LncRNA NEAT1/miR-146a-5p Axis Restores Normal Angiogenesis in Diabetic Foot Ulcers by Targeting mafG.

Non-healing lesions in diabetic foot ulcers are a significant effect of poor angiogenesis. Epigenetic regulators, mainly lncRNA and miRNA, are recognized for their important roles in disease progression. We deciphered the regulation of lncRNA NEAT1 through the miR-146a-5p/mafG axis in the progression of DFU. A lowered expression of lncRNA NEAT1 was associated with dysregulated angiogenesis through the reduced expression of mafG, SDF-1α, and VEGF in chronic ulcer subjects compared to acute DFU. This was validated by silencing NEAT1 by SiRNA in the endothelial cells which resulted in the transcriptional repression of target genes. Our in silico analysis identified miR-146a-5p as a potential target of lncRNA NEAT1. Further, silencing NEAT1 led to an increase in the levels of miR-146a-5p in chronic DFU subjects. This research presents the role of the lncRNA NEAT1/miR-146a-5p/mafG axis in enhancing angiogenesis in DFU.

MicroRNA-125b regulates vitamin D resistance by targeting CYP24A1 in the progression of gestational diabetes mellitus.

Vitamin D deficiency is prevalent in pregnancy and has been associated with increased occurrences of preeclampsia, cesarean delivery, neonatal bacterial vaginosis, and gestational diabetes. CYP24A1, recognized as a key factor in vitamin D metabolism homeostasis, encodes 24-hydroxylase responsible for converting 25(OH)D3 and 1,25(OH)2D3 into inactive metabolites. Recently, we have reported CYP24A1 overexpression in patients with gestational diabetes mellitus (GDM) and trophoblast cells exposed to hyperglycemia. In this study, we explored miRNA-mediated regulation of CYP24A1 in GDM progression, validating our findings through silencing experiments in a trophoblast cell line. In silico tools identified miR-125b-5p as a putative target of CYP24A1. Expression analysis revealed downregulation of miR-125b-5p in blood samples from early GDM and GDM compared to healthy pregnant women, positively correlating with vitamin D levels. Hyperglycemic exposure in human trophoblastic cell lines (BeWo) decreased miR-125b-5p expression, concomitant with an increase in CYP24A1. To confirm the regulatory role of miR-125b on CYP24A1, we transfected BeWo cells with antimiR-125b or miR-125b mimic. AntimiR-125b transfection heightened CYP24A1 levels, while miR-125b mimic overexpression resulted in decreased CYP24A1 expression. These findings establish miR-125b as a regulator of CYP24A1. To explore the influence of miR-125b on vitamin D metabolism, trophoblast cells overexpressing miR-125b were treated with 0.1 and 1 µM calcitriol. Hyperglycemic conditions exhibited a reduction in CYP24A1 levels. Collectively, our results indicate that miR-125b may regulate vitamin D metabolism by targeting CYP24A1, contributing to GDM progression. These findings may pave the way for understanding vitamin D resistance in concurrent GDM development and identifying novel miRNAs targeting CYP24A1.

MicroRNA-27b Impairs Nrf2-Mediated Angiogenesis in the Progression of Diabetic Foot Ulcer.

Nuclear factor erythroid-2-related factor 2 (Nrf2) is a stress-activated transcription factor regulating antioxidant genes, and a deficiency thereof, slowing lymphangiogenesis, has been reported in diabetic foot ulcer (DFU). The mode of Nrf2 regulation in DFU has been less explored. Emerging studies on miRNA-mediated target regulation show miRNA to be the leading player in the pathogenesis of the disease. In the present study, we demonstrated the role of miR-27b in regulating Nrf2-mediated angiogenesis in DFU. A lower expression of mRNA targets, such as Nrf2, HO-1, SDF-1α, and VEGF, was observed in tissue biopsied from chronic DFU subjects, which was in line with miR-27b, signifying a positive correlation with Nrf2. Similarly, we found significantly reduced expression of miR-27b and target mRNAs Nrf2, HO-1, SDF-1α, and VEGF in endothelial cells under a hyperglycemic microenvironment (HGM). To confirm the association of miR-27b on regulating Nrf2-mediated angiogenesis, we inhibited its expression through RNA interference-mediated knockdown and observed disturbances in angiogenic signaling with reduced endothelial cell migration. In addition, to explore the role of miR-27b and angiogenesis in the activation of Nrf2, we pretreated the endothelial cells with two well-known pharmacological compounds-pterostilbene and resveratrol. We observed that activation of Nrf2 through these compounds ameliorates impaired angiogenesis on HGM-induced endothelial cells. This study suggests a positive role of miR-27b in regulating Nrf2, which seems to be decreased in DFU and improves on treatment with pterostilbene and resveratrol.

Modulation of transcription factors by small molecules in ß-cell development and differentiation.

Transcription factors regulate gene expression and play crucial roles in development and differentiation of pancreatic ß-cell. The expression and/or activities of these transcription factors are reduced when ß-cells are chronically exposed to hyperglycemia, which results in loss of ß-cell function. Optimal expression of such transcription factors is required to maintain normal pancreatic development and ß-cell function. Over many other methods of regenerating ß-cells, using small molecules to activate transcription factors has gained insights, resulting in ß-cells regeneration and survival. In this review, we discuss the broad spectrum of transcription factors regulating pancreatic ß-cell development, differentiation and regulation of these factors in normal and pathological states. Also, we have presented set of potential pharmacological effects of natural and synthetic compounds on activities of transcription factor involved in pancreatic ß-cell regeneration and survival. Exploring these compounds and their action on transcription factors responsible for pancreatic ß-cell function and survival could be useful in providing new insights for development of small molecule modulators.

The role of circRNA-miRNA-mRNA interaction network in endothelial dysfunction.

Circular RNA (circRNA) is a neoteric researched transcript that involves gene regulation by serving as a micro-RNA (miRNA) sponge. This circRNA-miRNA-mRNA interaction is being recently explored, and its dysregulation is associated with disease pathogenesis and progression. Studies have demonstrated the involvement of this regulatory network in endothelium dysfunction-mediated regulation of pathology in vascular diseases. The disturbances or imbalance of vasodilation and vasoconstriction factors due to changes in oxidative stress, inflammatory markers, and nitric oxide signaling leads to endothelial dysfunction. These disturbances cause impermeability of blood through the endothelial barrier, thus developing atherosclerotic lesions. Advancements in high-throughput techniques like genome and RNA sequencing have made us understand this complex regulatory network causing endothelial dysfunction. In this review, we emphasize the network of interactions between circRNA, miRNA, and mRNA that mediates gene regulation and is linked to endothelial dysfunction in various pathological conditions.

Role of Long Non-Coding RNA in Regulating ER Stress Response to the Progression of Diabetic Complications.

Chronic hyperglycemia damages the nerves and blood vessels, culminating in other vascular complications. Such complications enhance cytokine, oxidative and endoplasmic reticulum (ER) stress. ER is the primary organelle where proteins are synthesised and attains confirmatory changes before its site of destination. Perturbation of ER homeostasis activates signaling sensors within its lumen, the unfolded protein response (UPR) that orchestrates ER stress and is extensively studied. Increased ER stress markers are reported in diabetic complications in addition to lncRNA that acts as an upstream marker inducing ER stress response. This review focuses on the mechanisms of lncRNA that regulate ER stress markers, especially during the progression of diabetic complications. Through this systemic review, we showcase the dysfunctional lncRNAs that act as a leading cause of ER stress response to the progression of diabetic complications.

Mangiferin alleviates hyperglycemia-induced endothelial impairment via Nrf2 signaling pathway.

The regulation of angiogenesis by nuclear factor erythroid 2-related factor 2 (Nrf2), a master redox transcription factor, is well established. In the present study, we aimed to activate Nrf2 by mangiferin (MG) and investigate its potential to regulate angiogenesis under a hyperglycemic (HG) environment in human endothelial cells. The mRNA expression of Nrf2 and its downstream targets HO-1, SOD-1, and CAT were observed to be decreased at 72 h of HG (33.3 mM glucose) exposure, and was ameliorated in MG (24 h) pretreated endothelial cells. ROS generation was assessed using an DCFDA assay, where we found the ROS generated at HG exposure was quenched by MG in a dose-dependent manner. The angiogenic markers HIF-1α and VEGF were also decreased in HG-induced endothelial cells, which significantly increased in the cells pretreated with MG. In addition, we have observed substantially more closed tube network formation in MG pretreated cells, which was low in HG-induced endothelial cells. The cell migration potential was monitored using a scratch assay, where the cells activated by MG showed a more significant movement than those under HG stress. Furthermore, to understand the role of Nrf2 in regulating angiogenesis, we knocked out the Nrf2 using CRISPR/Cas9 in endothelial cells. The wild-type endothelial cells exposed to MG alone showed a cytoprotective effect under a hyperglycemic environment. Our findings collectively demonstrated that MG helps regulate impaired angiogenesis under a hyperglycemic environment through Nrf2 signaling.

Histone deacetylase inhibitors as antidiabetic agents: Advances and opportunities.

The loss of function or dysfunction of ß-cells in the pancreas, attributed to the development of diabetes, involve alterations in genetic and epigenetic signatures. Recent evidences highlight the pathophysiological role of histone deacetylases (HDACs) in type 1 and type 2 diabetes. Indeed, most HDAC members have been linked to critical pathogenic events in diabetes, including redox imbalance, endoplasmic reticulum (ER) homeostasis perturbation, onset of oxidative stress and inflammation, which ultimately deteriorate ß-cell function. Accumulating evidence highlights the inhibition of HDACs as a prospective therapeutic strategy. Several chemically synthesized small molecules have been investigated for their specific ability to inhibit HDACs (reffered as HDAC inibitors) in various experimental studies. This review provides insights into the critical pathways involved in regulating different classes of HDACs. Further, the intricate signaling networks between HDACs and the stress mediators in diabetes are also explored. We exhaustively sum up the inferences from various investigations on the efficiency of HDAC inhibitors in managing diabetes and its associated complications.

Vitamin D resistant genes - promising therapeutic targets of chronic diseases.

Vitamin D is an essential vitamin indispensable for calcium and phosphate metabolism, and its deficiency has been implicated in several extra-skeletal pathologies, including cancer and chronic kidney disease. Synthesized endogenously in the layers of the skin by the action of UV-B radiation, the vitamin maintains the integrity of the bones, teeth, and muscles and is involved in cell proliferation, differentiation, and immunity. The deficiency of Vit-D is increasing at an alarming rate, with nearly 32% of children and adults being either deficient or having insufficient levels. This has been attributed to Vit-D resistant genes that cause a reduction in circulatory Vit-D levels through a set of signaling pathways. CYP24A1, SMRT, and SNAIL are three genes responsible for Vit-D resistance as their activity either lowers the circulatory levels of Vit-D or reduces its availability in target tissues. The hydroxylase CYP24A1 inactivates analogs and prohormonal and/or hormonal forms of calcitriol. Elevation of the expression of CYP24A1 is the major cause of exacerbation of several diseases. CYP24A1 is rate-limiting, and its induction has been correlated with increased prognosis of diseases, while loss of function mutations cause hypersensitivity to Vit-D. The silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) and its corepressor are involved in the transcriptional repression of VDR-target genes. SNAIL1 (SNAIL), SNAIL2 (Slug), and SNAIL3 (Smuc) are involved in transcriptional repression and binding to histone deacetylases and methyltransferases in addition to recruiting polycomb repressive complexes to the target gene promoters. An inverse relationship between the levels of calcitriol and the epithelial-to-mesenchymal transition is reported. Studies have demonstrated a strong association between Vit-D deficiency and chronic diseases, including cardiovascular diseases, diabetes, cancers, autoimmune diseases, infectious diseases, etc. Vit-D resistant genes associated with the aforementioned chronic diseases could serve as potential therapeutic targets. This review focuses on the basic structures and mechanisms of the repression of Vit-D regulated genes and highlights the role of Vit-D resistant genes in chronic diseases.

Emerging role of long non-coding RNAs in endothelial dysfunction and their molecular mechanisms.

Long non-coding RNAs (lncRNAs) are the novel class of transcripts involved in transcriptional, post-transcriptional, translational, and post-translational regulation of physiology and the pathology of diseases. Studies have evidenced that the impairment of endothelium is a critical event in the pathogenesis of atherosclerosis and its complications. Endothelial dysfunction is characterized by an imbalance in vasodilation and vasoconstriction, oxidative stress, proinflammatory factors, and nitric oxide bioavailability. Disruption of the endothelial barrier permeability, the first step in developing atherosclerotic lesions is a consequence of endothelial dysfunction. Though several factors interfere with the normal functioning of the endothelium, intrinsic epigenetic mechanisms governing endothelial function are regulated by lncRNAs and perturbations contribute to the pathogenesis of the disease. This review comprehensively addresses the biogenesis of lncRNA and molecular mechanisms underlying and regulation in endothelial function. An insight correlating lncRNAs and endothelial dysfunction-associated diseases can positively impact the development of novel biomarkers and therapeutic targets in endothelial dysfunction-associated diseases and treatment strategies.

Role of circRNA-miRNA-mRNA interaction network in diabetes and its associated complications.

The majority of the non-protein-coding RNAs are being identified with diversified functions that participate in cellular homeostasis. The circular RNAs (circRNAs) are emerging as noncoding transcripts with a key role in the initiation and development of many physiological and pathological conditions. The advancements in high-throughput RNA sequencing and bioinformatics tools help us to identify several circRNA regulatory pathways, one of which is microRNA (miRNA)-mediated regulation. Besides the direct influence over mRNA transcription, the circRNA can also control the target's expression via sponging miRNAs or the RNA-binding proteins. Studies have demonstrated the dysregulation of the circRNA-miRNA-mRNA interaction network in the pathogenesis of many diseases, including diabetes. This intricate mechanism is associated with the pathogenesis of diabetes and its complications. This review will focus on the circRNA-miRNA-mRNA interaction network that influences the gene expression in the progression of diabetes and its associated complications.

Targeting Nrf2/Keap1 signaling pathway by bioactive natural agents: Possible therapeutic strategy to combat liver disease.

BACKGROUND: Nuclear factor erythroid 2-related factor (Nrf2), a stress-activated transcription factor, has been documented to induce a defense mechanism against oxidative stress damage, and growing evidence considers this signaling pathway a key pharmacological target for the treatment of liver diseases. PURPOSE: The present review highlights the role of phytochemical compounds in activating Nrf2 and mitigate toxicant-induced stress on liver injury. METHODS: A comprehensive search of published articles was carried out to focus on original publications related to Nrf2 activators against liver disease using various literature databases, including the scientific Databases of Science Direct, Web of Science, Pubmed, Google, EMBASE, and Scientific Information (SID). RESULTS: Nrf2 activators exhibited promising effects in resisting a variety of liver diseases induced by different toxicants in preclinical experiments and in vitro studies by regulating cell proliferation and apoptosis as well as an antioxidant defense mechanism. We found that the phytochemical compounds, such as curcumin, naringenin, sulforaphane, diallyl disulfide, mangiferin, oleanolic acid, umbelliferone, daphnetin, quercetin, isorhamnetin-3-O-galactoside, hesperidin, diammonium glycyrrhizinate, corilagin, shikonin, farrerol, and chenpi, had the potential to improve the Nrf2-ARE signaling thereby combat hepatotoxicity. CONCLUSION: Nrf2 activators may offer a novel potential strategy for the prevention and treatment of liver diseases. More extensive studies are essential to identify the underlying mechanisms and establish future therapeutic potentials of these signaling modulators. Further clinical trials are warranted to determine the safety and effectiveness of Nrf2 activators for hepatopathy.

The pivotal role of Nrf2 activators in adipocyte biology.

Adipose tissue is instrumental in maintaining metabolic homeostasis by regulating energy storage in the form of triglycerides. In the case of over-nutrition, adipocytes favorably regulate lipogenesis over lipolysis and accumulate excess triglycerides, resulting in increased adipose tissue mass. An abnormal increase in hypertrophic adipocytes is associated with chronic complications such as insulin resistance, obesity, diabetes, atherosclerosis and nonalcoholic fatty liver disease. Experimental studies indicate the occurrence of oxidative stress in the pathogenesis of obesity. A common underlying link between increasing adipose tissue mass and oxidative stress is the Nuclear Factor Erythroid 2-related factor 2 (Nrf2), Keap1-Nrf2-ARE signaling, which plays an indispensable role in metabolic homeostasis by regulating oxidative and inflammatory responses. Additionally, Nrf2 also activates CCAAT/enhancer-binding protein α, (C/EBP-α), C/EBP-ß and peroxisome proliferator-activated receptor γ (PPARγ) the crucial pro-adipogenic factors that promote de novo adipogenesis. Hence, at the forefront of research is the quest for prospecting novel compounds to modulate Nrf2 activity in the context of adipogenesis and obesity. This review summarizes the molecular mechanism behind the activation of the Keap1-Nrf2-ARE signaling network and the role of Nrf2 activators in adipocyte pathophysiology.

Role of long non-coding RNAs on the regulation of Nrf2 in chronic diseases.

Studies have identified dysregulated long non-coding RNA (lncRNA) in several diseases at transcriptional, translational, and post-translational levels. Although our mechanistic knowledge on the regulation of lncRNAs is still limited, one of the mechanisms of action attributed is binding and regulating transcription factors, thus controlling gene expression and protein function. One such transcription factor is nuclear factor erythroid 2-related factor 2 (Nrf2), which plays a critical biological role in maintaining cellular homeostasis at multiple levels in physiological and pathophysiological conditions. The levels of Nrf2 were found to be down-regulated in many chronic diseases, signifying that Nrf2 can be a key therapeutic target. Few lncRNAs like lncRNA ROR, ENSMUST00000125413, lncRNA ODRUL, Nrf2-lncRNA have been associated with the Nrf2 signaling pathway in response to various stimuli, including stress. This review discusses the regulation of Nrf2 in different responses and the potential role of specific lncRNA in modulating its transcriptional activities. This review further helps to enhance our knowledge on the regulatory role of the critical antioxidant transcription factor, Nrf2.

Analysis of the Exonic Single Nucleotide Polymorphism rs182428269 of the NRF2 Gene in Patients with Diabetic Foot Ulcer.

BACKGROUND: The pivotal role of Nuclear factor erythroid-2-related factor 2 (NRF2) in redox homeostasis and wound healing has been well documented. However, the genetic mechanisms that regulate NRF2 in type 2 diabetes and diabetic foot ulcers remain unexplored. The present study investigated the association of single nucleotide polymorphism rs182428269 (-127 C/T) in subjects with type 2 diabetes and diabetic foot ulcers. METHODS: This cross-sectional study comprised 400 participants that included group I: normal glucose tolerant subjects (NGT, n = 150), group II: type 2 diabetes mellitus subjects (T2DM, n = 150) and group III: infected diabetic foot ulcer subjects (DFU, n = 100). The non-synonymous SNP rs182428269 was selected based on in silico analysis and genotyped by PCR-restriction fragment length polymorphism (RFLP) followed by bidirectional Sanger sequencing. In addition, the gene expression of NRF2 in patients with polymorphism was analyzed by qPCR to evaluate the functional impact of the SNP. RESULTS: NRF2 expression was significantly decreased among the T2DM and DFU subjects when compared to the NGT subjects. Of particular interest, the homozygous mutant (TT) genotype of rs182428269 polymorphism was significantly associated with an increased risk for the development of T2DM (OR = 1.95 (1.02-3.72), p = 0.04) and DFU (OR = 5.66 (2.98-10.76), p = 0.0001). Furthermore, a progressive decline in NRF2 expression was observed among the T2DM and DFU subjects with "TT" genotype compared to the "CC" and "CT" genotypes. CONCLUSION: NRF2 polymorphism rs182428269 is associated with the pathogenesis of T2DM and DFU.

Role of Nrf2 in MALAT1/ HIF-1α loop on the regulation of angiogenesis in diabetic foot ulcer.

Diabetic non healing wounds often result in significant morbidity and mortality. The number of effective targets to detect these wounds are meagre. Slow lymphangiogenesis is one of the complex processes involved in impaired healing of wounds. Long non coding RNAs (lncRNAs) have been importantly recognized for their role in pathological conditions. Multiple studies highlighting the role of lncRNAs in the regulation of several biological processes and complex diseases. Herein, we investigated the role of lncRNA Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in the progression of diabetic foot ulcer (DFU). We report a significant reduction in the expression of lncRNA MALAT1 in the infected DFU subjects which was positively correlated with the expression of angiogenic factors such as Nrf2, HIF-1α and VEGF. Further, expression of pro-inflammatory markers TNF-α and IL-6 were found to be increased while, the expression of anti-inflammatory marker IL-10 was decreased in infected DFU tissues. Involvement of lncRNA MALAT1 in angiogenesis in EA.hy926 cells was demonstrated by silencing the expression of Nrf2, HIF-1α, and VEGF through interference mediated by MALAT1. In addition, its inflammatory role was demonstrated by decreased expression of TNF-α, IL-6 and not affecting the expression of IL-10. Further, CRISPR-Cas9 knock out of Nrf2 decreased the expression of lncRNA MALAT1, HIF-1α and VEGF which revealed the association of Nrf2 in regulating MALAT1/HIF-1α loop through positive feedback mechanism. Collectively, our results suggested the role of Nrf2 on MALAT1/HIF-1α loop in the regulation of angiogenesis, which could act as a novel target in the treatment of diabetic wounds.

The pivotal role of nuclear factor erythroid 2-related factor 2 in diabetes-induced endothelial dysfunction.

Endothelial dysfunction (ED) is a key event in the onset and progression of vascular complications associated with diabetes. Regulation of endothelial function and the underlying signaling mechanisms in the progression of diabetes-induced vascular complications have been well established. Recent studies indicate that increased oxidative stress is an important determinant of endothelial injury and patients with hypertension display ED mediated by impaired Nitric Oxide (NO) availability. Further, oxidative stress is known to be associated with inflammation and ED in vascular remodeling and diabetes-associated hypertension. Numerous strategies have been developed to improve the function of endothelial cells and increasing number of evidences highlight the indispensable role of antioxidants in modulation of endothelium-dependent vasodilation responses. Nuclear factor Erythroid 2-related factor 2 (Nrf2), is the principal transcriptional regulator, that is central in mediating oxidative stress signal response. Having unequivocally established the relationship between type 2 diabetes mellitus (T2DM) and oxidative stress, the pivotal role of Nrf2/Keap1/ARE network, has taken the center stage as target for developing therapies towards maintaining the cellular redox environment. Several activators of Nrf2 are known to combat diabetes-induced ED and few are currently in clinical trials. Focusing on their therapeutic value in diabetes-induced ED, this review highlights some natural and synthetic molecules that are involved in the modulation of the Nrf2/Keap1/ARE network and its underlying molecular mechanisms in the regulation of ED. Further emphasis is also laid on the therapeutic benefits of directly up-regulating Nrf2-mediated antioxidant defences in regulating endothelial redox homeostasis for countering diabetes-induced ED.