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Implications of a genetically determined nitric oxide deficit for endothelial cell-leukocyte interaction and cardiovascular disease

Kadiyska, Ivelina

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Abstract

Blood flow-generated shear stress (FSS) is the major determinant of endothelial nitric oxide synthase (NOS-3) expression. In humans, a promoter variant of the NOS3 gene, the C-variant of the T-786C single nucleotide polymorphism, renders the gene insensitive to shear stress, resulting in a reduced endothelial cell (EC) capacity to generate nitric oxide (NO). Endothelial dysfunction, commonly associated with decreased NO bioavailability, may facilitate vascular inflammation. Consequently, individuals homozygous for the C-variant have an increased risk of developing cardiovascular (e.g., coronary heart disease (CHD)) and rheumatic diseases (e.g., rheumatoid arthritis (RA)).

However, there are at least two mechanisms by which insuffcient NO production can be counterbalanced in CC-genotype endothelial cells (ECs), one of which involves a multicomponent pathway leading to the increased release of the anti-inflammatory prostanoid 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2). Exposure of human ECs to physiological levels of FSS effectively reduced monocyte migration, not only through monolayers of TT- but most notably also of NO-deficient CC-genotype ECs. FSS up-regulated the expression of COX-2 and L-PGDS, the rate-limiting enzymes for 15d-PGJ2 synthesis, solely in CC-genotype ECs, and only these cells revealed an increased release of 15d-PGJ2 in response to FSS. Exogenously added 15d-PGJ2 significantly reduced the transmigration of monocytes through EC monolayers. In addition, pre-treatment with 15d-PGJ2 or exposure to FSS-pretreated CC-genotype ECs exerted a pronounced anti-inflammatory effect on the (transmigrated) monocytes, as demonstrated e.g. by an inhibitory effect on interleukin-1 beta (IL-1 beta) expression, a marker for monocyte pro-inflammatory activation. This inhibition occurs at the transcriptional level, as 15d-PGJ2 repressed tumor necrosis factor-alpha-induced IL-1 beta promoter activity in transiently transfected HEK293 cells.

The anti-inflammatory activity of 15d-PGJ2 in monocytes involves the Nrf2-antioxidant response element (ARE) pathway. Similar to 15d-PGJ2, constitutive activation of Nrf2 reduced the expression of IL-1 beta. Bioinformatic analysis revealed three putative Nrf2-responsive elements (i.e., AREs) in the human IL-1B promoter, suggesting that Nrf2 may act through an as yet unknown mechanism to repress transcription of the IL-1B gene. Chromatin immunoprecipitation showed a 15d-PGJ2-induced binding of Nrf2 to the promoter of the IL-1B gene. Deletion of two of the identified ARE motifs attenuated the inhibitory potency of 15d-PGJ2 toward IL-1B promoter activity, thereby corroborating Nrf2 as a downstream effector of this prostanoid’s transcriptional effects.

Given its powerful immunomodulatory properties, 15d-PGJ2 has been proposed to have anti-atherogenic potential. To evaluate its prognostic relevance, the relationship between plasma 15d-PGJ2 levels and disease severity and outcome in patients suffering from CHD, RA or both was investigated. The levels of 15d-PGJ2 were found to be significantly increased in the CHD group compared to age-matched controls, suggesting that 15d-PGJ2 may constitute a general defense mechanism to counteract the ongoing chronic inflammatory process in affected individuals. Moreover, pre-treatment with 15d-PGJ2 potently inhibited the in vitro transendothelial migration of interferon-gamma-producing human T helper type 1 cells, major players in atherosclerosis as well as various other chronic inflammatory disorders.

Despite an inadequate capacity to form NO, CC-genotype ECs maintain a robust anti-inflammatory phenotype by enhancing the shear stress-dependent synthesis of 15d-PGJ2. Its anti-inflammatory activity on human monocytes may ascribe a novel role to Nrf2 as a direct repressor of pro-inflammatory gene expression.

Item Type: Dissertation
Supervisor: Hecker, Prof. Dr. Markus
Date of thesis defense: 13 July 2016
Date Deposited: 25 Aug 2016 08:01
Date: 2016
Faculties / Institutes: Medizinische Fakultät Heidelberg > Institut fuer Physiologie und Pathophysiologie
Subjects: 500 Natural sciences and mathematics
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