NF-κB, A Potential Therapeutic Target in Cardiovascular Diseases

Cardiovascular diseases (CVDs) are the leading cause of death globally. Atherosclerosis is the basis of major CVDs – myocardial ischemia, heart failure, and stroke. Among numerous functional molecules, the transcription factor nuclear factor κB (NF-κB) has been linked to downstream target genes involved in atherosclerosis. The activation of the NF-κB family and its downstream target genes in response to environmental and cellular stress, hypoxia, and ischemia initiate different pathological events such as innate and adaptive immunity, and cell survival, differentiation, and proliferation. Thus, NF-κB is a potential therapeutic target in the treatment of atherosclerosis and related CVDs. Several biologics and small molecules as well as peptide/proteins have been shown to regulate NF-κB dependent signaling pathways. In this review, we will focus on the function of NF-κB in CVDs and the role of NF-κB inhibitors in the treatment of CVDs.


Introduction
In the past decades, great progress has been made in understanding the molecular mechanisms underlying the pathogenesis of cardiovascular diseases (CVDs). Some studies indicate that the activation of nuclear factor kappa B (NF-κB) is involved in the genesis and development of CVDs. In this review, we summarize the recent progress in regulatory mechanisms of NF-κB in CVDs and the potential of its inhibitors to treat CVDs.

NF-κB Family
NF-κB was discovered as an inducible transcription factor 30 years ago [1]. NF-κB in mammals is a family of five related proteins: RelAIp65, RelB, c-Rel, NF-κB1 (p50 and its precursor p105), and NF-κB2 (p52 and its precursor p100) ( Fig. 1-part I) [1]. These subunits share the N-terminal Rel homology domain (RHD) with the v-Rel oncogene and form dimers that can positively or negatively regulate gene expression ( Fig. 1-part I). NF-κB family contains two subfamilies: one subfamily includes p65, RelB, and c-Rel containing a C-terminal transcriptional transactivation (TA) domain, which promotes gene expression, and another one includes p50 and p52 without TA domain, which have a dual role. The C-terminal death domain (DD) in the C-terminal half of p100 and p105 is N-terminal to the inhibitory kinase (Ik) B kinase (IKK) phosphorylation site ( Fig. 1-part I). The main function of the DD motif is to promote protein-adapter interactions between receptors and corresponding adapters related to NF-κB and activator protein-1 (AP1) pathways and cell apoptosis.  1 N-terminus of the NF-κB family members contains a conserved 300 amino acid: Rel homology domain (RHD) and nuclear localization signal (NLS). RHD mediates DNA binding, dimerisation, IκBα interaction. Panel I: The members in the family are divided into two groups: C-terminal transactivation domain (TAD) contained Rel A (p65), c-Rel, and RelB; p50 and p52 lacking a TAD (TA). TAD is responsible for the transcriptional activity of dimers. p50 and p52 act as transcriptional repressors in their homodimeric form because they lack a TAD. p50 and p52 are derived from the limited proteasomal processing of precursors p105 and p100, respectively. p100 and p105 also share C-terminal ankyrin repeats

IκB Family
In unstimulated cells, inactive NF-κB dimers are sequestered in the cytoplasm due to the binding of "inhibitor of kB" (IkB) family [2]. IκB family members contain five to seven tandem ankyrin repeats (AnkRs), a set of 33 amino acid ankyrin-like protein-protein association domains. Classic IκB proteins include IκBα, IκBβ, and IκBε ( Fig. 1-part II). In resting cells, they sequester the NF-κB dimer to retain in the cytoplasm and then degrade it in an IKK-dependent manner after activation. Precursor IκB proteins include p105 and p100. The "precursor" IκB proteins p105 and p100 isolate the NF-κB subunit in the cytoplasm in an unstimulated state similar to classical IκB proteins. Proteasome-dependent limited proteolysis of p105 and p100 results in the release of NF-κB subunit p50 and p52, respectively ( Fig. 1-part II). The coordinative and post-transcriptional processing of p105 is thought to be primarily compositional and occurs in an ubiquitin-independent fashion. Additionally, there are four atypical IκB proteins: B-cell lymphoma 3 (BCL-3) and IκBζ, IκBNS, and IκBη ( Fig. 1-part II).

IKK Complex
For the canonical pathway, degradation of IκB proteins is led by phosphorylation induced by a kinase complex called IκB kinase (IKK) [3]. IKK consists of IKKα, IKKβ, and a regulatory scaffold subunit called NF-κB essential modulator (NEMO, also called IKKγ) ( Fig. 1-part III). IKKα and IKKβ both include a helix-loop-helix (HLH) that functions in modulating IKK kinase activity and a leucine zipper (LZ) that allows the kinase homo-or heterodimerization. IKKα contains a putative NLS, which is possibly linked to its nuclear activity. NEMO tethers IKKα and IKKβ into a regulatory complex, thereby causing ubiquitination of NEMO and phosphorylation of IKK that induce its kinase activity ( Fig. 1-part III). NEMO-containing canonical IKK complexes may consist of homodimers of either IKKα or IKKβ, or a heterodimer of the two. In non-stimulated cells, the HLH motif contacts a kinase domain (KD) of IKKs forming an activation loop. Once cells are stimulated, the KD domain of IKKβ is phosphorylated, IKKs are recruited to the IKK complex through NEMO, then the IKK complex is activated.

NF-κB Dependent Signaling Pathway
So far, there are two main NF-κB activating pathways involved in multiple physiological and pathological processes in inflammation and immunity-the canonical and the non-canonical signaling pathway (Fig. 2). The canonical signaling pathway is induced by a diversity of stimuli, especially pro-inflammatory factors, including tumor necrosis factor α (TNF-α), interleukin-1 (IL-1), and lipopolysaccharide (LPS) [4][5][6]. These ligands usually bind to their own receptors, leading to the conformational change or oligomerization of themselves, then initiating a series of signaling cascades and finally releasing p65/p50 dimer into nucleus for target gene regulation [6]. Otherwise, the noncanonical pathway is primarily triggered by CD40 ligand (CD40L) (Fig. 2, right panel) or lymphotoxin β (LTbeta) that are specific members of the TNF family, resulting in activation and nuclear translocation of the RelB/p52 dimer [7,8]. Importantly, as a crucial regulatory factor, the IKK complex exists in both two activating pathways, which predominantly affect NF-κB activity in an inducible manner. The IKK complex consists of two catalytical subunits IKKα and IKKβ, as well as one regulatory subunit NEMO [3]. In the canonical activating pathway, IKKβ and NEMO are essential for the IKK complex to phosphorylate IκBα, which is independent of IKKα's activity [9]. However, in the non-canonical activating pathway, IKKα is the only effector involved in phosphorylation-dependent activation of p100, which is further processed to p52, and this entire process is independent of both IKKβ and NEMO [10].
The canonical NF-κB pathway has been typically defined in signal transduction emitting from tumor necrosis factor receptor (TNFR) or patterns recognition receptors such as Toll-like receptor 4 (TLR4) [8,11]. TNF signals generally derive from two kinds of TNFR, TNFR1, and TNFR2 [12]. TNFR1 is traceable in almost all tissues and can be continuously expressed in physical and pathological situations; therefore, it is thought to be the dominant type of TNFR mediated in the TNF-associated signaling cascade [6]. Upon binding of TNF-α to TNFR1 (Fig. 2), a tail in the cytoplasmic side of TNFR1 appears, which contains a death domain that provides a binding site to recruit TNFRSF1A associated via death domain (TRADD) and receptor-interacting serine/ threonine kinase (RIP)1 [13]. Next, TRADD is essential for the recruitment of TNF receptor associated factor (TRAF) 2/5 that recruits E3-type ubiquitin ligases inhibitor of apoptotic proteins (cIAPs), leading to RIP1 conjugated with two linear lysine 63 (K63)-linked polyubiquitin chains [14]. Adapter protein TAB2/3 and its associated TGF-β-activated kinase-1 (TAK1) are thought to anchor together with one K63-linked polyubiquitin chain, which can phosphorylate and activate the IKK complex [15]. The linear ubiquitin chain assembly complex (LUBAC), another E3 ligase, is required for the formation of methionine 1 (M1)-linked polyubiquitin chain connected with RIP1, which mediates the interaction with downstream NEMO of the IKK complex and activates the IKK complex in another way [16]. The other canonical activating pathway is triggered by LPS as recognized by TLR4. Signals from TLR4 induce the recruitment of adaptor myeloid differentiation gene factor 88 (MyD88), followed by interleukin 1 receptor associated kinase (IRAK) 4 and TRAF6 [17].
The non-canonical NF-κB pathway is activated by several members of the TNFR family, including CD40, lymphotoxin β receptor (LTβ-R), B-cell activating factor receptor (BAFF-R), and receptor activation of NF-κB (RANK) [7]. Among those, however, CD40 is the most studied and the best-characterized receptor in non-canonical signaling (Fig. 2). Similar to the function of IKKβ in the canonical signaling pathway, IKKα servers as the major mechanism for activating NF-κB, which is independent of NEMO. Another important difference is that the activation of IKKα relies on the accumulation of NF-κB-inducing kinase (NIK) [18].

The Role of NF-κB in Various Cardiovascular Diseases
The activation of NF-κB is critical in the pathological processes of most CVDs. NF-κB mediates inflammation, cell survival, cell differentiation, and cell proliferation that contribute to the pathogenesis of CVDs such as hypertension, atherosclerosis, and related manifestations such as myocardial ischemia and infarction, cerebrovascular ischemia and strokes, heart failure and cardiac hypertrophy. NF-κB activation also regulates extracellular matrix (ECM) formation by affecting the production of matrix metalloproteinases (MMPs) and collagens in a variety of cells in the cardiovascular system (Fig. 3). Upon induction of NF-κB, inflammatory cytokines, chemokines as well as adhesion molecules are generated from innate and adaptive immune cells, such as macrophages, neutrophils, dendritic cells, T cell, and B cells. The subsequent inflammatory process leads to injury in tissues and organs of the cardiovascular system (Fig. 3).

Atherosclerosis
Atherosclerosis is a chronic inflammatory disorder characterized by the accumulation of lipid particles in arterial walls with pathological risk to heart attack or stroke [19,20]. The activated NF-κB signaling is involved in all stages of atherosclerosis development [19]. the degradation of IκBα are mediated by IKKβ activation, leading to the nuclear translocation of unbound p65/P50 heterodimer. On the other hand, the non-canonical NF-κB pathway is triggered by clustered differentiation 40 (CD40) or lymphotoxin beta receptor (LTβR) upon binding with their ligand, which leads to the upregulation and the aggregation of NF-κB-inducing kinase (NIK) via TRAF2/3-cIAPs complex-dependent way. NIK-mediated IKKα activation promotes the processing of p100 and liberates RELB/p52 heterodimer to the nucleus The initiation of atherosclerosis is mediated by endothelial cell (EC) activation and engulfment of oxidized lowdensity lipoproteins (ox-LDLs). Upon stimulation by various stimuli, vascular ECs express cytokines, chemokines, and cell adhesion molecules that facilitate the recruitment of circulating leukocytes and their migration to the subendothelial layer of arterial intima. The essential role of the NF-κB-mediated signaling cascade has been shown in this process [21]. Various pro-atherogenic molecules, including cytokines such as TNF-α, IL-1, bacterial and viral infections, ox-LDL, reactive oxygen species (ROS), and advanced glycation endproducts (AGEs), activate NF-kB [22]. NF-κB mediated downstream canonical and non-canonical signaling pathways are involved in different aspects of the atherosclerotic process. Subsequent induction of a large array of molecules in the ECs, such as intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), P-and E-selectins, TNF-α, IL-1, IL-6, IL-8, monocyte chemoattractant protein-1 (MCP-1), and MMPs, promote the recruitment of innate and adaptive immune cells to the vessel wall [23,24]. It is noteworthy that conditional deletion of NEMO or transgenic expression of dominant-negative IκBα leads to inhibition of expression of adhesion molecules in vascular ECs and impaired monocyte/macrophage recruitment to atherosclerotic plaques in ApoE KO mice fed a high-fat diet. Subsequently, there is a reduction in the severity of atherosclerosis [25].
Furthermore, ox-LDL-mediated TLR activation in vascular ECs results in the upregulation of NF-κB induced expression of proinflammatory cytokines and adhesion molecules, triggering atherosclerotic progress of the inner coating of the blood vessels. Importantly, the degree of atherosclerosis was shown to be inhibited by suppressing TLR2/4-MyD88 signaling with decreased expression of chemokine and macrophage recruitment [25].
Fluid mechanical force is another factor that regulates NF-κB expression and activity in ECs. It is shown that NF-κB activation was markedly elevated in human aortic ECs in response to the prolonged steady low shear stress as compared with high shear stress [26] (Fig. 4). The increased NF-κB activity in blood flow stressed ECs and susceptibility to atherosclerosis suggest that disturbed blood flow-induced NF-κB activation in vascular ECs might be involved in the early stages of atherogenesis by facilitating monocyte/ NF-κB target genes are involved in inflammation and development and progression of CVDs. After its activation, the transcription of inflammatory cytokines, chemokines, and adhesion molecules as well as growth, anti-apoptotic, and other stress factors increase and cell maturation, proliferation, apoptosis, morphogenesis, and differentiation occur macrophage recruitment and plaque formation [25,27]. In the early stage of atherogenesis when NF-κB is activated, ROS acts as a second messenger in response to extracellular stimuli such as ox-LDL and angiotensin II (Ang II). Ox-LDL mediated ROS generation results in reduced NO production with subsequent further increase in NF-κB activation [28]. Ang II is the most important mediator of the renin-angiotensin-aldosterone system (RAAS) under high blood pressure conditions. Ang II-ROS-NF-κB activation links hypertension to increased risk factor of atherosclerosis [29].
NF-κB is also involved in differentiation of monocytes into macrophages after their recruitment to the intima. The macrophages express surface scavenger receptors, such as lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), SR-A, and CD36, which facilitate binding and uptake the native and oxidized form of LDL as a protective reaction to eliminate the accumulation of these inflammatory components [31]. As a consequence, the damage caused by modified LDLs (mLDL) to vascular ECs and smooth muscle cells is reduced. CD36-mLDL interaction induces LDL absorption, oxidative stress, and the production of proinflammatory cytokines through NF-kB activation [32,33].
The various forms of LDL phagocytosis results in overconsumption of cholesterol in the cytoplasm and this cellular event converts the macrophages into foam cells. Foam cell formation and aggregation on the arterial vessels promotes the development of fatty streaks [34]. Enrichment of lipids, foam cells, and T-lymphocytes has been identified in early lesions. The NF-κB activation has also been characterized in smooth muscle cells, macrophages, and T-cells in progressing atherosclerotic lesions [35]. NF-κB mediated macrophage colony-stimulating factor (M-CSF) generation is involved in monocyte-to-macrophage differentiation [36]. NF-κB mediated MMP-9 expression contributes to the degradation of extracellular matrix, facilitating the migration of monocyte/macrophages into the target tissues [37]. Reduced macrophage infiltration and reduced vessel media damage and limited progression of atherosclerosis have been shown in MMP-9 and apoE dual deficient mice [38]. Short periods of stimulation of monocytes with ox-LDL activate NF-κB and its target genes, while longer-time stimulation reverse these responses. Ox-LDL-NF-κB mediated chemokines production strengthen inflammatory effect by promoting the resident macrophages proliferation and migration of Fig. 4 NF-κB signaling plays a pathogenic role in various CVDs, including atherosclerosis, myocardial infarction, reperfusion injury, heart failure, hypertrophy, and ischemic stroke; NF-κB inhibitors affect different therapeutic target in NF-κB-dependent signaling. For example, Vinpocetine suppresses atherosclerotic development in apoE -/mice through blocking IKKα/β, IκBα phosphorylation and subsequently NF-κB activity [30] new monocytes into lesion sites [39]. In addition, ox-LDL-NF-κB mediated pro-inflammatory cytokines expression promotes the binding of LDL to ECs and smooth muscle cells (SMCs) and upregulates the expression of CD36, leading to further inflammation [39]. Recently, proprotein convertase subtilisin/kexin type 9 (PCSK9) was identified as a contributor to atherogenesis and inflammation [40]. It was also shown that PCSK9 overexpression could mediate inflammation through promoting NF-κB translocation in ox-LDL-stimulated macrophages [41,42].
Atherosclerotic lesion development involves migration of SMCs from the vessel wall media into the intima from where proliferation of the SMCs and ECM formation occur. This event drives the early atherosclerotic lesion and fibrotic cap formation. NF-κB activation plays a critical role in this pathological process. Activated NF-kB p65 and p50 components have been identified in SMCs derived from human atherosclerotic lesions compared to SMCs from healthy tissues [43]. Also, several activated NF-κB components, including p65, p50, and c-Rel have been found in cells isolated from human atherosclerotic tissue [43]. Vascular SMCs convert from health contractile phenotype to pathological fibroblastlike synthetic phenotype, which is the major source of connective tissue in the lesion site. This switching progress is an important step in atherosclerotic pathology [44]. In vitro studies have shown that the production of TNF-α, IL-1, M-CSF, granulocyte-macrophage colony-stimulating factor (GM-CSF), or MCP-1 is regulated by NF-κB in syntheticstate SMCs [45][46][47]. These inflammatory molecules produced by SMCs, monocyte/macrophages, ECs, as well as lymphocytes induce the activation of NF-κB in an autocrine manner [46]. NF-κB signaling in ECs may promote recruitment and activation of inflammatory cells, whereas NF-κB activity in SMCs leads to their proliferation [48].
In later stages of atherosclerosis, programmed lipid-laden cells death (apoptosis) is an important feature; it controls the stability of the lesion and the generation of the necrotic core. Apoptosis plays an essential role in atherogenesis and its progression in all stages of atherosclerosis [49]. Clearance of dead cells derived from foam cells apoptosis by the innate and adaptive immune system in early lesions suppresses macrophage burden and inhibits atherosclerosis progression. However, in the late stages of atherosclerosis, impaired ability to clear dead foam cells facilitates the growth of lipid core, leading to inflammation, necrosis, and reduced stability of plaque. These pathological events increase the risk of rupture of an unstable atherosclerotic lesion, which causes acute vascular diseases such as stroke and acute myocardial infarction/ischemia [50].
In atherosclerosis, NF-κB pathway exhibits its dual effect in cell survival/apoptosis in the context of various activating stimuli [51,52]. NF-κB promotes the survival signal by inhibiting TNFRs mediated apoptosis signal, while it contributes to apoptosis through regulation of ROS generation and activation of the JNK-MAPK signal transduction [51,53]. Activation of NF-κB increases the expression of Fas ligand, which induces cell apoptosis via its receptor CD95. IκB kinase (IKK) promotes the NF-κB-induced antiapoptotic signal by mediating phosphorylation and degradation of the NF-κB inhibitory IκBα proteins [51], and inhibition of IKK-mediated NF-κB activation facilitates apoptosis in monocytes [51,54]. Deviant NF-κB-mediated inhibition of apoptosis might be involved in the initiation of atherosclerosis [51].
In atherosclerotic plaques, the secretion of MMP-1, -3 and -9, induces the destruction of the ECM and the loss of fibrous cap integrity, by reduction of collagen protein and might be important in plaque rupture; this release process is regulated by NF-κB [55]. However, the types of MMPs might be different in various cell type and stimulus [37,56]. Following the inhibition of NF-κB, decreased MMP-1 production in response to CD40L stimulation has been identified in healthy human macrophages. In addition, inhibition of NF-κB reduces MMP-1 and MMP-3 expression in cholesterol-feeding rabbits [57]. Ox-LDL stimulation in macrophages promote the expression MMP-9 with upregulated activation of NF-κB [37,56].

Myocardial Infarction
Rupture of the coronary atherosclerotic plaque results in localized clot formation and subsequently myocardial infarction. The clot may be spontaneously resolved or may be therapeutically dissolved. Unfortunately, reperfusion also results in the phenomenon of reperfusion injury. NF-κB plays a critical role in tissue responses to ischemia and reperfusion, including inflammation, regional cardiomyocyte death and myocardial infarction. TNFR and TLR-mediated NF-κB activation are induced following ischemia/reperfusion. Moreover, hypoxia downregulates the activity of prolyl hydroxylase 1 (PHD1) and enhances IKKβ expression as well as phosphorylation of IκBα for NF-κB activation following ischemia [58] (Fig. 4). Hypoxia also induces the expression of hypoxia-inducible factor 1α (HIF-1α) and a subsequent activation of NF-κB, which causes apoptosis of cardiomyocytes [59]. Inhibition of NF-κB activation could reduce cardiomyocyte apoptosis and deteriorate cardiac injury in rats with ischemia-reperfusion, meanwhile activating NF-κB using LPS could blunt the protective effects of p300/CBP-associated factor (PCAF) on myocyte apoptosis and cardiac injury [60,61]. NF-κB activation also induces the expression of hypertrophic genes atrial natriuretic factor (ANF) and c-myc in cardiomyocytes and myocyte hypertrophy [62]. Inhibition of NF-κB activaty using various drugs or substances such as arbutin, harmine, FGF13 and so on was also reported to alleviate cardiac hypertrophy in a series of animal studies [63][64][65]. However, NF-κB activation is required for heart regeneration in a regeneration animal model with zebrafish and inhibition of NF-κB blocks heart regeneration through decreasing cardiomyocyte proliferation and epicardial responses [66].
It is known that myocardial infarction induces ROS production, which in turn enhances cardiac injury [67]. The negative regulators of the NF-κB, including redox-sensitive enzymes, would be inhibited and NF-κB would be induced in response to the increase of ROS generation [68]. Myocardial ischemia/reperfusion induces NF-κB activation in several cell types, including ECs and resident immune cells in the heart. The production of pro-inflammatory molecules, including the adhesion proteins ICAM-1 and P-selectin, in these cells facilitates the migration of leukocyte to the infarct area [69]. NF-κB activation (p50/p65) with elevated expression of ICAM-1, TNF-α, and IL-1β has been characterized in human atrial tissue derived from patients following myocardial ischemia/reperfusion. The in vivo studies have shown that inhibiting NF-κB using pharmacological inhibitors or decoy oligonucleotides suppresses myocardial injury following ischemia [70][71][72]. Additionally, loss of NF-κB1 also leads to improved cardiac function and lower mortality after myocardial infarction [73].
Recently studies on the activation of NOD-like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome has been identified in CVDs, including myocardial injury [74,75]. It is known that TLR and NF-κB are the upstream signals NLRP3 inflammasome, which is the requisite for NLRP3 inflammasome activation [76]. The pyroptosis of myocytes mediated by NLRP3/caspase-1 activation has been proved as a key contributor to myocardial injury following ischemia [77,78]. Lei et al. reported that the ROS/NF-κB/NLRP3 and Gasdermin D (GSDMD) axis is a determinant for cardiomyocyte pyroptosis after myocardial infarction [79]. Recently, our study indicated that proprotein convertase subtilisin/Kexin type 9 (PCSK9) could also activate NLRP3 inflammasome, subsequently induce pyroptosis, and enhance myocardial injury in mice with myocardial ischemia [80]. Therefore, the inhibitors of NF-κB, NLRP3 inflammasome, and inflammation theoretically have the potential to intervene myocardial injury.
However, similar to its effect on cell survival/apoptosis, NF-κB also has dual effects on myocardial injury following ischemia. Plenty of studies have indicated that NF-κB also exerts protective function. It has been reported that a modest activation of NF-κB protects against myocardial injury following myocardial infarction and limits myocyte apoptosis [81,82]. A defect of NF-κB activation in the transgenic mice increases susceptibility to tissue injury after acute left anterior descending occlusion [83]. Here, the protective effects of NF-κB on cardiomyocytes are mediate by promoting expression of cytoprotective genes such as c-IAP1 and Bcl-2 . Diwan et al. reported that the remote renal preconditioning and erythropoietin preconditioning could attenuate myocardial injury caused by ischemia-reperfusion and present an obvious cardioprotective effect [84]. The activation of NF-kB plays an important role in this process, and inhibition of NF-kB using its selective inhibitor diethyldithiocarbamic acid attenuates cardioprotection of remote preconditioning and erythropoietin preconditioning [85]. Qiao et al. also found that delayed anesthetic preconditioning also protected the hearts from ischemia-reperfusion injury in rats, which is mediated by NF-κB activation and NF-κB DNA-binding activity [85].
The controvertible effects of NF-κB activation may result from the difference of intervention times and its varied effects on inflammatory response. Recently, our study indicated that the intensity of inflammation was increased and then decreased (after 1 week) following myocardial infarction and inhibition of inflammation (administration of TNF-α inhibitor) could worsen cardiomyocyte apoptosis instead of ameliorating myocardial injury [86]. Although many studies indicated that the activation of NF-κB-induced myocardial injury through stimulating inflammation, a few studies reported that appropriate activation of NF-κB activation could also reduce inflammation and protects against myocardial infarction. For instance, Qiao et al. reported that in the inflammatory factors TNF-α and IL-1β were markedly reduced with NF-κB activation in the hearts with ischemia/ reperfusion [85]. We think that the effect of NF-κB activation and inflammation on myocardial injury varies depending on the different periods of progress and the degree of inflammatory response or NF-κB activation.

Heart Failure
Long-term effects of ischemia cause cardiac damage and cardiomyocyte apoptosis, finally resulting in cardiac failure [87]. Several clinical studies have found a strong link between NF-κB activation and heart failure. Failed human hearts contain activated forms of NF-κB [75]. Improved cardiac function with decreased cardiac NF-κB activity was characterized in patients that received left ventricular assist devices [88]. Conditions such as hypertension result in cardiac hypertrophy, which is associated with NF-κB activation; however, the role of the NF-κB component p50 in cardiac hypertrophy is controversial. One study showed that loss of NF-κB led to a reduced cardiac hypertrophy (Fig. 4). However, another study showed that p50 deficiency enhanced cardiac dysfunction following myocardial infarction. The mechanism of discrepancy between the results of these two studies is unclear. However, p50 deficient mice display reduced cardiac hypertrophy in response to TNF-α and Ang II, suggesting that NF-κB might be a positive regulator of hypertrophy [73,89].
The in vitro investigations also showed that Ang II facilitated IκB degradation, as well as p65 nuclear translocation and transcriptional activity in cardiomyocytes [90,91]. Cardiac-specific overexpression of a non-degradable form of IκBα in mice attenuates cardiac hypertrophy phenotype and facilitates heart failure in response to Ang II and isoproterenol infusion [92]. Several other studies have shown the protective effect of NF-κB in mice. Conditional deletion of NEMO in heart blocks NF-κB activation and the mice showed enlargement of left atrium and eccentric hypertrophy. Moreover, the heart function was also significantly impaired. In another investigation, cardiac-specific IKKβdeficient mice exhibited normal heart morphology and function. However, heart failure, including cardiac dilation and dysfunction, was observed in these mice under acute pressure overload [93,94]. Channeling the mice with antioxidants reversed these phenotypes, indicating that NF-κB may perform its protective effect against cardiomyopathy by the induction of its downstream antioxidant genes.

Ischemic Strokes
Atherosclerotic plaque rupture in the carotid artery increases the risk of ischemic strokes with subsequent tissue injury, breakdown of the blood-brain barrier, and hemorrhage. In this process, NF-κB activation has been demonstrated in neurons, ECs, microglia, and astrocytes [95][96][97]. NF-κB is upregulated in the penumbra of human stroke patients and nuclear translocation of RelA has been identified in the brain samples from patients. Nuclear translocation of RelA has been observed in neurons derived from mouse models of both permanent and transient carotid occlusion. In addition, significantly smaller infarct size was found in p50 deficient mice of transient and permanent stroke models and selective deletion of NF-κB in neurons led to dramatically reduced infarct size [98]. Conditional knockout of RelA results in decreased brain infarct as compared to wild-type mice [99]. These data reveal the critical role of NF-κB in cerebral ischemia. The injurious effect of NF-κB in cerebral ischemia is mediated by its downstream target genes, such as TNF, IL-1 and IL-6, inducible nitric oxide synthase, ICAM-1, and MMP-9 [30]. Although the anti-apoptotic effect of NF-κB activation has been described in numerous studies, it seems to mainly contribute to prolonged cerebral ischemia in most investigations.

Inhibitors of NF-κB Dependent Signaling Pathway
The NF-κB pathway seems to serve as a link between inflammatory processes and CVDs, and may be an attractive target for development of drugs in ischemia-reperfusion injury treatment (Fig. 4). Thus, a variety of NF-κB inhibitors have been developed for treatment of CVDs. Table 1 summarizes the function and mechanisms of NF-κB inhibitors in CVDs. For example, Traf-stop inhibitor 6877002, a selective inhibitor of CD40-TRAF6 interaction, was shown to specifically inhibit the NF-κB pathway and maintain CD40-mediated immunity, thereby affecting the progression of atherosclerosis in mice [100].
Different plant extracts exhibit anti-atherosclerosis effects, which may be related to the inhibition of NF-κB activation. For example, the extracts of two herbs, Vinpocetine and Parthenolide, reduce the severity of atherosclerosis in mice through the regulation of NF-kB [27,101] (Fig. 4). Flavonoid is a large class of polyphenolic compounds extracted from a large variety of natural origins. These compounds are effective in the treatment of various CVDs. Epigallokatechingallat (EGCG), a typical flavonoid which is isolated from green tea, exerts its anti-atherosclerosis effect through activation of the Nrf-2/keap1 pathway, which inhibits the function of NF-κB in foam cells [120].
Some available drugs possibly exert their anti-atherosclerosis effect by inhibiting NF-κB. Atorvastatin, a lipidlowering drug, exerts an anti-inflammatory effect by inhibiting NLRP3 inflammasome through suppressing the TLR4/ MyD88/NF-κB pathway [121] and limiting infarct size [122]. Pioglitazone, a hypoglycemic drug, downregulates lipid levels as well as p65 expression and thereby slows the progression of atherosclerosis [123]. Benidipine, a dihydropyridine-Ca 2+ channel blocker, may exert an anti-atherosclerosis effect by its anti-inflammatory and anti-NF-κB effect [102]. Dilazep, an antiplatelet drug with antioxidative activity may potentially prevent atherosclerosis in diabetes mellitus through inhibition of NF-κB mediated MCP-1 expression following the stimulation of glycoxidized-LDL [103].
Several other NF-κB inhibitors have shown a cardio-protective effect in pathologic states. Muscone, the main active component of musk, has been shown to reduce inflammatory response and improve cardiac function in mice after myocardial infarction by inhibiting the activation of NF-κB and NLRP3 inflammasome [104]. Ophiopogonin D is a natural glycoside isolated from the tuber of Ophiopogonin. Studies have shown that it can inhibit NF-κB, reduce cell inflammation, and protect vascular endothelium from injurious stimuli [83]. Several flavonoids also show protection against impaired cardiac function. Curcumin, a natural polyphenol, was found to antagonize cardiomyocyte apoptosis and inflammatory infiltration after myocardial infarction by inhibiting the expression of NF-KB in cardiomyocytes [105]. IL-1β and TNF-α concentration as well as NF-κB activity are inhibited in patients with stable coronary artery disease (CAD) after the treatment of Quercetin [106]. Fisetin, Chrysin and Kaempferol, which are bioactive flavonoids widely found in fruits, vegetables and flowers, also suppresses the myocardial injury through inhibiting the activity of NF-κB in isoproterol-induced myocardial injury model [107][108][109][110]. In a rat myocardial infarction model, chrysin administration results in the decreased expression of MMP-2 and MMP-9 through the downregulated IκKβ phosphorylation and NFκB expression, protecting the heart from cardiac injury [107]. Another flavonoid rutin, inhibits myocardial hypertrophy by increasing the expression of IκB-α and decreasing the expression of NF-κB in carfilzomib-induced cardiotoxicity rat model administration. The NF-κB inhibitor pyrrolidine dithiocarbamate inhibits ox-LDL-induced lipid accumulation and improves myocardial hypertrophy and myocardial remodeling after acute myocardial infarction by inhibiting the activation of NF-κB [111,112].
NF-κB inhibitors also play an important role in blocking the occurrence and development of ischemic stroke. The inhibitors are divided into two subgroups, small-molecules and proteins (Fig. 4). Among the small molecules, after the stimulation by naloxone, a clinical opioid receptor antagonist, nerves are protected from ischemic brain injury via Pitavastatin Transient cerebral ischemia in gerbils ↓NF-кB, neuronal cell death [125] inhibition of the NIK/IKKα/IKBα pathway [124]. Pitavastatin prevents neuronal cell death after cerebral ischemia by inhibiting upregulations of NF-κB [125]. Oxypropoxybenzoic acid, a pyruvate and salicylate ester, has been shown to protect nerves after cerebral ischemia by inhibiting the canonical NF-KB pathway [113]. In another study, 4-methylcyclopentadecanone (4-MCPC) treatment was found to significantly reduce infarct size in a rat model of middle cerebral artery occlusion (MACO). This effect is thought to be achieved by inhibiting the activation of NF-κB [114]. In addition, a naturally occurring protein, ulinastatin, reduces infarct size in the middle cerebral artery occlusion rat model and protects neurological function by inhibiting NF-κB [115]. Neuregulin 1, a member of the epidermal growth factor family, was found to protect neural function and reduce infarct size in a rat model of MACO [116]. This effect may be related to the inhibition of NF-KB activity [117]. s-nitroso glutathione, also a natural product, was found to reduce infarct size in the MACO model and to reduce NF-κB activation [118]. Progranulin is a glycoprotein growth factor that inhibits the activation of inflammatory cells and protects nerves from cellular damage [119].
Although inhibition of NF-κB is advantageous to ameliorate CVDs, it also brings a few unexpected side effects, especially under the inflammation or cancer conditions [126]. For instance, systemic inhibition of NF-κB using etomidate could cause adrenal suppression and more deleterious sepsis, and finally increase the mortality of septic rats [127]. Therefore, the adverse effects of NF-κB inhibitors must be noticed when using them to treat CVDs, especially combined with other serious diseases.

Conclusion
NF-κB is a therapeutic target to treat several CVDs.
Although several studies have shown adverse effect of NF-κB signaling in a number of CVDs, some investigations have shown an opposite effect in the pathogenesis of CVDs. For example, NF-κB activity in ECs and macrophages may have contrasting effects. The effect of NF-κB activation might be cell-autonomous. Thus, the cell-type specific effects of NF-κB in CVDs still need to be determined, and this may facilitate the development of specific inhibitors of NF-κB to treat CVDs.

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Code Availability Not applicable.

Declarations
Ethical Approval and Consent to participate Not applicable.

Conflict of Interest
The authors declare that they have no conflict of interest.