Publications by authors named "Subrata Chakrabarti"

169 Publications

The Long Non-Coding RNA HOTAIR Is a Critical Epigenetic Mediator of Angiogenesis in Diabetic Retinopathy.

Invest Ophthalmol Vis Sci 2021 Mar;62(3):20

Department of Pathology and Laboratory Medicine, Western University, London, Ontario, Canada.

Purpose: Diabetic retinopathy (DR) remains a pressing issue worldwide. Abnormal angiogenesis is a distinct vascular lesion in DR, and research has established that vascular endothelial growth factor A (VEGF-A) is a primary mediator of such changes. However, limitations in current anti-VEGF therapies suggest that our understanding of molecular networks underlying ocular angiogenesis remains far from complete. Based on our long non-coding RNA (lncRNA) array analyses, HOX antisense intergenic RNA (HOTAIR) was identified as one of the top upregulated lncRNAs in high glucose-cultured human retinal endothelial cells (HRECs). Given the well-documented roles of HOTAIR in cancer, no studies have examined the epigenetic implications of HOTAIR in DR, and we investigated such relationships herein.

Methods: We used HRECs exposed to various glucose concentrations and epigenetic modulators to examine HOTAIR, angiogenic, and DR-related molecular markers. Oxidative stress, angiogenesis, and mitochondrial dysfunction were assessed. Retinal tissues of diabetic rodents and the vitreous humor and serum of patients with proliferative DR were also investigated.

Results: Hyperglycemia significantly augmented HOTAIR expression in HRECs and promoted angiogenesis, oxidative damage, and mitochondrial aberrations. Similarly, vitreous humor and serum from proliferative DR patients and retinas from diabetic animals demonstrated increased HOTAIR expression compared to non-diabetic controls. HOTAIR knockdown protected against glucose-induced increases of angiogenic and diabetes-associated molecules in the retina. Mechanistically, we showed that HOTAIR exerts its capabilities by preventing oxidative stress and modulating epigenetic pathways involving histone methylation, histone acetylation, DNA methylation, and transcription factors.

Conclusions: Our findings suggest that HOTAIR is a critical lncRNA in the pathogenesis of DR and may potentially be important for diagnostic and therapeutic targeting.
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http://dx.doi.org/10.1167/iovs.62.3.20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7980040PMC
March 2021

Role of long non‑coding RNAs and related epigenetic mechanisms in liver fibrosis (Review).

Int J Mol Med 2021 03 26;47(3). Epub 2021 Jan 26.

Department of Pathology and Laboratory Medicine, University of Western Ontario, London, ON N6A 5C1, Canada.

Liver fibrosis is one of the major liver pathologies affecting patients worldwide. It results from an improper tissue repair process following liver injury or inflammation. If left untreated, it ultimately leads to liver cirrhosis and liver failure. Long non‑coding RNAs (lncRNAs) have been implicated in a wide variety of diseases. They can regulate gene expression and modulate signaling. Some of the lncRNAs promote, while others inhibit liver fibrosis. Similarly, other epigenetic processes, such as methylation and acetylation regulate gene transcription and can modulate gene expression. Notably, there are several regulatory associations of lncRNAs with other epigenetic processes. A major mechanism of action of long non‑coding RNAs is to competitively bind to their target microRNAs (miRNAs or miRs), which in turn affects miRNA availability and bioactivity. In the present review, the role of lncRNAs and related epigenetic processes contributing to liver fibrosis is discussed. Finally, various potential therapeutic approaches targeting lncRNAs and related epigenetic processes, which are being considered as possible future treatment targets for liver fibrosis are identified.
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http://dx.doi.org/10.3892/ijmm.2021.4856DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7846421PMC
March 2021

Overexpression of Long Noncoding RNA HOTAIR Is a Unique Epigenetic Characteristic of Myxopapillary Ependymoma.

J Neuropathol Exp Neurol 2020 11;79(11):1193-1202

Department of Pathology and Laboratory Medicine, Western University.

Ependymomas are a heterogeneous group of central nervous system tumors. Despite the recent advances, there are no specific biomarkers for ependymomas. In this study, we explored the role of homeobox (HOX) genes and long noncoding RNA (LncRNA) HOTAIR in ependymomas along the neural axis. Bioinformatics analysis was performed on publicly available gene expression data. Quantitative RT-PCR was used to determine the mRNA expression level among different groups of ependymomas. RNA in situ hybridization (ISH) with probes specific to HOTAIR was performed on tumor tissue microarray (TMA) constructed with 19 ependymomas formalin-fixed paraffin-embedded tissue. Gene expression analysis revealed higher expression of posterior HOX genes and HOTAIR in myxopapillary ependymoma (MPE), in comparison to other spinal and intracranial ependymoma. qRT-PCR confirmed the high HOXD10 expression in spinal MPEs. There was a significant upregulation of HOTAIR expression in spinal MPE and elevated HOTAIR expressions were further confirmed by RNA ISH on the TMA. Intriguingly, HOXD10 and HOTAIR expressions were not elevated in nonependymoma spinal tumors. Our collective results suggest an important role for the lncRNA HOTAIR and posterior HOX genes in the tumorigenesis of spinal MPE. HOTAIR may also serve as a potential diagnostic marker for spinal MPE.
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http://dx.doi.org/10.1093/jnen/nlaa103DOI Listing
November 2020

Resident macrophages as potential therapeutic targets for cardiac ageing and injury.

Clin Transl Immunology 2020 26;9(8):e1167. Epub 2020 Aug 26.

International Genome Center Jiangsu University Zhenjiang China.

Cardiac-resident macrophages (CRMs) play critical roles in maintaining cardiac homoeostasis and removing senescent and dying cells. Recent preclinical data have re-energised the area of cardioimmunology and provided improved understanding of the modulation of compositional and functional phenotypes of CRMs. These data can aid in achieving improved cardiac regeneration, repair and functional remodelling following cardiac injury. In this review, we discuss the composition and renewal of various subsets of CRMs. Specific attention has been given to delineate the roles of various CRM subsets with respect to (1) facilitation of cardiac development and maintenance of physiological function such as electrical conduction and rhythm; (2) promotion of cardiac regeneration, inflammation resolution and functional remodelling following a cardiac injury; and (3) therapeutic potential. We have also highlighted the relationship between CRM replenishment and cardiomyocyte senescence as well as cardiovascular diseases development. Finally, we have addressed future perspectives and directions in basic research and potentially clinical applications of CRMs.
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http://dx.doi.org/10.1002/cti2.1167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7450172PMC
August 2020

Glucose-induced, duration-dependent genome-wide DNA methylation changes in human endothelial cells.

Am J Physiol Cell Physiol 2020 08 27;319(2):C268-C276. Epub 2020 May 27.

Department of Pathology and Laboratory Medicine, London Health Sciences Centre, London, Ontario, Canada.

DNA methylation, a critical epigenetic mechanism, plays an important role in governing gene expressions during biological processes such as aging, which is well known to be accelerated in hyperglycemia (diabetes). In the present study, we investigated the effects of glucose on whole genome DNA methylation in small [human retinal microvascular endothelial cells (HRECs)] and large [human umbilical vein endothelial cells (HUVECs)] vessel endothelial cell (EC) lines exposed to basal or high glucose-containing media for variable lengths of time. Using the Infinium EPIC array, we obtained 773,133 CpG sites (probes) for analysis. Unsupervised clustering of the top 5% probes identified four distinct clusters within EC groups, with significant methylation differences attributed to EC types and the duration of cell culture rather than glucose stimuli alone. When comparing the ECs incubated for 2 days versus 7 days, hierarchical clustering analyses [methylation change >10% and false discovery rate (FDR) <0.05] identified 17,354 and 128 differentially methylated CpGs for HUVECs and HRECs, respectively. Predominant DNA hypermethylation was associated with the length of culture and was enriched for gene enhancer elements and regions surrounding CpG shores and shelves. We identified 88 differentially methylated regions (DMRs) for HUVECs and 8 DMRs for HRECs (all FDR <0.05). Pathway enrichment analyses of DMRs highlighted involvement of regulators of embryonic development (i.e., genes) and cellular differentiation [transforming growth factor-β (TGF-β) family members]. Collectively, our findings suggest that DNA methylation is a complex process that involves tightly coordinated, cell-specific mechanisms. Such changes in methylation overlap genes critical for cellular differentiation and embryonic development.
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http://dx.doi.org/10.1152/ajpcell.00011.2020DOI Listing
August 2020

Fibroblast transdifferentiation promotes conversion of M1 macrophages and replenishment of cardiac resident macrophages following cardiac injury in mice.

Eur J Immunol 2020 06 25;50(6):795-808. Epub 2020 Feb 25.

International Genome Center, Jiangsu University, Zhenjiang, China.

Resident cardiac macrophages play important roles in homeostasis, maintenance of cardiac function, and tissue repair. After cardiac injury, monocytes infiltrate the tissue, undergo phenotypic and functional changes, and are involved in inflammatory injury and functional remodelling. However, the fate of cardiac infiltrating/polarized macrophages and the relationship between these cells and resident cardiac macrophage replenishment following injury remain unclear. Our results showed that angiotensin II induces cardiac fibroblast transdifferentiation into cardiac myofibroblasts (MFBs). In cocultures with MFBs and murine macrophages, the MFBs promoted macrophage polarization to M1 phenotype, followed by selective apoptosis, which was associated with TNF/TNFR1 axis and independent of NO production. Surprisingly, after 36 h of coculture, the surviving macrophages were converted to M2 phenotype and settled in heart, which was dependent on leptin produced by MFBs or polarized macrophages via the PI3K or Akt pathway. CCR2 CD45.2 cells adoptively transferred into CD45.1 mice with viral myocarditis, differentiated into CD45.2 CCR2 CX3CR1 M2 cells during the resolution of inflammation and settled within the heart. Our data highlight a novel mechanism related to the renewal or replenishment of cardiac resident macrophages following cardiac injury; and suggest that transdifferentiation of cardiac fibroblasts may promote the resolution of inflammation.
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http://dx.doi.org/10.1002/eji.201948414DOI Listing
June 2020

Glucose-induced oxidative stress and accelerated aging in endothelial cells are mediated by the depletion of mitochondrial SIRTs.

Physiol Rep 2020 02;8(3):e14331

Department of Pathology and Laboratory Medicine, Western University, London, ON, Canada.

Diabetic complications cause significant morbidity and mortality. Dysfunction of vascular endothelial cells (ECs), caused by oxidative stress, is a main mechanism of cellular damage. Oxidative stress accelerates EC senescence and DNA damage. In this study, we examined the role of mitochondrial sirtuins (SIRTs) in glucose-induced oxidative stress, EC senescence, and their regulation by miRNAs. Human retinal microvascular endothelial cells (HRECs) were exposed to 5 mmol/L (normoglycemia; NG) or 25 mmol/L glucose (hyperglycemia; HG) with or without transfection of miRNA antagomirs (miRNA-1, miRNA-19b, and miRNA-320; specific SIRT-targeting miRNAs). Expressions of SIRT3, 4 and 5 and their targeting miRNAs were examined using qRT-PCR and ELISAs were used to study SIRT proteins. Cellular senescence was investigated using senescence-associated β-gal stain; while, oxidative stress and mitochondrial alterations were examined using 8-OHdG staining and cytochrome B expressions, respectively. A streptozotocin-induced diabetic mouse model was also used and animal retinas and hearts were collected at 2 months of diabetes. In HRECs, HG downregulated the mRNAs of SIRTs, while SIRT-targeting miRNAs were upregulated. ELISA analyses confirmed such downregulation of SIRTs at the protein level. HG additionally caused early senescence, endothelial-to-mesenchymal transition and oxidative DNA damage in ECs. These changes were prevented by the transfection of specific miRNA antagomirs and by resveratrol. Retinal and cardiac tissues from diabetic mice also showed similar reductions of mitochondrial SIRTs. Collectively, these findings demonstrate a novel mechanism in which mitochondrial SIRTs regulate glucose-induced cellular aging through oxidative stress and how these SIRTs are regulated by specific miRNAs. Identifying such mechanisms may lead to the discovery of novel treatments for diabetic complications.
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http://dx.doi.org/10.14814/phy2.14331DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7002531PMC
February 2020

Curcumin Analogs Reduce Stress and Inflammation Indices in Experimental Models of Diabetes.

Front Endocrinol (Lausanne) 2019 18;10:887. Epub 2019 Dec 18.

Department of Pathology and Laboratory Medicine, Western University, London, ON, Canada.

Chronic inflammation and oxidative stress lead to a multitude of adverse cellular responses in target organs of chronic diabetic complications. Curcumin, a highly investigated phytochemical, has been shown to exhibit both anti-inflammatory and antioxidant activities. However, the clinical application of curcumin has been greatly limited due to a poor pharmacokinetic profile. To overcome these limitations, we have generated analogs of curcumin to enhance bioavailability and offer a preferable pharmacokinetic profile. Here, we explored the effects of two mono-carbonyl curcumin analogs, L2H21 and L50H46, in alleviating indices of inflammation and oxidative stress in cell culture and mouse model of diabetic complications. Our results show that L2H21 and L50H46 normalize inflammatory mediators ( and α), extracellular matrix proteins ( and α), vasoactive factors ( and ) and a key transcriptional coactivator () in cultured human retinal microvascular endothelial cells (HRECs) and dermal-derived microvascular endothelial cells (HMVECs) challenged with high levels of glucose. These curcumin analogs also reduced glucose-induced oxidative DNA damage as evidenced by 8-OHdG labeling. We further show that treatment of streptozotocin-induced diabetic mice with curcumin analogs prevents cardiac and renal dysfunction. The preservation of target tissue function was associated with normalization of pro-inflammatory cytokines and matrix proteins. Collectively, our results show that L2H21 and L50H46 offer the anti-inflammatory and antioxidant activities as has been reported for curcumin, and may provide a clinically applicable therapeutic option for the treatment of diabetic complications.
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http://dx.doi.org/10.3389/fendo.2019.00887DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6930691PMC
December 2019

Diabetic Retinopathy, lncRNAs, and Inflammation: A Dynamic, Interconnected Network.

J Clin Med 2019 Jul 14;8(7). Epub 2019 Jul 14.

Department of Pathology and Laboratory Medicine, Western University, London, ON N6A5A5, Canada.

Diabetic retinopathy (DR) is reaching epidemic levels globally due to the increase in prevalence of diabetes mellitus (DM). DR also has detrimental effects to quality of life, as it is the leading cause of blindness in the working-age population and the most common cause of vision loss in individuals with DM. Over several decades, many studies have recognized the role of inflammation in the development and progression of DR; however, in recent years, accumulating evidence has also suggested that non-coding RNAs, especially long non-coding (lncRNAs), are aberrantly expressed in diabetes and may play a putative role in the development and progression of DR through the modulation of gene expression at the transcriptional, post-transcriptional, or epigenetic level. In this review, we will first highlight some of the key inflammatory mediators and transcription factors involved in DR, and we will then introduce the critical roles of lncRNAs in DR and inflammation. Following this, we will discuss the implications of lncRNAs in other epigenetic mechanisms that may also contribute to the progression of inflammation in DR.
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http://dx.doi.org/10.3390/jcm8071033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6678747PMC
July 2019

Safety of anti-VEGF treatments in a diabetic rat model and retinal cell culture.

Clin Ophthalmol 2019 1;13:1097-1114. Epub 2019 Jul 1.

Department of Pathology and Laboratory Medicine, Western University, London, ON, Canada.

Purpose: To analyze the safety of different concentrations of anti-VEGF on retinal cells.

Methods: Non-diabetic and streptozotocin (STZ)-induced diabetic rats received intravitreal rat anti-VEGF injections that had final vitreous concentrations of 0, 0.0625, 0.125 (clinical dose), and 0.25 mg/mL. Rats were also injected with the clinical dose of ranibizumab. TUNEL assay was performed on sectioned eyes to evaluate apoptotic cells. In vitro, rat retinal cell cultures were exposed to 0, 0.0625, 0.125 (clinical dose), and 0.25 mg/mL of ranibizumab for 48 and 72 hrs. Cellular metabolic activity was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, necrosis by lactate dehydrogenase (LDH), and apoptosis by cell death enzyme-linked immunosorbent assay (ELISA).

Results: Diabetic rats had a significant increase (<0.03) in apoptotic cell death at half the clinical dose, at the clinical dose, and at double the clinical dose. In vitro, MTT showed a significant decrease (<0.04) in cellular metabolic activity at the clinical dose and double the clinical dose compared to control at 48 and 72 hrs. LDH showed a significant increase (<0.04) in necrosis at the clinical dose and double the clinical dose compared to control at 48 and 72 hrs. ELISA showed a significant increase (<0.04) in apoptosis at half the clinical dose, at the clinical dose, and double the clinical dose, compared to control at 48 and 72 hrs.

Conclusions: Anti-VEGF treatment may be potentially detrimental to the retina by decreasing cellular metabolic activity and increasing cytotoxicity of retinal cells. The results provide a cautionary note to monitor both the retina and optic nerve status in patients undergoing frequent injections.
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http://dx.doi.org/10.2147/OPTH.S199771DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6616316PMC
July 2019

Two-year analysis of changes in the optic nerve and retina following anti-VEGF treatments in diabetic macular edema patients.

Clin Ophthalmol 2019 1;13:1087-1096. Epub 2019 Jul 1.

Department of Pathology and Laboratory Medicine, Western University, London, ON, Canada.

To evaluate long-term structural and functional changes that happen to the optic nerve and retina following ranibizumab (Lucentis) injections in diabetic macular edema (DME) patients. Patients with clinically significant DME requiring anti-VEGF injections underwent pre-injection baseline, 6, 12, and 24 month follow-up tests. The tests performed were optical coherence tomography (OCT), best-corrected visual acuity (BCVA), and visual field (VF). Wide-field fluorescein angiogram (IVFA) was performed to monitor the progression of diabetic ischemia. A total of 30 patients requiring anti-VEGF injections and 21 control patients not requiring anti-VEGF injections were enrolled in the study. From baseline, the average macular thickness significantly decreased (<0.0002) over the 24-month time period. Mean perfused ratio significantly increased (<0.0005) at 6, 12, and 24 months. Cup volume and vertical cup-to-disk ratio significantly increased (<0.0014) over the study period. This was verified by masked independent grading of patient optic nerve stereo-photographs by glaucoma specialists. BCVA significantly (<0.0006) improved over the study period. VFs showed a non-significant trend of deteriorating peripheral vision at 12 and 24 months. Clinically, anti-VEGF therapy appears to affect the optic nerve by increasing cup volume and increasing vertical cup/disk ratio over time. The results provide a cautionary note to monitor both the retina and optic nerve status in patients undergoing frequent injections.
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http://dx.doi.org/10.2147/OPTH.S199758DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6611706PMC
July 2019

Corrigendum to: "Preventive effects of North American ginseng (Panax quinquefolium) on diabetic nephropathy" [Phytomedicine 19 (2012) 494-505].

Phytomedicine 2019 Sep 1;62:152995. Epub 2019 Jul 1.

Department of Pathology and Department of Physiology and Pharmacology University of Western Ontario, Schulich School of Medicine and Dentistry, London, Ontario, Canada. Electronic address:

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http://dx.doi.org/10.1016/j.phymed.2019.152995DOI Listing
September 2019

CDX2 and Muc2 immunohistochemistry as prognostic markers in stage II colon cancer.

Hum Pathol 2019 08 21;90:70-79. Epub 2019 May 21.

Department of Pathology and Laboratory Medicine London Health Sciences Centre, London, Ontario, Canada N6A 5A5.

The treatment for colorectal cancer is largely surgical followed by adjuvant chemotherapy in high-risk cases. In patients with stage II cancer, there is no clear benefit for chemotherapy, and the current tools for assessment of risk are inadequate. A recent study identified that colorectal cancer with a gene signature similar to undifferentiated colonic stem cells was associated with a worse outcome. It was later shown that loss of CDX2 detected by immunohistochemistry (IHC) alone resulted in a worse prognosis and that this could be used to predict patients who would benefit from chemotherapy. Having observed that CDX2 expression can be patchy, we elected to validate these prior results for clinical practice using whole-slide IHC. The pathology of all cases was reviewed, and 3 blocks were selected for CDX2 IHC. We also expanded the panel beyond CDX2 to assess whether other markers in the gene signature including CDX1, Muc2, GPX2, and villin could better predict outcome. Among 210 cases, CDX2 expression was diffusely lost in 11% and focally lost in 23% of cases. There was no difference in survival based on CDX2 expression, but Muc2 loss was associated with reduced survival (hazard ratio, 3.32; 95% confidence interval, 1.20 to 9.20). No significant differences in outcome were identified based on CDX1, GPX2, or villin expression. In keeping with this, assessment of The Cancer Genome Atlas gene expression data demonstrated that decreased Muc2 expression was associated with reduced overall survival. Our results with whole-slide IHC are different from the previous studies and caution against the use of CDX2 in isolation as a prognostic marker in clinical practice. We have identified that loss of Muc2 is associated with reduced survival. This supports the use of the colonic differentiation gene expression signature to identify high-risk patients but cautions against the use of any one IHC-based marker in isolation.
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http://dx.doi.org/10.1016/j.humpath.2019.05.005DOI Listing
August 2019

Endothelin-1 traps potently reduce pathologic markers back to basal levels in an in vitro model of diabetes.

J Diabetes Metab Disord 2018 Dec 18;17(2):189-195. Epub 2018 Oct 18.

2Department of Pathology, University of Western Ontario, London, ON Canada.

Background: Diabetes mellitus is a group of metabolic disorders in which there are high blood glucose levels over a prolonged period. Diabetes is one of many diseases associated with pathologically elevated levels of endothelin (ET)-1. We have recently proposed the development of ET-traps, which are an antibody - based fusion protein that potently bind and sequester pathologically elevated levels of endothelin-1.

Methods: We constructed ET-traps that were found to be very potent binders to ET-1, with a K of 32.5ρM. We then treated human retinal microvascular endothelial cells (HRMECs), which are an in vitro model of glucose induced cellular damage, with 10 nM ET-1 or high glucose levels (25 mM).

Results: In this study, we investigated the effects of our ET-trap constructs on the expression levels of both collagen 4α1 and fibronectin, which are both important pathologic markers in diabetes. Treating HRMECs with 10 nM ET-1 or 25 mM glucose significantly induces the expression of the ECM proteins fibronectin and collagen 4α1, as is found in chronic diabetic complications; Incubation of the cells with the ET-traps significantly prevented the increased expression of fibronectin and collagen 4α1 back to basal levels. This was found with both mRNA and protein expression levels of the two ECM proteins.

Conclusion: Our results provide the first evidence of the efficacy of ET-traps in reducing pathologic markers in an in vitro model (of diabetes). Further research is warranted to determine the efficacy of ET-traps as a therapeutic tool for diabetes, which is a major public health burden around the world.
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http://dx.doi.org/10.1007/s40200-018-0360-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6405379PMC
December 2018

Increased Extracellular Matrix Protein Production in Chronic Diabetic Complications: Implications of Non-Coding RNAs.

Noncoding RNA 2019 Mar 22;5(1). Epub 2019 Mar 22.

Department of Pathology and Laboratory Medicine, Western University, London, ON N6A5A5, Canada.

Management of chronic diabetic complications remains a major medical challenge worldwide. One of the characteristic features of all chronic diabetic complications is augmented production of extracellular matrix (ECM) proteins. Such ECM proteins are deposited in all tissues affected by chronic complications, ultimately causing organ damage and dysfunction. A contributing factor to this pathogenetic process is glucose-induced endothelial damage, which involves phenotypic transformation of endothelial cells (ECs). This phenotypic transition of ECs, from a quiescent state to an activated dysfunctional state, can be mediated through alterations in the synthesis of cellular proteins. In this review, we discussed the roles of non-coding RNAs, specifically microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), in such processes. We further outlined other epigenetic mechanisms regulating the biogenesis and/or function of non-coding RNAs. Overall, we believe that better understanding of such molecular processes may lead to the development of novel biomarkers and therapeutic strategies in the future.
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http://dx.doi.org/10.3390/ncrna5010030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468528PMC
March 2019

MALAT1: A regulator of inflammatory cytokines in diabetic complications.

Endocrinol Diabetes Metab 2018 Apr 18;1(2):e00010. Epub 2018 Jan 18.

Department of Pathology and Laboratory Medicine Western University London ON Canada.

Objectives And Design: In this study, we examined the role of MALAT1, a highly conserved nuclear long non-coding RNA molecule, in chronic diabetic complications affecting the heart and kidneys using both in vitro and in vivo models: human endothelial cell culture and a Malat1 knockout mice model.

Results: Findings from our in vitro experiments demonstrated that MALAT1 was predominantly localized to nuclear speckles in endothelial cells and MALAT1 expression was significantly increased following incubation with high glucose in association with increased expression of inflammatory cytokines. As for our in vivo experiments, we used Malat1 knockout mice and wild-type controls with or without streptozotocin-induced diabetes over 2 months of follow-up, where all of our diabetic animals showed hyperglycaemia and polyuria. Examination of cardiac and renal tissues demonstrated altered MALAT1 RNA expression in wild-type diabetic animals. Such changes were associated with augmented production of downstream inflammatory molecules at the mRNA and protein levels. Diabetes-induced elevations of inflammatory markers were significantly decreased in Malat1 knockout diabetic animals. In addition to transcript and protein analyses, we examined functional changes in the heart and kidneys. Organ functions were affected in the wild-type diabetic mice but were rescued in Malat1 knockout mice.

Conclusions: Taken together, findings from this study will provide direct evidence and insight into the importance of MALAT1 in the pathogenesis of chronic diabetic complications involving the heart and kidneys.
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http://dx.doi.org/10.1002/edm2.10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6354803PMC
April 2018

lncRNA H19 prevents endothelial-mesenchymal transition in diabetic retinopathy.

Diabetologia 2019 03 5;62(3):517-530. Epub 2019 Jan 5.

Department of Pathology and Laboratory Medicine, Western University, Dental Science Building 4033, 1151 Richmond Street, London, ON, N6A 5C1, Canada.

Aims/hypothesis: The pathophysiology of diabetic retinopathy is linked to hyperglycaemia and its effect on retinal microvascular tissues. The resulting endothelial injury changes the endothelial cell phenotype to acquire mesenchymal properties (i.e. endothelial-mesenchymal transition [EndMT]). Such changes can be regulated by epigenetic mechanisms, including long non-coding RNAs (lncRNAs). lncRNA H19 may influence EndMT through TGF-β. We investigated the role of H19 in regulating EndMT during diabetic retinopathy.

Methods: H19 was overexpressed or silenced in human retinal endothelial cells exposed to various glucose levels. The cells were examined for H19, endothelial and mesenchymal markers. We then expanded the study to retinal tissues in a mouse model of diabetic retinopathy and also examined vitreous humour samples from individuals with proliferative diabetic retinopathy.

Results: Expression of H19 was downregulated in high glucose conditions (25 mmol/l). H19 overexpression prevented glucose-induced EndMT. Such changes appear to involve TGF-β through a Smad-independent mechanism. Diabetes caused downregulation of retinal H19. Using H19 knockout mice, we demonstrated similar EndMT in the retina. Examination of vitreous humour from individuals with proliferative diabetic retinopathy also reinforced the downregulation of H19 in diabetes.

Conclusions/interpretation: We therefore concluded that H19 regulates EndMT in diabetic retinopathy through specific mechanisms.

Data Availability: The results from our previous microarray can be found online using the GEO accession number GSE122189.
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http://dx.doi.org/10.1007/s00125-018-4797-6DOI Listing
March 2019

Molecular mechanisms and genetic regulation in atherosclerosis.

Int J Cardiol Heart Vasc 2018 Dec 25;21:36-44. Epub 2018 Sep 25.

Department of Biochemistry and Molecular Biology, University of South China, Hengyang, Hunan Province 421001, China.

Atherosclerosis (AS) manifested by lipid accumulation, extracellular matrix protein deposition, and calcification in the intima and media of the large to medium size arteries promoting arterial stiffness and reduction of elasticity. It has been accepted that AS leads to increased morbidity and mortality worldwide. Recent studies indicated that genetic abnormalities play an important role in the development of AS. Specific genetic mutation and histone modification have been found to induce AS formation. Furthermore, specific RNAs such as microRNAs and circular RNAs have been identified to play a crucial role in the progression of AS. Nevertheless, the mechanisms by which genetic mutation, DNA and histone modification, microRNAs and circular RNA induce AS still remain elusive. This review describes specific mechanisms and pathways through which genetic mutation, DNA and histone modification, microRNAs and circular RNA instigate AS. This review further provides a therapeutic strategic direction for the treatment of AS targeting genetic mechanisms.
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http://dx.doi.org/10.1016/j.ijcha.2018.09.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6161413PMC
December 2018

MALAT1: An Epigenetic Regulator of Inflammation in Diabetic Retinopathy.

Sci Rep 2018 04 25;8(1):6526. Epub 2018 Apr 25.

Department of Pathology and Laboratory Medicine, Western University, London, Ontario, Canada.

Despite possessing limited protein-coding potential, long non-coding RNAs (lncRNAs) have been implicated in a myriad of pathologic conditions. Most well documented in cancer, one prominent intergenic lncRNA known as MALAT1 is notorious for its role in impacting epigenetic mechanisms. In this study, we established a novel epigenetic paradigm for MALAT in diabetic retinopathy (DR) by employing siRNA-mediated MALAT1 knockdown in human retinal endothelial cells (HRECs), a Malat1 knockout animal model, vitreous humor from diabetic patients, pharmacological inhibitors for histone and DNA methylation, RNA immunoprecipitation, western blotting, and a unique DNA methylation array to determine glucose-related alterations in MALAT1. Our findings indicated that MALAT1 is capable of impacting the expressions of inflammatory transcripts through its association with components of the PRC2 complex in diabetes. Furthermore, the vitreous humors from diabetic patients revealed increased expressions of MALAT1, TNF-α, and IL-6. Intriguingly, our DNA methylation array demonstrated that transient high glucose exposure in HRECs does not contribute to significant methylation alterations at CpG sites across the MALAT1 gene. However, global inhibition of DNA methyltransferases induced significant increases in MALAT1 and associated inflammatory transcripts in HRECs. Our findings collectively demonstrate the importance of MALAT1 in inflammation and epigenetic regulation in DR.
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http://dx.doi.org/10.1038/s41598-018-24907-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5916949PMC
April 2018

LncRNAs: Proverbial Genomic "Junk" or Key Epigenetic Regulators During Cardiac Fibrosis in Diabetes?

Front Cardiovasc Med 2018 4;5:28. Epub 2018 Apr 4.

Department of Pathology and Laboratory Medicine, Western University, London, ON, Canada.

Long non-coding RNAs (lncRNAs) are critical regulators in a multitude of biological processes. Recent evidences demonstrate potential pathogenetic implications of lncRNAs in diabetic cardiomyopathy (DCM); however, the majority of lncRNAs have not been comprehensively characterized. While the precise molecular mechanisms underlying the functions of lncRNAs remain to be deciphered in DCM, emerging data in other pathophysiological conditions suggests that lncRNAs can have versatile features such as genomic imprinting, acting as guides for certain histone-modifying complexes, serving as scaffolds for specific molecules, or acting as molecular sponges. In an effort to better understand these features of lncRNAs in the context of DCM, our review will first summarize some of the key molecular alterations that occur during fibrosis in the diabetic heart (extracellular proteins and endothelial-to-mesenchymal transitioning), followed by a review of the current knowledge on the crosstalk between lncRNAs and major epigenetic mechanisms (histone methylation, histone acetylation, DNA methylation, and microRNAs) within this fibrotic process.
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http://dx.doi.org/10.3389/fcvm.2018.00028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5893820PMC
April 2018

Changes in the Cardiac GHSR1a-Ghrelin System Correlate With Myocardial Dysfunction in Diabetic Cardiomyopathy in Mice.

J Endocr Soc 2018 Feb 28;2(2):178-189. Epub 2017 Dec 28.

Imaging Research, Lawson Health Research Institute, London, Ontario N6A 4V2, Canada.

Ghrelin and its receptor, the growth hormone secretagogue receptor 1a (GHSR1a), are present in cardiac tissue. Activation of GHSR1a by ghrelin promotes cardiomyocyte contractility and survival, and changes in myocardial GHSR1a and circulating ghrelin track with end-stage heart failure, leading to the hypothesis that GHSR1a is a biomarker for heart failure. We hypothesized that GHSR1a could also be a biomarker for diabetic cardiomyopathy (DCM). We used two models of streptozotocin (STZ)-induced DCM: group 1, adult mice treated with 35 mg/kg STZ for 3 days; and group 2, neonatal mice treated with 70 mg/kg STZ at days 2 and 5 after birth. In group 1, mild fasting hyperglycemia (11 mM) was first detected 8 weeks after the last injection, and in group 2, severe fasting hyperglycemia (20 mM) was first detected 1 to 3 weeks after the last injection. In group 1, left ventricular function was slightly impaired as measured by echocardiography, and Western blot analysis showed a significant decrease in myocardial GHSR1a. In group 2, GHSR1a levels were also decreased as assessed by Cy5-ghrelin(1-19) fluorescence microscopy, and there was a significant negative correlation between GHSR1a levels and glucose tolerance. There were significant positive correlations between GHSR1a and ghrelin and between GHSR1a and sarcoplasmic reticulum Ca-ATPase 2a (SERCA2a), a marker for contractility, but not between GHSR1a and B-type natriuretic peptide, a marker for heart failure. We conclude that the subclinical stage of DCM is accompanied by alterations in the myocardial ghrelin-GHSR1a system, suggesting the possibility of a biomarker for DCM.
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http://dx.doi.org/10.1210/js.2017-00433DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5799831PMC
February 2018

Tuning the Optical Properties of Silicon Quantum Dots via Surface Functionalization with Conjugated Aromatic Fluorophores.

Sci Rep 2018 02 14;8(1):3050. Epub 2018 Feb 14.

Department of Chemical and Biochemical Engineering, Western University, London, Ontario, N6A 5B9, Canada.

Silicon Quantum Dots (SQDs) have recently attracted great interest due to their excellent optical properties, low cytotoxicity, and ease of surface modification. The size of SQDs and type of ligand on their surface has a great influence on their optical properties which is still poorly understood. Here we report the synthesis and spectroscopic studies of three families of unreported SQDs functionalized by covalently linking to the aromatic fluorophores, 9-vinylphenanthrene, 1-vinylpyrene, and 3-vinylperylene. The results showed that the prepared functionalized SQDs had a highly-controlled diameter by HR-TEM, ranging from 1.7-2.1 nm. The photophysical measurements of the assemblies provided clear evidence for efficient energy transfer from the fluorophore to the SQD core. Fӧrster energy transfer is the likely mechanism in these assemblies. As a result of the photogenerated energy transfer process, the emission color of the SQD core could be efficiently tuned and its emission quantum efficiency enhanced. To demonstrate the potential application of the synthesized SQDs for bioimaging of cancer cells, the water-soluble perylene- and pyrene-capped SQDs were examined for fluorescent imaging of HeLa cells. The SQDs were shown to be of low cytotoxicity.
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http://dx.doi.org/10.1038/s41598-018-21181-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5813013PMC
February 2018

Sex-specific analysis post-liver transplantation in hemochromatosis with aplastic anemia and hepatocellular carcinoma.

Hepatol Commun 2018 01 11;2(1):13-15. Epub 2017 Nov 11.

Department of Pathology University of Western Ontario London ON Canada.

A 42-year-old man with hemochromatosis and cirrhosis developed aplastic anemia. He underwent liver transplantation from a female donor and splenectomy, and his aplastic anemia spontaneously resolved. A bone marrow examination 6 months after the liver transplant showed 17.5% female cells. He did well for 13 years without the need for any blood product support but then developed bone pain and was found to have metastatic hepatocellular carcinoma in the vertebral bodies. Molecular analysis demonstrated that the tumor cells were from his original liver. No primary liver tumor was identified in the explant. The case demonstrates the application of fluorescent hybridization with X and Y chromosome-specific probes to study chimerism and tumor origin after liver transplantation between individuals of different sex. ( 2018;2:13-15).
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http://dx.doi.org/10.1002/hep4.1122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5776868PMC
January 2018

Effect of ginseng therapy on diabetes and its chronic complications: lessons learned.

J Complement Integr Med 2017 May 11;14(4). Epub 2017 May 11.

Ginseng played a significant role in the management of diabetes in China and in other Asian countries for a long period of time. It has a large number of pharmacological properties and is relatively free from adverse effects. As a part of Ontario Ginseng Research and Innovation Consortium, we investigated the effects of ginseng extract on diabetes and its complications. We demonstrated large number of beneficial effects of ginseng therapy and showed that these effects are possibly mediated through its antioxidant properties. Thus ginseng may lend itself as a relatively safe and inexpensive adjuvant treatment for diabetes and chronic diabetic complications.
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http://dx.doi.org/10.1515/jcim-2016-0166DOI Listing
May 2017

ANRIL regulates production of extracellular matrix proteins and vasoactive factors in diabetic complications.

Am J Physiol Endocrinol Metab 2018 03 7;314(3):E191-E200. Epub 2017 Nov 7.

Department of Pathology and Laboratory Medicine, Western University , London, Ontario , Canada.

noncoding RNAs (lncRNAs) have gained widespread interest due to their prevailing presence in various diseases. lncRNA ANRIL (a. k. a. CDKN2B-AS1) is located on human chromosome 9 (p21.3) and transcribed in opposite direction to the INK4b-ARF-INK4a gene cluster. It has been identified as a highly susceptible region for diseases such as coronary artery diseases and type 2 diabetes. Here, we explored its regulatory role in diabetic nephropathy (DN) and diabetic cardiomyopathy (DCM) in association with epigenetic modifiers p300 and polycomb repressive complex 2 (PRC2) complex. We used an ANRIL-knockout (ANRILKO) mouse model for this study. The wild-type and ANRILKO animals with or without streptozotocin-induced diabetes were monitored for 2 min. At the end of the time point, urine and tissues were collected. The tissues were measured for fibronectin (FN), type IV collagen (Col1α4), and VEGF mRNA and protein expressions. Renal function was determined by the measurement of 24-h urine volume and albumin/creatinine ratio at euthanasia. Renal and cardiac structures were investigated using periodic acid-Schiff stain and/or immunohistochemical analysis. Elevated expressions of extracellular matrix (ECM) proteins were prevented in ANRILKO diabetic animals. Furthermore, ANRILKO had a protective effect on diabetic mouse kidneys, as evidenced by lowering of urine volume and urine albumin levels in comparison with the wild-type diabetic animals. These alterations regulated by ANRIL may be mediated by p300 and enhancer of zeste 2 (EZH2) of the PRC2 complex. Our study concludes that ANRIL regulates functional and structural alterations in the kidneys and hearts in diabetes through controlling the expressions of ECM proteins and VEGF.
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http://dx.doi.org/10.1152/ajpendo.00268.2017DOI Listing
March 2018

Prevention of Diabetic Nephropathy by Modified Acidic Fibroblast Growth Factor.

Nephron 2017 29;137(3):221-236. Epub 2017 Jul 29.

Department of Pathology and Laboratory Medicine, Schulich School of Medicine and Dentistry, Western University, London, ON, Canada.

Background/aims: Oxidative stress (OS) contributes to all chronic diabetic complications, including diabetic nephropathy (DN). Acidic fibroblast growth factor (aFGF) has shown to confer protection from OS. However, it also has potent angiogenic activity. We hypothesized that a modified human aFGF (maFGF), with antioxidant properties but devoid of angiogenic activity, has preventative action in DN.

Methods: Streptozotocin-induced diabetic mice were treated with maFGF (intraperitoneally) daily for 1 or 6 months and were compared with untreated diabetic and non-diabetic controls. Microalbuminuria was assessed to determine functional damage. Renal cortical tissues were examined for multiple extracellular matrix proteins, vasoactive factors and OS markers. For mechanistic studies, immortalized mouse podocytes and human microvascular endothelial cells were exposed to high (25 mM) or low glucose (5 mM). OS, vasoactive factors, fibrosis and apoptosis-related gene expression were tested by real-time qPCR and Enzyme-Linked Immunosorbent Assay. Nitric oxide (NO) analyses were also performed.

Results: maFGF did not affect body weight and glycemia but prevented renal hypertrophy and functional changes in DN. It also prevented diabetes-induced DNA damage, nitrosative stress, vasoactive factors, angiotensinogen and endothelial NO synthase alterations. Although it failed to prevent transforming growth factor (TGF)-β1 mRNA upregulation, it prevented fibronectin production. Similar results were obtained in vitro. Decreased NO production in vivo and in vitro was also prevented by maFGF.

Conclusions: maFGF treatment prevents DN. This prevention probably involves NO production.
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http://dx.doi.org/10.1159/000478745DOI Listing
July 2018

Decrease in InsGlut2 β-cells with advancing age in mouse and human pancreas.

J Endocrinol 2017 06 27;233(3):229-241. Epub 2017 Mar 27.

Lawson Health Research InstituteSt Joseph Health Care, London, Ontario, Canada

The presence and location of resident pancreatic β-cell progenitors is controversial. A subpopulation of insulin-expressing but glucose transporter-2-low (InsGlut2) cells may represent multipotent pancreatic progenitors in adult mouse and in human islets, and they are enriched in small, extra-islet β-cell clusters (<5 β cells) in mice. Here, we sought to identify and compare the ontogeny of these cells in mouse and human pancreata throughout life. Mouse pancreata were collected at postnatal days 7, 14, 21, 28, and at 3, 6, 12, and 18 months of age, and in the first 28 days after β-cell mass depletion following streptozotocin (STZ) administration. Samples of human pancreas were examined during fetal life (22-30 weeks gestation), infancy (0-1 year), childhood (2-9), adolescence (10-17), and adulthood (18-80). Tissues were analyzed by immunohistochemistry for the expression and location of insulin, GLUT2 and Ki67. The proportion of β cells within clusters relative to that in islets was higher in pancreas of human than of mouse at all ages examined, and decreased significantly at adolescence. In mice, the total number of InsGlut2 cells decreased after 7 days concurrent with the proportion of clusters. These cells were more abundant in clusters than in islets in both species. A positive association existed between the appearance of new β cells after the STZ treatment of young mice, particularly in clusters and smaller islets, and an increased proportional presence of InsGlut2 cells during early β-cell regeneration. These data suggest that InsGlut2 cells are preferentially located within β-cell clusters throughout life in pancreas of mouse and human, and may represent a source of β-cell plasticity.
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http://dx.doi.org/10.1530/JOE-16-0475DOI Listing
June 2017

miR-146a regulates glucose induced upregulation of inflammatory cytokines extracellular matrix proteins in the retina and kidney in diabetes.

PLoS One 2017 16;12(3):e0173918. Epub 2017 Mar 16.

Dept. of Pathology and Laboratory Medicine, Western University, London ON, Canada.

Hyperglycemic damage to the endothelial cells (ECs) leads to increased synthesis of inflammatory cytokines. We have previously shown miR-146a downregulation in ECs and in the tissues of diabetic mice. Here we investigated the role of miR-146a, in the production of specific inflammatory cytokines and extracellular matrix (ECM) proteins in retina and kidneys in diabetes. We generated an endothelial specific miR-146a overexpressing transgenic mice (TG). We investigated these mice and wild type (WT) controls with or without streptozotocin (STZ) induced diabetes. Retinal and renal cortical tissues from the mice were examined for mRNAs for specific inflammatory markers, (ECM) proteins and inflammation inducible transcription factor by real time RT-PCR. Corresponding proteins, where possible, were examined using immunofluorescence or ELISA. In parallel, we examined ECs following incubation with various levels of glucose with or without miR-146a mimic transfection. In the retina and kidneys of WT mice with diabetes, increased expression of inflammatory markers (IL-6, TNFα, IL1β) in association augmented expression of ECM proteins (collagen 1αIV, fibronectin) and NF κB-P65 were observed, compared to WT non-diabetic controls. These changes were prevented in diabetic miR-146a TG mice along with retinal and renal functional and structural changes. In vitro studies showed similar changes in the ECs exposed to high glucose. Such changes were corrected in the cells following miR-146a mimic transfection. Further analyses of renal cortical tissues showed diabetes induced significant upregulation of two regulators of NFκB, namely Interleukin-1 associated Kinase 1 and tumour necrosis factor receptor associated factor. Such changes were prevented in diabetic TG animals. These data indicate that augmented production of inflammatory cytokines and ECM proteins in the retina and kidneys in diabetes are regulated through endothelium derived miR-146a. Identification of such novel mechanisms may potentially lead to the development of novel therapies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0173918PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5354466PMC
September 2017

miR-146a mediates inflammatory changes and fibrosis in the heart in diabetes.

J Mol Cell Cardiol 2017 04 6;105:70-76. Epub 2017 Mar 6.

Department of Pathology and Laboratory Medicine, Western University, London, ON, Canada. Electronic address:

Hyperglycemia induced endothelial injury is a key pathogenetic factor in diabetic cardiomyopathy. In diabetes, changes in pro-inflammatory cytokines are a key mechanism leading to cardiac fibrosis. We have previously demonstrated alteration of miR-146a in chronic diabetic complications. Here, we investigated the role of endothelial miR-146a in mediating inflammation and fibrosis in diabetic cardiomyopathy. To examine the effects of miR-146a on the inflammatory mediators, an endothelial specific miR-146a overexpressing transgenic mice (TG) using tie-2 promoter, was generated. We examined these mice and wild type littermate controls with or without STZ induced diabetes. Transthoracic echocardiography was performed. Cardiac tissues were examined for inflammatory cytokine mRNAs and proteins by real time RT-PCR or ELISA. Cardiac fibrosis was examined by histology staining. Human cardiac microvascular endothelial cells (HCMECs) and primary endothelial cells isolated from mice were used following incubation with various levels of glucose with or without miR-146a mimics or antagomir transfection. In hearts of wild type mice with diabetes, increased expression of inflammatory markers and extracellular matrix proteins (IL6, TNFα, IL-1β, MCP-1, NF-κB, Col1α1, Col4α1) were seen compared to wild type controls. These changes were prevented in the diabetic TG mice. In addition, WT diabetic mice showed cardiac functional abnormalities, which were improved in the diabetic TG mice. In vitro studies showed glucose induced increase the expressions of the above inflammatory cytokines and specific NF-κB regulators (IRAK1 &TRAF6). Such changes were corrected in the HCMECs following miR-146a mimic transfection. These data indicate that in diabetes, increased inflammatory cytokine and extracellular matrix protein productions and associated cardiac functional alterations are regulated by endothelial miR-146a. Identification of such mechanisms may potentially lead to the development of novel RNA based therapeutics.
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http://dx.doi.org/10.1016/j.yjmcc.2017.03.002DOI Listing
April 2017

ANRIL: A Regulator of VEGF in Diabetic Retinopathy.

Invest Ophthalmol Vis Sci 2017 01;58(1):470-480

Department of Pathology and Laboratory Medicine, Western University, London, Ontario, Canada.

Purpose: Long noncoding RNAs (lncRNAs) previously thought to be "dark matter" of the genome, play key roles in various biological processes. The lncRNA ANRIL is located at a genetic susceptibility locus for coronary artery diseases and type 2 diabetes. We examined the role of ANRIL in diabetic retinopathy, through study of its regulation of VEGF both in vitro and in vivo.

Methods: Human retinal endothelial cells (HRECs) were subjected to incubation in high glucose to mimic diabetes. ANRIL expression was measured with or without small interfering RNA (siRNA) knockdown in HRECs. ANRIL knockout mice, with or without streptozotocin-induced diabetes, were also investigated. Cell and tissues were measured for VEGF mRNA and protein expression. Functional alterations in VEGF were determined through tube formation, cell proliferation, and retinal vascular permeability assays. Vascular endothelial growth factor regulation through ANRIL's interactions with polycomb repressive complex 2 (PRC2) components and p300 were studied thorough PRC2 blocker, siRNA, and RNA immunoprecipitation (RNA-IP) assays.

Results: High glucose and diabetes caused ANRIL upregulation in HRECs and in the retina. Glucose-mediated elevation of ANRIL, on silencing, prevented VEGF expression. Such regulation involved ANRIL-mediated control of the PRC2 components p300 and miR200b. Direct binding of ANRIL to p300 and enhancer of zeste homolog 2 (EZH2; a PRC2 component) were elevated following exposure to high glucose levels.

Conclusions: Our results demonstrate for the first time that ANRIL regulates VEGF expression and function in diabetic retinopathy. This regulation is mediated by p300, miR200b, and EZH2 of the PRC2 complex.
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http://dx.doi.org/10.1167/iovs.16-20569DOI Listing
January 2017
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