Open Access
Review

Regulation of NOS expression in vascular diseases

Andrea Pautz1,*, Huige Li1, Hartmut Kleinert1
1
Department of Pharmacology, Johannes Gutenberg University Medical Center, Langenbeckstr. 1, 55131 Mainz, Germany
DOI: 10.52586/4926 Volume 26 Issue 5, pp.85-101
Submited: 14 October 2020 Accepted: 15 February 2021 Published: 30 April 2021
(This article belongs to the Special Issue Innate immune mechanisms in thrombosis and vascular biology)
*Corresponding Author(s):  
Andrea Pautz
E-mail:  
pautz@uni-mainz.de
Copyright: © 2021 The author(s). Published by BRI. This is an open access article under the CC BY 4.0 license (http://creativecommons.org/licenses/by/4.0/).
Abstract

Nitric oxide synthases (NOS) are the major sources of nitric oxide (NO), a small bioactive molecule involved in the regulation of many cellular processes. One of the most prominent functions of NO is regulation of vasodilatation and thereby control of blood pressure. Most important for vascular tone is NOS3. Endothelial NOS3-generated NO diffuses into the vascular smooth muscle cells, activates the soluble guanylate cyclase resulting in enhanced cGMP concentrations and smooth muscle cell relaxation. However, more and more evidence exist that also NOS1 and NOS2 contribute to vascular function. We summarize the current knowledge about the regulation of NOS expression in the vasculature by transcriptional, post-transcriptional and post-­translational mechanisms, in regard to inflammation and innate immune pathways.

Key words

NOS1; NOS2; NOS3; Nitric oxide; Gene regulation; Vascular inflammation; Innate immunity

2. Nitric oxide as bioactive molecule

Nitric oxide (NO), a small gas molecule, has been shown to act as bioactive substance. NO can be produced by a great number of organisms ranging from bacteria [1], yeast [2] and invertebrates [3] to mammals. NO as a simple gas molecule, controls important functions such as vascular tone, smooth muscle cell proliferation, platelet aggregation, leucocyte adhesion (see Fig. 1), and neurotransmission or the contraction of gastrointestinal organs. These broadly based regulation activities are performed mainly by the NO-dependent activation of soluble guanylyl cyclase [4]. Further, by activation or deactivation of transcription factors NO can affect gene transcription [5, 6] and mRNA translation (e.g., via iron-responsive elements) [7].

. NOS3 enzyme activity in endothelial cells (EC) can be stimulated by shear stress or several agonists, like bradykinin (BK) and vascular endothelial growth factor (VEGF). The EC-synthesized NO diffuses into the blood stream and inhibits platelet aggregation and adhesion. In addition, the EC related NO inhibits leukocyte adhesion to the vascular endothelium and leukocyte migration into the vascular wall. NO also diffuses into smooth muscle cells (SMCs). In SMCs NO stimulates vasodilation and prevents SMC proliferation. Reprinted by permission from Springer Science + Business Media New York: Li H, Xia N, Förstermann U. Chapter 16-Nitric Oxide Synthesis in Vascular Physiology and Pathophysiology. In “Endothelial Signaling in Development and Disease” Eds. Schmidt MH, Liebner S. COPYRIGHT 2015.

Figure 1: Antihypertensive, antithrombotic, and antiatherosclerotic effects of endothelial NOS3. NOS3 enzyme activity in endothelial cells (EC) can be stimulated by shear stress or several agonists, like bradykinin (BK) and vascular endothelial growth factor (VEGF). The EC-synthesized NO diffuses into the blood stream and inhibits platelet aggregation and adhesion. In addition, the EC related NO inhibits leukocyte adhesion to the vascular endothelium and leukocyte migration into the vascular wall. NO also diffuses into smooth muscle cells (SMCs). In SMCs NO stimulates vasodilation and prevents SMC proliferation. Reprinted by permission from Springer Science + Business Media New York: Li H, Xia N, Förstermann U. Chapter 16-Nitric Oxide Synthesis in Vascular Physiology and Pathophysiology. In “Endothelial Signaling in Development and Disease” Eds. Schmidt MH, Liebner S. COPYRIGHT 2015.

The NO radical reacts with multiple partners, a: the SH groups of cysteine in peptides or proteins, resulting in the formation of S-nitrosothiols. This modification is reversible and is important for the NO-related signaling functions in the immune system. b: superoxide anions (O-2) (a chemical reaction with one of the highest reaction speeds known) generating peroxynitrite (ONOO-) able to modify proteins by tyrosine nitration. c: Fe2+ (in heme groups or iron-sulfur clusters) or Zn2+ (in zinc–sulfur clusters), important for the regulation of several enzymes and transcription factors. d: nucleic acids, resulting in deamination leading to mutations. e: to unsaturated lipids, producing nitrolipids [8].

At high concentrations NO is known to kill bacteria, parasites and certain tumor cells by inhibiting iron-containing enzymes [9], either by direct NO-DNA interactions [10, 11], or by post-translational modifications of proteins (for example S-nitrosothiol adduct formation [12] or ADP-ribosylation [13]). At these high NO concentrations (mostly formed by NOS2) reactive nitrogen species (RNS) are formed that harm cell membranes, the endoplasmic reticulum, mitochondria, nucleic acids and proteins/enzymes, which result in necrosis and cell death [14].

3. Nitric oxide synthases

In mammals, three isoforms of nitric oxide synthase (NOS) exist. The cDNA, protein structures and genomic DNA loci have been characterized in different species (see Table 1 [4, 15, 16]). NOS1, first discovered in neurons of rat and porcine cerebellum [17, 18, 19], and NOS3, originally described in endothelial cells [20], are Ca2+-activated enzymes with relative low NO production whose physiological function is mainly signal transduction. NOS2, primarily detected in cytokine-induced macrophages [21, 22], is the high NO-producing isoform, able to produce toxic amounts of NO. Innate immune cells use this NO for their antimicrobial, antiparasitic and antineoplastic activities. NOS2 activity is mostly (human) or completely (mouse and rat) Ca2+-independent. All NOS enzymes are homodimers that oxidize a guanidino nitrogen of L-arginine, using molecular oxygen and NADPH as co-substrates, to produce NO. Therefore, limiting the substrate arginine by other arginine utilizing enzymes, as by arginase I or II, or modulation of arginine transport [23, 24] is able to regulate the activity of all three isoforms. For example, in several cardiovascular diseases the consumption of arginine by arginase leads to the dysfunction of the NOS3 enzyme (NOS3 uncoupling) converting it to a superoxide-producing enzyme, resulting in NOS3-dependent superoxide production [25]. All NOS isoforms contain the prosthetic groups FAD, FMN and heme iron and depend on BH4 as cofactor. Suboptimal concentrations of the essential cofactor BH4 result in NOS3 uncoupling and superoxide production by the enzyme. NOS1 and NOS2 are mostly soluble enzymes. In contrast, NOS3 due to its amino-terminal fatty acylation by myristic and palmitic acid is membrane bound [4, 26].

Table 1: Nomenclature of the different NOS isoforms.
Isoform descriptive name protein source cDNA source
NOS1 bNOS (for brain NOS); rat cerebellum porcine cerebellum human and rat brain
cNOS (for constitutive or Ca2+-regulated NOS);
bcNOS (for brain constitutive NOS);
nNOS (for neuronal NOS);
ncNOS (for neuronal constitutive NOS)
NOS2 iNOS (for inducible NOS); mouse RAW 264.7 macrophages, mouse macrophages, rat hepatocyte
macNOS (for macrophage NOS); rat peritoneal macrophages, human and liver, human A-172- and DLD-
hepNOS (for hepatocyte NOS) DLD-1 adenocarcinoma cells 1 cells, hepatocytes and articular
chondrocytes
NOS3 eNOS (for endothelial NOS); bovine lung endothelial cells bovine and human endothelium
cNOS (for constitutive or Ca2+-regulated NOS; overlap with nomenclature for NOS1);
ecNOS (for endothelial constitutive NOS)
The descriptive names are used in the literature, the protein sources and the cDNA sources are described. Summary from [27].

4. NOS genes

In humans, three different genes located on chromosomes 12, 17 and 7, respectively, encode the NOS isoforms 1, 2 and 3. Deduced from the cloned cDNAs, the amino acid sequences of the three human isozymes show less than 59% identity. Across tested mammalian species, amino acid sequences are more than 90% conserved for NOS1 and 3, and greater 80% identical for NOS2.

5. NOS isoform expression

In contrast to the often-used descriptive names, researches have shown by immunohistochemical and western blotting methods that NOS1 and NOS3 are expressed in a large number of different cell types. NOS1 is expressed for example in skeletal myocytes, in endothelial-, smooth muscle-, or epithelial cells (see [16, 28, 29] for reviews) as well as unprimed macrophages [30]. NOS3 is expressed in different cell types like endothelial cells, epithelial cells, neuronal cells, T cells, erythrocytes, perivascular adipose tissue and platelets (see [16, 31] for reviews). NOS1 and 3 are believed to be constitutively expressed. However, also expression of NOS1 and 3 is regulated by external stimuli [28]. For example estrogens (for NOS1 and 3), shear stress, TGF-ß1, and in certain endothelial cells high glucose (for NOS3) enhanced the expression of these enzymes. The expressional regulation of the “constitutive” NOS (as well of NOS2) is mediated by different mechanisms. These include changes in chromatin packaging, mediated by histone methylation/acetylation, and/or effects of long non-coding RNAs (ncRNAs), activation/inhibition of transcription factors and usage of different promoters (modulation of transcription), regulation of mRNA-splicing, -localization and -stability (post-transcriptional regulation by RNA-binding proteins-RNA-BP, or micro-RNAs-miRNAs) and modulation of protein-stability.

5.1 Transcriptional regulation of the NOS1 gene

For the human and rodent NOS1 gene, tissue-specific or developmentally regulated NOS1 mRNA transcripts (at least 12 different human NOS1 mRNA isoforms) have been reported. These different NOS1 mRNAs are produced by alternative promoter usage, alternative splicing (see below), and/or the usage of alternate polyadenylation signals [32, 33, 34, 35, 36, 37, 38]. The different promoters display a multitude of potential transcription factor binding sites [37], but their functionality has been tested only for a small number. For example, the cAMP-depending enhancement of NOS1 expression (mRNAs containing the exons ex1f and g) has been shown to depend on a CRE site in the respective promoter sequence [37].

5.2 Transcriptional regulation of the NOS3 gene

To analyze the differential activity pattern of chromatin-versus episome-based human NOS3 promoter Chan et al. examined the methylation status of 5’-regulatory sequences of the human NOS3 gene. The authors observed huge differences in the DNA methylation of the NOS3 promoter sequence between endothelial and non-endothelial cell types, like vascular smooth muscle cells (VSMCs). The same cell type-specific methylation pattern was observed at the native murine NOS3 promoter in vivo in endothelial cells (EC) and VSMCs of the mouse aorta. Transient transfection analyses showed that that methylated NOS3 promoter sequences exhibited a marked decrease in the action of Sp1, Sp3, and Ets1 on NOS3 promoter activity, an effect enhanced by methyl-CpG-binding protein 2 (MECP2). In addition, ChIP analyses showed the binding of Sp1, Sp3, and Ets1 to the NOS3 promoter in ECs but not VSMCs. Finally, NOS3 mRNA expression could be induced in non-ECs by inhibition of DNA methyltransferase activity with 5-azacytidine [39]. As described by Miao et al. the LEENE lncRNA stimulates the binding of RNA polymerase II to the NOS3 promoter upregulating NOS3 nascent RNA synthesis [40]. The nuclear located lncRNA spliced-transcript endothelial-enriched lncRNA (STEEL) also enhances RNA polymerase II loading at the proximal promoter of the NOS3 gene and enhances NOS3 transcription [41]. The placenta-specific expression of a placenta NOS3 mRNA isoform is described to be related to usage of an alternative Herv-LTR10A-related promoter upstream of the classical promoter sequences [31, 42, 43] used in non-placental tissues. The placenta-restricted expression was also determined to be associated with placenta-specific hypomethylation of the LTR10A element [44]. Analysis of the human NOS3 promoter revealed the functional importance of binding sequences for several transcriptional factors like the AP-1-, AP-2-, Elf-1-, Erg-, Ets1-, GATA-, HIF-, KLF2-, MAZ-, MZF-, NF-1-, p53-like, PEA3-, Smad2-, Sp1-, Sp1/Sp3-like, YY1-like-binding site. Also, acute phase reactant-, sterol-, and shear stress-regulated elements have been described (see [31, 45] for reviews). Also, the LTR10A-derived NOS3 promoter element important for placenta-specific NOS3 expression, contains several putative transcription factor binding sites, for example C/EBPdelta, FOXO4, NF-Y, and Sox-5 [44], but the functionality of these sequences have not been proved yet.

5.3 Post-transcriptional regulation of the NOS1 expression

The different 5’-UTRs of the multiple NOS1-mRNA isoforms (see above) are likely to regulate the translatability of these different NOS1 mRNAs [16]. Several miRNAs have been shown to directly [46, 47, 48, 49, 50, 51] or indirectly [52] modulate human NOS1 expression.

5.4 Post-transcriptional regulation of the NOS3 expression

Lorenz et al. detected three splice variants (NOS3-13A, NOS3-13B, and NOS3-13C) of the NOS3 mRNA in HUVEC with novel 3’ splice sites within intron 13. All variants use the same polyadenylation site located at the end of the novel exon, and all these NOS3 mRNA isoforms code for inactive NOS3 proteins. These mRNA isoforms are expressed in endothelial cells and various human tissues. By formation of heterodimers, expression of the full-length NOS3 with NOS3-13A diminished NOS3 enzyme activity in COS-7 cells [53].

Beside promoter activity regulation, TNF-α reduces NOS3 expression in endothelial cells of different species by destabilization of its mRNA [54, 55, 56, 57, 58, 59]. By RNA-protein interaction analyses different RNA-BPs (translation elongation factor 1-alpha 1 - eEF1A1 and polypyrimidine tract-binding protein 1 - PTB1) were found to bind to the 3’-UTR of the NOS3 mRNA [56, 57, 58]. In addition, TNF-α-dependent post-transcriptional regulation of NOS3 expression by different miRNAs binding to its mRNA has been described [59, 60, 61].

5.5 Post-translational regulation of the NOS1 protein

Several post-translational modifications, such as phosphorylation, ubiquitination, and sumoylation, of the NOS1 protein have been described [62]. NOS1 localization, enzymatic activity and protein stability is also regulated by protein-protein interactions with calmodulin (CaM), heat shock proteins (hsp90/hsp70), PDZ-domain containing proteins (syntrophin, PSD-95, or PSD-93), the Carboxy-Terminal Postsynaptic Density-95/Discs Large/Zona Occludens-1 Ligand of NOS1 (CAPON) (also named Nitric Oxide Synthase 1 Adaptor Protein - NOS1AP) [62] and PIN, a protein inhibitor of NOS1 acting by dissociation of NOS1 dimers into monomers [63, 64].

5.6 Post-translational regulation of the NOS3 protein

Post-translational modification of NOS3 has been shown to include acetylation (decreasing its activity), acylation (membrane targeting), glutathionylation (uncoupling, resulting in superoxide production), phosphorylation (regulation of enzyme activity) or S-nitrosylation (reducing its activity) [26]. Especially phosphorylation of different amino acids (Y81, S615, S633, S1177 activating; S114, T495, Y657 deactivating) by multiple kinases (Akt, AMPK, CaM-K-II, PKA, PKC, PKG, pp60src, PYK) modulates NOS3 activity by different signaling pathways [65].

Beside post-translational modifications there are numerous reports demonstrating the importance of proteins interacting with NOS3 and thereby stimulating or inhibiting NOS3 function. In addition to CaM, several proteins like caveolin-1, cell division cycle 37 (Cdc37), C-terminal hsp70-interacting protein (CHIP), connexin 37 and 40 (Cx37/40), G-protein-coupled receptor (GPCR) kinase interactor-1 (GIT1), hemoglobin alpha (Hbα), heat shock protein 90 (Hsp90), integrin-linked kinase (ILK), NOS3 interacting protein (NOSIP) and NOS3 traffic inducer (NOSTRIN), proviral integration site for Moloney murine leukemia virus 1 (Pim1), prolyl isomerase (Pin) 1, and stromal cell-derived factor 2 (SDF2), have been shown to interact and regulate NOS3 (see [66] for a recent review). In addition, the plasminogen activator inhibitor-1 (PAI-1) interacts with NOS3 and inhibits its activity [67].

Cytokine-dependent regulation of NOS1 and NOS3 by microbial products have been reported also. The differentiation and activity of immune cells in vitro is affected by NOS1 or 3. In addition, modulation of immune responses and inflammatory processes in vivo have been described [8].

5.7 Regulation of NOS2 expression

A “constitutive” expression of NOS2 has been described for epithelial cells of the colon and lungs, which is likely “induced” by the microbiota in these organs, and spinal tissue of the brain and for different human cancer cells (see [28] for a review).

NOS2 is mainly regulated at the expressional level (Fig. 2). LPS, cytokines, and several other compounds (mostly secreted by the innate immune system) are able to induce NOS2 synthesis in many cell types (see [68] for a review). Pathways involved in the NOS2 promoter activation seem to vary in different cells. However, activation of the transcription factors NF-κB and STAT-1α are believed as essential steps for NOS2 transcription in most cells. Beside transcriptional control NOS2 expression is intensively controlled by post-transcriptional regulation of NOS2 mRNA stability [68].

. NOS2 is mainly regulated by modulation of NOS2 expression. NOS2 promoter activity is regulated by modulation of the accessibility of the chromatin (CpG-Methylation, histone acetylation) and binding of transcription factors (NF-

Figure 2: Regulation of (human) NOS2 expression and NOS2-mediated NO production. NOS2 is mainly regulated by modulation of NOS2 expression. NOS2 promoter activity is regulated by modulation of the accessibility of the chromatin (CpG-Methylation, histone acetylation) and binding of transcription factors (NF-κB, STAT-1α). Also modulation of NOS2 mRNA stability is a major regulatory mechanism. RNA-BP (AUF1, HuR, KSRP, PTB, TTP) bind to the NOS2 mRNA and regulate stability by interaction with the exosome. In rodents, modulation of NOS2 mRNA stability by interaction with anti-NOS2-asRNA has been described. NOS2 protein stability is regulated by interacting proteins (Cav1, src) and the proteasome. Finally, enzyme activity of the NOS2 protein is modulated by interaction with several proteins (kalirin, NAP110, src, CaMKII, Rac2, hsp90). NOS2-mediated NO production depends on arginine (Arg) supply. Therefore, proteins that also use arginine as substrate (arginase) may regulate NOS2 activity by substrate competition. Membrane transporters important for arginine uptake into cells (cationic amino acid transporter, CAT) may have a role in the regulation of NOS2-dependent NO production.

5.8 Transcriptional regulation of the NOS2 gene

Buzzo et al. demonstrated that NOS2 expression in murine peritoneal macrophages, induced by purified flagellin from Bacillus subtillis, involves caspase-1 mediated cleavage of the chromatin regulator Poly [ADP-ribose] polymerase 1 (PARP1) to enhance the chromatin accessibility of the NF-κB binding sites located in the NOS2 promoter [69] (Fig. 2). In sharp contrast to murine macrophages, LPS- and IFN-γ-treated human alveolar macrophages express no NOS2 mRNA or protein. This unresponsiveness is related to epigenetic gene silencing (chromatin compaction, CpG methylation and histone modifications) [70].

In macrophages from Leishmania amazonensis patients binding of the inhibitory NF-κB p50/50 monomer leads to a recruitment of histone deacetylase 1 (HDAC1) to the human NOS2 promoter, preventing histone acetyltransferase (CBP/p300) binding to the NOS2 promoter and further acetylation of H3K9 [71] (Fig. 2).

LPS/cytokine induced NOS2 expression in the murine system depends on a promoter sequence with around 1000 bp [72, 73]. In sharp contrast, the 1000 bp human NOS2 promoter displays only basal activity not induced by cytokine stimulation [74, 75, 76]. Only if much longer DNA promoter fragments (up to 16 kb) are used in transfection experiments (transient or stable) with human A549, AKN or DLD-1 cells, a clear promoter induction (8-10-fold) was detected (see [68] for a review). Analyzation of the 16 kb human NOS2 promoter sequence with bioinformatic tools revealed a multitude of putative transcription factor binding sites. However, only a few of these binding sites have been shown to be functional important. The human 16 kb promoter contains a TATA-box and binding sites for AP-1, CAR, C/EBPβ, EGFR-STAT3, FKHRL1, HIF-1α, HMGA1, KLF6, NF-κB, NRF, Oct-1, RAR/RXR, PXR, STAT-1α, Tcf-4, TCF11/MafG, and YY1 (all proven to be functional at least in transfection experiments) [68, 77].

5.9 Post-transcriptional regulation of NOS2 expression

We and others have shown that the post-transcriptional regulation of the mammalian (especially human) NOS2 expression is quite complex (Fig. 2). Translational efficacy and non-sense mediated mRNA decay [78, 79] of the human NOS2 mRNA is regulated by a short μORF located in exon 1 of the human NOS2 gene [80]. Several RNA-BP have been shown to bind to the 3’-UTR of the human NOS2 mRNA and regulate mRNA-stability [81, 82, 83, 84, 85, 86, 87, 88]. In addition, different miRNAs (miR-26a, miR-146a miR-939) directly bind to the human NOS2 mRNA and regulate its translatability and stability [8, 59, 89]. In murine and rat cells natural antisense RNAs (NATs) are transcribed from the 3’-UTR of the NOS2 gene, which stabilize NOS2 mRNA by interacting with the mRNA 3’-UTR [90]. No such NATs were detected in the human system.

5.10 Post-translational regulation of NOS2 expression and activity

Post-translational modification of NOS2 seem to be important for NOS2 activity and intracellular localization (Fig. 2). Palmitoylation of NOS2 at the amino acid Cys-3 is essential for NO synthesis and intracellular localization [91]. In muscle of septic patients, tyrosine-nitration of NOS2 has been described, which reduces enzymatic activity [92]. Also, for the NOS2 protein several protein-protein-interactions have been published that enhance or reduce the activity of the NOS2 enzyme (α-actinin-4, ezrin/radizin/moesin-binding phosphoprotein 50-EBP50, kinase suppressor of Ras-1-Ksr1-adaptor-or scaffold-proteins; Hsp90, Rac2-allosteric activators; kalirin-dimerization inhibitor). Other protein interactions lead to proteasomal degradation of the NOS2 protein (e.g., Rpn13/ARDM1/NAP110, UCH37) (see [8] for a review).

6. Structure of the healthy vessel wall

Normal blood vessels are made of the tunica intima, the tunica media and the adventitia surrounded by the perivascular adipose tissue (PVAT) [93].

The tunica intima is composed of an EC monolayer attached to a basement membrane filled with extracellular matrix. EC are exposed to shear stress resulting from the blood flow [94]. Laminar shear stress up-regulates in EC the expression of vasculoprotective transcription factors such as KLF2 and Nrf2, which orchestrated the anti-inflammatory and antioxidant EC phenotype. However, disturbed shear stress induces the pleiotropic transcription factor NF-κB, leading to a pro-inflammatory and proatherogenic EC phenotype. As natural barrier of the blood vessel ECs prevent toxic molecules from penetration into the arterial vessel wall and inhibit platelet- and leukocyte adhesion. ECs are able to regulate the vascular tone by secretion of vasoactive substances, such as endothelium-derived hyperpolarizing factor (EDHF), NO, and prostaglandin I2 (PGI2), which are vasodilators. EC-derived NO is also regarded as a major anti-inflammatory factor in the vasculature [95]. On the other hand, EC are able to secrete endothelium-derived contracting factors (EDCFs) such as angiotensin II (Ang II), endothelin 1 (ET-1), thromboxane A2 (TXA2), and uridine adenosine tetra-phosphate (UP4A). Healthy EC promote the balance of pro- and anti-thrombotic mechanism by releasing anti- or pro-thrombotic substances and also regulate VSMC proliferation [96].

The tunica media contains a layer of smooth muscle cells (SMC), which secrete elastic and collagen fibers, and pericytes. Mature SMCs contain a unique set of contractile proteins (e.g., α-smooth muscle actin (αSMA) or smooth muscle myosin heavy chain (SM-MHC)), ion channels, and specific signaling molecules that are required for their contractile functions [97]. SMCs produce different components of the extracellular matrix (ECMs), namely cadherins, collagen, elastin, integrins, and proteoglycans that build up a major portion of the blood vessel mass [98].

The adventitia consists of fibroblasts, mesenchymal stem cells (MSCs), vasa vasorum, nerves and a small number of immune cells in connective tissue [99].

In addition, most vessels (e.g., aorta and coronary arteries) are embedded by perivascular adipose tissue (PVAT), which is an active endocrine tissue affecting the vasculature by secreting different mediators [100]. In addition, also cells of the immune system (like macrophages, T cells), fibroblasts and capillary EC are found in the PVAT [95, 101].

The above-described blood vessel structure is mainly preserved throughout the body. However, the vasculature in the different parts of the human body has unique functions depending on the needs of the different organs and tissues. For instance, the resistance vessels (arteries and arterioles), are in contact with shear stress resulting from the high pressure [102]. Towards the veins, the blood pressure and shear stress are stepwise reduced. Veins are exposed to a nearly 70-fold less pressure than arteries. As a result of this high pressure, arteries and arterioles possess a thick media layer with copious SMCs that provide elastic support. In sharp contrast, capillaries display only an intima layer covered with a basement membrane and are supported by pericytes.

7. NOS isoforms expressed in the healthy vasculature

The expression of the different isoforms of NOS1-3 has been published for nearly all cell types of the healthy vasculature.

NOS1 is expressed in vascular smooth muscle cells [103, 104] and vascular endothelium [105, 106]. This was shown by immunohistochemistry or western blot using isoform-specific antibodies. Research, often done in NOS3 deficient mice, showed a physiologically relevant role of NOS1 in modulating cardiac function [107], systemic arterial pressure [108], myogenic tone [109], and cerebral blood flow [110]. Also, inactivation of the NOS1 gene resulted in reduced acetylcholine-induced vasodilation [111] in the mouse aorta. There are clear data that NOS1-generated H2O2 [112] has an important impact on the regulation of the vascular tone.

By immunohistochemistry NOS2 protein expression has been described in normal aortas in the surrounding adventitia. NOS2 protein was detected also in neutrophils and monocytes enclosed in thrombi surrounding these vessels [113].

NOS3 expression in the vasculature has been shown for the EC (see [31] for a review) and the PVAT [114, 115, 116] by immunohistochemistry and western blot. Although NOS3 is mainly believed to be a constitutively expressed gene there are several reports showing induction of NOS3 expression. NOS3 expression has been described to be upregulated by fluid shear stress [117] and cyclic stretch [118] in cultured EC (see Fig. 1). This has been also observed in animals after exercise [119, 120].

In normal vessel NO synthesized by NOS3 is believed to be a major regulator of vascular tone and to be the most important anti-inflammatory mediator in the vessel (see Fig. 1).

By post-translational acylation NOS3 is localized to biological membranes such as the Golgi apparatus or plasmalemma caveolae. This subcellular localization permits optimal regulation by shear stress, calcium ions and kinases. Therefore, agonists enhancing intracellular calcium concentrations (e.g., bradykinin, histamine, VEGF), or modulating pathways leading to increased CaM binding or reduced CaM dissociation are able to activate NOS3-dependent NO release [121].

8. Innate immunity

In higher vertebrates the immune system is made up by two components: the non-specific innate immunity and the adaptive immunity, which is highly specific. As first level of reaction against anything foreign, the innate immune system have evolved conserved strategies to defend the body against a pathogen. These defense mechanisms comprise a magnitude of structures and mediators like the skin barrier, saliva, tears, various cytokines, complement proteins, lysozyme, bacterial flora, and numerous cells including neutrophils, basophils, eosinophils, monocytes, macrophages, reticuloendothelial system, natural killer cells (NK cells), epithelial cells, endothelial cells, red blood cells, and platelets.

The adaptive immune system (B- and T lymphocytes and their products) depends on antigen receptors, which are somatically generated and clonally selected. In contrast, the innate immune system senses pathogens by highly conserved, relatively invariant structural motifs. The “danger theory” published by Polly Matzinger in 1994 [122] described that the innate immune system responds to endogenous or exogenous “danger signals”. Pathogen-associated molecular patterns (PAMPs) are exogenous danger signals and consist of highly conserved motifs in microbial organisms. Endogenous danger signals, also named danger-associated molecular patterns (DAMPs), are proteins, cytokines, chemokines, and other molecules from distressed and injured cells. PAMPs and DAMPs stimulate innate immune cells by binding to pattern recognition receptors (PRRs), which then activate signaling pathways (e.g. MAPK-pathways), which result in the activation of transcription factors, like AP1, CREB, c/EBP, IRFs, NF-κB, and STATs, or RNA-BPs, involved in the regulation of mRNA-stability and translatability like HuR, and modulate ncRNA expression (miRNAs and lncRNAs) to initiate a wide array of responses against cell damage [123, 124, 125, 126, 127, 128]. Aberrant activation of innate immune signaling cascades can lead to a failure to regulate inflammatory events, resulting in considerable damage to host tissues and is involved in the pathophysiology of cardiometabolic diseases [129].

Innate immune cells (phagocytes) use NOS2-generated NO and NADPH Oxidase 2 (NOX2)-generated superoxide to kill invading microorganisms. A patient with genetic deficiency of NOS2 died by a fatal cytomegalovirus infection [130], demonstrating the importance of NOS2 for anti-viral innate immune processes. NOS2 expression in innate immune cells resulted in the modulation of cell-intrinsic capabilities and phenotypes, and regulatory effects on neighboring (immune) cells. For example, NOS2-generated NO modulates different important immune-relevant mechanisms like antigen presentation, cytokine production, expression of MHC class II and costimulatory molecules, phagocytosis, and survival as well as apoptosis of myeloid cells [8].

Beside classic innate immune cells (monocytes, macrophages, neutrophils, dendritic cells, and natural killer cells) other non-immune cells like cardiomyocytes, endothelial cells, and fibroblasts express these receptors and can actively contribute to immune response via PRR signaling [131, 132].

ECs can exert some innate immune functions that macrophages can also perform, for example cytokine secretion, phagocytic function, antigen presentation, pro-inflammatory immune-enhancing as well as anti-inflammatory and immunosuppressive actions. Therefore, Shao et al. have introduced ECs as multifunctional innate immune cells [133].

9. Vascular inflammation

Vascular inflammation can be induced by a multitude of stimuli. In microbial infections, the increased concentrations of pro-inflammatory cytokines and chemokines result in vascular inflammation. Also, alterations in blood flow and shear stress, hypoxia, metabolic dysregulation like increase of the low-density lipoprotein (LDL)-, fatty acid- or blood glucose-concentration as well as cardiovascular diseases like hypertension induce (and often result from) vascular inflammation [134, 135, 136, 137, 138, 139]. As in infections also in cardiometabolic diseases the important involvement of several cytokines, chemokines and adipokines (including IL-6, IL-1β, TNF-α, MCP1, and leptin) in the pathophysiologic process has been described [140]. In vascular inflammation circulating leukocytes (monocytes/macrophages as well as neutrophils, cells of the innate immune system) are allured to the site of injury and transmigrate into the intima. Their task is to clear the tissue from the source of inflammation and dead cells and ultimately resolve the inflammation. However, if the inflammation cannot be stopped and develops into a chronic state, this leads to pathologic situations through the development of vascular diseases like atherosclerosis. In these processes, enhanced generation of reactive oxygen/nitrogen species (ROS/RNS) by innate immune cells is central to the pathological mechanisms [139]. Since blood vessels play an important role in the maintenance of homeostasis, the dysregulation of vascular function in inflammation is central to numerous disorders such as atherosclerosis [134] and related complications (ischemia, myocardial infarction, stroke, and thrombosis [141]), as well as age-related cognitive decline [142], cancer [143], and neurodegeneration [144].

10. The intima in atherosclerosis and vascular inflammation

Endothelial dysfunction (ED) is the most important step in the development of atherosclerosis. Cardiovascular risk factors, such as aging, diabetes mellitus, hyperlipidemia, hypertension, obesity, and smoking induce endothelial cell damage, resulting in ED [145]. In contrast to the healthy situation, dysfunctional EC accelerate the generation of ROS and potentiate vascular inflammation [146]. The defect of the endothelium causes a disturbance of the balance between vasoconstriction and vasodilation. The increased EDCFs (especially ET-1) and reduced EDRFs (mainly NO) initiate pathophysiologic changes that stimulate or fortify atherosclerosis, like increased vascular permeability to lipoproteins and enhanced leukocyte adhesion, platelet aggregation, and generation of cytokines [147]. In addition, the enhanced concentrations of pro-inflammatory cytokines, (TNF-α, IL-1β, and IL-6), result in the endothelial expression of adhesion molecules (VCAM, ICAM), as well as MCP-1 and other chemokines, transforming it to an “inflamed endothelium”. This also leads to enhanced adherence and migration of monocytes [148, 149, 150, 151]. After immigration into the intima the monocytes develop to tissue macrophages with enhanced expression of scavenger receptor (SR) and increased internalization of (ROS)-modified lipoproteins [152]. In the end these cells become foam cells (FCs), a highlight of an early atherosclerotic lesion [153]. Atherosclerotic plaque rupture leads to an imbalance of thrombotic and anti-thrombotic substances. Here, EC-dysfunction leads to an increase of thrombotic substances (vWF, TXA2) and to reduced concentrations of antithrombotic substances. These effects result in thrombosis, causing devastating consequences [154].

11. The media in atherosclerosis and vascular inflammation

In the inflamed vessels SMCs have been shown to be crucially involved in the pathophysiological process of atherosclerosis [155]. In this process, SMCs migrate to the intima, proliferate, synthesize extracellular matrix (ECM) and deposit lipids. This facilitates arterial wall fibrosis and thickening and leads to luminal stenosis. Normally, SMC proliferation is inhibited by NO (and other factors) but, as described above, NO concentration decline in the inflamed vessel. Some of the ECMs released by SMCs contribute to stabilization of the fibrous cap of the atherosclerotic plaque and thereby help to protect against plaque rupture and thrombosis [156]. Several cytokines are produced by SMC (PDGF, TGF-β1, MIF, IFN-γ and MCP-1) which are involved in the inflammatory response to lipids [157].

12. The adventitia in atherosclerosis and vascular inflammation

Several data show that the adventitia displays an important role in the pathogenesis of atherosclerosis. Mainly activated by TGF-β1, fibroblasts in the adventitia could differentiate into myofibroblasts [158], resulting in increased expression of inflammatory cytokines and growth factors [159, 160]. In addition, the NAPDH oxidase (NOX)-generated ROS in adventitial fibroblasts has been described as sensors and messengers for the development of vascular diseases [161].

Also, lymphocytes (T and B cells) accumulate in the adventitia, the major site of inflammation in the arterial wall. These processes are related to lymphocyte infiltration in atherosclerotic arteries [162]. T helper 1 (Th1) cells, secreting proinflammatory cytokines such as IL-2, TNF-α, and IFN-γ, are believed to be proatherogenic cells. In contrast, by releasing anti-inflammatory cytokines (e.g., IL-4, IL-5, IL-9, IL-10, and IL-13) regulatory T (Treg) cells are atheroprotective. Th2 cells are mainly proatherogenic whereas Th17 cells are predominantly atheroprotective. Although the exact mechanisms are unclear, natural killer T (NKT) cells are regarded as proatherogenic cells. B-1 cells, commonly found in peripheral sites and not in spleen or lymphnodes, are involved in antibody response during an infection or vaccination. They exert anti-atherogenic activities via secreting IgM, inhibiting the formation of FCs. B-2 cells (also named as common B cells) stimulate Th1 cells and dendritic cells (DCs) to play a proatherogenic role. By secreting GM-CSF (acts on DCs), innate responsive activator (IRA) play proatherogenic roles [163].

13. The PVAT in atherosclerosis and vascular inflammation

PVAT acts as modulator of the vessel function by releasing adipokines, such as leptin, adiponectin, visfatin, resistin, and cytokines/chemokines, such as TNF-α, IL-6, IL-8, MCP-1, and other factors like plasminogen activator inhibitor 1 (PAI-1). Altogether, these factors contribute to SMC migration and proliferation [164], enhance neointimal formation and hyperplasia [165, 166], stimulate inflammation responses and oxidative stress [167], and regulate vascular tone [168]. All these factors exert important roles in atherosclerosis.

PVAT plays an essential role in the inflammatory response to atherosclerosis. For example, analyzing the EC-dependent, NO-mediated vasodilator response to acetylcholine in aortas isolated from high-fat diet treated male C57BL/6J mice, Xia et al. described normal vasodilation in PVAT-free samples. In sharp contrast, a decent reduction in the acetylcholine-induced vasodilator response was observed in aortas from obese mice with intact PVAT. By immunohistochemistry, the authors demonstrate that adipocytes in PVAT express NOS3. High-fat diet did not change NOS3 expression but resulted in reduced NO production due to NOS3-uncoupling. This was related to arginase induction and l-arginine deficiency observed in PVAT [169]. In addition, locally elevated levels of leptin in the PVAT seems to promote neointimal formation [166]. Finally, endovascular injury-induced neointimal formation is associated with a rapid phenotypic modification of PVAT with proinflammatory adipocytokines being upregulated, and adiponectin downregulated. TNF-α has been shown to play a central role in these changes in the PVAT [165].

14. Changes in NOS expression and activity in atherosclerosis and vascular inflammation

As stated above, in vascular cells expression of all NOS isoforms (1-3) is regulated by a number of different stimuli (e.g., cytokines, ROS, miRNAs). The mode of regulation is complex and comprises multiple epigenetic, transcriptional, post-transcriptional post-translational mechanisms as well as protein-protein-interactions.

14.1 Changes in NOS1 expression/activity

Both in early and advanced human atherosclerotic lesions NOS1 expression is up-regulated in ECs, macrophages and in the neointima [113]. As demonstrated in NOS1 knockout mice the inactivation of the NOS1 gene results in a worsening of neointimal formation and constrictive vascular remodeling [170]. In line with that, NOS1/apoE double knockout mice, compared to apoE-ko animals, displayed an accelerated atherosclerotic vascular lesion formation [171]. These data imply that NOS1 may also suppress atherosclerotic vascular lesion formation [172]. The upregulation of NOS1 expression is likely to have a compensatory role in case of reduced NOS3 expression/activity, as present in inflammation and atherosclerosis, to maintain vascular homeostasis. In addition, there are reports using immunohistochemically methods or western blot showing enhanced vascular NOS1 expression after stimulation with inflammatory/proliferative stimuli (angiotensin II, interleukin-1β, and platelet-derived growth factor), hypoxia, hypertensive situation, and statin treatment [103, 173, 174, 175, 176].

14.2 Changes in NOS2 expression/activity

In human atherosclerotic plaques, NOS2 expression was detected. Immunostaining and in situ hybridization localized NOS2 to (CD68-positive) macrophages, FC and VSMC [177]. In contrast to murine endothelial cells, cytokine incubation do not induce NOS2 expression in human endothelial cells (HUVEC). Dreger et al. indicated at least a partial role of the histone methyltransferase enhancer of zeste homolog 2 (Ezh2), which mediates trimethylation of histone 3 at lysine 27-H3K27me3, in the epigenetic suppression of NOS2 expression in human endothelial cells [178]. In septic patients high expression NOS2 is described in many organs or tissues, which results in an enhanced NO formation that are important for hypotension, vascular hyporeactivity to vasoconstrictors, organ injury, and organ dysfunction [179]. The marked hypotension in septic shock patients is attributed to the strong induction of NOS2 in the vessels as shown in different animal studies [180]. It seems that the major part of this enhancement could be attributed to enhanced NOS2 expression in VSMC [181].

14.3 Changes in NOS3 expression/activity

Regulation of NOS3 expression by treatment of EC with pro-inflammatory mediators activating the innate immune system or cytokines (like TNF-α) produced by these cells has been reported. In addition, hypoxia regulates NOS3 expression both on the transcriptional as well as on the post-transcriptional level.

TNF-α reduces human NOS3 promoter activity in pulmonary microvessel endothelial monolayers (PMEM) [182]. This decrease was related to TNF-α-induced modulation of the binding activity of the transcription factors GATA-4 and Sp3 to the promoter sequence.

Activators of the innate immune system like oxidized LDL (ox-LDL) [183] and LPS [184] as well as cytokines produced by innate immune cells like TNF-α [54, 55, 56, 57, 58, 59, 117] as well as hypoxia [185, 186] have been described to regulate NOS3 mRNA levels post-transcriptionally. Analyzing the factors involved in the TNF-α-mediated reduction of NOS3 mRNA, the RNA-BPs translation elongation factor 1-alpha 1 (eEF1A1) and polypyrimidine tract-binding protein 1 (PTB1) were found to interact with the 3’-UTR of the NOS3 mRNA and thereby destabilizing the mRNA [56, 57, 58]. In addition to RNA-BPs, also miRNAs have been shown to be involved in the TNF-α-mediated reduction of NOS3 mRNA expression. In HUVEC, TNF-α increased the expression of miR-155, an important regulator of the innate immune system [187], which directly binds to the 3’-UTR of the NOS3 mRNA and destabilize it. In addition, in human internal mammary artery rings adenoviral overexpression of miR-155 decreased both NOS3 expression and acetylcholine-induced endothelium-dependent vasodilation [60]. As shown by Lee et al. NF-κB is important for the TNF-α-mediated upregulation of miR-155 expression and the post-transcriptional downregulation of NOS3 mRNA expression [61]. Kim et al. reported that the NF-κB-regulated miR-31-5p is up-regulated in sera from patients with pre-eclampsia and in HUVECs treated with TNF-α. miR-31-5p downregulated human NOS3 mRNA expression by post-transcriptional destabilization [59].

Hypoxia regulates NOS3 expression both transcriptionally and post-transcriptionally [185]. Analyzing the effects of hypoxia on the NOS3 expression in human EC (HUVEC and HMEC cells) Coulet et al. described hypoxia-induced NOS3 mRNA expression. In transfection experiments a hypoxia regulated element (HRE) was identified (located at position -5382/-5356) in the human NOS3 promoter. Binding of the transcription factors HIF-1α/1β and 2 to this element was shown by supershift experiments [188]. Hypoxia is known to induce endothelial dysfunction (ED), in part, by reduction of NOS3 in ECs. Fish et al. showed that hypoxia reduced NOS3 transcription with parallel decreased histone acetylation and H3 lysine 4 methylation on NOS3 proximal promoter histones. In addition, the authors demonstrate that histones are quickly removed from the proximal promoter NOS3 in hypoxia. Longer duration of hypoxia leads to reincorporation of histone, lacking substantial histone acetylation. After reoxygenation of the ECs the chromatin remodeler BRG1 is involved in the reactivation of NOS3 expression [186]. Hypoxia-mediated downregulation of NOS3 mRNA and protein expression enhances the expression of a natural antisense transcript (NAT) ncRNA sONE, also known as NOS3AS or APG9L2, in HUVEC or rat aorta. sONE displays antisense homology to the 3’-UTR and part of the coding sequence of the NOS3 mRNA. Downregulation of sONE by siRNAs diminished hypoxia-induced reduction of NOS3 expression indicating NOS3 expressional regulation by sONE [189]. Hypoxia upregulates the expression of miR-134 in rat cardiomyoblast H9c2 cells. As miR-134 directly targets NOS3 mRNA and reduce NOS3 protein expression this post-transcriptional mechanisms seem to be part of the hypoxia related downregulation of NOS3 expression [190].

Cardiovascular diseases often are related to enhanced synthesis of reactive oxygen/nitrogen species (superoxide, hydrogen peroxide) as well as peroxynitrite or hypochlorous acid. In addition, the detoxification of theses reactive molecules by low molecular weight antioxidants or ROS degrading enzymes is often reduced [139, 191, 192, 193, 194]. As shown in several animal models and also in humans, the pathophysiology of vascular inflammation and ED depends on enhanced expression/activity of superoxide generating NOX enzymes resulting in enhanced production of ROS [193]. This excessive superoxide has been shown to react with NO to peroxynitrite and which in turn by oxidation of the essential NOS cofactor BH4 leads to NOS3 uncoupling converting it into a superoxide-producing enzyme. Beside cellular and animal studies, NOS3 uncoupling in ED has also shown in patients with hypercholesterolemia [195], diabetes mellitus [196], or essential hypertension [197].

EC express arginase II and its expression can be enhanced by different factors leading to ED. As NOS3 and arginase II compete for the substrate l-arginine the enhanced arginase II expression/activity also contributes to vascular dysfunction [193].

The endogenous NOS inhibitor asymmetric dimethyl-L-arginine (ADMA) is synthesized by the enzyme arginine N-methyltransferase (PRMT) and degraded by the enzyme dimethylarginine dimethylaminohydrolase (DDAH). Both enzymes are redox-sensitive, and ROS have been shown to upregulate PRMT- and downregulate DDAH activity. As a result, ROS-induced ADMA-levels may reduce NOS3-mediated NO synthesis or even uncouple the enzyme [193].

As described above, in healthy situations there are post-translational regulatory mechanisms of NOS3 activity and localization, such as modulation by interacting proteins like calcium/calmodulin, caveolin, HSP90 as well as protein modifications like phosphorylation, palmitoylation, and myristoylation. Different kinases (like PKB/Akt, AMPK) perform the stimulating phosphorylation at Ser1177. In the inflamed vessel dysregulation of NOS3 activity is related to the synthesis of redox-active species that initiate inhibitory phosphorylation by redox-active kinases at Thr495/Tyr657 (e.g., PKC and PYK-2), disruption of the zinc-sulfur-complex needed to stabilize the NOS3 dimer, S-glutathionylation, oxidative BH4 depletion, and ADMA depletion (enhanced activity of PRMT and reduced activity of DDAH) (reviewed in [26, 139, 194, 198]).

In summary, NOS1 and NOS3 are vasoprotective (see Fig. 3) whereas NOS2 has detrimental effects in the vasculature. During sepsis, NOS2 induction represents a major cause of hypotension (see Fig. 3). In addition, NO produced by NOS2 in inflammatory cells contributes to atherogenesis. In contrast, NOS3-derived NO is diminished during atherosclerosis. The reduced level of endothelial NO is mainly attributable to NOS3 uncoupling, reduced NOS3 enzymatic activity and enhanced NO inactivation by superoxide.

. Adiponectin stimulates NO production from PVAT and from endothelial cells (EC). By stimulating the leptin receptor (LepR) Leptin induces EC-dependent vasodilatation. The Leptin/LepR interaction results in NOS3 activation via the AMP-activated protein kinase (AMPK) and Akt pathway. PVAT- and EC-synthezised NO induce vasodilatation by activating soluble guanylate cyclase (sGC), leading to the synthesis of cyclic guanosine monophosphate (cGMP). NO from PVAT and EC can also induce/potentiate vascular smooth muscle cell (VSMC) hyperpolarization through K

Figure 3: PVAT-, EC- and immune cell-derived NO regulates vascular tone. Adiponectin stimulates NO production from PVAT and from endothelial cells (EC). By stimulating the leptin receptor (LepR) Leptin induces EC-dependent vasodilatation. The Leptin/LepR interaction results in NOS3 activation via the AMP-activated protein kinase (AMPK) and Akt pathway. PVAT- and EC-synthezised NO induce vasodilatation by activating soluble guanylate cyclase (sGC), leading to the synthesis of cyclic guanosine monophosphate (cGMP). NO from PVAT and EC can also induce/potentiate vascular smooth muscle cell (VSMC) hyperpolarization through KCa channels. Pro-inflammatory mediators, like LPS and TNF-α, can induce NOS2 expression in innate immune cells and thereby lead to the synthesis of high amounts of NO resulting in strong vasodilation. NOS1 expression has been detected in EC and VSCM and contribute to NO-mediated vasodilation. Modified from [116], an open access article under the terms of the Creative Commons Attribution-NonCommercial License.

15. Author contributions

AP and HK prepared the original draft. AP, HL and HK reviewed and edited the manuscript.

16. Ethics approval and consent to participate

Not applicable.

17. Acknowledgment

Not applicable.

18. Funding

Original works from the authors’ laboratory contributing to this review were supported by grants LI 1759/1-1 (to HK), KL-1020/10-1, PA1933/3-1, PA1933/2-3, LI-1042/1-1, LI-1042/3-1, LI-1042/5-1, XI 139/2-1 from the Deutsche Forschungsgemeinschaft (DFG), Bonn, Germany.

19. Conflict of interest

The authors declare no conflict of interest.

References
  • [1] Sudhamsu J, Crane BR. Bacterial nitric oxide synthases: what are they good for? Trends in Microbiology. 2009; 17: 212–218.
  • [2] Astuti RI, Nasuno R, Takagi H. Nitric oxide signaling in yeast. Applied Microbiology and Biotechnology. 2016; 100: 9483–9497.
  • [3] Choi SK, Choi HK, Kadono-Okuda K, Taniai K, Kato Y, Yamamoto M, et al. Occurrence of novel types of nitric oxide synthase in the silkworm, Bombyx mori. Biochemical and Biophysical Research Communications. 1995; 207: 452–459.
  • [4] Förstermann U, Closs EI, Pollock JS, Nakane M, Schwarz P, Gath I, et al. Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions. Hypertension. 1994; 23: 1121–1131.
  • [5] Kröncke KD, Fehsel K, Schmidt T, Zenke FT, Dasting I, Wesener JR, et al. Nitric oxide destroys zinc-sulfur clusters inducing zinc release from metallothionein and inhibition of the zinc finger-type yeast transcription activator LAC9. Biochemical and Biophysical Research Communications. 1994; 200: 1105–1110.
  • [6] Peunova N, Enikolopov G. Amplification of calcium-induced gene transcription by nitric oxide in neuronal cells. Nature. 1993; 364: 450–453.
  • [7] Weiss G, Goossen B, Doppler W, Fuchs D, Pantopoulos K, Werner-Felmayer G, et al. Translational regulation via iron-responsive elements by the nitric oxide/no-synthase pathway. The EMBO Journal. 1993; 12: 3651–3657.
  • [8] Bogdan C. Nitric oxide synthase in innate and adaptive immunity: an update. Trends in Immunology. 2015; 36: 161–178.
  • [9] Nathan CF, Hibbs JB. Role of nitric oxide synthesis in macrophage antimicrobial activity. Current Opinion in Immunology. 1991; 3: 65–70.
  • [10] Fehsel K, Jalowy A, Qi S, Burkart V, Hartmann B, Kolb H. Islet cell DNA is a target of inflammatory attack by nitric oxide. Diabetes. 1993; 42: 496–500.
  • [11] Wink DA, Kasprzak KS, Maragos CM, Elespuru RK, Misra M, Dunams TM, et al. DNA deaminating ability and genotoxicity of nitric oxide and its progenitors. Science. 1991; 254: 1001–1003.
  • [12] Laval F, Wink DA. Inhibition by nitric oxide of the repair protein, O6-methylguanine-DNA-methyltransferase. Carcinogenesis. 1994; 15: 443–447.
  • [13] Brüne B, Dimmeler S, Molina y Vedia L, Lapetina EG. Nitric oxide: a signal for ADP-ribosylation of proteins. Life Sciences. 1994; 54: 61–70.
  • [14] Perez-Torres I, Manzano-Pech L, Rubio-Ruiz ME, Soto ME, Guarner-Lans V. Nitrosative stress and its association with cardiometabolic disorders. Molecules. 2020; 2555.
  • [15] Förstermann U, Kleinert H. Nitric oxide synthase: expression and expressional control of the three isoforms. Naunyn-Schmiedeberg’S Archives of Pharmacology. 1995; 352: 351–364.
  • [16] Förstermann U, Boissel JP, Kleinert H. Expressional control of the ‘constitutive’ isoforms of nitric oxide synthase (NOS i and NOS III). FASEB Journal. 1998; 12: 773–790.
  • [17] Bredt DS, Snyder SH. Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme. Proceedings of the National Academy of Sciences of the United States of America. 1990; 87: 682–685.
  • [18] Mayer B, John M, Böhme E. Purification of a Ca2+/calmodulin-dependent nitric oxide synthase from porcine cerebellum. Cofactor-role of tetrahydrobiopterin. FEBS Letters. 1991; 277: 215–219.
  • [19] Schmidt HH, Murad F. Purification and characterization of a human no synthase. Biochemical and Biophysical Research Communications. 1991; 181: 1372–1377.
  • [20] Förstermann U, Schmidt HH, Pollock JS, Sheng H, Mitchell JA, Warner TD, et al. Isoforms of nitric oxide synthase. Characterization and purification from different cell types. Biochemical Pharmacology. 1992; 42: 1849–1857.
  • [21] Hevel JM, White KA, Marletta MA. Purification of the inducible murine macrophage nitric oxide synthase. Identification as a flavoprotein. The Journal of Biological Chemistry. 1992; 266: 22789–22791.
  • [22] Stuehr DJ, Cho HJ, Kwon NS, Weise MF, Nathan CF. Purification and characterization of the cytokine-induced macrophage nitric oxide synthase: an FAD- and FMN-containing flavoprotein. Proceedings of the National Academy of Sciences. 1991; 88: 7773–7777.
  • [23] Verrey F, Closs EI, Wagner CA, Palacin M, Endou H, Kanai Y. CATs and HATs: the SLC7 family of amino acid transporters. Pflügers Archiv European Journal of Physiology. 2004; 447: 532–542.
  • [24] Rochette L, Lorin J, Zeller M, Guilland J, Lorgis L, Cottin Y, et al. Nitric oxide synthase inhibition and oxidative stress in cardiovascular diseases: possible therapeutic targets? Pharmacology & Therapeutics. 2013; 140: 239–257.
  • [25] Pernow J, Jung C. Arginase as a potential target in the treatment of cardiovascular disease: reversal of arginine steal? Cardiovascular Research. 2013; 98: 334–343.
  • [26] Garcia V, Sessa WC. Endothelial NOS: perspective and recent developments. British Journal of Pharmacology. 2019; 176: 189–196.
  • [27] Forstermann U, Gath I, Schwarz P, Closs EI, Kleinert H. Isoforms of nitric oxide synthase. Properties, cellular distribution and expressional control. Biochemical Pharmacology. 1995; 50: 1321–1332.
  • [28] Mattila JT, Thomas AC. Nitric oxide synthase: non-canonical expression patterns. Frontiers in Immunology. 2014; 5: 478.
  • [29] Costa ED, Rezende BA, Cortes SF, Lemos VS. Neuronal nitric oxide synthase in vascular physiology and diseases. Frontiers in Physiology. 2016; 7: 206.
  • [30] Huang Z, Hoffmann FW, Fay JD, Hashimoto AC, Chapagain ML, Kaufusi PH, et al. Stimulation of unprimed macrophages with immune complexes triggers a low output of nitric oxide by calcium-dependent neuronal nitric-oxide synthase. The Journal of Biological Chemistry. 2012; 287: 4492–4502.
  • [31] Li H, Wallerath T, Förstermann U. Physiological mechanisms regulating the expression of endothelial-type no synthase. Nitric Oxide. 2002; 7: 132–147.
  • [32] Wang Y, Marsden PA. Nitric oxide synthases: gene structure and regulation. Advances in Pharmacology. 1995; 34: 71–90.
  • [33] Brenman JE, Xia H, Chao DS, Black SM, Bredt DS. Regulation of neuronal nitric oxide synthase through alternative transcripts. Developmental Neuroscience. 1997; 19: 224–231.
  • [34] Wang Y, Goligorsky MS, Lin M, Wilcox JN, Marsden PA. A novel, testis-specific mRNA transcript encoding an NH2-terminal truncated nitric-oxide synthase. The Journal of Biological Chemistry. 1997; 272: 11392–11401.
  • [35] Boissel JP, Schwarz PM, Förstermann U. Neuronal-type no synthase: transcript diversity and expressional regulation. Nitric Oxide. 1998; 2: 337–349.
  • [36] Wang Y, Newton DC, Marsden PA. Neuronal NOS: gene structure, mRNA diversity, and functional relevance. Critical Reviews in Neurobiology. 1999; 13: 21–43.
  • [37] Bros M, Boissel J, Gödtel-Armbrust U, Förstermann U. Transcription of human neuronal nitric oxide synthase mRNAs derived from different first exons is partly controlled by exon 1–specific promoter sequences. Genomics. 2006; 87: 463–473.
  • [38] Bros M, Boissel J, Gödtel-Armbrust U, Förstermann U. The untranslated region of exon 2 of the human neuronal nitric oxide synthase (NOS1) gene exerts regulatory activity. Gene. 2007; 405: 36–46.
  • [39] Chan Y, Fish JE, D’Abreo C, Lin S, Robb GB, Teichert A, et al. The cell-specific expression of endothelial nitric-oxide synthase. Journal of Biological Chemistry. 2004; 279: 35087–35100.
  • [40] Miao Y, Ajami NE, Huang T, Lin F, Lou C, Wang Y, et al. Enhancer-associated long non-coding RNA LEENE regulates endothelial nitric oxide synthase and endothelial function. Nature Communications. 2018; 9: 292.
  • [41] Man HSJ, Sukumar AN, Lam GC, Turgeon PJ, Yan MS, Ku KH, et al. Angiogenic patterning by STEEL, an endothelial-enriched long noncoding RNA. Proceedings of the National Academy of Sciences of the United States. 2018; 115: 2401–2406
  • [42] Karantzoulis-Fegaras F, Antoniou H, Lai SL, Kulkarni G, D’Abreo C, Wong GK, et al. Characterization of the human endothelial nitric-oxide synthase promoter. The Journal of Biological Chemistry. 1999; 274: 3076–3093.
  • [43] Laumonnier Y, Nadaud S, Agrapart M, Soubrier F. Characterization of an upstream enhancer region in the promoter of the human endothelial nitric-oxide synthase gene. Journal of Biological Chemistry. 2000; 275: 40732–40741.
  • [44] Huh J, Ha H, Kim D, Kim H. Placenta-restricted expression of LTR-derived NOS3. Placenta. 2008; 29: 602–608.
  • [45] Searles CD. Transcriptional and posttranscriptional regulation of endothelial nitric oxide synthase expression. American Journal of Physiology-Cell Physiology. 2006; 291: C803–C816.
  • [46] Wang Y, Veremeyko T, Wong AH, El Fatimy R, Wei Z, Cai W, et al. Downregulation of miR-132/212 impairs S-nitrosylation balance and induces tau phosphorylation in Alzheimer’s disease. Neurobiology of Aging. 2017; 51: 156–166.
  • [47] Reilly SN, Liu X, Carnicer R, Recalde A, Muszkiewicz A, Jayaram R, et al. Up-regulation of miR-31 in human atrial fibrillation begets the arrhythmia by depleting dystrophin and neuronal nitric oxide synthase. Science Translational Medicine. 2016; 8: 340ra74.
  • [48] Ding J, Tang Y, Tang Z, Zhang X, Wang G. A variant in the precursor of microRNA-146a is responsible for development of erectile dysfunction in patients with chronic prostatitis via targeting NOS1. Medical Science Monitor. 2017; 23: 929–937.
  • [49] Zhang X, Huo Q, Sun W, Zhang C, Wu Z, Xing B, et al. Rs2910164 in microRNA-146a confers an elevated risk of depression in patients with coronary artery disease by modulating the expression of NOS1. Molecular Medicine Reports. 2018; 18: 603–609.
  • [50] Guilbaud M, Gentil C, Peccate C, Gargaun E, Holtzmann I, Gruszczynski C, et al. MiR-708-5p and miR-34c-5p are involved in nNOS regulation in dystrophic context. Skeletal Muscle. 2018; 8: 15.
  • [51] Chen M, Li L, Liu C, Song L. Berberine attenuates Aβ-induced neuronal damage through regulating miR-188/NOS1 in Alzheimer’s disease. Molecular and Cellular Biochemistry. 2020; 474: 285–294.
  • [52] Tang Y, Li Y, Yu G, Ling Z, Zhong K, Zilundu PLM, et al. MicroRNA-137-3p protects PC12 cells against oxidative stress by downregulation of calpain-2 and nNOS. Cellular and Molecular Neurobiology. 2020.
  • [53] Lorenz M, Hewing B, Hui J, Zepp A, Baumann G, Bindereif A, et al. Alternative splicing in intron 13 of the human eNOS gene: a potential mechanism for regulating eNOS activity. FASEB Journal. 2007; 21: 1556–1564.
  • [54] Zhang J, Patel JM, Li YD, Block ER. Proinflammatory cytokines downregulate gene expression and activity of constitutive nitric oxide synthase in porcine pulmonary artery endothelial cells. Research Communications in Molecular Pathology and Pharmacology. 1997; 96: 71–87.
  • [55] Sumi D, Hayashi T, Jayachandran M, Iguchi A. Estrogen prevents destabilization of endothelial nitric oxide synthase mRNA induced by tumor necrosis factor alpha through estrogen receptor mediated system. Life Sciences. 2001; 69: 1651–1660.
  • [56] Lai PFH, Mohamed F, Monge J, Stewart DJ. Downregulation of eNOS mRNA expression by TNFalpha: identification and functional characterization of RNA-protein interactions in the 3’UTR. Cardiovascular Research. 2003; 59: 160–168.
  • [57] Yan G, You B, Chen S, Liao JK, Sun J. Tumor necrosis factor-alpha downregulates endothelial nitric oxide synthase mRNA stability via translation elongation factor 1–alpha 1. Circulation Research. 2008; 103: 591–597.
  • [58] Yi B, Ozerova M, Zhang G, Yan G, Huang S, Sun J. Post-transcriptional regulation of endothelial nitric oxide synthase expression by polypyrimidine tract-binding protein 1. Arteriosclerosis, Thrombosis, and Vascular Biology. 2015; 35: 2153–2160.
  • [59] Kim S, Lee K, Choi S, Kim J, Lee D, Park M, et al. NF-κB-responsive miRNA-31–5p elicits endothelial dysfunction associated with preeclampsia via down-regulation of endothelial nitric-oxide synthase. Journal of Biological Chemistry. 2018; 293: 18989–19000.
  • [60] Sun H, Zeng D, Li R, Pang R, Yang H, Hu Y, et al. Essential role of microRNA-155 in regulating endothelium-dependent vasorelaxation by targeting endothelial nitric oxide synthase. Hypertension. 2012; 60: 1407–1414.
  • [61] Lee K, Kim J, Kwak S, Lee K, Lee D, Ha K, et al. Functional role of NF-κB in expression of human endothelial nitric oxide synthase. Biochemical and Biophysical Research Communications. 2014; 448: 101–107.
  • [62] Sharma NM, Patel KP. Post-translational regulation of neuronal nitric oxide synthase: implications for sympathoexcitatory states. Expert Opinion on Therapeutic Targets. 2017; 21: 11–22.
  • [63] Jaffrey SR, Snyder SH. PIN: an associated protein inhibitor of neuronal nitric oxide synthase. Science. 1996; 274: 774–777.
  • [64] Sharma NM, Llewellyn TL, Zheng H, Patel KP. Angiotensin II-mediated posttranslational modification of nNOS in the PVN of rats with CHF: role for PIN. American Journal of Physiology. Heart and Circulatory Physiology. 2013; 305: H843–H855.
  • [65] Shu X, Keller TCS, Begandt D, Butcher JT, Biwer L, Keller AS, et al. Endothelial nitric oxide synthase in the microcirculation. Cellular and Molecular Life Sciences. 2015; 72: 4561–4575.
  • [66] Siragusa M, Fleming I. The eNOS signalosome and its link to endothelial dysfunction. Pflugers Archiv: European Journal of Physiology. 2016; 468: 1125–1137.
  • [67] Garcia V, Park EJ, Siragusa M, Frohlich F, Mahfuzul Haque M, Pascale JV, et al. Unbiased proteomics identifies plasminogen activator inhibitor-1 as a negative regulator of endothelial nitric oxide synthase. Proceedings of the National Academy of Sciences. 2020; 117: 9497–9507.
  • [68] Pautz A, Art J, Hahn S, Nowag S, Voss C, Kleinert H. Regulation of the expression of inducible nitric oxide synthase. Nitric Oxide. 2010; 23: 75–93.
  • [69] Buzzo CDL, Medina T, Branco LM, Lage SL, Ferreira LCDS, Amarante-Mendes GP, et al. Epigenetic regulation of nitric oxide synthase 2, inducible (Nos2) by NLRC4 inflammasomes involves PARP1 cleavage. Scientific Reports. 2017; 7: 41686.
  • [70] Gross TJ, Kremens K, Powers LS, Brink B, Knutson T, Domann FE, et al. Epigenetic silencing of the human NOS2 gene: rethinking the role of nitric oxide in human macrophage inflammatory responses. Journal of Immunology. 2014; 192: 2326–2338.
  • [71] Calegari-Silva TC, Vivarini ÁC, Pereira RDMS, Dias-Teixeira KL, Rath CT, Pacheco ASS, et al. Leishmania amazonensis downregulates macrophage iNOS expression via Histone Deacetylase 1 (HDAC1): a novel parasite evasion mechanism. European Journal of Immunology. 2018; 48: 1188–1198.
  • [72] Xie QW, Whisnant R, Nathan C. Promoter of the mouse gene encoding calcium-independent nitric oxide synthase confers inducibility by interferon gamma and bacterial lipopolysaccharide. Journal of Experimental Medicine. 1993; 177: 1779–1784.
  • [73] Lowenstein CJ, Alley EW, Raval P, Snowman AM, Snyder SH, Russell SW, et al. Macrophage nitric oxide synthase gene: two upstream regions mediate induction by interferon gamma and lipopolysaccharide. Proceedings of the National Academy of Sciences. 1993; 90: 9730–9734.
  • [74] de Vera ME, Shapiro RA, Nussler AK, Mudgett JS, Simmons RL, Morris SM, et al. Transcriptional regulation of human inducible nitric oxide synthase (NOS2) gene by cytokines: initial analysis of the human NOS2 promoter. Proceedings of the National Academy of Sciences of the United States of America. 1996; 93: 1054–1059.
  • [75] Linn SC, Morelli PJ, Edry I, Cottongim SE, Szabó C, Salzman AL. Transcriptional regulation of human inducible nitric oxide synthase gene in an intestinal epithelial cell line. The American Journal of Physiology. 1997; 272: G1499–G1508.
  • [76] Chu SC, Marks-Konczalik J, Wu HP, Banks TC, Moss J. Analysis of the cytokine-stimulated human inducible nitric oxide synthase (iNOS) gene: characterization of differences between human and mouse iNOS promoters. Biochemical and Biophysical Research Communications. 1998; 248: 871–878.
  • [77] Lee M, Wang C, Jin SW, Labrecque MP, Beischlag TV, Brockman MA, et al. Expression of human inducible nitric oxide synthase in response to cytokines is regulated by hypoxia-inducible factor-1. Free Radical Biology and Medicine. 2019; 130: 278–287.
  • [78] Nasif S, Contu L, Mühlemann O. Beyond quality control: the role of nonsense-mediated mRNA decay (NMD) in regulating gene expression. Seminars in Cell & Developmental Biology. 2018; 75: 78–87.
  • [79] Nickless A, Bailis JM, You Z. Control of gene expression through the nonsense-mediated RNA decay pathway. Cell & Bioscience. 2017; 7: 26.
  • [80] Gather F, Schmitz K, Koch K, Vogt L, Pautz A, Kleinert H. Regulation of human inducible nitric oxide synthase expression by an upstream open reading frame. Nitric Oxide. 2019; 88: 50–60.
  • [81] Rodriguez-Pascual F, Hausding M, Ihrig-Biedert I, Furneaux H, Levy AP, Förstermann U, et al. Complex contribution of the 3’-untranslated region to the expressional regulation of the human inducible nitric-oxide synthase gene. Involvement of the RNA-binding protein HuR. The Journal of Biological Chemistry. 2000; 275: 26040–26049.
  • [82] Fechir M, Linker K, Pautz A, Hubrich T, Kleinert H. The RNA binding protein TIAR is involved in the regulation of human iNOS expression. Cellular and Molecular Biology. 2005; 51: 299–305.
  • [83] Linker K, Pautz A, Fechir M, Hubrich T, Greeve J, Kleinert H. Involvement of KSRP in the post-transcriptional regulation of human iNOS expression-complex interplay of KSRP with TTP and HuR. Nucleic Acids Research. 2005; 33: 4813–4827.
  • [84] Bollmann F, Fechir K, Nowag S, Koch K, Art J, Kleinert H, et al. Human inducible nitric oxide synthase (iNOS) expression depends on chromosome region maintenance 1 (CRM1)- and eukaryotic translation initiation factor 4E (elF4E)-mediated nucleocytoplasmic mRNA transport. Nitric Oxide. 2013; 30: 49–59.
  • [85] Casper I, Nowag S, Koch K, Hubrich T, Bollmann F, Henke J, et al. Post-transcriptional regulation of the human inducible nitric oxide synthase (iNOS) expression by the cytosolic poly(a)-binding protein (PABP). Nitric Oxide. 2013; 33: 6–17.
  • [86] Söderberg M, Raffalli-Mathieu F, Lang MA. Inflammation modulates the interaction of heterogeneous nuclear ribonucleoprotein (hnRNP) i/polypyrimidine tract binding protein and hnRNP L with the 3′untranslated region of the murine inducible nitric-oxide synthase mRNA. Molecular Pharmacology. 2002; 62: 423–431.
  • [87] Söderberg M, Raffalli-Mathieu F, Lang MA. Identification of a regulatory cis-element within the 3’-untranslated region of the murine inducible nitric oxide synthase (iNOS) mRNA; interaction with heterogeneous nuclear ribonucleoproteins i and L and role in the iNOS gene expression. Molecular Immunology. 2007; 44: 434–442.
  • [88] Eshelman MA, Matthews SM, Schleicher EM, Fleeman RM, Kawasawa YI, Stumpo DJ, et al. Tristetraprolin targets Nos2 expression in the colonic epithelium. Scientific Reports. 2019; 9: 14413.
  • [89] Guo Z, Shao L, Zheng L, Du Q, Li P, John B, et al. MiRNA-939 regulates human inducible nitric oxide synthase posttranscriptional gene expression in human hepatocytes. Proceedings of the National Academy of Sciences of the United States of America. 2012; 109: 5826–5831.
  • [90] Yoshigai E, Hara T, Araki Y, Tanaka Y, Oishi M, Tokuhara K, et al. Natural antisense transcript-targeted regulation of inducible nitric oxide synthase mRNA levels. Nitric Oxide. 2013; 30: 9–16.
  • [91] Navarro-Lérida I, Corvi MM, Barrientos AA, Gavilanes F, Berthiaume LG, Rodríguez-Crespo I. Palmitoylation of inducible nitric-oxide synthase at Cys-3 is required for proper intracellular traffic and nitric oxide synthesis. The Journal of Biological Chemistry. 2004; 279: 55682–55689.
  • [92] Lanone S, Manivet P, Callebert J, Launay J, Payen D, Aubier M, et al. Inducible nitric oxide synthase (NOS2) expressed in septic patients is nitrated on selected tyrosine residues: implications for enzymic activity. Biochemical Journal. 2002; 366: 399–404.
  • [93] Bondareva O, Sheikh BN. Vascular homeostasis and inflammation in health and disease-lessons from single cell technologies. International Journal of Molecular Sciences. 2020; 21: 4688.
  • [94] Zhou J, Li Y, Chien S. Shear stress-initiated signaling and its regulation of endothelial function. Arteriosclerosis, Thrombosis, and Vascular Biology. 2014; 34: 2191–2198.
  • [95] Wang D, Wang Z, Zhang L, Wang Y. Roles of cells from the arterial vessel wall in atherosclerosis. Mediators of Inflammation. 2017; 2017: 8135934.
  • [96] Schwartz BG, Economides C, Mayeda GS, Burstein S, Kloner RA. The endothelial cell in health and disease: its function, dysfunction, measurement and therapy. International Journal of Impotence Research. 2010; 22: 77–90.
  • [97] Owens GK. Regulation of differentiation of vascular smooth muscle cells. Physiological Reviews. 1995; 75: 487–517.
  • [98] Margariti A, Zeng L, Xu Q. Stem cells, vascular smooth muscle cells and atherosclerosis. Histology and Histopathology. 2006; 21: 979–985.
  • [99] Moreno PR, Purushothaman K, Sirol M, Levy AP, Fuster V. Neovascularization in human atherosclerosis. Circulation. 2006; 113: 2245–2252.
  • [100] Kohlgrüber S, Upadhye A, Dyballa-Rukes N, McNamara CA, Altschmied J. Regulation of transcription factors by reactive oxygen species and nitric oxide in vascular physiology and pathology. Antioxidants & Redox Signaling. 2017; 26: 679–699.
  • [101] Szasz T, Webb RC. Perivascular adipose tissue: more than just structural support. Clinical Science. 2012; 122: 1–12.
  • [102] Papaioannou TG, Stefanadis C. Vascular wall shear stress: basic principles and methods. Hellenic Journal of Cardiology. 2005; 46: 9–15.
  • [103] Boulanger CM, Heymes C, Benessiano J, Geske RS, Lévy BI, Vanhoutte PM. Neuronal Nitric Oxide Synthase is Expressed in Rat Vascular Smooth Muscle Cells. Circulation Research. 1998; 83: 1271–1278.
  • [104] Brophy CM, Knoepp L, Xin J, Pollock JS. Functional expression of NOS 1 in vascular smooth muscle. American Journal of Physiology. Heart and Circulatory Physiology. 2000; 278: H991–H997.
  • [105] Bachetti T, Comini L, Curello S, Bastianon D, Palmieri M, Bresciani G, et al. Co-expression and modulation of neuronal and endothelial nitric oxide synthase in human endothelial cells. Journal of Molecular and Cellular Cardiology. 2004; 37: 939–945.
  • [106] Huang A, Sun D, Shesely EG, Levee EM, Koller A, Kaley G. Neuronal NOS-dependent dilation to flow in coronary arteries of male eNOS-KO mice. American Journal of Physiology. Heart and Circulatory Physiology. 2002; 282: H429–H436.
  • [107] DANSON E, CHOATE J, PATERSON D. Cardiac nitric oxide: emerging role for nNOS in regulating physiological function. Pharmacology & Therapeutics. 2005; 106: 57–74.
  • [108] Kurihara N, Alfie ME, Sigmon DH, Rhaleb NE, Shesely EG, Carretero OA. Role of nNOS in blood pressure regulation in eNOS null mutant mice. Hypertension. 1998; 32: 856–861.
  • [109] Fleming I. Brain in the Brawn. Circulation Research. 2003; 93: 586–588.
  • [110] Hagioka S, Takeda Y, Zhang S, Sato T, Morita K. Effects of 7-nitroindazole and N-nitro-l-arginine methyl ester on changes in cerebral blood flow and nitric oxide production preceding development of hyperbaric oxygen-induced seizures in rats. Neuroscience Letters. 2005; 382: 206–210.
  • [111] Cotter MA, Cameron NE, Nangle MR. An in vitro investigation of aorta and corpus cavernosum from eNOS and nNOS gene-deficient mice. PfluGers Archiv European Journal of Physiology. 2004; 448: 139–145.
  • [112] Capettini LSA, Cortes SF, Gomes MA, Silva GAB, Pesquero JL, Lopes MJ, et al. Neuronal nitric oxide synthase-derived hydrogen peroxide is a major endothelium-dependent relaxing factor. American Journal of Physiology Heart and Circulatory Physiology. 2008; 295: H2503–H2511.
  • [113] Wilcox JN, Subramanian RR, Sundell CL, Tracey WR, Pollock JS, Harrison DG, et al. Expression of multiple isoforms of nitric oxide synthase in normal and atherosclerotic vessels. Arteriosclerosis, Thrombosis, and Vascular Biology. 1997; 17: 2479–2488.
  • [114] Dashwood MR, Dooley A, Shi-Wen X, Abraham DJ, Souza DSR. Does periadventitial fat-derived nitric oxide play a role in improved saphenous vein graft patency in patients undergoing coronary artery bypass surgery? Journal of Vascular Research. 2007; 44: 175–181.
  • [115] Xia N, Förstermann U, Li H. Resveratrol and endothelial nitric oxide. Molecules. 2014; 19: 16102–16121.
  • [116] Xia N, Li H. The role of perivascular adipose tissue in obesity-induced vascular dysfunction. British Journal of Pharmacology. 2017; 174: 3425–3442.
  • [117] Nishida K, Harrison DG, Navas JP, Fisher AA, Dockery SP, Uematsu M, et al. Molecular cloning and characterization of the constitutive bovine aortic endothelial cell nitric oxide synthase. The Journal of Clinical Investigation. 1992; 90: 2092–2096.
  • [118] Awolesi MA, Sessa WC, Sumpio BE. Cyclic strain upregulates nitric oxide synthase in cultured bovine aortic endothelial cells. Journal of Clinical Investigation. 1995; 96: 1449–1454.
  • [119] Sessa WC, Pritchard K, Seyedi N, Wang J, Hintze TH. Chronic exercise in dogs increases coronary vascular nitric oxide production and endothelial cell nitric oxide synthase gene expression. Circulation Research. 1994; 74: 349–353.
  • [120] Fukai T, Siegfried MR, Ushio-Fukai M, Cheng Y, Kojda G, Harrison DG. Regulation of the vascular extracellular superoxide dismutase by nitric oxide and exercise training. The Journal of Clinical Investigation. 2000; 105: 1631–1639.
  • [121] Förstermann U, Sessa WC. Nitric oxide synthases: regulation and function. European Heart Journal. 2012; 33: 829–837.
  • [122] Matzinger P. Tolerance, danger, and the extended family. Annual Review of Immunology. 1994; 12: 991–1045.
  • [123] Frevel MAE, Bakheet T, Silva AM, Hissong JG, Khabar KSA, Williams BRG. P38 Mitogen-activated protein kinase-dependent and -independent signaling of mRNA stability of AU-rich element-containing transcripts. Molecular and Cellular Biology. 2003; 23: 425–436.
  • [124] Katsanou V, Papadaki O, Milatos S, Blackshear PJ, Anderson P, Kollias G, et al. HuR as a negative posttranscriptional modulator in inflammation. Molecular Cell. 2005; 19: 777–789.
  • [125] Hayden MS, West AP, Ghosh S. NF-κB and the immune response. Oncogene. 2006; 25: 6758–6780.
  • [126] Newton K, Dixit VM. Signaling in innate immunity and inflammation. Cold Spring Harbor Perspectives in Biology. 2012; 4: a006049.
  • [127] Asirvatham AJ, Gregorie CJ, Hu Z, Magner WJ, Tomasi TB. MicroRNA targets in immune genes and the Dicer/Argonaute and are machinery components. Molecular Immunology. 2008; 45: 1995–2006.
  • [128] Murphy MB, Medvedev AE. Long noncoding RNAs as regulators of Toll-like receptor signaling and innate immunity. Journal of Leukocyte Biology. 2016; 99: 839–850.
  • [129] Xu M, Liu PP, Li H. Innate immune signaling and its role in metabolic and cardiovascular diseases. Physiological Reviews. 2019; 99: 893–948.
  • [130] Drutman SB, Mansouri D, Mahdaviani SA, Neehus A, Hum D, Bryk R, et al. Fatal cytomegalovirus infection in an adult with inherited NOS2 deficiency. New England Journal of Medicine. 2020; 382: 437–445.
  • [131] Chen GY, Nuñez G. Sterile inflammation: sensing and reacting to damage. Nature Reviews. Immunology. 2010; 10: 826–837.
  • [132] Zhang Y, Huang Z, Li H. Insights into innate immune signalling in controlling cardiac remodelling. Cardiovascular Research. 2017; 113: 1538–1550.
  • [133] Shao Y, Saredy J, Yang WY, Sun Y, Lu Y, Saaoud F, et al. Vascular endothelial cells and innate immunity. Arteriosclerosis, Thrombosis, and Vascular Biology. 2020; 40: e138–e152.
  • [134] Gimbrone MA, García-Cardeña G. Endothelial cell dysfunction and the pathobiology of atherosclerosis. Circulation Research. 2016; 118: 620–636.
  • [135] Brandes RP. Endothelial dysfunction and hypertension. Hypertension. 2014; 64: 924–928.
  • [136] Higashi Y, Kihara Y, Noma K. Endothelial dysfunction and hypertension in aging. Hypertension Research. 2012; 35: 1039–1047.
  • [137] Sheikh BN, Guhathakurta S, Tsang TH, Schwabenland M, Renschler G, Herquel B, et al. Neural metabolic imbalance induced by MOF dysfunction triggers pericyte activation and breakdown of vasculature. Nature Cell Biology. 2020; 22: 828–841.
  • [138] Jain T, Nikolopoulou EA, Xu Q, Qu A. Hypoxia inducible factor as a therapeutic target for atherosclerosis. Pharmacology & Therapeutics. 2018; 183: 22–33.
  • [139] Daiber A, Steven S, Vujacic-Mirski K, Kalinovic S, Oelze M, Di Lisa F, et al. Regulation of vascular function and inflammation via cross talk of reactive oxygen and nitrogen species from mitochondria or NADPH oxidase-implications for diabetes progression. International Journal of Molecular Sciences. 2020; 21: 3405.
  • [140] Kwaifa IK, Bahari H, Yong YK, Noor SM. Endothelial dysfunction in obesity-induced inflammation: molecular mechanisms and clinical implications. Biomolecules. 2020; 10: 291.
  • [141] Hadi HAR, Carr CS, Al Suwaidi J. Endothelial dysfunction: cardiovascular risk factors, therapy, and outcome. Vascular Health and Risk Management. 2005; 1: 183–198.
  • [142] Iadecola C. The pathobiology of vascular dementia. Neuron. 2013; 80: 844–866.
  • [143] Zuazo-Gaztelu I, Casanovas O. Unraveling the role of angiogenesis in cancer ecosystems. Frontiers in Oncology. 2018; 8: 248.
  • [144] Kisler K, Nelson AR, Montagne A, Zlokovic BV. Cerebral blood flow regulation and neurovascular dysfunction in Alzheimer disease. Nature Reviews. Neuroscience. 2017; 18: 419–434.
  • [145] Hamburg NM, Keyes MJ, Larson MG, Vasan RS, Schnabel R, Pryde MM, et al. Cross-sectional relations of digital vascular function to cardiovascular risk factors in the Framingham Heart Study. Circulation. 2008; 117: 2467–2474.
  • [146] Park K, Park WJ. Endothelial dysfunction: clinical implications in cardiovascular disease and therapeutic approaches. Journal of Korean Medical Science. 2015; 30: 1213–1225.
  • [147] Ross R. Atherosclerosis-an inflammatory disease. The New England Journal of Medicine. 1999; 340: 115–126.
  • [148] Barton M. Prevention and endothelial therapy of coronary artery disease. Current Opinion in Pharmacology. 2013; 13: 226–241.
  • [149] Zernecke A, Weber C. Chemokines in the vascular inflammatory response of atherosclerosis. Cardiovascular Research. 2010; 86: 192–201.
  • [150] Brunetti ND, Salvemini G, Cuculo A, Ruggiero A, De Gennaro L, Gaglione A, et al. Coronary artery ectasia is related to coronary slow flow and inflammatory activation. Atherosclerosis. 2014; 233: 636–640.
  • [151] Luc G, Bard J, Juhan-Vague I, Ferrieres J, Evans A, Amouyel P, et al. C-reactive protein, interleukin-6, and fibrinogen as predictors of coronary heart disease: the PRIME Study. Arteriosclerosis, Thrombosis, and Vascular Biology. 2003; 23: 1255–1261.
  • [152] Chistiakov DA, Bobryshev YV, Orekhov AN. Macrophage-mediated cholesterol handling in atherosclerosis. Journal of Cellular and Molecular Medicine. 2016; 20: 17–28.
  • [153] Libby P. Inflammation in atherosclerosis. Nature. 2002; 420: 868–874.
  • [154] Nieswandt B, Pleines I, Bender M. Platelet adhesion and activation mechanisms in arterial thrombosis and ischaemic stroke. Journal of Thrombosis and Haemostasis. 2011; 9: 92–104.
  • [155] Ross R, Glomset JA. Atherosclerosis and the arterial smooth muscle cell: proliferation of smooth muscle is a key event in the genesis of the lesions of atherosclerosis. Science. 1973; 180: 1332–1339.
  • [156] Schwartz SM, Virmani R, Rosenfeld ME. The good smooth muscle cells in atherosclerosis. Current Atherosclerosis Reports. 2000; 2: 422–429.
  • [157] Doran AC, Meller N, McNamara CA. Role of smooth muscle cells in the initiation and early progression of atherosclerosis. Arteriosclerosis, Thrombosis, and Vascular Biology. 2008; 28: 812–819.
  • [158] Shi Y, O’Brien JE, Fard A, Zalewski A. Transforming growth factor-beta 1 expression and myofibroblast formation during arterial repair. Arteriosclerosis, Thrombosis, and Vascular Biology. 1996; 16: 1298–1305.
  • [159] Fujiwara M, Muragaki Y, Ooshima A. Keloid-derived fibroblasts show increased secretion of factors involved in collagen turnover and depend on matrix metalloproteinase for migration. the British Journal of Dermatology. 2005; 153: 295–300.
  • [160] Jabs A, Okamoto E, Vinten-Johansen J, Bauriedel G, Wilcox JN. Sequential patterns of chemokine- and chemokine receptor-synthesis following vessel wall injury in porcine coronary arteries. Atherosclerosis. 2007; 192: 75–84.
  • [161] Haurani MJ, Pagano PJ. Adventitial fibroblast reactive oxygen species as autacrine and paracrine mediators of remodeling: bellwether for vascular disease? Cardiovascular Research. 2007; 75: 679–689.
  • [162] Moos MPW, John N, Gräbner R, Nossmann S, Günther B, Vollandt R, et al. The lamina adventitia is the major site of immune cell accumulation in standard chow-fed apolipoprotein E-deficient mice. Arteriosclerosis, Thrombosis, and Vascular Biology. 2005; 25: 2386–2391.
  • [163] Ait-Oufella H, Sage AP, Mallat Z, Tedgui A. Adaptive (T and B cells) immunity and control by dendritic cells in atherosclerosis. Circulation Research. 2014; 114: 1640–1660.
  • [164] Miao C, Li Z. The role of perivascular adipose tissue in vascular smooth muscle cell growth. British Journal of Pharmacology. 2012; 165: 643–658.
  • [165] Takaoka M, Suzuki H, Shioda S, Sekikawa K, Saito Y, Nagai R, et al. Endovascular injury induces rapid phenotypic changes in perivascular adipose tissue. Arteriosclerosis, Thrombosis, and Vascular Biology. 2010; 30: 1576–1582.
  • [166] Schroeter MR, Eschholz N, Herzberg S, Jerchel I, Leifheit-Nestler M, Czepluch FS, et al. Leptin-dependent and leptin-independent paracrine effects of perivascular adipose tissue on neointima formation. Arteriosclerosis, Thrombosis, and Vascular Biology. 2013; 33: 980–987.
  • [167] Salgado-Somoza A, Teijeira-Fernández E, Fernández AL, González-Juanatey JR, Eiras S. Proteomic analysis of epicardial and subcutaneous adipose tissue reveals differences in proteins involved in oxidative stress. American Journal of Physiology. Heart and Circulatory Physiology. 2010; 299: H202–H209.
  • [168] Maenhaut N, Van de Voorde J. Regulation of vascular tone by adipocytes. BMC Medicine. 2011; 9: 25.
  • [169] Xia N, Horke S, Habermeier A, Closs EI, Reifenberg G, Gericke A, et al. Uncoupling of endothelial nitric oxide synthase in perivascular adipose tissue of diet-induced obese mice. Arteriosclerosis, Thrombosis, and Vascular Biology. 2016; 36: 78–85.
  • [170] Morishita T, Tsutsui M, Shimokawa H, Horiuchi M, Tanimoto A, Suda O, et al. Vasculoprotective roles of neuronal nitric oxide synthase. FASEB Journal. 2002; 16: 1994–1996.
  • [171] Kuhlencordt PJ, Hötten S, Schödel J, Rützel S, Hu K, Widder J, et al. Atheroprotective effects of neuronal nitric oxide synthase in apolipoprotein e knockout mice. Arteriosclerosis, Thrombosis, and Vascular Biology. 2006; 26: 1539–1544.
  • [172] Tsutsui M, Shimokawa H, Otsuji Y, Yanagihara N. Pathophysiological relevance of no signaling in the cardiovascular system: novel insight from mice lacking all no synthases. Pharmacology & Therapeutics. 2010; 128: 499–508.
  • [173] Ebrahimian T, Mathieu E, Silvestre JS, Boulanger CM. Intraluminal pressure increases vascular neuronal nitric oxide synthase expression. Journal of Hypertension. 2003; 21: 937–942.
  • [174] Nakata S, Tsutsui M, Shimokawa H, Tamura M, Tasaki H, Morishita T, et al. Vascular neuronal no synthase is selectively upregulated by platelet-derived growth factor. Arteriosclerosis, Thrombosis, and Vascular Biology. 2005; 25: 2502–2508.
  • [175] Ward ME, Toporsian M, Scott JA, Teoh H, Govindaraju V, Quan A, et al. Hypoxia induces a functionally significant and translationally efficient neuronal no synthase mRNA variant. The Journal of Clinical Investigation. 2005; 115: 3128–3139.
  • [176] Nakata S, Tsutsui M, Shimokawa H, Yamashita T, Tanimoto A, Tasaki H, et al. Statin treatment upregulates vascular neuronal nitric oxide synthase through Akt/NF-kappaB pathway. Arteriosclerosis, Thrombosis, and Vascular Biology. 2007; 27: 92–98.
  • [177] Buttery LD, Springall DR, Chester AH, Evans TJ, Standfield EN, Parums DV, et al. Inducible nitric oxide synthase is present within human atherosclerotic lesions and promotes the formation and activity of peroxynitrite. Laboratory Investigation. 1996; 75: 77–85.
  • [178] Dreger H, Ludwig A, Weller A, Baumann G, Stangl V, Stangl K. Epigenetic suppression of iNOS expression in human endothelial cells: a potential role of Ezh2-mediated H3K27me3. Genomics. 2016; 107: 145–149.
  • [179] Thiemermann C. Nitric oxide and septic shock. General Pharmacology. 1997; 29: 159–166.
  • [180] Nin N, El-Assar M, Sánchez C, Ferruelo A, Sánchez-Ferrer A, Martínez-Caro L, et al. Vascular dysfunction in sepsis: effects of the peroxynitrite decomposition catalyst MnTMPyP. Shock. 2011; 36: 156–161.
  • [181] Koide M, Kawahara Y, Tsuda T, Yokoyama M. Cytokine-induced expression of an inducible type of nitric oxide synthase gene in cultured vascular smooth muscle cells. FEBS Letters. 1993; 318: 213–217.
  • [182] Neumann P, Gertzberg N, Johnson A. TNF-alpha induces a decrease in eNOS promoter activity. American Journal of Physiology Lung Cellular and Molecular Physiology. 2004; 286: L452–L459.
  • [183] Liao JK, Shin WS, Lee WY, Clark SL. Oxidized low-density lipoprotein decreases the expression of endothelial nitric oxide synthase. The Journal of Biological Chemistry. 1995; 270: 319–324.
  • [184] Lu JL, Schmiege LM, Kuo L, Liao JC. Downregulation of endothelial constitutive nitric oxide synthase expression by lipopolysaccharide. Biochemical and Biophysical Research Communications. 1996; 225: 1–5.
  • [185] McQuillan LP, Leung GK, Marsden PA, Kostyk SK, Kourembanas S. Hypoxia inhibits expression of eNOS via transcriptional and posttranscriptional mechanisms. American Journal of Physiology-Heart and Circulatory Physiology. 1994; 267: H1921–H1927.
  • [186] Fish JE, Yan MS, Matouk CC, St. Bernard R, Ho JJD, Gavryushova A, et al. Hypoxic repression of endothelial nitric-oxide synthase transcription is coupled with eviction of promoter histones. Journal of Biological Chemistry. 2010; 285: 810–826.
  • [187] Alivernini S, Gremese E, McSharry C, Tolusso B, Ferraccioli G, McInnes IB, et al. MicroRNA-155-at the critical interface of innate and adaptive immunity in arthritis. Frontiers in Immunology. 2017; 8: 1932.
  • [188] Coulet F, Nadaud S, Agrapart M, Soubrier F. Identification of hypoxia-response element in the human endothelial nitric-oxide synthase gene promoter. The Journal of Biological Chemistry. 2003; 278: 46230–46240.
  • [189] Fish JE, Matouk CC, Yeboah E, Bevan SC, Khan M, Patil K, et al. Hypoxia-inducible expression of a natural cis-antisense transcript inhibits endothelial nitric-oxide synthase. The Journal of Biological Chemistry. 2007; 282: 15652–15666.
  • [190] Xiao J, Wang J, Sun L. Effect of miR-134 against myocardial hypoxia/reoxygenation injury by directly targeting NOS3 and regulating PI3K/Akt pathway. Acta Cirurgica Brasileira. 2019; 34: e201900802.
  • [191] Griendling KK, FitzGerald GA. Oxidative stress and cardiovascular injury: part II: animal and human studies. Circulation. 2003; 108: 2034–2040.
  • [192] Griendling KK, FitzGerald GA. Oxidative stress and cardiovascular injury: part I: basic mechanisms and in vivo monitoring of ROS. Circulation. 2003; 108: 1912–1916.
  • [193] Förstermann U, Münzel T. Endothelial nitric oxide synthase in vascular disease: from marvel to menace. Circulation. 2006; 113: 1708–1714.
  • [194] Daiber A, Xia N, Steven S, Oelze M, Hanf A, Kröller-Schön S, et al. New therapeutic implications of endothelial nitric oxide synthase (eNOS) function/dysfunction in cardiovascular disease. International Journal of Molecular Sciences. 2019; 20: 187.
  • [195] Stroes E, Kastelein J, Cosentino F, Erkelens W, Wever R, Koomans H, et al. Tetrahydrobiopterin restores endothelial function in hypercholesterolemia. The Journal of Clinical Investigation. 1997; 99: 41–46.
  • [196] Heitzer T, Krohn K, Albers S, Meinertz T. Tetrahydrobiopterin improves endothelium-dependent vasodilation by increasing nitric oxide activity in patients with Type II diabetes mellitus. Diabetologia. 2000; 43: 1435–1438.
  • [197] Higashi Y, Sasaki S, Nakagawa K, Fukuda Y, Matsuura H, Oshima T, et al. Tetrahydrobiopterin enhances forearm vascular response to acetylcholine in both normotensive and hypertensive individuals. American Journal of Hypertension. 2002; 15: 326–332.
  • [198] Schulz E, Wenzel P, Münzel T, Daiber A. Mitochondrial redox signaling: interaction of mitochondrial reactive oxygen species with other sources of oxidative stress. Antioxidants & Redox Signaling. 2014; 20: 308–324.
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Andrea Pautz, Huige Li, Hartmut Kleinert. Regulation of NOS expression in vascular diseases. Frontiers in Bioscience-Landmark. 2021. 26(5); 85-101.