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.
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].
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
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].
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 Ca
Isoform | descriptive name | protein source | cDNA source |
NOS1 | bNOS (for brain NOS); | rat cerebellum porcine cerebellum | human and rat brain |
cNOS (for constitutive or Ca | |||
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 Ca | |||
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]. |
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.
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.
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].
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.
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.
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-
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].
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 (Hb
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].
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-
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-
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-
In macrophages from Leishmania amazonensis patients binding of the inhibitory
NF-
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/EBP
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
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 (
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-
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.,
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.
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 H
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].
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-
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].
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-1
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-
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-
Several data show that the adventitia displays an important role in the
pathogenesis of atherosclerosis. Mainly activated by TGF-
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-
PVAT acts as modulator of the vessel function by releasing adipokines, such as
leptin, adiponectin, visfatin, resistin, and cytokines/chemokines, such as
TNF-
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-
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.
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-1
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].
Regulation of NOS3 expression by treatment of EC with pro-inflammatory mediators
activating the innate immune system or cytokines (like TNF-
TNF-
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-
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-1
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
BH
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 BH
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.
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 K
AP and HK prepared the original draft. AP, HL and HK reviewed and edited the manuscript.
Not applicable.
Not applicable.
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.
The authors declare no conflict of interest.