic acid-Schiff staining. Results: Our data showed that peptain-1 entered HRECs and blocked proinflammatory cytokine-mediated apoptosis. Intravitreally administered peptain-1 was distributed throughout the retinal vessels after 4 h. I/R injury caused retinal capillary degeneration. Unlike scrambled peptide, peptain-1 protected capillaries against I/R injury. Additionally, peptain-1 inhibited microglial activation and reduced proinflammatory cytokine levels in the retina following I/R injury. Discussion: Our study suggests that peptain-1 could be used as a therapeutic agent to prevent capillary degeneration and neuroinflammation in retinal ischemia.

Mechanisms contributing to inhibition of retinal ganglion cell death by cell permeable peptain-1 under glaucomatous stress.

This study assesses the neuroprotective potential of CPP-P1, a conjugate of an anti-apoptotic peptain-1 (P1) and a cell-penetrating peptide (CPP) in in vitro, in vivo, and ex vivo glaucoma models. Primary retinal ganglion cells (RGCs) were subjected to either neurotrophic factor (NF) deprivation for 48 h or endothelin-3 (ET-3) treatment for 24 h and received either CPP-P1 or vehicle. RGC survival was analyzed using a Live/Dead assay. Axotomized human retinal explants were treated with CPP-P1 or vehicle for seven days, stained with RGC marker RBPMS, and RGC survival was analyzed. Brown Norway (BN) rats with elevated intraocular pressure (IOP) received weekly intravitreal injections of CPP-P1 or vehicle for six weeks. RGC function was evaluated using a pattern electroretinogram (PERG). RGC and axonal damage were also assessed. RGCs from ocular hypertensive rats treated with CPP-P1 or vehicle for seven days were isolated for transcriptomic analysis. RGCs subjected to 48 h of NF deprivation were used for qPCR target confirmation. NF deprivation led to a significant loss of RGCs, which was markedly reduced by CPP-P1 treatment. CPP-P1 also decreased ET-3-mediated RGC death. In ex vivo human retinal explants, CPP-P1 decreased RGC loss. IOP elevation resulted in significant RGC loss in mid-peripheral and peripheral retinas compared to that in naive rats, which was significantly reduced by CPP-P1 treatment. PERG amplitude decline in IOP-elevated rats was mitigated by CPP-P1 treatment. Following IOP elevation in BN rats, the transcriptomic analysis showed over 6,000 differentially expressed genes in the CPP-P1 group compared to the vehicle-treated group. Upregulated pathways included CREB signaling and synaptogenesis. A significant increase in Creb1 mRNA and elevated phosphorylated Creb were observed in CPP-P1-treated RGCs. Our study showed that CPP-P1 is neuroprotective through CREB signaling enhancement in several settings that mimic glaucomatous conditions. The findings from this study are significant as they address the pressing need for the development of efficacious therapeutic strategies to maintain RGC viability and functionality associated with glaucoma.

Thiol-Mediated Enhancement of Nε-Acetyllysine Formation in Lens Proteins.

Lysine acetylation (AcK) is a prominent post-translational modification in eye lens crystallins. We have observed that AcK formation is preferred in some lysine residues over others in crystallins. In this study, we have investigated the role of thiols in such AcK formation. Upon incubation with acetyl-CoA (AcCoA), αA-Crystallin, which contains two cysteine residues, showed significantly higher levels of AcK than αB-Crystallin, which lacks cysteine residues. Incubation with thiol-rich γS-Crystallin resulted in higher AcK formation in αB-Crystallin from AcCoA. External free thiol (glutathione and N-acetyl cysteine) increased the AcK content in AcCoA-incubated αB-Crystallin. Reductive alkylation of cysteine residues significantly decreased (p < 0.001) the AcCoA-mediated AcK formation in αA-Crystallin. Introduction of cysteine residues within ∼5 Å of lysine residues (K92C, E99C, and V169C) in αB-Crystallin followed by incubation with AcCoA resulted in a 3.5-, 1.3- and 1.3-fold increase in the AcK levels when compared to wild-type αB-Crystallin, respectively. Together, these results suggested that AcK formation in α-Crystallin is promoted by the proximal cysteine residues and protein-free thiols through an S → N acetyl transfer mechanism.

Quantifying Protein Acetylation in Diabetic Nephropathy from Formalin-Fixed Paraffin-Embedded Tissue.

PURPOSE: Diabetic kidney disease (DKD) is a serious complication of diabetes mellitus and a leading cause of chronic kidney disease and end-stage renal disease. One potential mechanism underlying cellular dysfunction contributing to kidney disease is aberrant protein post-translational modifications. Lysine acetylation is associated with cellular metabolic flux and is thought to be altered in patients with diabetes and dysfunctional renal metabolism. EXPERIMENTAL DESIGN: A novel extraction and LC-MS/MS approach was adapted to quantify sites of lysine acetylation from formalin-fixed paraffin-embedded (FFPE) kidney tissue and from patients with DKD and non-diabetic donors (n = 5 and n = 7, respectively). RESULTS: Analysis of FFPE tissues identified 840 total proteins, with 225 of those significantly changing in patients with DKD. Acetylomic analysis quantified 289 acetylated peptides, with 69 of those significantly changing. Pathways impacted in DKD patients revealed numerous metabolic pathways, specifically mitochondrial function, oxidative phosphorylation, and sirtuin signaling. Differential protein acetylation in DKD patients impacted sirtuin signaling, valine, leucine, and isoleucine degradation, lactate metabolism, oxidative phosphorylation, and ketogenesis. CONCLUSIONS AND CLINICAL RELEVANCE: A quantitative acetylomics platform was developed for protein biomarker discovery in formalin-fixed and paraffin-embedded biopsies of kidney transplant patients suffering from DKD.

Advanced Glycation End Products Upregulate CD40 in Human Retinal Endothelial and Müller Cells: Relevance to Diabetic Retinopathy.

CD40 induces pro-inflammatory responses in endothelial and Müller cells and is required for the development of diabetic retinopathy (DR). CD40 is upregulated in these cells in patients with DR. CD40 upregulation is a central feature of CD40-driven inflammatory disorders. What drives CD40 upregulation in the diabetic retina remains unknown. We examined the role of advanced glycation end products (AGEs) in CD40 upregulation in endothelial cells and Müller cells. Human endothelial cells and Müller cells were incubated with unmodified or methylglyoxal (MGO)-modified fibronectin. CD40 expression was assessed by flow cytometry. The expression of ICAM-1 and CCL2 was examined by flow cytometry or ELISA after stimulation with CD154 (CD40 ligand). The expression of carboxymethyl lysine (CML), fibronectin, and laminin as well as CD40 in endothelial and Müller cells from patients with DR was examined by confocal microscopy. Fibronectin modified by MGO upregulated CD40 in endothelial and Müller cells. CD40 upregulation was functionally relevant. MGO-modified fibronectin enhanced CD154-driven upregulation of ICAM-1 and CCL2 in endothelial and Müller cells. Increased CD40 expression in endothelial and Müller cells from patients with DR was associated with increased CML expression in fibronectin and laminin. These findings identify AGEs as inducers of CD40 upregulation in endothelial and Müller cells and enhancers of CD40-dependent pro-inflammatory responses. CD40 upregulation in these cells is associated with higher CML expression in fibronectin and laminin in patients with DR. This study revealed that CD40 and AGEs, two important drivers of DR, are interconnected.

Aggrelyte-2 promotes protein solubility and decreases lens stiffness through lysine acetylation and disulfide reduction: Implications for treating presbyopia.

Aging proteins in the lens become increasingly aggregated and insoluble, contributing to presbyopia. In this study, we investigated the ability of aggrelyte-2 (N,S-diacetyl-L-cysteine methyl ester) to reverse the water insolubility of aged human lens proteins and to decrease stiffness in cultured human and mouse lenses. Water-insoluble proteins (WI) of aged human lenses (65-75 years) were incubated with aggrelyte-2 (500 µM) for 24 or 48 h. A control compound that lacked the S-acetyl group (aggrelyte-2C) was also tested. We observed 19%-30% solubility of WI upon treatment with aggrelyte-2. Aggrelyte-2C also increased protein solubility, but its effect was approximately 1.4-fold lower than that of aggrelyte-2. The protein thiol contents were 1.9- to 4.9-fold higher in the aggrelyte-2- and aggrelyte-2C-treated samples than in the untreated samples. The LC-MS/MS results showed Nε -acetyllysine (AcK) levels of 1.5 to 2.1 nmol/mg protein and 0.6 to 0.9 nmol/mg protein in the aggrelyte-2- and aggrelyte-2C-treated samples. Mouse (C57BL/6J) lenses (incubated for 24 h) and human lenses (incubated for 72 h) with 1.0 mM aggrelyte-2 showed significant decreases in stiffness with simultaneous increases in soluble proteins (human lenses) and protein-AcK levels, and such changes were not observed in aggrelyte-2C-treated lenses. Mass spectrometry of the solubilized protein revealed AcK in all crystallins, but more was observed in α-crystallins. These results suggest that aggrelyte-2 increases protein solubility and decreases lens stiffness through acetylation and disulfide reduction. Aggrelyte-2 might be useful in treating presbyopia in humans.

Promotion of Protein Solubility and Reduction in Stiffness in Human Lenses by Aggrelyte-1: Implications for Reversing Presbyopia.

With aging, human lenses lose the ability to focus on nearby objects due to decreases in accommodative ability, a condition known as presbyopia. An increase in stiffness or decrease in lens elasticity due to protein aggregation and insolubilization are the primary reasons for presbyopia. In this study, we tested aggrelyte-1 (S,N-diacetyl glutathione diethyl ester) for its ability to promote protein solubility and decrease the stiffness of lenses through its dual property of lysine acetylation and disulfide reduction. Treatment of water-insoluble proteins from aged human lenses (58-75 years) with aggrelyte-1 significantly increased the solubility of those proteins. A control compound that did not contain the S-acetyl group (aggrelyte-1C) was substantially less efficient in solubilizing water-insoluble proteins. Aggrelyte-1-treated solubilized protein had significant amounts of acetyllysine, as measured by Western blotting and LC-MS/MS. Aggrelytes increased the protein-free thiol content in the solubilized protein. Aged mouse (7 months) and human (44-66 years) lenses treated with aggrelyte-1 showed reduced stiffness accompanied by higher free thiol and acetyllysine levels compared with those treated with aggrelyte-1C or untreated controls. Our results suggested that aggrelyte-1 reduced lens stiffness through acetylation followed by disulfide reduction. This proof-of-concept study paves the way for developing aggrelyte-1 and related compounds to reverse presbyopia.

Topical ocular application of aggrelyte-2A reduces lens stiffness in mice.

Presbyopia is the progressive loss of the ability of the lens to focus on nearby objects due to its increased stiffness. It occurs in the mid-40s and continues to worsen until the mid-60s. The age-associated increase in protein cross-linking in the lens leads to protein aggregation and water insolubility, especially in the nuclear region, contributing to lens stiffness. This study reports the development of aggrelyte-2A (methyl S-acetyl-N-(3,3-dimethylbutanoyl) cysteinate, a derivative of our previously reported aggrelyte-2) for reversing the stiffness of aged lenses. Aggrelyte-2A showed minimal toxicity in cultured mouse lens epithelial cells (up to 2000 µM) and human lens epithelial cells (up to 250 µM). Lenses from aged mice (age: 24-25 months) treated with 1 mM aggrelyte-2A for 24 h, and human lenses (age: 47-67 years) treated with 250 µM aggrelyte-2A for 48 h showed 11-14% reductions in stiffness, accompanied by an increase in acetyllysine in lens proteins, and free-thiols in the lens. Topical application of aggrelyte-2A (40 mM, 5 µl twice daily for 4 weeks) on mouse eyes significantly reduced lens stiffness. The topical application showed no toxicity to the lens, cornea, or retina, as revealed by morphological examination, H&E staining, and optical coherence tomography. These data suggest that aggrelyte-2A could be developed as a presbyopia-reversing therapeutic.

AAV2-Mediated Expression of HspB1 in RGCs Prevents Somal Damage and Axonal Transport Deficits in a Mouse Model of Ocular Hypertension.

Purpose: Ocular hypertension is a significant risk factor for vision loss in glaucoma caused by the death of retinal ganglion cells (RGCs). We investigated whether small heat shock proteins (sHsps) expressed in RGCs protect those cells against ocular hypertension in mice. Methods: AAV2 vectors encoding genes for one of the following four human sHsps: HSPB1, HSPB4, HSPB5, or HSPB6 were constructed for RGC-specific expression. Ischemia/reperfusion was induced by elevating the intraocular pressure (IOP) to 120 mm Hg for one hour, followed by a rapid return to normal IOP. Microbeads (MB) were injected into the anterior chamber of mice to induce ocular hypertension. RGC death and glial activation were assessed by immunostaining for Brn3a, RBPMS, Iba1, and glial fibrillary acid protein in retinal flat mounts. RGC axonal defects were evaluated by anterograde transport of intravitreally injected cholera toxin-B. RGC function was assessed by pattern electroretinography. Results: Among the sHsps, HspB1 offered the best protection against RGC death from ischemia/reperfusion injury in the mouse retina. Intravitreal administration of AAV2-HSPB1 either two weeks before or one week after instituting ocular hypertension resulted in significant prevention of RGC loss. The MB-injected mice showed RGC axonal transportation defects, but AAV2-HSPB1 administration significantly inhibited this defect. AAV2-HSPB1 prevented glial activation caused by ocular hypertension. More importantly, a single injection of AAV2-HSPB1 protected RGCs long-term in MB-injected eyes. Conclusions: The administration of AAV2-HSPB1 inhibited RGC death and axonal transport defects and reduced glial activation in a mouse model of ocular hypertension. Translational Relevance: Our results suggested that the intravitreal delivery of AAV2-HSPB1 could be developed as a gene therapy to prevent vision loss on a long-term basis in glaucoma patients.

Peptains block retinal ganglion cell death in animal models of ocular hypertension: implications for neuroprotection in glaucoma.

Ocular hypertension is a significant risk factor for vision loss in glaucoma due to the death of retinal ganglion cells (RGCs). This study investigated the effects of the antiapoptotic peptides peptain-1 and peptain-3a on RGC death in vitro in rat primary RGCs and in mouse models of ocular hypertension. Apoptosis was induced in primary rat RGCs by trophic factor deprivation for 48 h in the presence or absence of peptains. The effects of intravitreally injected peptains on RGC death were investigated in mice subjected to retinal ischemic/reperfusion (I/R) injury and elevated intraocular pressure (IOP). I/R injury was induced in mice by elevating the IOP to 120 mm Hg for 1 h, followed by rapid reperfusion. Ocular hypertension was induced in mice by injecting microbeads (MB) or silicone oil (SO) into the anterior chamber of the eye. Retinal flatmounts were immunostained with RGC and activated glial markers. Effects on anterograde axonal transport were determined by intravitreal injection of cholera toxin-B. Peptain-1 and peptain-3a inhibited neurotrophic factor deprivation-mediated RGC apoptosis by 29% and 35%, respectively. I/R injury caused 52% RGC loss, but peptain-1 and peptain-3a restricted RGC loss to 13% and 16%, respectively. MB and SO injections resulted in 31% and 36% loss in RGCs following 6 weeks and 4 weeks of IOP elevation, respectively. Peptain-1 and peptain-3a inhibited RGC death; the loss was only 4% and 12% in MB-injected eyes and 16% and 15% in SO-injected eyes, respectively. Anterograde transport was defective in eyes with ocular hypertension, but this defect was substantially ameliorated in peptain-injected eyes. Peptains suppressed ocular hypertension-mediated retinal glial activation. In summary, our results showed that peptains block RGC somal and axonal damage and neuroinflammation in animal models of glaucoma. We propose that peptains have the potential to be developed as therapeutics against neurodegeneration in glaucoma.

Small Heat Shock Proteins in Retinal Diseases.

This review summarizes the latest findings on small heat shock proteins (sHsps) in three major retinal diseases: glaucoma, diabetic retinopathy, and age-related macular degeneration. A general description of the structure and major cellular functions of sHsps is provided in the introductory remarks. Their role in specific retinal diseases, highlighting their regulation, role in pathogenesis, and possible use as therapeutics, is discussed.

Advanced glycation end products in human diabetic lens capsules.

Advanced glycation end products (AGEs) accumulate with age in human lens capsules. AGEs in lens capsules potentiate the transforming growth factor beta-2-mediated mesenchymal transition of lens epithelial cells, which suggests that they play a role in posterior capsule opacification after cataract surgery. We measured AGEs by liquid chromatography-mass spectrometry in capsulorhexis specimens obtained during cataract surgery from nondiabetic and diabetic patients with and without established retinopathy. Our data showed that the levels of most AGEs (12 out of 13 measured) were unaltered in diabetic patients and diabetic patients with retinopathy compared to nondiabetic patients. There was one exception: glucosepane, which was significantly higher in diabetic patients, both with (6.85 pmol/µmol OH-proline) and without retinopathy (8.32 pmol/µmol OH-proline), than in nondiabetic patients (4.01 pmol/µmol OH-proline). Our study provides an explanation for the similar incidence of posterior capsule opacification between nondiabetic and diabetic cataract patients observed in several studies.

Transforming growth factor-ß2-mediated mesenchymal transition in lens epithelial cells is repressed in the absence of RAGE.

Transforming growth factor-ß2 (TGFß2)-mediated epithelial to mesenchymal transition (EMT) in lens epithelial cells (LECs) has been implicated in fibrosis associated with secondary cataracts. In this study, we investigated whether the receptor for advanced glycation end products (RAGE) plays a role in TGFß2-mediated EMT in LECs. Unlike in the LECs from wild-type mice, TGFß2 failed to elicit an EMT response in LECs from RAGE knockout mice. The lack of RAGE also diminished TGFß2-mediated Smad signaling. In addition, treatment with TGFß2 increased IL-6 levels in LECs from wild-type mice but not in those from RAGE knockout mice. Treatment of human LECs with the RAGE inhibitor FPS-ZM1 reduced TGFß2-mediated Smad signaling and the EMT response. Unlike that in wild-type lenses, the removal of fiber cell tissue in RAGE knockout lenses did not result in elevated levels of α-smooth muscle actin (α-SMA), fibronectin (FN), and integrin ß1 in capsule-adherent LECs. Taken together, these results suggest that TGFß2 signaling is intricately linked to RAGE. Targeting RAGE could be explored as a therapeutic strategy against secondary cataracts.

Glycation-mediated protein crosslinking and stiffening in mouse lenses are inhibited by carboxitin in vitro.

Proteins in the eye lens have negligible turnover and therefore progressively accumulate chemical modifications during aging. Carbonyls and oxidative stresses, which are intricately linked to one another, predominantly drive such modifications. Oxidative stress leads to the loss of glutathione (GSH) and ascorbate degradation; this in turn leads to the formation of highly reactive dicarbonyl compounds that react with proteins to form advanced glycation end products (AGEs). The formation of AGEs leads to the crosslinking and aggregation of proteins contributing to lens aging and cataract formation. To inhibit AGE formation, we developed a disulfide compound linking GSH diester and mercaptoethylguanidine, and we named it carboxitin. Bovine lens organ cultured with carboxitin showed higher levels of GSH and mercaptoethylguanidine in the lens nucleus. Carboxitin inhibited erythrulose-mediated mouse lens protein crosslinking, AGE formation and the formation of 3-deoxythreosone, a major ascorbate-derived AGE precursor in the human lens. Carboxitin inhibited the glycation-mediated increase in stiffness in organ-cultured mouse lenses measured using compressive mechanical strain. Delivery of carboxitin into the lens increases GSH levels, traps dicarbonyl compounds and inhibits AGE formation. These properties of carboxitin could be exploited to develop a therapy against the formation of AGEs and the increase in stiffness that causes presbyopia in aging lenses.

Transient elevation of temperature promotes cross-linking of α-crystallin-client proteins through formation of advanced glycation endproducts: A potential role in presbyopia and cataracts.

The chaperone activity of α-crystallin is important for maintaining the transparency of the human lens. αB-crystallin (αBC) is a long-lived protein in the lens that accumulates chemical modifications during aging. The formation of advanced glycation end products (AGEs) through glycation is one such modification. αBC is a small heat shock protein that exhibits chaperone activity. We have previously shown that αBC-client protein complexes can undergo AGE-mediated interprotein cross-linking. Here, we demonstrate that short-term (1 h) exposure to elevated temperatures and methylglyoxal (MGO) during the chaperoning of client proteins by αBC promotes AGE-mediated interprotein cross-linking. Liquid chromatography/mass spectrometry (LC-MS/MS) analyses revealed the rapid formation of AGEs by MGO. Interestingly, we found that despite protein cross-linking, the chaperone activity of αBC increased during the transient elevation of temperature in the presence of MGO. Together, these results imply that transient and subtle elevation of temperature in the lens of the eye can promote protein cross-linking through AGEs, and if this phenomenon recurs over a period of many years, it could lead to early onset of presbyopia and age-related cataracts.

Glycation-mediated inter-protein cross-linking is promoted by chaperone-client complexes of α-crystallin: Implications for lens aging and presbyopia.

Lens proteins become increasingly cross-linked through nondisulfide linkages during aging and cataract formation. One mechanism that has been implicated in this cross-linking is glycation through formation of advanced glycation end products (AGEs). Here, we found an age-associated increase in stiffness in human lenses that was directly correlated with levels of protein-cross-linking AGEs. α-Crystallin in the lens binds to other proteins and prevents their denaturation and aggregation through its chaperone-like activity. Using a FRET-based assay, we examined the stability of the αA-crystallin-γD-crystallin complex for up to 12 days and observed that this complex is stable in PBS and upon incubation with human lens-epithelial cell lysate or lens homogenate. Addition of 2 mm ATP to the lysate or homogenate did not decrease the stability of the complex. We also generated complexes of human αA-crystallin or αB-crystallin with alcohol dehydrogenase or citrate synthase by applying thermal stress. Upon glycation under physiological conditions, the chaperone-client complexes underwent greater extents of cross-linking than did uncomplexed protein mixtures. LC-MS/MS analyses revealed that the levels of cross-linking AGEs were significantly higher in the glycated chaperone-client complexes than in glycated but uncomplexed protein mixtures. Mouse lenses subjected to thermal stress followed by glycation lost resilience more extensively than lenses subjected to thermal stress or glycation alone, and this loss was accompanied by higher protein cross-linking and higher cross-linking AGE levels. These results uncover a protein cross-linking mechanism in the lens and suggest that AGE-mediated cross-linking of α-crystallin-client complexes could contribute to lens aging and presbyopia.

Kynurenic Acid Protects Against Ischemia/Reperfusion-Induced Retinal Ganglion Cell Death in Mice.

BACKGROUND: Glaucoma is an optic neuropathy and involves the progressive degeneration of retinal ganglion cells (RGCs), which leads to blindness in patients. We investigated the role of the neuroprotective kynurenic acid (KYNA) in RGC death against retinal ischemia/reperfusion (I/R) injury. METHODS: We injected KYNA intravenously or intravitreally to mice. We generated a knockout mouse strain of kynurenine 3-monooxygenase (KMO), an enzyme in the kynurenine pathway that produces neurotoxic 3-hydroxykynurenine. To test the effect of mild hyperglycemia on RGC protection, we used streptozotocin (STZ) induced diabetic mice. Retinal I/R injury was induced by increasing intraocular pressure for 60 min followed by reperfusion and RGC numbers were counted in the retinal flat mounts. RESULTS: Intravenous or intravitreal administration of KYNA protected RGCs against I/R injury. The I/R injury caused a greater loss of RGCs in wild type than in KMO knockout mice. KMO knockout mice had mildly higher levels of fasting blood glucose than wild type mice. Diabetic mice showed significantly lower loss of RGCs when compared with non-diabetic mice subjected to I/R injury. CONCLUSION: Together, our study suggests that the absence of KMO protects RGCs against I/R injury, through mechanisms that likely involve higher levels of KYNA and glucose.

Aspirin inhibits TGFß2-induced epithelial to mesenchymal transition of lens epithelial cells: selective acetylation of K56 and K122 in histone H3.

Posterior capsule opacification (PCO) is a complication after cataract surgery that can disrupt vision. The epithelial to mesenchymal transition (EMT) of lens epithelial cells (LECs) in response to transforming growth factor ß2 (TGFß2) has been considered an obligatory mechanism for PCO. In this study, we tested the efficacy of aspirin in inhibiting the TGFß2-mediated EMT of human LECs, LECs in human lens capsular bags, and lensectomized mice. In human LECs, the levels of the EMT markers α-smooth muscle actin (α-SMA) and fibronectin were drastically reduced by treatment with 2 mM aspirin. Aspirin also halted the EMT response of TGFß2 when introduced after EMT initiation. In human capsular bags, treatment with 2 mM aspirin significantly suppressed posterior capsule wrinkling and the expression α-SMA in capsule-adherent LECs. The inhibition of TGFß2-mediated EMT in human LECs was not dependent on Smad phosphorylation or MAPK and AKT-mediated signaling. We found that aspirin significantly increased the acetylation of K56 and K122 in histone H3 of human LECs. Chromatin immunoprecipitation assays using acetyl-H3K56 or acetyl-H3K122 antibody revealed that aspirin blocked the TGFß2-induced acetylation of H3K56 and H3K122 at the promoter regions of ACTA2 and COL1A1. After lensectomy in mice, we observed an increase in the proliferation and α-SMA expression of the capsule-adherent LECs, which was ameliorated by aspirin administration through drinking water. Taken together, our results showed that aspirin inhibits TGFß2-mediated EMT of LECs, possibly from epigenetic down-regulation of EMT-related genes.

Lysine malonylation and propionylation are prevalent in human lens proteins.

Acylated lysine residues represent major chemical modifications in proteins. We investigated the malonylation and propionylation of lysine residues (MalK, PropK) in the proteins of aging human lenses. Western blot results showed that the two modifications are present in human lens proteins. Liquid chromatography-mass spectrometry (LC-MS/MS) results showed 4-18 and 4-32 pmol/mg protein of MalK and PropK, respectively, in human lens proteins with no apparent changes related to aging. Mass spectrometry results revealed that MalK- and PropK-modified lysine residues are present in all major crystallins, other cytosolic proteins, and membrane and cytoskeletal proteins of the lens. Several mitochondrial and cytosolic proteins in cultured human lens epithelial cells showed MalK and PropK modifications. Sirtuin 3 (SIRT3) and sirtuin 5 (SIRT5) were present in human lens epithelial and fiber cells. Moreover, lens epithelial cell lysate deacylated propionylated and malonylated lysozyme. The absence of SIRT3 and SIRT5 led to higher PropK and MalK levels in mouse lenses. Together, these data suggest that MalK and PropK are widespread modifications in lens and SIRT3 and SIRT5 could regulate their levels in lens epithelial cells.

Correction: Systemically administered peptain-1 inhibits retinal ganglion cell death in animal models: implications for neuroprotection in glaucoma.

[This corrects the article DOI: 10.1038/s41420-019-0194-2.].