Exosomal miR-29b found in aqueous humour mediates calcium signaling in diabetic patients with cataract

AIM: To investigate the role of exosomal miR-29b and Ca2+ in regulating the function of human lens epithelial cells (HLECs). METHODS: Exosomes were isolated from human aqueous humour (AH) by ultracentrifugation, and visualized by nanoparticle tracking and transmission electron microscopy. Exosomal miRNA sequencing was performed to identify differentially expressed miRNAs between diabetes with cataracts (DMC) group and age-related cataracts (ARC) group. TargetScan was used to predict potential target of certain miRNA. The expression of CACNA1C mRNA was determined by quantitative real-time polymerase chain reaction and CACNA1C protein was determined by Western blotting. Concentration of Ca2+ in human AH and the culture supernatant of cells were detected by the calcium assay kit. Cell counting kit-8 was used to determine cell viability. RESULTS: Exosomes were isolated from human AH, which had a typical cup-shaped phenotype and a particle size distribution in accordance with micro extracellular vesicles. Exosomal miRNA sequencing revealed that miR-29b was significantly downregulated in DMC group compared with ARC. Ca2+ concentration of human AH in DMC was higher than that in ARC. The culture supernatant of cells transfected with miR-29b inhibitors had a higher concentration of Ca2+ than that transfected with miR-29b mimics. miR-29b reduced the viability of HLECs by upregulating CACNA1C expression. CONCLUSION: Exosomes isolated from human AH contains abundant miRNAs. A significantly expressed miRNA, miR-29b, can affect the concentration of Ca2+ and regulate HLEC processes by upregulating CACNA1C.

Download Full-text

Exosomal miR-29b found in aqueous humour mediates calcium signaling in diabetic patients with cataract

10.21203/rs.3.rs-34868/v4 ◽

2020 ◽

Author(s):

Chao Gao ◽

Xin Liu ◽

Fan Fan ◽

Jianing Yang ◽

Xiyue Zhou ◽

...

Keyword(s):

Aqueous Humour ◽

Culture Supernatant ◽

Cell Counting ◽

Human Lens ◽

Diabetic Patients ◽

Age Related ◽

Transmission Electron ◽

Mirna Sequencing ◽

Human Lens Epithelium ◽

Exosomal Mirna

Abstract Background: Ca2+ was supposed to play an important role in the cataract formation. Exosomal miR-29b from aqueous humour may play an important role in the mechanism of diabetes and cataracts. The purpose of this study was to investigate the role of exosomal miR-29b and Ca2+ in regulating the function of human lens epithelial cells. Methods: Exosomes were isolated from human aqueous humour by ultracentrifugation, and visualized by nanoparticle tracking and transmission electron microscopy. Exosomal miRNA sequencing was performed to identify differentially expressed miRNAs between diabetes and cataracts group (DMC) and age-related cataracts group (ARC). TargetScan was used to predict potential target of certain miRNA. The expression of CACNA1C mRNA was determined by quantitative real-time PCR and CACNA1C protein was determined by Western blotting. Concentration of Ca2+ in human aqueous humour and the culture supernatant of cells was detected by the Calcium Assay Kit. Cell Counting Kit-8 was used to determine cell viability. Results: Exosomes were isolated from human aqueous humour, which had a typical cup-shaped phenotype and a particle size distribution in accordance with micro extracellular vesicles. Exosomal miRNA sequencing revealed that miR-29b was significantly downregulated in diabetes and cataracts group (DMC) compared with age-related cataracts group (ARC). Ca2+ concentration of human aqueous humour in DMC was higher than that in ARC. The culture supernatant of cells transfected with miR-29b inhibitors had a higher concentration of Ca2+ than that transfected with miR-29b mimics. miR-29b reduced the viability of human lens epithelium cells (HLECs) by upregulating CACNA1C expression.Conclusions: Exosomes isolated from human aqueous humour contained abundant miRNAs. A significantly expressed miRNA, miR-29b, could affect the concentration of Ca2+ and regulate HLEC processes by upregulating CACNA1C.

Download Full-text

Exosomal miR-29b found in aqueous humour mediates calcium signaling in diabetic patients with cataract

10.21203/rs.3.rs-34868/v3 ◽

2020 ◽

Author(s):

Chao Gao ◽

Xin Liu ◽

Fan Fan ◽

Jianing Yang ◽

Xiyue Zhou ◽

...

Keyword(s):

Aqueous Humour ◽

Culture Supernatant ◽

Cell Counting ◽

Human Lens ◽

Diabetic Patients ◽

Age Related ◽

Transmission Electron ◽

Mirna Sequencing ◽

Human Lens Epithelium ◽

Exosomal Mirna

Download Full-text

Exosomal miR-29b found in aqueous humour mediates calcium signaling in diabetic patients with cataract

10.21203/rs.3.rs-34868/v2 ◽

2020 ◽

Author(s):

Chao Gao ◽

Xin Liu ◽

Fan Fan ◽

Jianing Yang ◽

Xiyue Zhou ◽

...

Keyword(s):

Cell Culture ◽

Aqueous Humour ◽

Culture Supernatant ◽

Cell Counting ◽

Cell Culture Supernatant ◽

Human Lens ◽

Diabetic Patients ◽

Age Related ◽

Mirna Sequencing ◽

Exosomal Mirna

Abstract Background: Ca2+ was supposed to play an important role in the formation of cataract. Exosomal miR-29b from aqueous humour may play an important role in the mechanism of diabetes and cataracts. The purpose of this study was to investigate the role of exosomal miR-29b and Ca2+ in regulating the function of human lens epithelial cells.Methods: Exosomes were isolated from human aqueous humour by ultracentrifugation, and visualized by nanoparticle tracking and transmission electron microscopy. Exosomal miRNA sequencing was performed to identify differentially expressed miRNAs between diabetes and cataracts group (DMC) and age-related cataracts group (ARC). TargetScan was used to predict potential target of certain miRNA. The expression of CACNA1C mRNA was determined by quantitative real-time PCR and CACNA1C protein was determined by Western blotting. Concentration of Ca2+ of human aqueous humour and cell culture supernatant was detected by the Calcium Assay Kit. Cell Counting Kit-8 was used to determine cell viability.Results: Exosomes were isolated from human aqueous humour, which had a typical cup-shaped phenotype and a particle size distribution in accordance with micro extracellular vesicles. Exosomal miRNA sequencing revealed that miR-29b was significantly downregulated in diabetes and cataracts group (DMC) compared with age-related cataracts group (ARC). Ca2+ concentration of human aqueous humour in DMC was higher than that in ARC. Cell culture supernatant transfected with miR-29b inhibitors had a higher concentration of Ca2+ than that transfected with miR-29b mimics. miR-29b reduced the viability of human lens epithelium cells (HLECs) by up-regulating CACNA1C expression.Conclusions: Exosomes isolated from human aqueous humour contained abundant miRNAs. A significantly expressed miRNA, miR-29b, could affect the concentration of Ca2+ and regulate HLEC processes by up-regulating CACNA1C.

Download Full-text

Exosomal miR-29b of Aqueous Humour Mediated Variation of Ca2+ in Diabetes and Cataract Patients

10.21203/rs.3.rs-34868/v1 ◽

2020 ◽

Author(s):

Chao Gao ◽

Xin Liu ◽

Fan Fan ◽

Jianing Yang ◽

Xiyue Zhou ◽

...

Keyword(s):

Cell Culture ◽

Aqueous Humour ◽

Culture Supernatant ◽

Cell Counting ◽

Cell Culture Supernatant ◽

Human Lens ◽

Age Related ◽

Mirna Sequencing ◽

Human Lens Epithelium ◽

Exosomal Mirna

Abstract Background: Ca2+ was supposed to play an important role in the formation of cataract. Considering our increasing knowledge of exosomes, exosomal miRNAs isolated from aqueous humour may play an important role in the mechanism of diabetes and cataracts. In this study, we aimed to investigate the role of exosomal miR-29b and Ca2+ in regulating the function of human lens epithelial cells. Methods: Exosomes were isolated from human aqueous humour by ultracentrifugation, and visualized by nanoparticle tracking and transmission electron microscopy. Exosomal miRNA sequencing was performed to identify differentially expressed miRNAs between diabetes and cataracts group (DMC) and age-related cataracts group (ARC). TargetScan was used to predict potential target of certain miRNA. The expression of CACNA1C mRNA was determined by quantitative real-time PCR and CACNA1C protein was determined by Western blotting. Concentration of Ca2+ of human aqueous humour and cell culture supernatant was detected by the Calcium Assay Kit. Cell Counting Kit-8 was used to determine cell viability. Results: Exosomes could be isolated from human aqueous humour, which had a typical cup-shaped phenotype and a particle size distribution in accordance with micro extracellular vesicles. Exosomal miRNA sequencing revealed that miR-29b was significantly downregulated in diabetes and cataracts group (DMC) compared with age-related cataracts group (ARC). Ca2+ concentration of human aqueous humour in DMC was higher than that in ARC. Cell culture supernatant transfected with miR-29b inhibitors had a higher concentration of Ca2+ than that transfected with miR-29b mimics. miR-29b reduced the viability of human lens epithelium cells (HLECs) by up-regulating CACNA1C expression.Conclusions: Exosomes isolated from human aqueous humour contained abundant miRNAs. A significantly expressed miRNA, miR-29b, could affect the concentration of Ca2+ and regulate HLEC processes by up-regulating CACNA1C.

Download Full-text

Pathogenesis of Age-related Cataract: a systematic review of proteomic studies

Current Proteomics ◽

10.2174/1570164617999201020205100 ◽

2020 ◽

Vol 17 ◽

Author(s):

Christina Karakosta ◽

Argyrios Tzamalis ◽

Michalis Aivaliotis ◽

Ioannis Tsinopoulos

Keyword(s):

Systematic Review ◽

Oxidant Stress ◽

Critical Role ◽

Human Lens ◽

Nuclear Cataract ◽

Cataract Formation ◽

Post Translational Modification ◽

Ability To Act ◽

Age Related

Background/Objective:: The aim of this systematic review is to identify all the available data on human lens proteomics with a critical role to age-related cataract formation in order to elucidate the physiopathology of the aging lens. Materials and Methods:: We searched on Medline and Cochrane databases. The search generated 328 manuscripts. We included nine original proteomic studies that investigated human cataractous lenses. Results:: Deamidation was the major age-related post-translational modification. There was a significant increase in the amount of αA-crystallin D-isoAsp58 present at all ages, while an increase in the extent of Trp oxidation was apparent in cataract lenses when compared to aged normal lenses. During aging, enzymes with oxidized cysteine at critical sites included GAPDH, glutathione synthase, aldehyde dehydrogenase, sorbitol dehydrogenase, and PARK7. Conclusion:: D-isoAsp in αA crystallin could be associated with the development of age-related cataract in human, by contributing to the denaturation of a crystallin, and decreasing its ability to act as a chaperone. Oxidation of Trp may be associated with nuclear cataract formation in human, while the role of oxidant stress in age-related cataract formation is dominant.

Download Full-text

A Narrative Review on Sarcopenia in Type 2 Diabetes Mellitus: Prevalence and Associated Factors

Nutrients ◽

10.3390/nu13010183 ◽

2021 ◽

Vol 13 (1) ◽

pp. 183

Author(s):

Anna Izzo ◽

Elena Massimino ◽

Gabriele Riccardi ◽

Giuseppe Della Pepa

Keyword(s):

Diabetes Mellitus ◽

Type 2 Diabetes ◽

Type 2 Diabetes Mellitus ◽

Elderly Population ◽

The Elderly ◽

Narrative Review ◽

Macrovascular Complications ◽

Diabetic Patients ◽

Age Related

Type 2 diabetes mellitus (T2DM) represents a major health burden for the elderly population, affecting approximately 25% of people over the age of 65 years. This percentage is expected to increase dramatically in the next decades in relation to the increased longevity of the population observed in recent years. Beyond microvascular and macrovascular complications, sarcopenia has been described as a new diabetes complication in the elderly population. Increasing attention has been paid by researchers and clinicians to this age-related condition—characterized by loss of skeletal muscle mass together with the loss of muscle power and function—in individuals with T2DM; this is due to the heavy impact that sarcopenia may have on physical and psychosocial health of diabetic patients, thus affecting their quality of life. The aim of this narrative review is to provide an update on: (1) the risk of sarcopenia in individuals with T2DM, and (2) its association with relevant features of patients with T2DM such as age, gender, body mass index, disease duration, glycemic control, presence of microvascular or macrovascular complications, nutritional status, and glucose-lowering drugs. From a clinical point of view, it is necessary to improve the ability of physicians and dietitians to recognize early sarcopenia and its risk factors in patients with T2DM in order to make appropriate therapeutic approaches able to prevent and treat this condition.

Download Full-text

Effect of Shuangdan Mingmu capsule, a Chinese herbal formula, on oxidative stress-induced apoptosis of pericytes through PARP/GAPDH pathway

BMC Complementary Medicine and Therapies ◽

10.1186/s12906-021-03238-w ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Fujiao Nie ◽

Jiazhao Yan ◽

Yanjun Ling ◽

Zhengrong Liu ◽

Chaojun Fu ◽

...

Keyword(s):

Oxidative Stress ◽

Nitric Oxide ◽

Oxidative Damage ◽

Damage Model ◽

B Cell Lymphoma ◽

Cell Counting ◽

Network Pharmacology ◽

Diabetic Patients ◽

Induced Apoptosis

Abstract Background Diabetic retinopathy (DR) has become a worldwide concern because of the rising prevalence rate of diabetes mellitus (DM). Despite much energy has been committed to DR research, it remains a difficulty for diabetic patients all over the world. Since apoptosis of retinal microvascular pericytes (RMPs) is the early characteristic of DR, this study aimed to reveal the mechanism of Shuangdan Mingmu (SDMM) capsule, a Chinese patent medicine, on oxidative stress-induced apoptosis of pericytes implicated with poly (ADP-ribose) polymerase (PARP) / glyceraldehyde 3-phosphate dehydrogenase (GAPDH) pathway. Methods Network pharmacology approach was performed to predict biofunction of components of SDMM capsule dissolved in plasma on DR. Both PARP1 and GAPDH were found involved in the hub network of protein-protein interaction (PPI) of potential targets and were found to take part in many bioprocesses, including responding to the regulation of reactive oxygen species (ROS) metabolic process, apoptotic signaling pathway, and response to oxygen levels through enrichment analysis. Therefore, in vitro research was carried out to validate the prediction. Human RMPs cultured with media containing 0.5 mM hydrogen oxide (H2O2) for 4 h was performed as an oxidative-damage model. Different concentrations of SDMM capsule, PARP1 inhibitor, PARP1 activation, and GAPDH inhibitor were used to intervene the oxidative-damage model with N-Acetyl-L-cysteine (NAC) as a contrast. Flow cytometry was performed to determine the apoptosis rate of cells and the expression of ROS. Cell counting kit 8 (CCK8) was used to determine the activity of pericytes. Moreover, nitric oxide (NO) concentration of cells supernatant and expression of endothelial nitric oxide synthase (eNOS), superoxide dismutase (SOD), B cell lymphoma 2 (BCL2), vascular endothelial growth factor (VEGF), endothelin 1 (ET1), PARP1, and GAPDH were tested through RT-qPCR, western blot (WB), or immunocytochemistry (ICC). Results Overproduction of ROS, high apoptotic rate, and attenuated activity of pericytes were observed after cells were incubated with media containing 0.5 mM H2O2. Moreover, downregulation of SOD, NO, BCL2, and GAPDH, and upregulation of VEGFA, ET1, and PARP1 were discovered after cells were exposed to 0.5 mM H2O2 in this study, which could be improved by PARP1 inhibitor and SDMM capsule in a dose-dependent way, whereas worsened by PARP1 activation and GAPDH inhibitor. Conclusions SDMM capsule may attenuate oxidative stress-induced apoptosis of pericytes through downregulating PARP expression and upregulating GAPDH expression.

Download Full-text

Nanoceria Prevents Glucose-Induced Protein Glycation in Eye Lens Cells

Nanomaterials ◽

10.3390/nano11061473 ◽

2021 ◽

Vol 11 (6) ◽

pp. 1473

Author(s):

Belal I. Hanafy ◽

Gareth W. V. Cave ◽

Yvonne Barnett ◽

Barbara K. Pierscionek

Keyword(s):

Protein Glycation ◽

Human Lens ◽

Lens Epithelial Cells ◽

Oxide Nanoparticles ◽

Lens Protein ◽

Oxygen Species ◽

Human Lens Epithelial Cells ◽

Age Related ◽

Age Related Changes

Cerium oxide nanoparticles (nanoceria) are generally known for their recyclable antioxidative properties making them an appealing biomaterial for protecting against physiological and pathological age-related changes that are caused by reactive oxygen species (ROS). Cataract is one such pathology that has been associated with oxidation and glycation of the lens proteins (crystallins) leading to aggregation and opacification. A novel coated nanoceria formulation has been previously shown to enter the human lens epithelial cells (HLECs) and protect them from oxidative stress induced by hydrogen peroxide (H2O2). In this work, the mechanism of nanoceria uptake in HLECs is studied and multiple anti-cataractogenic properties are assessed in vitro. Our results show that the nanoceria provide multiple beneficial actions to delay cataract progression by (1) acting as a catalase mimetic in cells with inhibited catalase, (2) improving reduced to oxidised glutathione ratio (GSH/GSSG) in HLECs, and (3) inhibiting the non-enzymatic glucose-induced glycation of the chaperone lens protein α-crystallin. Given the multifactorial nature of cataract progression, the varied actions of nanoceria render them promising candidates for potential non-surgical therapeutic treatment.

Download Full-text