Infammation Research (2019) 68:429–441 https://doi.org/10.1007/s00011-019-01229-9 Inflammation Research

REVIEW

Roles of eNOS in atherosclerosis treatment

Fen‑fang Hong1 · Xiao‑yu Liang1 · Wei Liu1 · Sha Lv1 · Shu‑jin He1 · Hai‑bin Kuang1 · Shu‑long Yang1

Received: 10 July 2018 / Revised: 16 March 2019 / Accepted: 18 March 2019 / Published online: 1 April 2019 © Springer Nature Switzerland AG 2019

Abstract Background Atherosclerosis (AS) is the main pathogeny of coronary heart disease, cerebral infarction and peripheral vascular disease. Endothelial dysfunction is one of the important pathogenesis of AS. As an important endothelium-derived relaxa- tion factor, (NO) plays a role in cardiovascular protection and anti-AS function; but in the pathological state, endothelial (eNOS) disorder causes an abnormal production of NO, which may damage endothelial function and trigger AS. This review summarized the research progresses in the treatment strategies for AS based on cor- recting the disordered eNOS/ NO signaling pathway. Main body According to the topic, select the search terms ‘atherosclerosis,’ ‘nitric oxide,’ ‘eNOS,’ ‘treatment,’ ‘manage- ment,’ ‘,’ ‘maintenance,’ ‘remission’. Using these terms, a structured literature search via multiple electronic databases was performed for the most recent trial evidence in recent years. We read and analyze these literatures carefully, classifed these literatures according to their content, and then summarized and outlined the common main points in these classifed literatures. Finally, literature data were organized to discuss these main points logically. We found that both aber- rant expression and dysfunction of eNOS are closely related to AS development, and some new treatment strategies aimed at eNOS have been proposed, including upregulation of eNOS expression and inhibition of eNOS uncoupling. The former one is mainly related to infammatory inhibition and protection of the PKB-eNOS signaling pathway; whereas the latter one is associated with the addition of the L- substrate of eNOS, arginase inhibition, and the supplement of tetrahydro- biopterin, which can elevate no level. Conclusions eNOS can be an important target for prevention and treatment of AS, and eNOS drugs may be another potent class of efective therapeutic treatment for AS following traditional lipid-lowering, anti-platelet, vasodilator drugs. But apply- ing these experimental results to clinic treatment still requires further studies and development of biotechnology.

Keywords Atherosclerosis · Endothelial nitric oxide synthase · Arginase · · Treatment

Introduction all large and medium-sized arteries and causes a series of cardio and cerebrovascular diseases such as coronary heart Cardio- and cerebro-vascular diseases caused by athero- disease, cerebral infarction and renal vascular hypertension. sclerosis (AS) have been the number one cause of death Furthermore, AS-associated vascular diseases also serve as in the world for many years [1]. AS is involved in almost the main harmful results of diabetes mellitus [2]. Endothelial dysfunction plays important roles in AS pathogenesis. Nitric oxide (NO) derived from endothelial Responsible Editor: John Di Battista. cells can dilate blood vessels and inhibit platelet adhesion Fen-fang Hong, Xiao-yu Liang, Wei Liu, Sha Lv and Shu-jin He and aggregation. It also prevents the adhesion between leu- are co-frst authors. kocytes and endothelial cells, as well as restrains the pro- liferation of smooth muscle cells [3], suggesting its anti- * Hai‑bin Kuang AS efects. NO is synthesized from arginine catalyzed by [email protected] nitric oxide synthase (NOS). There are three NOS subtypes, * Shu‑long Yang including endothelial nitric oxide synthase (eNOS), induc- [email protected] ible nitric oxide synthase (iNOS), and neuronal nitric oxide 1 Department of Physiology, College of Medicine, Nanchang synthase (nNOS). University, Nanchang 330006, China

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eNOS is a homodimer, and its C-terminal bonds nico- Therefore, correcting the disordered eNOS/NO pathway tinamide adenine dinucleotide phosphate(NADPH), favin for the treatment of AS and other related diseases is impor- mononucleotide (FMN), favin adenine dinucleotide (FAD) tant. Here, we reviewed the present treatment strategies for reductase and -binding region, whereas its amino ter- AS aimed at eNOS intervention after consulting a large minal bonds tetrahydrobiopterin4 (BH4), ­O2, the oxidase number of literatures. Our study procedure was shown in zone, and the medium binding sequences of Fig. 1. l-arginine (l-Arg) [4]. Under physiological conditions, most of NO is produced from endothelial cells in which eNOS oxidates the guanidino nitrogen of l-Arg end. Endothelium- derived NO (eNO) is a multifunctional signaling molecule eNOS expression upregulation increases that, as an efective endogenous vasodilator, inhibits the key NO content to treat AS process in vascular lesion formation. eNO reduces the pro- duction of reactive oxygen species(ROS) and lipid peroxida- eNOS is one of three kinds of NOS. A number of studies tion [5]. In addition, eNO also has the efect of inhibiting [8, 9] have found that, with the development of AS lesions, platelet adhesion and aggregation, inhibiting adhesion mol- the expression level of eNOS decreased gradually. This ecule and chemokine expression, as well as reducing infam- fnding demonstrates that eNOS abnormality has an impor- matory cell infltration and smooth muscle cells (SMCs) tant infuence on AS. For example, the downregulation migration and proliferation [6]. eNO signaling pathways of eNOS expressions, the decreased eNOS activity and mainly include the phosphatidylinositol 3-kinase (PI3K)/ eNOS inhibitors all resulted in the decrease of NO syn- serine threonine protein kinase B (AKT)/eNOS pathway [7]. thesis, which can accelerate the development of AS dis- Thus, abnormal eNOS expression or its dysfunction ease. Therefore, treatments aimed at eNOS have become may cause NO production disorder, in turn damaging an important target for the treatment of AS. endothelial function [8]. That is a pathogenesis of AS.

Fig. 1 Study procedure

1 3 431 Roles of eNOS in atherosclerosis treatment

Upregulation of eNOS expression by regulating ciglitazone. Of interest, inhibiting AMPK abolished cigl- ox‑LDL itazone-mediated eNOS function, NO synthesis and angi- ogenesis, but increased RMVECs’ aging and apoptosis. Oxidized low-density lipoprotein (ox-LDL)-induced Further experiments showed that inhibition of peroxisome endothelial cell injury is thought to be the frst step in the proliferator-activated receptors (PPAR) signifcantly sup- pathogenesis of AS. Ox-LDL has high cytotoxicity, injuring pressed AMPK phosphorylation, eNOS expression and NO endothelial cell by reducing eNOS expression and increas- production [9]. ing iNOS expression to induce apoptosis of endothelial Bao et al. [11] found that ox-LDL signifcantly decreased cells, which accelerates the progression of AS (Fig. 2). Yin endothelial cell viability, increased lactate dehydroge- et al. found that epigallocatechin-3-gallate protects against nase (LDH) and IL-8 levels, inhibited NO production and ox-LDL-induced endothelial dysfunction through the Jag- induced cyclooxygenase-2 (COX-2) expression. Further- ged-1/Notch signaling pathway [10] (Table 1). In Xu et al. more, ox-LDL also stimulated the expression of hemag- study, the data showed that eNOS and Akt phosphorylation, glutinin-like oxidized low-density lipoprotein receptor-1 vascular endothelial growth factor (VEGF) expression and (LOX-1), ROS overproduction, p38 mitogen-activated pro- NO production were signifcantly decreased; moreover, the tein kinase (p38MAPK) phosphorylation, protein kinase B aging and apoptosis of rat microvascular endothelial cells (PKB) dephosphorylation and nuclear factorκB (NF-κB) (RMVECs) increased after ox-LDL induction for 24 h. All activation as well as inhibitor nuclear factor-kappa B (IκB) these changes were efectively reversed by ciglitazone pre- degradation. But, these ox-LDL-induced efects were sup- treatment. Meanwhile, the phosphorylation of adenosine pressed by puerarin pretreatment. These results suggest that 5′-monophosphate (AMP)-activated protein kinase (AMPK) puerarin inhibits ox-LDL-induced endothelial cell injuries was suppressed by ox-LDL, which was also prevented by via inhibition of the LOX-1-mediated p38MAPK-NF-κB

Fig. 2 Regulating efects of oxidized low-density lipoprotein on endothelial dysfunction in atherosclerosis

1 3 432 F. Hong et al. [ 18 ] [ 24 ] [ 25 ] References [ 6 ] [ 7 ] [ 8 ] [ 10 ] [ 10 ] [ 16 ] [ 17 ] tive on atherosclerosis tive atherosclerotic lesions atherosclerotic aortic atherosclerosis sclerotic events sclerotic endothelial cell injuries - possible molecular mecha - CoQ10 pro which nisms by atherogenesis tects against pathways NO-related by therapeutic candidate for the therapeutic candidate for of atherosclerosis treatment tion in apoE (−/−) mice progression - the antioxida Demonstrated Signifcantly ameliorates ameliorates Signifcantly Prevent endothelial damage Prevent Relationship with atheroscle - Relationship rosis Inhibited the development of Inhibited the development Be efective in limiting athero - Be efective Inhibits ox-LDL-induced Provide new insight into the insight into new Provide XJP-1 is potentially a novel a novel is potentially XJP-1 - endothelial dysfunc Improved Reduces atherosclerotic lesion atherosclerotic Reduces SIRT1 the integrity of the vascular - activat endothelium by ing the PI3K/AKT/eNOS pathway ox-LDL-diminished cell adhesion activation suppresses LOX-1 LOX-1 suppresses activation theand moderates AMPK/ pathway eNOS diated p38MAPK-NF-κB diated p38MAPK-NF-κB infammatory and the PKB– signaling pathways eNOS induced generation of induced generation species and oxygen reactive the antioxidant improved capacity entry in an IκB-dependent as inhibited as well manner, induced MAPK activation LPS by dependent vasorelaxation dependent vasorelaxation and carbachol induced by basal cGMP renal increased production ability Inhibit the expression of Inhibit the expression WXD protects and maintains WXD protects Mechanism ↓ , ox-LDL-induced apoptosis Ciglitazone-mediated PPARγ Ciglitazone-mediated PPARγ - Inhibition of the LOX-1-me Attenuated the ox-LDL- Attenuated JP-1 blocked NF-κB nuclear blocked JP-1 Improved renal endothelium- renal Improved - bioavail of NO Restoration ↑ ↑ ↑ ↑ ↑ ↑ eNOS ↑ eNOS NO ↓ iNOS NO eNOS/NO levels eNOS/NO ↑ eNOS ↓ iNOS ↑ eNOS NO ↑ eNOS NO ↑ eNOS ↓ iNOS ↑ eNOS ↑ eNOS NO ↑ eNOS cells (RMVECs) Cell HUVECs Rat microvascular endothelial microvascular Rat Endothelial cells HUVEC HUVEC [apoE (−/−)] mice (ApoE−/−) mice fed a high- (ApoE−/−) mice fed (21%) diet fat High-fat diet rats diet High-fat Wistar rats Wistar Animal Apolipoprotein E-defcient Apolipoprotein Apolipoprotein E-defcient Apolipoprotein Mechanisms that treat atherosclerosis by the eNOS/NO pathway the eNOS/NO that by atherosclerosis treat Mechanisms (EGCG) romanone (XJP-1) romanone (1–7)] Atorvastatin (ATV) Atorvastatin Wen-Xin Decoction (WXD) Wen-Xin Pretreatment Epigallocatechin-3-gallate Epigallocatechin-3-gallate Ciglitazone Puerarin 1 Table Coenzyme Q10 (CoQ10) - 8-Dihydroxy-3-methyl-isoch Angiotensin-(1–7) [Ang- Angiotensin-(1–7) CGP42112

1 3 433 Roles of eNOS in atherosclerosis treatment References [ 26 ] [ 27 ] [ 28 ] [ 29 ] [ 30 ] [ 33 ] [ 48 ] [ 57 ] [ 64 ] endothelial function in dia - with rats blood glucose betic fuctuations aortic atherosclerosis aortic atherosclerosis decrease in blood pressure decrease sclerotic efects of GLP-1 efects of GLP-1 sclerotic agonists and removing stasis and removing induced VEA and vascular induced VEA and vascular stress oxidative Relationship with atheroscle - Relationship rosis Sal B is capable of improving Sal B is capable of improving Inhibited the development of Inhibited the development Inhibited the development of Inhibited the development Exhibited a dose-dependent The potential anti-athero - The potential Promoting blood circulation blood circulation Promoting Prevented diabetes mellitus- diabetes Prevented endothelial cell apoptosis endothelial cell apoptosis of eNOS and stimulation phosphorylation (Ser 1177) profle, regulating NOS, and NOS, regulating profle, thesuppressing vascular infammatory in the response aorta mice of ApoE KO oxidant proliferation, inhibited apop - proliferation, eNOS and increased tosis of aortaexpression to inhibit ET-1 expression expression inhibit ET-1 to and protein both at mRNA levels activity endothelial PI3K signal and of activation subsequent of NO and generation eNOS suppressed thesuppressed release adhesion of intercellular cell molecule-1 and vascular adhesion molecule-1 from ECs Mechanism Related to suppression of suppression to Related Improving the plasma lipid Improving Anti-infammatory and anti - Stimulated endothelial cell Stimulated Promote eNOS expression and expression eNOS Promote The inhibition of arginase The inhibition of arginase Through the activation of theThrough activation Restored eNOS activity and eNOS Restored ↑ ↑ eNOS/NO levels eNOS/NO ↑ eNOS ↓ iNOS ↑ eNOS ↑ eNOS ↑ eNOS ↑ eNOS NO NO rophage-like cells rophage-like isolated mice aorta Cell RAW264.7 mouse mac - RAW264.7 Endothelial cells of aorta HUVECs HUVECs, endothelium of Endothelial cell (EC) (ApoE KO) mice (ApoE KO) /NIH-corpulent (SHR/ rats NDmcr-cp) (apoE(−/−)) mice fed a (apoE(−/−)) mice fed (21%) diet high-fat Animal Diabetic rats Diabetic Apolipoprotein E knockout E knockout Apolipoprotein Spontaneously hypertensive hypertensive Spontaneously Apolipoprotein E-defcient Apolipoprotein Diabetic rats Diabetic (continued) pan) 3β(GSK-3β) Pretreatment Salvianolic acid B (Sal B) Salvianolic Perilla Oil (PO) Kaempferia parviforaKaempferia (KP) Phycocyanin 1 Table 1,25(OH)(2)D(3) Liraglutide Caesalpinia sappan (C. sap - hydrate Catechin Glycogen synthase kinase- Glycogen

1 3 434 F. Hong et al.

inflammatory and the PKB–eNOS signaling pathways. However, in the researches of Yu et al. [12], ox-LDL at low concentration could upregulate the phosphorylation of References [ 65 ] Akt, PI3K and eNOS. It also could promote in vitro angio- - genesis and increase NO synthesis through the PI3K/Akt/ eNOS pathway in Human Coronary Artery Endothelial Cell (HCAEC). Coenzyme Q10 (CoQ10), a potent antioxi- dant, has been reported to inhibit the progression of AS. It attenuated the ox-LDL-mediated downregulation of eNOS and upregulation of iNOS. In addition, CoQ10 suppressed ox-LDL-activated NF-kB and its downstream infammatory represents a new multi-tar a new represents the drug geted candidate for of atherosclerosis treatment Relationship with atheroscle - Relationship rosis The compound LASSBio-788 LASSBio-788 The compound mediators, including the expression of adhesion molecules, the release of proinfammatory cytokines, and the adher- ence of human monocytic cell line (THP-1) cells [13]. High- density lipoprotein (HDL) binding to B scavenger receptor family of type I(SR-BI) activates eNOS with the generation of NO, which may contribute to the positive cardiovascular efects [14].

Upregulation of eNOS expression by regulating

a decrease in contractile in contractile a decrease phenylephrine to response in and an improvement endothelium-dependent by response vasorelaxant the twofold increasing of eNOS expression the renin–angiotensin system Mechanism LASSBio-788 promoted promoted LASSBio-788

Numerous studies have indicated that the renin–angiotensin system is closely related to the development of AS. Angio- tensin II can cause vasoconstriction, increase blood pressure, eNOS/NO levels eNOS/NO ↑ eNOS cause oxidative stress, making a variety of infammatory responses more serious and increasing the production of infammatory factors, resulting in secondary development of AS [15]. The renin–angiotensin system can increase NO release and reduce the production of superoxide dismutase (SOD), which is related to nicotinamide adenine dinucleo- tide phosphate (NADPH) oxidase [16]. Angiotensin recep- tor blockers (ARBs) and angiotensin-converting inhibitors (ACEIs) can reduce oxidative stress and infam-

Cell mation in patients with AS, and prevent the formation of AS from many causes. Thus, these drugs may become a new class of anti-AS drugs [17]. 8-Dihydroxy-3-methyl-isoch- romanone (XJP-1), a novel ACEI, exhibited an inhibitory activity to lipopolysaccharide (LPS)-accelerated vascular infammation. XJP-1 also reduced endothelin-1 secretion, LPS-induced cytotoxicity and infammatory response, which may be a potential new therapy for AS [18]. In addition, the endogenous peptide, angiotensin-(1–7) [Ang-(1–7)], acting Animal Male Wistar rats Male Wistar through its specifc G protein-coupled receptor (GPCR), the meconium aspiration syndrome (Mas) receptor, has endothe- lium-dependent vasodilator properties. In D-Ala-Ang-(1–7), apolipoprotein E-defcient [apoE (−/−)] mice, chronic treat- ment with Ang-(1–7) improved endothelial dysfunction [19]. Kljajic et al. [20] found that the therapeutic dose of angio- tensin type II-2 receptor (ATIIR) doping CGP42112 signif- (continued) cantly increased endothelial function and the immunoreac- tivity and protein content of eNOS. Moreover, CGP42112 (N-Allyl (2-thienylidene)-3 (2-thienylidene)-3 (N-Allyl - ,4-methylenedioxybenzoyl hydrazine) Pretreatment The compound LASSBio-788 LASSBio-788 The compound 1 Table (1 µg/kg/min) signifcantly attenuated the progression of AS

1 3 435 Roles of eNOS in atherosclerosis treatment lesions and increased the stability of plaque. These fndings as compared to single ATV treatment. Reversely, ATV com- suggest that these efects were partly due to increasing eNOS bined with N′-nitro-l-Arg-methyl ester hydrochloride sig- content and recovering NO bioavailability. nifcantly increased serum TC, LDL-C, H2S, and CSE, and decreased NO and eNOS compared with the single ATV Upregulation of eNOS expression by statins [27].

Statins are inhibitors of the 3-hydroxy-3-two-CoA reduc- Chinese herbal medicine upregulates the eNOS tase (A), which is used to reduce the incidence of coronary expression artery disease (CAD) due to its pleiotropic efects [21], and it had the potential modulatory efects on endothelial eNOS, Wen‑Xin Decoction (WXD) a key enzyme involved in the regulation of cardiovascular function by generating endothelium-derived relaxing factor. Wen-Xin Decoction has been shown to relieve myocardial The upregulation of eNOS by statins is mediated through its ischemia reperfusion injury and prevent leukocyte adhesion inhibiting the synthesis of isoprenoids and subsequent pre- and invasion; in addition, it can accelerate angiogenesis and vention of the isoprenylation of small GTPase Rho, whereas prevent platelet activation and aggregation. Li et al. [28] -induced activation of eNOS is mediated through the found in rat control experiments that WXD signifcantly activation of phosphatidylinositol-3-kinase (PI3K)/protein upregulated the mRNA and protein expressions of PI3K, kinase B (PKB/Akt) signals. Moreover, the eNOS upregu- AKT, and eNOS and signifcantly increased the phospho- lation and its activation, in part, could play a fundamen- rylation of AKT and eNOS. These results suggest that tal role in the cardiovascular defensive potential of statins WXD protects and maintains the integrity of the vascular [22]. Combining a statin with arginine or citrulline can endothelium by activating the PI3K/AKT/eNOS pathway, increase NO production in endothelial cells by increasing decreasing iNOS expression, and promoting the release of eNOS protein levels [23]. Endothelial cell senescence leads physiological NO levels. Furthermore, WXD at a high dose to abnormal vascular function and AS; silent information regulated the NO/ET-1 ratio as efectively as atorvastatin. regulator 2 homolog 1(SIRT1) mediates vascular relaxation mediated by eNOS, and participates in multiple pathways Salvianolic acid B (Sal B) to inhibit endothelial cell senescence [24–26]. Kilic et al. [21] investigated the efects of the statins, atorvastatin and It is well known that vascular endothelial cell injury is an rosuvastatin on CAD patients by analyzing the associations initial event in AS. Sal B, a main bioactive component in the among gene variants, rs7069102C > G and rs2273773C > T, root of Salvia miltiorrhiza, has a vascular protective efect SIRT1/eNOS expressions, the status of total antioxidant and in diabetes (Fig. 3). Recent studies have shown that diabetic oxidant, as well as the oxidative stress index. They observed rats developed marked endothelial dysfunction as exhibited that compared with controls, SIRT1 expression was higher, by the impaired vasodilation induced by acetylcholine. Sup- whereas eNOS expression was lower in patients with CAD. plementation with Sal B resulted in an evident improvement Statin treatment reduced SIRT1 expression and increased of endothelial function. And, phosphorylation (Ser 1177) of eNOS expression in patients with CAD, which was inde- eNOS was signifcantly restored in Sal B-treated diabetic pendent from the studied gene variants. Furthermore, oxida- rats, accompanied by an evident recovery of NO metabo- tive stress parameters were signifcantly increased in patients lites. Sal B efectively reduced vascular endothelial cell with CAD, but were decreased by statin treatment, dem- apoptosis, with Bcl-2 protein upregulated and Bax protein onstrating the antioxidative efects of statins on AS. These downregulated markedly. This study indicates that Sal B is results suggest that statin treatment could produce its protec- capable of improving endothelial function in diabetic rats tive efect on cardiovascular disease through the inhibition with blood glucose fuctuations, of which the underlying of SIRT1 expression. mechanisms might be related to its suppression of endothe- Liu et al. [27] investigated the anti-AS efect of atorv- lial cell apoptosis and its stimulation of eNOS phosphoryla- astatin (ATV) in a rat AS model and found that ATV sig- tion (Ser 1177) [29]. nifcantly ameliorated AS lesions and enhanced the activity of the serum NO system. ATV also signifcantly decreased Perilla oil (PO) serum total cholesterol (TC), low-density lipoprotein cho- lesterol (LDL-C), as well as decreased serum NO and eNOS PO is a vegetable oil rich in alpha linolenic acid (ALA) and levels. However, it had no signifcant efects on serum hydro- n-3 polyunsaturated fatty acids (n-3 PUFA). It reduced the gen sulfde (H2S) and cystathionine-γ- (CSE). ATV formation of lipid striae in the aortic sinus of ApoE knock- combined with dl-propargylglycine signifcantly reduced out mice by reducing blood lipid and regulating NOS. Hong serum H2S and CSE, and increased serum NO and eNOS et al. observed that the expression of eNOS increased and

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Fig. 3 Protective efect of salvianolic acid on diabetes by improving endothelial dysfunc- tion

the expression of iNOS decreased in the PO group. Their long-term administration of phycocyanin may ameliorate the research suggests that PO inhibited the development of systemic blood pressure by enhancing eNOS expression in aortic AS by improving the plasma lipid profle, regulating the aorta that is stimulated by adiponectin. Phycocyanin may NOS, and suppressing the vascular infammatory response be benefcial for preventing endothelial dysfunction-related in the aorta of ApoE knockout mice [30]. diseases in metabolic syndrome [32].

Kaempferia parvifora (KP) Benefcial efects of other molecules or therapy on eNOS expression The rhizome of KP is used in traditional Thai medicine. Horigome et al. [31] found that KP extract and two of its Xiang et al. [33] found that in apolipoprotein E-defcient components [5,7-dimethoxyfavone (DMF) and 5-hydroxy- [apoE(−/−)] mice, 1,25(OH)(2)D(3) stimulated endothe- 3,7,3′,4′-tetramethoxyfavone (TMF)] mediated the mRNA lial cell proliferation, signifcantly increased the mRNA expression of eNOS in tumor necrosis factor-alpha (TNF- expressions of Bcl-2 and eNOS, but decreased Fas mRNA α)-stimulated human umbilical vein endothelial cells level. This fnding indicates that it inhibited endothelial (HUVECs) and downregulated the genes of various cell cell apoptosis and increased eNOS expression of the aorta adhesion molecules, infammatory mediators and elements in apoE(−/−) mice. In addition, mononuclear cell (MNC) related to endothelial function. KP extract, DMF and TMF therapy attenuates the progression of AS in the aortas of showed their potential efects of anti-infammatory, antioxi- apoE KO mice. The mechanism by which this attenuation dant and inhibiting the development and progression of AS occurs includes the homing of endothelial progenitor cells in those in vitro models. (EPCs), a decrease in oxidative stress and an upregulation of eNOS expression [34]. In addition, platelet endothelial Phycocyanin cell adhesion molecule-1(PECAM1), a key molecule in an endothelial mechanosensing complex, specifcally mediates Phycocyanin is a pigment found in the blue-green algae, Gab1 tyrosine phosphorylation and its downstream Akt and Spirulina, that possesses antihypertensive efects. It was eNOS activation in ECs on fow, showing its arterial protec- discovered that phycocyanin increased serum adiponectin tive function [35]. Moreover, the glucagon-like peptide-1 level in the adipose tissue of rats, and showed a signifcant receptor agonist, liraglutide, in a concentration-dependent and positive correlation between aortic eNOS expression manner, was also observed to promote eNOS expression but levels, a downstream target of the adiponectin receptor, and inhibit ET-1 expression both at the mRNA and protein lev- serum adiponectin levels. In addition, it was suggested that els, possibly inhibiting the development of AS [36].

1 3 437 Roles of eNOS in atherosclerosis treatment

Inhibition of eNOS uncoupling other diseases. Peroxynitrite plays a critical role in vascu- lar pathophysiology by increasing arginase activity and eNOS uncoupling refers to as eNOS dysfunction occurs, decreasing eNOS activity. 5-Amino-3-morpholinyl-1,2,3- in which eNOS no longer produces NO, but superox- oxadiazolium chloride (SIN-1) was used to produce argi- ide anion, resulting in reduced NO bioavailability and nase in the treatment of HUVECs, it increased arginase increased oxidative stress, and causing or aggravating activity in a time- and dose-dependent manner and reduced endothelial dysfunction [37]. the production of NO, whereas the addition of arginase l-Arg and tetrahydrobiopterin (BH4) are closely related inhibitor blood group system(ABH) or exogenous sup- to eNOS uncoupling. l-Arg is a substrate of eNOS, and ply of arginine can reverse the role of SIN-1 and increase when it is defcient, the electrons that should be trans- endothelial NO production. Therefore, evidence for the ferred to l-Arg are transferred to O­ 2 to produce O­ 2−, rather protection of vascular endothelial function by inhibition than producing NO. ­O2− changes the structure of eNOS of arginine or exogenous supplementation of arginine is and aggravates the decoupling of eNOS. It is known that still sufcient [44]. l-Arg was administered orally to rats Arg is the substrate of eNOS competing with asymmetric with a high-calorie diet in saline at 150 mg/kg daily for dimethyl-l-arginine (ADMA), and ADMA acts an endog- 12 weeks. The level of NF-κB and TNF-α decreased sig- enous inhibitor of NOS. In the cross-sectional study of nifcantly in the arginine group compared with the control Notsu et al., they discovered an association between the group. Obviously, AS, of which obesity and high-fat diet Arg/ADMA ratio and the maximal intima-media thick- as its risk factor, can be controlled by oral arginine [43]. ness (IMT) in the carotid artery. Imbalance of Arg and After the follow-up of Iranian adults for 4.7 years, a latest ADMA is independently involved in the progression of report [45] of a prospective cohort study found that l-argi- AS, and the Arg/ADMA ratio may be a sensitive marker nine originating from plants in our daily diets has a negative for AS [38]. BH4, an important of eNOS, pro- correlation with cardiovascular risks and coronary heart dis- motes the dimerization of eNOS and the synthesis of NO ease events, which exhibits a potential vascular protection. [39]. The synthesis of NO in vivo is closely related to the In contrast, excessive intake of l-arginine from animals will concentration of BH4 in blood vessels, which is mainly have adverse efects. This phenomenon may be attributed related to the regulation of eNOS by BH4 [40]. When to better utilization of l-arginine from plant than animal BH4 is absent, eNOS cannot catalyze the oxidation of l- sources. There is a higher proportion of in animal Arg into l-citrulline and NO, and also transfer electrons protein. In addition, lysine and l-Arg are competitive in the l from NADPH to ­O2 to produce ­O2−, causing oxidative process of intracellular transport; therefore, the lysine/ -Arg stress, and then leading to endothelial dysfunction [41]. ratio may indirectly afect arginine utilization, resulting in Experimental results from in vitro cell culture and ani- diferent efects in adults [46]. mal models confrmed the role of BH4 in restoring eNOS coupling [42]. The defciency or dysfunction of l-Arg and BH4 may be an important molecular mechanism of AS and Arginine production inhibition other relevant diseases. Focusing on improving the levels of l-Arg and BH4 will has a positive signifcance in the Both arginase-1 and arginase-2 can cause uncoupling of treatment of AS. eNOS. They compete against eNOS for the same enzyme substrate, l-Arg, to regulate eNOS and NO generation. Meanwhile, they enhance the generation of ROS by eNOS, l‑Arginine supplement leading to vascular oxidative stress and infammation reac- tion, and ultimately leading to cardiovascular disease [47]. It has been shown that arginine is the only substrate for Ryoo et al. [48] found that arginase inhibition can restore NO production, which plays a crucial role in the function the vascular endothelial function of the high blood choles- of the cardiovascular system. In the previous study, argi- terol in ApoE−/−mice and arginase II deletion (ArgII−/−) nine showed a moderating efects on the vascular homeo- mouse, reduce vascular stifness and AS plaque area. S-(2- stasis in healthy people and the patients with hypertension Boronoethyl)-l-cysteine (BEC), an arginase inhibitor, can and diabetes [43]. Peroxynitrite has a wide range of tissue also increase eNOS’s activity through the phosphorylation destruction. In vivo, it is produced from lipid peroxida- of its 1177 serine to reverse related pathological changes tion and deactivated some protein, enzyme and ion chan- in diabetic nephropathy. At the same time, BEC treat- nel through its oxidation and nitration on them, which is ment in diabetic nephropathy (DN) animals also resulted an important pathophysiological mechanism of diabe- in upregulation of NO level [49]. Using molecular docking tes, ischemia reperfusion, sepsis, infammation, AS, and technology, Pham et al. confrmed the arginine inhibition function of some chemical-synthesized cinnamic derivatives

1 3 438 F. Hong et al. in mammals. Nevertheless, concrete pathophysiological and reperfusion can quickly be relieved [41]. In addition, mechanisms have not been elucidated [50]. ACEI, angiotensin II type 1 receptor (AT1) receptor block- The ethyl acetate extract of medicinal plant stone lotus ers, statins, and resveratrol have been shown to (CLE) mainly inhibit arginase activity of HUVECs through inhibit eNOS uncoupling, stimulate eNOS activity at the enhancing the stability of eNOS two-dimers, thus increasing same time [59]. the production of NO. These results suggest that CLE may be of great value in the treatment of cardiovascular diseases associated with endothelial dysfunction [51]. Drugs to increase eNOS activity

Supplement of tetrahydrobiopterin Activating eNOS via the PI3K/Akt pathway

Researches have reported that BH4 is closely related to Catechin hydrate treatment can activate endothelial cells eNOS uncoupling. Therefore, associated factors that can phosphatidylinositol 3-kinase (PI3K) pathway and sub- increase the level of BH4 may be important targets in the sequently activate eNOS, increasing the production of treatment of AS. Pharmacological experiments showed that NO,which is of great signifcance in preventing endothelial BH4 vessel supplement could raise the bioavailability of NO dysfunction induced by diabetes, and for the early prevention and improve endothelial function. Thus, BH4 supplement of AS [60]. Adiponectin induces AKT-mediated phospho- may serve as an efective way to protect vascular endothelial rylation of eNOS [37].Through restoring eNOS coupling and function [52]. In a vitro study, Margaritis et al. [37] found improving the redox state of human blood vessels, it plays that adiponectin increased BH4 bioavailability, promoted an important anti-AS function. Uncarboxylated Osteocalcin eNOS coupling, and changed the redox state of human blood (ucOC) can also activate the PI3K/Akt signaling pathway vessels in the saphenous vein and internal mammary artery. to prevent endothelial cell apoptosis induced by free fatty In addition, they also found that statins can increase BH4 acids and increase vascular endothelial eNOS phosphoryla- bioavailability, improve eNOS coupling and reduce the level tion and NO production [61]. The efect of berberine on of ­O2− in the blood vessels, which play a positive role in palmitic acid-induced vascular endothelial cell injury may anti-AS function in general [51, 53]. It has been reported that be related to the upregulation of eNOS and NO generation integrin-linked kinase (ILK) can prevent eNOS uncoupling, as well [62]. and then regulate vasomotor function [54]. Conversely, ILK Go et al. [63] found that sodium hydrogen sulfde, as an deletion will cause uncoupling of eNOS, accompanied by H2S donor, can activate the Akt/eNOS pathway to gener- vascular protein nitration, extensive decreased BH4, and ate NO in response to oscillatory shear stress (OSS), and increased BH2. increase eNOS expression. However, the vascular H2S syn- Propofol can improve the dysfunction of endothelial cells thase, cystathionine-γ-lyase (CSE), was downregulated by induced by high glucose, and its mechanism may be owed OSS. That CSE downregulation leads to H­ 2S level decline is to the inhibition of ONOO­ −-mediated BH4 degradation and a key factor in OSS-associated AS, indicating that regulating prevention of eNOS uncoupling [55]. As an agonist of per- H2S is a potential target for the prevention of cardiovascular oxisome proliferator-activated receptor alpha (PPAR-alpha), diseases like AS. Dysfunction of CSE and the decrease of Fenofbrate signifcantly increased the level of BH4 and pro- endogenous H2S levels are closely related to the pathogen- moted eNOS complex coupling [56]. In addition, Chinese esis of AS, and have become a new target for the study of herbal extracts such as Guizhi active ingredient ethyl acetate AS mechanisms, treatment, and prevention [64]. A research and magnolin of Xinyi can inhibit the uncoupling of eNOS study reported that onion extract has anti-AS efect to some in a certain range of concentration [57]. Experiments have degree by upregulation of the endogenous CSE/H2S path- been found that delivery of eNOS cofactor BH4 by nanocar- way [65]. rier can reduce vascular stress in wild-type mice, and pre- vent plaque rupture in ApoE−/− mice. These results suggest Increasing eNOS activity through improving that targeting to regions that are prone to form AS through endothelial function functional nanocarriers will be a better protection measure from AS. It is suggested that the phage will better prevent Vascular endothelial cell senescence is an important factor the formation of AS by targeting the region of the circulation in the pathogenesis of AS. Hayashi et al. [66] found that the that is prone to AS through the functional nanocarrier [58]. can prevent the decrease To reduce the BH4 biodegradation and improve efciency, of telomerase activity in the SA-galactosidase-positive cells Xie et al. transferred BH4 into circulation via liposome. It in a high-glucose environment, and this is related to the was seen that eNOS uncoupling and dysfunction caused reduction of ROS and eNOS activation, representing a new by decreased level of BH4 during myocardial ischemia mechanism to prevent AS. C-reactive protein (CRP) will

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