The mechanisms by which macrophages mediate the enhanced inflammation associated with diabetes complications are not completely understood. We used RNA sequencing to profile the transcriptome of bone marrow macrophages isolated from diabetic db/db mice and identified 1,648 differentially expressed genes compared with control db/+ mice. Data analyses revealed that diabetes promoted a proinflammatory, profibrotic, and dysfunctional alternatively activated macrophage phenotype possibly via transcription factors involved in macrophage function. Notably, diabetes altered levels of several long noncoding RNAs (lncRNAs). Because the role of lncRNAs in diabetes complications is unknown, we further characterized the function of lncRNA E330013P06, which was upregulated in macrophages from db/db and diet-induced insulin-resistant type 2 diabetic (T2D) mice, but not from type 1 diabetic mice. It was also upregulated in monocytes from T2D patients. E330013P06 was also increased along with inflammatory genes in mouse macrophages treated with high glucose and palmitic acid. E330013P06 overexpression in macrophages induced inflammatory genes, enhanced responses to inflammatory signals, and increased foam cell formation. In contrast, small interfering RNA–mediated E330013P06 gene silencing inhibited inflammatory genes induced by the diabetic stimuli. These results define the diabetic macrophage transcriptome and novel functional roles for lncRNAs in macrophages that could lead to lncRNA-based therapies for inflammatory diabetes complications.

Objective—: Diabetes mellitus accelerates proatherogenic and proinflammatory phenotype of vascular smooth muscle cell (VSMC) associated with vascular complications. Evidence shows that microRNAs (miRNAs) play key roles in VSMC functions, but their role under diabetic conditions is unclear. We profiled miRNAs in VSMC from diabetic mice and examined their role in VSMC dysfunction. Approach and Results—: High throughput small RNA-sequencing identified 135 differentially expressed miRNAs in VSMC from type 2 diabetic db/db mice (db/dbVSMC) versus nondiabetic db/+ mice. Several of these miRNAs were known to regulate VSMC functions. We further focused on miR-504, because it was highly upregulated in db/dbVSMC, and its function in VSMC is unknown. miR-504 and its host gene Fgf13 were significantly increased in db/dbVSMC and in aortas from db/db mice. Bioinformatics analysis predicted that miR-504 targets including signaling adaptor Grb10 and transcription factor Egr2 could regulate growth factor signaling. We experimentally validated Grb10 and Egr2 as novel targets of miR-504. Overexpression of miR-504 in VSMC inhibited contractile genes and enhanced extracellular signal-regulated kinase 1/2 activation, proliferation, and migration. These effects were blocked by miR-504 inhibitors. Grb10 knockdown mimicked miR-504 functions and increased inflammatory genes. Egr2 knockdown-inhibited anti-inflammatory Socs1 and increased proinflammatory genes. Furthermore, high glucose and palmitic acid upregulated miR-504 and inflammatory genes, but downregulated Grb10 . Conclusions—: Diabetes mellitus misregulates several miRNAs including miR-504 that can promote VSMC dysfunction. Because changes in many of these miRNAs are sustained in diabetic VSMC even after in vitro culture, they may be involved in metabolic memory of vascular complications. Targeting such mechanisms could offer novel therapeutic strategies for diabetic complications.

Transforming growth factor-β1 (TGF-β)-induced fibrotic and inflammatory genes in renal mesangial cells (MCs) play important roles in glomerular dysfunction associated with diabetic nephropathy (DN). TGF-β regulates gene expression in MCs by altering key chromatin histone modifications at target gene promoters. However, the role of the repressive histone H3 lysine 27 trimethylation (H3K27me3) modification is unclear. Here we show that TGF-β reduces H3K27me3 at the Ctgf, Serpine1, and Ccl2 gene promoters in rat MCs (RMCs) and reciprocally up-regulates the expression of these pro-fibrotic and inflammatory genes. In parallel, TGF-β down-regulates Enhancer of Zeste homolog 2 (Ezh2), an H3K27me3 methyltransferase, and decreases its recruitment at Ctgf and Ccl2 but not Serpine1 promoters. Ezh2 knockdown with siRNAs enhances TGF-β–induced expression of these genes, supporting its repressive function. Mechanistically, Ezh2 down-regulation is mediated by TGF-β–induced microRNA, miR-101b, which targets Ezh2 3′-UTR. TGF-β also up-regulates Jmjd3 and Utx in RMCs, suggesting a key role for these H3K27me3 demethylases in H3K27me3 inhibition. In RMCs, Utx knockdown inhibits hypertrophy, a key event in glomerular dysfunction. The H3K27me3 regulators are similarly altered in human and mouse MCs. High glucose inhibits Ezh2 and increases miR-101b in a TGF-β–dependent manner. Furthermore, in kidneys from rodent models of DN, fibrotic genes, miR-101b, and H3K27me3 demethylases are up-regulated, whereas Ezh2 protein levels as well as enrichment of Ezh2 and H3K27me3 at target genes are decreased, demonstrating in vivo relevance. These results suggest that H3K27me3 inhibition by TGF-β via dysregulation of related histone-modifying enzymes and miRNAs augments pathological genes mediating glomerular mesangial dysfunction and DN.

Long noncoding RNAs (lncRNAs) are increasingly implicated in the pathology of diabetic complications. Here, we examined the role of lncRNAs in monocyte dysfunction and inflammation associated with human type 2 diabetes mellitus (T2D). RNA sequencing analysis of CD14 ⁺ monocytes from patients with T2D versus healthy controls revealed downregulation of antiinflammatory and antiproliferative genes, along with several lncRNAs, including a potentially novel divergent lncRNA diabetes regulated antiinflammatory RNA ( DRAIR ) and its nearby gene CPEB2 . High glucose and palmitic acid downregulated DRAIR in cultured CD14 ⁺ monocytes, whereas antiinflammatory cytokines and monocyte-to-macrophage differentiation upregulated DRAIR via KLF4 transcription factor. DRAIR overexpression increased antiinflammatory and macrophage differentiation genes but inhibited proinflammatory genes. Conversely, DRAIR knockdown attenuated antiinflammatory genes, promoted inflammatory responses, and inhibited phagocytosis. DRAIR regulated target gene expression through interaction with chromatin, as well as inhibition of the repressive epigenetic mark H3K9me2 and its corresponding methyltransferase G9a. Mouse orthologous Drair and Cpeb2 were also downregulated in peritoneal macrophages from T2D db/db mice, and Drair knockdown in nondiabetic mice enhanced proinflammatory genes in macrophages. Thus, DRAIR modulates the inflammatory phenotype of monocytes/macrophages via epigenetic mechanisms, and its downregulation in T2D may promote chronic inflammation. Augmentation of endogenous lncRNAs like DRAIR could serve as novel antiinflammatory therapies for diabetic complications.

Background: Obesity and diabetes are associated with elevated free fatty acids like palmitic acid (PA), which promote chronic inflammation and impaired inflammation resolution associated with cardiometabolic disorders. Long noncoding RNAs (lncRNAs) are implicated in inflammatory processes; however, their roles in PA-regulated inflammation and resolution are unclear. Methods: We performed RNA-sequencing analysis to identify PA-regulated coding genes and novel lncRNAs in CD14 ⁺ monocytes from healthy volunteers. We investigated the regulation and function of an uncharacterized PA-induced lncRNA PARAIL (PA- r egulated a nti- i nflammatory l ncRNA). We examined its role in inflammation resolution by employing knockdown and overexpression strategies in human and mouse macrophages. We also used RNA pulldown coupled with mass spectrometry to identify PARAIL interacting nuclear proteins and their mechanistic involvement in PARAIL functions in human macrophages. Results: Treatment of human CD14 ⁺ monocytes with PA-induced several lncRNAs and genes associated with inflammatory phenotype. PA strongly induced lncRNA PARAIL expressed near RIPK2 . PARAIL was also induced by cytokines and infectious agents in human monocytes/macrophages and was regulated by NF-κB (nuclear factor-kappa B). Time course studies showed PARAIL was induced during inflammation resolution phase in PA-treated macrophages. PARAIL knockdown with antisense oligonucleotides upregulated key inflammatory genes and vice versa with PARAIL overexpression. We found that PARAIL interacts with ELAVL1 (ELAV-like RNA-binding protein 1) protein via adenylate/uridylate–rich elements (AU-rich elements; AREs). ELAVL1 knockdown inhibited the anti-inflammatory functions of PARAIL . Moreover, PARAIL knockdown increased cytosolic localization of ELAVL1 and increased the stability of ARE-containing inflammatory genes. Mouse orthologous Parail was downregulated in macrophages from mice with diabetes and atherosclerosis. Parail overexpression attenuated proinflammatory genes in mouse macrophages. Conclusions: Upregulation of PARAIL under acute inflammatory conditions contributes to proresolution mechanisms via PARAIL -ELAVL1 interactions. Conversely, PARAIL downregulation in cardiometabolic diseases enhances ELAVL1 function and impairs inflammation resolution to further augment inflammation. Thus, inflammation-resolving lncRNAs like PARAIL represent novel targets to combat inflammatory cardiometabolic diseases.

Long non-coding RNAs (lncRNAs) play key roles in Angiotensin II (AngII) signaling but their role in chondrogenic transformation of vascular smooth muscle cells (VSMCs) is unknown. We describe a novel AngII-induced lncRNA Alivec (Angiotensin II-induced lncRNA in VSMCs eliciting chondrogenic phenotype) implicated in VSMC chondrogenesis. In rat VSMCs, Alivec and the nearby gene Acan, a chondrogenic marker, were induced by growth factors AngII and PDGF and the inflammatory cytokine TNF-α. AngII co-regulated Alivec and Acan through the activation of AngII type1 receptor signaling and Sox9, a master transcriptional regulator of chondrogenesis. Alivec knockdown with GapmeR antisense-oligonucleotides attenuated the expression of AngII-induced chondrogenic marker genes, including Acan, and inhibited the chondrogenic phenotype of VSMCs. Conversely, Alivec overexpression upregulated these genes and promoted chondrogenic transformation. RNA-pulldown coupled to mass-spectrometry identified Tropomyosin-3-alpha and hnRNPA2B1 proteins as Alivec-binding proteins in VSMCs. Furthermore, male rats with AngII-driven hypertension showed increased aortic expression of Alivec and Acan. A putative human ortholog ALIVEC, was induced by AngII in human VSMCs, and this locus was found to harbor the quantitative trait loci affecting blood pressure. Together, these findings suggest that AngII-regulated lncRNA Alivec functions, at least in part, to mediate the AngII-induced chondrogenic transformation of VSMCs implicated in vascular dysfunction and hypertension.