Jun Okabe

Hyperglycemia Induces a Dynamic Cooperativity of Histone Methylase and Demethylase Enzymes Associated With Gene-Activating Epigenetic Marks That Coexist on the Lysine Tail

OBJECTIVE Results from the Diabetes Control Complications Trial (DCCT) and the subsequent Epidemiology of Diabetes Interventions and Complications (EDIC) Study and more recently from the U.K. Prospective Diabetes Study (UKPDS) have revealed that the deleterious end-organ effects that occurred in both conventional and more aggressively treated subjects continued to operate >5 years after the patients had returned to usual glycemic control and is interpreted as a legacy of past glycemia known as “hyperglycemic memory.” We have hypothesized that transient hyperglycemia mediates persistent gene-activating events attributed to changes in epigenetic information. RESEARCH DESIGN AND METHODS Models of transient hyperglycemia were used to link NFκB-p65 gene expression with H3K4 and H3K9 modifications mediated by the histone methyltransferases (Set7 and SuV39h1) and the lysine-specific demethylase (LSD1) by the immunopurification of soluble NFκB-p65 chromatin. RESULTS The sustained upregulation of the NFκB-p65 gene as a result of ambient or prior hyperglycemia was associated with increased H3K4m1 but not H3K4m2 or H3K4m3. Furthermore, glucose was shown to have other epigenetic effects, including the suppression of H3K9m2 and H3K9m3 methylation on the p65 promoter. Finally, there was increased recruitment of the recently identified histone demethylase LSD1 to the p65 promoter as a result of prior hyperglycemia. CONCLUSIONS These studies indicate that the active transcriptional state of the NFκB-p65 gene is linked with persisting epigenetic marks such as enhanced H3K4 and reduced H3K9 methylation, which appear to occur as a result of effects of the methyl-writing and methyl-erasing histone enzymes.

NADPH Oxidase 1 Plays a Key Role in Diabetes Mellitus–Accelerated Atherosclerosis

Background— In diabetes mellitus, vascular complications such as atherosclerosis are a major cause of death. The key underlying pathomechanisms are unclear. However, hyperglycemic oxidative stress derived from NADPH oxidase (Nox), the only known dedicated enzyme to generate reactive oxygen species appears to play a role. Here we identify the Nox1 isoform as playing a key and pharmacologically targetable role in the accelerated development of diabetic atherosclerosis. Methods and Results— Human aortic endothelial cells exposed to hyperglycemic conditions showed increased expression of Nox1, oxidative stress, and proinflammatory markers in a Nox1-siRNA reversible manner. Similarly, the specific Nox inhibitor, GKT137831, prevented oxidative stress in response to hyperglycemia in human aortic endothelial cells. To examine these observations in vivo, we investigated the role of Nox1 on plaque development in apolipoprotein E–deficient mice 10 weeks after induction of diabetes mellitus. Deletion of Nox1, but not Nox4, had a profound antiatherosclerotic effect correlating with reduced reactive oxygen species formation, attenuation of chemokine expression, vascular adhesion of leukocytes, macrophage infiltration, and reduced expression of proinflammatory and profibrotic markers. Similarly, treatment of diabetic apolipoprotein E–deficient mice with GKT137831 attenuated atherosclerosis development. Conclusions— These studies identify a major pathological role for Nox1 and suggest that Nox1-dependent oxidative stress is a promising target for diabetic vasculopathies, including atherosclerosis.

Nox1 Plays a Key Role in Diabetes Accelerated Atherosclerosis

Background— In diabetes mellitus, vascular complications such as atherosclerosis are a major cause of death. The key underlying pathomechanisms are unclear. However, hyperglycemic oxidative stress derived from NADPH oxidase (Nox), the only known dedicated enzyme to generate reactive oxygen species appears to play a role. Here we identify the Nox1 isoform as playing a key and pharmacologically targetable role in the accelerated development of diabetic atherosclerosis. Methods and Results— Human aortic endothelial cells exposed to hyperglycemic conditions showed increased expression of Nox1, oxidative stress, and proinflammatory markers in a Nox1-siRNA reversible manner. Similarly, the specific Nox inhibitor, GKT137831, prevented oxidative stress in response to hyperglycemia in human aortic endothelial cells. To examine these observations in vivo, we investigated the role of Nox1 on plaque development in apolipoprotein E–deficient mice 10 weeks after induction of diabetes mellitus. Deletion of Nox1, but not Nox4, had a profound antiatherosclerotic effect correlating with reduced reactive oxygen species formation, attenuation of chemokine expression, vascular adhesion of leukocytes, macrophage infiltration, and reduced expression of proinflammatory and profibrotic markers. Similarly, treatment of diabetic apolipoprotein E–deficient mice with GKT137831 attenuated atherosclerosis development. Conclusions— These studies identify a major pathological role for Nox1 and suggest that Nox1-dependent oxidative stress is a promising target for diabetic vasculopathies, including atherosclerosis.

Epigenetic phenomena linked to diabetic complications

Diabetes mellitus (type 1 and type 2) and the complications associated with this condition are an urgent public health problem, as the incidence of diabetes mellitus is steadily increasing. Environmental factors, such as diet and exposure to hyperglycemia, contribute to the etiology of diabetes mellitus and its associated microvascular and macrovascular complications. These vascular complications are the main cause of the morbidity and mortality burden of diabetes mellitus. The DCCT–EDIC and UKPDS epidemiological studies correlated poor glycemic control with the development of vascular complications in patients with type 1 or type 2 diabetes mellitus. The findings of these studies suggest that early exposure to hyperglycemia predisposes individuals to the development of diabetic complications, a phenomenon referred to as metabolic memory or the legacy effect. The first experimental evidence for metabolic memory was reported >20 years ago and the underlying molecular mechanisms are currently being characterized. Interestingly, transient exposure to hyperglycemia results in long-lasting epigenetic modifications that lead to changes in chromatin structure and gene expression, which mediate these persistent metabolic characteristics.

Genetic Targeting or Pharmacologic Inhibition of NADPH Oxidase Nox4 Provides Renoprotection in Long-Term Diabetic Nephropathy

Diabetic nephropathy may occur, in part, as a result of intrarenal oxidative stress. NADPH oxidases comprise the only known dedicated reactive oxygen species (ROS)-forming enzyme family. In the rodent kidney, three isoforms of the catalytic subunit of NADPH oxidase are expressed (Nox1, Nox2, and Nox4). Here we show that Nox4 is the main source of renal ROS in a mouse model of diabetic nephropathy induced by streptozotocin administration in ApoE(-/-) mice. Deletion of Nox4, but not of Nox1, resulted in renal protection from glomerular injury as evidenced by attenuated albuminuria, preserved structure, reduced glomerular accumulation of extracellular matrix proteins, attenuated glomerular macrophage infiltration, and reduced renal expression of monocyte chemoattractant protein-1 and NF-κB in streptozotocin-induced diabetic ApoE(-/-) mice. Importantly, administration of the most specific Nox1/4 inhibitor, GKT137831, replicated these renoprotective effects of Nox4 deletion. In human podocytes, silencing of the Nox4 gene resulted in reduced production of ROS and downregulation of proinflammatory and profibrotic markers that are implicated in diabetic nephropathy. Collectively, these results identify Nox4 as a key source of ROS responsible for kidney injury in diabetes and provide proof of principle for an innovative small molecule approach to treat and/or prevent chronic kidney failure.

Analysis of the IGF2/H19 imprinting control region uncovers new genetic defects, including mutations of OCT-binding sequences, in patients with 11p15 fetal growth disorders

The imprinted expression of the IGF2 and H19 genes is controlled by the imprinting control region 1 (ICR1) located at chromosome 11p15.5. This methylation-sensitive chromatin insulator works by binding the zinc-finger protein CTCF in a parent-specific manner. DNA methylation defects involving the ICR1 H19/IGF2 domain result in two growth disorders with opposite phenotypes: an overgrowth disorder, the Beckwith-Wiedemann syndrome (maternal ICR1 gain of methylation in 10% of BWS cases) and a growth retardation disorder, the Silver-Russell syndrome (paternal ICR1 loss of methylation in 60% of SRS cases). Although a few deletions removing part of ICR1 have been described in some familial BWS cases, little information is available regarding the mechanism of ICR1 DNA methylation defects. We investigated the CTCF gene and the ICR1 domain in 21 BWS patients with ICR1 gain of methylation and 16 SRS patients with ICR1 loss of methylation. We identified four constitutional ICR1 genetic defects in BWS patients, including a familial case. Three of those defects are newly identified imprinting defects consisting of small deletions and a single mutation, which do not involve one of the CTCF binding sites. Moreover, two of those defects affect OCT-binding sequences which are suggested to maintain the unmethylated state of the maternal allele. A single-nucleotide variation was identified in a SRS patient. Our data extends the spectrum of constitutive genetic ICR1 abnormalities and suggests that extensive and accurate analysis of ICR1 is required for appropriate genetic counseling in BWS patients with ICR1 gain of methylation.

Distinguishing Hyperglycemic Changes by Set7 in Vascular Endothelial Cells

Rationale: Epigenetic changes are implicated in the persisting vascular effects of hyperglycemia. The precise mechanism whereby chromatin structure and subsequent gene expression are regulated by glucose in vascular endothelial cells remain to be fully defined. Objective: We have studied the molecular and functional mechanism whereby the Set7 methyltransferase associates with chromatin formation and histone methylation in vascular cells in response to current and previous exposure to glucose. Methods and Results: To characterize the molecular and functional identity of the Set7 protein, we used vascular cells overexpressing or lacking Set7. Chromatin fractionation for mono-methylation of lysine 4 on histone H3 identified methyltransferase activity. Immunofluorescence experiments strongly suggest that Set7 protein accumulates in the nucleus in response to hyperglycemia. Moreover, activation of proinflammatory genes by high glucose is dependent on Set7 but distinguished by H3K4m1 gene patterns. We show that transient hyperglycemia regulates the expression of proinflammatory genes in vascular endothelial cells in vitro and the persistent increase in glucose-induced gene expression in the aorta of nondiabetic mice. Conclusions: This study uncovers that the response to hyperglycemia in vascular endothelial cells involves the H3K4 methyltransferase, Set7. This enzyme appears to regulate glucose-induced chromatin changes and gene expression not only by H3K4m1-dependent but also H3K4m1-independent pathways. Furthermore, Set7 appears to be responsible for sustained vascular gene expression in response to prior hyperglycemia and is a potential molecular mechanism for the phenomenon of hyperglycemic memory.

Reactive Oxygen Species Can Provide Atheroprotection via NOX4-Dependent Inhibition of Inflammation and Vascular Remodeling

Objective— Oxidative stress is considered a hallmark of atherosclerosis. In particular, the superoxide-generating type 1 NADPH oxidase (NOX1) has been shown to be induced and play a pivotal role in early phases of mouse models of atherosclerosis and in the context of diabetes mellitus. Here, we investigated the role of the most abundant type 4 isoform (NOX4) in human and mouse advanced atherosclerosis. Approach and Results— Plaques of patients with cardiovascular events or established diabetes mellitus showed a surprising reduction in expression of the most abundant but hydrogen peroxide (H2O2)-generating type 4 isoform (Nox4), whereas Nox1 mRNA was elevated, when compared with respective controls. As these data suggested that NOX4-derived reactive oxygen species may convey a surprisingly protective effect during plaque progression, we examined a mouse model of accelerated and advanced diabetic atherosclerosis, the streptozotocin-treated ApoE −/− mouse, with (NOX4 −/−) and without genetic deletion of Nox4. Similar to the human data, advanced versus early plaques of wild-type mice showed reduced Nox4 mRNA expression. Consistent with a rather protective role of NOX4-derived reactive oxygen species, NOX4 −/− mice showed increased atherosclerosis when compared with wild-type mice. Deleting NOX4 was associated with reduced H2O2 forming activity and attenuation of the proinflammatory markers, monocyte chemotratic protein-1, interleukin-1&bgr;, and tumor necrosis factor-&agr;, as well as vascular macrophage accumulation. Furthermore, there was a greater accumulation of fibrillar collagen fibres within the vascular wall and plaque in diabetic Nox4 −/− ApoE −/− mice, indicative of plaque remodeling. These data could be replicated in human aortic endothelial cells in vitro, where Nox4 overexpression increased H2O2 and reduced the expression of pro-oxidants and profibrotic markers. Interestingly, Nox4 levels inversely correlated with Nox2 gene and protein levels. Although NOX2 is not constitutively active unlike NOX4 and forms rather superoxide, this opens up the possibility that at least some effects of NOX4 deletion are mediated by NOX2 activation. Conclusions— Thus, the appearance of reactive oxygen species in atherosclerosis is apparently not always a nondesirable oxidative stress, but can also have protective effects. Both in humans and in mouse, the H2O2-forming NOX4, unlike the superoxide-forming NOX1, can act as a negative modulator of inflammation and remodeling and convey atheroprotection. These results have implications on how to judge reactive oxygen species formation in cardiovascular disease and need to be considered in the development of NOX inhibitory drugs.

Vascular histone deacetylation by pharmacological HDAC inhibition

HDAC inhibitors can regulate gene expression by post-translational modification of histone as well as nonhistone proteins. Often studied at single loci, increased histone acetylation is the paradigmatic mechanism of action. However, little is known of the extent of genome-wide changes in cells stimulated by the hydroxamic acids, TSA and SAHA. In this article, we map vascular chromatin modifications including histone H3 acetylation of lysine 9 and 14 (H3K9/14ac) using chromatin immunoprecipitation (ChIP) coupled with massive parallel sequencing (ChIP-seq). Since acetylation-mediated gene expression is often associated with modification of other lysine residues, we also examined H3K4me3 and H3K9me3 as well as changes in CpG methylation (CpG-seq). RNA sequencing indicates the differential expression of ∼30% of genes, with almost equal numbers being up- and down-regulated. We observed broad deacetylation and gene expression changes conferred by TSA and SAHA mediated by the loss of EP300/CREBBP binding at multiple gene promoters. This study provides an important framework for HDAC inhibitor function in vascular biology and a comprehensive description of genome-wide deacetylation by pharmacological HDAC inhibition.

Testing for association between MeCP2 and the brahma-associated SWI/SNF chromatin-remodeling complex

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