Retinal Mitochondrial DNA Mismatch Repair in the Development of Diabetic Retinopathy, and Its Continued Progression After Termination of Hyperglycemia

Mishra, Manish; Kowluru, Renu A.

doi:10.1167/iovs.14-15020

Cited by 61 publications

(93 citation statements)

References 49 publications

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“…In the pathogenesis of diabetic retinopathy, mitochondria in retinal endothelium are swollen with partial crystolysis, and they are dysfunctional, with impaired respiration [39,41,[59][60][61]. The role of mitochondria in the development of diabetic retinopathy is further supported by our studies showing the protection of the development of diabetic retinopathy in mice overexpressing the gene encoding mitochondrial superoxide dismutase, MnSOD [39].…”

Section: Mitochondrial Damagementioning

confidence: 67%

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Oxidative stress, mitochondrial damage and diabetic retinopathy

Kowluru

Mishra

2015

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Diseas

272

246

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Diabetes has emerged as an epidemic of the 21st century, and retinopathy remains the leading cause of blindness in young adults and the mechanism of this blinding disease remains evasive. Diabetes-induced metabolic abnormalities have been identified, but a causal relationship between any specific abnormality and the development of this multi-factorial disease is unclear. Reactive oxygen species (ROS) are increased and the antioxidant defense system is compromised. Increased ROS result in retinal metabolic abnormalities, and these metabolic abnormalities can also produce ROS. Sustained exposure to ROS damages the mitochondria and compromises the electron transport system (ETC), and, ultimately, the mitochondrial DNA (mtDNA) is damaged. Damaged mtDNA impairs its transcription, and the vicious cycle of ROS continues to propagate. Many genes important in generation and neutralization of ROS are also epigenetically modified further increasing ROS, and the futile cycle continues to fuel in. Antioxidants have generated beneficial effects in ameliorating retinopathy in diabetic rodents, but limited clinical studies have not been encouraging. With the ongoing use of antioxidants for other chronic diseases, there is a need for a controlled trial to recognize their potential in ameliorating the development of this devastating disease.

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Section: Mitochondrial Damagementioning

confidence: 67%

“…In addition, the number of sequence variants is increased [60]. Although mtDNA encodes only 13 proteins, these are essential for proper functioning of the electron transport chain and for the mitochondrial homeostasis [65].…”

Section: Accepted Manuscriptmentioning

confidence: 99%

Oxidative stress, mitochondrial damage and diabetic retinopathy

Kowluru

Mishra

2015

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Diseas

272

246

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“…11,17 Complementary DNA was synthesized from 1 μg total RNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA), and transcript abundance was quantified by SYBR green–based quantitative real-time PCR (q-PCR) using gene- and species-specific primers (Table). The results were normalized to the cycle threshold (Ct) value from β-actin in the same sample, and the relative fold change in the expression of target genes was calculated using the delta Ct method.…”

Section: Methodsmentioning

confidence: 99%

“…The results were normalized to the cycle threshold (Ct) value from β-actin in the same sample, and the relative fold change in the expression of target genes was calculated using the delta Ct method. 11,15,20 …”

Section: Methodsmentioning

confidence: 99%

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The Role of DNA Methylation in the Metabolic Memory Phenomenon Associated With the Continued Progression of Diabetic Retinopathy

Mishra

Kowluru

2016

Invest. Ophthalmol. Vis. Sci.

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PurposeClinical and experimental studies have shown that diabetic retinopathy progression does not halt after termination of hyperglycemia, suggesting a “metabolic memory” phenomenon. DNA is highly dynamic, and cytosine methylation changes can last for several years. In diabetes, DNA methylation regulates expression of many genes associated with retinal mitochondrial homeostasis. Our aim was to investigate the role of DNA methylation in the metabolic memory.MethodsReversal of 4 days of 20 mM glucose by 4 to 8 days of 5 mM glucose, in the presence/absence of Dnmt inhibitor (5-aza-2′-deoxycytidine), was investigated on DNA methylation and its machinery in human retinal endothelial cells. The key parameters were confirmed in the retina from diabetic rats maintained in good glycemic control (glycated hemoglobin ∼6%) for 3 months after 3 months of poor control (glycated hemoglobin >10%).ResultsDNA methyltransferase 1 (Dnmt 1) remained active after 4 days of normal glucose that followed 4 days of high glucose, and mtDNA stayed hypermethylated with impaired transcription. Hydroxymethylating enzyme Tet2, and matrix metalloproteinase-9 (regulated by hydroxymethylation) also remained upregulated. But, 8 days of normal glucose after 4 days of high glucose ameliorated mtDNA methylation and MMP-9 hydroxymethylation. Direct Dnmt targeting by Aza during the reversal period benefited methylation status of mtDNA and MMP-9 DNA. Similarly, reinstitution of good control after 3 months of poor control in rats did not reverse diabetes-induced increase in retinal Dnmt1 and Tet2, and alter the methylation status of mtDNA and MMP-9.ConclusionsRetinal DNA methylation-hydroxymethylation machinery does not benefit immediately from reversal of hyperglycemia. Maintenance of good glycemic control for longer duration, and/or direct targeting DNA methylation ameliorates continuous mitochondrial damage, and could retard/halt diabetic retinopathy progression.

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Development of novel quality control material based on CRISPR/Cas9 editing and xenografts for MLH1 protein deficiency testing

Zhang

Tan

et al. 2021

Clinical Laboratory Analysis

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Background Mismatch repair deficiency (dMMR) status induced by MLH1 protein deficiency plays a pivotal role in therapeutic decision‐making for cancer patients. Appropriate quality control (QC) materials are necessary for monitoring the accuracy of MLH1 protein deficiency assays used in clinical laboratories. Methods CRISPR/Cas9 technology was used to edit the MLH1 gene of GM12878Cas9 cells to establish MLH1 protein‐deficient cell lines. The positive cell lines were screened and validated by Sanger sequencing, Western blot (WB), and next‐generation sequencing (NGS) and were then used to prepare formalin‐fixed, paraffin‐embedded (FFPE) samples through xenografting. These FFPE samples were tested by hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) for suitability as novel QC materials for MLH1 protein deficiency testing. Results We successfully cultured 358 monoclonal cells, with a survival rate of 37.3% (358/960) of the sorted monoclonal cells. Through Sanger sequencing, cell lines with MLH1 gene mutation were identified. Subsequently, two cell lines with MLH1 protein deficiency were identified by WB and named as GM12878Cas9_6 and GM12878Cas9_10. The NGS results further confirmed that the MLH1 gene mutation in these two cell lines would cause the formation of stop codons and terminate the expression of the MLH1 protein. The H&E staining and IHC results also verified the deficiency of the MLH1 protein, and FFPE samples from xenografts proved their similarity and consistency with clinical samples. Conclusions We successfully established MLH1 protein‐deficient cell lines. Followed by xenografting, we developed novel FFPE QC materials with homogenous, sustainable, and typical histological structures advantages that are suitable for the standardization of clinical IHC methods.

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Retinal Mitochondrial DNA Mismatch Repair in the Development of Diabetic Retinopathy, and Its Continued Progression After Termination of Hyperglycemia

Cited by 61 publications

References 49 publications

Oxidative stress, mitochondrial damage and diabetic retinopathy

Oxidative stress, mitochondrial damage and diabetic retinopathy

The Role of DNA Methylation in the Metabolic Memory Phenomenon Associated With the Continued Progression of Diabetic Retinopathy

Development of novel quality control material based on CRISPR/Cas9 editing and xenografts for MLH1 protein deficiency testing

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