Purpose:Prevalence of diabetic retinopathy closely depends on the duration of diabetes and severity of hyperglycemia. Experimental models have shown that high glucose initiates many metabolic, molecular and epigenetic changes in the retina before vascular histopathology is detectable. Several LncRNAs (RNAs with >200bp and no reading frame) are also aberrantly expressed in the retina in diabetes. These RNAs have high organ and cell specificity and show minimal gradual instability in plasma. Our goal was to examine the utility of aberrantly expressed retinal LncRNAs (nuclear DNA-encoded NEAT1, HOTAIR, HOTTIP, MALAT1, H19 and Meg3 and mitochondrial DNA-encoded CytB) in diabetes as possible biomarkers of diabetic retinopathy. Methods:Plasma LncRNAs were analyzed by qRT-PCR in diabetic patients with proliferative (PDR), or no retinopathy (No-DR), and in rats with streptozotocin-induced diabetes for two to six months. The results were confirmed in the retina from donors with diabetic retinopathy and from diabetic rats. Results:Plasma NEAT1, HOTAIR, MALAT1 and HOTTIP were upregulated and Meg3 and CytB were downregulated in No-DR and PDR groups and retina from human donors with diabetic retinopathy. Although these LncRNAs were also aberrantly expressed in plasma and retina within four to six months of diabetes in rats, NEAT1 and CytB had abnormal expression within two months of diabetes (without any retinal vascular histopathology). Conclusions:NEAT1 and CytB have consistent expression patterns in plasma from diabetic patients and plasma and retina from rodents. Translational Relevance:Plasma NEAT1 and CytB could serve as possible early noninvasive biomarkers of retinopathy in diabetic patients.

[This corrects the article DOI: 10.3389/fendo.2023.1160155.].

Introduction and Objective: Diabetes damages retinal mitochondria and impairs their removal by mitophagy, and these damaged mitochondria continue to accumulate. Diabetes also results in aberrant expression of many long noncoding RNAs (LncRNAs), the RNA transcripts with >200 nucleotides. These noncoding RNAs can interact with RNA, DNA and proteins, regulating many physiological processes. Among them, LncRNA HOTAIR, a highly conserved LncRNA implicated in oxidative stress, apoptosis and inflammation, is upregulated in the retina, vitreous and serum in diabetes. Our aim was to investigate the role of HOTAIR in mitophagy in diabetic retinopathy. Methods: Human retinal endothelial cells, untransfected or HOTAIR-siRNA transfected, incubated in 20mM D-glucose, were analyzed for mitophagy by quantifying mitophagy flux (flow cytometrically using Mtphagy Dye) and mitophagosome formation (LysoTraker staining for lysosome and MitoTracker green for mitochondria). Since mitophagy is closely associated with mtDNA biogenesis, mtDNA copy numbers (ratio of mtDNA-encoded CoxIV and nuclear DNA-encoded β-actin) were quantified. For mitochondrial function, mitochondrial membrane potential (JC-1 staining) and ROS levels (MitoSox red) were measured. Results: Compared to untransfected cells in normal glucose (5mM D-glucose), high glucose upregulated HOTAIR expression by ~75%. HOTAIR-siRNA, in addition to preventing glucose-induced increase in HOTAIR expression, ameliorated decrease in mitophagy flux and mitophagosome formation. In the same HOTAIR-siRNA transfected cells, glucose-induced decrease in mtDNA copy numbers, increase in mitochondrial ROS and impairments in mitochondrial potential were also improved. Conclusion: HOTAIR upregulation in diabetes impedes removal of the damaged mitochondria, and the dysfunctional mitochondria continue to produce damaging ROS. Thus, targeting HOTAIR to restore mitochondrial integrity could provide a fresh insight into the removal of the damaged mitochondria in diabetic retinopathy. Disclosure R. Kowluru: None. J. Kumar: None. P.B. Malaviya: None. Funding R01 014370R01 033516

Mitochondrial dysfunction plays a major role in diabetic retinopathy development and in its resistance to halt after the reversal of hyperglycemia (metabolic memory). Diabetes also upregulates many long noncoding RNAs, RNAs with >200 nucleotides with no reading frame, and several of them resist reversal after hyperglycemia cessation. Our aim was to investigate the role of LncRNA HOTAIR, a master regulator of chromatin dynamics, in mitochondrial biogenesis in diabetic retinopathy and in metabolic memory. Using retinal endothelial cells and Müller cells, incubated in high glucose (20 mM D-glucose), the effect of HOTAIR-siRNA on mitochondrial biogenesis was investigated by quantifying mitochondrial mass, copy numbers, and mtDNA replication, structure, and function. HOTAIR’s role in metabolic memory was investigated by analyzing mitochondrial biogenesis in HOTAIR-siRNA transfected cells incubated in high glucose for four days, followed by normal glucose (5 mM D-glucose) for four days. HOTAIR was upregulated in both retinal vascular and nonvascular cells, and HOTAIR-siRNA ameliorated decreases in mtDNA biogenesis and protected their mitochondria from structural/functional damage. Reversal of high glucose insult failed to ameliorate HOTAIR upregulation and impaired mtDNA biogenesis in both endothelial and Müller cells, but regulation of HOTAIR during high glucose incubation, which followed normal glucose, prevented a decrease in mitochondrial mass and mtDNA copies. Thus, HOTAIR has a major role in mitochondrial biogenesis and in the continued impaired biogenesis in both vascular and nonvascular cells. Regulating HOTAIR may provide a therapeutic option to inhibit the development/progression of diabetic retinopathy.

Mitochondria are dynamic in nature and depending on the energy demand they fuse and divide. This fusion-fission process is impaired in diabetic retinopathy and the promoter DNA of Mfn2, a fusion gene, is hypermethylated and its expression is downregulated. Long noncoding RNAs (RNAs with >200 nucleotides that do not encode proteins) can regulate gene expression by interacting with DNA, RNA, and proteins. Several LncRNAs are aberrantly expressed in diabetes, and among them, MALAT1 is upregulated in the retina, altering the expression of the genes associated with inflammation. Our aim was to investigate MALAT1’s role in mitochondrial dynamics in diabetic retinopathy. Using MALAT1-siRNA-transfected human retinal endothelial cells (HRECs) and human retinal Muller cells (RMCs) incubated in 20 mM D-glucose, Mfn2 expression and activity and its promoter DNA methylation were quantified. Mitochondrial integrity was evaluated by analyzing their fragmentation, ultrastructure, membrane potential, and oxygen consumption rate. Compared to normal glucose, high glucose upregulated MALAT1 expression and downregulated Mfn2 expression and activity in both HRECs and RMCs. MALAT1-siRNA ameliorated the glucose-induced increase in Mfn2 promoter DNA hypermethylation and its activity. MALAT1-siRNA also protected against mitochondrial fragmentation, structural damage, and reductions in the oxygen consumption rate. In conclusion, the upregulation of MALAT1 in diabetes facilitates Mfn2 promoter DNA hypermethylation in retinal vascular and nonvascular cells, leading to its suppression and the accumulation of the fragmented/damaged mitochondria. Thus, the regulation of MALAT1 has the potential to protect mitochondria and provide a possible new target to inhibit/prevent the blinding disease in diabetic patients.

Introduction and Objective: Mitochondrial dysfunction plays a central role in diabetic retinopathy. Depending on the energy demand, mitochondria constantly fuse and divide, but in diabetic retinopathy, this process is impaired and Mfn2, a fusion gene, is downregulated due to its promoter DNA hypermethylation. Long noncoding RNAs (RNAs with >200 nucleotides) do not have open reading frame, but can regulate gene expression by interacting with DNA, RNA, and proteins. Many LncRNAs are aberrantly expressed in diabetes, and among those, MALAT1 is upregulated in the retina, altering expression of genes associated with inflammation. However, the role of MALAT1 in mitochondrial dynamics is not clear. Our aim was to investigate the role of MALAT1 in mitochondrial stability in diabetic retinopathy. Methods: Using MALAT1-siRNA transfected retinal endothelial cells incubated in 20mM D-glucose, Mfn2 expression (transcripts and protein), and its promoter DNA methylation (5-methyl cytosine by MeDIP) and GTPase activity were quantified. Mitochondrial integrity was evaluated by analyzing mitochondrial fragmentation (MitoTracker green staining), structure (electron microscopy and membrane potential), mtDNA damage (extended length PCR and nucleoids by SYBR green staining), and function (oxygen consumption rate by SeaHorse) Results: Compared to normal glucose, high glucose upregulated MALAT1 expression, and MALAT1-siRNA ameliorated glucose-induced decrease in Mfn2 expression, activity and its promoter DNA hypermethylation. MALAT1-siRNA also protected glucose-induced increase in mitochondrial fragmentation, structural and DNA damage and reduction in oxygen consumption rate. Conclusion: Upregulation of MALAT1 in hyperglycemia hypermethylates Mfn2 promoter DNA and downregulates its transcription, resulting in impaired mitochondrial dynamics, and structural, genomic and functional instability. This opens an avenue to target MALAT1 and restore mitochondrial integrity in diabetic retinopathy. R. Kowluru: None. J. Kumar: None. NIH (R01 EY333516)

Diabetic retinopathy, a microvascular complication of diabetes, is the leading cause of blindness in adults, but the molecular mechanism of its development remains unclear. Retinal mitochondrial DNA is damaged and hypermethylated, and mtDNA-encoded genes are downregulated. Expression of a long noncoding RNA (larger than 200 nucleotides, which does not translate into proteins), encoded by mtDNA, cytochrome B (LncCytB), is also downregulated. This study aims to investigate the role of DNA methylation in the downregulation of LncCytB in diabetic retinopathy. Human retinal endothelial cells, incubated in 5mM (normal) or 20mM (high) D-glucose, in the presence/absence of Azacytidine (a DNA methyl transferase inhibitor) were analyzed for LncCytB DNA methylation by immunoprecipitation and methylation specific PCR techniques, and LncCytB transcripts by strand-specific PCR and RNA-FISH. Mitochondrial genomic stability was evaluated by quantifying protective mtDNA nucleoids by SYBR green staining and by flow cytometry, and functional stability by oxygen consumption rate using Seahorse analyzer. Results were confirmed in an in vivo model using retina from diabetic rat. While high glucose elevated 5mC and the ratio of methylated to unmethylated amplicons at LncCytB and downregulated its transcripts, azacytidine prevented LncCytB DNA hypermethylation and decrease in its expression. Azacytidine also ameliorated decrease in nucleoids and oxygen consumption rate. Similarly, azacytidine prevented increase in retinal LncCytB DNA methylation and decrease in its expression in diabetic rats. Thus, DNA hypermethylation plays a major role in the downregulation of retinal LncCytB in diabetes, resulting in impaired mitochondrial homeostasis, and culminating in the development of diabetic retinopathy.

Progression of diabetic retinopathy resists arrest even after institution of intensive glycemic control, suggesting a "metabolic memory" phenomenon, but the mechanism responsible for this phenomenon is still elusive. Gene expression and biological processes can also be regulated by long noncoding RNAs (LncRNAs), the RNAs with >200 nucleotides and no open reading frame for translation, and several LncRNAs are aberrantly expressed in diabetes. Our aim was to identify retinal LncRNAs that fail to reverse after termination of hyperglycemia. Microarray analysis was performed on retinal RNA from streptozotocin-induced diabetic rats in poor glycemic control for 8 months, followed by in good glycemic control (blood glucose >400 mg/dL), or for 4 months, with four additional months of good glycemic control (blood glucose <150 mg/dL). Differentially expressed LncRNAs and mRNAs were identified through Volcano filtering, and their functions were predicted using gene ontology and pathway enrichment analyses. Compared with age-matched normal rats, rats in continuous poor glycemic control had >1479 differentially expressed LncRNAs (710 downregulated, 769 upregulated), and among those, 511 common LncRNAs had similar expression in Diab and Rev groups (139 downregulated, 372 upregulated). Gene Ontology/pathway analysis identified limited LncRNAs in biological processes, but analysis based on biological processes/molecular function revealed >350 genes with similar expression in Diab and Rev groups; these genes were mainly associated with stress response, cell death, mitochondrial damage and cytokine production. Thus, identifying retinal LncRNAs and their gene targets that do not benefit from termination of hyperglycemia have potential to serve as therapeutic targets to slow down the progression of diabetic retinopathy.

Diabetic retinopathy is driven by oxidative stress-mitochondrial damage. Activation of ROS producing cytosolic NADPH oxidase 2 (Nox2) in diabetes precedes retinal mitochondrial damage, initiating a vicious cycle of free radicals. Elevated ROS levels peroxidize membrane lipids increasing damaging lipid peroxides (LPOs). While glutathione peroxidase 4 (GPx4) neutralizes LPOs, an imbalance in its generation-neutralization leads to ferroptosis, which is characterized by increased LPOs, free iron and decreased GPx4 activity. Mitochondria are rich in polyunsaturated fatty acids and iron and have mitochondrial isoform of GPx4. Our aim was to investigate mitochondrial ferroptosis in diabetic retinopathy, focusing on Nox2 mediated ROS production. Using human retinal endothelial cells, incubated in 5 mM or 20 mM Dglucose for 12-96 h, with or without Nox2 inhibitors (100 mu M apocynin, 5 mu M EHop-016 or 5 mu M Gp91 ds-tat), or ferroptosis inhibitors (1 mu M ferrostatin-1, 50 mu M deferoxamine) or activator (0.1 mu M RSL3), cytosolic and mitochondrial ROS, LPOs, iron, GPx4 activity, mitochondrial integrity (membrane permeability, oxygen consumption rate, mtDNA copy numbers) and cell death were quantified. High glucose significantly increased ROS, LPOs and iron levels and inhibited GPx4 activity in cytosol, and while Nox2 and ferroptosis inhibitors prevented glucose-induced increase in ferroptosis markers, mitochondrial damage and cell death, RSL3, further worsened them. Furthermore, high glucose also increased ferroptosis markers in the mitochondria, which followed their increase in the cytosol, suggesting a role of cytosolic ROS in mitochondrial ferroptosis. Thus, targeting Nox2-ferroptosis should help break down the self-perpetuating vicious cycle of free radicals, initiated by the damaged mitochondria, and could provide novel therapeutics to prevent/retard the development of diabetic retinopathy.

AIM:Hyperglycemia damages mitochondria and downregulates transcription of mtDNA-encoded genes and the long noncoding RNA LncCytB, causing mitochondrial genomic instability. The genes encoded by mtDNA are transcribed as large polycistronic transcripts, and the 5' ends of precursor tRNAs are processed by mitochondrial-targeted ribonuclease P (MRPPs). Our aim was to investigate the role of MRPP1 in the downregulation of LncCytB in diabetic retinopathy. METHODS:Using human retinal endothelial cells incubated in 20 mM D-glucose for 96 h, the gene expression and mitochondrial localization (immunofluorescence) of MRPP1 and the interaction between MRPP1 and LncCytB (determined by RNA-FISH and RNA immunoprecipitation) were quantified. The results were confirmed in retinal microvessels from streptozotocin-induced diabetic mice and from human donors with documented diabetic retinopathy. RESULTS:Compared to normal glucose, high glucose decreased mRNA and mitochondrial localization of MRPP1 and its interaction with LncCytB. While MRPP1 overexpression prevented glucose-induced decrease in MRPP1-LncCytB interaction, LncCytB expression and mitochondrial damage (reduction in protective nucleoids in mtDNA), MRPP1-siRNA further worsened them. Similar results were obtained from retinal microvessels from diabetic mice and from human donors with diabetic retinopathy. CONCLUSIONS:Downregulation of MRPP1 in diabetes suppresses LncCytB transcription, resulting in mitochondrial functional and genomic instability, ultimately leading to the development of diabetic retinopathy. Thus, preventing MRPP1 downregulation has the potential to inhibit retinopathy and prevent the fear of vision loss in diabetic patients.

In diabetic retinopathy, mitochondrial DNA (mtDNA) is damaged and mtDNA-encoded genes and long noncoding RNA cytochrome B (LncCytB) are downregulated. LncRNAs lack an open reading frame, but they can regulate gene expression by associating with DNA/RNA/protein. Double stranded mtDNA has promoters on both heavy (HSP) and light (LSP) strands with binding sites for mitochondrial transcription factor A (TFAM) between them. The aim was to investigate the role of LncCytB in mtDNA transcription in diabetic retinopathy. Using human retinal endothelial cells incubated in high glucose, the effect of regulation of LncCytB on TFAM binding at mtDNA promoters was investigated by Chromatin immunoprecipitation, and binding of LncCytB at TFAM by RNA immunoprecipitation and RNA fluorescence in situ hybridization. High glucose decreased TFAM binding at both HSP and LSP, and binding of LncCytB at TFAM. While LncCytB overexpression ameliorated decrease in TFAM binding and transcription of genes encoded by both H- and L- strands, LncCytB-siRNA further downregulated them. Maintenance of mitochondrial homeostasis by overexpressing mitochondrial superoxide dismutase or Sirtuin-1 protected diabetes-induced decrease in TFAM binding at mtDNA and LncCytB binding at TFAM, and mtDNA transcription. Similar results were obtained from mouse retinal microvessels from streptozotocin-induced diabetic mice. Thus, LncCytB facilitates recruitment of TFAM at HSP and LSP, and its downregulation in diabetes compromises the binding, resulting in the downregulation of polypeptides encoded by mtDNA. Regulation of LncCytB, in addition to protecting mitochondrial genomic stability, should also help in maintaining the transcription of mtDNA encoded genes and electron transport chain integrity in diabetic retinopathy.

Retinopathy fails to halt even after diabetic patients in poor glycemic control try to institute tight glycemic control, suggesting a “metabolic memory” phenomenon, and the experimental models have demonstrated that mitochondria continue to be damaged/dysfunctional, fueling into the vicious cycle of free radicals. Our aim was to investigate the role of removal of the damaged mitochondria in the metabolic memory. Using human retinal endothelial cells (HRECs), incubated in 20 mM D-glucose for 4 days, followed by 5 mM D-glucose for 4 additional days, mitochondrial turnover, formation of mitophagosome, and mitophagy flux were evaluated. Mitophagy was confirmed in a rat model of metabolic memory where the rats were kept in poor glycemic control (blood glucose ~ 400 mg/dl) for 3 months soon after induction of streptozotocin-induced diabetes, followed by 3 additional months of good control (BG < 150 mg/dl). Reversal of high glucose by normal glucose had no effect on mitochondrial turnover and mitophagosome formation, and mitophagy flux remained compromised. Similarly, 3 months of good glycemic control in rats, which had followed 3 months of poor glycemic control, had no effect on mitophagy flux. Thus, poor turnover/removal of the damaged mitochondria, initiated during poor glycemic control, does not benefit from the termination of hyperglycemic insult, and the damaged mitochondria continue to produce free radicals, suggesting the importance of mitophagy in the metabolic memory phenomenon associated with the continued progression of diabetic retinopathy.

Aims: Mitochondrial dysfunction is closely associated with the development of diabetic complications. In diabetic retinopathy, electron transport chain is compromised and mitochondrial DNA (mtDNA) is damaged, downregulating transcription of mtDNA-encoded cytochrome B (CYTB) and its antisense long noncoding RNA, long noncoding RNA cytochrome B (LncCytB). Our goal was to investigate the role of LncCytB in the regulation of CYTB and mitochondrial function in diabetic retinopathy. Methods: Using human retinal endothelial cells, genetically manipulated for LncCytB (overexpression or silencing), the effect of high glucose (20 mM d-glucose) on LncCytB-CYTB interactions (by chromatin isolation by RNA purification), CYTB gene expression (by real-time quantitative polymerase chain reaction), complex III activity, mitochondrial free radicals, and oxygen consumption rate (OCR, by Seahorse XF analyzer) was investigated. Key results were confirmed in the retinal microvessels from streptozotocin-induced diabetic mice. Results: High glucose decreased LncCytB-CYTB interactions, and while LncCytB overexpression ameliorated glucose-induced decrease in CYTB gene transcripts, complex III activity and OCR and increase in mitochondrial reactive oxygen species, LncCytB-siRNA further attenuated CYTB gene transcription, complex III activity, and OCR. Similar decrease in LncCytB-CYTB interactions and CYTB transcription was observed in diabetic mice. Furthermore, maintenance of mitochondrial homeostasis by overexpressing superoxide dismutase or sirtuin 1 in mice ameliorated diabetes-induced decrease in LncCytB-CYTB interactions and CYTB gene transcripts, and also improved complex III activity and mitochondrial respiration. Innovation and Conclusion: LncCytB downregulation in hyperglycemic milieu downregulates CYTB transcription, which inhibits complex III activity and compromises mitochondrial stability and OCR. Thus, preventing LncCytB downregulation in diabetes has potential of inhibiting the development of diabetic retinopathy, possibly via maintaining mitochondrial respiration. Antioxid. Redox Signal. 39, 817-828.

Mitochondria experience genomic and functional instability in diabetes, and mitochondrial dysfunction has a critical role in the development of diabetic retinopathy. Diabetes also alters expressions of many long noncoding RNAs (LncRNAs), the RNAs with >200 nucleotides and no open reading frame. LncRNAs are mainly encoded by the nuclear genome, but mtDNA also encodes three LncRNAs. Our goal was to investigate the effect of hyperglycemia on mtDNA-encoded LncRNA cytochrome B (LncCytB) in mtDNA stability in diabetic retinopathy. Retinal endothelial cells, transfected with LncCytB-overexpressing plasmids or siRNA, incubated in 5 mmol/L d-glucose (normal glucose [NG]) or 20 mmol/L d-glucose (high glucose [HG]) for 4 days, were analyzed for LncCytB expression by strand-specific PCR and its mitochondrial localization by RNA fluorescence in situ hybridization. Damage-sensitive mtDNA regions were examined by micrococcal nuclease (MNase) digestion sequencing and LncCytB occupancy at mtDNA by chromatin isolation by RNA purification. Protective nucleoids in mtDNA were analyzed by SYBR Green-MitoTracker Red staining and confirmed in isolated mitochondria by flow cytometry. Compared with NG, HG downregulated LncCytB by >50% but had no significant effect on the other mtDNA-encoded LncRNAs. mtDNA packaging was impaired, MNase sensitivity was increased, and LncCytB occupancy at mtDNA was decreased. While LncCytB overexpression ameliorated mtDNA damage and decrease in nucleoids and copy numbers, LncCytB-siRNA exacerbated damage and further reduced nucleoids. Retinal microvessels from streptozotocin-induced diabetic mice and human donors with diabetic retinopathy presented a similar decrease in LncCytB and mtDNA nucleoids. Thus, LncCytB has a major role in maintaining mitochondrial genomic stability, and its downregulation in the hyperglycemic milieu contributes to increased vulnerability of mtDNA to damage.ARTICLE HIGHLIGHTS:

Introduction:Mitochondrial dynamic plays a major role in their quality control, and the damaged mitochondrial components are removed by autophagy. In diabetic retinopathy, mitochondrial fusion enzyme, mitofusin 2 (Mfn2), is downregulated and mitochondrial dynamic is disturbed resulting in depolarized and dysfunctional mitochondria. Our aim was to investigate the mechanism of inhibition of Mfn2, and its role in the removal of the damaged mitochondria, in diabetic retinopathy. Methods:Using human retinal endothelial cells, effect of high glucose (20mM) on the GTPase activity of Mfn2 and its acetylation were determined. Role of Mfn2 in the removal of the damaged mitochondria was confirmed by regulating its acetylation, or by Mfn2 overexpression, on autophagosomes- autolysosomes formation and the mitophagy flux. Results:High glucose inhibited GTPase activity and increased acetylation of Mfn2. Inhibition of acetylation, or Mfn2 overexpression, attenuated decrease in GTPase activity and mitochondrial fragmentation, and increased the removal of the damaged mitochondria. Similar phenomenon was observed in diabetic mice; overexpression of sirtuin 1 (a deacetylase) ameliorated diabetes-induced inhibition of retinal Mfn2 and facilitated the removal of the damaged mitochondria. Conclusions:Acetylation of Mfn2 has dual roles in mitochondrial homeostasis in diabetic retinopathy, it inhibits GTPase activity of Mfn2 and increases mitochondrial fragmentation, and also impairs removal of the damaged mitochondria. Thus, protecting Mfn2 activity should maintain mitochondrial homeostasis and inhibit the development/progression of diabetic retinopathy.

Purpose: Diabetic retinopathy has a complex pathogenesis, and mitochondrial damage is considered to play a central role. Diabetes dysfunctions the mitochondria and damages their DNA (mtDNA) , decreasing the transcription of mtDNA-encoded genes including Cytochrome B (CYTB) , which is important for the functioning of complex III of the electron transport chain system (ETC) . This propagate the vicious cycle of free radicals. Diabetes also decreases mtDNA-encoded long non-coding RNA Cytochrome B (LncCytB) . Although long noncoding RNAs have no open reading frame for translation, they can bind to the DNA or RNA in a sequence specific manner and alter the gene expression. Our aim was to investigate the role of LncCytB in regulating CYTB-ETC system in diabetic retinopathy. Methods: Human retinal endothelial cells, incubated in 5mM (normal) or 20mM (high) D-glucose, were used to quantify the expressions of LncCytB (by strand-specific PCR) and CYTB (qRT-PCR) . The binding of LncCytB at CYTB promoter was determined by Chromatin isolation by RNA Purification method. Role of LncCytB in regulating CYTB expression and mitochondrial damage was performed in the cells overexpressing LncCytB. Results: Compared to cells in normal glucose, LncCytB and CYTB levels were decreased by over 50% in the cells exposed to high glucose, the binding of LncCytB at CYTB promoter was reduced by ∼40% and the activity of the complex III was inhibited. Overexpression of LncCytB ameliorated glucose-induced decrease in CYTB gene transcripts, and increase in mtDNA damage and in mitochondrial reactive oxygen species. Conclusions: Due to decreased interactions between LncCytB-CYTB in hyperglycemic milieu, CYTB transcripts are decreased, and this compromises the ETC system, resulting in increased mitochondrial ROS. Thus, regulating LncCytB levels in diabetes could halt the self-propagating cycle of free radicals, and prevent the development of diabetic retinopathy. Funding EY014370, EY017313, and EY022230