Simona-Adriana Manea

Regulation of Nox enzymes expression in vascular pathophysiology: Focusing on transcription factors and epigenetic mechanisms

NADPH oxidases (Nox) represent a family of hetero-oligomeric enzymes whose exclusive biological function is the generation of reactive oxygen species (ROS). Nox-derived ROS are essential modulators of signal transduction pathways that control key physiological activities such as cell growth, proliferation, migration, differentiation, and apoptosis, immune responses, and biochemical pathways. Enhanced formation of Nox-derived ROS, which is generally associated with the up-regulation of different Nox subtypes, has been established in various pathologies, namely cardiovascular diseases, diabetes, obesity, cancer, and neurodegeneration. The detrimental effects of Nox-derived ROS are related to alterations in cell signalling and/or direct irreversible oxidative damage of nucleic acids, proteins, carbohydrates, and lipids. Thus, understanding of transcriptional regulation mechanisms of Nox enzymes have been extensively investigated in an attempt to find ways to counteract the excessive formation of Nox-derived ROS in various pathological states. Despite the numerous existing data, the molecular pathways responsible for Nox up-regulation are not completely understood. This review article summarizes some of the recent advances and concepts related to the regulation of Nox expression in the vascular pathophysiology. It highlights the role of transcription factors and epigenetic mechanisms in this process. Identification of the signalling molecules involved in Nox up-regulation, which is associated with the onset and development of cardiovascular dysfunction may contribute to the development of novel strategies for the treatment of cardiovascular diseases.

C/EBP transcription factors regulate NADPH oxidase in human aortic smooth muscle cells

In atherosclerosis, oxidative stress‐induced vascular smooth muscle cells (SMCs) dysfunction is partially mediated by up‐regulated NADPH oxidase (Nox); the mechanisms of enzyme regulation are not entirely defined. CCAAT/enhancer‐binding proteins (C/EBP) regulate cellular proliferation and differentiation, and the expression of many inflammatory and immune genes. We aimed at elucidating the role of C/EBP in the regulation of Nox in SMCs exposed to pro‐inflammatory conditions. Human aortic SMCs were treated with interferon‐γ (IFN‐γ) for up to 24 hrs. Lucigenin‐enhanced chemiluminescence, real‐time PCR, Western blot, promoter‐luciferase reporter analysis and chromatin immunoprecipitation assays were employed to investigate Nox regulation. IFN‐γ dose‐dependently induced Nox activity and expression, nuclear translocation and up‐regulation of C/EBPα, C/EBPβ and C/EBPδ protein expression levels. Silencing of C/EBPα, C/EBPβ or C/EBPδ reduced significantly but differentially the IFN‐γ‐induced up‐regulation of Nox activity, gene and protein expression. In silico analysis indicated the existence of typical C/EBP sites within Nox1, Nox4 and Nox5 promoters. Transient overexpression of C/EBPα, C/EBPβ or C/EBPδ enhanced the luciferase level directed by the promoters of the Nox subtypes. Chromatin immunoprecipitation demonstrated the physical interaction of C/EBPα, C/EBPβ and C/EBPδ proteins with the Nox1/4/5 promoters. C/EBP transcription factors are important regulators of Nox enzymes in IFN‐γ‐exposed SMCs. Activation of C/EBP may induce excessive Nox‐derived reactive oxygen species formation, further contributing to SMCs dysfunction and atherosclerotic plaque development. Pharmacological targeting of C/EBP‐related signalling pathways may be used to counteract the adverse effects of oxidative stress.

High Glucose-Induced Increased Expression of Endothelin-1 in Human Endothelial Cells Is Mediated by Activated CCAAT/Enhancer-Binding Proteins

High glucose-induced endothelial dysfunction is partially mediated by the down-stream pathophysiological effects triggered by increased expression of endothelin-1 (ET-1). The molecular control mechanisms of ET-1 synthesis are yet to be discovered. Members of the CCAAT/enhancer-binding proteins (C/EBP) family are important regulators of key metabolic processes, cellular differentiation and proinflammatory genes. In this study, we aimed at elucidating the role of C/EBP in mediating the high glucose effect on ET-1 expression in human endothelial cells (EC). Human umbilical vein cells (EAhy926) and primary cultures of human aortic EC were exposed to high levels of glucose (16.5–25 mM). Real-time PCR, Western blot, enzyme-linked immunosorbent assay, ET-1 promoter-luciferase reporter analysis, and chromatin immunoprecipitation assays were employed to investigate ET-1 regulation. High glucose activated C/EBPα, C/EBPβ, and C/EBPδ in a dose-dependent manner. It also promoted significant increases in ET-1 gene and peptide expression. Chemical inhibition of JNK, p38MAPK and ERK1/2 diminished significantly the high glucose-induced nuclear translocation of C/EBP and ET-1 expression. Silencing of C/EBPα, C/EBPβ or C/EBPδ greatly reduced the high glucose-induced upregulation of ET-1 mRNA, pre-pro-ET-1, and ET-1 secretion. The expression of various C/EBP isoforms was selectively downregulated by siRNA-mediated gene silencing. In silico analysis indicated the existence of typical C/EBP elements within human ET-1 gene promoter. Transient overexpression of C/EBPα, C/EBPβ or C/EBPδ upregulated the luciferase level controlled by the ET-1 gene promoter. The direct interaction of C/EBPα, C/EBPβ or C/EBPδ proteins with the ET-1 promoter in high glucose-exposed EC was confirmed by chromatin immunoprecipitation assay. High glucose-induced ET-1 expression is mediated through multiple mechanisms. We present evidence that members of the C/EBP proinflammatory transcription factors are important regulators of ET-1 in high glucose-exposed human endothelial cells. High glucose-induced activation of C/EBP-related signaling pathways may induce excessive ET-1 synthesis, thus promoting vasoconstriction and dysfunction of the vascular wall cells in diabetes.

Identification of gene variants in NOS3, ET-1 and RAS that confer risk and protection against microangiopathy in type 2 diabetic obese subjects.

The study aim was to investigate NOS3 VNTR, NOS3 G894T, EDN1 C8002T, ACE I/D, AGT M235T and AGTR1 A1166C in nonobese and obese T2DM patients, and their interaction with the incidence of microangiopathy. T2DM subjects (n=250; 166 nonobese, and 84 obese) were genotyped for the gene variants by PCR/RFLP. The interaction of these polymorphisms with obesity and their contribution to microangiopathy were analyzed by multivariate regression analysis. A higher frequency of NOS3 4a allele was found in obese (P=0.027) vs. nonobese subjects. ACE D (P=0.009) and AGT 235T (P=0.026) alleles were associated with the reduced risk of diabetic nephropathy in nonobese and obese patients, respectively. In obese subjects, NOS3 4a (P=0.011) had a converse effect to NOS3 894T (P=0.043), and EDN1 8002T (P=0.035) on the prevalence of combined microangiopathy (neuropathy/retinopathy/nephropathy) vs. microangiopathy-negative subjects. The study indicates association of RAS variants with obesity and nephropathy, and an opposite effect of NOS3 VNTR and NOS3 G894T on the occurrence of combined microangiopathy.

Genetic Determinants of Microvascular Complications in Type 1 Diabetes

Diabetes mellitus is one of the most prevalent chronic diseases of modern societies and a major health problem in nearly all countries. Its prevalence has risen sharply worldwide during the past few decades (Amos et al., 1997; Shaw et al., 2010). Moreover, predictions show that diabetes prevalence will continue to rise, reaching epidemic proportions by 2030: 7.7% of world population, representing 439 million adults worldwide (Shaw et al., 2010). This increase is largely due to the epidemic of obesity and consequent type 2 diabetes (T2DM). However, the incidence of type 1 diabetes (T1DM) is also rising all over the world (DiaMond Project Group, 2006; Maahs et al., 2010). Recent data for Europe (Patterson et al., 2009) predict the doubling of new cases of T1DM between 2005 and 2020 in children younger than 5 years and an increase of 70% in children younger than 15 years, old. Despite major progresses in T1DM treatment during the past decades, mortality in T1DM patients continues to be much higher than in general population, with wide variations in mortality rates between countries. In Europe, these variations are not explained by the country T1DM incidence rate or its gross domestic product, but are greatly influenced by the presence of its chronic complications, especially diabetic renal disease (Groop et al., 2009; Patterson et al., 2007). In fact, much of the health burden related to T1DM is created by its chronic vascular complications, involving both large (macrovascular) and small (microvascular) blood vessels. Many genetic, metabolic and hemodynamic factors are involved in the genesis of diabetic vascular complications. However, major epidemiological and interventional studies showed that chronic hyperglycemia is the main contributor to diabetic tissue damage (DCCT Research Group, 1993). If the degree of metabolic control remains the main risk factor for the development of diabetic chronic complications, an important contribution can be attributed to genetic risk factors, some of them common for all microvascular complications (diabetic retinopathy, neuropathy, and renal disease) and some specific for each of them (Cimponeriu et al., 2010). Additional factors are represented by some accelerators such as hypertension and dyslipidemia. In the following pages, we present briefly the pathogenesis type 1 diabetes and its chronic microvascular complications. The main information of the genetic background in T1DM with particular focus on gene variants having strong impact on endothelial dysfunction as the key factor in the development of microvascular disorders are also summarized.

c-Src tyrosine kinase mediates high glucose-induced endothelin-1 expression.

Endothelin-1 (ET-1) plays an important role in the pathophysiology of diabetes-associated cardiovascular disorders. The molecular mechanisms leading to ET-1 upregulation in diabetes are not entirely defined. c-Src tyrosine kinase regulates important pathophysiological aspects of vascular response to insults. In this study, we aimed to elucidate whether high glucose-activated c-Src signaling plays a role in the regulation of ET-1 expression. Human endothelial cells EAhy926 (ECs) were exposed to normal or high levels of glucose for 24h. Male C57BL/6J mice were rendered diabetic with streptozotocin and then treated with a specific c-Src inhibitor (Src I1) or c-Src siRNA. Real-time PCR, Western blot, and ELISA, were used to investigate ET-1 regulation. The c-Src activity and expression were selectively downregulated by pharmacological inhibition and siRNA-mediated gene silencing, respectively. High glucose dose-dependently up-regulated c-Src phosphorylation and ET-1 gene and protein expression levels in human ECs. Chemical inhibition or silencing of c-Src significantly decreased the high-glucose augmented ET-1 expression in cultured ECs. In vivo studies showed significant elevations in the aortic ET-1 mRNA expression and plasma ET-1 concentration in diabetic mice compared to non-diabetic animals. Treatment with Src I1, as well as in vivo silencing of c-Src, significantly reduced the upregulated ET-1 expression in diabetic mice. These data provide new insights into the regulation of ET-1 expression in endothelial cells in diabetes. Pharmacological targeting of c-Src activity and/or expression may represent a potential therapeutic strategy to reduce ET-1 level and to counteract diabetes-induced deleterious vascular effects.

Epigenetic regulation of vascular NADPH oxidase expression and reactive oxygen species production by histone deacetylase-dependent mechanisms in experimental diabetes

Reactive oxygen species (ROS) generated by up-regulated NADPH oxidase (Nox) contribute to structural-functional alterations of the vascular wall in diabetes. Epigenetic mechanisms, such as histone acetylation, emerged as important regulators of gene expression in cardiovascular disorders. Since their role in diabetes is still elusive we hypothesized that histone deacetylase (HDAC)-dependent mechanisms could mediate vascular Nox overexpression in diabetic conditions. Non-diabetic and streptozotocin-induced diabetic C57BL/6J mice were randomized to receive vehicle or suberoylanilide hydroxamic acid (SAHA), a pan-HDAC inhibitor. In vitro studies were performed on a human aortic smooth muscle cell (SMC) line. Aortic SMCs typically express Nox1, Nox4, and Nox5 subtypes. HDAC1 and HDAC2 proteins along with Nox1, Nox2, and Nox4 levels were found significantly elevated in the aortas of diabetic mice compared to non-diabetic animals. Treatment of diabetic mice with SAHA mitigated the aortic expression of Nox1, Nox2, and Nox4 subtypes and NADPH-stimulated ROS production. High concentrations of glucose increased HDAC1 and HDAC2 protein levels in cultured SMCs. SAHA significantly reduced the high glucose-induced Nox1/4/5 expression, ROS production, and the formation malondialdehyde-protein adducts in SMCs. Overexpression of HDAC2 up-regulated the Nox1/4/5 gene promoter activities in SMCs. Physical interactions of HDAC1/2 and p300 proteins with Nox1/4/5 promoters were detected at the sites of active transcription. High glucose induced histone H3K27 acetylation enrichment at the promoters of Nox1/4/5 genes in SMCs. The novel data of this study indicate that HDACs mediate vascular Nox up-regulation in diabetes. HDAC inhibition reduces vascular ROS production in experimental diabetes, possibly by a mechanism involving negative regulation of Nox expression.

Lessons from Experimental-Induced Atherosclerosis: Valuable for the Precision Medicine of Tomorrow

Identifying, preventing, and treating the subjects under the risk of developing an acute ischemic attack remain the most provocative challenges in cardiovascular clinical care. In this chapter, we describe in brief some of the recent lessons learned from basic and preclinical studies on the molecular pathobiochemistry of atherosclerosis highlighting new mechanistic concepts of the implications of inflammation and oxidative stress and the emergent nanotechnologies meant to improve diagnosis and treatment.