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* Each chapter is written by one or more invited world-renowned experts * Information provided in handy reference tables and design charts* Numerous examples demonstrate how the theory outlined in the book is applied in the design of structuresTremendous strides have been made in the last decades in the advancement of offshore exploration and.

To examine whether the long non‐coding RNA (lncRNA) metastasis associated lung adenocarcinoma transcript 1 (MALAT1) is altered in the endothelial cells in response to glucose and the significance of such alteration. We incubated human umbilical vein endothelial cells with media containing various glucose levels. We found an increase in MALAT1 expression peaking after 12 hrs of incubation in high glucose. This increase was associated with parallel increase in serum amyloid antigen 3 (SAA3), an inflammatory ligand and target of MALAT1 and was further accompanied by increase in mRNAs and proteins of inflammatory mediators, tumour necrosis factor alpha (TNF‐α) and interleukin 6 (IL‐6). Renal tissue from the diabetic animals showed similar changes. Such cellular alterations were prevented following MALAT1 specific siRNA transfection. Results of this study indicate that LncRNA MALAT1 regulates glucose‐induced up‐regulation of inflammatory mediators IL‐6 and TNF‐α through activation of SAA3. Identification of such novel mechanism may lead to the development of RNA‐based therapeutics targeting MALAT1 for diabetes‐induced micro and macro vascular complications.

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.

Chronic diabetic complications affect multiple organs causing widespread organ damage. Although there are some commonalities, the phenotype of such changes show tissue specific variation. Given this, we examined whether differences in circular RNA (circRNA) mediated gene regulatory mechanisms contribute to changes in gene expression at the basal level and in diabetes. CircRNAs are single-stranded RNA with covalently closed loop structures and act as miRNA sponges, factors of RNA splicing, scaffolding for proteins, regulators of transcription, and modulators of the expression of parental genes, among other roles. We examined heart and retinal tissue from Streptozotocin-induced diabetic mice with established diabetes related tissue damage and tissue from non-diabetic controls. A custom array analysis was performed and the data were analysed. Two major circRNA mediated processes were uniquely upregulated in diabetic heart tissue, namely, positive regulation of endothelial cell migration and regulation of mitochondria: mitochondrial electron transport. In the retina, circRNAs regulating extracellular matrix protein production and endothelial to mesenchymal transition (EndMT) were found to be upregulated. The current study identified regulatory and potential pathogenetic roles of specific circRNA in diabetic retinopathy and cardiomyopathy. Understanding such novel mechanisms, may in the future, be useful to develop RNA based treatment strategies.

In diabetes, some of the cellular changes are similar to aging. We hypothesized that hyperglycemia accelerates aging-like changes in the endothelial cells (ECs) and tissues leading to structural and functional damage. We investigated glucose-induced aging in 3 types of ECs using senescence associated β-gal (SA β-gal) staining and cell morphology. Alterations of sirtuins (SIRTs) and their downstream mediator FOXO and oxidative stress were investigated. Relationship of such alteration with histone acetylase (HAT) p300 was examined. Similar examinations were performed in tissues of diabetic animals. ECs in high glucose (HG) showed evidence of early senescence as demonstrated by increased SA β-gal positivity and reduced replicative capacities. These alterations were pronounced in microvascular ECs. They developed an irregular and hypertrophic phenotype. Such changes were associated with decreased SIRT (1-7) mRNA expressions. We also found that p300 and SIRT1 regulate each other in such process, as silencing one led to increase of the others' expression. Furthermore, HG caused reduction in FOXO1's DNA binding ability and antioxidant target gene expressions. Chemically induced increased SIRT1 activity and p300 knockdown corrected these abnormalities slowing aging-like changes. Diabetic animals showed increased cellular senescence in renal glomerulus and retinal blood vessels along with reduced SIRT1 mRNA expressions in these tissues. Data from this study demonstrated that hyperglycemia accelerates aging-like process in the vascular ECs and such process is mediated via downregulation of SIRT1, causing reduction of mitochondrial antioxidant enzyme in a p300 and FOXO1 mediated pathway.

Diabetic retinopathy (DR) is a leading cause of blindness. Increased vascular endothelial growth factor (VEGF), promoting angiogenesis and increased permeability, is a key mechanistic abnormality in DR. We investigated microRNA (miRNA) alterations in DR with specific focus on miR-200b, and its downstream target, VEGF. miRNA expression profiling microarray was used to examine the retinas of streptozotocin-induced diabetic rats. Expressions of specific miRNAs were verified with PCR in the rat retina and in glucose-exposed endothelial cells. A target search, based on sequence complementarities, identified specific targets. We analyzed mRNA levels and protein expression in endothelial cells from large vessels and retinal capillaries and in the rat retina, with or without injection of miR-200b mimic or antagomir. Localization of miR-200b and its functional analysis in the rat and human retinas were performed. Alteration of several miRNAs, including downregulation of miR-200b, were observed in the retina in diabetes. Such downregulation was validated in the retina of diabetic rats and in endothelial cells incubated in glucose. In parallel, VEGF (target of miR-200b) mRNA and protein were elevated. In the retina, miR-200b was localized in neuronal, glial, and vascular elements. Transfection of endothelial cells and intravitreal injection of miR-200b mimic prevented diabetes-induced increased VEGF mRNA and protein. Also prevented were glucose-induced increased permeability and angiogenesis. Furthermore, transfection of miR-200b antagonists (antagomir) led to increased VEGF production. Similar alterations were seen in the human retina. These studies show a novel mechanism involving miR-200b in DR. Identification of such mechanisms may lead to the development of novel miRNA-based therapy.

Diabetic cardiomyopathy (DCM) is one of the most prevalent causes of morbidity and mortality in diabetic patients. Hyperglycemia induces increased expression/deposition of extracellular matrix (ECM) proteins including fibronectin (FN) and collagen (Col) and plays an important role in fibrosis in diabetic cardiomyopathy (DCM). The roles of RNAs including microRNA (miRNA) and long non-coding RNAs (lncRNA) have begun to be understood in many conditions. In this study, we investigated the role of a specific miRNA, miR-9, and its interactions with lncRNA ZFAS1 in mediating fibrosis in DCM. Treatment with 25 mM glucose (HG) decreased miR-9 expression and increased expressions of ZFAS1, ECM proteins and inflammatory markers, compared to 5 mM glucose (NG) in the HCMECs by using qRT-PCR. Glucose-induced upregulation of ECM proteins can be prevented by ZFAS1 siRNA or miR-9 mimic transfection. Luciferase assay was confirmed miR-9 binding to FN 3’-UTR. miR-9 expression can be regulated by ZFAS1 through polycomb repressive complex 2 (PRC2) components using RNA immunoprecipitation (RIP) and chromatin immunoprecipitation (ChIP) assays. In the in vivo experiment, hyperglycemia-induced the ECM production can be prevented by the miR-9 overexpression in the fibrosis in DCM. These studies showed a novel glucose-induced molecular mechanism in which ZFAS1 participates in the transcriptional regulation of ECM protein production in diabetes through miR-9.

Cardiac mast cells (CMCs) are multifarious immune cells with complex roles both in cardiac physiological and pathological conditions, especially in cardiac fibrosis. Little is known about the physiological importance of CMCs in cardiac homeostasis and inflammatory process. Therefore, the present review will summarize the recent progress of CMCs on origin, development and replenishment in the heart, including their effects on cardiac development, function and ageing under physiological conditions as well as the roles of CMCs in inflammatory progression and resolution. The present review will shed a light on scientists to understand cardioimmunology and to develop immune treatments targeting on CMCs following cardiac injury.

Endothelial cells and high glucose-induced endothelial dysfunction are the common origin of chronic diabetic complications such as retinopathy, nephropathy, and cardiomyopathy. Yet their common origins, the vascular manifestations of such complications are different. We examined the basal heterogeneity between microvascular endothelial cells(MECs) from the retina, kidneys, and heart, as well as their differential responses to hyperglycemia in diabetes. To this extent, we used a spatial transcriptomic approach to investigate gene expression differences across retinal, renal, and cardiac MECs in diabetic and non-diabetic mouse models. We validated MEC heterogeneity in vitro using human retinal and cardiac MECs. The spatial transcriptomic approach was also used to explore potential similarities in retinal MECs and neuronal cells in response to hyperglycemia. We found that MECs from different target organs of major diabetic complications were transcriptomically distinct at the basal state and respond differently to hyperglycemia. These findings were recapitulated in cell culture, with selected analytes. We found minimal similarities between retinal MECs and neuronal cells. Our findings show considerable heterogeneity across retinal, renal, and cardiac MECs, both at the basal state and in their responses to hyperglycemia in diabetes. These findings show that organ specific MEC heterogeneity can influence differential development of pathological changes across various target organs of chronic diabetic complications, and suggest that MEC heterogeneity may influence treatment target(s) and drug development.

Glucose-induced augmented vascular endothelial growth factor (VEGF) production is a key event in diabetic retinopathy. We have previously demonstrated that downregulation of miR-200b increases VEGF, mediating structural and functional changes in the retina in diabetes. However, mechanisms regulating miR-200b in diabetes are not known. Histone methyltransferase complex, Polycomb Repressive Complex 2 (PRC2), has been shown to repress miRNAs in neoplastic process. We hypothesized that, in diabetes, PRC2 represses miR-200b through its histone H3 lysine-27 trimethylation mark. We show that human retinal microvascular endothelial cells exposed to high levels of glucose regulate miR-200b repression through histone methylation and that inhibition of PRC2 increases miR-200b while reducing VEGF. Furthermore, retinal tissue from animal models of diabetes showed increased expression of major PRC2 components, demonstrating in vivo relevance. This research established a repressive relationship between PRC2 and miR-200b, providing evidence of a novel mechanism of miRNA regulation through histone methylation.