Re: 혈관내피세포 기능장애와 NO.. 2022년

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Crit Care Clin

. Author manuscript; available in PMC: 2022 Apr 18.

Published in final edited form as: Crit Care Clin. 2020 Apr;36(2):307–321. doi: 10.1016/j.ccc.2019.12.009

Nitric Oxide and Endothelial Dysfunction

Anthony R Cyr a, Lauren V Huckaby a, Sruti S Shiva b, Brian S Zuckerbraun c,*

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PMCID: PMC9015729  NIHMSID: NIHMS1797086  PMID: 32172815

The publisher's version of this article is available at Crit Care Clin

INTRODUCTION

The vascular endothelium is composed of a monolayer of specialized cells (endothelial cells), which form the interface between the underlying smooth muscle cells from the vascular lumen. Endothelial cells may exhibit significant plasticity in function depending on the milieu in which they exist; for example, the endothelium comprising the blood–brain barrier has significantly different functional characteristics than that lining the aorta. However, all endothelial cells share a common set of functions, including the regulation of hemostasis, maintenance of vascular permeability, mediation of both acute and chronic immune responses to various types of injury, and control of vascular tone.1 There are several molecular mechanisms that govern these critical processes, but none are as critical as the nitric oxide (NO) signaling pathway.2 NO is a small, soluble gas with strong vasodilatory, anti-inflammatory, and antioxidant properties that plays a central role in the maintenance of vascular homeostasis.3 Highlighting this, the concept of endothelial dysfunction (ED) is centrally linked to decreased NO production and sensitivity, which ultimately results in an imbalance in vascular homeostasis leading to a prothrombotic, proinflammatory, and less compliant blood vessel wall.2,4 In the present review, we highlight the mechanisms underpinning the regulatory effects of NO on ED and link these to currently understood clinical phenomena.

소개

혈관 내피는

혈관 내강에 있는 평활근 세포와 혈관 내피 사이의 경계면을 형성하는

특수 세포(내피 세포)의 단일층으로 구성되어 있습니다.

내피 세포는

존재하는 환경에 따라

기능에 상당한 가소성을 나타낼 수 있습니다.

예를 들어,

혈액-뇌 장벽을 구성하는 내피는

대동맥을 감싸고 있는 내피와 기능적 특성이 상당히 다릅니다.

그러나

모든 내피 세포는

지혈 조절,

혈관 투과성 유지,

다양한 유형의 손상에 대한 급성 및 만성 면역 반응의 매개,

혈관 긴장도 조절을 포함한 공통된 기능 집합을 공유합니다.1

이러한 중요한 과정을 관장하는

여러 가지 분자 메커니즘이 있지만,

산화질소(NO) 신호 전달 경로만큼 중요한 것은 없습니다. 2

NO는

혈관 확장, 항염, 항산화 작용을 하는 작은 가용성 기체로,

혈관 항상성 유지에 중요한 역할을 합니다. 3

이를 강조하면서,

내피 기능 장애(ED)의 개념은

NO 생산과 민감도의 감소와 밀접하게 연관되어 있으며,

이는 궁극적으로 혈관 항상성의 불균형을 초래하여

혈관벽의 응고, 염증, 순응성을 감소시킵니다.2,4

endothelial dysfunction (ED)

본 고찰에서는 ED에 대한 NO의 조절 효과를 뒷받침하는 메커니즘을 강조하고,

이를 현재 이해되고 있는 임상 현상과 연결합니다.

NITRIC OXIDE IN NORMAL ENDOTHELIAL FUNCTION

Nitric Oxide Synthesis and Nitric Oxide Synthase Enzyme Isoforms

NO is a highly reactive, readily diffusible gaseous free radical with strong intrinsic oxidant properties. It is synthesized by 3 distinct subtypes of the NO synthase (NOS) enzyme, each with unique expression‎ patterns and functional properties: neuronal NOS (nNOS, NOS1), inducible NOS (iNOS, NOS2), and endothelial NOS (eNOS, NOS3).5,6 Broadly, these proteins catalyze the production of NO and l-citrulline from l-arginine and O2, using electrons donated from dihydronicotinamide-adenine dinucleotide phosphate (NADPH). This process is tightly regulated requiring several key protein–protein interactions and multiple prosthetic groups and cofactors. In monomeric form, the NOS subtypes are incapable of binding to l-arginine, and subsequently can function primarily as weak NADPH oxidases resulting in the production of harmful superoxide radical anion (O2•−). When bound by the calcium signaling protein calmodulin (CaM), the transfer of electrons through a flavin adenine mononucleotide and flavin adenine dinucleotide domain is enhanced. Finally, in the presence of heme and tetrahydrobiopterin (BH4), NOS monomers form homodimers capable of using the donated NADPH electrons to catalyze the 2-step oxidation of l-arginine to l-citrulline and NO. In the first step, NOS promotes the hydroxylation of l-arginine to Nω-hydroxy-l-arginine, which remains bound by the enzyme. In the second step, NOS catalyzes the oxidation of Nω-hydroxy-l-arginine to l-citrulline, thereby releasing NO (Fig. 1).

정상적인 내피 기능에 있는 산화질소

산화질소 합성 및 산화질소 합성효소 이소형질

산화질소(NO)는

반응성이 매우 높고 확산이 쉬운 기체 형태의 자유 라디칼로서,

강력한 고유 산화 특성을 가지고 있습니다.

strong intrinsic oxidant properties

산화질소는

3가지 유형의 산화질소 합성효소(NOS)에 의해 합성되는데,

각각 고유한 발현 패턴과 기능적 특성을 가지고 있습니다:

신경성 산화질소(nNOS, NOS1),

유도성 산화질소(iNOS, NOS2),

내피성 산화질소(eNOS, NOS3). 5,6

대체로,

이 단백질들은

디하이드로니코틴아미드-아데닌 디뉴클레오티드 포스페이트(NADPH)로부터 기증된 전자를 사용하여,

l-아르기닌과 O2로부터

NO와 l-시트룰린의 생성을 촉진합니다.

이 과정은

여러 가지 핵심적인 단백질-단백질 상호작용과 여러 가지 보조 그룹과 보조 인자를 필요로 하는

엄격한 규제를 받습니다.

단량체 형태인 NOS 아형은 l-아르기닌에 결합할 수 없기 때문에,

주로 약한 NADPH 산화효소로서 기능하여

유해한 과산화물 라디칼 음이온(O2•−)을 생성할 수 있습니다.

칼슘 신호 전달 단백질인 칼모둘린(CaM)에 결합하면,

플라빈 아데닌 모노뉴클레오티드와 플라빈 아데닌 디뉴클레오티드 도메인을 통한

전자 전달이 강화됩니다.

마지막으로,

헴과 테트라하이드로비오테린(BH4)이 존재할 때,

NOS 단량체는 기증된 NADPH 전자를 사용하여

l-아르기닌을 l-시트룰린과 NO로 2단계 산화시키는 데 사용할 수 있는

동종 이량체를 형성합니다.

첫 번째 단계에서

NOS는

l-아르기닌의 하이드록실화를 촉진하여

효소에 의해 결합된 상태로 남아 있는 Nω-하이드록시-l-아르기닌을 생성합니다.

두 번째 단계에서

NOS는

Nω-하이드록시-l-아르기닌의 산화를 촉진하여

l-시트룰린으로 전환함으로써 NO를 방출합니다(그림 1).

Fig. 1.

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eNOS regulation, coupling, and uncoupling.

During coupled eNOS activity (blue side), in the presence of heme and tetrahydrobiopterin, electrons from NADPH are passed through a core of flavin adenine dinucleotide–flavin adenine mononucleotide in the reductase domain to the Heme prosthetic group on the oxygenase domain. Here, l-arginine and O2 are consumed to create l-citrulline and NO. In the uncoupled state (red side), electrons are passed directly from the flavin adenine dinucleotide (FAD)–flavin adenine mononucleotide (FMN) core of the reductase domain to O2, generating superoxide (O2•−), which can ultimately combine with locally produced NO to make peroxynitrite (ONOO−). Several of the effects of ONOO− and NO are listed here, as well as factors contributing to both coupling and uncoupling of eNOS activity. These are explained in greater detail in the body of the text. VEGF, vascular endothelial growth factor.

eNOS 조절, 결합, 그리고 결합 해제. 

결합된 eNOS 활동(파란색 부분) 동안, 

헴과 테트라하이드로비오테린이 존재할 때, 

NADPH의 전자는 환원효소 영역에 있는 

플라빈 아데닌 디뉴클레오티드-플라빈 아데닌 모노뉴클레오티드의 핵을 통과하여 

산소화 효소 영역에 있는 헴 보철 그룹으로 전달됩니다. 

여기서, 

l-아르기닌과 O2가 소비되어 

l-시트룰린과 NO가 생성됩니다. 

결합이 해제된 상태(빨간색 부분)에서는, 

플라빈 아데닌 디뉴클레오티드(FAD)에서 

플라빈 아데닌 모노뉴클레오티드(FMN)로 이어지는 환원효소 도메인의 핵심에서 

전자가 O2로 직접 전달되어, 

슈퍼옥사이드(O2•−)를 생성합니다. 

이 슈퍼옥사이드가 국소적으로 생성된 NO와 결합하여 

퍼옥시니트라이트(ONOO−)를 생성할 수 있습니다. 

ONOO−와 NO의 여러 가지 효과와 eNOS 활동의 결합과 분리에 기여하는 요인이 

여기에 나열되어 있습니다. 

이에 대한 자세한 설명은 본문에서 확인할 수 있습니다. 

NOS isoforms are differentially regulated through post-translational modifications as well as interactions with the associated scaffolding proteins. nNOS and eNOS, for example, are highly dependent on Ca2+-activated CaM for homodimerization and activity, whereas iNOS is minimally dependent on calcium concentration.6 These nuances have critical functional effects. In neurons, NO produced by nNOS signaling functions to modulate longer term synaptic transmission, leading to critical involvement in learning, memory, and neurogenesis.7 Additionally, some data suggest that nNOS may play a significant role in the maintenance of vascular tone through effects of nNOS-expressing end neurons that innervate smooth muscle in microvasculature.8 In contrast, iNOS is strongly induced in activated macrophages, in which it creates an NO burst representing the sentinel event in the acute inflammatory cascade. This often leads to significant collateral damage to bystander healthy tissue through the generation of downstream reactive oxygen species (ROS) and reactive nitrogen species.9 Herein, we focus on the regulation and function of eNOS, the native NOS of vascular endothelial cells.

NOS 이소효소는

번역 후 변형과 연관된 스캐폴딩 단백질과의 상호작용을 통해 차별적으로 조절됩니다.

예를 들어,

nNOS와 eNOS는

동종 이량체화와 활동에 있어 Ca2+ 활성화 CaM에 크게 의존하는 반면,

iNOS는

칼슘 농도에 최소한의 의존성을 보입니다.6

이러한 미묘한 차이는

중요한 기능적 영향을 미칩니다.

뉴런에서 nNOS 신호 전달에 의해 생성된 NO는

장기적인 시냅스 전달을 조절하는 기능을 수행하여

학습, 기억, 신경 발생에 중요한 역할을 합니다.7

또한, 일부 데이터에 따르면

nNOS는 미세혈관 내의 평활근을 자극하는 nNOS 발현 말초 뉴런의 효과를 통해

혈관 긴장도 유지에 중요한 역할을 할 수 있다고 합니다. 8

이와는 대조적으로,

iNOS는 활성화된 대식세포에서 강하게 유도되는데,

이때 급성 염증 캐스케이드에서 감시자 역할을 하는

NO 버스트가 생성됩니다.

이로 인해 하류 반응성 산소 종(ROS)과 반응성 질소 종의 생성을 통해

주변의 건강한 조직에 상당한 부수적 손상이 발생하는 경우가 많습니다.9

여기서는 혈관 내피 세포의 고유 NOS인 eNOS의 조절과 기능에 초점을 맞춥니다.

Endothelial Nitric Oxide Synthase Regulation and Function

eNOS is predominantly expressed in vascular endothelial cells, although its expression‎ has been detected in other specialized groups of cells with important circulatory roles. These include cardiac myocytes, platelets, certain neurons in the brain, placental cells, and kidney tubular epithelium.10 Nominally, NO production by eNOS increases substantially with increasing calcium concentrations secondary to its dependence on CaM. However, there are several alternative mechanisms for eNOS activation that lessen the importance of sustained increases in intracellular calcium for enzyme activity. The most well-studied of these is phosphorylation of the Ser1177 residue in the eNOS reductase domain, which leads to a higher flux of electrons and increased calcium sensitivity.6,11,12 This modification is the end result of multiple signaling cascades associated with a variety of different protein kinase activation pathways. Signals include estrogen and vascular endothelial growth factor, which activate protein kinase B (Akt); insulin, which is thought to function through both Akt and AMP-activated protein kinase; bradykinin, which signals via Ca2+/CaM-dependent protein kinase II; and mechanical shear stress, which activates protein kinase A as well as Akt.6,11,13 Another important residue is Thr495, which is phosphorylated by protein kinase C under resting conditions in endothelial cells. Phosphorylation of Thr495 limits CaM binding to eNOS, slowing electron transfer, and studies have demonstrated that Thr495 becomes dephosphorylated under stimuli that promote increases in intracellular Ca2+.11 However, this may be deleterious under certain circumstances, as dephosphorylated Thr495 also seems to favor uncoupling of eNOS activity.14 Several other sites of post-translational modification have been identified, and are reviewed elsewhere.15

eNOS function is critical for maintenance of appropriate vascular homeostasis. NO signaling directly leads to blood vessel dilation by stimulating soluble guanylyl cyclase, leading to an increase in cyclic guanosine monophosphate and subsequent relaxation of vascular smooth muscle.16–18 However, NO has multiple other distinct roles in vascular physiology. NO exerts antiplatelet effects through inhibition of platelet aggregation and adhesion; furthermore, eNOS-derived NO from platelets likely has both autocrine and paracrine inhibitory effects in a developing thrombus to limit pathologic clot formation.5 NO decreases the expression‎ of macrophage chemoattractant protein-1, which limits leukocyte trafficking to endothelium.19 NO additionally alters the functionality of CD11/CD18 proteins on leukocytes, further altering their ability to adhere to the endothelial wall.20 With regard to vascular remodeling, eNOS-derived NO plays a critical role in angiogenesis, and NO production is one of the final products of angiogenic signaling cascades. Studies have shown roles for eNOS in early neonatal lung capillary development, as well as for the appropriate development of collateral circulation and neovascularization following ischemic insult.21 Broadly, this may be associated with the mobilization of appropriate progenitor cells from the bone marrow, as demonstrated by murine studies in eNOS-deficient mice.22

내피 산화질소 합성효소 조절과 기능

eNOS는

주로 혈관 내피 세포에서 발현되지만,

중요한 순환 기능을 담당하는 다른 특수 세포 그룹에서도 발현이 확인되었습니다.

여기에는

심장 근육 세포, 혈소판, 뇌의 특정 뉴런, 태반 세포, 신장 세뇨관 상피 등이 포함됩니다.10

명목상,

eNOS에 의한 NO 생산은

CaM에 의존하기 때문에 칼슘 농도가 증가하면 상당히 증가합니다.

그러나

eNOS 활성화를 위한 몇 가지 다른 메커니즘이 있어,

효소 활동을 위한 세포 내 칼슘의 지속적인 증가의 중요성을 줄여줍니다.

이 중 가장 잘 연구된 것은

eNOS 환원효소 도메인의 Ser1177 잔기의 인산화인데,

이로 인해 전자 흐름이 증가하고

칼슘 민감도가 증가합니다.6,11,12

이 변형은

다양한 단백질 키나아제 활성화 경로와 관련된 여러 신호 전달 경로의 최종 결과입니다.

신호에는

단백질 키나아제 B(Akt)를 활성화하는 에스트로겐과 혈관 내피 성장 인자,

Akt와 AMP-활성화 단백질 키나아제를 통해 작용하는 것으로 여겨지는

인슐린, Ca2+/CaM 의존성 단백질 키나아제 II를 통해 신호를 보내는

브라디키닌, 그리고 단백질 키나아제 A와 Akt를 활성화하는 기계적 전단 응력이 포함됩니다. 6,11,13

또 다른 중요한 잔류물은 Thr495인데, 이것은 내피 세포의 휴식 상태에서 단백질 키나아제 C에 의해 인산화됩니다. Thr495의 인산화는 eNOS에 대한 CaM 결합을 제한하여 전자 전달을 늦추고, 연구에 따르면 Thr495는 세포 내 Ca2+의 증가를 촉진하는 자극 하에서 인산화가 제거된다는 사실이 입증되었습니다.11 그러나 인산화가 제거된 Thr495는 eNOS 활동의 결합 해제를 촉진하는 것으로 보이기 때문에 특정 상황에서는 해로울 수 있습니다. 14 번역 후 수정이 이루어지는 다른 사이트가 몇 군데 더 확인되었으며, 이 사이트들은 다른 곳에서 검토되고 있습니다.15

eNOS 기능은

적절한 혈관 항상성 유지에 매우 중요합니다.

NO 신호는

수용성 구아닐릴 사이클라제를 자극하여

혈관 확장을 직접 유도하고,

그 결과 순환 구아노신 모노포스페이트가 증가하여

혈관 평활근이 이완됩니다.16-18

그러나 NO는

혈관 생리학에서 여러 가지 다른 뚜렷한 역할을 수행합니다.

NO는

혈소판 응집과 부착을 억제함으로써

항혈소판 효과를 발휘합니다.

또한,

혈소판에서 유래된 eNOS-NO는

혈전 발생 과정에서 자가분비 및 파라크린 억제 효과를 발휘하여

병리학적 혈전 형성을 제한할 가능성이 있습니다. 5

NO는

대식세포의 화학주성 단백질-1의 발현을 감소시켜,

백혈구의 내피 세포로의 이동을 제한합니다.19

NO는 또한

백혈구에서 CD11/CD18 단백질의 기능을 변화시켜,

내피 세포벽에 부착하는 능력을 더욱 변화시킵니다.20 혈

관 리모델링과 관련하여,

eNOS에서 유래된 NO는 혈관 신생에 중요한 역할을 하며,

NO 생산은 혈관 신생 신호 전달의 최종 산물 중 하나입니다.

연구에 따르면

eNOS는

신생아 초기 폐 모세혈관 발달과 허혈성 손상 후

부수적 순환과 신생 혈관 형성의 적절한 발달에 중요한 역할을 하는 것으로 나타났습니다.21

eNOS가 결핍된 생쥐를 대상으로 한 연구에서 입증된 바와 같이,

이는 골수에서 적절한 전구세포를 동원하는 것과 광범위하게 연관될 수 있습니다.22

Endothelial Nitric Oxide Synthase in Vascular Pathophysiology

NO and its signaling functions, particularly in thrombosis and vascular tone, are central to the maintenance of vascular homeostasis. Highlighting this, ED is defined biochemically as a decreased amount of bioavailable NO in the vasculature. Broadly, the mechanisms underpinning ED can be placed into 2 basic categories: consumptive processes that transform bioavailable NO into other species and deficiencies in production of NO in the endothelium (Fig. 2).

혈관 병태 생리학에서의 내피 산화질소 합성 효소

NO와 그 신호 전달 기능,

특히 혈전증과 혈관 긴장도에서는

혈관 항상성 유지에 핵심적인 역할을 합니다.

이를 강조하면서, ED는 생화학적으로 혈관 내 생체 이용 가능한 NO의 양이 감소하는 것으로 정의됩니다. 대체로, ED의 기저 메커니즘은 두 가지 기본 범주로 나눌 수 있습니다: 생체 이용 가능한 NO를 다른 물질로 변환하는 소모성 과정과 내피에서 NO 생산의 결핍(그림 2).

Fig. 2.

The nitrate–nitrite–NO pathway. Increasingly, research is demonstrating that dietary nitrates and nitrites serve as an endogenous reservoir for noncanonically produced NO, particularly in hypoxic conditions when NOS may be less functional or predisposed to being uncoupled. A detailed explanation of this pathway can be found in the body of the text.

질산염-아질산염-NO 경로.

점점 더 많은 연구에서 식이성 질산염과 아질산염이 비정상적으로 생성된 NO의 내인성 저장소 역할을 한다는 사실이 밝혀지고 있습니다. 특히 NOS가 덜 기능적이거나 결합 해제되기 쉬운 저산소 상태일 때 더욱 그렇습니다. 이 경로의 자세한 설명은 본문의 본문에 나와 있습니다.

Because NO is a highly diffusible and reactive species with an unpaired electron, there are a variety of chemical fates that prevent appropriate signaling. A primary driver of this deficiency is ROS, and O2•− in particular. O2•− reacts readily with NO, forming peroxynitrite (ONOO−), a deleterious reactive nitrogen species that itself reacts readily with biological molecules both as a potent oxidant as well as a nitrating agent. Myriad acute and chronic pathologic states can potentiate the overproduction of ROS and subsequent ONOO− formation; a full discussion of these reactions is beyond the scope of this review.

In addition to the consumption of bioavailable NO as a mechanism to reduce NO signaling in ED, modifications to eNOS itself can also alter the production of NO at the source. As mentioned elsewhere in this article, eNOS requires dimerization in the presence of heme and BH4 for effective electron movement to l-arginine. If this relationship is disrupted, the end result is that eNOS functions as a weak NADPH oxidase, producing O2•− instead of NO—a situation referred to as eNOS uncoupling. Multiple mechanisms contribute to eNOS uncoupling, which enhances local oxidative stress in addition to eliminating the vasoprotective effects of NO signaling. One such pathway involves ONOO− produced by initial oxidative stressors and functional eNOS. ONOO− both disrupts a key zinc-thiolate cluster in eNOS and oxidizes BH4 to BH3•, which is biologically inactive and leads to eNOS uncoupling—creating a vicious cycle in which more ROS are produced instead of NO.23,24

Another potential mechanism for eNOS uncoupling involves the bioavailability of l-arginine or its inhibitor, asymmetric dimethyl-l-arginine (ADMA). l-Arginine supplementation has been shown to partially alleviate ED in various animal and human subject models.25–28 This does not seem to be associated with substantially altered global l-arginine levels, but instead with relative bioavailability at the endothelium. Endothelial cells and acute inflammatory cells such as macrophages can express arginases that locally decrease l-arginine pools and effectively starve eNOS of substrate.29–31 ADMA, in contrast, is an endogenous inhibitor of eNOS and its production is strongly governed by redox status. Both the production of ADMA by protein arginine N-methyltransferase type 1 and its subsequent degradation by dimethylarginine dimethylaminohydrolase are altered by oxidative stress. Protein arginine N-methyltransferase type 1 is more active under oxidative conditions and dimethylarginine dimethylaminohydrolase is less active, leading to increased steady-state concentrations of ADMA and eNOS inhibition and uncoupling.32

Post-translational modifications of eNOS may also contribute to pathologic uncoupling and reduction of NO production. As mentioned elsewhere in this article, dephosphorylation of Thr495 may lead to uncoupling and production of deleterious ROS. Another increasingly well-characterized modification is S-glutathionylation, which is involved in signaling under conditions of oxidative stress and may serve to protect redox-sensitive cysteine residues in the eNOS protein. S-glutathionylation, which is reversible under reducing conditions, is associated with reduced eNOS activity and enhanced production of O2•−.33,34

NO는

전자가 짝을 이루지 않는 확산성이 높고 반응성이 강한 물질이기 때문에

적절한 신호 전달을 방해하는 다양한 화학적 요인이 존재합니다.

이러한 결핍의 주요 원인은

특히 O2-와 같은 ROS입니다.

O2-는 NO와 쉽게 반응하여

생물학적 분자와 쉽게 반응하는

유해한 반응성 질소 종인 퍼옥시니트라이트(ONOO-)를 형성합니다.

수많은 급성 및 만성 병리학적 상태가

ROS의 과잉 생산과

그에 따른 ONOO− 형성을 촉진할 수 있습니다.

이러한 반응에 대한 전체적인 논의는 이 리뷰의 범위를 벗어납니다.

ED에서 NO 신호를 감소시키는 메커니즘으로서 생체 이용 가능한 NO의 소비 외에도,

eNOS 자체의 변형은

원천에서 NO의 생산을 변화시킬 수 있습니다.

이 기사의 다른 부분에서 언급했듯이, eNOS는 l-arginine으로의 효과적인 전자 이동을 위해 heme과 BH4가 존재하는 상태에서 이합체화가 필요합니다. 이 관계가 붕괴되면, 최종 결과는 eNOS가 약한 NADPH 산화효소로서 기능하여 NO 대신 O2-를 생성하는 것입니다. 이를 eNOS 분리라고 합니다. 여러 가지 메커니즘이 eNOS 분리에 기여하는데, 이로 인해 NO 신호 전달의 혈관 보호 효과가 사라지는 것 외에도 국소 산화 스트레스가 증가합니다. 이러한 경로 중 하나는 초기 산화 스트레스 요인과 기능적 eNOS에 의해 생성되는 ONOO-를 포함합니다. ONOO−는 eNOS의 핵심 아연-티올레이트 클러스터를 파괴하고 BH4를 생물학적으로 비활성인 BH3•로 산화시켜 eNOS의 결합 해제를 유발합니다. 이로 인해 NO 대신 더 많은 ROS가 생성되는 악순환이 발생합니다.23,24

eNOS의 결합 해제를 위한 또 다른 잠재적 메커니즘은 l-아르기닌 또는 그 억제제인 비대칭 디메틸-l-아르기닌(ADMA)의 생체 이용률과 관련이 있습니다. l-아르기닌 보충제는 다양한 동물 및 인간 대상 모델에서 발기부전을 부분적으로 완화하는 것으로 나타났습니다.25-28 이것은 실질적으로 변화된 전신 l-아르기닌 수치와 관련이 있는 것이 아니라, 내피에서의 상대적 생체 이용률과 관련이 있는 것으로 보입니다. 내피세포와 대식세포와 같은 급성 염증 세포는 국소적으로 l-아르기닌 풀을 감소시키고 eNOS의 기질을 효과적으로 고갈시키는 아르기나제를 발현할 수 있습니다.29-31 반면에, ADMA는 eNOS의 내인성 억제제이며, 그 생산은 산화 환원 상태에 의해 강력하게 조절됩니다. 단백질 아르기닌 N-메틸트랜스퍼라제 타입 1에 의한 ADMA의 생산과 그 이후의 디메틸아르기닌 디메틸아미노하이드롤라제에 의한 분해는 산화 스트레스에 의해 변화됩니다. 단백질 아르기닌 N-메틸트랜스퍼라제 1형은 산화 조건에서 더 활발하게 작용하고, 디메틸아르기닌 디메틸아미노하이드롤라제는 덜 활발하게 작용하여 ADMA의 정상 상태 농도가 증가하고 eNOS의 억제 및 결합 해제가 발생합니다.32

eNOS의 번역 후 변형도 병리학적 결합 해제와 NO 생산 감소에 기여할 수 있습니다. 이 기사의 다른 부분에서 언급했듯이, Thr495의 탈인산화는 결합 해제와 유해한 ROS의 생산으로 이어질 수 있습니다. 또 다른 특징적인 변형은 S-글루타티온화입니다. 이것은 산화 스트레스 조건에서 신호 전달에 관여하며, eNOS 단백질에서 산화 환원 민감성 시스테인 잔기를 보호하는 역할을 할 수 있습니다. 환원 조건에서 가역적인 S-글루타티온화는 eNOS 활동 감소 및 O2•− 생성 증가와 관련이 있습니다.33,34

ALTERNATIVE NITRIC OXIDE NO PRODUCTION: NITRATE–NITRITE–NITRIC OXIDE PATHWAY

NO produced enzymatically from l-arginine by NOS isoforms may not always be sufficient to maintain appropriate endothelial homeostasis, particularly under hypoxic conditions. Recently, more research has focused on the contributions of alternative routes to NO production—namely, from the reduction of dietary nitrates and nitrites to NO.35–38 Nitrate (NO3−) and nitrite (NO2−) were previously thought to be inert metabolites of NO, and their detection was considered a surrogate for NO production.38 However, more recent studies suggest that NO3−, NO2− and NO all exist in a complex equilibrium mediated by microenvironmental stimuli, the microbiome, and dietary nitrate/nitrite consumption. A full discussion of this pathway is beyond the scope of the present review, but a summary of the relevant material can be seen in Fig. 2.

Briefly, dietary NO3− is reduced by oral nitrate reductase-expressing commensal bacteria to NO2−. NO2− then has multiple fates that result in NO production, both via enzymatic and nonenzymatic means. For example, in the acidic environment of the stomach, NO2− is nonenzymatically converted to NO, which then exerts local effects. NO2− may also enter the bloodstream via gastrointestinal absorption. Here, under relative hypoxia, NO2− can be reduced by deoxyhemoglobin, releasing NO in local microcirculation. Xanthine oxidase and eNOS itself also likely reduce NO2− to NO under acidic or hypoxic conditions, such as tissue ischemia. Critically, circulating NO2− also may be oxidized to NO3−, approximately 25% of which is recirculated into the saliva by the salivary glands to maintain the signaling axis.36 The potential health benefits associated with modulating dietary NO2− and NO3− are currently under active investigation, and recent studies demonstrate that NO3− supplementation is beneficial in ischemia–reperfusion injury,39,40 pulmonary hypertension,41–43 and hypertension,44–46 among other conditions.

CLINICAL IMPLICATIONS OF ENDOTHELIAL NITRIC OXIDE AND NITRIC OXIDE REGULATION

NO signaling pathways, involving the regulation of NO production as well as downstream activation of second messengers, are heavily associated with a variety of pathologies. Here, we discuss broadly how the NO axis plays a role in both the multisystem organ dysfunction of critical illness and sepsis, as well as provide brief summaries of some of the data regarding NO signaling in specific organ pathology. This process is also summarized in Fig. 3.

Fig. 3.

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A brief overview of NO signaling in specific organ system pathophysiology. For more specific details and references, please refer to the body of the text.

Sepsis and Infectious Disease

Sepsis is characterized by a dysregulated immune response to an initial infectious insult, and has gained significant attention owing to its emergence as a leading cause of in-hospital mortality.47 ED is a key feature of sepsis and septic shock, resulting in microcirculatory dysfunction and end-organ hypoperfusion. Subsequently, NO has been studied extensively as a potential therapeutic target in treating sepsis.

NO mediates the inflammatory response to host pathogens through various mechanisms. Endogenous NO from iNOS expressed during the acute inflammatory response has been identified as a regulator of the NLRP3 inflammasome via stabilization of mitochondria.48 Using either genetic knockout (iNOS−/−) or pharmacologic inhibition of iNOS in a murine endotoxemia model, inflammatory cytokine production was significantly enhanced and associated with increased mortality.48 In another study, supplementation with l-arginine decreased neutrophil adhesion and increased rolling velocity, which was reversed with a nxxxxonselective NOS inhibitor but not a selective iNOS inhibitor.49 This result suggests that eNOS mediates these effects, although it is unclear what the relative contributions of endothelium-derived eNOS versus platelet-derived eNOS are in the regulation of the inflammatory response. Nevertheless, selective regulation of eNOS in relation to the deleterious impact of iNOS may attenuate the negative effects on microvascular tone.49

Despite its vasodilatory properties, administration of inhaled NO (iNO) has not been demonstrated to cause systemic hypotension. However, this treatment does increase nitrite levels, which might supply NO and promote beneficial vasodilatation in targeted microcirculatory beds. Because local shunting and imbalance in regional flow may contribute to sepsis-induced organ dysfunction, this paradigm has remained a therapeutic target. A randomized controlled trial of iNO compared with a sham treatment in severe sepsis was designed to address this issue. However, on eval‎uation the authors found no differences in microcirculatory flow, lactate clearance, or organ dysfunction, suggesting little value for NO in the later stages of sepsis.50

Methylene blue has also been studied for its potential therapeutic value in NO-mediated vasodilatation. In addition to being a powerful reducing agent, methylene blue inhibits guanylate cyclase and has been demonstrated to increase systemic vascular resistance in patients with shock. Its widespread use is currently limited owing to associations with renal failure and hyperbilirubinemia, in addition to broader questions about its efficacy.51

Although multisystem organ dysfunction is a deadly consequence of sepsis, isolated organ dysfunction can occur secondary to a wide array of noninfectious critical illnesses. Global tissue hypoperfusion results in hypoxia, accumulation of toxic metabolites, and subsequent ischemia–reperfusion injury, all of which may result in permanent organ damage or compound preexisting injuries to other organ systems. Regulation of microvascular flow mediated by NO in these circumstances may thus determine outcomes in critically ill patients. Several specific studied roles for NO and ED in the in the pathophysiology of various organ systems are discussed in the subsequent sections.

Cardiovascular System

NO has demonstrable direct inotropic effects in addition to its role in peripheral vasodilatation. Cardiomyocytes express both nNOS and eNOS constitutively, whereas iNOS expression‎ can be induced by inflammatory mediators. Abnormally high amounts of NO produced in response to acute inflammation following myocardial infarction may have negative inotropic effects, compounding circulatory dysfunction after myocardial infarction. Moreover, NOS uncoupling leads to additional ROS formation (as detailed elsewhere in this article), which further exacerbates cardiac injury after ischemic insult.52

Owing to these effects, NOS inhibition has been studied as a potential therapeutic option for cardiogenic shock.53 In a randomized trial in patients with refractory cardiogenic shock, N-γ-nitro-l-arginine methyl ester (l-NAME), a nonspecific NOS inhibitor, was associated with significantly improved mortality at 30 days (27% vs 67%).53 Additionally, l-NAME therapy was associated with increased mean arterial blood pressure, increased urine output, and decreased requirements for intra-aortic blood pump support and need for mechanical ventilation. Similar results were seen in a small exploratory trial with the nonspecific NOS inhibitor NG-monomethyl-l-arginine (L-NMMA).54 However, a larger randomized controlled trial eval‎uating L-NMMA in patients with myocardial infarction and refractory cardiogenic shock (TRIUMPH) was terminated early owing to a lack of efficacy after demonstrating no difference in 30-day mortality.55 Despite these conflicting findings, NOS inhibition-associated increases in blood pressure were present in all treated patients suggesting some biological effect. It is also important to note that these drugs carry a relatively safe risk profile.55 One question that remains is whether isoform-specific NOS inhibition may provide a more robust outcome, given the confluence of NO signaling from all 3 isoforms in cardiomyocytes.

Pulmonary Function and Nitric Oxide

iNO has theoretic benefit in the lungs owing to its ability induce selective pulmonary vasodilatation and improve ventilation–perfusion mismatch.56 Studies have demonstrated that iNO exerts a vasodilatory effect only in conditions of elevated pulmonary vascular tone and has little effect in normal pulmonary vasculature.57 The US Food and Drug Administration has approved iNO administration for neonatal hypoxic respiratory failure; at present, there is no US Food and Drug Administration approval for treatment in adults. However, there are several lung pathologies for which iNO is presently being investigated as a potential therapy.

The theoretic benefit of iNO in adult acute respiratory distress syndrome is 2-fold: immune modulation in the setting of exuberant inflammation, as well as alleviation pulmonary vasoconstriction. However, a Cochrane review from 2010 found no benefit to iNO administration in acute respiratory distress syndrome, despite the theoretic benefit of direct pulmonary administration.56 Indeed, iNO administration in acute respiratory distress syndrome was associated with harmful effects, most notably renal impairment.

In pulmonary arterial hypertension (PAH) with associated right ventricular dysfunction, localized ED of the pulmonary vasculature is a known entity. In PAH, pulmonary arterial eNOS is frequently uncoupled, favoring O2•− generation and leading to less production of NO.57 The NO pathway is 1 of 3 well-characterized molecular pathways in PAH, alongside prostacyclin and endothelin-1 signaling.58 Several therapies in current clinical use target downstream elements of the NO signaling pathway, including phosphodiesterase type 5 inhibitors (sildenafil) as well as direct soluble guanylate cyclase stimulators (riociguat).59 Collectively, these agents enhance cyclic guanosine monophosphate-based signaling, which comprises the second message portion of the NO signaling cascade. Other approaches have examined the production of NO itself: the PHACeT trial was designed as a phase I clinical trial to deliver eNOS-overexpressing endothelial progenitor cells to patients with refractory PAH.60 Endothelial cells were delivered via pulmonary artery catheter to 5 patients resulting in a transient improvement in pulmonary resistance with a sustained increase in 6-minute walk distance even at 6 months.60 This promising approach requires further study to examine efficacy.

Given the vasodilatory effect of iNO on the pulmonary vasculature and potential to alter shunt fraction, iNO therapy has theoretic benefit in patients with acute pulmonary embolism. In the recently published iNOPE trial, patients with submassive pulmonary embolism and right ventricular dysfunction were randomized to receive either iNO or O2. The authors demonstrated that iNO failed to normalize troponin or echocardiogram endpoints, but patients receiving iNO were more likely to display improvements in right ventricular hypokinesis and dilation on echocardiography.61 This area is another that demands additional study for the delineation of exact benefits of iNO on pulmonary physiology.

Hepatic Function and Nitric Oxide

NO has been implicated in the hyperdynamic circulation seen in acute liver failure, subsequent to presumed overproduction of NO in splanchnic circulation.62 In a devas cularized porcine model of acute liver failure, Sharma and colleagues62 measured plasma levels of l-arginine, citrulline, ornithine, NO, and ADMA, as well as plasma arginase activities in both sham and injured animals. Plasma arginine was significantly depleted 6 hours after injury, concomitant with an increase in plasma ADMA as well as enhanced plasma arginase activity. Despite this, systemic levels of NO were not significantly decreased, suggesting that in this model of acute liver failure NO production was not limited by plasma substrate or inhibitor bioavailability. However, other data suggest that hepatic clearance of ADMA may play a central role in the propagation of multiorgan failure in shock states. Hepatocytes are the primary expressors of dimethylarginine dimethylaminohydrolase, which metabolize ADMA, and liver dysfunction through a variety of means leads to increasing plasma ADMA levels. Increasing ADMA concentration, in turn, is an independent risk factor for multiorgan failure in critically ill patients, highlighting the importance of this NOS regulatory capacity.63

Hepatic ischemia–reperfusion injury studies have similarly provided a wealth of data that could be extrapolated to whole organism shock states and global hypoperfusion. These studies have been recently reviewed elsewhere by Zhang and colleagues.64 In summary, NO has multiple complex functions in hepatic ischemia–reperfusion injury, depending on the source of production and depending on the downstream pathways favored. For example, eNOS expression‎ in hepatic vasculature is protective against hepatic ischemia–reperfusion injury, but overexpression‎ can be detrimental. In contrast, inhibition of iNOS induced during the inflammatory cascade after reperfusion is also protective, suggesting that overproduction of NO leads to detrimental downstream effects. Clearly, NOS regulation involves a delicate homeostasis, and extreme overproduction or underproduction of NO after liver injury can lead to harmful downstream effects.

Renal Function and Nitric Oxide

NO broadly regulates renal hemodynamics and function, which has been extensively reviewed elsewhere.65 In shock states, as modeled by ischemic models of acute renal failure, targeting the NO pathway seems to be beneficial.66 In addition to its direct vasodilatory effects, NO also suppresses production of endothelin-1, a potent vasoconstrictor. In a rat model of renal ischemia-reperfusion injury, a key pathologic driver of acute kidney injury, pretreatment with the NO donor FK409 attenuated renal dysfunction, histologic damage, and endothelin-1 overproduction. Conversely, pretreatment with the NOS inhibitor l-NAME resulted in increased endothelin-1 production, demonstrating that the inhibition of NO production led to deleterious effects.66 The effects of NOS inhibition seems to be isoform specific, similar to what is seen in the liver. In a similar model system, selective iNOS inhibition attenuated the decrease in renal oxygen delivery and microvascular oxygen pressure seen with reperfusion injury; it also improved renal oxygen extraction and consumption.67 Taken together, these findings suggest that iNOS may be contributing to harmful pathways in ischemia–reperfusion events and that inhibition of iNOS in selected scenarios may be of clinical value.

Nitric Oxide and Coagulation

Studies in human patients have demonstrated activation of coagulation factors as well as platelets in severe sepsis and septic shock.68 This may represent a protective mechanism to contain pathogen spread that is exhausted in severe sepsis and septic shock. However, overexuberant coagulation with platelet activation leads to the consumptive pathology of disseminated intravascular coagulation, a devastating condition that is associated with significantly increased mortality.69 NO signaling plays a major role in regulating platelet aggregation, adhesion, and clot formation overall.5 NO produced by healthy endothelium nominally serves a local antiplatelet role by limiting platelet adhesion and activation through activation of the NO-soluble guanylate cyclase axis in platelets themselves. There is additionally a substantial amount of eNOS expressed by platelets, suggesting that both autocrine and paracrine NO signaling occurs within developing clots, thereby limiting progression. Indeed, there is some consideration that NO-enhancing drugs may provide both cardiovascular and antiplatelet benefit, and this area of research is constantly evolving.5

Endocrine Signaling and Nitric Oxide

Sepsis is marked by low cortisol levels and blunted response to adrenocorticotrophic hormone stimulation, which together lead to adrenal insufficiency. Because NO has been linked to organ dysfunction in various other tissues, studies have focused on NO-mediated effects in the adrenal glands. In an experimental endotoxemia model, both iNOS expression‎ and NO production were increased in the adrenal glands of mice.70 This was associated with the development of an adrenal insufficiency phenotype in these animals, as well as enhanced mitochondrial superoxide production in adrenal cortical cells. This effect was mitigated by iNOS inhibition, but was exacerbated by treatment with a systemic NO donor. Notably, increased expression‎ of iNOS was localized to the endothelium and resident macrophages rather than adrenocortical cells, suggesting that some degree of ED contributes to adrenal insufficiency in sepsis.70

SUMMARY

NO is a critically important gaseous transmitter with a large number of context-dependent functions, and its production and consumption are tightly regulated to limit potential damage. The endothelium is a major source of NO, as it constitutively expresses eNOS and modifies NO production profiles in response to a variety of endogenous and exogenous stressors. ED occurs when this signaling axis is perturbed, limiting the ability of the vasculature and local structures either to produce NO itself or to respond appropriately to its presence. This is a central pathologic process in a number of different acute and chronic conditions, and NO and its downstream signaling partners are thus a prime therapeutic target. Although enthusiasm regarding iNO as a therapy is partially hindered by its exorbitant cost, numerous pharmacologic approaches for NO delivery have been researched and more are certainly in development.

Underscoring this, there are currently 101 registered clinically trials in the United States involving NO that are actively recruiting subjects. These trials span a wide breadth of disease processes, echoing the diverse effects and widespread therapeutic potential of this gaseous mediator. With the increasing recognition of the roles of specific NOS isoforms in certain tissues and a focus on optimizing NO delivery given its intrinsic properties, future work stands poised to optimize this pharmacologic target and provide a helpful toolbox to mitigate ED associated with critical illness.

KEY POINTS.

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Footnotes

DISCLOSURE

The authors have nothing to disclose.

REFERENCES

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